Methods and compositions for generating oligodendrocyte precursor cells - Patents.com

Abstract

Methods for generating pre-oligodendrocyte precursor cells (pre-OPCs) and oligodendrocyte progenitor cells (OPCs) from human pluripotent stem cells are provided using chemically defined culture media that allow for the generation of pre-OPCs and OPCs in as few as three days. Culture media, isolated cell populations, and kits are also provided.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 63 / 168,065, filed March 30, 2021, the entire contents of which are incorporated herein by reference.

[0002] Government Licensed Rights This invention was made with Government support under Grant No. W911NF-17-3-0003 awarded by the US ARMY ACC-AGP-RTP. The United States Government has certain rights in the invention. [Background technology]

[0003] 2. Background of the Invention Oligodendrocytes (OLs) are a type of glial cell that synthesize the myelin sheath around axons. As such, they are crucial for nerve conduction in the central nervous system (CNS). A better understanding of oligodendrocyte biology will be crucial for developing treatments for neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), which may involve demyelination later in the disease process, as well as demyelination diseases such as multiple sclerosis and leukodystrophy. Furthermore, radiation therapy to the brain is associated with the side effect of oligodendrocyte depletion, which may lead to cognitive decline and / or impaired motor coordination.

[0004] Since mature human oligodendrocytes cannot be easily isolated from human subjects, human oligodendrocyte cell lines have been developed to allow the study of said cells. However, immortalized cell lines may not fully mimic the biology of natural cells and are not suitable for therapeutic use. Therefore, the ability to generate human oligodendrocytes in vitro, such as from stem cells, is highly desirable. Various protocols have been reported for the differentiation of oligodendrocytes from human pluripotent stem cells. However, these protocols remain inefficient and variable in terms of oligodendrocyte yield, and require very long differentiation periods to generate myelin basic protein (MBP) positive oligodendrocytes.

[0005] An early protocol used a four-step process (see also Hu et al. (2009) Nature Protocols 4:1614-1622; Wang et al. (2013) Cell Stem Cell 12:252-264). In this protocol, human embryonic stem cells (hESCs) were first induced to differentiate into neuroepithelial cells for 2 weeks to form neural tube-like rosettes, and then treated with retinoic acid (RA) and sonic hedgehog (SHH) for 10 days to induce progenitor cells expressing OLIG2. An additional 10 days of treatment with fibroblast growth factor (FGF2) led to conversion to pre-OPCs expressing OLIG2 and NKX2.2. Finally, the pre-OPCs were cultured in the absence of FGF2 for an additional 8-9 weeks to differentiate into OPCs expressing markers such as platelet-derived growth factor receptor alpha (PDGFRα), SOX10, and NG2. Thus, using this protocol, it took about 24 days to give rise to progenitor cells expressing OLIG2, about 34 days to give rise to pre-OPCs expressing OLIG2 and NKX2.2, and about 100 days to obtain mature OLs. A variation of this protocol was reported by Douvaras et al. (Stem Cell Reports (2014) 3:250-259), but it still required about 20 days to obtain pre-OPCs and about 50 days to obtain OPCs, and included culture with exogenously added growth factors PDGF, IGF-1, and HGF.

[0006] Alternative protocols have since been reported, but these still utilized a neural induction and patterning phase (also called neuralization) lasting approximately one week, followed by induction of cells expressing pre-OPC and OPC markers using medium 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] Recently, a protocol has been reported that first generates neural progenitor cells (NPCs) by neural induction of hESCs, followed by overexpression of SOX10 transcription factor in NPCs (by viral transduction) and proliferation in the presence of bFGF to generate MBP-positive oligodendrocytes in only about 20 days (Garcia-Leon et al. (2018) Stem Cell Reports 10:655-672 (Non-Patent Document 8)). Furthermore, it was found that transient and partial inhibition of SHH pathway transcription factor GLI1 in neural stem cells (generated by neurotization) by the small molecule inhibitor GANT61 generates OPCs that are more migratory and can differentiate earlier toward myelin-producing oligodendrocytes (Namchaiw et al. (2019) Stem Cell Res & Therapy 10:272 (Non-Patent Document 9)).

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

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[0009]

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Summary of the Invention

[0010] The present disclosure provides a method for generating human pre-OPCs and OPCs from pluripotent stem cells using a chemically defined culture medium that allows for the generation of OLIG2 and NKX2.2 positive pre-OPCs and OPCs in as little as three days of culture. The culture medium lacks serum and other exogenously added growth factors and contains small molecule agents that agonize or antagonize the activity of specific signaling pathways in pluripotent stem cells, thereby promoting differentiation along the OPC lineage leading to cell maturation and expression of OPC-associated biomarkers. The disclosed method has the advantage of allowing direct differentiation of pluripotent stem cells into pre-OPCs and OPCs, avoiding the neural induction step of prior art protocols, thereby significantly reducing the time required for the generation of pre-OPCs and OPCs. Furthermore, the use of small molecule agents in the culture medium allows for precise control of the culture components.

[0011] Thus, in one aspect, the present disclosure relates to a method of generating human pre-oligodendrocyte precursor cells (pre-OPCs) or oligodendrocyte precursor cells (OPCs), the method comprising the steps of: Culturing human pluripotent stem cells in a culture medium lacking exogenously added growth factors and containing a retinoic acid (RA) pathway agonist, an Akt pathway agonist and an mTOR pathway agonist, such that pre-OPCs or OPCs expressing OLIG2 are generated.

[0012] In one embodiment, the OLIG2-expressing pre-OPCs or OPCs are generated within 72 hours of initiating culture of human pluripotent stem cells in said culture medium. In one embodiment, the pre-OPCs or OPCs also express NKX2-2.

[0013] In another embodiment, the culture medium further comprises a WNT pathway antagonist. In another embodiment, the culture medium further comprises a SHH pathway agonist. In another embodiment, the culture medium further comprises a BMP pathway antagonist. In another embodiment, the culture medium further comprises a PKC pathway antagonist. In various embodiments, the pre-OPC or OPC also express OTX2 and / or FEZF2.

[0014] Thus, in another aspect, the present disclosure relates to a method of generating human pre-oligodendrocyte precursor cells (pre-OPCs) or oligodendrocyte precursor cells (OPCs), the method comprising the steps of: Culturing human pluripotent stem cells in a culture medium lacking exogenously added growth factors and 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, such that pre-OPCs or OPCs expressing OLIG2 are generated. In other embodiments, the pre-OPCs or OPCs also express NKX2-2, OTX2 and / or FEZF2.

[0015] Non-limiting examples of suitable agonist and antagonist agents, as well as their concentrations, are described in further detail herein.

[0016] 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.

[0017] In one embodiment, human pluripotent stem cells are attached to vitronectin-coated plates throughout the culture.

[0018] In another aspect, the present disclosure relates to a culture medium for obtaining pre-oligodendrocyte precursor cells (pre-OPCs) or oligodendrocyte precursor cells (OPCs), the culture medium comprises a retinoic acid (RA) pathway agonist, an Akt pathway agonist and an mTOR pathway agonist, and lacks exogenously added growth factors.In one aspect, the culture medium further comprises a WNT pathway antagonist.In one aspect, the culture medium further comprises a SHH pathway agonist.In one aspect, the culture medium further comprises a BMP pathway antagonist.In one aspect, the culture medium further comprises a PKC pathway antagonist.

[0019] In another aspect, the present disclosure relates to an isolated cell culture of pre-oligodendrocyte precursor cells (pre-OPCs) or oligodendrocyte precursor cells (OPCs), comprising pre-OPCs or OPCs expressing OLIG2, cultured in a culture medium that contains a retinoic acid (RA) pathway agonist, an Akt pathway agonist and an mTOR pathway agonist, and lacks exogenously added growth factors. In one embodiment, the culture medium further comprises a WNT pathway antagonist. In one embodiment, the culture medium further comprises a 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 other embodiments, the pre-OPCs or OPCs also express NKX2-2, OTX2 and / or FEZF2. In one embodiment of the cell culture, the pre-OPCs or OPCs are attached to a plate coated with vitronectin.

[0020] In another aspect, the present disclosure relates to a pre-oligodendrocyte precursor cell (pre-OPC) or oligodendrocyte precursor cell (OPC), for example, produced by the method of the present disclosure. In one aspect, the present disclosure provides a composition comprising a non-natural pre-oligodendrocyte precursor cell (pre-OPC) or oligodendrocyte precursor cell (OPC), wherein the pre-OPC or OPC expresses OLIG2, NKX2-2, OTX2 and FEZF2, and lacks expression of NESTIN.

[0021] In yet another aspect, the present disclosure provides a method for the preparation of a medicament comprising administering to a subject a medicament comprising administering to said subject a medicament ... 6The present invention relates to an isolated cell population of pre-OPCs or OPCs, comprising pre-oligodendrocyte precursor cells (pre-OPCs) or oligodendrocyte precursor cells (OPCs) that express OLIG2, the cell population lacking neural stem cells that express NESTIN. In other embodiments, the pre-OPCs or OPCs also express NKX2-2, OTX2 and / or FEZF2. In one embodiment of the isolated cell population, the pre-OPCs or OPCs are bound to at least one antibody that binds to at least one cell surface marker expressed by the pre-OPCs or OPCs.

[0022] In yet another aspect, the present disclosure relates to a method of isolating pre-oligodendrocyte precursor cells (pre-OPCs) or oligodendrocyte precursor cells (OPCs), the method comprising the steps of: contacting the OLIG2-expressing pre-OPCs or OPCs produced by the methods of the disclosure with at least one binding agent that binds to a cell surface marker expressed by the OLIG2-expressing pre-OPCs or OPCs; and Isolating cells that bind to the binding agent, thereby isolating OPCs.

[0023] In one embodiment, the binding agent is an antibody.

[0024] Other features and advantages of the invention will become apparent from the following detailed description, and from the claims. [Brief description of the drawings]

[0025] [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 top part of the model shows the predicted expression levels of 53 preselected genes when optimized for NKX2-2. The bottom part of the model shows the effectors tested in the model and their contribution to maximum expression of NKX2-2. The value column shows the concentration of each effector required to mimic the model. [Diagram 2] Figure 2 shows the results from the HD-DoE model of an eight-factor experiment optimized for maximum expression of PDGFRA. The top and bottom rows are as described for Figure 1. This condition highlights the effector PD0325901 with a factor contribution of 30.05 as a critical input for high expression of PDGFRA. [Diagram 3] Figure 3 shows the dynamic profile of expression levels of NKX2-2, OLIG1, OLIG2 and PDGFRA genes against the concentrations of the eight effectors tested. The positive effects of TTNPB, MHY1485 and PD0325901 on the expression of PDGFRA and their factorial contributions are indicated by the slope of the plot for each effector. The dotted box highlights the opposite effect of PD0325901 on NKX2-2 and OLIG2 compared to PDGFRA. [Figure 4] Figure 4 shows the results from a HD-DoE model of a 13-factor experiment optimized for maximum OTX2 expression, in which MK2206, PD0325901, CHIR99021, LDN193189, Go6983 and PD173074 were introduced as positive effectors on OTX2 expression. [Diagram 5] Figure 5 shows the results from a HD-DoE model of a 13-factor experiment optimized for maximum expression of FEZF2. In this model, the positive effects of LDN193189, MK2206 and PD0325901 on cell patterning were confirmed, and three other factors were introduced, including SC79, XAV939 and palmorfamine-500 nM. [Figure 6] Figure 6 shows the dynamic profile of OTX2 and FEZF2 expression levels versus the concentrations of the 13 factors tested in this model. XAV939 and palmorfamine-500 nM have a significant positive effect on FEZF2 expression and no significant negative effect on OTX2 expression. [Figure 7A]Figure 7A-D shows the dynamic profile analysis of the elimination process in an eight-factor modeling experiment and its impact on the expression of NKX2-2, OLIG2 and PDGFRA. [Figure 7B] See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 7D] See legend to Figure 7A. [Figure 8A] Figure 8A-D shows the dynamic profile analysis of the elimination process in a 13-factor modeling experiment and its impact on the expression of FEZF2 and OTX2. [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 a photograph of the image of cells cultured in optimized OPC differentiation medium after 3 days. 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 (neuronal and late OPC biomarkers, respectively) are absent. KI67 expression indicates the proliferative state of the progenitor cells. [Figure 10] Figure 10A-B shows RNA-seq data after 3 days of cells cultured in optimized OPC differentiation medium. Expression levels of stem cell genes NANOG and POU5F1 were decreased, whereas genes involved in early development of brain regions and oligodendrocyte lineages were increased. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Detailed Description of the Invention Described herein are methodologies and compositions that allow the generation of pre-OPCs and OPCs from human pluripotent stem cells under chemically defined culture conditions using a small molecule-based approach. The disclosed method has the advantage that the starting pluripotent stem cells do not go through neural induction, as employed by many prior art protocols. This allows the generation of pre-OPCs and OPCs in as little as three days, which is much shorter than current protocols that require an average of 10 days to generate pre-OPCs.

[0027] Using a High-Dimensional Design of Experiments (HD-DoE) approach, multiple process inputs (e.g., small molecule agonists or antagonists) were simultaneously tested for output responses such as gene expression, as described in Example 1. These experiments allowed the identification of chemically defined culture media containing agonists and / or antagonists of specific signaling pathways sufficient to generate pre-OPCs or OPCs in a very short time. The optimized culture media was further validated by factor criticality analysis, examining the effect of omitting individual agonist or antagonist drugs, as described in Example 2. The phenotype of cells generated by this differentiation protocol was further confirmed by immunohistochemistry, as described in Example 3.

[0028] Various aspects of the invention are described in further detail in the following subsections.

[0029] I. cell The starting cells used in the culture of the present disclosure are human pluripotent stem cells. As used herein, the term "human pluripotent stem cells" (abbreviated as hPSC) refers to human stem cells that have the ability to differentiate into a variety of different cell types. As used herein, the term "pluripotent" refers to cells that have the ability to differentiate into cell types characteristic of all three germ layers (endoderm, mesoderm and ectoderm) under different conditions. Pluripotent cells are primarily characterized by their ability to differentiate into all three germ layers, for example, using nude mice and teratoma formation assays. Although 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.

[0030] 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, 19-9-7 or 6-9-9 cells (e.g., as described in Yu, J. et al. (2009) Science 324:797-801). Non-limiting examples of human embryonic stem cell lines include ES03 cells (WiCell Research Institute) and H9 cells (Thomson, JA et al. (1998) Science 282:1145-1147). Human pluripotent stem cells (PSCs) express cell markers that can be used to identify cells as PSCs. Non-limiting examples of pluripotent stem cell markers include TRA-1-60, TRA-1-81, TRA-2-54, SSEA1, SSEA3, SSEA4, CD9, CD24, OCT3, OCT4, NANOG and / or SOX2. Because the methods of generating pre-OPCs and / or OPCs of the present disclosure are used to differentiate (mature) a starting pluripotent stem cell population, in various embodiments, the pre-OPC and / or OPC cell population generated by the methods of the present disclosure 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.

[0031] The pluripotent stem cells are exposed 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 to more mature (i.e., less primitive) cells, where mature cells typically exhibit phenotypic characteristics that are deeply committed to a particular cell lineage. Early precursor 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 precursor cells that express the type VI intermediate filament protein Nestin. The method of producing pre-OPCs and / or OPCs of the present disclosure does not generate NPCs to avoid the use of neural induction, and in various embodiments, the cell population produced by the method of the present disclosure lacks Nestin-positive cells.

[0032] 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 progenitor cell that expresses cell markers OLIG2 and NKX2.2. Pre-OPC may express additional markers, including but not limited to OTX2 (anterior neuroectodermal biomarker), FEZF2 (anterior ectodermal biomarker), and / or OLIG1.

[0033] In one embodiment, the cells produced by the method of the present disclosure are oligodendrocyte progenitor cells (OPCs), which are more differentiated (more mature) cells than pre-OPCs. As used herein, "oligodendrocyte progenitor cells" or "OPCs" refer to stem cell-derived progenitor cells that express cell markers OLIG2 and NKX2.2 as well as PDGFRa. OPCs may express additional markers, non-limiting examples of which include SOX10 (neural crest marker), OTX2 (anterior neuroectoderm biomarker), FEZF2 (anterior ectoderm biomarker), and / or OLIG1.

[0034] Pre-OPCs and OPCs generated by the methods of the present disclosure can be further cultured in vitro to generate mature oligodendrocytes (OLs). Markers of mature OLs include, but are not limited to, myelin basic protein (MBP) and O4.

[0035] II. Culture medium components The disclosed methods for generating pre-OPCs or OPCs include culturing human pluripotent stem cells in a culture medium lacking exogenously added growth factors and containing specific agonists and / or antagonists of cell signaling pathways.

[0036] As described in Example 1, the culture medium containing retinoic acid (RA) pathway agonist, Akt pathway agonist and mTOR pathway agonist was sufficient to generate pre-OPCs expressing OLIG2 and NKX2.2 in only 3 days. The inclusion of additional agents optimized the expression of other markers, such as PDGFRa as a marker of OPC differentiation. In other embodiments, the culture medium further comprises at least one additional agent selected from the group consisting of WNT pathway antagonist, SHH pathway agonist, BMP pathway antagonist and PKC pathway antagonist. In one embodiment, the culture medium further comprises a WNT pathway antagonist. In one embodiment, the culture medium further comprises a 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 a SHH pathway agonist, wherein the differentiated cells express OTX2 and FEZF2, in addition to OLIG2 and NKX2.2.

[0037] In one embodiment, the culture medium comprises a retinoic acid (RA) pathway agonist, an Akt pathway agonist, an mTOR pathway agonist, a WNT pathway antagonist, a 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 (and may express additional markers, such as OTX2, FEZF2, and / or OLIG1).

[0038] As used herein, an "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 the use of an agonist that activates a cell surface receptor involved in the signaling pathway (e.g., the agonist can be a receptor ligand). Additionally or alternatively, stimulation of cell signaling can be initiated intracellularly, for example, by the use of a small molecule agonist that interacts intracellularly with a component(s) of the signaling pathway.

[0039] As used herein, an "antagonist" of a cell signaling pathway is intended to refer to an agent that inhibits (downregulates) a cell signaling pathway. Inhibition of a cell signaling pathway can be initiated extracellularly, for example, by using an antagonist that blocks a cell surface receptor involved in the signaling pathway. Additionally or alternatively, inhibition of cell signaling can be initiated intracellularly, for example, by using a small molecule antagonist that interacts intracellularly with a component(s) of the signaling pathway.

[0040] Retinoic acid (RA) pathway agonist, Akt pathway agonist, mTOR pathway agonist, WNT pathway antagonist, SHH pathway agonist, BMP pathway antagonist and PKC pathway antagonist are known in the art and are commercially available.They are used in culture medium at effective concentrations to achieve desired results, such as producing pre-OPCs and / or OPCs that express markers of interest.Non-limiting examples of suitable agonist and antagonist drugs, as well as effective concentration ranges, are further described below.

[0041] Agonists of the RA pathway include drugs, molecules, compounds, or substances that can stimulate the retinoic acid receptors (RARs), which are activated by both all-trans and 9-cis retinoic acids. There are three RARs, RARα, RARβ, and RARγ, which are encoded by the RARA, RARB, and RARG genes, respectively. Various retinoic acid analogs have been synthesized that can activate the retinoic acid pathway. Non-limiting examples of such compounds include TTNPB (RARα, β and 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 RARβ and γ), isotretinoin (endogenous agonist of retinoic acid receptor; 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) retinoid compounds, ii) retinoid X receptor (RXR) agonists, and iii) 25 retinoic acid receptor (RAR) agonists. 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).

[0042] Thus, in one embodiment, the RA pathway agonist is selected from the group consisting of TTNPB, AM 580, CD 1530, CD 2314, Ch 55, BMS 753, tazarotene, isotretinoin, AC 261066, retinoic acid (RA), Sr11237, adapalene, EC23, 9-cis retinoic acid, 13-cis retinoic acid, 4-oxo retinoic acid, and all-trans retinoic acid (ATRA), or combinations thereof. In one embodiment, the RA pathway agonist is present in the culture medium at a concentration in the range of 5-500 mM, or 10-100 nM, or 25-75 nM. In one embodiment, the RA pathway agonist is TTNPB. In one embodiment, the RA pathway agonist is TTNPB and is present in the culture medium at a concentration in the range of 5-500 nM, or 10-100 nM, or 25-75 nM. In one embodiment, the RA pathway agonist is TTNPB and is present in the culture medium at a concentration of 50 nM.

[0043] Akt pathway agonists include agents, molecules, compounds, or substances that can stimulate (activate) one or more signaling pathways of serine / threonine kinase Akt family members, including Akt1 (also referred to as PKB or RacPK), Akt2 (also referred to as PKBβ or RacPK-β), and Akt3 (also referred to as PKBγ or thyoma viral proto-oncogene 3). In one embodiment, the Akt pathway agonist is a pan Akt activator. In one embodiment, the pan Akt activator is SC79. In one embodiment, the Akt pathway agonist is present in the culture medium at a concentration in the range of 0.1-10 μM. In one embodiment, the Akt pathway agonist is SC79. In one embodiment, the Akt pathway agonist is SC79 and is present in the culture medium at a concentration of 0.1-10 μM, or 0.5-5 μM, or 0.5-2.5 μM, or 0.5-1.5 μM. In one embodiment, the Akt pathway agonist is SC79 and is present in the culture medium at a concentration of 1 μM.

[0044] 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 that 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, and combinations thereof. In one embodiment, the mTOR pathway agonist is present in the culture medium at a concentration in the range of 0.1-10 μM, or 0.5-5 μM, or 0.5-2.5 μM, or 0.5-1.5 μM. In one embodiment, the mTOR pathway agonist is MHY1485. In one embodiment, the mTOR pathway agonist is MHY1485 and is present in the culture medium at a concentration of 0.1-10 μM, or 0.5-5 μM, or 0.5-2.5 μM, or 0.5-1.5 μM. In one embodiment, the mTOR pathway agonist is MHY1485 and is present in the culture medium at a concentration of 1 μM.

[0045] WNT pathway antagonists include drugs, molecules, compounds, or substances that can inhibit (downregulate) the canonical Wnt / β-catenin signaling pathway; biologically, the Wnt / β-catenin signaling pathway is 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, JW67, JW74, Cardionogen 1, NLS-StAx-h, TAK715, PNU 74654, iCRT3, WIF-1, DKK1, and combinations thereof. In one embodiment, the WNT pathway antagonist is present in the culture medium at a concentration in the range of 10-500 nM, 50-250 nM, or 50-150 nM. In one embodiment, the WNT pathway antagonist is XAV939. In one embodiment, the WNT pathway antagonist is XAV939 and 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 and is present in the culture medium at a concentration of 100 nM.

[0046] Agonists of the sonic hedgehog (SHH) pathway include drugs, molecules, compounds, or substances that can stimulate (activate) signaling through the SHH pathway; biologically, the SHH pathway involves binding of SHH to the Patched-1 (PTCH1) receptor and transduction through the Smoothened (SMO) transmembrane protein. In one embodiment, the SHH pathway agonist is selected from the group consisting of palmorphamin, GSA 10, SAG, and combinations thereof. In one embodiment, the SHH pathway agonist is present in the culture medium at a concentration in the range of 100-1000 nM, or 250-750 nM, or 400-600 nM. In one embodiment, the SHH pathway antagonist is palmorphamin. In one embodiment, the SHH pathway antagonist is palmorphamin and 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 palmorphamin and is present in the culture medium at a concentration of 500 nM.

[0047] Antagonists of the BMP (bone morphogenetic protein) pathway include drugs, molecules, compounds, or substances that can inhibit (downregulate) the BMP signaling pathway; biologically, the BMP signaling pathway is activated by the binding of BMP to a BMP receptor, which 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 within the range 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 and 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 and is present in the culture medium at a concentration of 250 nM.

[0048] Antagonists of the PKC (protein kinase C) pathway include drugs, molecules, compounds, or substances that can inhibit (downregulate) the PKC signaling pathway; the PKC signaling pathway 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 isoforms α, β1, β2, and γ. In one embodiment, the PKC pathway antagonist inhibits the activity of at least one (in other embodiments, at least two or three) PKC enzymes selected from PKCα, PKCβ1, PKCβ2, and PKCγ. In one embodiment, the PKC pathway antagonist is selected from the group consisting of Go 6983, sotrastaurin, enzastaurin, staurosporine, LY31615, Go 6976, GF 109203X, Ro 31-8220 mesylate, and combinations thereof. In one embodiment, the PKC pathway antagonist is present in the culture medium at a concentration in the range 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 and 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 and is present in the culture medium at a concentration of 110 nM.

[0049] III. Culture conditions In combination with the chemically defined and optimized culture medium described in subsection II above, the method of producing pre-OPCs and OPCs of the present disclosure utilizes standard culture conditions established in the art for cell culture. For example, cells can be cultured at 37° C. under conditions of 5% O2 and 5% CO2. Cells can be cultured in standard culture vessels or plates, such as 96-well plates. In certain embodiments, the starting pluripotent stem cells are attached to a plate, preferably coated with an extracellular matrix material such as vitronectin. In one embodiment, the stem cells are cultured on a vitronectin-coated culture surface (e.g., a vitronectin-coated 96-well plate).

[0050] Pluripotent stem cells can be cultured in commercially available medium prior to differentiation. For example, stem cells can be cultured in Essential 8 Flex medium (Thermo Fisher#A2858501) for at least 1 day before starting differentiation protocol. In a non-limiting exemplary embodiment, stem cells are subcultured on vitronectin (Thermo Fisher#A14700) coated 96-well plate at a density of 150,000 cells / cm2 and cultured in Essential 8 Flex medium for 1 day prior to differentiation.

[0051] To initiate the differentiation protocol, the medium in which the stem cells are cultured is replaced with basal differentiation medium, which is supplemented with agonists and / or antagonists of signaling pathways as described above in subsection II. Basal differentiation medium may, for example, comprise commercially available basal medium supplemented with additional standard media components necessary to maintain cell survival and proliferation, but lacking serum (basal differentiation medium is a serum-free medium) or other exogenously added growth factors, such as FGF2, PDGF, IGF, HGF, etc. In non-limiting exemplary embodiments, basal differentiation medium contains 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 μM 1-thioglycerol (Sigma #M6145), 0.7 μg / ml insulin (Sigma #11376497001), and 15 μg / ml transferrin (Sigma #10652202001).

[0052] The culture medium is typically replaced with fresh medium periodically, for example, in one embodiment, the medium is replaced every 24 hours.

[0053] To generate pre-OPCs and / or OPCs, the stem cells are cultured in the optimized culture medium for a time sufficient for the differentiation of the cells and the expression of pre-OPC or OPC-associated markers. As described in the Examples, it has been found that culturing the stem cells in the optimized culture medium for only 72 hours (3 days) is sufficient to differentiate them into pre-OPCs and OPCs. Thus, in one embodiment, the cells are cultured for at least 72 hours. In other embodiments, the cells are cultured for at least 60, 64, 68, 72, 76, 80, 84, 88, 92 or 96 hours. In other embodiments, the cells are cultured for at least 2.5, 3, 3.5, 4, 4.5 or 5 days.

[0054] IV. Purpose The disclosed methods and compositions for generating pre-OPCs and OPCs allow for efficient and robust utilization of these cell populations for a variety of applications. For example, these methods and compositions can be used to study oligodendrocyte development and biology to aid in understanding oligodendrocyte-related diseases and disorders. For example, pre-OPCs and / or OPCs generated using the disclosed methods can be further purified according to methods established in the art using agents that bind to surface markers expressed on the cells. Thus, in one aspect, the disclosure provides a method for isolating pre-oligodendrocyte precursor cells (pre-OPCs) or oligodendrocyte precursor cells (OPCs), the method comprising the steps of: contacting the OLIG2-expressing pre-OPCs or OPCs produced by the methods of the present disclosure with at least one binding agent that binds to a cell surface marker expressed by the pre-OPCs or OPCs; and Isolating cells that bind to the binding agent, thereby isolating pre-OPCs or OPCs.

[0055] In one embodiment, the binding agent is an antibody, for example, a monoclonal antibody (mAb) that binds to cell surface marker.Non-limiting examples of suitable OPC cell surface markers include PDGFRa, O4 and A2B5.The cells that bind to the antibody can be isolated by methods known in the art, including but not limited to fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS).

[0056] The progenitor cells of oligodendrocyte lineage are also intended to be used for treating various diseases and disorders by delivering the cells to subjects with oligodendrocyte-related diseases or disorders.Examples of oligodendrocyte-related diseases and disorders include but are not limited to multiple sclerosis (MS), progressive multifocal leukoencephalopathy, periventricular leukomalacia, certain types of cerebral leukodystrophy, and amyotrophic lateral sclerosis (ALS).

[0057] V. composition In other aspects, the disclosure provides compositions related to methods of generating pre-OPCs and OPCs, including culture media and cell cultures, as well as isolated progenitor cells and cell populations.

[0058] In one aspect, the present disclosure provides a culture medium for obtaining pre-OPCs or OPCs, the culture medium lacking exogenously added growth factors and comprising a retinoic acid (RA) pathway agonist, an Akt pathway agonist and an mTOR pathway agonist. In one embodiment, the culture medium further comprises a WNT pathway antagonist. In one embodiment, the culture medium further comprises a SHH pathway agonist. In one embodiment, the culture medium further comprises a WNT pathway antagonist and 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 BMP pathway antagonist and a PKC pathway antagonist. In one embodiment, the culture medium comprises a retinoic acid (RA) pathway agonist, an Akt pathway agonist, an mTOR pathway agonist, a WNT pathway antagonist, a SHH pathway agonist, a BMP pathway antagonist, and a PKC pathway antagonist.

[0059] In one aspect, the present disclosure provides an isolated cell culture of pre-OPCs or OPCs, comprising: pre-OPCs or OPCs expressing OLIG2, cultured in a culture medium containing a retinoic acid (RA) pathway agonist, an Akt pathway agonist and an mTOR pathway agonist, and lacking exogenously added growth factors. In various embodiments, the culture medium may also contain a WNT pathway antagonist, a SHH pathway agonist, a BMP pathway antagonist and / or a PKC pathway antagonist. In one embodiment, the pre-OPCs or OPCs also express NKX2.2. In other embodiments, the pre-OPCs or OPCs also express OTX2 and FEZF2. In one embodiment, the pre-OPCs or OPCs are attached to a vitronectin-coated plate.

[0060] In another aspect, the present disclosure provides pre-oligodendrocyte precursor cells (pre-OPCs) or oligodendrocyte precursor cells (OPCs) generated by the differentiation method of the present disclosure. In one embodiment, the present disclosure provides a composition comprising non-natural pre-OPCs or OPCs, wherein the pre-OPCs or OPCs express OLIG2, NKX2-2, OTX2 and FEZF2, and lack expression of NESTIN. In another embodiment, the present disclosure provides a composition comprising at least 1×10 6 In another aspect, the present invention provides an isolated cell population of pre-OPCs or OPCs comprising pre-OPCs or OPCs expressing OLIG2 and NKX2.2, wherein the cell population is devoid of neural stem cells expressing NESTIN. 6 pieces, 1×10 7 pieces, 5×10 7 pieces, 1×10 8 pieces, 5×10 8 pcs or 1×10 9 The pre-OPC or OPC expresses OLIG2 and NKX2.2. In one embodiment of the isolated cell population, the pre-OPC or OPC is bound to at least one antibody that binds to at least one cell surface marker expressed by the pre-OPC or OPC. Non-limiting examples of suitable OPC cell surface markers include PDGFRa, O4 and A2B5.

[0061] The present invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of the figures and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference. EXAMPLES

[0062] Example 1 : Development of a culture protocol for generating stem cell-derived oligodendrocyte precursor cells In this example, a culture medium recipe for generating oligodendrocyte precursor cells was developed; this medium recipe can induce human pluripotent stem cells to differentiate into oligodendrocyte precursor cells expressing NKX2-2 and OLIG2 after 3 days of culture. These cells can be further differentiated into mature oligodendrocytes.

[0063] In this example, we utilize a high-dimensional design of experiments (HD-DoE) method, as previously described in Bukys et al. (2020) Iscience 23:101346. This method employs computerized design geometry to simultaneously test multiple process inputs and provides mathematical modeling of deep effector / response space. This method allows for the finding of combinatorial signaling inputs that control complex processes such as cell differentiation. This allows for the testing of multiple plausible key process parameters, as such parameters affect output responses such as gene expression. Since gene expression provides a hallmark of the phenotype, for example of human cells, this method can be applied to identify and understand which signaling pathways control cell fate. In this example, the HD-DOE method was applied with the goal of finding conditions that induce genes expressed by oligodendrocyte precursor cells directly from a pluripotent stem cell state.

[0064] Specifically, to develop a novel method to generate oligodendrocytes, agonists and antagonists of multiple signaling pathways (herein referred to as effectors) were tested for their effects on the expression of two sets of 53 preselected genes after 3 days of treatment. These effectors are small molecules that are frequently used in the stepwise differentiation of stem cells to specific fates. The selection of effectors was based on the most recent literature on neural induction in the anterior ectoderm and differentiation of stem cells into oligodendrocytes.

[0065] HD-DoE #1 To test effectors, we designed an experiment with at least eight factors; this experiment allows us to evaluate the response of cells to more than 48 different combinations of effectors at a range of concentrations. To analyze this model, we focused on the expression of genes expressed during the early development of anterior neuroectoderm and oligodendrocytes, such as NKX2-2, OLIG2, OLIG1, and PDGFRA. The impact of each effector on gene expression levels is defined by a parameter called factor contribution, which is calculated for each effector during modeling.

[0066] As shown in the results summarized in Figure 1, one model clearly showed promising results in terms of upregulation of NKX2-2 and OLIG2 genes when optimized to maximize NKX2-2 expression with 12480.6. Eight factors were tested in this model: PD0325901, MK2206, TTNPB, SC79, MHY1485, ZM336372, AGN193109, and AZD3147.

[0067] Of the eight factors tested, three factors, namely TTNPB (agonist of the retinoic acid pathway), SC79 (agonist of the Akt signaling pathway), and MHY1485 (agonist of the mTOR signaling pathway), had a significant positive impact on the expression of target genes; TTNPB had the greatest impact with a factor contribution of 31.3, MHY1485 with a factor contribution of 13.8, and SC79 with a factor contribution of 1.47. These factors 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) that fit the gene expression pattern during oligodendrocyte differentiation.

[0068] As shown in the results summarized in Figure 2, the normalized expression of PDGFRA in this model could reach up to 832.9, the highest expression level among all models; therefore, this model was also optimized to maximize the expression of PDGFRA. This setting showed that TTNPB (agonist of the retinoic acid pathway) and MHY1485 (agonist of the mTOR signaling pathway) also had a positive effect on the upregulation of PGFRA, with factor contributions of 49.01 and 13.4, respectively. It was also observed that PD0325901 could have a significant positive effect on this gene, with a factor contribution of 30.6. As the factor contribution of ZM336372 was low (<1), this factor was not included in the medium recipe. In this condition, the average expression level of OLIG1 was 228.36, similar to the optimized condition of NKX2-2. One difference in this condition was that the OLIG2 gene was downregulated from 1049.4 in the previous condition to 241.9.

[0069] As shown in the results summarized in Figure 3, among the effectors with positive contributions to the expression levels of NKX2-2, OLIG2 or PDGFRA, two factors had negative effects on the expression levels of NKX2-2 and OLIG2. Therefore, these two factors, PD0325901 and ZM336372, were excluded from the candidate list of medium recipes for oligodendrocyte differentiation.

[0070] Thus, in this initial HD-DoE screen, culture medium lacking exogenously added growth factors and containing agonists of the retinoic acid pathway, the Akt signaling pathway and the mTOR signaling pathway was identified as sufficient to generate OLIG2-expressing OPCs from pluripotent stem cells after 3 days (72 hours) of culture.

[0071] HD-DoE #2 To further refine the conditions for oligodendrocyte differentiation from pluripotency, we performed additional HD-DoE experiments. We obtained additional gene regulation models, which we used to prepare the differentiation protocol. The basis for this was a 13-factor HD-DoE experiment focused on the initiation of cell differentiation into anterior neuroectoderm. In this model, the expression of FEZF2 and OTX2 was emphasized.

[0072] As shown in the results summarized in Figure 4, this model was optimized so that the expression of OTX2 was the highest at 12755.9. According to this high expression model of OTX2, seven effectors including MK2206, PD0325901, CHIR99021, LDN193189, Go6983, PD173074 and BLU9931 had positive contributions, with MK2206 having the highest factor contribution of 22.2 and PD173074 and BLU9931 having the lowest factor contribution of 1.7.

[0073] As shown in the results summarized in Figure 5, the model was optimized for the highest expression of FEZF2 at 4466. When the model was optimized for the highest expression level of FEZF2, three effectors were common with 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 introduced, including palmorfamine-500 nM, XAV939, and SC79.

[0074] As shown in the results summarized in Figure 6, we performed dynamic profiling analysis to fine-tune this recipe and find the optimal combination of factors to highly express both OTX2 and FEZF2. This analysis showed that XAV939 (an inhibitor of the WNT signaling pathway) and palmorfamine (an agonist of the SHH signaling pathway, known to ventralize cells during development of brain regions) had a significant positive effect on the expression of FEZF2 and had no negative effect on the expression level of OTX2. Therefore, these two factors were added to the optimized medium recipe.

[0075] In addition to including factors that promote the expression of OPC-associated surface markers, certain factors that inhibit the expression of such markers were excluded from the optimized medium recipe. CHIR99021, an agonist of the WNT signaling pathway, was excluded. MK2206 and PD0325901 were also excluded because, according to the eight-factor model, they had a negative effect on the expression of oligodendrocyte genes. PD173074 and BLU9931 were also excluded because of their low factor contribution of 1.7.

[0076] summary Considering both models, culture conditions maximizing differentiation of human induced pluripotent stem cells into cells with oligodendrocyte progenitor cell (OPC) identity leading to upregulation of OTX2, FEZF2, NKX2-2 and OLIG2 included the following effector inputs: TTNPB (RA pathway agonist), SC79 (Akt pathway agonist), MHY1485 (mTOR pathway agonist), palmorfamine (SHH pathway agonist), XAV939 (WNT pathway antagonist), LDN193189 (BMP pathway antagonist) and Go6983 (PKC pathway antagonist).

[0077] Example 2 : Factor importance analysis of culture conditions for inducing OPCs To evaluate the factor criticality of each component in the optimized culture medium described in Example 1, an in-silico predictive analysis of the results was performed under conditions where individual effectors were excluded and other effectors were present. To do so, we used a dynamic profile analysis at a set point, comparing the expression level of the gene of interest in the absence of each factor. This factor criticality analysis revealed the degree of importance of each input effector, since the expression of the gene of interest reveals whether the desired outcome can be reached.

[0078] Figures 7A-D summarize the results of factor importance analysis of 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 optimized for maximum expression of NKX2-2. As shown in Figure 7B, upon removal of TTNPB, the predicted expression levels of NKX2-2, OLIG2 and PDGFRA were significantly reduced from over 12000 to 4500 for NKX2-2, from 1000 to 400 for OLIG2 and from 350 to less than 100 for PDGFRA. This result implies that there is a significant negative impact on the expression of all desirable markers when the RA pathway agonists are removed. As shown in Figure 7C, removal of MHY1485 again reduced expression levels, but not as drastically as in the previous condition, and as shown in Figure 7D, only a slight shift in the plot was observed when SC79 was removed, suggesting that this factor is less important than TTNPB and MHY1485 for maximizing induction of OPC markers.

[0079] Figure 8A-D summarizes the results of factor importance analysis of the effectors parmorphamine (SHH pathway agonist), XAV939 (WNT pathway antagonist), LDN193189 (BMP pathway antagonist) and Go6983 (PKC pathway antagonist). To achieve the desired patterning of oligodendrocyte populations to anterior regions of the brain, these additional factor inputs were queried, such as FEZF2 and OTX2. We examined the expression levels of FEZF2 and OTX2 in the absence of either LDN193189, XAV, parmorphamine or Go6983 when the model was optimized for maximum expression of FEZF2. The most striking change was observed in the absence of LDN193189, where FEZF2 expression was reduced by almost 50% (from 4500 to 2500). OTX2 expression also decreased from 9000 to 7000, the lowest in all four exclusion processes. Upon removal of XAV939 and palmorfamine, FEZF2 expression levels decreased to 3000 and 3500, respectively, whereas OTX2 expression was only slightly higher in both cases. Upon removal of Go6983, no significant changes in expression levels of the genes of interest were observed, suggesting that Go6983 is optional in the regulation of FEZF2 and OTX2.

[0080] 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 and oligodendrocyte progenitor biomarkers. Biomarkers included OTX2 and oligodendrocyte progenitor biomarkers such as NKX2-2, OLIG2, and PDGF. To distinguish between neural stem cells and oligodendrocyte progenitor cells, an early neuronal marker, Nestin, was used. Ki67 was also used to confirm the proliferation of cells after induction. Representative immunohistochemistry results are shown in Figure 9. These immunocytochemistry images confirmed that most cells expressed OTX2. However, no trace of the neuronal biomarker Nestin was detected, confirming that the differentiated OPC population lacked neural stem cells. Expression of OLIG2 and NKX2-2 was also observed in more than 90% of the cells, thereby confirming the oligodendrocyte lineage of the cells. None of the cells expressed PDGFR, which was expected since this gene is expressed at a late stage of oligodendrocyte differentiation.

[0081] Example 4: RNA-seq validation of stem cell-derived pre-OPCs 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 3 days and RNA from the resulting cells was sequenced by standard RNA-seq analysis. Results in Figure 10A show normalized expression levels of selected genes representative of different brain regions, early oligodendrocyte identity (NKX2-2, OLIG2, PDGFRa), and stem cell state (NANOG, POU5F1) in three replicates at days 0 and 3. The results demonstrated that the levels of stem cell genes were decreased in pre-OPC cells, while the levels of oligodendrocyte genes were increased, thus validating the differentiation of hiPSCs into the oligodendrocyte lineage using the differentiation medium. Results in Figure 10B show differential expression and fold change of selected genes, with HOXA1 at the highest level (15), followed by OLIG2, NKX2-2, and PDGFRa at 5. This data demonstrates the ability of the developed recipe as a first-step medium in directing cells towards an oligodendrocyte identity.

[0082] 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 which equivalents are intended to be encompassed by the following claims.

Claims

1. 1. A method for generating human pre-oligodendrocyte precursor cells (pre-OPCs) or oligodendrocyte precursor cells (OPCs), comprising the steps of: Culturing human pluripotent stem cells in a culture medium lacking exogenously added growth factors and containing a retinoic acid (RA) agonist, an Akt agonist and an mTOR agonist, such that pre-OPCs or OPCs expressing OLIG2 are generated.

2. The method of claim 1, wherein the human pluripotent stem cells are induced pluripotent stem cells (iPSCs).

3. The method of claim 1, wherein the human pluripotent stem cells are embryonic stem cells.

4. The method of any one of claims 1 to 3, wherein pre-OPCs or OPCs expressing OLIG2 are generated within 72 hours of initiating culture of human pluripotent stem cells in said culture medium.

5. 5. The method of any one of claims 1 to 4, wherein the pre-OPCs or OPCs also express NKX2-2.

6. The method according to any one of claims 1 to 5, wherein the human pluripotent stem cells are attached to vitronectin-coated plates throughout the culture.

7. 7. The method of any one of claims 1 to 6, wherein the RA agonist is selected from the group consisting of TTNPB, AM 580, CD 1530, CD 2314, Ch 55, BMS 753, tazarotene, isotretinoin, AC 261066, retinoic acid (RA), Sr11237, adapalene, EC23, 9-cis retinoic acid, 13-cis retinoic acid, 4-oxo retinoic acid, all-trans retinoic acid (ATRA), and combinations thereof.

8. The method of claim 7, wherein the RA agonist is present in the culture medium at a concentration in the range of 10 to 100 nM.

9. The method of claim 8, wherein the RA agonist is TTNPB and is present in the culture medium at a concentration in the range of 10 to 100 nM.

10. The method of any one of claims 1 to 6, wherein the Akt agonist is SC79.

11. The method of claim 10, wherein SC79 is present in the culture medium at a concentration in the range of 0.1 to 10 μM.

12. The method of claim 10, wherein SC79 is present in the culture medium at a concentration in the range of 0.1 to 10 μM.

13. The method of any one of claims 1 to 6, wherein the mTOR agonist is selected from the group consisting of MHY1485, 3BDO, salidroside, L-leucine, NV-5138, and combinations thereof.

14. The method of claim 13, wherein the mTOR agonist is present in the culture medium at a concentration in the range of 0.1 to 10 μM.

15. The method of claim 14, wherein the mTOR agonist is MHY1485 and is present in the culture medium at a concentration in the range of 0.1 to 10 μM.

16. The method of any one of claims 1 to 15, wherein the culture medium further comprises a WNT antagonist and an SHH agonist.

17. 17. The method of claim 16, wherein the WNT 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, JW67, JW74, Cardionogen 1, NLS-StAx-h, TAK715, PNU 74654, iCRT3, WIF-1, DKK1, and combinations thereof.

18. The method of claim 17, wherein the WNT antagonist is present in the culture medium at a concentration in the range of 50 to 200 nM.

19. 19. The method of claim 18, wherein the WNT antagonist is XAV939 and is present in the culture medium at a concentration in the range of 50 to 200 nM.

20. 17. The method of claim 16, wherein the SHH agonist is selected from the group consisting of palmorfamine, GSA 10, SAG, and combinations thereof.

21. The method of claim 20, wherein the SHH agonist is present in the culture medium at a concentration in the range of 250 to 750 nM.

22. 22. The method of claim 21, wherein the SHH antagonist is palmorfamine and is present in the culture medium at a concentration in the range of 250 to 750 nM.

23. The method of any one of claims 1 to 22, wherein the culture medium further comprises a BMP antagonist.

24. 24. The method of claim 23, wherein the BMP antagonist is selected from the group consisting of LDN193189, DMH1, DMH2, dorsomorphin, K02288, LDN214117, LDN212854, follistatin, ML347, noggin, and combinations thereof.

25. 25. The method of claim 24, wherein the BMP antagonist is present in the culture medium at a concentration in the range of 100 to 500 nM.

26. 26. The method of claim 25, wherein the BMP antagonist is LDN193189 and is present in the culture medium at a concentration in the range of 100-500 nM.

27. The method of any one of claims 1 to 26, wherein the culture medium further comprises a PKC antagonist.

28. 28. The method of claim 27, wherein the PKC 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.

29. 29. The method of claim 28, wherein the PKC antagonist is present in the culture medium at a concentration in the range of 50 to 200 nM.

30. 30. The method of claim 29, wherein the PKC antagonist is Go6983 and is present in the culture medium at a concentration in the range of 50-200 nM.

31. 1. A method for generating human pre-oligodendrocyte precursor cells (pre-OPCs) or oligodendrocyte precursor cells (OPCs), comprising the steps of: Culturing human pluripotent stem cells in a culture medium lacking exogenously added growth factors and containing a retinoic acid (RA) agonist, an Akt agonist, an mTOR agonist, a WNT antagonist, an SHH agonist, a BMP antagonist, and a PKC antagonist, such that pre-OPCs or OPCs expressing OLIG2 are generated.

32. A culture medium for obtaining pre-oligodendrocyte precursor cells (pre-OPCs) or oligodendrocyte precursor cells (OPCs), comprising a retinoic acid (RA) agonist, an Akt agonist and an mTOR agonist, and lacking exogenously added growth factors.

33. 33. The culture medium of claim 32, further comprising a WNT antagonist and an SHH agonist.

34. 34. The culture medium of claim 33, further comprising a BMP antagonist and a PKC antagonist.

35. An isolated cell culture of pre-oligodendrocyte precursor cells (pre-OPCs) or oligodendrocyte precursor cells (OPCs), comprising pre-OPCs or OPCs expressing OLIG2, cultured in a culture medium comprising a retinoic acid (RA) agonist, an Akt agonist and an mTOR agonist and lacking exogenously added growth factors.

36. 36. The isolated cell culture of claim 35, wherein the culture medium further comprises a WNT antagonist and an SHH agonist, and the pre-OPCs or OPCs also express OTX2 and FEZF2.

37. 37. The isolated cell culture of claim 35 or 36, wherein the culture medium further comprises a BMP antagonist and a PKC antagonist.

38. 38. The isolated cell culture of any one of claims 35 to 37, wherein the pre-OPCs or OPCs are attached to a vitronectin-coated plate.

39. A pre-oligodendrocyte precursor cell (pre-OPC) or oligodendrocyte precursor cell (OPC) produced by the method of any one of claims 1 to 32.

40. A composition comprising non-natural pre-oligodendrocyte precursor cells (pre-OPCs) or oligodendrocyte precursor cells (OPCs), wherein the pre-OPCs or OPCs express OLIG2, NKX2-2, OTX2 and FEZF2, and lack expression of NESTIN.

41. At least 1×10 6 1. An isolated cell population of pre-oligodendrocyte precursor cells (pre-OPCs) or oligodendrocyte precursor cells (OPCs) that express OLIG2, wherein the isolated cell population is devoid of neural stem cells that express NESTIN.

42. 42. The isolated cell population of claim 41, wherein the pre-OPCs or OPCs also express NKX2-2, OTX2 and FEZF2.

43. 43. The isolated cell population of claim 41 or 42, wherein the pre-OPCs or OPCs are bound to at least one antibody that binds to at least one marker expressed by the pre-OPCs or OPCs.

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