METHODS FOR GENERATION OF CELLS THAT SECRET BRAIN-DERIVED NEUROTROPHIC FACTOR (BDNF), GLIA-DERIVED N

专利摘要:
method for generating mesenchymal stem cells that secrete neurotrophic factors, isolated population of cells, and pharmaceutical composition comprising an isolated population of cells. encompasses method for generating mscs that secrete neurotrophic factors (ntfs), comprising incubating a population of undifferentiated mesenchymal stem cells (mscs) in a differentiation medium comprising a basic fibroblast growth factor (bfgf), a derived growth factor of platelets (pdf), heregulin and camp.
公开号:BR112015001435B1
申请号:R112015001435-6
申请日:2013-08-04
公开日:2022-01-18
发明作者:Yael Gothelf；Yosef Levy；Alex Burshtein
申请人:Brainstorm Cell Therapeutics Ltd；
IPC主号:

专利说明:

FIELD OF APPLICATION AND HISTORY
[001] The present patent application, in some respective applications, relates to methods for generating cells from mesenchymal stem cells that secrete neurotrophic factors and methods for selecting the same.
[002] Amyotrophic lateral sclerosis (ALS| amyotrophic lateral sclerosis) is one of the most common neurodegenerative diseases in adults. It is a fatal progressive neurodegenerative disease characterized by cell death of motor neurons in the brain and spinal cord, accompanied by rapid loss of muscle function and eventually complete paralysis.
[003] Current experimental drugs against ALS are developed based on putative pathophysiological mechanisms, such as anti-glutamatergic agents, drugs targeting protein unevenness and accumulation, antioxidant therapy, immunomodulatory agents, and stem cells.
[004] Of the current experimental therapies, stem cell transplantation may have the greatest potential. In addition to replacing lost or damaged neurons, stem cell implant therapy may benefit ALS patients by an independent cytoprotective effect. In addition, there is the potential for stem cells to differentiate into supporting interstitial cells, including astrocytes and microglia, which can potentially produce neurotrophic factors, as well as enzymatic and paracrine mediators, which antagonize neurotoxicity. More experimental data have demonstrated that non-neuronal cell replacement can be a strategic therapy in promoting motor neuron survival and improving neuromuscular function (Corti S. et al. 2010).
[005] The use of stem cells as a cellular source in cell replacement therapies for additional neurodegenerative diseases, including Parkinson's disease and multiple sclerosis, has also been suggested.
[006] Neurotrophic factors (NTF I Neurotrophic factors) are small natural polypeptides that aid in the development and survival of neurons and thus have been considered in recent years as candidates for therapy options against different neurodegenerative diseases, including ALS. Studies in animal models with ALS have shown a delay in disease onset and/or progression following administration of various neurotrophic factors.
[007] However, clinical trials of intrathecal or systematic administration of recombinant growth factors to patients with ALS have not been effective, likely due in part to their short half-life, low concentration at target sites, and high incidence of effects. collaterals.
[008] Several studies have shown that mesenchymal stem cells (MSCs | mesenchymal stem cells), after exposure to different factors in vitro, change their phenotype and demonstrate neuronal and glial markers [Kopen, GC, et al., Proc Natl Acad USA . 96(19):10711-6, 1999; Sanchez-Ramos, et al. Neurol Exp. 164(2) 1247-56. 2000; Woodbury, D., J Neurosci Res. 61 (4):364-70,2000; Woodbury, D., et al., J Neurosci Res. 69(6):908-17, 2002 ; Black, I.B., Woodbury, D. Blood Cells Mol Dis. 27(3)1632-6, 2001; Kohyama, J., et al. Differentiation. 68 (4-5):235-44, 2001; Levy, Y.S. J Mol Neurosci. 21(2)1121-32, 2003, Blondheim N.R., Stem Cells & Dev. 151141-164, 2006 ].
[009] WO2006/134602 and WO2009/144718 instruct differentiation protocols for the generation of neurotrophic factor-secreting cells from mesenchymal stem cells.
[0010] WO2007/066338 instructs differentiation protocols for the generation of oligodendrocyte-like cells from mesenchymal stem cells.
[0011] WO2004/046348 instructs differentiation protocols for generating neuronal cells from mesenchymal stem cells.
[0012] Abbaszadeh et al [Iranian Biomedical Journal 17 (2): 62-70 (April 2013)] instructs a two-step differentiation protocol for the generation of oligodendrocytes from MSCs, where one of the media is composed of PDGF , heregulin, bFGF and triiodothyronine. SUMMARY
[0013] In accordance with an aspect of some applications of the present patent application, there is provided a method for generating cells that secrete neurotrophic factors (NTFs) comprising incubating a population of undifferentiated mesenchymal stem cells (MSCs) in a differentiation medium comprising a basic fibroblast growth factor (bFGF I basic fibroblast growth factor), a platelet derived growth factor (PDGF), heregulin and cAMP.
[0014] In accordance with an aspect of some applications of the present application, an isolated population of cells that secretes neurotrophic factors is provided, generated according to the methods described herein.
[0015] In accordance with one aspect of some applications of the present application, there is provided a method for treating a disease, in which administration of neurotrophic factors is beneficial to an individual in need thereof, comprising administering to the individual of a therapeutically effective amount of the isolated population of cells described herein, thereby treating the disease.
[0016] In accordance with an aspect of some applications of the present patent application, there is provided a method for selecting cells that secrete neurotrophic factors (NTFs) from a mixed population of MSCs, comprising: (a) analyzing the cells of the mixed population of cells on at least one of the following parameters: (i) cells expressing CD44 below a predetermined threshold; (ii) cells expressing CD73 above a predetermined threshold; and (b) selecting cells that are positive in at least one of the parameters, thereby selecting cells that secrete neurotrophic factors.
[0017] In accordance with one aspect of some applications of the present application, there is provided a pharmaceutical composition, comprising the isolated population of cells described herein, as an active agent and a pharmaceutically acceptable carrier.
[0018] In accordance with an aspect of some applications of the present patent application, there is provided a method of cell qualification useful for the treatment of a disease that has been differentiated ex vivo from MSCs, and that secretes neurotrophic factors, comprising analyzing cells by the expression of at least one protein selected from the group consisting of: Isobutyryl-CoA dehydrogenase, Chemokine 6 (CXC motif), Neuromodulin, Growth/differentiation factor 15, Hyaluronic acid synthase-1, Interleukin-1 beta , Interleukin-8, Inhibin beta A chain, Insulin receptor substrate 1, Integrin alpha-1, Laccase domain-containing proteins 1, Laminin alpha-4 subunit, Lumican, Collagenase 3, Esophagus-specific gene protein 1 normal mucosa , Pre-B-cell leukemia transcription factor interaction protein, Pleckstrin homology domain - family A, member 1 , Phosphatidylinositol 3,4, 5-trif-dependent RAC exchange protein Phosphate, Prostaglandin-endoperoxide synthase, Prostaglandin G/H synthase 2, Ras family-related Rab-27B protein, Rho-related GTP-binding protein RhoB, O-acetylesterase sialate, Monocarboxylate transporter 7, Tissue factor 2 pathway inhibitor , Transmembrane protein 65, Vam6/Vps39 type protein, 3-Oxo-5-beta-Steroidal 4-Dehydrogenase, Mitochondrial beta propionyl-CoA carboxylase chain, Interferon regulatory factor binding protein-2, Tissue alpha-L- fucosidase, Aldo-keto reductase - family 1, C2 member, Inositol 1,4,5-triphosphate receptor interacting protein, KIAA1199 Protein, Selenium Binding Protein, Phospholipase D3, Mitochondrial Phosphotransferase GTP:AMP, Wnt-5a Proteins ; Wπt Protein, Aldo-keto reductase - family 1, member C3, Nexins rank-9, GAP junction protein alpha-1, Mitochondrial Pyruvate carboxylase, Protein 2B containing SH3 and PX domain, Integrin alpha-2, Cytochrome P450 1B1, Protein -1 type quitinase-3, Nicotinamide phosphoribosyltransferase, Seprase, Superoxide dismutase, Aldoketo reductase - family 1, Cl member, Protein containing the FERM, RhoGEF and pleckstrin domains 1, Prolyl 4-hydroxylase alpha-3 subunit, M2 subunit B from ribonucleoside© diphosphate reductase, Histone macro-H2A.2 and Histoπa H2A from the nucleus, Choline transporter protein 1 and Niemann-Pick Protein Cl, Lysosomal alpha-glucosidase, characterized by increased expression of at least one protein, if compared to undifferentiated MSCs, be an indication that the cells are useful in treating a disease.
[0019] In accordance with an aspect of some applications of the present patent application, there is provided a cell qualification method useful for the treatment of a disease that has been differentiated ex vivo from MSCs, and that secretes neurotrophic factors, comprising analyzing cells by the expression of at least one protein selected from the group consisting of: ZQ-2 tight junction protein, Alpha-1,3-mannosyl-glooprotein 2-beta-N-acetylglucosaminyltransferase, Esmothelin, Homologous Protein-5 from echotropic P granules, BRCA1-associated ATM Activator 1, WD repeat-containing protein 32, SH3 binding domain protein 4, Type 1 protein, EH binding domain protein 1, Ras GTPase activation protein IQGAP3, Lysyl oxidase homolog 2, Alpha-tropomyosin isoform CRA_f 1, yolk-associated protein 5, Tripartite motif-containing protein 16, Connective tissue growth factor, Protein kinase originated from p-activated killer T cells or lymphokines, tetratricopeptide repeat protein 4, breast cancer anti-estrogen resistance protein L, ribonucleoside diphosphate reductase M2 subunit, ubiquitin-conjugated E2 C enzyme, neutrophil defensin 1, Cdc42 effector proteins 3, çondensin complex subunit 2, C region of the Ig kappa chain, Condensin 3 complex subunit, Syncoiln, Structural maintenance of chromosome 2 protein, Condensin 1 complex subunit, H4 heavy chain inter-alpha-trypsin inhibitor, Thymidyl synthase, Serotransferrin, H4 protein Pregnancy Zone, DNA Replication Licensing Factor MCM7, Hemopexins, DNA Mismatch Repair Msh6 Proteins, Ankyrin Repeat Domain-Containing 13A Proteins, Phosducin-like Protein 3, 1-Phosphatidylinositol 4,5-Bisphosphate Phosphodiesterase Beta- 3, Complement C3; DNA Replication Licensing Factor MCM3, CD97 Antigen; CD97 Antigen Alpha Subunit, DNA Replication Licensing Factor MCM6, DMA Replication Licensing Factor MCM4, Deactivated Homolog 2, KIAA0664 Protein, DNA Replication Licensing Factor MCM2, Protein-Lysine 6-Oxidase, Large Subunit of Ribonucleoside Diphosphate Reductase, Melanoma Associated D2 Antigen, lg Gamma-1 Chain C Region, Heparanase, Importin Alpha-2 Subunit, Asparagine Synthetase [Glutamine Hydrolysis], Alpha-2-Macroglobulin, Collagen Type (I) Alpha-1 Chain , Collagen type (V) alpha-1 chain, DnaJ homolog subfamily 3, member 4, Thrombospondin-1, Serum albumin and type (I) collagen alpha-2 chain, characterized by decreased expression of at least one protein with respect to undifferentiated cells being indicative that the cells are useful in treating a disease.
[0020] In accordance with one aspect of some applications of the present patent application, an isolated population of cells that secrete neurotrophic factors is provided, characterized in that cells express each of the following mesenchymal stem cell markers: CD44, CD73, CD90 and CD105, and do not express any of the following surface markers: CD3, CD14, CD19, CD34, CD45 and HLA-DR, as detected by flow cytometry.
[0021] In accordance with an aspect of some applications of the present patent application, there is provided a cell qualification method useful for the treatment of a disease that has been differentiated ex vivo from MSCs, and that secretes neurotrophic factors, comprising analyzing cells by expressing at least one miRNA selected from the group consisting of: miR-503-5p, miR-3659, miR-3529-3p, miR-320b, miR-424-5p, miR-320a, miR -222-3p, miR-3663-3p, miR-762, miR-4327, miR-3665, miR34a-5p, miR-4327, miRNA-3665 and miR132-3p; characterized by an increased expression of miR-3663-3p, miR-762, miR-4327, miR-3665, miR34a-5p, miR-4327, miRNA 3665 or miR132-3p compared to undifferentiated MSCs or a decreased expression of miR-503-5p, miR-3659, miR-3529-3p, miR-320b, miR-424-5p, miR-320a or miR-222-3p, compared to undifferentiated MSCs, is an indication that the cells are useful in the treatment of a disease.
[0022] According to some applications of the present application, the differentiation means is devoid of a phosphodiesterase inhibitor.
[0023] According to some applications of the present patent application, the differentiation medium is devoid of Triiodothyronine.
[0024] According to some applications of the present patent application, the phosphodiesterase inhibitor is composed of IBMX.
[0025] According to some applications of the present patent application, the means of differentiation is devoid of xenobiotic components.
[0026] According to some applications of the present patent application, the method also comprises the cultivation of the population of undifferentiated MSCs before incubation, characterized by the cultivation being carried out under conditions that do not promote cell differentiation.
[0027] According to some applications of the present patent application, cultivation is carried out for three days after sowing the undifferentiated MSCs.
[0028] According to some applications of the present patent application, sowing is carried out at a density of approximately 6000 to 8000 cm2.
[0029] According to some applications of the present patent application, the cultivation is carried out in a culture medium composed of platelet lysates.
[0030] According to some applications of the present patent application, the percentage of platelet lysate in the culture medium is about 10%.
[0031] According to some applications of the present patent application, the culture medium further comprises L-glutamine, sodium pyruvate and heparin.
[0032] According to some applications of the present patent application, the method further comprises analyzing an expression of CD44 and/or CD73 on a cell surface.
[0033] According to some applications of the present patent application, the method further comprises analyzing an expression of CD105 on the cell surface.
[0034] According to some applications of the present patent application, the method further comprises comparing the expression with an expression of CD44 and/or CD73 on a surface of undifferentiated MSCs.
[0035] In accordance with some applications of the present patent application, cells express each of the following mesenyard stem cell markers: CD44, CD73, CD90 and CD105, detected by flow cytometry.
[0036] According to some applications of the present patent application, cells do not manifest any of the following surface markers: CD3, CD14, CD19, CD34, CD45 and HLA-DR, detected by flow cytometry.
[0037] According to some applications of the present patent application, the cells are not genetically modified.
[0038] In accordance with some applications of the present application, cells are differentiated ex vivo from MSCs that are autologous to the individual.
[0039] In accordance with some applications of the present application, cells are differentiated ex vivo from MSCs that are homologous to the individual.
[0040] In accordance with some applications of the present patent application, cells are differentiated ex vivo from MSCs that are derived from the individual's bone marrow.
[0041] According to some applications of the present patent application, the disease is a neurodegenerative disease or an immune disease.
[0042] According to some applications of the present patent application, the neurodegenerative disease is selected from the group consisting of Parkinson's Disease, Multiple System Atrophy (MSA | multiple system atrophy), multiple sclerosis, epilepsy, amyotrophic lateral sclerosis ( ALS), stroke, autoimmune encephalomyelitis, diabetic neuropathy, glaucomatous neuropathy, Alzheimer's disease and Huntingdon's disease.
[0043] According to some applications of the present patent application, the neurodegenerative disease is ALS.
[0044] According to some applications of the present patent application, the immune disease is an autoimmune disease.
[0045] According to some applications of the present patent application, the autoimmune disease is myasthenia gravis.
[0046] According to some applications of the present patent application, the administration is carried out by the intramuscular and/or intrathecal route.
[0047] According to some applications of the present patent application, when the administration is carried out by the intramuscular route, the total amount of cells administered to a 70 kg individual must be between 20 to 100x10 6 cells.
[0048] According to some applications of the present patent application, when the administration is carried out intrathecally, an amount of MSC-NTFs administered to a 70 kg individual should be between 50 to 200x106 cells per administration.
[0049] According to some applications of the present patent application, when intrathecal and intramuscular administration is performed, a total amount of MSC-NTFs administered to a 70kg individual should be between 20 to 500x106 cells.
[0050] According to some applications of the present patent application, the method further comprises analyzing a level of CD105 on the cells and selecting cells that express CD105 below a predetermined level.
[0051] According to some applications of the present patent application, NTFs comprise a glial-derived neurotrophic factor (GDNF | glial derived neurotrophic factor) and a brain-derived neurotrophic factor (BDNF | brain derived neurotrophic factor).
[0052] According to some applications of the present patent application, the method further comprises incubating a population of undifferentiated MSCs in a differentiation medium in order to generate MSCs that secrete NTFs before analysis.
[0053] According to some applications of the present application, the pharmaceutically acceptable carrier maintains the number of cells in the composition for at least 48 hours.
[0054] In accordance with some applications of the present patent application, miRNAs are selected from the group consisting of miR-503-5p, miR-320b, miR424-5p, miR-132-3p and miR-34a- 5p.
[0055] According to some applications of the present patent application, neurotrophic factors are selected from the group consisting of BDNF, GDNF, VEGF and HGF.
[0056] According to some applications of the present patent application, neurotrophic factors comprise BDNF, GDNF, VEGF and HGF.
[0057] According to some applications of the present patent application, the cells are not genetically modified.
[0058] According to some applications of the present patent application, cells show an increased expression of a miRNA selected from the group consisting of miR34a-5p, míR-222-3p, miR762, miRNA 3663-3p or miR132-3p compared to undifferentiated MSCs.
[0059] According to some applications of the present patent application, cells show a decreased expression of at least one miRNAs selected from the group consisting of miR-503-5p, miR-320b, miR-424-5p, miR-320a or miR-222— 3p, compared to undifferentiated MSCs,
[0060] According to some applications of the present patent application, cells show an increased expression of one or more of: Isobutyryl-CoA dehydrogenase, Chemokine 6 (CXC motif), Neuromodulin, Growth/differentiation factor 15, Acid Hyaluronic synthase 1, Interleukin-1 beta, Interleukin-8, Inhibin beta A chain. Insulin receptor substrate 1, Integrin alpha-1, laccase domain-containing proteins 1, Laminin alpha-4 subunit, Lumican, Collagenase 3, Esophageal specific gene protein 1 normal mucosa, Esophageal leukemia transcription factor interaction protein pre-B cell 1, Pleckstrin homology domain - family A, member 1, Phosphatidylinositol 3,4,5-triphosphate-dependent RAC exchange protein, Prostaglandin-endoperoxide synthase, Prostaglandin G/H synthase 2, Rab-27B-related protein Ras family, Rho-related GTP-binding protein RhoB, O-acetylesterase sialate, Monocarboxylate transporter 7, Tissue factor pathway inhibitor 2, Transmembrane protein 65, Vam6/Vps39-like protein, 3-Oxo-5-beta- Steroid 4-dehydrogenase, Beta-propionyl-CoA carboxylase chain, mitochondrial, Interferon regulatory factor binding protein-2, Tissue alpha-L-fucosidase, Aldo-keto reductase - family 1, C2 member, Receptor-interacting protein i nositol 1,4,5-triphosphate, KIAA1199 Protein, Selenium Binding Protein, Phospholipase D3, Mitochondrial Phosphotransferase GTP:AMP, Wnt-5a Proteins; Wnt protein, Aldo-keto reductase - family 1, member C3, Rank-9 Nexins, GAP junction protein alpha-1, Pyruvate carboxylase, mitochondrial, Protein 2B containing SH3 and PX domain, Integrin alpha-2, Cytochrome P450 1B1, Chiitinase-3-like protein-1, Nicotinamide phosphoribosyltransferase, Seprase, Superoxide dismutase, Aldoketo reductase - family 1, member Cl, Protein containing domains FERM, RhoGEF and pleckstrin 1, Prolyl 4-hydroxylase alpha-3 subunit, Subunit Ribonucleoside diphosphate reductase M2 B, Histone macro-H2A.2 and Core Histone H2A, Choline Carrier Protein 1 and Niemann-Pick Protein Cl, Lysosomal alpha-glucosidase, compared to undifferentiated MSCs.
[0061] According to some applications of the present patent application, cells show decreased expression of one or more of: Tight junction protein ZO-2, Alpha-1, 3-mannosyl-glycoprotein 2-beta-N -acetylglucosaminyltransferase, Esmothelin, Echotropic P-granule homologous protein-5, BRCA1-associated ATM 1 Activator, WD repeat-containing protein 36, SH3 binding domain protein 4, Type 1 protein, EH binding domain protein 1, Ras protein of activation of GTPase IQGAP3, Homologous Lysyl Oxidase 2, Alpha-tropomyosin Isoform CRA_f, Yolk Associated Protein 5, Tripartite motif-containing Protein 16, Connective tissue growth factor, Protein kinase originated from lymphokine-activated killer T cells, Tetratricopeptide Repeat Protein 4, Breast Cancer Anti Estrogen Resistance Protein 1, Ribonucleoside© Diphosphate Reductase M2 Subunit, Ubiquitin-conjugated E2 C Enzyme, Neutrophil Defensin los 1, Cdc42 effector proteins 3, Condensin complex 2 subunit, C region of the kappa Ig chain, Condensin 3 complex subunit, Syncoilin, Structural maintenance of chromosome 2 protein, Condensin complex 1 subunit, Inter-alpha-inhibitor H4 heavy chain trypsin, Thymidyl synthase, Serotransferrin, Pregnancy zone protein, DNA replication licensing factor MCM7, Hemopexin, Msh6 DNA mismatch repair protein, Ankyrin repeat domain-containing 13A proteins, Phosducin-like protein 3,1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase beta-3, Complement C3; DNA Replication Licensing Factor MCM3, CD97 Antigen; CD97 Antigen Alpha Subunit, DNA Replication Licensing Factor MCM6, DNA Replication Licensing Factor MCM4, Deactivated Homolog 2, KIAA0664 Protein, DNA Replication Licensing Factor MCM2, Protein-Lysine 6-Oxidase, Large Subunit of Ribonucleoside Diphosphate Reductase, Melanoma Associated D2 Antigen, C Region Gamma-1 Chain Ig, Heparanase, Importin Alpha-2 Subunit, Asparagine Synthetase [Glutamine Hydrolysis], Alpha-2-Macroglobulin, Collagen Type (I.) Alpha-Chain 1, Collagen type (V) alpha-1 chain, DnaJ homolog subfamily B, member 4, Thrombospondin-1, Serum albumin and collagen type (I) alpha-2 chain, compared to undifferentiated MSCs.
[0062] A. Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present patent application belongs. Although methods and materials similar or equivalent to what is described herein may also be used in practice or in testing applications of the present patent application, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including its definitions, will prevail. Furthermore, the materials, methods and examples are illustrative only and are not intended to be necessarily limiting. BRIEF DESCRIPTION OF THE DRAWINGS
[0063] Some applications of the present patent application are described in this document by way of example only, with reference to the attached drawings. With specific reference to the drawings in detail, it is emphasized that the elements indicated are by way of example and for purposes of illustrative discussion of the applications of the present patent application. In this regard, the description taken with the drawings makes it evident to those skilled in the art how the applications of the present patent application can be practiced. IN THE DRAWINGS
[0064] Figures 1A-B are graphs illustrating the stability of the final product at concentrations suitable for the two routes of administration in this case, as measured by viable cell counts using Trypan blue dye exclusion. The stability of the final MSC-NTF product in ALS patients was evaluated at 2 to 8°C for up to 7 hours after harvest at two cell concentrations: 10x10s cells/ml (A) for the intramuscular transplant route (IM I intramuscular) and 35x106 cells/ml (B) for intrathecal (IT | intrathecal) transplantation. The cells were incubated for 5 hours in a 50ml tube and then transferred to 1ml and 5ml syringes respectively for another two hours.
[0065] Figure 2 is a graph illustrating the number of MSC cells derived from ALS patients after long-term propagation. The spread of bone marrow-derived MSC from seven patients with ALS is shown by 5 to 7 passages for up to 60 days. Cell numbers were determined at each passage and cumulative population doublings (PD [opulation doublings] were calculated. PD = LoglO (number of cells harvested at the end of the passage) - Log (number of cells seeded at the beginning of the passage) / Logí. The total number of PD corresponding to the addition of the PD for all passes. The first PDs were determined in relation to the number of cells after the first passage (Pl).
[0066] Figures 3A-I are photographs of MSCs from three donors (#60, #61 and #62) at passage 2, which were induced to differentiate into adipocytes (Figures 3A-C), osteocytes (Figures 3D-F ) and chondrocytes (Figures 3G-I).
[0067] Figures 4A-B are photos of the results of chromosome analysis as performed by the G-banding technique. MSCs grown from an ALS patient were harvested in an early passage - P2 (A) and in a passage final - P5 (B). Cells exhibited a normal karyotype at both passages.
[0068] Figures 5A-F are photographs of MSCs from the same patient with ALS at passage 3, where they were induced to differentiate into adipocytes, osteocytes and chondrocytes previously (Figures 5A-C) and cryopreservation following (Figures 5D-F)
[0069] Figures 6A-B are graphs illustrating the GDNF and BDNF productivity of MSC-NTF cells. The productivity of MSC-NTF cells (red) from 12 patients with ALS compared to the productivity of MSC (blue) from the same patient, analyzed in the same ELISA assay. GDNF secretion is induced 2 to 20 times or more in NTF-MSC compared to MSC from the same patient, and BDNF secretion is induced 1.5 to 5 times in NTF-MSC compared to MSC from the same patient .
[0070] Figure 7 is a graph illustrating the productivity of MSC BDNF and MSC-NTF cells from 23 different patients with ALS.
[0071] Figure 8 is a graph illustrating the productivity of MSC GDNF and MSC-NTF cells from 23 different patients with ALS.
[0072] Figure 9 is a graph illustrating VEGF productivity from MSC-NTF cells. The productivity of MSC-NTF cells (red) from 22 patients with ALS compared to the productivity of MSC (blue) from the same patient, analyzed in the same ELISA assay. VEGF secretion is induced 4.1+1.4 or more times in MSC-NTFs, as compared to MSCs from the same patient.
[0073] Figure 10 is a graph illustrating HGF productivity from MSC-NTF cells. The productivity of MSC-NTF cells (red) from 19 patients with ALS, compared to the productivity of MSC (blue) from the same patient, analyzed in the same ELISA assay. HGF secretion is induced 6.7 + 3.9 or more times in MSC-NTFs, as compared to MSCs from the same patient.
[0074] Figures 11A-B are graphs illustrating the production of GDNF and BDNF by MSC-NTF cells after harvest (time "0") and three days after culturing in "transplant" growth medium, mimicking an "in vivo". Results show the mean ±SD of 4 independent experiments with MSC-NTF cells from ALS patients.
[0075] Figures 12A-B are the results of the phenotypic characterization of MSC surface marker expression NTF from the ALS patient at the end of differentiation, as compared to MSCs from the same patient. The panel of surface markers characteristic of MSCs, analyzed by flow cytometry, included the positive markers (A) CD44, CD73, CD90 and CD105 and the negative markers (B) CD3, CD14, CD19, CD34, CD45 and HLA-DR . MSC and MSC NTF cells are in red and the isotype control in black. The initials ZH stand for ALS patient.
[0076] Figures 13A-B are histograms illustrating flow cytometry analysis of CD44 and CD73 expression on the surface of MSC (black) and MSC NTF cells (red) from the same patient at the end of differentiation. The dotted line to the left is for the isotype control.
[0077] Figures 14A-B are graphs illustrating flow cytometry analysis of CD105 expression of MSC and MSC-NTF cells during differentiation. Flow cytometric analysis of CD105 expression on the surface of MSC (black) and MSC NTF cells (red) from the same patient on days 2 and 3 of differentiation (n=16 and 22, respectively). The dotted line to the left is the isotype control.
[0078] Figures 15A-B are histograms illustrating cell cycle analyzes of MSC and MSC-NTF secreting cells. The distribution of cells in the Go/Gi, S and G2/M phases of the cell cycle is shown. The shift to G0/G1 is manifested in the MSC-NTF cell population.
[0079] Figures 16A-B compare the two cell types based on all 160 miRNAs detected, with cell type and donor identification shown. A - A representation of the miRNA profiles of the 8 different cell samples in a 3D PCA projection, including donor identification; B - A representation of the miRNA profiles of the 8 different cell samples as the delineation of a clustergram* from a heat map [*N.T. A chart for visualizing hierarchical and non-hierarchical clustering analysis after hierarchical clustering, including donor identification.
[0080] Figure 17 is an expression profile of the 19 key upregulated miRNAs in MSC-NTF vs. MSC on a log2 scale. The strongly induced/mostly highly upregulated miRNAs in MSC-NTFs are highlighted with red ovals. When miRNA expression falls below the detection level for the arrays, a nominal intensity value is given to these data points. This value is entered to avoid errors arising from uncomputable math operations during subsequent data analysis. From the normalization process, this then results in a normalized intensity value of 1.1375 for these miRNAs.
[0081] Figure 18 is an expression profile of the 22 key downregulated miRNAs in MSC-NTF vs MSC on a log2 scale. The miRNAs, mostly highly downregulated in MSC-NTFs, are highlighted with red ovals. When miRNA expression falls below the detection level for the arrays, a nominal intensity value is given to these data points. This value is entered to avoid errors arising from uncomputable math operations during subsequent data analysis. From the normalization process, this results in a normalized intensity value of 1.1375 for these miRNAs.
[0082] Figure 19 is a bar graph illustrating that miR-503 expression is downregulated in MSC-NTF vs MSC.
[0083] Figure 20 is a bar graph illustrating that miR132-3P expression is upregulated in MSC-NTF vs MSC.
[0084] Figure 21 is a schematic summarizing the profile of differentially expressed miRNA in MSC-NTFs, leading to a predicted enhanced pro-angiogenic capacity of these cells.
[0085] Figures 22A-B illustrate that miR-762 (A) and miR-34a-5p (B) expression are upregulated in MSC-NTF vs MSC. When miRNA expression falls below the detection level for the arrays, a nominal intensity value is given to these data points. This value is entered to avoid errors arising from non-computable math operations during subsequent data analyses. From the normalization process, this then results in a normalized intensity value of 2.2 for these miRNAs.
[0086] Figures 23A-E illustrate the expression profiles of highly discriminatory miRNAs, with no validated target mRNA that was DE in MSC-NTF vs MSC. When miRNA expression falls below the detection level for the arrays, a nominal intensity value is given to these data points. This value is entered to avoid errors arising from non-computable math operations during subsequent data analyses. From the normalization process, this then results in a normalized intensity value of 2.2 for these miRNAs on a linear scale.
[0087] Figures 24A-G are bar graphs comparing the expression of certain miRNAs in MSC samples and MSC-NTF samples. Figure 24A illustrates that hsamiR-503 is downregulated in MSC-NTFs as compared to MSCs. Figure 24B illustrates that hsa-miR-320b is downregulated in MSC-NTFs as compared to MSCs. Figure 24C illustrates that hsa-miR-424-5p is downregulated in MSC-NTFs as compared to MSCs. Figure 24D illustrates that hsa-miR-34a-5p is up-regulated in MSC-NTFs as compared to MSCs. Figure 24E illustrates that hsa-miR~132-3p is up-regulated in MSC-NTFs as compared to MSCs. Figure 24F illustrates that hsa-miR-320a is not significantly downregulated in MSC-NTFs compared to MSCs. Figure 24G illustrates that miR-222-3p is not significantly downregulated in MSC-NTFs compared to MSCs.
[0088] Figure 25 is a graph illustrating the effect of IT administration of MSC-NTFs on the ALS Functional Rating Scale (ALSFRS-R | ALS Functional Rating Score).
[0089] Figure 26 is a graph illustrating the effect of MSC-NTFs IT administration on FVC I forced vital capacity.
[0090] Figure 27 is a graph illustrating the effect of IT administration of MSC-NTFs on mean muscle circumference.
[0091] Figure 28A-C are bar graphs comparing the profitability obtained using a one- or two-step differentiation protocol on three samples from ALS patients. DESCRIPTION OF SPECIFIC APPLICATIONS
[0092] The present patent application, in some respective applications, relates to methods for generating cells from mesenchymal stem cells that secrete neurotrophic factors and methods for selecting the same.
[0093] Before explaining at least one administration of the present patent application in detail, it should be understood that the present patent application is not necessarily limited in its administration to the details set forth in the following descriptions or exemplified by the Examples. The present patent application is capable of other applications, or of being practiced or performed in various ways.
[0094] Neurotrophic factors (NTFs) are secreted proteins that regulate survival, functional maintenance and phenotypic development of neuronal cells. Changes in NTF levels are involved in triggering programmed cell death in neurons and thus contribute to the pathogenesis of Parkinson's disease and other neurodegenerative diseases.
[0095] However, the direct use of neurotrophic factors is not applicable as they do not pass the blood-brain barrier and do not distribute correctly after systemic injection. Therefore, other strategies must be developed to benefit from its therapeutic properties.
[0096] Protocols for differentiating human mesenchymal stem cells into neurotrophic factor-secreting cells are known in the art - see, for example, WO 2006/134602 and WO 2009/144718.
[0097] The present inventors have developed new methods that increase the secretion of neurotrophic factors from mesenchymal stem cells (MSCs). The method comprises for the first time the direct differentiation of undifferentiated MSCs in a single medium, composed of a basic fibroblast growth factor (bFGF), a platelet-derived growth factor (PDGF), heregulin and cAMP. The level of glial secretion derived from growth factor (GDNF), and brain-derived neurotrophic factor (BDNF), has been shown to be consistently up-regulated after the differentiation process, with GDNF being up-regulated for up to 20 times and BDNF by up to three times compared to the corresponding undifferentiated cell population, as exemplified in Figures 5A-B.
[0098] The present inventors characterized these unique cells by surface marker expression, as illustrated in Figures 7A-B.
[0099] Thus, in accordance with one aspect of the present application, there is provided a method for generating cells that secrete NTFs comprising incubating a population of undifferentiated MSCs in a differentiation medium comprising basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), heregulin and cAMP.
[00100] The term "mesenchymal stem cell" or "MSC" is used interchangeably for adult cells that are not terminally differentiated, that can divide to produce cells that are also stem cells, or that irreversibly differentiate to give rise to cells of a mesenchymal cell lineage (chondrocyte, osteocyte and adipocyte). The mesenchymal stem cells of the present patent application, at least in some applications, may be from a syngeneic or allogeneic source.
[00101] Populations of MSCs normally express specific markers on their cell surface. According to a given administration, undifferentiated MSCs express CD105, CD73, and CD90 on the cell surface (e.g., >95% positive) and lack expression (e.g., <2% positive) of CD3, CD14, CD19, CD34, CD45 and HLA-DR as determined by flow cytometry.
[00102] Exemplary antibodies that can be used to check for the presence of mesenchymal stem cells include: CD44 FITC conjugate, BD Biosciences, CD73 PE conjugate (BD Pharmingen), CD73 PE conjugate, BD Biosciences, CD90 PE-Cy5 conjugate (eBioscience ) CD90 PE conjugate, BD Biosciences, CD105 PE conjugate (Beckman Coulter) CD3 PerCP conjugate, BD Biosciences, CD14 FITC conjugate (eBioscience) CD14 FITC conjugate, BD Biosciences, CD19 PE-Cy5 conjugate (eBioscience), CD19 FITC conjugate, BD Biosciences , FITC conjugated CD34, BD Biosciences (Beckman Coulter), PE conjugated CD45 (eBioscience), PerCP conjugated CD45, BD Biosciences and PE-Cy5 conjugated HLA-DR (BD Pharmingen). Conjugated HLA-DR PerCP, BD Biosciences.
[00103] Another method to verify the presence of mesenchymal stem cells is by showing that the cells are capable of differentiating into various lineages, such as, for example, adipocytes, osteocytes and chondrocytes. This can be done using, for example, the Human Mesenchymal Stem Cell Functional Identification Kit (R&D Systems).
[00104] In accordance with a preferred administration of this aspect of the present application, mesenchymal stem cells are not genetically engineered (i.e., transformed with an expression construct) to generate the cells and cell populations described herein. document.
[00105] It will be appreciated that the cells of the present application, at least in some applications, may be derived from any stem cell, although preferably not embryonic stem (ES) cells.
[00106] Mesenchymal stem cells can be isolated from various tissues, including but not limited to bone marrow, peripheral blood, blood, placenta, and adipose tissue. A method of isolating mesenchymal stem cells from peripheral blood is described by Kassis et al. [Bone Marrow Transplant. May 2006; 37(10):967-76]. A method of isolating mesenchymal stem cells from placental tissue is described by Brooke G. et al. [Br J. Haematol. February 2009; 144(4):571-9].
[00107] Methods of isolation and cultivation of adipose tissue, placenta and mesenchymal stem cells from umbilical cord blood are described by Kern et al. [Stem Cells, 2006; 24:1294-1301].
[00108] In accordance with a preferred administration of this aspect of the present patent application, the mesenchymal stem cells are human.
[00109] Bone marrow can be isolated from the iliac crest or sternum of an individual by aspiration. Low density BM mononuclear cells (BMMNC | BM mononuclear cells) can be separated by FICOLL-PAQUE density gradient centrifugation. In order to obtain mesenchymal stem cells, a population of cells that includes the mesenchymal stem cells (e.g., BMMNC) can be grown in a proliferation medium capable of maintaining and/or expanding the cells in the presence of platelet lysate. . According to one administration, populations are plated on plastic surfaces (eg, in a volumetric flask) and mesenchymal stem cells are isolated by removing non-adherent cells. Alternatively, mesenchymal stem cells can be isolated by a fluorescence-activated cell sorter (FACS) using mesenchymal stem cell markers.
[00110] After isolation, cells are normally expanded by culturing in a proliferation medium capable of maintaining and/or expanding the isolated cells ex vivo in the presence of platelet lysate. The proliferation medium can be DMEM, alpha-MEM or DMEM/F12. Normally, the glucose concentration in the medium is about 0.5 to 3 grams/liter.
[00111] Cultivation can be done on any suitable surface, including plastic dishes and bioreactors suitable for culturing mesenchymal stem cells.
[00112] The platelet lysate can be prepared using any method known in the art. Platelet-rich plasma (PRP I platelet rich plasma) can be derived from blood bank donations determined to be free of infectious agents (ie HIV, HTLV, HCV, HBsAg). PRP-containing pouches can be stored at -80°C and thawed in a 37°C water bath. After thawing, platelet-rich plasma is typically centrifuged to remove platelet particles and membranes. The supernatant platelet lysate can be collected and frozen at -80°C until use. Platelet Lysate is tested for Endotoxin, Hemoglobin, pH, Total Proteins, Albumin, Osmolality, Sterility and Mycoplasma.
[00113] The proliferation medium may include additional components including, for example, L-glutamine, sodium pyruvate and heparin.
[00114] It will be appreciated that, preferably, when the mesenchymal stem cells are human, the platelet lysate is also obtained from human cells.
[00115] According to one administration, the proliferation/growth medium is devoid of xenobiotic contaminants, ie free of animal-derived components such as serum, animal-derived growth factors, and albumin. Thus, according to this administration, cultivation is carried out in the absence of xenobiotic contaminants.
[00116] An exemplary mesenchymal stem cell propagation and isolation protocol is presented in the Examples section below this document.
[00117] As mentioned, after propagation of mesenchymal stem cells in a medium containing platelet lysate and an adequate number of undifferentiated cells are obtained, the cells can be differentiated in a differentiation medium to generate cells useful in treating disease.
[00118] According to a given administration, cells are re-propagated in a fresh proliferation/growth medium (e.g. at a density of approximately 6000 to 8000 cells per cm2) for 1 day, 2 days, 3 days, 4 days or 5 days before addition of the differentiation medium.
[00119] The phrase "Undifferentiated MSCs" refers to MSCs that have not been cultured in a medium that induces differentiation. Thus, according to at least some applications of the present application, after proliferation, MSCs are contacted directly with the differentiation medium without any intermediate pre-differentiation steps.
[00120] For differentiation, the undifferentiated MSCs of the present patent application, in at least some applications, are incubated in a medium comprising a fibroblast growth factor (FGF), a platelet-derived growth factor (PDGF), heregulin and c-AMP. According to this administration, each fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), heregulin and c-AMP is mixed in a single medium and the culture is carried out in a single step.
[00121] According to one administration, the undifferentiated MSCs of the present patent application are not pre-incubated in the presence of epidermal growth factor (EGF I epidermal growth factor) and/or N2 supplement, before this step and after the expansion stage.
[00122] An exemplary concentration of bFGF, which is envisaged for the application differentiation means of the present patent application, is optionally somewhere between 5 to 50ng/ml, optionally, between 10 to 40ng/ml, optionally between 10 to 10 to 25ng/ml.
[00123] An exemplary concentration of PDGF-AA, which is envisaged for the application differentiation means of the present patent application, is optionally between 1 to 30ng/ml, optionally between 1 to 20ng/ml, optionally between 1 to 10ng/ml, optionally between 2.5 to 10ng/ml.
[00124] An exemplary concentration of βl heregulin, which is envisaged for the application differentiation means of the present patent application, is optionally somewhere between 5 to 100ng/ml, 10 to 90ng/ml, optionally between 25 to "75ng /ml and optionally between 40 to 60ng/ml.
[00125] An exemplary concentration of dbc-AMP, which is envisaged for the application differentiation means of the present patent application, is optionally somewhere between 0.5 to 10 mM, optionally between 0.5 to 5 mM, and optionally , between 0.5 and 2.5 mM.
[00126] According to one administration, the means of differentiating this aspect of the present application is devoid of a phosphodiesterase inhibitor (e.g. IBMX) that is, the cultivation is carried out in the absence of a phosphodiesterase inhibitor.
[00127] According to another administration, the means of differentiating this aspect of the present patent application is devoid of triiodothyronine, that is, the cultivation is carried out in the absence of triiodothyronine.
[00128] Optionally, any of these applications and sub-applications may be combined, so that, for example, the means of differentiation may optionally be devoid of a phosphodiesterase inhibitor and triiodothyronine.
[00129] Preferably, the MSCs are differentiated on the differentiation medium described above for at least one day, at least two days, or at least 3 days. Preferably, the differencing stage does not run for more than five days.
[00130] The differentiation medium used, according to this aspect of the present patent application, is preferably free of xenobiotics (devoid of serum) and devoid of any antibiotics, that is, the cultivation is carried out in the absence of xenobiotics- contaminants.
[00131] In accordance with one administration, the cells are produced in industrial quantities sufficient to be used in the treatment regimens described below.
[00132] Thus, for example, from a donor, it is anticipated that at least 20x106 cells are produced, more preferably, at least 50x106 cells are produced, more preferably, at least HOx106 cells are produced, more preferably, at least at least 200x106 cells are produced, more preferably at least 330x106 cells are produced, more preferably at least 500x10® cells are produced, more preferably at least 20x10® cells are produced, more preferably at least 600x10® cells are produced, more preferably, at least 700x10® cells are produced, more preferably, at least 800x10® cells are produced, more preferably, at least 900x10® cells are produced, more preferably, at least 100x10® cells are produced.
[00133] The present invention patent application also provides for storing differentiated stem cells in banks.
[00134] Each aliquot of differentiated stem cells can correspond to a particular donor. Alternatively, differentiated stem cells from more than one donor can be pooled and stored in a single aliquot. The bank may also contain one or more samples of human feeder cells and/or platelet lysates used to expand and/or differentiate MSC populations.
[00135] MSC populations are stored under appropriate conditions (usually by freezing) to keep stem cells alive and functioning. According to one administration, MSC populations are stored as cryopreserved populations. Other preservation methods are described in U.S. Patent Nos. 5,656,498, 5,004,681, 5,192,553, 5,955,257 and 6,461,645. Methods for storing stem cells in banks are described, for example, in US Patent Application Publication No. 2003/0215942.
[00136] According to an administration, the cell populations stored in the bank are characterized according to at least one predetermined characteristic - for example, amount of neurotrophic factor that is secreted. Additional predetermined traits include morphological features, e.g. differentiation profile, blood type, major histocompatibility complex, donor disease state, or genotypic information (e.g. single nucleotide polymorphisms, 'SNPs' (single nucleated polymorphisms) of a sequence specific nucleic acids associated with a genomic or mitochondrial gene or DNA) associated or not associated with the disease.
[00137] Cataloging may constitute the creation of a centralized record of the characteristics obtained for each cell population, such as, but not limited to, an assembled written record or a computer database with typed information itself. The stem cell bank facilitates the selection of a plurality of samples from a specific mesenchymal stem cell sample suitable for the needs of the researcher or clinician.
[00138] According to an administration, the mesenchymal stem cell bank described in this document is maintained by a computer unit of the stem cell database. Each computer unit comprises at least one processing module, respectively, for processing information. The computer unit can be connected by communicating with a display unit. Information targeting mesenchymal stem cell populations can be stored in a database computer that is transported to users over a network connection. This system provides the customer with the ability to assess mesenchymal stem cell populations to determine which ones are appropriate for their ongoing research and use, and also serves to facilitate the stem cell purchase transaction and appropriate shipment.
[00139] As will be appreciated by one skilled in the art, the applications of the present patent application may be incorporated as a device or system that includes a processing module, and/or a computer program product comprising at least one , program code module. Accordingly, the present application may take the form of a complete hardware administration or an administration combining software and hardware aspects. Furthermore, the present patent application may include a computer program product on a computer-usable storage medium having computer-usable program code means applied to the medium. Any suitable computer-readable medium may be used, including hard disks, CD-ROMs, DVDs, optical storage devices, or magnetic storage devices.
[00140] As mentioned, the cells generated, according to the applications of this method, secrete various neurotrophic factors.
[00141] As used herein, the phrase "neurotrophic factor" refers to a cellular factor that acts on the central nervous system, comprising growth, differentiation, functional maintenance and/or survival effects in neurons. Examples of neurotrophic factors include, but are not limited to, glial-derived neurotrophic factors (GDNF), GenBank accession No. L19063, L15306; brain-derived neurotrophic factor (BDNF), GenBank accession No. CAA62632; neurotrophins-3 (NT-3); neurotrophins-4/5; Neurturin (NTN), GenBank registration number NP_0C4549; neurotrophins-4, GenBank Registration No. M86528; Persephin, GenBank Registration No. AAC39640; brain-derived neurotrophic factor, (BDNF), GenBank accession No. CAA42761; artemin (ART), GenBank Registration No. AAD13110; ciliary neurotrophic factor (CNTF | ciliary neurotrophic factor), GenBank registration no. NP_000605; and Neublastin, GenBank registration number AAD21075.
[00142] According to another administration, the cells generated according to the applications of the present patent application, secrete a Hepatocyte Growth Factor (HGF I Hepatocyte Growth Factor), (GenBank Registration No. D90334.2 ). According to one administration, the cells secrete at least 2 times, at least 3 times, at least 4 times, at least 5 times or up to at least 6 times the amount of HGF as undifferentiated MSCs. Control undifferentiated MSCs are preferably from the same source (eg, same donor, same organ) as those used to generate the cells that secrete neurotrophic factors.
[00143] According to another administration, cells generated according to applications of the present patent application secrete a Vascular Endothelial Growth Factor (VEGF | Vascular endothelial growth factor). According to one administration, the cells secrete at least 2 times, at least 3 times, at least 4 times, at least 5 times or up to at least 6 times the amount of HGF as undifferentiated MSCs. Control undifferentiated MSCs are preferably from the same source (eg, same donor, same organ) as those used to generate the cells that secrete neurotrophic factors.
[00144] According to another administration, the cells generated, according to the applications of the present patent application, do not secrete the tumor necrosis factor 6 inducing protein (TSG-6 | Tumor Necrosis Factor-inducible Gene 6 protein). ), (Genbank Registration No. AJ421518.1 Gene ID: 7130).
[00145] According to another administration, the cells generated, in accordance with applications of the present patent application, do not secrete a nerve growth factor (NGFInerve growth factor), (Genbank Registration No. M57399.1) .
[00146] According to another administration, the cells generated, according to applications of the present patent application, do not secrete an insulin-like growth factor - I (IGF-I I insulin growth factor), (No. registration in GenBank. NP_000609).
[00147] According to an administration, at least 70%, at least 80%, at least 90% or more of a population of differentiated cells of the present application secrete GDNF.
[00148] Preferably, the amount of GDNF secreted by the cells of the present application is increased by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7 times, at least 8 times in the secretion of the same population of undifferentiated mesenchymal stem cells.
[00149] A typical concentration of GDNF is approximately 200 to 2000pg/106 cells.
[00150] According to an administration, at least 501, at least 60%, at least 70%, at least 80%, at least 90% or more of a population of differentiated cells of the present patent application secrete BDNF .
[00151] Preferably, the amount of BDNF secreted by the cells of the present patent application is increased by at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold in the secretion of the same population of undifferentiated mesenchymal stem cells.
[00152] A typical concentration of BDNF is approximately 500 to 5000pg/106 cells. a) Once differentiated and optionally isolated, cells can be tested (in culture) for their ability to secrete NTFs. For analysis of secreted NTFs, the supernatant is collected from cultures of MSCs or from NTF-secreting cells at the end of the differentiation process described above, and the cells are harvested and counted. Cells can also be analyzed during the differentiation process (e.g. after 1 day of differentiation or after two days of differentiation). The amount of NTFs, such as Glial-Derived Neurotrophic Factor (GDNF) or Brain-Derived Neurotrophic Factor (BDNF), in cell culture supernatants can be quantified by means of a GDNF assay or BDNF ELISA (GDNF DuoSet DY212 ; BDNF DuoSet DY248; R&D Systems), according to the manufacturer's protocol, for example and without limitation. The amount of IGF-1 can be quantified using an IGF ELISA assay (IGF-1 DuoSet Cat No.DY291; R&D System), by way of example and without limitation.
[00153] The amount of VEGF can be quantified using a VEGF ELISA assay (VEGF DuoSet R&D systems, Cat: DY293B) for example and without limitation. The amount of HGF can be quantified using an HGF ELISA assay (HGF DuoSet R&D systems, Cat: DY294) for example and without limitation.
[001541 Neurotrophic factor-secreting cells may express enhanced levels (eg, at least twice, or even at least three times) of alpha 1 integrin compared to undifferentiated MSCs.
[00155] In addition, neurotrophic factor-secreting cells may express enhanced levels (e.g., at least twice, or even at least three times) of TGF beta, stem cell factor (SCFlstem cell factor), macrophage colony stimulating factor (M-CSFI), IL-6, IL-8, IL-10, IL-12, IFN-γ, and/or prostaglandin E2 (PGE2) compared to undifferentiated MSCs.
[00156] According to yet another administration, neurotrophic factor-secreting cells do not express enhanced levels of oligodendrocyte markers such as PDGF receptor, sulfatide marker 04, galactocerebrosides (01, GalC), Nkx2.2, SoxlO, protein Oligodendrocyte specific protein (OSPIoligodendrocyte specific protein), myelin-associated glycoprotein (MAG I myelin-associated glycoprotein), 2',3'-Cyclic Nucleotide-nucleotide-3'-Phosphodiesterase (CNP|cyclic nucleotidephosphodiesterase), glutathione S-transferase (GST) glutathione-S-transferase), Adenomatous Polyposis of the Colon (APCI adenomatous polyposis coli); oligodendrocyte myelin glycoprotein (MOG I myelin oligodendrocyte glycoprotein), CNPase, MOSP and/or NS-1 oligodendrocytes, compared to undifferentiated MSCs.
[00157] According to yet another administration, neurotrophic factor-secreting cells do not express enhanced levels of neurotransmitters compared to undifferentiated MSCs. Examples of neurotransmitters that are not expressed in cells include: dopamine, noradrenaline and serotonin.
[00158] It will be appreciated that the cells of the present patent application, at least in some applications, are ex vivo differentiated from mesenchymal stem cells. As such, they still express mesenchymal stem cell markers such as: CD29, CD47, MSCA-1, CD44, CD90, CD73 and CD105, detected by flow cytometry.
[00159] In the same way as MSCs, the cells of the present patent application, at least in some applications, do not express the surface markers: CD3, CD14, CD19, CD34, CD45 and HLA-DR, detected by cytometry flow.
[00160] It will further be appreciated that while the cells described in this document retain certain characteristics of MSCs, they differ from MSCs in a number of ways, including, for example, in the secretion of neurotrophic factors and expression of particular miRNAs and proteins.
[00161] Thus, for example, the present inventors have shown that the cell surface markers in question are differentially expressed in cells secreting differentiated NTF as opposed to undifferentiated MSCs.
[00162] Surface markers that are differentially expressed by differentiated mesenchymal stem cells include, for example, CD44, which is down-regulated on differentiated cells, and CD73, which is up-regulated on differentiated cells, as determined by the mean intensity of fluorescence. For example, according to at least some applications, the mean fluorescence intensity of the CD44 positive population is lower in differentiated cells compared to undifferentiated cells - optionally at least 5% lower, optionally and preferably at least 10% lower. less, optionally and more preferably, at least 15%, optionally and more preferably, at least 20%, 25% to 30% or 40%, or even 50% less. Also as an example, according to at least some applications, the mean fluorescence intensity of the CD73 positive population is higher in differentiated cells compared to undifferentiated cells - optionally at least 5% higher, optionally and preferably at least 10 % greater, optionally and more preferably, at least 15%, optionally and more preferably, at least 20%, 25%, 30% or 40%, or even 50% greater.
[00163] miRNAs that are up-regulated in differentiated MSCs as compared to undifferentiated MSCs include, for example: miR-3663-3p (SEQ ID NO: 1) miR-132-3p (SEQ ID NO: : 2) miR-762 (SEQ ID NO: 3) miR-4327 (SEQ ID NO: 4) miR-3665 (SEQ ID NO: 5) miR-34a-5p (SEQ ID NO: : 6) miR-1915-3p (SEQ ID NO: 7) miR-34a-3p (SEQ ID NO: 8) miR-34b-5p (SEQ ID NO: 9) miR-874-3p ( SEQ ID NO: 10) miR-874-5p (SEQ ID NO: 11) miR-4281 (SEQ ID NO: 12) miR-1207-5p (SEQ ID NO: 13) miR- 30b-5p (SEQ ID NO: 14) miR-29b-3p (SEQ ID NO: 15) miR-199b-5p (SEQ ID NO: 16) miR-30e-5p (SEQ ID NO: 16) No: 17) miR-26a-5p (SEQ ID NO: 18) miR-4324 (SEQ ID No: 19)
[00164] miRNAs that are down-regulated in differentiated MSCs as compared to undifferentiated MSCs include, for example: miR-503-5p (SEQ ID NO: 20) miR-3659 (SEQ ID NO: 21 ) miR-3529-3p (SEQ ID NO: 22) miR-320b (SEQ ID NO: 23) miR-1275 (SEQ ID NO: 24) miR-3132 (SEQ ID NO: 25 ) miR-495-5p (SEQ ID NO: 26) miR-181b-5p (SEQ ID NO: 27) miR-424-5p (SEQ ID NO: 28) miR-4284 (SEQ ID NO: 28) No: 29) miR-574-5p (SEQ ID No: 30) miR-143-3p (SEQ ID No: 3D miR-106a-5p (SEQ ID No: 32) miR-455- 3p (SEQ ID NO: 33) miR-20a-5p (SEQ ID NO: 34) miR~145-5p (SEQ ID NO: 35) miR-324-3p (SEQ ID NO: 36) miR -130b-3p (SEQ ID NO: 37) miR-1305 (SEQ ID NO: 38) miR-140-3p (SEQ ID NO: 39).
[00165] Additional miRNAs that are down-regulated include: miR-320a (SEQ ID NO: 40) miR-222-3p (SEQ ID NO: 41).
[00166] According to another administration, the cell population expresses an increased level of miR-3663-3p, miR-762, miR-4327, miR-3665, miR34a-5p, miR-4327, miRNA 3665 or miR132-3p , compared to undifferentiated MSCs.
[00167] According to another administration, the cell population expresses an increased level of miR34a-5p and/or miR132-3p.
[00168] According to another administration, the cell population expresses a reduced level of 503-5p, miR-3659, miR-3529-3p, miR-320b, miR-424-5p, miR-320a and/or miR- 222-3p compared to undifferentiated MSCs.
[00169] According to another administration, the cell population expresses a reduced level of miR-503-5p, miR-320b, miR-424-5p, miR-320a and/or miR-222-3p.
[00170] According to another administration, the cell population expresses a reduced level of miR-150-3p.
[00171] According to another administration, the cell population expresses a reduced level of miR-503-5p, miR-320b and/or miR-424-5p.
[00172] All descriptions above the increased or decreased level of expression are in comparison to undifferentiated MSCs.
[00173] Proteins that have upregulated expression levels in differentiated cells, compared to undifferentiated cells, are detailed in Table 7 of the Examples section.
[00174] Proteins that have downregulated expression levels in differentiated cells compared to undifferentiated cells are detailed in Table 8 of the Examples section.
[00175] As mentioned, according to at least some applications of the present patent application, the cells and cell populations of the present patent application can be used to treat a certain disease or disorder. Cell populations can be used directly after differentiation, or they can be enriched for a particular phenotype as described below.
[00176] The cells generated, according to applications of this patent application, exhibit a particular expression pattern of cell surface markers. Thus, for example, after differentiation, cells normally show a single increased level of CD73 on their cell surface, compared to the same population of cells before differentiation. Furthermore, in the sequence of differentiation, cells typically show a single reduced level of CD44 on their cell surface, compared to the same cell population before differentiation.
[00177] Analysis of cell surface markers can be performed using any method known in the art, including, for example, flow cytometry, HPLC, immunohistochemistry, in situ PCR.
[00178] The present inventors propose that populations of MSCs can be enriched for MSCs secreting NTF by selecting cells that express these markers.
[00179] Thus, in accordance with another aspect of the present patent application, there is provided a method of selecting mesenchymal stem cells (MSCs) that secrete neurotrophic factors (NTFs) from a mixed population of MSCs, comp r ee n den do: (a) analyzing cells of the mixed population of cells by at least one of the following parameters: (i) cells expressing CD44 below a predetermined threshold; (ii) cells expressing CD73 above a predetermined threshold; and (b) selecting cells that are positive for at least one of the parameters, thereby selecting MSCs that secrete neurotrophic factors.
[00180] The classification is normally carried out 2 or 3 days from the beginning of the directed differentiation protocol.
[00181] It will be appreciated that the mixed cell population, from which NTF-secreting MSCs are selected, comprises MSCs in a different differentiation state, secreting NTFs at different levels, depending on the differentiation method used and the time allotted for the cells differentiate.
[00182] As mentioned, NTF-secreting MSCs are selected according to one of the following criteria: (i) cells that express CD44 below a predetermined threshold; (ii) cells expressing CD73 above a predetermined threshold.
[00183] Selecting cells that express CD73 is a process typically performed using an agent that specifically binds to CD73. Normally, cells express enough CD73 on their membrane that they are capable of being detected using methods such as FACS, MACS, and immunopanning [separation or purification of cells by immunolabeling techniques], as described in this document, later.
[00184] The selection of a predetermined threshold is normally performed for each individual mesenchymal stem cell population, as it is based on the amount of expression of that cell surface marker in an identical population of mesenchymal stem cells before the differentiation, as described in this document, later.
[00185] Normally, the selection is carried out using antibodies that are capable of specifically recognizing this cell surface protein, although the present patent application contemplates additional agents, such as polynucleotides or small molecules.
[00186] Antibodies that recognize CD73 or CD44 can be obtained according to methods known in the art or can be obtained from commercial sources.
[00187] If the CD73 antibody is attached to a magnetic medium (either directly or indirectly through a cognate binding molecule), the heterogeneous cell population can be enriched for cells that highly express CD73 by magnetic activated cell sorting.
[00188] If the CD73 antibody is attached to an affinity group, the heterogeneous cell population can be enriched for CD73+ cells through affinity purification with the cognate binding molecule. Thus, for example, if the CD73 antibody is attached to biotin, a heterogeneous cell population can be depleted of CD73+ cells by purification with streptavidin beads or columns. CD73* cells can later be recovered. If, for example, the CD73 antibody is attached to an antibody or an Fc of an antibody, the heterogeneous cell population can be depleted of CD73+ cells by purification with protein A beads or columns. CD73+ cells can later be recovered . If the CD73 antibody is attached to a fluorescent medium, the heterogeneous cell population can be enriched for CD73T cells through a FACS.
[00189] As used herein, the term "flow cytometry" refers to an assay in which the proportion of a material (e.g., kidney cells comprising a specific marker) in a sample is determined by labeling the material (e.g., binding a labeled antibody to the material), causing a flow of fluid containing the material to pass through a beam of light, separating the light emitted by the sample into constituent wavelengths by a series of filters and mirrors, detecting the light.
[00190] A multitude of cytometers are commercially available, including, for example: Becton Dickinson FACScan and FACScalibur (BD Biosciences, Mountain View, CA). Antibodies that can be used for FACS analysis are taught in Schlossman S, Boumell L, et al, [ Leucocyte Typing V. New York: Oxford University Press; 1995] and are widely available commercially.
[00191] It will be appreciated that when using a FACS classifier it is also possible to select for cells that have a particular level of surface markers.
[00192] The present patent application, in at least some applications, contemplates analyzing a level of CD73 in the undifferentiated MSC population and then selecting a population of cells from the corresponding differentiated MSC population that has an increase in the expression of at least 1.5, or at least 2 times or more.
[00193] Additionally, or alternatively, the present patent application, in at least some applications, contemplates analyzing a CD44 level in the undifferentiated MSC population and then selecting a population of cells from the differentiated MSC population counterpart that has a decrease in expression of at least 1.5, or at least 2-fold or more.
[00194] The present application, in at least some applications, also contemplates analyzing the expression of additional cell surface markers (such as CD105) throughout the differentiation protocol. CD105 expression is initially increased (after the second day) after differentiation, but at the time of maximum NTF secretion (after the third day), CD105 expression is decreased. Thus, the present patent application, in at least some applications, contemplates selecting NTF-secreting MSCs, selecting cells that have a decrease in CD105 expression, for example, at least 0.5-fold, at least 1-fold, or at least 2 times.
[00195] After the generation and optional surface analysis of the cell marker, the NTF-secreting MSCs can be further analyzed (eg, karyotypic analysis, morphology, cell number and viability, Gram stain, sterility).
[00196] The generated cell populations are normally removed from the culture plate using cell dispersing agents. Preferably, single cell populations are obtained. Examples of agents that can be used to disperse cells include, but are not limited to: collagenase, dispase, accutase, trypsin (e.g., trypsin-EDTA, non-animal trypsin substitutes such as TrypLE™), papain. Alternatively, or additionally, grinding can also be performed to increase cell dispersion.
[00197] An exemplary concentration of trypsin that can be used is 0.005 to 0.5% trypsin-EDTA. Cells can be incubated with the dispersing agent for about 5 to 30 minutes at a temperature of 37°C.
[00198] Cell harvesting is normally conducted in an appropriate medium, e.g. Hanks balanced salt solution (HBSS I Hanks balanced salt solution), Dulbecco's Modified Eagle Essential Medium (DMEM I Dulbecco Modified Eagle Medium) RPMI, PBS etc.
[00199] Optionally, cells are and can be preserved at this stage - eg frozen or cryopreserved. This may be relevant for repeated applications to patients.
[00200] Optionally, the cells can be qualified or characterized prior to cryopreservation, or alternatively, prior to administration to the subject. Once qualified, the cells can be labeled accordingly or can be administered directly to the individual.
[00201] Thus, according to another aspect of the present patent application, there is provided a method of classifying cells that secrete neurotrophic factors, comprising analyzing the cells by the expression of at least one miRNA, selected from the group consisting by: miR-503, miR-3659, miR-3529-3p, miR-320b, miR-424—5p, miR-320a, miR-222-3p, miR-3663-3p, miR-762, miR-4327, miR-3665, miR34a-5p, miR-4327, miRNA 3665 and miR132-3p; characterized by increased expression of miR-3663-3p, miR-762, miR-4327, miR-3665, miR34a-5p, miR-4327, miRNA 3665 or miR132-3p, compared to undifferentiated MSCs or a decreased expression of miR- 503, miR-3659, miR-3529-3p, míR-320b, miR-424-5p, miR-320a or miR-222-3p, compared to undifferentiated MSCs, be indicative of cells secreting neurotrophic factors.
[00202] In accordance with a given administration, miRNAs are selected from the group consisting of: miR-503, miR-320b, miR424-5p, miR-132-3p and miR-34a-5p.
[00203] Preferably, the change in expression in the miRNAs is a statistically significant amount.
[00204] Preferably, the control cells to which the differentiated cells are compared are the same cells that are used to generate the cells that secrete the neurotrophic factors (ie, undifferentiated MSCs from the same donor and the same organ).
[00205] Analysis of miRNA expression can be performed using any method known in the art, including miRNA data analysis, PCR analysis, etc.
[00206] Another method of cell qualification is by protein expression analysis.
[00207] Thus, in accordance with another aspect of the present patent application, there is provided a method of cell qualification that has been differentiated ex vivo from MSCs that secrete neurotrophic factors, comprising analyzing cells for the expression of at least , a protein selected from the group consisting of: Isobutyryl-CoA dehydrogenase, Chemokine 6 (CXC motif), Neuromodulin, Growth factor/differentiation 15, Hyalurcnic acid synthase 1, Interleukin-1 beta, Interleukin-8, Inhibin beta chain A, Insulin receptor substrate 1, Integrin alpha-1, laccase domain-containing proteins 1, Laminin subunit alpha-4, Lumican, Collagenase 3, Esophageal specific gene protein 1 normal mucosa, Esophageal leukemia transcription factor interaction protein pre-B cell 1, Pleckstrin homology domain - family A, member; 1, Phosphatidylinositol 3,4,5-triphosphate dependent RAC exchange protein, Prostaglandin-endoperoxide synthase, Prostaglandin G/H synthase 2, Ras family-related Rab-27B protein, Rho-related GTP-binding protein Rho, O-Sialate acetylesterase, Monocarboxylate transporter 7, Inhibitor of tissue factor 2 pathway, Transmembrane protein 65, Vam6/Vps39-like protein, 3-Oxo-5-beta-steroid 4-Dehydrogenase, Mitochondrial beta-propionyl-CoA carboxylase chain, interferon regulatory factor binding-2, Tissue alpha-L-fucosidase, Aldo-keto reductase - family 1, C2 member, Inositol 1,4,5-triphosphate receptor interaction protein, KIAA1199 Protein, Selenium Binding Protein 1, Phospholipase D3, Mitochondrial Phosphotransferase GTP:AMP, Wnt-5a Proteins; Writ Protein, Aldo-keto reductase - family 1, C3 member, Classification-9 Nexins, GAP junction protein alpha-1, Mitochondrial Pyruvate carboxylase, Protein 2B containing SH3 and PX domain, Integrin alpha-2, Cytochrome P450 1B1, Protein -1 type quitinase-3, Nicotinamide phosphoribosyltransferase, Seprase, Superoxide dismutase, Aldoket reductase - family 1, member Cl, Protein containing the FERM, RhoGEF and pleckstrin domains 1, Prolyl 4-hydroxylase alpha-3 subunit, M2 subunit B from Ribonucleoside Diphosphate Reductase, Histone Macro-H2A.2 and Core Histone H2A, Choline Carrier Protein 1 and Niemann-Pick Protein C1, Lysosomal Alpha Glycosidase;
[00208] characterized by an increase in the expression of at least one protein, compared to undifferentiated MSCs, being indicative that the cells secrete neurptofic factors.
[00209] According to another aspect of the present patent application, there is provided a method of cell qualification that has been differentiated ex vivo from MSCs that secrete neurotrophic factors, comprising analyzing the cells for the expression of at least one protein selected from the group consisting of: Tight junction protein ZO-2, Alpha-1, 3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase, Esmothelin, Echotropic P granule homologous protein-5, Activator ATM 1 associated with BRCA1, WD repeat-containing protein 32, SH3 binding domain Protein 4, Type 1 protein, EH binding domain protein 1, Ras GTPase activating protein IQGAP3, Homologous Lysyl Oxidase 2, Alpha-tropomyosin 1 isoform CRA_f, Protein yolk-associated 5, Tripartite motif-containing Protein 16, Connective tissue growth factor, Lymphokine-activated killer T cell-derived protein kinase, Tetratricopept repeat protein 4 ide, Breast Cancer Anti Estrogen Resistance Protein 1, Ribonucleoside Diphosphate Reductase M2 Subunit, Ubiquitin-conjugated E2 Enzyme C, Neutrophil Defensin 1, Cdc42 Effector Proteins 3, Condensin Complex Subunit 2, Ig Kappa Chain C Region , Condensin 3 complex subunit, Syncoilin, Chromosome protein structural maintenance 2, Condensin 1 complex subunit, H4 heavy chain inter-alpha-trypsin inhibitor, Thymidyl synthase, Serotransferrin, Pregnancy zone protein, MCM7 licensing factor of DNA replication, Hemopexin, Msh6 DNA mismatch repair protein, Ankyrin repeat domain-containing 13A proteins, Phosducin-like protein 3, 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase beta-3, Complement C3; DNA Replication Licensing Factor MCM3, CD97 Antigen; CD97 Antigen Alpha Subunit, DNA Replication Licensing Factor MCM6, DNA Replication Licensing Factor MCM4, Deactivated Homolog 2, KIAA0664 Protein, DNA Replication Licensing Factor MCM2, Protein-Lysine 6-Oxidase, Large Subunit of Ribonucleoside Diphosphate Reductase, Melanoma Associated D2 Antigen, C Region Gamma-1 Chain Ig, Heparanase, Importin Alpha-2 Subunit, Asparagine Synthetase [Glutamine Hydrolysis], Alpha-2-Macroglobulin, Collagen Type (I) Alpha-1 Chain , Collagen type (V) alpha-1 chain, DnaJ homologous subfamily B, member 4, Thrombospondin-1, Serum albumin and type (I) collagen alpha-2 chain, characterized by a decrease in the expression of at least one protein , in relation to undifferentiated cells being indicative that the cells secrete neurotrophic factors.
[00210] Preferably, the change in expression of the analyzed protein is a statistically significant amount (ie, a statistically significant increase or a statistically significant decrease).
[00211] Preferably, the control cells to which the differentiated cells are compared are the same cells that are used to generate the cells that secrete the neurotrophic factors (ie, undifferentiated MSCs from the same donor and the same organ).
[00212] Analysis of protein expression can be done using any method known in the art, including Western Blot, immunocytochemistry, mass spectrometry, radioimmunoassay, etc. According to a given administration, the analysis is performed using an antibody that specifically recognizes the protein.
[00213] As mentioned, the cells of the applications of this patent application can be used for the preparation of a medicament (alternately referred to as a pharmaceutical composition), characterized in that such medicament is formulated for the treatment of diseases that can be beneficially treated with cells that secrete neurotrophic factors.
[00214] Examples of such diseases include: neurodegenerative diseases and immune diseases (eg, autoimmune diseases) of the nervous system.
[00215] The term "neurodegenerative disease" is used herein to describe a disease that is caused by an injury to the central nervous system. Exemplary neurodegenerative diseases that can be treated using the cells and methods in accordance with the present application include, for example: Amyotrophic Lateral Sclerosis (ALS), Parkinson's Disease, Multiple System Atrophy (MSA), Huntington's Disease, Alzheimer's Disease, Rett's Syndrome, Lysosomal Storage Disorders ("white matter disease" or glial/demyelination disorders, as described, for example, by Folkerth, J. Neuropath. Exp. Neuro., Sept. 1999, 58:9 ), including Sanfilippo, Gaucher disease, Tay-Sachs disease (hexosaminidase beta deficiency), other genetic diseases, multiple sclerosis (MS), brain injury or trauma caused by ischemia, accidents, environmental, environmental insults, etc. spinal cord, ataxia. In addition, the present patent application may be used to reduce or eliminate the effects of a stroke on a patient's central nervous system that has otherwise been caused by lack of blood flow or ischemia to a site in the patient's brain or which has occurred from physical injury to the brain and/or spinal cord, Neurodegenerative disorders also include neurodevelopmental disorders including, for example, autism and related neurological disorders such as schizophrenia, among numerous others.
[00216] Autoimmune diseases of the nervous system that can be treated using the cells described herein include, for example, multiple sclerosis and myasthenia gravis, Guillain-Barré syndrome, multiple system atrophy (MSA; a sporadic, progressive, neurodegenerative disorder that occurs in adults, associated with varying degrees of parkinsonism, autonomic dysfunction, and cerebellar ataxia). Other autoimmune diseases are described in Kraker et al. , Curr Neuropharmacol. September 2011; 9(3): 400 to 408, the contents of which are incorporated herein by reference.
[00217] The cells of the present application can be administered to the treated individual using a variety of transplantation approaches, the nature of which depends on the site of implantation.
[00218] The term or phrase "transplant", "cell replacement" or "graft" injection are used interchangeably herein and refer to the introduction of the cells of the present patent application into the target tissue, such as the brain , gray matter etc. Cells can be derived from the recipient (allogeneic) or from a non-allogeneic or xenogeneic donor.
[00219] Cells can be transplanted directly into muscle (intramuscularly, such as the muscles of the upper arm or leg), into respiratory muscles, into muscles for swallowing, into the spinal cord (intrathecal), intravenously , directly into the brain or combinations thereof (eg, intramuscularly and intrathecally). Other modes of administration are also contemplated as systemic administration.
[00220] An exemplary dose of cells that can be administered intramuscularly is 1 to 20x106 cells/site. The number of applications per muscle can vary from 5 to 50, 10 to 30, 20 to 100, or 15 to 25 during the course of treatment. In accordance with one administration, the total number of cells administered is between 20 to 2000x106, more preferably between 20 to 1000x105', more preferably between 20 to 500x106, more preferably between 20 to 200x106, most preferably between 20 to 100x10 . According to a given administration, each administration comprises 1 x 106 cells with somewhere between 20 to 30 applications (e.g. 24), 1.5 x 106 cells with somewhere between 20 to 30 applications (e.g. 24), during the course of treatment, or 2x10e cells with somewhere between 20 to 30 applications (for example 24).
[00221] An exemplary dose of cells that can be administered intrathecally is 0.5 to 20 x 10 6 cells/kg body weight, more preferably 0.5 to 10 x 10 6 cells/kg body weight, more preferably 1 to 10x106 cells/kg of body weight, more preferably from 1 to 5x106 cells/kg of body weight, more preferably from 1 to 2.5x106 cells/kg of body weight and most preferably from 1 to 2x106 cells/kg of body weight.
[00222] For a combination of both intramuscular delivery and intrathecal delivery, the present application, in at least some applications, contemplates an intramuscular delivery of about 1 to 10x106 cells/site, more preferably somewhere between 1 at 5x106 cells/site and more preferably somewhere between 1 to 2.5x106 cells/site. The number of locations can vary from 5 to 50, 10 to 30, 20 to 100 or from 15 to 25 - for example 24; and 1 to 10x106 cells/kg of body weight and, more preferably, somewhere between 1 to 5x106 cells/kg of body weight for intrathecal delivery. According to one administration, a maximum number per administration of about 20 to 1400x10e cells per 70 kg patient is contemplated, more preferably, somewhere between 50 to 1000x10% cells per 70 kg patient is contemplated, more preferably, somewhere between 50 at 500x106 cells per 70 kg patient is contemplated, more preferably somewhere between 50 to 200x106 cells per 70 kg patient is contemplated. According to another administration, a maximum number of about 20 to 500x10 5 cells per 70 kg patient is contemplated. According to another administration, a maximum number of about 100 to 2000x10' cells per 70 kg patient is contemplated, and more specifically 200x10' cells per 70 kg patient is contemplated.
[00223] In any of the methods described herein, the cells may be administered per se or, preferably, as part of a pharmaceutical composition which further comprises a pharmaceutically acceptable carrier.
[00224] As used herein, a "pharmaceutical composition" refers to a preparation of one or more of the chemical conjugates described herein, with other chemical components, such as pharmaceutically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to a subject.
[00225] In the following, the term "pharmaceutically acceptable carrier" refers to a carrier or a diluent that does not cause significant irritation to an individual and does not abrogate the biological activity and properties of the applied compound. Examples, without limitation, of carriers: propylene glycol; saline; emulsions; tampons; culture media such as DMEM or REMI; hypothermic storage medium containing components that scavenge free radicals, provide pH buffering, oncotic/osmotic support, energy substrates and ionic concentrations that balance the intracellular state at low temperatures; and mixtures of organic solvents with water.
[00226] Typically, the pharmaceutical carrier preserves the cell number (eg, it is not reduced by more than 90%) in the composition for at least 24 hours, for at least 48 hours, or even for at least 96 hours.
[00227] In this document, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound and maintain cell viability at a predetermined temperature for an appropriate period of time. before transplantation/injection. Examples, without limitation, of excipients include: albumin, plasma, serum and cerebrospinal fluid (cerebrospinal fluid CSFI), antioxidants such as N-acetylcysteine (NAC | N-Acetylcysteine) or resveratrol.
[00228] According to a preferred administration of the present patent application, the pharmaceutical carrier is an aqueous buffer solution or a culture medium such as DMEM.
[00229] Techniques for formulating and administering drugs can be found in "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.
[00230] For any preparation used in the methods of the present application, the therapeutically effective amount or dose can be estimated initially from in vitro cell culture assays. Preferably, a dose is formulated in an animal model to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
[00231] The toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or in experimental animals. For example, rats or mice injured with 6-OHDA can be used as animal models of Parkinson's disease. Additionally, a sunflower test can be used to test for improvement in fine motor function by challenging the animals to open the sunflower seeds for a set period of time. Animal models for testing motor function improvement in MSA patients are disclosed, for example, in Stefanova et al., Trends Neurosci. September 2005; 28(9): 501-6.
[00232] The transgenic mice can be used as a model for Huntingdon's disease, comprising increasing the number of CAG repeats having intranuclear inclusions of huntingtin and ubiquitin in neurons of the striatum and cerebral cortex, but not in the brain stem, in the thalamus or spinal cord, near the sites of neuronal cell loss in the corresponding disease.
[00233] Transgenic mice can be used as a model for ALS disease that make up SOD-1 mutations - see, for example, Uccelli A et al. t Mol Med April 2, 2012.
[00234] The septo-hippocampal pathways, sectioned unilaterally by cutting the fimbria, mimic the cholinergic deficit of the loss of the septo-hippocampal pathway in Alzheimer's disease. Consequently, animal models comprising this lesion can be used to test the cells of the present patent application for the treatment of Alzheimer's.
[00235] The rotational and survival behavior (eg in a Rota Rod) of the animals can be analyzed after administration of the cells of the present patent application.
[00236] The data obtained from these in vitro cell culture assays, and animal studies, can be used in formulating a range of dosages for use in humans. More information can be obtained from clinical studies - see, for example, Salem HK et al., Stem Cells 2010; 28:585-96; and Uccelli et al. Lancet Neurol. 2011; 10:649-56): .
[00237] The dosage may vary depending on the dosage form employed and the route of administration used. The exact composition, route of administration and dosage can be chosen by the individual's physician, taking into account the patient's conditions, (see, for example, Fingi, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 pl). For example, patients with ALS can be monitored symptomatically for improved motor functions, indicating a positive response to treatment.
[00238] For an injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as: Hank's solution, Ringer's solution or physiological saline buffer and additional agents as described above.
[00239] The dosage amount and intervals can be individually adjusted to levels of the active ingredient, being sufficient to effectively regulate the synthesis of neurotransmitters by the implanted cells. The dosages necessary to achieve the desired effect will depend on individual characteristics and the route of administration.
[00240] Depending on the severity and responsiveness of the condition being treated, the dosage of cells can be from a single or a plurality of applications, with the course of treatment lasting from several days to several weeks, or even months, depending on when disease state abatement is achieved.
[00241] The amount of a composition to be administered will, of course, depend on the individual being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc. Dosage and timing of administration will be sensitive to careful and continuous monitoring of the individual's changing condition. For example, a patient with treated ALS will be given an amount of cells that is sufficient to alleviate the symptoms of the disease, based on monitoring indications.
[00242] The cells of the present patent application, in at least some applications, may be packaged in unit dosage forms in a ready-to-use syringe. The syringe can be labeled with the name of the cells and their source. Labeling may also include information related to cell function (eg, the amount of neurotrophic factors secreted from the cell). The syringe may be packaged in a package that is also labeled with information about the cells.
[00243] The cells of the present application, at least in some applications, can be co-administered together with therapeutic agents useful in the treatment of neurodegenerative diseases, such as: gangliosides; antibiotics, neurotransmitters, neurohormones, toxins, neuritis promoting molecules; molecular agents of small antimetabolites and precursors of neurotransmitter molecules such as L-DOPA. For ALS, for example, the cells of the present patent application can be co-administered with Rilutek® (Riluzol, Sanofi Aventis). Additionally, or alternatively, the cells of the present application, in at least some applications, may be co-administered with other cells capable of synthesizing a neurotransmitter. Such cells are described in US Patent Application No. 20050265983, incorporated herein by reference.
[00244] As used herein, the term "about" refers to ±10%.
[00245] Additional objects, advantages and innovative features of the present patent application will become apparent to those of ordinary skill in the art upon examining the following examples, which are not intended to be a limiting factor.
[00246] The terms: "comprises", "comprising" "includes", "including", "having", and their conjugates, mean: "including, but not limited to".
[00247] The term "comprising" means "including and limited to".
[00248] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
[00249] Throughout this application, various applications of the present application may be presented in the form of ranges. It is to be understood that the description in range format is for convenience and brevity only and should not be interpreted as an inflexible limitation on the scope of the present patent application. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges as well as the individual numerical values within that range. For example, describing a range such as 1 to 6 should be considered to have specifically disclosed subranges such as: 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, from 3 to 6, etc., as well as the individual numbers within the range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the range's amplitude.
[00250] As used in this document, the term "method" refers to the ways, means, techniques and procedures for carrying out a particular task, including, but not limited to, those known or readily developed ways, means, techniques and procedures. ways, means, techniques and procedures known to practitioners of chemical, pharmacological, biological, medical and biochemical techniques.
[00251] As used herein, the term "treatment" includes nullifying, substantially inhibiting, slowing or reversing the progression of a condition, substantially improving the clinical or aesthetic symptoms of a disease, or substantially preventing the onset of clinical or aesthetic symptoms of a disease. condition.
[00252] It is appreciated that certain features of the present application, which are, for clarity, described in the context of separate applications, may also be provided combined in a single administration. On the other hand, various features of the present application which are, for the sake of brevity, described in the context of a single administration, may also be provided separately or in any suitable sub-combination or as appropriate in any other described administration of the invention. present invention patent application. Certain features described in the context of various applications are not to be considered essential features of those applications unless the administration is inoperative without these elements.
[00253] Various applications and aspects of the present patent application, as outlined above and as claimed in the claims section below, find experimental support in the following examples. EXAMPLES
[00254] At this time, reference is made to the following examples which, together with the above descriptions, illustrate some applications of the present patent application in a non-limiting manner.
[00255] Generally, the nomenclature used in this document and the laboratory procedures used in the non-limiting description of some applications of the present patent application, include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are exhaustively explained in the literature. See, for example, the methodologies: "Molecular Cloning: A Laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R.M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); as defined in US Patent Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J.E., ed. (1994); "Culture of Animal Cells - A. Manual of Basic Technique" by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J.E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W.H. Freeman and Co., New York (1980); Available immunoassays are widely described in the patent and scientific literature, see, for example, US Patent No. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M.J., ed. (1984); "Nucleic Acid Hybridization" Hames, B.D., and Higgins S.J., eds. (1985); "Transcription and Translation" Hames, B.D., and Higgins S.J., eds. (1984); "Animal Cell Culture" Freshney, R.I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference, as if fully set forth, herein. Other generic references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All information contained therein is incorporated herein by reference. EXAMPLE 1: Generation of mesenchymal stem cells that secrete neurotrophic factors (MSC-NTF).
[00256] In this non-limiting example, the processes of producing neurotrophic factor-secreting mesenchymal clinical grade stromal cells (MSC-NTF) from the bone marrow involves the following main steps: 1. Bone marrow aspiration (BMA I Bone marrow aspiration) 2. Separation of mononuclear cells (MNC I mononuclear cells) 3. Enrichment and propagation of Multipotent Mesenchymal Stromal Cells (MSC | Multipotent Mesenchymal Stromal Cells) 4. Induction of differentiation in MSC-NTF cells 5. Product harvest Final. 6. Product packaging and labeling. 7. Launch of the transplant product.
[00257] Differentiation into NTF-MSC secreting cells can be induced between passages 2 to 6.
[00258] Bone Marrow Aspiration (BMA): Fresh bone marrow was aspirated, according to routine Medical Center procedure, from the patient's iliac crest under local anesthesia and sedation, by an anaesthetist. Bone marrow (30 to 60 ml) was aspirated using aspiration needles in tubes containing heparin. The bone marrow aspiration procedure is preceded by documentation showing negative results for HBV, HCV and HIV tests.
[00259] MNC Separation and MSC Enrichment: The first step of the production process involves the separation of mononuclear cells (MNC) from the whole bone marrow.
[00260] Human multipotent mesenchymal stromal cells (MSC), estimated to comprise 0.01% MNC of total bone marrow, are enriched in vitro from MNC by virtue of their ability to adhere to plastic.
[00261] Aspirated bone marrow was diluted 1:1 (v.v) in Hank's Balanced Salt Solution (HBSS), and MNC were separated from total bone marrow cells by Ficoll density gradient centrifugation.
[00262] MNC were counted and viability and cell number were determined by Trypan blue dye exclusion test. The yield of MNC recovered after density gradient centrifugation varied between donors and depends on the volume of bone marrow collected. The yield of MNC recovered from 30 to 50ml of bone marrow aspirated from ALS patients ranged from 70 to 400x10E MNC and was sufficient to isolate the number of MSCs needed for the entire production process.
[00263] The medium used for seeding the primary marrow mononuclear cells and propagating the MSCs throughout the production process was designated as Platelet Growth medium (PM | Platelets Growth). PM medium was used throughout the MSC production process (Pass 0 - Pass 6) [P0-P6] and contained low glucose DMEM, sodium L-glutamine pyruvate, heparin and platelet lysate.
[00264] MNCs were seeded at a density of 100,000 to -300,000 cells/cmz in PM/flask volumetric flasks and incubated overnight in a humidified CO2 incubator at 37°C/5%. The next day, the cell culture was examined under the microscope. At this stage, non-adherent mononuclear cells were floating in the cell culture supernatant and the MSCs from the adherent plastic were attached to the surface of the volumetric flask. The culture supernatant containing the adherent mononuclear cells was not removed, and the adherent cells were washed gently with DMEM. The DMEM was discarded and PM was added to each volumetric flask that contained the adherent plastic MSC cells. The process phase from the sowing of the MNC to the harvest of the MSC was designated as passage 0 (P0).
[00265] The P0 cells were incubated in a humidified CO2 incubator at 37°C/5% and the PM was replaced twice a week with fresh PM until the culture was sub-confluent.
[00266] After collection of Passage 1, the MSC cell population was characterized by flow cytometry by the expression (>95% positive) of CD73, CD90 and CD105 on the cell surface. To confirm the purity of the cell population and to exclude the presence of hematopoietic cell contamination, these cells had no expression (<2% positive) of CD3, CD14, CD19, CD34, CD45, and HLA-DR, as determined by flow cytometry. .
[00267] MSC Propagation: Primary cultures of MSC were grown in vitro as a single cell layer attached to a plastic substrate. Once the surface of the available substrate was covered with cells (a confluent culture), growth slowed and then ceased. Thus, to keep the cells healthy and actively growing, it was necessary to subculture them at regular intervals when the culture became subconfluent. Each subculture cycle is designated as Passage. Crops can be sub-cultivated up to 6 Passages. MSC cultures were continuously monitored by careful microscopic inspection throughout the production process and monitored for MSC plastic adhesion and characteristic morphological appearance.
[00268] The MSC culture was passaged at a density of 500 to 2000 cells/cm 2 .
[00269] For Passage of MSC, the culture supernatant was removed from the volumetric flask and a trypsin (Invitrogen) was added to each flask. The flask was incubated for several minutes at 37°C and the resulting cell suspension was collected from the flasks into centrifuge tubes and DMEM was added to each flask to dilute the trypsin and collect the remaining cells.
[00270] The cell suspension was centrifuged and again suspended in PM, counted and propagated again at a density of 500 to 2000 cells/cm2 in new culture vessels. The cultures were then incubated in a humidified CO2 incubator at 37°C/5%.
[00271] During the course of each Passage, PMs were replaced every 3-4 days, removing all culture supernatant and replacing it with the same volume of fresh PM.
[00272] Induction of Differentiation: Beginning at Passage 2 (but no later than Passage 6), once the culture was estimated to contain a sufficient number of cells, the MSCs were harvested and reseeded for induction of differentiation into secretory cells of NTF (MSC-NTF).
[00273] MSCs were seeded to induce differentiation into PM at a concentration of more than 6,000 to 8,000 cells/cm2. Three days later, differentiation was induced by replacing the PM with a differentiation medium (S2M) containing low glucose DMEM, supplemented with 1mM cyclic dibutyryl AMP (cAMP | Cyclic adenosine monophosphate), 20ng/ml Fibroblast Growth Factor Human Basic Fibroblast Growth Factor (hbFGF I human Basic Fibroblast Growth Factor), 5 ng/ml of human Platelet-Derived Growth Factor (PDGF-AA) and 50 ng/ml of human Heregulinaa βl. The culture was maintained in a differentiation medium for 3 days until harvest.
[00274] One day before the end of differentiation, the culture supernatant was sampled for analysis of GDNF and BDNF secretion by ELISA or HPLC and cells were harvested for analysis of cell surface markers.
[00275] Harvesting the final product for transplantation: At the end of the differentiation process, the NTF-secreting cells (MSC-NTF) were harvested for transplantation. MSC-NTF cells were washed in DMEM and cell viability and cell numbers were determined. Only cultures resulting in >80% cell viability were released for transplantation. Cells were resuspended at DMEM concentrations of 10x106 cells/ml for IM transplantation and at a concentration of 22.5 to 30x106 (since injection volume is constant, final cell concentration is based on patient weight ).
[00276] At the end of the differentiation process, the culture supernatant was collected and sampled for NTF (GDNF and BDNF) secretion.
[00277] Stability of the final product: The stability of the final product of MSC-NTFs in a medium or saline solution was evaluated at 2 to 8°C for up to 7 hours after collection in two cell concentrations: 10x106cells/ml, the cell concentration used for IM transplantation, and 35x106 cells/ml, the maximum cell concentration anticipated for IT transplantation. The cells were incubated at 2 to 8 °C for 5 hours in a 50 ml tube and then transferred to 1 ml and 5 ml syringes respectively for another two hours at the same temperature. The results indicate that the number of cells at both concentrations remained stable for a total of 7 hours and is in the range of 80 to 100% (Figures 1A-B).
[00278] Product Packaging and Labeling: Each treatment package consists of ready-to-injection syringe(s) containing cultured, freshly harvested bone marrow, neurotrophic factor-secreting autologous mesenchymal stromal cells (MSC-NTF) in accordance with dose defined for the appropriate route of administration in the clinical study protocol.
[00279] The primary label will be affixed to each syringe, which will be packaged in a Single Syringe Pack, the secondary label will be affixed to the syringe pack or Compartment Tray and transferred to the physician for transplantation.
[00280] MSC-NTF cells, harvested one day before transplantation, were analyzed by flow cytometry for cell surface expression of CD44 and CD73, compared to MSC expression from the same patient. In addition, MSC-NTFs were analyzed by ELISA for secretory neurotrophic factors (GDNF and BDNF). This assay was repeated on the culture supernatant harvested on the day of transplantation.
[00281] Sterility tests are performed on reservoir culture supernatant 3 days prior to transplantation. Mycoplasma and nPCR culture tests are performed from a volumetric flask randomly collected 1 or 2 days before the end of differentiation. Gram stain and endotoxin tests were performed on the final product on the day of transplantation.
[00282] The selection of MSC-NTF was performed according to the criteria defined in Table 1, in this document, below. Table 1

RESULTS
[00283] Isolation and Propagation of MSCs from ALS Patients: The yield of mononuclear cells separated from the bone marrow of ALS patients varied between patients and ranged from OT to 400xl06 cells. The number of MSCs enriched with mononuclear cells from patients with ALS was also variable in the range of 5 to 150xl06.
[00284] MSC from ALS patients were cultured and passaged in up to 7 Passages (population doubling 32) for a total of up to 60 days (Figure 2). The mean population doubling time was 0.5 days. Despite the patient-to-patient variability in MSC cell numbers, the cell propagation process was consistent and reproducible (Figure 2.).
[00285] Phenotypic Characterization of MSC: MSC cells from patients with ALS were characterized by flow cytometry analysis of surface antigen expression. It was found that MSCs from ALS patients expressed CD105, CD73 and CD90 on the cell surface (>95% positive) and lack of expression (<2% positive) of CD3, CD14, CD19, CD34, CD45 and HLA-DR, as determined by flow cytometry, which excludes the presence of hematopoietic cell contamination, as illustrated in Table 2, in this document, below. Table 2

[00286] Additional Characterization: Trilineage Differentiation, Morphological Analysis and Mutagenesis Analysis: To confirm the identity of MSC cells, during the development of the manufacturing process, MSCs were subjected to differentiation into adipocyte, osteocyte and chondrocyte lineages (Figure 3). Osteoblast formation was induced by culturing MSCs with dexamethasone (Dex), ascorbate, glycerophosphate and evaluated using osteocalcin antibody. Adipocytes were induced by culturing the MSCs with basic medium supplemented with hydrocortisone, isobutylmethylxanthine and indomethacin in 95% ethanol, and identified by the presence of neutral lipids labeled with oil red (Oil-Red-O) in the cytoplasm. Chondrocyte formation of MSCs was induced in dexamethasone, ascorbate phosphate, proline, pyruvate and TGF-β3 and determined by secretion of alkian blue labeled proteoglycan sulfate and Dapi counterstain (Figs. 3A-I). Cytogenetic analysis was performed on MSCs from ALS patients to confirm chromosome stability and a normal karyotype after five passages. At least 14 metaphase cells were analyzed in each expanded sample. No trisomy, tetraploidy, or chromosomal rearrangement was observed, as shown in Figures 4A-B»
[00287] As illustrated in Figures 5A-F, cryopreservation did not affect the ability of MSCs from ALS patients to differentiate into the adipocyte, osteocyte, and chondrocyte lineages.
[00288] MSC Differentiation into MSC-NT Secreting Cells: NTF Secretion: Differentiation into MSC-NTF Secreting Cells was induced in MSC Passage 3 cells in 12 ALS patients during the Phase I/II clinical study , using the means of differentiation. After differentiation, NTF secretion was measured using ELISA assays for GDNF and BDNF. The secretion of GDNF and BDNF from MSC-NTF cells from twelve different ALS patients in the Phase I/II clinical study is shown in Figures 6A-B. GDNF secretion was found to be induced on average 2 to 20-fold in MSC-NTF compared to MSC, and BDNF secretion was found to be induced 1.5- to 5-fold in NTF-MSC compared to with the MSC (n=10, Figures 6A-B). Specific productivity differences are the result of patient-to-patient variability.
[00289] On the day before the end of differentiation and organ transplantation, GDNF secretion from MSC-NTF cells was considered to be 54±12% of their secretion on the last day of differentiation, and GDNF secretion was considered to be BDNF from MSC-NTF cells was 64+21% of its secretion on the last day of differentiation.
[00290] The trial was repeated on a larger sample of patients. BDNF secretion from MSC-NTF cells from 23 different patients with ALS in phase I/II and phase II clinical studies is shown in Figure 7. BDNF secretion was considered to be induced on average 2.2±0 .7 times in MSC NTF compared to MSC (Figure 7).
[00291] GDNF secretion from MSC-NTF cells from 23 different patients with ALS in phase I/II and phase II clinical studies are shown in Figure 8. GDNF secretion was considered to be induced at >6, 6±2.4 fold in NTF-MSC compared to MSC (Figure 8). (Note: When GDNF expression has fallen below the detection level of the ELISA test, a nominal value equivalent to the lower limit of quantitation (23pg/ml) is given to allow for fold induction calculations. Results are therefore expressed , such as "more than" >),
[00292] On the day before the end of differentiation and transplantation, BDNF secretion from MSC-NTF cells was considered to be 76±23% of their secretion on the last day of differentiation, and GDNF secretion from cells was considered of MSC-NTF was 66+18% of its secretion on the last day of differentiation.
[00293] Secretion (TSG-6) of the induced protein 6 TNF-alpha was tested in supernatant samples from cultures of 13 MSC and MSC-NTF and using the ELISA kit from MyBioSource (USA).
[00294] All MSC and MSC-NTF culture supernatants tested were found to be negative for TSG-6.
[00295] More MSC-NTF culture supernatants were found to be negative for insulin growth factor-1 (IGF-1) and nerve growth factor (NGF) as measured by the ELISA assay using the Kit IGF-1 ELISA: Human IGF-1 DuoSet Cat No.DY291; R&D System.
[00296] VEGF and HGF secretion was measured in MSC-NTFs generated by differentiating MSCs from patients with ALS as described above. The ELISA assays for the respective cytokines were as follows (VEGF DuoSet R&D systems, Cat:DY293B and HGF DuoSet R&D systems, Cat:DY294). MSC-NTF cells were found to secrete high levels of VEGF and HGF with specific productivities in the range of 20 to 100ng/10 6 cells (Figure 9 and Figure 10).
[00297] VEGF secretion from MSC-NTF cells from 22 different patients with ALS in phase I/II and phase II clinical studies, are shown in Figure 9. VEGF secretion was found to be induced on average 4,111, 4-fold in MSC NTF compared to MSC (Figure 9).
[00298] HGF secretion from MSC-NTF cells from 19 different patients with phase I/II ALS, and phase II clinical studies, are shown in Figure 10. HGF secretion was found to be induced 6,713,9 times in NTF-MSC compared to MSC (Figure 10). On the day before the end of differentiation and transplantation, VEGF secretion from MSC-NTF cells was considered to be 69125% of their secretion on the last day of differentiation and HGF secretion from MSC-NTF cells was considered to be 79+31 % of its secretion on the last day of differentiation (n=8).
[00299] Post-transplantation stability: To assess the stability of NTFs secretion by MSC-NTF cells from 'post-transplant' ALS patients in vivo, MSC-NTF cells harvested at the end of differentiation were reseeded in a growth to simulate the "post-transplant" scenario in vivo.
[00300] At the end of the three day period, the cells were harvested and the secretion of NTF was compared to the secretion on the day the cells were initially harvested (after three days in Time "0" differentiation medium).
[00301] It was considered that MSC-NTF cells maintained the level of NTF secretion also after three days of culture in the growth medium. In four independent experiments using cells from ALS patients, the specific productivity of GDNF and BDNF from MSC NTF cells, three days "post-transplant", was found to be similar to that at harvest time (Figures 11A-B).
[00302] Phenotypic Characterization of MSC-NTF Secreting Cells; Phenotypic Characterization of MSC-NTF Secretory Cells indicated that at the end of differentiation (day 3), they expressed all the characteristic surface markers of MSC and did not express any of the negative markers of MSC (Figures 12A-B).
[00303] However, some of the characteristics of MSC surface markers were downregulated during differentiation into MSC-NTF cells. Downregulation of surface markers of MSC has previously demonstrated differentiation along osteogenic, chondrogenic and adipogenic lineages (Jeong JA et al, 2007, Lee HJ at al. 2009, Niehage C et al. 2011, Liu F at al. 2008 ). The expression of CD44 and CD73, characteristic MSC surface markers, was considered to be modulated on the surface of MSC-NTF cells during differentiation. CD4 4 expression, as determined by the Mean Fluorescence Intensity (IFM I Mean Fluorescence Intensity), was found to be down-regulated to 59% in MSC NTF cells at the end of the differentiation process (day 3), compared to their expression in MSCs analyzed by flow cytometry on the same day and under identical flow cytometer instrument settings (Figures 13A-B and Table 5). Using the same experimental approach, CD44 expression was found to be down-regulated on NTF-MSC compared to MSC, a similar measure also on the day before the end of differentiation (the MFI ratio of MSC/MSC-NTF is 0, 67 on day 2, Table 3). As determined by the IMF, CD73 expression was considered to be up-regulated by 80% on MSC-NTF cells at the end of the differentiation process (day 3), compared to its expression on MSCs, (Figures 13A-B and of Table 3). CD73 expression was considered to be up-regulated on NTF-MSCs to a similar extent, also on the day before the end of differentiation (76%, day 2, Table 3). The modulated expression of CD44 and CD73 on the surface of MSC-NTF cells, compared to their expression on MSC cells from the same patient, analyzed on the same day and under the same experimental conditions, is therefore a distinctive feature of the cells of MSC-NTF.
[00304] This expression pattern is characteristic of MSC-NTF cells, both at the end of differentiation (on the day of transplantation, day 3) as well as on the day before the end of differentiation (day 2, Table 3), with approximately the same measure (Table 3), allowing the use of these surface markers for identification of MSC-NTF cells for transplantation.
[00305] Table 3, indicated in this document, just below, summarizes the modulation of surface marker expression on the surface of NTF-MSC cells, compared to MSCs. Table 3

[00306] CD 105 expression followed a different pattern during MSC differentiation into MSC-NTF cells. On day 2, 95.8+4.2% (mean-standard deviation) of 105CD-expressed MSC-NTF cells and IFM of MSC-NTF cells were upregulated to a mean of 1.3610.26 (mean+deviation -default) , compared to MSC. On day 3 of differentiation, only 73.6113.8% (mean standard deviation) of MSC-NTF expressed 105CD during IMP of positive cells, decreased to a mean of 0.5010.22 (mean standard deviation, Figures 14A-B ) compared to its expression in MSC analyzed by flow cytometry on the same day and under identical flow cytometer settings.
[00307] Table 4 in this document, just below, summarizes the NTF-MSC cell surface expression of the additional surface markers compared to MSCs.

[00308] Cell cycle analysis: The cell cycle distribution of MSC-NTF secreting cells from ALS patients was analyzed by flow cytometry and in relation to the cell cycle distribution of MSC from the same patient. MSC-NTF-secreting cells were considered trapped in the Go/Gi phase of the cell cycle on the last day (day 3) of differentiation (Figures 15A-B), as well as the day before the end of differentiation (day 2, Table 5) , compared to MSC which showed a distribution characteristic of cyclic populations,. These results indicate that MSC-NTF cells are not a cyclic cell population. Table 5 % Go—Gj Mean±SD
EXAMPLE 2: MiRNA analysis of mesenchymal stem cells that secrete neurotrophic factors (MSC-NTF)
[00309] The objectives of the study were to perform microRNA-based fingerprinting (miRNA) to thus characterize MSC-NTF cells and bone marrow-derived MSCs from 4 samples from independent and compatible donors, identifying differences and similarities between miRNA expression profiles. and identifying the key miRNAs that define the differences between the two cell types and determine how they are represented (neural/astrocytic differentiation pathways, ie as well as BDNF, GDNF, and VEGF expression and signaling).
[00310] The study identified a total of 160 miRNAs that were reliably detected in all samples. Donor-to-donor variability was evident by creating a miRNA profile that was relatively low and Principal Component Analysis (PCA I Principal Component Analysis) revealed that the entire sample formed clusters of cells ("Cluster"). distinct, based on cell type.
[00311] Statistical comparisons of miRNA profiles for the two different cell types identified 41 differentially-expressed key miRs (DE I).
[00312] 19 were upregulated in MSC-NTFs, relative to MSCs;
[00313] 22 were downregulated in MSC-NTFs relative to MSCs.
[00314] Contextual analysis revealed that differentially expressed miRNAs targeted mRNA-encoding proteins with roles in VEGF regulation, signaling, neurogenesis and/or associated with a Nerve Precursor Cell phenotype. MATERIALS AND METHODS
[00315] Sample Processing and Quality Control: Total RNA was isolated from MSC and MSC-NTF matching pairs from 4 donor-independent samples. The RNA concentration was determined by Absorbance ratios (Abs I Absorbance) at 260/280nm and 260/230nm, which were also determined as indicators of sample yield and purity. For all samples, an additional RNA Quality Control was performed using the Agilent 2200 TapeStation Kit and ScreenTape R6K to determine the RIN I RNA Integrity Number.
[00316] Microarray Profiling: Samples were analyzed on the Agilent miRNA platform (using Agilent vl6 SurePrint G3 human microRNA 8x60K microarray slides; miRBase version 16.0). One hundred nanograms of total RNA, from a 50/pl active solution in nuclease-free water, was used as input for each microarray experiment. Each slide contains 8 individual arrays, each array represents 1349 microRNAs; 1205 human (1199 verified as real miRNAs in miRbase 18) and 144 viral.
[00317] The four main steps of the microarray process were: 1. Single color labeling of the RNA, based on the Cy3 reagent. 2. Hybridization of the labeled RNA samples to the microarray. 3. Washing steps. 4. Digitizing the slides, extracting the capture and data assets (array dots for the matching miRNA IDs), and checking the quality control of the resulting image files and data.
[00318] Data pre-processing and quality control: The microarray data were normalized using pre-processing and data quality control (QC | quality control) methods. Array quality control was performed using an extreme value test based on the following metrics: •average signal per array •average historical background per array • % present (% of miRNAs where expression is detected in each array) • principal components 1 to 3 of PCA (Jackson JE, 1991) of the complete set of the normalized sample.
[00319] In addition, a sample-to-sample correlation analysis was performed on the normalized dataset using Pearson's correlation metric. Extreme values were identified using the Grubbs Extreme Value Test (Grubbs, 1969) with significance named at p<0.0 5. DATA ANALYSIS
[00320] Arrest Calls Overview: Arrest calls (present or absent) for individual miRNAs were compared across samples. Detection calls were calculated using Feature Extraction (Agilent) (AFE) version 10.7.3.1. A detailed description of how these calls are made is available in the Feature Extraction reference guide on the Agilent website.
[00321] Where miRNA expression was below the detection level for the arrays, a nominal intensity value was given to these data points. This value (1.1375 on a log2 scale and 2.2000 on a linear scale) was assigned to each undetected miRNA and was calculated during the normalization process and was used to avoid errors arising from non-computable mathematical operations during subsequent data analysis. . Furthermore, the normalization methodology resulted in groups of miRNAs having very similar intensities of expression, being assigned the same normalized mean intensity.
[00322] Overview of miRNA expression data summary: A summary representation of expression data was produced using PCA. PCA extracts the main effects from high-dimensional data, such as microarray datasets, which for each sample have expression measurements of hundreds of miRNAs. These main effects (main components) can be displayed in a simplified graphical representation that retains the main properties of the data. The key point is that samples that have clusters of similar miRNA profiles in the same space in the PCA frame. In addition, a heat map was produced to visualize the expression levels and relationships in the sample. The clusters associated with the heatmap were derived from agglomerative hierarchical clustering using Euclidean distance with Ward binding.
[00323] Hypothesis tests: The identification of miRNAs expressed equivalently (EE I equivalently-expressed) and expressed differently (DE | differentially-expressed) between different groups of samples and functional analysis of DE miRNA sets. MiRNAs with equivalent expression levels (stably expressed invariant markers) were identified using the One/Two-Sided Tests (TOST I Two OneSided Tests) approach; seen, for example, (Barker LE et al. 2002) as paired tests. This approach is recommended for bioequivalence studies by the EDA (EDA guidance document, 2001). MiRNAs with max (pFDR) <0.05 from the upper and lower limits, respectively, were considered equivalently expressed. The range of expression levels (Δ) allowed for the equivalence corresponds to a one-time shift of 1.5<log2-space.
[00324] Differences in miRNA expression between each cell group were evaluated by performing paired Analysis of Variance (ANOVA | Analysis of Variance) between the different cell groups. The p-values generated from the ANOVA were adjusted for multiple test inflation using the Benjamini-Hochberg method (Benjamini Y and Hochberg Y, 1995) and are referred to as pFDR. MiRNAs with significant hypothesis testing differences at pFDR < 0.05 as well as having one-time absolute variance (FC) £1.5 were considered differentially expressed between a particular sample and the remaining samples. A 0.05 cutoff from the p-value is common practice when analyzing microarray data, and the use of the 1.5 modulation limit is based on the Agilent system's documented set-to-set variability.
[00325] Functional (contextual) analysis was performed by importing the list of differentially expressed (DE) miRNAs into GeneGo MetaCoreTM (v6.14) and mapping to their validated mRNA targets. In addition, a literature review was conducted for the selected DE miRNAs. RESULTS
[00326] The complete list of miRNAs detected across all 8 samples is given in Table 6, in this document, just below. Table 6





[00327] PCA and heat map view of the complete sample set: To get an overview of the donor-to-donor variability within each cell group, and the relationships between the different cell groups, a view of the set A complete data set was produced by PCA using all detected 160miRNAs. The PCA plot represents the information content (variance) of each single microRNA complete dataset over the plot, as a single point in the principal component projection (PC I principal component). The key point is the grouping of similar data sets.
[00328] This was initially done as a projection of the first 3 PCs (Figure 16A). An alternative visualization of the expression patterns for the miRNAs in each sample and the sample relationships was generated using a heat map based on agglomerative hierarchical clustering (Figure 16B).
[00329] The PCA and heatmap clustergram show that the sample set clearly separated, forming two distinct clusters, based on cell type.
[00330] Identification of differentially expressed miRNAs (DE)
[00331] Hypothesis tests of the differences between groups were performed using a paired ANOVA, with a so-called significant empFDR<0.05 and FC^1.5. The identification of DE miRNAs between groups was performed as described in this document, just above.
[00332] Statistical comparisons of miRNA profiles for the two cell types identified 41 key DE miRNAs (differently expressed)
[00333] 19 were upregulated when comparing MSC-NTF vs MSC
[00334] 22 were downregulated when comparing MSC-NTF vs MSC
[00335] A summary of the expression profiles of DE key miRNAs is shown in Figures 17 and 18. The key upregulated 19miRNAs in MSC-NTF vs MSC are shown in Figure 17 and 22 and the key downregulated miRNAs in MSC-NTF vs. MSC are shown in Figure 18.
[00336] Contextual analysis of selected DE miRNAs
[00337] To obtain an overview of pathways affected by DE miRNA profiling for MSC-NTFs, selected DE kmiRs™ were mapped to high-confidence experimentally verified mRNA targets using GeneGO's MetaCore™ and literature review.
[00338] Angiogenesis: A number of DE miRNAs have been identified to be involved in the regulation of VEGF signaling and/or angiogenesis. MiRNA-503 was the most prominent downregulated miRNA in MSC-NTFs (8.4-fold), with expression being reduced to below the detection limit in MSC-NTFs 3, 5 and 7. Only MSC-NTF 2 had a very low expression, although detectable - this is shown in Figure 19, where, as an aid to visualizing the modulations, the expression values have been converted to a linear scale.
[00339] In addition, a group of less deeply downregulated miRNAs (1.5 to 2.0 fold) was also identified as directly targeting VEGF A (miR-145a, 20a-5p, miR-320a & 424-5p) , VEGFR-2 (424-5, FGF2 (4245p), or being reported to be antiangiogenic (miR-222-3p) (Poliseno et al. 2006, Chamorro-Jorganes et al. 2011, Anand 2013, Kim et al. . 2013).
[00340] Furthermore, miR-132-3p was highly up-regulated, being strongly induced in MSC-NTFs (7.9-fold) - this is shown in Figure 20, where again, as an aid to visualizing the modulations, the expression values have been converted to a linear scale.
[00341] MiR-132-3p is pro-angiogenic, via inhibition of pl20RasGAP, a negative regulator of VEGF signaling. Furthermore, blocking miR-132-3p decreases angiogenesis (Anand 2013), In contrast, miR-34a~5P, an anti-angiogenic miRNA (Zhao et al 2010, Nalls et al 2011) is highly expressed in both cell types, but is also up-regulated on MSC-NTFs (4-fold), see Figure 17 and Figure 22B. However, miR-34a-5P also has a role in neuronal cell differentiation and this effect may dominate over the potential negative effects on angiogenesis discussed above.
[00342] Clearly, there are complex interaction pathways involving VEGF signaling in MSC-NTFs. On balance, however, considering these pooled data, especially the deep regulation of miR-503 and miR-132-3p, it is suggested that VEGF signaling would be up-regulated in MSC-NTFs leading to an enhancement of the pro-angiogenic capacity in these cells compared to MSCs - see Figure 21.
[00343] Neural Precursor Cells (NPCs)/Neurogenesis: A number of miRNAs have been identified to be enriched/up-regulated in NPCs and/or neurons, and to be involved in neurogenesis.
[00344] MiR-132-3p was highly upregulated and was strongly induced in MSC-NTFs (7.9 fold) - See Figure 17. MiR-132-3p plays an important role in neuronal development and maturation, and its expression is required for dendrite outgrowth, promoting dendritogenesis (in vitro and in vivo) by inhibiting p250GAP, a negative regulator of RAC and Cdc42 (Magill et al. 2010).
[00345] MiR-762 was also strongly up-regulated on MSC-NTFs (5.9-fold) - see Figure 22A. MiR-762 is an enriched neuronal miRNA and is up-regulated during NPC differentiation from embryonic stem cells and plays a key role in this process (Zhang et al. 2012).
[00346] MiR-34a-5p is highly expressed in both cell types and is upregulated on MSC-NTFs (4-fold), see Figure 22B. This miRNA is up-regulated in NPCs derived from bone marrow MSCs, where its elevation has been shown to promote natural axon outgrowth and is a key regulator of neuronal differentiation (Agustine et al. 2011, Chang et al. 2011).
[00347] Overall, the upregulation of these miRNAs in MSC-NTFs is consistent with these cells having a neuronal precursor phenotype with a pathway of differentiation to neurons.
[00348] Highly discriminating DE MiRNAs with no known biological function can be used as markers of surrogate candidate potency: A pool of highly discriminating miRNAs, with no currently validated target mRNA, was identified as being DE in MSC-NTFs (Figures 23A- AND). These key miRs represent candidate identity/potency markers for the MSC-NTFs. For miR-3659, expression levels were downregulated below the detection limit in 3 of the 4 donors, only donor 2 had low expression, although detectable. EXAMPLE 3: Quantitative PCR Validation Study
[00349] Sample processing and quality control: All total RNA samples were checked for RNA concentration, yield and quality. RNA QC was performed using Agilent 2200 TapeStation and R6K Screen Tapes and Reagents after systemic PCOS (SSOP2'7) to determine RIN.
[00350] The 8 samples used in Phase 1 were previously checked for RNA quantity and quality.
[00351] QPCR profile and data analysis: QPCR was performed using miRCURY LNA™ Universal RT microRNA PCR methodology and reagents (Exiqon A/S), following instruction manual v 5.1; Protocol A - Individual Assays. Briefly, cDNA was synthesized using 5ng/ul of a primer RNA template. An RNA LNA™ Control RNA was added to each sample. Expression levels of candidate miRNAs (kmiRs™) were measured in triplication techniques for all samples of interest, using primer sets* [*N.T. adhesion promoter] miRNA specific.
[00352] A positive control (measures the expression of the RNA-Spike-in LNA™ control) and the negative controls 'no RNA template' and 'no reverse transcriptase (no RT)' were included for each of the samples tested. A 'no cDNA template' negative control was included for each of the miRNAs tested.
[00353] QPCR was performed using LC480 LightCycler (Roche Ltd) and quantification cycle values (Cq) were calculated by performing absolute quantification analysis using the maximum second derivative method.
[00354] Standard curves and efficiency estimation: The efficiency and linearity of the miRNA amplification process were evaluated whenever necessary, using the standard curve approach (custom designed and non-experimentally validated primers only). Serial dilutions of the cDNA pooled from 8 samples of MSC-NTF were performed for hsa-miR-762 and hsa-miR-3663-3 in technical triplicate (three measurements per gene probe). The resulting Cq values were imported into Biogazelle gbase-j- version 2.5; [Hellemans J et al., 2007] to produce standard curves and calculate efficiency values.
[00355] Selection of the optimal miRNA normalization panel: The expression levels of candidate invariant miRNAs were measured in technical triplicate for all 8 samples (Phase I). The resulting Cq values were imported into Biogazelle gbase+, version 2.5. The selection of the most stable subset of invariant markers (ideal number of normalizers and their identity) was performed using the GeNorm algorithm [Vandesompele J et al., 2002], as implemented in qbase+. A subset of invariant genes was considered ideal and stable and therefore suitable for normalization if the geometric mean of their GeNorm expression stability value (M value) is <0.5, pairwise variation between 2 normalization factors sequential, containing an increasing number of genes (V value) <0.15 and coefficient of variation (Cv) <0.25. The normalization factor for each sample was then calculated as a geometric mean of the expression of the normalizers chosen in this sample.
[00356] Data pre-processing: MiRNA expression levels were measured in technical triplicate for all samples. The resulting Cq values were imported into Biogazelle qbase+ v 2.5. CQ values, where applicable, were corrected for differences in amplification efficiency using the Pfaffl method [Pfaffl MW, 2001] and normalized using sample-specific normalization factors [Hellemans J et al., 2007]. mean of the repetitions of techniques and a Normalized Relative Quantity (NRQI) was determined for each miRNA and sample by calculating the ratio between the mean value of Cq against the geometric mean of the selected invariant miRNAs (normalization factor) [Hellemans J et al., 2007].
[00357] Statistical analysis: Differences in miRNA expression levels between groups of samples were formally tested using paired t-tests. Differences were considered significant if the p-value of the t-test was less than 0.05. List of analyzed miRNAs: hsa-miR-22-3p; miR-19b-3p; hsa-miR-503, hsa-miR-320b, hsa-miR-424-5p, hsa-miR-34a-5p and hsa-miR-132-3p, hsa-miR-320a and miR-222-3p. RESULTS
[00358] Expression levels of hsa-miR-22-3p and hsa-miR-19b-3p were found to be identical in MSCs and MSC-NTFs. Figure 24A illustrates that hsa-miR-503-5p is downregulated in MSC-NTFs as compared to MSCs. Figure 24B illustrates that hsa-miR-320b is downregulated in MSC-NTFs as compared to MSCs. Figure 24C illustrates that hsa-miR-424-5p is downregulated in MSC-NTFs as compared to MSCs. Figure 24D illustrates that hsa-miR-34a-5p is up-regulated on MSC-NTFs compared to MSCs. Figure 24E illustrates that hsa-miR-132-3p is up-regulated on MSC-NTFs compared to MSCs. MSCs. Figure 24F illustrates that hsa-miR-320a is non-significantly down-regulated in MSC-NTFs compared to MSCs. Figure 24F illustrates that miR-222-3p is non-significantly down-regulated in MSC-NTFs compared to MSCs. EXAMPLE 4: Analysis of mesenchymal stem cell proteins that secrete neurotrophic factors (MSC-NTF) MATERIALS AND METHODS
[00359] Proteolysis: Proteins were extracted from cell granules in 9M Urea, 400mM ammonium bicarbonate and 10mM DTT and two cycles of sonication. 20μg of protein from each sample was reduced with 2.8mM DTT (60°C for 30 min), modified with 8.8mM iodoacetamide in 400mM ammonium bicarbonate (in the dark, at room temperature, for 30 min) and digested in 2M Urea, 25 mM trypsin-modified ammonium bicarbonate (Promega) in a 1:50 enzyme to substrate ratio, overnight at 37°C. A second additional trypsinization was performed for 4 hours.
[00360] Mass Spectrometry Analysis: The tryptic peptides were desalted using dry C18 (Harvard) tips and resuspended in. 0.1% formic acid.
[00361] The peptides were re-solved by reversed-phase chromatography in 0.075X180-mm (J&W) fused silica capillaries, packed with Reprosil reversed-phase material (Dr Maisch GmbH, Germany). Peptides were eluted with a 180 minute linear gradient at 5 to 28%, 5 minute gradient from 28 to 95%, and 25 minutes at 95% acetonitrile with 0.1% formic acid in water at flow rates of 0 .15μl/min. Mass spectrometry was performed by the Exactive Q (Thermo) mass spectrometer in a positive mode using full MS scan repeatedly, followed by collision induced dissociation (CID) of the 10 most dominant ions selected from the first scan of MS.
[00362] Mass spectrometry data from three biological repeats were analyzed using MaxQuant 1.3.0.5 software (Mathias Mann's group) vs human section of the üniprot database with 1% FDR. Data were quantified by free analysis of labels using the same software. Intensity data were transformed to log 2 in order to obtain a normal distribution. Missing values have been replaced by 10.
[00363] Q T-test with FDR based on permutation, (with random selection of 250, threshold value=0.05) between groups A and B, was done using Preseuse 1.3.0.4. The same software was used to correlate annotations and additional data. RESULTS
[00364] 3622 proteins were identified in the project with at least 2 peptides. Although there are great similarities between the samples, the correlation between the intensity profiles shows a greater correlation between samples from the same group.
[00365] Tables 7 and 8 included below, in this document, list the most differently expressed proteins. (P-value less than 0.05 with difference above 3 or below -3, and at least 2 peptides identified in at least two repeats). Negative values were replaced with 10. A = MSCs; B = differentiated MSCs. Table 7 - upregulated proteins in differentiated samples




EXAMPLE 5: A PHASE I/II OPEN CLINICAL STUDY TO EVALUATE THE SAFETY, TOLERABILITY AND THERAPEUTIC EFFECTS OF TRANSPLANTATION OF AUTOLOGOUS CULTURED MESENCHYMAL STRUM CELLS FROM THE NEUROTROPHIC FACTOR SECTORING (MSC-NTF) IN PATIENTS WITH LATERAL SCLEROSIS (AMIOTROPHIC SCLEROSIS) ALS)
[00366] Study Objectives: To assess the safety, tolerability, and therapeutic effects (preliminary efficacy) of injecting cultured autologous mesenchymal stromal cells from the neurotrophic factor-secreting bone marrow (MSC-NTF) as a treatment for patients with amyotrophic lateral sclerosis (ALS) ) in the early stages of progressive disease. Main Outcomes: 1. Assessment of the safety and tolerability of administering a single treatment of autologous mesenchymal stromal cells cultured from the neurotrophic factor-secreting bone marrow (MSC-NTF) by multiple intramuscular (IM) injections at 24 separate sites in the biceps and biceps muscles. triceps, with a total of ~24 x 106 cells, for patients with early-stage ALS. 2. Assessment of safety and tolerability of single intrathecal (IT) injection into cerebrospinal fluid (CSF | cerebrospinal fluid) of a total of ~60 x 10 6 cultured autologous bone marrow mesenchymal stromal cells that secrete neurotrophic factors (MSC-NTF), for patients with ALS in the progressive phase of the disease. Secondary Outcomes: •Change in the ALS Functional Rating Scale (ALS-FRS-R). •Changes in muscle strength classification (MVIC) by muscle group and, optionally, by adherence, •Changes in forced vital capacity (FVC % | forced vital capacity) (only in the group with the progressive phase of the disease). •Changes in muscle volume estimated by MRI of the upper and lower extremities. •Changes in upper and lower extremity circumference (cm) •Changes in EMG parameters •Need and time for tracheotomy or permanent assisted ventilation. •Overall survival, time to death calculation.
[00367] Number of Subjects: A total of 12 subjects - 6 in the early stage of ALS and 6 in the progressive stage of ALS disease.
[00368] Study Design: This is a two-patient, open-label, phase I/II perspective, one-group clinical study to assess the safety, tolerability, and preliminary efficacy of cultured autologous bone marrow-secreting mesenchymal mesenchymal cells. neurotrophic factors (MSC-NTF) as a potent treatment for patients with amyotrophic lateral sclerosis (ALS) in the early and progressive stages of the disease. This study is a single core test.
[00369] All enrolled patients will have a documented history of ALS disease prior to study enrollment. Patients are diagnosed with ALS in the early stage of the disease, lasting less than 6 months, and patients are also diagnosed with ALS in its progressive stage, lasting 6 to 12 months. Patients with ALS identified as "predisposed" will be approached and asked to sign an Informed Consent Form (ICF | Informed Consent Form). Overall, 12 patients will be recruited and assigned based on the severity of their ALS disease into 2 treatment groups: Group A - 6 patients with early stage ALS disease; Group B - 6 patients with the progressive phase of ALS disease.
[00370] The expected length of the patient screening period prior to enrollment for this study is anything from 2 weeks to 2 days prior to the day of study enrollment during visit 2 (verification of compliance with inclusion/exclusion criteria , including clinical laboratory results). Eligible patients will be enrolled in the study and will be observed every 2 weeks during a 3-month "run in period" to determine the rate of disease progression (allowing a time window of ±5 days for all visits). During the "run in period", approximately 6 weeks after enrollment, patients in both study groups will undergo a BMA I Bone Marrow Aspiration procedure and MSC-NTF cells will be produced at from bone marrow aspirate, based on the currently disclosed method. At the last "run in period" visit, patients from both study groups will undergo treatment and MSC-NTFs will be transplanted by IM or IT injection to patients with early and progressive ALS, respectively.
[00371] Following MSC-NTF transplantation, patients will be seen on a monthly basis for a 6-month post-treatment follow-up period (allowing a time window of ±5 days for all visits). Treatment safety, adverse effects and exploratory parameters to establish an assessment of the rate of ALS disease progression will be recorded throughout the duration of the "run on period" and the post-treatment follow-up period.
[00372] Duration of Study: In general, under the study protocol, each patient will undergo a total of 13 visits during the duration of the study of approximately 9 months.
[00373] Treatment Doses: As detailed in Table 9, in this document, just below Table 9
RESULTS
[00374] Treatment of MSC NTF by both IM and IT administration was safe and well tolerated during the 6-month follow-up visits. No significant treatment-related adverse events were observed in 12 patients treated by any route of administration. Two of six patients experienced bruising and fever after IM administration and three of six patients experienced headache, neck stiffness and fever after IT administration.
[00375] As illustrated in Figure 25, the ALS Functional Rating Score (ALSFRS-R 1 ALS Functional Rating Score) for patients treated with IT showed a more moderate monthly rate of decline after treatment than before treatment: slope improved from 1.5 over three months to 0.08 over six months.
[00376] As illustrated in Figure 26, Forced Vital Capacity (FVC) for patients treated with IT showed a more moderate monthly rate of decline after treatment than before treatment: the slope improved from -0.107 over three months to - 0.06 for six months.
[00377] As illustrated in Figure 27, muscle circumference also showed a similar positive trend. These differences did not reach statistical significance, probably due to the small number of patients. EXAMPLE 6: AN INCREASED DOSE, OPEN LABEL, PHASE II, CLINICAL STUDY TO EVALUATE THE SAFETY, TOLERABILITY AND THERAPEUTIC EFFECTS OF TRANSPLANTATION OF AUTOLOGOUS MESENCHYMAL STRUM CELLS CULTURED FROM NEUROTROPHIC FACTOR SECTOR BONE MARROW (NTF-MSC) IN PATIENTS WITH AMYOTROPHIC LATERAL SCLEROSIS (ALS).
[00378] Study Objectives: The study objectives are to assess the safety, tolerability, and therapeutic effects (preliminary efficacy) of intrathecally and intramuscularly co-administered injection of increasing doses of autologous bone marrow-secreting neurotrophic factor-secreting mesenchymal stromal cells (MSC- NTF), as a treatment for patients with Amyotrophic Lateral Sclerosis (ALS) in the early stage of the disease.
[00379] Main Outcomes: The evaluation of safety and tolerability of a single administration treatment of neurotrophic factor-secreting autologous mesenchymal stromal cells (MSC-NTF) cultured autologous mesenchymal cells, administered in a low, medium and high dose escalation (94xlQ6, 141xlO6, and 188xl05 respectively) by multiple intramuscular (IM) injections at 24 separate sites in the biceps and triceps, plus a single intrathecal (IT) injection into cerebrospinal fluid (CSF) to patients with early-stage amyotrophic lateral sclerosis.
[00380] ]Minor Outcomes: - Change in ALS Functional Rating Scale (ALS-FRS-R). - Change in muscle strength classification (MVIC) by grip. Changes in % Forced Vital Capacity (FVC). - Changes in muscle volume estimated by MRI of the upper extremities. - Changes in the circumference of the upper and lower extremities (cm). - Changes in EMG parameters.
[00381] Number of Subjects: A total of 12 subjects in early stage ALS disease.
[00382] Study Design: This is an increased dose, open-label, phase Ha-phase clinical study, done in a group of three patients, to assess the safety, tolerability, and preliminary efficacy of cultured autologous mesenchymal stromal cells from marrow neurotrophic factor-secreting bone (MSC-NTF) as a potent treatment for patients with amyotrophic lateral sclerosis (ALS) in the early stage of the disease. This study is a single core test.
[00383] All enrolled patients will have a documented history of ALS disease prior to study enrollment. Patients are diagnosed with ALS in the early stage of the disease, lasting less than 2 years. Patients with ALS identified as "predisposed" will be approached and asked to sign an Informed Consent Form (ICF). Overall, 12 patients will be recruited.
[00384] Treatment will start with the lowest dose (94xl06 cells) and the dose will be increased to the next medium and high doses (141xl06 and 188xl06 respectively) in the next patient group only after safety analysis.
[00385] The expected length of the patient screening period prior to enrollment for this study is anything from 2 weeks to 2 days prior to the day of study enrollment during visit 2 (verification of compliance with inclusion/exclusion criteria , including clinical laboratory results). Eligible patients will be enrolled in the study and will be observed each month during a 3-month "run in period" to determine the rate of disease progression (allowing a time window of ±5 days for all visits). During the "run in period", after approximately 6 weeks after enrollment, patients in both study groups will undergo a Bone Marrow Aspiration (EMA) procedure and MSC-NTF cells will be produced from the bone marrow, based on proprietary method Brainstorm Cell Therapeutics Ltd. At the last "run in period" visit, patients will undergo treatment and MSC-NTFs will be transplanted by IM+IT to the first patients with ALS.
[00386] Following MSC-NTF transplantation, patients will be seen on a monthly basis for a 6-month post-treatment follow-up period (allowing a time window of ±5 days for all visits). Treatment safety, adverse effects, and exploratory parameters to establish an assessment of the rate of ALS disease progression will be recorded throughout the duration of the "run on period" and the post-treatment follow-up period.
[00387] Duration of Study: In general, under the study protocol, each patient will undergo a total of 10 visits during the duration of the study of approximately 9 months.
[00388] Treatment Doses: As detailed in Table 10, in this document, below. Table 10

[00389] Efficacy Assessment: The preliminary efficacy assessment in the treatment of MSC-NTF will be based on the observation of the following variables over the post-treatment follow-up period of the study: The ALS Functional Rating Scale (ALS-FRS-R ), the muscle strength classification (MVIC) by adherence, the % forced vital capacity (FVC %), the muscle mass estimated by MRI of the upper extremities, the circumference of the upper and lower extremities (cm), and the EMG parameters .
[00390] Safety Assessment: The safety of the individual will be assessed after treatment by MSC-NTF, using measurements of the following variables: • Physical examination, • Vital Signs (HR, BP, RR, Body temperature), • Clinical laboratory parameters : • CBC - RBC with indices, WBC with differential and platelet count, hemoglobin (Hb) and hematocrit (Ht) • Coagulation functions - Prothrombin time (PT I ProThrombin time), INR, Partial thromboplastin time (PTT/ Partial thromboplastin time), Fibrinogen. • Blood chemistry for electrolytes (sodium, potassium, calcium, magnesium, chloride), glucose, total proteins, triglycerides (TG), total cholesterol, HDL, LDL. • Kidney function (urea, creatinine,) • Liver function (total bilirubin, AST(GOT), ALT(GPT), ALP) • Urine analysis (strip test) - Specific Gravity, pH, protein, glucose, ketones, blood . •Recording of adverse events and •Concomitant drugs
[00391] Statistical analysis: All data obtained in this study and documented in the CRFs will be listed and tabulated with descriptive statistics of the group (mean, standard deviation, maximum, minimum, number of valid cases), as appropriate. The calculation and statistical processing will be done in parallel for the three groups of patients. For discrete variables such as sex, status, number of adverse events (occurrence, severity, relationship with PI), etc., frequencies, percentages and distributions will be calculated. The results between the three groups will be compared and analyzed by statistical analysis.
[00392] A paired t-test will be used to compare changes in baseline efficacy parameters.
[00393] New data analysis will be done as appropriate. Each statistical test will be analyzed with a significance level of 0.05: p<0.05 means a significant result, p>0.05 does not mean a significant result. EXAMPLE 6: Comparing the yield of MSC-NTFs obtained using a one-step or two-step protocol MATERIALS AND METHODS
[00394] One-step protocol: as described in Example 1.
[00395] Two-step protocol: Human MSCs (12,000 cells/cm2) were seeded in PM containing low glucose DMEM, sodium pyruvate, L-glutamine, heparin and lysed platelets. Two days later the medium was replaced with low glucose DMEM supplemented with 2mM L-glutamine (Biological industries), 20ng/ml human epidermal growth factor (hEGF), 20ng/ml human basic fibroblast growth factor (hbFGF) (R&D systems) and N2 supplement (Invitrogen). After 72 hours, the medium was replaced with DMEM supplemented with 1mM dibutyryl-cyclic AMP (dbcAMP), 0.5mM isobutylmethylxanthine (IBMX) (Sigma-Aldrich), 5ng/ml human platelet-derived growth factor (PDGFIhuman platelet). derived growth factor), 50ng/ml human neuregulin 1-βl/HRGl-βl EGF domain and 20ng/ml hbFGF (all from R&D Systems) for an additional 3 days. RESULTS
[00396] As illustrated in Figures 28A-C, the one-step protocol resulted in a significantly higher yield of MSC-NTFs, compared to the yield obtained using the two-step protocol, as shown in three different patient samples, the which allowed to establish a viable production and manufacturing process capable of supporting the clinical trial in patients, with the doses described in Examples 1 and 6.
[00397] While the present application has been described in conjunction with specific applications thereof, it is evident that many alternatives, modifications and variations will be obvious to those skilled in the art. In this sense, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and vast scope of the added claims.
[00398] All publications, patents and patent applications mentioned in this specification are incorporated herein in their entirety by reference to the specification, in the same way as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated in this document by reference. Furthermore, citations or identifications of any reference in this application should not be construed as an admission that such reference is available as a prior art to the present application. To the extent that section titles are used, they should not be construed as limiting, necessarily. REFERENCES: Agostini M, Tucci P, Steinert JR, Shalom-Feuerstein R, Rouleau M, Aberdam D, Forsythe ID, Young KW, Ventura A, Concepcion CP, Han YC, Candi E, Knight RA, Mak TW, Melino G (2011) ) . microRNA-34a regulates neurite outgrowth, spinal morphology, and function. Proc Natl Acad Sci USA. 108(52):21099-104. Anand S (2013). A brief primer on microRNAs and their roles in angiogenesis. Vase Cell. 5(1):2. Barker LE, Luman ET, McCauley MM, Chu SY (2002). Assessing equivalence: an alternative to the use of difference tests for measuring disparities in vaccination coverage. Am J Epidemiol. 156(11):1056-61 Benjamin! Y and Hochberg Y (1995). Controlling the false discovery rate: a practical and powerful approach to multiple testing. J Royal Stat Soc B, 57:269-300 Caporali A, Emanuel! C (2011). MicroRNA-503 and the extended microRNA-16 family in angiogenesis. Trends Cardiovasc Med. 21(6):162-6. Chamorro-Jorganes A, Araldi E, Penalva LO, Sandhu D, Fernández-Hernando C, Suárez Y (2011). MicroRNA-16 and microRNA-424 regulate cell-autonomous angiogenic functions in endothelial cells via vascular targeting endothelial growth factor receptor-2 and fibroblast growth factor receptor-1. Arterioscler Thromb Vase Biol. 31(11):2595-606. Chang SJ, Weng SL, Hsieh JY, Wang TY, Chang MD, Wang HW (2011). MicroRNA-34a modulates genes involved in cellular motility and oxidative phosphorylation in neural precursors derived from human umbilical cord mesenchymal stem cells. BMC Med Genomics. 4:65. FDA guidance document "Statistical Approaches to Establishing Bioequivalence" (2001). www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatorylnfo rmation/Guidances/ucm070244.pdf Grubbs, F (1969). Procedures for Detecting Outlying Observations in Samples, Technometrics, 11:1-21. Jackson JE. (1991). A User's Guide to Principal Components, New York: John Wiley & Sons Kim J, Kang Y, Kojima Y, Lighthouse JK, Hu X, Aldred MA, McLean DL, Park H, Comhair SA, Greif DM, Erzurum SC, Chun HJ ( 2013). An endothelial apelin-FGF link mediated by miR-424 and miR-503 is disrupted in pulmonary arterial hypertension. Nat Med. 19(1):74-82. Leeper NJ, Cooke JP (2011). MicroRNA and mechanisms of impaired angiogenesis in diabetes mellitus. Circulation. 123(3):236-8. Magill ST, Cambronne XA, Luikart BW, Lioy DT, Leighton BH, Westbrook GL, Mandel G, Goodman RH (2010). microRNA-132 regulates dendritic growth and arborization of newborn neurons in the adult hippocampus. Proc Natl Acad Sci USA. 107 (47)-.20382-7. Nalls D, Tang SN, Rodova M, Srivastava RK, Shankar S (2011) . Targeting epigenetic regulation of miR-34a for treatment of pancreatic cancer by inhibition of pancreatic cancer stem cells. PLoS One. 6(8):e24099. Poliseno L, Tuccoli A, Mariani L, Evangelista M, Citti L, Woods K, Mercatanti A, Hammond S, Rainaldi G (2006). MicroRNAs modulate the angiogenic properties of HUVECs. Blood. 108(9):3068-71. Presta M, Dell'Era P, Mitola S, Moroni E, Ronca R, Rusnati M (2005). Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis. Cytokine Growth Factor Rev. 16(2):159-78. Zhang D, Zhao T, Ang HS, Chong P, Saiki R, Igarashi K, Yang H, Vardy LA (2012) . AMD1 is essential for ESC self-renewal and is translationally down-regulated on differentiation to neural precursor cells. Genes Dev. 26(5):461-73. Zhao T, Li J, Chen AF (2010). MicroRNA-34a induces endothelial progenitor cell senescence and prevents its angiogenesis via suppressing silent information regulator 1. Am J Physiol Endocrinol Metab. 299(1):E110-6.Zheng K, Li H, Huang H, Qiu M (2012). MicroRNAs and glial cell development. Neuroscientist. 18(2):114-8. Zhou 3, Ma R, Si W, Li S, Xu Y, Tu Xr Wang Q (2013). MicroRNA-503 targets FGF2 and VEGFA and inhibits tumor angiogenesis and growth. Cancer Lett. 333(2):159-69.

权利要求:
Claims (5)
[0001]
1. Method for generating cells that secrete brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) in which said cells do not secrete nerve growth factor (NGF), characterized by the fact that said cells express CD44, CD73, CD90 and CD105 and do not express CD3, CD14, CD19, CD34, CD45 and HLA-DR, in which said generation comprises: (a) culturing a population of undifferentiated human bone marrow-derived mesenchymal stem cells (MSCs) under conditions that do not promote cell differentiation for 2-6 passages, wherein said population of undifferentiated MSCs has not been preliminarily cultured in a differentiation; and (b) incubating said population of MSCs in a differentiation medium comprising 5-50 ng/ml basic fibroblast growth factor (bFGF), 1-20 ng/ml platelet-derived growth factor (PDGF), 5-100 ng/mL of heregulin and 0.5-10 mM of cAMP, wherein said incubation is carried out in a single step, in which said differentiation medium is devoid of isobutylmethylxanthine (IBMX), thus generating the cells.
[0002]
Method according to claim 1, characterized in that said differentiating means is devoid of triiodothyronine.
[0003]
Method according to claim 1, characterized in that said means of differentiation is devoid of components derived from xenobiotics.
[0004]
4. Method according to claim 1, characterized in that said cultivation is carried out in a culture medium composed of lysed platelets.
[0005]
Method according to claim 1, characterized in that it further comprises analyzing an expression of CD44 and/or CD73 on a surface of said cells after step (b).

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法律状态:
2018-08-07| B15I| Others concerning applications: loss of priority|

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2018-08-14| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2018-09-25| B12F| Other appeals [chapter 12.6 patent gazette]|

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2019-09-17| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2020-10-20| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2021-01-26| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-07-27| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-11-09| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2022-01-18| 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 04/08/2013, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201261679822P| true| 2012-08-06|2012-08-06|

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US61/679,822|2012-08-06|

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PCT/IL2013/050660|WO2014024183A1|2012-08-06|2013-08-04|Methods of generating mesenchymal stem cells which secrete neurotrophic factors|

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