Method and composition for producing oligodendrocyte progenitor cells

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 on 30 March 2021, the entire contents of which are incorporated herein by reference.

[0002] Government licensing rights This invention was made with government support under grant number W911NF-17-3-0003, awarded by the US Army ACC-AGP-RTP. The United States Government has certain rights to this invention. [Background technology]

[0003] Background of the Invention Oligodendrocytes (OLs) are a type of glial cell that synthesizes the myelin sheath surrounding axons. Therefore, they are crucial for nerve conduction in the central nervous system (CNS). A deeper understanding of oligodendrocyte biology is considered essential for developing treatments for neurodegenerative diseases, including demyelinating diseases such as multiple sclerosis and cerebral white matter atrophy, as well as amyotrophic lateral sclerosis (ALS), which can later lead to demyelination during the disease process. Furthermore, radiation therapy to the brain carries the side effect of oligodendrocyte depletion, which can 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 enable the study of these 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 from stem cells or other sources is highly desirable. Various protocols have been reported for differentiating 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 produce myelin basic protein (MBP)-positive oligodendrocytes.

[0005] The initial protocol employed a four-step process (see also Hu et al. (2009) Nature Protocols 4:1614-1622 (Non-Patent Literature 1); Wang et al. (2013) Cell Stem Cell 12:252-264 (Non-Patent Literature 2)). In this protocol, human embryonic stem cells (hESCs) were first differentiated into neuroepithelial cells over two weeks to form neural tube-like rosettes, followed by treatment with retinoic acid (RA) and sonic hedgehog (SHH) for 10 days to induce progenitor cells expressing OLIG2. Further treatment with fibroblast growth factor (FGF2) for 10 days resulted in conversion to pre-OPCs expressing OLIG2 and NKX2.2. Finally, pre-OPCs were cultured for a further 8-9 weeks in the absence of FGF2 to differentiate them into OPCs expressing markers such as platelet-derived growth factor receptor α (PDGFRα), SOX10, and NG2. Therefore, using this protocol, it took approximately 24 days to produce OLIG2-expressing progenitor cells, approximately 34 days to produce pre-OPCs expressing OLIG2 and NKX2.2, and approximately 100 days to obtain mature OLs. A variation of this protocol was reported by Douvaras et al. (Stem Cell Reports (2014) 3:250-259 (Non-Patent Literature 3)), but even that required approximately 20 days to obtain pre-OPCs and approximately 50 days to obtain OPCs, and included culture using externally added growth factors PDGF, IGF-1, and HGF.

[0006] Subsequently, alternative protocols were reported, but these protocols still utilized a neural induction and pattern formation phase (also called neuralization) lasting approximately one week, followed by induction of cells expressing pre-OPC and OPC markers using a culture medium containing externally added growth factors such as FGF2, PDGF, IGF-1, and / or HGF, depending on the protocol (see, for example, Piao et al. (2015) Cell Stem Cell 16:198-210 (Non-Patent Literature 4); Douvaras & Fossati (2015) Nature Protocols 10:1143-1154 (Non-Patent Literature 5); Livesey et al. (2016) Stem Cells 34:1040-1053 (Non-Patent Literature 6); and Yamashita et al. (2017) PLOS One 12: e0171947 (Non-Patent Literature 7)).

[0007] Recently, a protocol has been reported in which MBP-positive oligodendrocytes are produced in just about 20 days by first inducing neural progenitor cells (NPCs) from hESCs, then overexpressing the SOX10 transcription factor in the NPCs (by viral transduction), and growing them in the presence of bFGF (Garcia-Leon et al. (2018) Stem Cell Reports 10:655-672 (Non-Patent Literature 8)). Furthermore, it has been found that transient and partial inhibition of the SHH pathway transcription factor GLI1 in neural stem cells (generated by neurogenesis) with the small molecule inhibitor GANT61 generates OPCs that are more mobile and can differentiate earlier into myelin-producing oligodendrocytes (Namchaiw et al. (2019) Stem Cell Res & Therapy 10:272 (Non-Patent Literature 9)).

[0008] Therefore, although some progress has been made, there is still a need for efficient and robust methods and compositions for generating oligodendrocyte progenitor cells from human pluripotent stem cells. [Prior art documents]

Non-Patent Literature

[0009]

Non-Patent Literature 1

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Non-Patent Literature 9

[0011] Therefore, in one aspect, this disclosure relates to a method for producing human preoligodendrocyte progenitor cells (preOPCs) or oligodendrocyte progenitor cells (OPCs), the method comprising the following steps: A step of culturing human pluripotent stem cells in a culture medium lacking externally added growth factors and containing a retinoic acid (RA) pathway agonist, an Akt pathway agonist, and an mTOR pathway agonist, so that pre-OPCs or OPCs expressing OLIG2 are produced.

[0012] In one embodiment, pre-OPCs or OPCs expressing OLIG2 are produced within 72 hours of initiating the culture of human pluripotent stem cells in the culture medium. In one embodiment, 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 an 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, pre-OPC or OPC also expresses OTX2 and / or FEZF2.

[0014] Thus, in another aspect, the present disclosure relates to a method for generating human pre-oligodendrocyte progenitor cells (pre-OPCs) or oligodendrocyte progenitor cells (OPCs), the method comprising the following steps: 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, a 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 more 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, the human pluripotent stem cells are attached to plates coated with vitronectin throughout the culture.

[0018] In another aspect, the present disclosure relates to a culture medium for obtaining pre-oligodendrocyte progenitor cells (pre-OPCs) or oligodendrocyte progenitor cells (OPCs), the 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 one embodiment, the culture medium further contains a WNT pathway antagonist. In one embodiment, the culture medium further contains a SHH pathway agonist. In one embodiment, the culture medium further contains a BMP pathway antagonist. In one embodiment, the culture medium further contains a PKC pathway antagonist.

[0019] In another aspect, the disclosure relates to an isolated cell culture of pre-oligodendrocyte progenitor cells (pre-OPCs) or oligodendrocyte progenitor cells (OPCs), wherein the culture comprises pre-OPCs or OPCs expressing OLIG2, cultured in a culture medium containing retinoic acid (RA) pathway agonists, Akt pathway agonists, and mTOR pathway agonists, and lacking externally added growth factors. 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 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 Disclosure relates to pre-oligodendrocyte progenitor cells (pre-OPCs) or oligodendrocyte progenitor cells (OPCs), for example, prepared by the method of the Disclosure. In one embodiment, the Disclosure provides a composition comprising non-natural pre-oligodendrocyte progenitor cells (pre-OPCs) or oligodendrocyte progenitor cells (OPCs), wherein the pre-OPCs or OPCs express OLIG2, NKX2-2, OTX2, and FEZF2, and lack NESTIN expression.

[0021] In another aspect, this disclosure provides at least 1 × 10 6The present invention relates to an isolated cell population of pre-OPCs or OPCs, comprising 100 OLIG2-expressing pre-OPCs or oligodendrocyte progenitor cells (OPCs), wherein this cell population lacks neural stem cells expressing NESTIN. In other embodiments, pre-OPCs or OPCs also express NKX2-2, OTX2, and / or FEZF2. In one embodiment of the isolated cell population, pre-OPCs or OPCs are conjugated with at least one antibody bound to at least one cell surface marker expressed by pre-OPCs or OPCs.

[0022] In another aspect, this disclosure relates to a method for isolating pre-oligodendrocyte progenitor cells (pre-OPCs) or oligodendrocyte progenitor cells (OPCs), the method comprising the following steps: A step of contacting a pre-OPC or OPC expressing OLIG2, prepared by the method of the present disclosure, with at least one binder that binds to a cell surface marker expressed by the pre-OPC or OPC expressing OLIG2; and A step of isolating cells that bind to the binder, thereby isolating OPCs.

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

[0024] [Invention 1001] A method for producing human preoligodendrocyte progenitor cells (preOPCs) or oligodendrocyte progenitor cells (OPCs), comprising the following steps: A step of culturing human pluripotent stem cells in a culture medium lacking externally added growth factors and containing a retinoic acid (RA) pathway agonist, an Akt pathway agonist, and an mTOR pathway agonist, so that pre-OPCs or OPCs expressing OLIG2 are generated. [Invention 1002] The method of the present invention 1001, wherein human pluripotent stem cells are induced pluripotent stem cells (iPSCs). [Invention 1003] The method of the present invention 1001, wherein human pluripotent stem cells are embryonic stem cells. [Invention 1004] A method according to any one of the present invention 1001 to 1003, wherein a pre-OPC or OPC expressing OLIG2 is generated in the culture medium within 72 hours of initiating the culture of human pluripotent stem cells. [Invention 1005] A method according to any of the present invention 1001 to 1004, wherein pre-OPC or OPC also expresses NKX2-2. [Invention 1006] A method according to any one of the present invention 1001 to 1005, wherein human pluripotent stem cells are attached to a plate coated with vitronectin throughout the culture period. [Invention 1007] The method according to any one of the present invention 1001 to 1006, wherein 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-cisretinoic acid, 13-cisretinoic acid, 4-oxoletinoic acid, all-trans retinoic acid (ATRA), and combinations thereof. [Invention 1008] The method of the present invention 1007, wherein the RA pathway agonist is present in the culture medium at a concentration in the range of 10 to 100 nM. [Invention 1009] The method of the present invention 1008, wherein the RA pathway agonist is TTNPB, and is present in the culture medium at a concentration of 50 nM. [Invention 1010] Any method 1001 to 1006 of the present invention, wherein the Akt pathway agonist is SC79. [Invention 1011] The method of the present invention 1010, wherein SC79 is present in the culture medium at a concentration in the range of 0.1 to 10 μM. [Invention 1012] The method of the present invention 1010, wherein SC79 is present in the culture medium at a concentration of 1 μM. [Invention 1013] The method according to any one of the invention 1001 to 1006, wherein the mTOR pathway agonist is selected from the group consisting of MHY1485, 3BDO, thalidroside, L-leucine, NV-5138, and combinations thereof. [Invention 1014] The method of the present invention 1013, wherein the mTOR pathway agonist is present in the culture medium at a concentration in the range of 0.1 to 10 μM. [Invention 1015] The method of the present invention 1014, wherein the mTOR pathway agonist is MHY1485, and is present in the culture medium at a concentration of 1 μM. [Invention 1016] The method according to any one of the present invention 1001 to 1015, wherein the culture medium further comprises a WNT pathway antagonist and an SHH pathway agonist. [Invention 1017] The method of the present invention 1016, wherein 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, tryptonide, KYA1797K, JW55, JW67, JW74, Cardionogen 1, NLS-StAx-h, TAK715, PNU 74654, iCRT3, WIF-1, DKK1, and combinations thereof. [Invention 1018] The method of the present invention 1017, wherein a WNT pathway antagonist is present in the culture medium at a concentration in the range of 50 to 150 nM. [Invention 1019] The method of the present invention 1018, wherein the WNT pathway antagonist is XAV939, which is present in the culture medium at a concentration of 100 nM. [Invention 1020] The method of the present invention 1016, wherein the SHH pathway agonist is selected from the group consisting of palmorfamine, GSA 10, SAG, and combinations thereof. [Invention 1021] The method of the present invention 1020, wherein the SHH pathway agonist is present in the culture medium at a concentration in the range of 250 to 750 nM. [Invention 1022] The method of the present invention 1021, wherein the SHH pathway antagonist is palmorfamine, which is present in the culture medium at a concentration of 500 nM. [Invention 1023] Any method 1001 to 1022 of the present invention, wherein the culture medium further comprises a BMP pathway antagonist. [Invention 1024] The method of the present invention 1023, wherein a BMP pathway antagonist is selected from the group consisting of LDN193189, DMH1, DMH2, dolsomorphine, K02288, LDN214117, LDN212854, follistatin, ML347, noggin, and combinations thereof. [Invention 1025] The method of the present invention 1024, wherein a BMP pathway antagonist is present in the culture medium at a concentration in the range of 100 to 500 nM. [Invention 1026] The method of the present invention 1025, wherein the BMP pathway antagonist is LDN193189, which is present in the culture medium at a concentration of 250 nM. [Invention 1027] Any method of the present invention 1001 to 1026, wherein the culture medium further comprises a PKC pathway antagonist. [Invention 1028] The method of the present invention 1027, wherein 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. [Invention 1029] The method of the present invention 1028, wherein a PKC pathway antagonist is present in the culture medium at a concentration in the range of 50 to 150 nM. [Invention 1030] The method of the present invention 1029, wherein the PKC pathway antagonist is Go6983, which is present in the culture medium at a concentration of 110 nM. [Invention 1031] A method for producing human preoligodendrocyte progenitor cells (preOPCs) or oligodendrocyte progenitor cells (OPCs), comprising the following steps: A step of culturing human pluripotent stem cells in a culture medium lacking externally 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, so that pre-OPCs or OPCs expressing OLIG2 are produced. [Invention 1032] A culture medium for obtaining pre-oligodendrocyte progenitor cells (pre-OPCs) or oligodendrocyte progenitor cells (OPCs) that contains a retinoic acid (RA) pathway agonist, an Akt pathway agonist, and an mTOR pathway agonist, and lacks externally added growth factors. [Invention 1033] A culture medium according to the present invention 1032, further comprising a WNT pathway antagonist and an SHH pathway agonist. [Invention 1034] A culture medium according to the present invention 1033, further comprising a BMP pathway antagonist and a PKC pathway antagonist. [Invention 1035] Isolated cell cultures of pre-oligodendrocyte progenitor cells (pre-OPCs) or oligodendrocyte progenitor cells (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 externally added growth factors. [Invention 1036] The isolated cell culture according to Invention 1035, wherein the culture medium further comprises a WNT pathway antagonist and an SHH pathway agonist, and the pre-OPC or OPC also expresses OTX2 and FEZF2. [Invention 1037] An isolated cell culture according to the present invention 1035 or 1036, wherein the culture medium further comprises a BMP pathway antagonist and a PKC pathway antagonist. [Invention 1038] An isolated cell culture according to any of invention 1035-1037, wherein pre-OPC or OPC is attached to a plate coated with vitronectin. [Invention 1039] Pre-oligodendrocyte progenitor cells (pre-OPCs) or oligodendrocyte progenitor cells (OPCs) prepared by any of the methods described in 1001 to 1032 of this invention. [Invention 1040] A composition comprising non-natural pre-oligodendrocyte progenitor cells (pre-OPCs) or oligodendrocyte progenitor cells (OPCs), wherein the pre-OPCs or OPCs express OLIG2, NKX2-2, OTX2, and FEZF2, and lack expression of NESTIN. [Invention 1041] at least 1 × 10 6 An isolated cell population of pre-OPCs or OPCs, comprising 1 OLIG2-expressing pre-OPCs or oligodendrocyte progenitor cells (OPCs), wherein the isolated cell population lacks neural stem cells expressing NESTIN. [Invention 1042] An isolated cell population of the present invention 1041, in which pre-OPC or OPC also expresses NKX2-2, OTX2, and FEZF2. [Invention 1043] An isolated cell population according to the present invention 1041 or 1042, wherein pre-OPC or OPC is conjugated with at least one antibody bound to at least one marker expressed by pre-OPC or OPC. Other features and advantages of the present invention will become apparent from the following detailed description and claims. [Brief explanation of the drawing]

[0025] [Figure 1] Figure 1 shows the results from an HD-DoE model of an 8-factor experiment optimized for maximizing NKX2-2 expression. The upper panel of the model shows the predicted expression levels of 53 pre-selected genes when optimized for NKX2-2. The lower panel of the model shows the effectors tested in this model and their contributions to maximizing NKX2-2 expression. The value column indicates the concentration of each effector required to mimic the model. [Figure 2] Figure 2 shows the results from an 8-factor HD-DoE model optimized to maximize PDGFRA expression. The upper and lower panels are as described in Figure 1. Under these conditions, the effector PD0325901, with a factor contribution of 30.05, is emphasized as an important input for high PDGFRA expression. [Figure 3] Figure 3 shows the dynamic profiles of the expression levels of the NKX2-2, OLIG1, OLIG2, and PDGFRA genes against the concentrations of the eight effectors tested. The positive effects of TTNPB, MHY1485, and PD0325901 on PDGFRA expression, and their factor contributions, are indicated by the slope of the plot for each effector. The dotted boxes highlight the opposing effects of PD0325901 on NKX2-2 and OLIG2 compared to PDGFRA. [Figure 4] Figure 4 shows the results from a 13-factor HD-DoE model optimized to maximize OTX2 expression. In this model, MK2206, PD0325901, CHIR99021, LDN193189, Go6983, and PD173074 were introduced as positive effectors for OTX2 expression. [Figure 5]Figure 5 shows the results from an HD-DoE model of a 13-factor experiment optimized to maximize FEZF2 expression. In this model, the positive effects of LDN193189, MK2206, and PD0325901 on cell patterning were confirmed, and three other factors, including SC79, XAV939, and palmorfamine-500nM, were introduced. [Figure 6] Figure 6 shows the dynamic profiles of OTX2 and FEZF2 expression levels against the concentrations of the 13 factors tested in this model. XAV939 and palmorfamine-500nM had a significant positive effect on FEZF2 expression, but no significant negative effect on OTX2 expression. [Figure 7A] Figures 7A-D show the dynamic profile analysis of the exclusion process in the 8-factor modeling experiment and its effects on the expression of NKX2-2, OLIG2, and PDGFRA. [Figure 7B] See the explanation in Figure 7A. [Figure 7C] See the explanation in Figure 7A. [Figure 7D] See the explanation in Figure 7A. [Figure 8A] Figures 8A-D show the dynamic profile analysis of the exclusion process in the 13-factor modeling experiment and its effect on the expression of FEZF2 and OTX2. [Figure 8B] See the explanation in Figure 8A. [Figure 8C] See the explanation in Figure 8A. [Figure 8D] See the explanation in Figure 8A. [Figure 9]Figure 9 shows a photograph of cells cultured in optimized OPC differentiation medium after 3 days. Cells were stained with oligodendrocyte and neuronal biomarkers. The 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 the proliferation state of progenitor cells. [Figure 10] Figures 10A-B show RNA-seq data from cells cultured in optimized OPC differentiation medium after 3 days. While the expression levels of stem cell genes NANOG and POU5F1 decreased, genes involved in the early development of brain regions and oligodendrocyte lineages increased. [Modes for carrying out the invention]

[0026] Detailed description of the invention This specification describes methodologies and compositions that enable the production of pre-OPCs and OPCs from human pluripotent stem cells under chemically defined culture conditions using a small molecule-based approach. The method disclosed herein has the advantage that the starting pluripotent stem cells do not undergo neural induction, which is employed in many prior art protocols. This makes it possible to produce pre-OPCs and OPCs in as little as 3 days, which is significantly shorter than the average 10 days required for pre-OPC production in current protocols.

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

[0028] Various aspects of the present invention will be described in more detail in the following subsections.

[0029] I. cell The starting cells used in the cultures described herein are human pluripotent stem cells. As used herein, “human pluripotent stem cells” (abbreviated as hPSC) refers to human stem cells that have the ability to differentiate into various different cell types. As used herein, “pluripotency” refers to cells that, under different conditions, have the ability to differentiate 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, a 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., 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 cellular markers that can be used to identify cells as PSCs. Non-limiting examples of pluripotent stem cell markers include TRA-1-60, TRA-1-81, TRA-2-54, SSEA1, SSEA3, SSEA4, CD9, CD24, OCT3, OCT4, NANOG, and / or SOX2. Since the methods for producing pre-OPC and / or OPC according to this disclosure are used to differentiate (mature) a starting pluripotent stem cell population, in various embodiments, the pre-OPC and / or OPC cell population produced by the methods of this disclosure lacks the expression of one or more stem cell markers selected from the group consisting of TRA-1-60, TRA-1-81, TRA-2-54, SSEA1, SSEA3, SSEA4, CD9, CD24, OCT3, OCT4, NANOG, and / or SOX2.

[0031] Pluripotent stem cells are subjected to culture conditions that induce cell differentiation, as described herein. As used herein, “differentiation” refers to the development of cells from a more primitive stage to a more mature (i.e., less primitive) cell, which typically exhibits phenotypic features deeply associated with a particular cell lineage. Early progenitor cells that can be induced from human PSCs by neural induction (neuronization) are neural progenitor cells (NPCs). As used herein, “neural progenitor cell” or “NPC” refers to stem cell-derived progenitor cells expressing the type VI intermediate filament protein nestin. Since the methods for producing pre-OPCs and / or OPCs according to this disclosure do not generate NPCs to avoid the use of neural induction, in various embodiments, the cell populations produced by the methods of this disclosure lack nestin-positive cells.

[0032] In one embodiment, the cells produced by the method of this disclosure are pre-oligodendrocyte progenitor cells (pre-OPCs). As used herein, “pre-oligodendrocyte progenitor cells” or “pre-OPCs” refers to stem cell-derived progenitor cells expressing the cell markers OLIG2 and NKX2.2. Pre-OPCs may express additional markers, including but not limited to OTX2 (anterior neuroectoderm biomarker), FEZF2 (anterior ectoderm biomarker), and / or OLIG1.

[0033] In one embodiment, the cells produced by the method of this disclosure are oligodendrocyte progenitor cells (OPCs), which are more differentiated (more mature) than pre-OPCs. As used herein, “oligodendrocyte progenitor cells” or “OPC” refers to stem cell-derived progenitor cells that express PDGFRa in addition to the cell markers OLIG2 and NKX2.2. 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 produced by the methods of this disclosure can be further cultured in vitro to produce mature oligodendrocytes (OLs). Markers for mature OLs include, but are not limited to, myelin basic protein (MBP) and O4.

[0035] II. Components of culture medium A method for producing pre-OPC or OPC according to the present disclosure comprises the step of culturing human pluripotent stem cells in a culture medium that lacks externally added growth factors and contains specific agonists and / or antagonists of cell signaling pathways.

[0036] As described in Example 1, a culture medium containing a retinoic acid (RA) pathway agonist, an Akt pathway agonist, and an mTOR pathway agonist was sufficient to produce pre-OPCs expressing OLIG2 and NKX2.2 in just 3 days. Including 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 antagonists, SHH pathway agonists, BMP pathway antagonists, and PKC pathway antagonists. 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, in which case 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, an SHH pathway agonist, a BMP pathway antagonist, and a PKC pathway antagonist. In one embodiment, the differentiated cells are OPCs expressing at least OLIG2, NKX2.2, and PDGFRa (and may further express additional markers such as OTX2, FEZF2, and / or OLIG1).

[0038] As used herein, the term "agonist" of a cellular signaling pathway is intended to refer to a drug that stimulates (upregulates) a cellular signaling pathway. Stimulation of a cellular signaling pathway may be initiated extracellularly, for example, by the use of an agonist that activates a cell surface receptor involved in the signaling pathway (for example, the agonist may be a receptor ligand). Alternatively, stimulation of cellular signaling may 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, the term “antagonist” of a cellular signaling pathway is intended to refer to a drug that inhibits (downregulates) the cellular signaling pathway. Inhibition of a cellular signaling pathway may be initiated extracellularly, for example, by the use of an antagonist that blocks cell surface receptors involved in the signaling pathway. Alternatively, inhibition of cellular signaling may be initiated intracellularly, for example, by the use of a small molecule antagonist that interacts intracellularly with a component(s) of the signaling pathway.

[0040] Retinoic acid (RA) pathway agonists, Akt pathway agonists, mTOR pathway agonists, WNT pathway antagonists, SHH pathway agonists, BMP pathway antagonists, and PKC pathway antagonists are known and commercially available in the art. They are used in culture media at concentrations effective to achieve the desired outcome, such as the generation of pre-OPC and / or OPC expressing a marker of interest. A non-limiting list of suitable agonist and antagonist agents, as well as effective concentration ranges, will be discussed further below.

[0041] Agonists of the retinoic acid pathway include drugs, molecules, compounds, or substances that can stimulate retinoic acid receptors (RARs), which are activated by both total trans retinoic acid and 9-cis retinoic acid. There are three RARs: RARα, RARβ, and RARγ, encoded by the RARA, RARB, and RARG genes, respectively. Various retinoic acid analogs capable of activating the retinoic acid pathway have been synthesized. Non-limiting examples of such compounds include TTNPB (agonist of RARα, β, and γ), AM 580 (RARα agonist), CD 1530 (potent and selective RARγ agonist), CD 2314 (selective RARβ agonist), Ch 55 (potent RAR agonist), BMS 753 (RARα selective agonist), tazarotene (receptor-selective 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, RA signaling pathway agonists are 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-cisretinoic acid, 13-cisretinoic acid, 4-oxoletinoic acid, and total trans retinoic acid (ATRA).

[0042] Therefore, 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-cisretinoic acid, 13-cisretinoic acid, 4-oxoletinoic acid, and total trans retinoic acid (ATRA), or combinations thereof. In one embodiment, the RA pathway agonist is present in the culture medium at concentrations in the range of 5-500 mM, 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, present in the culture medium at a concentration in the range of 5-500 nM, 10-100 nM, or 25-75 nM. In another embodiment, the RA pathway agonist is TTNPB, present in the culture medium at a concentration of 50 nM.

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

[0044] Agonists of the mTOR (mammalian target of rapamycin) pathway include drugs, molecules, compounds, or substances that can stimulate (activate) signaling mediated by mTOR (a member of the PI3K-related kinase family, a core component of the mTORC1 and mTORC2 complex). In one embodiment, the mTOR pathway agonist is selected from the group consisting of MHY1485, 3BDO, thalidroside, L-leucine, NV-5138, and combinations thereof. In one embodiment, the mTOR pathway agonist is present in the culture medium at concentrations ranging from 0.1 to 10 μM, or 0.5 to 5 μM, or 0.5 to 2.5 μM, or 0.5 to 1.5 μM. In one embodiment, the mTOR pathway agonist is MHY1485. In one embodiment, the mTOR pathway agonist is MHY1485, present in the culture medium at concentrations of 0.1–10 μM, 0.5–5 μM, 0.5–2.5 μM, or 0.5–1.5 μM. In another embodiment, the mTOR pathway agonist is MHY1485, present in the culture medium at a concentration of 1 μM.

[0045] Antagonists of the WNT pathway include drugs, molecules, compounds, or substances that can inhibit (downregulate) the canonical Wnt / β-catenin signaling pathway; the Wnt / β-catenin signaling pathway is biologically activated when Wnt protein ligands bind 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, tryptonide, 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 concentrations 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, present in the culture medium at concentrations of 10-500 nM, 50-250 nM, or 50-150 nM. In another embodiment, the WNT pathway antagonist is XAV939, present in the culture medium at a concentration of 100 nM.

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

[0047] BMP (bone morphogenetic protein) pathway antagonists include drugs, molecules, compounds, or substances that can inhibit (downregulate) the BMP signaling pathway; the BMP signaling pathway is biologically activated by the binding of BMP to its BMP receptor, which is an activin receptor-like kinase (ALK) (including, but not limited to, ALK2 and ALK3, type I BMP receptors). In one embodiment, the BMP pathway antagonist is selected from the group consisting of LDN193189, DMH1, DMH2, dolsomorphine, K02288, LDN214117, LDN212854, follistatin, ML347, noggin, and combinations thereof. In one embodiment, the BMP pathway antagonist is present in the culture medium at concentrations in 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 concentrations 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] PKC (protein kinase C) pathway antagonists 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, a 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 concentrations ranging from 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 concentrations ranging from 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 The methods for preparing pre-OPCs and OPCs according to this disclosure, in combination with the chemically defined and optimized culture media described in subsection II above, utilize standard culture conditions established in the art with respect to 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, starting pluripotent stem cells are attached to a plate, 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).

[0050] Pluripotent stem cells can be cultured in commercially available media prior to differentiation. For example, stem cells may be cultured in Essential 8 Flex medium (Thermo Fisher #A2858501) for at least one day prior to the initiation of a differentiation protocol. In a non-limiting, exemplary embodiment, stem cells are subcultured 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.

[0051] To initiate the differentiation protocol, the culture medium in which the stem cells are cultured is replaced with basal differentiation medium, which is supplemented with agonists and / or antagonists of the signaling pathways described above in Subsection II. Basal differentiation medium may include, for example, commercially available basic media supplemented with additional standard media components necessary to maintain cell viability and proliferation, but lacking serum (basal differentiation medium is serum-free) or other externally added growth factors, such as FGF2, PDGF, IGF, HGF, etc. In a non-limiting, exemplary embodiment, the basal differentiation medium contains 1 × IMDM (Thermo Fisher #12440046), 1 × F12 (Thermo Fisher #11765047), 1 mg / ml of poly(vinyl alcohol) (Sigma #p8136), 1% of a lipid concentrate with a clearly defined chemical composition (Thermo Fisher #11905031), 450 μM of 1-thioglycerol (Sigma #M6145), 0.7 μg / ml of insulin (Sigma #11376497001), and 15 μg / ml of transferrin (Sigma #10652202001).

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

[0053] To produce pre-OPCs and / or OPCs, stem cells are cultured in an optimized culture medium for a sufficient amount of time for cell differentiation and expression of pre-OPC or OPC-related markers. As described in the examples, it has been found that culturing stem cells in an optimized culture medium for as little as 72 hours (3 days) is sufficient to differentiate them into pre-OPCs and OPCs. Therefore, in one embodiment, cells are cultured for at least 72 hours. In another embodiment, cells are cultured for at least 60, 64, 68, 72, 76, 80, 84, 88, 92, or 96 hours. In yet another embodiment, cells are cultured for at least 2.5, 3, 3.5, 4, 4.5, or 5 days.

[0054] IV. Purpose The methods and compositions of this disclosure for preparing pre-OPCs and OPCs enable the efficient and robust utilization of these cell populations for a variety of applications. For example, these methods and compositions can be used in the study of oligodendrocyte development and biology to aid in the understanding of oligodendrocyte-related diseases and disorders. For example, pre-OPCs and / or OPCs prepared using the methods of this disclosure 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 embodiment, this disclosure provides a method for isolating pre-oligodendrocyte progenitor cells (pre-OPCs) or oligodendrocyte progenitor cells (OPCs), the method comprising the following steps: A step of contacting a pre-OPC or OPC expressing OLIG2, prepared by the method of the present disclosure, with at least one binder that binds to a cell surface marker expressed by the pre-OPC or OPC; and A step of isolating cells that bind to the binder, thereby isolating pre-OPC or OPC.

[0055] In one embodiment, the conjugate is an antibody, for example, 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 bound to the antibody can be isolated by methods known in the art, including but not limited to fluorescence-activated cell sorting (FACS) and magnetically activated cell sorting (MACS).

[0056] Oligodendrocyte-derived progenitor cells are also intended for use in the treatment of various diseases and disorders by delivering them 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 white matter atrophy, and amyotrophic lateral sclerosis (ALS).

[0057] V. composition In other aspects, the present disclosure provides compositions related to methods for producing 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-OPC or OPC that lacks externally added growth factors and contains 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 an 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, an SHH pathway agonist, a BMP pathway antagonist, and a PKC pathway antagonist.

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

[0060] In another aspect, the present disclosure provides pre-oligodendrocyte progenitor cells (pre-OPCs) or oligodendrocyte progenitor cells (OPCs) produced by the differentiation methods of the present disclosure. In one aspect, 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 NESTIN expression. In another aspect, the present disclosure provides an isolated cell population of pre-OPCs or OPCs comprising pre-OPCs or OPCs that express at least 1×10 6 cells of OLIG2 and NKX2.2, wherein the cell population lacks neural stem cells that express NESTIN. In other aspects, the cell population comprises pre-OPCs or OPCs that express at least 5×10 6 cells, 1×10 7 cells, 5×10 7 cells, 1×10 8 cells, 5×10 8 cells, or 1×10 9 cells of OLIG2 and NKX2.2. In one aspect 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. 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 limitations. The contents of the drawings and all references, patents and published patent applications cited throughout this application are hereby expressly incorporated herein by reference.

Examples

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

[0063] This embodiment utilizes 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 a deep mathematical modeling of the effector / response space. This method makes it possible to find combinatorial signaling inputs that control complex processes such as cell differentiation. This allows testing of multiple valid and important process parameters, as such parameters influence output responses such as gene expression. Since gene expression provides prominent phenotypic features, for example, in human cells, this method can be applied to identify and understand which signaling pathways control cell fate. In this embodiment, the HD-DOE method was applied with the aim of finding the conditions that induce the expression of genes in oligodendrocyte progenitor cells directly from the pluripotent stem cell state.

[0064] Specifically, to develop a novel method for generating oligodendrocytes, the effects of agonists and antagonists (referred to herein as effectors) of multiple signaling pathways on the expression of 53 pre-selected genes in two sets after a 3-day treatment were tested. These effectors are small molecules commonly used to differentiate stem cells stepwise to specific fates. The selection of effectors was based on the latest literature on neural induction in the anterior ectoderm and differentiation from stem cells to oligodendrocytes.

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

[0066] As shown in the results summarized in Figure 1, one model clearly demonstrated promising results regarding the upregulation of the NKX2-2 and OLIG2 genes when optimized to maximize NKX2-2 expression at 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—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 their target genes; TTNPB had the largest factor contribution of 31.3, followed by MHY1485 at 13.8 and SC79 at 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 were consistent with the gene expression patterns during oligodendrocyte differentiation.

[0068] As shown in the results summarized in Figure 2, the normalized expression of PDGFRA in this model could reach 832.9, the highest expression level among all models; therefore, this model was also optimized to maximize PDGFRA expression. This setting also showed that TTNPB (a retinoic acid pathway agonist) and MHY1485 (an mTOR signaling pathway agonist) also had a positive effect on PGFRA upregulation, 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. Due to its low factor contribution (<1), ZM336372 was not included in the culture medium recipe. Under these conditions, the average expression level of OLIG1 was 228.36, similar to the optimization conditions for NKX2-2. One difference under these conditions is that the OLIG2 gene was downregulated from 1049.4 under the previous conditions to 241.9.

[0069] As shown in the results summarized in Figure 3, of the effectors that positively contribute to the expression levels of NKX2-2, OLIG2, or PDGFRA, two factors negatively affected the expression levels of NKX2-2 and OLIG2. Therefore, these two factors, PD0325901 and ZM336372, were excluded from the candidate list of oligodendrocyte differentiation culture medium recipes.

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

[0071] HD-DoE #2 To further enhance the conditions for oligodendrocyte differentiation from pluripotency, the inventors conducted additional HD-DoE experiments. They obtained an additional gene regulatory model, which they used to prepare the differentiation protocol. This was based on 13-factor HD-DoE experiments focusing on the initiation of cell differentiation into the anterior neuroectoderm. This model emphasized the expression of FEZF2 and OTX2.

[0072] As shown in the results summarized in Figure 4, this model was optimized to achieve the highest OTX2 expression 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 at 22.2, and PD173074 and BLU9931 having the lowest factor contributions at 1.7.

[0073] As shown in the results summarized in Figure 5, this model was optimized to maximize FEZF2 expression at 4466. When the model was optimized for maximum FEZF2 expression, three effectors—LDN193189 with a factor contribution of 19.5, PD0325901 with a factor contribution of 14.6, and MK2206 with a factor contribution of 12.3—were common to the previous condition, and three new effectors were introduced, including palmorphamine-500nM, XAV939, and SC79.

[0074] As shown in the results summarized in Figure 6, dynamic profiling analysis was performed to fine-tune this recipe 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 palmorfamine (an agonist of the SHH signaling pathway, known to ventrally diverge cells during brain region development) had a significant positive effect on FEZF2 expression and no negative effect on OTX2 expression levels. Therefore, these two factors were added to the optimized culture medium recipe.

[0075] In addition to including factors that promote the expression of OPC-related surface markers, specific factors that inhibit the expression of such markers were excluded from the optimized culture medium recipe. CHIR99021, an agonist of the WNT signaling pathway, was excluded. MK2206 and PD0325901 were also excluded because, according to the 8-factor model, they negatively affected the expression of oligodendrocyte genes. PD173074 and BLU9931 were also excluded due to their low factor contribution of 1.7.

[0076] summary Considering both models, the culture conditions that maximized the differentiation of human induced pluripotent stem cells into cells with oligodendrocyte progenitor (OPC) identity, leading to 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), 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 that induce OPC To evaluate the factor criticality of each component in the optimized culture medium described in Example 1, in-silico predictive analysis of the results was performed under conditions where individual effectors were excluded and other effectors were present. For this purpose, the inventors used dynamic profiling analysis at the set point, comparing the expression levels of the gene of interest in the absence of each factor. Since the expression of the gene of interest reveals whether the desired result can be achieved, this factor criticality analysis revealed the degree of importance of each input effector.

[0078] Figures 7A-D summarize the results of factor importance analysis for the effectors TTNPB (RA pathway agonist), SC79 (Akt pathway agonist), and MHY1485 (mTOR pathway agonist). Figure 7A shows the expression levels of target OPC genes in the presence of TTNPB, MHY1485, and SC79 when the model is optimized to maximize NKX2-2 expression. As shown in Figure 7B, when TTNPB is removed, the predicted expression levels of NKX2-2, OLIG2, and PDGFRA decrease significantly: NKX2-2 from over 12000 to 4500, OLIG2 from 1000 to 400, and PDGFRA from 350 to less than 100. This result indicates that the removal of RA pathway agonists has a significant negative impact on the expression of all desirable markers. As shown in Figure 7C, removing MHY1485 also reduced the expression level, but not as abruptly as under the previous conditions. As shown in Figure 7D, removing SC79 resulted in only a slight shift in the plot, suggesting that this factor is not as important as TTNPB and MHY1485 for maximizing the induction of OPC markers.

[0079] Figures 8A-D summarize the results of factor importance analysis for the effectors palmorfamine (SHH pathway agonist), XAV939 (WNT pathway antagonist), LDN193189 (BMP pathway antagonist), and Go6983 (PKC pathway antagonist). These additional factor inputs were queried to FEZF2 and OTX2, among others, in order to achieve the desired patterning of oligodendrocyte populations in the anterior brain region. When the model was optimized to maximize FEZF2 expression, the expression levels of FEZF2 and OTX2 were examined in the absence of either LDN193189, XAV, palmorfamine, or Go6983. The most significant change was observed in the absence of LDN193189, with FEZF2 expression decreasing by almost 50% (from 4500 to 2500). OTX2 expression also decreased from 9000 to 7000, the lowest in all four exclusion processes. Removal of XAV939 and palmorfamine reduced FEZF2 expression levels to 3000 and 3500, respectively, while OTX2 expression remained only slightly higher in both cases. Since no significant changes were observed in the expression levels of the target gene when Go6983 was removed, it was suggested that Go6983 is optional in relation to 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 the expression of biomarkers for anterior neuroectoderm and oligodendrocyte progenitor cells was evaluated using immunocytochemistry. Biomarkers included OTX2 and oligodendrocyte progenitor cell biomarkers such as NKX2-2, OLIG2, and PDGF. To distinguish neural stem cells from oligodendrocyte progenitor cells, nestin, an early neuronal cell marker, was used. Ki67 was also used to confirm cell proliferation after induction. Representative immunohistochemical results are shown in Figure 9. These immunocytochemical images confirmed that most cells expressed OTX2. However, no trace of nestin, a neuronal cell biomarker, was detected, confirming that the differentiated OPC population lacked neural stem cells. Expression of OLIG2 and NKX2-2 was also observed in over 90% of cells, thereby confirming the oligodendrocyte lineage of the cells. None of the cells expressed PDGFR, which was expected as this gene is expressed in the later stages of oligodendrocyte differentiation.

[0081] Example 4: RNA-seq validation of stem cell-derived pre-OPC 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. Figure 10A shows the normalized expression levels of selected genes representing various brain regions, early oligodendrocyte identity (NKX2-2, OLIG2, PDGFRa), and stem cell status (NANOG, POU5F1) in three replicate experiments on days 0 and 3. The results demonstrate that in pre-OPC cells, the levels of stem cell genes decreased while the levels of oligodendrocyte genes increased, thus validating the differentiation of hiPSCs into oligodendrocyte lines using the differentiation medium. Figure 10B shows the differential expression and pluripotency changes of selected genes, with HOXA1 at the highest level (15) and OLIG2, NKX2-2, and PDGFRa at 5. This data demonstrates the capability of the recipe developed as a first-step culture medium for inducing cells toward oligodendrocyte identity.

[0082] Equal portions Those skilled in the art will recognize, or confirm by routine experimentation, many equivalents of the specific embodiments of the present invention described herein. Such equivalents shall be covered by the following claims.

Claims

1. A method for producing human pre-oligodendrocyte progenitor cells (pre-OPCs) or oligodendrocyte progenitor cells (OPCs), comprising the following steps: The process involves culturing human pluripotent stem cells in a culture medium lacking externally added growth factors and containing a retinoic acid (RA) agonist, an Akt agonist, a mammalian target of rapamycin (mTOR) agonist, a sonic hedgehog (SHH) agonist, a wingless-related integration site (WNT) antagonist, a bone morphogenetic protein (BMP) antagonist, and a protein kinase C (PKC) antagonist, so that pre-OPCs or OPCs expressing OLIG2 are generated.

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

3. The method according to claim 1, wherein the human pluripotent stem cell is an embryonic stem cell.

4. The method according to any one of claims 1 to 3, wherein a pre-OPC or OPC expressing OLIG2 is generated in the culture medium within 72 hours of initiating the culture of human pluripotent stem cells.

5. The method according to any one of claims 1 to 4, wherein pre-OPC or OPC also expresses NKX2-2.

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

7. The method according to 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-cisretinoic acid, 13-cisretinoic acid, 4-oxoletinoic acid, total trans retinoic acid (ATRA), and combinations thereof.

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

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

14. The method according to 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 according to 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 according to claim 1, 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, tryptonide, KYA1797K, JW55, JW67, JW74, Cardionogen 1, NLS-StAx-h, TAK715, PNU 74654, iCRT3, WIF-1, DKK1, and combinations thereof.

17. The method according to claim 16, wherein the WNT antagonist is present in the culture medium at a concentration in the range of 10 to 500 nM.

18. The method according to claim 17, wherein the WNT antagonist is XAV939 and is present in the culture medium at a concentration in the range of 10 to 500 nM.

19. The method according to claim 1, wherein the SHH agonist is selected from the group consisting of palmorfamine, GSA 10, SAG, and combinations thereof.

20. The method according to claim 19, wherein the SHH agonist is present in the culture medium at a concentration in the range of 100 to 1000 nM.

21. The method according to claim 20, wherein the SHH antagonist is palmorfamine and is present in the culture medium at a concentration in the range of 100 to 1000 nM.

22. The method according to claim 1, wherein the BMP antagonist is selected from the group consisting of LDN193189, DMH1, DMH2, dolsomorphine, K02288, LDN214117, LDN212854, follistatin, ML347, noggin, and combinations thereof.

23. The method according to claim 22, wherein the BMP antagonist is present in the culture medium at a concentration in the range of 100 to 1000 nM.

24. The method according to claim 23, wherein the BMP antagonist is LDN193189 and is present in the culture medium at a concentration in the range of 100 to 1000 nM.

25. The method according to claim 1, 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.

26. The method according to claim 25, wherein the PKC antagonist is present in the culture medium at a concentration in the range of 10 to 500 nM.

27. The method according to claim 26, wherein the PKC antagonist is Go6983 and is present in the culture medium at a concentration in the range of 10 to 500 nM.

28. A culture medium for obtaining pre-OPC or OPC, comprising an RA agonist, an Akt agonist, an mTOR agonist, a WNT antagonist, an SHH agonist, a BMP antagonist, and a PKC antagonist, and lacking externally added growth factors.

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