AUTOMATED SYSTEM FOR INDUCED PLURIPOTENT STEM CELLS (IPSCS)

专利摘要:
automated system for the production of induced pluripotent stem cells or differentiated cells. The present invention relates to an automated system for the production of induced pluripotent stem cells (ipscs) from adult somatic cells. in addition, the system is used to produce differentiated adult stem cells. The inventive system is useful for isolating somatic cells from tissue samples, producing ipsc lines of differentiated adult cells by reprogramming such cells, identifying the reprogrammed pluripotent adult cells among other cells, and expanding and sorting identified reprogrammed cells.
公开号:BR112014013152B1
申请号:R112014013152-0
申请日:2012-11-30
公开日:2021-08-31
发明作者:Scott Noggle；Kevin Eggan；Stephen Chang；Susan L. Solomon
申请人:New York Stem Cell Foundation, Inc；
IPC主号:

专利说明:

FIELD OF THE INVENTION
[0001] The present invention generally refers to an automated system for the production of induced pluripotent stem cells (iPSC) from differentiated adult cells and more specifically to an automated system to isolate somatic cells from tissue samples, producing lines of iPSC from differentiated adult cells by reprogramming such cells, identifying the reprogrammed pluripotent adult cells among other cells, and expanding the identified reprogrammed cells. BACKGROUND OF THE INVENTION
[0002] Stem cells are non-specialized cells that self-renew for long periods through cell division and can be induced to differentiate into cells with specialized functions, that is, differentiated cells. These qualities give great promise to stem cells for use in therapeutic applications to replace tissue and cells damaged in various medical conditions. Embryonic stem (ES) cells are derived from the blastocyst of an early stage embryo and have the potential to become endoderm, ectoderm and mesoderm (the three germ layers) (ie they are "pluripotent"). In vitro, ES cells tend to spontaneously differentiate into various tissue types, and controlling their direction of differentiation can be a challenge. There are unresolved ethical concerns that are associated with destroying embryos to harvest human ES cells. These problems limit its availability for research and therapeutic applications.
[0003] Adult stem cells (AS) are found among the differentiated tissues. Stem cells obtained from adult tissues usually have the potential to form a more limited spectrum of cells (ie, "multipotent") and usually only differentiate into cell types from the tissues in which they are found, although recent reports have shown some plasticity in certain types of cells. They also often have limited proliferation potential.
[0004] Induced pluripotent stem cells (iPSCs or iPSCs) are produced by laboratory methods from differentiated adult cells. iPSCs are widely recognized as important tools, for example, for conducting medical research. Until then, the technology for producing iPSCs has been time-consuming and laborious. Differentiated adult cells, eg fibroblasts, are reprogrammed, cultivated and allowed to form individual colonies that represent the unique clones. Previously, identifying these cell types has been difficult because most cells are not fully reprogrammed iPSC clones. The pattern is that iPSC clones are selected on the basis of cell morphology, with desirable colonies having sharply demarcated boundaries containing cells with a high nuclear-to-cytoplasmic ratio. When clones are identified, they are hand picked by microfine glass tools and grown in "feeder" layers of cells typically, murine embryonic fibroblasts (MEF). This step is typically performed 14-21 days post-infection with a reprogramming vector. The clones are then expanded for another 14-21 days or more, before undergoing molecular characterization.
[0005] Others have focused on developing techniques to quickly and more accurately identify and characterize fully reprogrammed adult fibroblasts and their downstream differentiation potential (Bock et al., 2011, Cell 144: 439-452; Boulting et al. , 2011, Nat Biotechnol 29: 279-286). Also see, for example, co-ownership of US Ser. No. 13/159,030, filed June 13, 2011, describing the use of Fluorescence Activated Cell Separation (FACS) to identify and screen live unique subpopulations of s, as defined by unique surface protein expression patterns.
[0006] Thus, stem cells are an attractive source of cells for therapeutic applications, medical research, pharmaceutical testing and the like. However, in the art there is still a longstanding need for an automated system to rapidly produce and isolate reproducible iPSC cell lines under standard conditions to meet these and other needs. SUMMARY OF THE INVENTION
[0007] The invention provides a system for using somatic cells from adult tissue and producing induced pluripotent stem cells (iPSCs) from these somatic cells, for example, adult fibroblasts. In one embodiment, the system also uses previously isolated somatic cells as a starting point.
[0008] The invention provides an automated system for generating and isolating iPSCs, comprising: a somatic cell plating unit for placing somatic cells on a plate; and an induction unit for automated somatic cell reprogramming by contacting the somatic cells of the somatic cell plating unit with reprogramming factors to produce iPSCs.
[0009] In one embodiment, the system further comprises a screening unit to selectively screen and isolate the iPSCs produced by the induction unit, for example, by identifying specific iPSC markers, including, for example, surface markers on cells . In an illustrative example, somatic cells are fibroblasts.
[00010] Furthermore, in one embodiment, the invention provides an automated system for generating and isolating differentiated adult stem cell cells, for example, iPSCs, embryonic stem cells (ES) or mesenchymal stem cells (MS), comprising : a stem cell plating unit for placing cells on a plate; and an induction unit for automated cell reprogramming by contacting the stem cells of the stem cell plating unit with reprogramming factors to produce differentiated adult cells. In one embodiment, the system further comprises a screening unit to selectively screen and isolate the differentiated adult cells produced by the induction unit.
[00011] In one aspect, the invention provides an automated system for generating and isolating differentiated adult cells from induced pluripotent stem cells (iPSC), comprising: an iPSC plating unit for placing iPSCs on a plate; and an induction unit for automated reprogramming of iPSCs by putting them in contact with iPSCs in the iPSC plating unit with reprogramming factors to produce differentiated adult cells. In another modality, the system includes a screening unit for selective screening and isolating the differentiated adult cells produced by the induction unit.
[00012] The invention also provides iPSCs, differentiated or transdifferentiated cells produced by the system of the invention. Furthermore, an array composed of a population of cells obtained from iPSCs of the invention or differentiated cells are included in this document. For example, differentiated cells include hematopoietic cells, muscle cells, cardiac muscle cells, liver cells, cartilage cells, epithelial cells, urinary tract and neuronal cells. In another embodiment, a cell bank generated by the system of the invention is included. BRIEF DESCRIPTION OF THE FIGURES
[00013] Figure 1 shows the steps for acquiring a fibroblast cell bank.
[00014] Figure 2 shows the steps to obtain a stem cell matrix from a fibroblast bank.
[00015] Figure 3 is a flowchart showing the steps in a system for producing iPSCs.
[00016] Figures 4A-4C show an example of the flow of patient samples through multi-well tissue culture plates during the automated reprogramming process.
[00017] Figures 5A-5C illustrate an example of an equipment configuration required to perform the workflow.
[00018] Figures 6A-6C show the automated biopsy growth tracking system. In Figure 6A, biopsies or discarded tissue are plated into multiple wells of a 6-well plate and maintained by an automated system that feeds, images, passes and freezes fibroblast growths. Examples of the image analysis interface are shown as a typical example. Figure 6B: Cell number is extrapolated from confluence measurements based on linear regression of an independently generated standard curve. Figure 6C: An example of cell counts for a typical biopsy growth maintained in our automated system. Extrapolated cell numbers per patient sample are plotted for each well independently (top) allowing calculation of the total sample output (bottom).
[00019] Figures 7A-D show FACS analysis and graphs showing automated iPSC reprogramming. Expression levels of pluripotent surface markers in reprogrammed human fibroblasts were followed over a 3-week period to observe kinetic reprogramming and determine optimal time points at which to isolate defined cell populations. Figure 7A shows FACS channel scheme used for analysis. Figure 7B: A substantial proportion of cells co-expressing traditional pluripotency surface markers SSEA4 & TRA-1-60 retain the fibroblast marker CD13 at all times during reprogramming using viral vectors to introduce reprogramming factors such as Oct4, Sox2, Klf4 and c-Myc. Box diagrams indicating aggregated data from 131 experiments (retrovirus, n = 66, Sendai virus, n = 65) are shown. While Sendai-mediated reprogramming produces more SSEA4/TRA-1-60 double positive cells, (C) there is a delay in the clearance of CD13 from the surface. (D) Sample staining pattern of a patient cell line reprogrammed using Sendai/Cytotune system in our automated system. On both days 7 and 13 post-infection (dpi), more than half of the double positive cell SSEA4/TRA-1-60 cells lost CD13. Furthermore, at both time points analyzed, CD13 negative/Nanog positive cells are present in this fraction, suggesting that they can be isolated by negative selection against CD13.
[00020] Figures 8A-C show analyzes of pre-screening FACs and a part of the automated system demonstrates enrichment and selection of iPSC clones. Figure 8A shows that unreprogrammed cell populations can be removed from iPSC cultures by negative selection by a fibroblast marker. In the example, fibroblasts are efficiently removed from the culture containing 2% established iPSCs leaving TRA-1-60 positive iPSCs untouched. Figure 8B shows a Miltenyi MultiMACS system integrated into the Hamilton liquid handler that can classify 24 samples in parallel. Figure 8C is an illustration of the iPS-enriched fraction of the anti-fibroblast magnetic negative selection step that is plated onto 96-well imaging plates at limiting dilution. These plaques are selected using live cell staining for the pluripotency surface marker TRA-1-60 or TRA-1-81. Wells with positive TRA-1-60 iPSCs are identified by automated image analysis using Celigo software capable of single colony confirmation. Wells that meet both criteria of containing a single colony that is positive for the surface marker are selected for passage and expansion and QC.
[00021] Figures 9A-B provide an illustration for the scorecard of trials described here. The first phase of the quality control scan uses a panel of transgene markers and pluripotency differentiation to choose an initial set of three clones. Figure 9A shows transcription counts after normalization for HK gene expression for two HESC strains, Sendai positive control, fibroblast negative control and iPS strains derived by FACS screening analyzed in passages 5 and 10. All assays are performed compared to a panel of normal ESC and iPS lines maintained under similar conditions. Figure 9B illustrates the second stage of our quality control scan, which uses an additional 83 germline/germline markers to monitor differentiation capacity in embryonic body assays. Single EBs are generated and pooled to generate RNA for analysis of germ layer marker expression in the Embryonic Body Scorecard Assay. A clustered dendrogram analysis of gene expression in EBs collected from nine different embryonic stem cell lines is shown. After normalization, data generated from direct lysis of six EBs compare favorably to data generated from total RNA extracted and purified from EBs prepared from mass culture.
[00022] Figures 10A-B demonstrate high-throughput karyotyping of iPSCs based on nCounter Nanostring assays for CNVs. Figure 10A is an example of nCounter karyotype assay on BC1 iPSCs; Figure 10B is an example of the nCounter karyotype assay on 1016 fibroblasts with partial gain and loss of chromosome arms. Comparison with Affymetrix SNP 6.0 chip data demonstrating copy number gained in part of q-arm of Chr1 (upper range, 1q21.2 - 1q43) and loss of part of long arm of Chr6 (lower range, 6q16.3 - 6q26). DETAILED DESCRIPTION OF THE PREFERRED MODALITY
[00023] The present invention is based on the generation of an automated system to produce iPSCs and differentiated cells. The system of the invention greatly improves the efficiency and reproducibility of making standardized iPSC strains. Typically, researchers generate iPSCs by hand, which limits the cells' usefulness due to researcher variability and an inability to generate large numbers of cells. The system of the invention circumvents these problems with a fully automated system from tissue or cell sample reception to bank building of large stocks of well-defined iPSC strains. The system allows for consistency and invariability to generate large numbers of cells from multiple donors, which will facilitate the use of iPSC technology to discover treatments and cures for many diseases.
[00024] In one embodiment, the workflow system of the invention includes an automated system for generating and isolating iPSCs, comprising: a somatic cell plating unit, e.g., fibroblast, for placing somatic cells on a plate; and an induction unit for automated reprogramming of cells in contact with the plating unit cells with factor reprogramming to produce iPSCs. In another embodiment, the system of the invention includes a screening unit for selectively classifying and isolating iPSCs produced by the induction unit by identifying specific iPSC markers, including, for example, surface markers or green fluorescent proteins inserted by a transfection vector. Somatic cells can be obtained from cell lines, biopsy or other tissue samples, including blood and the like.
[00025] In another embodiment, the invention provides an automated system to generate and isolate adult cells differentiated from stem cells, for example, iPSCs, embryonic stem cells (ES) or mesenchymal stem cells (MS), comprising: a unit stem cell plating to place cells, eg iPSCs, ES or MS cells, on a plate; and an induction unit for automated cell reprogramming by contacting cells in the stem cell plating unit with reprogramming factors to produce differentiated adult cells. In one embodiment, the system further includes a screening unit for selective screening and isolating the differentiated adult cells produced by the induction unit by identifying specific markers for the differentiated adult cells.
[00026] In yet another embodiment, the invention provides an automated system to generate and isolate differentiated adult cells from induced pluripotent stem cells (iPSC), comprising: an iPSC plating unit for placing iPSCs on a plate; and an induction unit for automated reprogramming of iPSCs by putting them in contact with iPSCs in the iPSC plating unit with reprogramming factors to produce differentiated adult cells. In one embodiment, the system further includes a screening unit for selective screening and isolating the differentiated adult cells produced by the induction unit by identifying specific markers for the differentiated adult cells.
[00027] The invention provides an automated workflow system to produce iPSCs from differentiated adult cells. Generally speaking, the workflow system of the invention offers a new workflow system that starts with differentiated adult cells (eg tissue samples or isolates) and results in iPSCs or adult cells derived from pluripotent cells. In one embodiment, the differentiated adult cells are preferably fibroblasts obtained, for example, from skin biopsies. Adult fibroblasts are converted to induced pluripotent stem cells (iPSCs) by the invention's workflow, which incorporates automation and robotics. The invention's workflow system is capable of generating thousands of iPSCs in parallel, resulting in an accelerated period of time, in a period of months instead of years that would previously have been necessary. The creative workflow system can be adapted to any raw material cell isolation system and applied to direct or indirect reprogramming and transdifferentiation, for example. The inventive workflow system will allow production employing cell arrays of array cells 6, 24, 96, 384, 1536 in size, or larger. The inventive workflow system is flexible and will allow for multiple iterations and flexibility in cell and tissue type. The description here is shown with fibroblasts as an illustrative somatic cell. As noted in this document, other cell types are used in the system. The example is not to be limited in this way.
[00028] The Workflow System
[00029] The workflow system is divided into four independently operated units: Quarantine Somatic Cell Isolation and Growth (System 1); Quarantine Assay (System 2); Thawing, Infection and Identification (Systems 3, 4 and 5); and Maintenance, QC, Expansion and Freezing. (Systems 6, 7 and 8).
[00030] In addition, an automated -80 storage and retrieval system for storing fibroblasts and final clones in 1.4 mL Matrix screw cap tubes is part of the system. The systems, and the steps and operations that each unit will perform, will be described below. System 1, Part A: Quarantine Somatic Cell Isolation and Growth Workflow, Pre-Mycoplasma Biopsy Processing Test
[00031] Technician will plate 40 biopsies per week in 6-well plates; 6-well plates will be kept in a quarantine incubator with a capacity of 200 plates;
[00032] Periodic confluence checks are performed on an integrated Cyntellect Celigo Cytometer.
[00033] System components that can be used to perform these automated steps include, by way of example, STARlet Manual Load, a Modular Arm for 4/8/12 ch./MPH, 8 channels with 1000μl Pipetting Channels, and an iSWAP Plate Handler, all available from Hamilton Science Robotics. If centrifugation is required or desired, an Agilent VSpin Microplate Centerfuge can be used. The software can be Celigo API Software. The incubator can be a Cytomat incubator. For plate handling, a Cytomat 24 Barcode Reader, Cytomat 23mm Stackers, and a Cytomat 400mm transfer station can be used. For Tilt Plate, a MultiFlex Tilt Module can be used. The system controller can be a Dell PG with a Windows XP operating system. The carrier package can be a Q Growth Carrier Package. System 1, Part B: Quarantine Workflow, Mycoplasma Test
[00034] Retrieve from incubator to Quarantine Growth STARlet deck, remove media from plating wells for mycoplasma based ELISA test.
[00035] Manually transfer 96-well assay plates to Quarantine Assay STARlet. System 1, Part C: Quarantine Growth Workflow After Passing Mycoplasma Test
[00036] Expanded fibroblasts distributed in several cryogenic vials, capped, transferred to SAM - 80°C.
[00037] The system components that can be used to perform these automated steps can be selected from the same components used in the Quarantine Growth Workflow, except that a STARlet Auto Load can be used. A Spectramax L Reader can be used as a spectral acquisition device. System 2: Quarantine Test Workflow
[00038] Test using the glow luminescence method, Lonza MycoAlert.
[00039] Perform luminescence plate reading on spectral acquisition device.
[00040] System components that can be used to perform these automated steps include STARlet Manual Load, a Modular Arm for 4/8/12 ch./MPH, 8 channels with 1000μl Pipetting Channels, and an iSWAP Plate Handler, all available by Hamilton Science Robotics. For luminescence assays the BioTek Synergy HT Reader can be used. The system controller can be a Dell PG with a Windows XP operating system. The carrier package can be a Q Growth Carrier Package. Systems 3, 4 and 5: Thawing, Infection and Identification
[00041] Defrost Module & Infection Module
[00042] Recover SAM-80°C cryotubes (61, 190)
[00043] Defrosting in the heating block (122)
[00044] Decap (Hamilton Capper Decapper) (126)
[00045] Add media to dilute cryoprotectants (122)
[00046] Rotation (128)
[00047] Suspension again in plating data (122)
[00048] Plaque one sample per well of 6 wells (62, 122)
[00049] Move to incubator (130, 132)
[00050] Recover fibroblasts for about 3-4 days
[00051] Confluence check in Cyntellect Celigo Cytometer (124)
[00052] Fibroblast passage from all wells on the same day for reprogramming (122)
[00053] In batches, tryspin passage wells (122)
[00054] Cell count in Cyntellect Celigo Cytometer (124)
[00055] Plate a defined number per well in one-to-three wells of a 24-well plate consolidating samples into as few 24-well plates as possible (64, 122)
[00056] Return plates to incubator overnight (130, 132)
[00057] Recover plates and thaw virus in tube format and add to each well of fibroblasts in 24-well plates (130, 122)
[00058] Daily partial media changes (122)
[00059] Magnetic Screening Module
[00060] Harvesting cultures with accutase for single cell suspension (134)
[00061] Dilute staining buffer (134)
[00062] Staining with magnetic beads against fibroblast surface marker (134)
[00063] Washing step (134)
[00064] Apply magnet (for Dynal granules) or column (for Miltenyi system) (134, 136)
[00065] Recover the non-magnetic fraction for new wells (134)
[00066] Cell count in Cyntellect Celigo Cytometer (124)
[00067] Dilute appropriate cell density for delivery of 110 cells per well to the 96-well plate in the media passage (66, 134)
[00068] Recover new Matrigel or matrix coated 96-well plate from the 4°C incubator (142)
[00069] Distribute cells to 96-well matrix plates, number based on cell count, eg two per plate per infection (66, 134)
[00070] Return plates to incubator (132)
[00071] Daily partial media changes (122)
[00072] Colony Identification Module
[00073] Recover 96-well plates from the incubator for colony identification liquid handler (66, 132, 138)
[00074] Perform live cell staining with pluripotency surface marker (138)
[00075] Image in Cyntellect Celigo Cytometer (140)
[00076] Identify wells with a single marker positive colony that has a strong colony boundary (140)
[00077] Technicians analyze hits and select 6 per original sample for passage and retrieve positive plate and well IDs.
[00078] Choose finger wells with single positive colonies (138)
[00079] Recover new Matrigel or matrix coated 96-well plate from the 4°C incubator (68, 142)
[00080] Harvest selected wells and passage to a new 96-well matrix plate consolidating new clones into as few plates as possible and plating each one in the media passage (68, 138)
[00081] Daily partial media changes (122)
[00082] The system components that can be used to perform these automated steps can be selected from the same components used in the Quarantine Growth Workflow, with the addition of one or more model CORE 96 PROBEHEAD II 1000μl probe heads. Systems 6, 7 and 8: Maintenance, QC, Expansion and Freezing
[00083] Maintenance Module
[00084] Clones will be serially passaged 1:1 into new 96-well matrix coated plates until the colony density is high enough (68-72, 160)
[00085] Daily feeding of all plates with medium change ~ 75% with 96-tip head (160)
[00086] Periodic monitoring of growth rates and colony density in the Cyntellect Celigo Cytometer (166)
[00087] Plate replication to produce plates for clone QC (74-86, 160)
[00088] Purpose is to expand clones onto multiple plates for use in multiple QC assays to eliminate low performing clones until there are two or three high quality clones left per original sample
[00089] Clones will also be handpicked and re-arranged that go through the QC steps as low performing clones are eliminated to consolidate clones onto as few plates as possible (80, 86, 160)
[00090] Daily food throughout this process (160)
[00091] QC Module
[00092] Cell harvesting (74, 150)
[00093] Cell count (164)
[00094] Plate a defined cell number in v-bottom plates (range 5000-10000 cells/well) in 2-6 replications per lineage (84, 150)
[00095] Return to incubator - (1g aggregation) (172)
[00096] Change of media after two days (150)
[00097] Incubate an additional 12 days in the incubator (172)
[00098] Change of media after two days (150)
[00099] Transfer to nucleic acid preparation station to remove media from wells leaving embryo bodies in the well (84, 192)
[000100] Suspension again in RNA lysis buffer, combine and mix replicates for each sample and make plates available for analysis in the nCounter Nanostring Assay (84, 192)
[000101] Freezing Module
[000102] Starts with a 96-well plate, after an expansion pass (88)
[000103] Incubate 6 days in the incubator (172)
[000104] Media change after two days (154)
[000105] Remove the incubator plate (88, 162)
[000106] Remove media (needs to be complete) (154)
[000107] Add pre-freeze cooling media (matrigel diluted in growth media) (154)
[000108] Incubate in an incubator for 1h (172)
[000109] Remove media (needs to be complete) (154)
[000110] Cold Freeze Media Addition - Low Volume (154)
[000111] Seal plate (88, 164
[000112] Samples taken off-line for storage at -80°C for freezing (190)
[000113] Storage in Vapor Phase Liquid Nitrogen
[000114] Cryogenic Ampoule Storage
[000115] Starts with a 96-well plate, after an expansion pass (90)
[000116] Incubate 6 days (172)
[000117] Daily Partial Media Changes (154)
[000118] Pass 1:1 wells to a 24-well plate (92, 154)
[000119] Incubate 6 days (172)
[000120] Daily Partial Media Changes (154)
[000121] Pass 1:1 wells to a 6-well plate (94, 154)
[000122] Incubate 4-6 days (172)
[000123] Daily Partial Media Changes (154)
[000124] Remove the incubator plate (162)
[000125] Partial media exchange with pre-freeze media (154)
[000126] Incubate in an incubator for 1h (172)
[000127] Harvesting cells for freezing and normal passage (154)
[000128] Change to matrix tubes, two or three tubes per well (96, 154)
[000129] Rotation and Remove Media (168, 154)
[000130] Addition of cold freezing media (154)
[000131] Cover tubes (170)
[000132] Off-line samples taken for storage at -80°C (190)
[000133] The system components that can be used to perform these automated steps can be selected from the same components used in the Quarantine Growth Workflow.
[000134] In this document "adult" means post-fetal, that is, an organism from the neonate stage to the end of life and includes, for example, cells obtained from tissue delivered from placenta, amniotic fluid and/or cord blood umbilical.
[000135] As used herein, the term "adult differentiated cells" encompasses a wide range of differentiated cell types obtained from an adult organism, which are conducive to the production of iPSCs using the instant-described automation system. Preferably, the differentiated adult cell is a fibroblast"." Fibroblasts, also known as "fibrocytes" in their less active form, are derived from mesenchyme. Its function includes secreting precursors of extracellular matrix components, including, for example, collagen. Histologically, fibroblasts are highly branched cells, but fibrocytes are usually smaller and often described as spindle-shaped. Fibroblasts and fibrocytes derived from any tissue can be employed as the raw material for the automated workflow system in the invention.
[000136] As used herein, the term "induced pluripotent stem cells" or, iPSCs, means that stem cells are produced from differentiated adult cells that have been induced or altered, that is, reprogrammed into cells capable of differentiating. if in tissues of all three or germinal dermal layers: mesoderm, endoderm and ectoderm. The iPSCs produced do not refer to cells as they are found in nature.
[000137] Mammalian somatic cells useful in the present invention include, by way of example, adult stem cells, Sertoli cells, endothelial cells, granulosa epithelial cells, neurons, pancreatic islet cells, epidermal cells, epithelial cells, hepatocytes, cells hair follicle, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), erythrocytes, macrophages, monocytes, mononuclear cells, fibroblasts, cardiac muscle cells, other known muscle cells, and generally any living somatic cells. In particular embodiments, fibroblasts are used. The term somatic cells, as used herein, is also intended to include adult stem cells. An adult stem cell is a cell that is capable of generating all types of cells from a specific tissue. Exemplary adult stem cells include hematopoietic stem cells, neural stem cells, and mesenchymal stem cells.
[000138] An advantage of the present invention is that it provides an essentially unlimited supply of isogenic and syngeneic human cells suitable for transplantation, use in drug discovery assays or for disease modeling. iPSCs are tailored specifically for the patient, preventing immune rejection. Therefore, it will remove the significant problem associated with current methods of transplantation, such as transplanted tissue rejection, which can occur because of host versus graft or graft versus host rejection. When used for drug discovery, cells demonstrate each person's response to chemicals when used in drug discovery or their individual manifestation of disease in disease models. Various types of iPSCs or fully differentiated somatic cells prepared from human-derived somatic cell-derived iPSCs can be stored in an iPSC bank as a cell library, and one type or more types of iPSCs in the library can be used for the preparation of somatic cells, tissues or organs that are free from rejection by a patient undergoing stem cell therapy.
[000139] The iPSCs of the present invention can be differentiated into a number of different cell types to treat a variety of disorders by methods known in the art. For example, iPSCs can be induced to differentiate into hematopoietic stem cells, muscle cells, cardiac muscle cells, liver cells, cartilage cells, epithelial cells, urinary tract cells, neuronal cells, and the like. The differentiated cells can be transplanted back into a patient's body to prevent or treat a condition or used to advance medical research or to develop drug discovery trials. Thus, the methods of the present invention can be used as a treatment or to develop a treatment for an individual who is having a myocardial infarction, congestive heart failure, stroke, ischemia, peripheral vascular disease, alcoholic liver disease, cirrhosis, Parkinson's disease , Alzheimer's disease, diabetes, cancer, arthritis, wound healing, immunodeficiency, aplastic anemia, anemia, Huntington's disease, amyotrophic lateral sclerosis (ALS), lysosomal storage diseases, multiple sclerosis, spinal cord injuries, genetic disorders and diseases similar, where an increase or a replacement of a specific cell/tissue type or cell dedifferentiation is desirable.
[000140] The term "totipotency" refers to a cell with a developmental potential to produce all cells in the adult body as well as extraembryonic tissues, including the placenta. The fertilized egg (zygote) is totipotent, as are the cells (blastomeres) of the morula (up to the 16-cell stage after fertilization).
[000141] The term "pluripotent", as used in this document, refers to a cell with the potential to develop, under different conditions, to differentiate from cell types characteristic of all three layers of germ cells, ie, endoderm ( for example, gut tissue), mesoderm (including blood, muscle and vessels) and ectoderm (such as skin and nerves). A pluripotent cell has a lower development potential than a totipotent cell. The ability of a cell to differentiate into all three germ layers can be determined using, for example, a hairless mouse teratoma formation assay. In some embodiments, pluripotency may also be evidenced by the expression of embryonic stem cell (ES) cell markers, although the preferred test for pluripotency of a cell or cell population generated using the compositions and methods described in this document is the demonstration that a cell has the developmental potential to differentiate into cells from each of the three germ layers. In some embodiments, a pluripotent cell is called an "undifferentiated cell". In this sense, the terms "pluripotency" or "pluripotent state", as used herein, refer to the developmental potential of a cell that provides the ability for the cell to differentiate into all three embryonic germ layers (endoderm, mesoderm and ectoderm). Those skilled in the art are aware of the embryonic germ layer or lineage that gives rise to a particular cell type. A cell in a pluripotent state usually has the potential to divide in vitro for a long period of time, for example, greater than a year or more than 30 passages.
[000142] The term "multipotent", when used in reference to a "multipotent cell", refers to a cell that has the developmental potential to differentiate into cells of one or more germ layers, but not all three . Thus, a multipotent cell can also be called a "partially differentiated cell". Multipotent cells are well known in the art, and examples of multipotent cells include adult stem cells, such as, for example, hematopoietic stem cells and neural stem cells. "Multipotent" indicates that a cell can form multiple types of cells in a given lineage, but not cells from other lineages. For example, a multipotent hematopoietic cell can form the many different types of blood cells (red, white, platelets, etc.), but it cannot form neurons. In this sense, the term "multipotency" refers to a state of a cell with a degree of developmental potential that is less than totipotent and pluripotent.
[000143] The terms "stem cell" or "undifferentiated cell", as used herein, refer to a cell in an undifferentiated or partially differentiated state that has the property of self-renewal and has the developmental potential to differentiate into multiple cell types, with no specific implied meaning in relation to developmental potential (ie, totipotent, pluripotent, multipotent, etc.). A stem cell is capable of proliferating and giving rise to more of these stem cells while maintaining their developmental potential. In theory, self-renewal can occur by either of two main mechanisms. Stem cells can divide asymmetrically, which is known as mandatory asymmetric differentiation, with one daughter cell maintaining the developmental potential of the originating stem cells and the other daughter cell expressing some other distinct specific function, phenotype and/ or potential for the development of the cell of origin. The daughter cells can themselves be induced to proliferate and produce progeny that further differentiate into one or more mature cell types, while also maintaining one or more cells with a potential for parental development. A differentiated cell can be derived from a multipotent cell, which in turn is derived from a multipotent cell, and so on. While each of these multipotent cells can be considered stem cells, the variety of cell types each of these stem cells can give rise to, that is, their developmental potential can vary considerably. Alternatively, some of the stem cells in a population can divide symmetrically into two stem cells, known as stochastic differentiation, thus keeping some stem cells in the population as a whole, while other cells in the population give rise to just differentiated progeny. In this sense, the term "stem cell" refers to any subset of cells that have the developmental potential, under particular circumstances, to differentiate into a differentiated or more specialized phenotype, and that retain the ability, under certain circumstances, to to proliferate without substantially differentiating. In some embodiments, the term stem cell generally refers to a naturally occurring cell of origin whose descendants (progeny cells) often specialize in different directions by differentiation, eg, acquiring completely individual characters, such as it occurs in the progressive diversification of embryonic cells and tissues. Some differentiated cells also have the ability to give rise to cells with greater potential for development. This ability can be natural or it can be artificially induced after treatment with various factors. Cells that start out as stem cells can proceed towards a differentiated phenotype, but then can be induced to "reverse" and re-express the stem cell phenotype, a term often referred to as "dedifferentiation" or "reprogramming" or "retrodifferentiation" by those skilled in the art.
[000144] The term "embryonic stem cell" as used herein refers to naturally occurring pluripotent stem cells from the inner cell mass of the embryonic blastocyst (see, for example, US Pat. Nos. 5,843,780; 6,200,806; 7,029,913; 7,584,479, which are incorporated herein by reference). Such cells can similarly be obtained from the inner cell mass of blastocysts derived from somatic cell nuclear transfer (see, for example, US Pat. Nos. 5,945,577, 5,994,619, 6,235,970, which are incorporated in this document for reference). Embryonic stem cells are pluripotent and give rise during development to all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. In other words, they can develop in each of the more than 200 cell types in the adult body, when given enough stimulation and needed for a specific cell type. They do not contribute to extraembryonic membranes or the placenta, that is, they are not totipotent.
[000145] As used in this document, the distinctive characteristics of an embryonic stem cell define an "embryonic stem cell phenotype". In this sense, a cell has the phenotype of an embryonic stem cell if it possesses one or more of the unique characteristics of an embryonic stem cell, such that this cell can be distinguished from other cells that do not have the embryonic stem cell phenotype. Distinctive phenotypic characteristics of exemplary stem cells include, without limitation, expression of specific intracellular or cell surface markers, including protein and microRNAs, gene expression profiles, methylation profiles, deacetylation profiles, proliferative capacity, differentiation capacity, karyotype, responsiveness to particular and similar cultural conditions. In some embodiments, the determination of whether a cell has an "embryonic stem cell phenotype" is made by comparing one or more characteristics of the cell to one or more characteristics of an embryonic stem cell line grown within the same laboratory .
[000146] The term "somatic stem cell" is used herein to refer to any pluripotent or multipotent stem cell derived from non-embryonic tissue, including fetal, juvenile and adult tissue. Natural somatic stem cells have been isolated from a wide variety of adult tissues including blood, bone marrow, brain, olfactory epithelium, skin, pancreas, skeletal muscle and cardiac muscle. Each of these somatic stem cells can be characterized based on gene expression, factor responsiveness, and cultured morphology. Exemplary naturally occurring somatic stem cells include, but are not limited to, neural stem cells, neural crest stem cells, mesenchymal stem cells, hematopoietic stem cells, and pancreatic stem cells. In some aspects described in this document, a "somatic pluripotent cell" refers to a somatic cell, or a cell from the progeny of the somatic cell, that has had its developmental potential altered, that is, increased, to that of a pluripotent state by contact with, or by inducing, one or more reprogramming factors using the compositions and methods described herein.
[000147] The term "progenitor cell" is used in this document to refer to cells that have greater potential for development, that is, a cell phenotype that is more primitive (eg, is at an earlier stage along a pathway of development or progression) with respect to a cell which it can give rise to by differentiation. Progenitor cells often have significant or very high proliferative potential. Progenitor cells can give rise to several distinct cells having less potential for development, that is, differentiated cell types, or to a single differentiated cell type, depending on the pathway of development and the environment in which the cells develop and differentiate.
[000148] As used herein, the term "somatic cell" refers to any cell other than a germ cell, a cell present in or obtained from a pre-implantation embryo, or a cell resulting from the proliferation of such a cell in vitro. In other words, a somatic cell refers to any cell that makes up the body of an organism, as opposed to a germline cell. In mammals, germline cells (also known as "gametes") are the sperm and eggs that fuse during fertilization to produce a cell called the zygote, from which the entire mammalian embryo develops. Any other cell type in the mammal's body -- besides sperm and eggs, the cells they're made of (gametocytes) and pluripotent, undifferentiated embryonic stem cells -- is a somatic cell: internal organs, skin, bones, blood and connective tissue are all made up of somatic cells. In some embodiments, the somatic cell is a "non-embryonic somatic cell", by which is meant a somatic cell that is not present in or obtained from an embryo and does not result from the proliferation of that cell in vitro. In some embodiments, the somatic cell is an "adult somatic cell", by which is meant a cell that is present in or obtained from an organism other than an embryo or fetus or results from the proliferation of such a cell in vitro. Unless otherwise indicated, the compositions and methods for reprogramming a somatic cell described herein can be performed both in vivo and in vitro (where in vivo is practiced when a somatic cell is present within an individual, and where in vitro is practiced using an isolated somatic cell maintained in culture).
[000149] The term "differentiated cell" encompasses any somatic cell that is not, in its native form, pluripotent, as that term is defined in this document. Thus, the term a "differentiated cell" also encompasses cells that are partially differentiated, such as multipotent cells, or cells that are stable, partially reprogrammed, or partially differentiated non-pluripotent cells, generated using any of the compositions and methods described herein. . In some embodiments, a differentiated cell is a cell that is a stable intermediate cell, such as a non-pluripotent cell, partially reprogrammed. It should be noted that placing multiple primary cells in culture may lead to some loss of fully differentiated features. Thus, simply culturing these somatic or differentiated cells does not represent these cells as undifferentiated cells (eg, undifferentiated cells) or pluripotent cells. The transition from a differentiated cell (including partially reprogrammed stable, non-pluripotent cell intermediates) to pluripotency requires a reprogramming stimulus in addition to stimuli that lead to partial loss of differentiated character upon placement in culture. Reprogrammed cells and, in some modalities, partially reprogrammed, also have the characteristic of having the ability to undergo prolonged passage without loss of growth potential, compared to parent cells that have a lower development potential, which generally have the capacity to only a limited number of divisions in culture. In some embodiments, the term "differentiated cell" also refers to a cell of a more specialized cell type (ie, decreased developmental potential), derived from a cell of a less specialized cell type (ie, increased developmental potential ) (for example, from an undifferentiated cell or from a reprogrammed cell) where the cell has undergone a process of cell differentiation.
[000150] The term "reprogramming", as used herein, refers to a process that reverses the developmental potential of a cell or population of cells (eg, a somatic cell). In other words, reprogramming refers to the process of leading a cell to a state with greater potential for development, that is, regressing to a less differentiated state. The cell to be reprogrammed can be partially or terminally differentiated prior to reprogramming. In some embodiments of the aspects described herein, reprogramming encompasses a complete or partial reversal of the state of differentiation, that is, an increase in the developmental potential of a cell, to that of a cell having a pluripotent state. In some modalities, reprogramming involves bringing a somatic cell into a pluripotent state, such that the cell has the developmental potential of an embryonic stem cell, that is, an embryonic stem cell phenotype. In some embodiments, reprogramming also encompasses a partial reversal of the state of differentiation or a partial increase in the developmental potential of a cell, such as a somatic cell or a unipotent cell, to a multipotent state. Reprogramming also encompasses the partial reversion of a cell's differentiation state to a state that makes the cell more susceptible to complete reprogramming to a pluripotent state when subjected to additional manipulations, such as those described in this document. Such manipulations can result in the endogenous expression of specific genes by cells, or by cell progeny, the expression of which contributes to or maintains reprogramming. In certain embodiments, reprogramming a cell using the modified synthetic RNAs and methods thereof described in this document causes the cell to assume a multipotent state (eg, it is a multipotent cell). In some embodiments, reprogramming a cell (eg, a somatic cell) using the modified synthetic RNAs and methods thereof described in this document causes the cell to assume a pluripotent state or an embryonic stem cell phenotype. The resulting cells are referred to herein as "reprogrammed cells", "somatic pluripotent cells" and "RNA-induced somatic pluripotent cells". The term "partially reprogrammed somatic cell", as referred to herein, refers to a cell that has been reprogrammed from a cell with lower developmental potential by the methods as disclosed herein, such that the partially reprogrammed cells have not been fully reprogrammed to a pluripotent state, but rather to a non-pluripotent stable intermediate state. Such a partially reprogrammed cell may have a developmental potential less than that of a pluripotent cell, but greater than that of a multipotent cell, as these terms are defined in this document. A partially reprogrammed cell may, for example, differentiate into one or two of the three germ layers, but it cannot differentiate into all three of the germ layers.
[000151] The term a "reprogramming factor", as used herein, refers to a developmental potential alteration factor, as that term is defined herein, such as a gene, protein, RNA, DNA or small molecule , the expression of which contributes to the reprogramming of a cell, eg a somatic cell, to a less differentiated or undifferentiated state, eg to a cell of a pluripotent or partially pluripotent state. A reprogramming factor can be, for example, transcription factors that can reprogram cells to a pluripotent state, such as SOX2, OCT3/4, KLF4, NANOG, LIN-28, c-MYC and the like, including any gene, protein, RNA or small molecule, which can replace one or more of these in an in vitro cell reprogramming method. In some embodiments, exogenous expression of a reprogramming factor, using the modified synthetic RNAs and respective methods described in this document, induces endogenous expression of one or more reprogramming factors, such that exogenous expression of one or more reprogramming factors does not it is most needed for the stable maintenance of the cell in the reprogrammed or partially reprogrammed state. "Reprogramming to a pluripotent state in vitro" is used herein to refer to in vitro reprogramming methods that do not require and/or do not include nuclear or cytoplasmic transfer or cell fusion, for example, with oocytes, embryos, germ cells or cells pluripotent. A reprogramming factor may also be termed a "differentiation factor", which refers to a factor altering developmental potential, as the term is defined herein, such as a protein or RNA, which induces a cell to dedifferentiate from a less differentiated phenotype, that is, a dedifferentiation factor increases the potential for development of a cell.
[000152] As used in this document, the term "differentiation factor" refers to a potential developmental alteration factor, as the term is defined herein, such as protein, RNA or small molecule, which induces a cell to seize. differentiating to a desired cell type, that is, a differentiating factor reduces a cell's potential for development. In some embodiments, a differentiating factor may be a cell-type specific polypeptide, however this is not necessary. Differentiation of a specific cell type may require simultaneous and/or successive expression of more than one differentiating factor. In some aspects described herein, the developmental potential of a cell or a population of cells is first enhanced by reprogramming or partial reprogramming using modified synthetic RNAs, as described herein, and then the progeny cells or cells thereof produced by such reprogramming is induced to undergo differentiation by contact with, or introduction of, one or more modified synthetic RNAs encoding differentiation factors, such that the cell or cells of their progeny have diminished developmental potential.
[000153] In the context of cell ontogeny, the term "differentiate" or "differentiation" is a relative term that refers to a developmental process by which a cell progressed further along a developmental pathway than its immediate precursor cell. Thus, in some embodiments, a reprogrammed cell, as the term is defined in this document, can differentiate into a lineage-restricted precursor cell (such as a mesodermal stem cell), which in turn can differentiate into other precursor cell types. further down the pathway (such as a specific tissue precursor, eg a cardiomyocyte precursor) and then to a differentiated end-stage cell, which plays a characteristic role in a particular tissue type and may or may not maintain the ability to proliferate further.
[000154] As used herein, the term "without the formation of an intermediate pluripotent cell" refers to cell transdifferentiation from one cell type to another cell type, preferably, in a single step; thus, a method that modifies the differentiated phenotype or developmental potential of a cell without the formation of an intermediate pluripotent cell does not require that the cell be first dedifferentiated (or reprogrammed) and then differentiated into another cell type. Instead, the cell type is merely "switched" from one cell type to another without going through a less differentiated phenotype. In this sense, cell transdifferentiation refers to a change in the developmental potential of a cell, whereby the cell is induced to become a different cell with a similar developmental potential, for example, a liver cell to a pancreatic cell, a pancreatic alpha cell into a pancreatic beta cell, etc. The system and methods of the invention are suitable for transdifferentiating cells.
[000155] The term "expression" refers to cellular processes involved in the production of RNA and proteins and, as appropriate, secretion of proteins, including, where applicable, but not limited to, for example, transcription, translation, folding, modification and processing. "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translating mRNA transcribed from a gene. In some embodiments, an expression product is transcribed from a sequence that does not encode a polypeptide, such as a microRNA.
[000156] As used herein, the term "transcription factor" refers to a protein that binds to specific pieces of DNA using DNA binding domains and is part of the system that controls the transcription of genetic information from DNA to the RNA.
[000157] As used herein, the term "small molecules" refers to a chemical agent which may include, but is not limited to, a peptide, a peptidomimetic, an amino acid, an amino acid analog, a polynucleotide, an analog of polynucleotide, an aptamer, a nucleotide, a nucleotide analogue, an organic or inorganic compound (for example, including hetero-organic and organometallic compounds) having a molecular weight less than approximately 10,000 grams per mol, inorganic or organic compounds having a molecular weight less than approximately 5,000 grams per mol, inorganic or organic compounds having a molecular weight less than approximately 1,000 grams per mol, inorganic or organic compounds having a molecular weight less than approximately 500 grams per mol, and salts, esters and other pharmaceutically acceptable forms of such compounds.
[000158] The term "exogenous", as used herein, refers to a nucleic acid (eg a modified synthetic RNA encoding a transcription factor), or a protein (eg a transcription factor) that has been introduced by a process that involves human interference with a biological system, such as a cell or organism in which it is not normally found, or in which it can be found in smaller quantities. A factor (for example, a modified synthetic RNA encoding a transcription factor, or a protein, for example, a polypeptide) is considered exogenous if it is introduced into an immediate precursor cell or a progeny cell that inherits the substance. In contrast, the term "endogenous" refers to a factor or expression product that is native to the biological or cellular system (for example, the endogenous expression of a gene, such as, for example, SOX2 refers to the production of a SOX2 polypeptide by the endogenous gene in a cell). In some embodiments, the introduction of one or more exogenous factors into a cell, for example, a developmental potential altering factor, using the compositions and methods comprising modified synthetic RNAs described herein, induces endogenous expression in cells or cell(s). ) of the progeny of a factor or gene product necessary for the maintenance of the progeny cell or cell(s) at new developmental potential.
[000159] The term "isolated cell", as used herein, refers to a cell that has been taken from an organism in which it was originally found, or a descendant of such a cell. Optionally, the cell was cultured in vitro, for example, in the presence of other cells. Optionally, the cell is later introduced into a second organism or reintroduced into the organism from which that cell (or the cell or population of cells from which it descends) was isolated.
[000160] The term "isolated population" in relation to an isolated population of cells, as used herein, refers to a population of cells that has been taken out and separated from a mixed or heterogeneous cell population. In some embodiments, an isolated population is a "substantially pure" population of cells compared to the heterogeneous population from which the cells were isolated or enriched. In some embodiments, the isolated population is an isolated population of pluripotent cells that comprises a substantially pure population of pluripotent cells, as compared to a heterogeneous population of somatic cells from which the pluripotent cells are derived.
[000161] As used herein, the terms "modified synthetic RNA" or "modified RNA" refer to an RNA molecule produced in vitro, which comprises at least one modified nucleoside, as that term is defined below. Methods of the invention do not require modified RNA. The composition of modified synthetic RNA does not cover mRNAs that are isolated from natural sources, such as cells, tissues, organs, etc., having these modifications, but only modified synthetic RNAs that are synthesized using in vitro techniques. The term "composition" as applied to the terms "modified synthetic RNA" or "modified RNA" encompasses a plurality of different modified synthetic RNA molecules (eg, at least 2, at least 3, at least 4, at least 5 , at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least at least 18, at least 19, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 90, at least 100 modified synthetic RNA molecules or more). In some embodiments, a modified synthetic RNA composition can further comprise other agents (e.g., an inhibitor of interferon expression or activity, a transfection reagent, etc.). Such a plurality may include modified synthetic RNA of different sequences (e.g., encoding different polypeptides), modified synthetic RNAs of the same sequence with different modifications, or any combination thereof.
[000162] As used herein, the term "polypeptide" refers to a polymer of amino acids comprising at least 2 amino acids (for example, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000 amino acids or more). The terms "protein" and "polypeptide" are used interchangeably in this document. As used herein, the term "peptide" refers to a relatively short polypeptide, typically between approximately 2 and 60 amino acids in length.
[000163] Microarrays and especially "cell arrays" are currently needed for scanning large libraries of biomolecules, such as RNAs, DNAs, proteins and small molecules, in relation to their biological functions and for fundamental investigation of cell and gene functions . Many research facilities in academia and industry need advanced high-density arrays to improve their scanning efficiency, speed, and quality. Many scans will first become possible or significantly more accessible with the development of the next generation of microarrays and cell arrays, respectively. Typically, an array of the invention must fit into a common sized microtiter plate to ensure the usability of conventional microplate handling robots and microscopes. Ideally arrays can be any set of cell lines that needs to be analyzed as a unit under identical conditions, but where the only variable is the genotype of the cell lines. An example could be a collection of disease-specific or normal iPSC strains or their differentiated derivatives placed in microtiter plates in wells adjacent to each other. This would allow researchers to investigate the activity of a single factor (eg, small molecule) on multiple genotypes simultaneously to discover the genotype-specific effects of that factor using the appropriate assays.
[000164] In one embodiment, the system of the invention can also be used to obtain cell populations enriched in fully reprogrammed cells, among cells that have undergone differentiation into established iPSC cell lines that were cultured under a murine embryonic fibroblast feeder layer ( MEF) as well as feeder conditions. The inventive system further allows for live screening of defined subpopulations of fully reprogrammed or differentiated iPSC cells in 96-well plates for use in high-throughput scanning campaigns.
[000165] Figure 1 shows the steps performed by System 1, including the plating of a biopsy (2), development and passage (4) (rolling production in the liquid handling robot), QC (6) (automated tests of mycoplasma) and (8) automated freezing in the liquid handling robot.
[000166] Figure 2 shows the steps performed by Systems 2, 3 and 4. Fibroblasts are plated by the automated system (10), reprogramming factors are introduced by the automated system (12), iPSCs are isolated by automated screening and isolation (14 ), desired clones are selected and expanded by automated system (16), automated quality checks (QC) for pluripotent status by marker assays and embryonic body assays (18), followed by automated freezing and storage of desired cells (20) .
[000167] Figure 3 is a flowchart showing the steps (22) to (60) involved in System 1.
[000168] Figure 3 illustrates an example decision tree and workflow for producing fibroblasts from biopsies. The workflow is divided into Clean Phases (60) and Quarantine (58). As biopsies enter the enclosure, a technician plates biopsies into 6-well plates (22) and records the plates in the automated incubator (24). After allowing time for the biopsies to attach to the plate, the liquid handling robot retrieves the plates from the automated incubator to feed and verify the confluence of the growths under an automated microscope (26). Plates are returned to the incubator and allowed to grow (28). The liquid handler removes the plate from the incubator and switches media to antibacterial and antibiotic free media (30). The robot moves the plate to the incubator for another five days (32). The robot then removes the plate and retrieves media for daughter plates for mycoplasma testing (34). The daughter plates are moved to the Quarantine Assay System for mycoplasma testing (36). A choice is then made based on a positive sign from the assay (38). If all wells of a 6-well plate fail with a positive mycoplasma test result (40), they are discarded. If all wells of a 6-well plate are negative and mycoplasma free, they are transferred from quarantine to the clean growth system (46). If some wells are positive and some wells are negative, the negative wells are quarantined (42). The negative wells are passed (44) to new plates, transferred to the incubator, and the source plates containing positive wells are discarded. These cultures proceed through retest steps for mycoplasma (24, 26, 28, 30, 32, 34, 36, 38). Clean cultures are monitored for growth (50), passed (52) and frozen in cryogenic vials (54, 56).
[000169] Figures 4A, 4B1, 4B2 and 4C illustrate an example of the flow of patient samples through multi-well tissue culture plates during the automated reprogramming process. At the top of each diagram, a flowchart describes the flow of procedures performed at each step of the workflow (70, 88, 98). At the bottom of each diagram, multi-well cell culture plates are shown with plate maps for example samples represented by shaded wells or groups of wells marked with sample tags (61-68, 72-86, 88-96) . Transferring a sample from plate-to-plate or well-to-well through the procedure is shown from left to right as indicated by the arrows. As indicated in Figure 4A, the automated iPSC derivation process begins when patient samples and fibroblast control samples (61) are plated into individual wells of a 6-well plate (62). These are passed in defined cell numbers to individual wells of a 24-well plate (64) for infection using virus-encoding reprogramming factors or other means of introducing reprogramming factors into the cells. In the next step, reprogrammed samples are depleted of unreprogrammed cells by cell sorting, or, as is preferred, using magnetic bead based enrichment and plated at clonal density in multiple wells in 96-well plates (66). Two such cards are shown in this example. In this example, 6 wells, as indicated by the wells with a dot in the middle (66) are identified, containing a single clone positive for a pluripotency surface marker as analyzed by immunofluorescence analysis in an automated imager. These clones are passed and hand-picked to reformat the clones in a minimum number of 96-well plates (68). The example figure shows six clones for each starting sample and indicates that starting 16 sample clones can be arranged in a 96-well plate. To facilitate plate processing, this hand-picking step can be performed over several passes to consolidate clones into a minimal number of plates. As shown in Figures 4B1 and 4B2, these clones are serially passaged until confluence of stem cell colonies within a well is reached for each initial sample (72). Each plate sample is then replicated in duplicate plates (74-86), to allow for quality control (6) and selection of clones that demonstrate appropriate stem cell characteristics. To initiate the QC process, a plate is generated by the system for a pluripotency quality control assay needed to determine the pluripotency status of individual clones (74) and a plate is generated to carry forward on subsequent passages (76). The board that is taken forward is re-swiped in three boards (78, 80, 82) for even more quality control and expansion. A plate is harvested for QC assays to characterize Karyotype and genetic diversity (78). A second plate (82) is passed into v-bottom plates to form embryo bodies (84) for a QC assay that assesses the ability of iPS clones to differentiate. The end plate (80) is carried forward for further expansion. Individual clones that do not pass quality control from previous pluripotency QC assays are not carried forward as shown by the "X" in the wells, indicated in Figure 4. In the example shown in Figure 4B2, the consolidated plate (86) will contain iPS strains (or differentiated strains) of up to 32 individuals represented by 3 iPS clones per individual in a single 96-well plate or up to 96 individuals if represented by a single clone each. Remaining clones are consolidated onto as few plates as possible until one to three clones remain (86-92). As shown in Figure 4C, these are expanded for Cryopreservation while fixed to the plate (88) or further expanded (92-94) and cryopreserved with cryovials (96). Any or all of the information from the pluripotency marker screen indicated in figure 4A (70), and the quality control assays, shown in figure 4B1, can be used alone or in combination to decide which clones to select for consolidation and disposition in the automated process.
[000170] Methods for transfection and transformation or reprogramming adult cells to form iPSC lineages are generally known, eg Takahashi et al., 2007 Cell, 131:861-872, 2007, Yu et al., 2007, Science, vol. 318, pp. 1917-1920. iPSC are induced from somatic cells with reprogramming factors. Reprogramming factors are contemplated to include, for example, transcription factors. The method for reprogramming adult cells includes, for example, introducing and expressing a combination of specific transcription factors, for example, a combination of Oct3/4, Sox2, Klf4 and c-Myc genes. Others have shown that other transcription factors can be used to transform or reprogram adult cells. Such other transcription factors include, for example, Lin28, Nanog, hTert and SV40 large T antigen as described, for example, by Takahashi et al., 2006 Cell, 126: 663-676 and Huiqun Yin, et al. 2009, Front. Agricultural China 3(2): 199208, incorporated herein by reference.
[000171] In another aspect, iPSCs can be generated using the direct introduction of RNAs into a cell, which, when translated, provides a desired protein or proteins. Higher eukaryotic cells have evolved cellular defenses against foreign, "non-self" RNA, which ultimately result in overall inhibition of cellular protein synthesis, resulting in cellular toxicity. This response involves, in part, the production of type I interferons or type II interferons and is commonly referred to as the "interferon response" or the "cellular innate immune response." Cellular defenses normally recognize synthetic RNAs as foreign and induce this innate cellular immune response. In certain aspects, where the ability to achieve prolonged or repeated expression of an exogenously targeted protein using RNA is hampered by the induction of that innate immune response, it is desirable to use synthetic RNAs that are modified in a way that prevents or reduces the response. Prevention or reduction of the innate immune response allows for sustained expression of exogenously introduced RNA needed, for example, to modify a cell's developmental phenotype. In one aspect, sustained expression is achieved by repeatedly introducing modified, synthetic RNAs into a target cell or its progeny. Inventive methods include natural or synthetic RNAs.
[000172] Synthetic RNAs, natural or modified in one aspect, can be introduced into a cell in order to induce exogenous expression of a protein of interest in a cell. The ability to direct exogenous expression of a protein of interest using the modified, synthetic RNAs described in this document is useful, for example, in the treatment of diseases caused by an endogenous genetic defect in a cell or organism that impairs or impedes the capacity of that cell. or organism to produce the protein of interest. Accordingly, in some embodiments, compositions and methods comprising the RNAs described in this document can be used for gene therapy purposes.
[000173] The RNAs described can advantageously be used in altering cell fate and/or developmental potential. The ability to express a protein from an exogenous RNA allows the alteration or reversal of a cell's developmental potential, that is, the reprogramming of the cell, and the directed differentiation of a cell to a more differentiated phenotype. A critical aspect of altering a cell's developmental potential is the requirement for sustained and prolonged expression of one or more potential developmental factors, altering factors in the cell or its immediate progeny. Traditionally, such sustained expression has been achieved by introducing DNA or viral vectors into a cell. These approaches have limited therapeutic utility due to the potential for insertional mutagenesis.
[000174] One of the areas that can most benefit from the ability to express a desired protein or proteins over a sustained period of time from exogenous RNAs as described in this document is the generation of pluripotent or multipotent cells from cells initially having a more differentiated phenotype. In this aspect, RNAs encoding a reprogramming factor or factors are used to reprogram cells to a less differentiated phenotype, that is, to have a greater potential for development.
[000175] One of the main goals of stem cell technology is to make the stem cells differentiate into a desired cell type, ie, directed differentiation or producing cells via transdifferentiation. Not only are the compositions and methods described herein useful for reprogramming cells, they are also applicable to this targeted differentiation and cell transdifferentiation to a desired phenotype. That is, the same technology described in this document for reprogramming is directly applicable to the differentiation of the reprogrammed cell, or any other stem cell or precursor cell, for that matter, for a desired cell type.
[000176] In some embodiments of this aspect and all of those aspects described herein, the modified synthetic RNA molecule comprises at least two modified nucleosides. In such an embodiment, the two modified nucleosides are selected from the group consisting of 5-methylcytidine (5mC), N6-methyladenosine (m6A), 3, 2'-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2 ' fluoruridine, pseudouridine, 2'-O-methyluridine (Um), 2' deoxy uridine (2' dU), 4-thiouridine (s4U), 5-methyluridine (m5U), 2'-O-methyladenosine (m6A), N6 , 2'-O-dimethyladenosine (m6Am), N6, N6, 2'-O-trimethyladenosine (m62Am), 2'-O-methylcytidine (Cm), 7-methylguanosine (m7G), 2'-O-methylguanosine (Gm ), N2, 7-dimethylguanosine (m2.7G), N2, N2, 7-trimethylguanosine (m2,2.7G) and inosine (I). In such an embodiment of this aspect and all such aspects described herein, the at least two modified nucleosides are 5-methylcytidine (5mC) and pseudouridine. (See, for example, Rossi U.S. 2012/0046346, incorporated herein by reference).
[000177] Genes, proteins or RNA used in the methods of the invention include, but are not limited to OCT4, SOX1, SOX 2, SOX 3, SOX15, SOX 18, NANOG, KLF1, KLF 2, KLF 4, KLF 5, NR5A2, c-MYC, 1-MYC, n-MYC, REM2, TERT, and LIN28.
[000178] It has also been shown that a single transcription factor can be employed in reprogramming adult fibroblasts from iPSCs with the addition of certain small molecule pathway inhibitors. Such pathway inhibitors include, for example, the transforming growth factor-beta (TGFb) pathway inhibitors, SB431542 (4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl) -1H-imidazol-2-yl]-benzamide) and A-83-01 [3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazol-1-car - botioamide], extracellular signal-regulated kinases (ERK) and microtubule-associated protein kinase pathway inhibitor (MAPK/ERK) PD0325901 (N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro- 2-[(2-fluoro-4-iodophenyl)amino]-benzamide), the GSK3 inhibitor CHIR99021 [6-((2-((4-(2,4-Dichlorophenyl)-5-(4-methyl-1H)) -imidazol-2-yl)pyrimidin-2-yl)amino)ethyl)amino)nicotinonitrile], which activates Wnt signaling by stabilizing beta-catenin, the lysine-specific demethylase1, Parnate (also known as tranylcypromine), the activator of PS48 small molecule dependent on 3'-phosphonoisotide kinase-1(PDK1) [(2Z)-5-(4-Chlorophenyl)-3-phenyl-2-pentenoic acid)], histone deacetylase (HDAC) inhibitors, butyrate in sodium and valproic acid, small molecules that modulate mitochondrial oxidation (eg, 2,4-dinitrophenol), glycolytic metabolism (fructose 2,6-bisphosphate and oxalate), activation of the HIF pathway (N-oxaloylglycine and quercetin) Zhu et al., 2010, Cell Stem Cell 7: 651-655, incorporated by reference herein in their entirety. Zhu et al showed that Oct4 combined with Parnate and CHIR99021 were sufficient to reprogram adult human epidermal keratinocytes.
[000179] Although individual protocols differ, a general reprogramming protocol consists of expanding differentiated adult cells from tissue samples, eg skin biopsies, and contacting them with reprogramming factors as discussed above, eg infecting them, or that is, transfecting with, for example, expression vectors, such as viral constructs, containing transcripts for pluripotent transcription factors. Fibroblasts are obtained by methods known in the art, for example, by mechanically disrupting tissue followed by enzymatic dissociation to release the fibroblasts, and culturing fibroblasts by methods known in the art, for example, as described by Dimos et. al., 2008, Science Vol. 321 (5893): 1218-1221.
[000180] While illustrative aspects of the invention use vectors, for example, viral vectors, plasmid vectors, in some aspects vectors are not necessary for transfection techniques, including those that transfer mRNA molecules into cells.
[000181] Transfection of the fibroblasts with an expression vector is performed according to the instructions provided with the desired vector. After a time (eg, ranging from about 2 to about 10 days post-transfection, cells are dissociated and brought into contact with high fluorescently labeled antibodies against the surface markers CD13NEG, SSEA4POS and Tra-1-60POS. The dissociated and antibody-labeled cells are then resuspended in a phosphate buffered saline solution and moved for automated sorting and isolation of iPSC clones Surface marker positive cells are sorted by label color or its absence directly into sterile tubes , containing tissue culture medium or multi-well tissue culture plates (6-96 wells) coated with MEFs or cell-free biological matrices and grown until visible colony formation occurs.
[000182] Colonies are then further confirmed as iPSC by light microscopic inspection of the resulting clones, or optionally by microscopic inspection of the fluorescence of the clones labeled with fluorescently labeled antibodies. Optionally, in certain embodiments, one or more of the vectors also inserts a green fluorescence protein expression marker (GFP) for convenience in classification and identification. Several individual colonies, having morphological characteristics consistent with pluripotent ES cell lines are taken from cultures and individually expanded to form monoclonal cultures.
[000183] In a preferred embodiment of the inventive system, treated cells are subjected to genetic analysis to provide early confirmation and identification of iPSCs. Preferably, genetic analysis is conducted by Southern blot, but other methods known in the art can be employed which include but are not limited to MicroArray, NanoString, quantitative real-time PCR (qPCR), whole genome sequencing, immunofluorescence microscopy , flow cytometry. Detection of alkaline phosphatase enzymatic activity, positive expression of cell membrane surface markers SSEA3, SSEA4, Tra-1-60, Tra-1-81 and expression of transcription factors KLF4, Oct3/4, Nanog, Sox2 in fibroblasts reprogrammed humans confirms that a clone is an iPSC. Preferably all markers are present.
[000184] Any transfection vector known in the art can be employed as a reprogramming factor, including, for example, an RNA such as mRNA, microRNA, siRNA, antisense RNA and combinations thereof. Other expression vectors that may be employed include, for example, a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, a herpes virus, a Sindbis virus, a smallpox virus, a baculovirus, a bacterial phage, a virus Sendai and its combinations. Preferably, a vector employed is a non-replicating vector such as, for example, Sendai virus vectors designed to be non-replicative. The preferential Sendai virus vector, although incapable of replication, remains capable of productive expression of proteins encoding vector-loaded nucleic acids, thus preventing any potential uncontrolled spread to other cells or within the body of a vaccinated person. This type of Sendai vector is commercially available as CytoTuneTM-iPSC Sendai viral vector kit (DNAVEC, DV-0301).
[000185] Any transfection method known in the art can be employed to insert such vectors into adult fibroblasts, including, for example, electroporation, gene injector and the like. Chemical transfection is optionally conducted by means of a transfecting agent, eg a polymer, calcium phosphate, a cationic lipid eg for lipofection and the like. Cell-penetrating peptides are also optionally employed to transport vectors or other agents into adult fibroblast cells. In summary, cell penetration peptides include those derived from proteins, e.g. protein transduction domains and/or amphiphilic peptides which can transport vectors or other agents into the cell include peptides. The subject of cell penetration peptides has been reviewed, for example, by Heitz et al., 2009 British Journal of Pharmacology, 157: 195-206, incorporated by reference herein in their entirety. Other cell-penetrating peptides are known in the art and are disclosed by Heitz, Id. Other cell penetration technologies including, for example, liposomes and nanoparticles, are also contemplated to be employed in the methods of the present invention. Liposomes and nanoparticles are also described by Heitz, Id.
[000186] Antibodies can be employed in order to identify transformed cells. Four antibodies against stem cell-specific surface proteins are used to identify and characterize human pluripotent stem cell populations; SSEA3, SSEA4, Tra-1-60 and Tra-1-81. Embryonic Stage Specific Antigens 3 and 4 (SSEA3 and SSEA4) are two monoclonal antibodies that recognize sequential regions of a ganglioside present in human 2102Ep cells (Henderson et al., 2002 Stem Cells 20: 329-337; Kannagi et al., 1983, Embo J 2: 2355-2361). Antibodies Tra-1-60 and Tra-1-81 were originally raised against human embryonic carcinoma (EC) cells (PW et al., 1984, Hybridoma 3:347-361) and have been shown to specifically recognize an epitope of carbohydrates on a sulfated keratan glycoprotein, identified as podocalixin, a member of the CD34-related family of sialomucins (Badcock et al., 1999, Cancer Research 59: 4715-4719; Nielsen et al., 2007, PLoS ONE 2: e237; Schopperle and DeWolf, 2007, Stem Cells 25: 723-730). Several other surface markers have been shown to be expressed on ES cells and include CD326 or EpCam (Sundberg et al., 2009, Stem Cell Res 2: 113-124), CD24 (Heat Stable Antigen) and CD133 (Barraud et al., 2007, Journal of Neuroscience Research 85, 250-259) (Gang et al., 2007, Blood 109: 1743-1751). Chan et al., 2009, Id. reported that identification of genuine IPSc of fibroblasts undergoing reprogramming via four-factor retroviral transduction can be achieved by imaging live cells and by observing, over time, that fibroblasts lose expression of cell surface markers CD13 and D7Fib, gain expression of pluripotent stem cell markers SSEA4 and Tra-1-60 (Chan et al.2009, Id.).
[000187] Also envisaged within the scope of the invention are compositions comprising iPSCs, for example compositions used as research tools, or as pharmaceutical compositions comprising effective amounts of iPSCs prepared by the automated inventive system.
[000188] The invention further relates to the treatment of a disease or disorder in an animal or person in need thereof by administering iPSCs, e.g. methods of tissue/organ treatment and/or repair by administering produced iPSCs by the automated inventive system, or differentiated cells derived therefrom. Suitable differentiated cells (from ectodermal, mesodermal or endodermal lineage) can be derived from iPSCs produced by the inventive methods. The mode of administration can be determined by a person skilled in the art depending on the type of organ/lesion to be treated. For example, iPSCs, or differentiated cells derived from them, can be administered by injection (as a suspension) or implanted in a biodegradable matrix.
[000189] In addition, the invention relates to methods of controlling pharmaceutical products, contacting iPSCs, transdifferentiated or differentiated cells derived therefrom, for example, with one or more pharmaceutical agents of interest and then detecting the effect of the agent(s) pharmacist(s) applied to the contacted cells. For efficiency, pharmaceutical agent(s) is (are) applied to a battery of iPSCs, or differentiated cells derived therefrom. Cells can vary in tissue source, differentiated cell type, or allelic source, to allow identification of cell types or tissues that react favorably or unfavorably to one or more pharmaceutical agents of interest.
[000190] In addition, iPSCs produced by the automated inventive system can be used as a vehicle for introducing genes to correct genetic defects such as osteogenesis imperfecta, diabetes mellitus, neurodegenerative diseases such as, for example, Alzheimer's disease , Parkinson's disease, the various neuronal motor diseases (MND), e.g., amyotrophic lateral sclerosis (ALS), primary lateral sclerosis (PLS), progressive muscle atrophy (PMA) and the like.
[000191] iPSCs produced by the automated inventive system can also be employed to provide specific cell types for biomedical research, as well as directly, or as precursors, to produce specific cell types for cell-based assays, eg for studies of cell toxicity (to determine the effect of test compounds on cellular toxicity), to determine teratogenic or carcinogenic effects of test compounds by treating cells with the compound and observing and/or noting the effects of the compound on cells, eg, effect on cell differentiation.
[000192] The present invention can be better interpreted by referring to the following non-limiting examples. The following examples are presented to more fully illustrate the preferred embodiments of the invention. They are in no way to be considered, however, as limiting the broad scope of the invention. EXAMPLE 1
[000193] Figures 5A, 5B, 5C illustrate an example of the equipment configuration required to perform the workflow. Figure 5A shows a system setup for the automated expansion and quality control of a fibroblast bank. Figure 5B shows a system setup for automatic thawing of patient samples such as fibroblast, automated introduction of reprogramming factors with patient samples such as fibroblast, automated cell sorting with MultiMACS, and automated colony identification and reformatting. Figure 5C shows a system setup for automated expansion of iPS clones, automated Embryonic Body production, and automated freezing. Automated Derivation of a Fibroblast Cell Bank
[000194] As an example, the hardware configuration used to perform the derivation of a fibroblast bank consists of a Hamilton STARlet liquid handling robot (100) connected to the following hardware components: a Cytomat 24C GLS automated incubator (108) that allows incubation of cell cultures, a Cyntellect Celigo cytometer (102) for automated image analysis and acquisition, an Agilent V-Spin automated centrifuge (106) for centrifuging cells in plates or tubes, and a Hamilton Capper DeCapper (104 ) for the automated capping and uncapping of cryotubes. These components are further controlled by programmable software (118) on a PC that communicates with all instruments and controls the handling of cell culture materials and cells between the hardware components. The controller software further communicates with the scheduling software (120) to link system interactions. The Hamilton STARlet (100) is equipped with a Modular Arm for 4/8/12 channel pipetting, 8 pipetting channels, iSWAP plate handler, CO-RE Gripper clamp for plate and cap manipulation, MultiFlex tilt module for tilt plates during media changes, Hamilton Heated Shaker 2.0, as well as a Carrier Package for flexible liquid handling platform layout with plate and lid stops, pipette stackers, daughter plate stackers, and media holding rails. Cyntellect Celigo (102) comprises an imaging unit and programmable software on a PC for image acquisition control and image analysis. Celigo is preferred because it does not move cell culture plates during imaging, thus reducing the agitation of plated biopsies. The Hamilton Capper Decapper (104) and Agilent V-Spin Centrifuge (106) are contained within the Hamilton STARlet within a NuAire BSL II (110) biosafety cabinet to maintain a sterile operating environment when handling cell culture plates .
[000195] To control plate handling in the automated system, MICROLAB STAR VENUS TWO Base Pack 4.3 software (118) with VENUS Dynamic Scheduler 5.1 (120) are used in conjunction with individual hardware component drivers connected to the centrifuge (106) , Capper Decapper (104), Celigo (102) and Cytomat 24 (108) and Cytomat transfer station to integrate system operation. The following methods programmed using the supplied controller software (118) are required for system functionality and can be combined in a defined sequence to perform the derivation of fibroblast strains from patient skin biopsies:
[000196] Load 6-well biopsy plates (22, 24) into STARlet (100) and transfer to Cytomat incubator (108).
[000197] Confluence check (26, 28) in Celigo (102) and a media switch in STARlet (100).
[000198] Confluence check (28) in Celigo (102).
[000199] Media change (30) in STARlet (100) for complete media change.
[000200] Preparation of assay plate (34) on STARlet (100) and Agilent V-Spin centrifuge (106).
[000201] Pass (44) in the star (100).
[000202] Swipe and finger pick (42) on STARlet (100).
[000203] Passage, collection and freezing in STARlet (100).
[000204] Recover plates (46, 40), for the STARlet (100) from the Cytomat (108). Automated Mycoplasma Test in Quarantine Assay System
[000205] A standalone hardware configuration is used to perform the mycoplasma test of a fibroblast bank and consists of a Hamilton STARlet liquid handling robot (112) connected to a BioTek Synergy HT Reader (114). These components are further controlled by programmable software (116) on a PC that communicates with all instruments and controls the handling of cell culture materials and cells between the hardware components. The Hamilton STARlet (112) is equipped with a Modular Arm for 4/8/12 channel pipetting, 8 channel pipetting, iSWAP plate handler, CO-RE Gripper clamp for plate and cap manipulation, as well as a Carrier Package for flexible liquid handling platform layout with plate and lid stops, pipette stackers, daughter plate stackers, and plate and chute stops to hold reagents needed for the assay.
[000206] To control the board handling in the automated system, MICROLAB STAR VENUS TWO Base Pack 4.3 software (116) is used in conjunction with the connected hardware component drivers for BioTek Synergy HT Reader (114) to integrate the operation of the system. A method is programmed using this software that allows the MycoAlert Mycoplasma Detection assay to run (36) and data analysis to determine the assay result (38). Automated System for Thawing, Infection and Identification of Reprogrammed Cells
[000207] The hardware configuration required to thaw fibroblasts, infect fibroblasts with reprogramming viruses, reprogrammed magnetic-type cells, and identification of stem cell colonies is comprised of three Hamilton STAR liquid handling units (122, 136, 138 ), two Cytomat 48C incubators (132), one Cytomat 2C 425 incubator (142), two Cyntellect Celigo cytometers (124, 140), Hamilton Capper DeCapper (126), Agilent V-Spin (128), Miltenyi MultiMACS magnetic separation device (136). The liquid handlers, a Celigo, Hamilton Capper Decapper and Agilent V-Spin are all connected by a Hamilton Rack Runner robotic rail (130). Each Hamilton STAR is equipped with a Modular Arm for channel pipetting 4/8/12, 8 pipetting channels, iSWAP plate handler, CO-RE Gripper clamp for plate and lid manipulation, MultiFlex tilt module for tilting plates during media exchanges, one or more Hamilton Heated Shaker 2.0, as well as Carrier Packages for flexible liquid handling platform layout with plate and lid stops, pipette stackers, daughter plate stackers, and media holding rails. One of the Hamilton STAR liquid handlers (122) is also equipped with a 96-well pipetting head. A Celigo (140) and Cytomat 2C incubator (142) are directly connected to one of the Hamilton STARs (138) to facilitate automated cell classification. Hamilton STARs are contained within NuAire BSL II biosafety cabinets (144, 146,148) to maintain a sterile operating environment when handling cell culture plates. The remaining components are placed in a Hepa filtered hood to maintain a sterile operating environment during transport of cell culture plates between devices. The Cytomat 48C incubator (132) is connected to other components by the Rack Runner transport rail (130).
[000208] To control card handling in the automated system, MICROLAB STAR VENUS TWO Base Pack 4.3 (150, 152, 154) with VENUS Dynamic Scheduler 5.1 (156) software drivers are used in conjunction with individual hardware component drivers for all the Hamilton STARs (122, 134, 138), the stripper/stripper (104), the two Celigo (140, 124), the Rack Runner (130) and Cytomat 24 (132), the CYTOMA 2C (142) and associated Cytomat transfer to integrate system operation. The following methods programmed using the supplied controller software (150, 152, 154) are required for system functionality and can be combined in a defined sequence to perform the derivation of iPS colony lines from fibroblasts:
[000209] Load the mycoplasma free 6-well plates (48) into the STAR (122) and transfer to the Cytomat incubator (132) under clean growth conditions (60).
[000210] Confluence check (50) in Celigo (124) and a media switch in STARlet (122).
[000211] Passage, collection (52) and freezing (54, 56) at STAR (122).
[000212] A thawing method whereby cryotubes containing fibroblasts (61) are loaded and thawed in the STAR (122), followed by uncapping tubes (126) and washing the fibroblast, followed by resuspending cells in the plating and plating media of fibroblasts in 6-well plates (62) and transfer to Cytomat incubator (132).
[000213] Media change in STARlet (122) for complete media change.
[000214] Confluence check in Celigo (124).
[000215] Passage and propagation of fibroblasts in 24-well plates (64) at STARlet (122).
[000216] A method for the infection of fibroblasts (64) in STARlet (122).
[000217] A method to add a defined volume of media to wells in STAR (122, 138, 144).
[000218] A method for performing a halfway media switch in STAR (122, 138, 144).
[000219] A method for magnetic classification, dilution and plating (66) at STAR (144) attached to MultiMACS Miltenyi (136) and Celigo (124).
[000220] A method for performing a halfway media switch in STAR (122, 138, 144).
[000221] A method for performing an immunocytochemical stain on live colonies followed by automated imaging of the colonies (66) using a STAR (138) and Celigo (140).
[000222] A method for collecting, handpicking and replacing colonies (68) from selected wells in a STAR (138).
[000223] Retrieve plates for the STARlet (122, 138, 144) from the Cytomat (132). Automated System for Quality Control, Expansion and Freezing of reprogrammed cells
[000224] The hardware configuration required to expand reprogrammed stem cell colonies, generate colony plates for quality control assays, and generate cryogenic storage plates and tubes is comprised of three Hamilton STAR liquid handling units (150, 154 , 160), Cytomat 24C incubator (172), Cytomat 2C 425 incubator (174), Cyntellect Celigo cytometer (166), Hamilton Capper DeCapper (170), Agilent V-Spin (168), and Agilent PlateLoc plate sealer (164 ). Liquid handlers, a Celigo, Hamilton Capper Decapper, Agilent V-Spin Agilent PlateLoc Plate Sealant are all connected by a Hamilton Rack Runner robotic rail (162). The Hamilton STARs and STARlet (100) are equipped with Modular Arms for 4/8/12 channel pipetting, 8 channel pipetting, iSWAP plate handlers, CO-RE Gripper fasteners for plate and cap manipulation, MultiFlex tilt modules for tilt plates during media changes, one or more Hamilton Heated Shaker 2.0, as well as Carrier Packages for flexible layout of liquid handling platforms with plate and lid stops, pipette stackers, daughter plate stackers, and media holding chutes. One of the STARs (160) also has a 96-channel multi-channel pipetting head to facilitate media changes and pass-through. The Cytomat 2C and Cytomat 24C incubators are linked to the Hamilton STARs by a Hamilton Rack Runner transport rail (162) to facilitate media exchanges. Hamilton STARs are contained within a NuAire BSL II biosafety cabinet (176, 178, 180) to maintain a sterile operating environment when handling cell culture plates. The remaining components are placed in a Hepa filtered hood to maintain a sterile operating environment during transport of cell culture plates between devices.
[000225] To control card handling in the automated system, MICROLAB STAR VENUS TWO Base Pack 4.3 software controllers (182, 184, 186) with two VENUS Dynamic Scheduler 5.1 (188) are used in conjunction with connected hardware individual component drivers for the centrifuge, plate seal stripper, Celigo, and Cytomat incubators, and Cytomat transfer station to integrate system operation. The following methods are required for system functionality and can be combined in a defined sequence to expand plate cell cultures for quality control assays and freezing in cryogenic plates or vials:
[000226] A method of loading onto the STAR (160) to receive plates (68) from the previous stage into the Cytomat incubator (172).
[000227] Media Exchange on STAR (150, 154, 160) for complete media exchanges using tilt modules and 8-channel pipetting arms.
[000228] Confluence check in Celigo (166) with associated methods for transporting plates to and from STARs (150, 154, 160) and Cytomat incubator (172).
[000229] A method for passage and propagation of iPSCs in 96-well plates (68-90) in the STARs (150, 154, 160).
[000230] A method for performing a partial media exchange on STARs (150, 154, 160).
[000231] A method for collecting, hand-picking and re-plating selected 96-well wells to new 96-well plates (80, 82, 86, 88) on a STAR (150, 154, 160).
[000232] A method for harvesting, hand-picking and re-plating colonies from selected 96-well wells to new 24-well plates (90, 92) on a STAR (154).
[000233] A method for collecting, hand-picking and replating colonies from selected 24-well wells to new 6-well plates (92, 94) in a STAR (154).
[000234] Passage, collection and distribution of cells in freezing plates (88) at STAR (154).
[000235] Passage, collection and distribution of cells in cryotubes (96) at STAR (154).
Plaque recovery on the STARs (150, 154, 160) of Cytomat 24C (172) or Cytomat 2C (174). EXAMPLE 2 Production of a Fibroblast Bank for Reprogramming
[000237] The first step in the workflow to derive iPSCs from patient samples is to obtain and expand the adult cells. This is accomplished, for example, by taking a skin biopsy (punch) or discarded dermal tissue, and then isolating and expanding fibroblast cultures from the discarded tissue. In our workflow, this is done by the automated system composed of Systems 1 and 2. The automated components of Systems 1 and 2 (100-120) and System 3 (122-132, 154, 190) perform the necessary steps to derive a fibroblast bank stored in cryotubes (61) of patient samples, including plating of a patient biopsy (2, 22-24), growth and passage (4, 2632) (rolling production in liquid handling robot) , QC (6, 34-46) (automated test for mycoplasma) and automated freezing in liquid handling robot (8,48-56). For example, the decision tree and workflow for producing fibroblasts from biopsies is divided into Clean Phases (60) and Quarantine (58). As biopsies enter the room, a technician plates biopsies into 6-well plates (22) and registers the plates in the automated incubator (24) to initiate the quarantine workflow. After allowing time for the biopsies to attach to the plate, the liquid handling robot retrieves the plates from the automated incubator to feed and verify the confluence of adult fibroblast growths from the plated tissue under an automated microscope (26). Plates are returned to the incubator and allowed to continue growing (28). The liquid handler removes the plate from the incubator and switches media to antibacterial and antibiotic free media (30) to prepare for mycoplasma testing. The robot moves the plate to the incubator for another five days (32). The robot then removes the plate and retrieves media for daughter plates for mycoplasma testing (34). The daughter plates are moved to the Quarantine Assay System for mycoplasma testing (36). A choice is then made based on a positive sign from the assay (38). If all wells of a 6-well plate fail with a positive mycoplasma test result (40), they are discarded. If all wells of a 6-well plate are negative and mycoplasma free, they are transferred from quarantine to the clean growth system provided by Systems 3, 4, 5 (46). If some wells are positive and some wells are negative, the negative wells are quarantined (42). The negative wells are passed (44) to new plates, transferred to the incubator, and the source plates containing positive wells are discarded. These cultures proceed through retest steps for mycoplasma (24-38). Clean cultures are monitored for growth (50), passed (52) and frozen in cryogenic ampoules (54, 56, 61). Production of Stem Cell Matrices
[000238] To produce iPSCs, fibroblasts in cryotubes (61) are plated by the automated system (10), reprogramming factors are introduced by the automated system (12), iPSCs are isolated by automated classification and isolation in the System (14), desired clones are selected by the automated system (16) and expanded by the automated system (16), automated quality checks by the automated system (QC) for pluripotent status by marker assays and embryonic body assays (18), followed by automated freezing and storage of cells desired by the automated system (20). These steps are performed in automated systems, 3, 4, 5, 6, 7 and 8 (122-192).
[000239] For example, the automated iPSC derivation process begins when 96 patient samples and fibroblast control samples (61) are plated into individual wells of a 6-well plate (62). These are passed in defined numbers of cells to individual wells of a 24-well plate for infection using virus-encoding reprogramming factors (64). In the next step, reprogrammed samples are depleted of unreprogrammed cells by cell sorting, or, as is preferred, using magnetic bead based enrichment, and plated at clonal density in multiple wells in 96-well plates (66). In this example, 6 wells (66) are identified containing a single clone positive for a pluripotency surface marker. These clones are handpicked and consolidated into a minimum number of 96-well plates (68). These clones are serially passaged until confluence is reached within a well for each initial sample (72). Samples from each plate are then replicated in duplicate plates (74, 76), one plate for a pluripotency quality control assay needed to determine the pluripotency status of individual clones (74) and one more plate to carry forward in passages. subsequent events (76). The plate that is carried forward is passed again in three plates (78, 80, 82). One plate is harvested for the QC assay which assesses karyotype and genetic diversity (78), one plate (82) is passed in v-bottom plates to form embryo bodies (84) for a QC assay which assesses the ability to differentiation of the iPS clones and the end plate (80) is carried forward for further expansion. Individual clones that do not pass quality control from previous pluripotency QC assays are not carried forward as shown by the "X" in the wells in Figures 4B2 and 4C (80, 82, 90). Remaining clones are consolidated onto as few plates as possible until one to three clones remain (86). These clones are expanded for cryopreservation while fixed to the plate (88) or further expanded (92-94) and cryopreserved with cryogenic vials (96).
[000240] Embryonic Stem Cells (ES) are also intended to be used with the automated system of the invention to generate differentiated adult cells. ES cells are derived from the blastocyst of an early stage embryo and have the potential to become endoderm, ectoderm and mesoderm (the three germ layers) (ie they are "pluripotent"). In vitro, ES cells tend to spontaneously differentiate into various tissue types, and controlling their direction of differentiation can be a challenge. However, some progress has been made in the targeted differentiation of ES cells from specific types of differentiated daughter cells. For example, it is now possible to direct the differentiation of human ES cells to functional midbrain dopaminergic neurons using defined factors added to cell cultures at defined stages of their gradual differentiation (see, for example, Kriks et al., 2011 Nature, Nov 6. doi: 10.1038/nature10648 (Epub)). As the differentiation is not homogeneous, it remains necessary to isolate populations of interest for further study or manipulation. The process and instrumentation described here can be used to first derive and expand pluripotent embryonic stem cells, and also isolate subpopulations of their differentiated derivatives by automated methods, including automated magnetic cell isolation.
[000241] For example, entire human blastocysts can be plated in arrays in multi-well plates amenable to the automated process. Growths from these plated blastocysts can be isolated using the same automated magnetic isolation procedures performed by Robotic Instrumentation and methods described for the isolation of induced pluripotent stem cells. The resulting human embryonic stem cell lines can be expanded, selected by quality control assays, and frozen using the same automated procedures described in this document.
[000242] Furthermore, using pluripotent stem cells, either derived from blastocysts or induced by defined factors or by somatic cell nuclear transfer, differentiated derivatives can be isolated using the described workflow and instrumentation. Differentiated derivatives can be obtained by defined application of defined factors necessary to induce a cell fate change or after spontaneous differentiation. For example, inhibitors of the TGF beta pathway can be used to induce neural cell fates from pluripotent stem cells. Neural cells can be further isolated from non-neural cells by magnetic bead immunostaining of surface antigens such as NCAM. The workflow and instrumentation described can be used to magnetically isolate, select, grow and expand differentiated cells such as neurons. This process is also applicable to other differentiated cell types, such as cardiac cells, for which there are antibodies that recognize cell surface antigens specific for the cell type of interest.
[000243] Multipotent stem cells are also envisioned to be used with the automated systems of the invention to generate differentiated adult cells. In particular, mesenchymal stem cells (MS) can be employed to generate differentiated adult cells using the automated systems of the invention. MS cells are formative pluripotent blasts or embryonic-like cells found in bone marrow, blood, periosteum, dermis and placenta that are capable of differentiating into specific types of mesenchymal or connective tissues, including adipose, bone, cartilaginous, elastic, muscular and fibrous connective tissue. The specific differentiation pathway these cells enter depends on various influences of mechanical influences and/or endogenous bioactive factors, such as growth factors, cytokines and/or local microenvironmental conditions established by host tissues. Examples include the differentiation of MS cells into differentiated cells with the properties of chondrocytes for cartilage repair, for example, see U.S. Patent No. 8,048,673. Chromosomal Test
[000244] In some respects, the Nanostring nCounter Plex2 Assay Kit is used to target the 400 genomic loci, generally known to be invariant across the population, allows for integrated molecular karyotype analysis along with "fingerprint" tracking of cell lineage identity . Molecular karyotype analysis uses an average of 8 probes per chromosome arm to verify genomic stability during the course of cell culture derivation and expansion of iPSC strains. Identity analysis will also be performed on all lines based on 30 common copy number variations (CNVs) of polymorphic loci, which allows unambiguous identification of individual genomes. pluripotency analysis
[000245] In one aspect, surface marker staining is performed to show that cells are positive for the surface marker Tra-1-60, which is monitored, for example, with the automated imager Celigo. PSC strains must show a significant level of pluripotency genes. In one example, we used a probe set of 100 gene markers (described below) that includes the six pluripotency markers (Oct4, Klf4, cMyc, Nanog, Lin28, ZFP42 and Sox2). To perform this analysis we lyse a cell sample and collect RNA. We used the nCounter Plex2 Assay Kit to analyze expression levels in multiple samples and hundreds of gene targets simultaneously, enabling the high-throughput approach to PSC characterization. As nCounter gene expression assays are quantitative, selection criteria are based on expression levels within a range relative to a control panel of established hESC lines analyzed in growth under identical conditions. Lineages that pass genetic pluripotency expression criteria will be further expanded and differentiated in vitro in embryonic body (EB) assays. EB Formation Gene Expression Assay
[000246] It has been shown that epigenetic and transcriptional variation is common among human pluripotent cell lines and that this variation can have a significant impact on the usefulness of a cell line. In an illustrative example, the gene marker panels include:
[000247] 83 different gene markers, selected from each of the 3 germ layers (83)
[000248] 5 retrovirus transgenes (4 factors with single detection probe, 1 probe)
[000249] 5 sendai transgenes (4 factors + vector only, 5)
[000250] Oct4, Klf4, cMyc, Nanog, Lin28, ZFP42 (pluripotency, Sox2 is in the germ layer group, 6 probes)
[000251] SRY, XIST sex markers (2) - donor sex must match or strains will be rejected.
[000252] Constitutive genes, ACTB, POLR2A, ALAS1 (3 probes).
[000253] Storage and expansion of hPSC lineage
[000254] Automated expansion:
[000255] Cell lines are expanded by plating the starting cells in 2 separate wells of a 6-well plate, then placing them inside a CO2 incubator and allowing them to grow to a maximum of 95% confluence.
[000256] Storage:
[000257] The vials are placed first into the SAM -80 freezer to perform the initial slow cooling. This system has automated temperature monitoring and records of the time the system is accessed.
[000258] The vials are then placed in LN2 for long-term storage. Quality control for monitoring is detailed later in this proposal. Each bottle is individually marked with a unique 2D barcode and inventory is tracked within the LIMS. hPSC lineage characterization
[000259] The set of EB and iPSC gene expression analysis probes covering the lineage differentiation assay record card (100 genes) to monitor germ layer differentiation in EB assays, pluripotency markers, sex markers and transgene expression.
[000260] Freeze-thaw analysis Cells are counted after recovery and plated in one well of a 6-well plate. Colonies are photographed on the first day of appearance and 5 days later, colonies must exhibit a doubling time of no more than 36 hours.
[000261] Marker Surface Analysis:
[000262] Perform marker surface analysis using the automated system using high-content images from Tra-1-60 staining the Celigo automated image producer.
[000263] EB and iPSC gene expression analysis:
[000264] Pluripotency gene expression - iPSC clones should show a significant level of pluripotency genes. We used a probe set of 100 gene markers (described below) that includes the six pluripotency markers (Oct4, Klf4, cMyc, Nanog, Lin28, ZFP42 and Sox2). To perform this analysis we lysed a cell sample for each of the selected clones and collected RNA. We utilize the nCounter Plex2 Assay Kit to analyze expression levels in multiple samples and hundreds of gene targets simultaneously, enabling the high-throughput approach to iPSC characterization. As nCounter gene expression assays are quantitative, selection criteria are based on expression levels within a range relative to a control panel of established hESC lines analyzed in growth under identical conditions. Selected clones that pass genetic pluripotency expression criteria will be further expanded and differentiated in vitro in embryonic body (EB) assays.
[000265] EB Formation Gene Expression Assay - In order to firmly establish the nature and magnitude of epigenetic variation that exists among human pluripotent stem cell lines, three genomic assays were applied to 20 established embryonic stem cell lines (ESC) and 12 iPSC strains that have been recently derived and functionally characterized. As a step towards reducing the experimental burden of comprehensive cell lineage characterization and to improve the accuracy of existing standard assays, all data from these studies are combined using the three genomic assays in a bioinformatics record card, which allows for high-yield prediction of the quality and usefulness of any pluripotent cell line. We used this record card to analyze gene expression data from the EBs formed from each clone of our iPSC strains. To test the differentiation potential, we use the automated system to generate EBs in 96-well v-bottom plates and finally collect RNA for the Nanostring nCounter Plex2 Assay Kit.
[000266] 83 different gene markers, selected from each of the 3 germ layers (83)
[000267] 5 retrovirus transgenes (4 factors with single detection probe, 1 probe)
[000268] 5 sendai transgenes (4 factors + vector only, 5)
[000269] Oct4, Klf4, cMyc, Nanog, Lin28, ZFP42 (pluripotency, Sox2 is in germ layer group, 6 probes) SRY, XIST sex markers (2)
[000270] Constitutive genes, ACTB, POLR2A, ALAS1 (3 probes).
[000271] Karyotype and identity analysis
[000272] Prior to accepting a lineage and at the end of each expansion, we utilize the nCounter Nanostring Plex2 Assay Kit to target the 400 genomic loci allowed for integrated molecular karyotype analysis along with "fingerprint" tracking of cell lineage identity. Molecular karyotype analysis uses an average of 8 probes per chromosome arm to verify genomic stability during the course of cell culture derivation and expansion of iPSC strains. The tracking identity "fingerprint" identity will depend on a combinatorial signature based on 30 common copy number variations (CNVs) of polymorphic loci, which allows unambiguous identification of individual genomes. To further avoid misidentification, tissue donors that are known to be related will not be processed in the same batch, as it is theoretically possible that they have similar CNVs. Data from the identity analysis will be cross-referenced with the initial CNV data to ensure that our LIMS system correctly tracked all cell lines. Freeze-thaw analysis
[000273] Freeze-thaw analysis: a vial is thawed after cryopreservation. Cells are counted after recovery and plated in one well of a 6-well plate. Cultures are observed daily. Colonies are photographed on the first day of appearance and after 5 days. Colonies must at least double in diameter within 5 days of the first observation.
[000274] Automated biopsy growth tracking. Using the system of the invention, one can control the growth of biopsies as well as other tissue sources by automated and traceable image analysis. As shown in Figure 6, images and growth rates are tracked during the production process. In Figure 6A, biopsies or discarded tissue are plated into multiple wells of a 6-well plate and maintained by an automated system that feeds, images, passes and freezes fibroblast growths. Examples of the image analysis interface are shown as a typical example. A single plate is used per donated sample to minimize cross contamination. (B) Number of cells is extrapolated from confluence measurements based on linear regression of an independently generated standard curve. (C) An example of cell counts for a typical biopsy growth maintained in our automated system. Extrapolated cell numbers per patient sample are plotted for each well independently (top) allowing calculation of the total sample output (bottom).
[000275] Figure 7 shows FACS analysis and graphs showing automated iPSC reprogramming. Expression levels of pluripotent surface markers in reprogrammed human fibroblasts were followed over a 3-week period to observe kinetic reprogramming and determine optimal time points at which to isolate defined cell populations. (A) FACS channel scheme used for analysis. (B) A substantial proportion of cells co-expressing traditional pluripotency surface markers SSEA4 & TRA-1-60 retain the fibroblast marker CD13 at all times during reprogramming using Sendai or retroviral vectors to introduce Oct4, Sox2 reprogramming factors, Klf4 and c-Myc. Box diagrams indicating aggregated data from 131 experiments (Retrovirus, n=66, Sendai virus, n=65) are shown. While Sendai-mediated reprogramming produces more SSEA4/TRA-1-60 double positive cells, (C) there is a delay in the clearance of CD13 from the surface. (D) Sample staining pattern of a patient cell line reprogrammed using Sendai/Cytotune system in our automated system. At both dpi 7 and 13, more than half of the double positive cell SSEA4/TRA-1-60 cells lost CD13. Furthermore, at both time points analyzed, CD13 negative/Nanog positive cells are present in this fraction, suggesting that they can be isolated by negative selection against CD13.
[000276] Figure 8 shows analyzes of pre-screening FACs and a part of the automated system demonstrates enrichment and selection of iPSC clones. (A) Non-reprogrammed cell populations can be removed from iPSC cultures by negative selection by a fibroblast marker. This strategy leaves iPSCs untouched. In the example, fibroblasts are efficiently removed from the culture containing 2% established iPSCs leaving TRA-1-60 positive iPSCs untouched. (B) A Miltenyi MultiMACS system integrated in the Hamilton liquid handler can classify 24 samples in parallel. (C) An example of a colony of newly derived iPSCs, derived by negative selection using anti-fibroblast antibody conjugated to magnetic beads in the MultiMACS system. Contrast phase, nuclear stain by Sytox, surface marker stain by TRA-1-60, and nuclear Nanog staining (not shown). (D) The iPS-enriched fraction from the antifibroblast magnetic negative selection step is plated on 96-well imaging plates at limiting dilution. These plaques are selected using live cell staining for the pluripotency surface marker TRA-1-60 or TRA-1-81. Wells with positive TRA-1-60 iPSCs are identified by automated image analysis using Celigo software capable of single colony confirmation. Wells that meet both criteria of containing a single colony that is positive for the surface marker are selected for passage and expansion and QC. (E) (Not shown) - Colonies produced by automated Sendai infection of adult fibroblasts.
[000277] Induction of iPSC has also been demonstrated by automated transfection of modified mRNA. BJ fibroblast iPSC colonies were efficiently recovered after 10 days of automated delivery of a transfection mixture containing modified mRNA. After an additional two days culture, the same well was stained with TRA-1-60 to identify undifferentiated cells. iPSCs in the well demonstrate that these are undifferentiated iPSCs. IPSC colonies isolated by purification away from non-reprogrammed cells using magnetic bead depletion in the automated system were efficiently retrieved.
[000278] High-throughput registry card assays for gene expression were generated. The first phase of our quality control scan uses a panel of transgene markers and pluripotency differentiation to choose an initial set of three clones. Figure 9A shows transcription counts after normalization for HK gene expression for two HESC strains, Sendai positive control, fibroblast negative control and iPS strains derived by FACS screening analyzed in passages 5 and 10. All assays are performed compared to a panel of normal HESC and iPS lines maintained under similar conditions. Not shown was an example image of an embryonic body generated in the system in v-bottom 96-well plates. The arrow points to the EB. Figure 9C illustrates the second stage of our quality control scan, which uses 83 germline/germline markers to monitor differentiation capacity in embryonic body assays. Single EBs are generated and pooled to generate RNA for analysis of germ layer marker expression in the Embryonic Body Registry Card Assay. A clustered dendrogram analysis of gene expression in EBs collected from nine different embryonic stem cell lines is shown. After normalization, data generated from direct lysing of six EBs compares favorably to data generated from total RNA extracted and purified from EBs prepared from mass culture.
[000279] Figure 10 demonstrates high-throughput karyotyping of iPSCs based on nCounter Nanostring assays for CNVs. Figure 10A is an example of nCounter karyotype assay on BC1 iPSCs; Figure 10B is an example of the nCounter karyotype assay on 1016 fibroblasts with partial gain and loss of chromosome arms. Comparison with Affymetrix SNP 6.0 chip data demonstrating copy number gained in part of q-arm of Chr1 (upper range, 1q21.2 - 1q43) and loss of part of long arm of Chr6 (lower range, 6q16.3 - 6q26).
[000280] While preferred embodiments of the invention have been described, the invention is not limited to these embodiments, and the scope of the invention is defined by means of appended claims.

权利要求:
Claims (21)
[0001]
1. An automated system for generating induced pluripotent stem cells (iPSCs) comprising: an automated cell plating unit for placing the cells on a plate; an automated induction unit to contact the cells in the automated plating unit with reprogramming factors to produce iPSCs, characterized in that the automated cell plating unit and an automated induction unit are contained within a liquid thawing handler and infection; and an automated colony identification liquid handler provided with a first automated microscope and an automated cell separation liquid handler unit for selectively separating and isolating the produced iPSCs, comprising a magnetic separation unit, in which the liquid handlers are connected with a robotic transport rail which is additionally connected to a first incubator, a second incubator, a second automated microscope, an automated centrifuge and an automated decapper, and wherein the automated system comprises a controller program.
[0002]
2. Automated system according to claim 1, characterized in that the magnetic separation unit selectively separates and isolates the iPSCs produced by the automated induction unit through the identification of specific iPS markers.
[0003]
3. Automated system according to claim 2, characterized in that it further comprises: an expansion unit to expand the isolated iPSCs and select the expanded iPSCs.
[0004]
4. Automated system according to claim 1, characterized in that it further comprises: a freezing unit to freeze the isolated iPSCs.
[0005]
5. Automated system according to claim 1, characterized in that it further comprises: a confluence checking unit that checks the confluence of the iPSCs to determine whether the iPSCs have a confluence characteristic.
[0006]
6. Automated system according to claim 5, characterized in that the confluence verification unit periodically checks the confluence of the iPSCs.
[0007]
7. Automated system according to claim 5, characterized in that the confluence verification unit periodically checks the confluence of the iPSCs on a more frequent basis over time.
[0008]
8. Automated system according to claim 5, characterized in that the confluence verification unit checks whether the confluence characteristic is within the range of 70% to 100%.
[0009]
9. Automated system according to claim 1, characterized in that the automated induction unit uses a viral vector to initiate reprogramming
[0010]
10. Automated system according to claim 9, characterized in that the automated induction unit uses a retrovirus or a Sendai virus to initiate reprogramming.
[0011]
11. Automated system according to claim 1, characterized in that the automated induction unit uses small molecules, peptides, proteins or nucleic acids to initiate reprogramming.
[0012]
12. Automated system according to claim 1, characterized in that it further comprises an electroporation unit to electroporate the cells.
[0013]
13. Automated system according to claim 1, characterized in that it further comprises a cell bank unit for obtaining the cells used by the automated cell plating unit.
[0014]
14. Automated system according to claim 13, characterized in that the cell bank unit comprises: a biopsy plating unit for placing biopsies on a plate; a growth and passage unit for cell culture; and a mycoplasma testing unit to test for the presence of mycoplasma.
[0015]
15. Automated system according to claim 14, characterized in that the test unit for mycoplasma comprises a luminescence testing device with glow.
[0016]
16. Automated system according to claim 14, characterized in that it further comprises: a distribution unit to distribute the expanded cells; and a storage and retrieval system for storing the cells.
[0017]
17. Automated system according to claim 16, characterized in that the storage and retrieval system freezes the cells.
[0018]
18. Automated system according to claim 3, characterized in that the expansion unit automatically expands and selects clones of superior quality.
[0019]
19. Automated system according to claim 1, characterized in that the cells are somatic cells.
[0020]
20. Automated system according to claim 19, characterized in that somatic cells are fibroblasts.
[0021]
21. Automated system according to claim 18, characterized in that the superior quality clones are consolidated to fewer plates than before consolidation.

类似技术:

---

公开号 | 公开日 | 专利标题

---

US10968435B2|2021-04-06|Automated system for producing induced pluripotent stem cells or differentiated cells

---

Dong et al.2020|Derivation of trophoblast stem cells from naïve human pluripotent stem cells

---

US11136556B2|2021-10-05|Systems and methods for producing stem cells and differentiated cells

---

US20160222355A1|2016-08-04|Systems and methods for producing stem cells differentiated cells, and genetically edited cells

---

US20190316075A1|2019-10-17|Microfluidic system and method of use thereof

---

US20210102188A1|2021-04-08|Production and Therapeutic Uses of Epinul Cells and Differentiated Cells Derived Therefrom

---

Sharma et al.2018|Human Induced Pluripotent Stem Cell Production and Expansion from Blood using a Non‐Integrating Viral Reprogramming Vector

---

EP3013941B1|2018-10-31|Improved systems and methods for producing stem cells and differentiated cells

---

BR122020005735B1|2021-12-28|SYSTEM FOR THE GENERATION OF DIFFERENTIATED ADULT CELLS FROM INDUCED PLURIPOTENT STEM CELLS |

---

De Kock et al.2012|Focus on stem cells as sources of human target cells for in vitro research and testing

---

WO2020204851A1|2020-10-08|Method for induced stem cell production

同族专利:

---

公开号 | 公开日

---

EP3653698A1|2020-05-20|

---

AU2018286561A1|2019-01-24|

---

IL289822D0|2022-03-01|

---

KR20190124326A|2019-11-04|

---

JP6257520B2|2018-01-10|

---

US10968435B2|2021-04-06|

---

BR112014013152A2|2017-06-13|

---

ZA201403945B|2018-08-29|

---

EP2785829A1|2014-10-08|

---

CA2857295A1|2013-06-06|

---

CA3118842A1|2013-06-06|

---

AU2021212081A1|2021-08-26|

---

US10273459B2|2019-04-30|

---

AU2012345676B2|2018-10-04|

---

US20210332330A1|2021-10-28|

---

DK2785829T3|2020-01-27|

---

WO2013082509A1|2013-06-06|

---

ES2773863T3|2020-07-15|

---

KR102039202B1|2019-10-31|

---

EP2785829A4|2015-04-29|

---

IN2014CN04920A|2015-09-18|

---

KR20140101393A|2014-08-19|

---

CA2857295C|2021-06-29|

---

EP2785829B1|2019-10-16|

---

CN110724636A|2020-01-24|

---

JP2015502747A|2015-01-29|

---

CN104080906A|2014-10-01|

---

KR20210096700A|2021-08-05|

---

IL232869D0|2014-07-31|

---

US20130345094A1|2013-12-26|

---

IL232869A|2022-02-01|

---

US20190352613A1|2019-11-21|

---

AU2012345676A1|2014-06-19|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

US5843780A|1995-01-20|1998-12-01|Wisconsin Alumni Research Foundation|Primate embryonic stem cells|

---

US5994619A|1996-04-01|1999-11-30|University Of Massachusetts, A Public Institution Of Higher Education Of The Commonwealth Of Massachusetts, As Represented By Its Amherst Campus|Production of chimeric bovine or porcine animals using cultured inner cell mass cells|

---

US5945577A|1997-01-10|1999-08-31|University Of Massachusetts As Represented By Its Amherst Campus|Cloning using donor nuclei from proliferating somatic cells|

---

EP0996736A1|1997-08-11|2000-05-03|Chiron Corporation|Methods for genetically modifying t cells|

---

US8026096B1|1998-10-08|2011-09-27|Protein Sciences Corporation|In vivo active erythropoietin produced in insect cells|

---

US20110171185A1|1999-06-30|2011-07-14|Klimanskaya Irina V|Genetically intact induced pluripotent cells or transdifferentiated cells and methods for the production thereof|

---

US6429013B1|1999-08-19|2002-08-06|Artecel Science, Inc.|Use of adipose tissue-derived stromal cells for chondrocyte differentiation and cartilage repair|

---

US6959438B2|2000-12-06|2005-10-25|Microsoft Corporation|Interface and related methods for dynamically generating a filter graph in a development system|

---

BR0309131A|2002-04-08|2005-02-01|Millenium Biologix Inc|Automated Tissue Creation System|

---

EP1445320A1|2003-02-05|2004-08-11|ARTEMIS Pharmaceuticals GmbH|Automated gene-targeting using non-toxic detectable markers|

---

US20070238175A1|2006-04-06|2007-10-11|Chi Alfred L|Standardization of processes for culturing primary cells|

---

GB0704406D0|2007-03-07|2007-04-18|Stem Cell Sciences Uk Ltd|Automated culture of stem cells|

---

JP2008307007A|2007-06-15|2008-12-25|Bayer Schering Pharma Ag|Human pluripotent stem cell induced from human tissue-originated undifferentiated stem cell after birth|

---

EP2173863B1|2007-06-29|2018-10-10|FUJIFILM Cellular Dynamics, Inc.|Automated method and apparatus for embryonic stem cell culture|

---

US10047346B2|2008-08-08|2018-08-14|Mayo Foundation For Medical Education And Research|Method of treating heart tissue using induced pluripotent stem cells|

---

US9045737B2|2008-12-13|2015-06-02|Dnamicroarray, Inc.|Artificial three-dimensional microenvironment niche culture|

---

CA2747398A1|2008-12-17|2010-07-08|The Scripps Research Institute|Generation and maintenance of stem cells|

---

WO2010096746A1|2009-02-20|2010-08-26|Cellular Dynamics International, Inc.|Methods and compositions for the differentiation of stem cells|

---

JP5816100B2|2009-02-27|2015-11-18|セルラー ダイナミクス インターナショナル， インコーポレイテッド|Pluripotent cell differentiation|

---

CN101580816B|2009-04-23|2012-02-29|中国科学院广州生物医药与健康研究院|Novel serum-free culture medium for inducing fast and efficient production of pluripotent stem cells and use method thereof|

---

US20110020814A1|2009-06-05|2011-01-27|Ipierian, Inc.|Methods and compositions for selection of stem cells|

---

EP2438160B1|2009-06-05|2015-12-23|Cellular Dynamics International, Inc.|Reprogramming t cells and hematopoietic cells|

---

US20120252697A1|2009-09-01|2012-10-04|Mcmaster University|Transformed human pluripotent stem cells and associated methods|

---

EP2558571A4|2010-04-16|2014-09-24|Immune Disease Inst Inc|Sustained polypeptide expression from synthetic, modified rnas and uses thereof|

---

US20110306516A1|2010-06-15|2011-12-15|The New York Stem Cell Foundation|Methods for producing induced pluripotent stem cells|

---

WO2012087965A2|2010-12-22|2012-06-28|Fate Therapauetics, Inc.|Cell culture platform for single cell sorting and enhanced reprogramming of ipscs|

---

EP2481795A1|2011-01-28|2012-08-01|Universität für Bodenkultur Wien|Method of generating induced pluripotent stem cells and differentiated cells|

---

BR112014013152B1|2011-12-01|2021-08-31|New York Stem Cell Foundation, Inc|AUTOMATED SYSTEM FOR INDUCED PLURIPOTENT STEM CELLS |US10428309B2|2011-12-01|2019-10-01|New York Stem Cell Foundation, Inc.|Systems and methods for producing stem cells and differentiated cells|

---

BR112014013152B1|2011-12-01|2021-08-31|New York Stem Cell Foundation, Inc|AUTOMATED SYSTEM FOR INDUCED PLURIPOTENT STEM CELLS |

---

WO2014201412A1|2013-06-13|2014-12-18|The New York Stem Cell Foundation Inc.|Pluripotent stem cells derived from non-cryoprotected frozen tissue and methods for making and using the same|

---

WO2014210533A1|2013-06-27|2014-12-31|The New York Stem Cell Foundation|Improved systems and methods for producing stem cells and differentiated cells|

---

JP6495281B2|2013-08-12|2019-04-03|インビボサイエンシーズ インコーポレイテッド|Automated cell culture system and method|

---

US20160222355A1|2014-12-26|2016-08-04|New York Stem Cell Foundation, Inc.|Systems and methods for producing stem cells differentiated cells, and genetically edited cells|

---

US9828634B2|2015-01-22|2017-11-28|Regenerative Medical Solutions, Inc.|Markers for differentiation of stem cells into differentiated cell populations|

---

JP2018526992A|2015-08-31|2018-09-20|アイ ピース，インコーポレイテッド|Pluripotent stem cell production system, stem cell induction method, stem cell suspension culture method, stem cell suspension incubator, method for producing induced pluripotent stem cells, and method for producing specific somatic cells from animal cells|

---

CN105567642B|2016-02-01|2019-07-12|中国科学院生物物理研究所|A kind of preparation method of xeroderma pitmentosum human pluripotent stem cells|

---

JP2019521715A|2016-07-21|2019-08-08|セリアドＣｅｌｙａｄ|Method and apparatus for automatically and independently batch processing cells in parallel|

---

US11259520B2|2016-08-04|2022-03-01|Fanuc Corporation|Stem cell manufacturing system, stem cell information management system, cell transport apparatus, and stem cell frozen storage apparatus|

---

EP3556842A4|2017-01-20|2020-01-15|FUJIFILM Corporation|Cell culture apparatus and cell culture method|

---

WO2018155607A1|2017-02-24|2018-08-30|剛士 田邊|Cell treatment device, suspension culture vessel, and stem cell induction method|

---

CN110392733B|2017-02-27|2021-02-26|田边刚士|Somatic cell production system|

---

SG11201907406WA|2017-02-27|2019-09-27|Koji Tanabe|Cell processing system and cell processing device|

---

GB2564437B|2017-07-10|2020-11-04|Univ Brunel|Method of testing a gene therapy vector using induced pluripotent stem cells|

---

JP6530577B1|2019-03-20|2019-06-12|剛士 田邊|Cell processing system and cell processing apparatus|

---

JP2019088335A|2019-03-20|2019-06-13|剛士 田邊|Cell processing system and cell processing device|

---

JP6530874B1|2019-03-20|2019-06-12|剛士 田邊|Cell processing system and cell processing apparatus|

---

WO2021067797A1|2019-10-04|2021-04-08|New York Stem Cell Foundation, Inc.|Imaging system and method of use thereof|

---

WO2021104453A1|2019-11-28|2021-06-03|The University Of Hong Kong|Mesenchymal stromal cells as a reprogramming source for ipsc induction|

法律状态:
2018-07-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

---

2018-07-10| B25D| Requested change of name of applicant approved|Owner name: NEW YORK STEM CELL FOUNDATION, INC. (US) |

---

2019-08-06| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

---

2021-06-01| B350| Update of information on the portal [chapter 15.35 patent gazette]|

---

2021-06-22| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

---

2021-08-31| 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 30/11/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

---

申请号 | 申请日 | 专利标题

---

US201161565818P| true| 2011-12-01|2011-12-01|

---

US61/565,818|2011-12-01|

---

US201161580007P| true| 2011-12-23|2011-12-23|

---

US61/580,007|2011-12-23|

---

US201261700792P| true| 2012-09-13|2012-09-13|

---

US61/700,792|2012-09-13|

---

PCT/US2012/067417|WO2013082509A1|2011-12-01|2012-11-30|Automated system for producing induced pluripotent stem cells or differentiated cells|

---