METHOD TO OBTAIN A NEURAL STEM CELL

专利摘要:
methods for cell reprogramming and uses thereof The present invention describes methods for cell dedifferentiation and transformation, as well as eukaryotic cell reprogramming. cells, cell lines and tissues that can be transplanted into a patient after in vivo dedifferentiation and in vitro reprogramming steps are also described. in particular embodiments, the cells are stem-like cells (slcs), including neural stem-like cells (nslcs). methods for generating such cells from human somatic cells and other cell types are also described. the invention further provides compositions and methods of using the cells generated in therapy for humans and in other areas.
公开号:BR112012009921B1
申请号:R112012009921-3
申请日:2010-11-01
公开日:2021-06-29
发明作者:Rouwayda Elayoubi；Jan-Eric Ahlfors
申请人:New World Laboratories Inc；
IPC主号:

专利说明:

Related order
[0001] This application claims priority from provisional US application No. 61/256,967 filed October 31, 2009, which is incorporated herein by reference in its entirety. Field of Invention
[0002] The present invention relates to the field of eukaryotic cell reprogramming, and particularly to cell dedifferentiation. This invention also relates to methods for generating stable Neural Stem Cells (NSLCs) from human somatic cells (and other cells) and the use of the cells thus generated in human therapy. Fundamentals of the Invention Cellular reprogramming
[0003] There is a desire in the medical, scientific and diagnostic fields to reprogram an easily obtainable cell into a cell that is generally more difficult to obtain, or to reprogram a cell so that it has new or different functionalities without fusing or exchanging material with an oocyte or other stem cell.
[0004] According to a first mechanism, a stem cell can naturally divide or differentiate into another stem cell, progenitor, precursor, or somatic cell. According to a second mechanism, the somatic cell can sometimes transiently change its phenotype or express certain markers when placed under certain conditions, and then revert when placed back to the original conditions. According to a third mechanism, the phenotype of many cells can be altered through the forced expression of certain genes (eg, stably transfecting the c-myc gene into fibroblasts transforming them into immortal cells having neuroprogenitor characteristics), however since this forced gene expression is removed, the cells slowly return to their original state. Consequently, none of the three mechanisms above should be considered true reprogramming: the first is considered natural differentiation which is part of a cellular program that is already in place (going from an undifferentiated state to a more differentiated state), the second is a transient phenotypic change, and the third is a constantly forced cell type. A true stem cell (i) self-renews almost "indefinitely" (for significantly longer than a somatic cell), (ii) is not a cancer cell, (iii) is not artificially maintained by forced gene expression or similar means (must also be able to be maintained in standard stem cell medium), (iv) can differentiate from parent, precursor, somatic or other more differentiated cell type (of the same lineage), and (v) has all characteristics of a stem cell and not just certain markers or gene expression or morphological appearance.
[0005] Despite numerous scientific and patent publications claiming successful reprogramming or dedifferentiation, usually in a stem cell, almost all of these publications do not disclose true reprogramming because they fall under one of the mechanisms mentioned above. For example, Bhasin (WO2010/088735), Cifarelli et al. (US2010/0003223), Kremer et al. (US2004/0009595), and Winnier et al. (US2010/0047908) all refer to reprogramming, dedifferentiation, and/or obtained stem cells (or progenitors) as phenotypic cell changes based only on a change in cell surface markers after culture in different media with supplements, with no evidence of true reprogramming or a true stem cell (non-cancerous self-renewal with stem cell markers and no differentiation markers). The same goes for Benneti (WO2009/079007) who used the augmented expression of Oct4 and Sox2. Others, such as Akamatsu et al. (WO2010/052904) and You et al. (WO2007/097494, US2009/0246870), refer to having created stem cells, but these arose through constant retrovirus-transmitted artificial gene induction (similar to cMyc) with no evidence of true stem cells that are not immortal/tumorigenic, and stable rather than transient. Others, such as Chen et al. (US2005/0176707) and You et al. (US2009/0227023), created “multipotent cells” but not stem cells. Furthermore, these so-called multipotent cells were not stable (in the case of You et al. the cells could not even proliferate) and both used constant medium supplements and conditions to force the phenotypic change. Still others, such as Oliveri et al. (WO 2009/018832) and Zahner et al. (US2002/0136709), claimed the creation of pluripotent, totipotent, multipotent, and/or unipotent cells automatically through pangenomic DNA demethylation and histone acetylase, but without evidence of a true stable, non-cancerous cell lineage.
[0006] True reprogramming appears to have been achieved with pluripotent stem cells (iPS cells) created independently by the Yamanaka group (Takahashi et al., 2007) and the Thomson group (Yu et al., 2007), and potentially by others before them, and although it was later discovered that many of these cells were cancerous, some of them were not. These cells can be induced by true reprogramming as long as they subsequently demonstrate that they can also be induced by transient transfection of non-gene integration (Soldner et al., 2009; Woltjen et al., 2009; Yu et al., 2009) as well as by RNA (Warren et al., 2010) or protein (Kim et al., 2009; Zhou et al., 2009) alone or by small molecules (Lyssiotis et al., 2009), and by similar methods. However, these cells are essentially identical to embryonic stem cells and have the same problems of uncontrolled growth, teratoma formation, and potential tumor formation.
[0007] A more desirable option is to have multipotent stem cells or pluripotent-type stem cells whose lineage and differentiation potential is more restricted so that they do not readily form teratomas and uncontrolled growth. There is therefore a need for methods to create multipotent stem cells, multipotent stem-like cells, and stem-like cells and a method for reprogramming or transforming readily obtainable cells into highly desirable multipotent stem cells, multipotent stem-like cells, and stem-like cells . Neural Stem Cells (NSLCs)
[0008] Repairing the central nervous system (CNS) is one of the frontiers that medical science has yet to conquer. Problems such as Alzheimer's disease, Parkinson's disease, and stroke can have devastating consequences for those afflicted. A fundamental hope for these problems is the development of cell populations that can reconstitute the neural network, and reorganize the functions of the central nervous system. For this reason, there is a great interest developed in neural stem and progenitor cells. To date, it has been generally believed that multipotent neural progenitor cells undergo early on the path of differentiation into both neural-restricted and glial-restricted cells.
[0009] Neural stem cells hold promise for tissue regeneration from a disease or injury, however, these therapies require precise control over cell function to create the necessary cell types. There is not yet a complete understanding of the mechanisms that regulate cell proliferation and differentiation, and it is therefore difficult to fully explore the plasticity of the population of neural stem cells derived from any given region of the brain or developing fetus.
[0010] The CNS, traditionally believed to have limited regenerative capacities, maintains a limited number of neural stem cells in adulthood, particularly in the dentate gyrus of the hippocampus and the subventricular zone that replenishes olfactory bulb neurons (Singec I et AL., 2007 ; Zielton R, 2008). The availability of precursor cells is a key prerequisite for transplant-based repair of mature nervous system defects. Thus, donor cells for neural transplants are largely derived from the fetal brain. This creates huge ethical problems, in addition to immunorejection, and it is questionable whether such an approach can be used to treat a large number of patients, since neural stem cells can lose some of their potency with each cell division.
[0011] Neural stem cells offer promising therapeutic potential for cell replacement therapies in neurodegenerative diseases (Mimeault et al., 2007). To date, numerous therapeutic transplants have been performed exploiting various types of human fetal tissue as a source of donor material. However, ethical and practical considerations and its inaccessibility limit its availability as a source of cells for transplant therapies (Ninomiy M et AL., 2006).
[0012] To overcome barriers and limitations for deriving patient-specific cells, one approach has been to use skin cells and induce trans-differentiation to neural stem cells and/or to neurons (Levesque et al., 2000 ). Trans-differentiation has received increasing attention over the past few years, and trans-differentiation of mammalian cells has been achieved in co-culture or by manipulating cell culture conditions. Cell fate alteration can be artificially induced in vitro by treating cell cultures with microfilament inhibitors (Shea et al., 1990.), hormones (Yeomans et al., 1976.) and calcium ionophores (Shea, 1990; Sato et al., 1991). Mammalian epithelial cells can be induced to acquire muscle-like shape and function (Paterson and Rudland, 1985), exocrine pancreatic duct cells can acquire an insulin-secreting endocrine phenotype (Bouwens, 1998a, b), and bone marrow stem cells they can be differentiated into liver cells (Theise et al., 2000) and into neuronal cells (Woodbury et al., 2000). Others such as Page et al. (US 2003/0059939) transdifferentiated somatic cells to neuronal cells by somatic cell culture, in the presence of acetylation and cytoskeletal methylation inhibitors, but after withdrawal of the initiating agent, neuronal morphology and established synapses lasted for not much longer. than a few weeks in vitro, and complete conversion to a fully functional and stable type of neuron has never been demonstrated. These are, thus, transient cell phenotypes. Complete conversion to a fully functional and stable type of neuroprogenitor or neural stem cell has also never been demonstrated. Acquisition of a stable phenotype after transdifferentiation has been one of the main challenges faced in the field.
Thus, there is a need in the biomedical field for potent and stable stable preferably autologous neural stem cells, neural progenitor cells, as well as neurons and glial cells for use in the treatment of various neurological disorders and diseases. The same is true for many other cell types. Recently, evidence has been obtained that basic Helix-Loop-Helix (bHLH) class genes are important regulators of various stages in neural lineage development, and overexpression of various bHLH neurogenic factors results in the conversion of undetermined ectoderm to neuronal tissue. Proneural bHLH proteins control differentiation into progenitor cells and their progression through the neurogenic program throughout the nervous system (Bertrand et al., 2002). MASH1, NeuroD, NeuroD2, MATH1 -3, and Neurogenin 1 - 3 are bHLH transcription factors expressed during mammalian neuronal determination and differentiation (Johnson et al., 1990; Takebyashi et al, 1997; McCormick et al, 1996; Akazawa et al., 1995). Selective disruptions of MASH1, Ngn1, Ngn2 or NeuroD in mice led to the loss of specific subsets of neurons (Guillemot et al, 1993; Fode et al, 1998; Miyata et al, 1999).
[0014] US patent 6,087,168 (Levesque et al.) describes a method for converting or transdifferentiating epidermal basal cells into viable neurons. In one example, the method comprises transfecting epidermal cells with one or more expression vector(s) containing at least one cDNA encoding a neurogenic transcription factor responsible for neuronal differentiation. Suitable cDNAs include basic helix-loop-helix activators such as NeuroDI, NeuroD2, ASH1 and zincfinger-type activators such as Zic3 and MyT1. The transfection step step was followed by the addition of at least one antisense oligonucleotide known to suppress neuronal differentiation in the growth medium, such as the human MSX1 gene and/or the human (or non-human, HES1 gene) counterparties). Finally, transfected cells were cultured in the presence of a retinoid and at least one neurotrophin one or cytokine, such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin 3 (NT-3) , or neurotrophin 4 (NT-4). This technology produces 26% of neuronal cells, however, neither the functionality nor the stability of these cells has been established. Furthermore, neural stem cells or neuroprogenitor cells are not produced according to this method.
[0015] A later process (Levesque et al., 2005; US patent 6,949,380) mentions the conversion of the epidermal basal cell into a neural progenitor cell, neuronal or glial cell, exposing the epidermal basal cell to a morphogenetic protein antagonist bone (BMP) and growing the cell in the presence of at least one antisense oligonucleotide that comprises a segment of an MSX1 gene and/or HES1 gene. However, there is no evidence or examples that any neural progenitor cells or glial cells were produced according to this method, let alone any details or evidence that the morphological, physiological or immunological characteristics of neuronal cells were achieved. Furthermore, since there is also no information about the functionality, stability, expansion, and performance of cells that may or may not have been produced, it is possible that these cells are actually skin-derived precursor cells (Fernandes et al., 2004 ) that have been differentiated into neuronal cells.
[0016] In view of the above, there is thus a need for preferentially autologous, stable and potent neural stem cells, neural stem cells, neural progenitor cells, neurons and glial cells, as well as other cell types, stem cells and progenitor cells . There is also a need for methods that can result in true cell dedifferentiation and cell reprogramming.
[0017] The present invention addresses these needs and provides various types of stem-like cells and progenitor-like cells and cells derived or differentiated from these stem or progenitor cells, as well as methods that can result in true cell dedifferentiation and reprogramming of the cell.
[0018] Additional features of the invention will be apparent from an analysis of the description, figures of the present invention. Invention Summary
[0019] The present invention relates to stem-like cells and progenitor-like cells and cells derived or differentiated from these stem or progenitor cells. The invention further relates to methods for cell dedifferentiation and cell reprogramming. The invention further includes compositions and methods that are useful for reprogramming cells and their therapeutic compositions and methods.
[0020] A particular aspect relates to the development of a technology to reprogram a somatic cell or non-neuronal cell to a cell having one or more physiological, morphological, and/or immunological characteristics of a neural stem cell and having the ability to differentiate along neuronal and glial lineages. According to some embodiments, the invention is more particularly related to methods of generating stable Neural-Type Stem Cells (NSLCs) from human somatic cells, human progenitor cells and/or human stem cells, as well as cells, cell lines. cells and tissues obtained using these methods. The invention further relates to compositions and methods for inducing the dedifferentiation of human somatic cells into neural stem cells expressing specific neural stem cell markers. According to the present invention it is possible to carry out the conversion of cells into various types of differentiated neuronal cells that can be created from a single type of cells taken from an individual donor and then reprogrammed and transplanted into the same individual . After induction the cells according to the invention express specific neural-type stem cell markers and become neural-type stem cells.
[0021] According to a particular aspect, the invention relates to a method of transforming a cell of a first type into a desired cell of a different type. The method comprises i) obtaining a first type cell, ii) transiently increasing in the first type cell the intracellular levels of at least one reprogramming agent, whereby the transient increase directly or indirectly induces endogenous expression of at least one regulatory gene; iii ) placing the cell in conditions to support the growth and/or transformation of the desired cell and maintain intracellular levels of the reprogramming agent for a period of time sufficient to allow stable expression of the regulatory gene in the absence of the reprogramming agent, and iv) maintaining the cell under culture conditions that support the growth and/or transformation of the desired cell. Such conditions are maintained for a period of time sufficient to allow stable expression of a plurality of secondary genes. According to the invention, the expression of one or more of the secondary genes is characteristic of the phenotypic and functional properties of the desired cell while not being characteristic of the phenotypic and functional properties of an embryonic stem cell. In this way, at the end of the time period, the desired cell of a different type is obtained.
[0022] According to another particular aspect, the invention relates to a method of transforming a cell of a first type into a cell of a second different type. The method comprises contacting the cell of a first type with one or more agents capable of increasing within said cell the levels of at least one reprogramming agent and directly or indirectly remodeling the cell's chromatin and/or DNA. The reprogramming agent is selected to directly or indirectly induce the expression of morphological and functional characteristics of a desired cell of a different type or different cell lineage.
[0023] According to another particular aspect, the invention relates to a method of transforming a cell of a first type to a cell of a second different type. The method comprises contacting the chromatin and/or DNA of a cell of a first type with an agent capable of remodeling the chromatin and/or DNA of said cell; and increasing intracellular levels of at least one reprogramming agent. The reprogramming agent is selected to directly or indirectly induce the expression of morphological and functional characteristics of a desired cell of a different type or cell lineage.
[0024] A further aspect of the invention relates to a method of transforming a cell of a first type to a cell of a desired cell of a different type, which comprises increasing intracellular levels of at least one drug. reprogramming, in which the reprogramming agent is selected to directly or indirectly induce the expression of morphological and functional characteristics of a desired second cell type; and maintaining the cell of a first type under culture conditions to support transformation of the desired cell for a period of time sufficient to allow stable expression of a plurality of secondary genes whose expression is characteristic of the phenotypic and functional properties of the desired cell; where at least one of the secondary genes is not characteristic of the phenotypic and functional properties of an embryonic stem cell. At the end of that time period, the desired cell of a different type is obtained and the obtained cell is further characterized by a stable repression of a plurality of genes expressed in the first type of cell.
[0025] Another aspect of the invention relates to a process in which a cell of a first type is reprogrammed to a desired cell of a different type, the process comprising: - a transient increase in intracellular levels of at least one agent of reprogramming, wherein the reprogramming agent induces a direct or indirect endogenous expression of at least one gene regulator, and wherein endogenous expression of said gene regulator is necessary for the desired cell of a different type to exist;- an expression stable expression of said gene regulator; - stable expression of a plurality of secondary genes, wherein stable expression of secondary genes is the result of stable expression of the gene regulator, and wherein: (i) stable expression of the plurality of genes secondary genes is characteristic of phenotypic and/or functional properties of the desired cell, (ii) the stable expression of at least one of said secondary genes is not characteristic of the p. phenotypic and functional properties of an embryonic stem cell, and where (i) and (ii) are indicative of the successful reprogramming of the cell of the first type into the desired cell of the different type.
In particular embodiments, the reprogramming agent in the process is an Msi1 polypeptide, or a Ngn2 polypeptide together with an MDB2 polypeptide. In particular embodiments, the gene regulator is Sox2 Msi1, or both. In further embodiments, the gene regulator may be one or more of the genes listed in Table A, for Neural-Type Stem Cells.
[0027] According to another aspect, the invention relates to a method of obtaining a Stem-Type Cell (SLC), comprising: i) providing a cell of a first type; ii) transiently increasing the intracellular levels of cells of at least one reprogramming agent, through which the transient increase directly or indirectly induces the endogenous expression of at least one gene regulator; iii) put the cell in a position to support transformation into stem-like cells and maintain intracellular levels of the reprogramming agent for a period of time sufficient to allow stable expression of the gene regulator in the absence of the reprogramming agent; iv) maintain the cell in culture conditions to support transformation into stem-like cells for a sufficient period of time to allow the stable expression of a plurality of secondary genes whose expression is characteristic of the phenotypic and/or functional properties of the stem cell, where p at least one of the secondary genes is not characteristic of the phenotypic and functional properties of an embryonic stem cell. At the end of said period of time a stem cell is obtained.
[0028] According to another aspect, the invention relates to a method of obtaining a Stem Cell. The method comprises increasing the intracellular levels of at least one polypeptide specific for the desired type of stem cell that is capable of directly or indirectly driving the transformation of the cell of the first type into the Stem-Type Cell. To increase the yield or type of Stem Cell, the method may further comprise contacting chromatin and/or DNA from a first type cell with a histone acetylator, a histone deacetylation inhibitor, a DNA demethylator, and/or a DNA methylation inhibitor; and/or increasing intracellular levels of at least one other polypeptide specific for the desired type of stem cell that is capable of directly or indirectly driving the transformation of the cell of the first type into a Stem-Type Cell.
[0029] According to another aspect, the invention relates to a method of obtaining a neural-like stem cell (NSLC). The method comprises increasing intracellular levels of at least one specific polypeptide of the neural stem cell that is capable of directly or indirectly driving the transformation of the cell of the first type into an NSLC. To increase the yield or type of NSLC, the method further comprises contacting the chromatin and/or DNA of a cell of a first type, with a histone acetylator, a histone deacetylation inhibitor, a DNA demethylator, and/or an inhibitor of DNA methylation; and/or increasing intracellular levels of at least one other neural stem cell specific polypeptide that is capable of directly or indirectly driving the transformation of the first cell type into an NSLC.
[0030] Another aspect of the invention concerns a method of obtaining a neural-like stem cell (NSLC). In one embodiment, the method comprises transfecting a skin cell with a polynucleotide encoding Musashi1, Musashi1 and Neurogenin 2, Musashi1 and Methyl-CpG which binds domain protein 2 (MBD2), or Neurogenin 2 and Methyl-CpG which binds protein domain 2, thus reprogramming the skin cell into an NSLC. In another embodiment, the method comprises exposing a skin cell to: (i) a histone deacetylation inhibitor, (ii) a DNA methylation inhibitor, (iii) a histone acetylator, and/or, (iv) ) a DNA demethylator such as an MBD2 polypeptide and/or transfect with a polynucleotide encoding an MBD2 polypeptide; and further transfecting the cell (either simultaneously, before or after) with a polypeptide encoding Musashi1 and/or with a polypeptide encoding NGN2, thereby reprogramming the skin cell for an NSLC. Some other cells, such as keratinocytes and CD34+ cells, can also be used and reprogrammed.
[0031] In a particular embodiment, the method of obtaining a neural-type stem cell (NSLC), comprises:- providing a cell of a first type;- introducing into the cell one or more polynucleotides capable of transient expression of one or more of the following polypeptides: Musashi1 (Msi1); one Musashi1 (Msi1) and one Neurogenin 2 (Ngn2); a Musashi1 (Msi1) and Methyl-CpG binding domain protein 2 (MBD2); and Neurogenin 2 (Ngn2) and Methyl-CpG which binds domain protein 2 (MBD2) (MBD2); e- place the cell in culture conditions that support transformation into an NSLC for a period of time sufficient to allow stable expression of a plurality of genes whose expression is characteristic of the phenotypic and functional properties of an NSLC.-
[0032] At the end of the time period an NSLC is obtained and the NSLC obtained is further characterized by a stable repression of a plurality of genes expressed in the first cell type.
[0033] According to another embodiment, the method of obtaining a neural-type stem cell (NSLC), comprises:- providing a cell of a first type that is not an NSLC;- increasing intracellular levels of at least one of the polypeptides specific to neural stem cells, where the polypeptide is able to directly or indirectly drive the transformation of the first cell type into an NSLC; and - contacting the chromatin and/or DNA of the cell of a first type, with a histone acetylator, a histone deacetylation inhibitor, a DNA demethylation, and/or a chemical inhibitor of DNA methylation.
[0034] According to another embodiment, the method of obtaining a neural-type stem cell (NSLC) comprises:- obtaining a non-NSLC cell;- co-transfecting the non-NSLC cell with a first polynucleotide encoding a MBD2 polypeptide and with at least one second polynucleotide encoding a MUSASHI1 polypeptide and/or encoding an NGN2 polypeptide; - placing the co-transfected cell under culture conditions to support NSLC transformation until said NSLC is obtained.
[0035] Certain aspects of the invention relate to isolated cells, cell lines, compositions, the 3D assembly of cells and tissues comprising cells obtained using the methods described herein. Additional aspects concern the use of such isolated cells, cell lines, compositions, the 3D assembly of cells and tissues for medical treatment, and methods of regenerating a mammalian tissue or organ.
[0036] Still, a further aspect concerns a method for repairing or regenerating tissue in an individual. In one embodiment, the method comprises administering a reprogrammed cell as defined herein to an individual in need thereof, wherein the administration provides a dose of reprogrammed cells sufficient to enhance or support a biological function of a given tissue or organ, improving the individual's condition. .
[0037] The benefits of the present invention are significant and include lower cost of cell therapy, eliminating the need for immunosuppressive agents, no need for embryos or fetal tissue, thus eliminating ethical and time constraints, lower production cost, and no health risks due to the possibility of transmission of virus or other disease. Also, since cells are created fresh, they tend to be more potent than cells that have undergone multiple passages.
[0038] Additional aspects, advantages and characteristics of the present invention will become more apparent from reading the non-limiting description of preferred embodiments, which are exemplary and should not be construed as limiting the scope of the invention. Brief Description of Figures
[0039] Figure 1 is a panel of light micrograph (10X) showing changes in cell morphology of non-transfected and Msi1 and MBD2 transfected cells at various time points.
[0040] Figure 2 is a panel of photomicrographs obtained using Cellomics™ (10x) and revealing NCAM positive cells in cells transfected with Msi1 or Ngn2 in the presence of MBD2. HFFs were pretreated with cytochalasin B (10µg/ml) and transfected with pCMV6-XL5-Msi1 and pCMV6-XL5-MBD2 or pCMV6-XL4-Ngn2 and pCMV6-XL5-MBD2. 24 hours after transfection, the medium was changed and cells were cultured in proliferation medium (NPBM, Lonza) supplemented with EGF (20ng/ml) and bFGF (20ng/ml) for one week. Differentiation was induced by changing the medium to NbActive (BrainBitsTM) supplemented with NGF (20ng/ml), bFGF (20ng/ml), ATRA (5μM) and Forskolin (10μM). Cells were incubated at 37°C, 5% CO 2 , 5% O 2 for 26 days.
[0041] Figure 3 is a panel of photomicrographs obtained using CellomicsTM (10x) and revealing MAP2b positive cells in cells transfected with Msi1 or Ngn2 in the presence of MBD2. MAP2b positive cells were undetectable in untransfected cells and Pax6/MBD2 transfected cells. HFFs were pretreated with cytochalasin B (10μg/ml) and transfected with pCMV6-XL5-Msi1, pCMV6-XL4-Ngn2 or pCMV6-XL5-Pax6 and pCMV6-XL5-MBD2. 24 hours after transfection, the medium was changed and cells were cultured in proliferation medium (NPBM, Lonza) supplemented with EGF (20ng/ml, Peprotech) and bFGF (20ng/ml, Peprotech) for one week. Differentiation was induced by changing the medium to NbActive (BrainBitsTM) supplemented with NT-3 (20ng/ml), bFGF (20ng/ml), ATRA (5μM) and Forskolin (10μM). Cells were incubated at 37°C, 5% CO 2 , 5% O 2 for 2 weeks.
[0042] Figure 4A is a panel of photographs showing that neurospheres formed by NSLCs from Example V were completely dissociated into single cell suspensions using Accutase and a single cell was monitored over time to reveal the ability of neurosphere formation ( A, light microscope observation). Neurospheres scored positively for Sox2.
Figure 4B is a panel of photographs of immunohistochemistry results obtained using CellomicsTM Immunohistochemistry was performed on day 20 to detect neurosphere markers and compared to neurosphere expression levels formed by normal cells human neuroprogenitors (hNPC, Lonza). In addition to Sox2, the cells stained positively for neural stem cell markers Musashi, CD133, Nestin and GFAP. The cells also stained positive for βIII-tubulin (a marker for neurons), O4 (an oligodendrocyte marker), and GFAP (an astrocyte marker), indicating the tri-potent differentiation potential of both sets of cells (NSLC and hNPC), and negative for NGFrec and NeuN (markers for differentiated neurons), indicating that the cells were not terminally differentiated.
[0044] Figure 5 is a panel photomicrograph of the immunohistochemical results obtained using CellomicsTM. Immunohistochemistry was performed on HFFs and NSLCs, and hNPCs to detect the expression of markers for fibroblasts as well as for neural stem cells (Sox2, Nestin, GFAP) in adherent cultures (which prevented cells from floating and forming neurospheres). Nuclei were stained with Hoechst (top level images). HFFs expressed fibroblast markers while NSLCs created from these HFFs did not. In comparison, NSLCs expressed neural stem cell markers similarly to hNPCs whereas HFFs did not express any of these markers.
[0045] Figure 6 is a photomicrograph of the panel showing Human NSLCs. Human NSLCs were induced to differentiate into neuronal lineages in the presence of NS-A differentiation medium (StemCell Technologies) in the presence of BDNF (20ng/ml, Peprotech) and bFGF (40ng/ml, Peprotech) for three weeks. At a different time point of differentiation, immunostaining using CellomicsTM (10x) revealed cell differentiation, as shown by the decrease in Sox2 positive cells and increase in the number and intensity of staining of p75, βIII -tubulin and GABA positive cells as well. as the differentiated morphology, while the total number of cells increased, as shown by Hoechst tagging.
[0046] Figure 7 is another panel of photomicrographs. HFF, keratinocytes and CD34+ cells were transfected with pCMV6-Msi1-Ngn2 and pCMV6-XL5-MBD2. 24 hours after transfection, the medium was changed to proliferation medium (StemCell Technologies) supplemented with EGF (20ng/ml, Peprotech) and bFGF (20ng/ml, Peprotech) for two weeks and then analyzed. Photomicrographs using CellomicsTM (10x) show that NSLCs created from all three cell types are positive for Nestin, Sox2 and GFAP (markers for neural stem cells), whereas the original HFFs are not.
[0047] Figure 8 is a panel of photomicrographs showing the effect of the CDM medium on the trans-differentiation of HFF towards neurons. HFF were pretreated with cytochalasin B (10μg/ml) and histone deacetylation inhibitor (VPA, 4 mM) and DNA methylation inhibitor (5-Aza, 5μM and cultured in CDM medium containing a 3:1 ratio of medium Dulbecco's modified Eagle's broth (DMEM, high glucose (4.5 g/L) with L-glutamine and sodium pyruvate) and Ham's F-12 medium supplemented with the following components: EGF (4.2x10-10M), bFGF (2.8x10-10M), ITS (8.6x10-5M), dexamethasone (1.0x10-7M), L-ascorbic acid phosphate magnesium salt n-hydrate (3.2x10-4M), L- 3,3',5-triiodothyronine (2.0x10-10M), ethanolamine (10-4M), GlutaMAX™ (4x10-3M), glutathione (3.3x10-6M) After 24 hours the culture medium was replaced with 75 % CDM media and 25% Neuronal Proliferation media (Lonza, Cat #CC-3210); for the next 3 days, the proportion of media was changed to 50%:50%, 25%:75%, and, then 100% of the Neuronal Proliferation medium until day 3. Photomicrographs were taken by CellomicsTM (10X) after immunostaining. βIII-tubulin (neuronal marker) and Hoechst (to stain nuclei) cells at different time points. Cells started trans-differentiation within a few days and trans-differentiated cells were positive for βIII-tubulin, however, after one week a spontaneous reversion to fibroblast form and loss of βIII-tubulin expression was observed.
[0048] Figure 9 is panel photomicrographs showing the characterization of cells reprogrammed within the CDM at different time points after transfection with Msi1 and Ngn2. The transfected cells were treated with Cytochalasin B (10 μg/ml), VPA (4 mM) and 5-AZA (5μM), resulting in microfilament rupture and cell rounding and chromatin loosening. 3-Dimensional CDM immunohistochemistry was performed after one and two weeks using CellomicsTM (10X). Cells were positive for a mature neuronal marker, such as MAP2b, but were absent in the untransfected control CDM.
[0049] Figure 10 is another panel of photomicrographs. Cells within the CDM on Day 4 were lipotransfected with the two vectors pCMV6-XL5-Msi1 and pCMV6-XL4-Ngn2 individually or together, in combination with pCMV-XL5-MBD2 for a period of 6 hours. In parallel, transfection was performed on fresh HFFs after 6 hours using Nucleofection, and these fresh HFFs were placed on top of the CDM while the lipofectamine medium was changed to fresh CDM medium after 6 hours. After 24 hours the medium was changed to Neural Proliferation Medium (NPBM, Lonza) with the presence of Noggin (50ng/ml, Peprotech), recombinant hFGF (20ng/ml, Peprotech), and recombinant hEGF (20ng/ml, Peprotech) for a week. Differentiation was induced on day 7 by addition of NS-A differentiation medium (StemCell Technologies) for 24 days. Immunohistochemistry was performed at various time points using CellomicsTM (10X). The CDM was labeled with a specific antibody against Nestin (a marker for neural stem cells), and cells within the CDM expressed Nestin at all time points tested (Day 8, 15 and 21) after transfection. Cells within the untransfected control CDM did not express any Nestin.
[0050] Figure 11 is a panel showing an image of a polyacrylamide gel electrophoresis. NSLCs grown as adherent cultures or suspension cultures (such as neurospheres) both expressed telomerase (which is expressed in all stem cells, but not in normal differentiated somatic cells). Both early (p5) and late (p27) passage NSCLs expressed telomerase. (The original HFFs from which the NSLCs were created did not express telomerase.) The samples (NSLCs) were centrifuged and the protein concentration of the supernatant was determined using the BCA assay. 900ng of protein from each cell extract was added directly to the TRAP reaction mix containing TRAP reaction buffer, dNTP, the template substrate (TS) primer, the TRAP primer mix and Taq polymerase. The reaction mixtures were incubated at 30°C for 30 minutes for template synthesis, followed by a PCR procedure (95°C/15 min for initial denaturation, 94°C/30 sec, 59°C/30 sec, 72 °C/1 min for 32 cycles) for the amplification of the extended telomerase products. To detect telomerase activity, polyacrylamide gel electrophoresis (PAGE) was performed for the reaction products on a non-denaturing 10% TBE gel. After electrophoresis, the gel was stained with SYBR® Green I Nucleic Acid Gel Stain for 30 minutes, followed by image capture using the Gel-Documentation System (Alfa Innotech). All 4 samples were telomerase positive (as indicated by the TRAP product ladder).
[0051] Figure 12 is a panel showing an image displaying Southern blot analysis of two different NSLC samples analyzed for Msi1 and Ngn2 gene integration two weeks after transient transfection. The Dig-labeled PCR probe revealed distinct signals in the positive control samples where plasmid DNA Msi1/Ngn2 was enriched in HFF genomic DNA for equivalence of 1, 10 or 100 pergenome integrations. There were some faint and identical bands that appeared in restriction enzyme digested genomic DNA from untransfected HFF and NSLC samples #1 and #2, suggesting that there was no integration of plasmid DNA into the genomic DNA of NSLCs. These weak bands may represent the endogenous Ngn2 gene as the 1.2 kb Dig-labeled PCR probe contains a small part of the Ngn2 gene. There were positive signals in the kb ladder band of DNA as the bands belong to a series of plasmids digested for action with the appropriate restriction enzymes (NEB). These data show that none, or only a small number of NSLCs had plasmid integration into the host genome after transient transfection, and that transiently transfected genes were only present in the cells for a short period of time (less than two weeks).
[0052] Figure 13 is a panel with a line graph and a bar graph showing improvement and significantly better clinical outcomes in EAE mice treated with NSLCs. Eight-week-old female C57BL/6 mice were immunized with MOG35-55 (Sheldon Biotechnology Center McGill University) in CFA containing 5 mg/ml of dissected Mycobacterium tuberculosis H37Ra (dead and dried) (Difco, Inc), at two sites in the back, and injected with 200 ng of pertussis toxin (List Biological Laboratories), Inc) in PBS intraperitoneally on days 0 and 2. Once the rats began to show symptoms of EAE (on day 13 post-immunization), they were injected with 200μl of NSLC (1 million cells), hNPC (1 million cells), saline, or saline with cyclosporine. All mice except the saline control group received daily cyclosporine injections. Mice were scored daily for clinical disease, data represent mean daily scores. Mice that received a single injection of NSLCs had a significantly lower disease severity than mice that received hNPCs or cyclosporine alone.
[0053] Figure 14 is a line graph showing the results of the rota-rod evaluations according to Example XVII part 2. Rats were trained on the rota-rod before the start of the experiment. Rats were placed on a stationary rotating rota-rod (rotating at 20 rpm) and the amount of time rats spent walking on the rota-rod before falling was monitored. Measurements were taken before (pre-surgery) and after (post-surgery) surgical ablation of the left hemisphere of the brain and treatment. Data points represent the average number of falls for each animal during each 60-second test session performed at a constant speed of 20 rpm. Each group consisted of eight rats.
[0054] Figure 15 is a line graph showing the results of walking beam assessments according to Example XVII part 2. Rats were measured on their ability to traverse a 100 cm beam after surgical ablation of the hemisphere left brain and treatment. Two days after surgery, all groups failed the test, and animals were not able to remain balanced on the beam. One week after surgery, all animals show an improvement in their ability to walk, but no significant difference was noted between the different treated groups. From week 4 through week 26, animals treated with NSLCs show a significant improvement in their ability to walk compared to the other groups.
[0055] Figure 16 is a panel displaying photographs of ADSCs transiently transfected with various pluripotent vectors using nucleofector equipment as described in Example XIX. After transfection cells were cultured in 6-well plates in suspension with a 50:50 mixture of ADSC complete medium (StemPro™-43) and embryonic stem cell medium (mTeSRI™, StemCell Technologies). After two days in culture, cells were re-transfected with the same plasmids and plated on Matrigel™ coated 96-well plates (BD Biosciences) in the presence of mTeSR1™ complete medium supplemented with thiazovivin (0.5μM), an ALK inhibitor -5 (SB341542, Stemgent, 2μM)), and a MEK inhibitor (PD0325901, Stemgent, 0.5μM). The medium was changed every day and cells were cultured for 22 days at 37°C, 5% CO 2 , 5% O 2 followed by AP labeling and immunohistochemistry to analyze the expression of pluripotency markers. The cells formed colonies and were found to express both Oct4 and AP pluripotency markers after transfection of cells with pEF-Rex1-EF-Oct4-2A-KLF4-2A-RFP.
Figure 17 is a panel showing photographs of ADSCs transiently transfected with pCMV6-XL5-Rex1/pCMV6-XL5-Klf4 and pCMV6-XL5-Rex1/pCMV6-XL4-Oct4. After the second transfection, ADSCs were cultured in MatrigelTM coated 96-well plates for 24 days in the presence of mTeSR1™ medium supplemented with SB341542 and PD0325901 at 37°C, 5% CO 2 , 5% O 2 . In order to characterize cell subpopulations after transfection, live labeling, immunohistochemistry and AP labeling were used. 1% -5 of whole cells or cells transfected with Rex1/Oct4 Rex1/Klf4 showed a phenotype for SSEA-4+ and TRA-1-81+ (first markers of pluripotency). Observation over time showed that the phenotype of these colonies moved earlier from the SSEA-4+ phenotype to a later Oct4+/Sox2/Nanog+ phenotype starting on day 22, which was closer to the reprogrammed end state and a pluripotent cell type .
[0057] Figure 18 is a panel showing photographs of ADSCs transiently transfected with various pluripotent vectors. After transfection cells were plated in StemPro™ MSC SFM medium in MatrigelTM coated 24-well plates (BD Biosciences) and incubated at 37°C, 5% CO 2 , 5% O 2 . On day 1, media was changed to a medium mixture of 75% StemPro™ MSC and 25% hES cells; the percentage of StemPro™ MSC SFM medium was decreased every day for four days to have 100% hES cell medium on day 4. Then the medium was changed every other day. The hES cell medium consisted of Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen) supplemented with Knockout™ Serum Replacemen Serum (KSR, Invitrogen), 1 mM GlutaMAX™, 100 µM non-essential amino acids, 100 µM β-mercaptoethanol and 10 ng/ml of FGF-2. In order to characterize cell subpopulations after transfection staining, live marker, immunohistochemistry and AP marker were used. Transfected cells transfected with Oct4/UTF1/MBD2, Oct4/Dppa4/MBD2, FoxD3/Dppa4/MBD2, Oct4/FoxD3/Dppa4, eSox2/FoxD3/UTF1 were positive for SSEA-4+, TRA1-60, and TRA phenotype -181+ (first pluripotency markers) on day 14.
[0058] Figure 19 is a panel showing photographs of transiently transfected HFFs. HFFS were transiently transfected using Nucleofector® II Device (Lonza), following the procedure described in Example II with the exception that 1 μg of each of the following 3 DNA plasmids were used: pCMV-Oct4nuc-IRES2-Sox2nuc, pCMV-Klf4nuc -IRES2-Cmycnuc and pCMV-Nanognuc-IRES2-Lin28. Cells were pretreated with and without VPA and 5-Aza. After transfection, cells were plated in fibroblast medium, supplemented with and without VPA (2 mM) and 5-AZA (2.5μM) in 6-well plates coated with MatrigelTM (BD Biosciences) and incubated at 37°C, 5% CO2. On Day 1 and 2, media were changed to 100% mTeSRITM medium (StemCell Technologies) supplemented with and without VPA and 5-AZA. On Day 3 and 6, cells were re-transfected as above and plated onto Matrigel™ coated plates in TeSRITM medium supplemented with and without VPA and 5-AZA. Means were changed daily as above. The medium was supplemented with Y27632 (Stemgent, 10μM]) from day 7 to day 14 to promote viability and clonal expansion of potential reprogrammed cells. Cells were analyzed on day 20 using the alkaline phosphatase detection kit (Millipore) and by immunohistochemical analysis. Some cells scored positive for the pluripotency markers AP, SSEA-4 and TRA-1-81 (similar to human embryonic stem cell lines Mel2 (positive control)). These clones were obtained only under the condition that they did not contain the inhibitors (ie: VPA and 5-AZA). No clones were observed for the condition treated with these inhibitors.
[0059] Figure 20 is a panel showing photographs of transfected NSLCs and BG-01. NSLCs and BG-01 NS were transfected as described above in Example II by two episomal vectors, pEF-Oct4nuc-IRES2-MBD2 (NC1) or pCMV-FOXD3-2A-Oct4-2A-Klf4 (F72). After transfection cells were collected and plated in uncoated Petri dishes in the presence of Proliferation medium and medium mTeSRITM (50:50) under proliferation conditions at 37°C, 5% CO2. After 48 hours, cells were re-transfected by the same plasmid and plated in 96-well plates coated with MatrigelTM and cultured in the presence of mTeSRITM medium supplemented by small molecules BIX01294 (Stemgent, 2μM) and BayK8644 (Stemgent, 2μM), at 37°C °C, 5% O2 for 22 days, after live labeling and immunohistochemistry were performed to characterize cell subpopulations for pluripotency markers. Cells formed colonies positive for both TRA-1-81 and SSEA-4 indicative of pluripotent-type cells.
[0060] Figure 21 is a panel displaying brightfield images on day 17 of fibroblasts transfected with Msi1/Ngn2 and pCMV6-XL5-MBD2 placed in different media conditions and showing different morphologies and degree of differentiation. (a) cells in neural proliferation medium from day 1 to day 12, and then in neural differentiation medium with cytokines from day 12 to 17. (b) cells in neural proliferation medium from day 12 to 17. day 1 to day 12, and then in NbActive4 medium with cytokines from day 12 to 17. (c) cells in neural differentiation medium with cytokines plus Fgf-2 from day 1 to day 12, and then in the same medium but without Fgf-2 from day 12 to 17. (d) cells in NbActive4 medium with the cytokines plus Fgf-2 from day 1 to day 12, and then the same medium but without Fgf-2 from day 12 to 17. (e) cells in CDM II medium with cytokines plus Fgf-2 from day 1 to day 12, and then the same medium but without Fgf-2 from day 12 to 17.
Figure 22 is a panel displaying images of immunochemistry results on day 17 of fibroblasts transfected with Msi1/Ngn2 and pCMV6-XL5-MBD2 in Figure 21. Figs. 22A and 22B: cells were in NS-A Proliferation medium from day 1 to day 12, and then in NS-A Differentiation medium (A) or NBActive4 medium (B) with cytokines from day 12 to 17. There was more cells in B, but differentiation from day 12-17 was too short to induce βIII-tubulin expression in both cases. Figs. 22C-E: cells were in NS-A Differentiation medium (C) or NbActive4 medium (D) from day 1-17 (FGF-2 supplementation from day 1-12), or CDM II medium from from day 1-12 and then NS-A Differentiation medium from day 12-17 (E). There were a large number of cells in C and a much smaller number of cells in D and E. The cells were immunopositive for both GFAP and βIII-tubulin in all cases and placing the cells in differentiating or non-proliferating media since day 1 onwards seems to have induced a more direct transformation in neurons and glia, with more intensity of βIII-tubulin than GFAP positive cells in E.
[0062] Figure 23 is a panel showing two heat maps that provides an overview of the comparison of gene expression between NSLC vs. HFF (Group 1), or NSLC vs. hNPC (Group 2). NSLC has a distinct gene expression profile when compared to HFF or hNPC. Based on intensity (the greater the intensity, the greater the relative change in expression), NSLC is much more similar to hNPC than to HFF.
[0063] Figure 24 is a panel showing images of NSLCs. NSLCs were tested to determine if they are a population of Skin Derived Precursor Cells (SKPs). SKPs capable of proliferating in response to EGF and bFGF, express nestin and fibronectin, and can differentiate into both neuronal and mesodermal progeny, including adipocytes. For this purpose, a standard protocol to transform SKPs into adipocytes was performed, in which adipocyte-derived stem cells (ADSCs) and NSLCs were cultured in StemProTM proliferation medium and differentiation to adipocytes were induced by culturing these cells in differentiation medium consisting of DMEM/F12 (50:50), ITS (1:100), HEPES (1:100), GlutaMAX™ (1:100), T3 (0.2 nM), Rosiglitazone (0.5 μg/ml) , IBMX (100μM) and Dexamethasone (1μM). Three days later, IBMX and Dexamethasone were removed from the medium. On day 10, cells were fixed with 4% formaldehyde solution for 10 min and stained with oil red O marking solution (Oil Red O, Invitrogen). Fat cells appeared red with lipid droplets (bright white spots in left frame) specifically stained with Oil Red O, however NSLCs scored negative and there was no presence of fat droplets in the cells, but instead adopted the cell morphology neuronals. These results confirm that NSLCs are not a population of Skin Derived Precursor Cells (SKPs). Detailed Description of the Invention
[0064] The present invention relates to methods for cell dedifferentiation and cell reprogramming. A significant aspect of the present invention is that it allows the use of the patient's own cells to develop different types of cells that can be transplanted, after the in vitro dedifferentiation and in vitro reprogramming steps. Thus, this technology eliminates the problems associated with non-host cell transplantation, such as immune rejection and the risk of disease transmission. Furthermore, since cells are "newly created", they have the potential to be more potent than alternative sources of natural cells that have already been divided several times. Definitions
[0065] As used herein and in the appended claims, the singular forms "a", and "the", include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "a cell" includes one or more such cells or a cell lineage derived from such cell, reference to "an agent" includes one or more such agent, and reference to "the method " includes reference to equivalent steps and methods known to those skilled in the art which may be modified or substituted for the methods described herein.
As used herein, "polynucleotide" refers to any DNA or RNA sequence or molecule comprising coding nucleotide sequences. The term is intended to include all polynucleotides whether naturally occurring or non-naturally occurring, in particular, a cell, tissue or organism. This includes DNA and its fragments, RNA and its fragments, cDNAs and its fragments, expressed sequence tags, artificial sequences, including randomized artificial sequences.
As used herein, the term "polypeptide" refers to any amino acid sequence having a desired functional biological activity (eg DNA demethylation). The term includes whole proteins, fragments thereof, fusion proteins and the like, including carbohydrates or lipid chains or compositions.
[0068] "Trans-differentiation" refers to a direct change from an already differentiated cell to another differentiated cell type.
[0069] "Dedifferentiation" refers to the loss of phenotypic characteristics of a cell differentiated by activating or deactivating genes or metabolic pathways.
[0070] "Marker" refers to a gene, polypeptide, or biological function that is characteristic of a particular cell type or cell phenotype.
[0071] "Genetically modified DNA sequence" means a DNA sequence, in which the component elements of the DNA sequence sequence are arranged within the DNA sequence in a manner not found in nature.
"Signal sequence" refers to a nucleic acid sequence which, when incorporated into a nucleic acid sequence encoding a polypeptide, directs secretion of the translated polypeptide from cells expressing said polypeptide, or permits the polypeptide easily crosses the cell membrane in a cell. The signal sequence is preferably located at the 5' end of the nucleic acid sequence encoding the polypeptide, such that the polypeptide sequence encoded by the signal sequence is located at the N-terminus of the translated polypeptide. "Signal peptide" means the peptide sequence resulting from translation of a signal sequence.
"Ubiquitous Promoter" refers to a promoter that directs the expression of a polypeptide or peptides encoded by nucleic acid sequences to which the promoter is operably linked. Preferred ubiquitous promoters include human cytomegalovirus (CMV); simian virus 40 (SV40) promoter; Rous sarcoma virus (RSV), or the adenovirus major late promoter.
[0074] "Gene expression profile" means an assay that measures the activity of several genes at once, creating a global view of cell function. For example, these profiles can distinguish between human neural stem cells and somatic cells that actively divide or differentiate.
"Transfection" refers to a method of gene delivery that introduces exogenous nucleotide sequences (e.g. DNA molecules) into a cell preferably by a non-viral method. In preferred embodiments according to the present invention exogenous DNA is introduced into a cell by transient transfection of an expression vector encoding a polypeptide of interest, where exogenous DNA is introduced but eliminated over time by the cell and during mitosis. where the introduced expression vectors and the vector-encoded polypeptide are not permanently integrated into the host cell genome, or anywhere in the cell, and therefore may be eliminated from the host cell or its progeny over time. polypeptides, or other compounds, can also be delivered to the cell using transfection methods.
[0076] "Neuroprogenitor cell" refers to an immature cell of the nervous system, which can differentiate into neurons and glia (oligodendrocytes and astrocytes). "Neural stem cell" is a germ layer of the ectoderm derived from multipotent stem cells having, as a physiological characteristic, an ability to form neuroprogenitor cells and under physiological conditions that favor differentiation to form neurons and glia. "Neural-like stem cell" or "NSLC" refers to any cell-derived multipotent stem cell having, as a physiological characteristic, an ability to form other neural-like stem cells and neuroprogenitor-like cells and under physiological conditions that favor differentiation to form neuron-like cells and glial-like cells.
[0077] "Neurosphere" refers to a cellular aggregate of neural stem cells and neuroprogenitor cells that form a floating sphere formed as a result of the proliferation of neural stem cells and neuroprogenitor cells under suitable proliferative conditions. NSLCs also form neurospheres consisting of aggregates of NSLCs and neuroprogenitor-like cells.
[0078] "Reprogrammed cell" refers to a cell that has undergone stable trans-differentiation, de-differentiation, or transformation. Some reprogrammed cells can subsequently be induced to re-differentiate. The reprogrammed cell stably expresses a specific cell marker or set of markers, morphology and/or biological function that was not characteristic of the original cell. "Reprogrammed somatic cell" refers to a process that alters or reverses the state of differentiation of a somatic cell, which can be either the complete or partial conversion of the differentiated state to a less differentiated state or a new differentiated state.
[0079] "Regeneration" refers to the ability to contribute to the repair or de novo construction of a cell, tissue or organ.
[0080] "Differentiation" refers to the developmental process of compromised cell lineage. Differentiation can be assayed by measuring an increase in one or more specific cell markers for differentiation relative to expression of the undifferentiated cell markers.
[0081] "Lineage" refers to a pathway of cell development, in which a more undifferentiated cell undergoes progressive physiological changes to become a more differentiated cell type having a characteristic function (for example, neurons and glia are of a neuroprogenitor lineage, which is an ectodermal lineage that formed from blastocysts and embryonic stem (ES) cells).
[0082] "Tissue" refers to a set of cells (identical or not) and an extracellular matrix (ECM) that together perform a specific function or set of functions.
"CDM" means a living tissue equivalent or matrix, a living support or cell-derived matrix. cell transformation
[0084] Some aspects of the invention relate to methods and cells to transform or reprogram a given somatic cell into a pluripotent, multipotent, and/or unipotent cell. Some aspects of the invention relate to methods for conditioning a somatic cell for reprogramming to a pluripotent, multipotent, or unipotent cell.
[0085] The terms "transform" or "reprogram" are used interchangeably to refer to the phenomenon in which a cell is dedifferentiated or transdifferentiated to become pluripotent, multipotent, and/or unipotent. The de-differentiated cell could subsequently be re-differentiated to a different type of cell. Cells can be reprogrammed or converted to varying degrees. For example, it is possible that only a small portion of cells are converted or that an individual cell is reprogrammed to be multipotent but not necessarily pluripotent. Thus, the terms "transformation" or "reprogramming" methods can refer to methods where it is possible to reprogram a cell in such a way that the "new" cell exhibits morphological and functional characteristics of a specific new or different cell lineage (eg the transformation of fibroblast cells into neuronal cells).
[0086] As used herein, the term "somatic cells" refers to any differentiated cell forming the body of an organism, in addition to stem cells, progenitor cells, and germline cells (i.e., ovogonia and spermatogonia) and cells derived from them (eg oocytes, sperm). For example, internal organs, skin, bones, blood and connective tissue are all made up of somatic cells. Somatic cells according to the invention may be isolated adult differentiated cells or they may be fetal somatic cells. Somatic cells are obtained from animals, preferably human subjects, and cultured according to standard cell culture protocols available to those of ordinary skill in the art.
[0087] As used herein, "Stem cells" refers to those cells that retain the ability to renew themselves through mitotic cell division and that can differentiate into a diverse range of specialized cell types. It includes both embryonic stem cells that are found in blastocysts, and adult stem cells that are found in adult tissue. "Totipotent cells" refers to cells that have the ability to develop into cells derived from all three embryonic germ layers (endoderm, mesoderm and ectoderm) and an entire organism (eg, human if placed in a woman's uterus in the human beings). Totipotent cells can give rise to an embryo, extra embryonic membranes and all post-embryonic tissues and organs. The term "pluripotent", as used herein, means the ability of a cell to give rise to differentiated cells from all three embryonic germ layers. "Multipotent cells" refers to cells that can produce only cells from a closely related family of cells (eg, hematopoietic stem cells differentiate into red blood cells, white blood cells, platelets, etc.). "Unipotent Cells" refers to cells that have the ability to develop/differentiate into only one tissue type/cell type (eg skin cells).
[0088] The present invention allows the reprogramming of any cell to a different type of cell. Although the present application mainly focuses on the preparation of stem-like cells, especially neural stem cells (NSLCs), the invention is not so limited because many different types of cells can be generated in accordance with the principles described herein. Likewise, while the Examples section describes embodiments in which fibroblasts, keratinocytes, CD34+, adipose tissue-derived stem cells (ADSCs), neural stem cells (including NSLCs), and cells within a cell-derived matrix ( CDM) are reprogrammed, the invention is not limited to such cells. The invention can be used to reprogram any cell of interest.
[0089] Therefore, a general aspect of the invention relates to a method of transforming a cell of a first type to a cell of a different second type. As used herein, examples of cells of a first type include, but are not limited to, germ cells, embryonic stem cells and derivatives thereof, adult stem cells and derivatives thereof, progenitor cells and derivatives thereof, derived cells from the mesoderm, endoderm or ectoderm, and a cell from the mesoderm, endoderm or ectoderm lineage, such as an adipose tissue-derived stem cell (ADSC), mesenchymal stem cell, hematopoietic stem cell (CD34+ cell), precursor cell derived from skin, hair follicle cell, fibroblast, keratinocyte, epidermal cell, endothelial cell, epithelial cell, epithelial granulosa cells, melanocyte, adipocyte, chondrocyte, hepatocyte, lymphocyte (B and T lymphocytes), granulocyte, macrophages, monocytes, mononuclear cell , pancreatic islet cell, sertoli cells, neuron, glial cell, cardiac muscle cell, and other muscle cells.
As used herein, examples of cells of a second type include, but are not limited to, germ cells, embryonic stem cells and derivations thereof, adult stem cells and derivations thereof, progenitor cells and derivations thereof, cells derived from mesoderm, endoderm or ectoderm, and a cell from the mesoderm, endoderm or ectoderm lineage, such as one of the adipose tissue derived stem cells, mesenchymal stem cells, hematopoietic stem cells, skin-derived precursor cells, follicle cells hair, fibroblasts, keratinocytes, epidermal cell, endothelial cells, epithelial cells, epithelial granulosa cells, melanocyte, adipocyte, chondrocytes, hepatocytes, lymphocytes (B and T lymphocytes), granulocytes, macrophages, monocytes, mononuclear cells, pancreatic islet cells , Sertoli cells, neuron, glia cells, cardiac muscle cell, and other muscle cells. In addition, each of the above “type” cells (a cell that has similar but not completely identical characteristics to the known natural type of cell) is also included in the examples of cells of a second type.
[0091] According to a particular aspect, the method of transforming a cell of a first type into a cell of a second different type comprises the steps of: i) providing a cell of a first type; ii) transiently increasing in the cell of a first type the intracellular levels of at least one reprogramming agent, through which the transient increase directly or indirectly induces the endogenous expression of at least one gene regulator; iii) put the cell in a position to support cell transformation desired and maintain intracellular levels of the reprogramming agent for a period of time sufficient to allow stable expression of the gene regulator in the absence of the reprogramming agent; and, iv) maintaining the cell in a condition to support transformation of the desired cell for a period of time sufficient to allow stable expression of a plurality of secondary genes whose expression is characteristic of the phenotypic and/or functional properties of the desired cell. At least one of the secondary genes expressed is not characteristic of the phenotypic and functional properties of an embryonic stem cell. At the end of said time period the cell of the first type was transformed into the desired cell of a different type. Preferably, the cell of a different type obtained after the transformation is further characterized by a stable repression of a plurality of genes expressed in the first cell type.
[0092] According to various embodiments, step (iii) may be carried out consecutively to step (ii), simultaneously with step (ii), or before step (ii).
[0093] According to a related aspect, the invention relates to a process in which a cell of a first type is reprogrammed to a desired cell of a different type, the process comprising:- a transient increase in intracellular levels of, at least one reprogramming agent, wherein the at least one reprogramming agent induces a direct or indirect exogenous expression of at least one gene regulator, where endogenous expression of the gene regulator is necessary for the cell's existence desired of a different type;- a stable expression of said at least one gene regulator;- stable expression of a plurality of secondary genes, wherein the stable expression of the plurality of secondary genes is the result of the stable expression of the gene regulator, and wherein: (i) the stable expression of the plurality of secondary genes is characteristic of the phenotypic and/or functional properties of the desired cell, (ii) the stable expression of at least one of the genes. Secondary nes is not characteristic of the phenotypic and functional properties of an embryonic stem cell, and where (i) and (ii) are indicative of the successful reprogramming of the cell of the first type into the desired cell of the different type.
[0094] As used herein, "transiently increasing" refers to an increase that is not necessarily permanent and therefore may decrease or disappear over time. For example, when referring to transiently increasing intracellular levels of at least one reprogramming agent in a cell, this means that the increase is present for a period of time sufficient to cause particular cellular events to occur (eg, induction of expression endogenous gene of a regulatory gene). Typically, a transient increase is not permanent and is not associated with, for example, genome integration of an expression vector.
[0095] As used herein the "reprogramming agent" refers to a compound that is capable of directly or indirectly inducing the expression of morphological and/or functional characteristics of the desired cell of a different type. Preferred compounds include those capable of directing directly or indirectly the transformation of the cell of the first type into the desired cell of a different type. In the preferred embodiment, the reprogramming agent is selected to induce a direct or indirect endogenous expression of at least one gene regulator as defined herein. There are many compounds that can be useful in reprogramming a cell in accordance with the invention and these compounds can be used alone or in combinations. In various embodiments, the reprogramming agent is a polynucleotide or polypeptide selected in accordance with Table A:








[0096] In some embodiments, the reprogramming agent is a polypeptide that shares at least 75%, 80%, 85%, 90%, 95%, 97%, 99% or more of the functionality or sequence identity of either of the reprogramming agents in the table above.
[0097] Identify the "sufficient period of time" to allow stable expression of the gene regulator in the absence of the reprogramming agent and the "sufficient period of time" in which the cell is to be maintained in culture conditions that support transformation of the desired cell to cell is within the skill of those skilled in the art. The sufficient or suitable period of time will vary according to a number of factors including, but not limited to, the particular type and epigenetic status of the cells (eg, the first type cell and the desired cell), the amount of starting material (for example, the number of cells to be transformed), the amount and type of reprogramming agent(s), the gene regulator(s), the culture conditions, the presence of compounds that accelerate reprogramming ( eg compounds that increase cell cycle renewal, modify epigenetic status, and/or improve cell viability), etc. In various embodiments the period of time sufficient to allow stable expression of the gene regulator in the absence of the reprogramming agent is about 1 day, about 2-4 days, about 4-7 days, about 1-2 weeks , about 2-3 weeks or about 3-4 weeks. In various embodiments the period of time sufficient for which cells must be maintained in culture conditions to support transformation of the desired cell and allow for stable expression of a plurality of secondary genes is about 1 day, about 2-4 days , about 4-7 days, or about 1-2 weeks, about 2-3 weeks, about 3-4 weeks, about 4-6 weeks, or about 6-8 weeks. In preferred embodiments, at the end of the transformation period, the number of desired cells transformed is substantially equivalent or even greater than an amount of cells of a first type given at the beginning.
[0098] The present invention encompasses several types of compounds that are suitable for increasing in a cell of a first type the intracellular levels of at least one reprogramming agent. Preferably, the compound should also be capable of directly or indirectly remodeling the cell's chromatin and/or DNA, thereby directly or indirectly resulting in the expression of morphological and functional characteristics of the desired cell of a different type. Preferred compounds are reprogramming agents, as defined herein or any other compound with a similar activity, and having the ability to activate or increase expression of the endogenous version of genes listed in the table of reprogramming agents hereinbefore presented that are capable of direct or indirectly direct the transformation of the cell of the first type into the desired cell of a different type.
[0099] As will be explained below, the increase in intracellular levels of the reprogramming agent can be achieved by several means. In preferred embodiments the reprogramming agent is a polypeptide and increased intracellular levels of such polypeptides include transfection (or co-transfection) of an expression vector having a polynucleotide (eg DNA or RNA) encoding the polypeptide(s) ), or by an intracellular delivery of polypeptide(s). According to the invention, transient expression is generally preferable. Additional suitable compounds may include compounds capable of increasing the expression of the endogenous version of genes listed in the table of reprogramming agents and gene regulators, including, but not limited to, the reprogramming factors listed in Table B.


[0100] According to the principles of the invention, increased intracellular levels of at least one reprogramming agent should induce a direct or indirect endogenous expression of at least one gene regulator. As used herein, "gene regulator" refers to a polynucleotide or polypeptide whose expression is associated with a series of intracellular events leading to the transformation of a given cell of a first type into a pluripotent, multipotent and/or cell. unipotent. Typically the expression of a gene regulator directly or indirectly activates the genes necessary for the phenotypic and functional trait of pluripotent, multipotent, and/or unipotent cells, while repressing the genes of the first cell type. The gene regulator can be the same or different than the reprogramming agent. Examples of gene regulators in accordance with the invention include, but are not limited to, polynucleotides and polypeptides listed hereinbefore in Table A.
[0101] In some embodiments, the gene regulator is a polypeptide that shares at least 75%, 80%, 85%, 90%, 95%, 97%, 99% or more of the functionality or sequence identity of either of the gene regulators provided here in Table A.
[0102] As used herein, "conditions that support growth" or "conditions that support transformation" when referring to a desired cell refers to various appropriate culture conditions (pH, temperature, O2 tension, cells, factors, compounds, growth substrate (eg laminin, collagen, fibronectin, MatrigelTM, low-binding surface, nanostructured or charged surface, etc.), 3D environment, etc.), favoring growth of the type of desired cell and/or favoring transformation to that desired cell type. Those skilled in the art know that the growth or transformation of particular cell types is stimulated, under specific conditions, while inhibited by others, and it is within their ability to select the appropriate conditions (eg culture conditions) to favor growth or transformation of desired cell types.
[0103] The terms "phenotypic and functional properties", when referring to a desired cell or an embryonic stem cell, mean the biological, biochemical, physiological and visual characteristics of a cell, including expression of certain genes and cell surface markers , which can be measured or evaluated to confirm your identity or function(s).
[0104] An example of a suitable reprogramming agent according to preferred embodiments of the invention is MUSASHI1. In some embodiments this polypeptide is preferred for targeting a first cell, such as a fibroblast, into a neural-like stem cell (NSLC). In other embodiments, the reprogramming agent whose intracellular levels are increased is (are) Musashi1 (Msi1) alone; Musashi1 (Msi1) and Neurogenin 2 (Ngn2); Musashi1 (Msi1) and methyl-CpG binding domain protein 2 (MBD2); or Neurogenin 2 (Ngn2) and Methyl-CpG which binds domain protein 2 (MBD2), Adequate intracellular levels of these polypeptides are preferred as they tend to be expressed throughout an entire cell lineage, hence from embryonic stem cells (or even earlier) for pre-somatic (or even later) cells.
[0105] MBD2 is a member of a methyl-CpG-binding protein family that has been reported to be both a transcriptional repressor and a DNA demethylase (dMTase). As used herein, the term "MBD2" generally refers to Methyl-CpG which binds human domain protein 2. The Human MBD2 GeneBankTM accession number (NCBI) is N_003927.3/AF072242, the UniGeneTM accession number is NP-003918/Q9UBB5 and the UnigeneTM accession number is Hs.25674.
[0106] As used herein, the term "Msi1" generally refers to human musashi homolog 1. The GeneBankTM (NCBI) accession number of human Msi1 is NM_002442.2/AB012851, the UniProtTM accession number is NP -002433/O43347 and the UniGeneTM accession number is Hs.158311.
[0107] As used herein, the term "Ngn2" generally refers to human neurogenin 2 . The GeneBankTM (NCBI) accession number of human Ngn2 is NM_024019.2/BC036847, the UniProtTM accession number is NP-076924/Q9H2A3 and the UniGeneTM accession number is Hs.567563.
[0108] According to further aspects, the method of transforming a cell of a first type to a desired cell of a different type comprises the steps of either: (1) contacting the cell of a first type with one or more compounds capable of increasing intracellular levels of at least one reprogramming agent within the cell and directly or indirectly remodeling the cell's chromatin and/or DNA; or (2) contacting the chromatin and/or DNA of a cell of a first type with an agent capable of remodeling the chromatin and/or DNA of the cell, and increasing intracellular levels of a reprogramming agent.
[0109] According to various embodiments, step (2) can be performed consecutively to step (1), simultaneously with step (1), or before step (1).
[0110] According to a particular aspect, the invention relates to a method for obtaining a neural-like stem cell (NSLC), comprising:- providing a cell of a first type that is not an NSLC; - increasing intracellular levels of at least one neural stem cell-specific polypeptide, where the polypeptide is able to directly or indirectly drive the transformation of the first cell type into an NSLC; e- contacting the chromatin and/or DNA of a cell of a first type, with a histone acetylator, a histone deacetylation inhibitor, a DNA demethylation, and/or a chemical inhibitor of DNA methylation.
[0111] With respect to the second step, the term "chromatin and/or DNA remodeling" refers to dynamic structural changes in chromatin. These changes can range from local changes needed for transcription regulation, to global changes needed for opening the chromatin structure or chromosomal segregation to allow the transcription of the new set of genes characteristic of the desired cell of a different type to close down the chromatin structure or chromosomal segregation to prevent the transcription of certain genes that are not characteristic of the desired cell of a different type. In some embodiments, chromatin backbone opening refers more specifically to histone acetylation, and DNA demethylation, while chromatin backbone closing more specifically refers to histone deacetylation, and DNA methylation.
[0112] As used herein, "compound" refers to a compound capable of effecting a desired biological function. The term includes, but is not limited to, DNA, RNA, protein, polypeptides, and other compounds, including growth factors, cytokines, hormones, or small molecules. As used herein, compounds capable of chromatin and/or DNA remodeling include, but are not limited to, histone acetylators, histone deacetylation inhibitors, DNA demethylators, DNA methylation inhibitors, and combinations thereof.
[0113] "DNA Methylation Inhibitor" refers to an agent that can inhibit DNA methylation. DNA methylation inhibitors have demonstrated the ability to restore expression of the deleted gene. Suitable agents for inhibiting DNA methylation include, but are not limited to, 5-azacytidine, 5-aza-2-deoxycytidine, 1-β-D-arabinofuranosyl-5-azacytosine, and dihydro-5-azacytidine, and zebularin (ZEB), BIX (lysine histone methyltransferase inhibitor), and RG108.
[0114] "Histone deacetylation inhibitor" refers to an agent that prevents the removal of the acetyl groups from the lysine residues of histones that would otherwise lead to the formation of a transcriptionally silenced and condensed chromatin. Hyatone deacetylase inhibitors fall into several groups, including: (1) hydroxamic acids such as trichostatin (A), (2) cyclic tetrapeptides, (3) benzamides, (4) electrophilic ketones, and (5) aliphatic acid group of compounds such as phenylbutyrate and valporic acid. Suitable agents for inhibiting histone deacetylation include, but are not limited to, valporic acid (VPA), Trichostatin A phenylbutyrate (TSA), Na-butyrate and banzamides. VPA promotes neuronal fate and inhibits glial fate simultaneously through the induction of neurogenic transcription factors including NeuroD.
[0115] "Histone Acetylator" refers to an agent that inserts acetyl groups to the lysine residues of histones that opens the chromatin and turns it into a transcriptionally active state. Suitable Histone acetylating agents include, but are not limited to, Polyamine, CREB (cAMP element binding protein), and BniP3.
[0116] "DNA Demetilator" refers to an agent that removes methyl groups from DNA and has the ability to inhibit hypermethylation and restore expression of the deleted gene. A demethylase is expected to activate the genes by removing the repressing methyl residues. Suitable DNA demethylators include, but are not limited to, MBD2 and Gadd45b.
[0117] In some embodiments, the reprogramming agent has one or more of the following functions: it decreases the expression of one or more cell markers of the first type (for example, see Table C), and/or increases the expression of one or more desired cell markers of different type (eg see Table A). Cells that display a selectable marker for the desired cell of a different type are then selected and evaluated for characteristics of the desired cell of a different type.
[0118] According to the invention, the transformation to desired cells results in the stable expression of a plurality of secondary genes whose secondary expression is characteristic of the phenotypic and/or functional properties of the desired cell. Genes whose expression is characteristic of the phenotypic and/or functional properties of the desired cell include, but are not limited to those listed in Table A.
[0119] In some embodiments, the expression of secondary genes whose expression is characteristic of the phenotypic and functional properties of desired cells results in the expression of defined markers according to the following table:

[0120] In some embodiments, transformation of a cell from a first type of desired cells results in a stable repression of a plurality of genes normally expressed in the cell of the first type. Examples of such deleted genes include, but are not limited to those defined in Table C: Table C: Examples of deleted genes



[0121] In preferred embodiments, stable repression of any one or more of the genes listed in Table C expressed in the first cell type is also characterized by a disappearance of the corresponding markers (see Table C).
[0122] Those skilled in the art will understand that there are many alternative steps to facilitate cell reprogramming. These include destabilizing the cell's cytoskeleton structure (eg, by exposing the cell to cytochalasin B), loosening the cell's chromatin structure (eg, through agents such as 5-azacytidine (5-Aza) and valproic acid (VPA) or DNA demethylating agents such as MBD2), transfection of the cell with one or more expression vector(s) containing at least one cDNA encoding a neurogenic transcription factor (eg, Msi1 or Ngn2), using a medium suitable for the desired cell of a different type and a suitable differentiation means to induce the commitment of differentiation of the desired cell of a different type, inhibition of repressive pathways that negatively affect the induction in the commitment of the desired cell of a different type, grow the cells on a suitable substrate for the desired cell of a different type (eg, the laminin of NSLCs or a low-binding surface for culturing floating neurospheres), and growing the cells in an environment that the desired cell of a different type (or "type" cell) would normally be exposed "in vivo" such as the proper temperature, pH, and low oxygen atmosphere (eg, about 2-5 % O2). In various embodiments, the invention encompasses these and other related methods and techniques for facilitating cell reprogramming.
[0123] Therefore, the method of transforming a cell of a first type into a cell of a different second type may comprise additional optional steps. In one embodiment, the method of transforming a cell further comprises the step of pre-treating the cell of a first type with a cytoskeleton disruptor. As used herein "cytoskeleton" refers to the filamentous network of F-actin, myosin light and heavy chain, microtubules, and intermediate filaments (IFs) composed of one of three chemically distinct subunits, actin, tubulin, or a of various classes of IF protein. Thus, the term "cytoskeleton disruptor" refers to any molecules that can inhibit the cell's cytoskeleton to destabilize the cell and, consequently, eliminate the feedback mechanisms between cell shape and cell and nuclear function. Suitable cytoskeletal disruptors according to the invention include, but are not limited to, the cytochalasin family of cytoskeletal actin inhibitors such as cytochalasin B or D, and myosin inhibitors such as 2,3-butanedione monoxime. Such pretreatment can reinforce reprogramming. In a preferred embodiment, the cell is cultured in the presence of at least one cytoskeletal inhibitor one day before, during or after the introduction of neurogenic transcription factor(s).
[0124] Placing the cell in conditions to support the transformation of the desired cell, and/or maintaining the cell in culture conditions that support the transformation of the desired cell may comprise culturing the cell in a medium comprising one or more factors suitable for inducing the expression of the morphological and functional characteristics of the desired cell of a different type. In some embodiments the one or more factors are reprogramming factors useful in reprogramming a cell and these reprogramming factors can be used alone or in combinations.
[0125] In other embodiments, the step of culturing the cell in a medium comprising one or more factors suitable for inducing the expression of the morphological and functional characteristics of the desired cell of a different type is performed subsequent to or simultaneously with steps (iii) or (iv), or subsequent to or concurrently with steps (1) or (2), as defined above. Those skilled in the art are aware of many different types of media and many reprogramming factors that can be useful in reprogramming a cell and these reprogramming factors can be used alone or in combinations. In various embodiments, the reprogramming factor is selected in accordance with Table B.
[0126] In some embodiments, the reprogramming factors have one or more of the following functions: decreasing the expression of one or more markers of the first cell type and/or increasing the expression of one or more markers of the desired cell. Cells that exhibit a selectable marker for the desired cell are then selected and evaluated for unipotency, multipotency, pluripotency, or similar characteristics (as appropriate).
[0127] In particular embodiments, cells are cultured in serum-free medium before, during or after any of steps (i) to (iv) as defined hereinbefore, or during or after steps (1) or ( 2) as defined hereinbefore. Obtaining neural stem cells (NSLCs)
[0128] According to preferred embodiments for the creation of neural stem cells (NSLCs), the methods of the invention are carried out such that the cells are treated with selected agents, compounds and factors to promote reprogramming and/or dedifferentiation in towards the stem cells (SLCs). Such somatic reprogrammed cells can then be further treated with agents and/or cultured under suitable conditions to promote reprogramming to neural stem cells (NSLCs), and long-term expansion of the NSLCs. NSLCs according to the invention have the potential to differentiate neuronal and/or glial type cells, as well as neuronal cells and/or glial cells, for potential treatment of neurological diseases and injuries, such as Parkinson's disease and spinal cord injury. spinal. The methods described herein are also useful for producing histocompatible cells for cell therapy.
[0129] Consequently, some aspects of the present invention relate to the generation of neurons from an individual patient, thus making possible autologous transplants, as a treatment modality for many neurological conditions, including neurotrauma, stroke, neurodegenerative diseases, such as multiple sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease. Thus, the invention provides neurological therapies to treat the disease or trauma of interest.
[0130] Therefore, another aspect of the invention concerns a method of obtaining a neural stem cell type (NSLC), comprising either: (1) contacting the cell of a first type with one or more neural stem cell regulatory polypeptides capable of increasing intracellular levels of neural stem cell-specific polypeptides within said cell and directly or indirectly remodeling the cell's chromatin and/or DNA and directly or indirectly driving the transformation of the first-type cell into an NSLC; or (2) contacting the chromatin and/or DNA of a cell of a first type with a histone acetylator, a histone deacetylation inhibitor, a DNA demethylation inhibitor, and/or a DNA methylation inhibitor, and increasing the intracellular levels of at least one specific neural stem cell-specific polypeptide leading directly or indirectly to the transformation of the cell of the first type into an NSLC.
[0131] In preferred embodiments, step (1) comprises increasing intracellular levels of a Musashi1 polypeptide. As will be explained below, this can be accomplished by various means, including, but not limited to, transient expression of the Musashi1 polypeptide, preferably by transfection of an expression vector encoding the polypeptide.
[0132] In preferred embodiments, step (2) comprises increasing intracellular levels of an MBD2 polypeptide or treating the cells with VPA and 5-AZA. As will be explained below this can be achieved by various means, including, but not limited to, transient expression of the MBD2 polypeptide, preferably by transfection of an expression vector encoding the polypeptide(s), and/or pre- -treatment and/or treatment of cells with VPA and 5-AZA.
[0133] In a particular embodiment, the reprogramming of a cell of a first type to another type of cell that presents at least two selectable markers for neural stem cells requires transfection of the cell of a first type, with a vector containing a cDNA that codes for a neurogenic transcription factor and a DNA demethylator. To improve dedifferentiation, cells are exposed or pre-exposed to an agent(s) that inhibits DNA methylation, inhibits histone deacetylation, and/or disrupts the cell's cytoskeleton. For example, dedifferentiation can be increased by pre-treating the cells with an agent that disrupts the cell's cytoskeleton followed by transfection of the cells with one or more vector(s) containing two neurogenic transcription factors, in the presence of a DNA demethylator and/or inhibitor of DNA methylation and histone deacetylation. Histone deacetylator, histone deacetylation inhibitor, DNA demethylation, and/or a DNA methylation inhibitor are as defined above.
[0134] As defined above, the method may further comprise a preliminary step of pre-treatment of the cell of a first type, with a cytoskeleton disruptor, as defined above, and/or culturing the cell in a medium comprising one or more suitable reprogramming factors for the appearance and maintenance of the morphological and functional characteristics of the NSLCs as defined above (eg a retinoid compound, a neurotrophic factor, bFGF, EGF, SHH, Wnt 3a, neuropeptide Y, Estrogen). In some embodiments, the method further comprises inhibiting BMP cell signaling pathways (e.g., NOGGIN, fetuin, or follistatin).
[0135] In preferred embodiments, generating an NSLC from a first cell comprises the use of one or more reprogramming agents. Suitable agents include, but are not limited to, Musashi-1 (Msi1) and Neurogenin 2 (Ngn2). Other potential agents are listed in Table A and B.
[0136] The present invention is also directed to the use of DNA expression vectors that encode a protein or transcript that increases the expression of neurogenesis. The genetically engineered DNA sequence encoding a reprogramming agent defined as Msi1 and Ngn2 can be introduced into cells using mono-, bi-, or poly-cistronic vectors. The expression of an endogenous multipotent gene indicates that the cDNA encodes a protein whose expression in the cell results, directly or indirectly, in dedifferentiation of the cell. Newly differentiated mammalian cells are able to re-differentiate to neuronal lineages to regenerate said mammalian cells, tissues and organs.
[0137] The present invention is further directed to a method to generate NSLCs by introducing a genetically engineered DNA sequence into human somatic cells through transient transfection. Since the DNA introduced in the transfection process is not inserted into the nuclear genome, the exogenous DNA decreases over time and when cells undergo mitosis. Non-viral vectors remain in a non-replicating form, have low immunogenicity, and are easy and safe to prepare and use. Furthermore, plasmids can accommodate large DNA fragments.
[0138] In a particular embodiment, the method starts with obtaining cells from the individual, and reprogramming the cells in vitro to generate NSLCs. The significant aspect of the present invention is the stable reprogramming of a somatic cell or non-neuronal cell into an NSLC which can give rise to different types of neuronal or glial cells (including neuronal or glial type cells). These can then be implanted back into the same patient from which the cells were obtained, thus making an autologous treatment modality for many neurological conditions, including neurotrauma, stroke, possible neurodegenerative disease, and possible. These can also be implanted into a different individual from which the cells were obtained. Therefore, the cells and methods of the present invention can be useful to treat, prevent, or stabilize a neurological disease, such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, or spinal cord injury. This technology provides a broad source of neural stem cells, neuroprogenitor cells, neurons and glia for clinical treatment, which can be performed by implantation of NSLCs in vivo or in vitro differentiation induction and implantation of neuroprogenitor cells or specific neurons or glia in vivo .
[0139] In another embodiment, the method comprises isolating somatic cells or non-neuronal cells and exposing the cells to one or more agents that change the cell morphology and chromatin structure, and transfection of the cells with one or more genes that contain at least one cDNA encoding a neurogenic transcription factor. The gene transfection step can be replaced with alternative agents that induce the expression of the neurogenic transcription factor(s) in the cell. Induction of epigenetic modifications to DNA and histones (especially DNA demethylation and an open chromatic structure) facilitates actual cell reprogramming. In another embodiment, cells are incubated in an atmosphere of low oxygen, for example 5% O2, thus aiding in cell reprogramming.
[0140] This methodology allows the reprogramming of a cell in an NSLC. The further course of development and expansion of the reprogrammed cells depends on the in situ environment to which they are exposed. Embodiments of the invention further include growing the reprogrammed cell in an appropriate proliferation medium to expand the generated NSLC, for example Neural Progenitor Proliferation Medium (StemCell Technologies) with the presence of epidermal growth factor (EGF) and growth factor of basic fibroblast (bFGF), to promote the proliferation of neural stem cells.
[0141] The NSLCs obtained according to the invention can be differentiated into neuron, astrocyte and/or oligodendrocyte lineages in suitable differentiation medium, for example NS-A differentiation medium (StemCell, Technologies) or NbActive medium (BrainBitsTM), including a retinoid compound, such as all-trans-retinoic acid or vitamin A, and BDNF, to induce differentiation of NSLCs into neuronal and/or glial cells. Neuronal cells include cells that display one or more neurospecific morphological, physiological, functional and/or immunological characteristics associated with a type of neuronal cell. Useful criteria features include: morphological features (eg, long processes or neurites), physiological and/or immunological features such as the expression of a set of neuronal-specific markers or antigens, synthesis of neurotransmitter(s) such as dopamine or gamma aminobutyric acid (GABA), and functional characteristics such as ion channels or potential actions characteristic of neurons.
[0142] According to the method, reprogrammed cells can be selected based on differential adhesion properties compared to non-transfected cells, for example, reprogrammed cells can form floating neurospheres or grow in treated plates in wells on laminin while fibroblasts do not transfected cells attach and grow in well-treated plates with regular cell culture. Reprogrammed cells include cells that exhibit one or more specific neural stem cell markers and morphology and loss of some or all of the specific markers related to the original cells. Furthermore, some of the functionalities of neural-like cells (NLCs) can be assessed at different time points, for example, by PatchClamp technique, immunostaining for synaptophysin and MAP2b, and by immunochemical means such as enzyme-linked immunosorbent assay (ELISA).
[0143] In certain embodiments, the present invention provides NSLCs that are capable of initiating and directing central nervous system regeneration at a site of tissue damage and can be customized for individual patients using their own cells as donor cells or starting cell . The present invention can be used to generate cells from an individual patient, thus making autologous transplants possible as a treatment modality for many neurological conditions. Thus, this technology eliminates the problems associated with non-host cell transplants, such as immune rejection and risk of communicable disease. The great advantage of the present invention is that it provides a substantial unlimited supply for autologous grafts suitable for transplantation. Therefore, it will avoid some significant problems associated with current source materials and transplantation methods. Polynucleotide Distribution
[0144] In certain embodiments, the invention relates to the use of polynucleotides, for example, a polynucleotide encoding an MBD2 polypeptide, a Musashi1 polypeptide and/or a Ngn2 polypeptide. Means for introducing polynucleotides into a cell are well known in the art. Methods of transfection of a cell, such as nucleofection and/or lipofection, or other types of transfection methods can be used. For example, a polynucleotide encoding a desired polypeptide can be cloned into intermediate vectors for transfection into eukaryotic cells for replication and/or expression. Intermediate vectors for nucleic acid storage or manipulation or protein production can be prokaryotic vectors, (eg plasmids), shuttle vectors, insect vectors, or viral vectors, for example. A desired polypeptide can also be encoded by a fusion nucleic acid.
[0145] To obtain expression from a cloned nucleic acid, it is typically subcloned into an expression vector that contains a promoter to direct transcription. Suitable bacterial and eukaryotic promoters are well known in the art and described, for example, in Sambrook and Russell (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press). The promoter used to direct expression of a nucleic acid of choice depends on the particular application. For example, a strong constitutive promoter is commonly used for expression and purification. In contrast, when a dedifferentiating protein or compound is to be used in vivo, either a constitutive or an inducible promoter or compound is used, depending on the particular use of the protein. Furthermore, a weak promoter can be used, such as HSV TK or a promoter having similar activity. The promoter may also typically include elements that are sensitive to transactivation, for example, hypoxia response elements, Ga14 response elements, lac repressor response element, and small molecule control systems such as tet-regulated and o RU-486 system.
[0146] In addition to a promoter, an expression vector typically contains a transcription unit or expression cassette that contains additional elements necessary for the expression of the nucleic acid in host cells, either prokaryotic or eukaryotic. A typical expression cassette thus contains a promoter operably linked, for example, to the nucleic acid sequence, and the necessary signals, for example, for efficient polyadenylation of the transcript, transcription termination, ribosome binding, and/or translation termination. Additional elements of the cassette may include, for example, heterologous “spliced” intronics and enhancers.
[0147] Expression vectors containing regulatory elements from eukaryotic viruses are often used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus. Other examples of eukaryotic vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, pDSVE baculovirus, and any other vector that allows expression of proteins under the direction of the SV40 early promoter, SV40 late promoter, the SV40 promoter. metallothionein, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters showing efficacy for expression in eukaryotic cells.
[0148] Standard transfection methods can be used to produce bacteria, yeast, insect or other cell lines that express large amounts of dedifferentiation proteins, which can be purified, if desired, using standard techniques. Transformation of eukaryotic and prokaryotic cells is performed according to standard techniques.
[0149] Any procedure for introducing exogenous nucleotide sequences into host cells can be used. These include, but are not limited to, use of calcium phosphate transfection, DEAE-dextran mediated transfection, polybrene, protoplast fusion, electroporation, lipid-mediated delivery (e.g., liposomes), microinjection, particle bombardment, a introduction of denatured DNA, plasmid vectors, viral vectors (both episomal and integrative) and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other exogenous genetic material into a host cell (see, eg, Sambrook et al., supra). It is only necessary that the particular genetic engineering procedure be able to successfully introduce at least one gene into the host cell capable of expressing the protein of choice.
[0150] Conventional virus and non-virus based gene transfer methods can be used to introduce the nucleic acids into mammalian cells or target tissues. Such methods can be used to deliver nucleic acids encoding reprogramming polypeptides to cells in vitro. Preferably, the nucleic acids are administered for in vivo or ex vivo gene therapy uses. Non-viral vector delivery systems include DNA plasmids, denatured nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.
Non-viral nucleic acid delivery methods include lipofection, microinjection, ballistics, virosomes, liposomes, immunoliposomes, polication or lipid-nucleic acid conjugates, denatured DNA, artificial virions, and increased uptake of DNA agent. Lipofection reagents are sold commercially (eg, TransfectamTM and LipofectinTM). Cationic and neutral lipids suitable for efficient polynucleotide receptor-recognition lipofection are known. Nucleic acid can be delivered to cells (ex vivo administration) or target tissues (in vivo administration). The preparation of lipid:nucleic acid complexes, including target liposomes such as immunolipid complexes, is well known to those skilled in the art.
[0152] The use of RNA or DNA virus-based systems for the delivery of nucleic acids takes advantage of highly evolved processes to target a virus to specific cells in the body and transit the viral load to the nucleus. Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro, where the modified cells are administered to patients (ex vivo). Conventional viral-based systems for delivery include retroviral, lentiviral, poxviral, adenoviral, adeno-associated viral, vesicular stomatitis viral, and herpesviral vectors, although integration into the host genome is possible with certain viral vectors, including retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long-term expression of the inserted transgene. Furthermore, high transduction efficiencies have been observed in many different types of cells and target tissues.
[0153] pLASN and MFG-S are examples of retroviral vectors that have been used in clinical trials. In applications for which transient expression is preferred, adenoviral-based systems are useful. Adenovirus-based vectors are capable of very high transduction efficiency in many cell types and are able to infect and therefore deliver nucleic acid to both divided and non-divided cells. With such vectors, high titers and expression levels were obtained. Adenovirus vectors can be produced in large quantities in a relatively simple system.
[0154] Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (eg, intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application. Alternatively, vectors can be delivered to ex vivo cells, such as cells explanted from an individual patient (eg, lymphocytes, bone marrow aspirates, tissue biopsy) or hematopoietic stem cells from universal donors, followed by reimplantation of the cells into a patient , usually after selecting cells that have been reprogrammed.
[0155] Ex vivo transfection of cells for diagnosis, research, or for gene therapy (for example, by re-infusion of the transfected cells into the host organism) is well known to those skilled in the art. In a preferred embodiment, cells are isolated from the individual's body, transfected with a nucleic acid (gene or cDNA), and reinfused back into the individual's body (e.g., the patient). Various cell types suitable for ex vivo transfection are well known to those skilled in the art.
[0156] Vectors (for example, retroviruses, adenoviruses, liposomes, etc.) containing therapeutic nucleic acids can also be directly administered to the body for transfection of cells in vivo. Alternatively, denatured DNA can be administered. Administration is by any of the routes normally used to introduce a molecule into final contact with blood cells or tissue. Suitable methods of administering such nucleic acids are available and well known to those skilled in the art, and although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate reaction. and more effective than another route.
[0157] Pharmaceutically acceptable carriers are determined, in part, by the particular composition being administered, as well as the particular method used to administer the composition. Therefore, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention. Distribution of polypeptides
[0158] In most, if not all of the methods described here, an alternative possibility is to bypass the use of a polynucleotide and contact a cell of a first cell type directly with a compound (eg, a polypeptide) for which it is An increase in the intracellular level is desired. In other embodiments, for example, in certain in vitro situations, cells are cultured in a medium containing one or more functional polypeptides.
[0159] An important factor in administering polypeptides is ensuring that the polypeptide has the ability to cross the plasma membrane of a cell, or the membrane of an intracellular compartment such as the nucleus. Cell membranes are composed of lipid-protein bilayers that are freely permeable to small, nonionic lipophilic compounds and are inherently impermeable to polar compounds, macromolecules, and therapeutic or diagnostic agents. However, proteins, lipids and other compounds have been described which have the ability to translocate polypeptides across a cell membrane. For example, "membrane translocation polypeptides" have amphiphilic or hydrophobic amino acid subsequences that have the ability to act as membrane translocation vehicles. Polypeptides for which an increase in the intracellular level is desired, according to the invention, can be linked to suitable peptide sequences to facilitate their absorption into cells. Other suitable chemical moieties that provide enhanced cellular uptake can also be linked, either covalently or non-covalently, to the polypeptides. Other suitable vehicles that are capable of transporting polypeptides across cell membranes can also be used. A desired polypeptide can also be introduced into an animal cell, preferably a mammalian cell, via liposomes and liposome derivatives, such as immunoliposomes. The term "liposome" refers to vesicles made up of one or more concentrically ordered lipid bilayers that encapsulate an aqueous phase. The aqueous phase usually contains the compound to be released into the cell. In certain embodiments, it may be desirable to label a liposome using labeled moieties that are specific to a particular cell type, tissue, and the like. Labeled liposomes, which utilize a variety of labeled moieties (eg, ligands, receptors, and monoclonal antibodies) have been described previously. Cells and cell lines
[0160] The invention encompasses cells, cell lines, stem cells and purified cell preparations derived from any of the methods described herein. In some embodiments, the cells, cell lines, stem cells and purified cell preparations of the invention are of mammalian origin, including but not limited to humans, primates, rodents, dog, cat, horse, cow, sheep, or. In preferred embodiments, they originate from a human.
[0161] Therefore, another aspect of the invention relates to modified cells, cell lines, pluripotent, pluripotent, multipotent or unipotent cells and purified cell preparations, wherein any of these cells comprises an exogenous polynucleotide encoding Musashi1 (Msi1); Msi1 and Ngn2; Msi1 and MBD2; and Ngn2 and MBD2; Msi1, Ngn2 and MBD2; Msi1, Ngn2, nestin and MBD2; and other possible combinations from Table A, preferably including Msi1 and Ngn2 and MBD2. In preferred embodiments, the cell according to the invention is a stem cell, more preferably a neural stem cell (NSLC), the cell having one or more of the following characteristics: - Expression of one or more neural stem cell marker selected from the group consisting of Sox2, Nestin, GFAP, Msi1, and Ngn2; - Decreased expression of one or more cell-specific genes that NSLC was obtained (eg see Table C); - Form neurospheres in the assay of neurosphere colony formation; - Able to be cultivated in suspension or as an adherent culture; - Able to proliferate without the presence of an exogenous reprogramming agent for more than 1 month, preferably more than 2 months, in addition to 3 months, more than 5 months and even for more than a year; - Able to divide every 36 hours at low passage; - Positive for telomerase activity; - Able to differentiate into a neuronal type cell, astrocyte type cell, oligodendrocyte type cell and their combinations; - Decreased expression of telomerase and one or more neural stem cell markers after differentiation; - It has one or more neurite-like morphological processes (axons and/or dendrites) greater than one cell diameter in length after differentiation in a neuronal-like cell;- Expression of at least one neural-specific antigen selected from the group consisting of specific neural tubulin, microtubule-associated protein 2, NCAM, and marker for a neurotransmitter after differentiation into a neuronal-like cell;- Expression of a or more neural functional markers (eg synapsin) after differentiation into a neuronal-like cell; - Able to release one or more neurotrophic factors (eg, BDNF) after differentiation into a neuronal-like cell; - Negative in a neuronal-forming assay tumor colony;- Negative for tumor growth in SCID mice;- Negative for teratoma growth in SCID mice;- Able to improve itself significantly one or more functional measurements after placing an adequate number of NSLCs in the void in a brain ablation model; - Able to significantly improve or maintain one or more functional measurements after injecting an adequate number of NSLCs into an EAE model, and - able to improve one or more functional measurements more significantly than hNPCs in CNS lesion or models neurodegeneratives.-
[0162] Examples of all of the above can be found in the Examples of this application.-
[0163] In preferred embodiments, an NSLC according to the inventions has all of the following characteristics:- Ability to self-renew much longer than a somatic cell;- It is not a cancer cell;- It is stable and not artificially maintained by forced gene expression or similar means and may be maintained in standard neural stem cell media; - May differentiate from a parent, precursor, somatic cell, or other more differentiated cell type of the same lineage; - It has the characteristics of a stem cell, not just certain markers or gene expression or morphological appearance, and- Does not show uncontrolled growth, teratoma formation, and tumor formation in vivo.
[0164] In a particular embodiment, the reprogrammed cells (NSLCs) according to the invention are able to proliferate for several months without losing their neural stem cell markers and their ability to differentiate into neuron-like, astrocyte-like cells, and oligodendrocyte type. The generation of neural lineages is characterized based on morphology, phenotypic changes and functionality.
[0165] In some embodiments, the cells of the invention may have one or more of the following characteristics and properties: self-renewal, in vitro and in vivo multilineage differentiation, clonogenicity, a normal karyotype, extensive proliferation in vitro under culture conditions well defined, and the ability to be frozen and thawed, as well as any of the commonly known and/or desired properties or typical characteristics of stem cells. Cells of the invention may further express multipotent or pluripotent cell molecular markers (i.e. genes, surface markers as defined above).
[0166] Another aspect of the invention relates to the production of autologous stem cell (own of the individual) tissue-specific and/or progenitor cells. These stem cells and/or progenitor cells can be used in cell therapy applications to treat cell degeneration diseases. Cellular degeneration diseases include, for example, neurodegenerative diseases such as stroke, Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, macular degeneration, osteolytic diseases such as osteoporosis, osteoarthritis, bone fissures, bone fractures , diabetes, liver damage, degenerative diseases, myocardial infarction, burns and cancer. It is anticipated that cells according to the invention can be implanted or transplanted into a host. An advantage of the invention is that large numbers of autologous progenitor cells can be produced for implantation without the risk of immune-mediated rejection. These cells can lead to the production of tissue suitable for transplantation in the individual. Since the tissue is derived from the transplant recipient, it should not stimulate an immune response as it would if the tissue were from an unrelated donor. Such transplants may consist of tissues (eg, vein, artery, skin, muscle), solid organ transplants (eg, heart, liver, kidney), neuronal cell transplants, or bone marrow transplants, as they are. used in the treatment of various malignancies, such as, for example, leukemias and lymphomas. Neural, neuroprogenitor, or neuronal stem cell transplants (as well as NSLCs and derivations thereof) can also be used in the treatment of, for example, neurological disorders, stroke, spinal cord injury, Parkinson's disease, and the like, as well as potentially in some non-neurological disorders, such as a cardiac infarction.
[0167] Another aspect of the invention relates to a method for producing engineered tissues ex vivo for subsequent implantation or transplantation into a host, wherein the cellular components of said engineered tissues comprise cells according to the invention, or cells derived therefrom. For example, expanded cultures of the cells of the invention can be differentiated by in vitro treatment with growth factors and/or morphogens. The differentiated cell populations are then implanted into the recipient host near the site of injury or damage, or cultured in vitro to generate engineered tissues, as described.
[0168] The methods and cells of the invention described herein can be used to immortalize cells, for example, to generate a cell lineage. Using the methods described herein, a somatic cell can be transformed into one having a dedifferentiated phenotype, thus facilitating the generation of cell lines from a variety of tissues. Therefore, the invention encompasses such immortalized cells.
[0169] In addition, the cell derivation methods according to the invention may be useful in scientific and therapeutic applications, including, but not limited to, (a) scientific discovery and research involving cell development and genetic research (by example, uses in place of human stem cells as a model cell line to study the differentiation, dedifferentiation, or reprogramming of human cells), (b) drug development and discovery (eg, screening for efficacy and toxicity of certain candidate drugs and chemicals, selection of potential drugs or agents that mediate differentiation, dedifferentiation, or reprogramming of cells), (c) gene therapy (eg, as a delivery device for gene therapy), and (d) treatment of injuries, trauma, diseases and disorders, including, but not limited to, Parkinson's, Alzehimer, Huntington, Tay-Sachs disease, Gauchers, spinal cord injury, stroke, burns and other skin damage, heart disease, diabetes, lupus, osteoarthritis, liver disease, hormonal disorders, kidney disease, leukemia, lymphoma, multiple sclerosis, rheumatoid arthritis, Duchenne muscular dystrophy, Ontogenesis Imperfecto, birth defects, infertility, abortion, and others types of cancer, degenerative and other diseases and disorders.
[0170] Additional aspects relate to therapeutic methods, methods of treatment and methods of tissue or organ regeneration in a mammal (eg, a human subject). A particular method concerns a method of regenerating a mammalian tissue or organ which comprises contacting the tissue or organ to be regenerated with an SLC, NSLC, or other desired cell or artificial tissue construct as defined herein. The SLC, NSLC, desired cell or artificial tissue construct can be placed in close proximity to the tissue or organ to be regenerated by administration to the subject using any suitable route (eg, by injecting the cell intrathecally, directly into the tissue or organ, or within the bloodstream).
[0171] Another method for repairing or regenerating a tissue or organ in a subject in need thereof comprises administering to the individual a compound that induces a direct or indirect endogenous expression of at least one gene regulator in cells of the tissue or organ and/or a compound that induces a direct or indirect endogenous expression of at least one gene regulator in cells capable of transformation or dedifferentiation in vivo in the individual. Therefore, expression of the at least one gene regulator reprograms the cells into desired cells of a different type (e.g. neural-type stem cells), and these cells of a different type are effective in repairing or regenerating said tissue or organ. .
[0172] Another method comprises obtaining cells or tissue from a patient (eg hematopoietic stem cells, fibroblasts, or keratinocytes), reprogramming a plurality of such cells or tissue, and reintroducing the reprogrammed cells or tissue into the patient. A related aspect pertains to pharmaceutical compositions comprising a desired plurality of cell, SLC and/or neural-like stem cell (NSLC) or reprogrammed tissue as defined herein.
[0173] The therapeutic methods of the invention may be applicable to the regeneration or repair of various tissues and organs, including, but not limited to, brain, spinal cord, heart, eyes, retina, cochlea, skin, muscles, intestines, pancreas (including beta cells), kidneys, liver, lungs, bones, bone marrow, cartilage, cartilage discs, hair follicles, teeth, blood vessels, endocrine glands (including and exocrine glands), ovaries, reproductive organs, mammary and breast tissue .
[0174] A related aspect pertains to pharmaceutical compositions comprising a plurality of desired cells, SLC and/or neural-like stem cell (NSLC) as defined herein. fabrics
[0175] Another aspect of the invention concerns a tissue containing reprogrammed cells, as defined herein, which can be implanted in an individual in need. In some embodiments of the present invention envisions the reprogramming of cells within a tissue, for example a tissue construct produced in vitro in 3D comprising cells and extracellular matrix produced by these cells. Furthermore, transfected cells can be seeded on top of these 3D tissue constructs which can be made completely autologous, thus preventing host rejection, making it completely immunocompatible and as a vehicle for reprogrammed cells to be transplanted in vivo. Advantageously, these newly created cells can be used in their undifferentiated and/or differentiated state within these tissues for in vitro diagnostic purposes or transplanted into a patient in need of such a construct cell therapy/tissue replacement approaches.
[0176] The invention further encompasses 3D neuronal type multilayer tissue. Cells within CDM reprogrammed to Neural-like stem cells according to the invention readily differentiate into neuronal-like cells, astrocyte-like cells, and oligodendrocyte-like cells within the CDM. Thus, it is possible to use the CDM and reprogramming methods of the invention to reprogram the cells within the CDM to form 3D neuronal-like multilayer tissue (up to >30 layers of cells). Such 3D tissue comprises neurons (or specifically, neuron-like cells), astrocytes (or specifically, astrocyte-like cells), and oligodendrocytes (or specifically, oligodendrocyte-like cells) and can be made completely autologously, can be handled and manually implanted with relative ease , or can be used as an in vitro tissue model in CNS.
[0177] A particular aspect concerns an artificial tissue construct, which comprises a 3D set of in vitro cultured cells and extracellular matrix produced by these cells. The cells can be desired cells, SLC and/or a plurality of Neural-like stem cells (NSLC) obtained by any of the methods described herein. Screening Methods
[0178] Another aspect of the invention concerns methods for identifying new compounds (eg, small molecules, drugs, etc.), capable of transforming a cell of a first type into a desired cell of a different type. These new compounds may be useful for research purposes or as medicines for use in tissue repair or regeneration in an individual.
[0179] The Examples section provides principles, methods, and techniques useful for screening and identifying such desirable active compounds. For example, those skilled in the art will understand that it is conceivable to screen for compounds that will induce the transformation of a cell of a first type to an NSLC by replacing the "induction" or "biological activity" provided by the transient increase of Musashi1, NGN2 or MBD2 in the cell by a candidate compound to be tested (eg, a library of small molecules or compounds) and measure the activity or efficacy of the candidate compound in generating NSLC. Single active compounds or mixtures of compounds would be selected if they have the same activity and/or if they can provide the same or similar effects as these polypeptides (eg cell transformation and/or the appearance of any desirable markers or desirable characteristics as defined herein before). For example, a compound or mixture of compounds capable of transforming a fibroblast into an NSLC could be identified by: (i) Adjusting, culturing and transforming fibroblasts into NSLC as in Example 1, (ii) Screening a compound library, replacing Msi1 , Ngn2 and/or MBD2 with each candidate compound from a different well; (iii) identify a "successful" compound when the candidate compound is capable of transforming fibroblasts into approximately NSLCs, as well as substituted Msi1, Ngn2 and/or MBD2; (iv) if the compound from part (iii) does not replace all of Msi1, Ngn2 and MBD2, and is not able to transform fibroblasts into NSLCs by itself, then by including the compound from (iii) in each well, screen a compound library by replacing Msi1, Ngn2 and/or MBD2 that was not removed in part (ii) with each candidate compound in a different well; (v) identify a "successful" compound when the candidate compound is able to transform , together with the compound of the pair te (iii), the fibroblasts in approximately NSLCs, as well as replaced Msi1, Ngn2 and/or MBD2; (vi) if the compound of part (v) does not replace all of Msi1, Ngn2 and/or MBD2, and is not able to transform the fibroblasts in NSLCs along with the compound from part (iii), then, by including the compound from (iii) and (v) in each well, screen a library of compounds by replacing the Msi1, Ngn2 or MBD2 that was not removed in part (ii) and (iv) with each candidate compound in a different well; (vii) Identify a "successful" compound when the candidate compound is capable of transforming, together with the compound in part (iii) and (v) , fibroblasts into NSLCs approximately as well as substituted Msi1, Ngn2 or MBD2; (viii) a combination of the compounds of part (iii), (v), and (vii) will be able to transform fibroblasts into NSLC; modifications to these compounds can be made and further screened to identify more effective or safer versions of these compounds.
[0180] The same principles are applicable to other desired types of stem-like cells including pluripotent cells, mesendoderm-like cells, pancreatic progenitor-like cells, etc. Tables A and B, and the Examples section, provide, for each of these cell types, a list of potential genes and/or compounds to be considered in these screening methods.
[0181] Therefore, the present invention encompasses these and all equivalent screening methods where candidate compounds are tested for their effectiveness with respect to transforming a cell of a first type to a desired cell of a different type when compared to the effectiveness of the gene reprogramming factor and/or regulator as defined herein. Distribution of neurotrophic factors
[0182] Local distribution of neurotrophic factors has been suggested as a method for the treatment of various neurological conditions. Strategies using neurotrophic molecules focus on preventing the progressive loss of neurons, maintaining neuronal connections and function (neuroprotection), and inducing additional regenerative responses in neurons, such as increased neurotransmitter volume and/or axonal emergence (neuroregeneration). To date, several therapeutic strategies to deliver neurotrophic factors in animal models have been explored, but until now testing the effects of growth factors on the brain and nervous system has been limited to direct peripheral injection of large doses of these factors, which entails a significant risk of side effects. Therefore, a related aspect of the invention relates to overcoming these problems using NSLC cells and cell lines according to the invention, which can stably express and secrete growth factors of potential interest after transplantation.
[0183] To summarize, the present invention provides an abundant source of neural stem cells, neuron-like cells, astrocyte-like cells and oligodendrocyte-like cells for potential clinical treatments that require the transplantation of neural stem cells, neurons, astrocytes and oligodendrocytes (1 ) to compensate for the loss of host cells (eg neurons), or (2) as vehicles to deliver genetically based drugs. In addition, the invention provides a new neurological tool for use in baseline drug research and screening.
[0184] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims and examples described herein. Such equivalents are considered to be within the scope of the present invention and covered by the appended claims. The invention is further illustrated by the following examples, which are not to be construed as limiting. Examples
[0185] The examples set forth herein below provide exemplary methods for obtaining reprogrammed and dedifferentiated cells, including neural stem cells (NSLCs). Exemplary protocols, molecular tools, probes, primers, and techniques are also provided. EXAMPLE IPreparation of Human Fibroblast Cells
[0186] Human Foreskin Fibroblast (HFF), cells were purchased from the American Type Culture Collection (ATCC, Manassas, Va) and expanded in cell culture flasks with Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% heat-inactivated fetal calf serum (FCS, Hyclone Laboratories), 0.1 mM non-essential amino acids, and 1.0 mM sodium pyruvate (Invitrogen), at 37°C, 5% CO 2 . The medium was changed twice a week. Cells were trypsinized using 0.25% trypsin for 4 minutes at 37°C, followed by addition of trypsin inhibitor solution, pelletizing the cells by centrifugation, washing the cells once with PBS, and plating the cells at a rate of 1:2 in tissue culture flasks until an adequate number of cells is reached.
[0187] Cells were then trypsinized and plated (8x104 cells/well) in cell culture plates pre-coated with laminin (10µ/ml, Sigma) in two different compositions of CDM medium: CDM 1 medium consisting of a ratio of 3:1 Dubelcco's modified Eagle's medium (DMEM, high glucose (4.5 g/L) with L-glutamine and sodium pyruvate) and Ham's F-12 medium supplemented with the following components: EGF (4.2x10 -10M), bFGF (2.8x10-10M), ITS (8.6x10-5M), dexamethasone (1.0x10-7M), L-3,3',5-triiodothyronine (2.0x10-10M), ethanolamine (10-4M), GlutaMAXTM (4x10-3M), and glutathione (3.3x10-6M), but without the presence of L-ascorbic acid.
[0188] CDM II medium consisting of a 3:1 ratio of Dubelcco's modified Eagle's medium (DMEM, high glucose (4.5 g/L) with L-glutamine and sodium pyruvate) and Ham's F-12 medium supplemented with the following components: EGF (2.5 ng/ml), bFGF (10ng/ml), ethanolamine (2.03mg/ml), insulin (10mg/ml), Selenious acid (2.5μg/ml), dexamethasone (19.7 μg/ml), L-3,3',5-triiodothyronine (675 ng/ml), GlutaMAXTM (4x10-3M), and glutathione (3.3x10-6M). Transient transfection of HFF by Lipofectamine using constructed vectors
[0189] After two days in culture, cells were transfected with pCMV6-XL5-MBD2 (2μg) (a DNA dedmethylator) using lipofectamine reagent (Invitrogen) according to the manufacturer's protocol. The DNA-lipid complex was added to the cells and incubated for 24 h at 37°C, 5% CO 2 . After 24 hours of transfection with the desmethylator fr DNA, the medium was changed and the cells were transfected by pCMV6-XL5-Musashi1 (2μg, Origene) or pCMV6-XL4-Ngn2 (2μg, Origene) for 24 h. After 24 hours, the medium was changed to Basal Progenitor Neural Medium (NPBM, Lonza) supplemented with Noggin (20 ng/ml, Peprotech), EGF (20ng/ml, Peprotech), and bFGF (20ng/ml, Peprotech) and cultured in this means of proliferation. Cells were retransfected after three days and incubated at 37°C, 5% CO2 and 5% O2. After 7 days, under proliferating conditions, 50% of the Proliferation Medium was changed to Differentiation Medium (NbActive, BrainBitsTM) supplemented with Forskolin (10μM, Tocris), an all-trans-retinoic acid (ATRA, 5μM, Spectrum), bFGF (20ng/ml, Peprotech), NGF (20ng/ml, Peprotech), and BDNF (20ng/ml, Peprotech); the medium was changed each day by increasing the percentage of Differentiation Medium over Proliferation Medium, and cells were cultured for 20 days.
[0190] Visual observation of the reprogrammed cells was performed by microscopic observation of light every day after transfection using bright field with a magnification of 10X. Samples were collected at different times (6, 12 and 20 days) to analyze neuronal gene expression and protein levels by genetic matrix and immunohistochemistry. After transfection, the reprogramming cells exhibited a rapid change in cell morphology within 3 days after transfection (Figure 1). The cells were more rounded and cell cytoplasm retracted towards the nucleus, forming contracted cell bodies with long cytoplasmic extensions and exhibiting a pericarial neuronal appearance on days 6 and 12, which was maintained until day 20. However, this morphology was not observed in untransfected cells on days 6 and 12. Gene Matrix Analysis
[0191] Characterization of newly engineered cells after transfection was performed using a neuronal gene array containing 48 partial cDNAs encoding these genes and controls.
[0192] RNA was isolated from samples using QIAshredderTM (Qiagen) and RNeasyTM Plus mini kit (Qiagen), as per the manufacturer's instructions. DNase I treatment was performed in RNeasyTM Column to remove transfected plasmid DNA using RNase DNase kit (Qiagen). RNA was eluted in 35μl of RNase-free water. Prior to cDNA synthesis, all RNA samples were quantified using the 1000TM NanoDrop (ThermoScientific). cDNA was prepared using the High Capacity cDNA Archive Kit (Applied Biosystems) per manufacturer's instructions. 400 ng of RNA was used in each 50μl of RT reaction. The resulting cDNA samples were used immediately for TLDA analysis. For each TaqmanTM Low Density Matrix (TLDA) card, there are eight distinct charge holes that feed 48 separate wells for a total of 384 wells per card. Each 2μl contains specific user-defined primers and probe, capable of detecting a single gene. In this study, a customized 2 TLDA neuronal marker was configured on eight identical sets of 48 genes, ie, 1 loading hole for each set of 48 genes. Genes were chosen based on the literature. Each set of 48 genes also contains three reference genes: ACTIN, GAPDH and PPIA.
[0193] A specific master sample mix was made for each sample by mixing cDNA (160 ng from each loading hole), 2X TaqmanTM Gene Expression Master Mix (Applied Biosystems) and Nuclease Free Water (USB) to a total of 100μl per charging hole. After gentle mixing and centrifugation, the mixture was then transferred to a loading hole on a TLDA card. The matrix was centrifuged twice for 1 minute, each at 1200 rpm to distribute samples from the loading hole in each well. The card was then sealed and PCR amplification was performed using Applied Biosystems' 7900HTTM Fast real-time PCR system. Thermocycler conditions were as follows: 2 minutes at 50°C, 10 minutes at 94.5°C, and 30 seconds at 97°C, 1 minute at 59.7°C for 40 cycles. 1 TLDA was prepared for 8 samples.
[0194] Relative expression values were calculated using the Comparative CT method. Briefly, this technique uses the formula 2-ΔΔCT to calculate the expression of target genes, normalized to a calibrator. The threshold cycle (CT) indicates the number of cycles in which the amount of amplified target reaches a fixed threshold. CT values range from 0 to 40 (the latter represents the cycle number of the upper-limit standard PCR, which defines not detecting a signal). ΔCT values [ΔCT = CT (target gene) - CT (mean of 3 cleansing genes)] were calculated for the HFF control and later used as a calibrator for the respective samples. All gene expression values have been assigned a relative value of 1.00 for the calibrator, which is used to determine the comparative gene expression such that ΔΔCT = ΔCT (Treated) - ΔCT (HFF Control). The relative expression is calculated using the formula 2-ΔΔCT
[0195] Quantitative comparison of astrocyte, neuron, and oligodendrocyte gene expression allowed the identification of most genes that are differentially expressed in reprogrammed cells. The data in Table 1 was analyzed using a significance analysis algorithm to identify genes that were reproducibly found to be enriched in reprogrammed cells compared to untransfected cells. After transfection with Msi1 or Ngn2 in the presence of MBD2, the expression of oligodendrocyte progenitors such as NKx2.2 and olig2, and MAG and two markers for astrocytes (GFAP and AQP4) were highly increased. Furthermore, several early neuronal cell markers were improved after HFF transfection. TDLA data revealed a notable increase in interneuron-specific markers such as somatostatin and calbindin1. Induction of Doublecortin (DCX), which is expressed by migration of immature cells during development, and acetylcholine mRNA (ACHE), an early marker of neuronal cells, were highly expressed in reprogrammed cells (Table 1). The transfection increased the expression of dihydropyrimidase-type 3 (DPYSL3), an early marker of newborn neurons, five-fold with Msi1 and seven-fold with Ngn2. The expression of Microtubule-Associated Protein Type 2 (MAP2), an essential marker for the development and maintenance of early neuronal morphology and neuronal cell adhesion molecule, was highly expressed with Msi1 and Ngn2 (Table 1). The expression of enolase-2, a marker of mature neurons, was 20 times higher by Msi1 and Ngn2. The NeuroD1 member of the NeuroD family was highly expressed after transfection with Msi1 84 fold and 34 fold by Ngn2.
[0196] Gene expression of growth factors such as IGF-1, IGF2, NPY and CSF-3 was also increased in reprogrammed cells. VEGF and GDNF gene expression were up-regulated to about five-fold and seven-fold by Msi1 and Ngn2, respectively. However, the expression of BDNF, EGF, and bFGF were not activated and even down-regulated compared to untransfected cells. Growth-associated protein expression (GAP-43), a marker associated with neuron growth and regeneration, and netrin expression, implicated in neuronal development and orientation, were highly enriched in reprogrammed cells. The expression of receptors for growth factors and neurotrophin was increased, such as type III receptor tyrosine kinase, neurotrophic tyrosine kinase, and neurotrophic receptor tyrosine kinase. Vimentin and fibronectin, markers for fibroblasts, were repressed in reprogrammed cells compared to untransfected control fibroblast cultures.Table 1: Msi1/MBD2 and Ngn2/MBD2 transfected human fibroblast cell gene matrix. Gene array was performed on samples after two weeks of differentiation. Expression values are given relative to untransfected fibroblasts.


Immunohistochemical analysis
[0197] Cells were fixed with a 4% formaldehyde/PBS solution for 10 minutes at room temperature and subsequently permeabilized for 5 min with 0.1% Triton X-100TM in 4% formaldehyde/PBS. After two brief washes with PBS, nonspecific antibody binding was blocked by a 30 min incubation with 5% normal goat serum in PBS. Then, primary antibodies were added in 5% normal goat serum / PBS as follows: from mouse anti-nestin (1:100, BD) as an intermediate microfilament present in neural stem cells and from mouse anti-NCAM (1: :100, Neuromics) as a neuronal adhesion molecule. After a 2 h incubation cells were washed 4 times for 5 min each with 0.1% Tween™/PBS. Appropriate fluorescence-labeled secondary antibody was used for visualization; was used goat anti-mouse 546 (1:200, Invitrogen) prepared in 5% normal goat serum / PBS. After incubation for one hour, cells were washed in 0.1% TweenTM / PBS three times for 5 min each. The DNA marker Hoechst33342 (Invitrogen) was used as a nuclei marker (1:5000 dilution in PBS, 10min incubation). Fluorescence images were taken with a CellomicsTM ArrayScan HCS reading microscopy system. To determine an estimate of the percentage of cells adopting neuronal or glial phenotypes, random fields were selected and for each field the total number of cells (as determined by Hoechst-labeled nuclei count) and the total number of positive cells were determined. for neuronal or glial markers.
[0198] To confirm that these cells exhibited neuronal lineage markers, the cells were immunostained for nestin and NCAM. This analysis revealed that the reprogrammed cells express both proteins. As shown in Figure 2, NCAM was present in the cells during 6 days after transfection and increases on days 12 and 20 after differentiation, whereas the reverse pattern was observed for nestin staining.
[0199] This study showed the ability to reprogram HFF cells using a neurogenic transcription factor with the presence of a DNA demethylator for cells that expressed neuronal genes and specific proteins for neural stem cells and neuronal cells. These reprogrammed cells were stable in culture for at least 2 weeks. EXAMPLE IIComparison of Reprogramming Efficiencies of Three Different Neurogenic Genes
[0200] HFF cells were cultured as described in Example I and plated in CDM I medium. Cells were transfected using an Amaxa NucleofectorTM Device (Lonza). HFFs were collected with TrypLETM (Gibco), resuspended in CDM medium and centrifuged for 10 min at 90xg (1x106 cells/tube). The supernatant was discarded and gently resuspended in 100μl of Basic NucleofectorTM solution (Basic NucleofectorTM kit for primary mammalian fibroblasts, Lonza). Each 100μl of cell suspension was combined with a different mixture of plasmid DNA (eg sample 1 was mixed with 2μg pCMV6-XL5-Pax6 and 2μg pCMV6-XL5-MBD2). Cell suspension was transferred to a certified cuvette and transfected with the appropriate program (U023). The sample was transferred, without any additional resuspension, to a LAS-Lysine/Alanine coated culture plate (BrainBitsTM, 50 µg/ml) and the cells were incubated at 37°C, 5% CO 2 . These steps were repeated for each sample that was transfected. After 24 hours, the medium was changed to proliferation medium. After two days, cells were retransfected using lipofectamine as described in Example I and incubated at 37°C, 5% CO2 and 5% O2. After 6 days, differentiation was induced with Differentiation Medium which gradually replaced the proliferation medium for several days. Cells were collected on day 14 for RT-PCR and immunohistochemical analysis. gene expression analysis
[0201] RNA isolation and quantification were performed as described above in Example I. cDNA was prepared using the High Capacity RT cDNA kit (Applied Biosystems) as per manufacturer's instructions, with a final cDNA concentration of 2ng/μl . Real-time PCR was then performed for each gene of interest using the FAST PCR master mix (Applied Biosystems) and TaqmanTM Gene Expression Assays (Applied Biosystems) listed below:

[0202] The FAST reaction in 96 wells was performed with 8 ng cDNA per well in a 10 μl reaction with 40 cycles. The Thermocycler conditions were as follows: 20 seconds at 95°C, and one second at 95°C, and 20 seconds at 60°C for 40 cycles.
[0203] Relative expression values were calculated as previously described in Example I, except the mean of 2 reference genes (GAPDH and PPIA) was used for normalization, instead of the mean of 3 reference genes. The identification of neuronal lineage genes was investigated after transfection with three independent vectors containing Msi1, Ngn2, and Pax6.
[0204] As shown in Table 2, after 14 days following transfection, the relative expression of neuronal lineage mRNA was not detected in untransfected cells (HFF), whereas cells transfected with Msi1 or Ngn2 in the presence of MBD2 expressed markers of neural stem cells (nestin and Sox2), however, Sox2 expression was much more highly expressed than nestin after transfection with Ngn2 or Msi1.
[0205] Neuronal and astrocyte-specific genes (βIII-Tubulin, MAP2b, GFAP, and ACHE) were also increased. The level of tripotent gene mRNAs-genes associated with βIII-Tubulin, MAP2b, aceticolin, and GFAP were undetectable in transfected PAX6 cells, indicating that Pax6 alone did not involve the reprogramming process for neuronal lineage. Table 2: Relative expression of different neuronal lineage gene expression performed by RT-PCR after HFF transfection by Msi1, Ngn2, or Pax6 in the presence of MBD2 and cultured for 14 days.
Immunohistochemical analysis
[0206] Fluorescent immunohistochemical staining was performed as previously described in Example I. According to the RT-PCR data, immunohistochemical analysis of these cultures revealed that the reprogrammed cells (with Msi1 or Ngn2) generated morphologically complex neurons that were positive for MAP2b, indicating the differentiation of NSLCs to neuron-like cells (NLCs) (Figure 3). However, positive staining for these markers was undetectable after transfection with Pax6/MBD2. Furthermore, newly formed neurons expressed the markers and developed long neurites with growth cones at their ends, expressed specific neural genes, and stopped proliferating when exposed to differentiating conditions. EXAMPLE III HFF transfection by various vector combinations and cell cytoskeleton disruption
[0207] Various combinations of neurogenic regulators and cytokines for epigenetic modifications have been tested to determine their effect on reprogramming efficiency. Starting one day before transfection, cells were treated with or without cytochalasin B (Calbiochem), with the concentration decreased each day for five days during media changes (starting with 10 μg/ml cytochalasin B on day 1 to 7.5 μg/ ml, 5 μg/ml, 2.5 μg/ml, and 0 μg/ml over the subsequent four days) in order to investigate the effect of disruption of the cell cytoskeleton on the reprogramming process. Cells were transiently transfected as described in Example II with one or two vectors containing a neurogenic transcription factor by nucleofection. Cells were co-transfected with either two DNA demethylators, such as MBD2 or GAdd45B, (eg 2x106 cells were transfected with pCMV6-XL5Msi1 (2μg) and pCMV6-XL5-MBD2 (2μg). After 24 hours, the medium was removed. switched to Neural Proliferation Medium (NeuroCult™ Proliferation Kit, StemCell Technologies) consisting of DMEM/F12 (1:1), glucose (0.6%), sodium bicarbonate (0.1%), glutamine (20 mM) , HEPES (5 mM), insulin (230 μg/ml), transferrin (100 μg/ml), progesterone (200 nM), putrescine (90 μg/ml) and sodium selenite (300nM) and supplemented with Noggin (20 ng /ml, Peprotech), recombinant hEGF (20ng/ml, Peprotech), and recombinant hEGF (20ng/ml, Peprotech) and the cells were cultured for two weeks at 37°C, 5% CO2 and 5% O2. were then analyzed for neural stem cell markers. gene expression analysis
[0208] Gene expression analysis was performed for neural stem cell specific markers (Sox2, nestin, GFAP) and a fibroblast specific marker (Col5A2) by RT-PCR as described above in Example I. The analysis of RT- PCR showed that the relative expression of Sox2, nestin and GFAP was improved after transfection of cells with transcription factors of neural origin. As shown in Table 3, transfection of cells with a transcription factor Msi1 in the presence of Gadd45b was associated with up-regulation of the relative expression of Sox2 (22.3 + 5.26) and GFAP (10.14 + 0.15 ) and the expression of these genes was highly increased when transfection of cells with Ngn2 doubled 20-fold and 10-fold, respectively. The combination of the two neurogenic factors (Msi1 and Ngn2) with Gadd45b further improved the expression of Sox2 and GFAP. Transfection of cells with a transcription factor (Msi1 or Ngn2) in the presence of MBD2 was associated with up-regulation of the relative expression of Sox2, nestin, and GFAP and down-regulation of Col5A2, while co-tranfection with Gadd45b was not increased nestin expression and Col5A2 expression was not regulated. Increased relative expression of neural stem cells was observed when transfection of cells with two neurogenic genes in combination with MBD2; a small increase in expression was noted in the presence of cytochalasin B under certain conditions. An increase in the relative expression of neural stem cell specific markers (Sox2, nestin, GFAP) and a decrease in the fibroblast specific gene (COL5A2) was observed after transfection with Msi1/Ngn2/MBD2, Msi1/Ngn2/Gadd45b, Msi1/ MBD2 or Ngn2/MBD2 (Table 3). This study demonstrated that MBD2 increased reprogramming efficiency more than GDA45b and showed that cytochalasin B had no effect of its own in control cultures.

Immunohistochemical analysis
[0209] Fluorescent immunohistochemistry staining was performed as previously described in Example I. Table 4 showed the percentage of nestin and Sox2 in each condition, with the highest percentage of Sox2 positive cells (38.18 + 1.75%) and nestin (28.18 + 2.77%) observed after transfection of cells simultaneously with both neurogenic transcription factors and in the presence of a DNA demethylator and cytochalasin B. A slight increase in SOx2 positive cells (10.42 + 10, 27%) and nestin positive cells (4.85 + 1.10%) were detected after transfection with a transcription factor Msi1 and MBD2. The same trend of nestin and Sox2 positive cells was observed after transfection with Ngn2 MBD2. Disruption of the citiesskeleton of cells with cytochalasin B significantly increased reprogramming, but had no reprogramming effect on its own (Table 4). or without the presence of cytochalasin B. After transfection, cells were cultured in proliferation medium (StemCell Technologies) supplemented with EGF (20ng/ml, Peprotech) and FGF (20ng/ml, Peprotech) for two weeks at 37°C/5 % CO2/5% O2. The percentage of immunopositive cells was determined by CellomicsTM and represented as mean + SD (n=3-5).

[0210] Several DNA demethylators have been tested as well for their effect on reprogramming efficiency. Cells were co-transfected with a vector (MSI1/NGN2) containing two neurogenic factors pCMV6-Msi1-Ngn2 with various DNA demethylators. Simultaneously another neurogenic factor was tested for its effect on dedifferentiating cells towards NSCs, pCMV-XL-nestin alone or in combination with pCMV-Msi1-Ngn2, pCMV-XL5-Msi1, or pCMV-XL4-Ngn2 in the presence of MBD2 as previously described in Example II.
[0211] Cells were co-transfected pCMV-Msi1-Ngn2 with different DNA demethylators (MBD1, MBD2, MBD3, MBD4, MeCP2, AICDA). Another trial was performed to assess the effect of nestin on reprogramming efficiency; therefore, cells were transfected with nestin individually or in combination with a vector containing either a neurogenic factor (Msi1 or Ngn2) or both neurogenic factors, in the presence of MBD2. Cells were cultured following transfection, in the presence of proliferation medium supplemented with EGF (20ng/ml), EGF (20ng/ml), and Noggin (20ng/ml) with and without VPA (1 mM) treatment for 12 days at 37°C, 5% CO2 and 5% O2.
[0212] Gene expression analysis and immunohistochemistry was performed to analyze neural specific gene and protein expression (βIII-tubulin, GFAP, Sox2, Nestin) as described in Example II. Cells transfected with Msi1 and Ngn2 in the presence of several DNA demethylators revealed and confirms previous data, showing that among several DNA demethylators used in this study, MBD2 promotes the expression of neural stem genes (Sox2, GFAP, nestin), as shown in Table 5. Furthermore, cells transfected with nestin with and without the presence of a neurogenic factor had no effect on reprogramming efficiency in neural stem cells. However co-transfection with nestin and Msi1/Ngn2/MBD2 increased the expression of neural stem cell genes and this increase was more pronounced in the presence of VPA.Table 5: RT-PCR analysis analysis of relative expression of neuronal precursor cell markers , such as nestin, Sox2, βIII-tubulin and GFAP after transfection of fibroblast cells with various combinations of pCMV-Msi1 -Ngn2 (MSI 1/NGN2), pCMV-XL5-Msi1, pCMV-XL4-Ngn2, pCMV-XL -nestine with different combinations of DNA demethylators, with and without VPA co-treatment.


[0213] Immunohistochemical analysis performed in parallel with RT-PCR data indicated that Sox2 positive cells were undetectable when transfection of cells with Msi1/Ngn2 in the presence of MBD1, MBD3, MBD4, MeCP1, or AICADA (Table 6 ) and that, among the different types of DNA demethylating genes tested, only MBD2 plays a significant positive role in the efficiency of HFF reprogramming for NSLCs when using the above neurogenic genes. Immunohistochemical analysis revealed a small increase in SOx2 immunopositive cells (89.49 + 3.18) after co-transfection of cells with nestin and Msi1/Ngn2 in the presence of MBD2 (Table 6). Table 6: Percentage of Sox2 positive cells after transfection of fibroblast cells with different expression vectors, with or without the presence of different DNA demethylators. After transfection, cells were cultured in proliferation medium (StemCell Technologies) supplemented with EGF (20ng/ml, Peprotech) and FGF (20ng/ml, Peprotech) for two weeks at 37°C / 5% CO2 / 5% O2 . The percentage of immunopositive cells was determined by Cellomics™ and represented as mean +SD (n = 3-5).

[0214] Another study was designed to test the effect of various neurogenic genes on reprogramming efficiency for neural stem cells. HFF cells were cultured as described in Example I, and transfected using a 96-well Shuttle® Nucleofector™ Device (Lonza) as per the procedure described in Example IV, except for the untreated HFF control and the untransfected HFF control (to determine the effect of complete media and compound treatments on cells). Cells that had been pretreated with VPA and 5-Aza and untreated cells were transfected with the DNA mixtures as described in Table 7. Cells were plated on laminin coated plates and incubated at 37°C, 5% CO2. The medium was changed daily according to Table 7. Cells were analyzed on day 3, 7, 12 by immunohistochemical analysis and on day 9 by gene array for multipotent and pluripotent gene expression. Gene Matrix Analysis
[0215] An additional batch of cells treated according to 0a and 1a in Table 7 was analyzed at day 9, along with HFFs and hNPCs, and NSLCs at passage 5 (frozen from previous experiments from Example III) by the Pluripotency Gene Matrix (ABI) (Tables 8a and b) and a gene pool (Table 8c) to determine the pluripotency gene expression profile of selected genes from the ectoderm, endoderm, mesoderm and neural lineage in passages 1 and 5 of NSLCs compared to HFFs (from which they were created) and normal human neuroprogenitor cells (hNPCs).
[0216] The results in Table 8 indicate that all genes related to neural stem cells (some of the significantly expressed pluripotency markers and mesendoderm markers are also expressed in neural stem cells) and neuronal lineage were significantly expressed in NSLCs as opposed to to HFFS, and the expression pattern was somewhat different from hNPCs indicating that NSLCs are similar, but not identical, to the tested hNPCs. Passage 5 of 5 NSLCs had a higher expression of stemness genes than passage 1 of NSLCs. The hNPCs had a higher expression of neuronally more compromised genes than NSLCs, proving their neuroprogenitor status versus the higher tronquicity status of NSLCs.








[0217] In another part of the experiment, another batch of cells that were transfected with Msi1/Ngn2 + pCMV6-XL5-MBD2 were plated in Poly-Ornithine (30 min in RT) and laminin (1 h in RT) on plates coated with CDM II medium in 5 different wells. On day 1, the medium in two of the wells was changed to the same medium as in condition 1a (Table 7) until day 12. The medium was changed daily until day 12, at which time it was changed to NS- Differentiation Medium A (StemCell Technologies) or NbActive4 medium (BrainBitsTM) which were both supplemented with BDNF (20 ng/ml), NT-3 (20 ng/ml), NGF (20 ng/ml), retinoic acid (5 μM), Noggin (20 ng/ml) and Forskolin (10 µM). These cells showed typical neural-type stem cell morphology by day 7, and proliferated by day 12. During exposure to one of the two differentiation media, these NSLC change to a more neuronal and glial phenotype, as shown in the bright field images (Figure 21), but expressed only GFAP by day 17 (Figure 22).
[0218] For the other three wells, on day 1, the medium was changed to NS-A Differentiation Medium (StemCell Technologies), NbActive4 medium (BrainBits), or CDM II medium; these two first were supplemented with the same cytokines as described above, but with the addition of FGF-2 (20 ng/ml). On day 12, FGF-2 was removed from the first two differentiation media, while cells in CDM II medium were switched to NS-A Differentiation Medium (StemCell Technologies) supplemented with cytokines without Fgf-2. Between the 12th and the 17th, medium was changed every two to three days. During the first 12 days of culture, cells in all 3 media developed into a mixture of more spindle-shaped cells compared to untransfected fibroblasts and some into cells with an NSLC morphology; after removal of Fgf-2 the cell morphology turned into a more pronounced neuronal form, as did glial cells with an established network between cells, as shown in the bright field images (Figure 21) that expressed GFAP and βIII- tubulin around day 17 (Figure 22).
[0219] An additional study was designed to assess the effect of Msi1, Ngn2 and MBD2 on their endogenous protein levels in reprogrammed cells. Cells were transfected with the MSI1/NGN2 and MBD2 vector as described above and grown under proliferative conditions at 37°C, 5% CO2 and 5% O2. Samples were collected at various time points from day 2-10 and analyzed by RT-PCR to investigate endogenous gene expression and the expression of neural stem cells and neuronal genes at different time points. RT-PCR revealed a gradual loss of total gene expression of Msi1, Ngn2 and MBD2 from day 2 to day 10, with the increase in the relative expression of MBD2 for the control having been almost completely lost by day 5. This decrease was associated with a significant activation of endogenous Msi1 and Ngn2 on day 5, with another jump in endogenous gene expression on day 9 (Table 9). A significant increase in Sox2 expression was detected at day 4, and expression of this ectoderm/neural stem cell/neuronal gene continued to increase with each subsequent time point (Table 10). GFAP (a neural stem cell and astrocyte marker) was slightly elevated already from day 2 onwards, but increased significantly on day 5 with a big jump in gene expression on day 7 and remained at this level of expression for the rest of the study period. The expression of the neural stem cell marker Nestin also began to increase slowly from day 5 onwards. Expression of the neuronal genes βIII-tubulin (TUBB3) and Map2b were slightly elevated already from day 2 onwards, but increased significantly from day 5 onwards. Expression of a marker for acetylcholine receptors (found in neurons), acetylcholine esterase (ACHE), was also slightly elevated from day 2 onwards, but did not increase significantly until day 7 onwards. It should be noted that, among the neural stem cell markers that were analyzed, the relative expression of Sox2 was high and early expressed which could then be directly or indirectly interacted with exogenous Msi/Ngn2 and/or other genes in nestin activation , GFAP, and endogenous Msi1 and Ngn2 and other genes that promote reprogramming and change cell fate, as well as activation of neuronal genes such as βIII-tubulin (TUBB3), Map2b, and ACHE.

EXAMPLE IV Comparison Nucleofector™ II Device and 96-well Nucleofector Shuttle® Nucleofector™ Device on HFF reprogramming in NSLC under sticky and floating conditions.
[0220] HFFS were cultured as described in Example I, and transfected using Nucleofector™ II Device (Lonza) as previously described in Example II or using 96-well Nucleofector Shuttle® Nucleofector™ Device. HFF cells were harvested with TrypLETM (Gibco), and 1x106 cells/Nucleofector™ II Device transfection for 10 min at 90g and 6x105 cells/transfection with the 96-well Nucleofector Shuttle® Nucleofector™ Device for 5 min at 80xg. After centrifugation, the cell pellet was gently resuspended in either 100 µl of Nucleofector™ Basic Solution for the Nucleofector™ II or in 20 µl of SE solution (SE cell line kit, Lonza) for the Nucleofector Shuttle® Nucleofector™ Device from 96 wells. For the Nucleofector™ II Device, each 100 μl of cell suspension was combined with 2 different mixtures of plasmid DNA (sample 1 was mixed with 2 μg pCMV6-XL5-Msi1 and 2 μg pCMV6-XL5-MBD2, and sample 2 with 2 µg Msi1/Ngn2 and 2 µg pCMV6-XL5-MBD2). Each cell suspension was transferred to an Amaxa certified cuvette and transfected with the appropriate program (U-023). Soon after transfection, 900 µl of warm CDM1 medium was added to each cuvette and the sample was transferred to a laminin-coated culture dish (Stemgent, 10 µg/ml) at a cell density of 1x105 to 1.5x105 cells per cm2 or in untreated cell culture Petri dishes for neurosphere formation. Cells were incubated at 37°C, 5% CO 2 overnight. However, for the 96-well Shuttle® Nucleofector™ Device, the steps described above were similar with the following exceptions: the cell suspension was mixed with 0.6 μg of each DNA from the same 2 DNA mixes, the cell suspension was transferred to a well of a 96-well NucleoplateTM (Lonza) and transfected with the FF-130™ program. After transfection, 80 µl of warm CDM1 medium was added to each well and the samples were left for 10 min in the incubator before being transferred to a laminin coated plate or plates with untreated cell culture at the same cell density as before. mentioned. For both devices, these steps were repeated for each sample that was transfected. Prior to transfection cells were cultured in CDM1 as described in Example I. After 24 hours, the medium was changed to a mixture of 75% CDM medium and 25% Proliferation Medium which was supplemented with EGF (20 ng/ml ), FGF-2 (20 ng/ml), Noggin (20 ng/ml) and cytochalasin B (10 μg/ml) and cells were incubated at 37°C, 5% CO2 and 5% O2. The medium was changed daily with a higher proportion of Neural Proliferation Medium up to 100% by day 4 and a lower proportion of cytochalasin B, which was completely omitted by day 5. Forskolin (10 µM) was added to the medium from day 4 on. Cells under floating conditions were pelleted by centrifugation and their medium changed daily as described for the adherent condition. Cells were harvested on days 3, 7 and 12 for immunohistochemical analysis.
[0221] Fluorescence images were taken with a Cellomics™ ArrayScan HCS Reader™ microscopy system to determine an estimate of the percentage of cells positive for Sox2, neural stem cell marker. This analysis revealed that in untransfected controls and 3 days after transfection, no staining for nuclear Sox2 was detectable. However, on day 7 and day 12 the percentage of Sox2 positive cells progressively increased under all transfection conditions with the exception of pCMV6-XL5-Musashi and pCMV6-XL5-MBD2 Nucleofector™® II condition. The highest percentage on day 12 was obtained with Msi1/Ngn2 and pCMV6-XL5-MBD2 transfected with the 96-well Shuttle® Nucleofector™ Device (~80%). The same combination transfected with Nucleofector™ II resulted in only ~35% positive cells. pCMV6-XL5-Musashi and pCMV6-XL5-MBD2 with the Shuttle® produced ~20% positive cells, although substantially none were seen with the Nucleofector™ II. The percentage of positive cells varied strongly between wells. The staining indicated that the cell population was not homogeneous, as densely arranged Sox2 positive cell fields and complete fields with only negative cells could be found in all cases. In general, Shuttle® was initially more toxic to cells than Nucleofector™® II, however, at least in the case of Msi1/Ngn2 and pCMV6-XL5-MBD2 shuttle vector, the population is positive for Sox2 quickly expanded from day 7 to day 12 to have cells twice as many as Sox2 positive cells when compared to Nucleofector™® II. Cells under floating conditions did not form spheres during the 12-day experiment under either condition, suggesting that neurosphere formation requires both the generation of neural-like stem cells first under more often adherent conditions.
[0222] Table 11 shows the percentage of Sox2 positive cells with a typical neural stem cell morphology using both the Nucleofector™ II Device and the 96-well Shuttle® Nucleofector™ Device. The latter has the advantages that it requires less starting material (needed fewer cells and less DNA) and, in addition, it has given rise to a greater number of cells positive for Sox2. In addition, a small population of Sox2 positive cells was observed with the Shuttle® Device only upon transfection with only one neurogenic transcription factor (MSI) in the presence of the MBD2 DNA demethylator. Table 11: Percentage of Sox2 Positive Cells After the transfection of fibroblast cells with different expression vectors. After transfection, cells were cultured in proliferation medium (StemCell Technologies) supplemented with EGF (20ng/ml, Peprotech) and FGF (20ng/ml, Peprotech) for two weeks at 37°C / 5% CO2 / 5% O2 . Percentage of immunopositive cells was determined by CellomicsTM and represented as mean +SD (n = 3-5).
EXAMPLE V Neurosphere Formation Assay and Cell Differentiation Analysis
[0223] Based on previous studies showing that greater proportional reprogramming is achieved through the transfection of two neurogenic genes, this study was designed to assess the number of reprogramming cells using the Msi1/Ngn2 vector containing two neurogenic transcription factors (Msi1 and Ngn2) and the role of DNA demethylation or DNA methylation inhibitor (5 - azacitidine) and histone deacetylation inhibitor (VPA) in the reprogramming process.
[0224] HFFS were cultured and treated with cytochalasin B as described in Example III, and treated simultaneously with VPA (1 mM) and 5-azacytidine (0.5 μM). After two days of treatment, cells were transfected by Nucleofection as described in Example II with the constructed vector Msi1/Ngn2. After preparing the cells, they were mixed with 2 μg of total DNA (Msi1/Ngn2) and cells that had not been treated with chemical inhibitors (VPA and 5-Aza) were co-transfected with MBD2 (2 μg) using the appropriate program (U023). Samples were transferred to a laminin coated culture plate (10 µg/ml, Sigma) and incubated in a humidified incubator at 37°C / 5% O2/ 5% CO 2 . The medium was changed to basal proliferation media, Neural proliferation medium (NeuroCultTM proliferation kit, StemCell Technologies), with the presence of Noggin (20 ng/ml, Peprotech), recombinant hFGF (20ng/ml, Peprotech), and Recombinant hEGF (20ng/ml, Peprotech). After 6 days of transfection, cells were harvested using AccutaseTM (Millipore), centrifuged (300xg, 5 min, RT) and plated on uncoated cell culture plates in NSC NeuroCult(TM) proliferation medium to investigate the ability to grow suspension cells as neurospheres or on laminin coated plates for adherent culture. To avoid the loss of floating beads during media changes, cells were pelleted by centrifugation at 150 x g for 3 minutes at room temperature (RT). The pellet was then resuspended in fresh medium and plated onto new low binding uncoated plates. Cultures were incubated at 37°C, 5% CO 2 , 5% O 2 and were fed daily for at least two months.
[0225] To investigate whether a single cell of human neuronal precursor cells (hNPCs) and human NSLCs was able to generate a neurosphere (a standard test to prove that a cell is a neural stem cell), the neurospheres were dissociated into individual cells and these individual cells were isolated and cultured in suspension proliferation medium, and neurosphere formation was monitored by obtaining brightfield images using light microscopy (Nikon, 10X) and by CellomicsTM. These cells proliferated and grew as spheres from day 6 to day 10 (Figure 4A). Immunohistochemical analysis of these beads (Table 12 and Figure 4) on day 20 revealed immunopositive staining for neural stem cell markers Sox2, Musashi, CD133, nestin, and GFAP. The cells also stain positive for βIII-tubulin (a marker for neurons), O4 (an oligodendrocyte marker), and GFAP (an astrocyte marker), indicating the tri-potent differentiation potential of both sets of cells (NSLC and hNPC), and negative for NGFrec and NeuN (markers for differentiated neurons), indicating that the cells were not terminally differentiated.Table 12: Percentage of cells positive for neural stem cells, and neuronal lineage markers, astrocytes and oligodendrocytes in formed neurospheres from individual NSLCs and hNPCs cultured in proliferation medium (StemCell Technologies) supplemented with EGF (20ng/ml, Peprotech) and FGF (20ng/ml, Peprotech) for 20 days at 37°C / 5% CO 2 / 5% O2. The percentage of positive cells was determined by Cellomics™ and represented as mean ± SD.

[0226] HFF cells were cultured as described in Example I, and transfected using the Nucleofector™ II device (Lonza), as described in Example II. Cells were co-transfected with pCMV6-XL5-Msi/pCMV6-XL4-Ngn2, pCMV-Msi1-Ngn2 with MBD2 or pretreated with VPA/5aza. Cells were grown in proliferation medium as suspension or adherent cultures. Gene expression analysis in 8 samples was performed as previously described in Example I with the customized 2 TLDA neuronal markers (Table 13), which profiled the expression of 48 genes (including three reference genes: ACTIN, GAPDH and PPIA) in four main categories: (1) fibroblast-specific genes; (2) neuronal lineage-specific genes; (3) specific neural stem cell marker genes; and, (4) Genes for growth factors and their receptors.


[0227] As shown in Table 14, fibroblast-specific genes (Col3A1, Lox, S100A4) were repressed in reprogrammed cells, indicating the loss of fibroblast-specific genes following transfection (note that not all cells were transfected and reprogrammed and Therefore, the presence of fibroblast-specific gene expression in the cultures is mostly due to the unscheduled fibroblasts left in the culture). The expression of these genes is observed to increase when HFFs were transfected in the absence of DNA demethylation or DNA methylation inhibitor, indicating that reduced expression of differentiated fibroblast cell markers requires DNA demethylation. The expression of these ectodermal genes, such as Msi1, Sox2, and nestin was remarkably increased after transfection in conjunction with DNA demethylation. The expression of neuronal markers such as synaptogamin1 (a synaptic vesicle protein) and NeuroD1 was activated in cells transfected with Msi1/Ngn2/MBD2, and slightly increased in cells transfected with Msi1/Ngn2 VPA and 5-AZA. The three selected oligodendrocyte markers were detected in cells transfected with a strong increase in Olig2. Two markers for astrocytes, GFAP and ALDH1L1, were increased after transfection. The results support the idea that neurospheres are composed of heterogeneous parent subtypes.
[0228] Among neurotrophic factors, CNTF expression was slightly increased in the reprogrammed cells. The expression of GAP-43 and neuropeptide Y (NPY) were the most noted genes. GAP-43 has long been recognized to play a crucial role in axonal plasticity and is used as a marker of regenerated neurite outgrowth and synaptogenesis, both in embryonic development and in neuronal regeneration in the injured brain and spinal cord. The expression of receptors for growth factors and neurotrophic was increased, as was the expression of neurotrophic receptor tyrosine kinase.



[0229] Further analysis and quantification of the adherent population of NSLCs showed that cells stained positively for Sox2 (93.43+1.9%), nestin (60.76+5.7%), and GABA (37, 48 +4.9), whereas these markers were undetectable in untransfected cells (Figure 5, Table 15). Furthermore, these cells stained positive for p75NTR (31.15 +1.6), βIII-tubulin (37.55 + 0.6%) and GFAP (6.47 +0.9). However, untransfected HFFs only stained positive for HFF markers (Figure 5), such as fibronectin and fibroblast protein marker, whereas these markers were undetectable in reprogrammed cells, demonstrating that the reprogrammed cells lost markers from the original cells and adopted morphology and neural stem cell markers and a neuronal lineage.Table 15: The percentage of cells stained positive for neural stem cell markers and fibroblast markers in untransfected cells and cells transfected with pMsi1/Ngn2/MBD2. Transfected cells (NSLCs) have a high percentage of neural stem markers, but a very low percentage of fibroblast markers compared to non-transfected cells. The percentage of immunopositive cells was determined by CellomicsTM and represented as mean +SD (n=5).


[0230] This study also showed that NSLCs have the ability to proliferate in culture and exhibit stable morphology, genes and protein expression that were maintained throughout the study period, which was for more than five months in culture (Table 16). Table 16: NSLC doubling time over serial passes. NSLCs were maintained under proliferating conditions for 35 passages in an incubator at 37°C, 5% CO2 and 5% O2 incubator. The time required for the cell population to double (g) was calculated for each passage, and was defined as g = (In2) / k, where k was the number of generations that occurred per unit of time (t) defined as, k = (ln Nf- ln No) / t, where Nf was the final number of cells and No the initial number of seeded cells. The average generation time was 25.4h over the 35 passages.

Gene expression microarrays
[0231] Expression analysis using microarrays was performed to obtain an overview to compare the NSLC passage 7 gene expression profile for both the HFF (the cells from which the NSLC was created) and the hNPCs. NSLC (n = 3), HFF (n = 2), and hNPC (n = 3) were resuspended in RNAIaterTM (Qiagen) and sent to Genotypics (India) where the samples were processed and the Gene Expression Microarray performed.
[0232] In summary, Genotypics extracted RNA from the samples and performed quality control using an Agilent BioanalyzerTM Labeling was done using Agilent's Quick Amp™ kit (DNA synthesis and in vitro transcription), followed by QC labeling. Hybridization was then performed using an 8 x 60K array, and scanning was done using an Agilent high-throughput scanner with SureScanTM technology. The “Agilent Feature Extraction” computer program was used for automatic data extraction, followed by raw QC and QC image. Advanced data analysis was then performed, including Network and Gene Ontology analysis using GeneSpring Agilent GXTM v10.0 and Genotypic's Biointerpreter computer program. NSLC samples were compared to HFF samples (Set 1) and hNPC samples (Set 2). The NSLC samples had an overall gene expression pattern that was much closer to the hNPCs than to the HFFs from which the NSLCs were created (Figure 23). Pearson's correlation analysis revealed that NSLCs are closely related to hNPCs, including, in terms of neuronal lineage markers, regenerative genes and migration genes. These data confirm that NSLCs are similar but not identical to hNPCs.
[0233] Microarray analysis revealed an increase in neuronal precursor genes in the NSLC samples compared to the HFF samples. ACTL6A and PHF10, which both belong to the neural progenitor-specific chromate remodeling complex (npbaf complex) and are required for neural progenitor proliferation, were increased by 2.9-fold and 2.3-fold, respectively. MSI2, which plays a role in the proliferation and maintenance of stem cells in the central nervous system, was increased 6-fold (Table X1). Glia genes increased in NSLC samples compared to HFF samples. GFAP, a neural stem cell and astrocyte-specific marker that, during development of the central nervous system, distinguishes astrocytes from other glial cells, is highly increased in the NSLC specimen compared to HFF (690-fold). OLIG1, which promotes the formation and maturation of oligodendrocytes, especially within the brain, is also highly increased in the NSLC sample compared to HFF (370-fold) (Table X2).
[0234] Table X3 lists a subset of regenerative genes that are enhanced in NSLC samples compared to HFF samples. SOX2, a gene essential for early embryogenesis and for pluripotency of embryonic stem cells as well as neural stem cells, is highly increased in NSLC samples compared to HFF samples (5000-fold). CCND2, which is essential for cell cycle control in the G1/S transition (early), is also increased in NSLC samples (70-fold compared to HFF samples). As shown in Table X4, numerous fibroblast genes were repressed in the NSLC samples compared to the HFF samples. This shows that NSLC loses the expression of numerous fibroblast genes as it is reprogrammed from HFF to NSLC.
[0235] Table X5 shows that neuronal precursor genes were also increased in NSLC samples compared to hNPC samples. BDNF, which promotes the survival and differentiation of certain neuronal populations of the peripheral and central nervous systems during development, is even more highly expressed in NSLC samples than in hNPC samples (34-fold increase). Table X6 shows that a subset of Glia genes is also increased in NSLC samples compared to hNPC samples. GFAP, a neural stem cell and astrocyte-specific marker that, during central nervous system development, distinguishes astrocytes from other glial cells, is more highly expressed in NSLC samples than in hNPC samples (13-fold). PLP1, the important central nervous system myelin protein, which plays an important role in the formation or maintenance of the multilamellar structure of myelin, is also more highly expressed in NSLC samples than in hNPC samples (20-fold).
[0236] Regeneration genes were also increased in NSLC samples compared to hNPC samples (Table X7). BMP2, a neural crest marker, but which induces especially cartilage growth and bone formation and BMP4, which in turn induces cartilage and bone formation and acts in mesoderm induction, tooth development, limb formation and repair of fractures, but also in neural stem cells, were both more highly expressed in NSLC samples than in hNPC samples (18-fold and 20-fold, respectively). GAP43, which is an important component of the mobile growth cones that form the tips of elongated axons, was more highly expressed in NSLC samples than in hNPC samples (4-fold). This suggests the regenerative potential of NSLC. HOXB4, a transcription factor that is involved in the development and expansion of neural stem cells as well as hematopoietic stem cells and progenitor cells in vivo and in vitro making it a potential therapeutic candidate for stem cell expansion, was also more highly expressed in NSLCs than in hNPCs. These data indicate that NSLCs are more similar to stem cells or have more stemness than hNPCs.Table X1: Augmented Neural Precursor Genes (NSLC VS HFF)

1 Folding shift represents gene augmentation in NSLC samples compared to HFF samples. (N = 2 for HFF samples, n = 3 for NSLC samples). Table X2: Enhanced Glia Genes (NSLC vs. HFF)








[0237] In order to investigate the differentiation potential of NSLCs, neuronal lineages (neurons, astrocytes and oligodendocytes), neurospheres were dissociated and plated in laminin/poly-D-lysine (10 μg/ml; Sigma) in differentiation medium during differentiation during differentiation during two weeks. Differentiation for neuronal lineage was performed using two different media: NbActive medium (BrainBitsTM) supplemented with Brain Derived Neurotrophin Factor (BDNF, 20ng/mL, Peprotech), all-trans retinoic acid (ATRA, 5 μM Spectrum,), and bFGF (40ng/ml, Peprotech) or NeuroCultTM differentiation medium (NeuroCultTM Differentiation kit, StemCell Technologies), supplemented with BDNF (20 ng/ml, Peprotech) and bFGF (40ng/ml, Peprotech). After two weeks in culture, cells were stained with the neuronal marker βIII-tubulin, astrocyte markers GFAP and S100 β, marker and oligodendrocyte marker CNPase. Cells were fixed with 4% formaldehyde and primary antibodies were added in 5% normal goat serum/PBS as follows: mouse antibody βIII-tubulin (1:200, Abeam), rabbit antibody S100β (1: 100, Abeam), and chicken CNPase antibody (1:50, Abeam). Secondary antibodies are added in 5% normal goat serum / PBS as follows: goat anti-mouse Alexa546TM (1:200, Invitrogen), goat anti-rabbit Alexa488TM (1:200, Invitrogen), and de anti-chicken made on cy5 goat (1:100, Jackson ImmunoResearch Labs).
[0238] Immunohistochemical analysis showed that the NbActive medium promoted equally the neuronal differentiation (48.66 +14.07%, βIII-tubulin) and potential early oligodendrocyte lineages (50.01% +4.04, CNPase ) and for a lower percentage of astrocyte cells (2.68 + 0.13%, S100β), while the NS-A differentiation medium induced differentiation, mainly for neurons (64.89 + 4.11%, βIII- tubulin) and astrocytes (35.94 + 4.04%, S100β), and a low percentage of potential initial oligodendrocyte cells (8.68 + 2.71%, CNPase). NSC-A medium was selected over NbActive for further differentiation studies. Differentiation of cells in NS-A differentiation medium promotes differentiation of hNPC and NSLC similarly as shown in Table 17 by decreasing the percentage of sox2, musashi and nestin positive cells. NSLCs were differentiated from neuronal (74.3 + 0.1, GABA) lineage, from astrocyte (65.6 + 0.0, S100beta) and a lower percentage of oligodendrocyte cells (5.2 + 0.6, CNPase) . The same pattern of tripotent lineage differentiation was observed with hNPCs (Table 17).Table 17: The percentage of stained cells positive for neural stem cell and neuronal lineage markers in transfected and untransfected cells. NSLCs and hNPCs were cultured in NS-A differentiation medium supplemented with BDNF (20ng/ml) and FGF (40 ng/ml), cultures were incubated at 37°C, 5% CO2, 5% O2 for three weeks. The percentage of immunopositive cells was determined by Cellomics™ and represented as mean +SD (n=5).

[0239] Several additional antibodies to neuronal antigens have been used to further characterize the nature of differentiated cells. Antibodies against microtubule associated protein (MAP2b), NCAM, and synaptophysin were used as recommended by the antibody manufacturer. After three weeks in differentiation medium, there was a reduction in differentiation induced in precursor cell markers and an increase in mature neuronal markers. The percentage of neuronal precursor markers such as Sox2 decreased during differentiation, whereas p75NTR, βIII-tubulin and GABA were increased with elongation of differentiation time (Figure 6), however, O4 positive cells were very low after 3 weeks of differentiation of hNPCs (6.4+2.9) and NSLCs (8.2+0.6). Synaptophysin, an antibody used to identify functional neuronal cells, was increased after 2 and 3 weeks of differentiation, indicating the maturity of neuronal cells. GABA and aceticoline markers were increased after 2 weeks of differentiation and decreased at week 3.
[0240] The morphological changes and expression of a number of neuronal antigens and genes show that the above method results in normal cells and viable neuronal cells. Furthermore, newly formed neuronal cells have the morphological criteria of neurons. In addition to the above markers, differentiated cells were evaluated characterizing the morphological markers of neurite differentiation. Neuron-like cells (cells strongly expressing βIII-tubulin) showed the formation of neurites after differentiation, including an increase in the mean number of neurites per neuron (from, for example, 1.38 +0.1). The same pattern was observed in βIII-tubulin positive cells. Therefore, the mean neurite length (118.3 +3.5 μm) and the number of branch points (3.28 +0.3) per neurons also increased. Differentiated neuron-like cells developed long neurites that were greater than three cell diameters in length, with a growth cone at the end, expressed neuron-specific genes, and stopped proliferation after inducing differentiation.
[0241] Further differentiation was performed using an optimized medium that promoted differentiation to oligodendrocyte lineage. NSLCs and hNPCs were cultured in NS-A differentiation medium as described above supplemented with FGF-2 (10ng/ml, Peprotech) and hedgehogsonic (SHH, 100ng/ml, Peprotech) for 4 days. After 4 days the medium was changed to NS-A differentiation medium supplemented with T3 (60 ng/ml, Peprotech), IGF1 (10ng/ml, Peprotech), NT-3 (10ng/ml, Peprotech), and PDGF (10ng/ml ml, Peprotech). Cells were cultured for 20 days at 37°C, 5% CO2. Table 18: Percentage of stained cells positive for neural stem cells and neuronal lineage markers in transfected and untransfected cells. NSLCs and hNPCs were grown in differentiation medium supplemented with SHH (100ng/ml, Peprotech), T3 (60ng/ml, Peprotech), IGF1 (10ng/ml, Peprotech), NT-3 (10ng/ml, Peprotech), and PDGF (10ng/ml, Peprotech) to induce differentiation to oligodendrocytes. The percentage of immunopositive cells was determined by Cellomics™ and represented as mean +SD (n = 5).

[0242] Quantification of the differentiation of hNPCs and NSLCs revealed a population of cells that stained positively for O4. As shown in Table 18, the percentage of O4 positive cells was more pronounced in differentiated hNPC (40.1+6.4%) compared to differentiated NSLCs (8.5%+0.6) when using the differentiation protocol above.
[0243] This study showed that transfection of cells with one or two neurogenic transcription factors in the presence of a DNA demethylator or small molecules for epigenetic modification achieves stable reprogrammed cells (NSLCs). As a DNA demethylator, epigenetic modification (inhibition of acetylation and methylation) is sometimes helpful in stimulating the reprogramming process. These cells possess and retain the properties of neural stem cells as determined by: (1) the expression of neural stem cell genes and proteins, (2) the ability to generate and grow as neurospheres from a single cell, and (3 ) differentiate to neuronal lineages under differentiating conditions. When differentiated to neurons, cells exhibit one or more neural-specific morphological, physiological, and/or immunological aspects associated with a neuronal cell type. Useful criteria include morphological features (long processes or neurites), physiological and/or immunological aspects, such as the expression of a set of neuronal-specific markers or antigens. Furthermore, NSLCs readily transform into a tripotent-like precursor cell with the potential to differentiate to a high percentage of neuronals, astrocytes and a lower percentage of oligodendrocyte populations. EXAMPLE VIIBMP signaling pathway implication in CNFP reprogramming
[0244] This study was designed to assess the role of Noggin in the dedifferentiation process from HFFs to NSLCs. HFFS were cultured and treated with cytochalasin B as described in Example III. After two days of treatment, cells were transfected by Nucleofection as described in Example II with the constructed vector Msi1/Ngn2. Briefly, after cell preparation, they were mixed with 2 pg of total DNA (Msi1/Ngn2) and were co-transfected with MBD2 (2μg) by Amaxa's Nucleofector™ according to the manufacturer's protocol. The samples were then transferred to a laminin (10 μg/ml, Sigma) coated culture plate and cultured in the presence of neural proliferation medium (NeuroCultTM proliferation kit, StemCell Technologies) with recombinant hFGF (20ng/ml, Peprotech), recombinant hEGF (20ng/ml, Peprotech), and with or without the presence of Noggin (20ng/ml, Peprotech). Samples were collected at different time points (1, 3, 4, 6 and 8 days) to analyze neuronal gene expression by RT-PCR and protein expression levels by immunohistochemistry.
[0245] Fluorescent staining for immunohistochemistry was performed on samples after 4 days of transfection, as described above in Example I. Transfected cells were stained and analyzed for expression of Sox2, the percentage of Sox2 was 33.3 +1, 00% in the presence of Noggin compared to 27.5 +0.50% without the presence of Noggin on day 4. RT-PCR analysis of relative expression of neuronal precursor cell marker such as nestin and Sox2 after transfection of HFFs with pCMV-Msi1-2A-Ngn2 and pCMV6-XL5-MBD2 with or without the presence of Noggin (20ng/mL) was associated with an increase in nestin and Sox2 from day 3 and maintained until day 8 (Table 19) . No difference was noted in expression in the absence of Noggin. Inhibition of the BMP signaling pathway by Noggin thus increased reprogramming, but had no effect on the reprogramming effect itself.Table 19: RT-PCR analysis of relative expression of neuronal precursor cell markers such as nestin and Sox2 after transfection of HFF with pCMV-Msi1-2A-Ngn2 and pCMV6-XL5-MBD2 with or without Noggin (20ng/ml). The relative expression of Sox2, and nestin was increased after transfection with and without Noggin.
EXAMPLE VIINSLCs created from HFF cells are not skin-derived precursors (SKPs)
[0246] It is known that cells called skin-derived precursors (SKPs) can reside in adult human skin (Fernandes et al., 2004). These cells are able to proliferate in response to EGF and bFGF and express nestin, versican and fibronectin, and can differentiate into both neuronal and mesodermal progenies. In order to verify whether NSLCs are distinct from SKPs, differentiation to adipocyte cells was performed. Adipose tissue-derived stem cells (ADSC) were maintained in StemProTM MSC serum-free medium (Invitrogen) in CellStart™ coated flasks (Invitrogen). CellStartTM was diluted 1:100 in dPBS/Ca2+/Mg2+ and the vial incubated for 2 hours at 37°C. Cells are passaged every 3 to 4 days using AccutaseTM and the medium was changed every 2 days. Three to four days before starting differentiation, ADSCs and NSLCs were seeded in 6-well plates on CellStartTM tissue culture re-coated plates (1:100 in dPBS / Ca2+/Mg2+/2 hours at 37°C. cells reached confluence (after 3 to 4 days), proliferation media was replaced by differentiation media consisting of DMEM/F12 (50:50), ITS (1:100), HEPES (1:100), GlutamaxTM (1 :100), T3 (0.2 nM), Rosiglitazone (0.5 μg/ml), IBMX (100 μM) and Dexamethasone (1 μM) Three days later, IBMX and dexamethasone were removed from the differentiation medium. 10, cells were fixed with a 4% formaldehyde solution for 10 min and stained with Oil Red O staining solution (Invitrogen) for 15 min. Staining was removed and cells washed twice with PBS. with lipid droplets specifically stained with Oil Red O, however NSLCs were stained negative, with no presence of lipid droplets in the cells, and the cells adopted the morphology of neuronal cells.
[0247] Immunohistochemical analysis confirmed that NSLCs are distinct from SKPs (Figure 24): NSLCs scored positive for p75NTR and negative for fibronectin and versican, while SKPs expressed fibronectin and versican and did not express p75NTR (Fernandes et al, 2004). This study indicates that NSLCs represent a tripotent-like precursor cell and they are not a subpopulation of SKPs. EXAMPLE VIII Release of BDNF from Neural Stem Cells (NLCs)
[0248] Neural-like stem cells (NSLCs) differentiated into neuronal and glial cells, were kept in culture for 55 days, and BDNF released into the conditioning medium was measured by antigen capture ELISA at different time points and compared with release in mature neurons (ScienCell), undifferentiated normal human neural precursor cells (NHNP, Lonza), as well as for undifferentiated NSLCs and untransfected cells (HFF). Conditioned medium from each group was collected, centrifuged and then stored at -80°C until assayed. BDNF concentrations were measured by ELISA kits (BDNF Immunoassay System Emax, Promega Corporation, USA) according to the manufacturer's instructions. Briefly, 96-well ELISA immunoplates were coated with anti-BDNF (CatNb # G700B) diluted 1/1000 in carbonate buffer (pH 9.7) and incubated at 4°C overnight. The next day, all wells were washed with 0.5% TBS-TweenTM prior to incubation with the Block / 1X sample buffer at room temperature for one hour without shaking. After blocking, standards and samples were added to the plates and incubated and shaken (450 +100 rpm) for 2 h at room temperature. Subsequently, after washing with TBS-TweenTM wash buffer, plates were incubated for 2h with anti-human BDNF Pab (1:500 dilution in Block and 1X Sample buffer) at 4°C. After incubation, plates were washed five times with 0.5% TBS-TweenTM wash buffer and 100 µl of diluted Anti-IgYHRP conjugate was added to each well (1:200 dilution in Block and 1X Sample buffer) and incubated for 1 hour at room temperature with stirring (450 + 100 rpm). Then, the plates were washed five times with 0.5% TBS-TweenTM wash buffer solution and 100 µl of TMB One Solution was added to each well. After 10 minutes of incubation at room temperature with shaking (450 +100 rpm) for the BDNF plate, a blue color formed in the wells. After stopping the reaction by adding 100 μl of 1N hydrochloric acid, the absorbance was read at 450 nm in a microplate reader (Synergy 4TM) within 30 minutes of stopping the reactions. The concentration of BDNF released in supernatants was determined according to standard curves.
[0249] ELISA results revealed that BDNF was released at the same concentration as differentiated neural-type cells (NLCs differentiated from NSLCs) and normal cells from human neurons from day 11 and remained until day 55 (Table 20), while none BDNF (except for small amounts in the untransfected HFF group) was released in other groups.Table 20: Quantification of BDNF release by Neural-Type Cells (NLCs) that had been differentiated for 55 days from Neural-Type Cells (NSLCs) that had been created from transfected HFFs. BDNF released from the NLCs in the medium at different time points was measured by antigen capture ELISA and compared to BDNF release from normal mature human neurons (ScienCell).

[0250] In addition, adopting neuronal morphology criteria, NLCs were functional and possessed the ability to release neurotrophic factor (BDNF). The generation of reprogrammed neuronal-like cell lines that can locally deliver these neurotrophic factors could be used as a method for treating various neurological conditions and could offer crucial advantages in the regeneration and functional recovery of brain and other injuries. EXAMPLE IXReprogramming of different cell types for NSLCs: This study was carried out to investigate the capacity of keratinocytes (Invitrogen), human adipocyte-derived stem cells (ADSCs, Invitrogen) and hematopoietic stem cells (CD34+, Invitrogen) in neural stem cells Preparation of Human CD34+ Cells, Human ADSC and Human Keratinocytes: CD34+ cells from mobilized human peripheral blood were purchased from StemCell Technologies and expanded as a floating culture in Petri dishes in complete serum-free StemPro™®-34 medium (Invitrogen) supplemented with stem cell factor (SCF, 150 g/ml, Peprotech), granulocyte colony-stimulating factor (GM-CSF, 37.5 ng/ml, Peprotech) and IL-3 (75 ng/ml, Peprotech). Cytokine-supplemented medium was changed every 2-3 days after centrifugation of the cell suspension at 300xg for 10 min. Every two days cytokines were added directly to the culture, without changing the media. Cells were incubated at 37°C, 5% CO 2 . For their passage, the cells were centrifuged, resuspended in the above medium plus cytokines and placed in adequate numbers in Petri dishes.
[0251] Human adipose tissue-derived stem cells (ADSC) were purchased from Invitrogen and expanded in serum-free MSC StemPro™ complete medium (Invitrogen) in CellStartTM coated flasks (Invitrogen) (diluted 1:100 in PBS containing Ca2+/Mg2+) at a cell density of 1x104 cells/cm2. The medium was replaced every other day with pre-warmed fresh complete StemPro™ MSC SFM. Cells were incubated at 37°C, 5% CO 2 Cells were sub-passed when confluent to 80% by incubation for 3-5 min in prewarmed TrypLETM (Invitrogen) and then harvested in StemPro™ MSC medium. After centrifugation at 1500 rpm for 5 min, cells were seeded into CellStart™ coated flasks as described above.
[0252] Primary human keratinocytes were purchased from Invitrogen and expanded in defined serum-free keratinocyte medium in coated (Invitrogen) matrix coating (Invitrogen) flasks with a cell density of 5x103 cells/cm2. Cells were incubated at 37°C, 5% CO 2 . The media were replaced with fresh, complete growth media every two to three days until subculture. Once the cells had reached 70-80% confluence, the media was removed and the cells were incubated in Versene™ (Invitrogen) for 3-5 minutes at room temperature. VerseneTM was removed, and pre-warmed 0.05% trypsin-EDTA (Invitrogen) was added to the vials. After the 5-10 min incubation, growth medium containing soy trypsin inhibitor (Invitrogen) was added to the flasks and the cells gently ground. After centrifugation at 100xg for 10 min, cells were resuspended in the desired volume of pre-warmed complete growth medium in coated flasks as described above.
[0253] Prior to transfection, cells were trypsinized and transiently co-transfected with pCMV-Msi1-Ngn2 and pCMV6-XL5-MBD2 as previously described in Example IV, using the shuttle vector and plated on a laminin-coated culture plate (Sigma , 10 µg/ml). Starting one day after transfection, cells were treated with VPA (1 mM) for 4 days and the medium was gradually changed to proliferation medium supplemented with FGF (20ng/ml) and EGF (20ng/ml) and were cultured for 18 days at 37°C, 5% CO2 and 5% O2. Cells were then analyzed for neural stem cell markers by RT-PCR and immunohistochemistry.
[0254] Further analysis and quantification of the reprogrammed cells revealed a population of NSLCs engendered from keratinocytes and CD34+ cells. RT-PCR analysis revealed an increased relative expression of neural stem cell markers such as Sox2, nestin, GFAP, and βIII-tubulin after transfection of keratinocytes and CD34+ by Msi1 and Ngn2. The relative expression of nestin and GFAP was increased in NSLCs created from keratinocytes and CD34+ cells compared to NSLCs from HFFs, however, the reverse was true for Sox2 and ACHE expression. βIII-tubulin (TUBB3) and Map2b expression was highest in NSLCs created from CD34+ cells, followed by NSLCs created from HFF (Table 21). These data show that different types of NSLCs with different gene expression profiles (and characteristics) can be created from different starting/source cell types (and at the same has been observed for the creation of some other type cell types trunk discussed in this application). The data are also intriguing, as it was not expected that keratinocytes (which are derived from the ectoderm such as endogenous neural stem cells) would have a lower expression than HFFs for all analyzed genes except nestin (it was expected that keratinocytes would be the easiest to reprogram into NSLCs since they are derived from the ectoderm).

[0255] Immunohistochemistry revealed positive staining for GFAP, Sox2, and nestin as shown in Figure 7. NSLCs developed from HFF produced a higher percentage of positive staining for Sox2 and GFAP (55.8±3.8 and 78.1±2.4) compared to CD34+ cells (42.8±2.7 and 24.2±4.4), and keratinocytes (47.1±2.1 and 43.4±8.9) . The percentage of nestin positive cells was high in keratinocytes (77.6±10.7) and HFF (68.45±12.9) and lower in CD34+ cells (15.5±2.7) (Table 22). Positive staining for Sox2 and nestin was not detectable in ADSCs.Table 22: The percentage of positive cells for Sox2 and nestin for neural stem cell markers after transfection of fibroblasts, keratinocytes, and CD34+ cells with pCMV-Msi1-Ngn2 in presence of MBD2 and VPA. Cells were grown in culture dishes coated in proliferation medium (StemCell Technologies) supplemented with EGF (20ng/ml) and FGF (20ng/ml) for 18 days. Untransfected cells were considered as a negative control. The percentage of immunopositive cells was determined by Cellomics TM and represented as mean ± SD (n=5).

[0256] NSLCs generated from keratinocytes and CD34+ cells were tested for tripotent capacity. Other differentiation studies were performed to induce the differentiation of these NSLCs to neuronal lineage, using the NeuroCult TM differentiation medium (NeuroCult TM Differentiation Kit, StemCell Technologies) supplemented with BDNF (20ng/mL, Peprotech) and bFGF (40ng/ml, Peprotech) as described in Example V. NSLCs generated from HFFS and hNPCs were used as controls, cultures were incubated at 37°C, 5% CO2, 5% O2 for three weeks. Samples were collected or fixed on Day 14 and 28 after differentiation for further analysis. RT-PCR analysis revealed a decrease in undifferentiated genes (nestina and Sox2) and an increase in differentiated genes (Map2, βIII - tubulin, CNPase, and GFAP), as shown in Tables 23A, 23B, 23C and 23D.




[0257] Fluorescent immunohistochemical staining was performed on samples after 14 days and 28 days of differentiation. The expression of Sox2 and Nestin was time-dependent decreased in differentiated cells (HFF, keratinocytes and CD34+ cells). This decrease was associated with an increase in differentiated markers on day 28, such as GFAP (68.51±11.87 for HFF-NC, 59.55±9.12 for NC keratinocyte, and 61.70±1.48 for CD34+ - NC). A high percentage of βIII-tubulin positive cells were generated from differentiated NSLCs generated from HFF (57.83±4.49), compared to βIII-tubulin positive cells generated from keratinocytes (23.27± 2.91) and CD34+ cells (39.15±7.99). (Table 24) Table 24: The percentage of cells stained positive for neural stem cell markers and neuronal lineage markers in hNPCs (LONZA) and transfected keratinocytes, HFF, and CD34+ cells with pMsi1/Ngn2/MBD2. Transfected cells (NSLCs) were cultured in proliferation medium or medium differentiation medium for 28 days at 37°C, 5% CO2, 5% 02. The percentage of immunopositive cells (Sox2, Nestin, GFAP, S100beta, and βIII -tubulin) was determined by Cellomics (TM) and represented as mean ± SD (n = 5).
nd = not determined; ± = standard deviation CD34+-NC: neuronal cells generated after differentiation from NSLCs generated from CD34+ cells. Each data point represents the analysis of at least 1000 cells from at least 8 images.
[0258] The % of Sox2 positive cells decreased more rapidly, the % of Nestin positive cells in general decreased more slowly, and the % of cells expressing one of the differentiation markers (S100β, βIII-tubulin, GFAP) in general increased more slowly in hNPCs than in NSLCs during differentiation. Of the three types of NSLC lines created, the % of cells expressing one of the differentiation markers (S100β, βIII-tubulin, GFAP) in general increased more slowly increased in NSLCs created from keratinocytes and more rapidly in NSLCs created from HFFs .
[0258] This study indicates that NSLCs can be created from keratinocytes and CD34+ blood cells, and these cells share morphology and markers similarly to NSLCs generated from HFF. Similar to hNPCs, NSLCs created from keratinocytes, CD34+ cells and HFFs had a tendency to differentiate more towards an astrocyte lineage than a neuronal lineage (except the NSLCs created from HFFs had an almost equal number of βIII-tubulin positive and GFAP positive cells) as shown by the high percentage of GFAP positive cells during differentiation, which was confirmed by S100beta staining. However, the proportion of astrocyte and neuronal cells generated from hNPCs was lower under identical culture conditions, indicating that NSLCs generated from HFF, keratinocytes, and CD34+ cells can give rise to a greater number of neuronal cells and of astrocytes compared to hNPCs. NSLCs, whether created from HFFS, keratinocytes or CD34+ cells (or potentially some other cell), are tripotent cells and have the ability to differentiate from neurons, astrocytes, and oligodendrocytes in a similar way to hNPCs. However, RT-PCR and immunohistochemistry analysis of transfected ADSCs did not reveal any significant expression of neural stem cell genes, indicating a need to optimize conditions for transforming ADSCs into NSLCs or to investigate the effect of other neurogenic factors that could turn them into NSLCs. EXAMPLE X3D Extracellular Matrix Fabrication (CDM)
[0259] Fibroblast cells were cultured in DMEM medium, in the presence of 10% FCS as described in Example I, followed by seeding in 12 wells pre-coated with laminin (10μg/ml) at a concentration of 2x106 cells/ml in defined CDM medium consisting of a 3:1 ratio of Dulbecco's modified Eagle medium (DMEM, high glucose (4.5 g/L) with L-glutamine and sodium pyruvate) and HAM F-12 medium supplemented with the following components: EGF (4.2x10-10 M), bFGF (2.8x10-10 M), ITS (8.6x10-5 M), dexamethasone (1.0x10-7 M), L-ascorbic acid phosphate magnesium salt n -hydrate (3.2x10-4 M), L-3, 3',5-triiodothyronine (2.0x10-10 M), ethanolamine (10-4 M), GlutaMAX TM (4x10-3 M), glutathione (3.3x10- 6 M), and 1% penicillin/streptomycin/amphotericin B. Culturing fibroblast cells at hyperconfluent density in this chemically defined medium completely causes them to enter a high synthetic phase with a deceleration of proliferation, leading to the production of and a living tissue equivalent (LTE) consisting of several layers of fibroblasts within the de novo 3D extracellular matrix (CDM) that is completely synthesized by the fibroblasts themselves. Trans-differentiation and reprogramming of cells within CDM
[0260] Day 14 CDM samples were treated with cytochalsin B (10μg/ml, Calbiochem), with the cytochalsin B concentration reduced from 10μg/ml to 0μg/ml (none) over 5 days, while changing the CDM Medium to NbActive Medium. Samples were grown for an additional 12 days at 37°C, 5% CO2, and the medium was changed every day.
[0261] Samples were fixed to perform immunohistochemistry as described above to detect neuronal markers. The following antibodies were used: mouse anti-nestin 647 (1:100, BD) and anti-βIII-tubulin (1:200, Neuromics). No clear change in cell morphology was observed under CDM and immunohistochemical analysis failed to detect βIII-tubulin positive cells. Thus, the induction of cell trans-differentiation using only cytochalasin B and chemically defined neural medium was not enough to reprogram the cells.
[0262] Next, CDM samples from Day 6 cultured in plates pre-coated with LAS at 37°C and 5% CO2 were simultaneously exposed to cytocahlasin B (10μg/ml) for 5 days, histone deacetylation inhibitor ( VPA, 4mm, Calbiochem) and DNA methylation inhibitor (5-azacytidine, 5μM, Sigma). Four days later, the medium was changed to differentiation medium which consists of a 3:1 ratio of CDM medium without the presence of EGFand NbActive medium (BrainBits TM) supplemented with NT-3 (20ng/ml, Peprotech) and BDNF (20ng/ml, Peprotech). The proportion of differentiation medium was gradually increased day after day until reaching 100% complete differentiation medium. After two weeks of treatment, cells were fixed for immunohistochemical analysis to investigate cell identity. Figure 18 shows cells immunostained with βIII-tubulin on day 7, indicating dedifferentiation of cells from fibroblasts to neurons. However, one week later, these trans-differentiated cells reverted to fibroblast cells and βIII -tubulin expression was lost (Figure 8). The loss of βIII-tubulin morphology and expression after removal of initiating agents indicates that complete conversion to functional and stable reprogrammed cells did not occur.
[0263] Then CDM was treated with VPA (4 mM), 5-Aza (5μM) and cytochalasin B (10μg/ml) as described above. After 2 days of chemical treatment, fibroblast cells within CDM were transfected with DNA using Lipofectamine reagent (Invitrogen) according to the manufacturer's protocol. 15 µg of eukaryotic DNA expression vectors pCMV6-XL5-PAX6 and pCMV6-XL5-Msi1 and pCMV6-XL4-Ngn2 (Origene) were used to transfect the cells. 24 hours later, the medium was changed to Basal Neural Progenitor Medium (Lonza) supplemented with Noggin (50ng/ml), EGF (20ng/ml), and bFGF (20ng/ml), and the cells were cultured at 37°C , 5% CO2and 5% 02, and the medium was changed every day. On day 6, differentiation was initiated by the gradual addition of NBActive medium (BrainBits TM) supplemented with NT-3 (20ng/mL, Peprotech), an all-trans-retinoic acid (ATRA, 5μM, Spectrum), BDNF (20ng/ ml, Peprotech), and bFGF (40ng/ml, Peprotech). To characterize the reprogrammed cells, immunohistochemical analysis and RT-PCR were performed at various time points according to the methods described in Example II using primers for nestin, βIII-tubulin, GFAP, MAP2b, and ACHE. According to previous studies, untransfected cells and cells transfected with Pax6 did not express genes specific for neuronal lineages (Table 25). On the other hand, after transfection with Msi1, nestin and ACHE levels were increased by 4-fold and 8-fold, respectively, and this expression was maintained during the 12-day period. Also GFAP mRNA levels were time-dependently increased by approximately 14-fold. Likewise, the same pattern was observed in Ngn2 transfected cells. Although the expression of βIII-tubulin and MAP2b were modestly increased after transfection with a neurogenic transcription factor, the regulation of gene expression after transfection of cells with two neurogenic factors, Msi1 or Ngn2 with Pax6, did not further increase the expression of neuronal genes. Figure 19 shows that the expression of these genes was increased when cells were transfected with Msi1 and Ngn2, with βIII-tubulin increased by almost six times a day 12.


[0264] The same gene expression pattern was observed when transfection of cells with three transcription factors (Msi1, Ngn2, and Pax6), but expression was less pronounced than in transfection cells with only Msi1 and Ngn2. In terms of immunohistochemical analysis, after 12 days of transfection, cells showed neuronal markers after transfection with Msi1 or Ngn2, as indicated by the expression of nestin and MAP2b (Figure 9). Cells transfected with pCMV-XL-PAX6 did not stain for nestin and MAP2b.
[0264] This study shows that transfection of cells within CDM with only one neurogenic factor (Msi1 or Ngn2) induces morphological changes and expression of one or more markers of neural stem cells and neuronal cells. Since the reprogrammed cells express a key neurogenic factor, a neuronal precursor marker, and a mature neuronal marker in a low percentage (10%), this suggests that cells within CDM were transformed into NSLCs and then began to differentiate through the various stages. of the neuronal differentiation determination program induced in neural stem cells. EXAMPLE XIA Gene expression analysis of reprogrammed cells in CDM
[0265] This study was designed to test the effect of transfection of cells with Msi1 and Ngn2 in the presence of MBD2 in the reprogramming process. Cells were transfected after two days of cytocahlasin B pretreatment with the DNA expression vectors using Lipofectamine reagent as described in Example X. 15 µg eukaryotic DNA expression vectors pCMV6-XL5-Musashi or pCMV6-XL4-Ngn2 , and pCMV6-XL5-MBD2 (Origene), were used to co-transfect the cells. After 24 hours, the media was changed to CDM: Neural Progenitor Maintenance Medium (1:1) supplemented with Noggin (50 ng/ml), EGF (20ng/ml), and bFGF (20ng/ml). The medium was changed every day, increasing the percentage of NPBM and decreasing the CDM medium. Cells were cultured for 6 days at 37°C, 5% CO2 and 5% O2. After one week, differentiation was started by gradually supplementing NPBM Medium with NT-3 (20ng/mL, Peprotech), all-trans-retinoic acid (ATRA, 5μM Spectrum), BDNF (20ng/mL, Peprotech) , and bFGF (40ng/ml, Peprotech). Samples were collected at the end of the study (day 14) and data were analyzed by gene ordering to identify genes that were reproducibly considered to be specific for neuronal lineages. gene expression analysis
[0266] Gene expression analysis in 8 samples was performed as previously described in Example I with the customized 2 TLDA neuronal markers in order to identify the expression of genes related to neural stem cells, neuronal cells and glial cells, and factors of growth expressed by cells after transfection. The expression of oligodendrocyte genes, such as NKx2.2 and olig2, and MAG was increased in Msi1 and Ngn2; however, the increase was more pronounced for Msi1 compared to Ngn2 (Table 26). Two markers for astrocytes (GFAP and AQP4) were highly expressed after transfection with Msi1 and Ngn2 in the presence of the DNA demethylator BD2. Interestingly, several early neuronal cell markers were increased; 12 days after transfection, TDLA data revealed increases in interneuron-specific markers such as somatostatin and Doublecortin calbindin.l (DCX), which is expressed by immature cell migration during development and acetylcholine (ACHE), an early marker of neuronal cells, were highly expressed in reprogrammed cells (Table 26). Transfection with Msi1 or Ngn2 increased the expression of type-dihydropyrimidase 3 (DPYSL3), an early marker of newborn neurons five-fold with Msi1 and seven-fold with Ngn2. The expression of microtubule-associated protein 2 (MAP2), an essential marker for the development and maintenance of early neuronal morphology, and neuronal cell adhesion molecule (NCAM) were highly expressed with Msi1 and Ngn2. The expression of enolase-2, a marker of mature neurons, was increased 20-fold by Msi1 and Ngn2. A member of the NeuroD family, NeuroD1 was highly expressed after transfection with Msi1 84.22 times and 34.27 times by Ngn2.
[0267] Gene expression of growth factors such as IGF-1, IGF-2, NPY and CSF-3 was increased after transfection with Msi1 or Ngn2. The expression of VEGF and GDNF genes was increased to almost five-fold and seven-fold by Msi1 and Ngn2, respectively. However, in transfected cells, expression of BDNF, EGF, and bFGF was not activated and even down-regulated compared to non-transfected cells. Expression of growth associated protein (GAP-43), a marker associated with growth and regeneration of neurite extension, and netrin expression, implicated in neuronal development and orientation, was highly expressed in transfected cells (Table 26). The expression of receptors for growth factors and neurotrophic was increased, such as type III receptor tyrosine kinase, Neurotrophic receptor tyrosine kinase, and neurotrophic tyrosine kinase. The fibroblast-specific markers vimentin and fibronectin were down-regulated in the reprogrammed cells.
[0268] HFF transfection with only Msi1 and Ngn2 in the presence of MBD2 increased the expression of glial cells and neuronal cell markers. Table 26: Ordering of pMsil and pNgn2 transfected CDM gene after pretreatment with cytochalasin B (10μg/ml), VPA (4mM) and 5-azacytidine (5μM). Transfected cells were cultured in differentiation medium (NbActive, BrainBits TM) supplemented with ATRA (5 µM), bFGF (40ng/ml) and BDNF (20ng/ml).

EXAMPLE XIIReprogramming of cells within CDM by Lipofectamine and nucleofection
[0269] This study was designed to improve CDM transfection by combining lipofectamine and nucleofection and using two vectors pCMV6-XL5-Msi1 and pCMV6-XL4-Ngn2 alone or in combination with pCMV-XL5-MBD2. Day 4 CDM cells were lipotransfected for 6 hours with Msi1/MBD2, Ngn2/MBD2 or Msi/Ngn2/MBD2 after 2 days of pretreatment with or without cytochalasin B. In parallel, transfection was performed in fresh HFFS after the 6 hours using Nucleofection as described in Example II, and transferred onto CDM when the lipofectamine media was changed to fresh CDM media. After 24 hours, the medium was changed to Basal Neural Progenitor Medium (NPBM, Lonza) with the presence of Noggin (50 ng/ml, Peprotech), recombinant hFGF (20ng/ml, Peprotech), and recombinant hEGF (20ng/ml , Peprotech). Differentiation was induced on day 7 by addition of NSA-A differentiation medium (StemCell Technologies) for 21 days. Gene expression analysis
[0270] Samples were collected on days 8, 15, and 21 to assess the nature of newly formed cells by analyzing the expression of various neuronal marker genes using RT-PCR according to the methods previously described in Example I. As shown in Table 27, cells transfected with a neurogenic transcription factor (Msi1 or Ngn2) express high levels of nestin and βIII-tubulin on day 8. The same pattern of expression was observed on days 15 and 21, whereas the expression was slightly reduced in the absence of cytochalasin B in Ngn2 transfected cells. The expression of all genes, with the exception of the mature neuronal marker MAP2b, was markedly increased in cells transfected with both neurogenic transcription factors. The up-regulation of these genes was slightly reduced in the absence of cytochalasin B, indicating its role in increasing reprogramming.

Immunohistochemical Analysis
[0271] Samples were collected on days 4, 8, 14, and 21 to assess the nature of any reprogrammed cells by analyzing the expression of various neural markers using immunohistochemical analysis according to the methods previously described in Example I. Immunohistochemical analysis at various time points revealed that within the first 8 days nestin expression was induced in a large proportion of cells and decreased independently of time after differentiation induction (Figure 10).
[0272] This study indicates that upon transfection of cells with one or two neurogenic genes in the presence of cytochalasin B and MBD2, the reprogrammed cells were stable in culture, responded to environmental changes (proliferation vs differentiation), and expressed neuronal markers during at least 24 days in culture. EXAMPLE XIIIA telomerase activity of NSLCs
[0273] Telomerase is active in neural precursor cells and suggests that its regulation is an important parameter for cell proliferation to occur in the mammalian brain (Caporaso GL et, 2003). This study was carried out to evaluate telomerase activity in cell extracts from adherent NSLCs (NSLCs grown on laminin-coated plates) as well as in floating neurosphere NSLCs (NSLCs grown on plates with a low binding surface) in early passage (P7) and late (P27). The telomerase activity of the 4 samples was measured by the PCR-based telomer repeat amplification protocol (TRAP) using the TRApeze® Telomerase Detection Kit (Chemicon). Briefly, cells were cultured in 24-well plates, washed in PBS, and homogenized for 30 min on ice in buffer containing 10 mM Tris-HCl, pH 7.5, 1 mM MgCl 2 , 1 mM EGTA, 0.1 mM Benzamidine, 5mM β-mercaptoethanol, 0.5% CHAPS and 10% glycerol (1X CHAPS lysis buffer, included in kit) and RNase Inhibitor. Samples were centrifuged and the protein concentration of the supernatant determined using the BCA assay. 900ng of protein from each cell extract was added directly to the TRAP reaction mix containing TRAP reaction buffer, dNTPs, template substrate primer (TS), the TRAP primer mix and Taq polymerase. Reaction mixes were incubated at 30°C for 30 minutes for template synthesis, followed by a PCR procedure (95°C/15 min for initial denaturation, 94°C/30 sec, 59°C/30 sec, 72°C / 1 min for 32 cycles) for the amplification of the extended telomerase products. To detect telomerase activity, polyacrylamide gel electrophoresis (PAGE) was performed for the reaction products on a 10% non-denaturing TBE gel. After electrophoresis, the gel was stained with SYBR® Nucleic Acid I Green Stain Gel for 30 minutes, followed by image capture using a Gel Documentation System (Alfa Innotech).
[0274] All 4 samples were positive for telomerase (as indicated by the TRAP product ladder) as shown in Figure 11. As expected, the Heat treated control (ΔH) did not show any telomerase activity (Negative Control). A 36bp internal control band (S-IC) is used to monitor PCR amplification (to distinguish false negative results). This S-IC band was observed for all samples except for the test samples. This may have been due to excessively high telomerase activity in the test samples; amplification of TRAP products and the S-IC control band are semi-competitive. All controls gave the expected results (non-TRAP products for CHAPS CTRL and TRAP ladder products for the positive control cells and the TSR8 control). EXAMPLE XIV Tumor Formation Assay
[0275] Malignantly transformed cells have reduced requirements for extracellular growth-promoting factors, are not restricted by cell-cell contact, and are often immortal. Anchorage-independent growth and proliferation is one of the main features of malignant transformation, which is considered the most accurate and rigorous in vitro assay for detection of malignant transformation of cells.
[0276] Adherent and early- and late-passing neurosphere NSLCs (P7 and P25), as well as normal human neuroprogenitor cells (hNPCs), have been investigated for anchorage-independent growth. HFFS were used as a negative control and cervical carcinoma HeLa cells were used as a positive control. Cells were pelleted by centrifugation at 150 xg for 3 minutes at room temperature (RT). The assay was performed using the CytoSelect (TM) 96-well CytoSelect (TM) Cell Transformation Assay (CellBiolabs). The base agar layer (1.2%) was dissolved in 2X DMEM / 20% PBS solution and 50 µl of the agar solution was added to the plate and incubated for 30 min at 4°C to solidify. Prior to addition of the cell agar layer, the plate was allowed to warm up for 15 minutes at 37°C. Cells were resuspended at a different density (20,000 and 5,000 cells/well) except hNPCs were resuspended with only 5000 cells/well due to lack of sufficient cells. The cells were mixed with the 1.2% agar solution, 2X DMEM / 20% PBS, and cell suspension (1: 1:1), and 75 μl of the mixture was transferred to the wells already containing the layer of base agar solidified, and was then placed at 4°C for 15 minutes to allow the cell agar layer to solidify. 100μl of proliferation medium (StemCell Technologies) was added and the plate was incubated for 8 days at 37°C and 5% CO2, before being solubilized, lysed and detected by CyQuant (TM) GR dye in a plate reader. fluorescence. Fluorescence measurement was performed using the Flexstation(TM) (Molecular Devices) with a 485/538 nm filter. Table 28: Fluorescence measurements (Relative Fluorescence Unit, RFU) indicate that under the same conditions only HeLa cells from carcinoma grow as an anchorage-independent colony, while both hNPCs and NSLCs (adherent and floating neurospheres) were negative for tumor growth in the standard agar plate tumor formation assay (CytoSelect(TM) cell transformation kit, Cell Biolabs Inc.).

[0277] As shown in Table 28, the fluorescence measurement indicated that under the same conditions only HeLa carcinoma cells grew and significantly proliferated as anchor-independent colonies, while both hNPCs and NSLCs (adherent and floating neurospheres) were negative for the Tumor growth (same value as HFFS (negative control) for 5,000 and 10,000 cells) in the standard agar plate tumor formation assay by visual observation of the cells by microscopic observation using 10X brightfield light confirm the Fluorescence measurement. Thus, the transient transfection method and genes used allow the reprogramming of cells without the neoplastic transformation that usually occurs with stable transfection or certain genes through a series of genetic and epigenetic alterations that produce a population of cells that is capable of independently proliferating of both external and internal signs that normally restrict growth. EXAMPLE XVI No genomic integration of plasmid DNA into transient transfection NSLCs
[0278] The plasmid DNA Msi1/Ngn2 (designed and built in house) was used in transient transfection for the generation of NSLCs together with MBD2 (for example 1), or 5-Aza and VPA (for example 2). Two weeks after transfection, a Southern blot was performed to test for possible integration of the plasmid DNA genome. 3μg of genomic DNA extracted from NSLC samples, as well as from HFF (a human fibroblast cell line) used as a negative control, was digested with various restriction enzymes, including BgIII, Pstl and Stul, subjected to electrophoresis on a gel 1% agarose and transferred to a positively charged nylon membrane (Roche). The membrane was hybridized in DIG Easy HYB(TM) buffer (Roche) at 42°C overnight with a 1.2 kb Dig-labeled PCR probe amplified from plasmid DNA using a set of primers. The membrane was washed twice at room temperature with 2 x SSC, 0.1% SDS for 5 min per wash, twice with 0.5 x SSC, 0.1% SDS at 65°C for 15 min per washing. Membrane hybridization signals were detected using the substrate CDP-Star(TM) (Roche). The membrane was exposed to X-ray film for analysis. Signals were removed from the membrane using extraction buffer (0.2 M NaOH, 0.1% SDS). The membrane was re-hybridized with a 0.9 kb Dig-labeled PCR probe amplified from plasmid DNA using a set of primers.
[0279] Southern blot analysis (Figure 12) with the 1.2 kb Dig-labelled PCR probe revealed distinct signals in the positive control samples, where plasmid DNA Msi1/Ngn2 was embedded in HFF genomic DNA for equivalence of integrations 1 , 10 or 100 per genome. There were some faint and identical bands that appeared in restriction enzyme digested genomic DNA from HFF, NSLC samples #1 and #2, suggesting that there is no integration of plasmid DNA into the genomic DNA of NSLCs. These bands can represent the endogenous Ngn2 gene since the 1.2 kb Dig-labelled PCR probe contains a small part of the Ngn2 gene. These data show that none, or only a small number of, NSLCs had plasmid integration into the host genome after transient transfection, and that transfected genes are only present in the cell for a short period of time (less than two weeks). EXAMPLE XVII Neuroprotective effect of transplanted hNSLCs in: 1) Animal model of multiple sclerosis.
[0280] Multiple Sclerosis (MS) is an incurable demyelinating inflammatory disease of the central nervous system (CNS) (Frohman EM et al 2006). Therapies for MS depend on manipulation of the immune system, but often with modest efficacy in reducing clinical episodes or permanent neurological disability, requiring frequent injections, and with sometimes significant side effects (Langer-Gould A et al, 2004). Experimental Allergic Encephalomyelitis (EAE) is an animal model of MS commonly used to study disease mechanisms and test potential therapies. EAE can be induced in a variety of animal species and strains [mice, rats, marmoset monkey, rhesus monkey] using various CNS antigens [Myelin Oligodendrocyte Glycoprotein (MOG), proteolipid protein (PLP) and myelin basic protein (MBP)].
[0280] After obtaining all appropriate animal approvals for the experiments, 7 to 8 week old female C57BL/6 mice were purchased from Charles Rivers, and housed in a MISPRO animal facility for one week prior to experimentation for adaptation to the new environment. C57BL/6 mice were injected s.c. with 100 µg of MOG 35-55 in CFA (Sheldon Biotechnology, McGill University) containing 5 mg/ml of Mycobacterium tuberculosis H37Ra (Disco, Inc), at 2 sites on the back. All mice received 200 ng of pertussis toxin (List Biological Laboratories, Inc) i.p. on days 0 and 2, while clinical scores were calculated blindly daily over a period of 43 days, according to the 0-5 scale as follows: 1 soft tail or waddle gait with the tonicity of the tail; 2, waddling gait with a soft tail (ataxia); 2.5, ataxia with partial limb paralysis; 3, complete limb paralysis; 3.5, complete paralysis of one limb with a partial paralysis of the second limb, 4, total paralysis of 2 limbs; 4.5, moribund, and 5, death. Treatment of an animal model of EAE with and without cells:
[0281] hNSLC and hNPCs (1.5 x106 cells in 200 μl PBS/each mouse) were given by a single intravenous injection via the tail vein, when the animals began to show symptoms of EAE (day 13 i.v). Both groups of animals received cyclosporine (10mg/kg/day) one day before cell injection and daily from the day of transplantation, to avoid any rejection of human cells. Mice sham-treated with age, sex, and strain matched i.p. injected with PBS alone, were used as a control. All groups of animals were observed for 43 days. Animals were sacrificed at 43 days p.t., brains and spinal cord were collected in 30% sucrose in PBS. Statistical analysis of clinical outcomes revealed that clinical signs of EAE were significantly attenuated in NSLC-injected animals compared to control and hNPC-injected animals. Cumulative scores were significantly reduced in NSLC-transplanted animals (Figure 13) and treatment had no effect on body weight.2) Hemiplegic animal model (unilateral ablation of the left sensorimotor cortex in adult rats).
[0282] After obtaining all appropriate animal approvals for the experiments, 8 rats per group (Sprague-Dawley, 250-300g, Charles River) were anesthetized with ketamine (Bimeda-MTC)/xylazine (50/10 mg/kg , Novopharm) and placed on a stereotaxic structure. A midline incision of the skull was made with a sterile surgical scalpel blade, the skullcap exposed and the bregma identified. The skull above the sensorimotor cortex was opened and the area of the sensorimotor cortex [0.5 - 4.0 mm caudal to bregma and 1.8-3.8 mm lateral to the midline (Paxinos and Watson 1986)] was carefully vacuumed. After ablation, treatments (Alginate, Alginate + hNPC, Alginate + NSLCs, RMX + NSLCs, RMX Only, fibrin gel, or saline) were applied directly to the brain after ablation. The opening in the skull was then filled with bone wax. In the case of a hemorrhage, small pieces of sterile homeostatic tissue were inserted into the lesion in order to stop the hemorrhage. The sutures were performed using Ethicon (TM) 1/2 needle-shaped monofilament suture in a circle. Surgeries were performed in sterile clean rooms, and topical antibiotics (Cicatrin(R), GlaxoSmithKline) were applied to the exposed skull and scalp to limit local infection. Rats were immunosuppressed by daily ip injection of starting cyclosporine A (10 mg/kg/day) starting the day before surgery, until the end of the study period. The aim of injecting cyclosporin A was to reduce the rat's immune response to the treatment. Immunosuppression was maintained until the end of the study to ensure that any potential failure to regenerate (if any) was not due to the immune reaction against the treatment. Functional scores were taken weekly in all groups and sensorimotor impairment was assessed based on behavioral tests as described below.Rotarod Test: Rotarod speed was manually calibrated for speeds of 10 and 20 RPM before all tests procedures. The animals had to perch on the stationary rod for 30 seconds to acclimate to the environment. During this time, if any animal fell, it was placed back on the rod until it had reached stationary capacities for a period of 30 seconds. The animals went through 3 tests. Animals that were comfortable on the stationary rod for 30 seconds could run at a constant speed of 10 and 20 RPM for 60 seconds, and the number of falls was recorded electronically. hind limbs, through the distance traveled through the beam of 100 cm (2.3 cm in diameter, 48 cm from the ground). Rats were systematically trained to walk along the raised beam from start to finish in order to complete the task. A safe place, ie a flat box, is placed at the end of the beam so that the mouse is motivated to complete the task. Scale used to assess the performance of walking on the beam

[0283] Prior to surgery, all animals fell at least once from the rotarod, not because they had a problem with walking or coordination, but because the speed was high. After surgery (2 days), all animals showed signs of significant walking and coordination problems, which leads to an increase in the number of rotarod falls. Three weeks after surgery, the number of falls was clearly reduced for animals that received NSLCs as treatment compared to controls (Figure 14).
[0284] The animals passed the beam walk test before surgery without any difficulty. The rats crossed the 100 cm beam and reached the safe place without falling off the beam. Two days after surgery, all groups completely failed to pass the test, and the animals were not able to remain balanced on the beam. One week after surgery, all animals showed some improvement in their ability to walk, but no significant difference was noted between the different treated groups. From week 4 through week 26, animals treated with NSLCs as well as RMX showed significant improvements in their ability to walk compared to controls (Figure 15). EXAMPLE XVIIITransfection of HFF by Various Combinations of Genes Using the Shuttle® Device and Treatment with Different Small Molecule Reprogramming for Mesendoderm-Like Cells.
[0285] HFF cells were cultured as described in CDM II medium as described in Example I, with only modification of EGF (5ng/ml) and FGF (10ng/ml), and transfection using the Shuttle® Device (Lonza) from 96-well Nucleofector (TM) ® following the procedure described in Example IV. Cells were transfected with various combinations of cDNA clones as described in Table 29. After transfection, cells were plated on plates coated with 0.1% gelatin and incubated at 37°C, 5% CO2, 5% 02. The medium was changed every two days according to Table 30. Cells were analyzed on day 4 by Real-Time Quantitative PCR.Table 29: Various combinations of plasmids with potential to transfect cells into the mesendoderm lineage.
1 where Oct4=pCMV6-XL4-Oct4, FoxD3=pCMV6-XL5-FoxD3, MBD2=pCMV6-AC-MBD2, T=pCMV6-XL5-T, Mixl1=pCMV6-XL5-MIXL1, Sox17=pCMV6-XL4-SOX17, FoxA2= PCMV6-XL5-FOXA2. All clones were purchased from Origene and prepared using the EndoFree Plasmid Maxi Kit (Qiagen). Table 30: Composition of medium from day -2 to day 10
Supplements added to media at the following concentrations: Activin A (Peprotech, 30ng/ml), HSA (Baxter, 0.5%), NEAA (Gibco, 1X), ITS (Gibco, 1X), EGF (Peprotech, 5ng/ml) , bFGF (Peprotech, 10ng/ml), CHIR99021 (Stemgent, 2um), VPA (Stemgent, 1mM), 5-Aza (Sigma, 0.5uM), BMP4 (Peprotech, 10ng/ml)
[0286] Cells were collected on day 4, being detached with TrypLE (TM), followed by centrifugation at 80xg for 5 minutes. The supernatant was aspirated and the cell pellet was frozen at -86°C until ready for RNA isolation. RNA isolation and quantification were performed as previously described in Example I. cDNA was prepared and quantitative real-time PCR was performed as previously described in Example II, with the exception that the following Taqman® Gene Expression Assays (Applied Biosystems) were used:
Table 31: Relative FoxA2, Sox17, and CXCR4 expression after transfection of HFFs once with various gene combinations with potential to reprogram cells into mesoendoderm-like cells. The exact values are not significantly accurate due to low RNA production, however, an upward trend was detected for FoxA2, Sox17, and CXCR4.

Table 32: Expression of GATA4, CDH1 (E-cadherin), p63 and SOX2 versus untreated control HFF 4 days after transfection of HFF cells with various gene combinations with potential to reprogram cells to Mesendoderm-like cells.


[0287] Identification of gene combinations that can induce the formation of Mesendoderm-like cells was investigated by transfection with combinations of Oct4 and Sox17 and FOXD3, T, and Mixl1 FoxA2, and MBD2. As shown in Table 25 and 26, the Relative Expression of CXCR4 and GATA4, both Mesendoderm/Endoderm/Mesoderm markers, appear to be up-regulated in various combinations, most notably in FoxD3/Mixl1/MBD2 and FoxD3/Sox17/MBD2. Likewise, FOXA2, a marker for Endoderm and Mesoderm, was up-regulated FoxD3/Sox17-transfected sample, although expression is still very low. Four days after transfection, SOX17 is still highly expressed in the SOX17-transfected samples (50,000 to 400,000 times compared to the untreated HFF sample). SOX17 gene expression represents residual plasmid DNA (exogenous SOX17) that still continues 4 days after transfection, and any endogenous SOX17 expression that may have been induced. Ectoderm CDH1, p63 and Sox2 markers were also up-regulated in some samples (eg Oct4/FoxD3/MBD2, Oct4/Sox17/MBD2). Reprogramming HFFs in Progenitor-type pancreatic cells: HFF cells were cultured as described in Example I, and transfected using the 96-well Shuttle® (Lonza) Device Nucleofector (TM)®, following the procedure described in Example IV. Cells were transfected with various combinations of cDNA clones as described in Table 27. After transfection, cells were plated on Fibronectin-coated collagen gels and incubated at 37°C, 5% CO2, 5% O2 . Fibronectin-coated collagen gel plates were prepared prior to transfection. Rat Tail Collagen I (Gibco) was diluted to 1.125mg/ml using 10X PBS and distilled water, where 125µl was added to each well of a 24-well plate and incubated at 37°C for 40 minutes. After rinsing with 1X PBS, Fibronectin (BD Biosciences) was added to the top of the gel at a concentration of 1.9ug/well. Media were changed every other day according to Table 33. Cells were analyzed on Day 7 by Real-Time Quantitative PCR.Table 33: Plasmids and media composition from day 0 to day 14
where FOXD3 = pCMV6-XL5-FOXD3, Sox17 = pCMV6-XL4-SOX17, Mixll = pCMV6-XL5-MIXL1, Pdx1 = pCMV6-XL5-PdX1, and Ngn3 = pCMV6-XL5-Ngn3. All clones were purchased from Origene and prepared using the EndoFree Plasmid Maxi Kit (Qiagen). Supplements added to media at the following concentrations: Activin A (Peprotech, 30ng/ml), HSA (Baxter, 0.5%), NEAA (Gibco, 1X), ITS (Gibco , 1X), B27 (Gibco, 1%), EGF (Peprotech, 5ng/ml), bFGF (Peprotech, 10ng/ml), CHIR99021 (Stemgent, 2um), Na-butyrate (Stemgent, 1 mM), VPA (Stemgent, 1 mM), 5-Aza (Sigma, 0.5U), retinoic acid (Sigma, 2um), FGF10 (Peprotech, 50ng/mL), cyclopamine (Stemgent, 2.5uM), Noggin (Peprotech, 50ng/mL)
[0287] Cells were collected on day 7 and RNA isolation and quantification were performed as previously described in Example I. cDNA was prepared and quantitative real-time PCR was performed as previously described in Example II, except that the following Assays Taqman expression (Applied Biosystems) (TM) ® were used:

[0288] Identification of gene combinations that can induce the formation of Progenitor-like pancreatic cells was investigated by transfection with combinations of FOXD3 and Sox17 and Pdx1 and Ngn3 and Mixl1, and MBD2. FoxA2, a marker for endoderm and mesoderm, was slightly up-regulated for the FoxD3/Sox17/Ngn3/MBD2-transfected sample compared to the mock-transfected GFP control sample. Likewise, CXCR4, also a marker for both endoderm and mesoderm, was slightly up-regulated (3-fold compared to GFP-ctrl) for the FoxD3/Mixl1/Ngn3/MBD2-transfected sample. 7 days after transfection, SOX17 could still be detected for samples transfected with SOX17 at varying levels (from 4 to 570-fold up-regulation compared to GFP-ctrl).
[0289] The highest up-regulation of SOX17 expression is detected for the sample transfected with Sox17/Mixl1/Pdx1/Ngn3 (570-fold compared to GFP-ctrl), which may suggest that this combination of genes may increase the amount of SOX17 RNA in cells. EXAMPLE XIX Reprogramming of Human Adipocyte-Derived Stem Cells (ADSC) to Pluripotent-Type Stem Cells (PLSC):
[0290] ADSCs (Invitrogen Corporation) were grown in cell culture flasks with StemProTM -43 complete medium medium (Invitrogen) at 37°C, 5% CO2 and the medium was changed 3 times a week. After 3 days in culture cells (passage 5) were trypsinized and counted to be transfected. Cells were transiently transfected with a plasmid: pCMV6-Oct4-2A-KLF4-2A-Nanog, pCMV-Sall4-2A-Oct4-2A-KLF4-2A-Nanog, pCMV-DAX1-2A-Oct4-2A-KLF4, pCMV - FOXD3-2A-Oct4-2A-KLF4, pCMV-Oct4-2A-KLF4-2A-Sall4, pCMV-MBD2-2A-Oct4-2A-KLF4-2A, pCMV-AGR2-2A-Oct4-2A-KLF4-2A , or Rex1-EF-Oct4-2A-KLF4 (2μg), or by two plasmids: pEF-Oct4nuc-IRES2-MBD2 with pCMV-Sox2nuc-IREC-Lin28 or pCMV-Klf4nuc-IRES2-Tpt1 nuc or pEF-Stella-IRES2 - NPM2, using Nucleofector (TM) as described in Example II. After the transfection cells are cultured in 6-well plates in suspension with a ratio of 50:50 complete adipocyte medium (StemPro(TM)-43) and embryonic stem cell medium (mTeSRI). After two days in culture, cells were re-transfected with the same plasmids listed above and cells were plated in Matrigel (TM) coated 96-well plates (BD Biosciences) in the presence of complete mTesR medium supplemented with thiazovivin (0, 5 [mu] [MU]), an ALK-5 inhibitor (SB 341542, Stemgent, 2 μM), and MEK inhibitor (PD0325901, Stemgent, 0.5 μM). The medium was changed every day and cells were cultured for 22 days at 37°C, 5% CO 2 , 5% O 2 . The Alkaline Phosphatase Detection Kit (AP, Millipore) and immunohistochemistry were performed to analyze the expression of pluripotency markers. ALP staining was performed using the AP detection kit (Millipore) according to the manufacturer's instructions.
[0291] Visual observation of the reprogrammed cells was performed by Cellomics(TM) using a live stain for SSEA-4647 (BD Biosciences) and TRA-1-81.555 (BD Biosciences) starting on Day 6 after transfection and every 5 days thereafter. Reprogrammed colonies of PLSCs, positively colored with SSEA-4 and TRA1-81, were only observed with plasmid pCMV-Sall4-2A-Oct4-2A-KLF4-2A-Nanog, pEF-Rex1 -EF-Oct4-2A-KLF4- 2A -RFP, pEF-Oct4nuc-IRES1-MBD2 with pCMV-Sox2nuc-IRES1-Lin28 and pEF-Oct4nuc-IRES1-MBD2 with pCMV-Klf4nuc-IRES2-Tpt1 nuc. These colonies emerged around day 6 and maintained in culture until the end of the study period (Day 22) with a stable morphology. Among the plasmids mentioned above, pCMV-Sall4-2A-Oct4-2A-KLF4-Nanog and pEF-Rex1-EF-Oct4-2A-Klf4-2A-RFP had the highest number of colonies. Live staining showed that these colonies express typical pluripotency markers, including SSEA-4 and -81 TRA1, and further analysis of these colonies demonstrated that the colonies also express other ESC markers, such as alkaline phosphatase and Oct4 (Figure 16). When cultures were treated with PD0325901 and SB431542 within 22 days, a 4-fold improvement in efficiency over the conventional method was obtained following transfection of ADSCs with pCMV-Sall4-2A-Oct4-2A-Klf4-Nanog and pEF- Rex1-EF-Oct4-2A-Klf4-2A-RFP.
[0292] Based on the previous study, the greatest reprogramming efficiency was observed using pEF-Rex1-EF-Oct4-2A-Klf4-2A-RFP and pCMV-Sall4-2A-Oct4-2A-Klf4-2A-Nanog. Another study was designed to determine the effect of pEF-Rex1-EF-Oct4-2A-Klf4-2A-RFP on reprogramming efficiency and to investigate the effect of individual pluripotent genes Rex1, Oct4, and Klf4 in different combinations. ADSCs were transfected as above with pEF-Rex1 - EF-Oct4-2A-Klf4-2A-RFP, pCMV6-XL5-Rex1, pCMV6-XL4-Oct4/pCMV6-XL5-Klf4, pCMV6-XL5-Rex1/pCMV6-XL4- Oct4, or pCMV6-XL5-Rex1/pCMV6-XL5-Klf4. After the second transfection, ADSC were grown in Matrigel (TM) coated 96-well plates for 24 days in the presence of mTeSRI medium supplemented with SB341542 and PD 0.325901 at 37°C, 5% CO2, 5% O2. In order to characterize cell subpopulations after transfection, live staining, immunohistochemistry, and AP staining were used to follow the shift of pluripotent markers. 1 - 5% of the total cells transfected with Rex1/Oct4 or Rex1/Klf4 showed an SSEA4 + and TRA-1 -81 + phenotype, and this pattern was stable until the end of the study period (Day 22). Observation over time showed that the phenotype of these colonies moved from an initial SSEA-4 + phenotype to a later Oct4 +/ Sox2/Nanog + phenotype by Day 22, which is closer to the final reprogrammed state of a cell -pluripotent-type trunk (Figure 17).
[0293] Several genes have been tested for their effect on reprogramming efficiency for pluripotent-like cells. ADSC cells were cultured as described in Example IX with 2 days of pretreatment with VPA and 5-aza (1 mM and 0.5 µM, respectively) in StemPro(TM) MSC SFM medium. Cells were transfected using Nucleofector (TM) ® 96-well Shuttle Device ® (Lonza) following the procedure described in Example IV and using the EW-104 transfection program with the DNA mixtures described in Table 34. After transfection the cells were plated in StemPro(TM) SFM MSC medium described in example A in Matrigel(TM) (BD Biosciences) coated on 24 well plates and incubated at 37°C, 5% CO 2 , 5% O 2 . On Day 1, the media was changed to a mixture of 75% StemPro(TM) MSC and 25% CTeh cell media; the percentage of StemPro(TM) MSC was decreased each day for four days to obtain 100% hES cell medium on Day 4. Thereafter the medium was changed every other day. The hES cell medium consisted of Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen) supplemented with 20% Knockout Serum Replacement (TM) (KSR, Invitrogen), 1 mM Glutamax (TM), 100 µM non-essential amino acids, 100 μM β-mercaptoethanol and 10 ng/ml FGF-2. Different inhibitors and growth factors were added during the course of the experiment; these are listed in Table 34. Cells were analyzed on Day 7 and Day 14 by immunohistochemical analysis and on Day 7 by RT-PCR.Table 34: Plasmids and media composition from Day 1 to Day 14.




[0294] In order to characterize cell subpopulations after transfection, live staining, immunohistochemistry, and AP staining were performed to follow the shift of pluripotent markers. Cells transfected with Oct4/UTF1/MBD2, Oct4/Dppa4/MBD2, FoxD3/Dppa4/MBD2, Oct4/FoxD3/Dppa4, or Sox2/FoxD3/UTF1 showed positive colonies for TRA1-60, TRA1-81, and SSEA4. This observation indicated that MBD2 generally had no effect by itself on reprogramming for pluripotent-like cells, except in the case of the Oct4/FoxD3/MBD2 transfection. Colonies began to form on Day 7 and continued to form through Day 14 (Figure 18) (at the end of the study period). These colonies were positive for AP as well.
[0295] These results were confirmed by RT-PCR analysis showing up-regulation of Oct4 expression as shown in Table 35. Relative expression for SOX2 was also slightly up-regulated on Day 7 after transfection of cells with Oct4/Foxd3/ MBD2. There is also a tendency to up-regulate Sox2 after transfection with Oct4/Sox2/Foxd3 and Oct4/Foxd3/Utf1. Table 35: The relative expression of pluripotent genes after ADSC transfection with various combinations of vectors as described in Table 34.
Reprogramming efficiency of defined pluripotency factors in HFF after triple transfection (one transfection every 3 days)
[0296] HFF cells were cultured as described in Example I, with the exception of the concentrations of VPA and 5-AZA which were respectively 2 mM and 2.5μM. Cells were transfected using a Nucleofector(TM)® Device II (Lonza) following the procedure described in Example II, with the exception of the amount of DNA: 1 µg of each of the 3 DNA plasmids was used. Cells that had been pretreated with VPA and 5-Aza and untreated cells were both transfected with a mixture of pCMV-Oct4nuc-IRES2-Sox2nuc, pCMV-Klf4nuc-IRES2-Cmycnuc or pCMV-Nanognuc-IRES2-Lin28. After transfection cells were plated in the fibroblast medium described in Example I, supplemented with or without VPA and 5-aza in Matrigel (TM) (BDBiosciences) coated in 6-well plates and incubated at 37°C, 5% CO2. On Day 1 and 2, media was changed to 100% mTeSRI media (StemCell Technologies) supplemented with or without VPA and 5-aza. On Day 3 and Day 6, cells from each condition were detached by incubation in TrypLE (TM) for 5 min, counted and centrifuged. Cells were retransfected as above and plated onto Matrigel (TM) coated plates in mTeSRI medium supplemented with or without VPA and 5-aza. Media were changed daily as described for day 1 and 2. The medium was supplemented in Y27632 (Stemgent, 10 µM) from day 7 to day 14 to promote viability and clonal expansion of potential reprogrammed cells. Cells were analyzed on day 20 using the Alkaline Phosphatase Detection Kit (Millipore) and by immunohistochemical analysis.
[0297] This analysis revealed that, after three transfections, three clones were considered positive for alkaline phosphatase activity and showed a complete cell/colony morphology. Antibody staining against the embryonic stem cell (ES) markers SSEA-4 and TRA-1-81 confirmed that these clones were pluripotent-like (Figure 19). Surrounding HFF cells were negative for these markers. These clones were obtained only under the condition that they did not contain inhibitors (ie: VPA and 5-AZA). Unexpectedly, no clone was observed for the condition treated with these inhibitors. Reprogramming of NSLCs in pluripotency
[0298] NSLC and neural stem cells derived from BG-01, a human ES cell line that expresses markers that are characteristic of ES cells including SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and Oct-3/4, were reprogrammed into pluripotency. BG-01 cells had previously been cultured under conditions to induce differentiation to neural stem cells as described by Chambers SM et al., 2009. NSLCs and BG-01-NSC were cultured in proliferation medium supplemented with FGF (20ng). µg/ml) and EGF (20ng/ml). NSLCs and BG-01-NSCs were transfected as described above in Example II by two episomal vectors, PFE-Oct4nuc-IRES2-MBD2 (NC1) or pCMV-FOXD3-2A-Oct4-2A-KLF4 (F72). After transfection, cells were collected and plated in uncoated petri dishes in the presence of Proliferation medium and mTeSRI medium (50:50) under proliferation conditions at 37°C, 5% CO2. After 48 hours, cells were re-transfected by the same plasmid and plated in Matrigel (TM) coated 96-well plates and cultured in the presence of mTeSRI medium supplemented by small molecules BIX0 294 (Stemgent, 2 μM) and BayK8644 (Stemgent, 2 µM) at 37°C, 5% 02 for 22 days. Live staining and immunohistochemistry were performed to characterize cell subpopulations for pluripotency markers.
[0299] NSLCs and BG-01-NSCs were positively stained with SSEA-4 starting on Day 7 and maintained for 22 days in culture (at the end of the study) (Figure 20). Within ten days, cells that were morphologically similar to ESCs were observed and expressed a broad panel of pluripotency markers, including SSEA-4, TRA1-81, Nanog and Oct4 (Figure 20). This study identified another way to obtain pluripotent-like cells from somatic cells through Neural Stem Cells-Like (NSLCs). The utility of NSLCs could offer multiple advantages for reprogramming to pluripotent-like cells. For example, obviating the need for tumorigenic genes like c-Myc reduces the risk of cancer cell induction. For neuroregenerative and neurodegenerative applications these cells could represent a valuable source of cells to further investigate the induction of human pluripotent cells, and also represent a potential source of cells to derive patient-specific multipotent stem cells and pluripotent stem cells for modeling of human diseases. EXAMPLE XX Teratoma Formation Assay in SCID mice
[0300] The transplantation of human pluripotent stem cells (SC) into "severely compromised immuno-deficient" (SCID) mice leads to the formation of differentiated tumors comprising all three germ layers to pluripotent stem cells, resembling teratomas spontaneous humans, and specialized tissue for multipotent stem cells. These assays are considered standards in the literature to demonstrate potential for differentiation of pluripotent stem cells and promise as a standard to assess the safety of SC-derived cell populations destined for therapeutic applications.
[0301] After all appropriate animal approvals for the experiment had been obtained, 24 mice were purchased from Charles Rivers, and housed in MISPRO vivarium for one week without any kind of experimentation to adapt to the new environment. One million human NSLCs, normal human neuroprogenitor cells (hNPCs), or human embryonic stem cells (ES) in 100 µl of calcium and magnesium free phosphate buffered saline (CMF-PBS) were injected with a needle 21- G intramuscularly in the right hind limb of 4-week-old male SCID-beige mice under ketamine/xylazine anesthesia (8 mice per group). After injection, the syringe was aspirated up and down a couple of times into a culture dish containing medium to verify that the cells were injected and not trapped inside the syringe.
[0302] The mice were kept for 12 weeks and monitored for clinical signs and any tumor growth regularly. Any specialized tissue growth or teratoma was monitored by external examination and an increase in muscle size relative to the same muscle on the left hind limb. When a specialized tissue or teratoma was identified, the location and size of the growth were measured (using gauges) and recorded. Specialized tissue or teratoma is usually identified primarily as a small increase in muscle size compared to left muscle control. Animals were monitored weekly until the onset of any tumor growth, and daily after tumors had appeared. After 12 weeks, mice were sacrificed by CO2 euthanasia. Each entire animal was observed for any tumor growth anywhere in the animal, and the injected muscle and comparable left muscle control were measured (with measuring gauges) (see results in table below) and then removed and stored in 4% paraformaldehyde solution for histological analysis. Muscle sizes were as follows:

[0303] Values represent the mean of 8 mice + - the standard error
[0304] Measurement of muscle size revealed that all muscles injected with human embryonic stem cells were larger than comparable left muscle controls, indicating teratoma growth in muscles injected with ES cells. About half of all human neuroprogenitor cell-injected muscles were larger than comparable left muscle controls, whereas NSLC-injected mice did not show any difference between muscles (cell-treated or not). Mice injected with NSLC did not show any evidence of tumor or teratoma growth.ReferenciasZeitlow R, Lane EL, Dunnet SB, Rosser AE.Human stem cells for CNSrepair.Cell Tissue Res.2008;331(1):301-22.Mimeault , M., Hauke, R. & Batra, SK 2007. Stem cells: a revolution in therapeutics-recent advances in stem cell biology and their therapeutic applications in regenerative medicine and cancer therapies. Clin Pharmacol Ther,82, 252-64.Levesque, MF and Neuman T. Transdiffentiation of transfected epidermal basal cells into neural progenitor cells, neuronal cells and/or glial cells. Patent, filling date 2000.Shea TB. Neuritogenesis in mouse NB2a/d1 neuroblastoma cells: triggering by calcium influx and involvement of actin and tubulin dynamics. Cell Biol Int Rep. 1990;14(11):967-79. Yeomans ND, Trier JS, Moxey PC, and Markezin ET. Maturation and differentiation of cultured fetal stomach. Effects of corticosteroids, pentagastrin, and cytochalasin B. Gasteroenterology 1976;71(5):770-7. Paterson FC, Rudland PS. Microtubule-disrupting drugs increase the frequency of conversion of a rat mammary epithelial stem cell line to elongated, myoepithelial-like cells in culture. J Cell Physiol. 1985;125(1):135-50.Bouwens L. Transdifferentiation versus stem cell hypothesis for the regeneration of islet beta-cells in the pancreas. Micro Res Tech. 1998;43(4):332-6.Bouwens L. Cytokeratins and cell differentiation in the pancreas. J Pathol. 1998b;184(3):234-9.Theise ND, Nimmakayalu M, Gardner R, Illei PB, Morgan G, Teperman L, Henegariu O, Krause DS. Liver from bone marrow in humans. Hepatology 2000;32(1):11-6.Woodbury D, Schwarz EJ, Prockop DJ, Black IB. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res. 2000;61(4):364-70.Brunet, JF; Ghysen, A. Deconstructing cell determination: proneural genes and neuronal identity. Bioessays. 1999;21:313-318.Bertrand N, Castro DS, and Guillemot F. Proneural genes and the specification of neural cell types. Nat Rev Neurosci. 2002;3(7):517-30.McCormick MB, Tamimi RM, Snider L, Asakura A, Bergstrom D, Tapscott SJ. NeuroD2 and neuroD3: distinct expression patterns and transcriptional activation potentials within the neuroD gene family. Mol Cell Biol. 1996;16(10):5792-800.Guillemot F, Lo LC, Johnson JE, Auerbach A, Anderson DJ, Joyner Al. Cell 1993;75(3):463-76. Fuck C, Gradwohl G, Morin X, Dierich A, LeMeur M, Goridis C, Guillemot F. The bHLH protein NEUROGENIN 2 is a determination factor for epibranchal placode-derived sensory neurons. Neuron 1998;20(3):483-94. Fernandes KJL, McKenzie IA, Mill P, Smith KM, Akhavan M, Barnabas-Heider F, Biernaskie J, Junek A, et al. A dermal niche for multipotent adult skin-derived precursor cells. Nature Cell Biology 2004;6:1082-1093. Jacobsen F, Hirsch T, Mittler D, Schulte M, Lehnhardt M, Druecke D, Homann HH, Steinau HU, Steinstraesser L. Polybrene improves transfection efficacy of recombinant replication-deficient adenovirus in cutaneous cells and burned skin. J Gene Med. 2006;8(2):138-46.Kearns CM, Gash DM. GDNF protects nigral dopamine neurons against 6-hydroxydopamine in vivo. Brain Res. 1995;672(1-2):104-11.Gash DM, Zhang Z, Ovadia A, Cass WA, Yi A, Simmerman L, Russell D, Martin D, Lapchak PA, Collins F, Hoffer BJ, Gerhardt GA. Functional recovery in parkinsonian monkeys treated with GDNF.Nature 1996;380(6571):252-5.Lindner MD, Winn SR, Baetge EE, Hammang JP, Gentile FT, Doherty E, McDermott PE, Frydel B, Ullman MD, Schallert T et al. Implantation of encapsulated catecholamine and GDNF-producing cells in rats with unilateral dopamine depletions and parkinsonian symptoms. Neurol Exp. 1995;132(1):62-76. Kordower JH, Emborg ME, Bloch J, Ma SY, Chu Y, Leventhal L, McBride J, Chen EY, Palfi S, Roitberg BZ, Brown WD, Holden JE, et al. Neurodegeneration prevented by lentiviral vector delivery of GDNF in primate models of Parkinson's disease. Science 2000;290(5492):767-73.Martinez-Serrano A, Bjorklund A. Immortalized neural progenitor cells for CNS gene transfer and repair. Trends Neurosci. 1997;20(11):530-8.Chambers SM, Fasano CA, Papapetrou EP, Tomishima M, Sadelain M, Studer L. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol. 2009;27(3):275-80.
[0305] Headers are includes here for reference purposes and to aid in locating certain sections. These headings are not to limit the scope and concepts described herein and these concepts may be applied in other sections of this description. Thus, the present invention is not to be limited to the embodiments shown herein, but is to be in accord with a broad scope consistent with the principles and novel aspects described herein.
[0306] It is to be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes based thereon will arise to those skilled in the art and are to be included within the scope of the claims.

权利要求:
Claims (7)
[0001]
1. Method for obtaining a neural-type stem cell (NSLC), characterized by: 1) placing a histone acetylator, a histone deacetylation inhibitor, a DNA demethylation inhibitor and/or a DNA methylation inhibitor in contact with DNA and/or chromatin from a cell of a first type that is not an NSCL;2) increasing intracellular levels in the cell of a first type of a Musashi1 polypeptide (Msi1) and/or Neurogenin 2 polypeptide (Ngn2); e3) place the cell of step (2) in culture conditions that support the transformation of the cell of a first type into an NSLC for a period of time sufficient to allow a stable expression of a plurality of genes whose expression is characteristic of the phenotypic and /or functional properties of an NSLC; wherein at the end of said time period an NSLC is obtained and wherein said obtained NSLC is defined by a stable repression of a plurality of genes expressed in the cell of a first type and wherein the NSCL expresses one or more of Sox2. Nestin, GFAP, Msi1 and Ngn2.
[0002]
2. Method according to claim 1, characterized in that the increase in intracellular levels of one or more polypeptides comprises the transient transfection of the cell of a first type with an expression vector that allows the expression of a polypeptide Musashi1 (Msi1) and /or Neurogenin 2 (Ngn2); a Musashi 1 polypeptide (Msi1) and methyl-CpG binding domain protein 2 (MBD2); a Neurogenin 2 (Ngn2) and CpG-methyl-binding domain protein 2 (MBD2) polypeptide; and, a Musashi1 polypeptide (Msi1), a Neurogenin 2 (Ngn2) polypeptide, and CpG-methyl-binding domain protein 2 (MBD2) polypeptide.
[0003]
3. Method according to claim 1 or 2, characterized in that the cell of a first type is selected from the group consisting of: embryonic stem cells, adult stem cells, progenitor cells, cells derived from mesoderm, endoderm or ectoderm, stem cell adipose-derived, mesenchymal stem cell, hematopoietic stem cell, skin-derived precursor cell, hair follicle cell, fibroblast, keratinocyte, epidermal cell, endothelial cell, epithelial cell, granular epithelial cell, melanocyte, adipocyte, chondrocyte, hepatocyte, B lymphocyte , T lymphocyte, granulocyte, macrophage, monocyte, mononuclear cell, pancreatic islet cell, sertoli cell, neuron, glial cell, cardiac muscle cell, and other muscle cells.
[0004]
Method according to any one of the preceding claims, characterized in that the DNA methylation inhibitor is selected from the group consisting of 5-azacytidine, 5-aza-2-deoxycytidine, 1-β-D-arabinofuranosyl-5-azacytosine , dihydro-5-azacytidine, zebularin, and RG108; the histone deacetylation inhibitor be selected from the group consisting of valproic acid, phenylbutyrate, Trichostatin A, Na-butyrate, 5-benzamides, and cyclic tetrapeptides; and the DNA demethylator is methyl-CpG binding domain protein 2 (MBD2).
[0005]
Method according to any one of the preceding claims, characterized in that the NSLC thus obtained has all the following characteristics: (i) ability to self-regenerate or proliferate; (ii) not being a cancer cell; (iii) being stable and not artificially maintained by forced gene expression or similar means and can be maintained in a standard neural stem cell medium; (iv) can differentiate to at least one progenitor cell, a precursor cell, a somatic cell and another type of cell plus differentiated from the same lineage, unless the cell is a terminally differentiated somatic cell;(v) does not exhibit uncontrolled growth, teratoma formation, and tumor formation in vivo.
[0006]
Method according to any one of claims 1 to 5, characterized in that the NSLC thus obtained has one or more of the following characteristics: - decreasing expression of one or more cell-specific genes of the first type; - forms neurospheres in the test of neurosphere colony formation; - capable of being cultivated in suspension or as an adherent culture; - capable of proliferating without the presence of an endogenous reprogramming agent for more than one month; - able to divide every 36 hours in slow passage; - positive for telomerase activity; - able to differentiate into a neuron-like cell, astrocyte-like cell, oligodendrocyte-like cell and combinations thereof; - decreasing telomerase expression and a or more neural stem cell markers; - having one or more neurite-like morphological processes (axons and/or dendrites) greater than the diameter of a cell in extension; - expression of at least one neural-specific antigen selected from the group consisting of of neural-specific tubulin, microtubule-associated protein 2, NCAM and a marker for a neurotransmitter; - expression of one or more functional neural markers; - able to release one or more neurotrophic factors; - negative in a tumor colony formation test ;- negative for tumor growth in SCID mice;- negative for teratoma growth in SCID mice;- able to significantly improve one or more functional measurements after placement o of an adequate number of NSLCs in the vacuum of a cerebral ablation model;- viii) capable of significantly improving or maintaining one or more functional measures after injection of an adequate number of NSLCs in an EAE model; and- ix) able to improve one or more functional measures more significantly than hNPCs in CNS damage or neurodegenerative models.
[0007]
Method according to any one of the preceding claims characterized in that a plurality of NSLCs are obtained and wherein said plurality of NSLCs are organized within a three-dimensional structure.

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法律状态:
2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-06-04| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2019-12-17| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2020-02-04| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-12-08| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-04-13| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-06-29| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 01/11/2010, OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF, , QUE DETERMINA A ALTERACAO DO PRAZO DE CONCESSAO. |

优先权:

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申请号 | 申请日 | 专利标题

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US25696709P| true| 2009-10-31|2009-10-31|

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US61/256,967|2009-10-31|

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PCT/CA2010/001727|WO2011050476A1|2009-10-31|2010-11-01|Methods for reprogramming cells and uses thereof|

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