SOX9 derived oligodendrocyte progenitor cells

Abstract

Provided is a differentiation agent consisting essentially of SOX9 for generating oligodendrocyte progenitor cells (OPCs) from pluripotent stem cells (PSCs). Also provided are methods of generating PSCs and methods of using PSCs to generate OPCs and oligodendrocytes.SOLUTION: A method comprising culturing pluripotent stem cells (PSCs) to produce a population of cells comprising oligodendrocyte progenitor cells (OPCs), wherein the PSCs express engineered nucleic acids encoding SOX9 at a level sufficient to differentiate the PSCs into OPCs in the absence of other transcription factors encoded by the engineered nucleic acids.SELECTED DRAWING: Figure 2C

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 USC §119(e) of U.S. Provisional Patent Application No. 62 / 855,135, filed May 31, 2019, which is incorporated herein by reference in its entirety.

[0002] Federally supported research This invention was made with government support under grants AG048056, HG008525 and MH103910 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] background Oligodendrocytes are a subtype of glial cell in the central nervous system that originate from oligodendrocyte progenitor cells (OPCs). OPCs account for approximately 5% of the cells in the central nervous system. Oligodendrocytes help support and protect axons by producing myelin. The myelin sheath in the central nervous system is composed of long oligodendrocyte plasma membranes. Mature oligodendrocytes are unable to self-renew, but OPCs repopulate oligodendrocytes after injury to the central nervous system in healthy individuals. Summary of the Invention [Means for solving the problem]

[0004] overview The present disclosure unexpectedly provides experimental data demonstrating that overexpression of the transcription factor SOX9 is sufficient to generate oligodendrocyte progenitor cells (OPCs) (e.g., O4-positive OPCs) from induced pluripotent stem cells (iPSCs) in as little as four days (e.g., without optimizing cell growth and / or differentiation culture conditions). This data was particularly surprising given the implication of SOX9 as a fundamental master regulator of chondrocyte development. Nevertheless, the data described herein demonstrate that chondrocyte morphology was not observed after SOX9 overexpression; rather, SOX9 overexpression was sufficient to induce oligodendrocyte differentiation.

[0005] Additionally, in some embodiments, the oligodendrocytes generated are "immunoprotective" - ​​they secrete immunosuppressive factors (eg, anti-inflammatory cytokines).

[0006] Accordingly, some aspects of the present disclosure provide methods comprising contacting pluripotent stem cells (PSCs) with a transcription factor consisting essentially of SOX9. In some embodiments, the transcription factor consists essentially of one or more engineered nucleic acids encoding SOX9. In some embodiments, the method further comprises introducing at least one engineered nucleic acid encoding interleukin-10 (IL-10) and / or interferon beta (IFNβ) into the pluripotent stem cells. In some embodiments, the method further comprises culturing the OPCs to generate oligodendrocytes.

[0007] Yet another aspect of the present disclosure provides pharmaceutical compositions comprising OPCs or oligodendrocytes produced by the methods described herein. These pharmaceutical compositions can be used, for example, to treat (e.g., ameliorate) demyelinating disorders such as multiple sclerosis, transverse myelitis, and other congenital and non-congenital demyelinating disorders. [Brief explanation of the drawings]

[0008] BRIEF DESCRIPTION OF THE DRAWINGS [Figures 1A-1D]1A-1D show that SOX9 overexpression induces hiPSCs to differentiate into OPCs, but not into chondrocytes. [Figure 1A] FIG. 1A shows human induced pluripotent stem cells (hiPSCs) exhibiting a colony-like morphology with close cell-cell packing and distinct, well-defined colony boundaries. [Figure 1B] FIG. 1B shows that SOX9-transfected cells differentiate into a branched and spread morphology 4 days after induction. [Figure 1C] FIG. 1C shows that SOX9-transfected cells differentiate into branched and spread morphologies 4 days after induction. [Figure 1D] Figure ID shows the percentage of O4-positive, GalC-negative cells in various populations generated by transfecting different amounts of DNA: 5 μg, 10 μg, and 20 μg. These graphs show the percentage of O4-positive, GalC-negative cells after 4 days of doxycycline treatment. [Figures 2A-2D] 2A-2D show that certain SOX9 stem cell clones can achieve near-complete OPC differentiation. [Figure 2A-2B] Figures 2A-2B show the morphological changes in individual colonies after 4 days. [Figure 2A] FIG. 2A shows that control cells in the absence of doxycycline had no stem cell differentiation after 4 days. [Figure 2B] FIG. 2B shows that in the presence of doxycycline, the cells differentiate, leaving behind iPSC colonies. [Figure 2C] Figure 2C shows that SOX9 rapidly and efficiently induced hiPSCs into induced oligodendrocytes at 4 dpi. Flow cytometry bar graph of O4 oligodendrocyte marker compared to uninduced cells. [Figure 2D] Figure 2D shows that SOX9 rapidly and efficiently induces hiPSCs into induced oligodendrocytes at 4 dpi. Flow cytometry bar graph of NG2 oligodendrocyte marker compared to uninduced cells. [Figure 3A-3B]Figures 3A-3B show that SOX9-induced OPCs form compact myelin around hiPSC-derived neurons and express mature markers in human cerebral organoids. [Figure 3A] Figure 3A shows a co-culture of OPCs and hiPSC-derived neurons after 4 weeks. The co-culture was sectioned vertically and imaged using transmission electron microscopy. A layer of myelin (M) wraps around the axons (A), demonstrating the functionality of the differentiated OPCs. [Figure 3B] Figure 3B shows that differentiated OPCs can myelinate human cerebral organoids. SOX9 overexpression induced cerebral organoids mixed with inducible OPCs to differentiate and stained positive for the mature myelin marker MOG. [Figure 4] Figure 4 shows that OPCs can be engineered to secrete anti-inflammatory cytokines. An ELISA assay was used to detect the secretion of the anti-inflammatory cytokine IL10 from differentiated OPCs engineered with various amounts of transfected DNA. [Figure 5A-5B] 5A-5B contain data showing that SOX9-induced OPCs can myelinate hypomyelinated axons in shiverer mice. [Figure 5A] Figure 5A shows a TEM image of a brain section from a treatment cohort in which mice received SOX9-induced OPCs. White arrows indicate aggregated myelination. Magnification 9300x. [Figure 5B] Figure 5B shows a TEM image of a brain section from the control group in which mice received PBS injections, magnification 9300x. [Figure 6] Figure 6 shows that shiverer mice transplanted with SOX9-inducing cells have significantly more myelinated axons than PBS-injected animals. Quantification of the number of myelinated axons from PBS injection or SOX9-inducing cell transplantation into shiverer mice. [Figure 7]Figure 7 shows that SOX9-induced cells resemble primary oligodendrocytes in terms of transcriptome. Principal component (PC) analysis of RNA-seq samples from SOX9-induced cells reveals transcriptomes similar to those of oligodendrocyte progenitor cells (OPCs), overlapping with samples from primary mature oligodendrocytes (OLs), and clearly separating them from newly formed oligodendrocytes (OLs) and PGP1 hiPSCs. [Figure 8] Figure 8 shows a schematic diagram of parallel programming in which engineered human induced pluripotent stem cells (hiPSCs) that inducibly express differentiation transcription factors (TFs) are co-cultured in a dish. TFs are induced to generate multiple cell types in the same medium. This schematic demonstrates that parallel programming utilizes cell-autonomous fate specification to enable simultaneous differentiation of multiple cell types in the same dish. [Figure 9] Figure 9 shows a schematic of orthogonal programming in which engineered hiPSCs for TF-induced differentiation are incorporated into developmentally activated cerebral organoids during development to synthetically accelerate myelination. [Figure 10A] FIG. 10A shows that quantification of the G ratio for myelin compaction is within the physiological range. [Figure 10B] Figure 10B shows that quantification of the G ratio for myelin compaction in cerebral organoids exhibits physiological similarity. ***P<0.001. [Figure 11] 11 is a graph showing that transfection of more SOX9 PIGGYBAC™ DNA increases the amount of SOX9 PIGGYBAC™ vector integrated into the genome. The X-axis shows the amount of transfected DNA. The Y-axis shows the population average copy number of the exogenous SOX9 plasmid. [Figure 12] FIG. 12 is a graph showing that electroporation of increasing amounts of SOX9 DNA up to 2.5 μg improved OPC differentiation efficiency as measured by flow cytometry for the percentage of O4+ cells. DETAILED DESCRIPTION OF THE INVENTION

[0009] Detailed Description Induced pluripotent stem cells (iPSCs) can be reprogrammed from differentiated adult cells to develop various phenotypes. iPSCs can be obtained from patients and transformed into any cell type needed to improve a specific condition, enabling patient-specific autologous clinical applications while minimizing the risk of immune response or rejection and enabling the scalable generation of allogeneic cells for similar clinical applications. The use of stem cell therapy for clinical applications such as neurodegenerative and myelin-degenerative diseases, myocardial infarction, and bone defect repair has shown promise, but has significant limitations related to uncontrolled proliferation, low cell survival, negative immune responses, differentiation into unwanted cell types, inconsistency, and long treatment times. For example, neural stem cells can be used to generate oligodendrocytes, but this process is inefficient, and the cells often form neurons or astrocytes that lack the ability to produce myelin. In contrast, O-4-positive OPCs appear to be primarily committed to developing into oligodendrocytes. Furthermore, OPCs have been demonstrated to integrate into the CNS and remyelinate a mouse model of congenital dysmyelination (Najm et al., Nat. Biotechnol. 31, 426-433 (2013)). However, these protocols remain largely impractical for application due to long culture times, complex culture conditions, and low differentiation efficiency. For example, one of the most technically successful protocols required over 200 days of differentiation and seven media conditions to induce approximately 12% of cells to express the surface marker O4 antigen (Wang et al., Cell Stem Cell. 12, 252-264 (2013)). More recent studies have shortened the timeline to 44–75 days and simplified the regimen to 4–5 steps, achieving O4 expression efficiencies of approximately 30%–70% (Douvaras et al., Nat. Protoc. 10, 1143–54 (2015); Ehlrich et al., Proc Natl Acad Sci, 114(11):E2243–E2252 (2017)).

[0010] The technology provided herein overcomes many of these limitations. The present disclosure provides methods for SOX9-mediated direct reprogramming of stem cells, e.g., iPSCs, to generate desired cell types in as few as 1-8 days, in some cases with high efficiency (e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of cells expressing markers of differentiation). For example, cells can be reprogrammed within 2-8, 3-8, or 4-8 days. In some embodiments, cells can be reprogrammed using the methods described herein in less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days, or less than 1 day. In some embodiments, cells can be reprogrammed (e.g., express differentiation markers) within 1 day. In some embodiments, cells can be reprogrammed (e.g., express differentiation markers) within 4 days. The present disclosure also provides methods for protecting desired cell types from immune (inflammatory) responses.

[0011] Autoimmune diseases resulting from a dysfunctional immune system may be suitable for stem cell therapy. Multiple sclerosis (MS), which affects approximately 2.5 million people worldwide, is an autoimmune disease that is often targeted by mesenchymal stem cell (MSC) therapy. However, success to date has been limited, primarily due to the aforementioned limitations. Existing strategies for overcoming stem cell hurdles remain largely impractical due to long culture times, complex culture conditions, and low efficiency.

[0012] The data provided herein demonstrate that, in some embodiments, SOX9-mediated direct reprogramming of iPSCs can be used to generate OPCs (e.g., O4-positive OPCs) without the need for media regimen optimization. This approach is significantly faster and more efficient than existing reprogramming methods. The data provided herein also demonstrate that these iPSCs and cells generated from these iPSCs can be programmed to secrete proinflammatory cytokines, thus rendering these cells immunoprotective.

[0013] The present disclosure is based, at least in part, on unexpected results demonstrating that SOX9 is sufficient to induce the formation of OPCs, which can be used to rebuild damaged myelin sheaths around axons, for example, in subjects with demyelinating disorders.

[0014] As used herein, a differentiation agent is a substance that causes differentiation (a process by which a cell changes from one cell type to another, e.g., herein, a PSC can differentiate into an OPC). Lineage-specifying genes encode differentiation agents. Examples of differentiation agents used herein include transcription factors (and nucleic acids encoding transcription factors), such as OLIG transcription factors (e.g., OLIG1, OLIG2, OLIG3 and OLIG4), NKX homeobox transcription factors (e.g., NKX2.1, NKX2.2, NKX6.1, NKX6.2 and NKX6.3), OCT transcription factors (e.g., OCT1, OCT2, OCT4 and OCT6), SOX transcription factors (e.g., SRY, SOX1, SOX2, SOX3, SOX4, SOX5, SOX6, SOX7, SOX8, SOX9, SOX10, SOX11, SOX12, SOX13, SOX14, SOX15, SOX17, SOX18, SOX21 and SOX30), and ASCL transcription factors (e.g., ASCL1 and ASCL2). As used herein, a transcription factor is a protein that regulates RNA transcription by binding to DNA in the promoter or enhancer region of a gene and interacting with RNA polymerase or other transcription factors. See, for example, International Publication No. 2018 / 049382, published March 15, 2018, entitled "TRANSCRIPTION FACTORS CONTROLLING DIFFERENTIATION OF STEM CELLS," International Publication No. 2019 / 108894, published June 6, 2019, entitled "METHODS AND COMPOSITIONS FOR THE PRODUCTION OF OLIGODENDROCYTE PROGENITOR CELLS," and U.S. Patent Application Publication No. 20200063105, published February 27, 2020, entitled "TRANSCRIPTION FACTORS CONTROLLING DIFFERENTIATION OF STEM CELLS."

[0015] In some embodiments, the methods described herein comprise (a) contacting PSCs (e.g., iPSCs) with a differentiation agent, where the differentiation agent consists essentially of SOX9 or one or more copies of a nucleic acid encoding SOX9, and (b) culturing the PSCs to generate a population of cells comprising OPCs. In another embodiment, the methods described herein comprise (a) contacting PSCs (e.g., iPSCs) with a transcription factor, where the transcription factor consists essentially of SOX9 or one or more copies of a nucleic acid encoding SOX9, and (b) culturing the PSCs to generate a population of cells comprising OPCs.

[0016] In some embodiments, the PSCs of the present disclosure do not include any exogenous transcription factors or exogenous nucleic acids encoding transcription factors other than SOX9. Accordingly, it should be understood that any PSCs consisting essentially of SOX9 of the present disclosure do not include any exogenous OLIG, NKX, or OCT transcription factors or exogenous nucleic acids encoding any one or more of the OLIG, NKX, or OCT transcription factors. It should also be understood that none of the PSCs consisting essentially of SOX9 of the present disclosure comprise exogenous SRY, SOX1, SOX2, SOX3, SOX4, SOX5, SOX6, SOX7, SOX8, SOX10, SOX11, SOX12, SOX13, SOX14, SOX15, SOX17, SOX18, SOX21 or SOX30 or exogenous nucleic acid encoding any one or more of SRY, SOX1, SOX2, SOX3, SOX4, SOX5, SOX6, SOX7, SOX8, SOX10, SOX11, SOX12, SOX13, SOX14, SOX15, SOX17, SOX18, SOX21 or SOX30.

[0017] Oligodendrocyte precursor cells and oligodendrocytes Oligodendrocyte progenitor cells (OPCs) are a subtype of glial cells in the central nervous system characterized by expression of the proteoglycans PDGFRA and NG2 (CSPG4). They are the precursors of oligodendrocytes, which function to support and protect axons by producing the myelin sheath wrapping. Mature oligodendrocytes are unable to self-renew, but OPCs can repopulate oligodendrocytes after injury to the central nervous system in healthy individuals.

[0018] In some embodiments, the OPCs or populations thereof are expressing or expressing any of the following proteins: myelin basic protein (MBP), NK2 homeobox 1 (NKX2-1), myelin oligodendrocyte glycoprotein (MOG), myelin-associated oligodendrocyte basic protein (MOBP), oligodendrocyte transcription factor 2 (OLIG2), chondroitin sulfate proteoglycan 4 (CSPG4), oligodendrocyte transcription factor 1 (OLIG1), SRY-box transcription factor 8 (SOX8), SRY-box transcription factor 10 (SOX10), myelin regulatory factor (MYRF), platelet-derived growth factor receptor alpha (PDGFRA), matrix metalloproteinase (MTCR ... The cells express at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, or at least sixteen oligodendrocyte genes selected from the group consisting of metallopeptidase 15 (MMP15), proteolipid protein 1 (PLP1), transmembrane protein 88B (TMEM88B), ectonucleotide pyrophosphatase / phosphodiesterase 6 (ENPP6), and neurofascin (NFASC).

[0019] In some embodiments, at least one gene is downregulated in OPCs or populations thereof, hi some embodiments, at least one, at least two, at least three, or at least four of the genes selected from the group consisting of myelin regulatory factor (MYRF), POU class 5 homeobox 1 (POU5F1), Nanog homeobox (NANOG), and SRY-box transcription factor 2 (SOX2) are downregulated in OPCs or populations thereof.

[0020] As stem cells develop into oligodendrocytes, each developmental stage can be characterized by specific cell surface markers. For example, membrane chondroitin sulfate proteoglycan NG2 (CSPG4) can be used as a marker for early proliferative OPCs. The oligodendrocyte marker O4 can be used as an indicator of mid- to late-stage OPCs (Jackman, N et al., Physiology (24):290-7 (2009)). In some embodiments, OPCs generated by the methods of the present disclosure are mid- to late-stage OPCs that express O4. Myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG) are expressed upon terminal differentiation of OPCs into oligodendrocytes. Both are oligodendrocyte-specific genes and can be used as markers for mature oligodendrocyte formation. MOG is a membrane protein found on the surface of oligodendrocyte cells and on the outer layer of the myelin sheath. GalC is a galactosylceramidase enzyme found on the oligodendrocyte membrane and can be used as a marker for late OPCs (postmitotic) and early mature oligodendrocytes. GalC can also be used as a marker for terminal differentiation. In some embodiments, oligodendrocytes generated by the methods of the present disclosure can be mature oligodendrocytes that express MOG and / or GalC. Additional markers of oligodendrocyte developmental stages (e.g., markers for early OPCs, intermediate and late OPCs, and mature oligodendrocytes) are known in the art and can be used as described herein. See, e.g., Jackman, N et al., Physiology (24):290-7 (2009).

[0021] In some embodiments, the oligodendrocytes express at least one, at least two, at least three, at least four, at least five, or at least six biomarkers selected from the group consisting of 2',3'-cyclic nucleotide 3' phosphodiesterase (CNPase), proteolipid protein 1 (PLP1), galactosylceramidase (GALC), myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), and myelin-associated glycoprotein (MAG).

[0022] In some embodiments, the OPCs are engineered to express SOX9 and / or contain an engineered nucleic acid encoding SOX9.

[0023] pluripotent stem cells Provided herein are methods for reprogramming pluripotent stem cells to generate OPCs (e.g., O4-positive OPCs). Pluripotent stem cells are cells that have the ability to self-renew by division and to develop into the three primary germ layers of the early embryo, thus giving rise to all cells of the adult body but not to extraembryonic tissues such as the placenta. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are multipotent stem cells. ESCs are obtained from the undifferentiated inner cell mass of an embryo and can differentiate into all derivatives of the three primary germ layers: ectoderm, endoderm, and mesoderm. iPSCs can be generated directly from adult cells (Takahashi, K; Yamanaka, S. Cell 126(4):663-76, 2006). In some embodiments, the pluripotent stem cells are ESCs. In some embodiments, the pluripotent cells are iPSCs. In some embodiments, the pluripotent stem cells are human ESCs. In some embodiments, the pluripotent cells are iPSCs. In some embodiments, the pluripotent cells are human iPSCs.

[0024] Pluripotent stem cells, such as iPSCs, can be engineered to express SOX9. In some embodiments, a differentiation agent consisting essentially of SOX9 or a nucleic acid encoding SOX9 is introduced into the pluripotent stem cells. In some embodiments, the only differentiation agent introduced into the pluripotent stem cells (e.g., iPSCs) is SOX9 or a nucleic acid encoding SOX9. In some embodiments, the only transcription factor introduced into the pluripotent stem cells (e.g., iPSCs) is SOX9 or a nucleic acid encoding SOX9.

[0025] The stem cells of the present disclosure are engineered. Engineered cells are cells that contain at least one engineered (e.g., recombinant or synthetic) nucleic acid or are otherwise modified so that they are structurally and / or functionally different from their natural counterparts. Thus, cells that contain an exogenous nucleic acid sequence are considered to be engineered cells.

[0026] In some embodiments, the engineered cells of the present disclosure are engineered pluripotent stem cells (e.g., induced pluripotent stem cells), oligodendrocyte precursor cells (OPCs), or oligodendrocytes. As used herein, unless otherwise indicated, cells engineered to express SOX9 can be engineered to constitutively express SOX9 or engineered to inducibly express SOX9.

[0027] SRY-Box 9 (SOX9) In some embodiments, pluripotent stem cells engineered to generate OPCs contain SRY-box 9 (SOX9). SOX9 is a member of the SOX (SRY-related HMG-box) family of transcription factors, which is characterized by a high mobility group (HMG) box DNA sequence. The HMG box is a DNA-binding domain that is highly conserved across eukaryotic species. The Sox family does not have a single function; many members have the ability to regulate several different aspects of development. SOX9 has been implicated in chondrogenesis. See, e.g., Bi et al., Nat Genet. 1999 May;22(1):85-9.

[0028] As used herein, SOX9 or a homolog or variant thereof can be human or other mammalian SOX9. Other SOX9 transcription factors (e.g., from other species) are known, and nucleic acids encoding SOX9 can be found in publicly available gene databases such as GenBank. In some embodiments, a nucleic acid encoding wild-type human SOX9 is at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 100%) identical to the open reading frame of the nucleic acid set forth in NCBI RefSeq under accession number Z46629.

[0029] In some embodiments, the nucleic acid encoding SOX9 is at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 100%) identical to SEQ ID NO: 1. In some embodiments, the amino acid sequence encoding SOX9 is at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 100%) identical to SEQ ID NO:2.

[0030] The SOX9 described herein may contain one or more amino acid substitutions relative to its wild-type counterpart. Mutants can be prepared according to methods for modifying polypeptide sequences well known to those skilled in the art, such as those found in references compiling such methods, for example, Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative amino acid substitutions include substitutions between amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0031] In some embodiments, the differentiation agent consists solely of SOX9 or one or more copies of a nucleic acid encoding SOX9.

[0032] How to generate an OPC Provided herein are methods of generating OPCs. In some embodiments, the methods comprise introducing at least one engineered nucleic acid encoding SOX9 into pluripotent stem cells and culturing the pluripotent stem cells to generate OPCs. In some embodiments, the OPCs express O4, NG2, or a combination thereof.

[0033] OPCs of the present disclosure can further comprise anti-inflammatory cytokines. For a discussion of anti-inflammatory cytokines, see, e.g., Opal et al., Chest. 2000 (4):1162-72 and Benveniste et al., Sci STKE. 2007 (416):pe70. OPCs in the body do not naturally express anti-inflammatory cytokines (Cannella B, Raine CS. Ann. Neurol. 2004 Jan;55(1):46-57). For example, OPCs of the present disclosure can comprise a nucleic acid encoding an anti-inflammatory cytokine. In some embodiments, the anti-inflammatory cytokine reduces the ability of OPCs to activate the immune system (e.g., suppress T cell activation). In some embodiments, the level of pro-inflammatory cytokines (e.g., cytokines that activate the immune system, including IL7 and IFNγ) secreted by T cells in the presence and absence of OPCs can be used to determine the ability of OPCs to activate the immune system. In other embodiments, in vitro cell proliferation assays can be used to assess the effect of cytokines (e.g., IL10 and IFNβ) expressed by engineered OPCs on T cell proliferation. Cytokine levels can be determined using assays known in the art, including enzyme-linked immunosorbent assay (ELISA) assays (see Example 3 in the Examples section below). In some embodiments, OPCs harboring an anti-inflammatory cytokine reduce T cell activation by at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold (e.g., as measured by reduced levels of secreted pro-inflammatory cytokines) compared to OPCs not harboring the anti-inflammatory cytokine under the same or substantially the same conditions.

[0034] Anti-inflammatory cytokines suitable for the present disclosure include, but are not limited to, interferons and interleukins. For example, cells can be engineered to express interferon beta (IFNβ) and / or interleukin 10 (IL10). In some embodiments, engineered OPCs express / contain IFNβ (e.g., also referred to herein as IFNβ1). In some embodiments, engineered OPCs express / contain IL10. In some embodiments, engineered OPCs express / contain both IFNβ and IL10. Sequences for interferons and cytokines can be obtained from publicly available databases, such as GenBank at the National Center for Biotechnology Information. An exemplary interferon beta 1 (IFNβ) sequence is set forth under GenBank accession identifier NM_002176. An exemplary IL10 sequence is set forth under GenBank accession identifier NM_000572. It should be understood that in some embodiments, only open reading frames are used to express SOX9 and the cytokines described herein.

[0035] The OPCs may secrete an anti-inflammatory cytokine or promote the secretion of another anti-inflammatory cytokine. For example, the anti-inflammatory cytokine may promote the secretion of IL10. In some embodiments, OPCs harboring the anti-inflammatory cytokine secrete at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 1,500-fold, at least 2,000-fold, at least 3,000-fold, at least 4,000-fold, at least 5,000-fold, at least 6,000-fold, at least 7,000-fold, at least 8,000-fold, at least 9,000-fold, or at least 10,000-fold more IL10 than control counterparts not harboring the anti-inflammatory cytokine.

[0036] In some embodiments, OPCs harboring an anti-inflammatory cytokine secrete at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 1,500-fold, at least 2,000-fold, at least 3,000-fold, at least 4,000-fold, at least 5,000-fold, at least 6,000-fold, at least 7,000-fold, at least 8,000-fold, at least 9,000-fold, or at least 10,000-fold more IFNβ than control counterparts that do not harbor the anti-inflammatory cytokine.

[0037] In some embodiments, iPSCs are engineered to express IL-10 and SOX9. In some embodiments, the engineered iPSCs differentiate into OPCs (e.g., O4+ cells) within 1-10 days of induction of IL-10 and SOX9 expression. In some embodiments, the engineered cells (e.g., iPSCs or OPCs) secrete IL-10. In some embodiments, the engineered cells (e.g., engineered iPSCs or engineered OPCs) secrete IL-10 at levels that are at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 1,500-fold, at least 2,000-fold, at least 3,000-fold, at least 4,000-fold, at least 5,000-fold, at least 6,000-fold, at least 7,000-fold, at least 8,000-fold, at least 9,000-fold, or at least 10,000-fold higher than control cells. The control cells can be naturally occurring iPSCs or OPCs, engineered iPSCs or OPCs that have not been specifically modified to express IL-10, or iPSCs or OPCs that have been engineered to inducibly express IL-10 but are cultured in the absence of the inducer (i.e., IL-10 expression is not induced). In some embodiments, the control cells are the same type of cell (e.g., iPSCs or OPCs) as the engineered cells. In some embodiments, the iPSCs or OPCs secrete IL-10 at levels at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold higher than the control cells within 1 to 10 days of induction of SOX9 expression.

[0038] In some embodiments, the engineered cells secrete IL-10 (e.g., steadily / continuously) over a culture period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days (e.g., in the presence or absence of an inducer).

[0039] In some embodiments, iPSCs or iPSC-derived cells are engineered to express IL10 and / or IFNβ.

[0040] In some embodiments, iPSCs are engineered to express IFNβ and SOX9. In some embodiments, the engineered iPSCs differentiate into OPCs (e.g., O4+ cells) within 1-10 days of induction of IFNβ and SOX9 expression. In some embodiments, the engineered cells (e.g., engineered iPSCs or engineered OPCs) secrete IL-10. In some embodiments, the engineered cells (e.g., engineered iPSCs or engineered OPCs) secrete IL-10 at levels that are at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 1,500-fold, at least 2,000-fold, at least 3,000-fold, at least 4,000-fold, at least 5,000-fold, at least 6,000-fold, at least 7,000-fold, at least 8,000-fold, at least 9,000-fold, or at least 10,000-fold higher than control cells. Control cells include naturally occurring iPSCs or OPCs, engineered iPSCs or OPCs that have not been specifically modified to express IFNβ, or iPSCs or OPCs that have been engineered to inducibly express IFNβ but are cultured under conditions that lack the inducing agent. In some embodiments, the engineered iPSCs or OPCs secrete IL-10 at levels that are at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold higher than control cells within 1 to 10 days of induction of IFNβ and SOX9 expression.

[0041] In some embodiments, iPSCs are engineered to express IFNβ, IL-10, and SOX9. In some embodiments, the engineered iPSCs differentiate into OPCs (e.g., O4+ cells) within 1-10 days of induction of IFNβ, IL-10, and SOX9 expression. In some embodiments, the engineered iPSCs or engineered OPCs secrete IL-10. In some embodiments, the engineered cells (e.g., engineered OPCs or engineered iPSCs) secrete IL-10 at levels that are at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 1,500-fold, at least 2,000-fold, at least 3,000-fold, at least 4,000-fold, at least 5,000-fold, at least 6,000-fold, at least 7,000-fold, at least 8,000-fold, at least 9,000-fold, or at least 10,000-fold higher than control cells. Control cells include naturally occurring iPSCs or OPCs, engineered iPSCs or OPCs that have not been specifically modified to express IFNβ and IL-10, and iPSCs or OPCs that have been engineered to inducibly express IFNβ and IL-10 but are cultured under conditions that lack the inducing agent. In some embodiments, the engineered iPSCs or OPCs secrete IL-10 at levels at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold higher than control cells within 1 to 10 days of induction of IFNβ, IL-10, and SOX9 expression.

[0042] A nucleic acid is generally at least two nucleotides covalently linked to each other and, in some cases, may include a phosphodiester bond (e.g., a phosphodiester "backbone"). A nucleic acid is considered to be "engineered" if it does not occur in nature. Examples of engineered nucleic acids include recombinant nucleic acids and synthetic nucleic acids. In some embodiments, the engineered nucleic acid encodes SOX9.

[0043] Nucleic acids encoding SOX9, IL-10, IFNβ, or a combination thereof described herein can be introduced into pluripotent stem cells using any known method, including, but not limited to, chemical transfection, viral transduction (e.g., using lentiviral vectors, adenoviral vectors, Sendai virus, and adeno-associated viral vectors), and electroporation. For example, methods that do not require genomic integration include transfection of mRNA encoding SOX9 and / or cytokines (e.g., IL-10, IFNβ, or a combination thereof) and introduction of episomal plasmids. In some embodiments, an episomal vector (e.g., an episomal plasmid) is used to deliver nucleic acids (e.g., mRNA) to pluripotent stem cells. In other embodiments, a nucleic acid encoding a transcription factor can be integrated into the genome of a cell to reprogram pluripotent stem cells. Genomic integration methods are known, any of which may be used herein, including the use of the PIGGYBAC™ transposon system, the Sleeping Beauty system, lentivirus, adeno-associated viral systems, and CRISPR gene editing systems.

[0044] In some embodiments, the engineered nucleic acid is present on a PIGGYBAC™ transposon vector, which contains PIGGYBAC™ inverted terminal repeats flanking a nucleotide sequence encoding SOX9, and / or a cytokine (e.g., IL-10, IFNβ, or a combination thereof), and / or an anti-inflammatory cytokine of the present disclosure. PIGGYBAC™ transposase is an enzyme that recognizes the PIGGYBAC™ inverted terminal repeats on either side of an inserted sequence (e.g., a sequence encoding SOX9 and / or a cytokine (e.g., IL-10, IFNβ, or a combination thereof), removes the inserted sequence, and inserts the removed element into another nucleic acid. PIGGYBAC™ transposase can insert the removed sequence into a target site containing the sequence TTAA. An exemplary sequence encoding PIGGYBAC™ transposase is set forth in GenBank Accession No. EF587698.

[0045] In some embodiments, SOX9 and / or cytokines (e.g., IL-10, IFNβ, or a combination thereof) are cloned into a PIGGYBAC™ transposon vector and then nucleofected into iPSCs at high copy number to co-deliver PIGGYBAC™ transposase and integrate into the genome (e.g., an average of 10 copies per cell). In some embodiments, the high copy number integrated into the genome is 5-50 copies (inclusive) of nucleotide sequences encoding SOX9 and / or cytokines (e.g., IL-10, IFNβ, or a combination thereof) per cell (e.g., an average of 5-50 copies, inclusive, or exactly 5-50 copies, inclusive). In some embodiments, the cells have at least 5, 10, 15, 20, 25, or 50 copies of nucleotide sequences encoding SOX9 and / or cytokines (e.g., IL-10, IFNβ, or a combination thereof). In some embodiments, the cells have at least 15 copies of a nucleotide sequence encoding SOX9 and / or a cytokine (eg, IL-10, IFNβ, or a combination thereof).In some embodiments, the cells (or a population of cells, on average) contain 1 to 20 copies, 2 to 20 copies, 3 to 20 copies, 4 to 20 copies, 5 to 20 copies, 6 to 20 copies, 7 to 20 copies, 8 to 20 copies, 9 to 20 copies, 11 to 20 copies, 12 to 20 copies, 13 to 20 copies, 14 to 20 copies, 5 to 6 copies, 5 to 7 copies, 5 to 8 copies, 5 to 9 copies, 5 to 10 copies, 5 to 20 copies, 5 to 30 copies, 5 to 40 copies, 10 to 20 copies, 1 to 3 ... The gene has 0 to 11 copies, 10 to 12 copies, 10 to 13 copies, 10 to 14 copies, 10 to 15 copies, 10 to 16 copies, 10 to 17 copies, 10 to 18 copies, 10 to 19 copies, 15 to 16 copies, 15 to 17 copies, 15 to 18 copies, 15 to 19 copies, 10 to 30 copies, 10 to 40 copies, 10 to 50 copies, 15 to 20 copies, 15 to 25 copies, 15 to 30 copies, 15 to 35 copies, 15 to 40 copies, 15 to 45 copies, 15 to 50 copies, 20 to 30 copies, 20 to 40 copies, 20 to 50 copies, 30 to 40 copies, 30 to 50 copies, or 40 to 50 copies. In some embodiments, the cell (or a population of cells, on average) has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 copies of a nucleotide sequence encoding SOX9 and / or a cytokine (e.g., IL-10, IFNβ, or a combination thereof). In some cases, the copy number refers to the average copy number of at least one nucleotide sequence encoding SOX9 and / or a cytokine per cell in a population of cells (e.g., in a polyclonal population of cells). In some cases, the copy number refers to the exact copy number of at least one nucleotide sequence encoding SOX9 and / or a cytokine in all cells in the cell or population of cells (e.g., in a clonal population of cells).

[0046] In some embodiments, the population of pluripotent stem cells has, on average, fewer than 20 copies (but at least 1 copy) of the engineered nucleic acid encoding SOX9 per cell. In some embodiments, the population of pluripotent stem cells has, on average, fewer than 19 copies, fewer than 18 copies, fewer than 17 copies, fewer than 16 copies, fewer than 15 copies, fewer than 14 copies, fewer than 13 copies, fewer than 12 copies, fewer than 11 copies, fewer than 10 copies, fewer than 9 copies, fewer than 8 copies, fewer than 7 copies, fewer than 6 copies, fewer than 5 copies, fewer than 4 copies, fewer than 3 copies, or fewer than 2 copies (but at least 1 copy) of the engineered nucleic acid encoding SOX9 per cell.

[0047] In some embodiments, high copy number can be achieved by introducing a high concentration of DNA into a population of cells, hi some embodiments, the high concentration of DNA is a concentration of 1,000 ng or more of DNA per million cells, more than 2,000 ng of DNA per million cells, more than 5,000 ng of DNA per million cells, or more than 10,000 ng of DNA per million cells.

[0048] In some embodiments, the low copy number is achieved by introducing a low concentration of DNA into a population of cells. In some embodiments, the low concentration of DNA is less than 1,000 ng of DNA per million cells, less than 500 ng of DNA per million cells, less than 400 ng of DNA, or less than 300 ng of DNA per million cells.

[0049] In some embodiments, the PSCs comprise an engineered nucleic acid encoding SOX9 at a level sufficient to differentiate the PSCs into oligodendrocyte progenitor cells (OPCs) in the absence of other transcription factors encoded by the engineered nucleic acid. As disclosed herein, oligodendrocyte progenitor cells can be identified by one or more markers, such as, but not limited to, O4 and other OPC-specific markers. In some embodiments, the average copy number of SOX9 in a population of PSC cells (e.g., 1-5, 1-10, 1-15, or 1-20 copies) is sufficient to differentiate at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the PSCs into OPCs.

[0050] A particular copy number or range of copy numbers can result in a percentage of cells expressing O4 and / or other OPC-specific markers. For example, at least 10% of a population of pluripotent stem cells may express O4 and / or other OPC-specific markers in a population of pluripotent stem cells having a population average of 10 copies of SOX9. In some embodiments, at least 15% of a population of pluripotent stem cells may express O4 and / or other OPC-specific markers in a population of pluripotent stem cells having a population average of 15 copies of SOX9. In some embodiments, at least 20% of a population of pluripotent stem cells may express O4 and / or other OPC-specific markers in a population of pluripotent stem cells having a population average of 20 copies of SOX9. In some embodiments, at least 20% of a population of pluripotent stem cells may express O4 and / or other OPC-specific markers in a population of pluripotent stem cells having a population average of 20 copies of SOX9. In some embodiments, at least 10-25% of a population of pluripotent stem cells may express O4 and / or other OPC-specific markers in a population of pluripotent stem cells that has a population average of 10-20 copies of SOX9.

[0051] In some embodiments, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of a population of pluripotent stem cells may express O4 and / or other OPC-specific markers in a population of pluripotent stem cells having a population average of 5-10, 5-15, or 5-20 copies of SOX9. In some embodiments, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of a population of pluripotent stem cells may express O4 and / or other OPC-specific markers in a population of pluripotent stem cells having a population average of 10-15 or 10-20 copies of SOX9. In some embodiments, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of a population of pluripotent stem cells may express O4 and / or other OPC-specific markers in a population of pluripotent stem cells having a population average of 10-15 or 10-20 copies of SOX9. In some embodiments, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of a population of pluripotent stem cells may express O4 and / or other OPC-specific markers in a population of pluripotent stem cells having a population average of 15-20 copies of SOX9.

[0052] The plasmids can be engineered to be, for example, antibiotic resistant and / or inducible (e.g., doxycycline inducible) to allow selection of SOX9- and / or cytokine-integrated cells and / or to control transcription.

[0053] In some embodiments, the PIGGYBAC™ transposon vector comprises PIGGYBAC™ inverted terminal repeats flanking a nucleotide sequence encoding SOX9. In some embodiments, the PIGGYBAC™ transposon vector comprises PIGGYBAC™ inverted terminal repeats flanking a nucleotide sequence encoding at least one cytokine. In some embodiments, the PIGGYBAC™ transposon vector comprises PIGGYBAC™ inverted terminal repeats flanking a nucleotide sequence encoding SOX9 and a cytokine. In some embodiments, the nucleotide sequences encoding SOX9 and at least one cytokine are in the same cassette flanked by PIGGYBAC™ inverted terminal repeats. The at least two cytokines or SOX9 and at least one cytokine in this cassette may or may not be separated to generate unlinked proteins, for example, by separating the nucleic acids encoding the proteins of interest (e.g., SOX9, cytokine, or a combination thereof) by a polypeptide cleavage signal such as an internal ribosome entry site (IRES) or a 2A sequence. In some embodiments, the nucleotide sequence encoding IFNβ and / or IL-10 is flanked by PIGGYBAC™ inverted terminal repeats.

[0054] In some embodiments, the engineered nucleic acid is present on an expression plasmid, which is introduced into the pluripotent stem cells. In some embodiments, the expression plasmid includes a selection marker, such as an antibiotic resistance gene (e.g., bsd, neo, hygB, pac, ble, or Sh bla) or a gene encoding a fluorescent protein (e.g., RFP, BFP, YFP, or GFP). In some embodiments, the antibiotic resistance gene encodes a puromycin resistance gene. In some embodiments, the selection marker allows for the selection of cells expressing the protein of interest. In some cases, the protein of interest is SOX9 or a cytokine.

[0055] Any of the engineered nucleic acids described herein can be produced using conventional methods. For example, recombinant or synthetic techniques can be used to produce nucleic acids encoding SOX9, cytokines, or combinations thereof described herein. Conventional cloning techniques can be used to insert SOX9, cytokines, or combinations thereof into a PIGGYBAC™ transposon vector.

[0056] In some embodiments, the engineered nucleic acid (optionally present on an expression plasmid) comprises a nucleotide sequence encoding SOX9, a cytokine, or a combination thereof operably linked to a promoter (promoter sequence). In some embodiments, the promoter is an inducible promoter (e.g., comprising a tetracycline regulatory sequence). The inducible promoter allows, for example, temporal and / or spatial control of expression of SOX9, a cytokine, or a combination thereof.

[0057] A promoter is a regulatory region of a nucleic acid sequence that controls the initiation and rate of transcription of the remaining nucleic acid sequence. A promoter may also contain subregions to which regulatory proteins and molecules bind, such as RNA polymerase and other transcription factors. A promoter may be constitutive, inducible, activatable, repressible, tissue-specific, or any combination thereof. A promoter drives the expression or transcription of a nucleic acid sequence that it regulates. As used herein, a promoter is considered to be "operably linked" when it is in the correct functional location and orientation with respect to the nucleic acid sequence that it regulates for initiation of transcription and / or control ("drive") the expression of that nucleic acid sequence.

[0058] An inducible promoter is one that is characterized by initiating or enhancing transcriptional activity in the presence of, affected by, or contacted with an inducing agent, which may be an endogenous or, more commonly, an exogenous condition, compound, or protein, that is contacted with the engineered active nucleic acid to induce transcriptional activity from the inducible promoter.

[0059] Inducible promoters for use in accordance with the present disclosure include any inducible promoter described herein or known to those of skill in the art. Examples of inducible promoters include, but are not limited to, chemically / biochemically regulated and physically regulated promoters, such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems, including tetracycline repressor protein (tetR), tetracycline operator sequence (tetO), and tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., rat glucocorticoid receptor, human estrogen receptor, galactosidase inhibitor ... These promoters include promoters based on the (moth) ecdysone receptor and promoters from the steroid / retinoid / thyroid 25 receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (a protein that binds and sequesters metal ions) genes from yeast, mouse, and human), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene, or benzothiadiazole (BTH)), temperature / heat-inducible promoters (e.g., heat shock promoters), and light-regulated promoters (e.g., light-responsive promoters from plant cells).

[0060] Preparations of pluripotent stem cells (e.g., expressing SOX9) can be cultured under standard stem cell culture conditions. For example, the pluripotent stem cells can be cultured in any commercially available feeder-free maintenance medium for human ESCs and iPSCs, such as mTeSR™1 medium. In some embodiments, the pluripotent stem cells are cultured in commercially available stem cell medium without the addition of nutrients or growth factors.

[0061] Preparations of pluripotent stem cells (e.g., expressing SOX9) can, in some embodiments, generate OPCs (e.g., O4-positive OPCs) after only 4-10 days of culture. In some embodiments, the pluripotent stem cells are cultured for 4-10 days, 4-9 days, 4-8 days, 4-7 days, 4-6 days, or 4-5 days. In some embodiments, the pluripotent stem cells generate OPCs (e.g., O4-positive OPCs) after being cultured for less than 4 days (e.g., 1, 2, and 3 days). In some embodiments, at least 10% (at least 20%, 30%, 40%, 50%, 60%, 70%, or 80%) of the cells in the preparation express O4 after only 4-10 days (e.g., 4, 5, 6, 7, 8, 9, or 10 days) of culture.

[0062] Preparations of pluripotent stem cells (e.g., expressing SOX9) can be prepared, e.g., at 10 4 ~10 10 In some embodiments, the preparation of pluripotent stem cells may comprise 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 or 10 10 Contains cells.

[0063] Expression of O4 and other OPC-specific markers can be assessed based on protein or nucleic acid expression using known methods. Additional OPC lineage markers include, but are not limited to, Sox1, Pax6, Nestin, Islet1, A2B5, Sox10, Olig2, Olig1, PDGFRa, NG2, RIP, O1, PLP1, CNPase, GalC, MBP, MAG, and MOG. In some embodiments, OPCs do not express OCT4, Nanog, and / or SOX2. Exemplary methods include immunofluorescence using a fluorophore-conjugated anti-O4 antibody, Western blot analysis using an anti-O4 antibody, quantitative polymerase chain reaction using primers targeting O4, and fluorescence-activated cell sorting (FACS) using an anti-O4 antibody. In some embodiments, the method further includes sorting for O4-positive OPCs (e.g., using FACS).

[0064] In some embodiments, the method further comprises culturing the OPCs to generate oligodendrocytes. Culturing the OPCs may include using medium containing factors that induce oligodendrocyte differentiation, including growth hormones (e.g., fibroblast growth factor). Oligodendrocytes may be characterized by cell surface markers (e.g., MOG and GalC). Oligodendrocytes may also be characterized by, or alternatively, the ability to form myelination. As exemplified below, cells engineered by the methods disclosed herein can be co-cultured (e.g., in polyethylene glycol molds) with iPSC-derived neurons for a specified period of time (e.g., several weeks) and assessed for myelination (e.g., myelination can be assessed by fixing, embedding in resin, sectioning, staining, and imaging the co-culture using transmission electron microscopy). In some embodiments, the engineered nucleic acids of the present disclosure increase the number of myelinated axons by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold. In some embodiments, the extent of myelination can be calculated by determining the g-ratio. The axonal g-ratio is the ratio of the inner diameter to the outer diameter of the myelin sheath. In some embodiments, the axonal g-ratio from a subject administered any of the engineered cells of the present disclosure is similar to the g-ratio of myelin from a control (e.g., the subject prior to administration of the engineered cells or a healthy subject). In some embodiments, the axonal g-ratio from a subject administered any of the engineered cells of the present disclosure is similar to the axonal g-ratio from a control (e.g., the subject prior to administration of the engineered cells or a healthy subject) if the axonal g-ratio of myelin from the control is, on average, between 0.6 and 0.8. See, e.g., Mohammadi et al., Front Neurosci. 2015 Nov 27;9:441.

[0065] In some embodiments, the OPCs described herein are engineered to express IL-10 and / or IFNβ. In some embodiments, the OPCs are cultured to express IL-10 and / or IFNβ (e.g., using an inducer when cytokine expression is controlled by an inducible promoter). Without being bound by theory, OPCs engineered to express IL-10 and / or IFNβ may be useful in treating subjects with myelin degenerative diseases (e.g., multiple sclerosis). Without being bound by theory, immune-tolerant cells, such as engineered OPCs capable of secreting IL10 and IFNβ, can not only regenerate and remyelinate axons, but also protect themselves and neighboring cells from immune attack. In some embodiments, OPCs engineered to express IL-10 and / or IFNβ can reduce or suppress the immune response of a host receiving the OPCs, promoting regeneration.

[0066] In some embodiments, the engineered OPCs are cultured with other cells (e.g., neurons) to generate myelinated organoids. In some embodiments, the myelinated organoids are transplanted into a subject in need thereof (e.g., a subject with a demyelination disorder). In some embodiments, the organoids are cerebral organoids (e.g., partial or complete cerebral organoids). In some embodiments, the engineered myelinated organoids are used for compound screening (e.g., therapeutic agents) or disease modeling.

[0067] In some embodiments, the differentiation agent in the engineered stem cells (eg, iPSCs) and / or OPCs consists essentially of SOX9.

[0068] Without being bound by any particular theory, limitations of established cell programming methods stem from attempts to mimic the long-term timeline and inherent complexity of developmental biology. During in vivo development, the cell-type specification process is intertwined with other regulatory events that spatially and temporally arrange the correct cell type for a defined population size. As demonstrated herein, in some embodiments, this limitation can be addressed by using synthetic biology-based cell programming, which allows for the decoupling of developmental processes. To achieve the engineered goal of fully controlling cell identity in a cell-autonomous manner, the differentiation process is decoupled from other developmental events. Current protocols rely on external signals, such as soluble factors or mechanical cues. Therefore, independently controlling the development of different cell types within the same culture has been extremely challenging. Without being bound by any particular theory, this is particularly important because tissues are composed of various lineages and germ layers. As an example, the brain is composed of neural cells from the ectodermal lineage, but is vascularized by endothelial cells derived from the mesoderm. Without being bound by theory, current approaches using external cues are impractical for simultaneously inducing both lineages due to incompatible induction conditions for each specialized cell type. However, in some embodiments, the cell programming methods described herein can provide the tools necessary to achieve cell identity independent of external cues.

[0069] In some embodiments, pluripotent stem cells (e.g., iPSCs) consisting essentially of SOX9 or one or more nucleic acids encoding SOX9 are co-cultured with one or more other cell types. In some embodiments, the other cell types are pluripotent stem cells that have not been engineered to express specific differentiation agents for orthogonal programming (i.e., unmodified pluripotent stem cells). In some embodiments, the unmodified pluripotent stem cells are human iPSCs (hiPSCs). Without being bound by theory, co-culturing another type of pluripotent stem cell with a pluripotent stem cell consisting of SOX9 or one or more copies of a nucleic acid encoding SOX9 can be useful for incorporating myelinating oligodendrocytes into the organoid to generate myelinated organoids. Non-limiting examples of organoids include whole brain organoids, spinal cord organoids, blood-brain barrier and brain region-specific organoids (e.g., forebrain, midbrain, cerebellum and hypothalamus brain organoids).

[0070] The nucleic acid encoding SOX9 can be operably linked to an inducible promoter, and SOX9 expression can be induced for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, or at least 20 days after co-culture of iPSCs consisting essentially of the nucleic acid encoding SOX9 with one or more other cell types. For example, a differentiation medium can be used during the co-culture to induce the formation of neurons by the other cell type (e.g., unmodified pluripotent stem cells).

[0071] In some embodiments, pluripotent stem cells (e.g., iPSCs) consisting essentially of SOX9 or one or more copies of a nucleic acid encoding SOX9 are co-cultured with pluripotent stem cells engineered to express at least one differentiation agent other than SOX9 for parallel programming. Pluripotent stem cells (e.g., iPSCs) consisting essentially of SOX9 or one or more copies of a nucleic acid encoding SOX9 can be cultured with another cell type for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, or at least 20 days. In some embodiments, expression of SOX9 and expression of the other differentiation agent are inducible. Expression of SOX9 and / or expression of other differentiation agents can be induced for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, or at least 20 days.

[0072] In some embodiments, a first population of pluripotent stem cells consisting essentially of SOX9 or one or more copies of a nucleic acid encoding SOX9 is cultured with 1 to 50 other PSC populations to generate a plurality of cell types. In some embodiments, a first population of pluripotent stem cells consisting essentially of SOX9 or one or more copies of a nucleic acid encoding SOX9 is cultured with at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 other PSC populations to generate a plurality of cell types. The other PSC populations comprise engineered nucleic acids encoding lineage-specific genes. In some embodiments, a plurality of 1 to 50 cell types is generated. In some embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 multiple cell types are generated.

[0073] Pharmaceutical compositions and uses thereof Also provided herein are pharmaceutical compositions comprising pluripotent stem cells (e.g., induced pluripotent stem cells), OPCs, and / or oligodendrocytes generated by any of the methods described herein. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier (e.g., nanocarriers) or excipient. Hydrogels may also be used as pharmaceutically acceptable carriers. Non-limiting examples of pharmaceutically acceptable excipients include water, saline, dextrose, glycerol, ethanol, and combinations thereof. Pharmaceutically acceptable excipients may include phosphate-buffered saline, bicarbonate solution, preservatives, stabilizers, emulsifiers (e.g., phospholipid emulsifiers), stabilizers (e.g., surfactants), or binders. Excipients may be selected based on the method and route of administration and standard pharmaceutical practice.

[0074] General considerations regarding the formulation and / or manufacture of pharmaceuticals, such as compositions comprising any of the engineered cells disclosed herein, can be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005, which is incorporated herein by reference in its entirety.

[0075] Any of the pharmaceutical compositions disclosed herein can be administered to a subject (e.g., a human subject). Other exemplary subjects include, but are not limited to, mice, rats, rabbits, horses, dogs, cats, goats, sheep, and other animals. The subject can have a demyelinating disorder. Demyelinating disorders include disorders in which the myelin surrounding axons is lost or damaged. Magnetic resonance imaging (MRI) can be used to diagnose demyelinating disorders. Examples of demyelinating disorders include disorders in which myelin in the central nervous system is damaged. In some embodiments, demyelinating disorders include damage to OPCs. Examples of demyelinating disorders include multiple sclerosis, transverse myelitis, and leukodystrophies (e.g., metachromatic leukodystrophy (MLD) and adrenoleukodystrophy (ALD)).

[0076] As used herein, the term "effective amount" or "therapeutically effective amount" refers to the amount of OPCs and / or oligodendrocytes required to confer a therapeutic effect on a subject, alone or in combination with at least one other active agent. Effective amounts will vary depending on the route of administration, the use of excipients, and the simultaneous use of other active agents, as will be recognized by those skilled in the art. The amount to be administered will depend on the subject being treated, including, for example, the strength of the individual's immune system or genetic predisposition. Suitable dosages can be readily determined by those skilled in the art and can be in milligrams of the polypeptide of the present disclosure. Dosages of the preparations disclosed herein can depend on the route of administration and will also vary depending on the size of the subject.

[0077] Suitable routes of administration include parenteral routes, such as intravenous, intrathecal, parenchymal or intraventricular routes.Suitable routes of administration include parenteral routes, such as intravenous, intrathecal, parenchymal or intraventricular injection. [Example]

[0078] Example Example 1: Identification of SOX9 as an agent that efficiently differentiates pluripotent stem cells (iPSCs) into oligodendrocyte progenitor cells (OPCs) To determine the effect of SOX9 on human induced pluripotent stem cell (hiPSC) differentiation, we overexpressed SOX9 in hiPSCs. SOX9 has been implicated as a fundamental master regulator of chondrocyte development. Surprisingly, chondrocyte morphology was not observed after SOX9 overexpression. Instead, cells reminiscent of oligodendrocytes were detected. This striking observation was confirmed by staining SOX9-induced differentiated cells with an antibody targeting O4 (an OPC marker) and performing flow cytometry on differentiation (Figures 1A-1C).

[0079] Indeed, up to 60% of the population was detected as O4+ cells (Figure 1D), which was confirmed after transfecting the cells with various amounts of DNA, suggesting differentiation into OPCs. Although the entire population was selected for cells with genomic integration of SOX9, O4 marker expression was observed in only a portion of the population. To determine whether specific expression levels of SOX9 may be required to achieve robust differentiation, heterologous transfection was used to isolate clones with various numbers of SOX9 integrations and, therefore, various SOX9 expression levels. Individual clones were screened to identify several clones that achieved robust OPC differentiation. After SOX9 overexpression, significant morphological changes were observed in some clones, which were attributed to near-complete differentiation. This change in morphology was not observed in control cells in which the transcription factor (TF) was not induced (Figures 2A-2B).

[0080] To characterize these clones, cells were fixed and stained for flow cytometry for early and late OPCs, selected based on the developmental stage at which they are normally expressed. High levels of O4 and NG2 expression were observed in these clones, but not in untransfected controls, confirming the differentiation potential of SOX9-induced OPCs (Figures 2C-2D).

[0081] Example 2: Use of SOX9-induced OPCs to form myelination in vitro To evaluate the ability of SOX9-induced OPCs to mature and generate myelin, we used an in vitro assay in which SOX9-induced OPCs were cocultured with hiPSC-derived neurons. To facilitate electron microscopy, we promoted the alignment of axon bundles by micropatterning microgrooves (which promote axonal fasciculation on neurons and maintain the health of such long-term cultures). After 4 weeks of coculture, vertical sections were performed, and the interaction between the two cell types was assessed by transmission microscopy. Surprisingly, robust myelination was observed in the coculture (Figure 3A). Multiple layers of aggregated myelin could be seen wrapped around the axons.

[0082] To demonstrate the ability of programmed OPCs to form myelin even in a more physiological context, we incorporated them into a human brain organoid model. This model best mimics in vivo phenomena due to its complex structure and 3D organization, including various human brain cell types. Unmodified hiPSCs were mixed with inducible SOX9 iPSCs. After 8 weeks, organoids were sectioned and stained for mature myelin markers, and then compared to controls without SOX9 overexpression. Figure 3B shows MOG-stained organoids, where MOG expression can be observed in the induced organoids but not in those without SOX9 overexpression. Myelination in this in vivo-like model is confirmation of the functionality of programmed OPCs.

[0083] Example 3: Generation of immunosuppressive OPCs To generate immunosuppressive OPCs, we engineered iPSCs to overexpress SOX9, IL10, and IFNβ1, generating stable cell lines. IL-10 and IFNβ1 are well known for the therapeutic benefits they confer to MS patients, but they are not normally secreted by OPCs. Furthermore, it is unclear whether OPCs possess the secretory machinery to effectively produce these cytokines. In contrast to adding growth factors to regulate OPC secretion, we determined whether genetic manipulation could achieve high levels of IL-10 and IFNβ1 secretion in a non-native context within OPCs. Expression of these genes was induced in these stable cell lines by adding doxycycline to the culture medium, and the ability of these cells to release cytokines was measured. OPCs were found to exhibit cytokine release 1,000-fold higher than uninduced control cells. Notably, increased IL10 secretion (which also induces IFNβ1 secretion as its downstream target) was observed after 4 days of induction with doxycycline. IL10 was consistently secreted after transfection with various amounts of DNA (Figure 4). IL10 was not released in the absence of doxycycline (control).

[0084] Example 4: Use of SOX9-induced OPCs to form myelination in vivo To confirm myelination in vivo, SOX9-induced OPCs were transplanted into shiverer mice, a congenital genetic model in which the myelin basic protein (MBP) gene is disrupted and expression is impaired. These mice do not form aggregated myelin. Because the MBP gene is disrupted, any observed aggregated myelin formation would be the result of engrafted transplanted cells. Newborn homozygous shiverer mice (1-3 days old) were cryoanesthetized and intracranially injected with 50,000 purified O4+ cells using the freehand technique. A control group underwent the same procedure, but instead of cells, they were injected with PBS containing 0.1% trypan blue. Compared to the control group, more aggregated myelination was detected in the OPC-injected mice, confirming that these cells are capable of engraftment and are functionally capable of myelination (Figure 5).

[0085] We also detected engraftment of SOX9-induced cells in immunostained brain sections 10 weeks after transplantation based on the presence of MBP, which can only be expressed in donor cells (data not shown). MBP was not observed in the control group. Furthermore, based on TEM of brain cross-sections, we observed aggregated myelin in mice transplanted with SOX9-induced cells, but little in the control group (data not shown). Quantification revealed a significantly increased number of myelinated axons in mice transplanted with cells compared with the control group (Figure 6). Taken together, these results demonstrate that induction of SOX alone is sufficient to autonomously program hiPSC cells into induced oligodendrocytes and that engineered stem cell-derived OPCs can form myelin where myelin deficiency exists.

[0086] As shown herein, these cells can be further engineered to confer additional characteristics, such as the ability to secrete anti-inflammatory cytokines. Collectively, these synthetic OPCs are promising candidates for diseases such as MS, in which the immune system degenerates oligodendrocytes (OLs).

[0087] Example 5: Parallel and Orthogonal Programming Using a cell-autonomous differentiation approach, we addressed one of the major limitations of organoid engineering: the long developmental timeline, which delays the emergence of specific key cell types. For example, the complexity of cerebral organoids is limited, in part, by their slow myelin development, requiring 103–210 days to form mature myelin. To address the lack of a transcription factor (TF)-mediated, mediator-independent protocol for differentiating myelin-producing oligodendrocytes, we utilized a human TFome library to identify individual TFs for synthetically programming oligodendrocytes to accelerate myelination. We identified 15 TFs involved in oligodendrocyte development and queried their rank-ordering in a screen, yielding SOX9 as the top hit. SOX9-induced cells expressed the signature oligodendrocyte precursor marker O4 (82 ± 6%) at 4 dpi without additional lineage-specific cues (Figure 2C, data not shown). Induced SOX9 cells were also positive for NG2 (Figure 2D, immunostaining data not shown). The oligodendrocyte transcriptome signature was found to be similar to that of primary oligodendrocytes based on unbiased PCA (Figure 7), upregulation of key oligodendrocyte genes, such as MBP, NKX2-1, MOG, MOBP, OLIG2, CSPG4, OLIG1, SOX8, SOX10, MYRF, PDGFRA, MMP15, PLP1, TMEM88B, ENPP6, and NFASC (data not shown), and transcriptome comparison of highly variable genes (data not shown). Expression of oligodendrocyte markers CSPG4 (NG2) and MYRF by scRNA-seq was also detected (data not shown). Pluripotency genes, including MYRF, POU5F1, NANOG, and SOX2, were downregulated by both scRNA-seq and bulk RNA-seq (data not shown).

[0088] To address one of the major challenges in tissue engineering—constructing complex physiological tissues—we introduced the concept of parallel programming, linking a set of three engineered hiPSC lines based on TFs discovered using the Human TFome. In this approach, multiple lines co-generate simultaneously in the same dish to form synthetic tissues in a medium-independent manner (Figure 8). To evaluate the myelination capacity of SOX9-inducible cells, we applied the parallel programming approach to generate synthetic oligo-neuron co-cultures. Inducible SOX9 hiPSCs were combined with well-characterized hiPSC-derived induced neurons (Busskamp, ​​V. et al. Mol Syst Biol 10, 760, (2014)), which project long axons upon differentiation. TF expression was then activated, and at 3 dpi, oligodendrocytes were observed contacting axons and initializing covering processes without the addition of external culture-specific factors (data not shown). After 30 days of co-culture in the optically micropatterned microchannels, robust myelin sheaths were observed around the axons by TEM (data not shown). The G-ratio, a measure of aggregated myelin, was calculated to be 0.56 ± 0.02 (Figure 10A), which is comparable to that of physiological myelin (Stikov, N. et al. NeuroImage 118, 397-405, (2015)). These results confirmed the in vitro myelin-forming functionality of SOX9-induced oligodendrocytes.

[0089] To synthetically accelerate myelination in cerebral organoids and build a more accurate model of human brain tissue, we introduced the concept of orthogonal cell programming by incorporating the extensively validated SOX9-induced oligodendrocytes (Figure 9). In this orthogonal approach, cell-autonomous TF overexpression, along with external induction conditions, was used to establish an alternative differentiation mode (data not shown). To achieve this, inducible SOX9 hiPSCs were combined with unmodified hiPSCs to form embryoid bodies. After 4 days, doxycycline was added to induce SOX9 expression in the orthogonally programmed organoids, compared with the control group without SOX9 induction. At 40 dpi, myelin oligodendrocyte glycoprotein (MOG) was observed in immunostained sections of the orthogonally programmed organoids (data not shown), but not in the control. TEM was performed on these sections to determine the presence of aggregated myelin. Robust and cohesive myelination was observed in orthogonally programmed organoids (data not shown), confirming the acceleration of myelin maturation through the orthogonal integration of SOX9-induced oligodendrocytes. To compare myelinated axons in these organoids with those in vivo, a G ratio of 0.52 ± 0.04 was calculated (Figure 10B), demonstrating physiological similarity. Here, we introduce the concept of orthogonal programming to synthetically accelerate myelination in cerebral organoids, which, overall, may be a critical step toward achieving the integrity of each engineered tissue with a complete cell-type repertoire.

[0090] Parallel programming-based in vitro myelination assay SOX9-induced oligodendrocytes were co-cultured with iNGN hiPSC-derived neurons to assess myelination (Theodorou, E. et al. Genes Dev 23, 575-588, (2009)). To facilitate the preparation of cross-sections of myelinated axons, these cells were co-cultured in microchannel molds that promote unidirectional axonal alignment along the channels. Microchannel molds were constructed by adding 10% (w / v) PEG-diacrylate (Mn 1000; Polysciences Inc., Warrington, PA) and 0.5% (w / v) Irgacure 2959 in PBS solution to collagen-coated transwells (Theodorou, E. et al. Genes Dev 23, 575-588, (2009); Bhatia-Gaur, R. et al. Genes Dev 13, 966-977, (1999); Dutta, A. et al. Science 352, 1576-1580, (2016)). After creating microchannels using a negative mask, the photosensitive medium was irradiated with 181 mW / cm2 UV light for 30 seconds. SOX9 and iNGN hiPSCs were then seeded into the microchannels and TF induction was induced with doxycycline. After maintaining the co-cultures in mTeSR1 for the first 4 days, the medium was replaced and long-term oligo-neuron cultures were maintained with the following components: DMEM-F12 containing 1:200 N2 supplement, 1:100 B27 supplement lacking vitamin A, 1% penicillin / streptomycin / glutamine, 60 ng / mL T3, 10 ng / mL NT3, 10 ng / mL IGF-I, 200 μM AA, 1:1000 trace element B, 2 ng / mL BDNF, and 2 ng / mL GDNF. After 4 weeks of co-culture, the constructs were fixed, embedded in resin, sectioned, stained, and imaged using transmission electron microscopy as described above for angiogenesis assays.

[0091] Orthogonal Cellular Programming in Brain Organoids Cerebral organoids were generated as previously described (Liang, CC, Park, AY & Guan, JL Nature Protocols 2, 329-333, (2007)) with minor modifications. To orthogonally program induced oligodendrocytes within cerebral organoids, inducible SOX9 hiPSCs and unmodified hiPSCs were dissociated with TrypLE Express (Life Technologies, 12604013), counted using an automated cell counter (Countess II, AMQAX1000, ThermoFisher Scientific), and then mixed at a 1:1 ratio in Aggrewell medium (STEMCELL Technologies, 05893). Subsequently, single-cell suspensions were transferred to Aggrewell 400 plates (STEMCELL Technologies, 27945) for embryoid body formation. 600,000 cells were seeded into Aggrewell plates containing Aggrewell medium with 10 μM Y-27632 ROCK inhibitor (Millipore, 688001). Plates were spun at 100 × g for 3 minutes and then placed in a tissue culture incubator overnight. The following day (day 1 of the protocol), embryoid body formation was confirmed by brightfield microscopy, and the medium was replaced with neural induction medium (DMEM / F12, GlutaMAX (Invitrogen, 11330-032), non-essential amino acids (Gibco, 11140-050) containing HEPES and N2 supplement (Gibco, A13707-01)). Half of the medium was replaced daily with neural induction medium on days 1–3. On day 4, embryoid bodies were harvested by gentle pipetting with a wide-bore tip, removed from the Aggrewell, and individually embedded in a drop of undiluted Matrigel (Corning, 354277). Starting on day 4, 0.5 μg / mL doxycycline was added to the medium daily to induce expression of TFs for orthogonal programming.On day 8, the medium was replaced with neural differentiation medium consisting of a 1:1 mixture of DMEM / F12 containing HEPES and GlutaMAX (Invitrogen, 11330-032) and Neurobasal medium (Invitrogen, 12348-017) containing non-essential amino acids (Gibco, 11140-050), N2 supplement (A13707-01), and vitamin A-free B27 supplement (Gibco, 12587-010). The medium was replaced every other day. Organoids were harvested, sliced, mounted on charged glass slides, and stored at -20°C until use. For staining, samples were warmed to room temperature and outlined with a wax pen. They were washed three times with MAXwash Washing Medium (ActiveMotif, 15254) to remove any residual OCT, then blocked with MAXblock Blocking medium (ActiveMotif, 15252) for 1 hour and washed with MAXwash Washing Medium. Primary antibody in binding buffer (ActiveMotif, 15251) was added and stained overnight. Samples were washed three times with wash buffer and then with secondary antibody in binding buffer for 5 hours. Samples were washed with buffer, stained with DAPI, and mounted for imaging using VectaShield mounting medium.

[0092] Example 6: Long-term study of oligodendrocyte maturation An experimental design was developed to conduct a long-term study to explore cell maturation and SOX9-induced OPC differentiation. In this design, oligodendrocyte differentiation was first induced in stem cell medium, followed by culturing the cells in oligodendrocyte differentiation or maintenance medium. This study was used to determine the effect of ideal conditions on cell stability and maturation. Therefore, this experiment was designed as a time course to monitor intermediate steps of differentiation and maturation.

[0093] Microscopic images from various culture conditions showed that oligodendrocytes had branched after 15 days of culture (data not shown). Microscopic images were taken from the same experiment after 31 days of culture. Oligodendrocytes exhibited a highly branched morphology and were attempting to reach axons to begin the myelinizing process (data not shown).

[0094] Immunohistochemistry was also performed on these cells for various markers. After 18 days of culture, the cells were found to be CNPase-, PLP1-, GALC-, MOG-, MBP-, and MAG-positive. MOG is a mature myelin protein. MBP is a signature myelin marker. MAG is another mature myelin marker. Branched morphology of oligodendrocytes was also observed.

[0095] These results support the robustness and reproducibility of the SOX9-induced OPC differentiation process.

[0096] Example 7: Determining the effect of SOX9 copy number To determine whether SOX9-induced OPC differentiation could be improved simply by transfecting more SOX9 DNA, we transfected increasing amounts of SOX9 DNA into hiPSCs and generated stable cell lines. Transfection of 1.5 μg, 2.5 μg, and 5 μg of SOX9 DNA confirmed that increasing the amount of transfected DNA resulted in more genomic integration of the SOX9 PIGGYBAC™ plasmid, as determined by quantifying the population-average copy number of exogenous SOX9 using digital droplet PCR (ddPCR) on extracted genomic DNA (Figure 11).

[0097] Next, we evaluated whether the amount of genomically integrated SOX9 improves OPC differentiation efficiency. OPC differentiation efficiency was assessed by staining for O4 and quantifying the percentage of O4-expressing cells using flow cytometry. Cells transfected with 2.5 μg of SOX9 DNA were found to have higher differentiation efficiency compared to cells transfected with 1.5 μg. Surprisingly, 5 μg of SOX9 DNA did not further improve OPC differentiation efficiency, despite the higher genome copy number (Figure 12).

[0098] These results indicate that less than 5 μg of SOX9 DNA is required to achieve high levels of OPC differentiation, which translates to a population average of less than 20 SOX9 copies per cell for these cells.

[0099] array Non-limiting examples of nucleic acid and amino acid sequences encoding SOX9 are set forth below. The nucleic acid sequences set forth below were used in the preceding examples.

[0100] Nucleic acid sequence encoding SOX9: [ka] [ka] The amino acid sequence encoding SOX9 is: [ka]

[0101] All references, patents, and patent applications disclosed herein are hereby incorporated by reference with respect to the subject matter for which each is cited, which may include the entire document.

[0102] The indefinite articles "a" and "an," as used in this specification and claims, should be understood to mean "at least one," unless a contrary intention is clearly indicated.

[0103] It should also be understood that, unless expressly indicated to the contrary, in any method claimed herein that includes more than one step or action, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are described.

[0104] In the claims and this specification, all transitional phrases such as "comprise," "include," "carry," "have," "contain," "involve," "hold," "consisting of," and the like, should be understood to mean open-ended, i.e., inclusive but not limited. Only the transitional phrases "consisting of" and "consisting essentially of" may be closed or partially closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0105] The terms "about" and "substantially" preceding a numerical value mean ±10% of the stated numerical value.

[0106] Where a range of numerical values ​​is described, each numerical value between the upper and lower limits of the range is specifically contemplated and described herein.

Claims

1. 1. A method comprising culturing pluripotent stem cells (PSCs) to generate a population of cells comprising oligodendrocyte progenitor cells (OPCs), wherein the PSCs express an engineered nucleic acid encoding SOX9 at a level sufficient to differentiate the PSCs into OPCs in the absence of other transcription factors encoded by the engineered nucleic acid.

2. 2. The method of claim 1, wherein the engineered nucleic acid encoding SOX9 is the only transcription factor encoded by an engineered nucleic acid expressed by the PSC.

3. 3. The method of claim 1 or 2, wherein at least 30%, at least 40%, at least 50% or at least 60% of the population of cells express O4 and / or NG2.

4. 4. The method of any one of claims 1 to 3, further comprising transfecting said PSCs with said engineered nucleic acid.

5. 5. The method of claim 4, wherein the engineered nucleic acid is a PiggyBac vector.

6. 6. The method of any one of claims 1 to 5, wherein the PSCs have a population average of less than 20 copies of the engineered nucleic acid encoding SOX9 per cell.

7. 6. The method of any one of claims 1 to 5, wherein the PSCs have a population average of 5 to 20, 10 to 20, or 15 to 20 copies of the engineered nucleic acid encoding SOX9 per cell.

8. The OPC population of cells contains myelin basic protein (MBP), NK2 homeobox 1 (NKX2-1), myelin oligodendrocyte glycoprotein (MOG), myelin-associated oligodendrocyte basic protein (MOBP), OLIG2 (oligodendrocyte transcription factor 2), chondroitin sulfate proteoglycan 4 (CSPG4), oligodendrocyte transcription factor 1 (OLIG1), SRY-box transcription factor 8 (SOX8), SRY-box transcription factor 10 (SOX1 8. The method of claim 1, wherein the oligodendrocytes express at least one oligodendrocyte gene selected from the group consisting of: myelin regulatory factor (MYRF), platelet-derived growth factor receptor alpha (PDGFRA), matrix metallopeptidase 15 (MMP15), proteolipid protein 1 (PLP1), transmembrane protein 88B (TMEM88B), ectonucleotide pyrophosphatase / phosphodiesterase 6 (ENPP6), and neurofascin (NFASC).

9. 9. The method of any one of claims 1 to 8, wherein at least one of the following pluripotency genes is downregulated in OPCs of said population of cells: myelin regulatory factor (MYRF), POU class 5 homeobox 1 (POU5F1), Nanog homeobox (NANOG), and SRY-box transcription factor 2 (SOX2).

10. The method of any one of claims 1 to 9, wherein the PSCs are human PSCs.

11. The method of any one of claims 1 to 10, wherein the PSCs are induced PSCs (iPSCs).

12. 12. The method of any one of claims 1 to 11, wherein the PSCs further express an engineered nucleic acid encoding a cytokine selected from IL-10 and IFNβ.

13. 13. The method of claim 12, wherein the PSCs further express an engineered nucleic acid encoding IL-10 and an engineered nucleic acid encoding IFNβ.

14. The method of claim 12 or 13, wherein the OPCs of the population of cells express IL-10 and / or IFNβ.

15. The method of claim 14, wherein the OPCs secrete IL-10 and / or IFNβ.

16. 16. The method of any one of claims 1 to 15, further comprising culturing OPCs of said population of cells to generate oligodendrocytes.

17. 17. The method of claim 16, wherein the oligodendrocytes express at least one biomarker selected from the group consisting of 2',3'-cyclic nucleotide 3' phosphodiesterase (CNPase), proteolipid protein 1 (PLP1), galactosylceramidase (GALC), myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), and myelin associated glycoprotein (MAG).

18. A population of cells produced by the method of any one of claims 1 to 17.

19. A pharmaceutical composition comprising OPCs produced by the method of any one of claims 1 to 17.

20. 18. A pharmaceutical composition comprising oligodendrocytes produced by the method of claim 16 or 17.

21. 21. A method comprising administering to a subject having a demyelinating disorder the pharmaceutical composition of claim 19 or 20.

22. 22. The method of claim 21, wherein the demyelinating disorder is selected from multiple sclerosis, transverse myelitis, metachromatic leukodystrophy (MLD) and adrenoleukodystrophy (ALD).

23. A pluripotent stem cell (PSC) comprising an engineered nucleic acid encoding SOX9 at a level sufficient to differentiate the PSC into an oligodendrocyte progenitor cell (OPC) in the absence of other transcription factors encoded by the engineered nucleic acid.

24. 24. The PSC of claim 23, wherein the engineered nucleic acid encoding SOX9 is the only transcription factor encoded by an engineered nucleic acid expressed by the PSC.

25. 25. A method comprising culturing a first population of PSCs of claim 23 or 24 with at least one other population of PSCs to generate a plurality of cell types, wherein the at least one other population of PSCs comprises an engineered nucleic acid encoding a lineage-specific gene.

26. 26. The method of claim 25, wherein the engineered nucleic acid encoding SOX9 and / or the engineered nucleic acid encoding a lineage-specific gene is operably linked to an inducible promoter.

27. 27. The method of claim 26, comprising culturing the first population of PSCs with at least one other population of PSCs and inducing expression of the engineered nucleic acid encoding SOX9 and / or the engineered nucleic acid encoding a lineage-specific gene.

28. 25. A method comprising culturing a first population of PSCs of claim 23 or 24 with at least one other population of PSCs to generate myelinated organoids, wherein the at least one other population of PSCs comprises unmodified PSCs.

29. 29. The method of claim 28, wherein the engineered nucleic acid encoding SOX9 is operably linked to an inducible promoter.

30. 27. The method of claim 26, comprising culturing the first population of PSCs with at least one other population of PSCs and inducing expression of the engineered nucleic acid encoding SOX9.

31. 1. A method comprising: (a) contacting pluripotent stem cells (PSCs) with a transcription factor, wherein the transcription factor consists essentially of SOX9 or one or more copies of a nucleic acid encoding SOX9; and (b) culturing the PSCs to generate a population of cells comprising oligodendrocyte progenitor cells (OPCs).

32. 1. A method comprising: (a) contacting pluripotent stem cells (PSCs) with a differentiation agent, wherein the differentiation agent consists essentially of SOX9 or one or more copies of a nucleic acid encoding SOX9; and (b) culturing the PSCs to generate a population of cells comprising oligodendrocyte progenitor cells (OPCs).

33. 33. The method of claim 31 or 32, wherein the population of cells comprises at least 70% OPCs.

34. 34. The method of any one of claims 31 to 33, wherein the PSCs of (a) are present in a culture medium.

35. The method of any one of claims 31 to 33, wherein the PSCs are human PSCs.

36. 35. The method of any one of claims 31 to 34, wherein the PSCs are induced PSCs (iPSCs).

37. The method of any one of claims 31 to 35, wherein the OPCs express O4 and / or NG2.

38. 37. The method of any one of claims 31 to 36, wherein an engineered nucleic acid encoding a cytokine selected from IL-10 and IFNβ is introduced into the PSCs of (a).

39. 38. The method of claim 37, wherein an engineered nucleic acid encoding IL-10 and an engineered nucleic acid encoding IFNβ are introduced into the pluripotent stem cells.

40. 39. The method of claim 38, wherein IL-10 and IFNβ are encoded by the same engineered nucleic acid.

41. 40. The method of any one of claims 31 to 39, wherein the PSCs of (b) are cultured for 1 to 10 days.

42. 42. The method of any one of claims 38-41, further comprising culturing the OPCs for 31 to 10 days.

43. The method of any one of claims 38 to 42, wherein the OPCs secrete IL-10.

44. 14. The method of claim 13, wherein the level of IL-10 secreted by the OPCs is at least 50-fold higher, at least 100-fold higher, or at least 1,000-fold higher than control cells, wherein the control cells are (i) naturally occurring OPCs, (ii) OPCs that have not been engineered to express IL-10, and / or (iii) OPCs that have been engineered to express IL-10 under an inducible promoter and are cultured in the absence of an inducer.

45. The method of any one of claims 31 to 44, wherein the differentiation agent consists essentially of 1 to 50 copies of a nucleotide sequence encoding SOX9.

46. 46. ​​The method of any one of claims 31 to 45, wherein the one or more engineered nucleic acids are operably linked to a promoter.

47. 47. The method of claim 46, wherein the promoter is an inducible promoter.

48. 48. The method of claim 47, wherein the PSCs of (b) are cultured in the presence of a substance that induces the inducible promoter to activate transcription of the transcription factor and / or cytokine.

49. 49. The method of any one of claims 31-48, wherein the one or more engineered nucleic acids are present on a PIGGYBAC™ transposon-based vector.

50. 50. The method of Claim 49, wherein said PIGGYBAC™ transposon-based vector comprises PIGGYBAC™ inverted terminal repeats flanking at least one nucleotide sequence encoding at least one of said transcription factors.

51. 51. The method of claim 49 or 50, further comprising introducing PIGGYBAC™ transposase or an engineered nucleic acid encoding PIGGYBAC™ transposase into the PSCs.

52. 52. The method of any one of claims 31-51, further comprising culturing the OPCs to generate oligodendrocytes.

53. A population of cells produced by the method of any one of claims 31 to 52.

54. 52. A pharmaceutical composition comprising OPCs produced by the method of any one of claims 31 to 51.

55. 53. A pharmaceutical composition comprising oligodendrocytes produced by the method of claim 52.

56. 56. A method comprising administering to a subject having a demyelinating disorder the pharmaceutical composition of claim 54 or 55.

57. 57. The method of claim 56, wherein the demyelinating disorder is selected from multiple sclerosis, transverse myelitis, metachromatic leukodystrophy (MLD), and adrenoleukodystrophy (ALD).

58. 58. The method of any one of claims 31 to 57, wherein the differentiation agent consists of SOX9 or one or more copies of a nucleic acid encoding SOX9.

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