Large-scale generation of oligodendrocytes

Abstract

A method of generating oligodendroglial progenitor cells and pre-myelinating oligodendrocytes is disclosed. Uses of the cell populations are further described.

Description

[0001] LARGE-SCALE GENERATION OF OLIGODENDROCYTES

[0002] RELATED APPLICATION

[0003] This application claims the benefit of priority of U. S. Provisional Patent Application No. 63 / 682,829 filed on August 14, 2024, the contents of which are incorporated herein by reference in their entirety.

[0004] FIELD AND BACKGROUND OF THE INVENTION

[0005] The present invention, in some embodiments thereof, relates to a large-scale method of generating oligodendrocytes and, for use of the cells for the treatment of de and dys-myelinating diseases.

[0006] Multiple sclerosis (MS) is a chronic immune mediated disease of the central nervous system (CNS), which is a leading cause for neurological disability in young adults. The pathological process of MS includes immune cell infiltrations, oligodendrocyte death, demyelination and axonal damage. Spontaneous remyelination is a regular feature at early stages of lesion formation in some MS cases. Nevertheless, the remyelination process eventually fails due to environmental factors and intrinsic properties of progenitor cells. Chronic demyelination leads to axonal degeneration, and several pathological and imaging studies indicate that the chronic disability is attributed mainly to irreversible axonal damage. Especially, an ongoing process of axonal degeneration occurs in chronically demyelinated plaques but not in remyelinated axons. Therefore, remyelination is a major therapeutic target in MS, and given the failure of intrinsic repair mechanisms, transplantation of myelin-forming cells is an attractive option for therapy.

[0007] The potential of embryonic stem (ES) cells to differentiate into oligodendroglial cells was demonstrated both with mouse and human ES cells (hESCs). Moreover, the potential of ES cells-derived neural progeny to remyelinate in genetic models of hypo / dysmyelination and in models of focal demyelination was shown.

[0008] Background art includes Nistor et al, 2005, Glia, 49(3):385-96; Izrael et al., Mol Cell Neurosci. 2007 Mar; 34(3):310-23; International Patent Application WO2015186128; International Patent Application WO 2004111210; Hu et al., Nat Protoc, 2009, 4(11): 1614-22; Gupta et al., 2011, ISBN: 978-953-307-632-4; Wang et al., 2013, Cell Stem Cell, 12(2):252-64; International Patent Application WO2014124087, US Patent Application No. US20040029269; Douvaras et al, Stem Cell Reports, 2014, 3(2):250-9; Douvaras et al, Nat Protoc, 2015, 10(8): 1143-54; Livesey et al, 2016, Stem Cells, 34(4): 1040-5, Marton et al., Nat Neurosci, 2019, 22(3):484-491; Gao et al, 2022, Stem Cells, Review and Reports, 18:3033-3049; US Patent No. 9,862,925 B2, International Patent Application W02020 / 250232. SUMMARY OF THE INVENTION

[0009] According to an aspect of the present invention, there is provided a method of generating oligodendroglial progenitor cells (OPCs) comprising:

[0010] (a) culturing human pluripotent stem cells or human formative pluripotent stem cells in a first medium comprising FGF2, a BMP antagonist in suspension under conditions that generate spheres which co-express at least two markers of neural precursor cells, as measured by FACs;

[0011] (b) culturing the spheres in suspension in a second medium comprising FGF2, retinoic acid, a SHH agonist and optionally a BMP antagonist, under conditions that generate spheres expressing Olig2, as measured by FACS; and

[0012] (c) culturing cells of the spheres expressing Olig2 in a third medium comprising an SHH agonist, FGF2, NT3 and ascorbic acid under conditions that generate a population of cells that comprises OPCs which co-express Olig2 and Nkx2.2, as measured by FACS, thereby generating the OPCs.

[0013] According to an embodiment of the invention, the OPCs are 04- and O1-, as measured by immunostaining.

[0014] According to an embodiment of the invention, the OPCs co-express Olig2 and SOX 10, as measured by immunostaining.

[0015] According to an embodiment of the invention, the second medium is the first medium supplemented with the retinoic acid and the SHH agonist.

[0016] According to an embodiment of the invention, the method further comprises culturing the human pluripotent stem cells in a pre-differentiating medium comprising FGF2, a BMP antagonist and a rock inhibitor for 12-72 hours prior to step (a).

[0017] According to an embodiment of the invention, the first medium is devoid of a rock inhibitor. According to an embodiment of the invention, the first medium is devoid of an activin / TGFB antagonist.

[0018] According to an embodiment of the invention, the first medium comprises an activin / TGFB antagonist.

[0019] According to an embodiment of the invention, step (a) is effected for at least one week. According to an embodiment of the invention, the BMP antagonist is selected from the group consisting of LDN and noggin.

[0020] According to an embodiment of the invention, the markers of neural precursor cells are selected from the group consisting of PSA-NCAM and A2B5.

[0021] According to an embodiment of the invention, the SHH agonist is purmorphamine.

[0022] According to an embodiment of the invention, step (b) is effected for about 3 weeks. According to an embodiment of the invention, step (c) is effected on an adherent surface. According to an embodiment of the invention, step (c) is effected as a suspension culture. According to an embodiment of the invention, the first medium comprises NUTRISTEM™.

[0023] According to an embodiment of the invention, the second medium, the third medium comprises BNN.

[0024] According to an embodiment of the invention, the first and second media comprise NSC. According to an embodiment of the invention, the third medium comprises BNN or ASTO. According to an embodiment of the invention, steps (a) and (b) are carried out under dynamic culture conditions.

[0025] According to an embodiment of the invention, step (c) is carried out under dynamic culture conditions.

[0026] According to an embodiment of the invention, the dynamic culture conditions are effected using a rotating bioreactor.

[0027] According to an embodiment of the invention, the pluripotent stem cells comprise embryonic stem cells.

[0028] According to an embodiment of the invention, the human formative pluripotent stem cells are differentiated ex vivo from pluripotent stem cells.

[0029] According to an embodiment of the invention, the pluripotent stem cells comprise induced pluripotent stem cells (iPSCs).

[0030] According to an embodiment of the invention, the population of cells further comprises neurons and astrocytes.

[0031] According to an embodiment of the invention, the majority of the cells of the population are neural cells.

[0032] According to an embodiment of the invention, about 10 % of the cells of the population are OPCs.

[0033] According to an embodiment of the invention, the method further comprises isolating the OPCs from the population of cells following step (c).

[0034] According to another aspect of the invention, there is provided a method of generating premyelinating human oligodendrocytes comprising:

[0035] (a) generating a population of cells that comprise oligodendroglial progenitor cells according to the method described herein; and

[0036] (b) culturing the oligodendroglial progenitor cells in a fourth medium comprising ascorbic acid, NT3, T3, IGF1 and optionally a BMP antagonist under conditions that generate cells co- expressing at least two markers of pre-myelinating oligodendrocytes, as measured by FACS, thereby generating the pre-myelinating human oligodendrocytes.

[0037] According to an embodiment of the invention, the culturing the oligodendroglial progenitor cells is effected on an adherent surface.

[0038] According to an embodiment of the invention, the at least two markers of pre-myelinating oligodendrocytes comprise 04 and 01.

[0039] According to an embodiment of the invention, the BMP antagonist comprised in the fourth medium is noggin.

[0040] According to an embodiment of the invention, the cells co-expressing at least two markers of pre-myelinating oligodendrocytes do not express markers of myelinating oligodendrocytes.

[0041] According to an embodiment of the invention, the markers of myelinating oligodendrocytes are selected from the group consisting of MBP.

[0042] According to an embodiment of the invention, the fourth medium comprises BNN or ASTO.

[0043] According to still another aspect of the invention, there is provided an isolated population of cells obtainable according to the methods described herein.

[0044] According to yet another aspect of the invention, there is provided a pharmaceutical composition comprising the isolated population of cells disclosed herein and a pharmaceutically acceptable carrier.

[0045] According to an embodiment of the invention, the isolated population of cells is for use in treating a demyelinating disease or disorder.

[0046] According to still another aspect of the invention, there is provided method of treating a demyelinating disease or disorder in a subject in need thereof, comprising transplanting to the subject a therapeutically effective amount of the isolated population of cells disclosed herein, thereby treating the demyelinating disease or disorder.

[0047] According to an embodiment of the invention, the demyelinating disease is selected from the group consisting of multiple sclerosis (MS), spinal cord injury, stroke, white matter stroke, neuromyelitis optica, MOG-antibody associated demyelinating disease (MOGAD), guillain-barre syndrome, diffuse disseminated encephalomyelitis, acute disseminated encephalomyelitis, concentric sclerosis, diffuse sclerosis, leukodystrophy, leukoencephalopathy caused by ischemiahypoxia, central pontine myelination, acute inflammatory demyelinating polyneuropathy, chronic inflammatory demyelinating polyneuropathy, subacute combined degeneration caused by nutritional deficiency diseases, subacute sclerosing panencephalitis or progressive multifocal leukoencephalopathy caused by viral infection, diabetic neuropathy, neuropathy of systemic lupus erythematosus, leukoablative diseases, vascular and age-related white matter loss of small vessel disease (commonly associated with subcortical dementias of advanced age), Alexander disease, vanishing white matter disease, adrenoleukodystrophy and perlescent-merzbacher disease, lysosomal storage disorders, Huntington's disease, frontotemporal dementia, childhood-onset schizophrenia, Radiation and chemotherapy-induced CNS injury.

[0048] According to an embodiment of the invention, the demyelinating disease is MS.

[0049] According to an embodiment of the invention, the transplanting is via the cerebral white matter tracts.

[0050] According to still another aspect of the invention, there is provided a culture medium comprising:

[0051] NUTRISTEM™ (minus GF), human serum albumin, N2 and B27.

[0052] According to an embodiment of the invention, the culture medium further comprises biotin and forskolin.

[0053] 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 invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0054] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0055] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0056] In the drawings:

[0057] FIG. 1: Oligodendrocyte differentiation protocol and the expression of specific markers along the process.

[0058] Undifferentiated hESCs cultured on laminin and FACS analysis show that the majority of the cells express Tra-1-60, a marker of undifferentiated hESCs (A.) In the first step, clusters of hESCs are cultured in suspension in the presence of FGF2, BMP and activin / TGFβ antagonist and form spheres enriched for neural precursor cells expressing PSA-NCAM and A2B5 (B.). At step two, the spheres are cultured in the presence of retinoic acid and SHH agonist in addition to FGF2 and BMP antagonist. After 3 weeks the cells express Olig2 (C.). Upon further propagation, at step 3, in the presence of FGF2, SHH agonist, NT3 and ascorbic acid the cells co-express Olig2 and Nkx2.2 (D.). Upon plating and further differentiation (Step 4) in the presence of ascorbic acid, NT3, T3, IGF1 and BMP antagonist (in some experiments), the cells express the preoligodendrocyte markers 04 (E.) and 01 (not shown).

[0059] FIG. 2. Comparison of the percentage of cells co-expressing PSA-NCAM and A2B5 after step 1 and expressing Olig2 after step 2 when cultured with or without activin / TGFβ inhibitor, SB431542. Histogram presentation of FACS analysis of the percentage of cells co-expressing PSA-NCAM and A2B5 after 2 weeks and expressing Olig2 after five weeks of differentiation of clusters of hESCs that were cultured for the first 3 days in the presence or absence of SB431542 (SB). The percentage of cells co-expressing PSA-NCAM and A2B5 after step 1 and expressing Olig2 after step 2 was similar.

[0060] FIG. 3. Comparison of the percentage of neural progenitors co-expressing PSA-NCAM and A2B5 after step 1 that is initiated from undifferentiated hESCs or human formative pluripotent stem cells. Histogram presentation of FACS analysis of the percentage of cells co-expressing PSA-NCAM and A2B5 after two weeks of differentiation of clusters of hESCs and human formative pluripotent stem cells. The percentage of cells co-expressing PSA-NCAM and A2B5 after step 1 was similar.

[0061] FIG. 4. Comparison of the percentage of cells co-expressing Olig2 and Nkx2.2 after step 2 that is initiated from undifferentiated hESCs or human formative pluripotent stem cells. Histogram presentation of FACS analysis of the percentage of cells co-expressing Olig2 and Nkx2.2 after five weeks of differentiation of clusters of hESCs and human formative pluripotent stem cells. The percentage of cells co-expressing Olig2 and Nkx2.2 after step 2 was similar.

[0062] FIG. 5. Comparison of the percentage of cells co-expressing Olig2 and Nkx2.2 after step 3 that is initiated from undifferentiated hESCs or human formative pluripotent stem cells. Histogram presentation of FACS analysis of the percentage of cells co-expressing Olig2 and Nkx2.2 after nine weeks of differentiation initiated from hESCs and human formative pluripotent stem cells. The percentage of cells co-expressing Olig2 and Nkx2.2 after step 3 was similar.

[0063] FIG. 6. Comparison of the percentage of cells co-expressing Olig2 and Nkx2.2 after step 4 that is initiated from undifferentiated hESCs or human formative pluripotent stem cells. Histogram presentation of FACS analysis of the percentage of cells co-expressing Olig2 and Nkx2.2 after 13 weeks of differentiation initiated from hESCs and human formative pluripotent stem cells. The percentage of cells co-expressing Olig2 and Nkx2.2 after step 4 was similar. FIG. 7. Comparison of the percentage of cells expressing 04 and 01 after step 4 that is initiated from undifferentiated hESCs or human formative pluripotent stem cells. Histogram presentation of FACS analysis of the percentage of cells expressing 04 and 01 after 13 weeks of differentiation initiated from hESCs and human formative pluripotent stem cells. The percentage of cells expressing 04 and 01 after step 4 was similar.

[0064] FIGs. 8A-C. Differentiation of hESCs into Neural Precursors comparing various rotation velocities in a dynamic culture system. 8A. Phase contrast images of the spheres formed after seeding an initial hESC density of 300,000 cells / mL and a starting rotation velocity of 20, 35 or 50 revolutions per minute (rpm). 8B. FACS analysis at the end of step 1 showing that in all conditions the percentage of cells expressing PSA-NCAM / A2B5 was similar, however, the percentage of live cells was significantly higher at 35 and 50 rpm. 8C. Histogram presenting the cumulative folds of increase of cell number from the number of starting undifferentiated population at the end of step 1 at the different rotation velocities.

[0065] FIG. 9. Comparison of phenotype, viability and expansion after step 2, following passage as single cells or spheres at the end of step 1. Cells cultured in PBS wheel bioreactor were split at the end of step 1 by two approaches. In the first approach, the cells were dissociated into single cells (SC) and reseeded at 300,000 cells / ml. In the second approach, spheres (sph) were split at 1:8 ratio providing a similar cell density per ml. The percentage of live cells and Olig2+ cells did not differ significantly. The fold increase in the number of cells was significantly higher after sphere splitting. p<0.05.

[0066] FIGs. 10A-10D. Comparison of the differentiation in static and dynamic cultures. 10A. Scheme representing the culture of cells as free-floating clusters for the first 3 steps and as adherent for step 4. 10B. FACS analysis of cells after cultivation in static or dynamic culture conditions showing that in both conditions the percentage of cells expressing PSA-NCAM / A2B5, Olig2, Olig2 / Nkx2.2 (ON) and 04 was similar after each step of differentiation. IOC. FACS analysis of the percentage of live cells after each step showing that the viability of the cells is significantly higher in dynamic culture conditions. 10D. Histogram presenting the cumulative folds of increase of cell number at the end of step 2. Cell number was monitored after 5 weeks of differentiation and the cumulative fold increase in cell number from the number of starting undifferentiated population was calculated. p<0.01.

[0067] FIG. 11. The percentage of live cells is significantly higher in the Olig2+ population compared to the total population of cells at the end of step 2. FACS analysis of cells after cultivation in suspension in PBS-wheel bioreactor at steps 1 and 2 showing higher viability of the Olig2+ compared to the total cell population. p<0.01. FIG. 12. Comparison of the expression of 04 in adherent cultures started from steps 2, 3 or 4. Histogram presentation of FACS analysis of the percentage of cells expressing 04 after step 4 (weeks 13-15). The cells were cultured in the adherent conditions starting from steps 2,3 and 4 of differentiation. The percentage of 04+ cells was similar when cells were plated from steps 3 and 4 and significantly higher compared to cells plated from step 2. p<0.001.

[0068] FIGs. 13A-13B. Significant increase in the expansion and viability of cells when cultured at step 3 as monolayer compared with floating sphere culture conditions. 13A. FACS analysis at the end of step 3 after cultivation in suspension or monolayer culture conditions showing that the percentage of live cells is significantly higher and more reproducible after monolayer culture condition. The experiments were performed using two different media BNN and SATO. 13B. Proliferation curve showing the fold increase in the number of cells cultivated in suspension (=bioreactor) or monolayer (=flat) culture conditions. Cell number was monitored every two weeks and the cumulative fold increase in cell number from the starting population was calculated. p<0.01 FIGs. 14A-14B. Novel media formulation, named ASTO and BNN can efficiently substitute the commonly used neural stem cell medium (NSC) and SATO (as specified herein). Cells were cultured in NUTRISTEM™ (minus growth factors medium) (Nut-) at step 1, in NSC or BNN media at step 2 and in the media SATO, BNN or ASTO at steps 3-4. Histogram presentation of FACS analysis of the percentage of cells expressing Olig2 after step 2 (14A), and 04 after step 4 (14B).

[0069] FIGs. 15A-15B. Medium NSC used at steps 1 and 2 of oligodendrocytic cell differentiation process can substitute Nut- and BNN media. HESCs or human formative pluripotent stem cells were cultured in NUTRISTEM™ minus growth factors medium (Nut-) at step 1 and in medium BNN at steps 2-4. Alternatively, the cells were cultured in NSC medium at steps 1 and 2 of the differentiation protocol followed by BNN medium at steps 3-4. Histogram presentation of FACS analysis of the percentage of cells expressing Olig2 after step 2, and 04 and 01 after step 4 by differentiated hESCs (15A) and differentiated human formative pluripotent stem cells (15B).

[0070] FIGs. 16A-16H: Transplantation and differentiation of human oligodendrocytic cells in the neonatal murine brain. 16A-16F: a coronal view at Bregma -2 at age 6 weeks (16A), indicating vast migration of transplanted cells, stained for human specific mitochondria (h.mit). h.mit+ cells were found in white and gray matter and acquired typical morphology of local oligodendrocytes, respectively (16B-16F). 16B, Fimbria; 16C, Medullary Lamina; 16D, Thalamus; 16E, Medial Habenula, 16F, Dentate Gyrus. 16G-16H: High resolution confocal microscopy shows close association of h.mit+ cellular extensions with MBP+ myelin covering axons (see arrows). FIGs. 17A-17H: hESC-OPC migration and differentiation in the chronic EAE brain. 17A: Longitudinal view of mouse brain showing the site of human oligodendrocytic cell transplantation.

[0071] 17B: Timing of transplantation after the 2ndrelapse and into the chronic phase of the Biozzi EAE model. 17C: Dissemination of human (h-mit+) cells in the transplanted brain. 17D-17F: Close association of h.mit+ cellular extension and MBP+ myelin. 17G: High resolution confocal microscopy shows close association of h.mit+ cellular extensions with MBP+ myelin covering axons. 17H: Association of h.mit+ cellular extension and NF+ axons.

[0072] FIGs. 18A- 18B: Stably-transfected human oligodendrocytic cells expressing RFP under the MBP promoter (RFP-MBP) transplantation to Biozzi EAE. 18 A: RFP+ cellular extensions in the transplanted EAE brains were identified, indicating the differentiation of transplanted oligodendrocytic cells into MBP-expressing (mature) oligodendrocytes. 18B: High resolution confocal microscopy identified close association of cytoplasmic RFP+ cellular extension and MBP+ myelin. White arrows: myelinated (MBP+) Axons associated with transplanted RFP+ human oligodendrocytic cell extension. Yellow arrows: Non-associated Axons.

[0073] FIG. 19: Transplanted oligodendrocytic cells reduce the burden of clinical disease: Human oligodendrocytic cells were transplanted at the chronic progressive phase of EAE in Biozzi mice into the Cerebellar peduncles and medial Lemniscus. EAE mice exhibited similar disease progression until the day of transplantation (Tx). After oligodendrocytic cell transplantation there was significant decrease in clinical scores, when compared to the sham group, and a significantly lower burden of disease score.

[0074] FIGs. 20A-20G illustrate that OPCs express Olig2 and Nkx2.2 and do not express 04 and 01. (20A-20F) Immunostaining images showing OPCs (step 3) and pre-myelinating oligodendrocytes (step 4) expressing Olig2 and Nkx2.2. (20A-20B), 04 (20C-20D) and 01 (20E-20F) Scale bars: 100mm; Representative Olig2 and Nkx2.2 co-expressing cells are marked by arrows. (20G) Quantitative immunostaining analyses of Olig2, Olig2 and Nkx2.2, and 04 positive cells. Data are mean ± SEM for three independent experiments.

[0075] FIGs. 21A-21D: OPCs and pre-myelinating oligodendrocytes express SOXIO. OPCs (step 3) and pre-myelinating OLs (step 4) co-express Olig2 and SOX10. The percentages of Olig2 and SOX10 co-expressing cells are similar to Olig2 and Nkx2.2 co-expressing cells. (21A-21B) Immunostaining images showing OPCs and pre-myelinating OLs co-expressing Olig2 and SOX10. Scale bars: 100mm (21A) and 200mm (21B); Representative co-expressing cells are marked by arrows. (21C-21D) Quantitative immuno staining and FACS analyses of Olig2 and Nkx2.2, and Olig2 and SOX10 positive cells. Data are mean ± SEM for three independent experiments. FIGs. 22A-22C: OPC and pre-myelinating cell populations are heterogeneous, and include also astrocytes and neurons. (22A-22B) Immuno staining images showing OPC (step 3) (22A) and pre-myelinating oligodendrocyte (step 4) (22B) enriched cell populations, expressing GFAP, an astrocyte marker, and P-III-tubulin, a neuronal marker. Scale bars: 200mm. (22C) Quantitative immunostaining analyses of Olig2, GFAP, and P-III-tubulin positive cells. Data are mean ± SEM for three independent experiments.

[0076] FIG. 23: Comparison of the percentage of OPCs and pre-myelinating OLs expressing 04 at two steps of OL differentiation protocol. Quantitative FACS and immunostaining analyses of the percentage of cells expressing 04, showing different results when analyzed by FACS or immunostaining. Data are mean ± SEM for three independent experiments.

[0077] FIG. 24: Expression of neural markers at different steps of the differentiation protocol. Quantitative FACS analysis of the percentages of cells co-expressing PSA-NCAM and A2B5, at different stages along OL differentiation, showing that the majority of the cells are from neural origin throughout the protocol. Data are mean ± SEM for three independent experiments.

[0078] FIGs. 25A-25E are photographs of immunofluorescence- stained mouse brains, carried out 6 weeks following transplantation of OPCs (Figures 25A-25C) and PMOs (Figures 25D-25E).

[0079] FIGs. 26A-26G are photographs of immunofluorescence-stained mouse brains, carried out 6 weeks following transplantation of OPCs into brain of 2-day old mice. Figure 26A: DAPI staining in coronal view at bregma 0 (scale bar = 200 pm), with boxes of areas in which images of zooming in is shown in Figures 26B-26G. Figures 26B-26G: Immunofluorescent identification of Human Mitochondria-i- (H. Mit) (a human specific mitochondrial marker) of stage 3 Olig2 NKx2.2 GPC found throughout the bregma 0 coronal section with labeled anatomical locations (scale bar = 30 pm). 26B, corpus Callosum; 26C, Cortex; 26D, Septofimbrial Nucleus; 26E, Caudate Putamen; 26F, Fornix; 26G, Preoptic Nuclei.

[0080] FIGs. 27A-27F are photographs of immunofluorescence- stained mouse brains, carried out 6 weeks following transplantation of PMOs into brain of 2-day old mice. Figure 27 A: DAPI staining in coronal view at bregma 0 (scale bar = 200 pm), with boxes of areas in which images of zooming in is shown in Figures 27B-27F. Figures 27B-27F: Immunofluorescent identification of Human Mitochondria-i- (H. Mit) (a human specific mitochondrial marker) of stage 404 PMO found throughout the bregma 0 coronal section with labeled anatomical locations (scale bar = 30 pm).

[0081] 26B, Cortex; 26C, Corpus Callosum; 26D, Septofimbrial Nucleus; 26E, Fornix; 26F, Caudate Putamen. DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0082] The present invention, in some embodiments thereof, relates to a large-scale method of generating oligodendrocytes and, for use of the cells for the treatment of demyelinating diseases.

[0083] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0084] Oligodendrocytes (OCs) exclusively produce myelin in the CNS, and their dysfunction or loss is central to numerous de- and dys -myelinating diseases affecting both children and adults. Cell therapy with oligodendrocyte lineage cells for regeneration of myelin in demyelinated CNS regions has become an attractive goal for cell-based therapeutic strategies in Multiple Sclerosis, and a wide range of dysmyelinating disorders. This prompted the development of methods to generate myelinogenic OCs from human pluripotent stem cells (hPSCs), as a scalable and renewable standardized cell source. Mass generation of oligodendrocytes is critical for therapeutic use, and has proved to be a difficult challenge, requiring development of novel approaches.

[0085] The present inventors set out to uncover novel methodologies to produce bulk cultures of oligodendrocytes, preferably under clinical-compliant conditions. Specifically, the present inventors utilized dynamic bioreactor systems and showed that they enhance the survival of Olig2+ cells (Figure 11). The viability of the cells was significantly increased when a combination of adherent and non-adherent cell culture systems were used. Specifically, the combination of dynamic culture conditions within a dynamic bioreactor system at early stages of the differentiation protocol together with monolayer 2D culture at later stages of the differentiation protocol, enabled bulk production of viable pre-myelinating O4+ / O1+ oligodendrocytes (Figures 13A-13B).

[0086] Whilst reducing the present invention to practice the present inventors, the present inventors performed transplantation studies into both neonate mice and mouse models of Multiple Sclerosis (MS) to determine the potential of the pre-myelinating oligodendrocytes (at various stages) to migrate, mature and exert a functional therapeutic effect. Following cell transplantation to neonatal mice vast OC migration was observed in their brains after 6 weeks (Figure 16A-13F). The transplanted human cells acquired differentiated oligodendrocyte morphology and their extensions were closely associated with MBP+ myelinated axons (Figure 16G-16H). Following cell transplantation at the chronic progressive stage of experimental autoimmune encephalomyelitis (EAE) in adult Biozzi ABH mice, the most authentic model of MS (Figure 17A-17B), vast migration of human oligodendroctyes was observed in the cerebellum, brainstem and spinal cord (Figure 17C). The human cells acquired differentiated oligodendrocyte morphology and their extensions were associated with MBP+ myelinated axons, and neurofilament+ axons (Figure 17D-17H). Furthermore, transplanted cells that reported the expression of MBP by RFP, also migrated in the brain and expressed RFP-MBP, indicating a mature oligodendrocyte state (Figure 18A-18B). Clinical scoring showed significant improvement in oligodendrocyte cell-transplanted chronic EAE mice, as compared to sham-transplanted mice (Figure 19).

[0087] Thus, according to an aspect of the invention there is provided a method of generating premyelinating human oligodendrocytes (PMOs) comprising:

[0088] (a) culturing human pluripotent stem cells or human formative pluripotent stem cells in a first medium comprising FGF2, a BMP antagonist in suspension under conditions that generate spheres which co-express at least two markers of neural precursor cells, as measured by FACs;

[0089] (b) culturing the spheres in suspension in a second medium comprising FGF2, a BMP antagonist, retinoic acid and a SHH agonist under conditions that generate spheres expressing Olig2, as measured by FACs;

[0090] (c) culturing cells of the spheres expressing Olig2 in a third medium comprising an SHH agonist, FGF2, NT3 and ascorbic acid under conditions that generate a population of cells that comprise oligodendroglial progenitor cells (OPCs) which co-express Olig2 and Nkx2.2, as measured by FACs; and

[0091] (d) culturing the oligo progenitor cells co-expressing Olig2 and Nkx2.2 in a fourth medium comprising ascorbic acid, NT3, T3, IGF1 and a BMP antagonist under conditions that generate cells co-expressing at least two markers of pre-myelinating oligodendrocytes, as measured by FACs, thereby generating pre-myelinating human oligodendrocytes.

[0092] According to another aspect of the invention there is provided a method of generating pre-oligodendroglial progenitor cells (OPCs) comprising:

[0093] (a) culturing human pluripotent stem cells or human formative pluripotent stem cells in a first medium comprising FGF2, a BMP antagonist in suspension under conditions that generate spheres which co-express at least two markers of neural precursor cells, as measured by FACS;

[0094] (b) culturing the spheres in suspension in a second medium comprising FGF2, retinoic acid, a SHH agonist and optionally a BMP antagonist, under conditions that generate spheres expressing Olig2, as measured by FACS; and

[0095] (c) culturing cells of the spheres expressing Olig2 in a third medium comprising an SHH agonist, FGF2, NT3 and ascorbic acid under conditions that generate oligodendroglial progenitor cells (OPCs) co-expressing Olig2 and Nkx2.2, as measured by FACS.

[0096] The term “pre-myelinating oligodendrocytes (PMOs)” refers to a population of oligodendrocyte cells being at a particular stage of development whereby they are no longer classified as oligodendrocyte progenitor cells, but have not yet started to form myelin sheaths. More specifically. PMOs may be distinguished from OPCs, in that the former express 04 and 01, whereas the latter do not significantly express 04 and 01 (e.g., as measured by immunofluorescence).

[0097] Examples of pre-myelinating oligodendrocyte markers expression include, but are not limited to 04 sulfatide marker, 01, with or without 01ig2, NKx2.2, NG2, PDGFRa, SOXIO or other oligodendroglial markers. An absence of terminal differentiation markers is also evident (e.g. MBP expression and / or myelin-associated glycoprotein (MAG)).

[0098] Pluripotent stem cells include embryonic stem cells and induced pluripotent stem cells. The phrase “embryonic stem cells” refers to embryonic cells which are capable of differentiating into cells of all three embryonic germ layers (i.e., endoderm, ectoderm and mesoderm), or remaining in an undifferentiated state. The phrase “embryonic stem cells” may comprise cells which are obtained from the embryonic tissue formed after gestation (e.g., blastocyst) before implantation of the embryo (i.e., a pre-implantation blastocyst), extended blastocyst cells (EBCs) which are obtained from a post-implantation / pre-gastrulation stage blastocyst (see W02006 / 040763), embryonic germ (EG) cells which are obtained from the genital tissue of a fetus any time during gestation, preferably before 10 weeks of gestation, and cells originating from an unfertilized ova which are stimulated by parthenogenesis (parthenotes).

[0099] The embryonic stem cells of some embodiments of the invention can be obtained using well-known cell-culture methods. For example, human embryonic stem cells can be isolated from human blastocysts. Human blastocysts are typically obtained from human in vivo preimplantation embryos or from in vitro fertilized (IVF) embryos. Alternatively, a single cell human embryo can be expanded to the blastocyst stage. For the isolation of human ES cells the zona pellucida is removed from the blastocyst and the inner cell mass (ICM) is isolated by immuno-surgery, in which the trophectoderm cells are lysed and removed from the intact ICM by gentle pipetting. The ICM is then plated in a tissue culture flask containing the appropriate medium which enables its outgrowth. Following 9 to 15 days, the ICM derived outgrowth is dissociated into clumps either by a mechanical dissociation or by an enzymatic degradation and the cells are then re-plated on a fresh tissue culture medium. Colonies demonstrating undifferentiated morphology are individually selected by micropipette, mechanically dissociated into clumps, and re-plated. Resulting ES cells are then routinely split every 4-7 days. For further details on methods of preparation human ES cells see Thomson et al., [U. S. Pat. No. 5,843,780; Science 282: 1145, 1998; Curr. Top. Dev. Biol.

[0100] 38: 133, 1998; Proc. Natl. Acad. Sci. USA 92: 7844, 1995]; Bongso et al., [Hum Reprod 4: 706, 1989]; Gardner et al., [Fertil. Steril. 69: 84, 1998]; Reubinoff et., Nature Biotech. 18(4): 399, 2000],

[0101] It will be appreciated that commercially available stem cells can also be used according to some embodiments of the invention. Human ES cells can be purchased from the NIH human embryonic stem cells registry [www(dot)grants (dot) nih (dot) gov / stem_cells / registry / current (dot) htm]. Non-limiting examples of commercially available embryonic stem cell lines are HAD-C100, HAD-C102, HAD-C103, HAD-C104, HAD-C105, HAD-C106, HAD-C107, BG01, BG02, BG03, BG04, CY12, CY30, CY92, CY10, TE03, TE32, CHB-4, CHB-5, CHB-6, CHB-8, CHB-9, CHB-10, CHB-11, CHB-12, HUES 1, HUES 2, HUES 3, HUES 4, HUES 5, HUES 6, HUES 7, HUES 8, HUES 9, HUES 10, HUES 11, HUES 12, HUES 13, HUES 14, HUES 15, HUES 16, HUES 17, HUES 18, HUES 19, HUES 20, HUES 21, HUES 22, HUES 23, HUES 24, HUES 25, HUES 26, HUES 27, HUES 28, CyT49, RUES3, WA01, UCSF4, NYUES1, NYUES2, NYUES3, NYUES4, NYUES5, NYUES6, NYUES7, UCLA 1, UCLA 2, UCLA 3, WA077 (H7), WA09 (H9), WA13 (H13), WA14 (H14), HUES 62, HUES 63, HUES 64, CT1, CT2, CT3, CT4, MA135, Eneavour-2, WIBR1, WIBR2, WIBR3, WIBR4, WIBR5, WIBR6, HUES 45, Shef 3, Shef 6, BJNheml9, BJNhem20, SA001, SA001.

[0102] In addition, ES cells can be obtained from other species as well, including mouse (Mills and Bradley, 2001), golden hamster [Doetschman et al., 1988, Dev Biol. 127: 224-7], rat [lannaccone et al., 1994, Dev Biol. 163: 288-92] rabbit [Giles et al. 1993, Mol Reprod Dev. 36: 130-8; Graves & Moreadith, 1993, Mol Reprod Dev. 1993, 36: 424-33], several domestic animal species [Notarianni et al., 1991, J Reprod Fertil Suppl. 43: 255-60; Wheeler 1994, Reprod Fertil Dev. 6: 563-8; Mitalipova et al., 2001, Cloning. 3: 59-67] and non-human primate species (Rhesus monkey and marmoset) [Thomson et al., 1995, Proc Natl Acad Sci U S A. 92: 7844-8; Thomson et al., 1996, Biol Reprod. 55: 254-9].

[0103] Extended blastocyst cells (EBCs) can be obtained from a blastocyst of at least nine days post fertilization at a stage prior to gastrulation. Prior to culturing the blastocyst, the zona pellucida is digested [for example by Tyrode’s acidic solution (Sigma Aldrich, St Louis, MO, USA)] so as to expose the inner cell mass. The blastocysts are then cultured as whole embryos for at least nine and no more than fourteen days post fertilization (z.e., prior to the gastrulation event) in vitro using standard embryonic stem cell culturing methods.

[0104] Another method for preparing ES cells is described in Chung et al., Cell Stem Cell, Volume 2, Issue 2, 113-117, 7 February 2008. This method comprises removing a single cell from an embryo during an in vitro fertilization process. The embryo is not destroyed in this process. Induced pluripotent stem cells (iPS; embryonic-like stem cells), are cells obtained by dedifferentiation of adult somatic cells which are endowed with pluripotency (z.e., being capable of differentiating into the three embryonic germ cell layers, i.e., endoderm, ectoderm and mesoderm). According to some embodiments of the invention, such cells are obtained from a differentiated tissue (e.g., a somatic tissue such as skin or blood cells) and undergo de-differentiation by genetic manipulation which re-program the cell to acquire embryonic stem cells characteristics. According to some embodiments of the invention, the induced pluripotent stem cells are formed by inducing the expression of Oct-4, Sox10, Kfl4 and c-Myc / LIN28 in a somatic stem cell.

[0105] According to one embodiment the method is effected by expressing in the cells at least one polypeptide belonging to the Oct family or the Sox family.

[0106] According to another embodiment, the method is effected by expressing in the cells at least two polypeptides - one belonging to the Oct family and one to the Sox family.

[0107] Examples of polypeptides belonging to the Oct family include, for example, Oct3 / 4 (NM_013633, mouse and NM_002701, human), Oct1A (NM_198934, mouse and NM_002697, human), Oct6 (NM_011141, mouse and NM_002699, human), and the like. Oct3 / 4 is a transcription factor belonging to the POU family, and is reported as a marker of undifferentiated cells (Okamoto et al., Cell 60:461-72, 1990). Oct3 / 4 is also reported to participate in the maintenance of pluripotency (Nichols et al., Cell 95:379-91, 1998).

[0108] Examples of polypeptides belonging to the Sox (SRY-box containing) family include, for example Sox1 (NM_009233, mouse and NM_005986, human), Sox3 (NM_009237, mouse and NM_005634, human), Sox7 (NM_011446, mouse and NM_031439, human), Sox15 (NM_009235, mouse and NM_006942, human), Sox17 (NM_011441, mouse and NM_022454, human) and Sox18 (NM_009236, mouse and NM_018419, human), and a preferred example includes Sox2 (NM_011443, mouse and NM_003106, human).

[0109] According to yet another embodiment, the method is effected by expressing in the cells four polypeptides - one belonging to the Oct family, one belonging to the Sox family, Nanog and LIN28.

[0110] According to yet another embodiment, the method is effected by expressing in the cells four polypeptides - one belonging to the Oct family, one belonging to the Sox family, KLF4 and LIN28.

[0111] Alternatively, the method is effected by expressing in the cells four polypeptides - one belonging to the Oct family, one belonging to the Sox family, KLF-4 and c-MYC.

[0112] Expressing the dedifferentiating factors described herein above in somatic cells may be performed by genetic manipulation - example using expression constructs. Various methods can be used to introduce the expression vectors of the present invention into the pancreatic beta cells. Such methods are generally described in, for instance: Sambrook, J. and Russell, D. W. (1989, 1992, 2001), Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York; Ausubel, R. M. et al., eds. (1994, 1989). Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989); Chang, P. L., ed. (1995). Somatic Gene Therapy, CRC Press, Boca Raton, Fla.; Vega, M. A. (1995). Gene Targeting, CRC Press, Boca Raton, Fla.; Rodriguez, R. L. and Denhardt, D. H. (1987). Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworth-Heinemann, Boston, Mass; and Gilboa, E. et al. (1986). Transfer and expression of cloned genes using retro-viral vectors. Biotechniques 4(6), 504-512; and include, for example, stable or transient transfection, lipofection, electroporation, and infection with recombinant viral vectors. In addition, see U. S. Pat. Nos. 5,464,764 and 5,487,992 for positivenegative selection methods.

[0113] Introduction of the expression constructs of the present invention into somatic cells by viral infection offers several advantages over other methods such as lipofection and electroporation offering higher efficiency of transformation and propagation. According to a particular embodiment, expressing the dedifferentiating factors described herein above in the somatic cells is performed by retroviral transduction.

[0114] Methods of inducing iPS cells without viral integration are also contemplated - see for example Stadtfeld et al., 2008, [Science 322, 945-949] and Okita et al., 2008, [Science 322, 949-953],

[0115] Alternatively, somatic cells may be transfected with mRNAs encoding the dedifferentiating factors [Givol et al., BBRC 394(2010): 189-193; Warren et al., Cell Stem Cell, Volume 7, Issue 5, 5 November 2010, Pages 549-550] or by introduction of the proteins themselves (see for example Kim, D. et al. Cell Stem Cell doi: 10.1016 / j.stem.2009.05.005 (2009) and Zhou, H. Et al. Cell Stem Cell 4, 381-384, ( 2009).

[0116] Pluripotent stem cells are typically cultured in an expansion medium that allows propagation of the cells.

[0117] As used herein the phrase “expansion medium” refers to a liquid substance used to support the growth and maintain the viability of pluripotent stem cells and ensures that they are maintained in their pluripotent state.

[0118] The culture medium used by the invention according to some embodiments can be a waterbased medium which includes a combination of substances such as salts, nutrients, minerals, vitamins, amino acids, nucleic acids, proteins such as cytokines, growth factors and hormones, all of which are needed for cell proliferation and are capable of maintaining the pluripotent stem cells in an undifferentiated state. For example, a culture medium according to an aspect of some embodiments of the invention can be a synthetic tissue culture medium such as the Ko-DMEM (Gibco-Invitrogen Corporation products, Grand Island, NY, USA), DMEM / F12 (Biological Industries, Biet HaEmek, Israel), Mab ADCB medium (HyClone, Utah, USA), NUTRISTEM™ (Biological Industries, Beit HaEmek, Israel; also known as STEMEDIA™ NUTRISTEM™ XF / FF Culture Medium, STEMGENT, USA), TESR™ (StemCell Technologies) and TESR2™ (StemCell Technologies), Essential 8 (Thermo Fisher Scientific supplemented with the necessary additives as is further described herein under.

[0119] According to some embodiments of the invention, the expansion medium (and also the differentiation media described below) are serum free.

[0120] As used herein the phrase “serum-free” refers to being devoid of a human or an animal serum.

[0121] It should be noted that the function of serum in culturing protocols is to provide the cultured cells with an environment similar to that present in vivo (i.e., within the organism from which the cells are derived, e.g., a blastocyst of an embryo). However, the use of serum, which is derived from either an animal source (e.g., bovine serum) or a human source (human serum), is limited by the significant variations in serum components between the donor individuals (from which the serum is obtained) and the risk of having xeno contaminants (in case of an animal serum is used).

[0122] According to some embodiments of the invention, the serum-free culture medium does not comprise serum or portions thereof.

[0123] According to some embodiments of the invention, the serum-free culture medium of the invention is devoid of serum albumin (e.g., albumin which is purified from human serum or animal serum).

[0124] According to some embodiments of the invention the culture medium comprises serum replacement.

[0125] As used herein the phrase “serum replacement” refers to a defined formulation, which substitutes the function of serum by providing pluripotent stem cells with components needed for growth and viability.

[0126] Various serum replacement formulations are known in the art and are commercially available.

[0127] For example, GIBCO™ Knockout™ Serum Replacement (Gibco-Invitrogen Corporation, Grand Island, NY USA, Catalogue No. 10828028) is a defined serum-free formulation optimized to grow and maintain undifferentiated ES cells in culture. It should be noted that the formulation of GIBCO™ Knockout™ Serum Replacement includes Albumax (Bovine serum albumin enriched with lipids) which is from an animal source (International Patent Publication No. WO 98 / 30679 to Price, P. J. et al). However, a recent publication by Crook et al., 2007 (Crook JM., et al., 2007, Cell Stem Cell, 1: 490-494) describes six clinical-grade hESC lines generated using FDA-approved clinical grade foreskin fibroblasts in cGMP-manufactured Knockout™ Serum Replacement (Invitrogen Corporation, USA, Catalogue No. 04-0095).

[0128] Another commercially available serum replacement is the B-27™ supplement without vitamin A which is available from Gibco-Invitrogen, Corporation, Grand Island, NY USA, Catalogue No. 12587-010. The B-27™ supplement is a serum-free formulation which includes d-biotin, fatty acid free fraction V bovine serum albumin (BSA), catalase, L-camitine HC1, corticosterone, ethanolamine HC1, D-galactose (Anhyd.), glutathione (reduced), recombinant human insulin, linoleic acid, linolenic acid, progesterone, putrescine-2-HCl, sodium selenite, superoxide dismutase, T-3 / albumin complex, DL alpha-tocopherol and DL alpha tocopherol acetate. However, the use of B-27™ supplement is limited since it includes albumin from an animal source.

[0129] According to some embodiments of the invention, the serum replacement is devoid of (completely free of) animal contaminants. Such contaminants can be pathogens which can infect human cells, cellular components or a-cellular components (e.g., fluid) of animals.

[0130] It should be noted that when an animal-contaminant-free serum replacement is used to culture human cells, then the serum replacement is referred to as being “xeno-free”.

[0131] The term “xeno” is a prefix based on the Greek word "Xenos", z.e., a stranger. As used herein the phrase “xeno-free” refers to being devoid of any components / contaminants which are derived from a xenos (z.e., not the same, a foreigner) species.

[0132] For example, a xeno-free serum replacement for use with human cells (i.e., an animal contaminant-free serum replacement) can include a combination of insulin, transferrin and selenium. Additionally or alternatively, a xeno-free serum replacement can include human or recombinantly produced albumin, transferrin and insulin.

[0133] Non-limiting examples of commercially available xeno-free serum replacement compositions include the premix of ITS (Insulin, Transferrin and Selenium) available from Invitrogen corporation (ITS, Invitrogen, Catalogue No. 51500-056); Serum replacement 3 (SR3; Sigma, Catalogue No. S2640) which includes human serum albumin, human transferring and human recombinant insulin and does not contain growth factors, steroid hormones, glucocorticoids, cell adhesion factors, detectable Ig and mitogens; KnockOut™ SR XenoFree [Catalogue numbers A10992-01, A10992-02, part Nos. 12618-012 or 12618-013, Invitrogen GIBCO] which contains only human- derived or human recombinant proteins. A particular example of a culture media which may be used whilst inducing and culturing iPS cells include DMEM, DMEM / F12, or DME culture solutions (these culture solutions may further appropriately contain serum (e.g. 10-15 %), LIF, antibiotics, L-glutamine, nonessential amino acids, beta-mercaptoethanol, or the like) or commercially available culture solutions e.g., a culture solution for culturing mouse ES cells (TX-WES culture solution, Thromb-X).

[0134] In one embodiment, the embryonic stem cells or iPSCs are cultured under conditions to generate formative pluripotent stem cells.

[0135] Formative pluripotent stem cells refers to pluripotent cells in a formative state.

[0136] The term “formative state” refers to a state that is a stage between a naive state and a primed state.

[0137] Formative pluripotent stem cells may be generated from embryonic stem cells or iPSCs. According to a specific embodiment of the invention, the formative hPSCs are positive for one or more marker.

[0138] Positive is also abbreviated by (+). Positive for a marker means that at least about 70 %, 80 %, 85 %, 90 %, 95 %, or 100 % of the cells in the population present detectable levels of the marker (e.g., FGF5) assayed by a method known to those of skill in the art (e.g. immunofluorescence, FACS).

[0139] Thus, for example, the formative hPSCs stain positively with anti FGF5 antibody (e.g. by immunofluorescence or immunohistochemistry using the FGF5 antibody (goat polyclonal IgG anti-human FGF5 (1:250; Cat. No. AF-237-NA, R& D Systems, Minneapolis, MN, USA). The FGF5-positive cells according to this embodiment, may stain negatively to one or more marker e.g., SOX17. Negative is also abbreviated by (-). Negative for a marker means that no more than about 5 %, 10 %, 20 %, 25 %, or 30 % of the cells in the population present detectable levels of the marker (e.g., SOX17) assayed by a method known to those of skill in the art such as immunostaining. Such a marker presentation either of a single cell or an isolated population of cells is also referred to as a signature.

[0140] Methods of generating formative pluripotent stem cells and methods of further distinguishing the formative state from the naiive and primed state are further described in International Application No. WO2020 / 234888, the contents of which are incorporated herein by reference.

[0141] In one embodiment, expansion of human pluripotent stem cells is effected under conditions such that they retain TRA-1-60+, TRA-1-81+, Oct4+ marker expression. Expansion is typically effected under static conditions (e.g. in an adherent culture). Preferably, the solid surface on which the cells are cultured are coated with a protein that is known to enhance adherence of pluripotent stem cells. For example, the solid surface may be coated with an extracellular protein (or fragments thereof) such as collagen I, collagen IV, fibronectin or laminin.

[0142] In one embodiment, the coating comprises a single protein. Preferably, the coating comprises no more than 2, 3 or 4 extracellular proteins.

[0143] In another embodiment, the coating consists of a single extracellular protein (e.g. recombinant laminin).

[0144] According to a particular embodiment, the extracellular protein (e.g. laminin) is not comprised in Matrigel™.

[0145] The coating (e.g. mouse laminin 111) is present in an amount of about 20 μg per cm2. In an exemplary embodiment, the coating (e.g. mouse laminin 111) comprises between 50-500 μg of laminin per 9.6 cm2well.

[0146] According to a particular embodiment, the laminin is recombinant laminin.

[0147] Any laminin is contemplated by the present inventors including, but not limited to laminin 111, laminin 211, laminin 221, laminin 511, laminin 521, laminin 332 and recombinant E8 fragment of laminin 511 or laminin 521.

[0148] According to a particular embodiment the laminin is mouse laminin (e.g. mouse laminin 111.

[0149] According to a particular embodiment the laminin is human laminin (e.g. human recombinant laminin 521.

[0150] In one embodiment, human embryonic stem cells are expanded in a feeder-independent medium on Laminin (e.g. Laminin 521) in an appropriate culture medium which includes growth factors such as FGF2 and TGF-β, as known in the art.

[0151] In another embodiment human embryonic stem cells are expanded in a feeder- independent medium on Laminin (e.g. Laminin 521) in an appropriate culture medium containing N2 / B-27™ and bFGF (e.g. 20 ng / ml), so as to expand formative pluripotent stem cells. An appropriate medium for culturing formative pluripotent stem cells is NSC medium (DMEM / F12 medium, containing N2 / B-27™, 1 mM glutamine antibiotics and 20 ng / ml bFGF).

[0152] Passaging of cells can be effected after 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.

[0153] As mentioned, the first step in the differentiation process comprises culturing the human pluripotent stem cells or formative pluripotent stem cells in a first medium comprising FGF2 and a BMP antagonist in suspension under conditions that generate spheres which co-express at least two markers of neural precursor cells, as measured by FACS.

[0154] Basic fibroblast growth factor (also known as bFGF, FGF2 or FGF-β) is a member of the fibroblast growth factor family. bFGF [(e.g., human bFGF polypeptide GenBank Accession No. NP_001997.5; human bFGF polynucleotide GenBank Accession No. NM_002006.4 can be obtained from various commercial sources such as Cell Sciences®, Canton, MA, USA (e.g., Catalogue numbers CRF001A and CRF001B), Invitrogen Corporation products, Grand Island NY, USA (e.g., Catalogue numbers: PHG0261, PHG0263, PHG0266 and PHG0264), ProSpec-Tany TechnoGene Ltd. Rehovot, Israel (e.g., Catalogue number: CYT-218), and Sigma, St Louis, MO, USA (e.g., catalogue number: F0291).

[0155] The concentration of bFGF in the culture medium is typically between 1-100 ng / ml, more preferably between 5-50 ng / ml, for example about 20 ng / ml.

[0156] Examples of BMP antagonists include LDN-193189 (4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline; Yu et al (2008) Nat Chem Biol 4 33-41)), GDF3, Noggin (e.g. 100 ng / ml), and dorsomorphin (6-[4-[2-(1-Piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine; Yu et al (2008) Nat Chem Biol 4 33-41)). Preferably the BMP antagonist is LDN-193189 (e.g. between 50-200 nM, about 100 nM).

[0157] The first medium is typically devoid of ROCK inhibitor (i.e. the first medium is not supplemented with ROCK inhibitor).

[0158] The first medium may or may not comprise an activin / TGFB antagonist (e.g. SB-431542, LY2157299, Trabedersen, follistatin). In a particular embodiment, the first medium is devoid of an activin TGFB antagonist.

[0159] The medium used for this stage may be of the same type as used for the expansion (as further described herein above). In one embodiment, the medium is NUTRISTEM™. In another embodiment, the medium is NSC medium (as described herein above).

[0160] In order to culture cells in suspension, they are removed from the adherent surface. According to a particular embodiment, the cells are cultured under dynamic conditions.

[0161] Culturing cells under dynamic conditions typically involves providing an environment where cells experience movement, fluid flow, or mechanical forces.

[0162] In one embodiment, the cells are cultured in bioreactors, including for example stirred tank bioreactors, wave bioreactors, rotatory bioreactors and air-lift bioreactors.

[0163] In one embodiment the bioreactor comprises a chamber for culturing the cells and a chamber drive capable of rotating the chamber. For example, the chamber drive may be capable of rotating the chamber about an arc of rotation (e.g. rotating wall vessels). In another embodiment, the bioreactor comprises a rotating vertical propeller. An exemplary bioreactor with a rotating vertical propeller is the PBS-Vertical-Wheel bioreactor. The speed of rotation may be adapted according to the size of the sphere required. High rotation velocity that is associated with increased shear forces is required to maintain small sized cell spheres. However, high shear forces may adversely affect cell survival. Hence, an optimal rotation velocity may be selected that balances between size and survival of cell spheres.

[0164] As used herein, the term “sphere” refers to a 3D aggregate or cluster of cells (e.g. in a sphere-like structure).

[0165] Culturing is carried out for a length of time that ensures expression of neural precursor cells. Neural precursor cells express markers of neural precursor cells including but not limited to PSA-NCAM and A2B5. An exemplary length of time is between 1 week - 2 weeks (e.g. about 10 days).

[0166] Methods of analyzing whether cells express particular markers are known in the art. The methods may be carried out on the protein level or the nucleic acid level. Exemplary methods for determining expression of proteins include Western blot, FACS analysis, immunohistochemistry.

[0167] Optionally, prior to culturing in the first medium, the pluripotent stem cells are cultured (in a non-adherent culture system, as described herein above) in a pre-differentiation medium for between 12-72 hours. This pre-differentiation culturing step may also be carried out under dynamic conditions, as described herein above.

[0168] The pre-differentiation medium comprises FGF2, a BMP antagonist (examples of which are provided herein above) and a rock inhibitor (e.g. Y-27632). In one embodiment, culturing in the presence of the rock inhibitor is no longer than 48 hours or no more than 24 hours. The predifferentiation medium is replaced by the first differentiation medium.

[0169] Following differentiation in the first medium (and optionally the pre-differentiating medium), the cell clusters are cultured in a second medium.

[0170] The second medium may replace the first medium.

[0171] Alternatively, additional components may be added to the first medium in order to generate the second medium.

[0172] An exemplary medium that can be used for the second differentiation medium is BNN (NUTRISTEM™, human serum albumin, Pen / Strep, N2 and B-27™).

[0173] Another medium that can be used in the second step is NSC medium (as described herein above).

[0174] The second medium comprises FGF2, retinoic acid, a SHH agonist and optionally a BMP antagonist.

[0175] Exemplary concentrations of retinoic acid are between 0.05-10 pM (e.g. about IpM) under conditions that generate spheres expressing Olig2, as measured by FACs. Sonic Hedgehog (SHH) agonists may be small molecule agonists such as purmorphamine at concentrations between 0.1-10 pM (e.g. about IpM) or SAG (smoothened agonist) or recombinant sonic hedgehog protein itself.

[0176] If added, the BMP antagonist may be the same antagonist as used in the first differentiation medium (e.g. at the same concentration). Alternatively, the BMP antagonist may be a different antagonist as used in the first differentiation medium.

[0177] FGF2 is typically used at the same concentration as used in the first differentiation medium. An exemplary medium that can be used for the second differentiation medium is BNN (NUTRISTEM™ human serum albumin, Pen / Strep, N2 and B-27™).

[0178] The spheres are cultured in the second medium under conditions (e.g. at least one, two, three, or four weeks under non-adherent, optionally dynamic conditions) that allow for generation of cells that express markers of pre-oligodendrocyte progenitor cells (e.g. express Olig2, as measured by FACS). Optionally, the cells also express Nkx2.2, PDGF-receptor and / or NG2 (Neural / Glial antigen 2 proteoglycan, homologous to human Chondroitin sulfate proteoglycan 4, CSPG4). In one embodiment, this differentiation step is effected under dynamic culturing conditions (as further described herein above).

[0179] The cells generated according to the first or second step of the protocol do not significantly co-express Olig2 and Nkx2.2 and do not express markers of pre-myelinating oligodendrocytes, (as measured by immunofluorescence or FACs).

[0180] The next step of the culture protocol comprises culturing the cells obtained from the previous step (i.e. those expressing Olig2) in a third medium comprising an SHH agonist at concentrations between 0.1-10 pM (e.g. about 0.5pM), FGF2, NT3 (e.g. between 1-20 g / ml, about 5 ng / ml) and ascorbic acid (50-500 pM, about 200 pM) under conditions that generate cells coexpressing Olig2 and Nkx2.2, as measured by FACS and / or immunofluorescence. Typically, the OPCs also co-express Olig2 and SOX 10, as measured by FACs or immunofluorescence. Optionally, the cells also express PDGF-receptor and / or NG2 (CSPG4). In addition, the cells also express (e.g. co-express) PSA-NCAM and A2B5. This step may be effected under flat, adherent culture conditions, non-adherent floating spheres under static or dynamic (within bioreactor) culturing conditions for at least two weeks, at least three weeks, at least four weeks, five weeks, 6 weeks or longer. In one embodiment, this step of the culturing protocol does not exceed 10 weeks. In order to culture the cells under flat conditions, the cells of the spheres are typically dissociated (e.g. using TrypLE Select™) and re-plated as a monolayer.

[0181] An exemplary medium that can be used for the third differentiation medium is BNN (NUTRISTEM™, human serum albumin, Pen / Strep, N2 and B-27™). Another exemplary medium that can be used for the third differentiation medium is SATO medium (DMEM / F12, antibiotic, Glutamine, BSA (e.g.lOOpg / ml), Biotin (e.g. lOng / ml), Forskolin (e.g. 2pg / ml), Insulin (e.g. 20pg / ml), Progesterone (e.g. 60ng / ml), Putrescine (e.g. 16pg / ml), Sodium Selenite (e.g. 5ng / ml), N-acetyl cysteine (e.g. 60pg / ml) and Transferrin (e.g. lOOpg / ml).

[0182] Another exemplary medium that can be used for the third differentiation medium is ASTO (NUTRISTEM™, human serum albumin, antibiotic, N2, B-27™, Biotin (e.g. lOng / ml) and Forskolin (e.g. 2pg / ml).

[0183] The cells at the end or middle of the third differentiation step may be used to treat the demyelinating disease or disorder or differentiated further (as detailed below) or frozen and subjected to further differentiating steps post thawing.

[0184] The cells at the end or middle of the third differentiation step may comprise a mixture of cells wherein at least 10 %, 15 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 % co-express Olig2 and Nkx2.2 (as measured by immunofluorescence or FACs). In addition, at least 50 %, 60 %, 70 %, 80 %, 90 %, 100 % of the cells express (e.g. co-express) PSA-NCAM and A2B5 (as measured by FACs).

[0185] The mixture of cells obtained after the third differentiation step typically comprises neural cells, including for example neurons and astrocytes as well as OPCs

[0186] The present inventors contemplate purifying the cells at the end of the third differentiation step so as to increase the percentage of cells which co-express Olig2 and Nkx2.2. For example, the cells can be purified by sorting for surface markers such as NG2 or PDGFRa or using a reporter of intracellular markers such as OLIG2, NKX2.2.

[0187] The fourth step of the culture protocol comprises culturing the cells (under static conditions, e.g. flat, adherent culture including for example laminin coated plates) obtained from the previous step (i.e. those expressing Olig2 and Nkx2.2) in a fourth medium comprising ascorbic acid (50-500 pM, about 200 pM), NT3 (e.g. between 1-20 g / ml, about 5 ng / ml), T3 (e.g. between 10-200 ng / ml, about 60 ng / ml), IGF1 (e.g. between 1-100 ng / ml, about 10 ng / ml) and optionally a BMP antagonist (e.g. noggin at a concentration between 10-100 ng / ml, about 50 ng / ml) under conditions (e.g. at least one, two, three, four, five, six, seven, eight, nine or ten weeks) that generate cells coexpressing at least two markers of pre-myelinating oligodendrocytes, as measured by (as measured by immunofluorescence or FACS. Cells from the previous step are dissociated (for example into single cells).

[0188] Markers of pre-myelinating oligodendrocytes include, but are not limited to 04 and 01 (e.g., as measured by immunofluorescence). The cells may optionally also express at least one, two, three, four or all of the following markers, Olig2, Nkx2.2, PDGFRa, SOX10 and NG2. An exemplary medium that can be used for the fourth differentiation medium is BNN (NUTRISTEM™ minus growth factors, human serum albumin, Pen / Strep, N2 and B-27™).

[0189] Another exemplary medium that can be used for the fourth differentiation medium is SATO medium (DMEM / F12, antibiotic, Glutamine, Biotin (e.g. lOng / ml), Insulin (e.g. 20pg / ml), Progesterone (e.g. 60ng / ml), Putrescine (e.g. 16pg / ml), Sodium Selenite (e.g. 5ng / ml), N-acetyl cysteine (e.g. 60pg / ml) and Transferrin (e.g. lOOpg / ml).

[0190] Another exemplary medium that can be used for the fourth differentiation medium is ASTO (NUTRISTEM™ minus growth factors, human serum albumin, antibiotic, N2, B-27™, Biotin (e.g. lOng / ml) and Forskolin (e.g. 2pg / ml).

[0191] Pre-myelinating oligodendrocytes may have a structural phenotype which includes, but is not limited to elongated, bipolar or multipolar morphology.

[0192] Examples of a pre-myelinating oligodendrocyte functional phenotype include, but are not limited to migratory capacities as well as the potential to differentiate into a myelinating phenotype to effect myelination in vivo and in vitro.

[0193] The pre-myelinating oligodendrocytes generated according to the methods described herein may be directly subjected to further differentiation steps as described in W02020 / 250232, the contents of which are incorporated herein, so as to generate mature oligodendrocytes. Alternatively, the pre-myelinating oligodendrocytes may be banked at this stage and stored for further use (e.g. frozen).

[0194] Mature oligodendrocytes may be distinguished from pre-myelinating oligodendrocytes both by structural and functional phenotypes.

[0195] Examples of a mature oligodendrocyte functional phenotype include, but are not limited to one or more, marker expression such as proteolipid protein (PLP), MBP expression, myelin-associated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein (MOG).

[0196] Examples of mature oligodendrocyte structural phenotype include, but are not limited to, a branched and ramified phenotype and formation of myelin membranes.

[0197] Once differentiated and optionally isolated, the cells may be tested (in culture) for their phenotype. The cultures may be comparatively analyzed for a phenotype of interest (e.g., O4 / O1 expression, expansion, migration), either in vitro and / or in vivo using biochemical analytical methods such as immunostaining, cell expansion assays (e.g., MTT), migration assays, Western blot and Real-time PCR. Cells of the present invention may be further cloned and cell-lines of interest may be generated. Cells generated according to the teachings of the present invention (described hereinabove) and in the Examples section which follows may be used in a myriad of clinical and research applications.

[0198] Thus, according to another aspect of the present invention there is provided a method of treating a de- or dys-myelinating disease in a subject-in-need-thereof. The method comprising administering to the subject a therapeutically effective amount of the cells of the present Invention (according to the intended use, as further described hereinbelow.

[0199] Subjects treated in accordance with the teachings of the present invention are preferably human subjects.

[0200] Exemplary diseases that may be treated include, but are not limited to multiple sclerosis (MS), spinal cord injury, stroke, white matter stroke, neuromyelitis optica, MOG-antibody associated demyelinating disease (MOGAD), guillain-barre syndrome, diffuse disseminated encephalomyelitis, acute disseminated encephalomyelitis, concentric sclerosis, diffuse sclerosis, leukodystrophy, leukoencephalopathy caused by ischemia-hypoxia, central pontine myelination, acute inflammatory demyelinating polyneuropathy, chronic inflammatory demyelinating polyneuropathy, subacute combined degeneration caused by nutritional deficiency diseases, subacute sclerosing panencephalitis or progressive multifocal leukoencephalopathy caused by viral infection, diabetic neuropathy, neuropathy of systemic lupus erythematosus, leukoablative diseases, vascular and age-related white matter loss of small vessel disease (commonly associated with subcortical dementias of advanced age), Alexander disease, vanishing white matter disease, Adrenoleukodystrophy and Pelizaeus-Merzbacher disease, lysosomal storage disorders, Huntington's disease, frontotemporal dementia, childhood-onset schizophrenia, Radiation and Chemotherapy CNS Injury.

[0201] In this respect, it should be noted that pre ■myelinating oligodendrocytes may be adventitiously used over mature oligodendrocytes as it is probably the mitotic and migratory capacity of these cells (in contrast to mature cells) which are vital prerequisites for successful remyelination. Mature differentiated oligodendrocytes may still be useful as myelinating cells in vivo (Duncan et al. 1992 Dev. Neurosci. 14:114-122). Differentiated and mature human oligodendrocytes may have important applications for testing drugs that can protect oligodendrocytes from toxic or other pathogenic injuries as further described hereinbelow.

[0202] The cells of the present invention can be administered to the treated subject using a variety of transplantation approaches, the nature of which depends on the site of implantation. ’The term or phrase “transplantation”, “cell replacement” “cell therapy” or “grafting” are used interchangeably herein and refer to the introduction of the cells of the present invention to target tissue. The cells can be derived from the recipient or from an allogeneic or xenogeneic donor.

[0203] For example, the cells can be grafted into the central nervous system or into the ventricular cavities or subdural and sub-arachnoid spaces onto the surface of a host brain or in the spinal cord. Conditions for successful transplantation include: (i) viability of the implant and sufficient number of cells; (ii) migration (e.g., pre-myelinating oligodendrocytes) of the cells within the nervous tissue to the lesions in accordance with the selected population of cells; and (iii) minimum amount of pathological reaction, Methods for transplanting various nerve tissues, for example embryonic brain tissue, into host brains have been described in: “Neural grafting in the mammalian CNS”, Bjorklund and Stenevi, eds. (1985); Freed et al., 2001; Olanow et al., 2003). These procedures include intraparenchymal transplantation, i.e. within the host brain (as compared to outside the brain or extra-parenchymal transplantation) achieved by injection or deposition of tissue within the host brain so as to be opposed to the brain parenchyma at the time of transplantation.

[0204] Intraparenchymal transplantation can be effected using two approaches: (i) injection of cells into the host brain parenchyma or (ii) preparing a cavity by surgical means to expose the host brain parenchyma and then depositing the graft into the cavity. Both methods provide parenchymal deposition between the graft and host brain tissue at the time of grafting, and both facilitate anatomical integration between the graft and host brain tissue. ’This is of importance if it is required that the graft becomes an integral part of the host brain and survives for the life of the host.

[0205] Alternatively, the graft may be placed in a ventricle, e.g. a cerebral ventricle or subdural and sub-arachnoid spaces, i.e. on the surface of the host brain where it is separated from the host brain parenchyma by the intervening pia mater or arachnoid and pia mater. Grafting to the ventricle may be accomplished by injection of the donor cells or by embedding the cells in a substrate such as 3% collagen to form a plug of solid tissue which may then be implanted into the ventricle to prevent dislocation of the graft. For subdural grafting, the cells may be injected around the surface of the brain after making a slit in the dura. Injections into selected regions of the host brain may be made by drilling a hole and piercing the dura to permit the needle of a micro-syringe to be inserted. The micro-syringe is preferably mounted in a stereotaxic frame and three dimensional stereotaxic coordinates are selected for placing the needle into the desired location of the brain or spinal cord. The cells may also be introduced into the putamen, nucleus basalis, hippocampus cortex, striatum, substantia nigra or caudate regions of the brain, as well as the spinal cord.

[0206] ’The cells may also be transplanted to a healthy region of the tissue. In some cases the exact location of the damaged tissue area may be unknown and the cells may be inadvertently transplanted to a healthy region. In other cases, it may be preferable to administer the cells to a healthy region, thereby avoiding any further damage to that region. Whatever the case, following transplantation, the cells preferably migrate to the damaged area.

[0207] For transplanting, the cell suspension is drawn up into the syringe and administered to anesthetized transplantation recipients. Multiple injections may be made using this procedure.

[0208] The cellular suspension procedure thus permits grafting of the cells to any predetermined site in the brain or spinal cord, is relatively non-traumatic, allows multiple grafting simultaneously in several different sites or the same site using the same cell suspension, and permits mixtures of cells from different anatomical regions. Multiple grafts may consist of a mixture of cell types, and / or a mixture of transgenes inserted into the cells. Preferably from approximately 104to approximately 10scells are introduced per graft.

[0209] For transplantation into cavities, which may be preferred for spinal cord grafting, tissue is removed from regions close to the external surface of the central nerve system (CNS) to form a transplantation cavity, for example as described by Stenevi et al. (Brain Res. 114:1-20, 1976), by removing bone overlying the brain and stopping bleeding with a material such a gelfoam. Suction may be used to create the cavity. The graft is then placed in the cavity. More than one transplant may be placed in the same cavity using injection of cells or solid tissue implants. Preferably, the site of implantation is dictated by the CNS disorder being treated.

[0210] For example, in treating multiple sclerosis transplantation into a small number of carefully chosen lesions, for example, the optic nerves, the spinal cord, or the superior cerebellar peduncle can be effected.

[0211] In other inherited disorders of myelin metabolism, a systemic mode of administration may be used to exploit the migratory capacity of oligodendroglial progenitor cells (OPCs) or premyelinating oligodendrocytes and both the circulation of the brain and the blood. In these cases disruption of the blood-brain barrier and / or supplementation with growth factor infusion or growth / trophic factor secreting cells.

[0212] Since non-autologous cells are likely to induce an immune reaction when administered to the body, several approaches have been developed to reduce the likelihood of rejection of non-autologous cells. These include either suppressing the recipient immune system or encapsulating the non-autologous cells in immuno-isolating, semipermeable membranes before transplantation.

[0213] Additionally, or alternatively, the cells (at any stage along their differentiation) may be genetically modified so as to reduce immunogenicity (e.g. deletion of HLA and forced expression of molecules such as checkpoint inhibitors, anti-inflammatory cytokines, Fas Ligand, CD47 etc.). ’The cells may be provided together with immunosuppressive agents. Examples of immunosuppressive agents inchide, but are not limited to, methotrexate, cyclophosphamide, cyclosporine, cyclosporin A, chloroquine, hydroxychloroquine, sulfasalazine (sulphasalazopyrine), gold salts, D-penicillamine, leflunomide, azathioprine, anakinra, infliximab (REMICADE. sup. R), etanercept, TNF alpha blockers, a biological agent that targets an inflammatory cytokine, and Non-Steroidal Anti-Inflammatory Drug (NSAIDs). Examples of NSAIDs include, but are not limited to acetyl salicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors and tramadol.

[0214] In any of the methods described herein, the cells can be administered either per se or, preferably as a part of a pharmaceutical composition that further comprises a pharmaceutically acceptable carrier.

[0215] 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.

[0216] Hereinafter, the term “pharmaceutically acceptable carrier” refers to a carrier or a diluent that does not cause significant irritation to a subject and does not abrogate the biological activity and properties of the administered compound. Examples, without limitations, of carriers are propylene glycol, saline, emulsions and mixtures of organic solvents with water.

[0217] Herein the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

[0218] According to a preferred embodiment of the present invention, the pharmaceutical carrier is an aqueous solution of saline.

[0219] Techniques for formulation and administration of drugs may be found in “Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference.

[0220] Suitable routes of administration include direct administration into the tissue or organ of interest. Thus, for example the cells may be administered directly into a specific region of the brai n or to the spinal cord. For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. Preferably, a dose is formulated in an animal model to achieve a. desired concentration or titer. Such information cart be used to more accurately determine useful doses in humans.

[0221] Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. For example, animal models of demyelinating diseases include shiverer (shi / shi, MBP deleted) mouse, MD rats (PLP deficiency), Jimpy mouse (PLP mutation), dog shaking pup (PLP mutation), twitcher mouse (galactosylceramidase defect, as in human Krabbe disease), trembler mouse (PMP-22 deficiency). Virus induced demyelination model comprise use of Theiler's virus and mouse hepatitis virus. Autoimmune EAE is a possible model for multiple sclerosis.

[0222] The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient’s condition, (see e.g., Fingl. et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p.

[0223] 1). For example, MS patient can be monitored symptomatically for improved functions indicating positive response to treatment, as well as radiologically for myelin and axon integrity, and electrophy siologically for speed of electrical impusles.

[0224] For 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 salt buffer.

[0225] Dosage amount and interval may be adjusted individually to levels of the active ingredient which are sufficient to effectively regulate the neurotransmitter synthesis by the implanted cells. Dosages necessary to achieve the desired effect will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

[0226] Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or diminution of the disease state is achieved.

[0227] The amount of a composition to be administered will, of course, be dependent on the individual being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc. The dosage and timing of administration will be responsive to a careful and continuous monitoring of the indi vidual changing condition. For example, a treated MS patient will be administered with an amount of cells which is sufficient to alleviate the symptoms of the disease, relevant for the specific site of cell delivery, and based on the monitoring indications.

[0228] The cells of the present invention may be co-administered with therapeutic agents useful in treating demyelinating diseases or disorders, such as gangliosides; antibiotics, neurotransmitters and their precursors, neurohormones, toxins, neurite promoting molecules, and antimetabolites. Additionally, the cells of the present invention may be co -administered with other cells.

[0229] Following transplantation, the cells of the present invention preferably survive in the diseased area for a period of time (e.g. at least 6 months), such that a therapeutic effect is observed. As described in the Example section below, the cells of the present invention were shown to migrate and myelinate an EAE mouse brain.

[0230] Generally, any method known in the art can be used to monitor success of transplantation. For example, MRI can be used for visualizing brain white matter and studying the burden of demyelinating lesions as currently practiced for monitoring MS patients. Magnetization transfer contrast and other MR-based quantitative measurements of myelin can be used to monitor remyelination (Deloire -Grassin 2000 J. Neurol, Sci, 178:10-16). Magnetic resonance spectroscopy measurement of N-acetyl-aspartate levels can be used to assess impact on local neuron / axon survival. IJ sing paramagnetic particles to label cells before transplantation enabling their dispersion to be tracked by MRI. Serial neurophysiology is useful for monitoring conduction. The optic nerve has particular advantages in this respect.

[0231] Other approaches to more generalized neurophysiological assessment are described in Leocani et al. Neurol Sci. 2000; 21(4 Suppl 2): S889-91 which may be useful for interventions aimed at multifocal or more diffuse myelin repair. Notwithstanding, it is appreciated that clinical improvement may also be assessed. Demyelination causes alterations of stature (trembling, shivering) and locomotion. Children with leukodystrophies have motor and intellectual retardation. Electrophysiological measures of sensory and motor nerve conductivity, for example nerve conduction velocity and other measures such as H- waves, are classical method used in monitoring neuropathies linked to demyelinating peripheral lesions (Lazzarini et al, eds (2004) Myelin biology and disorders, Elsevier Academic Press, San Diego, Calif.). The latency and size of visual evoked potentials in optic pathways, sensory- and motor- evoked potential in spinal pathways, and brainstem evoked potentials nay serve to monitor de- and -re-myelination in the central nervous system.

[0232] . As mentioned above, cells of the present invention can be used as an imperative tool for in vitro screening of drugs.

[0233] Thus, according to yet another aspect of the present invention there is provided a method of determining an effect of a treatment on neural cell functionality, the method comprising subjecting a cell of the present invention (e.g., pre-myelinating oligodendrocyte) to the treatment (e.g., drug, condition such as electrical treatment and an irradiation treatment); and determining at least one of a structural or functional phenotype of the treated cell as compared to an untreated cell, thereby determining an effect of the treatment on neural cell functionality.

[0234] Qualifying the effect of a treatment of interest on the cells of the present invention can be used to identify and optimize treatments capable of restoring the neural function, and hence can be used to identify and optimize drugs suitable for treating neural disorders (e.g., including treatment methods envisaged by the present invention).

[0235] Furthermore, qualifying the effect of a treatment (either directed to diseases of the CNS or any other tissue) on neural functionality can be used to assess the toxicity of such clinical treatments.

[0236] Thus, this aspect of the present invention can be preferably utilized to determine the therapeutic and toxic effects of various treatments, such as drug treatments, and electrical treatments, on neural function.

[0237] Hence the method of the present invention can be used to screen and / or test drugs.

[0238] This aspect of the present invention can be also utilized to obtain gene expression profiles and changes thereof in cells of the present invention subjected to a treatment. ’Thus, the method according to this aspect of the present invention can be used to determine, for example, gene expression pattern changes in response to a treatment.

[0239] As mentioned, the present inventors have uncovered novel media formulations for the production of pre-myelinating oligodendrocytes.

[0240] Such media include:

[0241] ASTO medium (this medium is particularly useful for the third and fourth differentiating step). The contents of the medium are as follows:

[0242] NUTRISTEM™ (minus GF)

[0243] human serum albumin

[0244] N2

[0245] B-27™

[0246] Biotin (e.g. lOng / ml)

[0247] Forskolin (e.g. 2pg / ml)

[0248] BNN medium (this medium is particularly useful for the second, third and fourth differentiating step). The contents of the medium are as follows:

[0249] NUTRISTEM™ (minus GF)

[0250] human serum albumin N2

[0251] B-27™

[0252] The media described herein typically include antibiotics (e.g. pen / strep).

[0253] In one embodiment, the media are devoid of additional growth factors.

[0254] As used herein the term “about” refers to ±10%.

[0255] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

[0256] The term “consisting of’ means “including and limited to”.

[0257] 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.

[0258] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0259] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0260] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0261] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts. As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

[0262] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0263] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0264] EXAMPLES

[0265] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

[0266] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "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); methodologies as set forth in U. S. Pat. 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 extensively described in the patent and scientific literature, see, for example, U. S. Pat. Nos. 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" Hanies, 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 general 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 the information contained therein is incorporated herein by reference.

[0267] EXAMPLE 1

[0268] MATERIALS AND METHODS

[0269] Pluripotent stem cell culture

[0270] Human ESC (HADC100) were cultured in a feeder- independent system on Laminin 521 (Biolamina, Sundbyberg, Sweden) in medium Nutristem plus growth factors (Biological Industries, Beit Haemek, Israel) and 50 units / ml penicillin / 50 pg / ml streptomycin (Gibco, Invitrogen, Gaithersburg, MD). Alternatively, hESCs were cultured on Laminin 521-coated plates as formative pluripotent stem cells in NSC medium: DMEM / F12 medium (Biological Industries), containing N2 / B-27™ (Gibco), 1 mM glutamine (Biological Industries), 50 units / ml penicillin / 50 p.g / ml streptomycin (Gibco) and 20 ng / ml bFGF (PeproTech, Cranbury, NJ). HESCs were weekly passaged with TrypLE Select (Thermo Fisher Scientific, Waltham, MA). Human formative pluripotent stem cells were weekly passaged with ReLeSR™ (Stemcell Technologies, Vancouver, BC).

[0271] Differentiation into oligodendrocyte lineage cells

[0272] Homogeneous size spheres comprised of 104cells were generated from a single-cell suspension of hESC cells or human formative pluripotent stem cells using AggreWell™ 800 24-well culture plates (Stemcell Technologies). Otherwise, a single-cell suspension of undifferentiated hESCs were seeded on a PBS-Mini Vertical- Wheel Bioreactor (PBS Biotech, Camarillo, CA) at a density of 300,000 cells / ml, and were set at a starting rotation velocity of 20 to 50 rpm. The rotation velocity was gradually increased along the differentiation protocol up to 80 rpm to avoid overgrowth of diameter of cell aggregates. The differentiation protocol was divided into four developmental stages. At the first stage, the cells were cultured in suspension for 2 weeks in Nutristem medium (minus growth factors) and 50 units / ml penicillin / 50 pg / ml streptomycin (Nut-) supplemented with 20ng / ml bFGF and lOOnM LDN (Stemcell Technologies) with or without 5pM SB431542 (Tocris Bioscience, Bristol, Avon, United Kingdom) during the first 3 days. Rock inhibitor (Y-27632, lOpM; BioGems, Westlake Village, CA) was present for the first 24h. During the second stage, the cells were cultured for 3 weeks in NSC or BNN media supplemented with 20ng / ml bFGF, lOOnM LDN, IpM purmorphamine (Stemcell Technologies), and IpM retinoic acid (Tretinion; Spectrum Chemical, New Brunswick, NJ). For the third stage of differentiation, the cells were cultured as spheres or, alternatively, 5xl05-7xl05single cells were plated per well on Laminin-521 -coated 6-well plates. The cells were cultured either in SATO or BNN or ASTO media. The medium was supplemented with 20ng / ml bFGF, 0.5pM purmorphamine, 200pM ascorbic acid (AA; Sigma-Aldrich, Saint Louis, MO), and 5ng / ml NT-3 (Stemcell Technologies). When cultured in 2D cultures, the cells were passaged every 2 weeks by dissociating the monolayer with TrypLE Select and re-plated as described below. The length of culture period of the third differentiation stage was 4-6 weeks. Further differentiation into preoligodendrocytes (step 4) was performed after dissociation into single cells by TrypLE Select and plating 5xl05cells per well on Laminin-521 -coated 6-well plates. The cells were cultured in either SATO or BNN or ASTO media supplemented with 200pM ascorbic acid (AA), lOng / ml IGF-1 (PeproTech), 60ng / ml T3 (Stemcell Technologies), 5ng / ml NT-3, and 50ng / ml noggin (in some experiments; PeproTech). Cell clusters cultured in the PBS vertical-wheel bioreactor were passaged at the end of step 1 and 2 by two methods. In one approach, the cell aggregates were split into a 1:8 and 1: 10 ratio, respectively, providing a calculated cell density of ~3×105cells / ml. In an alternative approach, the aggregates were dissociated with TrypLE Select enzyme (Thermo Fisher Scientific) for 10 min at 37°C followed by gentle trituration to a single-cell suspension. Inactivation was performed by the addition of medium and further incubation with Pulmozyme (Roche, Vienna, Austria; 10 min at 37°C). The single cells were seeded at 3×105cells / ml density for further propagation and differentiation in the PBS vertical- wheel bioreactor.

[0273] FACS analysis

[0274] The clusters of cells were dissociated with TrypLE Select enzyme for 10 min at 37°C, followed by gentle trituration to a single-cell suspension, inactivation by the addition of medium and further incubation with Pulmozyme (10 min at 37°C). The same treatment was used to dissociate attached cells. The cells were then washed with PBS supplemented with 1% BSA and 0.05% sodium azide (both from Sigma, Saint Louis MO). The cells were incubated with anti-PSA-NCAM (1:50, PE conjugated, Miltenyi Biotech, Gaithesburg, MD), anti-A2B5 (1:50, APC conjugated, Miltenyi Biotech), anti-04 and anti-01 (both 1:150, monoclonal mouse IgM, R& D Systems, Inc., Minneapolis, MN). Control cells were stained with respective isotype control antibodies. Non-conjugated primary antibodies were detected using secondary Allophycocyanin (APC)-labeled goat anti-mouse Ig (1: 150, Jackson ImmunoResearch, West Grove, PA). Propidium iodide (PI; Sigma) was added (final concentration of 4 pg / ml) for the gating of live cells. In addition, cells stained for nuclear markers were dissociated as before, and stained with FVS-780 (BD Biosciences, San Jose, CA) to determine viability of the cells. The cells were then fixated and immunostained using the eBioscience FOXP3 / Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) according to the manufacturer instructions. The cells were stained with anti-Olig2 (1:150, mouse monoclonal IgG2a, EMD Millipore Corp., Temecula, CA) and anti-Nkx2.2 (1:50, rabbit, Novus Biologicals, Centennial, CO) antibodies. Control cells were stained with respective isotype control antibodies. Primary antibodies were detected using APC-labeled goat anti-mouse Ig and fluorescein isothiocyanate (488)-labeled goat anti-rabbit Ig (both 1: 150, Jackson ImmunoResearch). FACS analysis was performed using the CytoFLEX Platform (Beckman Coulter, Brea, CA).

[0275] Immunofluorescent staining of the cells differentiated towards the oligodendroglial linage

[0276] To characterize the expression of markers at different steps of the differentiation protocol by immunofluorescence, the differentiated cells were plated on glass coverslips pretreated with poly-D-lysine (30-70KDa, lOmg / ml) and laminin (4 mg / ml; both from Sigma, St. Louis, MO) and cultured for 3-7 days. The cells were then fixed with 4% paraformaldehyde and incubated with mouse anti-01ig2 Ab (1:150, Millipore), rabbit anti-Nkx2.2 Ab (1:50, Novus Biologicals), rabbit anti-SoxlO Ab (1:150, Cell Signaling Technology), mouse anti-GFAP Ab (1:200, Thermo Fisher Scientific), rabbit anti-P-tubulin III Ab (1:200, Covance) and rat anti-MBP Ab (1:200, Millipore). For the immuno staining with mouse anti-04 and mouse anti-Ol Abs (both 1:150, R& D) fixation with 4% paraformaldehyde was performed after the incubation with the primary Abs. 555-labeled Donkey anti-mouse Ab (1:150, Invitrogen), 488-labeled donkey anti-rabbit Ab, 488-labeled donkey anti-mouse IgM, 488-labeled donkey anti-rat (all 1:150; Jackson ImmunoResearch Laboratories) were used for detection. Nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA). The specimens were visualized with Olympus BX61 fluorescent microscope (Olympus, Hamburg, Germany).

[0277] Generation of RFP-MBP OC

[0278] A reporter system was generated for endogenous MBP expression, using CRISPR / Cas9 gene targeting technology. The Cas9 nuclease and single-guided oligo designed to target the last exon of the human MBP gene was employed to stimulate homology-directed repair with a donor construct encoding 2A-Tomato and neomycin expression cassette flanked by homology arms of the MBP locus. Tomato is one of the family of red fluorescent proteins (RFP) and hence is termed as RFP in the Results section and throughout the text.

[0279] Transplantation

[0280] In neonate mice, approximately 100,000 oligodendroglial progenitor cells (OPCs) were transplanted to cerebral white matter tracts at Bregma 0 in 3 days old wild type mice. The mice were sacrificed for histopathology at age 6 weeks. In adult mice, experimental autoimmune encephalomyelitis was induced in 2-3 months old Biozzi ABH mice, by subcutaneous injection at days 0 and 7 in both hind flanks with an emulsion containing Img spinal cord homogeneate, incomplete Freund's adjuvant and 60 pg Mycobacterium tuberculosis H37Ra and M. butyricum at 8:1 ratio. OCs were transplanted at around day 40 post induction, during the resolution of the second clinical relapse, entering the chronic phase of disease. Transplantation was performed using a stereotactic device into the Cerebellar peduncles and medial Lemniscus. EAE mice were monitored and scored daily as follows: 0= normal, l=limp tail, 2= impaired righting reflex, 3= hind-limb paresis; 4= complete hind-limb paralysis and 5= moribund / death. Mice were sacrificed after 5-6 weeks for histopathology.

[0281] Histopathology

[0282] Animals were anesthetized with a lethal dose of pentobarbital and brain and spinal cords were perfused via the ascending Aorta with ice-cold PBS, followed by cold 4% paraformaldehyde in PBS and processed for serial 8pm frozen sections. Immune-fluorescent staining was performed with mouse anti-human specific mitochondria (Millipore MAB 1273, 1:1250), rat anti- myelin basic protein (Sigma MAB386, 1:150), mouse anti-neurofilament M (Sigma SAB4200747, 1:100). Alexa-fluor 488 / 555 conjugated secondary antibodies were used as appropriate.

[0283] Statistical analysis

[0284] Data is presented as mean ± SD. The significance of differences between treatments was calculated using two-tailed t-test.

[0285] RESULTS

[0286] A differentiation protocol was developed, which is divided into several developmental stages. Flow Cytometry Analysis (FACS) was used to analyze the expression of specific markers along the differentiation process (Figure 1). The development of NPs and subsequently OCs with bFGF and LDN without activin / TGFp inhibitor SB431542 (SB)) at the first 3 days of differentiation was evaluated. A similar percentage of cells co-expressing PSA-NCAM and A2B5 after step 1 and expressing Olig2 after step 2 with or without SB was shown (Figure 2). Using the differentiation protocol detailed herein, OCs were derived from either undifferentiated hESCs or, alternatively, from human formative pluripotent stem cells. With both starting cell populations, after 2 weeks of differentiation, more than 90% of the cells within spheres expressed the neural progenitor markers PSA-NCAM and A2B5 (Figure 3). A similar expression of Olig2, Nkx2.2 and co-expression of these two markers after steps 2, 3, and 4 of differentiation (Figures 4, 5 and 6, respectively) was found. In addition, comparing the two starting pluripotent stem cells populations, the present inventors showed similar expression of the pre-oligodendrocyte markers 04 and 01 after step 4 of the differentiation protocol (Figure 7). The protocol yields above 80% 04+ cells at the end of step 4. Moreover, the present inventors showed for the first time, the induction of OC differentiation from human formative pluripotent stem cells. The yield of OCs from human formative pluripotent stem cells was similar to hESCs.

[0287] Large scale production of OCs is required for the development of clinical transplantation therapy products. In order to increase the number and survival of the OCs, a methodology was developed for induction of OC differentiation using dynamic bioreactor systems. Specifically, the PBS Vertical-Wheel Bioreactor was utilized. An advantage of dynamic culture systems is their ability to control the size of cell aggregates avoiding numerous steps of manual manipulation (such as seeding in microwells for aggregate formation, aggregate size selection, etc.) that are needed in static cultures. A small diameter of aggregates enables uniform exposure of the cells within them to growth- or differentiation-inducing factors. In the PBS Wheel system, the velocity of the wheel rotation controls the aggregates' size. High rotation velocity that is associated with increased shear forces is required to maintain small sized cell aggregates. However, high shear forces may adversely affect cell survival. Hence optimal rotation velocity should be identified balancing between size and survival of cell aggregates.

[0288] In order to determine the optimal initial rotation velocity, 3×105undifferentiated hESCs / ml were seeded in the PBS Vertical- Wheel Bioreactor, and the first step of the differentiation protocol was carried out at different rotation velocities. At low velocity (20 rpm), the initial size of the aggregates that were formed after one day was relatively large and the aggregates clustered to form a very large aggregate at the end of the two weeks step. The percentage of live cells within the aggregate was relatively low (20.6%). At higher velocities (35 & 50 rpm), small clusters were composed after one day but at 50 rpm clusters of dead cells were observed after 10 days (Figure 8A). The percentage of live cells was improved at higher rotation velocities (35 and 50 rpm), while the percentage of cells expressing neural precursor markers was similar in all velocities (Figure 8B). In addition, expansion of cell number was not observed at 20 rpm, while ~8 fold increase in cell numbers was obtained at 35 and 50 rpm (Figure 8C). Based on these results, the optimal starting rotation velocity was set at 35 rpm.

[0289] The size of clusters gradually increased during differentiation within the dynamic culture conditions. Growth of diameter of the clusters after 10 days dynamic culture is demonstrated in Figure 8A. To avoid overgrowth of the cell clusters and to maintain the efficiency of differentiation, the rotation velocity was gradually increased along the differentiation protocol. During stage 1 of differentiation, the rotation velocity was unchanged or was slightly increased by 5rpm.

[0290] The cells were split at the end of step 1 and 2 by two methodologies. In the first approach the cells were dissociated into single cells and reseeded at 3×105cells / ml. In the second approach split of the spheres at 1:8 and 1:10 ratio was performed at the end of step 1 and 2, respectively. The phenotype, viability and expansion of the cells was compared at the end of step 2 after their splitting by the two approaches at the end of step 1. The percentage of live cells and Olig2+ cells did not differ significantly. The fold increase in the number of cells was significantly higher after sphere splitting (Figure 9). It should be noted that during stage 2, a stepwise increase of the rotation velocity by 5-10 increments was needed to prevent overgrowth of the aggregates size. With the single-cell passaging method the starting rotational velocity was 35rpm and was increased to 40-45rpm at the end of the stage. With the aggregates splitting approach the initial rotational velocity was 40-45rmp and it was increased to 50-55 at the end of stage 2.

[0291] Marker expression, survival and extent of expansion of cells cultured in PBS Vertical-Wheel Bioreactor versus static floating clusters culture was compared (Figure 10B-D). There were no differences in the percentage of cells expressing differentiation markers at the end of steps 1-4. The survival of the cells within the spheres was significantly higher in the dynamic compared to the static culture conditions (44.8+13.9 vs 17.8+1.3 % of live cells, respectively). The expansion of cell numbers was also significantly increased with dynamic vs static culture conditions (89.3+45.1 vs 1.54+0.48 folds of increase, respectively). At the end of step 2, the percentage of live cells was higher among Olig2+ cells compared to the total cell population (76.1+8.9 vs 47.0+16.8, respectively; Figure 11). Hence the dynamic culture conditions specifically promoted the survival of Olig2+ cells. This observation may promote the efficiency of a differentiation protocol towards the OC lineage.

[0292] Next, the efficacy of the oligodendroglia cell differentiation protocol implemented on attached monolayer 2D cultures was analyzed. There are several potential advantages to differentiation in monolayer cultures compared with floating aggregates, including uniform exposure of the cultured cells to growth and differentiation factors, and higher cell viability. The cells were plated as monolayers on Laminin 521 -coated plates starting from stages 2,3, or 4 of the protocol. To analyze the efficacy of using 2D cultures at different steps of our differentiation protocol, the percentage of cells expressing the oligodendroglial marker, 04 at the end of stage 4 was monitored (Figure 12). It was found that culturing the cells in adherent cultures from steps 3 and 4 increased the expression of 04, compared to adherent cultures plated from step 2 (62.3+18.9, 74.1+11.8, and 40.0+9.5, respectively). The viability of the cells at the end of step 3 was significantly increased when the cells were cultured in adherent cultures at step 3, compared to static and dynamic floating cultures (Figures 13A-B). The combination of dynamic culture conditions within PBS Vertical- Wheel Bioreactor at stages 1-2, with monolayer 2D culture at stage 3, enables bulk production of oligodendroglia cells. In the absence of culture vessel space or surface limitations, initiation of the differentiation process from one T75 flask of undifferentiated hESCs (21×106cells) would give rise to ~2×1012oligodendroglia cells at the end of stage 3.

[0293] Previous studies showing the derivation of oligodendroglia cells were done using researchgrade materials that were not suitable for human clinical transplantation. To translate the researchgrade differentiation protocol into a clinical-grade one that utilizes mainly animal-free, GMP-grade reagents, we substituted the NSC and basic non-GMP SATO differentiation media used at steps 2, and 3-4, respectively, with simpler media: GMP-medium BNN at steps 2-4 and medium ASTO at steps 3-4. Efficiency of the differentiation into Olig2+ cells after step 2 in medium BNN was similar to that in medium NSC (Figure 14A). Efficiency of the differentiation into 04+ cells after step 4 was comparable in cells cultured in media SATO, ASTO, or BNN during steps 3-4, as well as in cells cultured in medium BNN during steps 2-4 (Figure 14B). It was also found that NSC medium may be used as the basic medium for steps 1 and 2 of the differentiation protocol both when the starting population was hESCs or human formative pluripotent stem cells (Figure 15A-B). The efficiency of the differentiation into Olig2+ cells after step 2 and into 04+ and 01+ after step 4 was similar after differentiation in NSC for steps 1 and 2, followed by BNN medium for steps 3 and 4 of the protocol compared with Nut- / BNN.

[0294] The present inventors further demonstrated by immuno staining and FACS analysis that the cells co-express Olig2 and SoxlO by the end of step 3 and 4 (Figure 21). Similarly with the coexpression of Olig2 and Nkx2.2 there is a significant increase in the expression of these markers by the end of step 4 compared to the end of step 3. Using immunocytochemical studies, it was shown that after step 3 (OPCs), only sparse cells expressed 04 and 01, however, at the end of step 4 the expression of these markers was significantly increased (Figure 20C-20F). Based on these results, OPCs were defined as cells positive for the expression of Olig2 and Nkx2.2, and negative for 04 and 01. Moreover, it was shown by immunostaining that OPCs and pre-myelinating cell populations are heterogeneous, including also astrocytes and neurons (Figure 22). In addition, expression of 04 was shown to be significantly different if analyzed by immunocytochemical staining or analyzed by FACS (Figure 23). Finally, the co-expression of PSA-NCAM and A2B5 was analyzed at all steps (step 1-4) of the differentiation protocol using FACS, demonstrating that the majority of the cells along the differentiation process are from neural origin (Figure 24).

[0295] Transplantation studies were performed to determine the potential of OPCs to migrate, mature and exert a functional therapeutic effect. Following OPC transplantation to neonatal mice vast OPC migration was observed in their brains after 6 weeks (Figure 16A-16F). The transplanted human cells acquired differentiated oligodendrocyte morphology and their extensions were closely associated with MBP+ myelinated axons (Figure 16G-16H). Following OPC transplantation at the chronic progressive stage of experimental autoimmune encephalomyelitis (EAE) in adult Biozzi ABH mice, the most authentic model of MS (Figure 17A-17B), vast migration of human OPC was observed in the Cerebellum, brainstem and spinal cord (Figure 17C). The human cells acquired differentiated oligodendrocyte morphology and their extensions were associated with MBP+ myelinated axons, and neurofilament+ axons (Figure 17D-17H). Furthermore, transplanted OPC that reported the expression of MBP by RFP, also migrated in the brain and expressed RFP-MBP, indicating a mature oligodendrocyte state (Figure 18A-18B). Clinical scoring showed significant improvement in OPC-transplanted chronic EAE mice, as compared to sham-transplanted mice (Figure 19).

[0296] In an additional experiment, stage 3 OPCs and stage 4 premyelinating oligodendrocytes (PMOs) were transplanted to newborn mice at age 2 days, and immune-fluorescence staining was performed after 6 weeks.

[0297] A shown in Figure 25A, mature oligodendrocyte morphology of human mitochondria and cells in gray matter are evident. The image shows a section from a stage 3 OPC -transplanted brain. Similar images were obtained from stage 4 PMO -transplanted brains. As shown in Figure 25B, a mature oligodendrocyte morphology of human mitochondria + cells in white matter are evident. Note the typical chain of oligodendrocytes and typical directionality of cells. The image shows a section from a stage 3 OPC -transplanted brain. Similar images were obtained from stage 4 PMO -transplanted brains. As illustrated in Figure 25C, no human mitochondria and cells stained for the Astrocytic marker GFAP.

[0298] Human mitochondria and cell extensions associated with MBP and myelin, suggesting these are myelinating human oligodendrocytes (Figures 25D-25E). These images were obtained from stage 4 PMO -transplanted brains, and similar images were obtained from stage 3 OPC -transplanted brains. In another experiment, OPCs and PMOs were transplanted into 2-day old neonatal Biozzi ABH mice. Two transplantations were performed of 1 pl to each side, depth 1mm, injected on either side between eye location and bregma 0. DAPI staining in coronal view at bregma 0 (scale bar = 200 pm), with boxes of areas in which images of zooming in is shown in Figure 26A for OPCs. Immunofluorescent identification of Human Mitochondria+ (H. Mit) (a human specific mitochondrial marker) of stage 3 Olig2 NKx2.2 GPC found throughout the bregma 0 coronal section with labeled anatomical locations (scale bar = 30 pm) as shown in Figures 26B-F.

[0299] DAPI staining in coronal view at bregma 0 (scale bar = 200 pm), with boxes of areas in which images of zooming in is shown in Figure 27A for PMOs. Immunofluorescent identification of Human Mitochondria+ (H. Mit) (a human specific mitochondrial marker) of stage 4 04 PMO found throughout the bregma 0 coronal section with labeled anatomical locations (scale bar = 30 pm), as shown in Figures 27B-27F.

[0300] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0301] It is the intent of the applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is / are hereby incorporated herein by reference in its / their entirety.

[0302] In addition, any priority document(s) of this application is / are hereby incorporated herein by reference in its / their entirety.

Claims

WHAT IS CLAIMED IS:

1. A method of generating oligodendroglial progenitor cells (OPCs) comprising: (a) culturing human pluripotent stem cells or human formative pluripotent stem cells in a first medium comprising FGF2, a BMP antagonist in suspension under conditions that generate spheres which co-express at least two markers of neural precursor cells, as measured by FACs;(b) culturing said spheres in suspension in a second medium comprising FGF2, retinoic acid, a SHH agonist and optionally a BMP antagonist, under conditions that generate spheres expressing Olig2, as measured by FACS; and(c) culturing cells of said spheres expressing Olig2 in a third medium comprising an SHH agonist, FGF2, NT3 and ascorbic acid under conditions that generate a population of cells that comprises OPCs which co-express Olig2 and Nkx2.2, as measured by FACS, thereby generating the OPCs.

2. The method of claim 1, wherein said OPCs are 04- and O1-, as measured by immunostaining.

3. The method of claim 1, wherein said OPCs co-express Olig2 and SOXIO, as measured by immuno staining.

4. The method of claim 1, wherein said second medium is said first medium supplemented with said retinoic acid and said SHH agonist.

5. The method of claim 1, further comprising culturing said human pluripotent stem cells in a pre-differentiating medium comprising FGF2, a BMP antagonist and a rock inhibitor for 12-72 hours prior to step (a).

6. The method of claim 1, wherein said first medium is devoid of a rock inhibitor.

7. The method of claim 1, wherein said first medium is devoid of an activin / TGFB antagonist.

8. The method of claim 1, wherein said first medium comprises an activin / TGFB antagonist.

9. The method of any one of claims 1-6, wherein step (a) is effected for at least one week.

10. The method of any one of claims 1-9, wherein said BMP antagonist is selected from the group consisting of LDN and noggin.

11. The method of any one of claim 1-10, wherein said markers of neural precursor cells are selected from the group consisting of PSA-NCAM and A2B5.

12. The method of any one of claims 1-11, wherein said SHH agonist is purmorphamine.

13. The method of any one of claims 1-12, wherein step (b) is effected for about 3 weeks.

14. The method of any one of claims 1-13, wherein step (c) is effected on an adherent surface.

15. The method of any one of claims 1-13, wherein step (c) is effected as a suspension culture.

16. The method of any one of claims 1-15, wherein said first medium comprises NUTRISTEM™.

17. The method of any one of claims 1-15, wherein said second medium, said third medium comprises BNN.

18. The method of any one of claims 1-15, wherein said first and second media comprise NSC.

19. The method of any one of claims 1-15 and 18, wherein said third medium comprises BNN or ASTO.

20. The method of any one of claims 1-19, wherein steps (a) and (b) are carried out under dynamic culture conditions.

21. The method of claim 15, wherein step (c) is carried out under dynamic culture conditions.

22. The method of claims 20 or 21, wherein said dynamic culture conditions are effected using a rotating bioreactor.

23. The method of any one of claims 1-21, wherein said pluripotent stem cells comprise embryonic stem cells.

24. The method of any one of claims 1-21, wherein said human formative pluripotent stem cells are differentiated ex vivo from pluripotent stem cells.

25. The method of any one of claims 1-21, wherein said pluripotent stem cells comprise induced pluripotent stem cells (iPSCs).

26. The method of any one of claims 1-25, wherein said population of cells further comprises neurons and astrocytes.

27. The method of any one of claims 1-26, wherein a majority of the cells of said population are neural cells.

28. The method of any one of claims 1-27, wherein about 10 % of the cells of said population are OPCs.

29. The method of any one of claims 1-28, further comprising isolating said OPCs from said population of cells following step (c).

30. A method of generating pre-myelinating human oligodendrocytes comprising: (a) generating a population of cells that comprise oligodendroglial progenitor cells according to the method of any one of claims 1-25; and(b) culturing said oligodendroglial progenitor cells in a fourth medium comprising ascorbic acid, NT3, T3, IGF1 and optionally a BMP antagonist under conditions that generate cells co-expressing at least two markers of pre-myelinating oligodendrocytes, as measured by FACS, thereby generating the pre-myelinating human oligodendrocytes.

31. The method of claim 30, wherein said culturing said oligodendroglial progenitor cells is effected on an adherent surface.

32. The method of any one of claims 30-31 wherein said at least two markers of premyelinating oligodendrocytes comprise 04 and 01.

33. The method of any one of claims 30-32, wherein said BMP antagonist comprised in said fourth medium is noggin.

34. The method of any one of claims 30-33, wherein said cells co-expressing at least two markers of pre-myelinating oligodendrocytes do not express markers of myelinating oligodendrocytes.

35. The method of claim 34, wherein said markers of myelinating oligodendrocytes are selected from the group consisting of MBP.

36. The method of any one of claims 30-35, wherein said fourth medium comprises BNN or ASTO.

37. An isolated population of cells obtainable according to the method of any one of claims 1-36.

38. A pharmaceutical composition comprising the isolated population of cells of claim 37 and a pharmaceutically acceptable carrier.

39. The isolated population of cells of claim 37 for use in treating a demyelinating disease or disorder.

40. A method of treating a demyelinating disease or disorder in a subject in need thereof, comprising transplanting to the subject a therapeutically effective amount of the isolated population of cells of claim 37, thereby treating the demyelinating disease or disorder.

41. The method of claim 40, wherein said demyelinating disease is selected from the group consisting of multiple sclerosis (MS), spinal cord injury, stroke, white matter stroke, neuromyelitis optica, MOG-antibody associated demyelinating disease (MOGAD), guillain-barre syndrome, diffuse disseminated encephalomyelitis, acute disseminated encephalomyelitis, concentric sclerosis, diffuse sclerosis, leukodystrophy, leukoencephalopathy caused by ischemiahypoxia, central pontine myelination, acute inflammatory demyelinating polyneuropathy, chronic inflammatory demyelinating polyneuropathy, subacute combined degeneration caused by nutritional deficiency diseases, subacute sclerosing panencephalitis or progressive multifocal leukoencephalopathy caused by viral infection, diabetic neuropathy, neuropathy of systemic lupus erythematosus, leukoablative diseases, vascular and age-related white matter loss of small vessel disease (commonly associated with subcortical dementias of advanced age), Alexander disease, vanishing white matter disease, adrenoleukodystrophy and perlescent-merzbacher disease, lysosomal storage disorders, Huntington's disease, frontotemporal dementia, childhood-onset schizophrenia, Radiation and chemotherapy-induced CNS injury.

42. The method of claim 40, wherein said demyelinating disease is MS.

43. The method of any of claims 40-42, wherein said transplanting is via the cerebral white matter tracts.

44. A culture medium comprising:NUTRISTEM™ (minus GF), human serum albumin, N2 and B27.

45. The culture medium of claim 44, further comprising biotin and forskolin.

Images