Methods and compositions for generating oligodendrocyte precursor cells - Patent Application 20070122997

Abstract

Methods are provided for generating pre-oligodendrocyte precursor cells (pre-OPCs), oligodendrocyte precursor cells (OPCs), and pre-myelinating oligodendrocytes (pre-OLs) from human pluripotent stem cells using chemically defined culture media, which allow for the generation of pre-OPCs in as little as 3 days, SOX10+ OLIG2+ NKX2-2+ OPCs in as little as 12 days, and CD9+ A2B5+ O4+ CNPase+ pre-OLs in as little as 18 days. Two alternative culture protocols for generating OPCs are provided. Culture media, isolated cell populations, and kits are also provided.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 396,073, filed August 8, 2022, and U.S. Provisional Patent Application No. 63 / 400,222, filed August 23, 2022, the entire contents of which are incorporated herein by reference in their entirety.

[0002] Granted Government Rights This invention was made with government support under Grant Number: W911NF-17-3-0003 awarded by the US ARMY ACC-AGP-RTP. The government has certain rights in this invention. [Background technology]

[0003] Background of the Invention Oligodendrocytes (OLs) are a type of glial cell that form myelin sheaths around axons. Oligodendrocytes are therefore important for nerve conduction in the central nervous system (CNS). A deeper understanding of the biology of oligodendrocytes is likely to be crucial for the development of therapies to treat neurodegenerative disorders, including demyelinating diseases such as multiple sclerosis and leukodystrophies, as well as amyotrophic lateral sclerosis (ALS), which may involve demyelination later in the disease process. Additionally, brain radiation therapy can be associated with the side effect of oligodendrocyte depletion, which can lead to cognitive decline and / or impaired motor coordination.

[0004] Because mature human oligodendrocytes cannot be easily isolated from human subjects, human oligodendrocyte cell lines have been developed to enable the study of these cells. However, immortalized cell lines may not fully mimic the biological properties of natural cells and are not suitable for therapeutic applications. Therefore, there is a strong need for techniques to generate human oligodendrocytes in vitro, for example, from stem cells. Various protocols have been reported for differentiating oligodendrocytes from human pluripotent stem cells. However, these protocols remain inefficient and vary in oligodendrocyte yield, and require very long differentiation times to generate myelin basic protein (MBP)-positive oligodendrocytes.

[0005] The initial protocol used a four-step process (see also Hu et al. (2009) Nature Protocols 4:1614-1622 (Non-Patent Document 1); Wang et al. (2013) Cell Stem Cell 12:252-264 (Non-Patent Document 2)). The protocol involved first inducing human embryonic stem cells (hESCs) to differentiate into neuroepithelial cells for 2 weeks to form neural tube-like rosettes, followed by 10 days of treatment with retinoic acid (RA) and sonic hedgehog (SHH) to generate progenitor cells expressing OLIG2. An additional 10 days of treatment with fibroblast growth factor (FGF2) resulted in conversion to pre-OPCs expressing OLIG2 and NKX2.2. Finally, the pre-OPCs were cultured for an additional 8–9 weeks in the absence of FGF2 to differentiate into OPCs expressing markers such as platelet-derived growth factor receptor α (PDGFRα), SOX10, and NG2. Therefore, using this protocol, approximately 24 days are required to generate progenitor cells expressing OLIG2, and approximately 34 days are required to generate pre-OPCs expressing OLIG2 and NKX2.2, resulting in approximately 100 days to obtain mature OLs. A variant of this protocol, reported by Douvaras et al. (Stem Cell Reports (2014) 3:250–259), involving culture with exogenously added growth factors PDGF, IGF-1, and HGF, still requires approximately 20 days to obtain pre-OPCs and approximately 50 days to obtain OPCs.

[0006] Although other protocols have been reported since then, these protocols still utilized a period of approximately one week of neural induction and patterning (also referred to as neuralization) followed by induction of cells expressing markers of pre-OPCs and OPCs using media containing exogenously added growth factors, such as FGF2, PDGF, IGF-1, and / or HGF, depending on the 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).

[0007] In a more recently reported protocol, hESCs were first neurally induced to generate neural progenitor cells (NPCs). Subsequently, the NPCs were transduced with the transcription factor SOX10 via viral transduction and expanded in the presence of bFGF, resulting in the generation of MBP-positive oligodendrocytes in only approximately 20 days (Garcia-Leon et al. (2018) Stem Cell Reports 10:655-672). Furthermore, transient and partial inhibition of the SHH pathway transcription factor GLI1 in neural stem cells (produced by neural induction) with the small molecule inhibitor GANT61 was found to generate OPCs that were more migratory and capable of earlier differentiation into myelin-producing oligodendrocytes (Namchaiw et al. (2019) Stem Cell Res & Therapy 10:272).

[0008] Thus, despite some progress, there remains a need for efficient and stable methods and compositions for generating oligodendrocyte precursor cells from human pluripotent stem cells. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Hu et al. (2009) Nature Protocols 4:1614-1622 [Non-patent document 2] Wang et al. (2013) Cell Stem Cell 12:252-264 [Non-patent document 3] Douvaras et al. (Stem Cell Reports (2014) 3:250-259) [Non-patent document 4] Piao et al. (2015) Cell Stem Cell 16:198-210 [Non-patent document 5] Douvaras & Fossati (2015) Nature Protocols 10:1143-1154 [Non-patent document 6] Livesey et al. (2016) Stem Cells 34:1040-1053 [Non-Patent Document 7] Yamashita et al. (2017) PLOS One 12: e0171947 [Non-patent document 8] Garcia-Leon et al. (2018) Stem Cell Reports 10:655-672 [Non-Patent Document 9] Namchaiw et al. (2019) Stem Cell Res & Therapy 10:272 Summary of the Invention

[0010] The present disclosure provides a method for generating human oligodendrocyte progenitor cells (OPCs) from pre-OPCs using a chemically defined culture medium, which enables the generation of SOX10+ OLIG2+ NKX2.2+ OPCs in as little as nine days of culture starting from pre-OPCs. Pre-OPCs can be obtained by culturing pluripotent stem cells in the chemically defined culture medium for three days, thereby providing a total 12-day protocol for generating OPCs from pluripotent stem cells. The present disclosure also provides a method for generating CD9+A2B5+O4+CNPase+ pre-myelinating oligodendrocytes from OPCs by further differentiating the OPCs for six days in another chemically defined culture medium of the present disclosure.

[0011] The present disclosure provides two interchangeable culture protocols for generating OPCs from pre-OPCs, referred to herein as Version 1 and Version 2. Each of these protocols includes two stages (Stage 2 and Stage 3), while the starting protocol for generating pre-OPCs from pluripotent stem cells includes only one stage (Stage 1), resulting in a three-stage protocol for generating OPCs overall. Non-limiting, representative protocols for generating OPCs are shown schematically in Figures 42 and 43. The present disclosure further provides a culture protocol for generating premyelinating oligodendrocytes (pre-OLs) from OPCs using Stage 4 culture medium. A representative Stage 4 protocol for generating pre-OLs is shown schematically in Figure 44.

[0012] Each culture medium for the different stages contains a small molecule agent that either agonizes or antagonizes specific signaling pathway activity in pluripotent stem cells, thereby promoting differentiation along the OPC lineage and achieving cell maturation and expression of OPC-associated biomarkers. The disclosed method has the advantage of avoiding the neural induction step present in prior art protocols and allowing direct differentiation of pluripotent stem cells into pre-OPCs and OPCs, thereby significantly shortening the time required to generate pre-OPCs and OPCs. Furthermore, the use of small molecule agents in the culture medium allows for precise control of the components in the culture.

[0013] Thus, in one aspect, the present disclosure relates to a method of generating SOX10+ OLIG2+ NKX2-2+ human oligodendrocyte progenitor cells (OPCs), the method comprising the steps of: (a) culturing OLIG2+ human pre-oligodendrocyte precursor cells (pre-OPCs) 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 from day 0 to day 3 to obtain a cell population; and (b) 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 for days 3 to 9 to generate SOX10+ OLIG2+ NKX2-2+ OPCs.

[0014] The above-described methods correspond to the Stage 2 and Stage 3 culture media of the Version 1 protocol. In another embodiment, the method further comprises culturing human pluripotent stem cells 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 from day -3 to day 0 to obtain OLIG2+ human pre-OPCs (i.e., a 3-day culture to obtain pre-OPCs from pluripotent stem cells prior to differentiation of the pre-OPCs to OPCs). This corresponds to the Stage 1 culture medium to obtain pre-OPCs.

[0015] 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 at a concentration of 10 ng / ml in step (a) and step (b). Other suitable FGFR pathway agonists and concentration ranges are disclosed herein.

[0016] 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.

[0017] 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.

[0018] In one embodiment, the Akt pathway antagonist is MK2206. In one embodiment, the Akt pathway antagonist is selected from the group consisting of MK2206, which is present in the culture medium at a concentration of 125 nM in steps (a) and (b). Other suitable Akt pathway antagonists and concentration ranges are disclosed herein.

[0019] 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.

[0020] 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.

[0021] In one embodiment, the PDGFR pathway agonist is PDGF-AA, hi one embodiment, the PDGFR pathway agonist is PDGF-AA, which is present in the culture medium at a concentration of 10 ng / ml.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] In another aspect, the present disclosure provides a culture medium for obtaining oligodendrocyte progenitor cells (OPCs), the 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 Stage 2 medium in Version 1). In another aspect, the present disclosure provides a culture medium for obtaining oligodendrocyte progenitor cells (OPCs), the 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 Stage 3 medium in Version 1). Also provided is an isolated cell culture comprising OLIG2+ OPCs cultured in one of the above-described culture media.

[0026] In yet another aspect, the present disclosure relates to a method of generating SOX10+ OLIG2+ NKX2-2+ human oligodendrocyte progenitor cells (OPCs), the method comprising the steps of: (a) culturing OLIG2+ human pre-oligodendrocyte precursor 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 from day 0 to day 6 to obtain a cell population; and (b) culturing the cell population from step (a) in a culture medium containing an FGFR pathway agonist, an IGF-1 pathway agonist, and a retinoic acid (RA) pathway agonist for days 6 to 9 to generate SOX10+ OLIG2+ NKX2-2+ OPCs.

[0027] The above-described method corresponds to the Stage 2 and Stage 3 culture media of the Version 2 protocol. In another embodiment, the method further comprises culturing human pluripotent stem cells 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 from day -3 to day 0 to obtain OLIG2+ human pre-OPCs (i.e., a 3-day culture to obtain pre-OPCs from pluripotent stem cells prior to differentiation of the pre-OPCs to OPCs). This corresponds to the Stage 1 culture medium to obtain pre-OPCs.

[0028] 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 at a concentration of 10 ng / ml in step (a) and step (b). Other suitable FGFR pathway agonists and concentration ranges are disclosed herein.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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 of 100 nM. Other suitable RA pathway antagonists and concentration ranges are disclosed herein.

[0034] In another aspect, the present disclosure provides a culture medium for obtaining oligodendrocyte progenitor cells (OPCs), the culture medium comprising an FGFR pathway agonist, an mTOR pathway antagonist, an SHH pathway agonist, and a WNT pathway agonist (corresponding to Stage 2 medium of Version 2). In another aspect, the present disclosure provides a culture medium for obtaining oligodendrocyte progenitor cells (OPCs), the culture medium comprising an FGFR pathway agonist, an IGF-1 pathway agonist, and a retinoic acid (RA) pathway agonist (corresponding to Stage 3 medium of Version 2). Also provided is an isolated cell culture comprising OLIG2+ OPCs cultured in one of the above-described culture media.

[0035] In another aspect, the present disclosure relates to compositions and methods for a stage 4 protocol for generating premyelinating oligodendrocytes (pre-OLs) from OPCs. In one embodiment, the present disclosure relates to a method for generating CD9+A2B5+O4+CNPase+ premyelinating oligodendrocytes (pre-OLs), the method comprising the steps of culturing SOX10+ OLIG2+ NKX2-2+ OPCs in a culture medium containing an IGF1R pathway agonist, a TrkC pathway agonist, a PDGFR pathway agonist, a thyroid hormone receptor agonist, and an insulin receptor agonist, so as to generate CD9+A2B5+O4+CNPase+ pre-OLs. In one embodiment, the OPCs are cultured in the culture medium for 6 days to generate pre-OLs.

[0036] In one embodiment, the IGF1R pathway agonist is selected from the group consisting of IGF-1, IGF-2, insulin, Rg5, IGF-1 30-41, demethylasteriquinone 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.

[0037] In one embodiment, the TrkC pathway agonist is selected from the group consisting of neurotrophin-3 (NT-3), a peptidomimetic 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.

[0038] 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.

[0039] In one embodiment, the thyroid hormone receptor agonist is selected from the group consisting of T3, T4, resmetirom, TRb agonist 3 (compound 3), sobetirom, tiratricol, 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.

[0040] In one embodiment, the insulin receptor agonist is selected from the group consisting of insulin, IGF-1, IGF-2, demethylasteriquinone B1, MK-5160, MK-1092, and combinations thereof. In one embodiment, the insulin receptor agonist is insulin. In one embodiment, the insulin is present in the culture medium at a concentration of 20 μg / ml.

[0041] In another aspect, the present disclosure relates to a stage 4 culture medium for producing pre-OLs. In one embodiment, the stage 4 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 some embodiments, 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 medium comprises 10 ng / ml IGF-1, 10 ng / ml NT-3, 10 ng / ml PDGF-AA, 50 nM T3, and 20 μg / ml insulin.

[0042] To generate pre-OLs from pre-OPCs, Stage 2 / Stage 3 medium (Version 1 or Version 2) can be combined with Stage 4 medium. Thus, in one embodiment relating to the Version 1 protocol, the present disclosure relates to a method for generating CD9+A2B5+O4+CNPase+ human pre-myelinating oligodendrocytes (pre-OLs), the method comprising the steps of: (a) culturing OLIG2+ human pre-oligodendrocyte precursor cells (pre-OPCs) 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 from day 0 to day 3 to obtain a cell population; (b) 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 for days 3 to 9 to generate SOX10+ OLIG2+ NKX2-2+ OPCs; and (c) culturing the cell population from step (b) in a culture medium containing an IGF1R pathway agonist, a TrkC pathway agonist, a PDGFR pathway agonist, a thyroid hormone receptor agonist, and an insulin receptor agonist for days 9 to 15 to generate CD9+A2B5+O4+CNPase+ pre-OLs.

[0043] In one embodiment relating to the Version 2 protocol, the present disclosure relates to a method of generating CD9+A2B5+O4+CNPase+ human premyelinating oligodendrocytes (pre-OLs), the method comprising the steps of: (a) culturing OLIG2+ human pre-oligodendrocyte precursor 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 from day 0 to day 6 to obtain a cell population; (b) culturing the cell population from step (a) in a culture medium containing an FGFR pathway agonist, an IGF-1 pathway agonist, and a retinoic acid (RA) pathway agonist for days 6 to 9 to generate SOX10+ OLIG2+ NKX2-2+ OPCs; and (c) culturing the cell population from step (b) in a culture medium containing an IGF1R pathway agonist, a TrkC pathway agonist, a PDGFR pathway agonist, a thyroid hormone receptor agonist, and an insulin receptor agonist for days 9 to 15 to generate CD9+A2B5+O4+CNPase+ pre-OLs.

[0044] In yet another embodiment, the above-described method combining Stage 2, Stage 3, and Stage 4 further comprises culturing human pluripotent stem cells 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 from day -3 to day 0 to obtain OLIG2+ human pre-OPCs (i.e., a 3-day culture to obtain pre-OPCs from pluripotent stem cells prior to differentiation of the pre-OPCs into OPCs). This corresponds to the Stage 1 culture medium for obtaining pre-OPCs, thereby providing a method combining the Stage 1, Stage 2, Stage 3, and Stage 4 protocols for generating pre-OLs.

[0045] 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.

[0046] In one embodiment, the human pluripotent stem cells are adhered to vitronectin-coated plates during culture.

[0047] Other features and advantages of the invention will become apparent from the following detailed description and claims. [Brief explanation of the drawings]

[0048] [Figure 1]Figure 1 shows the results from the HD-DoE model of an eight-factor experiment optimized for maximum expression of NKX2-2. The upper column of the model shows the predicted expression levels of 53 preselected genes when optimized for NKX2-2. The lower column of the model shows the effectors tested in this model and their contribution to maximum expression of NKX2-2. The "Value" column indicates the concentration of each effector required to mimic the model. [Figure 2] Figure 2 shows the results from the HD-DoE model of an eight-factor experiment optimized for maximal expression of PDGFRA. The top and bottom columns are as described in Figure 1. This condition highlights the effector PD0325901, with a factor contribution of 30.05, as a critical input for high expression of PDGFRA. [Figure 3] Figure 3 shows the dynamic profiles of gene expression levels for NKX2-2, OLIG1, OLIG2, and PDGFRA versus the concentrations of the eight effectors tested. The positive effects of TTNPB, MHY1485, and PD0325901 on PDGFRA expression, as well as their factorial contributions, are indicated by the slope of the plot for each effector. The dotted box highlights the antagonistic effect of PD0325901 on NKX2-2 and OLIG2, which contrasts with its effect on PDGFRA. [Figure 4] Figure 4 shows the results from a HD-DoE model of a 13-factor experiment optimized for maximum OTX2 expression, which identified MK2206, PD0325901, CHIR99021, LDN193189, Go6983, and PD173074 as positive effectors of OTX2 expression. [Figure 5]Figure 5 shows results from a HD-DoE model of a 13-factor experiment optimized for maximal expression of FEZF2. The model confirmed the positive effects of LDN193189, MK2206, and PD0325901 on cell patterning, and derived three other factors, including SC79, XAV939, and 500 nM purmorphamine. [Figure 6] Figure 6 shows the dynamic profile of OTX2 and FEZF2 expression levels versus the concentrations of 13 factors tested in this model. XAV939 and 500 nM purmorphamine had a significant positive effect on FEZF2 expression and no significant negative effect on OTX2 expression. [Figure 7A] 7A-7D show the dynamic profile analysis of the deletion process and its effect on the expression of NKX2-2, OLIG2, and PDGFRA in an eight-factor modeling experiment. [Figure 7B] See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 7D] See legend to Figure 7A. [Figure 8A] 8A-8D show the dynamic profile analysis of the deletion process and its effect on the expression of FEZF2 and OTX2 in a 13-factor modeling experiment. [Figure 8B] See legend to Figure 8A. [Figure 8C] See legend to Figure 8A. [Figure 8D] See legend to Figure 8A. [Figure 9]Figure 9 shows photographs of cells after 3 days of culture in optimized OPC differentiation medium. Cells were stained with oligodendrocyte and neural biomarkers. Cells express anterior neuroectoderm biomarkers, including OTX2 and NKX2-2, along with the OPC-specific biomarker OLIG2. Nestin and PDGFRa, biomarkers for neurons and late OPCs, respectively, are absent. KI67 expression indicates progenitor cells in a proliferative state. [Figure 10] Figures 10A-10B show RNA sequencing data for cells after 3 days of culture in the optimized OPC differentiation medium. Expression levels of stem cell genes NANOG and POU5F1 were decreased, while genes involved in early development of brain regions and genes involved in the oligodendrocyte lineage were increased. [Figure 11] Figure 11 shows results from an HD-DoE model of a 12-factor experiment optimized for maximal OLIG2 expression. The top column of the model shows the predicted expression levels of 53 preselected genes when optimized for OLIG2. The bottom column of the model shows the effectors tested in the model and their contribution to maximal OLIG2 expression. The "Value" column indicates the concentration of each effector required to mimic the model. [Figure 12] Figure 12 shows the dynamic profile analysis of the expression levels of NKX2-2, OLIG2, SOX10, OLIG1, and PDGFRa versus the concentration of 12 effectors. The positive effects of FGF-2 and MK2206 and their factorial contributions to OLIG2 expression are indicated by the slope of the plot for each effector. [Figure 13]Figure 13 shows results from an HD-DoE model of a 12-factor experiment optimized for maximal OLIG2 expression. The top and bottom columns and the "Value" column are as in Figure 11. The model highlights two effectors, purmorphamine with a factor contribution of 13.03 and AGN193109 with a factor contribution of 18.3, as critical inputs for maximal and minimal OLIG2 expression, respectively. [Figure 14] Figure 14 shows results from an HD-DoE model of a 12-factor experiment applied to stage 1 pre-OPCs to generate a composition for stage 2 differentiation. The top and bottom columns and the "Value" column are as in Figure 11. The model was optimized for maximum expression of OLIG2. The model highlights the role of purmorphamine, with a factor contribution of 16.9, in OLIG2 expression. [Figure 15] FIG. 15 is a plot of the effect of factors showing the expression levels of NKX2-2, OLIG2, OLIG1, and SOX10 versus the concentration of purmorphamine in two separate 12-factor models. [Figure 16] Figure 16 shows the results of an HD-DoE model of an eight-factor experiment applied to early stage 2 oligodendrocyte progenitor cells to generate a composition for stage 3 differentiation. The top and bottom columns and the "Value" column are as in Figure 11. The model highlights the positive role of activin A and TTNPB, with factor contributions of 50.2 and 19.1, respectively, in OLIG1 expression. [Figure 17]Figure 17 shows the results of the HD-DoE model of the eight-factor experiment applied to Stage 2 early OPCs. The top and bottom columns and the "Value" column are as in Figure 11. The model was optimized for maximal expression of PDGFRa. The model highlights two effectors, TTNPB with a factor contribution of 71.6 and linoleic acid with a factor contribution of 22.1, as critical inputs for maximal expression of PDGFRa. [Figure 18] Figure 18 shows the results from the HD-DoE model of the eight-factor experiment applied to early stage 2 OPCs. The top and bottom columns and the "Value" column are as in Figure 11. The model highlights the positive role of TTNPB, with a factor contribution of 8, in SOX10 expression, while all other factors have negative regulatory effects. [Figure 19] Figure 19 shows the dynamic profile analysis of the expression levels of OLIG1, PDGFRa, and SOX10 versus the concentrations of eight factors. The positive effect of TTNPB on PDGFRa and OLIG1 expression and its factor contribution are indicated by the slope of the plot for each effector. [Figure 20] Figure 20 shows results from an HD-DoE model of a 12-factor experiment applied to stage 2 primary OPCs to generate a composition for stage 3 differentiation. The top and bottom columns and the "Value" column are as in Figure 11. The model is optimized for maximum expression of SOX10. The model highlights the role of MK2206 and MHY1485, with factor contributions of 16.2 and 11.5, respectively, in SOX10 expression. [Figure 21]Figure 21 shows results from an HD-DoE model of a 12-factor experiment applied to stage 2 primary OPCs to generate a composition for stage 3 differentiation. The top and bottom columns and the "Value" column are as in Figure 11. The model is optimized for maximum expression of CSPG4. The model highlights the role of TTNPB and activin A, with factor contributions of 15.9 and 9.3, respectively, in CSPG4 expression. [Figure 22] Figure 22 shows the dynamic profile analysis of the expression levels of OLIG1, PDGFRa, and SOX10 versus concentrations of four of the 12 factors. The positive effects of MK2206 and DBZ and their factor contributions on the expression level of SOX10 are indicated by the slope of the plot for each effector. [Figure 23] Figure 23 shows a plot of the effect of factors, depicting the expression levels of OPC genes, including OLIG1, PDGFRa, and SOX10, and neuronal genes, including NEUROD1, NEUROG1, and NEUROG2, versus DBZ concentration. As DBZ concentration increases, the expression of both gene groups also increases. [Figure 24] Figure 24 shows a plot of the effect of factors, depicting the expression levels of late OPC genes, including BCAN, CNP, and ID2, versus the concentration of AICAR. As the concentration of AICAR increases, the expression of both gene groups also increases. [Figure 25A] Figures 25A-25C show the dynamic profiles of expression levels of NKX2-2, OLIG2, SOX10, and OLIG1 compared with the concentrations of four final effectors in the composition of stage 2 differentiation medium. Figure 25A shows the expression level of the gene of interest in the presence of all final effectors. Figure 25B shows the expression level of the gene of interest in the absence of one final effector at a time but the presence of the others. Figure 25C shows the expression level of the gene of interest in the presence and absence of the remaining final effector, purmorphamine. [Figure 25B] See legend to Figure 25A. [Figure 25C] See legend to Figure 25A. [Figure 26] Figures 26A-26B show the dynamic profiles of expression levels of OLIG1, PDGFRa, and SOX10 versus concentrations of two of the final effectors in the composition of stage 3 differentiation medium. Figure 26A shows the expression level of the gene of interest in the presence of both effectors. Figure 26B shows the expression level of the gene of interest in the absence of one final effector but the presence of the other at one time. [Figure 27A] Figures 27A-27B show the dynamic profiles of expression levels of OLIG1, PDGFRa, SOX10, and SOX8 versus the concentration of four of the final effectors in the composition of stage 3 differentiation medium. Figure 27A shows the expression levels of the gene of interest in the presence of the four final effectors. Figure 27B shows the expression levels of the gene of interest in the absence of one final effector at a time but the presence of the others. [Figure 27B] See legend to Figure 27A. [Figure 28] Figure 28 shows a photograph of a fluorescent image of hiPSC-derived early oligodendrocyte progenitor cells at the end of stage 2. 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, cells were positive for all markers predicted for the early progenitor state. [Figure 29] Figure 29 shows a photograph of a fluorescent image of oligodendrocyte precursor cells at the end of stage 3. Cells were stained with OPC markers, including OLIG2, PDGFRa, SOX10, NG2, and A2B5. At this stage, cells were positive for all expected biomarkers, confirming their identity as oligodendrocytes. [Figure 30] Figure 30 shows results from an HD-DoE model of an eight-factor experiment optimized for maximal OLIG2 expression. The top column of the model shows the predicted expression levels of 53 preselected genes when optimized for OLIG2. The bottom column of the model shows the effectors tested in the model and their contribution to maximal OLIG2 expression. The "Value" column indicates the concentration of each effector required to mimic the model. [Figure 31] Figure 31 shows results from an HD-DoE model of an eight-factor experiment optimized for maximal expression of OLIG1. The model highlights two effectors, AZD3147 with a factor contribution of 26.76 and purmorphamine with a factor contribution of 22.75, as critical inputs for maximal expression of OLIG1. [Figure 32] Figure 32 shows the dynamic profile analysis of the expression levels of NKX2-2, OLIG2, SOX10, and OLIG1 versus the concentration of eight effectors. The positive effects of AZD3147 and CHIR99021 and their factorial contributions to OLIG2 expression are indicated by the slope of the plot for each effector. [Figure 33] Figure 33 shows results from an HD-DoE model of an eight-factor experiment optimized for maximal SOX10 expression. The model highlights two effectors, TTNPB with a factor contribution of 17.98 and FGF-2 with a factor contribution of 15.3, as important inputs for maximal SOX10 expression. [Figure 34] Figure 34 shows results from an HD-DoE model of an eight-factor experiment optimized for maximal expression of PDGFRa. The model highlights two effectors, purmorphamine with a factor contribution of 20.61 and IGF-1 with a factor contribution of 11.2, as important inputs for maximal expression of SOX10. [Figure 35]Figure 35 shows plots of the effect of factors, showing the expression levels of SOX10 versus the concentrations of IGF-1, FGF-2, and AGN193109 in the 12-factor model (left model), and versus the concentrations of IGF-1 and FGF-2 in the 8-factor model (right model). [Figure 36] Figure 36 shows a contour plot modeling OLIG1 expression levels versus the concentrations of IGF-1, FGF-2, TTNPB, and AGN193109 in an eight-factor model, excluding all other factors. AGN193109 concentration is shown on the top axis, TTNPB concentration on the right axis, IGF-1 concentration on the left axis, and FGF-2 concentration on the bottom axis. OLIG1 expression levels are represented as a spectrum progressing from red at the highest level to blue at the lowest level. The space in the lower right corner indicates the highest expression level in the presence of IGF-1, FGF-2, and AGN193109. [Figure 37] Figure 37 shows a plot of the effect of factors, depicting the expression levels of late OPC genes, including BCAN, CSPG4, ID2, and FYN, and neuronal genes, including NEUROG1 and NEUROD1, versus the concentration of IGF-1. As the concentration of IGF-1 increases, the expression levels of OPC genes of interest increase, while the expression levels of neuronal genes decrease. [Figure 38] Figure 38 shows a plot of the effect of factors, depicting the expression levels of late OPC genes, including CSPG4, ID2, IGFBP2, and SOX8, and neuronal genes, including NEUROG1 and NEUROD1, versus the concentration of FGF-2. As the concentration of FGF-2 increases, the expression levels of OPC genes of interest increase, while the expression levels of neuronal genes decrease. [Figure 39]Figure 39 shows a plot of the effect of factors, depicting the expression levels of late OPC genes, including BCAN, ID2, FYN, CNP, PLP1, and SOX8, and neuronal genes, including NEUROG1, NEUROG2, and NEUROD1, versus the concentration of TTNPB. As the concentration of TTNPB increases, the expression levels of OPC genes decrease, while the expression levels of neuronal genes increase. [Figure 40] Figure 40 shows a photograph of a fluorescent image of hiPSC-derived early oligodendrocyte progenitor cells at the end of stage 2. Cells were stained with OPC biomarkers, including NKX2-2, OLIG2, SOX10, A2B5, and PDGFRa, and the proliferation marker KI67. At this stage, cells were positive for all markers predicted for the early progenitor state. [Figure 41] Figure 41 shows a photograph of a fluorescent image of oligodendrocyte precursor cells at the end of stage 3. Cells were stained with OPC biomarkers, 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, confirming their identity as oligodendrocytes. [Figure 42] FIG. 42 is a schematic diagram of a representative example of the three-stage protocol for obtaining oligodendrocyte precursor cells from pluripotent stem cells described herein, where stages 2 and 3 use version 1 of the protocol. [Figure 43] FIG. 43 is a schematic diagram of a representative example of the three-stage protocol for obtaining oligodendrocyte precursor cells from pluripotent stem cells described herein, where version 2 of the protocol is used for stages 2 and 3. [Figure 44] FIG. 44 is a schematic diagram of a representative example of a stage 4 protocol for generating premyelinating OLs from OPCs. [Figure 45] Figure 45 shows the results from the HD-DoE model of an eight-factor experiment optimized for maximum CNP expression. The upper column of the model shows the predicted expression levels of 53 preselected genes when optimized for CNP. The lower column of the model shows the effectors tested in this model and their contribution to the maximum expression of CNP. The "Value" column indicates the concentration of each effector required to mimic the model. [Figure 46] Figure 46 shows the results from the HD-DoE model of a 12-factor experiment optimized for maximum CNP expression. The conditions highlight two effectors, NT-3 with a factor contribution of 13.3 and biotin with a factor contribution of 16.5, as important inputs for maximum and minimum CNP expression, respectively. [Figure 47] Figure 47 shows the results from the HD-DoE model of the 12-factor experiment optimized for maximum CNP expression. When CNP is expressed at maximum, several other oligodendrocyte genes, including APOD, BCAN, MYT1, and PLP1, are also upregulated, and these are shown in red boxes. At the same time, ID2 and MKI67, which are expressed at the oligodendrocyte progenitor stage, are downregulated, and these are shown in blue boxes. [Figure 48] Figure 48 shows results from an HD-DoE model of a 12-factor experiment optimized for maximal expression of PLP1. The conditions highlight two effectors, insulin with a factor contribution of 13.2 and AICAR with a factor contribution of 11.8, as important inputs for maximal and minimal PLP1 expression, respectively. [Figure 49]Figure 49 shows the dynamic profile analysis of expression levels of CNP, PLP1, NeuroD1, NeuroG1, and NeuroG2 versus the concentrations of 12 effectors at maximum PLP1 expression. The positive effect of GSI-XX and its factorial contribution to neuronal gene expression is indicated by the slope of the plot for each effector. [Figure 50] Figure 50 shows a plot of the effect of factors, showing the expression levels of CNP, PLP1, PTGDS, MBP, KLK6, and NeuroG1 versus the concentration of T3 in a 12-factor model. The positive effect of T3 on oligodendrocytes and the negative effect of T3 on neuronal genes are indicated by the slope of the plot. [Figure 51] Figure 51 shows a photograph showing a fluorescent image of hiPSC-derived premyelinating oligodendrocytes at the end of stage 4. 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 small proportion of the population 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 precursor cells in the culture. [Figure 52] Figure 52 shows flow cytometry-based detection of CD9+ cells in hiPSC-derived premyelinating cells at differentiation day 18. CD9+ cells comprise 81.5% of the total cell population. DETAILED DESCRIPTION OF THE INVENTION

[0049] Detailed Description of the Invention Described herein are methodologies and compositions that enable the generation of SOX10+ OLIG2+ NKX2-2+ OPCs from human pre-OPCs, which themselves can be generated from human pluripotent stem cells under chemically defined culture conditions using a small molecule-based approach. Using the methodologies and compositions described herein, OPCs can be further differentiated under chemically defined culture conditions to generate CD9+A2B5+O4+CNPase+ pre-OLs. While many prior art protocols use neural induction, the disclosed method has the advantage that the starting pluripotent stem cells do not undergo this neural induction. This allows the generation of OLIG2+ pre-OPCs in as little as three days, significantly shorter than the average 10 days required for current protocols to generate pre-OPCs. Pre-OPCs can be further differentiated into SOX10+ OLIG2+ NKX2-2+ OPCs in as little as an additional 9 days, resulting in a total 12-day protocol for generating OPCs from pluripotent stem cells. Pre-OLs can be obtained from OPCs within an additional 6 days of culture in appropriate medium, making it possible to obtain pre-OLs from pluripotent stem cells in as little as 18 days, compared to the significantly longer time periods required by other approaches (e.g., 50-70 days).

[0050] As described in Examples 1, 5, 8, and 10, a high-dimensional design of experiments (HD-DoE) approach was used to simultaneously test multiple process inputs (e.g., small molecule agonists or antagonists) to an output response, such as gene expression. These experiments enabled the identification of chemically defined culture media containing agonists and / or antagonists of specific signaling pathways that were sufficient to generate pre-OPCs, OPCs, or pre-OLs in a very short time. The optimized culture media were further validated by factor fatality analysis, which investigated the effect of removing individual agonist or antagonist agents, as described in Examples 2, 6, and 10. The phenotype of cells generated by the differentiation protocols described in Examples 3, 7, 9, and 11 was further confirmed by immunohistochemistry.

[0051] Pre-OPCs can be differentiated into OPCs using one of two interchangeable protocols described herein. These interchangeable protocols are referred to herein as Version 1 (further described in Example 5) and Version 2 (further described in Example 8), and each protocol includes two stages. As used herein, a first culture medium that generates pre-OPCs from pluripotent stem cells over a three-day period is referred to as Stage 1 medium, and a second and third culture medium that generate OPCs from pre-OPCs over an additional nine days are referred to as Stage 2 medium and Stage 3 medium.

[0052] An exemplary three-stage culture protocol for generating OPCs from pluripotent stem cells using the Version 1 protocol for Stages 2 and 3 is shown schematically in Figure 42. As shown, Stage 1 medium is used for three days (days 0-3), Stage 2 medium is used for three days (days 3-6), and Stage 3 medium is used for six days (days 6-12).

[0053] An exemplary three-stage culture protocol for generating OPCs from pluripotent stem cells using the Version 2 protocol for Stages 2 and 3 is shown schematically in Figure 43. As shown, Stage 1 medium is used for 3 days (days 0-3), Stage 2 medium is used for 6 days (days 3-9), and Stage 3 medium is used for 3 days (days 9-12).

[0054] OPCs generated by either the Version 1 protocol for Stages 2 and 3 or the Version 2 protocol for Stages 2 and 3 can be further differentiated into pre-OLs in the Stage 4 protocol. An exemplary Stage 4 culture protocol for generating pre-OLs from OPCs is shown schematically in Figure 44. As shown, Stage 1 medium is used for 3 days (days 0-3), Stage 2 medium and Stage 3 medium are used for 9 days (days 3-12), and Stage 4 medium is used for 6 days (days 12-18).

[0055] Various aspects of the invention are described in more detail in the following subsections.

[0056] I. Cell The starting cells used to generate pre-OPCs in the culture of the present disclosure are human pluripotent stem cells. As used herein, the term "human pluripotent stem cells" (abbreviated as hPSCs) refers to human stem cells that have the ability to differentiate into a wide variety of cell types. The term "pluripotency," as used herein, refers to cells that have the ability to differentiate under various conditions into cell types characteristic of all three germ layers (endoderm, mesoderm, and ectoderm). Pluripotent cells are primarily characterized by their ability to differentiate into all three germ layers, for example, using nude mice and teratoma formation assays. While pluripotency can also be demonstrated by the expression of embryonic stem (ES) cell markers, the preferred test for pluripotency is to demonstrate the ability to differentiate into cells of each of the three germ layers.

[0057] Human pluripotent stem cells include, for example, induced pluripotent stem cells (iPSCs), and human embryonic stem cells, such as ES cell lines. Non-limiting examples of induced pluripotent stem cells (iPSCs) include 19-11-1 cells, 19-9-7 cells, or 6-9-9 cells (e.g., those 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. Because the disclosed methods of generating pre-OPCs and / or OPCs are used to differentiate (maturate) a starting pluripotent stem cell population, in various embodiments, the pre-OPC and / or OPC cell populations generated by the disclosed methods lack 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.

[0058] The starting cells used to generate OPCs in the cultures of the present disclosure are human pre-OPCs, e.g., pre-OPCs generated from pluripotent stem cells, as described herein.

[0059] Pluripotent stem cells and pre-OPCs are subjected to culture conditions that induce cell differentiation, as described herein. As used herein, the term "differentiation" refers to the development of cells from a more primitive stage toward more mature cells (i.e., less primitive cells), which typically exhibit phenotypic characteristics of commitment to a specific cell lineage. Early progenitor cells that can be derived from human PSCs by neural induction (neuralization) are neural progenitor cells (NPCs). As used herein, "neural progenitor cells" or "NPCs" refer to stem cell-derived progenitor cells that express the type VI intermediate filament protein, nestin. Because the disclosed methods of producing pre-OPCs and / or OPCs do not use neural induction and therefore do not produce NPCs, in various embodiments, the cell populations produced by the disclosed methods lack nestin-positive cells.

[0060] In one embodiment, the cell produced by the method of the present disclosure is a pre-oligodendrocyte precursor cell (pre-OPC).As used herein, " pre-oligodendrocyte precursor cell" or "pre-OPC" refers to a stem cell-derived precursor cell, which expresses cell markers OLIG2 and NKX2.2.Pre-OPC may also express additional markers, including but not limited to OTX2 (anterior neuroectoderm biomarker), FEZF2 (anterior ectoderm biomarker), and / or OLIG1.

[0061] In one embodiment, the cell produced by the method of the present disclosure is an oligodendrocyte progenitor cell (OPC), which is a more differentiated (more mature) cell than pre-OPC.As used herein, "oligodendrocyte progenitor cell" or "OPC" refers to a stem cell-derived progenitor cell, which expresses cell markers SOX10, OLIG2, and NKX2.2, and PDGFRa.OPC may also express additional markers, non-limiting examples of which include OTX2 (anterior neuroectoderm biomarker), FEZF2 (anterior ectoderm biomarker), and / or OLIG1.

[0062] In one embodiment, the cells produced by the methods of the present disclosure are premyelinating oligodendrocytes (pre-OLs), which are more differentiated (more mature) cells than OPCs. As used herein, "premyelinating oligodendrocytes" or "pre-OLs" refer to cells of the oligodendrocyte lineage that express the cell markers CD9, A2B5, O4, and CNPase.

[0063] Pre-OLs produced by the methods of the present disclosure can be further cultured in vitro to produce mature oligodendrocytes (OLs), markers of which include, but are not limited to, myelin basic protein (MBP) and O4.

[0064] II. Culture Medium Components The disclosed methods for generating pre-OPCs, OPCs, or pre-OLs include culturing human pluripotent stem cells in a culture medium containing a specific agonist and / or a specific antagonist of a cell signaling pathway.

[0065] As described in Example 1 (Stage 1 Protocol), culture medium containing a retinoic acid (RA) pathway agonist, an Akt pathway agonist, and an mTOR pathway agonist was sufficient to generate pre-OPCs expressing OLIG2 and NKX2.2 in as little as three days. The inclusion of additional agents optimized the expression of other markers, including PDGFRa as a marker of differentiation into OPCs. In other embodiments, the culture medium further comprises at least one additional agent selected from the group consisting of a WNT pathway antagonist, an SHH pathway agonist, a BMP pathway antagonist, and a PKC pathway antagonist. In one embodiment, the culture medium further comprises a WNT pathway antagonist. In one embodiment, the culture medium further comprises an SHH pathway agonist. In one embodiment, the culture medium further comprises a BMP pathway antagonist. In one embodiment, the culture medium further comprises a PKC pathway antagonist. In one embodiment, the culture medium further comprises a WNT pathway antagonist and an SHH pathway agonist, and the differentiated cells express OTX2 and FEZF2, in addition to OLIG2 and NKX2.2.

[0066] In one embodiment, the stage 1 culture medium for generating pre-OPCs comprises 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. In one embodiment, the differentiated cells are OPCs that express at least OLIG2, NKX2.2, and PDGFRa (in addition, may express additional markers, such as OTX2, FEZF2, and / or OLIG1).

[0067] Pre-OPCs can be further differentiated into SOX10+ OLIG2+ NKX2-2+ OPCs by further culturing in Stage 2 and Stage 3 medium according to either Version 1 or Version 2 protocols (schematically shown in Figures 42 and 43).

[0068] As described in Examples 5 and 8, two interchangeable protocols for generating human OPCs from pre-OPCs have been developed, designated Version 1 and Version 2, with each version consisting of two stages designated Stage 2 and Stage 3. The Stage 2 and 3 protocols can be combined with the Stage 1 protocol for generating pre-OPCs from pluripotent stem cells, described herein, thereby enabling the generation of OPCs from human pluripotent stem cells in as little as 12 days.

[0069] As described in Example 5 (version 1 protocol for stages 2 and 3), culturing pre-OPCs (i) in 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., culture days 0-3), followed by culturing the resulting cells (ii) in 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., culture days 3-9), was sufficient to generate SOX10+ OLIG2+ NKX2-2+ OPCs.

[0070] As described in Example 8 (Version 2 Protocol for Stages 2 and 3), culturing pre-OPCs (i) in culture medium containing an FGFR pathway agonist, an mTOR pathway antagonist, an SHH pathway agonist, and a WNT pathway agonist for 6 days (e.g., culture days 0-6), followed by culturing the resulting cells (ii) in culture medium containing an FGFR pathway agonist, an IGF-1 pathway agonist, and a retinoic acid (RA) pathway antagonist for 3 days (e.g., culture days 6-9), was sufficient to generate SOX10+ OLIG2+ NKX2-2+ OPCs.

[0071] OPCs can be further differentiated into CD9+A2B5+O4+CNPase pre-OLs by further culturing in Stage 4 medium (schematically shown in Figure 44).

[0072] As described in Examples 10 and 11, culturing SOX10+ OLIG2+ NKX2-2+ OPCs in culture medium containing an IGF1R agonist, a TrkC agonist, a PDGFR agonist, a thyroid hormone receptor agonist, and an insulin receptor agonist was sufficient to generate CD9+ A2B5+ CNPase+ O4+ pre-OLs.

[0073] As used herein, the "agonist" of a cell signaling pathway is intended to refer to an agent that stimulates (upregulates) the cell signaling pathway.Stimulation of a cell signaling pathway can be initiated extracellularly, for example, by using an agonist that activates a cell surface receptor involved in the signaling pathway (for example, the agonist can be a receptor ligand).In addition to or instead of the above, stimulation of a cell signaling pathway can also be initiated intracellularly, for example, by using a small molecule agonist that interacts with a component of the signaling pathway intracellularly.

[0074] As used herein, the "antagonist" of cell signaling pathway is intended to refer to the agent that inhibits (downregulates) the cell signaling pathway.The inhibition of cell signaling pathway can be initiated outside the cell, for example, by using an antagonist that blocks the cell surface receptor involved in signaling pathway.In addition to or instead of the above, the inhibition of cell signaling can also be initiated inside the cell, for example, by using a small molecule antagonist that interacts with the component of signaling pathway inside the cell.

[0075] Retinoic acid (RA) pathway agonist, Akt pathway agonist, mTOR pathway agonist, WNT pathway antagonist, SHH pathway agonist, BMP pathway antagonist, PKC pathway antagonist, FGFR pathway agonist, WNT pathway agonist, IGF-1 pathway agonist, mTOR pathway antagonist, RA pathway antagonist, Akt pathway antagonist, activin receptor (AR) pathway agonist, PDGFR pathway agonist and AMPK pathway agonist are known in the art and commercially available.They are used in culture medium at effective concentration to achieve desired outcome, where desired outcome is, for example, generating pre-OPC and / or OPC that expresses interested marker.Non-limiting examples of suitable agonist and antagonist agonist and their effective concentration range are further described below.

[0076] RA pathway agonists are used in stage 1 medium and version 1 stage 3 medium, and include agents, molecules, compounds, or substances that can stimulate retinoic acid receptors (RARs), which are activated by both all-trans retinoic acid and 9-cis retinoic acid. There are three types of RARs: RAR-α, RAR-β, and RAR-γ, which are encoded by the RARA gene, RARB gene, and RARG gene, respectively. A variety of retinoic acid analogs have been synthesized that can activate the retinoic acid pathway. Non-limiting examples of such compounds include: TTNPB (RAR-α, RAR-β, and RAR-γ agonist), 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 retinoid; binds to RAR-β and RAR-γ), isotretinoin (endogenous agonist for 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) a retinoid compound, (ii) a retinoid X receptor (RXR) agonist, and (iii) a 25 retinoic acid receptor (RAR) agonist. In certain embodiments, 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).

[0077] Thus, 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, isotretinoin, tretinoin, 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 within the range of 5 to 500 mM, or within the range of 10 to 100 nM, or within the range of 25 to 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 in the range of 5 to 500 nM, or in the range of 10 to 100 nM, or in the range of 25 to 75 nM. In one embodiment, the RA pathway agonist is TTNPB, which is present in the culture medium at a concentration of 50 nM in Stage 1 medium, Stage 3 medium (version 1), or both.

[0078] Akt pathway agonists include agents, molecules, compounds, or substances capable of stimulating (activating) the signaling pathway of one or more members of the Akt family of serine / threonine kinases, including Akt1 (also called PKB or RacPK), Akt2 (also called PKBβ or RacPK-β), and Akt 3 (also called PKBγ or thymoma viral 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, demethylasteriquinone B1, resilisib, N-oleoylglycine, NSC45586 sodium, periplosin, CHPG sodium salt, bilobalide, 6-hydroxyflavone, musk ketone, SEW2871, 8-prenylnaringenin, 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 within the range of 0.1 to 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 Stage 1 culture medium at a concentration of 1 μM. In one embodiment, the Akt pathway agonist is SC79, which is present in the Stage 2 culture medium (version 1) at a concentration of 2 μM.

[0079] Agonists of the mTOR (mammalian target of rapamycin) pathway include agents, molecules, compounds, or substances that can stimulate (activate) signaling through mTOR, a member of the PI3K-related kinase family, which is a core component of the mTORC1 and mTORC2 complexes. In one embodiment, the mTOR pathway agonist is selected from the group consisting of 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, 1-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 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 stage 1 culture medium at a concentration of 1 μM. In one embodiment, the mTOR pathway agonist is MHY1485, which is present in the stage 3 culture medium (version 1) at a concentration of 2 μM.

[0080] WNT pathway antagonists include agents, molecules, compounds, or substances that can inhibit (downregulate) 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 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, triptonide, KYA1797K, JW55, JW 67, 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 in the range of 10 to 500 nM, in the range of 50 to 250 nM, or in the range of 50 to 150 nM. 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 10-500 nM, 50-250 nM, or 50-150 nM. In one embodiment, the WNT pathway antagonist is XAV939, which is present in the stage 1 culture medium at a concentration of 100 nM.

[0081] Agonists of the SHH (Sonic Hedgehog) pathway include agents, molecules, compounds, or substances capable of stimulating (activating) signal transduction through the SHH pathway, which is biologically involved in the binding of SHH to its receptor, Patched-1 (PTCH1), and in transduction through the transmembrane protein Smoothened (SMO). In one embodiment, the SHH pathway agonist is selected from the group consisting of purmorphamine, GSA 10, SHH, SAG, and combinations thereof. In one embodiment, the SHH pathway agonist is present in the culture medium at a concentration within the range of 100-1000 nM, or within the range of 250-750 nM, or within the range of 400-600 nM. In one embodiment, the SHH pathway antagonist is purmorphamine. In one embodiment, the SHH pathway antagonist is purmorphamine, which is present in the culture medium at a concentration of 100-1000 nM, or 250-750 nM, or 400-600 nM. In one embodiment, the SHH pathway antagonist is purmorphamine, which is present in the Stage 1 culture medium, the Stage 2 culture medium (version 1), and / or the Stage 2 culture medium (version 2) at a concentration of 500 nM.

[0082] 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 BMP binding 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, dorsomorphin, K02288, LDN214117, LDN212854, follistatin, ML347, noggin, and combinations thereof. In one embodiment, the BMP pathway antagonist is present in the culture medium at a concentration of 100-1000 nM, 150-750 nM, 100-500 nM, or 150-350 nM. 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 100-1000 nM, 150-750 nM, 100-500 nM, or 150-350 nM. In one embodiment, the BMP pathway antagonist is LDN193189, which is present in the culture medium at a concentration of 250 nM.

[0083] PKC (protein kinase C) pathway antagonists include agents, molecules, compounds, or substances capable of inhibiting (down-regulating) the PKC signaling pathway, which is biologically mediated by members of the PKC family. The PKC family of serine / threonine kinases includes 15 isozymes, including the "classical" PKC subcategory, which includes α, β1, β2, and γ isoforms. In one embodiment, the PKC pathway antagonist inhibits the activity of at least one PKC enzyme selected from PKCα, PKCβ1, PKCβ2, and PKCγ (and in other embodiments, inhibits the activity of at least two or three). 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 of 10-500 nM, 50-300 nM, 50-150 nM, or 75-150 nM. In one embodiment, the PKC pathway antagonist is Go 6983. In one embodiment, the PKC pathway antagonist is Go 6983, which is present in the culture medium at a concentration of 10-500 nM, 50-300 nM, 50-150 nM, or 75-150 nM. In one embodiment, the PKC pathway antagonist is Go 6983, which is present in the culture medium at a concentration of 110 nM.

[0084] Agonists of the FGFR pathway include agents, molecules, compounds, or substances capable of stimulating (upregulating) the fibroblast growth factor receptor signaling pathway, which is biologically activated by FGF binding to FGFR. In one embodiment, the FGFR agonist is FGF2, SUN11602, or a combination thereof. In one embodiment, the FGFR pathway agonist is present in the culture medium at a concentration of 1-20 ng / ml, 5-15 ng / ml, 7.5-12.5 ng / ml, 9-11 ng / ml, or 10 ng / ml. In one embodiment, the FGFR agonist is FGF2 (e.g., human recombinant FGF2). In one embodiment, the FGFR agonist is FGF2, which is present in the Stage 1 culture medium, and / or in the Stage 2 culture medium (versions 1 and 2), and / or in the Stage 3 culture medium (versions 1 and 2) at a concentration in the range of 1 to 20 ng / ml, in the range of 5 to 15 ng / ml, in the range of 7.5 to 12.5 ng / ml, in the range of 9 to 11 ng / ml, or at a concentration of 10 ng / ml.

[0085] 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 a Wnt protein ligand to a Frizzled family receptor. 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-acetoxime, AZD1080, WNT3A, alsterpaullone, indirubin-3-oxime, 1-azakempaullone, kenpaullone, TC-G 24, TDZD 8, TWS 119, 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 within the 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 within the 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 at a concentration of 1.0 μM.

[0086] IGF-1 (insulin-like growth factor 1) pathway agonists include agents, molecules, compounds, or substances capable of stimulating (activating) 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, demethylasteriquinone 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 within the range 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. 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 culture medium (version 2) 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.

[0087] Antagonists of the mTOR (mammalian target of rapamycin) pathway include agents, molecules, compounds, or substances capable of inhibiting (downregulating) the mTOR signaling pathway, where mTOR is a member of the PI3K-related kinase family, which is 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, dactolisib, rapamycin, everolimus, AZD 8055, temsirolimus, PI-103, NU7441, BC-LI-0186, eCF 309, ETP 45658, niclosamide, omipalisib, PF 04691502, PF 05212384, Torin 1, Torin 2, WYE 687, XL 388, STK16-IN-1, PP 242, torkinib, ridaforolimus, sapanisertib, voxtalisib, and combinations thereof. In one embodiment, the mTOR pathway antagonist is present in the culture medium at a concentration in the range of 5-200 nM, 10-150 nM, or 15-100 nM. 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 in the range of 5-200 nM, 10-150 nM, or 15-100 nM. In one embodiment, the mTOR pathway antagonist is AZD 3147, which is present in the culture medium at a concentration of 15 nM in the Stage 2 culture medium (version 1). In one embodiment, the mTOR pathway antagonist is AZD 3147, which is present in the Stage 2 culture medium (version 2) at a concentration of 100 nM.

[0088] Retinoic acid (RA) pathway antagonists include agents, molecules, compounds, or substances capable of inhibiting (downregulating) the RA signaling pathway. In one embodiment, the RA pathway antagonist is selected from the group consisting of AGN193109, BMS 195614, 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 within the range of 50-300 nM, 75-250 nM, 100-200 nM, or 90-110 nM. 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 in the range of 50-300 nM, 75-250 nM, 100-200 nM, or 90-110 nM. In one embodiment, the RA pathway antagonist is AGN193109, which is present in the Stage 3 culture medium (version 2) at a concentration of 100 nM.

[0089] Antagonists of the AKT pathway include agents, molecules, compounds, or substances capable of inhibiting (downregulating) the signal transduction 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 proto-oncogene 3). In one embodiment, the AKT pathway antagonist is MK2206, GSK690693, Perifosine (KRX-0401), Ipatasertib (GDC-0068), Capivasertib (AZD5363), PF-04691502, AT 7867, Triciribine (NSC154020), ARQ751, Miransertib (ab235550), Borussertib, Cerisertib, Akti1 / 2, CCT128930, A 674563, PHT 427, Miltefosine, AT 13148, ML 9, BAY 1125976, Oridonin, TIC10, Pectolinarin, 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 of 25-300 nM, 50-250 nM, 75-200 nM, or 100-150 nM. 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 of 25-300 nM, 50-250 nM, 75-200 nM, or 100-150 nM. In one embodiment, the AKT pathway antagonist is MK2206, which is present in the culture medium at a concentration of 125 nM in the stage 2 culture medium (version 1) and / or the stage 3 culture medium (version 1).

[0090] Agonists of the activin receptor (AR) pathway include agents, molecules, compounds, or substances capable of stimulating (activating) signal transduction through the AR pathway. In one embodiment, the AR pathway agonist is selected from the group consisting of activin A, alantolactone, and combinations thereof. In one embodiment, the AR pathway agonist 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 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 2-20 ng / ml, 5-15 ng / ml, or 7.5-12.5 ng / ml. In one embodiment, the AR pathway agonist is activin A, which is present in the culture medium at a concentration of 10 ng / ml.

[0091] PDGFR (platelet-derived growth factor receptor) pathway agonists include agents, molecules, compounds, or substances capable of stimulating (activating) 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 of 2-20 ng / ml, 5-15 ng / ml, or 7.5-12.5 ng / ml. In one embodiment, the PDGFR pathway agonist is PDGF-AA, 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 PDGFR pathway agonist is PDGF-AA, which is present in the culture medium at a concentration of 10 ng / ml in stage 3 culture medium (version 1). In one embodiment, the PDGFR pathway agonist is PDGF-AA, which is present in the stage 4 culture medium at a concentration of 10 ng / ml.

[0092] Agonists of the AMPK (5' AMP-activated protein kinase) pathway include agents, molecules, compounds, or substances that can stimulate (activate) signal transduction through the AMPK pathway. In one embodiment, the AMPK pathway agonist is AICAR, metformin, cadinol B, malein, amarogentin, A 769662, PF 06409577, metformin hydrochloride, ZLN 024, ZLN 024 hydrochloride, nilotinib, phenformin, nilotinib hydrochloride monohydrate, adenosine 5'-monophosphate monohydrate, hispidulin, MK 8722, euphorbia steroid, ASP4132, GSK621, EX229 (compound 991), trans-ferulic acid, O-304, MK 3903, BAM 15, ligstroflavone, ETC-1002, BC1618, IMM-H007, IM156, chikusetsusaponin IVa, polychoric acid A, 7-methoxyisoflavone, urolithin B, danthron, demethylene berberine, AMPK activator 1, AMPK activator 2, AMPK activator 4, malvidin-3-O-arabinoside chloride, RSVA 405, etilefrine, COH-SR4, buformin, buformin hydrochloride, PT1, bempedoic acid, 3a-hydroxymogrol, ampkinone, and combinations thereof. In one embodiment, the AMPK pathway agonist 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 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 stage 3 culture medium (version 1) at a concentration of 200 μM.

[0093] Agonists of the TrkC (tropomyosin-related kinase receptor C) pathway include agents, molecules, compounds, or substances capable of stimulating (activating) signal transduction through the TrkC pathway. In one embodiment, the TrkC pathway agonist is selected from the group consisting of neurotrophin-3 (NT-3), peptidomimetics 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, the TrkC pathway agonist is present in the culture medium at a concentration in the range of 2 to 20 ng / ml, in the range of 5 to 15 ng / ml, or in the range of 7.5 to 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, or 7.5-12.5 ng / ml. In one embodiment, the TrkC pathway agonist is NT-3, which is present in the stage 4 culture medium at a concentration of 10 ng / ml.

[0094] Thyroid hormone receptor (THR) agonists include agents, molecules, compounds, or substances capable of stimulating (activating) signal transduction through the thyroid hormone receptor pathway. In one embodiment, the thyroid hormone receptor agonist is selected from the group consisting of T3, T4, resmetirom, TRb agonist 3 (compound 3), sobetirom, tiratricol, and combinations thereof. In one embodiment, the THR agonist is present in the culture medium at a concentration of 10-100 nM, or 25-75 nM, or 40-60 nM, or 50 nM. In one embodiment, the THR agonist is T3. In one embodiment, the THR agonist is T3, which is present in the culture medium at a concentration of 10-100 nM, or 25-75 nM, or 40-60 nM, or 50 nM. In one embodiment, the THR agonist is T3, which is present in the stage 4 culture medium at a concentration of 50 nM.

[0095] Insulin receptor (IR) agonists include agents, molecules, compounds, or substances capable of stimulating (activating) 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, demethylasteriquinone B1, MK-5160, MK-1092, and combinations thereof. In one embodiment, the insulin receptor agonist is present in the culture medium at a concentration of 4-40 μg / ml, or 10-30 μg / ml, or 15-25 μg / ml, or 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 stage 4 culture medium at a concentration of 20 μg / ml.

[0096] When a given agonist or antagonist is used in more than one step of the method, in one embodiment, the same particular agonist or antagonist is used for each step in which the agent is present in the culture medium, hi another embodiment, different agonists or antagonists that affect the same signal transduction pathway are used in different steps of the method.

[0097] When an agonist or antagonist is used in more than one step of the method, in one embodiment, the same agonist or antagonist is used at the same concentration for each step in which the agent is present in the culture medium, hi another embodiment, the same agonist or antagonist is used at different concentrations in different steps of the method.

[0098] III. Culture conditions The disclosed methods for producing pre-OPCs, OPCs, and pre-OLs utilize standard culture conditions for cell culture established in the art in combination with the chemically defined and optimized culture media described in subsection "II" above. For example, cells can be cultured at 37 degrees and 5% CO. Cells can be cultured in standard culture vessels or plates, such as 96-well plates. In some embodiments, the starting pluripotent stem cells are attached to plates, preferably coated with an extracellular matrix material, such as vitronectin. In one embodiment, stem cells are cultured on a vitronectin-coated culture surface (e.g., a vitronectin-coated 96-well plate).

[0099] Pluripotent stem cells can be cultured in commercially available media prior to differentiation. For example, stem cells can be cultured in Essential 8 Flex medium (Thermo Fisher, # A2858501) for at least one day prior to the start of the differentiation protocol. In a non-limiting exemplary embodiment, stem cells are passaged at a density of 150,000 cells / cm2 in a 96-well plate coated with vitronectin (Thermo Fisher, # A14700), and cultured in Essential 8 Flex medium for one day prior to differentiation.

[0100] To initiate the differentiation protocol, the medium in which the stem cells were cultured is replaced with a basal differentiation medium supplemented with a signaling pathway agonist and / or antagonist, as described above in subsection "II." The basal differentiation medium can include, for example, commercially available basal media supplemented with additional standard culture media components necessary to maintain cell viability and proliferation, but without serum (basal differentiation medium is serum-free medium) and without 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 poly(vinyl alcohol) (Sigma, #p8136), 1% chemically defined lipid concentrate (Thermo Fisher, #11905031), 450 uM 1-thioglycerol (Sigma, #M6145), 0.7 ug / ml insulin (Sigma, #11376497001), and 15 ug / ml transferrin (Sigma, #10652202001). In one embodiment, this basal medium is supplemented with Albumax II.

[0101] Typically, the culture medium is replaced with fresh medium periodically, for example, in one embodiment, the medium is replaced every 24 hours.

[0102] To generate pre-OPCs, OPCs, and / or pre-OLs, stem cells are cultured in optimized culture medium for a time sufficient for the cells to differentiate and express markers associated with pre-OPCs, OPCs, or pre-OLs. As described in Example 1, it has been discovered that culturing stem cells for as short a period as 72 hours (3 days) in optimized Stage 1 culture medium is sufficient for differentiation into pre-OPCs. Thus, 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.

[0103] As described in Example 5, it has been discovered that culturing pre-OPCs for as short a period as 72 hours (3 days) in optimized Stage 2 culture medium is sufficient for differentiation of pre-OPCs. Accordingly, in one embodiment, pre-OPCs are cultured in Stage 2 culture medium (version 1) for at least 72 hours. In other embodiments, pre-OPCs are cultured in Stage 2 culture medium (version 1) for at least 60 hours, 64 hours, 68 hours, 72 hours, 76 hours, 80 hours, 84 hours, 88 hours, 92 hours, or 96 hours. It has further been discovered that further culturing of cells for as short a period as 6 days (144 hours) in optimized Stage 3 medium is sufficient for differentiation into OPCs. Accordingly, in one embodiment, cells are cultured in Stage 3 culture medium (version 1) for at least 144 hours. In other embodiments, cells are cultured in Stage 3 culture medium (version 1) for at least 132 hours, 136 hours, 140 hours, 144 hours, 150 hours, 154 hours, or 158 hours.

[0104] As described in Example 8, it has been discovered that culturing pre-OPCs for as short a period as 6 days (144 hours) in optimized Stage 2 culture medium is sufficient for differentiation of the cells. Accordingly, in one embodiment, pre-OPCs are cultured in Stage 2 culture medium (version 2) for at least 144 hours. In other embodiments, pre-OPCs are cultured in Stage 2 culture medium (version 2) for at least 132, 136, 140, 144, 150, 154, or 158 hours. It has further been discovered that further culturing of the cells for as short a period as 72 hours (3 days) in optimized Stage 3 medium is sufficient for differentiation into OPCs. Accordingly, in one embodiment, cells are cultured in Stage 3 culture medium (version 2) for at least 72 hours. In other embodiments, cells are cultured in Stage 3 culture medium (version 2) for at least 60, 64, 68, 72, 76, 80, 84, 88, 92, or 96 hours.

[0105] As described in Example 10, it has been discovered that culturing OPCs for as short a period as 6 days (144 hours) in optimized Stage 4 culture medium is sufficient for cell differentiation into pre-OLs. Thus, in one embodiment, OPCs are cultured in Stage 4 culture medium for at least 144 hours. In other embodiments, OPCs are cultured in Stage 4 culture medium for at least 132 hours, 136 hours, 140 hours, 144 hours, 150 hours, 154 hours, or 158 hours.

[0106] IV. Application The disclosed methods and compositions for producing pre-OPCs, OPCs, and pre-OLs enable these cell populations to be efficiently and stably utilized in a variety of applications. For example, the methods and compositions can be used to study the development and biological characteristics of oligodendrocytes, aiding in the understanding of diseases and disorders associated with oligodendrocytes. For example, pre-OPCs, OPCs, and / or pre-OLs produced using the disclosed methods can be further purified using agents that bind to surface markers expressed on the cells, according to techniques established in the art. Thus, in one embodiment, the present disclosure provides a method for isolating pre-oligodendrocyte precursor cells (pre-OPCs), oligodendrocyte precursor cells (OPCs), or pre-myelinating oligodendrocytes (pre-OLs), the method comprising the steps of: contacting OLIG2-expressing pre-OPCs or OPCs, or CD9+ pre-OLs, produced by the methods of the present disclosure, with at least one binding agent, wherein the binding agent binds to a cell surface marker expressed by the pre-OPCs, OPCs, or pre-OLs; and isolating cells that bind to the binding agent, thereby isolating pre-OPCs, OPCs, or pre-OLs.

[0107] In one embodiment, the binding agent is an antibody, such as a monoclonal antibody (mAb) that binds to a cell surface marker. Non-limiting examples of suitable OPC cell surface markers include PDGFRa, O4, and A2B5. Cells that bind to the antibody can be isolated by techniques known in the art, including, but not limited to, fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS).

[0108] Oligodendrocyte lineage progenitor cells are also intended to be used in the treatment of various diseases and disorders associated with oligodendrocytes by delivering the cells to subjects with the diseases or disorders. Examples of oligodendrocyte-associated diseases and disorders include, but are not limited to, multiple sclerosis (MS), progressive multifocal leukoencephalopathy, periventricular leukomalacia, some leukodystrophies, and amyotrophic lateral sclerosis (ALS).

[0109] V. Composition In other aspects, the present disclosure provides compositions related to methods of producing pre-OPCs and OPCs, including culture media and cell cultures, as well as isolated progenitor cells and cell populations thereof.

[0110] In one aspect, the present disclosure provides a culture medium for obtaining oligodendrocyte progenitor cells (OPCs), the 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 stage 2 medium in version 1). In another aspect, the present disclosure provides a culture medium for obtaining oligodendrocyte progenitor cells (OPCs), the 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 stage 3 medium in version 1).

[0111] In another aspect, the present disclosure provides a culture medium for obtaining oligodendrocyte progenitor cells (OPCs), the culture medium comprising an FGFR pathway agonist, an mTOR pathway antagonist, an SHH pathway agonist, and a WNT pathway agonist (corresponding to stage 2 medium in version 2). In another aspect, the present disclosure provides a culture medium for obtaining oligodendrocyte progenitor cells (OPCs), the culture medium comprising an FGFR pathway agonist, an IGF-1 pathway agonist, and a retinoic acid (RA) pathway agonist (corresponding to stage 3 medium in version 2).

[0112] In another aspect, the present disclosure provides a culture medium for obtaining premyelinating oligodendrocytes (pre-OLs), the 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.

[0113] In one aspect, the disclosure provides an isolated cell culture comprising OPCs cultured in one of the culture media disclosed herein. In one embodiment, the disclosure provides an isolated cell culture comprising OLIG2+ OPCs cultured in a culture medium (corresponding to Stage 2 medium of Version 1) comprising an FGFR pathway agonist, an mTOR pathway antagonist, an SHH pathway agonist, an AKT pathway antagonist, and an AKT pathway agonist. In another embodiment, the disclosure provides an isolated cell culture comprising OLIG2+ OPCs cultured in a culture medium (corresponding to Stage 3 medium of Version 1) 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.

[0114] In another embodiment, the disclosure provides an isolated cell culture comprising OLIG2+ OPCs, the cell culture being 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 Stage 2 medium of Version 2). In another embodiment, the disclosure provides an isolated cell culture comprising OLIG2+ OPCs, the cell culture being cultured in a culture medium comprising an FGFR pathway agonist, an IGF-1 pathway agonist, and a retinoic acid (RA) pathway agonist (corresponding to Stage 3 medium of Version 2).

[0115] In another aspect, the disclosure provides an isolated cell culture comprising CD9+ pre-OLs, the cell culture being 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.

[0116] The present invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of all figures and references, patents, and patent publications cited throughout this application are expressly incorporated herein by reference. [Example]

[0117] Example 1: Development of a culture protocol for generating stem cell-derived oligodendrocyte precursor cells In this example, a culture medium composition for generating oligodendrocyte precursor cells was developed that can induce human pluripotent stem cells to differentiate into oligodendrocyte precursor cells expressing NKX2-2 and OLIG2 after 3 days in culture, which can be further differentiated into mature oligodendrocytes.

[0118] This example utilizes high-dimensional experimental design (HD-DoE), as previously described in Bukys et al. (2020) Iscience 23:101346. This approach utilizes computational design geometry to simultaneously test multiple process inputs and provides deep spatial mathematical modeling of effectors / responses. This approach allows for the discovery of combinatorial signaling inputs that control complex processes, such as during cell differentiation. This approach allows for the testing of multiple plausible key process parameters when they affect the output response, such as gene expression. Because gene expression provides distinctive phenotypic characteristics, such as for human cells, this approach can be applied to identify and understand which signaling pathways control cell fate. In this example, HD-DOE was applied to identify conditions that induce genes expressed in oligodendrocyte precursor cells directly from a pluripotent stem cell state.

[0119] Specifically, to develop a novel method for generating oligodendrocytes, the effects of agonists and antagonists (herein referred to as effectors) of multiple signaling pathways on the expression of two sets of 53 preselected genes were examined after three days of treatment. These effectors are small molecules commonly used in the stepwise differentiation of stem cells toward specific fates. The selection of effectors was based on current literature on neural induction in the anterior ectoderm and differentiation of stem cells into oligodendrocytes.

[0120] HD-DoE #1 To test effectors, experiments were designed using at least eight factors, allowing for the evaluation of cellular responses to over 48 different combinations of effectors at a range of concentrations. To analyze the model, we focused on the expression of genes expressed during early development of 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.

[0121] One model in particular showed promising results for upregulating the NKX2-2 and OLIG2 genes when optimized for the highest expression of NKX2-2, 12480.6, as shown in the results summarized in Figure 1. Eight factors were tested in this model: PD0325901, MK2206, TTNPB, SC79, MHY1485, ZM336372, AGN193109, and AZD3147.

[0122] Three of the eight tested factors, namely, TTNPB (a retinoic acid pathway agonist), SC79 (an Akt signaling pathway agonist), and MHY1485 (an mTOR signaling pathway agonist), had a significant positive effect on the expression of the targeted genes, with TTNPB having the greatest effect (factor contribution: 31.3), MHY1485 (factor contribution: 13.8), and SC79 (factor contribution: 1.47). These factors were able to significantly increase the expression of NKX2-2 and OLIG2. OLIG1 and PDGFRA had average expression levels (129.9 and 346.45, respectively), which is consistent with the gene expression pattern during oligodendrocyte differentiation.

[0123] As shown in Figure 2, the normalized expression of PDGFRA in this model could reach a maximum of 832.9, which was the highest expression level in all models, so this model was also optimized for maximum PDGFRA expression. This setting showed that TTNPB (a retinoic acid pathway agonist) and MHY1485 (an mTOR signaling pathway agonist) also had a positive effect on the upregulation of PDGFRA, 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. ZM336372 had a low factor contribution (<1), so it was not included in the composition. Under these conditions, OLIG1 had an average expression level of 228.36, similar to the optimized conditions for NKX2-2. One difference in this condition was the downregulation of the OLIG2 gene, which went from 1049.4 in the previous condition to 241.9.

[0124] As shown in Figure 3, among the effectors that positively contributed to the expression levels of NKX2-2, OLIG2, or PDGFRA, two had negative effects on the expression levels of NKX2-2 and OLIG2. Therefore, these two factors, PD0325901 and ZM336372, were removed from the list of candidate compositions for oligodendrocyte differentiation.

[0125] Thus, this first HD-DoE screen identified that culture medium lacking exogenously added growth factors but containing agonists of the retinoic acid pathway, the Akt signaling pathway, and the mTOR signaling pathway was sufficient to result in the production of OLIG2-expressing OPCs from pluripotent stem cells after 3 days (72 hours) in culture.

[0126] HD-DoE #2 To further enhance the conditions for differentiation from pluripotent to oligodendrocytes, we conducted additional HD-DoE experiments. We obtained additional gene regulation models and used them to prepare differentiation protocols. The basis of this model was a 13-factor HD-DoE experiment that focused on the initiation of cell differentiation toward anterior neuroectoderm. In this model, we focused on the expression of FEZF2 and OTX2.

[0127] As shown in the results summarized in Figure 4, the model was optimized for the highest expression of OTX2, 12755.9. According to the high expression model of OTX2, seven effectors, including MK2206, PD0325901, CHIR99021, LDN193189, Go6983, PD173074, and BLU9931, had positive contributions, with the largest factor contribution of 22.2 for MK2206 and the smallest factor contributions of 1.7 for PD173074 and BLU9931.

[0128] As shown in the results summarized in Figure 5, the model was optimized for the highest expression of FEZF2, 4466. When the model was optimized for the maximum expression level of FEZF2, three effectors were common to the previous condition, 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, and three new effectors were derived, including 500 nM purmorphamine, XAV939, and SC79.

[0129] As shown in Figure 6, dynamic profiling analysis was performed to fine-tune the composition and find the optimal combination of factors for high expression of both OTX2 and FEZF2. This analysis revealed that XAV939 (an inhibitor of the WNT signaling pathway) and purmorphamine (an agonist of the SHH signaling pathway, known to mediate ventralization of cells during brain development) had a significant positive effect on FEZF2 expression and no negative effect on OTX2 expression. Therefore, these two factors were added to the optimized culture composition.

[0130] In addition to including factors that promoted the expression of OPC-associated surface markers, several factors that inhibited the expression of such markers were removed from the optimized culture composition. CHIR99021, an agonist of the WNT signaling pathway, was removed. MK2206 and PD0325901 were also removed because, according to the eight-factor model, they have a negative effect on oligodendrocyte gene expression. PD173074 and BLU9931 were also removed due to their low factor contribution of 1.7.

[0131] overview Considering both models, culture conditions that maximized differentiation of human induced pluripotent stem cells into cells with an identity as oligodendrocyte progenitor cells (OPCs) and resulted in increased 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), purmorphamine (SHH pathway agonist), XAV939 (WNT pathway antagonist), LDN193189 (BMP pathway antagonist), and Go6983 (PKC pathway antagonist).

[0132] Example 2: Factor criticality analysis of culture conditions for inducing OPCs To assess the factor criticality of each component in the optimized culture medium described in Example 1, we performed an in silico predictive analysis of outcome when effectors were removed one by one while other factors were present. To do this, we used a set-point dynamic profile analysis comparing the expression levels of genes of interest in the absence of each factor. This factor criticality analysis revealed the degree of importance of each effector input, since the expression of the genes of interest reveals whether the desired outcome is achievable.

[0133] Figures 7A-7D summarize the results of factor fatality analyses for the effectors TTNPB (RA pathway agonist), SC79 (Akt pathway agonist), and MHY1485 (mTOR pathway agonist). Figure 7A shows the expression levels of OPC genes of interest in the presence of TTNPB, MHY1485, and SC79, when the model was otherwise optimized for maximal expression of NKX2-2. As shown in Figure 7B, upon removal of TTNPB, the predicted expression levels of NKX2-2, OLIG2, and PDGFRA significantly decreased from over 12,000 to 4,500 for NKX2-2, from 1,000 to 400 for OLIG2, and from 350 to less than 100 for PDGFRA. This outcome demonstrates a significant negative impact on the expression of all desired markers when the RA pathway agonists are removed. As shown in Figure 7C, when MHY1485 was removed, expression levels again decreased, but not as dramatically as in the previous condition. As shown in Figure 7D, when SC79 was removed, only a small shift in the plot was observed, suggesting that this factor is not as important as TTNPB and MHY1485 for achieving maximal induction of OPC markers.

[0134] Figures 8A-8D summarize the results of factor lethality analyses for the effectors purmorphamine (an SHH pathway agonist), XAV939 (a WNT pathway antagonist), LDN193189 (a BMP pathway antagonist), and Go6983 (a PKC pathway antagonist). To achieve the desired patterning of oligodendrocyte populations to anterior brain regions, these additional factor inputs were investigated, for example, with respect to FEZF2 and OTX2. When the model was optimized for maximal expression of FEZF2, the expression levels of FEZF2 and OTX2 were examined in the absence of either LDN193189, XAV, purmorphamine, or Go6983. The most significant change was observed in the absence of LDN193189, which resulted in a nearly 50% decrease in FEZF2 expression (from 4500 to 2500). OTX2 expression also decreased from 9000 to 7000, the lowest among all four deletion processes. When XAV939 and purmorphamine were removed, FEZF2 expression levels decreased to 3000 and 3500, respectively, while OTX2 expression increased slightly in both cases. When Go6983 was removed, we did not observe any significant changes in the expression levels of the genes of interest, suggesting that Go6983 is optional for regulating FEZF2 and OTX2.

[0135] Example 3: Immunocytochemical verification of stem cell-derived OPCs To further validate the optimized culture medium described in Example 1, cells were cultured in the optimized medium for 3 days, and immunocytochemistry was used to evaluate the expression of anterior neuroectoderm biomarkers and oligodendrocyte progenitor biomarkers. Biomarkers included OTX2, as well as oligodendrocyte progenitor biomarkers including NKX2-2, OLIG2, and PDGF. The early neuronal marker Nestin was used to distinguish neural stem cells from oligodendrocyte progenitor cells. Ki67 was also used to confirm cell proliferation after induction. Representative immunohistochemistry results are shown in Figure 9. These immunocytochemistry images confirmed that the majority of cells expressed OTX2. However, no trace of the neuronal biomarker Nestin was detected, confirming that this differentiated OPC population lacked neural stem cells. Expression of OLIG2 and NKX2-2 was also observed in more than 90% of the cells, thereby confirming the oligodendrocyte lineage. As expected, none of the cells expressed PDGFR, since this gene is expressed at a late stage of differentiation into oligodendrocytes.

[0136] Example 4: Validation of stem cell-derived pre-OPCs by RNA sequencing RNA sequencing was used to obtain gene profiles of cells cultured in the differentiation medium detailed in Examples 1 and 2. hiPSCs were cultured in the medium for three days, and the RNA of the resulting cells was sequenced by standard RNA sequencing analysis. The results in Figure 10A show the normalized expression levels of selected genes in three replicates on day 0 and three replicates on day 3, representing various brain regions, genes representing early oligodendrocyte identity (NKX2-2, OLIG2, PDGFRa), and genes representing stem cell status (NANOG, POU5F1). The results demonstrated that the levels of stem cell genes decreased in pre-OPC cells, while the levels of oligodendrocyte genes increased, demonstrating the differentiation of hiPSCs into the oligodendrocyte lineage using the differentiation medium. The results in Figure 10B show differential expression and fold change of selected genes, with HOXA1 at the highest level (15) and OLIG2, NKX2-2, and PDGFRa at 5. This data demonstrates the ability of the developed composition as a Stage 1 medium to direct cells towards an oligodendrocyte identity.

[0137] Example 5: Development of a culture protocol to generate stem cell-derived oligodendrocyte progenitor cells expressing SOX10, OLIG2, and NKX2-2 In this example, a stepwise differentiation protocol for generating oligodendrocyte precursor cells was developed, which can induce human pluripotent stem cells into precursor cells expressing SOX10, NKX2-2, and OLIG2 after 12 days in culture. The differentiated cells also express other markers of oligodendrocyte precursor cells, including PDGFRa and NG2. After treatment with Stage 1 pre-OPC medium for 3 days as described in Example 1, the cells are cultured in Stage 2 medium for 3 days, followed by Stage 3 medium for 6 days. The cells can then be further differentiated into mature oligodendrocytes. The entire three-stage protocol is shown diagrammatically in Figure 42.

[0138] To develop a formulation for oligodendrocyte differentiation, the effects of various agonists and antagonists (effectors) on pre-OPC differentiation were investigated using HD-DoE. These effectors were selected based on the then-available literature on developmental biology and stem cell differentiation, as well as single-cell RNA sequencing data from mouse and human oligodendrocytes.

[0139] These experiments led to the development of compositions for Stage 2 shown in Table 1 below and compositions for Stage 3 shown in Table 2 below.

[0140] (Table 1) Verified factors in the Stage 2 composition TIFF2025526018000001.tif41128

[0141] Table 2: Verified factors in the Stage 3 composition TIFF2025526018000002.tif54148

[0142] To design a composition for stage 2 differentiation, cells were first cultured in the stage 1 medium described in Example 1 and then treated with combinations of 8 or 12 factors for 3 days, and the gene expression of the cells in each condition was modeled. At this point, due to the short duration of culture (only 6 days in vitro), we decided to focus on initiating expression of SOX10, OLIG1, and PDGFRa while maximally expressing OLIG2 and maintaining high expression of NKX2-2 to direct 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).

[0143] In one 12-factor model, the individual and combined effects of the following agents were tested: SUN 11602, an agonist of the FGF signaling pathway; FGF2; activin A, an activator of a TGF-β superfamily receptor; A 8301, an inhibitor of the TGF-β pathway; CHIR99021, a WNT activator; AGN193109, an RA antagonist; MK2206, an AKT inhibitor; purmorphamine, an agonist of the SHH signaling pathway; AZD3147, an antagonist of the mTOR pathway; MHY1485, an agonist of the mTOR pathway; SC79, an agonist of the AKT pathway; and GO6983, an inhibitor of the PKC pathway. This model demonstrated the ability to modulate pre-OPCs to upregulate OLIG2, NKX2-2, and PDGFRa. When optimized for maximum OLIG2 expression, which was 1841.6, multiple factors demonstrated positive regulatory effects, including FGF-2, with a maximum 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 of less than 3 ( FIG. 11 ). AGN193109, MHY1485, and GO6983 had the greatest negative effects on OLIG2 expression, with factor contributions of 23.2, 22.5, and 10.2, respectively. Within the specifications for achieving 80% maximum expression of OLIG2, this complex media composition had a Cpk value (process capability index) of 0.43, corresponding to a risk of failure of 9.6%.

[0144] Dynamic profile analysis was used to evaluate the expression levels of other genes of interest, such as NKX2-2, OLIG1, and SOX10, when the model was maximized for OLIG2. Activin A and SUN 11602 were reduced to zero due to their very low factor contributions. When this model was optimized, we observed that the expression levels were 3000 for NKX2-2, 1000 for PDGFRa, 80 for OLIG1, and 15 for SOX10 (FIG. 12). According to this analysis, four compounds, including FGF2, MK2206, SC79, and AZD3147, showed positive effects on OLIG2 with factor contributions greater than 3, and either acted positively on other genes or had no significant effect on the expression levels of other genes. Therefore, we did not exclude any of these four factors from the candidate compositions. We also observed that purmorphamine and GO6983 had a negative effect on OLIG2 (factor contributions of 7.2 and 10.22), but they positively regulated the expression of SOX10. Therefore, the effects of these two compounds were investigated on other selected genes, and it was concluded that GO6983 had a significant negative effect on PDGFRA and should not be included in the composition.

[0145] To obtain more information about the effect of purmorphamine on the differentiation of pre-OPCs into more committed cell populations, we tested this factor in two other models. In one of the 12-factor models, we included eight new inputs, including TTNPB, biotin, insulin, propionate, DBZ, Yhhu3792, LDN193189, and Y-27632, along with purmorphamine, FGF2, AGN193109, and CHIR99021. When optimized for OLIG2, 3299.4, purmorphamine exhibited a strong positive effect, with a factor contribution of 13 (Figure 13). Within the specifications for achieving 80% maximal expression of OLIG2, this complex medium composition had a Cpk value (process capability index) of 0.68, corresponding to a risk of failure of 0.1%. In another 12-factor model, purmorphamine was tested with A8301, CHIR99021, dexamethasone, T3+T4, PD0325901, ZM336372, DBZ, BMP7, PD173074, SANT-1, and PDGF-AA. When optimized for OLIG2, which had a factor contribution of 644.6, purmorphamine had the greatest positive effect on OLIG2 expression, with a factor contribution of 16.9 (Figure 14). Within the specifications for achieving 80% maximum expression of OLIG2, this complex media composition had a Cpk value (process capability index) of 0.95, corresponding to a risk of failure of 0.22%.

[0146] We also investigated the individual effects of factors on the rest of the selected genes in both models and observed positive trends for SOX10, OLIG1, and NKX2-2 (FIG. 15).

[0147] Thus, considering the above model, a candidate composition for stage 2 was created consisting of FGF2, purmorphamine, MK2206, SC79, and AZD3147. This composition can maximize cell differentiation toward stable and elevated expression of OLIG2, NKX2-2, and early expression of SOX10 and PDGFRa. This composition was further validated by immunocytochemical assay (see Example 7).

[0148] To further differentiate cells into their oligodendrocyte progenitor identity, additional HD-DoE experiments were performed on cells cultured in stage 1 and stage 2 differentiation media. After 3 days, gene expression in cells in various combination conditions was examined. We focused on maximal expression of oligodendrocyte progenitor signature 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 eight-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 for maximum OLIG1 expression at 256, activin A and TTNPB had the largest positive effects, with factor contributions of 50.2 and 19.11, respectively (Figure 16). LDN193189 and SUN 11602 also positively regulated OLIG1 expression, with factor contributions of 10.8 and 3.7. Within the specification to achieve 80% maximum expression of OLIG1, this complex media composition had a Cpk value (process capability index) of 0.46, which corresponded to a risk of failure of 8.1%.

[0149] The same model was also optimized for maximum expression of PDGFRa, which was 3510.9. Specifically, two factors, TTNPB and linoleic acid, were determined to have factor contributions of 71.6 and 22.1, respectively, on the expression level of PDGFRa (Figure 17). Within the specifications for achieving 80% maximum expression of PDGFRa, this complex medium composition had a Cpk value (process capability index) of 0.54, which corresponded to a risk of failure of 5.2%. This model was also optimized for maximum expression of SOX10, which was 18.8. In this optimization, TTNPB was the only input capable of increasing the expression levels of selected genes (Figure 18).

[0150] Dynamic profiling analysis was used to identify combinations that could optimize the expression of all three selected genes. Among all effectors, only TTNPB had a positive effect on all three genes (Figure 19). Because activin A is the primary compound regulating the expression level of Olig1, we examined the changes in the plot after adding activin A and observed that the expression level of SOX10 remained within the target range and that PDGFRa was also expressed at high levels (2520). Adding other compounds reduced the expression levels of two of the three genes, so only activin A and TTNPB were added to the candidate compositions for stage 3.

[0151] In another model, the effects of activin A and TTNPB on the gene profile of differentiating cells were examined together with the effects of LDN193189, MHY1485, PDGF-AA, AICAR, FGF2, CHIR99021, DBZ, MK2206, purmorphamine, and T3+T4. When this model was optimized for the maximum expression of SOX10, which was 54.7, the inventors observed that activin A again had a positive regulatory effect on SOX10 expression, with a factor contribution of 4.7. Other positive factors included: MK2206, with a maximum factor contribution of 16.2; MHY1485, with a factor contribution of 11.49; CHIR99021, with a factor contribution of 9.6; DBZ, with a factor contribution of 9.3; FGF2, with a factor contribution of 3.6; and PDGF-AA, with a factor contribution of less than 3 (Figure 20). Within the specifications to achieve 80% maximal expression of SOX10, this complex media composition had a Cpk value (process capability index) of 0.47, which corresponded to a risk of failure of 7.7%.

[0152] In this model, we also investigated 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 for maximum expression of CSPG4, which was 38.6, five factors demonstrated positive regulatory behavior, including TTNPB, with a maximum factor contribution of 15.96, and purmorphamine, 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 (Figure 21). Within the specifications to achieve 80% maximum expression of CSPG4, this complex media composition had a Cpk value (process capability index) of 0.69, which corresponded to a risk of failure of 1.8%.

[0153] Dynamic profiling analysis was used to further evaluate the effects of factors on the genes we selected and identify combination sets that could optimize their expression (Figure 22). This analysis showed that activin A had a positive effect on SOX10 but reduced the levels of PDGFRa and OLIG1. However, previous models indicated that activin A could also act positively on OLIG1, depending on other factors present in the composition. Furthermore, SOX10, along with OLIG2 and NKX2-2, is one of the master transcription factors for oligodendrocytes, demonstrating its ability to commit stem cells to oligodendrocytes in reprogramming efforts (Wang et al. (2014) Proc Natl Acad Sci USA 111:E2885-94; Garcia-Leon et al. (2018) Stem Cell Reports 10:655-672). DBZ demonstrated a positive effect on all three genes; however, it also increased neuronal genes (Figure 23). One of the major challenges in differentiating cells toward an oligodendrocyte fate is controlling the mixed population and increasing the purity of the culture by committing more cells to oligodendrocytes rather than neuronal identity. Therefore, DBZ was excluded from the final composition. MK2206, MHY1485, FGF2, and PDGF-AA are positive regulators of SOX10 and were included in the final composition because they either positively increased the expression levels of PDGFRa and OLIG1 or had no significant negative effect on the expression of PDGFRa and OLIG1.

[0154] At this point, we investigated the expression profiles of a larger group 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, the expression of these genes was 1900, 660, and 44, respectively. AICAR was identified as a positive regulator of this group of genes (Figure 24).

[0155] Thus, considering the above model, a candidate composition for stage 3 was created consisting of activin A, TTNPB, FGF2, PDGF-AA, MK2206, MHY1485, and AICAR. This composition should be able to maximize cell differentiation associated with stable and elevated expression of SOX10, PDGFRa, and OLIG1. This composition was further verified by immunocytochemical assay (see Example 7).

[0156] Example 6: Factor criticality analysis of culture conditions for inducing stem cell-derived oligodendrocyte precursor cells To assess the effect of deleting each of the validated factors, we again used dynamic profile analysis and compared the expression levels of the gene of interest when one of the final effectors was absent but the others were present. This factor fatality analysis revealed the degree of importance of each input effector, as the expression level of the gene of interest revealed whether the desired outcome was achievable.

[0157] In the Stage 2 formulations of Example 5, one final factor was removed from each model while the other factors were present, and the expression levels of the genes of interest were evaluated compared to when all factors were present. The results are shown in Figures 25A-25B. When FGF-2 was omitted 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 from 15 to 17. We observed a similar trend when AZD3147 was removed, showing that OLIG2 and NKX2-2 levels decreased to 1400 and 1800, respectively, while SOX10 and OLIG1 levels increased slightly to 17 and 100, respectively. When SC79 was omitted from the formulation, NKX2-2, SOX10, and OLIG1 levels decreased to 2800, 7, and 25, respectively. We noted that OLIG2 levels increased from 1600 to 1700.

[0158] In another model, the lethality of including purmorphamine in the formulation was determined by tracking the expression levels of genes of interest in the presence and absence of the compound. When purmorphamine was removed, OLIG1 and OLIG2 levels decreased from 60 to 20 and from 430 to 105, respectively (Figure 25C).

[0159] In the stage 3 formulation, the final factors were omitted one by one while the others were present, and the expression levels of SOX10, PDGFRa, and OLIG1 were evaluated compared to when all factors were present. In one model, in the absence of TTNPB, PDGFRa and OLIG1 levels dramatically decreased from 2600 to 400 and from 210 to 150. When activin A was omitted from the formulation, OLIG1 levels decreased to 120, while PDGFRa levels remained unchanged. In the absence of activin A, we observed an increase in SOX10 expression from 14 to 19, and when TTNPB was removed, a slight change in SOX10 expression from 14 to 15 was observed (Figures 26A-26B).

[0160] In another model, to determine the effect of the combination of MK2206, MHY1485, FGF-2, and PDGF-AA on the gene profile of cells in the presence of other factors in the final composition, the expression levels of OLIG1, PDGFRa, SOX8, and SOX10 were compared to 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, from 70 to 40, and from 30 to 20, respectively. OLIG1 levels remained unchanged. Removal of MK2206 resulted in an increase in OLIG1 levels from 130 to 170, while the expression levels of SOX10 and PDGFRa decreased to 15 and 750, respectively. SOX8 levels remained unchanged. 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 omitted from the formulation, the level of SOX8 decreased to 60, while the other three genes had only minor changes.

[0161] In summary, the factor lethality analysis demonstrated the importance of including each compound in the composition of the Stage 2 and Stage 3 differentiation media described in Example 5.

[0162] Example 7: Immunocytochemical verification of stem cell-derived oligodendrocyte precursor cells expressing SOX10 and PDGFRa To further validate the optimized culture 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, after which standard immunocytochemical assays were used to assess the expression of early oligodendrocyte progenitor biomarkers at the end of stage 2 and late oligodendrocyte progenitor biomarkers at the end of stage 3. To determine the homogeneity of the cultures, 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.

[0163] Immunocytochemistry images of cells at the end of stage 2 (day 6 in culture) confirmed the expression of OLIG2 and NKX2-2, as well as the early expression of SOX10 and PDGFRa in differentiated cells. KI67 expression in the cells indicated that the majority of cells were still proliferating, as expected at the progenitor stage (Figure 28). TUBB3 was also detected in less than 50% of the cells, indicating that the cultures were not 100% glial.

[0164] Immunocytochemical assays of hiPSC-derived cells at the end of stage 3 (day 12 in culture) confirmed the expression of SOX10 and PDGFRa in differentiated cells (Figure 29). The cells were able to maintain expression of OLIG2, and we also detected NG2 (CSPG4) and A2B5 in a portion of the cells.

[0165] By the end of stage 3 differentiation, SOX10 and PDGFRa were detected in differentiated cells, confirming the stable and highly converting ability of the staged composition described in Example 5 to differentiate human induced pluripotent stem cells into oligodendrocyte precursor cells after 12 days in vitro.

[0166] Example 8: Development of a culture protocol to generate stem cell-derived oligodendrocyte precursor cells expressing SOX10, OLIG2, and NKX2-2 In this example, a stepwise differentiation protocol for generating oligodendrocyte progenitor cells, alternative to that described in Example 5, was developed that can induce human pluripotent stem cells into progenitor cells that express SOX10, NKX2-2, and OLIG2 after 12 days in culture. The differentiated cells also express other markers of oligodendrocyte progenitor cells, 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.

[0167] For the protocol, cells are treated with Stage 1 pre-OPC medium (described in Example 1) for 3 days, then cultured in Stage 2 medium for 6 days, followed by culture in Stage 3 medium for 3 days. The cells can then be further differentiated into mature oligodendrocytes. This entire three-stage protocol is shown diagrammatically in Figure 43.

[0168] To develop a formulation for oligodendrocyte differentiation, the effects of various agonists and antagonists (effectors) on pre-OPC differentiation were investigated using HD-DoE. These effectors were selected based on the then-available literature on developmental biology and stem cell differentiation, as well as single-cell RNA sequencing data from mouse and human oligodendrocytes.

[0169] These experiments led to the development of compositions for Stage 2 shown in Table 3 below and compositions for Stage 3 shown in Table 4 below.

[0170] Table 3: Verified factors in the Stage 2 composition TIFF2025526018000003.tif36129

[0171] Table 4: Verified factors in the Stage 3 composition TIFF2025526018000004.tif28129

[0172] To design compositions for stage 2 differentiation, cells were first cultured in the stage 1 medium described in Example 1, and then 48 or 96 different combinations of effectors were systematically prepared using D-optimization experimental design compression. Effector combinations were prepared in basal medium and then added to cells, allowing the cells to subsequently differentiate. After three days, RNA extraction was performed, and gene expression was obtained using quantitative PCR analysis. Data were normalized and modeled using partial least squares regression analysis of the effector design, resulting in gene-specific models that, after being adjusted for maximum predictive power, provided descriptions of the effectors' ability to combinatorially and individually regulate the expression of individual genes. Solutions within the tested space could then be investigated for desirability.

[0173] At this point, due to the short duration of culture (only 6 days in vitro), we decided to focus on initiating expression of SOX10, OLIG1, and PDGFRa while maximally expressing OLIG2 and maintaining high expression of NKX2-2 to direct 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 eight-factor model, the individual and combined effects of the following agents on further cell differentiation were tested: CHIR99021 (WNT pathway agonist), AGN193109 (RA pathway antagonist), FGF-2 (FGFR agonist), purmorphamine (SHH pathway agonist), AICAR (AMPK agonist), GW3965 (LXR pathway agonist), GW590735 (PPAR-α agonist), and AZD3147 (mTOR antagonist). This model demonstrated the ability to modulate pre-OPCs to overexpress OLIG2, OLIG1, NKX2-2, and PDGFRa. When the model was optimized for maximum OLIG2 expression at 3325, multiple factors showed positive regulatory effects, including AZD3147, with the largest factor contribution of 30.2, and CHIR99021, purmorphamine, FGF-2, and GW590735, with factor contributions of 17.96, 7.39, 5.2, and 5.4, respectively (Figure 30). AGN193109 had the largest negative effect on OLIG2 expression, with a factor contribution of 25.8. Within the specification to achieve 80% maximum expression of OLIG2, this complex media composition had a Cpk value (process capability index) of 0.58, corresponding to a 4% risk of failure.

[0174] This model was also optimized for maximum OLIG1 expression, which was 454.7, and we observed that, similar to the optimization of OLIG2, AZD3147 had the most positive effect on OLIG1 regulation, with a factor contribution of 26.7, and AGN193109 had the most negative effect, with a factor contribution of 25.3 (Figure 31). Other factors with positive effects included CHIR99021, FGF-2, and GW590735, with factor contributions of 22.8, 9.9, and 3.1, respectively. Within the specification to achieve 80% maximum expression of OLIG1, this complex media composition had a Cpk value (process capability index) of 0.42, which corresponded to a risk of failure of 9.9%.

[0175] Dynamic profile analysis was used to assess the expression levels of other genes of interest, such as NKX2-2 and SOX10, when the model was maximized for OLIG2. When the model was optimized, the expression levels were 2000 for NKX2-2, 440 for OLIG1, and 15 for SOX10 (Figure 32). This analysis showed that four of the five compounds, including FGF-2, purmorphamine, CHIR99021, and AZD3147, had a positive effect on OLIG2 expression, and these also had a positive effect on NKX2-2 expression, while FGF-2, CHIR99021, AZD3147, and GW590735 had a positive effect on OLIG1 expression. Although purmorphamine did not significantly improve Olig1 expression, it was a significant positive regulator of both Olig2 and NKX2-2, target genes in stage 2. Therefore, purmorphamine was included as a candidate for the differentiation medium composition for stage 2 as a common positive regulator of these two genes. Because GW590735 has a negative effect on the expression of SOX10 and NKX2-2, we decided to exclude it from the final composition.

[0176] Thus, considering the above model, a candidate composition for stage 2 was created consisting of FGF2, purmorphamine, CHIR99021, and AZD3147. This composition should be able to maximize cell differentiation with stable and elevated expression of OLIG2, NKX2-2, and early expression of SOX10. This composition was further validated by immunocytochemical assay (see Example 9).

[0177] To further differentiate cells toward their oligodendrocyte progenitor identity, additional HD-DoE experiments were performed on cells cultured in Stage 1 and Stage 2 differentiation media. After 3 days, gene expression in cells in various combinations was examined. We focused on maximal expression of oligodendrocyte progenitor signature genes, such as SOX10, OLIG1, and PDGFRa (Goldman and Kuypers (2015) Development 142:3983-95; Marques et al. (2016) Science 352:1326-1329). In one eight-factor model, the individual and combined effects of the following were tested to induce cells toward a SOX10 / PDGFRa-positive population: TTNPB (RA agonist), CHIR99021, FGF-2, IGF-1, AGN193109, purmorphamine, MHY1485 (mTOR pathway agonist), and SC79 (AKT pathway agonist). When optimized for SOX10, which has a β-kappa factor of 41.04, TTNPB and FGF-2 exhibited the most positive effects, with factor contributions of 17.98 and 15.3, respectively. CHIR99021 also had a positive effect, with a factor contribution of 8.1 (Figure 33). SC79 had the most negative effect, with a factor contribution of 30.2. Within the specifications to achieve 80% maximal expression of SOX10, this complex media composition had a Cpk value (process capability index) of 0.47, which corresponded to a risk of failure of 7.3%.

[0178] When the model was optimized for maximum expression of PDGFRa, which was 853.4, purmorphamine had the greatest positive effect, 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 of less than 1 (Figure 34). Within the specification to achieve 80% maximum expression of PDGFRa, this complex media composition had a Cpk value (process capability index) of 0.45, which corresponded to a risk of failure of 8.6%.

[0179] According to literature, proteins such as FGF-2, IGF-1, PDGF-AA, and NT-3 play important roles in oligodendrocyte differentiation (Goldman and Kuypers (2015) Development 142:3983-95). In this first model, FGF-2 only had a positive regulatory effect on the expression level of SOX10, and IGF-1 also had a similar effect on PDGF-Ra. Therefore, to further understand the effects of IGF-1 and FGF-2 on cell differentiation and confirm their positive role, we included these factors in the other two models and optimized the models for maximum SOX10 expression. This gene, along with OLIG2 and NKX2-2, is one of the master transcription factors for oligodendrocytes, demonstrating its ability to commit stem cells to oligodendrocytes in reprogramming efforts (Wang et al. (2014) Proc Natl Acad Sci USA 111:E2885-94; Garcia-Leon 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 (Figure 35). AGN193109 also demonstrated a positive effect on increasing the level of SOX10, with a factor contribution of 16.

[0180] Because TTNPB positively regulated one of the OPC genes in the previous model and 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 (Figure 36). In this analysis, the highest and lowest expression levels are shown in a spectrum from red to blue, and each of the four axes represents one compound. OLIG1 expression was maximized at the lowest concentration of TTNPB and the highest concentrations of AGN193109 and FGF-2, and IGF-1, although it demonstrated a weaker effect on OLIG1 expression, still positively regulated OLIG1 levels.

[0181] To confirm the effects of TTNPB, FGF-2, and IGF-1 on a larger scale, we examined the expression profiles of a larger group of genes in the presence of IGF-1, FGF-2, or TTNPB: 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. Figure 37 shows a factor plot of various genes in the presence of IGF-1. With increasing concentrations of IGF-1, we observed increased expression levels of OPC genes, while decreased expression levels of neuronal genes. This supports the positive effect of IGF-1 on committing cells to an oligodendrocyte fate rather than a neuronal fate. Figure 38 shows the same analysis for FGF-2. We observed that the presence of FGF-2 increased expression levels of OPC genes, while decreased expression levels of neuronal genes. However, in the presence of TTNPB, the predicted behavior of the genes changed, and opposing trends were observed in the plot, demonstrating a negative effect of TTNPB on OPC genes and a positive effect on neuronal genes (Figure 39).

[0182] Thus, considering the above model, a candidate composition for stage 3 consisting of FGF-2, IGF-1, and AGN193109 was generated. This composition could maximize cell differentiation toward stable and elevated expression levels of SOX10, PDGFRa, and OLIG1. This composition was further validated by immunocytochemical assays (see Example 9).

[0183] Example 9: Immunocytochemical verification of stem cell-derived oligodendrocyte precursor cells expressing SOX10 and PDGFRa 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, after which standard immunocytochemical assays were used to assess the expression of early oligodendrocyte progenitor biomarkers at the end of stage 2 and late oligodendrocyte progenitor biomarkers at the end of stage 3. To determine the homogeneity of the cultures, 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).

[0184] Immunocytochemistry images of cells at the end of stage 2 (day 9 in culture) confirmed the expression of OLIG2, NKX2-2, and SOX10, as well as early expression of PDGFRa in differentiated cells (Figure 40). Expression of KI67 in the cells indicated that the majority of cells were still proliferating, as expected at the progenitor stage.

[0185] Immunocytochemical assays 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 early expression of O4 was observed in some cells in the culture (Figure 41). TUBB3 was also detected in less than half of the cultures, indicating heterogeneity in the cultures.

[0186] By the end of stage 3 differentiation, expression of SOX10 and PDGFRa was detected in differentiated cells, confirming the stable and highly converting ability of the staged composition described in Example 8 to differentiate human induced pluripotent stem cells into oligodendrocyte precursor cells after 12 days in vitro.

[0187] Example 10: Development of a culture protocol to generate premyelinating oligodendrocytes derived from stem cells that express CNP, CD9, and O4 In this example, a culture protocol for generating premyelinating oligodendrocytes from pre-OPCs, referred to herein as the Stage 4 protocol, has been developed. As described in the previous examples, a stepwise, three-stage differentiation protocol has been developed that induces human pluripotent stem cells into progenitor cells expressing SOX10, NKX2-2, and OLIG2 after 12 days in culture. The differentiated cells also express other markers of oligodendrocyte progenitors, including PDGFRa and NG2. The cells can be further matured into premyelinating oligodendrocytes expressing CNP, CD9, and O4 after 6 days in culture in the Stage 4 differentiation medium described herein. Early expression of the myelinating marker MBP is also observed in the cell population.

[0188] To develop a stage 4 formulation for oligodendrocyte differentiation, the effects of various agonists and antagonists (effectors) on pre-OPC differentiation were investigated using the HD-DoE method described in the Examples above. These effectors were selected based on the then-available literature on developmental biology and stem cell differentiation, as well as single-cell RNA sequencing data from mouse and human oligodendrocytes.

[0189] These experiments led to the development of the composition for Stage 4 shown below in Table 5. A representative schematic of the Stage 4 culture protocol is shown in Figure 44.

[0190] Table 5: Verified factors in the Stage 4 composition TIFF2025526018000005.tif41128

[0191] To design compositions for stage 4 differentiation, 48 or 96 different combinations of effectors were systematically prepared using D-optimization experimental design compression. Effector combinations were prepared in basal medium and then added to cells, which were treated with stage 1, stage 2, and stage 3 differentiation media to generate OPCs as described in the previous examples. The resulting OPCs were then replated in 96-well plates. After 3–7 days, RNA extraction was performed, and gene expression was obtained using quantitative PCR analysis. Data were normalized and modeled using partial least squares regression analysis of the effector design to generate gene-specific models that, after adjusting for maximum Q2 (i.e., maximum predictive power), provided a description of the effectors' ability to combinatorially and individually regulate the expression of individual genes. Solutions within the tested space could then be investigated for desirability.

[0192] To induce differentiation of OPCs into oligodendrocytes, we focused on the expression of postmitotic genes, such as CNP and PLP1 (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, purmorphamine, cAMP, and 2-phospho-ascorbic acid on the gene profile of differentiating cells were examined. When optimized for maximum expression of CNP at 1339, multiple inputs showed positive regulatory effects on CNP expression (Figure 45), including: NT-3 with a factor contribution of 24, T3 with a factor contribution of 18, purmorphamine 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 of less than 1. Biotin and ascorbic acid were the only factors with negative effects, with factor contributions of 12 and 17. Within the specifications to achieve 80% maximum expression of CNP, this complex media composition had a Cpk value (process capability index) of 0.43, which corresponded to a risk of failure of 9%.

[0193] Based on previous results, the effects of the eight tested factor combinations were investigated along with four additional factors predicted to promote oligodendrocyte differentiation (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, purmorphamine, biotin, ascorbic acid, Albumax, cAMP, propionate, AICAR, and gamma-secretase inhibitor-XX (GSI-XX), a Notch pathway inhibitor, on OPC differentiation were tested after 6 days in culture. When optimized for CNP at 1790, NT-3, IGF-1, insulin, and GSI-XX had positive effects on maximizing CNP expression, with factor contributions of 13, 5, 3, and 7, respectively (Figure 46). Biotin and cAMP had the largest negative effects on maximizing CNP expression, both with factor contributions of 16. T3 also showed a negative effect; however, its factor contribution was relatively low, at 1.2. Within the specifications for achieving 80% maximal expression of CNP, this complex media composition had a Cpk value (process capability index) of 0.46, corresponding to a risk of failure of 8.3%.

[0194] When the model was optimized for CNP expression, 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 (Figure 47). This gene profile demonstrated the ability of this formulation to stably differentiate cells toward an oligodendroglial fate.

[0195] Since the expression level of PLP1 was above 2000, the optimization conditions for this marker, which was finally expressed in the model, were investigated. Under the optimized conditions, the expression level of PLP1 increased to 3600, with only two positive regulators: NT-3 and insulin. Insulin had the largest factor contribution of 13, and NT-3 had 7 (Figure 48). AICAR, Albumax, and Purmorphamine had the largest negative effects, with factor contributions of 12, 11, and 10, respectively. Within the specifications for achieving 80% maximum expression of PLP1, this complex medium composition had a Cpk value (process capability index) of 0.77, which corresponded to a risk of failure of 1.2%.

[0196] Because GSI-XX and IGF-1 had opposing effects on CNP and PLP1, dynamic profiling analysis was used to further investigate the effects of GSI-XX and IGF-1 on additional genes in the model (Figure 49). GSI-XX was observed to positively regulate the expression of neuronal genes, such as NeuroG1, NeuroG2, and NeuroD1, as well as the expression of CNP. Therefore, to minimize culture heterogeneity, it was decided to remove GSI-XX from the final composition. Because both HD-DoE experiments showed that IGF-1 had a positive regulatory effect on CNP, it was decided to add IGF-1 to the final composition.

[0197] The results of the previous HD-DoE experiment indicated that T3 had a positive regulatory effect on the expression of CNP, with a factor contribution of 18. Therefore, the effect of T3 on other genes in this model was further investigated. This model showed that T3 could upregulate other OL genes, such as PLP1, PTGDS, KLK6, and MBP, while downregulating the neuronal gene NeuroG1 (Figure 50). This observation led to the inclusion of T3 in the final stage 4 composition.

[0198] Finally, PDGF-AA was added to the final stage 4 composition without further HD-DoE modeling, as its ability to promote oligodendrocyte differentiation has been established in the literature.

[0199] Thus, a composition for stage 4 differentiation was created having the components shown in Table 5 above. This composition can maximize cell differentiation with stable and elevated expression of CNP and O4 and early expression of PLP1 and MBP. This composition was validated by immunocytochemical assays as described in Example 11.

[0200] Example 11: Immunocytochemical validation of a culture protocol for stem cell-derived premyelinating oligodendrocytes expressing CNP and O4 To validate the Stage 4 composition 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 in Stage 4 composition for 6 days. At this time, immunocytochemistry assays were used to assess the expression of late oligodendrocyte precursor biomarkers and early oligodendrocyte biomarkers in differentiated cells. To determine the homogeneity of the cultures, biomarkers included OPC-specific markers such as OLIG2, NKX2-2, PDGFR, and A2B5, oligodendrocyte markers including CNP, O4, MBP, and PLP1, as well as the pan-neuronal marker TUBB3.

[0201] Immunocytochemistry images of cells at the end of stage 4 (day 18 in culture) confirmed the expression of OLIG2, NKX2-2, CNP, and O4, as well as early expression of MBP and PLP1 in differentiated cells (Figure 51). Expression of KI67 and PDGFRa in a subset of cells indicates that some cells were still proliferating. TUBB3 was detected in less than 50% of cells, indicating that the cultures were not 100% glial.

[0202] CD9 expression levels were also measured using flow cytometry at the end of stage 4. CD9 is a surface marker expressed on committed oligodendrocyte precursor cells and premyelinating oligodendrocytes (Goldman and Kuypers (2015) Development 142:3983-95). As shown in Figure 52, 81.5% of the cells were CD9 positive, indicating that the majority of the cultures were progressing toward an oligodendrocyte fate.

[0203] By the end of stage 4 differentiation, CNP, CD9, and O4 were detected in differentiated cells, confirming the stable and highly converting capacity of the four-stage composition described in the Examples to differentiate human induced pluripotent stem cells into premyelinating oligodendrocytes after 18 days in vitro.

[0204] equivalent 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, and it is intended that the appended claims encompass such equivalents.

Claims

1. A method for producing CD9+A2B5+O4+CNPase+ pre-myelin-forming oligodendrocytes (preOL), This step involves culturing SOX10+ OLIG2+ NKX2-2+ oligodendrocyte progenitor cells (OPCs) in a culture medium containing an insulin-like growth factor 1 receptor (IGF1R) pathway agonist, a tropomyosin-related kinase C (TrkC) pathway agonist, a platelet-derived growth factor receptor (PDGFR) pathway agonist, a thyroid hormone receptor agonist, and an insulin receptor agonist, in order to produce CD9+A2B5+O4+CNPase+ pre-OL. The method, including the method described above.

2. The method according to claim 1, wherein the OPC is cultured in a culture medium for at least 132 hours.

3. (a) The IGF1R pathway agonist is selected from the group consisting of IGF-1, IGF-2, insulin, Rg5, IGF-1 30-41, demethylasteriquinone B1, IGF1-Ado, X10, mecasermin, and combinations thereof; (b) The TrkC pathway agonist is selected from the group consisting of neurotrophin-3 (NT-3), NT-3 β-turn-based peptidomimetic, LM22B 10, GNF 5837, and combinations thereof; (c) The PDGFR pathway agonist is PDGF-AA; (d) The thyroid hormone receptor agonist is selected from the group consisting of T3, T4, resmethylome, TRb agonist 3 (compound 3), sovethyrom, tilatrichol, and combinations thereof; and / or (e) The insulin receptor agonist is selected from the group consisting of insulin, IGF-1, IGF-2, demethylasteriquinone B1, MK-5160, MK-1092, and combinations thereof. The method according to claim 1.

4. (a) An IGF1R pathway agonist is present in the culture medium at a concentration of 2 to 20 ng / ml; (b) The TrkC pathway agonist is present in the culture medium at a concentration of 2–20 ng / ml; (c) The PDGFR pathway agonist is present in the culture medium at a concentration of 2–20 ng / ml; (d) A thyroid hormone receptor agonist is present in the culture medium at a concentration of 10–100 nM; and / or (e) The insulin receptor agonist is present in the culture medium at a concentration of 4–40 μg / ml. The method according to claim 3.

5. A method for preparing CD9+A2B5+O4+CNPase+PreOL, (a) To obtain a certain cell population, OLIG2+ human preoligodendrocyte progenitor cells (preOPCs) are cultured for at least 60 hours in a culture medium containing a fibroblast growth factor receptor (FGFR) pathway agonist, a mammalian target of rapamycin (mTOR) pathway antagonist, a sonic hedgehog (SHH) pathway agonist, an Akt pathway antagonist, and an Akt pathway agonist; (b) A step of culturing the cell population from step (a) for at least 132 hours in a culture medium containing an FGFR pathway agonist, a PDGFR pathway agonist, an Akt pathway antagonist, and a retinoic acid (RA) pathway agonist so that SOX10+ OLIG2+ NKX2-2+ OPCs are produced; and (c) The cell population from step (b) is cultured for at least 132 hours in a culture medium containing an IGF1R pathway agonist, a TrkC pathway agonist, a PDGFR pathway agonist, a thyroid hormone receptor agonist, and an insulin receptor agonist, so that CD9+A2B5+O4+CNPase+ pre-OL is produced. The method, including the method described above.

6. The method according to claim 5, wherein human pluripotent stem cells are cultured for at least 60 hours in a culture medium containing an RA pathway agonist, an Akt pathway agonist, an mTOR pathway agonist, a Wingless-related integration site (WNT) pathway antagonist, an SHH pathway agonist, a bone morphogenetic protein (BMP) pathway antagonist, and a protein kinase C (PKC) pathway antagonist to obtain OLIG2+ human pre-OPC of step (a).

7. (a) the human pluripotent stem cells are induced pluripotent stem cells (iPSCs) or embryonic stem cells; and / or (b) Human pluripotent stem cells adhere to a plate coated with vitronectin during culture. The method according to claim 6.

8. (a) The FGFR pathway agonist is FGF2, SUN11602, or a combination thereof; (b) The mTOR pathway antagonist is selected from the group consisting of AZD 3147, dactricib, rapamycin, everolimus, AZD 8055, temsirolimus, PI-103, NU7441, BC-LI-0186, eCF 309, ETP 45658, niclosamide, omiparisib, PF 04691502, PF 05212384, Trin-1, Trin-2, WYE 687, XL 388, STK16-IN-1, PP 242, tolkinib, ridahorolimus, sapanicertib, voxtalisib, and combinations thereof; (c) The SHH pathway agonist is selected from the group consisting of purmorphamine, GSA 10, SHH, SAG, and combinations thereof; (d) Akt pathway antagonists include MK2206, GSK690693, Perifosin (KRX-0401), Ipatasertib (GDC-0068), Capivasertib (AZD5363), PF-04691502, AT 7867, Trisirivine (NSC154020), ARQ751, Miransertib (ab235550), Borussertib, Cerisertib, Akti1 / 2, CCT128930, A 674563, PHT 427, Miltefosine, AT 13148, ML 9, BAY 1125976, Oridonin, TIC10, Pectolinalin, Akti IV, 10-DEBC, API-1, SC 66, FPA Selected from the group consisting of 124, API-2, urolithin A, and combinations thereof; (e) The Akt pathway agonist is selected from the group consisting of Sc79, demethylcocrawrine, LM22B-10, YS-49, YS-49 monohydrate, demethylasteriquinone B1, resilicib, N-oleoylglycine, NSC45586 sodium, periprosin, CHPG sodium salt, bilovalide, 6-hydroxyflavone, musk ketone, SEW2871, 8-prenylnaringenin, razuprotafib, and combinations thereof; (f) The PDGFR pathway agonist is PDGF-AA; and / or (g) 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, isotretinoin, tretinoin, tamibarotene, ATRA, AC 261066, AC 55649, retinoic acid (RA), Sr11237, adapalene, EC23, 9-cisretinoic acid, 13-cisretinoic acid, 4-oxoletinoic acid, and all-trans retinoic acid (ATRA), and combinations thereof. The method according to claim 5.

9. (a) An FGFR pathway agonist is present in the culture medium at a concentration of 1 to 20 ng / ml; (b) mTOR pathway antagonists are present in the culture medium at concentrations of 5–200 nM; (c) SHH pathway agonists are present in the culture medium at concentrations of 100–1000 nM; (d) Akt pathway antagonists are present in the culture medium at concentrations of 25–300 nM; (e) The Akt pathway agonist is present in the culture medium at a concentration of 0.1–10 μM; (f) The PDGFR pathway agonist is present in the culture medium at a concentration of 2–20 ng / ml; and / or (g) The RA pathway agonist is present in the culture medium at a concentration of 10–100 nM. The method according to claim 8.

10. (a) comprising an FGFR pathway agonist, an mTOR pathway antagonist, an SHH pathway agonist, an Akt pathway antagonist, and an Akt pathway agonist; or (b) Including FGFR pathway agonists, PDGFR pathway agonists, Akt pathway antagonists, and RA pathway agonists, Culture medium for obtaining OPC.

11. Culture medium for obtaining CD9+A2B5+O4+CNPase+PreOL, containing IGF1R pathway agonists, TrkC pathway agonists, PDGFR pathway agonists, thyroid hormone receptor agonists, and insulin receptor agonists.

12. A method for producing CD9+A2B5+O4+CNPase+Human Preol, (a) To obtain a certain cell population, OLIG2+ human pre-OPCs are cultured for at least 132 hours in a culture medium containing an FGFR pathway agonist, an mTOR pathway antagonist, an SHH pathway agonist, and a WNT pathway agonist; and (b) A step of culturing the cell population from step (a) for at least 60 hours in a culture medium containing an FGFR pathway agonist, an IGF-1 pathway agonist, and an RA pathway agonist so that SOX10+ OLIG2+ NKX2-2+ OPCs are produced; and (c) The cell population from step (b) is cultured for at least 132 hours in a culture medium containing an IGF1R pathway agonist, a TrkC pathway agonist, a PDGFR pathway agonist, a thyroid hormone receptor agonist, and an insulin receptor agonist, so that CD9+A2B5+O4+CNPase+ pre-OL is produced. The method, including the method described above.

13. The method according to claim 12, wherein human pluripotent stem cells are cultured for at least 60 hours in a culture medium containing an 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 to obtain OLIG2+ human pre-OPC in step (a).

14. (a) the human pluripotent stem cell is an iPSC or an embryonic stem cell; and / or (b) Human pluripotent stem cells adhere to a plate coated with vitronectin during culture. The method according to claim 13.

15. (a) The FGFR pathway agonist is FGF2, SUN11602, or a combination thereof; (b) The mTOR pathway antagonist is selected from the group consisting of AZD 3147, dactricib, rapamycin, everolimus, AZD 8055, temsirolimus, PI-103, NU7441, BC-LI-0186, eCF 309, ETP 45658, niclosamide, omiparisib, PF 04691502, PF 05212384, Trin-1, Trin-2, WYE 687, XL 388, STK16-IN-1, PP 242, tolkinib, ridahorolimus, sapanicertib, voxtalisib, and combinations thereof; (c) The SHH pathway agonist is selected from the group consisting of purmorphamine, GSA 10, SHH, SAG, and combinations thereof; (d) 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-acetoxime, AZD1080, WNT3A, Alsterpaulon, Indirubin-3-oxime, 1-Azakenpaulon, Kenpaulon, TC-G 24, TDZD 8, TWS 119, NP 031112, AT 7519, KY 19382, AZD2858, and combinations thereof; (e) The IGF-1 pathway agonist is selected from the group consisting of IGF-1, IGF-2, insulin, Rg5, IGF-1 30-41, demethylasteriquinone B1, IGF1-Ado, X10, mecasermin, and combinations thereof; and / or (f) The RA pathway antagonist is selected from the group consisting of AGN193109, BMS 195614, CD 2665, ER 50891, LE 135, LY 2955303, MM11253, and combinations thereof. The method according to claim 12.

16. (a) An FGFR pathway agonist is present in the culture medium at a concentration of 1 to 20 ng / ml; (b) mTOR pathway antagonists are present in the culture medium at concentrations of 5–200 nM; (c) SHH pathway agonists are present in the culture medium at concentrations of 100–1000 nM; (d) The WNT pathway agonist is present in the culture medium at a concentration of 0.3–3.0 μM; (e) IGF-1 pathway agonists are present in the culture medium at a concentration of 2–20 ng / ml; and / or (f) The RA pathway antagonist is present in the culture medium at a concentration of 10–100 nM. The method according to claim 15.

17. (a) including FGFR pathway agonists, mTOR pathway antagonists, SHH pathway agonists, and WNT pathway agonists; or (b) Including FGFR pathway agonists, IGF-1 pathway agonists, and RA pathway agonists, Culture medium for obtaining OPC.

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