Methods and compositions for producing oligodendrocyte progenitor cells

Abstract

Provided are methods of producing preoligodendrocyte progenitor cells (pre-OPCs), oligodendrocyte progenitor cells (OPCs), and preOLs) from human pluripotent stem cells using a chemically defined culture medium, which allow the production of pre-OPCs in only 3 days, the production of SOX10 + OLIG2 + NKX2-2 + OPCs in only 12 days, and the production of CD9 + A2B5 + O4 + CNPase + preOLs in only 18 days. Two alternative culture protocols for producing OPCs are provided. Media, isolated cell populations, and kits are also provided.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Application Nos. 63 / 396,073, filed on August 8, 2022, and 63 / 400,222, filed on August 23, 2022, the entire contents of the prior applications are incorporated herein by reference.

[0003] Government licensing rights

[0004] This application was made with government support under Grant No. W911NF-17-3-0003 awarded by the U.S. Army ACC-AGP-RTP. The government has certain rights in this invention. Background Art

[0005] Oligodendrocytes (OLs) are a type of glial cell that synthesizes the myelin sheath around axons. They are therefore essential for neurotransmission in the central nervous system (CNS). A greater understanding of oligodendrocyte biology may be important for developing therapeutics to treat neurodegenerative conditions, including demyelinating diseases such as multiple sclerosis and leukodystrophies, as well as amyotrophic lateral sclerosis (ALS), which may involve demyelination in later stages of the disease. In addition, radiation therapy to the brain may be associated with side effects of oligodendrocyte depletion, leading to cognitive decline and / or impaired motor coordination.

[0006] Since mature human oligodendrocytes are not easy to be separated from human subjects, human oligodendrocyte cell lines have been developed to allow the study of these cells. However, immortalized cell lines may not be able to simulate the entire biology of natural cells and are not suitable for therapeutic use. Therefore, the ability to produce (e.g., from stem cells) human oligodendrocytes in vitro is highly anticipated. Various schemes for differentiating oligodendrocytes from human pluripotent stem cells have been reported. However, these schemes are still inefficient and variable in terms of oligodendrocyte yield, and require a long differentiation time to produce oligodendrocytes positive for myelin basic protein (MBP).

[0007] Early protocols used a four-step approach (Hu et al. (2009) Nature Protocols 4:1614-1622; see also Wang et al. (2013) Cell Stem Cell 12:252-264). The protocol first involved inducing human embryonic stem cells (hESCs) to differentiate into neuroepithelial cells for two weeks, forming neural tube-like rosettes, and then treating them with retinoic acid (RA) and sonic hedgehog (SHH) for 10 days to become progenitor cells expressing OLIG2. Treatment with fibroblast growth factor (FGF2) for another 10 days resulted in conversion into pre-OPCs expressing OLIG2 and NKX2.2. Finally, pre-OPCs were cultured for an additional 8-9 weeks in the absence of FGF2 to differentiate into OPCs, which express markers such as platelet-derived growth factor receptor alpha (PDGFRα), SOX10, and NG2. Thus, using this protocol, it takes about 24 days to generate progenitor cells expressing OLIG2, and about 34 days to generate pre-OPCs expressing OLIG2 and NKX2.2, and about 100 days to obtain mature Ols. Douvaras et al. reported a variation of this protocol (Stem Cell Reports (2014) 3: 250-259), but it still takes about 20 days to obtain pre-OPCs and about 50 days to obtain OPCs, including culturing with exogenously added growth factors PDGF, IGF-1, and HGF.

[0008] Subsequently, alternative protocols were reported, but these still used a neural induction and patterning phase (also called neuralization) of approximately one week, followed by the induction of cells expressing pre-OPC and OPC markers using culture medium supplemented with exogenous growth factors (such as FGF2, PDGF, IGF-1 and / or HGF, depending on the experimental protocol) (see, e.g., Piao et al. (2015) Cell Stem Cell 16:198-210; Douvaras & Fossati (2015) Nature Protocols 10:1143-1154; Livesey et al. (2016) Stem Cells 34:1040-1053; and Yamashita et al. (2017) PLOS One 12:e0171947).

[0009] Recently, it was reported that a protocol that first generates neural progenitor cells (NPCs) by neural induction of hESCs, then overexpresses the SOX10 transcription factor in NPCs (via viral transduction) and expands them in the presence of bFGF, leads to the generation of MBP-positive oligodendrocytes in only about 20 days (Garcia-Leon et al. (2018) Stem Cell Reports 10:655-672). In addition, transient and partial inhibition of the SHH pathway transcription factor GLI1 in neural stem cells (generated by neuralization) by the small molecule inhibitor GANT61 was found to generate OPCs that are more migratory and can differentiate into myelin-producing oligodendrocytes earlier (Namchaiw et al. (2019) Stem Cell Res & Therapy 10:272).

[0010] Therefore, despite some progress, there remains a need for efficient and robust methods and compositions for generating oligodendrocyte progenitor cells from human pluripotent stem cells. Summary of the invention

[0011] The present application provides a method for producing human oligodendrocyte progenitor cells (OPCs) from pre-OPCs using a chemically defined medium, which allows the production of SOX10+OLIG2+NKX2.2+OPCs in only 9 days of culture starting from pre-OPCs. Pre-OPCs can be obtained from pluripotent stem cells in a chemically defined medium for three days, thereby providing a total of twelve days for the production of OPCs from pluripotent stem cells. The present application also provides a method for producing CD9+A2B5+O4+CNPase+ myelinating preoligodendrocytes from OPCs by further differentiating OPCs for six days in a chemical medium defined separately in the present application.

[0012] The present application provides two alternative culture protocols for generating OPCs from pre-OPCs, referred to herein as Version 1 and Version 2. Each of these protocols includes two stages (Stages 2 and 3); however, the initial protocol for generating pre-OPCs from pluripotent stem cells only includes a single stage (Stage 1); taken together, the entire process for generating OPCs is divided into three stages. Fig.42 and Fig.43 A non-limiting representative scheme for generating OPCs is schematically shown in . The present application also provides a culture scheme for generating premyelinating oligodendrocytes (preOLs) from OPCs using stage 4 culture medium. Fig.44 A representative stage 4 scheme for generating preOLs is shown schematically in FIG.

[0013] Each culture medium at different stages contains small molecule agents that can excite or antagonize the activity of specific signaling pathways in pluripotent stem cells, thereby promoting the differentiation of the OPC lineage, leading to cell maturation and the expression of OPC-related biomarkers. The methods of the present application have the advantage that they bypass the neural induction step of the prior art solutions and allow pluripotent stem cells to directly differentiate into pre-OPCs and OPCs, thereby significantly shortening the time required to generate pre-OPCs and OPCs. In addition, the use of small molecule agents in the culture medium allows precise control of the composition of the culture medium.

[0014] Therefore, in one aspect, the present application relates to a method for generating human SOX10+OLIG2+NKX2-2+ oligodendrocyte progenitor cells (OPCs), comprising:

[0015] (a) on days 0-3, culturing human OLIG2+ pre-oligodendrocyte progenitor cells (pre-OPCs) in a culture medium to obtain a cell population, wherein the culture medium comprises an FGFR pathway agonist, an mTOR pathway antagonist, an SHH pathway agonist, an AKT pathway antagonist, and an AKT pathway agonist; and

[0016] (b) On days 3-9, culturing the cell population from step (a) in a culture medium containing an FGFR pathway agonist, an activin receptor (AR) pathway agonist, a PDGFR pathway agonist, an AKT pathway antagonist, a retinoic acid (RA) pathway agonist, an AMPK pathway agonist, and an mTOR pathway agonist to produce SOX10+OLIG2+NKX2-2+OPCs.

[0017] The above method corresponds to the culture medium of stage 2 and stage 3 of the version 1 protocol. In another embodiment, the method further comprises culturing human pluripotent stem cells in a culture medium comprising a retinoic acid (RA) pathway agonist, an Akt pathway agonist, an mTOR pathway agonist, a WNT pathway antagonist, an SHH pathway agonist, a BMP pathway antagonist, and a PKC pathway antagonist, and obtaining human OLIG2+pre-OPCs on days -3 to 0 (i.e., culturing pluripotent stem cells for three days to obtain pre-OPCs, and then differentiating these pre-OPCs into pre-OPCs). This corresponds to the stage 1 culture medium for obtaining pre-OPCs.

[0018] In one embodiment, the FGFR pathway agonist is FGF2. In one embodiment, the FGFR pathway agonist is FGF2, which is present in the culture medium of step (a) and step (b) at a concentration of 10 ng / ml. Other suitable FGFR pathway agonists and concentration ranges are disclosed herein.

[0019] In one embodiment, the mTOR pathway antagonist is AZD 3147. In one embodiment, the mTOR pathway antagonist is AZD 3147, which is present in the culture medium at a concentration of 15 nM. Other suitable mTOR pathway antagonists and concentration ranges are disclosed herein.

[0020] In one embodiment, the SHH pathway agonist is Purmorphamine. In one embodiment, the SHH pathway agonist is Purmorphamine, which is present in the culture medium at a concentration of 500 nM. Other suitable SHH pathway agonists and concentration ranges are disclosed herein.

[0021] In one embodiment, the Akt pathway antagonist is MK2206. In one embodiment, the Akt pathway antagonist is MK2206, which is present in the culture medium of step (a) and step (b) at a concentration of 125 nM. Other suitable Akt pathway antagonists and concentration ranges are disclosed herein.

[0022] In one embodiment, the Akt pathway agonist is Sc79. In one embodiment, the Akt pathway agonist is Sc79, which is present in the culture medium at a concentration of 2 μM. Other suitable Akt pathway agonists and concentration ranges are disclosed herein.

[0023] In one embodiment, the AR pathway agonist is activin A. In one embodiment, the AR pathway agonist is activin A, which is present in the culture medium at a concentration of 10 ng / ml. Other suitable AR pathway agonists and concentration ranges are disclosed herein.

[0024] In one embodiment, the PDGFR pathway agonist is PDGF-AA. In one embodiment, the PDGFR pathway agonist is PDGF-AA, which is present in the culture medium at a concentration of 10 ng / ml.

[0025] In one embodiment, the RA pathway agonist is TTNPB. In one embodiment, the RA pathway agonist is TTNPB, which is present in the culture medium at a concentration of 50 nM. Other suitable RA pathway agonists and concentration ranges are disclosed herein.

[0026] In one embodiment, the AMPK pathway agonist is AICAR. In one embodiment, the AMPK pathway agonist is AICAR, which is present in the culture medium at a concentration of 200 μM. Other suitable AMPK pathway agonists and concentration ranges are disclosed herein.

[0027] In one embodiment, the mTOR pathway agonist is MHY1485. In one embodiment, the mTOR pathway agonist is MHY1485, which is present in the culture medium at a concentration of 2 μM. Other suitable mTOR pathway agonists and concentration ranges are disclosed herein.

[0028] On the other hand, the present application provides a culture medium for obtaining oligodendrocyte progenitor cells (OPCs), which comprises an FGFR pathway agonist, an mTOR pathway antagonist, an SHH pathway agonist, an AKT pathway antagonist, and an AKT pathway agonist (corresponding to the culture medium of stage 2 of version 1). On the other hand, the present application provides a culture medium for obtaining oligodendrocyte progenitor cells (OPCs), which comprises an FGFR pathway agonist, an activin receptor (AR) pathway agonist, a PDGFR pathway agonist, an AKT pathway antagonist, a retinoic acid (RA) pathway agonist, an AMPK pathway agonist, and an mTOR pathway agonist (corresponding to the culture medium of stage 3 of version 1). Also provided is a separate cell culture of OLIG2+OPCs cultured in any of the above-mentioned culture media.

[0029] In another aspect, the present application relates to a method for generating human SOX10+OLIG2+NKX2-2+ oligodendrocyte progenitor cells (OPCs), comprising:

[0030] (a) on days 0-6, culturing human OLIG2+ pre-oligodendrocyte progenitor cells (pre-OPCs) in a culture medium containing an FGFR pathway agonist, an mTOR pathway antagonist, an SHH pathway agonist, and a WNT pathway agonist to obtain a cell population; and

[0031] (b) on day 6-9, culturing the cell population from step (a) in a culture medium comprising an FGFR pathway agonist, an IGF-1 pathway agonist, and a retinoic acid (RA) pathway agonist to produce SOX10+OLIG2+NKX2-2+OPCs.

[0032] The above method corresponds to the culture medium of stage 2 and stage 3 of the version 2 protocol. In another embodiment, the method further comprises obtaining human OLIG2+pre-OPCs from day -3 to day 0 (i.e., culturing pluripotent stem cells for three days to obtain pre-OPCs, and then differentiating these pre-OPCs into pre-OPCs) by culturing human pluripotent stem cells in a culture medium comprising a retinoic acid (RA) pathway agonist, an Akt pathway agonist, an mTOR pathway agonist, a WNT pathway antagonist, an SHH pathway agonist, a BMP pathway antagonist, and a PKC pathway antagonist. This corresponds to the stage 1 culture medium for obtaining pre-OPCs.

[0033] In one embodiment, the FGFR pathway agonist is FGF2. In one embodiment, the FGFR pathway agonist is FGF2, which is present in the culture medium of step (a) and step (b) at a concentration of 10 ng / ml. Other suitable FGFR pathway agonists and concentration ranges are disclosed herein.

[0034] In one embodiment, the mTOR pathway antagonist is AZD 3147. In one embodiment, the mTOR pathway antagonist is AZD 3147, which is present in the culture medium at a concentration of 100 nM. Other suitable mTOR pathway antagonists and concentration ranges are disclosed herein.

[0035] In one embodiment, the SHH pathway agonist is purple morphine. In one embodiment, the SHH pathway agonist is purple morphine, which is present in the culture medium at a concentration of 500 nM. Other suitable SHH pathway agonists and concentration ranges are disclosed herein.

[0036] In one embodiment, the WNT pathway agonist is CHIR99021. In one embodiment, the WNT pathway agonist is CHIR99021, which is present in the culture medium at a concentration of 1 μM. Other suitable WNT pathway agonists and concentration ranges are disclosed herein.

[0037] In one embodiment, the IGF-1 pathway agonist is IGF-1. In one embodiment, the IGF-1 pathway agonist is IGF-1, which is present in the culture medium at a concentration of 10 ng / ml. Other suitable IGF-1 pathway agonists and concentration ranges are disclosed herein.

[0038] In one embodiment, the RA pathway antagonist is AGN 193109. In one embodiment, the RA pathway antagonist is AGN 193109, which is present in the culture medium at a concentration of 100 nM. Other suitable RA pathway antagonists and concentration ranges are disclosed herein.

[0039] On the other hand, the present application provides a culture medium for obtaining oligodendrocyte progenitor cells (OPCs), which comprises an FGFR pathway agonist, an mTOR pathway antagonist, an SHH pathway agonist, and a WNT pathway agonist (corresponding to the culture medium of version 2 stage 2). On the other hand, the present application provides a culture medium for obtaining oligodendrocyte progenitor cells (OPCs), which comprises an FGFR pathway agonist, an IGF-1 pathway agonist, and a retinoic acid (RA) pathway agonist (corresponding to the culture medium of version 2 stage 3). Also provided is a separate cell culture of OLIG2+OPCs cultured in any of the above-mentioned culture media.

[0040] On the other hand, the present application relates to compositions and methods of a stage 4 protocol for producing premyelinating oligodendrocytes (preOLs) from OPCs. In one embodiment, the present application relates to a method for producing CD9+A2B5+O4+CNPase+ premyelinating oligodendrocytes (preOLs), comprising culturing SOX10+OLIG2+NKX2-2+OPCs in a culture medium to produce CD9+A2B5+O4+CNPase+preOLs, wherein the culture medium comprises an IGF1R pathway agonist, a TrkC pathway agonist, a PDGFR pathway agonist, a thyroid hormone receptor agonist, and an insulin receptor agonist. In one embodiment, the OPCs are cultured in the culture medium for six days to produce preOLs.

[0041] In one embodiment, the IGF1R pathway agonist is selected from the group consisting of IGF-1, IGF-2, insulin, Rg5, IGF-1 30-41, demethyl chrysanthemum quinone B1, IGF1-Ado, X10, mecasermin, and combinations thereof. In one embodiment, the IGF1R pathway agonist is IGF-1. In one embodiment, IGF-1 is present in the culture medium at a concentration of 10 ng / ml.

[0042] In one embodiment, the TrkC pathway agonist is selected from the group consisting of neurotrophin-3 (NT-3), peptide mimetics based on the β-turn of NT-3, LM22B 10, GNF 5837, and combinations thereof. In one embodiment, the TrkC pathway agonist is NT-3. In one embodiment, NT-3 is present in the culture medium at a concentration of 10 ng / ml.

[0043] In one embodiment, the PDGFR pathway agonist is PDGF-AA. In one embodiment, PDGF-AA is present in the culture medium at a concentration of 10 ng / ml.

[0044] In one embodiment, the thyroid hormone receptor agonist is selected from the group consisting of T3, T4, Resmetirom, TRb agonist 3 (Compound 3), Sobetirome, Tilatracol, and combinations thereof. In one embodiment, the thyroid hormone receptor agonist is T3. In one embodiment, T3 is present in the culture medium at a concentration of 50 nM.

[0045] In one embodiment, the insulin receptor agonist is selected from the group consisting of insulin, IGF-1, IGF-2, demethyl chrysanthemum quinone B1, MK-5160, MK-1092, and combinations thereof. In one embodiment, the insulin receptor agonist is insulin. In one embodiment, insulin is present in the culture medium at a concentration of 20 μg / ml.

[0046] On the other hand, the present application relates to a culture medium for stage 4 for producing preOLs. In one embodiment, the culture medium for stage 4 comprises an IGF1R pathway agonist, a TrkC pathway agonist, a PDGFR pathway agonist, a thyroid hormone receptor agonist, and an insulin receptor agonist. In one embodiment, the IGF1R pathway agonist is IGF-1, the TrkC pathway agonist is NT-3, the PDGFR pathway agonist is PDGF-AA, the thyroid hormone receptor agonist is T3, and the insulin receptor agonist is insulin. In one embodiment, the culture medium comprises 10 ng / ml of IGF-1, 10 ng / ml of NT-3, 10 ng / ml of PDGF-AA, 50 nM of T3, and 20 μg / ml of insulin.

[0047] The stage 4 medium can be used in combination with the stage 2 / stage 3 medium (version 1 or version 2) to produce preOLs from preOPCs. Therefore, in one embodiment of the version 1 scheme, the present application relates to a method for producing human CD9+A2B5+O4+CNPase+ premyelinating oligodendrocytes (preOLs), comprising:

[0048] (a) On days 0-3, human OLIG2+ pre-oligodendrocyte progenitor cells (pre-OPCs) were cultured in a culture medium containing an FGFR pathway agonist, an mTOR pathway antagonist, an SHH pathway agonist, an AKT pathway antagonist, and an AKT pathway agonist to obtain a cell population;

[0049] (b) on days 3-9, culturing the cell population from step (a) in a culture medium comprising an FGFR pathway agonist, an activin receptor (AR) pathway agonist, a PDGFR pathway agonist, an AKT pathway antagonist, a retinoic acid (RA) pathway agonist, an AMPK pathway agonist, and an mTOR pathway agonist to produce SOX10+OLIG2+NKX2-2+OPCs; and

[0050] (c) on days 9-15, culturing the cell population from step (b) in a culture medium comprising an IGF1R pathway agonist, a TrkC pathway agonist, a PDGFR pathway agonist, a thyroid hormone receptor agonist, and an insulin receptor agonist to produce CD9+A2B5+O4+CNPase+preOLs.

[0051] In one embodiment of the scheme of version 2, the present application relates to a method for generating human CD9+A2B5+O4+CNPase+ premyelinating oligodendrocytes (preOLs), comprising:

[0052] (a) On days 0-6, human OLIG2+ pre-oligodendrocyte progenitor cells (pre-OPCs) were cultured in a culture medium containing an FGFR pathway agonist, an mTOR pathway antagonist, an SHH pathway agonist, and a WNT pathway agonist to obtain a cell population;

[0053] (b) on day 6-9, culturing the cell population from step (a) in a culture medium comprising an FGFR pathway agonist, an IGF-1 pathway agonist, and a retinoic acid (RA) pathway agonist to produce SOX10+OLIG2+NKX2-2+OPCs; and

[0054] (c) on days 9-15, culturing the cell population from step (b) in a culture medium comprising an IGF1R pathway agonist, a TrkC pathway agonist, a PDGFR pathway agonist, a thyroid hormone receptor agonist, and an insulin receptor agonist to produce CD9+A2B5+O4+CNPase+preOLs.

[0055] In another embodiment, the above method combining stages 2, 3 and 4 further comprises obtaining human OLIG2+ pre-OPCs by culturing human pluripotent stem cells in a culture medium from day -3 to day 0, the culture medium comprising a retinoic acid (RA) pathway agonist, an Akt pathway agonist, an mTOR pathway agonist, a WNT pathway antagonist, an SHH pathway agonist, a BMP pathway antagonist and a PKC pathway antagonist (i.e., culturing pluripotent stem cells for three days to obtain pre-OPCs, and then differentiating these pre-OPCs into pre-OPCs). This corresponds to the stage 1 culture medium for obtaining pre-OPCs, thereby providing a method for combining the schemes of stages 1, 2, 3 and 4 to produce preOLs.

[0056] In one embodiment, the human pluripotent stem cells are induced pluripotent stem cells (iPSCs). In another embodiment, the human pluripotent stem cells are embryonic stem cells.

[0057] In one embodiment, the human pluripotent stem cells attach to a vitronectin-coated plate during culture.

[0058] Other features and advantages of the present application will become apparent from the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 Results of the HD-DoE model for an 8-factor experiment optimized for maximum NKX2-2 expression are shown. The upper part of the model shows the predictions for the expression levels of the 53 genes preselected for NKX2-2 optimization. The lower part of the model shows the effectors tested in the model and their contribution to the maximum expression of NKX2-2. The value columns refer to the concentration of each effector required to simulate the model. Figure 2 Results of the HD-DoE model for an 8-factor experiment optimized for maximizing PDGFRA expression are shown. Figure 1 This situation emphasizes that the effector PD0325901 with a factor contribution of 30.05 is an important input for high PDGFRA expression.

[0060] Figure 3 Dynamic profiles of NKX2-2, OLIG1, OLIG2, and PDGFRA gene expression levels relative to the concentrations of the eight effectors tested are shown. The positive effects of TTNPB, MHY1485, and PD0325901 on PDGFRA expression and their factorial contributions are represented by the slope of the plot for each effector. The dashed boxes emphasize the opposite effects of PD0325901 on NKX2-2 and OLIG2 compared to PDGFRA.

[0061] Figure 4 Results of a HD-DoE model of a 13-factor experiment optimized for maximizing OTX2 expression are shown. The model incorporates MK2206, PD0325901, CHIR99021, LDN193189, Go6983, and PD173074 as positive effectors of OTX2 expression.

[0062] Figure 5 Results of a HD-DoE model of a 13-factor experiment optimized for maximizing FEZF2 expression are shown. The model confirmed the positive effects of LDN193189, MK2206, and PD0325901 on cell patterning and introduced three additional factors, including SC79, XAV939, and purple morphine-500nM.

[0063] Figure 6Shown are dynamic profiles of OTX2 and FEZF2 expression levels relative to the concentrations of the 13 factors tested in this model. XAV939 and viomorphin-500 nM had significant positive effects on FEZF2 expression and no significant negative effects on OTX2 expression.

[0064] Figures 7A-7D Dynamic profiling of the elimination process in the 8-factor modeling experiment and its effects on the expression of NKX2-2, OLIG2, and PDGFRA are shown.

[0065] Figures 8A-8D Shown is a dynamic profiling analysis of the elimination process in the 13-factor modeling experiment and its effects on FEZF2 and OTX2 expression.

[0066] Fig. 9 Images of cells cultured in optimized OPC differentiation medium after three days are shown. Cells were stained with oligodendrocyte and neural biomarkers. Cells expressed anterior neuroectoderm biomarkers including OTX2 and NKX2-2 and OPC-specific biomarker OLIG2. NESTIN and PDGFRa, neuronal and late OPC biomarkers, respectively, were not expressed here. Expression of KI67 shows the proliferative state of precursor cells.

[0067] Figures 10A-10B RNA-seq data from cells cultured in optimized OPC differentiation medium after three days are shown. Expression levels of stem cell genes NANOG and POU5F1 are reduced, while expression levels of genes involved in early development of brain regions and the oligodendrocyte lineage are increased.

[0068] Fig.11 Results of a HD-DoE model for a 12-factor experiment optimized for maximizing OLIG2 expression are shown. The upper part of the model shows the predictions for the expression levels of the 53 genes preselected for OLIG2 optimization. The lower part of the model shows the effectors tested in the model and their contribution to the maximum expression of OLIG2. The value column refers to the concentration of each effector required to simulate the model.

[0069] Fig.12 Dynamic plots of the expression levels of NKX2-2, OLIG2, SOX10, OLIG1, and PDGFRa relative to the concentrations of the 12 effectors are shown. The positive effects of FGF-2 and MK2206 on OLIG2 expression and their factorial contributions are indicated by the slope of the plot for each effector.

[0070] Fig.13 Results of the HD-DoE model for a 12-factor experiment optimized for maximizing OLIG2 expression are shown. The upper and lower panels, and the values ​​are listed as Fig.11The model highlights the effectors purplemorphine, with a factor contribution of 13.03, and AGN193109, with a factor contribution of 18.3, as two important inputs for the maximum and minimum expression of OLIG2, respectively.

[0071] Fig.14 Results of a HD-DoE model applied to a 12-factor experiment on stage 1 pre-OPCs to generate a stage 2 differentiation recipe are shown. Upper and lower panels, and values ​​are listed as Fig.11 As shown. The model was optimized for maximum expression of OLIG2. The model emphasized the role of morpholino in OLIG2 expression with a factor contribution of 16.9.

[0072] Fig.15 is a factor effect plot showing the expression levels of NKX2-2, OLIG2, OLIG1, and SOX10 relative to purple morphine concentration in two separate 12-factor models.

[0073] Fig.16 Results of an HD-DoE model applied to stage 2 early oligodendrocyte progenitor cells to generate a stage 3 differentiation recipe are shown. Upper and lower panels, and values ​​are listed as Fig.11 The model highlights the positive effects of Activin A and TTNPB on OLIG1 expression, with factor contributions of 50.2 and 19.1, respectively.

[0074] Fig.17 The results of the HD-DoE model applied to an 8-factor experiment of the early OPCS in Phase 2 are shown. The upper and lower parts, and the values ​​are listed as Fig.11 The model is optimized for maximum expression of PDGFRa. The model highlights the effectors TTNPB, with a factor contribution of 71.6, and linoleic acid, with a factor contribution of 22.1, as two important inputs for maximum expression of PDGFRa.

[0075] Fig.18 Results of the HD-DoE model applied to an 8-factor experiment on early OPCs in Phase 2 are shown. The upper and lower panels, and the values ​​are listed as Fig.11 The model highlights the positive effect of TTNPB on SOX10 expression with a factor contribution of 8, whereas all other factors have negative regulatory influences.

[0076] Fig.19 Dynamic plot analysis of the expression levels of OLIG1, PDGFRa, and SOX10 relative to the concentrations of the eight factors is shown. The positive effects of TTNPB on the expression of PDGFRa and OLIG1 and their factor contributions are indicated by the slope of the plot for each effector.

[0077] Fig. 20Results of a HD-DoE model for a 12-factor experiment applied to early stage 2 OPCs to generate a recipe for stage 3 differentiation are shown. Upper and lower panels, and values ​​are listed as Fig.11 The model is optimized for maximum expression of SOX10. The model highlights the role of MK2206 and MHY1485 in SOX10 expression, with factor contributions of 16.2 and 11.5, respectively.

[0078] Fig.21 Results of a HD-DoE model for a 12-factor experiment applied to early stage 2 OPCs to generate a recipe for stage 3 differentiation are shown. Upper and lower panels, and values ​​are listed as Fig.11 As shown. The model is optimized for maximum expression of CSPG4. The model highlights the role of TTNPB and Activin A in CSPG4 expression, with factor contributions of 15.9 and 9.3, respectively.

[0079] Fig. 22 A kinetic plot analysis of the expression levels of OLIG1, PDGFRa and SOX10 versus the concentration of 4 of the 12 factors is shown. The positive effects of MK2206 and DBZ on SOX10 expression levels and their factor contributions are indicated by the slope of the plot for each effector.

[0080] Fig.23 A factor effect diagram is shown, showing the expression levels of OPC genes (including OLIG1, PDGFRa, SOX10) and neuronal genes (including NEUROD1, NEUROG1, and NEUROG2) relative to DBZ concentration. As DBZ concentration increased, the expression of both groups of genes also increased.

[0081] Fig.24 A factor influence diagram is shown, which shows the expression level of late OPC genes (including BCAN, CNP and ID2) relative to AICAR concentration. As AICAR concentration increases, the expression of both groups of genes also increases.

[0082] Figures 25A-25C Shown are dynamic profiles of the expression levels of NKX2-2, OLIG2, SOX10, and OLIG1 relative to the concentrations of the four final effectors in the stage 2 differentiation medium formulation. Fig.25A The expression levels of target genes in the presence of all final effectors are shown. Fig.25B Shown are the expression levels of target genes in the absence of one final effector at a time and in the presence of the other effectors. Fig.25C The expression levels of target genes in the presence and absence of the remaining final factor (purple morphine) are shown.

[0083] Figures 26A-26BShown are dynamic profiles of OLIG1, PDGFRa, and SOX10 expression levels relative to the two final effector concentrations in the stage 3 differentiation medium formulation. Fig.26A The expression levels of the target genes in the presence of both effectors are shown. Fig.26B The expression levels of target genes in the absence of one final effector at a time and in the presence of another are shown.

[0084] Figures 27A-27B Shown are dynamic profiles of the expression levels of OLIG1, PDGFRa, SOX10, and SOX8 relative to the four final effector concentrations in the stage 3 differentiation medium formulation. Fig.27A The expression levels of target genes in the presence of four final effectors are shown. Fig.27B The expression levels of target genes in the absence of one final effector at a time and in the presence of the other effectors are shown.

[0085] Fig.28 A photograph showing a fluorescent image of hiPSC-derived early oligodendrocyte progenitor cells at the end of stage 2. The cells were stained with OPC biomarkers including NKX2-2, OLIG2, SOX10, and PDGFRa, the pan-neuronal biomarker TUBB3, and the proliferation marker KI67. At this stage, the cells were positive for all markers expected for an early progenitor state.

[0086] Fig.29 A photograph showing a fluorescent image of oligodendrocyte progenitor cells at the end of stage 3. The cells were stained with OPC markers including OLIG2, PDGFRa, SOX10, NG2, and A2B5. At this stage, the cells were positive for all expected biomarkers and their oligodendrocyte identity was confirmed.

[0087] Fig.30 Results of an HD-DoE model for an 8-factor experiment optimized for maximal OLIG2 expression are shown. The upper part of the model shows the predictions for the expression levels of 53 genes preselected for OLIG2 optimization. The lower part of the model shows the effectors tested in the model and their contribution to maximal OLIG2 expression. The Value column refers to the concentration of each effector required to simulate the model.

[0088] Fig.31 Results of an HD-DoE model for an 8-factor experiment optimized for maximizing OLIG1 expression are shown. The model highlights the effectors AZD3147, with a factor contribution of 26.76, and purple morphine, with a factor contribution of 22.75, as two important inputs for maximal OLIG1 expression.

[0089] Fig.32A dynamic plot analysis of the expression levels of NKX2-2, OLIG2, SOX10, and OLIG1 relative to the concentrations of the eight effectors is shown. The positive effects of AZD3147 and CHIR99021 on OLIG2 expression and their factorial contributions are represented by the slope of the plot for each effector.

[0090] Fig.33 Results of an HD-DoE model for an 8-factor experiment optimized for maximizing SOX10 expression are shown. The model highlights the effectors TTNPB, with a factor contribution of 17.98, and FGF-2, with a factor contribution of 15.3, as two important inputs for maximal expression of SOX10.

[0091] Fig.34 Results of an HD-DoE model for an 8-factor experiment optimized for maximizing PDGFRa expression are shown. The model highlights the effectors morphine, with a factor contribution of 20.61, and IGF-1, with a factor contribution of 11.2, as two important inputs for maximal expression of SOX10.

[0092] Fig.35 Factor effect plots showing the relationship between the expression level of SOX10 and the concentrations of IGF-1, FGF-2, and AGN193109 in the 12-factor model (left model) and the concentrations of IGF-1 and FGF-2 in the 8-factor model (right model) are shown.

[0093] Fig.36 Contour plots of simulated OLIG1 expression levels relative to IGF-1, FGF-2, TTNPB, and AGN193109 concentrations in the 8-factor model excluding all other factors are shown. The concentration of AGN193109 is shown on the upper axis, TTNPB on the right axis, IGF-1 on the left axis, and FGF-2 on the lower axis. The expression level of OLIG1 is shown in a spectrum from the highest level in red to the lowest level in blue. The space at the bottom right shows the highest expression level in the presence of IGF-1, FGF-2, and AGN193109.

[0094] Fig.37 Factor effect plots are shown, showing the expression levels of late OPC genes (including BCAN, CSPG4, ID2, and FYN) and neuronal genes (including NEUROG1 and NEUROD1) relative to IGF-1 concentration. As IGF-1 concentration increases, the expression levels of target OPC genes increase, while the expression levels of neuronal genes decrease.

[0095] Fig.38Factor effect plots are shown, showing the expression levels of late OPC genes (including CSPG4, ID2, IGFBP2, and SOX8) and neuronal genes (including NEUROG1 and NEUROD1) relative to FGF-2 concentration. As FGF-2 concentrations increased, the expression levels of target OPC genes increased, while the expression levels of neuronal genes decreased.

[0096] Fig.39 Factor effect plots showing the expression levels of late OPC genes (including BCAN, ID2, FYN, CNP, PLP1, and SOX8) and neuronal genes (including NEUROG1, NEUROG2, and NEUROD1) relative to TTNPB concentrations are shown. As TTNPB concentrations increased, OPC gene expression levels decreased, while neuronal gene expression levels increased.

[0097] Fig.40 A photograph showing fluorescent images of hiPSC-derived early oligodendrocyte progenitor cells at the end of stage 2 is shown. The cells were stained with OPC biomarkers including NKX2-2, OLIG2, SOX10, A2B5, and PDGFRa and the proliferation marker KI67. At this stage, the cells were positive for all markers expected for an early progenitor state.

[0098] Fig.41 A photograph showing a fluorescent image of oligodendrocyte progenitor cells at the end of stage 3 is shown. Cells were stained with OPC markers including OLIG2, NKX2-2, PDGFRa, SOX10, NG2, CNP, and O4 and the pan-neuronal marker TUBB3. At this stage, cells were positive for all expected biomarkers and their oligodendrocyte identity was confirmed.

[0099] Fig.42 is a schematic diagram of a representative example of the three-stage protocol described herein for deriving oligodendrocyte progenitor cells from pluripotent stem cells, where version 1 of the protocol is used for stages 2 and 3.

[0100] Fig.43 is a schematic diagram of a representative example of the three-stage protocol described herein for deriving oligodendrocyte progenitor cells from pluripotent stem cells, wherein the version 2 protocol is used for stages 2 and 3.

[0101] Fig.44 is a schematic diagram of a representative example of the stage 4 protocol for generating premyelinating OLs from OPCs.

[0102] Fig.45Results of an HD-DoE model for an 8-factor experiment optimized for maximum CNP expression are shown. The upper part of the model shows the predictions for the expression levels of 53 genes preselected for CNP optimization. The lower part of the model shows the effectors tested in the model and their contribution to maximum expression of CNP. The Value column refers to the concentration of each effector required to simulate the model.

[0103] Fig.46 Results of a HD-DoE model for a 12-factor experiment optimized for maximizing CNP expression are shown. This case highlights the effectors NT-3, with a factor contribution of 13.3, and biotin, with a factor contribution of 16.5, as two important inputs for maximum and minimum expression of CNP, respectively.

[0104] Fig.47 Results from a HD-DoE model of a 12-factor experiment optimized for maximizing CNP expression are shown. When CNP is maximally expressed, multiple other oligodendrocyte genes including APOD, BCAN, MYT1, and PLP1 are also upregulated, as shown in the red boxes. At the same time, ID2 and MKI67, which are expressed at the oligodendrocyte progenitor stage, are downregulated, as shown in the blue boxes.

[0105] Fig.48 Results of a HD-DoE model for a 12-factor experiment optimized for maximizing PLP1 expression are shown. This case highlights the effectors insulin, with a factor contribution of 13.2, and AICAR, with a factor contribution of 11.8, as two important inputs for maximum and minimum expression of PLP1, respectively.

[0106] Fig.49 Dynamic plots of the expression levels of CNP, PLP1, NeuroD1, NeuroG1, and NeuroG2 relative to the concentrations of the 12 effectors are shown when PLP1 is maximally expressed. The positive effects of GSI-XX on neuronal gene expression and their factor contributions are represented by the slope of the plot for each effector.

[0107] Fig.50 A factor effect plot is shown showing the expression levels of CNP, PLP1, PTGDS, MBP, KLK6, and NeuroG1 relative to T3 concentration in the 12-factor model. The slope of the plot indicates the positive effect of T3 on oligodendrocytes and its negative effect on neuronal genes.

[0108] Fig.51is a photograph showing fluorescent images of hiPSC-derived premyelinating oligodendrocytes at the end of stage 4. The cells were stained with OL biomarkers including A2B5, CNP, O4, PLP1, MBP, OLIG2, and PDGFRa, the pan-neuronal biomarker TUBB3, and the proliferation marker KI67. A few cells expressed the myelinating markers MBP and PLP1, while the majority of cells expressed CNP, O4, and A2B5. PDGFRa and KI67 were detected in some cells, indicating the presence of some oligodendrocyte progenitors in the culture.

[0109] Fig.52 Shown is the detection of CD9+ cells in hiPSC-derived myelinating pro-cells based on flow cytometry at differentiation day 18. CD9+ cells accounted for 81.5% of the total cell population. DETAILED DESCRIPTION OF THE INVENTION

[0111] Described herein are methods and compositions that allow the generation of SOX10+OLIG2+NKX2-2+OPCs from human pre-OPCs (which themselves can be generated by human pluripotent stem cells) using small molecule-based methods under chemically defined culture conditions. The OPCs can be further differentiated using the methods and compositions described herein to generate CD9+A2B5+O4+CNPase+preOLs under chemically defined culture conditions. The method of the present application has the advantage that the starting pluripotent stem cells do not undergo neural induction used by many prior art schemes. This allows the generation of OLIG2+pre-OPCs in only three days, which is much shorter than current schemes, which require an average of 10 days to generate pre-OPCs. Pre-OPCs can be further differentiated into SOX10+OLIG2+NKX2-2+OPCs in only 9 days, resulting in a total of 12 days required to generate OPCs from pluripotent stem cells. After 6 days of culture in appropriate culture media, preOLs can be obtained from OPCs, thereby obtaining preOLs from pluripotent stem cells in only 18 days, while other methods require significantly longer times (e.g., 50-70 days).

[0112] As described in Examples 1, 5, 8 and 10, a high-dimensional experimental design (HD-DoE) approach was used to simultaneously test the effects of multiple process inputs (e.g., small molecule agonists or antagonists) on output responses (e.g., gene expression). These experiments allow the identification of chemically defined culture media that contain agonists and / or antagonists of specific signaling pathways sufficient to produce pre-OPCs, OPCs or preOLs in a very short period of time. As described in Examples 2, 6 and 10, the optimized culture media were further validated by factor importance analysis, which examined the effect of eliminating a single agonist or antagonist. As described in Examples 3, 7, 9 and 11, immunohistochemistry further validated the phenotype of the cells produced by the differentiation strategy.

[0113] Using one of two alternatives described herein, pre-OPCs can be differentiated into OPCs. These alternatives are referred to herein as Version 1 (further described in Example 5) and Version 2 (further described in Example 8), and each scheme includes two stages. As used herein, the first culture medium for producing pre-OPCs from pluripotent stem cells in three days is referred to as Stage 1 culture medium, and the second and third culture medium for producing OPCs from pre-OPCs in another nine days is referred to as Stage 2 culture medium and Stage 3 culture medium respectively.

[0114] Fig.42 An exemplary 3-stage culture protocol for generating OPCs from pluripotent stem cells using version 1 protocol for stages 2 and 3 is schematically shown in FIG. As shown, stage 1 culture medium is used for three days (days 0-3), stage 2 culture medium is used for three days (days 3-6), and stage 3 culture medium is used for six days (days 6-12).

[0115] Fig.43 An exemplary 3-stage culture protocol for generating OPCs from pluripotent stem cells using version 2 protocol for stages 2 and 3 is schematically shown in FIG. As shown, the culture medium for stage 1 is used for three days (days 0-3), the culture medium for stage 2 is used for six days (days 3-9), and the culture medium for stage 3 is used for three days (days 9-12).

[0116] OPCs generated by the version 1 protocol or the version 2 protocol for phases 2 and 3 can be further differentiated into preOLs in the phase 4 protocol. Fig.44 An exemplary stage 4 culture protocol for generating preOLs from OPCs is schematically shown in Figure 1. As shown, stage 1 culture medium is used for three days (days 0-3), stage 2 and 3 culture medium is used for nine days (days 3-12), and stage 4 culture medium is used for six days (days 12-18).

[0117] Various aspects of the present application are described in further detail in the following subsections.

[0118] I. cell

[0119] The starting cell for producing pre-OPCs in the culture of the present application is a human pluripotent stem cell. As used herein, the term "human pluripotent stem cell" (abbreviated as hPSC) refers to a human stem cell with the ability to be differentiated into various different cell types. As used herein, the term "multipotent" refers to a kind of cell, which has the ability to differentiate into all three germ layers (endoderm, mesoderm and ectoderm) characteristic cell types under different conditions. The main feature of pluripotent cells is that they can, for example, be differentiated into all three germ layers by using nude mice and teratoma formation experiments. Pluripotency can also be shown by the expression of embryonic stem cell (ES) markers, although the preferred test of pluripotency is to prove the ability of the cell that is differentiated into each of the three germ layers.

[0120] Human pluripotent stem cells include, for example, induced pluripotent stem cells (iPSC) and human embryonic stem cells, such as ES cell lines. Non-limiting examples of induced pluripotent stem cells (iPSC) include 19-11-1, 19-9-7 or 6-9-9 cells (e.g., as described in Yu, J. et al. (2009) Science 324:797-801). Non-limiting examples of human embryonic stem cell lines include ES03 cells (WiCell Research Institute) and H9 cells (Thomson, JA et al. (1998) Science 282:1145-1147). Human pluripotent stem cells (PSCs) express cell markers that can be used to identify cells as PSCs. Non-limiting examples of pluripotent stem cell markers include TRA-1-60, TRA-1-81, TRA-2-54, SSEA1, SSEA3, SSEA4, CD9, CD24, OCT3, OCT4, NANOG and / or SOX2. Since the method of producing pre-OPCs and / or OPCs of the present application is used to differentiate (mature) the initial pluripotent stem cell population, in various embodiments, the pre-OPC and / or OPC cell population produced by the method of the present disclosure lacks the expression of one or more stem cell markers selected from the group consisting of TRA-1-60, TRA-1-81, TRA-2-54, SSEA1, SSEA3, SSEA4, CD9, CD24, OCT3, OCT4, NANOG and / or SOX2.

[0121] The starting cells used to generate OPCs in the culture of the present application are human pre-OPCs, such as pre-OPCs generated from pluripotent stem cells as described herein.

[0122] The pluripotent stem cells and pre-OPCs are subjected to the culture conditions of inducing cell differentiation as described herein. As used herein, the term "differentiation" refers to the cell development process from a more primitive stage to a more mature (i.e., not so primitive) cell, which generally shows the phenotypic characteristics of being committed to a specific cell lineage. An early progenitor cell that can be derived from people's PSC by neural induction (neuralization) is a neural precursor cell (NPC). As used herein, "neural precursor cell" or "NPC" refers to a stem cell-derived progenitor cell expressing VI type intermediate filament protein nestin. Due to the method for producing pre-OPCs and / or OPCs of the application, neural induction is avoided, and therefore NPCs are not produced. In various embodiments, the cell mass produced by the method of the application lacks nestin-positive cells.

[0123] In one embodiment, the cell produced by the method of the present application is a pre-oligodendrocyte progenitor cell (pre-OPCs). As used herein, "pre-oligodendrocyte progenitor cell" or "pre-OPC" refers to a stem cell-derived progenitor cell expressing cell markers OLIG2 and NKX2.2. Pre-OPC may express other markers, including but not limited to: OTX2 (previous neuroectodermal biomarker), FEZF2 (previous ectodermal biomarker) and / or OLIG1.

[0124] In one embodiment, the cell produced by the method of the present application is an oligodendrocyte progenitor cell (OPCs), which is a more differentiated (more mature) cell than pre-OPCs. As used herein, "oligodendrocyte progenitor cells" or "OPCs" refer to stem cell-derived progenitor cells expressing cell markers SOX10, OLIG2 and NKX2.2 and PDGFRa. OPCs may express other markers, non-limiting examples of which include OTX2 (previous neuroectodermal biomarker), FEZF2 (previous ectodermal biomarker) and / or OLIG1.

[0125] In one embodiment, the cell produced by the method of the present application is a premyelinating oligodendrocyte (preOLs), which is a more differentiated (more mature) cell than OPCs. As used herein, "premyelinating oligodendrocyte" or "preOL" refers to an oligodendrocyte lineage cell expressing cell markers CD9, A2B5, O4 and CNPase.

[0126] The pre-OLs produced by the method of the present application can be further cultured in vitro to produce mature oligodendrocytes (OLs). Markers of mature OLs include but are not limited to myelin basic protein (MBP) and O4.

[0127] II. Medium composition

[0128] The method for producing pre-OPCs, OPCs or preOLs of the present application comprises culturing human pluripotent stem cells in a culture medium containing a specific agonist and / or antagonist of a cell signaling pathway.

[0129] As described in Example 1 (Phase 1 Protocol), a culture medium comprising a retinoic acid (RA) pathway agonist, an Akt pathway agonist, and an mTOR pathway agonist is sufficient to generate pre-OPCs expressing OLIG2 and NKX2.2 in only three days. Other agents that can optimize the expression of other markers are added, including PDGFRa as an OPC differentiation marker. In other embodiments, the culture medium further comprises at least one additional agent selected from the group consisting of a WNT pathway antagonist, a SHH pathway agonist, a BMP pathway antagonist, and a PKC pathway antagonist. In one embodiment, the culture medium also comprises a WNT pathway antagonist. In one embodiment, the culture medium also comprises a SHH pathway agonist. In one embodiment, the culture medium also comprises a BMP pathway antagonist. In one embodiment, the culture medium also comprises a PKC pathway antagonist. In one embodiment, the culture medium also comprises a WNT pathway antagonist and a SHH pathway agonist, wherein the differentiated cells express OLIG2 and NKX2.2, as well as OTX2 and FEZF2.

[0130] In one embodiment, the culture medium for stage 1 of producing pre-OPCs includes a retinoic acid (RA) pathway agonist, an Akt pathway agonist, an mTOR pathway agonist, a WNT pathway antagonist, an SHH pathway antagonist, a BMP pathway antagonist, and a PKC pathway antagonist. In one embodiment, the differentiated cells are OPCs that express at least OLIG2, NKX2.2, and PDGFRa (and may express other markers such as OTX2, FEZF2, and / or OLIG1).

[0131] Pre-OPCs can be further differentiated into SOX10+OLIG2+NKX2-2+ OPCs (e.g., Fig.42 and Fig.43 Schematically shown).

[0132] As described in Examples 5 and 8, two alternative culture protocols have been developed for generating human OPCs from pre-OPCs, referred to as Version 1 and Version 2, each consisting of two stages, referred to as Stage 2 and Stage 3. The Stage 2 and Stage 3 protocols can be combined with the Stage 1 protocol described herein for generating pre-OPCs from pluripotent stem cells, thereby allowing the generation of human OPCs from pluripotent stem cells in only 12 days.

[0133] As described in Example 5 (version 1 protocol for stages 2 and 3), pre-OPCs are (i) cultured in a culture medium containing an FGFR pathway agonist, an mTOR pathway antagonist, an SHH pathway agonist, an AKT pathway antagonist, and an AKT pathway agonist for three days (e.g., days 0-3 of culture), and then (ii) the resulting cells are cultured in a culture medium containing an FGFR pathway agonist, an activin receptor (AR) pathway agonist, a PDGFR pathway agonist, an AKT pathway antagonist, a retinoic acid (RA) pathway agonist, an AMPK pathway agonist, and an mTOR pathway agonist for six days (e.g., days 3-9 of culture), sufficient to produce SOX10+OLIG2+NKX2-2+OPCs.

[0134] As described in Example 8 (version 2 protocol for stages 2 and 3), pre-OPCs (i) are cultured in a culture medium containing a FGFR pathway agonist, an mTOR pathway antagonist, a SHH pathway agonist, and a WNT pathway agonist for six days (e.g., days 0-6 of culture), and then (ii) the resulting cells are cultured in a culture medium containing a FGFR pathway agonist, an IGF-1 pathway agonist, and a retinoic acid (RA) pathway antagonist for three days (e.g., days 6-9 of culture), sufficient to produce SOX10+OLIG2+NKX2-2+OPCs.

[0135] By further culturing in stage 4 medium, OPCs can be further differentiated into CD9+A2B5+O4+CNPase preOLs (e.g. Fig.44 Schematically shown in ).

[0136] As described in Examples 10 and 11, culturing SOX10+OLIG2+NKX2-2+OPCs in a medium containing an IGF1R agonist, a TrkC agonist, a PDGFR agonist, a thyroid hormone receptor agonist, and an insulin receptor agonist is sufficient to generate CD9+A2B5+CNPase+O4+pre-OLs.

[0137] As used herein, an "agonist" of a cell signaling pathway refers to an agent that stimulates (upregulates) a cell signaling pathway. Stimulation of a cell signaling pathway can be initiated extracellularly, such as by using an agonist that activates a cell surface receptor involved in the signaling pathway (e.g., an agonist can be a receptor ligand). Additionally or alternatively, stimulation of cell signaling can be initiated intracellularly, such as by using a small molecule agonist that interacts with a signaling pathway component intracellularly.

[0138] As used herein, an "antagonist" of a cell signaling pathway refers to an agent that inhibits (downregulates) a cell signaling pathway. Inhibition of a cell signaling pathway can be initiated extracellularly, such as by using an antagonist that blocks a cell surface receptor involved in the signaling pathway. Additionally or alternatively, inhibition of cell signaling can be initiated intracellularly, such as by using a small molecule antagonist that interacts with a signaling pathway component intracellularly.

[0139] Retinoic acid (RA) pathway agonists, Akt pathway agonists, mTOR pathway agonists, WNT pathway antagonists, SHH pathway agonists, BMP pathway antagonists, PKC pathway antagonists, FGFR pathway agonists, WNT pathway agonists, IGF-1 pathway agonists, mTOR pathway antagonists, RA pathway antagonists, Akt pathway antagonists, activin receptor (AR) pathway agonists, PDGFR pathway agonists and AMPK pathway agonists are known in the art and are commercially available. They are used in culture medium with effective concentrations to achieve desired results, for example, to produce pre-OPCs and / or OPCs expressing target markers. Suitable agonists and antagonists and non-limiting examples of effective concentration ranges will be further described below.

[0140] Agonists of the RA pathway are used in the Stage 1 medium and the Version 1 Stage 3 medium and include agents, molecules, compounds or substances that are capable of stimulating the retinoic acid receptor (RAR), which is activated by all-trans retinoic acid and 9-cis retinoic acid. There are three RARs: RAR-α (alpha), RAR-β (beta), and RAR-γ (gamma), encoded by the RARA, RARB, and RARG genes, respectively. Different retinoic acid analogs have been synthesized that can activate the retinoic acid pathway. Non-limiting examples of such compounds include TTNPB (agonist of RARα, β and γ), AM 580 (RARα agonist), CD 1530 (potent and selective RARγ agonist), CD 2314 (selective RARβ agonist), Ch 55 (potent RAR agonist), BMS 753 (RARα selective agonist), tazarotene (receptor selective retinoic acid; binds RARβ and γ), isotretinoic acid (endogenous agonist of retinoic acid receptors; inducer of neuronal differentiation), and AC 261066 (RARβ2 agonist). In some embodiments, the RA signaling pathway agonist is selected from the group consisting of i) retinoic acid compounds, ii) retinoic acid X receptor (RXR) agonists, and iii) 25 retinoic acid receptor (RARs) agonists. In a specific embodiment, the RA pathway agonist is selected from the group consisting of retinoic acid, Sr11237, adapalene, EC23, 9-cis retinoic acid, 13-cis retinoic acid, 4-oxo retinoic acid, and all-trans retinoic acid (ATRA).

[0141] Therefore, in one embodiment, the RA pathway agonist is selected from the group consisting of TTNPB, AM 580, CD 1530, CD 2314, CD 437, Ch 55, BMS 753, BMS 961, tazarotene, isotretinoic acid, retinoic acid, tamibarotene, ATRA, AC 261066, AC 55649, retinoic acid (RA), Sr11237, adapalene, EC23, 9-cis retinoic acid, 13-cis retinoic acid, 4-oxo retinoic acid and all-trans retinoic acid (ATRA), and combinations thereof. In one embodiment, the RA pathway agonist is present in the culture medium at a concentration range of 5-500 mM, or 10-100 nM or 25-75 nM. In one embodiment, the RA pathway agonist is TTNPB. In one embodiment, the RA pathway agonist is TTNPB, which is present in the culture medium at a concentration range of 5-500 nM, or 10-100 nM, or 25-75 nM. In one embodiment, the RA pathway agonist is TTNPB, which is present in the culture medium of stage 1, the culture medium of stage 3 (version 1), or both at a concentration of 50 nM.

[0142] Agonists of the Akt pathway include agents, molecules, compounds or substances that can stimulate (activate) the signaling pathway of one or more members of the serine / threonine kinase Akt family, including Akt1 (also known as PKB or RacPK), Akt2 (also known as PKBβ or RacPK-β) and Akt3 (also known as PKBγ or thymoma virus proto-oncogene 3). In one embodiment, the Akt pathway agonist is a pan-Akt activator. In one embodiment, the Akt pathway agonist is selected from the group consisting of Sc79, demethylcoclaurine, LM22B-10, YS-49, YS-49 monohydrate, demethylchrysanthemine B1, Recilisib, N-oleylglycine, NSC45586 sodium, periwinkle, CHPG sodium salt, bilobalide, 6-hydroxyflavone, musk ketone, SEW2871, 8-isopentenylnaringenin, Razuprotafib and combinations thereof. In one embodiment, the Akt pathway agonist is Sc79. In one embodiment, the Akt pathway agonist is present in the culture medium at a concentration range of 0.1-10 μM. In one embodiment, the Akt pathway agonist is SC79. In one embodiment, the Akt pathway agonist is Sc79, which is present in the culture medium at a concentration of 0.1-10 μM, or 0.5-5 μM, or 0.5-3.0 μM, or 0.5-2.5 μM. In one embodiment, the Akt pathway agonist is SC79, which is present in the culture medium of stage 1 at a concentration of 1 μM. In one embodiment, the Akt pathway agonist is SC79, which is present in the culture medium of stage 2 (version 1) at a concentration of 2 μM.

[0143] Agonists of the mTOR (mammalian target of rapamycin) pathway include agents, molecules, compounds or substances that are capable of stimulating (activating) signaling through mTOR, a member of the PI3K-related kinase family and a core component of the mTORC1 and mTORC2 complexes. In one embodiment, the mTOR pathway agonist is selected from the following group: MHY1485, 3BDO, salidroside, L-leucine, NV-5138, testosterone; 3-benzyl-5-((2-nitrophenoxy)methyl)-dihydrofuran-2(3H)-one (3BDO); ​​NV-5138 hydrochloride, NV-5138, L-leucine-d1, L-leucine-2-13C, 15N, leucine-13C6, L-leucine-d7, L-leucine-d10, L-leucine d2, L-leucine-d3, L-leucine-18O2, L-leucine-13C, L-leucine-2-13C, L-leucine-13C6-15N, L-leucine-15N, L-leucine-1-13C, 15N, and combinations thereof. In one embodiment, the mTOR pathway agonist is present in the culture medium at a concentration range of 0.1-10 μM, or 0.5-5 μM, or 0.5-3.0 μM, or 0.5-2.5 μM. In one embodiment, the mTOR pathway agonist is MHY1485. In one embodiment, the mTOR pathway agonist is MHY1485, which is present in the culture medium at a concentration of 0.1-10 μM, or 0.5-5 μM, or 0.5-3.0 μM, or 0.5-2.5 μM. In one embodiment, the mTOR pathway agonist is MHY1485, which is present in the culture medium of stage 1 at a concentration of 1 μM. In one embodiment, the mTOR pathway agonist is MHY1485, which is present in the culture medium of stage 3 (version 1) at a concentration of 2 μM.

[0144] Antagonists of the WNT pathway include agents, molecules, compounds or substances that can inhibit (downregulate) the typical Wnt / β-catenin signaling pathway, which is biologically activated by the binding of Wnt protein ligands to Frizzled family receptors. In one embodiment, the WNT pathway antagonist is selected from the group consisting of XAV939, ICG001, capmatinib, endo-IWR-1, IWP-2, IWP-4, MSAB, CCT251545, KY02111, NCB-0846, FH535, LF3, WIKI4, triptolide, KYA1797K, JW55, JW67, JW74, Cardionogen 1, NLS-StAx-h, TAK715, PNU 74654, iCRT3, WIF-1, DKK1, and combinations thereof. In one embodiment, the WNT pathway antagonist is present in the culture medium at a concentration range of 10-500nM, 50-250nM, or 50-150nM. In one embodiment, the WNT pathway antagonist is XAV939. In one embodiment, the WNT pathway antagonist is XAV939, which is present in the culture medium at a concentration of 10-500nM, 50-250nM, or 50-150nM. In one embodiment, the WNT pathway antagonist is XAV939, which is present in the culture medium at stage 1 at a concentration of 100nM.

[0145] Agonists of the SHH (Sonic Hedgehog) pathway include agents, molecules, compounds or substances that can stimulate (activate) signal transduction through the SHH pathway, which biologically involves the binding of SHH to the Patched-1 (PTCH1) receptor and transduction through the Smoothened (SMO) transmembrane protein. In one embodiment, the SHH pathway agonist is selected from the group consisting of purple morphine, GSA 10, SHH, SAG, and combinations thereof. In one embodiment, the SHH pathway agonist is present in the culture medium at a concentration range of 100-1000nM, 250-750nM or 400-600nM. In one embodiment, the SHH pathway agonist is purple morphine. In one embodiment, the SHH pathway agonist is purple morphine, which is present in the culture medium at a concentration of 100-1000nM, 250-750nM or 400-600nM. In one embodiment, the SHH pathway agonist is virulent, which is present in the stage 1, stage 2 (version 1) and / or stage 2 (version 2) culture medium at a concentration of 500 nM.

[0146] Antagonists of the BMP (bone morphogenetic protein) pathway include agents, molecules, compounds or substances that can inhibit (downregulate) the BMP signaling pathway, which is biologically activated by binding of BMP to a BMP receptor, wherein the BMP receptor is an activin receptor-like kinase (ALK) (e.g., type I BMP receptor, including but not limited to ALK2 and ALK3). In one embodiment, the BMP pathway antagonist is selected from the group consisting of LDN193189, DMH1, DMH2, Dorsopmorphin, K02288, LDN214117, LDN212854, folistatin, ML347, noggin and combinations thereof. In one embodiment, the BMP pathway antagonist is present in the culture medium at a concentration range of 100-1000nM, 150-750nM, 100-500nM or 150-350nM. In one embodiment, the BMP pathway antagonist is LDN193189. In one embodiment, the BMP pathway antagonist is LDN193189, which is present in the culture medium at a concentration of 100-1000nM, 150-750nM, 100-500nM or 150-350nM. In one embodiment, the BMP pathway antagonist is LDN193189, which is present in the culture medium of stage 1 at a concentration of 250nM.

[0147] Antagonists of the PKC (protein kinase C) pathway include agents, molecules, compounds or substances that can inhibit (downregulate) the PKC signaling pathway, which is biologically mediated by members of the PKC family. The PKC family of serine / threonine kinases includes fifteen isozymes, including the "classical" PKC subclass, which contains subtypes α, β1, β2 and γ. In one embodiment, the PKC pathway antagonist inhibits the activity of at least one (in other embodiments, at least two or three) PKC enzyme selected from PKCα, PKCβ1, PKCβ2 and PKCγ. In one embodiment, the PKC pathway antagonist is selected from the group consisting of Go 6983, sotrastaurin, enzastaurin, staurosporine, LY31615, Go 6976, GF 109203X, Ro 31-8220 mesylate and combinations thereof. In one embodiment, the PKC pathway antagonist is present in the culture medium at a concentration range of 10-500nM, 50-300nM, 50-150nM, or 75-150nM. In one embodiment, the PKC pathway antagonist is Go6983. In one embodiment, the PKC pathway antagonist is Go 6983, which is present in the culture medium at a concentration of 10-500nM, 50-300nM, 50-150nM, or 75-150nM. In one embodiment, the PKC pathway antagonist is Go6983, which is present in the culture medium at a concentration of 110nM.

[0148] The agonist of FGFR pathway includes reagents, molecules, compounds or substances that can stimulate (upregulate) fibroblast growth factor receptor signaling pathway, which is biologically activated by the combination of FGF and FGFR. In one embodiment, the FGFR pathway agonist is FGF2, SUN11602, or a combination thereof. In one embodiment, the FGFR pathway agonist is present in the culture medium at a concentration range of 1-20ng / ml, 5-15ng / ml, 7.5-12.5ng / ml, 9-11ng / ml or at a concentration of 10ng / ml. In one embodiment, the FGFR pathway agonist is FGF2 (e.g., recombinant human FGF2). In one embodiment, the FGFR pathway agonist is FGF2, which is present in the culture medium of stage 1 and / or stage 2 (versions 1 and 2) and / or stage 3 (versions 1 and 2) at a concentration range of 1-20 ng / ml, 5-15 ng / ml, 7.5-12.5 ng / ml, 9-11 ng / ml, or at a concentration of 10 ng / ml.

[0149] Agonists of the WNT pathway include agents, molecules, compounds or substances that can stimulate (upregulate) the canonical Wnt / β-catenin signaling pathway, which is biologically activated by the binding of Wnt protein ligands to Frizzled family receptors. In one embodiment, the WNT pathway agonist is a glycogen synthase kinase 3 (Gsk3) inhibitor. In one embodiment, the WNT pathway agonist is selected from the group consisting of CHIR99021, CHIR98014, SB 216763, SB 415286, LY2090314, 3F8, A 1070722, AR-A 014418, BIO, BIO-acetone oxime, AZD1080, WNT3A, Atepolone, Indirubin-3-oxime, 1-azacamparolone, camparolone, TC-G 24, TDZD 8, TWS119, NP 031112, AT 7519, KY 19382, AZD2858, and combinations thereof. In one embodiment, the WNT pathway agonist is present in the culture medium at a concentration range of 0.3-3.0 μM, 0.5-2.0 μM, 0.75-1.5 μM, or 0.9-1.1 μM. In one embodiment, the WNT pathway agonist is CHIR99021. In one embodiment, the WNT pathway agonist is CHIR99021, which is present in the culture medium at a concentration range of 0.3-3.0 μM, 0.5-2.0 μM, 0.75-1.5 μM or 0.9-1.1 μM. In one embodiment, the WNT pathway agonist is CHIR99021, which is present in the culture medium of stage 2 (version 2) at a concentration of 1.0 μM.

[0150] Agonists of the IGF-1 (insulin-like growth factor 1) pathway include agents, molecules, compounds or substances that can stimulate (activate) signal transduction through the IGF-1 pathway. In one embodiment, the IGF-1 pathway agonist is selected from the group consisting of IGF-1, IGF-2, insulin, Rg5, IGF-1 30-41, demethyl chrysanthemum quinone B1, IGF1-Ado, X10, mecasermin, and combinations thereof. In one embodiment, the IGF-1 pathway agonist is present in the culture medium at a concentration range of 2-20 ng / ml or 5-15 ng / ml or 7.5-12.5 ng / ml. In one embodiment, the IGF-1 pathway agonist is IGF-1. In one embodiment, the IGF-1 pathway agonist is IGF-1, which is present in the culture medium at a concentration of 2-20 ng / ml, 5-15 ng / ml or 7.5-12.5 ng / ml. In one embodiment, the IGF-1 pathway agonist is IGF-1, which is present in the stage 3 (version 2) culture medium at a concentration of 10 ng / ml. In one embodiment, the IGF-1 pathway agonist is IGF-1, which is present in the stage 4 culture medium at a concentration of 10 ng / ml.

[0151] Antagonists of the mTOR (mammalian target of rapamycin) pathway include agents, molecules, compounds or substances that are capable of inhibiting (downregulating) the mTOR signaling pathway, wherein mTOR is a member of the PI3K-related kinase family and a core component of the mTORC1 and mTORC2 complexes. In one embodiment, the mTOR pathway antagonist is selected from the group consisting of AZD 3147, Daclis, Rapamycin, Everolimus, AZD 8055, Temsirolimus, PI-103, NU7441, BC-LI-0186, eCF 309, ETP45658, Niclosamide, Omilide, PF 04691502, PF 05212384, Torin 1, Torin 2, WYE 687, XL388, STK16-IN-1, PP 242, Torkinib, Ridaforolimus, Sapaseti, Voxtalisib, and combinations thereof. In one embodiment, the mTOR pathway antagonist is present in the culture medium at a concentration range of 5-200 nM, 10-150 nM, or 15-100 nM. In one embodiment, the mTOR pathway antagonist is AZD3147. In one embodiment, the mTOR pathway antagonist is AZD 3147, which is present in the culture medium at a concentration range of 5-200nM, 10-150nM or 15-100nM. In one embodiment, the mTOR pathway antagonist is AZD 3147, which is present in the culture medium at a concentration of 15nM in stage 2 (version 1). In one embodiment, the mTOR pathway antagonist is AZD 3147, which is present in the culture medium at a concentration of 100nM in stage 2 (version 2).

[0152] Antagonists of the retinoic acid (RA) pathway include agents, molecules, compounds or substances that can inhibit (downregulate) the RA signaling pathway. In one embodiment, the RA pathway antagonist is selected from the group consisting of AGN193109, BMS195614, CD 2665, ER 50891, LE 135, LY 2955303, MM11253, and combinations thereof. In one embodiment, the RA pathway antagonist is present in the culture medium at a concentration range of 50-300nM, 75-250nM, 100-200nM or 90-110nM. In one embodiment, the RA pathway antagonist is AGN193109. In one embodiment, the RA pathway antagonist is AGN193109, which is present in the culture medium at a concentration range of 50-300nM, 75-250nM, 100-200nM or 90-110nM. In one embodiment, the RA pathway antagonist is AGN 193109, which is present in the stage 3 (version 2) culture medium at a concentration of 100 nM.

[0153] Antagonists of the AKT pathway include agents, molecules, compounds or substances that are capable of inhibiting (downregulating) the signaling pathway of one or more members of the AKT family of serine / threonine kinases, including AKT1 (also known as PKB or RacPK), AKT2 (also known as PKBβ or RacPK-β), and AKT3 (also known as PKBγ or thymoma viral oncogene 3). In one embodiment, the Akt pathway antagonist is selected from the group consisting of MK2206, GSK690693, perifosine (KRX-0401), patasertib (GDC-0068), capasertib (AZD5363), PF-04691502, AT 7867, triciribine (NSC154020), ARQ751, milanthate (ab235550), Borussertib, Cerisertib, Akti1 / 2, CCT128930, A 674563, PHT 427, miltefosine, AT 13148, ML 9, BAY 1125976, oridonin, TIC10, salidroside, Akti IV, 10-DEBC, API-1, SC 66, FPA 124, API-2, urolithin A, and combinations thereof. In one embodiment, the Akt pathway antagonist is present in the culture medium at a concentration range of 25-300nM, 50-250nM, 75-200nM, or 100-150nM. In one embodiment, the Akt pathway antagonist is MK2206. In one embodiment, the Akt pathway antagonist is MK2206, which is present in the culture medium at a concentration range of 25-300nM, 50-250nM, 75-200nM, or 100-150nM. In one embodiment, the Akt pathway antagonist is MK2206, which is present in the culture medium at a concentration of 125nM in stage 2 (version 1) and / or stage 3 (version 1).

[0154] Agonists of the activin receptor (AR) pathway include agents, molecules, compounds or substances that can stimulate (activate) signal transduction through the AR pathway. In one embodiment, the AR pathway agonist is selected from the group consisting of activin A, scutellariae lactone, and combinations thereof. In one embodiment, the AR pathway agonist is present in the culture medium at a concentration range of 2-20ng / ml, 5-15ng / ml or 7.5-12.5ng / ml. In one embodiment, the AR pathway agonist is activin A. In one embodiment, the AR pathway agonist is activin A, which is present in the culture medium at a concentration of 2-20ng / ml, 5-15ng / ml or 7.5-12.5ng / ml. In one embodiment, the AR pathway agonist is activin A, which is present in the culture medium at a concentration of 10ng / ml in stage 3 (version 1).

[0155] Agonists of the PDGFR (platelet-derived growth factor receptor) pathway include agents, molecules, compounds or substances that can stimulate (activate) signal transduction through the PDGFR pathway. In one embodiment, the PDGFR pathway agonist is PDGF-AA. In one embodiment, the PDGFR pathway agonist is present in the culture medium at a concentration range of 2-20ng / ml, 5-15ng / ml or 7.5-12.5ng / ml. In one embodiment, the PDGFR pathway agonist is PDGF-AA, which is present in the culture medium at a concentration of 2-20ng / ml, 5-15ng / ml or 7.5-12.5ng / ml. In one embodiment, the PDGFR pathway agonist is PDGF-AA, which is present in the culture medium of stage 3 (version 1) at a concentration of 10ng / ml. In one embodiment, the PDGFR pathway agonist is PDGF-AA, which is present in the culture medium of stage 4 at a concentration of 10ng / ml.

[0156] AMPK (5'AMP-activated protein kinase) pathway agonists include agents, molecules, compounds or substances that can stimulate (activate) signal transduction through the AMPK pathway. In one embodiment, the AMPK pathway agonist is selected from the following group: AICAR, metformin, dendrobium alcohol B, mari glycosides, gentian ester glycosides, A769662, PF 06409577, metformin hydrochloride, ZLN 024, ZLN 2024 hydrochloride, nilotinib, phenformin, nilotinib hydrochloride monohydrate, 5'-adenosine monophosphate monohydrate, homophyll, MK 8722, sterol, ASP4132, GSK621, EX229 (Compound 991), trans-feluric acid, O-304, MK 3903, BAM 15, Ligustrum lucidum, ETC-1002, BC1618, IMM-H007, IM156, Panax notoginseng saponin IVa, Pachymic acid A, 7-methoxy isoflavone, Urolithin B, Danthron, Desmethyl berberine, AMPK activator 1, AMPK activator 2, AMPK activator 4, malvidin-3-O-arabinoside, RSVA 405, etilefrine, COH-SR4, buformin, buformin hydrochloride, PT1, bepedic acid, 3a-Hydrocymogrol, 2-hydroxyestradiol, and combinations thereof. In one embodiment, the AMPK pathway agonist is present in the culture medium at a concentration range of 50-500 μM, 100-300 μM, 150-250 μM, or 175-225 μM. In one embodiment, the AMPK pathway agonist is AICAR, which is present in the culture medium at a concentration of 50-500 μM, 100-300 μM, 150-250 μM or 175-225 μM. In one embodiment, the AMPK pathway agonist is AICAR, which is present in the culture medium of stage 3 (version 1) at a concentration of 200 μM.

[0157] Agonists of the TrkC (tropomyosin-related kinase receptor C) pathway include agents, molecules, compounds or substances that can stimulate (activate) signal transduction through the TrkC pathway. In one embodiment, the TrkC pathway agonist is selected from the group consisting of neurotrophin-3 (NT-3), peptide mimetics based on NT-3's β-turn, LM22B 10, GNF 5837, and combinations thereof. In one embodiment, the TrkC pathway agonist is NT-3. In one embodiment, the TrkC pathway agonist is present in the culture medium at a concentration range of 2-20 ng / ml, 5-15 ng / ml, and 7.5-12.5 ng / ml. In one embodiment, the TrkC pathway agonist is NT-3, which is present in the culture medium at a concentration of 2-20 ng / ml, 5-15 ng / ml, and 7.5-12.5 ng / ml. In one embodiment, the TrkC pathway agonist is NT-3, which is present in the culture medium at a concentration of 10 ng / ml in stage 4.

[0158] Thyroid hormone receptor (THR) agonists include reagents, molecules, compounds or substances that can stimulate (activate) signal transduction through thyroid hormone receptor pathways. In one embodiment, the thyroid hormone receptor agonist is selected from the group consisting of T3, T4, resmetirol, TRb agonist 3 (compound 3), sobetirome, tiratracol and combinations thereof. In one embodiment, the THR agonist is present in a culture medium at a concentration range of 10-100nM, or 25-75nM or 40-60nM or at a concentration of 50nM. In one embodiment, the THR agonist is T3. In one embodiment, the THR agonist is T3, which is present in a culture medium at a concentration of 10-100nM, 25-75nM, 40-60nM or 50nM. In one embodiment, the THR agonist is T3, which is present in a culture medium at a concentration of 50nM.

[0159] Insulin receptor (IR) agonists include agents, molecules, compounds or substances that can stimulate (activate) signal transduction through the insulin receptor pathway. In one embodiment, the insulin receptor agonist is selected from the group consisting of insulin, IGF-1, IGF-2, demethyl chrysanthemum quinone B1, MK-5160, MK-1092, and combinations thereof. In one embodiment, the insulin receptor agonist is present in the culture medium at a concentration range of 4-40 μg / ml, 10-30 μg / ml or 15-25 μg / ml or at a concentration of 20 μg / ml. In one embodiment, the insulin receptor agonist is insulin, which is present in the culture medium at a concentration of 4-40 μg / ml, 10-30 μg / ml or 15-25 μg / ml. In one embodiment, the insulin receptor agonist is insulin, which is present in the culture medium at a concentration of 20 μg / ml.

[0160] When an agonist or antagonist is used in multiple steps of the method, in one embodiment, the same specific agonist or antagonist is used for each step in which the agent is present in the culture medium. In another embodiment, different agonists or antagonists that affect the same signaling pathway are used in different steps of the method.

[0161] When using an agonist or antagonist in multiple steps of the method, in one embodiment, the same specific agonist or antagonist is used at the same concentration in each step in which the agent is present in the culture medium. In another embodiment, different concentrations of the same agonist or antagonist are used in different steps of the method.

[0162] III. Culture conditions

[0163] In conjunction with the chemically defined and optimized culture medium described in subsection II above, the method for producing pre-OPCs, OPCs and preOLs of the present application utilizes the standard culture conditions established in the field of cell culture. For example, cells can be cultured under 37° C. and 5% CO2 conditions. Cells can be cultured in standard culture containers or plates (such as 96-well plates). In certain embodiments, the starting pluripotent stem cells adhere to the plate, preferably coated with extracellular matrix materials, such as vitronectin. In one embodiment, the stem cells are cultured on a culture surface coated with vitronectin (e.g., a 96-well plate coated with vitronectin).

[0164] Pluripotent stem cells can be cultured in a commercially available culture medium prior to differentiation. For example, the stem cells can be cultured in Essential 8Flex Medium (Thermo Fisher #A2858501) for at least one day prior to the start of the differentiation protocol. In a non-limiting exemplary embodiment, the stem cells are cultured at 150,000 cells / cm 2 Cells were passaged at a density of 1:1 onto 96-well plates coated with vitronectin (Thermo Fisher #A14700) and cultured in Essential 8Flex medium for one day prior to differentiation.

[0165] To start the differentiation protocol, the culture medium of the stem cells to be cultured is changed to a basal differentiation medium supplemented with a signaling pathway agonist and / or antagonist, as described in Section II above. The basal differentiation medium may include, for example, a commercially available matrix supplemented with additional standard culture medium components required to maintain cell survival and growth, but lacks serum (basal differentiation medium is a serum-free medium) or any other exogenously added growth factors, such as FGF2, PDGF, IGF or HGF. In a non-limiting exemplary embodiment, the basal differentiation medium comprises 1x IMDM (Thermo Fisher #12440046), 1x F12 (Thermo Fisher #11765047), 1 mg / ml polyvinyl alcohol (Sigma #p8136), 1% chemically defined lipid concentrate (Thermo Fisher #11905031), 450uM 1-thioglycerol (Sigma #M6145), 0.7ug / ml insulin (Sigma #11376497001), 15ug / ml transferrin (Sigma #10652202001). In one embodiment, the basal medium is supplemented with Albumax II.

[0166] The culture medium is usually replaced with fresh culture medium regularly. For example, in one embodiment, the culture medium is replaced once every 24 hours.

[0167] In order to produce pre-OPCs, OPCs and / or preOLs, stem cells are cultured in an optimized culture medium for a sufficient time to differentiate cells and express markers associated with pre-OPC, OPC and / or preOL. As described in Example 1, it has been found that culturing stem cells in the optimized culture medium of stage 1 for only 72 hours (3 days) is sufficient for pre-OPC differentiation. Therefore, in one embodiment, stem cells are cultured for at least 72 hours. In other embodiments, stem cells are cultured for at least 60, 64, 68, 72, 76, 80, 84, 88, 92 or 96 hours.

[0168] As described in Example 5, it has been found that in the culture medium of the stage 2 of optimization, it is only enough to carry out pre-OPC differentiation for 72 hours (3 days) to cultivate pre-OPCs. Therefore, in one embodiment, pre-OPCs are cultivated in the culture medium of stage 2 (version 1) for at least 72 hours. In other embodiments, pre-OPCs are cultivated in the culture medium of stage 2 (version 1) for at least 60, 64, 68, 72, 76, 80, 84, 88, 92 or 96 hours. It is further found that it is only enough to make OPC differentiation for 6 days (144 hours) to further cultivate cells in the culture medium of the stage 3 of optimization. Therefore, in one embodiment, cells are cultivated in the culture medium of stage 3 (version 1) for at least 144 hours. In other embodiments, the cells are cultivated in the culture medium of stage 3 (version 1) for at least 132, 136, 140, 144, 150, 154 or 158 hours.

[0169] As described in Example 8, it has been found that in the culture medium of the stage 2 of optimization, it is only enough to carry out cell differentiation to cultivate pre-OPCs for 6 days (144 hours). Therefore, in one embodiment, pre-OPCs are cultivated in the culture medium of stage 2 (version 2) for at least 144 hours. In other embodiments, pre-OPCs are cultivated in the culture medium of stage 2 (version 2) for at least 132,136,140,144,150,154 or 158 hours. It is further found that it is only enough to make OPC differentiation to further cultivate cells in the culture medium of the stage 3 of optimization for 72 hours (3 days). Therefore, in one embodiment, cells are cultivated in the culture medium of stage 3 (version 2) for at least 72 hours. In other embodiments, the cells are cultivated in the culture medium of stage 3 (version 2) for at least 60,64,68,72,76,80,84,88,92 or 96 hours.

[0170] As described in Example 10, it has been found that culturing OPCs in an optimized stage 4 medium for only 6 days (144 hours) is sufficient for cell differentiation into preOLs. Thus, in one embodiment, OPCs are cultured in a stage 4 medium for at least 144 hours. In one embodiment, OPCs are cultured in a stage 4 medium for at least 132, 136, 140, 144, 150, 154, or 158 hours.

[0171] IV. use

[0172] The method and composition for producing pre-OPCs, OPCs and preOLs of the present application allow these cell groups to be used for various purposes effectively and robustly. For example, these methods and compositions can be used for the study of oligodendrocyte development and biology, to help understand the diseases and disorders related to oligodendrocytes. For example, pre-OPCs, OPCs and / or preOLs produced using the method of the present application can be further purified using reagents combined with surface markers expressed on cells according to the method established in the art. Therefore, in one embodiment, the application provides a method for separating pre-oligodendrocyte progenitor cells (pre-OPCs), oligodendrocyte progenitor cells (OPCs) or pre-myelinating oligodendrocytes (preOLs), the method comprising: pre-OPCs or OPCs expressing OLIG2 produced by the method of the present application, or CD9+preOLs are contacted with at least one binding agent, the binding agent is bound to the cell surface markers expressed by pre-OPCs, OPCs or preOLs; and separation of cells combined with binding agents to separate pre-OPCs, OPCs or preOLs.

[0173] In one embodiment, the binding agent is an antibody that binds to a cell surface marker, such as a monoclonal antibody (mAb). Non-limiting examples of suitable OPC cell surface markers include PDGFRa, O4, and A2B5. Antibody-bound cells can be separated by methods known in the art, including but not limited to fluorescence activated cell sorting (FACS) and magnetic activated cell sorting (MACS).

[0174] Progenitor cells of the oligodendrocyte lineage are also contemplated for use in treating various oligodendrocyte-related diseases and disorders by delivering the cells to a subject suffering from the disease or disorder. Examples of oligodendrocyte-related diseases and disorders include, but are not limited to, multiple sclerosis (MS), progressive multifocal leukoencephalopathy, periventricular leukomalacia, certain leukodystrophies, and amyotrophic lateral sclerosis (ALS).

[0175] V. Composition

[0176] In other aspects, the present application provides compositions related to methods of generating pre-OPCs and OPCs, including culture media and cell cultures, and isolated progenitor cells and cell populations.

[0177] On the one hand, the present application provides a culture medium for obtaining oligodendrocyte progenitor cells (OPCs), which comprises an FGFR pathway agonist, an mTOR pathway antagonist, an SHH pathway agonist, an AKT pathway antagonist, and an AKT pathway agonist (corresponding to the culture medium of stage 2 of version 1). On the other hand, the present application provides a culture medium for obtaining oligodendrocyte progenitor cells (OPCs), which comprises an FGFR pathway agonist, an activin receptor (AR) pathway agonist, a PDGFR pathway agonist, an AKT pathway antagonist, a retinoic acid (RA) pathway agonist, an AMPK pathway agonist, and an mTOR pathway agonist (corresponding to the culture medium of stage 3 of version 1).

[0178] On the other hand, the present application provides a culture medium for obtaining oligodendrocyte progenitor cells (OPCs), which comprises an FGFR pathway agonist, an mTOR pathway antagonist, an SHH pathway agonist, and a WNT pathway agonist (corresponding to the culture medium of version 2 stage 2). On the other hand, the present application provides a culture medium for obtaining oligodendrocyte progenitor cells (OPCs), which comprises an FGFR pathway agonist, an IGF-1 pathway agonist, and a retinoic acid (RA) pathway agonist (corresponding to the culture medium of version 2 stage 3).

[0179] In another aspect, the present application provides a culture medium for obtaining premyelinating oligodendrocytes (preOLs), which comprises an IGF1R pathway agonist, a TrkC pathway agonist, a PDGFR pathway agonist, a thyroid hormone receptor agonist, and an insulin receptor agonist.

[0180] On the one hand, the present application provides an isolated cell culture comprising OPCs cultured in one of the culture media disclosed herein. In one embodiment, the present application provides an isolated cell culture comprising OLIG2+OPCs cultured in a culture medium comprising an FGFR pathway agonist, an mTOR pathway antagonist, an SHH pathway agonist, an AKT pathway antagonist, and an AKT pathway agonist (corresponding to the culture medium of version 1 stage 2). In another embodiment, the present application provides an isolated cell culture comprising OLIG2+OPCs cultured in a culture medium comprising an FGFR pathway agonist, an activin receptor (AR) pathway agonist, a PDGFR pathway agonist, an AKT pathway antagonist, a retinoic acid (RA) pathway agonist, an AMPK pathway agonist, and an mTOR pathway agonist (corresponding to the culture medium of version 1 stage 3).

[0181] In another embodiment, the present application provides an isolated cell culture comprising OLIG2+OPCs cultured in a culture medium comprising an FGFR pathway agonist, an mTOR pathway antagonist, an SHH pathway agonist, and a WNT pathway agonist (corresponding to a culture medium of version 2, stage 2). In another embodiment, the present application provides an isolated cell culture comprising OLIG2+OPCs cultured in a culture medium comprising an FGFR pathway agonist, an IGF-1 pathway agonist, and a retinoic acid (RA) pathway agonist (corresponding to a culture medium of version 2, stage 3).

[0182] In another embodiment, the present application provides an isolated cell culture comprising CD9+preOLs cultured in a culture medium comprising an IGF1R pathway agonist, a TrkC pathway agonist, a PDGFR pathway agonist, a thyroid hormone receptor agonist, and an insulin receptor agonist.

[0183] The present application is further illustrated by the following examples, which should not be construed as further limiting.The contents of the figures and all references, patents and published patent applications cited in this application are expressly incorporated herein by reference. Example

[0184] Example 1 :Development of a culture protocol for generating stem cell-derived oligodendrocyte progenitor cells

[0185] In this example, a culture medium formulation for producing oligodendrocyte progenitor cells was developed, which can guide human pluripotent stem cells to differentiate into oligodendrocyte progenitor cells expressing NKX2-2 and OLIG2 after 3 days of culture. These cells can be further differentiated into mature oligodendrocytes.

[0186] As previously described by Bukys et al. (2020) Iscience 23:101346, this embodiment uses a high-dimensional experimental design (HD-DoE) approach. The method uses computerized design geometries to test multiple process inputs simultaneously and provides mathematical modeling of deep effector / response space. The method allows for finding combined signal inputs that control complex processes, such as in cell differentiation processes. This allows for testing multiple reasonable key process parameters because these parameters affect output responses, such as gene expression. Since gene expression provides, for example, hallmark features of human cell phenotypes, this method can be used to identify and understand which signaling pathways control cell fate. In the current embodiment, the HD-DOE method is applied to find conditions for inducing oligodendrocyte progenitor cell expression of genes directly from the pluripotent stem cell state.

[0187] Specifically, in order to develop a new method for producing oligodendrocytes, after 3 days of treatment, the effects of agonists and antagonists of multiple signaling pathways (referred to herein as effectors) on the expression of two groups of 53 pre-selected genes were tested. These effectors are small molecules commonly used in the process of stem cell differentiation into a specific fate. The selection of effectors is based on the current literature on preectoderm neural induction and stem cell differentiation into oligodendrocytes.

[0188] HD-DoE#1

[0189] To test effectors, experiments containing at least eight factors were designed, allowing the evaluation of cellular responses to 48 or more combinations of effectors at different concentration ranges. To analyze these models, we focused on genes expressed during early development of the anterior neuroectoderm and oligodendrocytes, including NKX2-2, OLIG2, OLIG1, and PDGFRA. The effect of each effector on gene expression levels is defined by a parameter called the factor contribution, which is calculated for each effector during the modeling process.

[0190] like Figure 1 One model showed particularly promising results in upregulation of the NKX2-2 and OLIG2 genes when optimized for maximizing NKX2-2 expression at 12480.6, as shown by the results summarized in . This model tested eight factors: PD0325901, MK2206, TTNPB, SC79, MHY1485, ZM336372, AGN193109, and AZD3147.

[0191] Among the eight factors tested, three of them, namely TTNPB (agonist of retinoic acid pathway), SC79 (agonist of Akt signaling pathway), and MHY1485 (agonist of mTOR signaling pathway), had significant positive effects on the expression of target genes, with TTNPB having the largest effect with a factor contribution of 31.3, MHY1485 with a factor contribution of 13.8, and SC79 with a factor contribution of 1.47. These factors can significantly increase the expression of NKX2-2 and OLIG2. OLIG1 and PDGFRA have average expression levels (129.9 and 346.45, respectively), which match the gene expression pattern during oligodendrocyte differentiation.

[0192] like Figure 2As shown in the results summarized in , the normalized expression of PDGFRA in this model can reach 832.9, which is the highest expression level among all models, and therefore, this model is also optimized for maximum PDGFRA expression. This setting shows that TTNPB (agonist of the retinoic acid pathway) and MHY1485 (agonist of the mTOR signaling pathway) also have a positive effect on the upregulation of PGFRA, with factor contributions of 49.01 and 13.4, respectively. PD0325901 was also observed to have a significant positive effect on this gene, with a factor contribution of 30.6. Due to the low factor contribution of ZM336372 (<1), this factor was not included in the recipe. Under this condition, the average expression level of OLIG1 was 228.36, which is similar to the optimized condition of NKX2-2. One difference under this condition is that the OLIG2 gene was downregulated from the previous 1049.4 to 241.9.

[0193] like Figure 3 As shown in the results summarized in, among the effectors that positively affected the expression levels of NKX2-2, OLIG2, or PDGFRA, two factors negatively affected the expression levels of NKX2.2 and OLIG2. Therefore, these two factors, PD0325901 and ZM336372, were removed from the list of candidates for the oligodendrocyte differentiation recipe.

[0194] Therefore, the first HD-DoE screen identified a culture medium lacking exogenously added growth factors and containing retinoic acid pathway agonists, Akt signaling pathway agonists, and mTOR signaling pathway agonists as sufficient to generate OLIG2-expressing OPCs from pluripotent stem cells after 3 days (72 hours) of culture.

[0195] HD-DoE#2

[0196] To further improve the conditions for oligodendrocyte differentiation from pluripotency, we performed additional HD-DoE experiments. We obtained other gene regulation models for the preparation of differentiation protocols. The basis of this is the 13-factor HD-DoE experiment, which focuses on initiating the differentiation of cells towards the anterior neuroectoderm. In this model, we focused on the expression of FEZF2 and OTX2.

[0197] like Figure 4As shown in the results summarized in , the model was optimized for the highest expression of OTX2 at 12755.9. According to the high expression model of OTX2, seven effectors including MK2206, PD0325901, CHIR99021, LDN193189, Go6983, PD173074, and BLU9931 had positive effects, among which MK2206 had the highest factor contribution of 22.2, and PD173074 and BLU9931 had the lowest factor contribution of 1.7.

[0198] like Figure 5 As shown in the results summarized in , the model was optimized for the highest expression of FEZF2 at 4466. When the model was optimized for the maximum expression level of FEZF2, three effectors, including LDN193189, with a factor contribution of 19.5; PD0325901, with a factor contribution of 14.6; and MK2206, with a factor contribution of 12.3, were the same as before, and three new effectors, including purple morphine-500nM, XAV939, and SC79, were introduced.

[0199] like Figure 6 As shown in the results summarized in , a dynamic profiling analysis was performed to fine-tune the recipe and find the best combination of factors for high expression of OTX2 and FEZF2. According to this analysis, XAV939 (a WNT signaling pathway inhibitor) and Vibriomorphin (an SHH signaling pathway agonist known for ventralizing cells during development of brain regions) had a significant positive effect on the expression of FEZF2 and had no negative effect on the expression level of OTX2. Therefore, these two factors were added to the optimized culture recipe.

[0200] In addition to including factors that promote the expression of OPC-associated surface markers, certain factors that inhibit the expression of these markers were eliminated from the optimized culture formulation. CHIR99021, a WNT signaling pathway agonist, was eliminated. MK2206 and PD0325901 were also eliminated because they negatively affected the expression of oligodendrocyte genes according to the 8-factor model. PD173074 and BLU9931 were also eliminated due to low factor contributions of 1.7.

[0201] Summarize

[0202] Considering both models, culture conditions that maximized differentiation of human induced pluripotent stem cells into cells with oligodendrocyte progenitor cell (OPC) properties, resulting in elevated expression of OTX2, FEZF2, NKX2-2, and OLIG2, included the following effector inputs: TTNPB (RA pathway agonist), SC79 (Akt pathway agonist), MHY1485 (mTOR pathway agonist), violacein (SHH pathway agonist), XAV939 (WNT pathway antagonist), LDN193189 (BMP pathway antagonist), and Go6983 (PKC pathway antagonist).

[0203] Embodiment 2: Analysis of the importance of factors in OPC induction culture conditions

[0204] To assess the factor importance of each component in the optimized culture medium described in Example 1, we performed a computer prediction analysis of the results under conditions where individual effectors were eliminated while retaining other effectors. To do this, we used dynamic profiling at set points and compared the expression levels of target genes when each factor was missing. Since the expression of the target gene reveals whether the expected result can be achieved, this factor importance analysis reveals the degree of importance of each input effector.

[0205] Figures 7A-7D The results of factor importance analysis for the effectors TTNPB (RA pathway agonist), SC79 (Akt pathway agonist), and MHY1485 (mTOR pathway agonist) are summarized. Fig. 7A Shown is the expression level of the OPC gene of interest in the presence of TTNPB, MHY1485, and SC79 when the model is otherwise optimized to maximize NKX2-2 expression. Figure 7B As shown, after removal of TTNPB, there was a significant decrease in the predicted expression levels of NKX2-2, OLIG2, and PDGFRA, with NKX2-2 decreasing from over 12,000 to 4,500, OLIG2 decreasing from 1,000 to 400, and PDGFRA decreasing from 350 to less than 100. This result suggests that when RA pathway agonists are removed, there is a significant detrimental effect on the expression of all desired markers. Figure 7C As shown in Figure 2, when MHY1485 was removed, the expression level decreased again, however, not as drastically as in the previous case. Fig.7D As shown, only a small shift in the plot was observed when SC79 was removed, suggesting that this factor is less important than TTNPB and MHY1485 for obtaining maximal induction of OPC markers.

[0206] Figures 8A-8DThe results of factor importance analysis for the effectors viomorphine (SHH pathway agonist), XAV939 (WNT pathway antagonist), LDN193189 (BMP pathway antagonist), and Go6983 (PKC pathway antagonist) are summarized. To obtain an ideal model of oligodendrocyte populations in the anterior brain region, these additional factor inputs, such as FEZF2 and OTX2, were questioned. When the model was optimized for maximizing the expression of FEZF2, the expression levels of FEZF2 and OTX2 were examined in the absence of LDN193189, XAV, viomorphine, or Go6983. The most significant changes were observed in the absence of LDN193189, which resulted in a nearly 50% reduction in the expression of FEZF2 (from 4500 to 2500). The expression of OTX2 was also reduced from 9000 to 7000, which was the lowest among all four elimination processes. When XAV939 and purple morpholino were removed, the expression level of FEZF2 dropped to 3000 and 3500 respectively, while the expression of OTX2 was only slightly higher in both conditions. When G06983 was removed, we did not observe any significant changes in the expression levels of the target genes, thus indicating that G06983 is optional for the control of FEZF2 and OTX2.

[0207] Example 3 : Immunocytochemical Validation of Stem Cell-Derived OPCs

[0208] To further validate the optimized culture medium described in Example 1, cells were cultured in the optimized culture medium for 3 days and the expression of anterior neural ectoderm and oligodendrocyte progenitor cell biomarkers was evaluated using immunocytochemistry. Biomarkers include OTX2 and oligodendrocyte precursor biomarkers, including NKX2-2, OLIG2 and PDGF. Nestin is an early neuronal marker for distinguishing neural stem cells from oligodendrocyte progenitor cells. Ki67 is also used to confirm the proliferation of cells after induction. Fig. 9 Representative immunohistochemistry results are shown in . These immunocytochemistry images confirmed that the majority of cells expressed OTX2. However, no trace of the neuronal biomarker nestin was detected, confirming that the differentiated OPC population lacked neural stem cells. Expression of OLIG2 and NKX2-2 was also observed in more than 90% of the cells, confirming the oligodendrocyte lineage of the cells. No cells expressed PDGFR, which was expected because this gene is expressed at later stages of oligodendrocyte differentiation.

[0209] Example 4: RNA-seq Validation of Stem Cell-Derived Pre-OPCs

[0210] RNA sequencing was used to obtain the gene profile of cells cultured in the differentiation medium detailed in Examples 1 and 2. hiPSCs were cultured in the medium for 3 days and the RNA of the resulting cells was sequenced by standard RNA-seq analysis. Fig. 10A The results in Figure 4 show the normalized expression levels of selected genes representing early oligodendrocyte identity (NKX2-2, OLIG2, PDGFRa) and stem cell state (NANOG, POU5F1) in different brain regions from triplicate experiments at day 0 and day 3. The results show a decrease in the levels of stem cell genes and an increase in the levels of oligodendrocyte genes in pre-OPC cells, validating the use of differentiation medium to differentiate hiPSCs into the oligodendrocyte lineage. Fig. 10B The results showed differential expression and fold changes of selected genes, with HOXA1 at the highest level (15), and OLIG2, NKX2-2, and PDGFRa at 5. These data demonstrate the ability of the developed formulation to serve as a stage 1 medium in directing cells toward an oligodendrocyte identity.

[0211] Example 5 :Development of a culture protocol for the generation of stem cell-derived oligodendrocyte progenitor cells expressing SOX10, OLIG2, and NKX2-2

[0212] In this embodiment, a stepwise differentiation protocol for producing oligodendrocyte progenitor cells was developed, which can guide human pluripotent stem cells to progenitor cells expressing SOX10, NKX2-2 and OLIG2 after 12 days of culture. Differentiated cells also express other oligodendrocyte progenitor cell markers, including PDGFRa and NG2. As described in Example 1, after treating cells with the pre-OPC medium of stage 1 for 3 days, the cells were cultured in the medium of stage 2 for 3 days and then cultured in the medium of stage 3 for 6 days. These cells can be further differentiated into mature oligodendrocytes. Fig.42 The complete three-stage scheme is shown schematically in .

[0213] To develop a recipe for oligodendrocyte differentiation, we used an HD-DoE approach to investigate the effects of various agonists and antagonists (effectors) on pre-OPCs differentiation. These effectors were selected based on existing literature in developmental biology and stem cell differentiation, as well as mouse and human single-cell RNA-seq data on oligodendrocytes at that time.

[0214] These experiments resulted in the Phase 2 formulation shown in Table 1 below and the Phase 3 formulation shown in Table 2 below.

[0215] Table 1. Proven factors in the Phase 2 formulation

[0216] Effector effect concentration FGF2 FGF pathway activators 10ng / ml Purple Morphine SHH agonists 500nM MK2206 AKT antagonists 125nM SC79 AKT agonists 2uM AZD3147 mTOR antagonists 15nM

[0217] Table 2. Proven factors in the Phase 3 formulation

[0218]

[0219]

[0220] To design the formulation for differentiation stage 2, cells were first cultured in the stage 1 medium described in Example 1, then treated with a combination of 8 or 12 factors for 3 days, and the gene expression of cells under each condition was modeled. At this point, due to the short culture time (only 6 days in vitro), we decided to focus on the maximum expression of OLIG2 and the maintenance of high expression of NKX2-2, while initiating the expression of SOX10, OLIG1, and PDGFRa to guide the cells toward an oligodendrocyte fate (Emery and Lu (2015). Cold Spring Harb Perspect Biol. 7:a020461; Perlman et al. (2020). Glia 68:1291–1303; Goldman and Kuypers (2015) Development 142:3983-95).

[0221] In a 12-factor model, the individual and combined effects of SUN 11602 (agonist of FGF signaling), FGF2, Activin A (activator of one of the TGF-b superfamily receptors), A8301 (TGF-b pathway inhibitor), CHIR99021 (WNT activator), AGN193109 (RA antagonist), MK2206 (AKT inhibitor), Vibriomorphin (SHH signaling pathway agonist), AZD3147 (mTOR pathway antagonist), MHY1485 (mTOR pathway agonist), SC79 (AKT pathway agonist), and GO6983 (PKC pathway inhibitor) were tested. This model demonstrated the potential to modulate pre-OPCs to highly express OLIG2, NKX2-2, and PDGFRa. When optimized for maximizing OLIG2 expression at 1841.6, multiple factors showed positive regulatory effects, including FGF-2, with the highest factor contribution of 12.9; MK2206 and AZD3147, with factor contributions of 5.3 and 5.2, respectively; SC79, with a factor contribution of 3.6; and SUN 11602 and Activin A, both with factor contributions less than 3 ( Fig.11). AGN193109, MHY1485 and GO6983 had the greatest negative impact on OLIG2 expression, with factor contributions of 23.2, 22.5 and 10.2, respectively. Within the specification range of achieving 80% of the maximum expression of OLIG2, the Cpk value (process capability index) of this complex medium composition was 0.43, corresponding to a risk of failure of 9.6%.

[0222] Dynamic profiling was used to assess the expression levels of other target genes, such as NKX2-2, OLIG1, and SOX10, and the model was maximized for OLIG2. Activin A and SUN 11602 were reduced to zero due to their very low factor contributions. We observed that when the model was optimized, the expression levels of NKX2-2 were 3000, PDGFRa was 1000, OLIG1 was 80, and SOX10 was 15 ( Fig.12 ). Based on the analysis, four compounds that showed a positive effect on OLIG2 with a factor contribution greater than 3, including FGF2, MK2206, SC79, and AZD3147, were positive for other genes or did not have any significant effect on their expression levels. Therefore, we did not exclude any of these four factors from the candidate formulation. We also observed that although purple morphine and GO6983 had a negative effect on OLIG2 (factor contribution of 7.2 and 10.22), they positively regulated the expression of SOX10. Therefore, the effects of these two compounds on the other selected genes were investigated, and it was concluded that GO6983 should not be included in the formulation due to its significant negative effect on PDGFRA.

[0223] To gain more information on the effect of violacein on the differentiation of pre-OPCs into more committed cell populations, we tested this factor in two additional models. In the 12-factor model, we included violacein, FGF2, AGN193109, and CHIR99021, as well as eight new inputs including TTNPB, biotin, insulin, propionate, DBZ, Yhhu3792, LDN193189, and Y-27632. When optimized for OLIG2 at 3299.4, violacein showed a strong positive effect with a factor contribution of 13 ( Fig.13). Within the specification range of achieving 80% of the maximum expression of OLIG2, the Cpk value (process capability index) of this complex medium composition was 0.68, corresponding to a risk of failure of 0.1%. In another 12-factor model, purple morphine was tested together with A8301, CHIR99021, dexamethasone, T3+T4, PD0325901, ZM336372, DBZ, BMP7, PD173074, SANT-1 and PDGF-AA. When OLIG2 was optimized at 644.6, purple morphine had the greatest positive impact on its expression, with a factor contribution of 16.9 ( Fig.14 ). Within the specification range of achieving 80% of the maximum expression of OLIG2, the Cpk value (process capability index) of this complex medium composition is 0.95, corresponding to a risk of failure of 0.22%.

[0224] We also investigated the single effect of this factor on other selected genes in both models and observed positive trends for SOX10, OLIG1, and NKX2-2 ( Fig.15 ).

[0225] Therefore, considering these models, a candidate formulation of stage 2 consisting of FGF2, purple morphine, MK2206, SC79 and AZD3147 was prepared. This formulation was able to maximize the differentiation of cells, resulting in stable and high expression of OLIG2, NKX2-2, and initial expression of SOX10 and PDGFRa. This formulation was further validated by immunocytochemical analysis (see Example 7).

[0226] To further differentiate the cells toward oligodendrocyte progenitor identity, additional HD-DoE experiments were performed on cells cultured in stage 1 and stage 2 differentiation media. After 3 days, the gene expression of the cells under different combinations of conditions was investigated. At this point, we focused on the maximum expression of hallmark oligodendrocyte progenitor genes such as SOX10, OLIG1, and PDGFRa (Goldman and Kuypers (2015) Development 142:3983-95; Marques et al. (2016) Science 352:1326-1329). In an 8-factor model, the individual and combined effects of LDN193189, SUN 11602, Activin A, Biotin, TTNPB, Isoproterenol, Linoleic Acid, and T3+T4 were tested. When the model was optimized at 256 for maximizing the expression of OLIG1, Activin A and TTNPB had the highest positive impact, with factor contributions of 50.2 and 19.11, respectively ( Fig.16). LDN193189 and SUN 11602 also positively regulated its expression with a factor contribution of 10.8 and 3.7. Within the specification range of achieving 80% of the maximum expression of OLIG1, the Cpk value (process capability index) of this complex medium composition was 0.46, corresponding to a risk of failure of 8.1%.

[0227] The same model was also optimized to maximize the expression of PDGFRa at 3510.9. Specifically, two factors, TTNPB and linoleic acid, determine the expression level of PDGFRa, with factor contributions of 71.6 and 22.1 ( Fig.17 ). Within the specification range of achieving 80% of the maximum expression of PDGFRa, this complex medium composition has a Cpk value (process capability index) of 0.54, corresponding to a risk of failure of 5.2%. The model also optimized for the highest expression of SOX10 at 18.8. In this optimization, TTNPB was the only input that could increase the expression level of the selected gene ( Fig.18 ).

[0228] Dynamic profiling was used to explore combinatorial sets that could optimize the expression of all three selected genes. Among all effectors, TTNPB was the only factor that had a positive effect on all three genes ( Fig.19 ). Since Activin A is the main compound regulating OLIG1 expression levels, we investigated the changes in the graph after adding Activin A and observed that the expression level of SOX10 remained within the target range, and PDGFRa was also expressed at a high level (2520). Addition of other compounds resulted in a decrease in the expression level of two-thirds of the genes, therefore, only Activin A and TTNPB were added to the candidate formulation for Phase 3.

[0229] In another model, the effects of Activin A and TTNPB were tested along with LDN193189, MHY1485, PDGF-AA, AICAR, FGF2, CHIR99021, DBZ, MK2206, Violet Morphine, and T3+T4 on the gene profile of differentiated cells. When the model was optimized for maximizing SOX10 expression at 54.7, we observed that Activin A again had a positive regulatory influence on its expression with a factor contribution of 4.7. Other positive factors included MK2206, with the highest factor contribution of 16.2; MHY1485 with 11.49; CHIR99021 with 9.6; DBZ with 9.3; FGF2 with 3.6; and PDGF-AA with a factor contribution of less than 3 ( Fig. 20 Within the specification range of 80% of the maximum expression of SOX10, the Cpk value (process capability index) of this complex medium composition was 0.47, corresponding to a risk of failure of 7.7%.

[0230] In this model, we also studied the expression conditions of another important OPC gene CSPG4 (Goldman and Kuypers (2015) Development 142:3983-95; van Tilborg et al. (2018) Glia 66:221-238). When the model was optimized at 38.6 to maximize the expression of CSPG4, five factors showed positive regulatory behavior, including TTNPB, with the highest factor contribution of 15.96; as well as purple morphine, activin A, MK2206 and PDGF-AA, with factor contributions of 10.3, 9.3, 5.2 and 2.9, respectively. In this model, LDN193189 had the largest negative effect, with a factor contribution of 16.9 ( Fig.21 Within the specification range of achieving 80% of the maximum expression of CSPG4, the Cpk value (process capability index) of this complex medium composition was 0.69, corresponding to a risk of failure of 1.8%.

[0231] Dynamic profiling was used to better assess the impact of factors on our selected genes and to find combinatorial sets that could optimize their expression ( Fig. 22 ). According to this analysis, although activin A has a positive effect on SOX10, it decreases levels of PDGFRa and OLIG1. However, previous models suggest that activin A may also be positive for OLIG1, depending on the other factors present in the recipe. In addition, SOX10 is one of the master transcription factors for oligodendrocytes and, like OLIG2 and NKX2-2, has shown potential to convert stem cells into oligodendrocytes in reprogramming efforts (Wang et al. (2014) Proc Natl Acad Sci USA 111:E2885-94; García-León et al. (2018) Stem Cell Reports 10:655-672). DBZ showed positive effects on all three genes, but it also increased the neuronal gene ( Fig.23 ). One of the main challenges in differentiating cells towards an oligodendrocyte fate is to control the mixed population and to increase the purity of the culture by directing more cells towards an oligodendrocyte rather than a neuronal identity. Therefore, DBZ was excluded from the final formulation. MK2206, MHY1485, FGF2, and PDGF-AA are positive regulators of SOX10 that either positively increase the expression levels of PDGFRa and OLIG1 or have no significant negative effect on their expression and were therefore included in the final formulation.

[0232] At this point, we investigated the expression profile of a larger set of genes that define the oligodendrocyte progenitor population at later stages, including ID2, CNP, and BCAN (Perlman et al. (2020) Glia 68:1291–1303; Goldman and Kuypers (2015) Development 142:3983-95). In this model, these genes are expressed at 1900, 660, and 44, respectively. AICAR was identified as a factor that positively regulates this set of genes ( Fig.24 ).

[0233] Therefore, with these models in mind, we created a candidate formulation for phase 3 consisting of Activin A, TTNPB, FGF2, PDGF-AA, MK2206, MHY1485, and AICAR. This formulation should maximize differentiation of cells, as this is associated with robust and high expression of SOX10, PDGFRa, and OLIG1. This formulation was further validated by immunocytochemical analysis (see Example 7).

[0234] Example 6 :Analysis of the importance of factors in the induction and culture conditions of oligodendrocyte progenitor cells derived from stem cells

[0235] To assess the impact of eliminating each validation factor, we again used dynamic profiling and compared the expression levels of the target gene in the absence of each final factor while retaining the others. As the expression level of the target gene reveals whether the expected outcome can be achieved, this factor importance analysis reveals the degree of importance of each input effector.

[0236] In the stage 2 formulation of Example 5, each final factor was removed from the respective model, and the expression level of the target gene was evaluated in the presence of other factors and compared with the presence of all factors. Figures 25A-25BWhen FGF-2 was excluded from the formulation, the expression levels of OLIG2 and SOX10 decreased from 1600 to 1000 and from 15 to 0, respectively. The levels of NKX2-2 and OLIG1 also decreased from 3100 to 2800 and from 75 to 65, respectively. When MK2206 was removed, the expression level of OLIG2 decreased from 1600 to 1400, and the level of NKX2-2 decreased significantly from 3100 to 2400. The expression of OLIG1 and SOX10 increased slightly from 75 to 90 and 15 to 17, respectively. When AZD3147 was removed, we observed a similar trend, showing that the levels of OLIG2 and NKX2-2 decreased to 1400 and 1800, respectively, while the levels of SOX10 and OLIG1 increased slightly to 17 and 100, respectively. When SC79 was excluded from the formulation, the levels of NKX2-2, SOX10, and OLIG1 decreased to 2800, 7, and 25, respectively. We noticed that the level of OLIG2 increased from 1600 to 1700.

[0237] In another model, the importance of including olivomorphin in the formulation was determined by tracking the expression levels of target genes in the presence and absence of the compound. When olivomorphin was removed, levels of OLIG1 and OLIG2 dropped from 60 to 20 and 430 to 105, respectively. Fig.25C ).

[0238] In the phase 3 formulation, the expression levels of SOX10, PDGFRa, and OLIG1 were evaluated in the absence of each finalized factor while retaining the others, compared to the presence of all factors. In one model, in the absence of TTNPB, the levels of PDGFRa and OLIG1 dropped dramatically from 2600 to 400, and from 210 to 150. When activin A was excluded from the formulation, the level of OLIG1 dropped to 120, while the level of PDGFRa did not change. In the absence of activin A, the expression level of SOX10 increased from 14 to 19, and when TTNPB was removed, we observed a slight change in its expression from 14 to 15 ( Figures 26A-26B ).

[0239] In another model, to determine the combined effects of MK2206, MHY1485, FGF-2, and PDGF-AA on the cellular gene profile in the presence of the other factors in the final formulation, the expression levels of OLIG1, PDGFRa, SOX8, and SOX10 were compared in the absence of each factor ( Figures 27A-27B). When FGF-2 was removed, the levels of PDGFRa, SOX8, and SOX10 decreased from 920 to 860, 70 to 40, and from 30 to 20, respectively. The level of OLIG1 did not change. Removal of MK2206 caused the level of OLIG1 to increase from 130 to 170, while the expression levels of SOX10 and PDGFRa decreased to 15 and 750, respectively. The level of SOX8 did not change. When MHY1485 was removed, the expression levels of SOX8, SOX10, PDGFRa, and OLIG1 decreased to 60, 15, 850, and 110, respectively. When PDGF-AA was excluded from the recipe, the level of SOX8 decreased to 60, while the other three genes changed slightly.

[0240] In summary, the factor importance analysis demonstrated the importance of incorporating each compound into the Stage 2 and Stage 3 differentiation media formulations described in Example 5.

[0241] Example 7 :Immunocytochemical validation of stem cell-derived oligodendrocyte progenitor cells expressing SOX10 and PDGFRa

[0242] To further validate the optimized medium described in Example 5, cells were treated with stage 1 differentiation medium for 3 days, stage 2 differentiation medium for 3 days, and stage 3 differentiation medium for 6 days, and then standard immunocytochemistry was used to assess the expression of biomarkers for early oligodendrocyte progenitors at the end of stage 2 and late oligodendrocyte blasts at the end of stage 3. The biomarkers tested included OPC-specific markers such as SOX10, OLIG2, NKX2-2, PDGFR, NG2 (CSPG4), and A2B5, as well as the pan-neuronal marker TUBB3 to determine the homogeneity of the culture.

[0243] Immunocytochemical images of cells at the end of stage 2 (day 6 in culture) confirmed the expression of OLIG2 and NKX2-2 in differentiated cells and the initial expression of SOX10 and PDGFRa. Expression of KI67 in cells showed that most cells were still proliferating, which is expected at the progenitor stage ( Fig.28 ). TUBB3 was also detected in less than 50% of the cells, indicating that the cultures were not 100% glial.

[0244] Immunocytochemical analysis of hiPSC-derived cells at the end of stage 3 (culture day 12) confirmed the expression of SOX10 and PDGFRa in differentiated cells ( Fig.29 ). The cells were able to maintain the expression of OLIG2, and we also detected NG2 (CSPG4) and A2B5 in some cells.

[0245] Detection of SOX10 and PDGFRa in differentiated cells at the end of stage 3 of differentiation confirmed the robustness and high conversion potential of the staged recipe described in Example 5 for differentiating human induced pluripotent stem cells into oligodendrocyte progenitor cells after 12 days of in vitro culture.

[0246] Example 8 :Development of a culture protocol for the generation of stem cell-derived oligodendrocyte progenitor cells expressing SOX10, OLIG2, and NKX2-2

[0247] In this example, an alternative stepwise differentiation protocol was developed for generating oligodendrocyte progenitor cells, which, unlike the method described in Example 5, can direct human pluripotent stem cells to differentiate into progenitor cells expressing SOX10, NKX2-2, and OLIG2 after 12 days of culture. Differentiated cells also express other oligodendrocyte progenitor cell markers, including PDGFRa and NG2. The two-stage protocol described in this example is referred to as version 2, while the two-stage protocol described in Example 5 is referred to as version 1.

[0248] For this protocol, after treating the cells for 3 days with stage 1 pre-OPC medium (as described in Example 1), the cells are cultured for 6 days in stage 2 medium and then for 3 days in stage 3 medium. These cells can be further differentiated into mature oligodendrocytes. Fig.43 The complete three-stage scheme is shown schematically in .

[0249] To develop a recipe for oligodendrocyte differentiation, the HD-DoE approach was used to investigate the effects of various agonists and antagonists (effectors) on pre-OPCs differentiation. These effectors were selected based on existing literature on developmental biology and stem cell differentiation, as well as mouse and human single-cell RNA-seq data from oligodendrocytes at that time.

[0250] These experiments resulted in the Phase 2 formulation shown in Table 3 below and the Phase 3 formulation shown in Table 4 below.

[0251] Table 3. Validated factors in Phase 2 formulation

[0252] Effector effect concentration FGF-2 FGF pathway activators 10ng / ml Purple Morphine SHH agonists 500nM CHIR99021 WNT agonists 1uM AZD3147 mTOR antagonists 100nM

[0253] Table 4. Proven factors in the Phase 3 formulation

[0254] Effector effect concentration FGF-2 FGF pathway activators 10ng / ml IGF-1 IGFR activators 10ng / ml AGN193109 RA antagonists 100nM

[0255] To design the formulation for differentiation stage 2, cells were first cultured in the culture medium of stage 1 described in Example 1, and 48 or 96 different combinations of effectors generated by D-optimization were compressed by the robot using the Design-of-Experiment. The effector combinations were prepared in the basal medium, which was then added to the cells, and then the cells were differentiated. RNA extraction was performed three days later, and gene expression was obtained using quantitative PCR analysis. The data were normalized and modeled using partial least squares regression analysis of the effector design to generate gene-specific models. After the model was adjusted to obtain maximum predictive power, the analysis provided an explanation of the effector combination and the ability to control the expression of individual genes alone. Solutions can then be explored within the test range to address the desirability issue.

[0256] At this time, due to the short culture time (only 6 days in vitro), we decided to focus on maximal expression of OLIG2 and maintenance of high expression of NKX2-2, while initiating expression of SOX10, OLIG1, and PDGFRa to guide the cells toward an oligodendrocyte fate (Emery and Lu (2015) Cold Spring Harb Perspect Biol. 7:a020461; Perlman et al. (2020). Glia 68:1291–1303; Goldman and Kuypers (2015) Development 142:3983-95). In an 8-factor model, CHIR99021 (WNT pathway agonist), AGN193109 (RA pathway antagonist), FGF-2 (FGFR agonist), purple morphine (SHH pathway agonist), AICAR (AMPK agonist), GW3965 (LXR pathway agonist), GW590735 (PPAR-a agonist), and AZD3147 (mTOR antagonist) were tested for their individual and combined effects on further cell differentiation. The model demonstrated the potential to regulate pre-OPCs to highly express OLIG2, OLIG1, NKX2-2, and PDGFRa. When the model was optimized at 3325 to maximize the expression of OLIG2, multiple factors showed positive regulatory effects, including AZD3147 (whose factor contribution was the highest at 30.2); the factor contributions of CHIR99021, purple morphine, FGF-2, and GW590735 were 17.96, 7.39, 5.2, and 5.4, respectively ( Fig.30 ). AGN193109 had the greatest negative impact on its expression, with a factor contribution of 25.8. Within the specification range of achieving 80% of the maximum expression of OLIG2, the Cpk value (process capability index) of this complex medium composition was 0.58, corresponding to a risk of failure of 4%.

[0257] The model was also optimized for maximizing OLIG1 expression at 454.7, and we observed that, similar to the optimization of OLIG2, AZD3147 had the largest positive impact on its regulation, with a factor contribution of 26.7, and AGN193109 had the largest negative impact, with a factor contribution of 25.3 ( Fig.31 ). Other factors with positive impact include CHIR99021, FGF-2 and GW590735, with factor contributions of 22.8, 9.9 and 3.1, respectively. Within the specification range of achieving 80% of the maximum expression of OLIG1, the Cpk value (process capability index) of this complex medium composition is 0.42, and the corresponding risk of failure is 9.9%.

[0258] Dynamic profiling was used to assess the expression levels of other target genes, such as NKX2-2 and SOX10, and the model was optimized for OLIG2. When the model was optimized, the expression level of NKX2-2 was 2000, OLIG1 was 440, and SOX10 was 15 ( Fig.32 ). Based on the analysis, four compounds out of the five showed positive effects on the expression of OLIG2, including FGF-2, purple morphine, CHIR99021, AZD3147, which also had a positive effect on the expression of NKX2-2, while FGF-2, CHIR99021, AZD3147, and GW590735 had positive effects on the expression of OLIG1. Although purple morphine did not significantly improve the expression of OLIG1, it was a significant positive regulator of both OLIG2 and NKX2-2, which are stage 2 target genes, so positive co-factors of these two genes were included in the candidate formulation of the stage 2 differentiation medium. The negative effects of GW590735 on the expression of SOX10 and NKX2-2 led us to exclude it from the final formulation.

[0259] Therefore, considering these models, a candidate formulation of stage 2 consisting of FGF2, purple morphine, CHIR99021 and AZD3147 was made. This formulation should maximize the differentiation of cells to achieve robust and high expression of OLIG2, NKX2-2 and initial expression of SOX10. This formulation was further validated by immunocytochemical analysis (see Example 9).

[0260] To further differentiate cells toward oligodendrocyte progenitor identity, additional HD-DoE experiments were performed on cells cultured in stage 1 and stage 2 differentiation media. After 3 days, the gene expression of cells under different combinations of conditions was investigated. At this point, we focused on the maximum expression of hallmark oligodendrocyte progenitor genes such as SOX10, OLIG1, and PDGFRa (Goldman and Kuypers (2015) Development 142:3983-95; Marques et al. (2016) Science 352:1326-1329). In an 8-factor model, the individual and combined effects of TTNPB (RA agonist), CHIR99021, FGF-2, IGF-1, AGN193109, purple morphine, MHY1485 (mTOR pathway agonist), and SC79 (AKT pathway agonist) were tested to guide cells toward the SOX10 / PDGFRa positive population. When optimized for SOX10 at 41.04, TTNPB and FGF-2 showed the highest positive impact, with factor contributions of 17.98 and 15.3, respectively. CHIR99021 was also positive, with a factor contribution of 8.1 ( Fig.33 SC 79 had the highest negative impact with a factor contribution of 30.2. Within the specification range of 80% of the maximum expression of SOX10, this complex medium composition had a Cpk value (process capability index) of 0.47, corresponding to a risk of failure of 7.3%.

[0261] When the model was optimized for maximizing PDGFRa expression at 853.4, purple morphine had the largest positive impact, with a factor contribution of 20.6. Other positive factors were IGF-1, with a factor contribution of 11.2; CHIR99021, with a factor contribution of 8.5; and TTNPB, with a factor contribution less than 1 ( Fig.34 Within the specification range of 80% of the maximum expression of PDGFRa, the Cpk value (process capability index) of this complex medium composition was 0.45, corresponding to a risk of failure of 8.6%.

[0262] According to the literature, proteins such as FGF-2, IGF-1, PDGF-AA and NT-3 play an important role in oligodendrocyte differentiation (Goldman and Kuypers (2015) Development 142: 3983-95). In the first model, FGF-2 had a positive regulatory effect on the expression levels of SOX10 and IGF-1 only on PDGFRa. Therefore, in order to further understand the effects of IGF-1 and FGF-2 on cell differentiation and confirm their positive effects, we included these factors in two other models and optimized the model with the maximum expression of SOX10. This gene is one of the main transcription factors for oligodendrocytes, and like OLIG2 and NKX2-2, OLIG2 and NKX2-2 have shown the potential to differentiate stem cells into oligodendrocytes during reprogramming (Wang et al. (2014) Proc Natl Acad Sci USA 111:E2885-94; García-León et al. (2018) Stem Cell Reports 10:655-672). In both models, IGF-1 and FGF-2 had a positive effect on the expression level of SOX10, with expression levels of 95 and 25 ( Fig.35 AGN193109 was also shown to have a positive effect on increasing the levels of SOX10, with a contribution of a factor of 16.

[0263] In the previous model, TTNPB was observed to positively regulate one of the OPC genes, and since TTNPB and AGN193109 have opposing functions in the RA signaling pathway, we decided to investigate the expression levels of another OPC gene, OLIG1, in the presence of AGN193109, TTNPB, FGF-2, and IGF-1 ( Fig.36 ). In this analysis, the highest and lowest expression levels are shown on a spectrum from red to blue, with each of the four axes representing a compound. OLIG1 expression was maximal when TTNPB concentration was lowest and AGN193109 and FGF-2 concentrations were highest, and although IGF-1 showed less effect on OLIG1 expression, it still positively regulated its levels.

[0264] To confirm the effects of TTNPB, FGF-2, and IGF-1 on a larger scale, we investigated the expression profiles of a large panel of genes that define late oligodendrocyte progenitor populations, such as ID2, CSPG4, FYN, SOX8, PLP1, and BCAN (Perlman et al. (2020) Glia 68:1291–1303; Goldman and Kuypers (2015) Development 142:3983-95); van Tilborg et al. (2018) Glia 66:221-238), and neuronal genes such as NEUROD1, NEUROG1, and NEUROG2, in the presence of IGF-1, FGF-2, or TTNPB. Fig.37 Factor plots of various genes in the presence of IGF-1 are shown, and it was observed that the expression levels of OPC genes increased with increasing IGF-1 concentrations, while the expression levels of neuronal genes decreased. This confirms the positive effect of IGF-1 on the differentiation of cells towards oligodendrocyte rather than neuronal fate. Fig.38 The same analysis for FGF-2 is shown, and it is observed that the presence of FGF-2 increases the expression levels of OPC genes while decreasing the expression levels of neuronal genes. However, in the presence of TTNPB, the predicted behavior of the genes changes, and opposite trends are observed in the graph, indicating a negative effect of TTNPB on OPC genes and its positive effect on neuronal genes ( Fig.39 ).

[0265] Therefore, taking these models into consideration, we created a phase 3 candidate formulation consisting of FGF-2, IGF-1, and AGN 193109. This formulation was able to maximize cell differentiation to stably and highly express SOX10, PDGFRa, and OLIG1. This formulation was further validated by immunocytochemical analysis (see Example 9).

[0266] Example 9 :Immunocytochemical validation of stem cell-derived oligodendrocyte progenitor cells expressing SOX10 and PDGFRa

[0267] To further validate the optimized culture medium described in Example 8, cells were treated with stage 1 differentiation medium for 3 days, stage 2 differentiation medium for 6 days, and stage 3 differentiation medium for 3 days, and then standard immunocytochemistry was used to assess the expression of biomarkers for early oligodendrocyte progenitor cells at the end of stage 2 and late oligodendrocyte progenitor cells at the end of stage 3. The biomarkers tested included OPC-specific markers such as SOX10, OLIG2, NKX2-2, PDGFR, and NG2 (CSPG4), as well as the pan-neuronal marker β-III tubulin TUBB3 to determine the homogeneity of the culture.

[0268] Immunocytochemical images of cells at the end of stage 2 (culture day 9) confirmed the expression of OLIG2, NKX2-2, and SOX10 in differentiated cells and the initial expression of PDGFRa ( Fig.40 ). The expression of KI67 in the cells indicated that most of the cells were still proliferating, which is expected at the progenitor stage.

[0269] Results from immunocytochemical analysis of hiPSC-derived cells at the end of stage 3 (day 12 in culture) confirmed the expression of SOX10, PDGFRa, and NG2 (CSPG4) in differentiated cells. The cells were able to maintain expression of OLIG2. In addition, the late OPC marker CNP was detected in more than half of the cells, and initial expression of O4 was observed in some cells in culture ( Fig.41 ). TUBB3 was also detected in less than half of the cultures, suggesting that the cultures were heterogeneous.

[0270] Detection of SOX10 and PDGFRa expression in differentiated cells at the end of differentiation stage 3 confirmed the robustness and high translational potential of the staged formulation described in Example 8 for differentiating human induced pluripotent stem cells into oligodendrocyte progenitor cells after 12 days of in vitro culture.

[0271] Example 10 :Development of a culture protocol for the generation of stem cell-derived premyelinating oligodendrocytes expressing CNP, CD9, and O4

[0272] In this example, a culture protocol was developed for producing pre-myelinating oligodendrocytes from pre-OPCs, referred to herein as the stage 4 protocol. As described in the previous examples, a stepwise 3-stage differentiation protocol was developed that guided human pluripotent stem cells to progenitor cells expressing SOX10, NKX2-2, and OLIG2 after 12 days of culture. Differentiated cells also express other oligodendrocyte progenitor cell markers, including PDGFRa and NG2. After 6 days of culture in the stage 4 differentiation medium described herein, these cells can further mature into pre-myelinating oligodendrocytes expressing CNP, CD9, and O4. In the cell population, the initial expression of the myelinating marker MBP was also observed.

[0273] To develop a stage 4 oligodendrocyte differentiation recipe, the effects of various agonists and antagonists (effectors) on pre-OPCs differentiation were investigated using the HD-DoE approach described in the previous examples. These effectors were selected based on existing literature in developmental biology, stem cell differentiation, and mouse and human single-cell RNA-seq data from oligodendrocytes at that time.

[0274] These experiments resulted in the Stage 4 formulation shown in Table 5 below. Fig.44 A representative schematic diagram of the stage 4 culture protocol is shown in .

[0275] Table 5. Proven factors in the Phase 4 formulation

[0276] Effector effect concentration IGF-1 IGF-1 receptor activators 10ng / ml PDGF-AA PDGFR pathway 10ng / ml NT-3 TrkC agonists 10ng / ml T3 Thyroid hormone receptor agonists 50nM insulin Insulin receptor agonists 20ug / ml

[0277] To design a recipe for stage 4 differentiation, 48 or 96 different combinations of effectors generated by D-optimization were prepared by the robot using experimental design compression. The effector combination was prepared in basal medium and then added to the cells, which were treated with differentiation medium for stages 1, 2, and 3 as described in the previous examples to produce OPCs. The resulting OPCs were then re-plated on 96-well plates. RNA extraction was performed after 3 to 7 days, and gene expression was obtained using quantitative PCR analysis. The data was normalized and modeled using partial least squares regression analysis of the effector design to generate a gene-specific model, and after the model was adjusted to obtain maximum Q2 predictive power, the analysis provided an explanation of the effector combination and the ability to control the expression of a single gene alone. Solutions can then be explored within the test range to address the desirability issue.

[0278] To guide the differentiation of OPCs into oligodendrocytes, the expression of postmitotic genes, such as CNP and PLP1, has been studied (Goldman and Kuypers (2015) Development 142:3983-95; Emery and Lu (2015) Cold Spring Harb Perspect Biol. 7:a020461; Tiane et al. (2019) Cells 8:1236). In one model, the effects of NT-3, T3, insulin, biotin, IGF-1, purple morphine, cAMP, and ascorbic acid 2-phosphate on the gene profile of differentiated cells were examined. When the expression of CNP was optimized at 1339 to maximize its expression, multiple inputs showed positive regulatory effects on its expression ( Fig.45 ), including NT-3, with a factor contribution of 24; T3, with a factor contribution of 18; morphine, with a factor contribution of 10; insulin, with a factor contribution of 10; and cAMP, with a factor contribution of 7. IGF-1 also had a positive regulatory effect, but with a factor contribution less than 1. Biotin and ascorbic acid were the only factors with a negative effect, with factor contributions of 12 and 17, respectively. Within the specification range of achieving 80% of the maximum expression of CNP, the Cpk value (process capability index) of this complex medium composition was 0.43, corresponding to a risk of failure of 9%.

[0279] Based on the preliminary results, the combined effects of the eight factors tested were investigated, and four additional factors that are expected to promote oligodendrocyte differentiation were included (Wang et al. (1998) Neuron 21:63-75; Weng et al. (2017) Sci Rep. 7:1705; Benarroch (2009) Neurology 72:1779-1785; Shi et al. (2019) Exp Ther Med. 18:1258-1266). In this HD-DoE model, the effects of IGF-1, NT-3, T3, insulin, purple morphine, biotin, ascorbic acid, Albumax, cAMP, propionate, AICAR, and Notch pathway inhibitor gamma secretase inhibitor XX (GSI-XX) on OPCs differentiation were tested after 6 days of culture. When CNP was optimized at 1790, NT-3, IGF-1, insulin, and GSI-XX had a positive impact on maximizing its expression, with factor contributions of 13, 5, 3, and 7, respectively ( Fig.46 ). Biotin and cAMP, with a combined factor contribution of 16, had the largest negative impact on CNP maximization. T3 also showed a negative impact; however, its factor contribution was relatively low, at 1.2. Within the specification range of achieving 80% of the maximum expression of CNP, the Cpk value (process capability index) of this complex medium composition was 0.46, corresponding to a risk of failure of 8.3%.

[0280] When the model was optimized for the expression of CNP, other oligodendrocyte genes, such as PLP1, MYT1, APOD, and BCAN, were also upregulated, with expression levels of 2800, 157, 40, 189, and 72, respectively, while the proliferation marker MKI67 was minimized ( Fig.47 ). The genetic profile showed that the formula had the potential to strongly induce cell differentiation toward an oligodendrocyte fate.

[0281] Since the expression level of PLP1 exceeded 2000, the optimization conditions for this terminal expression marker in the model were studied. Under the optimized conditions, the expression level of PLP1 increased to 3600, and NT-3 and insulin were its only two positive regulators. Insulin had the highest factor contribution of 13, and NT-3 had 7 ( Fig.48 ). AICAR, Albumax and Purple Morphine had the largest negative impact, with factor contributions of 12, 11 and 10, respectively. Within the specification range of 80% of the maximum expression of PLP1, the Cpk value (process capability index) of this complex medium composition was 0.77, corresponding to a risk of failure of 1.2%.

[0282] Since GSI-XX and IGF-1 have opposite effects on CNP and PLP1, dynamic profiling analysis was used to further investigate their effects on other genes in the model ( Fig.49 ). It was observed that GSI-XX positively regulated the expression of neuronal genes such as NeuroG1, NeuroG2, and NeuroD1, as well as CNP. Therefore, it was decided to remove it from the final formulation to minimize the heterogeneity of the cultures. Since both experiments from the HD-DoE showed that IGF-1 had a positive regulatory effect on CNP, it was decided to add it to the final formulation.

[0283] Previous HD-DoE experimental results showed that T3 had a high positive regulatory effect on the expression of CNP, with a factor contribution of 18. Therefore, the effect of T3 on other genes in the model was further investigated. The model showed that T3 could upregulate other OL genes, such as PLP1, PTGDS, KLK6, and MBP, while downregulating the neuronal gene NeuroG1 ( Fig.50 ). This observation led to the inclusion of T3 in the final Phase 4 formulation.

[0284] Finally, PDGF-AA has been established in the literature to promote oligodendrocyte differentiation, so it was added to the final stage 4 formulation without the need for additional HD-DoE modeling.

[0285] Accordingly, a stage 4 differentiation formula was prepared with the ingredients shown in Table 5 above. This formula was able to maximize the differentiation of cells to achieve robust and high expression of CNP and O4, as well as initial expression of PLP1 and MBP. This formula was validated by immunocytochemical analysis as described in Example 11.

[0286] Embodiment 11 Immunocytochemical validation of a culture protocol for stem cell-derived premyelinating oligodendrocytes expressing CNP and O4

[0287] To validate the stage 4 formulation described in Example 10, cells were treated with stage 1, stage 2, and stage 3 differentiation media for a total of 12 days and then cultured for 6 days in the stage 4 formulation. At this point, immunocytochemistry was used to assess the expression of late oligodendrocyte progenitor and early oligodendrocyte biomarkers in differentiated cells. Biomarkers included OPC-specific markers such as OLIG2, NKX2-2, PDGFR, and A2B5, oligodendrocyte markers including CNP, O4, MBP, and PLP1, and pan-neuronal marker TUBB3 to determine the homogeneity of the culture.

[0288] Immunocytochemical images of cells at the end of stage 4 (culture day 18) confirmed the expression of OLIG2, NKX2-2, CNP, and O4 in differentiated cells and the initial expression of MBP and PLP1 ( Fig.51 ). Expression of KI67 and PDGFRa in a subset of cells showed that some cells were still proliferating. TUBB3 was detected in less than 50% of the cells, indicating that the cultures were not 100% glial.

[0289] At the end of stage 4, flow cytometry was also used to measure the expression level of CD9. CD9 is a surface marker expressed in oligodendrocyte progenitor cells and premyelinating oligodendrocytes with differentiation tendency (Goldman and Kuypers (2015) Development 142:3983-95). Fig.52 As shown, 81.5% of the cells were CD9 positive, indicating that the majority of the culture was developing toward an oligodendrocyte fate.

[0290] Detection of CNP, CD9 and O4 in differentiated cells at the end of differentiation stage 4 confirmed that the 4-stage formulation described in the example can differentiate human induced pluripotent stem cells into premyelinating oligodendrocytes after 18 days of in vitro culture, demonstrating its robustness and high conversion capacity.

[0291] Equivalent

[0292] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A method for producing CD9+A2B5+O4+CNPase+ premyelinating oligodendrocytes (preOLs), comprising culturing SOX10+OLIG2+NKX2-2+OPCs in a culture medium to produce CD9+A2B5+O4+CNPase+preOLs, wherein the culture medium contains an IGF1R pathway agonist, a TrkC pathway agonist, a PDGFR pathway agonist, a thyroid hormone receptor agonist, and an insulin receptor agonist.

2. The method according to claim 1, wherein the OPCs are cultured in the culture medium for six days.

3. The method according to claim 1 or 2, wherein the IGF1R pathway agonist is selected from the group consisting of IGF-1, IGF-2, insulin, Rg5, IGF-1 30-41, demethyl chrysanthemumquinone B1, IGF1-Ado, X10, mecasermin, and combinations thereof. The method of claim 3 , wherein the IGF1R pathway agonist is IGF-1. The method according to claim 4 , wherein IGF-1 is present in the culture medium at a concentration of 10 ng / ml.

6. The method of claim 1 or 2, wherein the TrkC pathway agonist is selected from the group consisting of neurotrophin-3 (NT-3), peptide mimetics based on the β-turn of NT-3, LM22B 10, GNF 5837, and combinations thereof.

7. The method of claim 6, wherein the TrkC pathway agonist is NT-3.

8. The method according to claim 7, wherein NT-3 is present in the culture medium at a concentration of 10 ng / ml.

9. The method of claim 1 or 2, wherein the PDGFR pathway agonist is PDGF-AA.

10. The method of claim 9, wherein PDGF-AA is present in the culture medium at a concentration of 10 ng / ml.

11. The method of claim 1 or 2, wherein the thyroid hormone receptor agonist is selected from the group consisting of T3, T4, Resmetirom, TRb agonist 3 (Compound 3), Sobetirome, Tilatracol, and combinations thereof.

12. The method of claim 11, wherein the thyroid hormone receptor agonist is T3.

13. The method of claim 12, wherein T3 is present in the culture medium at a concentration of 50 nM.

14. The method according to claim 1 or 2, wherein the insulin receptor agonist is selected from the group consisting of insulin, IGF-1, IGF-2, demethyl chrysanthemumquinone B1, MK-5160, MK-1092, and combinations thereof.

15. The method of claim 14, wherein the insulin receptor agonist is insulin.

16. The method of claim 15, wherein insulin is present in the culture medium at a concentration of 20 μg / ml.

17. A method for producing CD9+A2B5+O4+CNPase+ premyelinating oligodendrocytes (preOLs), comprising: (a) On days 0-3, human OLIG2+ pre-oligodendrocyte progenitor cells (pre-OPCs) were cultured in a culture medium containing an FGFR pathway agonist, an mTOR pathway antagonist, an SHH pathway agonist, an AKT pathway antagonist, and an AKT pathway agonist to obtain a cell population; (b) on days 3-9, culturing the cell population from step (a) in a culture medium comprising an FGFR pathway agonist, an activin receptor (AR) pathway agonist, a PDGFR pathway agonist, an AKT pathway antagonist, a retinoic acid (RA) pathway agonist, an AMPK pathway agonist, and an mTOR pathway agonist to produce SOX10+OLIG2+NKX2-2+OPCs; and (c) on days 9-15, culturing the cell population from step (b) in a culture medium comprising an IGF1R pathway agonist, a TrkC pathway agonist, a PDGFR pathway agonist, a thyroid hormone receptor agonist, and an insulin receptor agonist to produce CD9+A2B5+O4+CNPase+preOLs.

18. The method of claim 17, wherein the human OLIG2+ pre-OPCs are obtained by culturing human pluripotent stem cells from day -3 to day 0 in a culture medium containing a retinoic acid (RA) pathway agonist, an Akt pathway agonist, an mTOR pathway agonist, a WNT pathway antagonist, an SHH pathway agonist, a BMP pathway antagonist, and a PKC pathway antagonist.

19. The method of claim 18, wherein the human pluripotent stem cells are induced pluripotent stem cells (iPSCs).

20. The method of claim 18, wherein the human pluripotent stem cells are embryonic stem cells.

21. The method according to any one of claims 18 to 20, wherein the human pluripotent stem cells attach to a vitronectin-coated plate during culture.

22. The method of any one of claims 17-21, wherein the FGFR pathway agonist is FGF2, SUN11602, or a combination thereof.

23. The method of claim 22, wherein the FGFR pathway agonist is FGF2.

24. The method of claim 22, wherein the FGFR pathway agonist is FGF2, which is present in the culture medium of step (a) and step (b) at a concentration of 10 ng / ml.

25. The method of any one of claims 17-21, wherein the mTOR pathway antagonist is selected from the group consisting of AZD3147, Daclis, Rapamycin, Everolimus, AZD 8055, Temsirolimus, PI-103, NU7441, BC-LI-0186, eCF 309, ETP 45658, Niclosamide, Omelisse, PF 04691502, PF 05212384, Torin 1, Torin 2, WYE 687, XL 388, STK16-IN-1, PP 242, Torkinib, Ridaforolimus, Sapacerti, Voxtalisib, and combinations thereof.

26. The method of claim 25, wherein the mTOR pathway antagonist is AZD 3147.

27. The method of claim 25, wherein the mTOR pathway antagonist is AZD 3147, which is present in the culture medium at a concentration of 15 nM.

28. The method of any one of claims 17-21, wherein the SHH pathway agonist is selected from the group consisting of purple morphine, GSA 10, SHH, SAG, and combinations thereof.

29. The method of claim 28, wherein the SHH pathway agonist is morphine.

30. The method of claim 28, wherein the SHH pathway agonist is morphine, which is present in the culture medium at a concentration of 500 nM.

31. The method according to any one of claims 17-21, wherein the Akt pathway antagonist is selected from the group consisting of MK2206, GSK690693, perifosine (KRX-0401), patasertib (GDC-0068), capasertib (AZD5363), PF-04691502, AT 7867, triciribine (NSC154020), ARQ751, miranthide (ab235550), bortezomib-resistant, Cerisertib, Akti1 / 2, CCT128930, A 674563, PHT 427, miltefosine, AT 13148, ML 9, BAY1125976, oridonin, TIC10, salsaponin, Akti IV, 10-DEBC, API-1, SC 66, FPA 124. API-2, urolithin A, and combinations thereof.

32. The method of claim 31, wherein the Akt pathway antagonist is MK2206.

33. The method of claim 31, wherein the Akt pathway antagonist is selected from the group consisting of MK2206, which is present in the culture medium of step (a) and step (b) at a concentration of 125 nM.

34. The method of any one of claims 17-21, wherein the Akt pathway agonist is selected from the group consisting of Sc79, demethylcoclaurine, LM22B-10, YS-49, YS-49 monohydrate, demethylchrysanthemumquinone B1, Recilisib, N-oleylglycine, NSC45586 sodium, periwinkle, CHPG sodium salt, bilobalide, 6-hydroxyflavone, musk ketone, SEW2871, 8-isopentenylnaringenin, Razuprotafib, and combinations thereof.

35. The method of claim 34, wherein the Akt pathway agonist is Sc79.

36. The method of claim 34, wherein the Akt pathway agonist is Sc79, which is present in the culture medium at a concentration of 2 μM.

37. The method of any one of claims 17-21, wherein the AR pathway agonist is selected from the group consisting of activin A, inulin, and combinations thereof.

38. The method of claim 37, wherein the AR pathway agonist is activin A.

39. The method of claim 37, wherein the AR pathway agonist is activin A, which is present in the culture medium at a concentration of 10 ng / ml.

40. The method of any one of claims 17-21, wherein the PDGFR pathway agonist is PDGF-AA.

41. The method of claim 40, wherein the PDGFR pathway agonist is PDGF-AA, which is present in the culture medium at a concentration of 10 ng / ml.

42. The method of any one of claims 17-21, wherein the RA pathway agonist is selected from the group consisting of TTNPB, AM 580, CD 1530, CD 2314, CD 437, Ch 55, BMS 753, BMS 961, tazarotene, isotretinoic acid, retinoic acid, tamibarotene, ATRA, AC 261066, AC 55649, retinoic acid (RA), Sr11237, adapalene, EC23, 9-cis retinoic acid, 13-cis retinoic acid, 4-oxo retinoic acid, and all-trans retinoic acid (ATRA), and combinations thereof.

43. The method of claim 42, wherein the RA pathway agonist is TTNPB.

44. The method of claim 42, wherein the RA pathway agonist is TTNPB, which is present in the culture medium at a concentration of 50 nM.

45. The method according to any one of claims 17-21, wherein the AMPK pathway agonist is selected from the group consisting of AICAR, metformin, arborvitae B, mariglycoside, gentian ester glycoside, A 769662, PF 06409577, metformin hydrochloride, ZLN 024, ZLN 2024 hydrochloride, nilotinib, phenformin, nilotinib hydrochloride monohydrate, 5'-adenosine monophosphate monohydrate, homoplanin, MK 8722, sterol, ASP4132, GSK621, EX229 (Compound 991), trans-feluric acid, O-304, MK 3903, BAM 15. Ligustrum lucidum, ETC-1002, BC1618, IMM-H007, IM156, Panax notoginseng saponin IVa, Pachymic acid A, 7-methoxyisoflavone, Urolithin B, Danthronone, Desmethylberberine, AMPK activator 1, AMPK activator 2, AMPK activator 4, Malvaceae chloride-3-O-arabinoside, RSVA 405, Etilefrine, COH-SR4, Buformin, Buformin hydrochloride, PT1, Bepedic acid, 3a-Hydrocymogrol, 2-hydroxyestradiol, and combinations thereof.

46. ​​The method of claim 45, wherein the AMPK pathway agonist is AICAR.

47. The method of claim 45, wherein the AMPK pathway agonist is AICAR, which is present in the culture medium at a concentration of 200 μM.

48. The method of any one of claims 17-21, wherein the mTOR pathway agonist is MHY1485, 3BDO, salidroside, L-leucine, NV-5138, testosterone, 3-benzyl-5-((2-nitrophenoxy)methyl)-dihydrofuran-2(3H)-one (3BDO), NV-5138 hydrochloride, NV-5138, L-leucine-d1, L-leucine-2-13C, 15N, leucine-13C6, L-leucine-d7, L-leucine-d10, L-leucine d2, L-leucine-d3, L-leucine-18O2, L-leucine-13C, L-leucine-2-13C, L-leucine 13C6-15N, L-leucine-15N, L-leucine-1-13C, 15N, and combinations thereof.

49. The method of claim 48, wherein the mTOR pathway agonist is MHY1485.

50. The method of claim 48, wherein the mTOR pathway agonist is MHY1485, which is present in the culture medium at a concentration of 2 μM.

51. A culture medium for obtaining oligodendrocyte progenitor cells (OPCs), comprising an FGFR pathway agonist, an mTOR pathway antagonist, an SHH pathway agonist, an AKT pathway antagonist and an AKT pathway agonist.

52. A culture medium for obtaining oligodendrocyte progenitor cells (OPCs), comprising an FGFR pathway agonist, an activin receptor (AR) pathway agonist, a PDGFR pathway agonist, an AKT pathway antagonist, a retinoic acid (RA) pathway agonist, an AMPK pathway agonist and an mTOR pathway agonist.

53. A culture medium for obtaining CD9+A2B5+O4+CNPase+ premyelinating oligodendrocytes (preOLs), comprising an IGF1R pathway agonist, a TrkC pathway agonist, a PDGFR pathway agonist, a thyroid hormone receptor agonist and an insulin receptor agonist.

54. A method for producing human CD9+A2B5+O4+CNPase+ premyelinating oligodendrocytes (preOLs), comprising: (a) On days 0-6, human OLIG2+ pre-oligodendrocyte progenitor cells (pre-OPCs) were cultured in a culture medium containing an FGFR pathway agonist, an mTOR pathway antagonist, an SHH pathway agonist, and a WNT pathway agonist to obtain a cell population; as well as (b) on day 6-9, culturing the cell population from step (a) in a culture medium comprising an FGFR pathway agonist, an IGF-1 pathway agonist, and a retinoic acid (RA) pathway agonist to produce SOX10+OLIG2+NKX2-2+OPCs; as well as (c) on days 9-15, culturing the cell population from step (b) in a culture medium comprising an IGF1R pathway agonist, a TrkC pathway agonist, a PDGFR pathway agonist, a thyroid hormone receptor agonist, and an insulin receptor agonist to produce CD9+A2B5+O4+CNPase+preOLs.

55. The method of claim 54, wherein the human OLIG2+ pre-OPCs are obtained by culturing human pluripotent stem cells from day -3 to day 0 in a culture medium comprising a retinoic acid (RA) pathway agonist, an Akt pathway agonist, an mTOR pathway agonist, a WNT pathway antagonist, an SHH pathway agonist, a BMP pathway antagonist, and a PKC pathway antagonist.

56. The method of claim 55, wherein the human pluripotent stem cells are induced pluripotent stem cells (iPSCs).

57. The method of claim 55, wherein the human pluripotent stem cells are embryonic stem cells.

58. The method of any one of claims 54-57, wherein the human pluripotent stem cells attach to a vitronectin-coated plate during culture.

59. The method of any one of claims 54-58, wherein the FGFR pathway agonist is FGF2, SUN11602, or a combination thereof.

60. The method of claim 59, wherein the FGFR pathway agonist is FGF2.

61. The method of claim 59, wherein the FGFR pathway agonist is FGF2, which is present in the culture medium of step (a) and step (b) at a concentration of 10 ng / ml.

62. The method of any one of claims 54-58, wherein the mTOR pathway antagonist is selected from the group consisting of AZD3147, Daclis, Rapamycin, Everolimus, AZD 8055, Temsirolimus, PI-103, NU7441, BC-LI-0186, eCF 309, ETP 45658, Niclosamide, Omelisse, PF 04691502, PF 05212384, Torin 1, Torin 2, WYE 687, XL 388, STK16-IN-1, PP 242, Torkinib, Ridaforolimus, Sapacerti, Voxtalisib, and combinations thereof.

63. The method of claim 62, wherein the mTOR pathway antagonist is AZD 3147.

64. The method of claim 62, wherein the mTOR pathway antagonist is AZD 3147, which is present in the culture medium at a concentration of 100 nM.

65. The method of any one of claims 54-58, wherein the SHH pathway agonist is selected from the group consisting of purple morphine, GSA 10, SHH, SAG, and combinations thereof.

66. The method of claim 65, wherein the SHH pathway agonist is morphine.

67. The method of claim 65, wherein the SHH pathway agonist is purple morphine, which is present in the culture medium at a concentration of 500 nM.

68. The method of any one of claims 54-58, wherein the WNT pathway agonist is selected from the group consisting of CHIR99021, CHIR98014, SB 216763, SB 415286, LY2090314, 3F8, A1070722, AR-A 014418, BIO, BIO-acetone oxime, AZD1080, WNT3A, atopron, indirubin-3-oxime, 1-azacamprolone, camparolone, TC-G 24, TDZD 8, TWS119, NP 031112, AT 7519, KY 19382, AZD2858, and combinations thereof.

69. The method of claim 68, wherein the WNT pathway agonist is CHIR99021.

70. The method of claim 68, wherein the WNT pathway agonist is CHIR99021, which is present in the culture medium at a concentration of 1 μM.

71. The method of any one of claims 54-58, wherein the IGF-1 pathway agonist is selected from the group consisting of IGF-1, IGF-2, insulin, Rg5, IGF-1 30-41, demethyl chrysanthemumquinone B1, IGF1-Ado, X10, mecasermin, and combinations thereof.

72. The method of claim 71, wherein the IGF-1 pathway agonist is IGF-1.

73. The method of claim 71, wherein the IGF-1 pathway agonist is IGF-1, which is present in the culture medium at a concentration of 10 ng / ml.

74. The method of any one of claims 54-58, wherein the RA pathway antagonist is selected from the group consisting of AGN193109, BMS195614, CD 2665, ER 50891, LE 135, LY 2955303, MM11253, and combinations thereof.

75. The method of claim 74, wherein the RA pathway antagonist is AGN 193109.

76. The method of claim 74, wherein the RA pathway antagonist is AGN 193109, which is present in the culture medium at a concentration of 100 nM.

77. A culture medium for obtaining oligodendrocyte progenitor cells (OPCs), comprising an FGFR pathway agonist, an mTOR pathway antagonist, an SHH pathway agonist, and a WNT pathway agonist.

78. A culture medium for obtaining oligodendrocyte progenitor cells (OPCs), comprising an FGFR pathway agonist, an IGF-1 pathway agonist, and a retinoic acid (RA) pathway agonist.

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