Cellular compositions and methods of use thereof

Abstract

The present disclosure generally relates to methods and systems for producing Schwann cells from pluripotent stem cells under fully defined conditions. Schwann cells produced by the disclosed methods have applications as a model of the enteric nervous system, as a tool for high-throughput screening of potential therapeutic agents for the treatment of enteric neuropathies, and in regenerative medicine. TIFF2025531671000362.tif55128

Description

[Technical Field]

[0001] Statement Regarding Federally Sponsored Research This invention was made with government support under grant numbers DP2NS116769 and R01DK121169 awarded by the National Institutes of Health (NIH). The government has certain rights in this invention.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 63 / 398,470, filed August 16, 2022, the entire contents of which are incorporated herein by reference.

[0003] Sequence Listing The contents of the attached electronic sequence listing (UCAL-031-PCT_Seq List.xml; size: 158,489 bytes, and created on August 15, 2023) are incorporated herein by reference in their entirety.

[0004] Technical Field The present disclosure generally relates to methods for culturing pluripotent stem cells under defined conditions and inducing the differentiation of the pluripotent stem cells into Schwann cells. The compositions comprising Schwann cells are suitable for screening potential therapeutic agents for Charcot-Marie-Tooth disease, schwannomatosis, Guillain-Barré syndrome, and diabetic peripheral neuropathy (DPN) in vitro, and for applications in regenerative medicine, either by direct administration or transplantation, independently. [Background technology]

[0005] background Schwann cells (SCs) are a key component and the primary glial cell of the peripheral nervous system (PNS). They are important for nerve growth, structural maintenance, and function and exhibit a remarkable ability to promote nerve repair after injury (Jessen and Mirsky, 2005; Lavdas et al., 2008). SCs support axons by forming insulating myelin sheaths and Remak bundles and provide essential neurotrophic factors. Schwann cells develop from the neural crest (NC) via a highly proliferative and migratory Schwann cell precursor (SCP) intermediate. SCPs further differentiate into immature SCs that ultimately give rise to mature myelinating or nonmyelinating SCs. In addition to SCs, SCPs can give rise to other derivatives (SCPDs), such as melanocytes (Adameyko et al., 2009; Bonnamour et al., 2021; Nitzan et al., 2013). SC deficiencies are involved in inherited and acquired PNS disorders such as Charcot-Marie-Tooth disease, schwannomatosis, Guillain-Barre syndrome and diabetic peripheral neuropathy (DPN), for which there are currently no faithful disease models or effective therapies.

[0006] Understanding SC development and function and their role in PNS health and disease has broad fundamental and translational implications. However, access to authentic models of large-scale human SCs remains a major challenge. Summary of the Invention

[0007] overview The present disclosure relates to methods for differentiating at least one or more stem cells into Schwann cells. The present disclosure relates to compositions comprising one or more Schwann cells, wherein the Schwann cells comprise CD98 or a functional fragment thereof comprising at least about 70% of the sequence relative to CD98. In some embodiments, the cells are derived from neural crest (NC) cells. In some embodiments, the cells are cultured for more than about 35 days. In some embodiments, the cells are cultured for more than about 58 days.

[0008] In some embodiments, the composition further comprises one or a combination of S100, myelin binding protein (MBP), and GFAP.

[0009] In some embodiments, the cell further comprises one or a combination of SOX10, POU3F2, GAP43, or functional fragments thereof comprising at least about 70% sequence identity to SOX10, POU3F2, and GAP43. In some embodiments, the cell further comprises an mRNA transcript encoding SOX10, POU3F2, GAP43, or one or a combination of functional fragments thereof comprising at least about 70% sequence identity to SOX10, POU3F2, and GAP43, respectively.

[0010] In some embodiments, the cells further comprise one or a combination of PMP22, SOX10, POU3F2, GAP43, NGFR, MP2, CD46, CD146, CD147, CD166, ERBB3, GDNF, or functional fragments thereof, any one or combination of such functional fragments comprising at least about 70% sequence identity to PMP22, SOX10, POU3F2, GAP43, NGFR, MP2, CD46, CD146, CD147, CD166, ERBB3, and GDNF. In some embodiments, the cells comprise mRNA transcripts encoding one or a combination of PMP22, SOX10, POU3F2, GAP43, NGFR, MP2, CD46, CD146, CD147, CD166, ERBB3, GDNF, or functional fragments thereof.

[0011] In some embodiments, the cell further comprises one or a combination of FOX01, TBX19, MATN2, PLAT, or functional fragments thereof comprising at least about 70% sequence identity to FOX01, TBX19, MATN2, and PLAT, hi some embodiments, the cell comprises an mRNA transcript encoding one or a combination of FOX01, TBX19, MATN2, PLAT, or functional fragments thereof.

[0012] In some embodiments, the cells further comprise one or a combination of PMP22, POU3F2, GAP43, NGFR, MP2, CD46, CD146, CD147, CD166, ERBB3, GDNF, CD9, CD49e, CD171, or a functional fragment thereof comprising at least about 70% sequence identity to one of PMP22, POU3F2, GAP43, NGFR, MP2, CD46, CD146, CD147, CD166, ERBB3, GDNF, CD9, CD49e, and CD 171. In some embodiments, the cells comprise an mRNA transcript encoding one or a combination of PMP22, POU3F2, GAP43, NGFR, MP2, CD46, CD146, CD147, CD166, ERBB3, GDNF, CD9, CD49e, CD171, or a functional fragment thereof.

[0013] In some embodiments, the Schwann cells comprise MPZ, MAG, PMPP22, PLLP, or functional fragments thereof that comprise at least about 70% sequence identity to MPZ, MAG, PMPP22, or PLLP, hi some embodiments, the cells comprise mRNA transcripts encoding one or a combination of MPZ, MAG, PMPP22, PLLP, or functional fragments thereof.

[0014] In some embodiments, the Schwann cells comprise POU6F2, CD44, CD81, or functional fragments thereof that comprise at least about 70% sequence identity to POU6F2, CD44, CD81, or hi some embodiments, the cells comprise mRNA transcripts encoding one or a combination of POU6F2, CD44, CD81, or functional fragments thereof.

[0015] The present disclosure relates to cell lines or compositions comprising any of the cells identified above. In some embodiments, the present disclosure relates to pharmaceutical compositions comprising any of the disclosed cells and a pharmaceutically acceptable carrier. In some embodiments, the compositions disclosed herein comprise greater than about 70% Schwann cells. In some embodiments, the compositions disclosed herein comprise greater than about 80% Schwann cells. In some embodiments, the compositions disclosed herein comprise greater than about 90% Schwann cells. In some embodiments, the cells are derived from human pluripotent stem cells. In some embodiments, the cells are cultured for at least about 2 weeks.

[0016] The present disclosure also relates to a system comprising any of the disclosed cells and tissue culture medium. In some embodiments, the system further comprises a solid substrate, such as plastic, to which the cells adhere. In some embodiments, the cells are cultured for more than about two weeks. In some embodiments, the present disclosure relates to an animal or patient comprising any one or more of the cells disclosed herein. The present disclosure also relates to a tissue culture system comprising a composition of cells described in the present disclosure and tissue culture medium. In some embodiments, the system further comprises a solid substrate on which the cells are disposed.

[0017] The present disclosure also relates to a pharmaceutical composition comprising a pharmaceutically effective amount of cells as described in this disclosure and a pharmaceutically acceptable carrier or excipient.

[0018] The present disclosure also relates to a method for differentiating pluripotent stem cells into Schwann cells, the method comprising exposing neural crest cells to an effective amount of FGF2 or a functional fragment thereof for a time sufficient to differentiate the neural crest into Schwann cells. The present disclosure also relates to a method for enriching Schwann cells in a cell culture, the method comprising exposing a composition of pluripotent stem cells to FGF2 or a functional fragment thereof for a time sufficient to cause the pluripotent stem cells to become neural crest cells, and subsequently exposing the neural crest cells to FGF2 or a functional fragment thereof for a time sufficient to cause the neural crest cells to express one or more mRNAs encoding SOX10 or functional fragments thereof. In some embodiments, the neural crest cells express an amino acid sequence comprising SOX10 or a functional fragment thereof.

[0019] In some embodiments, the method further comprises exposing the composition of neural crest cells to a WNT pathway activator for a time sufficient for the neural crest cells to express SOX10 or a functional fragment thereof.

[0020] In some embodiments, the method further comprises exposing the neural crest cell composition to SB431542 and / or dbcAMP for a time sufficient to cause the neural crest cells to express mRNA encoding POU3F1, PMP22, MBP, MPZ, AQP4, or functional fragments thereof, or one or a combination of amino acids comprising the same. In some embodiments, exposing the neural crest cell composition to SB431542 and / or dbcAMP comprises exposing for a time sufficient to cause the neural crest cells to differentiate into Schwann cells. In some embodiments, the one or more exposure steps are cumulatively performed for more than about 19 days. In some embodiments, the method further comprises observing cell morphology and / or performing polymerase chain reaction (PCR) and / or immunohistochemistry to confirm that differentiation of the neural crest cells into Schwann cells has occurred. In some embodiments, the cells are cultured in the form of crestspheres or spheroids.

[0021] The present disclosure also relates to methods of culturing one or more Schwann cells, the methods comprising exposing one or more neural crest cells to tissue culture medium containing FGF2, SB431542, and / or dbcAMP, or derivatives or functional fragments thereof. In some embodiments, the one or more exposure steps are cumulatively performed for more than about 19 days. In some embodiments, the method further comprises differentiating human pluripotent stem cells into neural crest cells prior to the step of exposing the neural crest cells to the Schwann cells. In some embodiments, the Schwann cells are cultured for about 20, 30, 40, 50, 60, 70, 80, 90 days or more, or for about 100 days.

[0022] The present disclosure also relates to a method for screening one or more agents for neuromodulatory activity, the method comprising: i) culturing any one of the disclosed cell compositions in a tissue culture system containing one or more healthy or dysfunctional neuronal cells; ii) exposing the composition to one or more agents; iii) monitoring the composition for neuromodulatory activity; and iv) identifying the one or more agents as being toxic to healthy cells of the nervous system if the neuromodulatory activity of the agent blocks, prevents, or reduces neuronal cell viability compared to the neuromodulatory activity of the neuronal cells in the absence of the one or more agents, or identifying the one or more agents as inducing neuronal repair if the neuromodulatory activity of the dysfunctional neuronal cells improves or restores function compared to the neuromodulatory activity of the dysfunctional neuronal cells in the absence of the one or more agents.

[0023] The present disclosure also relates to methods of transplanting a Schwann cell population into a subject in need thereof by administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of any one or combination of the disclosed Schwann cells and a pharmaceutically acceptable carrier.

[0024] The present disclosure also relates to a method of treating spinal cord injury in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a drug or a pharmaceutical composition comprising a therapeutically effective amount of any one or combination of the disclosed Schwann cells and a pharmaceutically acceptable carrier.

[0025] The present disclosure also relates to a method of treating diabetic peripheral neuropathy in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agent or a pharmaceutical composition comprising a therapeutically effective amount of any one or combination of the disclosed Schwann cells and a pharmaceutically acceptable carrier. In some embodiments, the agent is selected from the agents in Table S4. In some embodiments, the agent is selected from the agents in Table S4 or pharmaceutically acceptable salts or derivatives thereof. In some embodiments, the agent is selected from the agents in Table S4 or pharmaceutically acceptable salts or derivatives thereof comprising a Z-score greater than 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or greater than 2.0. [Brief explanation of the drawings]

[0026] [Figure 1-1]Figures 1A-1J. [Figure 1A] Schwann cell induction from hPSCs. Schematic diagram of the protocol for inducing outgrowth precursor and Schwann cell (SC) cultures from hPSC-derived neural crest (NC). [Figure 1B] Schwann cell induction from hPSCs. SOX10::GFP expression at days 11, 25, and 35 of differentiation. Scale bar = 100 μm in B left and B middle panels, 25 μm in B right panels. [Figure 1C] Schwann cell induction from hPSCs. Representative immunofluorescence images of hPSC-derived SCs for Schwann lineage markers at day 60. Scale bar = 25 μm. [Figure 1D] Schwann cell induction from hPSCs. Quantification of markers in (D). [Figure 1E] Schwann cell induction from hPSCs. UMAP visualization of scRNA-seq data for low-passage (day 38) and high-passage (day 58) SC cultures. SCPD is derived from Schwann cell precursors, SCP is Schwann cell precursors, and SC is Schwann cell. [Figure 1F] Derivation of Schwann cells from hPSCs. Dot plots of the scaled mean expression of SC differentiation and myelination (left) and neural support (right) markers in single-cell RNA-seq data of low- and high-passage Schwann cells. [Figure 1G] Derivation of Schwann cells from hPSCs. Dot plots of the scaled mean expression of the top 15 primary mouse myelinating (mySC) and nonmyelinating (nmSC) Schwann cell markers in single-cell RNA-seq data of low- and high-passage Schwann cell cultures. [Figure 1H] Derivation of Schwann cells from hPSCs. Module scoring of the top 100 high-passage (HP) Schwann cell type-specific DE marker genes in low-passage (LP) Schwann cell types (left). Visualization of features (left) and dot plots (right) are shown. [Figure 1I] Schwann cell induction from hPSCs. Module scoring of the top 100 low-passage (LP) Schwann cell type-specific DE marker genes in high-passage (HP) Schwann cell types (left). Feature (left) and dot plot (right) visualizations are shown. [Figure 1J] Schwann cell induction from hPSCs.Principal component analysis (PCA) of NC cells, outgrowth precursors, human primary Schwann cells, and hPSC-derived SC cultures at days 50 and 100 of differentiation compared to central nervous system (CNS) precursors. [Figure 1-2] See description of Figure 1-1. [Figure 1-3] See description of Figure 1-1. [Figure 1-4] See description of Figure 1-1. [Figure 2-1]Figures 2A-2J. [Figure 2A] hPSC-derived Schwann cells myelinate hPSC-derived sensory neurons and engraft into injured rat sciatic nerves. Feature plots of mature SC clusters isolated from low-passage (top) and high-passage (bottom) single-cell RNA-seq data. Darker colors indicate SCs identified as myelinating (mySCs). Bar graphs indicate the relative abundance of mySCs. [Figure 2B] hPSC-derived Schwann cells myelinate hPSC-derived sensory neurons and engraft into injured rat sciatic nerves. Pathway enrichment analysis of the top 250 DE genes of myelinating mature SCs in low-passage (left) and high-passage (right) cells. The top 50 pathways from the combined GO BP, Reactome, and KEGG analyses are shown. Scale bar = 100 μm in the left panel of Figure 2B and 20 μm in the right panel of B. [Figure 2C] hPSC-derived Schwann cells myelinate hPSC-derived sensory neurons and transplant into injured rat sciatic nerves. Schematic diagram of coculture of hPSC-SCs with hPSC-derived sensory or motor neurons. Scale bars = 0.2 μm and 100 μm in Figure 2C. [Figure 2D] hPSC-derived Schwann cells myelinate hPSC-derived sensory neurons and transplant into injured rat sciatic nerves. Physical association of hPSC-SCs with hPSC-derived sensory neurons. [Figure 2E] hPSC-derived Schwann cells myelinate hPSC-derived sensory neurons and transplant into injured rat sciatic nerves. Physical association of hPSC-SCs with hPSC-derived motor neurons. [Figure 2F] hPSC-derived Schwann cells myelinate hPSC-derived sensory neurons and transplant into injured rat sciatic nerves. Schematic diagram of hPSC-SC transplantation in the adult rat sciatic nerve. RFP+ hPSC-derived Schwann cells were injected into the injury site (adult Cyclosporin-A-treated SD rats) after nerve crush. Scale bar = 20 µm in Figure 2F. [Figure 2G] hPSC-derived Schwann cells myelinate hPSC-derived sensory neurons and engraft into the injured rat sciatic nerve. Immunofluorescent staining of the transplanted sciatic nerve for the human-specific nuclear marker SC101 8 weeks after transplantation. Scale bar = 20 µm in Figure 2G. Figures 2J-2L.[Figure 2H] hPSC-derived Schwann cells myelinate hPSC-derived sensory neurons and engraft into injured rat sciatic nerves. Confocal analysis of free sciatic nerve fibers for RFP (transplanted human cells), an axon marker (NFH), and DAPI. Scale bar = 10 μm in Figure 2H. [Figure 2I] hPSC-derived Schwann cells myelinate hPSC-derived sensory neurons and engraft into injured rat sciatic nerves. Confocal analysis of free nerve fibers for RFP (transplanted human cells), myelin markers MAG and PO, and DAPI. Scale bar = 10 μm in Figure 2I. [Figure 2J] hPSC-derived Schwann cells myelinate hPSC-derived sensory neurons and engraft into injured rat sciatic nerves. Confocal analysis of free nerve fibers for RFP and the nodal marker Pan-Na+ (sodium channel, arrow, Figure 2H). Scale bar = 10 μm in Figure 2J. [Fig. 2K] Confocal analysis of free nerve fibers for RFP and the nodal marker CASPR (arrow, Fig. 2I). [Fig. 2L] Confocal analysis of free nerve fibers for RFP and the nodal marker Kv1.2 (K+ channel, arrow, Fig. 2J). [Figure 2-2] See description of Figure 2-1. [Figure 2-3] See description of Figure 2-1. [Figure 3-1]Figures 3A-3P. [Figure 3A] Schwann cells are selectively vulnerable to high glucose exposure. Schematic of the experimental paradigm for modeling diabetic nerve damage in hPSC-derived cell types. [Figure 3B] Schwann cells are selectively vulnerable to high glucose exposure. Lactate dehydrogenase (LDH) cytotoxicity analysis of hPSC-derived SCs and sensory neurons in response to exposure to different glucose concentrations using an LDH activity assay. [Figure 3C] Schwann cells are selectively vulnerable to high glucose exposure. Oxidative stress measurements of hPSC-derived SCs exposed to increasing glucose concentrations. Statistical analysis was performed using one-way ANOVA comparing values ​​to the low glucose (5 mM) condition. ns not significant, p values ​​*p<0.05, **p<0.01. [Figure 3D] Schwann cells are selectively vulnerable to high glucose exposure. Schematic of a high-throughput drug screen for identifying compounds that enhance the survival of high-glucose-treated hPSC-SCs. [Figure 3E] Schwann cells are selectively vulnerable to high glucose exposure. Distribution of library compounds by their corresponding normalized viability z-scores. [Figure 3F] Schwann cells are selectively vulnerable to high glucose exposure. Gene set enrichment analysis using iPAGE for library compound targets identifies GO terms associated with hits for improved and worsened SC viability. [Figure 3G] Schwann cells are selectively vulnerable to high glucose exposure. P-value correlation plot to identify genes most likely to be targets of effective treatments. Normalized z-scores from all treatments associated with a gene are combined. Furthermore, a Fisher's exact test is performed to assess the enrichment of individual genes among those that are targets of treatments with increased z-scores. The plot shows the correlation between p-values. [Figure 3H] Schwann cells are selectively vulnerable to high glucose exposure. One-sided volcano plot showing the mean z-score versus the logarithm of the p-value for all genes with positive z-scores. Genes that pass the statistical thresholds of combined z-score FDR<0.25 and Fisher's p-value<0.1 are marked in gold. [Figure 3I] Schwann cells are selectively vulnerable to high glucose exposure.The identified target genes (marked in gold in H) are ranked by their combined z-scores. [Figure 3J] Schwann cells are selectively vulnerable to high glucose exposure. This is a protein-protein interaction network of the identified target genes (listed in I) constructed using the STRING database. The minimum required interaction score was set to 0.4, and the edge thickness indicates the degree of data support. [Figure 3K] Schwann cells are selectively vulnerable to high glucose exposure. Predicted targets of hits in (K) compiled from the following databases: BindingDB (Liu et al., 2007), Carlsbad (Mathias et al., 2013), Dinies (Yamanishi et al., 2014), PubChem bioassays (Kim et al., 2019), SEA (Keiser et al., 2007), Superdrug2 (Siramshetty et al., 2018), and SwissTargetPrediction (Gfeller et al., 2014). [Figure 3L] Schwann cells are selectively vulnerable to high glucose exposure. Schematic of unbiased metabolite and transcriptional profiling of differentially treated SCs. [Figure 3M] Schwann cells are selectively vulnerable to high glucose exposure. Pathway enrichment analysis of genes upregulated by high glucose and downregulated upon BP treatment. [Figure 3N] Schwann cells are selectively vulnerable to high glucose exposure. Glycerolipid metabolic enzymes were upregulated under high glucose conditions. [Figure 3O] Schwann cells are selectively vulnerable to high glucose exposure. A schematic diagram of glycerolipid metabolism adopted from KEGG shows the changes in enzymes and metabolites in response to high glucose and BP treatment in SCs. HighGluc is high glucose, LowGluc is low glucose, HighGluc.Bup is high glucose + bupropion, and LowGluc.Bup is low glucose + bupropion. [Figure 3P] Schwann cells are selectively vulnerable to high glucose exposure. PTGER4 KO in SCs rescues SCs from high glucose treatment.CRISPR-Cas9-mediated knockout of PTGER4 in SCs protects them against increased levels of cleaved caspase 3 (a marker of apoptosis) under high glucose conditions, as measured by flow cytometry. p-values ​​are *p<0.05, **p<0.01, ***p<0.001. [Figure 3-2] See description of Figure 3-1. [Figure 3-3] See description of Figure 3-1. [Figure 4-1] Figures 4A-4F. [Figure 4A] Bupropion treatment prevents diabetic nerve damage in mice. Schematic diagram modeling diabetes and bupropion treatment in mice. [Figure 4B] Bupropion treatment prevents diabetic nerve damage in mice. Heat sensitivity test measuring hind paw withdrawal latency in normal mice and mice treated with STZ and bupropion. [Figure 4C] Bupropion treatment prevents diabetic nerve damage in mice. TUNEL staining in sciatic nerves from normal mice and mice treated with STZ and bupropion. [Figure 4D] Bupropion treatment prevents diabetic nerve damage in mice. Quantification in sciatic nerves from normal mice and mice treated with STZ and bupropion. [Figure 4E] Bupropion treatment prevents diabetic nerve damage in mice. Electron transmission microscopy of damaged myelin structure in the sciatic nerves of normal mice and mice treated with STZ and bupropion. [Figure 4F] Bupropion treatment prevents diabetic nerve damage in mice. Quantification of damaged myelin structure in the sciatic nerves of normal mice and mice treated with STZ and bupropion. p values: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Scale bars = 100 μm in Figure 4C and 5 μm in Figure 4E. BP, bupropion HCl. [Figure 4-2] See description of Figure 4-1. [Figure 5A]Figures 5A-5B. [Figure 5A] Characterization of hPSC-derived SC lineages. qRT-PCR of a panel of Schwann cell and progenitor markers at different time points of the differentiation protocol. Log2 fold change relative to D0 of differentiation is shown. [Figure 5B] Characterization of hPSC-derived SC lineages. qRT-PCR of a panel of Schwann lineage markers involved in Schwann cell differentiation and myelination (left) and neural interaction and support (right). [Figure 5B] See legend to Figure 5A. [Figure 6] Figures 6A-6D. [Figure 6A] Subtype-specific features of hPSC-derived SC lineages. Dot plots of the scaled mean expression of the top 10 differentially expressed (DE) genes for each low- and high-passage Schwann cell type. SCPD, Schwann cell precursor derived, SCP, Schwann cell precursor, and SC, Schwann cell. [Figure 6B] Subtype-specific features of hPSC-derived SC lineages. Low- and high-passage cell type-specific neurotrophic factors. All gene sets were pre-filtered to include genes expressed in at least 25% of cells of either cell type in low- and high-passage SC culture data. [Figure 6C] Subtype-specific features of hPSC-derived SC lineages. Neurotransmitter receptors and postsynaptic signaling. All gene sets were pre-filtered to include genes expressed in at least 25% of cells of either cell type in low- and high-passage SC culture data. [Figure 6D] Subtype-specific signatures of hPSC-derived SC lineages. Dot plots of scaled mean expression of transcription factors. All gene sets were pre-filtered to include genes expressed in at least 25% of cells of either cell type in low-passage (LP) and high-passage (HP) SC culture data. [Figure 7-1]Figures 7A-7H. [Figure 7A] Molecular changes in SC cultures after long-term maintenance. Feature plots (left) and cell cycle phase distributions (right) in low-passage Schwann cell cultures. [Figure 7B] Molecular changes in SC cultures after long-term maintenance. Feature plots (left) and cell cycle phase distributions (right) in high-passage Schwann cell cultures. [Figure 7C] Molecular changes in SC cultures after long-term maintenance. UMAP visualization of merged low- and high-passage Schwann cell datasets. [Figure 7D] Molecular changes in SC cultures after long-term maintenance. Feature plots showing module scoring of the top 100 low-passage (top) and high-passage (bottom) cell-type-specific DE marker genes in the merged dataset. [Figure 7E] Molecular changes in SC cultures after long-term maintenance. UMAP visualization of low- and high-passage merged datasets showing subclusters of primary SCs and SCPD. [Figure 7F] Molecular changes in SC cultures after long-term maintenance. Figure 7G: Distribution of cell cycle phases in the merged dataset of low and high passages showing subclusters of primary SCs and SCPD. [Figure 7G]: Molecular changes in SC cultures after long-term maintenance. [Figure 7H]: Molecular changes in SC cultures after long-term maintenance. [Figure 7H]: Ontology of Biological Process (GO BP) pathway enrichment analysis of the top 250 differentially expressed genes in low and high passage SCPD. [Figure 7-2] See description of Figure 7-1. [Figure 8-1]Figures 8A-8D. [Figure 8A] Antibody screening identifies novel surface markers for human SCs. Schematic diagram of the human antibody screening paradigm. [Figure 8B] Antibody screening identifies novel surface markers for human SCs. Primary screening identifies novel surface markers for hPSC-SCs. [Figure 8C] Antibody screening identifies novel surface markers for human SCs. Dot plots of scaled mean expression of low- and high-passage cell-type-specific surface markers. Surface markers identified in the human antibody screen are highlighted in red. Gene sets were pre-filtered to include genes expressed in at least 25% of cells of either cell type in low- and high-passage SC culture data. [Figure 8D] Antibody screening identifies novel surface markers for human SCs. Immunocytochemical (left) and flow cytometry-based (right) validation of surface marker expression at different stages of SC differentiation. [Figure 8-2] See description of Figure 8-1. [Figure 9-1] Figures 9A-9C. [Figure 9A] Molecular signature of myelinating SCs. Dot plots of the scaled mean expression of specific neurotrophic factors in myelinating SCs (mySc) and other cells in low- and high-passage cultures. [Figure 9B] Molecular signature of myelinating SCs. Dot plots of the scaled mean expression of specific neurotransmitter receptors in myelinating SCs (mySc) and other cells in low- and high-passage cultures. [Figure 9C] Molecular signature of myelinating SCs. Dot plots of the scaled mean expression of specific cell adhesion molecules in myelinating SCs (mySc) and other cells in low- and high-passage cultures. All gene sets were pre-filtered to include genes expressed in at least 25% of cells of either cell type in low- (LP) and high- (HP) passage SC culture data. [Figure 9-2] See description of Figure 9-1. [Figure 10]Figures 10A-10B. [Figure 10A] hPSC-SCs accelerate the functional maturation of hPSC-motoneurons in coculture. Calcium imaging of a motor neuron monoculture and a motor neuron-Schwann coculture at day 40 of motor neuron differentiation. [Figure 10B] hPSC-SCs accelerate the functional maturation of hPSC-motoneurons in coculture. Calcium imaging of a motor neuron monoculture and a motor neuron-Schwann coculture at day 70 of motor neuron differentiation. [Figure 11] 1 shows hPSC-SC expression profiles related to differentiation factors. 2 shows expression plots of various expression patterns of genes related to CMT in cells with different maturity levels. [Figure 12] Bupropion treatment is associated with lower odds of neuropathy in diabetic patients (left and center panels) Schematic (left panel) and Venn diagram (center panel) of a cohort of diabetic individuals derived from health records. (Right panel) Association of bupropion with neuropathy in diabetic patients in a multivariate logistic model adjusted for age, diabetes duration, sex, smoking, and antidepressant treatment. [Figure 13]Figures 13A-13D. [Figure 13A] Association of BP and other medications with diabetic neuropathy. Schematic diagram of a cohort of diabetic individuals filtered for age (>40 years) and diabetes duration (>10 years) from the University of California, San Francisco (UCSF) de-identified health records. [Figure 13B] Association of BP and other medications with diabetic neuropathy. Venn diagram of a cohort of diabetic individuals filtered for age (>40 years) and diabetes duration (>10 years) from the University of California, San Francisco (UCSF) de-identified health records. Incidence of neuropathy is assessed in individuals with gabapentin & pregabalin (green), bupropion (blue), fluoxetine (SSRI) (light red), and amitriptyline (TCA) (orange). Neuropathy is highlighted. [Figure 13C] Association of BP and other medications with diabetic neuropathy. Association of neuropathy with bupropion, fluoxetine, gabapentin & pregabalin, and amitriptyline in diabetic individuals in a multivariate logistic model adjusted for age, diabetes duration, sex, and smoking. [Figure 13D] Association of BP and other drugs with diabetic neuropathy. Antidepressants and antipsychotics in the FDA-approved drug library ordered according to their z-scores for the effect on SC survival when co-treated with 30 mM glucose. DETAILED DESCRIPTION OF THE INVENTION

[0027] Detailed Description of the Embodiments The disclosed methods and compositions may be more readily understood by reference to the following detailed description of specific embodiments and the examples contained therein, as well as the figures and their foregoing and subsequent descriptions. It is understood that the disclosed methods and compositions are not limited to particular synthetic methods, specific analytical techniques, or particular reagents, unless otherwise specified, and as such may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0028] This disclosure describes a hPSC differentiation strategy (a method for differentiating cells) for the efficient derivation of SCs that recapitulate the molecular characteristics and functions of primary SCs. Using a combination of imaging and high-resolution transcriptomics profiling, we characterized the diversity of cell types in our cultures and identified novel markers and molecular signatures for human SC subtypes. We further validated the engraftment potential of these cells upon transplantation into a rat model of peripheral neuropathy. Finally, we utilized hPSC-derived SCs to model the most common cause of peripheral neuropathy, namely diabetic peripheral neuropathy (DPN), which affects 30–60% of diabetic patients (Callaghan et al., 2012) and is the leading cause of diabetes-related hospitalizations and non-traumatic lower limb amputations (Boulton et al., 2005). The pathogenesis of DPN is complex, involving vascular disease, hyperglycemia, hypoxia, and oxidative stress, which results in peripheral nerve cytotoxicity and progressive degeneration (Simmons and Feldman, 2002). Although symptoms arise from neuronal dysfunction, it is unclear whether sensory neuron damage is the primary event in DPN; there is evidence that SC degeneration and peripheral demyelination may be contributing factors (Eckersley, 2002). Given the involvement of various non-cell-autonomous factors, including systemic vascular abnormalities, dissecting cell-type-specific mechanisms using current animal models of DPN is challenging. Using hPSC-derived SCs and sensory neurons, we have determined cell-type-specific vulnerability to high glucose, established alternative human-based models of DPN, and identified potential therapeutic candidates. Therefore, novel protocols for the induction of enteric neurons (ENs) from hPSCs and a basis for modeling EN development and the contribution of specific lineages to ENS disease are needed.

[0029] The present disclosure relates to compositions comprising SCs. The disclosure further relates to tissue culture systems. The disclosure also relates to methods for differentiating pluripotent stem cells into SCs, methods for enriching SCs in cell culture systems, and methods for culturing SCs. It should be understood that such methods have application, for example, in investigating genetic contributions to ENS pathogenesis using induced pluripotent stem cell (iPSC) lines. In some embodiments, the cells are derived from stem cells from patients suffering from enteric neuropathy. Disease phenotypes can be modeled by in vitro differentiation and addressed through genetic or molecular perturbation strategies. Accordingly, the present disclosure also relates to methods for screening compounds, methods for transplanting SCs, and methods for treating spinal cord injury and peripheral nerve injury by administering a pharmaceutical composition comprising any one or a combination of the disclosed cells and a pharmaceutically acceptable carrier.

[0030] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994), provides those of ordinary skill in the art with a general guide to many of the terms used in this application. In addition, the practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Such techniques are fully explained in, for example, "Molecular Cloning: A Laboratory Manual", 2nd edition (Sambrook et al., 1989), "Oligonucleotide Synthesis" (M.J. Gait, ed., 1984), "Animal Cell Culture" (R.I. Freshney, ed., 1987), "Methods in Enzymology" (Academic Press, Inc.), "Handbook of Experimental Immunology", 4th edition (D.M. Weir & C.C. Blackwell, eds., Blackwell Science Inc., 1987), "Gene Transfer Vectors for Mammalian Cells" (J.M. Miller & M.P. Calos, eds., 1987), "Current Protocols in Molecular Biology" (F.M. Ausubel et al., eds., 1987), and "PCR: The Polymerase Chain Reaction" (Mullis et al., eds., 1994).

[0031] As used in this disclosure and claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. Thus, for example, reference to "a nucleic acid sequence" includes a plurality of nucleotides present, and reference to "the nucleic acid sequence" is a reference to one or more nucleic acid sequences and equivalents thereof known to those of skill in the art.

[0032] Where an embodiment is described herein using the language "comprising," it is understood that other similar embodiments described using the terms "consisting of" and / or "consisting essentially of" are also provided. Where an embodiment is described herein using the language "consisting essentially of," it is understood that other similar embodiments described using the term "consisting of" are also provided.

[0033] The term "and / or" as used herein in phrases such as "A and / or B" is intended to include both A and B, A or B, A alone, and B alone. Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone.

[0034] As used herein, the term "substantially free" refers to a composition having only trace or negligible amounts of the referenced substance. In some embodiments, substantially free means that the composition contains only about 0.1%, 0.2%, 0.3%, 0.4%, or 0.5% of the referenced substance. In some embodiments, substantially free means that the composition contains less than about 1.0% of the referenced substance, relative to the number or weight of the substances in the composition, and does not impart a biological effect to the composition.

[0035] Ranges may be expressed herein as "about" one particular value and / or to "about" another particular value. When such ranges are expressed, ranges from the one particular value and / or to the other particular value are considered to be specifically contemplated and disclosed, unless the context dictates otherwise. Similarly, when values ​​are expressed as approximations, it will be understood that by using the antecedent "about," the particular value forms another specifically contemplated embodiment that should be considered disclosed, unless the context dictates otherwise. Further, it will be understood that each of the endpoints of a range is significant both in relation to the other endpoint, and independently of the other endpoint, unless the context dictates otherwise. The term "about," as used herein when referring to measurable values, such as amounts, time durations, etc., is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or ±0.1% from the specified value, where such variations are appropriate for performing the disclosed methods.

[0036] "Contacting" is used according to its plain and ordinary meaning to refer to the process of allowing at least two different species (e.g., chemical compounds, including biomolecules or cells) to come into sufficient proximity to react, interact, or physically touch. However, it should be understood that the resulting reaction product can be produced directly from the reaction between added reagents or from an intermediate from one or more added reagents that may be produced in the reaction mixture. The term "contacting" can include allowing two species to react, interact, or physically touch, and the two species can be a compound described herein and a cell (e.g., a Schwann cell). In some embodiments, contacting includes allowing a compound described herein to interact with a protein or enzyme involved in a signal transduction pathway (e.g., PDGFR).

[0037] As used herein, the term "culture vessel" is defined as any vessel suitable for growing, culturing, cultivating, expanding, or otherwise similarly manipulating cells. A culture vessel may also be referred to herein as a "culture insert." In some embodiments, the culture vessel is made from biocompatible plastic and / or glass. In some embodiments, the plastic is a thin layer of plastic containing one or more pores that allow proteins, nucleic acids, nutrients (such as heavy metals and hormones), antibiotics, and other cell culture media components to diffuse through the pores. In some embodiments, the pores are about 0.1, 0.5, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 microns or less in width. In some embodiments, the culture vessel is a hydrogel matrix and does not include a substrate or any other structure. In some embodiments, the culture vessel is designed to contain a hydrogel or hydrogel matrix and various culture media. In some embodiments, the culture vessel consists of, or consists essentially of, a hydrogel or hydrogel matrix. In some embodiments, the only plastic components of the culture vessel are those components that make up the sidewalls and / or bottom of the culture vessel, which separate the well or cell growth zone volume from points outside the culture vessel. In some embodiments, the culture vessel comprises a hydrogel and one or more isolated Schwann cells. In some embodiments, the culture vessel comprises a hydrogel and one or more isolated pluripotent stem cells, or is seeded with one or more neuronal cells.

[0038] As used herein, the term "exposing" refers to contacting, directly or indirectly, a disclosed compound with a cell, target receptor, or other biological entity in such a way that the disclosed compound can affect the activity of the cell (e.g., receptor, cell, etc.). This can occur directly, by physical contact between the disclosed compound and the cell, receptor, or other entity, i.e., by contacting the target or cell itself, or it can occur indirectly, by interacting with another molecule, cofactor, factor, or protein on which the activity of the cell depends. In some embodiments, the activity of a cell in response to a compound or molecule is differentiation. In some embodiments, the compound is one or more differentiation factors.

[0039] "Analogs" or "derivatives" of the compounds disclosed herein, when used interchangeably, refer to pharmaceutically acceptable salts, prodrugs, deuterated forms, radioactively labeled forms, isomers, solvates, and combinations thereof. In this context, a "combination" refers to a derivative that falls into at least two of the following groups: pharmaceutically acceptable salts, prodrugs, deuterated forms, radioactively labeled forms, isomers, and solvates. Examples of radioactively labeled forms include compounds labeled with tritium, phosphorus-32, iodine-129, carbon-11, fluorine-18, and the like. The compounds described herein may exist in the form of pharmaceutically acceptable salts. When used in pharmaceuticals, salts of the compounds described herein refer to non-toxic "pharmaceutically acceptable salts." Pharmaceutically acceptable salt forms include pharmaceutically acceptable acidic / anionic or basic / cationic salts. Suitable pharmaceutically acceptable acid addition salts of the compounds described herein include, for example, salts of inorganic acids (such as hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, and sulfuric acid), and salts of organic acids (such as acetic acid, benzenesulfonic acid, benzoic acid, methanesulfonic acid, and p-toluenesulfonic acid). Examples of pharmaceutically acceptable base addition salts include, for example, sodium, potassium, calcium, ammonium, organic amino, or magnesium salts.

[0040] As used herein, the term "salt" refers to an acid or base salt of a compound used in the methods of the present disclosure. Illustrative examples of acceptable salts are salts of mineral acids (such as hydrochloric acid, hydrobromic acid, phosphoric acid, etc.), salts of organic acids (such as acetic acid, propionic acid, glutamic acid, citric acid, etc.), and salts of quaternary ammonium (such as methyl iodide, ethyl iodide, etc.).

[0041] As used herein, the term "pharmaceutically acceptable excipient, carrier, or diluent" refers to an excipient, carrier, or diluent that can be administered to a subject together with a drug, does not destroy its pharmacological activity, and is not toxic when administered in a dose sufficient to deliver a therapeutic amount of the drug. As used herein, the term "pharmaceutically acceptable salt" of a nucleic acid can be an acid or base salt generally considered in the art to be suitable for use in contact with human or animal tissues without undue toxicity, irritation, allergic response, or other problems or complications. Such salts include inorganic and organic acid salts of basic residues such as amines, and alkali or organic salts of acidic residues such as carboxylic acids. Specific pharmaceutical salts include, but are not limited to, salts of acids such as hydrochloric acid, phosphoric acid, hydrobromic acid, maleic acid, glycolic acid, fumaric acid, sulfuric acid, sulfamic acid, suifanilic acid, formic acid, toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethanedisulfonic acid, 2-hydroxyethylsulfonic acid, nitric acid, benzoic acid, 2-acetoxybenzoic acid, citric acid, tartaric acid, lactic acid, stearic acid, salicylic acid, glutamic acid, ascorbic acid, pamoic acid, succinic acid, fumaric acid, maleic acid, propionic acid, hydroxymaleic acid, hydroiodic acid, phenylacetic acid, alkanoic acids such as acetic acid, HOOC-(CH)-COOH (n is 0-4), etc. Similarly, pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium. Those skilled in the art will recognize from this disclosure and knowledge in the art additional pharmaceutically acceptable salts for the pooled virus-specific antigens or polynucleotides provided herein, including those listed in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, p. 1418 (1985). In general, pharmaceutically acceptable acid or base salts can be synthesized from a parent compound that contains a basic or acidic moiety by any conventional chemical method.Briefly, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in an appropriate solvent.

[0042] As used herein, the term "progenitor cells" is defined as cells that are pluripotent cells exposed to a cell culture medium containing differentiation factors but remain at least partially undifferentiated and pluripotent. In some embodiments, progenitor cells comprise WNT2B+. In some embodiments, progenitor cells comprise PAX6+.

[0043] As used herein, the term "pluripotent stem cells" is defined as cells that are capable of self-renewal and development into cells and tissues of the three major germ layers. Pluripotent stem cells include embryonic and induced pluripotent cells as defined herein. Contemplated pluripotent stem cells are derived from mammals such as humans, mice, rats, monkeys, horses, goats, sheep, dogs, and cats.

[0044] The term "induced pluripotent stem cells" (iPSCs) refers to a type of pluripotent cell created by reprogramming somatic cells to have the same properties as embryonic stem cells, i.e., the ability to self-renew and differentiate into the three major germ layers. In some embodiments, iPSCs comprise mammalian cells, such as human, mouse, rat, monkey, horse, goat, sheep, dog, or cat, that have been reprogrammed to express Oct4, Nanog, Sox2, and optionally c-Myc. In some embodiments, iPSCs comprise reprogrammed primary cell lines. In some embodiments, iPSCs are obtained from the Coriell Institute for Medical Research (e.g., catalog IDs GM25256 (WTC-11), GM25430, GM23392, GM23396, GM24666, GM27177, GM24683), the California Institute for Regenerative Medicine: California's Stem Cell Agency (e.g., CW60261, CW60354, CW60359, CW60480, CW60335, CW60280, CW60594, CW60083, CW60086, CW60087, CW60167, CW60186), and the American Type Culture Collection (ATCC®) (e.g., ATCC-DYR0530 Human Induced Pluripotent Stem (IPS) Cells may be obtained from repositories such as ATCC® ACS-1012™, ATCC® ACS-1011™, ATCC® Number: ACS-1024™, ATCC® Number: ACS-1028™, ATCC® Number: ACS-1031™, ATCC® Number: ACS-1004™, ATCC® Number: ACS-1029™, ATCC® Number: ACS-1020™, ATCC® Number: ACS-1007™, ATCC® Number: ACS-1030™).Induced pluripotent stem cells can be derived from cell types such as fibroblasts harvested from the skin, lungs, or veins of apparently healthy or diseased subjects. In some embodiments, iSPCs are isolated from subjects suffering from an indication disclosed herein, such as diabetic peripheral neuropathy.

[0045] As defined herein, the terms "inhibition," "inhibit," "inhibiting," and the like, in reference to the interaction of a protein inhibitor (e.g., an antagonist), mean to adversely affect (e.g., decrease) the activity or function of a protein relative to the activity or function of the protein in the absence of the inhibitor. In embodiments, inhibition refers to the reduction of a disease or disease symptoms. In embodiments, inhibition refers to the reduction of the activity of a signal transduction pathway or a signaling pathway. Thus, inhibition includes, at least in part, partially or completely blocking a stimulus, reducing, preventing, or delaying activation, or inactivating, desensitizing, or downregulating signal transduction or enzymatic activity or the amount of a protein.

[0046] As used herein, the term "embryonic stem cell line" is defined as cells derived from the inner cell mass of a preimplantation blastocyst that are capable of self-renewal and differentiation into the three major germ layers. In some embodiments, the embryonic stem cell line is selected from the group consisting of: CHB-1, CHB-2, CHB-3, CHB-4, CHB-5, CHB-6, CHB-8, CHB-9, CHB-10, CHB-11, CHB-12, RUES1, RUES2, HUES1, HUES2, HUES3, HUES4, HUES5, HUES6, HUES7, HUES8, HUES9, HUES10, HUES11, HUES12, HUES13, HUES14, HUES15, HUES16, HUES17, HUES18, HUES19, HUES20, HUES21, HUES22, HUES23, HUES24, HUES26, HUES27, HUES 28, CyT49, RUES3, WA01(H1), UCSF4, NYUES1, NYUES2, NYUES3, NYUES4, NYUES5, NYUES6, NYUES7, MFS5, HUES 48, HUES 49, HUES 53, HUES 65, HUES 66, UCLA 1, UCLA 2, UCLA 3, WA07(H7), WA09(H9), WA13(H13), WA14(H14), HUES 62, HUES 63, HUES 64, CT1, CT2, CT3, CT4, MA135, Endeavor-2, WIBR1, WIBR2, HUES 45, Shef 3, Shef 6, WIBR3, WIBR4, WIBR5, WIBR6, BJNhem19, BJNhem20, SA001, SA002, UCLA 4, UCLA 5, UCLA 6, HUES PGD 13, HUES PGD 3, ESI-014, ESI-017, HUES PGD 11, HUES PGD 12, WA15, WA16, WA17, WA18, WA19, etc. In some embodiments, the embryonic stem cells comprise a gene or genes associated with a disease or disorder.

[0047] The term "enteric neural crest cells" or "neural crest cells" refers to cells produced by inducing differentiation of pluripotent stem cells, wherein the enteric neural crest cells express SOX10, PHOX2B, EDNRB, TFAP2A, BRN3A, ISL1, and / or ASCL1. In some embodiments, the enteric neural crest cells comprise FOX3D. In some embodiments, the neural crest cells are present in embryoid bodies or neural rosettes. In some embodiments, the neural crest cells express the vagus nerve markers HOXB2, HOXB3, and / or HOXB5. In some embodiments, the neural crest cells express p75 and HNK1. In some embodiments, the neural crest cells express HOXB2, HOXB3, HAND2, and EDNRB. In some embodiments, the neural crest cells or enteric neural crest cells are isolated from primary stem cells or induced pluripotent stem cells. In some embodiments, the neural crest cells are any of those cells identified as "neural crest cells" in PCT / US2021 / 024244 or PCT / US2019 / 068447, both of which are incorporated by reference in their entireties. In some embodiments, the Schwann cells are any of those cells identified as Schwann cells in WO / 2018 / 090002 or WO / 2018 / 090006, both of which are incorporated by reference in their entireties.

[0048] The term "enteric neuron" refers to cells that exhibit downregulation of SOX10, sustained expression of EDNRB, ASCL1 and PHOX2B, and upregulation of TUJ1 and TRKC. In some embodiments, enteric neurons express cholinergic neuronal markers, The enteric neurons express neural subtype-specific markers, including choline acetyltransferase (CHAT), serotonin (5-HT) receptors, gamma-aminobutyric acid (GABA), and neuronal nitric oxide synthase (nNOS). In some embodiments, CHAT expression indicates the presence of cholinergic neurons. In some embodiments, NOS1 expression indicates the presence of nitrergic neurons. In some embodiments, the enteric neurons comprise glial cells that express glial fibrillary acidic protein (GFAP) and SOX10. In some embodiments, the enteric neurons are produced by inducing differentiation of enteric neural crest cells. In some embodiments, the enteric neurons express SOX10, persistently express EDNRB, ASCL1, and PHOX2B, and upregulate TUJ1 and TRKC.

[0049] The term "enteric glial cells" refers to cells that exhibit expression of SOX10 and GPAP and / or PMP22. In some embodiments, enteric glial cells exhibit expression of SOX10 and PMP22. In some embodiments, enteric glial cells are produced by inducing differentiation of enteric neural crest cells.

[0050] The term "Schwann cell" refers to a cell that is a nerve-related cell derived from a nerve crest from the peripheral nervous system. In some embodiments, the Schwann cell interacts with and supports the function of neurons when placed in an animal or patient. In some embodiments, the Schwann cell expresses a nucleic acid sequence encoding CD98, SOX10, POU3F2, NGFR, and / or GFAP43, or a functional fragment thereof, or an amino acid sequence comprising at least about 90% sequence identity thereto. In some embodiments, the cell exhibits expression of one or a combination of proteins or nucleic acid sequences encoding the proteins identified in any one of claims 1-10.

[0051] The term "rho kinase inhibitor" refers to a compound that reduces the activity of rho kinase. In some embodiments, the rho kinase inhibitor is N-[(3-hydroxyphenyl)methyl]-N'-[4-(4-pyridinyl)-2-thiazolyl]urea dihydrochloride (RKI-1447), (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride (Y-27632), fasudil (HA-1077), hydroxyfasudil (HA 1100 hydrochloride), thiazovivin, GSK429286A, narciclasine, and / or (+)-(R)-trans-4-(1-aminoethyl)-N-(1H-pyrrolo[2,3-b]pyridin-4-yl)cyclohexanecarboxamide dihydrochloride (Y-30141).

[0052] The term "hydrogel," as used herein, is defined as any water-insoluble, crosslinked, three-dimensional network of polymer chains that is water-filled or has voids between the polymer chains that can be filled with water. The term "hydrogel matrix," as used herein, is defined as any three-dimensional hydrogel construct, system, device, or similar structure. In some embodiments, the hydrogel or hydrogel matrix comprises one or more proteins and / or glycoproteins. In some embodiments, the hydrogel or hydrogel matrix comprises one or more of the following proteins: collagen, gelatin, elastin, titin, laminin, fibronectin, fibrin, keratin, silk fibroin, and any derivative or combination thereof. In some embodiments, the hydrogel or hydrogel matrix comprises Matrigel® or vitronectin. In some embodiments, the hydrogel or hydrogel matrix can be solidified into various shapes, for example, branched shapes designed to mimic the neural tube. In some embodiments, the hydrogel or hydrogel matrix comprises poly(ethylene glycol) dimethacrylate (PEG). In some embodiments, the hydrogel or hydrogel matrix comprises Puramatrix. In some embodiments, the hydrogel or hydrogel matrix comprises glycidyl methacrylate-dextran (MeDex). In some embodiments, two or more hydrogels or hydrogel matrices are used simultaneously in a cell culture vessel. In some embodiments, two or more hydrogels or hydrogel matrices are used simultaneously in the same cell culture vessel, but the hydrogels are separated by walls that create independently addressable microenvironments within the tissue culture vessel, such as wells. In multiple tissue culture vessels, some embodiments can include any number of such wells or independently addressable locations within the cell culture vessel, such that the hydrogel matrix in one well or location is different or the same as the hydrogel matrix in another well or location of the cell culture vessel.

[0053] The term "Matrigel®" refers to a solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma that contains ECM proteins including laminin, collagen IV, heparin sulfate proteoglycans, entactin / nidogen, and other growth factors. In some embodiments, Cultrex® BME (Trevigen, Inc.) or Geltrex® (Thermo-Fisher Inc.) can be used in place of Matrigel®.

[0054] As used herein, the term "two-dimensional culture" is defined as the culture of cells on a flat hydrogel, including Matrigel® and vitronectin, placed within a culture vessel.

[0055] As used herein, "spheroid" or "cell spheroid" means any group of cells in a three-dimensional shape that generally corresponds to an ellipse or circle rotated about one of its major axes, the major or minor axis, and includes three-dimensional ovoid, oblate and prolate spheroids, spheres, and substantially equivalent shapes.

[0056] As used herein, the term "subject" refers to any animal (e.g., mammal), including, but not limited to, humans, non-human primates, dogs, cats, rodents, etc. In some embodiments, the subject is a human subject. The terms "subject," "individual," and "patient" are used interchangeably herein. Thus, the terms "subject," "individual," and "patient" encompass individuals with cancer (e.g., breast cancer), including individuals who have undergone or are candidates for resection (surgery) to remove cancerous tissue.

[0057] As used herein, the term "diagnosed" means having undergone a physical examination by a skilled artisan, e.g., a physician, and found to have a condition that can be diagnosed or treated by the compounds, compositions, or methods disclosed herein. In some embodiments of the disclosed methods, the subject has been diagnosed as needing treatment for nerve injury or DNP prior to the administering step. As used herein, phrases such as "identified as needing treatment for a disorder" refer to selecting a subject based on need for treatment for a disorder. It is contemplated that the identification, in some embodiments, may be performed by a person different from the person making the diagnosis. In further embodiments, it is also contemplated that the administration may be performed by the person who subsequently performed the administration.

[0058] The term "associated with" or "related to" in the context of a substance or substance activity or function associated with a disease (e.g., a symptom associated with a protein-related disease, nerve repair, or dysfunction) refers to the substance or substance activity or function that causes (in whole or in part) the symptoms associated with the disease (e.g., diabetic peripheral neuropathy) or the symptoms of the disease (in whole or in part). For example, the symptoms of a condition can be symptoms resulting (in whole or in part) from spinal cord injury or DNP. As used herein, something that is described as associated with a disease can be a target for the treatment of the disease if it is a causative agent.

[0059] As used herein, the term "administering" refers to oral administration, administration as a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intracranial, intranasal, or subcutaneous administration, or implantation of a device containing the disclosed cells into a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like. By "co-administered" is meant that the compositions described herein are administered simultaneously with, immediately before, or immediately after the administration of one or more additional therapies (e.g., a cardiomyopathy therapy, e.g., an angiotensin-converting enzyme inhibitor (e.g., enalipril, lisinopril), an angiotensin receptor blocker (e.g., losartan, valsartan), a beta-blocker (e.g., Lopressor, Toprol-XL), digoxin, or a diuretic (e.g., Lasix, or a Parkinson's disease therapy, e.g., including levodopa, a dopamine agonist (e.g., bromocriptine, pergolide, pramipexole, ropinirole, piribedil, cabergoline, apomorphine, lisuride), an MAO-B inhibitor (e.g., selegiline or rasagiline), amantadine, an anticholinergic, an anti-inflammatory (e.g., clozapine), a cholinesterase inhibitor, modafinil, or a nonsteroidal anti-inflammatory drug).

[0060] As used herein, the terms "subject," "individual," "host," and "patient" are used interchangeably herein and refer to a vertebrate, particularly a human, including, but not limited to, a mammal or human, for whom diagnosis, treatment, or therapy is desired. Mammals include, but are not limited to, mice, monkeys, humans, livestock, cattle, pigs, goats, sheep, horses, dogs, sport animals, rats, and pets. Tissues, cells, and their progeny obtained in vivo or cultured in vitro are also encompassed within the definition of the term "subject." The methods described herein are applicable to both human therapeutic applications and veterinary applications. In some examples within the description of this disclosure, the term "patient" refers to a human patient suffering from a particular disease or disorder. In some embodiments, the subject may be a human suspected of or identified as being at risk for developing peripheral neuropathy. In some embodiments, the subject may be diagnosed with DPN or may be diagnosed as being at risk for developing DPN. In some embodiments, the subject is a mammal; in other embodiments, the subject is a human. In some embodiments, the subject is a non-human vertebrate.

[0061] As used herein, the term "therapeutic agent" means an agent utilized to treat, combat, mitigate, prevent, or ameliorate an unwanted condition or disease in a patient.

[0062] A "therapeutically effective amount" or "effective amount" of a composition is a predetermined amount calculated to achieve a desired effect, i.e., to treat, combat, alleviate, prevent, or ameliorate one or more symptoms of a viral infection. The activity contemplated by the present method includes both medical and / or prophylactic treatment, as appropriate. The specific dose of a compound administered in accordance with the present disclosure to achieve a therapeutic and / or prophylactic effect will, of course, be determined by the particular circumstances surrounding the case, including, for example, the compound administered, the route of administration, and the condition being treated. It will be understood that the effective amount to be administered will be determined by a physician in light of the relevant circumstances, including the condition being treated, the choice of compound to be administered, and the selected route of administration. Therefore, the dosage ranges set forth above are not intended to limit the scope of the present disclosure in any way. A therapeutically effective amount of a compound of an embodiment of the present disclosure is typically an amount sufficient to achieve an effective systemic or local concentration in tissues when administered in a physiologically acceptable excipient composition. As used herein, the terms "treating," "treatment," or "treat" refer to a therapeutic procedure that cures, alleviates, relieves symptoms of, and / or halts progression of a diagnosed pathological condition or disorder.

[0063] As used herein, the terms "preventing" or "prevention" or "prevent" refer to protective or prophylactic measures that prevent or delay the onset of the targeted pathological condition or disorder. Those in need of treatment include those already diagnosed with the disorder, those prone to having the disorder, and those in whom the disorder is to be prevented.

[0064] The "percent identity" or "percent homology" of two polynucleotide or two polypeptide sequences is determined by comparing the sequences using the GAP computer program (part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) using its default parameters. As used herein, "identical" or "identity" in the context of two or more nucleic acid or amino acid sequences can mean that the sequences have a specified percentage of residues that are the same over a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over a specified region, determining the number of positions at which identical residues occur in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to obtain the percentage sequence identity. If the two sequences have different lengths or the alignment results in one or more staggered ends, and a specified comparison region contains only a single sequence, the residues of the single sequence are included in the denominator rather than the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be determined manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0. Briefly, the BLAST algorithm stands for Basic Local Alignment Search Tool and is suitable for determining sequence similarity. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov). This algorithm involves first identifying short words of length W, which, when aligned with words of the same length in database sequences, identify high-scoring sequence pairs (HSPs), and obtaining a query sequence that matches or meets a positive threshold score T. T is referred to as the neighborhood word score threshold (Altschul et al., supra).These initial neighborhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is stopped when: 1) the cumulative alignment score drops by a quantity X from its maximum achieved value; 2) the accumulation of one or more negative-scoring residue alignments causes the cumulative score to fall below 0; or 3) the end of either sequence is reached. The Blast algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The Blast program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 10915-10919, incorporated herein by reference in its entirety), alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands. The BLAST algorithm (Karlin et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 5873-5787, incorporated herein by reference in its entirety) and Gapped BLAST perform statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide sequences would occur by chance. For example, a nucleic acid is considered to be similar to another nucleic acid if the minimum sum probability in a comparison of the test nucleic acid with the other nucleic acid is less than about 1, less than about 0.1, less than about 0.01, and less than about 0.001. Two single-stranded polynucleotides are "complements" of each other if their sequences can be aligned in an antiparallel orientation such that every nucleotide in one polynucleotide is opposite its complementary nucleotide in the other polynucleotide, without introducing gaps and without unpaired nucleotides at the 5' or 3' end of either sequence. A polynucleotide is "complementary" to another polynucleotide if the two polynucleotides are capable of hybridizing to one another under moderately stringent conditions.Thus, a polynucleotide can be complementary to another polynucleotide without being its complement.

[0065] The term "functional fragment" refers to any portion of a polypeptide or nucleic acid sequence to which a full-length polypeptide or nucleic acid, respectively, is related, and is of sufficient length and has sufficient structure to confer at least a similar or substantially similar biological effect as the full-length polypeptide or nucleic acid on which the fragment is based. In some embodiments, a functional fragment is a portion of a full-length or wild-type nucleic acid sequence encoding any one of the nucleic acid sequences disclosed herein, the portion encoding a polypeptide of a particular length and / or structure less than the full length, but still encoding a domain that is biologically functional compared to the full-length or wild-type protein. In some embodiments, a functional fragment can have reduced, approximately the same, or enhanced biological activity compared to the wild-type or full-length polypeptide sequence on which the fragment is based. In some embodiments, a functional fragment is derived from a sequence of an organism, e.g., a human. In such embodiments, a functional fragment may retain 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity with the wild-type human sequence from which it is derived, hi some embodiments, a functional fragment may retain 85%, 80%, 75%, 70%, 65%, or 60% sequence identity with the wild-type sequence from which it is derived.

[0066] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule, which portion preferably contains at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or about 90% of the entire length of the reference nucleic acid molecule or polypeptide. Fragments can contain about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more nucleotides or amino acids.

[0067] "Variant" is intended to mean a substantially similar sequence. For nucleic acid molecules, variants include nucleic acid molecules having deletions (i.e., truncations) at the 5' and / or 3' ends, deletions and / or additions of one or more nucleotides at one or more internal sites within the naturally occurring polynucleotide, and / or substitutions of one or more nucleotides at one or more sites within the naturally occurring polynucleotide. As used herein, a "naturally occurring" nucleic acid molecule or polypeptide includes a nucleotide sequence or amino acid sequence, respectively, that occurs in nature. For nucleic acid molecules, conservative variants include sequences that, due to the degeneracy of the genetic code, encode the amino acid sequence of one of the polypeptides of the present disclosure. Variant nucleic acid molecules also include synthetically derived nucleic acid molecules, such as those generated by using site-directed mutagenesis, yet still encode proteins of the present disclosure. Generally, variants of particular nucleic acid molecules of the present disclosure have at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to that particular polynucleotide, as determined by sequence alignment programs and parameters as described elsewhere herein. Variants of particular nucleic acid molecules of the present disclosure (i.e., nucleic acids encoding any amino acid in Tables 1, 2, or 3) can also be assessed by comparing the percent sequence identity between the polypeptide encoded by the variant nucleic acid molecule and the polypeptide encoded by the reference nucleic acid molecule. The percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters as described elsewhere herein. When any given pair of nucleic acid molecules of the disclosure is assessed by comparing the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.In some embodiments, the term "variant" protein is intended to mean a protein derived from a native protein by the deletion of one or more amino acids at the N-terminus and / or C-terminus of the native protein (so-called truncation), the deletion and / or addition of one or more amino acids at one or more internal sites in the native protein, or the substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present disclosure are biologically active, i.e., retain the desired biological activity of the native protein described herein. Such variants may result, for example, from genetic polymorphism or from human manipulation. Biologically active variants of proteins of the present disclosure have at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the amino acid sequence of the native protein, as determined by sequence alignment programs and parameters described elsewhere herein. Biologically active variants of proteins of the disclosure can differ from the protein by 1 to 15 amino acid residues, 1 to 10, e.g., 6 to 10, 5, 4, 3, 2, or even only 1 amino acid residue. Proteins or polypeptides of the disclosure can be modified in a variety of ways, including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants and fragments of proteins can be prepared by mutation of a nucleic acid sequence recombinantly encoding the amino acid sequence. In some embodiments, the disclosure relates to cells containing those amino acid variants (or nucleic acid sequences encoding the amino acid sequences) identified in Tables 1, 2, or 3.

[0068] "Optionally" or "optionally" means that the subsequently described event, circumstance, or substance may or may not occur or exist, and the description includes cases where the event, circumstance, or substance occurs or exists, as well as cases where it does not occur or exist.

[0069] In some embodiments, the present disclosure relates to a system comprising a culture vessel containing a hydrogel and one or more isolated stem cells and / or neural crest cells. In some embodiments, the culture vessel contains Schwann cells. In some embodiments, the culture vessel contains Schwann cells that have differentiated in culture for about 12 to about 20 days. In some embodiments, the culture vessel contains a hydrogel, one or more isolated pluripotent stem cells, and tissue culture medium containing FGF2 or a functional fragment or variant thereof.

[0070] Composition of Matter The present disclosure relates to Schwann cells (SCs), and in some embodiments, compositions comprising same. In some embodiments, the SC cells of the present disclosure express CD98. In some embodiments, the cells comprise a nucleic acid sequence encoding or an amino acid sequence comprising CD98, an amino acid sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:1, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:1.

[0071] In some embodiments, the SC cells of the present disclosure express S100. In some embodiments, the SC cells comprise a nucleic acid sequence encoding or an amino acid sequence comprising S100, an amino acid sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:2, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:2.

[0072] In some embodiments, the SC cells of the present disclosure express MBP. In some embodiments, the SC cells comprise a nucleic acid sequence encoding or an amino acid sequence comprising MBP, an amino acid sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:3, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:3.

[0073] In some embodiments, the SC cells of the present disclosure express GFAP. In some embodiments, the SC cells comprise a nucleic acid sequence encoding or an amino acid sequence comprising GFAP, an amino acid sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:4, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:4.

[0074] In some embodiments, the SC cells of the present disclosure express PMP22. In some embodiments, the SC cells comprise a nucleic acid sequence encoding or an amino acid sequence comprising PMP22, an amino acid sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:5, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:5.

[0075] In some embodiments, the SC cells of the present disclosure express CD6. In some embodiments, the SC cells comprise a nucleic acid sequence encoding or an amino acid sequence comprising CD6, an amino acid sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:6, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:6.

[0076] In some embodiments, the SC cells of the present disclosure express CD9. In some embodiments, the SC cells comprise a nucleic acid sequence encoding or an amino acid sequence comprising CD9, an amino acid sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:7, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:7.

[0077] In some embodiments, the SC cells of the present disclosure express CD44. In some embodiments, the SC cells comprise a nucleic acid sequence encoding or an amino acid sequence comprising CD44, an amino acid sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:8, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:8.

[0078] In some embodiments, the SC cells of the present disclosure express CD46. In some embodiments, the SC cells comprise a nucleic acid sequence encoding or an amino acid sequence comprising CD46, an amino acid sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:9, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:9.

[0079] In some embodiments, the SC cells of the present disclosure express CD49e. In some embodiments, the SC cells comprise a nucleic acid sequence encoding or an amino acid sequence comprising CD49e, an amino acid sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 10, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 10.

[0080] In some embodiments, the SC cells of the present disclosure express CD81. In some embodiments, the SC cells comprise a nucleic acid sequence encoding or an amino acid sequence comprising CD81, an amino acid sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:11, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:11.

[0081] In some embodiments, the SC cells of the present disclosure express CD 146. In some embodiments, the SC cells comprise a nucleic acid sequence encoding or an amino acid sequence comprising CD146, an amino acid sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 12, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 12.

[0082] In some embodiments, the SC cells of the present disclosure express CD 147. In some embodiments, the SC cells comprise a nucleic acid sequence encoding or an amino acid sequence comprising CD147, an amino acid sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 13, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 13.

[0083] In some embodiments, the SC cells of the present disclosure express CD 166. In some embodiments, the SC cells comprise a nucleic acid sequence encoding or an amino acid sequence comprising CD166, an amino acid sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 14, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 14.

[0084] In some embodiments, the SC cells of the present disclosure express CD 171. In some embodiments, the SC cells comprise a nucleic acid sequence encoding or an amino acid sequence comprising CD 171, an amino acid sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 15, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 15.

[0085] In some embodiments, the SC cells of the present disclosure express NGFR. In some embodiments, the SC cells comprise a nucleic acid sequence encoding or an amino acid sequence comprising NGFR, an amino acid sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 16, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 16.

[0086] In some embodiments, the SC cells of the present disclosure express SOX10. In some embodiments, the SC cells comprise a nucleic acid sequence encoding or an amino acid sequence comprising SOX10, an amino acid sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 17, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 17.

[0087] In some embodiments, the SC cells of the present disclosure express POU3F2. In some embodiments, the SC cells comprise a nucleic acid sequence encoding or an amino acid sequence comprising POU3F2, an amino acid sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 18, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 18.

[0088] In some embodiments, the SC cells of the present disclosure express MPZ. In some embodiments, the SC cells comprise a nucleic acid sequence encoding or an amino acid sequence comprising MPZ, an amino acid sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 19, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 19.

[0089] In some embodiments, the SC cells of the present disclosure express GAP43. In some embodiments, the SC cells comprise a nucleic acid sequence encoding or an amino acid sequence comprising GAP43, an amino acid sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:20, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:20.

[0090] In some embodiments, the SC cells of the present disclosure express ERBB3. In some embodiments, the SC cells comprise a nucleic acid sequence encoding or an amino acid sequence comprising ERBB3, an amino acid sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:21, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:21.

[0091] In some embodiments, the SC cells of the present disclosure express GDNF. In some embodiments, the SC cells comprise a nucleic acid sequence encoding GDNF, or an amino acid sequence comprising same, an amino acid sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:22, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:22.

[0092] In some embodiments, the SC cells of the present disclosure express MAG. In some embodiments, the SC cells comprise a nucleic acid sequence encoding or an amino acid sequence comprising MAG, an amino acid sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:23, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:23.

[0093] In some embodiments, the SC cells of the present disclosure express PLLP. In some embodiments, the SC cells comprise a nucleic acid sequence encoding or an amino acid sequence comprising PLLP, an amino acid sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:24, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:24.

[0094] In some embodiments, the SC cells of the present disclosure express POU6F2. In some embodiments, the SC cells comprise a nucleic acid sequence encoding or an amino acid sequence comprising POU6F2, an amino acid sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:25, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:25.

[0095] In some embodiments, the SC cells of the present disclosure express PLXNB3. In some embodiments, the SC cells comprise a nucleic acid sequence encoding or an amino acid sequence comprising PLXNB3, an amino acid sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:26, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:26.

[0096] In some embodiments, the SC cells of the present disclosure express ERBB3, an amino acid sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:27, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:27.

[0097] In some embodiments, the SC cells comprise a nucleic acid sequence encoding or an amino acid sequence comprising one or a combination of proteins identified in FIG. 11, an amino acid sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to one or a combination of nucleic acids encoding one or a combination of amino acids identified in FIG. 11, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to one or a combination of nucleic acids encoding one or a combination of amino acids identified in FIG. 11.

[0098] Glial fibrillary acidic protein (GFAP) is a class III intermediate filament. During central nervous system development, GFAP is a cell-specific marker that distinguishes astrocytes from other glial cells. A non-limiting example of GFAP is GFAP from humans (Homo sapiens, UniProt accession number P14136) having the following sequence: MERRRITSAARRSYVSSGEMMVGGLAPGRRLGPGTRLSLAARMPPPLPTRVDFSLAGALNAGFKETRASERAEMMELNDRFASYIEKVRFLEQQNKALAAELNQLRAKEPTKLADVYQAELRELRLRLDQLTANSARLEVERDNLAQDLATVRQKLQDETNLRLEAENNLAAYRQEADEATLARLDLERKIESLEEEIRFLRKIHEEEVRELQEQLARQQV HVELDVAKPDLTAALKEIRTQYEAMASSNMHEAEEWYRSKFADLTDAAARNAELLRQAKHEANDYRRQLQSLTCDLESLRGTNESLERQMREQEERHVREAASYQEALARLEEEGQSLKDEMARHLQEYQDLLNVKLALDIEIATYRKLLEGEENRITIPVQTFSNLQIRETSLDTKSVSEGHLKRNIVVKTVEMRDGEVIKESKQEHKDVM (SEQ ID NO: 53).

[0099] Another non-limiting example of GFAP is GFAP from rat (Rattus norvegicus; UniProt accession number P47819) having the following sequence: MERRRITSARRSYASSETMVRGHGPTRHLGTIPRLSLSRMTPPLPARVDFSLAGALNAGFKETRASERAEMMELNDRFASYIEKVRFLEQQNKALAAELNQLRAKEPTK LADVYQAELRELRLRLDQLTTNSARLEVERDNLTQDLGTLRQKLQDETNLRLEAENNLAVYRQEADEATLARVDLERKVESLEEEIQFLRKIHEEEVRELQEQLAQQQVH VEMDVAKPDLTAALREIRTQYEAVATSNMQETEEWYRSKFADLTDVASRNAELLRQAKHEANDYRRQLQALTCDLESLRGTNESLERQMREQEERHARESASYQEALARLEEEGQSLKEEMARHLQEYQDLLNVKLALDIEIATYRKLLEGEENRITIPVQTFSNLQIRETSLDTKSVSEGHLKRNIVVKTVEMRDGEVIKESKQEHKDVM (SEQ ID NO: 54).

[0100] A further non-limiting example of GFAP is GFAP from mouse (Mus musculus; UniProt accession number P03995) having the following sequence: MERRRITSARRSYASETTVVRGLGPSRQLGTMPRFSLSRMTPPLPARVDFSLAGALNAGFKETRASERAEMMELNDRFASYIEKVRFLEQQNKALAAELNQLRAKEPTKL ADVYQAELRELRLRLDQLTANSARLEVERDNFAQDLGTLRQKLQDETNLRLEAENNLAAYRQEADEATLARVDLERKVESLEEEIQFLRKIYEEEVRELREQLAQQQVHV EMDVAKPDLTAALREIRTQYEAVATSNMQETEEWYRSKFADLTDAASRNAELLRQAKHEANDYRRQLQALTCDLESLRGTNESLERQMREQEERHARESASYQEALARLEEEGQSLKEEMARHLQEYQDLLNVKLALDIEIATYRKLLEGEENRITIPVQTFSNLQIRETSLDTKSVSEGHLKRNIVVKTVEMRDGEVIKDSKQEHKDVVM (SEQ ID NO: 55).

[0101] Thus, in some embodiments, GFAP comprises at least about 70% sequence identity to SEQ ID NO:53, SEQ ID NO:54, or SEQ ID NO:55, or a functional fragment thereof. In some embodiments, GFAP comprises at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:53, SEQ ID NO:54, or SEQ ID NO:55, or a functional fragment thereof. In some embodiments, GFAP comprises SEQ ID NO:53, SEQ ID NO:54, or SEQ ID NO:55, or a functional fragment thereof.

[0102] Enteric neural crest cells express the transcription factor SOX10, which signals neural crest cells to direct the activity of other genes to become more specific cell types, including enteric neurons. A non-limiting example of SOX10 is SOX10 from humans (Homo sapiens, UniProt accession number P56693), which has the following sequence: MAEEQDLSEVELSPVGSEEPRCLSPGSAPSLGPDGGGGGSGLRASPGPGELGKVKKEQQDGEADDDKFPVCIREAVSQVLSGYDWTLVPMPVRVNGASKSKPHVKRPMNAFMVWAQAA RRKLADQYPHLHNAELSKTLGKLWRLLNESDKRPFIEEAERLRMQHKKDHPDYKYQPRRRKNGKAAQGEAECPGGEAEQGGTAAIQAHYKSAHLDHRHPGEGSPMSDGNPEHPSGQSHG PPTPPTTPKTELQSGKADPKRDGRSMGEGGKPHIDFGNVDIGEISHEVMSNMETFDVAELDQYLPPNGHPGHVSSYSAAGYGLGSALAVASGHSAWISKPPGVALPTVSPPGVDAKAQVKTETAGPQGPPHYTDQPSTSQIAYTSLSLPHYGSAFPSISRPQFDYSDHQPSGPYYGHSGQASGLYSAFSYMGPSQRPLYTAISDPSPSGPQSHSPTHWEQPVYTTLSRP (SEQ ID NO: 56).

[0103] Another non-limiting example of SOX10 is SOX10 from rat (Rattus norvegicus; UniProt accession number O55170) having the following sequence: MAEEQDLSEVELSPVGSEEPRCLSPSSAPSLGPDGGGGGSGLRASPGPGELGKVKKEQQDGEADDDKFPVCIREAVSQVLSGYDWTLVPMPVRVNGASKSKPHVKRPMNAFMVWAQAA RRKLADQYPHLHNAELSKTLGKLWRLLNESDKRPFIEEAERLRMQHKKDHPDYKYQPRRRKNGKAAQGEAECPGGETDQGGAAAIQAHYKSAHLDHRHPEEGSPMSDGNPEHPSGQSHG PPTPPTTPKTELQSGKADPKRDGRSLGEGGKPHIDFGNVDIGEISHEVMSNMETFDVTELDQYLPPNGHPGHVGSYSAAGYGLSSALAVASGHSAWISKPPGVALPTVSPPAVDAKAQVKTETTGPQGPPHYTDQPSTSQIAYTSLSLPHYGSAFPSISRPQFDYSDHQPSGPYYGHAGQASGLYSAFSYMGPSQRPLYTAISDPSPSGPQSHSPTHWEQPVYTTLSRP (SEQ ID NO: 57).

[0104] A further non-limiting example of SOX10 is SOX10 from mouse (Mus musculus; UniProt accession number Q04888) having the following sequence: MAEEQDLSEVELSPVGSEEPRCLSPGSAPSLGPDGGGGGSGLRASPGPGELGKVKKEQQDGEADDDKFPVCIREAVSQVLSGYDWTLVPMPVRVNGASKSKPHVKRPMNAFMVWAQAA RRKLADQYPHLHNAELSKTLGKLWRLLNESDKRPFIEEAERLRMQHKKDHPDYKYQPRRRKNGKAAQGEAECPGGEAEQGGAAAIQAHYKSAHLDHRHPEEGSPMSDGNPEHPSGQSHG PPTPPTTPKTELQSGKADPKRDGRSLGEGGKPHIDFGNVDIGEISHEVMSNMETFDVTELDQYLPPNGHPGHVGSYSAAGYGLGSALAVASGHSAWISKPPGVALPTVSPPGVDAKAQVKTETTGPQGPPHYTDQPSTSQIAYTSLSLPHYGSAFPSISRPQFDYSDHQPSGPYYGHAGQASGLYSAFSYMGPSQRPLYTAISDPSPSGPQSHSPTHWEQPVYTTLSRP (SEQ ID NO: 58).

[0105] Thus, in some embodiments, SOX10 comprises at least about 70% sequence identity to SEQ ID NO:56, SEQ ID NO:57, or SEQ ID NO:58, or a functional fragment thereof. In some embodiments, SOX10 comprises at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18, or a functional fragment thereof. In some embodiments, SOX10 comprises SEQ ID NO:56, SEQ ID NO:57, or SEQ ID NO:58, or a functional fragment thereof.

[0106] (Table 1) TIFF2025531671000002.tif168167TIFF2025531671000003.tif137167TIFF2025531671000004.tif192167TIFF2025531671000005.tif238167TIFF2025531671000006.tif217167TIFF2025531671000007.tif187167TIFF2025531671000008.tif202167TIFF2025531671000009.tif238167TIFF2025531671000010.tif208167TIFF2025531671000011.tif218167TIFF2025531671000012.tif238167TIFF2025531671000013.tif213167TIFF2025531671000014.tif192167TIFF2025531671000015.tif238167TIFF2025531671000016.tif157167

[0107] In some embodiments, compositions of the present disclosure comprise greater than about 25% Schwann cells. In some embodiments, compositions of the present disclosure comprise greater than about 30% Schwann cells. In some embodiments, compositions of the present disclosure comprise greater than about 35% Schwann cells. In some embodiments, compositions of the present disclosure comprise greater than about 40% Schwann cells. In some embodiments, compositions of the present disclosure comprise greater than about 45% Schwann cells. In some embodiments, compositions of the present disclosure comprise greater than about 50% Schwann cells. In some embodiments, compositions of the present disclosure comprise greater than about 55% Schwann cells. In some embodiments, compositions of the present disclosure comprise greater than about 60% Schwann cells. In some embodiments, compositions of the present disclosure comprise greater than about 65% Schwann cells. In some embodiments, compositions of the present disclosure comprise greater than about 70% Schwann cells. In some embodiments, compositions comprise greater than about 75% Schwann cells. In some embodiments, compositions comprise greater than about 80% Schwann cells. In some embodiments, compositions comprise greater than about 85% Schwann cells. In some embodiments, the composition comprises greater than about 90% Schwann cells, in some embodiments, greater than about 95% Schwann cells, in some embodiments, greater than about 99% Schwann cells.

[0108] In some embodiments, the compositions of the present disclosure comprise SCs derived from pluripotent stem cells. In some embodiments, the compositions of the present disclosure are passaged for at least about 1 week, at least about 2 weeks, or at least about 3 weeks while maintaining expression of one or a combination of CD98, S100, MBP, GFAP, PMP22, or functional fragments or variants thereof.

[0109] In some embodiments, the composition of the present disclosure comprises a spheroid. The spheroid of the present invention can have any suitable width, length, thickness, and / or diameter. In some embodiments, the spheroid has a diameter of about 100 μm to about 50,000 μm, or any range therein, such as, but not limited to, about 100 μm to about 900 μm, about 100 μm to about 700 μm, about 300 μm to about 600 μm, about 400 μm to about 500 μm, about 500 μm to about 1,000 μm, about 600 μm to about 1,000 μm, or about 700 μm to about 1,000 μm. , about 800 μm to about 1,000 μm, about 900 μm to about 1,000 μm, about 750 μm to about 1,500 μm, about 1,000 μm to about 5,000 μm, about 1,000 μm to about 10,000 μm, about 2,000 to about 50,000 μm, about 25,000 μm to about 40,000 μm, or about 3,000 μm to about 15,000 μm. In some embodiments, the spheroids may have a width, length, thickness, and / or diameter of about 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1,000 μm, 5,000 μm, 10,000 μm, 20,000 μm, 30,000 μm, 40,000 μm, or about 50,000 μm. In some embodiments, a plurality of spheroids are generated, each of the plurality of spheroids having a width, length, thickness, and / or diameter that varies by less than about 20%, e.g., less than about 15%, 10%, or 5%. In some embodiments, each of the plurality of spheroids may have a different width, length, thickness, and / or diameter within any of the above ranges.

[0110] Cells within a spheroid may have a specific orientation. In some embodiments, a spheroid may comprise an inner core and an outer surface. In some embodiments, a spheroid may be hollow (i.e., may not contain cells inside). In some embodiments, the inner core cells and outer surface cells are different cell types. In some embodiments, a spheroid comprises neural crest cells and at least one Schwann cell.

[0111] In some embodiments, the spheroids may be composed of one, two, three, or more different cell types, including one or more neuronal cell types and / or one or more stem cell types. In some embodiments, the inner core cells may be composed of one, two, three, or more different cell types. In some embodiments, the outer surface cells may be composed of one, two, three, or more different cell types. In some embodiments, the spheroids comprise Schwann cells.

[0112] In some embodiments, the spheroids comprise at least two types of cells, and at least one type of cell is a SC.

[0113] In some embodiments, the hydrogel or hydrogel matrix can have a variety of thicknesses. In some embodiments, the hydrogel or hydrogel matrix has a thickness of about 100 μm to about 800 μm. In some embodiments, the hydrogel or hydrogel matrix has a thickness of about 150 μm to about 800 μm. In some embodiments, the hydrogel or hydrogel matrix has a thickness of about 200 μm to about 800 μm. In some embodiments, the hydrogel or hydrogel matrix has a thickness of about 250 μm to about 800 μm. In some embodiments, the hydrogel or hydrogel matrix has a thickness of about 300 μm to about 800 μm. In some embodiments, the hydrogel or hydrogel matrix has a thickness of about 350 μm to about 800 μm. In some embodiments, the hydrogel or hydrogel matrix has a thickness of about 400 μm to about 800 μm. In some embodiments, the hydrogel or hydrogel matrix has a thickness of about 450 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 500 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 550 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 600 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 650 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 700 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 750 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 750 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 700 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 650 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​from about 100 μm to about 600 μm.In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 550 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 500 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 450 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 400 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 350 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 300 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 250 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 200 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 150 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 300 μm to about 600 μm, hi some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 400 μm to about 500 μm.

[0114] In some embodiments, the hydrogel or hydrogel matrix can have a variety of thicknesses. In some embodiments, the hydrogel or hydrogel matrix has a thickness of about 10 μm to about 3000 μm. In some embodiments, the hydrogel or hydrogel matrix has a thickness of about 150 μm to about 3000 μm. In some embodiments, the hydrogel or hydrogel matrix has a thickness of about 200 μm to about 3000 μm. In some embodiments, the hydrogel or hydrogel matrix has a thickness of about 250 μm to about 3000 μm. In some embodiments, the hydrogel or hydrogel matrix has a thickness of about 300 μm to about 3000 μm. In some embodiments, the hydrogel or hydrogel matrix has a thickness of about 350 μm to about 3000 μm. In some embodiments, the hydrogel or hydrogel matrix has a thickness of about 400 μm to about 3000 μm. In some embodiments, the hydrogel or hydrogel matrix has a thickness of about 450 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 500 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 550 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 600 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 650 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 700 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 750 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 800 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 850 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​from about 900 μm to about 3000 μm, hi some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​from about 950 μm to about 3000 μm.In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 1000 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 1500 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 2000 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 2500 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 2500 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 2000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 1500 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 1000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 950 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 900 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 850 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 750 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 700 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 650 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 600 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 550 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​from about 100 μm to about 500 μm, hi some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​from about 100 μm to about 450 μm.In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 400 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 350 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 300 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 250 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 200 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 150 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 300 μm to about 600 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 400 μm to about 500 μm.

[0115] In some embodiments, the hydrogel or hydrogel matrix comprises one or more synthetic polymers, hi some embodiments, the hydrogel or hydrogel matrix comprises one or more of the following synthetic polymers: polyethylene glycol (polyethylene oxide), polyvinyl alcohol, poly-2-hydroxyethyl methacrylate, polyacrylamide, silicone, and any derivative or combination thereof.

[0116] In some embodiments, the hydrogel or hydrogel matrix comprises one or more synthetic and / or natural polysaccharides, hi some embodiments, the hydrogel or hydrogel matrix comprises one or more of the following polysaccharides: hyaluronic acid, heparin sulfate, heparin, dextran, agarose, chitosan, alginate, and any derivative or combination thereof.

[0117] In some embodiments, the hydrogel or hydrogel matrix comprises one or more proteins and / or glycoproteins, ie, one or more of the following proteins: collagen, gelatin, elastin, titin, laminin, fibronectin, fibrin, keratin, polyornithine, silk fibroin, and any derivative or combination thereof.

[0118] In some embodiments, one or more cells are stimulated with a differentiation factor or agent. The differentiation factor may include one or any combination of the following: BMP4 MIPGNRMLMV VLLCQVLLGG ASHASLIPET GKKKVAEIQG HAGGRRSGQSHELLRDFEAT LLQMFGLRRR PQPSKSAVIP DYMRDLYRLQ SGEEEEEQIHSTGLEYPERP ASRANTVRSF HHEEHLENIP GTSENSAFRF LFNLSSIPENEVISSAELRL FREQVDQGPD WERGFHRINI YEVMKPPAEV VPGHLITRLLDTRLVHHNVT RWETFDVSPA VLRWTREKQP NYGLAIEVTH LHQTRTHQGQ HVRISRSLPQ GSGNWAQLRP LLVTFGHDGR GHALTRRRRA KRSPKHHSQRARKKNKNCRR HSLYVDFSDV GWNDWIVAPP GYQAFYCHGD CPFPLADHLN STNHAIVQTL VNSVNSSIPK ACCVPTELSA ISMLYLDEYD KVVLKNYQEMVVEGCGCR [SEQ ID NO: 59] FGF2 MVGVGGGDVE DVTPRPGGCQ ISGRGARGCN GIPGAAAWEA ALPRRRPRRHPSVNPRSRAA GSPRTRGRRT EERPSGSRLG DRGRGRALPG GRLGGRGRGRAPERVGGRGR GRGTAAPRAA PAARGSRPGP AGTMAAGSIT TLPALPEDGGSGAFPPGHFK DPKRLYCKNG GFFLRIHPDG RVDGVREKSD PHIKLQLQAEERGVVSIKGV CANRYLAMKE DGRLLASKCV TDECFFFERL ESNNYNTYRSRKYTSWYVAL KRTGQYKLGS KTGPGQKAIL FLPMSAKS [SEQ ID NO: 60] NRG1 MEIYSPDMSE VAAERSSSPS TQLSADPSLD GLPAAEDMPE PQTEDGRTPG LVGLAVPCCA CLEAERLRGC LNSEKICIVP ILACLVSLCL CIAGLKWVFV DKIFEYDSPT HLDPGGLGQD PIISLDATAA SAVWVSSEAY TSPVSRAQSE SEVQVTVQGD KAVVSFEPSA APTPKNRIFA FSFLPSTAPS FPSPTRNPEV RTPKSATQPQ TTETNLQTAP KLSTSTSTTG TSHLVKCAEK EKTFCVNGGE CFMVKDLSNP SRYLCKCPNE FTGDRCQNYV MASFYSTSTP FLSLPE [SEQ ID NO: 61] TIFF2025531671000017.tif191128

[0119] The systems or methods disclosed herein may include tissue culture media containing any one or combination of differentiation factors, or functional fragments, salts, or derivatives thereof. In any of the methods or systems disclosed herein, the differentiation factor used may be a functional fragment or variant of the polypeptide disclosed above having at least about 70% sequence identity to the above sequence. In any of the methods or systems disclosed herein, the differentiation factor used may be a functional fragment or variant of the polypeptide disclosed above having at least about 80% sequence identity to the above sequence. In any of the methods or systems disclosed herein, the differentiation factor used may be a functional fragment or variant of the polypeptide disclosed above having at least about 85% sequence identity to the above sequence. In any of the methods or systems disclosed herein, the differentiation factor used may be a functional fragment or variant of the polypeptide disclosed above having at least about 90% sequence identity to the above sequence. In any of the methods or systems disclosed herein, the differentiation factor used may be a functional fragment or variant of the polypeptide disclosed above having at least about 95% sequence identity to the above sequence. In any of the methods or systems disclosed herein, the differentiation factor used can be a functional analog of a small molecule disclosed above. The disclosed methods involve sequentially exposing a culture of cells to two or more different tissue culture media. In some embodiments, the systems disclosed herein include one or more of the differentiation factors identified above, or functional fragments or derivatives thereof. In some embodiments, the cells disclosed herein are exposed to an amount of a differentiation factor for a time sufficient to differentiate the cell or cells into another cell type, such as SCs. In some embodiments, the methods disclosed herein include exposing neural crest cells to one or a combination of differentiation factors or salts thereof for a time sufficient to differentiate the neural crest cells into SCs. In such methods, the exposure step can be for about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days or more.In some embodiments, the methods of the present disclosure comprise exposing Schwann cells to segments of gastrointestinal tissue in culture.

[0120] Pharmaceutical Composition In some embodiments, the composition is a pharmaceutical composition.

[0121] In some embodiments, the compositions, spheroids, or pharmaceutical compositions comprising SCs are administered at a desired dosage, and in some aspects, comprise a desired dose or number of cells and / or a desired ratio of neuronal cell subpopulations. In some embodiments, the dosage of cells is based on the total number of cells (or number per m2 of body surface area or per kg of body weight) and the desired ratio of individual populations or subtypes. In some embodiments, the dosage of cells is based on the desired total number of cells (or number per m2 of body surface area or per kg of body weight) in each population or each cell type. In some embodiments, the dosage is based on a combination of such characteristics, such as the desired number of total cells in each population, the desired ratio, and the desired total number of cells.

[0122] In some embodiments, compositions, spheroids, or pharmaceutical compositions comprising SCs are administered at or within a range of acceptable variations in a desired dose of total cells, e.g., a desired dose of a subtype of neuronal cells, such as Schwann cells. In some aspects, the desired dose is a desired number of cells, a desired number of cells per unit of body surface area, or a desired number of cells per unit of body weight of the subject to whom the cells are administered, e.g., cells / m2 or cells / kg. In some aspects, the desired dose is equal to or exceeds a minimum number of cells per unit of body surface area or body weight. In some aspects, of the total cells administered at a desired dose, individual populations or subtypes are present at or near a desired output ratio as described herein, e.g., within a particular tolerance or error of such ratio.

[0123] In some embodiments, the cells are administered at or within a desired dose tolerance. In some aspects, the desired dose is expressed as a desired number of cells, or a desired number of such cells per unit of body surface area or body weight of the subject to whom the cells are administered, e.g., cells / m 2 or cells / kg. In some embodiments, the desired dose is equal to or greater than the minimum number of cells in the population, or the minimum number of cells in the population per unit of body surface area or body weight.

[0124] Thus, in some embodiments, dosage is based on a desired fixed dose and desired ratio of total cells, and / or based on a desired fixed dose of each of two or more, e.g., individual neuronal subpopulations. Thus, in some embodiments, dosage is based on a desired fixed dose or minimum dose of a neuronal subpopulation and a desired ratio thereof.

[0125] In certain embodiments, the composition, spheroid, or pharmaceutical composition comprising SCs contains about 1 million to about 100 billion cells, e.g., about 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), e.g., about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, or a range defined by any two of the foregoing values). The subject may be administered a range of cells from about 100 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), in some cases, from about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells), or any value therebetween.

[0126] In some embodiments, the dose of total cells and / or the dose of individual neuronal subpopulations of cells is less than 10 cells. 5 ~10 6 Approximately 10 cells per square meter of body surface area 4 ~about 10 9 cells / meter² (m²) of body surface area, e.g., approximately 1 × 10 cells 5 pcs / m2, cells 1.5×10 5 pcs / m2, cells 2×10 5 cells / m², or 1 x 10 cells 6 For example, in some embodiments, the cells are in the range of about 10 cells / m of body surface area. 5 ~10 6 pieces / m 2 of body surface area, and approximately 10 nerve cells 4 ~about 10 9 Pieces / meter 2 (m 2 ) body surface area, e.g., 1 × 10 SC cells 5 pieces / m 2 , SC cells 1.5 × 10 5 pieces / m 2 , SC cells 2 x 10 5 pieces / m 2 , or 1 × 10 SC cells 6 pieces / m 2 The dose is administered within a certain range of body surface area tolerance.

[0127] In some embodiments, the cells are cells 10 5 ~10 6 pieces / m 2 The weight of the cells is approximately 10 4 ~about 10 9 Pieces / meter 2 (m2) body weight, e.g., approximately 1 × 10 cells 5 pieces / m 2 , cells 1.5 x 10 5 pieces / m 2 , cells 2 x 10 5 cells / kg, or 1 x 10 cells 6 pieces / m 2 The dose is administered within a certain range of body surface area tolerance.

[0128] Pharmaceutical compositions provided by the present disclosure include compositions containing an active ingredient (e.g., a cell described herein, including an embodiment or example) in a therapeutically effective amount, i.e., an amount effective to achieve its intended purpose. The actual amount effective for a particular application will depend, among other things, on the condition being treated. When administered in a method of treating a disease, such a composition will contain an amount of cells effective to achieve the desired result, e.g., modulate the activity of the subject (e.g., increase the number of Schwann cells in the subject) and / or reduce, eliminate, or slow the progression of disease symptoms (e.g., symptoms of peripheral neuropathy). Determination of a therapeutically effective amount of a compound of the present disclosure is well within the capabilities of one of ordinary skill in the art, especially in light of the detailed disclosure provided herein.

[0129] Pharmaceutical compositions can be formulated according to the mode of administration used. Injectable pharmaceutical compositions can be sterile, pyrogen-free, and particle-free. Isotonic preparations or solutions can be used as pharmaceutically acceptable carriers. Additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol, and lactose. Isotonic solutions can include phosphate-buffered saline. Pharmaceutical compositions can further include stabilizers, including gelatin and albumin. Stabilization can allow formulations such as LGS, polycations, or polyanions for pharmaceutical composition formulations to remain stable for extended periods at room or ambient temperature.

[0130] system The present disclosure also relates to a system comprising: (i) a cell culture vessel optionally comprising a hydrogel; (ii) one or more stem cells or Schwann cells in suspension or attached to a solid substrate; and (iii) one or more differentiation factors.

[0131] The present disclosure also relates to a system comprising: (i) a cell culture vessel optionally comprising a hydrogel; (ii) one or more stem cells or Schwann cells in suspension or as a component of a spheroid; and (iii) one or more differentiation factors.

[0132] In some embodiments, the system further comprises one or a combination of culture media disclosed herein. The present disclosure also relates to a method for culturing Schwann cells in a system, the system comprising (i) a cell culture vessel, optionally comprising a hydrogel; (ii) one or more stem cells or neural crest cells in suspension or as components of spheroids; and (iii) one or more differentiation factors. In some embodiments, the system further comprises one or a combination of culture media disclosed herein. In some embodiments, the method involves (i) exposing one or more stem cells to a first cell culture medium for a period sufficient to differentiate the one or more stem cells into neural crest cells, with successive medium changes, and (ii) exposing one or more neural crest cells to a second cell culture medium for a period sufficient to differentiate the one or more neural crest cells into Schwann cells, with the medium changed at least once during a culture period of about 12 to about 21 days.

[0133] In some embodiments, the system includes a solid substrate. As used herein, the term "solid substrate" refers to any material that is a solid support free of or substantially free of cytotoxins. In some embodiments, the solid substrate includes one or a combination of silica, plastic, and metal. In some embodiments, the solid substrate includes pores of a size and shape sufficient to allow diffusion or inert transport of proteins, nutrients, and gases through the solid substrate in the presence of cell culture medium. In some embodiments, the pore size is less than or equal to about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 micron in diameter. One skilled in the art can determine the necessary pore size based on the content of the cell culture medium and the exposure of cells grown on the solid substrate in a particular microenvironment. For example, one skilled in the art can observe whether any cultured cells in a system or device can survive under conditions involving solid substrates containing pores of various diameters. In some embodiments, the solid substrate includes a base having a predetermined shape that defines the shape of its outer and inner surfaces. In some embodiments, the base comprises one or a combination of silica, plastic, ceramic, or metal, and the base is cylindrical or substantially cylindrical in shape, such that the first polymer coats the interior surface of the base and defines a cylindrical or substantially cylindrical interior chamber, with the opening located at one end of the cylinder. In some embodiments, the base comprises one or more pores of a size and shape sufficient to allow proteins, nutrients, and oxygen to diffuse through the solid substrate in the presence of cell culture medium. In some embodiments, the solid substrate comprises a plastic base having pores of about 1 micron or less in diameter and includes at least one layer of a hydrogel matrix, and the solid substrate comprises at least one compartment defined at least in part by the shape of the interior surface of the solid substrate and accessible by the opening from a point outside the solid substrate, optionally located at one end of the solid substrate.In embodiments in which the solid substrate includes a hollow interior portion defined by at least one interior surface, cells in suspension or tissue explants can be seeded by placing the cells in or near the opening so that the cells can attach to at least a portion of the interior surface of the solid substrate before growth. The at least one compartment or hollow interior of the solid substrate allows for the confinement of cells in a specific three-dimensional shape defined by the shape of the interior surface. In some embodiments, the solid substrate promotes directional growth of cells away from the opening. In the case of neural cells, the degree and shape of the confinement of the at least one compartment favors axonal growth from cell bodies located within the at least one compartment and at or near the opening.

[0134] In some embodiments, the solid substrate is coated with polyornithine (“PO”), laminin (“LM”), and / or fibronectin (“FN”).

[0135] The present disclosure provides devices, methods, and systems for the production, maintenance, and physiological investigation of neural cells in microengineered structures designed to mimic the anatomy of native neural tissue. Another object of the present disclosure is to provide a medium-throughput assay of neurological function for screening the pharmacological and / or toxicological properties of chemical and biological agents. In some embodiments, the agent is a cell, such as any of the cell types disclosed herein, or an antibody, such as an antibody used to treat a clinical disease. In some embodiments, the agent is any drug or agent used to treat a human disease, such that toxicity, efficacy, or neuromodulation can be compared between the proposed new agent as a mammalian treatment and an existing treatment for the human disease. In some embodiments, the new agent for treating a human disease is a treatment for a neurodegenerative disease and is compared to an existing treatment for the neurodegenerative disease.

[0136] Similarly, information gleaned from imaging can determine quantitative metrics about the degree of cytotoxicity, providing further insight into the toxicity and neuroprotective mechanisms of various agents or compounds of interest. In some embodiments, the at least one agent comprises a small chemical compound. In some embodiments, the at least one agent comprises at least one environmental or industrial pollutant. In some embodiments, the at least one agent comprises one or a combination of small chemical compounds selected from chemotherapeutic agents, analgesics, cardiovascular modulators, cholesterol, neuroprotective agents, neuromodulators, immunomodulators, anti-inflammatory agents, and antimicrobial agents.

[0137] In some embodiments, the at least one agent is actinomycin, alitretinoin, all-trans retinoic acid, azacitidine, azathioprine, bexarotene, bleomycin, bortezomib, capecitabine, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, erlotinib, etoposide, fluorouracil, gefitinib, gemcitabine, hydroxyurea, idarubicin, imatinib, irinotecan ... The present invention also includes one or a combination of chemotherapeutic agents selected from notecan, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, nitrosoureas, oxaliplatin, paclitaxel, pemetrexed, romidepsin, tafluposide, temozolomide (oral dacarbazine), teniposide, tioguanine (formerly thioguanine), topotecan, tretinoin, valrubicin, vemurafenib, vinblastine, vincristine, vindesine, vinorelbine, vismodegib, and vorinostat. In some embodiments, the at least one agent comprises one or a combination of analgesics selected from paracetamol, a nonsteroidal anti-inflammatory drug (NSAID), a COX-2 inhibitor, an opioid, flupirtine, a tricyclic antidepressant, carbamazepine, gabapentin, and pregabalin.

[0138] In some embodiments, the at least one agent comprises one or a combination of cardiovascular modulating agents selected from nepicastat, cholesterol, niacin, scutellaria, prenylamine, dehydroepiandrosterone, monatepir, esketamine, niguldipine, asenapine, atomoxetine, flunarizine, milnacipran, mexiletine, amphetamine, thiopental sodium, flavonoids, bretylium, oxazepam, and honokiol.

[0139] In some embodiments, the at least one agent comprises one or a combination of neuroprotective and / or neuromodulatory agents selected from tryptamine, galanin receptor 2, phenylalanine, phenethylamine, N-methylphenethylamine, adenosine, kyotorphin, substance P, 3-methoxytyramine, catecholamines, dopamine, GABA, calcium, acetylcholine, epinephrine, norepinephrine, and serotonin. In some embodiments, the at least one agent comprises one or a combination of immunomodulatory agents selected from clenolizimab, enoticumab, ligelizumab, simtuzumab, batelizumab, palsatuzumab, imgatuzumab, tregalizumab, pateclizumab, namulumab, perakizumab, faralimomab, patritumab, atinumab, ublituximab, futuximab, and durigotumab.

[0140] In some embodiments, the at least one agent comprises one or a combination of anti-inflammatory agents selected from ibuprofen, aspirin, ketoprofen, sulindac, naproxen, etodolac, fenoprofen, diclofenac, flurbiprofen, ketorolac, piroxicam, indomethacin, mefenamic acid, meloxicam, nabumetone, oxaprozin, ketoprofen, famotidine, meclofenamate, tolmetin, and salsalate. In some embodiments, the at least one agent comprises one or a combination of antimicrobial agents selected from antibacterial agents, antifungal agents, antiviral agents, anthelmintics, heat, radiation, and ozone.

[0141] Table 2 lists biomarkers specific to one or more cells disclosed in the present application. Biomarkers may be expressed as proteins on the surface of cells. In some embodiments, biomarkers are expressed as mRNAs encoding proteins identified in Table 2 or functional fragments thereof. Biomarkers in figures, including Figure 5, are disclosed in Figures 5E and 5F and correspond to the cell types disclosed in those panels. When a cell type corresponds to a gene name, it is understood that the cell type comprises a protein or expresses a nucleic acid sequence encoding the disclosed nucleic acid sequence or an amino acid associated with that nucleic acid sequence. Compositions of the present disclosure relate to compositions comprising low-passage mature SCs, high-passage mature SCs, myelinating SCs, and SCPDs. In some embodiments, the composition consists of one of low-passage mature SCs, high-passage mature SCs, myelinating SCs, and SCPDs. In some embodiments, the composition comprises one or a combination of low-passage mature SCs, high-passage mature SCs, myelinating SCs, and SCPDs. In some embodiments, the composition comprises greater than about 20% low-passage mature SCs, high-passage mature SCs, myelinating SCs, or SCPDs. In some embodiments, the composition comprises greater than about 30% low-passage mature SCs, high-passage mature SCs, myelinating SCs, or SCPDs. In some embodiments, the composition comprises greater than about 40% low-passage mature SCs, high-passage mature SCs, myelinating SCs, or SCPDs. In some embodiments, the composition comprises greater than about 50% low-passage mature SCs, high-passage mature SCs, myelinating SCs, or SCPDs. In some embodiments, the composition comprises greater than about 60% low-passage mature SCs, high-passage mature SCs, myelinating SCs, or SCPDs. In some embodiments, the composition comprises greater than about 70% low-passage mature SCs, high-passage mature SCs, myelinating SCs, or SCPDs. In some embodiments, the composition comprises greater than about 80% low-passage mature SCs, high-passage mature SCs, myelinating SCs, or SCPDs. In some embodiments, the composition comprises greater than about 90% low passage mature SCs, high passage mature SCs, myelinating SCs, or SCPDs.

[0142] In some embodiments, the cell types disclosed in the accompanying figures express an RNA associated with an accession number in Table 3. In some embodiments, the cell types disclosed in the accompanying figures express a protein associated with an accession number in Table 3.

[0143] (Table 2) TIFF2025531671000018.tif142167

[0144] Table 3: Biomarker RNA sequences TIFF2025531671000019.tif232170TIFF2025531671000020.tif227170TIFF2025531671000021.tif167170TIFF2025531671000022.tif227170TIFF2025531671000023.tif227170TIFF2025531671000024.tif227170TIFF2025531671000025.tif227170TIFF2025531671000026.tif227170TIFF2025531671000027.tif227170TIFF2025531671000028.tif227170TIFF2025531671000029.tif227170TIFF2025531671000030.tif227170TIFF2025531671000031.tif227170TIFF2025531671000032.tif227170TIFF2025531671000033.tif227170TIFF2025531671000034.tif227170TIFF2025531671000035.tif227170TIFF2025531671000036.tif227170TIFF2025531671000037.tif227170TIFF2025531671000038.tif227170TIFF2025531671000039.tif227170TIFF2025531671000040.tif227170TIFF2025531671000041.tif187170TIFF2025531671000042.tif227170TIFF2025531671000043.tif227170TIFF2025531671000044.tif227170TIFF2025531671000045.tif227170TIFF2025531671000046.tif227170TIFF2025531671000047.tif227170TIFF2025531671000048.tif227170TIFF2025531671000049.tif232170TIFF2025531671000050.tif217170TIFF2025531671000051.tif227170TIFF2025531671000052.tif227170TIFF2025531671000053.tif227170TIFF2025531671000054.tif227170TIFF2025531671000055.tif227170TIFF2025531671000056.tif227170TIFF2025531671000057.tif227170TIFF2025531671000058.tif227170TIFF2025531671000059.tif187170TIFF2025531671000060.tif227170TIFF2025531671000061.tif227170TIFF2025531671000062.tif227170TIFF2025531671000063.tif227170TIFF2025531671000064.tif227170TIFF2025531671000065.tif227170TIFF2025531671000066.tif227170TIFF2025531671000067.tif227170TIFF2025531671000068.tif227170TIFF2025531671000069.tif227170TIFF2025531671000070.tif227170TIFF2025531671000071.tif227170TIFF2025531671000072.tif227170TIFF2025531671000073.tif227170TIFF2025531671000074.tif227170TIFF2025531671000075.tif227170TIFF2025531671000076.tif227170TIFF2025531671000077.tif227170TIFF2025531671000078.tif227170TIFF2025531671000079.tif227170TIFF2025531671000080.tif227170TIFF2025531671000081.tif232170TIFF2025531671000082.tif237170TIFF2025531671000083.tif237170TIFF2025531671000084.tif227170TIFF2025531671000085.tif227170TIFF2025531671000086.tif227170TIFF2025531 671000087.tif227170TIFF2025531671000088.tif227170TIFF2025531671000089.tif227170T IFF2025531671000090.tif227170TIFF2025531671000091.tif227170TIFF2025531671000092. tif227170TIFF2025531671000093.tif227170TIFF2025531671000094.tif227170TIFF2025531 671000095.tif227170TIFF2025531671000096.tif227170TIFF2025531671000097.tif227170T IFF2025531671000098.tif227170TIFF2025531671000099.tif227170TIFF2025531671000100. tif227170TIFF2025531671000101.tif227170TIFF2025531671000102.tif227170TIFF2025531 671000103.tif227170TIFF2025531671000104.tif227170TIFF2025531671000105.tif112170.

[0145] In some embodiments, the cell types disclosed in the accompanying figures express an RNA associated with an accession number in Table 4. In some embodiments, the cell types disclosed in the accompanying figures express a protein associated with an accession number in Table 4.

[0146] (Table 4) Genbank accession numbers TIFF2025531671000106.tif220167TIFF2025531671000107.tif231167TIFF2025531671000108.tif231167TIFF2025531671000109.tif231167TIFF2025531671000110.tif231167TIFF2025531671000111.tif231167TIFF2025531671000112.tif231167TIFF2025531671000113.tif231167TIFF2025531671000114.tif238167TIFF2025531671000115.tif238167TIFF2025531671000116.tif231167TIFF2025531671000117.tif231167TIFF2025531671000118.tif231167TIFF2025531671000119.tif231167TIFF2025531671000120.tif231167TIFF2025531671000121.tif231167TIFF2025531671000122.tif231167TIFF2025531671000123.tif231167TIFF2025531671000124.tif231167TIFF2025531671000125.tif231167TIFF2025531671000126.tif231167TIFF2025531671000127.tif231167TIFF2025531671000128.tif231167TIFF2025531671000129.tif231167TIFF2025531671000130.tif231167TIFF2025531671000131.tif231167TIFF2025531671000132.tif231167TIFF2025531671000133.tif231167TIFF2025531671000134.tif231167TIFF2025531671000135.tif231167TIFF2025531671000136.tif223167TIFF2025531671000137.tif234167TIFF2025531671000138.tif231167TIFF2025531671000139.tif231167TIFF2025531671000140.tif231167TIFF2025531671000141.tif231167TIFF2025531671000142.tif231167TIFF2025531671000143.tif231167TIFF2025531671000144.tif225167TIFF2025531671000145.tif230167TIFF2025531671000146.tif231167TIFF2025531671000147.tif231167TIFF2025531671000148.tif231167TIFF2025531671000149.tif231167TIFF2025531671000150.tif231167TIFF2025531671000151.tif231167TIFF2025531671000152.tif235167TIFF2025531671000153.tif234167TIFF2025531671000154.tif230167TIFF2025531671000155.tif231167TIFF2025531671000156.tif231167TIFF2025531671000157.tif238167TIFF2025531671000158.tif240167TIFF2025531671000159.tif238167TIFF2025531671000160.tif234167TIFF2025531671000161.tif234167TIFF2025531671000162.tif234167TIFF2025531671000163.tif231167TIFF2025531671000164.tif231167TIFF2025531671000165.tif231167TIFF2025531671000166.tif231167TIFF2025531671000167.tif231167TIFF2025531671000168.tif231167TIFF2025531671000169.tif231167TIFF2025531671000170.tif231167TIFF2025531671000171.tif238167TIFF2025531671000172.tif238167TIFF2025531671000173.tif241167TIFF2025531671000174.tif234167TIFF2025531671000175.tif234167TIFF2025531671000176.tif240167TIFF2025531671000177.tif231167TIFF2025531671000178.tif231167TIFF2025531671000179.tif231167TIFF2025531671000180.tif231167TIFF2025531671000181.tif231167TIFF2025531671000182.tif231167TIFF2025531671000183.tif231167TIFF2025531671000184.tif231167TIFF2025531671000185.tif231167TIFF2025531671000186.tif231167TIFF2025531671000187.tif231167TIFF2025531671000188.tif231167TIFF2025531671000189.tif231167TIFF2025531671000190.tif231167TIFF2025531671000191.tif231167TIFF2025531671000192.tif231167TIFF2025531671000193.tif231167TIFF2025531671000194.tif243167TIFF2025531671000195.tif231167TIFF2025531671000196.tif234167TIFF2025531671000197.tif234167TIFF2025531671000198.tif235167TIFF2025531671000199.tif243167TIFF2025531671000200.tif243167TIFF2025531671000201.tif231167TIFF2025531671000202.tif231167TIFF2025531671000203.tif231167TIFF2025531671000204.tif231167TIFF2025531671000205.tif231167TIFF2025531671000206.tif231167TIFF2025531671000207.tif231167TIFF2025531671000208.tif231167TIFF2025531671000209.tif231167TIFF2025531671000210.tif231167TIFF2025531671000211.tif231167TIFF2025531671000212.tif231167TIFF2025531671000213.tif231167TIFF2025531671000214.tif231167TIFF2025531671000215.tif231167TIFF2025531671000216.tif231167TIFF2025531671000217.tif231167TIFF2025531671000218.tif231167TIFF2025531671000219.tif243167TIFF2025531671000220.tif238167TIFF2025531671000221.tif231167TIFF2025531671000222.tif231167TIFF2025531671000223.tif231167TIFF2025531671000224.tif216167TIFF2025531671000225.tif234167TIFF2025531671000226.tif231167TIFF2025531671000227.tif231167TIFF2025531671000228.tif231167TIFF2025531671000229.tif231167TIFF2025531671000230.tif231167TIFF2025531671000231.tif231167TIFF2025531671000232.tif231167TIFF2025531671000233.tif231167TIFF2025531671000234.tif231167TIFF2025531671000235.tif231167TIFF2025531671000236.tif231167TIFF2025531671000237.tif231167TIFF2025531671000238.tif231167TIFF2025531671000239.tif231167TIFF2025531671000240.tif231167TIFF2025531671000241.tif225167TIFF2025531671000242.tif234167TIFF2025531671000243.tif230167TIFF2025531671000244.tif231167TIFF2025531671000245.tif231167TIFF2025531671000246.tif231167TIFF2025531671000247.tif231167TIFF2025531671000248.tif231167TIFF2025531671000249.tif231167TIFF2025531671000250.tif231167TIFF2025531671000251.tif231167TIFF2025531671000252.tif231167TIFF2025531671000253.tif231167TIFF2025531671000254.tif231167TIFF2025531671000255.tif231167TIFF2025531671000256.tif231167TIFF2025531671000257.tif231167TIFF2025531671000258.tif231167TIFF2025531671000259.tif231167TIFF2025531671000260.tif231167TIFF2025531671000261.tif231167TIFF2025531671000262.tif231167TIFF2025531671000263.tif231167TIFF2025531671000264.tif231167TIFF2025531671000265.tif231167TIFF2025531671000266.tif231167TIFF2025531671000267.tif231167TIFF2025531671000268.tif231167TIFF2025531671000269.tif231167TIFF2025531671000270.tif231167TIFF2025531671000271.tif231167TIFF2025531671000272.tif231167TIFF2025531671000273.tif231167TIFF2025531671000274.ti f231167TIFF2025531671000275.tif231167TIFF2025531671000276.tif238167TIFF2025531671000277.tif231167TIFF2025531671000278.tif22 3167TIFF2025531671000279.tif218167TIFF2025531671000280.tif234167TIFF2025531671000281.tif231167TIFF2025531671000282.tif2311 67TIFF2025531671000283.tif231167TIFF2025531671000284.tif231167TIFF2025531671000285.tif231167TIFF2025531671000286.tif198167.

[0147] method The present disclosure also relates to a method for differentiating pluripotent stem cells into Schwann cells, the method comprising exposing the pluripotent stem cells to an effective amount of a differentiation agent for a time sufficient to cause the cells to differentiate into Schwann cells.

[0148] The present disclosure also relates to methods of culturing Schwann cells with a TGFP inhibitor. The present disclosure also relates to methods of culturing Schwann cells, comprising exposing one or more cells to one or more tissue culture media disclosed in any of the disclosed tables. In some embodiments, the method comprises exposing the cells to one or more tissue culture media for about 1 day to about 20 days.

[0149] The present disclosure also relates to a method for culturing Schwann cells in a system comprising: (i) a cell culture vessel, optionally comprising a hydrogel; (ii) one or more stem cells in suspension or as components of spheroids; and (iii) one or more differentiation factors. In some embodiments, the system further comprises one or a combination of culture media disclosed herein. In some embodiments, the method involves exposing the one or more stem cells to the cell culture medium for a period sufficient to differentiate the one or more stem cells into Schwann cells, with at least one change of medium during a culture period of about 12 to about 21 days. In some embodiments, the system is free of, or substantially free of, feeder cells.

[0150] This disclosure provides an improved method for inducing enteric neural progenitors from human pluripotent stem cells (22). Many laboratories in the stem cell field no longer rely on feeder cell support and have adopted the use of defined basal media, such as mTeSR™ 1 (Stemcell Tech, 85850) or Essential 8 (Life Technologies, A2858501), for the maintenance of hPSC lines. Nevertheless, previous ENC induction methods generally involve media containing serum replacement factors, i.e., knockout serum replacement (KSR), as in Comparative Example 2 (14, 20). To reduce inconsistencies and quality control measures that undefined conditions can introduce into protocols, we sought to optimize ENC induction in minimally chemically defined conditions.

[0151] Recent studies have demonstrated an alternative strategy for general NC induction using hPSCs: a free-floating embryoid body-based approach. The resulting migratory cells from the embryoid bodies and subsequent neural rosette formation were shown to be positive for neural crest-specific markers SOX10, TFAP2A, BRN3A, ISL1, and ASCL1, and a subset was found to be positive for the region-specific vagus nerve markers HOXB2 and HOXB5, even in the absence of RA (23). Overall neural crest induction efficiency was assessed by FACS for p75 and HNK1 double-positive cells, a strategy used to isolate NC cells in a previous protocol (Lee et al. 2007). Results showed induction efficiencies of over 60% in the ES cell line H9 and across independent hiPSC lines. The enriched NC population was then cocultured with primary intestinal explants in a Transwell system to promote ENC identity enriched for HOXB2, HOXB3, HAND2, and EDNRB. Specifically, this method incorporates brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), nerve growth factor (NGF), and neurotrophin-3 (NT3) into the culture conditions. A similar embryoid body approach incorporated a brief exposure to RA during NC induction before ultimately combining hPSC-derived NC cells with hPSC-derived intestinal organoids (HIOs) (24).

[0152] The present disclosure relates to methods for differentiating stem cells into neural crest cells and then differentiating NC cells into Schwann cells. In some embodiments, the method for culturing or differentiating SCs includes exposing NCs to BDNF, GDNF, NGF, and / or NT3 for a period sufficient to differentiate the stem cells into NCs. Some embodiments further include exposing one or more NCs to a differentiation agent identified above for a period sufficient to differentiate them into SCs. In some embodiments, the periods are sequential periods involving two or more cell culture media disclosed herein, where the cells are exposed to a first cell culture medium for a first period, a second cell culture medium for a second period, and a third cell culture medium for a third period. As an example, in some embodiments, the cells are sequentially exposed first to stem cells and then to Schwann cells in cell culture media under the conditions and for the periods described in Example 2.

[0153] In some embodiments, the cells are cultured in first and second steps, wherein the first or second step comprises two different cell culture media, and the first exposure to the first cell culture medium is for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or about 10 days or more. In some embodiments, the second exposure to the second cell culture medium is for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or about 10 days or more after exposure to the first cell culture medium.

[0154] In some embodiments, the cells are cultured in first, second, and third steps, the first, second, and third exposure steps comprising three different cell culture media (one per step), and the first exposure to the first cell culture medium is for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or about 10 days or more. In some embodiments, the second exposure to the second cell culture medium is for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or about 10 days or more after exposure to the first cell culture medium. In some embodiments, the second exposure to the third cell culture medium is for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or about 10 days or more after exposure to the second cell culture medium.

[0155] Screening Method The present disclosure also relates to a method of assessing the toxicity of a drug, the method comprising: (a) culturing one or more neuronal cells or Schwann cells on any of the devices disclosed herein; (b) exposing the one or more neuronal cells or Schwann cells to at least one drug; (c) measuring and / or observing one or more metrics of neuronal cell or Schwann cell health; and (d) correlating the one or more metrics of the one or more neuronal cells with the toxicity of the drug, whereby the drug is characterized as toxic if the metric indicates decreased cell viability, or the drug is characterized as non-toxic if the metric indicates unchanged or increased cell viability, wherein step (c) optionally comprises observing one or more morphometric changes in the one or more neuronal cells or Schwann cells; and step (d) optionally comprises correlating the one or more morphometric changes in the one or more neuronal cells or Schwann cells with the toxicity of the drug, whereby the drug is characterized as toxic if the change indicates decreased cell viability, or the drug is characterized as non-toxic if the change indicates unchanged or increased cell viability. In some embodiments, the neuronal cell is any of the cells disclosed herein.

[0156] In some embodiments, the at least one agent comprises a small chemical compound. In some embodiments, the at least one agent comprises at least one environmental or industrial pollutant. In some embodiments, the at least one agent comprises one or a combination of small chemical compounds selected from chemotherapeutic agents, analgesics, cardiovascular modulators, cholesterol level regulators, neuroprotective agents, neuromodulators, immunomodulators, anti-inflammatory agents, and antimicrobial agents such as bacterial antibiotics. In some embodiments, the at least one agent comprises a therapeutically effective amount of an antibody, e.g., a clinically relevant monoclonal antibody such as Tysabri.

[0157] The present disclosure also relates to a method for measuring the amount or extent of myelination or demyelination of one or more axons of one or more neural cells and / or one or more tissue explants, the method comprising: (a) culturing one or more neural cells and / or one or more tissue explants on any of the devices disclosed herein for a time and under conditions sufficient to grow at least one axon; (b) measuring and / or observing one or more morphometric changes in the one or more neural cells; and (c) correlating the one or more morphometric changes in the one or more neural cells with quantitative or qualitative changes in myelination of the neural cells or tissue explants.

[0158] The present disclosure also relates to a method for measuring myelination or demyelination of one or more axons of one or more neuronal cells, the method comprising: (a) culturing one or more neuronal cells on any of the devices disclosed herein for a time and under conditions sufficient to grow at least one axon; (b) measuring and / or observing one or more physiological metrics of the one or more neuronal cells and / or one or more tissue explant cells; and (c) correlating the one or more metrics of the one or more neuronal cells and / or one or more tissue explant cells with quantitative or qualitative changes in neuronal myelination, wherein step (b) optionally comprises observing one or more morphological changes in the one or more neuronal cells, and step (c) optionally comprises correlating the one or more morphological changes in the one or more neuronal cells with quantitative or qualitative changes in neuronal myelination.

[0159] The present disclosure also relates to methods for measuring myelination or demyelination of one or more axons of one or more neural cells and / or one or more tissue explants, the methods comprising: (a) culturing one or more neural cells and / or one or more tissue explants on any of the devices disclosed herein for a time and under conditions sufficient to grow at least one axon; and (b) detecting the amount of myelination on one or more axons of the one or more neural cells and / or one or more tissue explants. In each of the above embodiments, the steps should include one or more SCs cultured with the neural cells or explants.

[0160] In some embodiments, detecting the amount of myelination on one or more axons of one or more neural cells and / or one or more tissue explants comprises exposing the cells to an antibody that binds to myelin.

[0161] In some embodiments, the method further comprises: (i) exposing the one or more neuronal cells and / or one or more tissue explants to at least one agent in the presence of SCs after steps (a) and (b); (ii) measuring and / or observing one or more expression patterns of the cells, measuring and / or observing one or more morphometric changes, and / or detecting a quantitative amount of myelin from the one or more neuronal cells and / or one or more tissue explants; (iii) calculating a change in the measured, observed, and / or quantitative amount of myelin from the one or more neuronal cells and / or one or more tissue explants in the presence and absence of the agent; and (iv) correlating the change in the measured, observed, and / or quantitative amount of myelin from the one or more neuronal cells and / or one or more tissue explants in the presence or absence of the agent.

[0162] In some embodiments, the at least one agent comprises at least one environmental or industrial pollutant. In some embodiments, the at least one agent comprises one or a combination of small chemical compounds selected from chemotherapeutic agents, analgesics, cardiovascular regulators, cholesterol level regulators, neuroprotective agents, neuromodulators, immunomodulators, anti-inflammatory agents, and antimicrobial agents.

[0163] The present disclosure also relates to methods for measuring myelination or demyelination of one or more axons of one or more neuronal cells and / or one or more tissue explants, the methods comprising: (a) culturing one or more neuronal cells and / or one or more tissue explants on any of the devices disclosed herein for a time and under conditions sufficient to grow at least one axon; (b) inducing compound action potentials in such one or more neuronal cells and / or one or more tissue explants; (c) measuring the compound action potentials; and (d) quantifying the level of myelination of such one or more neuronal cells based on the presence of SCs in the culture. In some embodiments, the methods further comprise exposing the one or more neuronal cells and / or one or more tissue explants to an agent. In some embodiments, the at least one agent comprises at least one environmental or industrial pollutant.

[0164] In some embodiments, the at least one agent comprises one or a combination of small chemical compounds selected from chemotherapeutic agents, analgesics, cardiovascular regulators, cholesterol level regulators, neuroprotective agents, neuromodulators, immunomodulators, anti-inflammatory agents, and antimicrobial agents.

[0165] In some embodiments, the at least one agent comprises a small chemical compound. In some embodiments, the at least one agent comprises at least one environmental or industrial pollutant. In some embodiments, the at least one agent comprises one or a combination of small chemical compounds selected from chemotherapeutic agents, analgesics, cardiovascular modulators, cholesterol, neuroprotective agents, neuromodulators, immunomodulators, anti-inflammatory agents, and antimicrobial agents.

[0166] In some embodiments, the at least one agent is actinomycin, alitretinoin, all-trans retinoic acid, azacitidine, azathioprine, bexarotene, bleomycin, bortezomib, capecitabine, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, erlotinib, etoposide, fluorouracil, gefitinib, gemcitabine, hydroxyurea, idarubicin, imatinib, irinotecan ... The present invention also includes one or a combination of chemotherapeutic agents selected from notecan, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, nitrosoureas, oxaliplatin, paclitaxel, pemetrexed, romidepsin, tafluposide, temozolomide (oral dacarbazine), teniposide, tioguanine (formerly thioguanine), topotecan, tretinoin, valrubicin, vemurafenib, vinblastine, vincristine, vindesine, vinorelbine, vismodegib, and vorinostat.

[0167] In some embodiments, the at least one agent comprises one or a combination of analgesics selected from paracetamol, a nonsteroidal anti-inflammatory drug (NSAID), a COX-2 inhibitor, an opioid, flupirtine, a tricyclic antidepressant, carbamazepine, gabapentin, and pregabalin.

[0168] In some embodiments, the at least one agent comprises one or a combination of cardiovascular modulating agents selected from nepicastat, cholesterol, niacin, scutellaria, prenylamine, dehydroepiandrosterone, monatepir, esketamine, niguldipine, asenapine, atomoxetine, flunarizine, milnacipran, mexiletine, amphetamine, thiopental sodium, flavonoids, bretylium, oxazepam, and honokiol.

[0169] In some embodiments, the at least one agent comprises one or a combination of neuroprotective and / or neuromodulatory agents selected from tryptamine, galanin receptor 2, phenylalanine, phenethylamine, N-methylphenethylamine, adenosine, kyotorphin, substance P, 3-methoxytyramine, catecholamines, dopamine, GAB A, calcium, acetylcholine, epinephrine, norepinephrine, and serotonin.

[0170] In some embodiments, the at least one agent comprises one or a combination of immunomodulatory agents selected from clenolizimab, enoticumab, ligelizumab, simtuzumab, batelizumab, palsatuzumab, imgatuzumab, tregalizumab, pateclizumab, namulumab, perakizumab, faralimomab, patritumab, atinumab, ublituximab, futuximab, and durigotumab.

[0171] In some embodiments, the at least one agent comprises one or a combination of anti-inflammatory agents selected from ibuprofen, aspirin, ketoprofen, sulindac, naproxen, etodolac, fenoprofen, diclofenac, flurbiprofen, ketorolac, piroxicam, indomethacin, mefenamic acid, meloxicam, nabumetone, oxaprozin, ketoprofen, famotidine, meclofenamate, tolmetin, and salsalate.

[0172] In some embodiments, the at least one agent comprises one or a combination of antimicrobial agents selected from antibacterial agents, antifungal agents, antiviral agents, antiparasitic agents, heat, radiation, and ozone.

[0173] The present disclosure also relates to methods for detecting and / or quantifying neuronal proliferation viability, the methods comprising: (a) quantifying one or more neuronal cells; (b) culturing one or more neuronal cells on any of the systems disclosed herein; and (c) calculating the number of neuronal cells in the composition after culturing for a period sufficient to allow growth of the one or more cells. In some embodiments, step (c) comprises detecting internal and / or external recordings of one or more neuronal cells after culturing such one or more neuronal cells and correlating the recordings with measurements of the same recordings corresponding to a known or control number of cells. In any of the methods, the system comprising one or more neuronal cells also comprises one or a combination of low-passage mature SCs, high-passage mature SCs, myelinating SCs, or SCPDs.

[0174] In some embodiments, the method further comprises contacting one or more neural cells with one or more agents. In some embodiments, the method further comprises (i) measuring intracellular and / or extracellular expression of a nucleic acid sequence or protein before and after contacting one or more neural cells and / or SCs with one or more agents, and (ii) correlating the difference in expression before contacting one or more neural cells and / or SCs with the one or more agents and after contacting one or more neural cells or SCs with the one or more agents with a change in cell number. Expression profiling can be completed by quantitative or semi-quantitative PCR.

[0175] Treatment method The present disclosure also relates to methods of transplanting a composition comprising Schwann cells into a subject in need thereof by administering the pharmaceutical compositions described herein.

[0176] In another aspect, the present disclosure relates to a method of treating spinal cord injury or peripheral neuropathy in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of Schwann cells disclosed herein.

[0177] In another aspect, the present disclosure relates to a method of treating diabetic peripheral neuropathy (DPN), Charcot-Marie-Tooth disease (CMT), amyotrophic lateral sclerosis (ALS), neuroinflammation, Guillain-Barré syndrome, radiation-induced nerve damage, and / or chemotherapy-induced nerve damage, the method comprising administering to a subject in need thereof a therapeutically effective amount of an agent identified by any of the screening methods disclosed herein. In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of one or a combination of cell compositions disclosed herein. In some embodiments, the cell composition comprises one or more low-passage Schwann cells, high-passage Schwann cells, SCPD cells, or myelinating Schwann cells. In some embodiments, the cells are derived from pluripotent stem cells and / or neural crest cells.

[0178] In another aspect, the present disclosure relates to a subject comprising any one of any of the compositions of Schwann cells disclosed herein. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0179] The present disclosure also relates to a method of treating diabetic peripheral neuropathy in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agent or a pharmaceutical composition comprising a therapeutically effective amount of any one or combination of the disclosed Schwann cells and a pharmaceutically acceptable carrier. In some embodiments, the agent is selected from the agents in Table S4 or Figures 13A-13D. In some embodiments, the agent is selected from the agents in Table S4 or pharmaceutically acceptable salts or derivatives thereof. In some embodiments, the agent is selected from the agents in Table S4 or pharmaceutically acceptable salts or derivatives thereof comprising a Z-score greater than 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or greater than 2.0.

[0180] The present disclosure also relates to a method of treating Charcot-Marie-Tooth disease (CMT) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agent or a pharmaceutical composition comprising a therapeutically effective amount of any one or combination of the disclosed Schwann cells and a pharmaceutically acceptable carrier. In some embodiments, the agent is selected from the agents in Table S4. In some embodiments, the agent is selected from the agents in Table S4 or pharmaceutically acceptable salts or derivatives thereof. In some embodiments, the agent is selected from the agents in Table S4 or pharmaceutically acceptable salts or derivatives thereof comprising a Z-score greater than 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or greater than 2.0.

[0181] The present disclosure also relates to a method of treating amyotrophic lateral sclerosis (ALS) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agent or a pharmaceutical composition comprising a therapeutically effective amount of any one or combination of the disclosed Schwann cells and a pharmaceutically acceptable carrier. In some embodiments, the agent is selected from the agents in Table S4. In some embodiments, the agent is selected from the agents in Table S4 or pharmaceutically acceptable salts or derivatives thereof. In some embodiments, the agent is selected from the agents in Table S4 or pharmaceutically acceptable salts or derivatives thereof comprising a Z-score greater than 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or greater than 2.0.

[0182] The present disclosure also relates to a method of treating neuroinflammation in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agent or a pharmaceutical composition comprising a therapeutically effective amount of any one or combination of the disclosed Schwann cells and a pharmaceutically acceptable carrier. In some embodiments, the agent is selected from the agents in Table S4. In some embodiments, the agent is selected from the agents in Table S4 or pharmaceutically acceptable salts or derivatives thereof. In some embodiments, the agent is selected from the agents in Table S4 or pharmaceutically acceptable salts or derivatives thereof comprising a Z-score greater than 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or greater than 2.0.

[0183] The present disclosure also relates to a method of treating Guillain-Barré syndrome in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agent or a pharmaceutical composition comprising a therapeutically effective amount of any one or combination of the disclosed Schwann cells and a pharmaceutically acceptable carrier. In some embodiments, the agent is selected from the agents in Table S4. In some embodiments, the agent is selected from the agents in Table S4 or pharmaceutically acceptable salts or derivatives thereof. In some embodiments, the agent is selected from the agents in Table S4 or pharmaceutically acceptable salts or derivatives thereof comprising a Z-score greater than 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or greater than 2.0.

[0184] The present disclosure also relates to a method of treating radiation-induced nerve damage and / or chemotherapy-induced nerve damage in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agent or a pharmaceutical composition comprising a therapeutically effective amount of any one or combination of the disclosed Schwann cells and a pharmaceutically acceptable carrier. In some embodiments, the agent is selected from the agents in Table S4. In some embodiments, the agent is selected from the agents in Table S4 or pharmaceutically acceptable salts or derivatives thereof. In some embodiments, the agent is selected from the agents in Table S4 or pharmaceutically acceptable salts or derivatives thereof comprising a Z-score greater than 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or greater than 2.0.

[0185] In some embodiments, the treatment method utilizes one or more active agents or pharmaceutically acceptable salt(s) thereof listed in Table S4. In some embodiments, the treatment method utilizes an active agent or pharmaceutically acceptable salt(s) thereof having a Z-score of 2.0 or greater. In some embodiments, the active agent is bupropion or a pharmaceutically acceptable salt thereof. In some embodiments, the active agent is cyclopenthiazide or a pharmaceutically acceptable salt thereof. In some embodiments, the active agent is niridazole or a pharmaceutically acceptable salt thereof. In some embodiments, the active agent is gliquidone or a pharmaceutically acceptable salt thereof. In some embodiments, the active agent is chonderphin or a pharmaceutically acceptable salt thereof. In some embodiments, the active agent is furaltadone or a pharmaceutically acceptable salt thereof. In some embodiments, the active agent is furaltadone hydrochloride. In some embodiments, the active agent is captopril or a pharmaceutically acceptable salt thereof. In some embodiments, the active agent is nimesulide or a pharmaceutically acceptable salt thereof. In some embodiments, the active agent is nafronyl or a pharmaceutically acceptable salt thereof. In some embodiments, the active agent is nafronyl oxalate. In some embodiments, the active agent is tolbutamide or a pharmaceutically acceptable salt thereof. In some embodiments, the active agent is perindopril or a pharmaceutically acceptable salt thereof. In some embodiments, the active agent is trolox or a pharmaceutically acceptable salt thereof. In some embodiments, the active agent is spaglumic acid or a pharmaceutically acceptable salt thereof. In some embodiments, the active agent is (d,l)-tetrahydroberberine or a pharmaceutically acceptable salt thereof. In some embodiments, the active agent is ribavirin or a pharmaceutically acceptable salt thereof. In some embodiments, the active agent is triamcinolone or a pharmaceutically acceptable salt thereof.

[0186] The dosage and frequency (single or multiple doses) administered to a mammal can vary depending on a variety of factors, such as whether the mammal is suffering from another disease and the route of administration, the recipient's size, age, sex, health, weight, body mass index, and diet, the nature and extent of symptoms of the disease being treated (e.g., symptoms of intestinal motility disorders), types of concurrent treatments, complications from the disease being treated, or other health-related problems. Other therapeutic regimens or agents can be used in conjunction with the methods and compounds of Applicant's disclosure. Adjustment and manipulation of established dosages (e.g., frequency and duration) is well within the ability of one of ordinary skill in the art.

[0187] The Schwann cells of the present disclosure can be administered to a patient alone or simultaneously. Coadministration is intended to include simultaneous or sequential administration of compounds individually or in combination (more than one compound or agent). Thus, the preparations can also be combined with other active substances (e.g., to reduce metabolic degradation) if desired. The compositions of the present disclosure can be delivered transdermally, topically, or formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols. Liquid formulations include solutions, suspensions, and emulsions, such as water or water / propylene glycol solutions. The compositions of the present disclosure can further include components that provide sustained release and / or comfort. Such components include high molecular weight, anionic mucus-mimetic polymers, gelling polysaccharides, and finely divided drug carrier matrices. These components are discussed in more detail in U.S. Patent Nos. 4,911,920, 5,403,841, 5,212,162, and 4,861,760. The entire contents of these patents are incorporated herein by reference in their entirety for all purposes. The compositions of the present disclosure can also be delivered as microspheres for sustained release in the body. For example, the microspheres can be administered via intravenous injection of drug-containing microspheres for subcutaneous sustained release (see Rao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995) or as biodegradable injectable gel formulations (see, e.g., Gao Pharm. Res. 12:857-863, 1995). In some embodiments, formulations of the disclosed compositions can be delivered by the use of liposomes that fuse with or are endocytosed by cell membranes, i.e., by using receptor ligands attached to the liposomes that bind to cell surface membrane protein receptors, resulting in endocytosis. The use of liposomes can target the delivery of the disclosed compositions to target cells in vivo, particularly when the liposome surface carries receptor ligands specific to the target cells or is otherwise preferentially directed to a particular organ.(See, e.g., Al-Muhammed, J. Microencapsul. 13:293-306, 1996; Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp. Pharm. 46, 1576-1587, 1989.) The compositions of the present disclosure can also be delivered as nanoparticles.

[0188] The present disclosure also relates to a method of treating DPN in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of buproprion, or a pharmaceutically acceptable salt or derivative thereof, and a pharmaceutically acceptable carrier. Buproprion has the following formula: Includes TIFF2025531671000287.tif56128.

[0189] Other embodiments are described in the following non-limiting examples. Various publications, including patents, published applications, GenBank accession numbers, technical papers, and scientific papers, are cited throughout this specification. Each of these cited publications is incorporated herein by reference in its entirety. [Example]

[0190] Examples 1 and 2 were carried out in a manner including, but not limited to, the following.

[0191] Example 1 Derivation and potential isolation of SC lineages from hPSCs We have previously established hPSC differentiation protocols to access various NC lineages, including enteric and sensory neurons (Barber et al., 2019; Chambers et al., 2012; Fattahi et al., 2016; Tchieu et al., 2017). However, there is currently no method to efficiently induce bona fide Schwann cells from hPSCs. Previous efforts to induce SCs relied on long-term culture of NC-enriched precursor cells for 2–3 months to obtain a small proportion of gliogenic cells (Lee et al., 2007). Although many studies have reported on the derivation of SC-like cells from hPSCs, they failed to demonstrate molecular authenticity by gene expression profiling and functional myelination (Huang et al., 2017; Kim et al., 2017; Liu et al., 2012; Ziegler et al., 2011). Although the mechanism of SC specification during human development remains unclear, SCs are thought to arise from SOX10+ NC cells in a stepwise process. Based on studies in mouse and chick embryos, NCs first give rise to SC precursors capable of associating with developing nerve fiber bundles. Associated neurons produce NRG1, which promotes the survival and further differentiation of SC precursors (SCPs) by activating the ERBB3 receptor (Newbern and Birchmeier, 2010). By E13.5 of mouse development, SC precursors give rise to immature SCs that express lineage-specific markers such as GFAP, S100, and POU3F1 while maintaining SOX10 expression. Terminal differentiation of SCs toward myelinating and nonmyelinating fates continues for a long period of time and is completed only after birth (Jessen et al., 2015).

[0192] The first hPSC-based NC differentiation protocols relied on delamination of putative NC cells from neuroepithelial lineages combined with the prospective isolation of p75+ and / or HNK1+ NC precursors (Bajpai et al., 2010; Lee et al., 2007). While these protocols yielded a variety of NC-derived lineages, levels of SOX10 expression were generally low. In contrast, more directed NC induction protocols based on timed exposure to activators of WNT signaling show robust induction of SOX10 in the majority of cells by day 11 of differentiation (Barber et al., 2019; Fattahi et al., 2016; Menendez et al., 2011; Mica et al., 2013; Tchieu et al., 2017). Upon further culture, these hPSC-derived NC cells can direct SOX10+ melanocytes (Mica et al., 2013), but can also give rise to SOX10- mesenchymal and neural precursors (Fattahi et al., 2016; Lee et al., 2007; Mica et al., 2013; Tchieu et al., 2017). Because SOX10 is a key marker for SC lineage (Finzsch et al., 2010), we first screened for conditions that maintain its expression in NC precursors in culture. We determined the percentage of SOX10+ cells in 2D or 3D NC cultures in the presence of modulators of EGF, FGF, WNT, Notch, TGFβ, BMP, and endothelin-3 signaling. We observed that activation of WNT signaling through CHIR99021 exposure and treatment with FGF2 resulted in the maintenance of SOX10 expression in 3D aggregates, which we termed developing progenitors (Figures 1A and B).

[0193] Further treatment of the progenitors with Schwann cell medium (SCM) containing FGF2, SB431542, and dbcAMP allowed the induction of additional SC markers, such as GFAP, POU3F1, PMP22, MBP, AQP4, and MPZ, as well as the upregulation of genes involved in glial-neuronal interaction and support, including GDNF, ERBB3, and GAP43, among others (Figure 5A and B). These cultures could be passaged and maintained for several weeks while retaining the expression of key SC markers S100, MBP, GFAP, and PMP22 (Figure 1C and D).

[0194] To determine the cellular diversity of our hPSC-derived SC cultures, we performed single-cell RNA-sequencing (scRNA-seq) at two differentiation time points: low passage (LP, day 38) and high passage (HP, day 58). Unbiased clustering of both the LP and HP datasets revealed four transcriptionally distinct cell types: SCPs, early SCs, mature SCs, and SCP derivatives (SCPDs) (Figure 1E and Figure S2A). These cell types differentially expressed defined markers of SC differentiation and function (Figure 1F). For example, mature SCs in both LP and HP cultures expressed higher levels of myelinating and nonmyelinating SC markers, such as PMP22 and NGFR. Neural supportive markers and neurotrophic factors, including ERBB3, GDNF, NGF, BDNF, and GAP43, were also enriched in mature SCs, particularly in HP cultures (Figure 1F and Figure S2B). We modularly scored LP and HP cell types for multiple SC functional gene sets, including neurotrophins, neurotransmitter receptors, and transcription factors (Table S1) and detected differential expression of many neurotransmitter receptor and postsynaptic signaling genes by our Schwann cell types (Figure S2C). We also identified transcription factors differentially expressed by each population in LP and HP cultures. For example, early SCs were enriched for E2F7 and E2F8, whereas mature SCs more specifically expressed SOX10, FOXO1, POU3F2, and TBX19. POU6F2 was a commonly enriched transcription factor in LP and HP SCPDs (Figure S2D).

[0195] (Table S1) TIFF2025531671000288.tif213160TIFF2025531671000289.tif214160TIFF2025531671000290.tif213160TIFF2025531671000291.tif221160

[0196] To verify the authenticity of our hPSC-derived SCs, we evaluated the expression of the top 15 differentially expressed myelinating and nonmyelinating Schwann cell-specific genes derived from a primary mouse single-cell transcriptome dataset previously published by Segal and colleagues (Tasdemir-Yilmaz et al., 2021) (Figure 1G, Table S2). We detected the expression of these markers in both our LP and HP cultures using cell-type-specific expression patterns (Figure 1G). Some markers, such as MPZ and MATN2, were specifically expressed in a single cluster, i.e., by mature SCs. However, the majority of other genes showed differential transcription levels between cell types but were not exclusively expressed in a single population (Figure 1G). Interestingly, our LP and HP mature SC populations were highly enriched for both myelinating and nonmyelinating markers, indicating that our hPSC-derived SCs reliably express markers of authentic SCs.

[0197] (Table S2) TIFF2025531671000292.tif157161TIFF2025531671000293.tif220161TIFF2025531671000294.tif222161 TIFF2025531671000295.tif221161TIFF2025531671000296.tif221161TIFF2025531671000297.tif220161 TIFF2025531671000298.tif222161TIFF2025531671000299.tif221161TIFF2025531671000300.tif219161 TIFF2025531671000301.tif222161TIFF2025531671000302.tif221161TIFF2025531671000303.tif220161

[0198] The proliferative capacity of our SC cultures allows for their expansion and expansion. To characterize the proliferative potential of our cell types, we determined the proportional distribution of cell cycle duration within the individual LP and HP populations (Figures 7A and 7B). As cultures transition from low to high passage, all cell types progressively exit the cell cycle. This is particularly evident in SCPs and early SCs, which cycle primarily at low passages (Figures 7A and 7B). This is consistent with the slower proliferation rate of our cultures as they age (not shown).

[0199] To assess whether our cultures retained their identity after long-term expansion, we assessed the lineage relationship between LP and HP cell types by modularly scoring the transcriptional signatures of LP cell types in HP cell types, and vice versa (Figure 1H and I). Notably, each cell type signature was most similar to its corresponding cell type in the other dataset (Figure 1H and I). The similarity between corresponding LP and HP clusters was further demonstrated when we performed clustering on the merged dataset and obtained the same cell populations (Figure 7C and D). Bulk transcript analysis of cultures at different time points demonstrated that hPSC-derived developmental precursors were closely related to early NC cells, whereas SC cultures, especially at higher passages, displayed gene expression patterns closely consistent with primary adult human SCs (Figure 1J).

[0200] To characterize the diversity in these cultures at a higher resolution, we performed further subclustering, revealing two early SC and two SCPD populations (Figure S3E). Early SC1 and SC2 were separated solely based on their cell cycle phase distribution (Figure S3F), whereas SCPD subclusters were primarily LP or HP specific (Figure S3G). To determine whether LP and HP SCPDs were functionally distinct, we performed gene ontology (GO) enrichment analysis of their top 250 differentially expressed genes. Interestingly, despite high levels of expression of canonical melanocyte genes such as MITF, MLANA, and PMEL, LP SCPDs were enriched for myelin production items such as cholesterol and lipid metabolism, suggesting a dual melanocyte-SC identity (Figures S2A and S3H). On the other hand, HP SCPDs showed enrichment for melanin synthesis and pigmentation, indicating that SCPDs become more melanocytic as the culture progresses (Figure S3H).

[0201] Promising isolation strategies enable the generation of pure, high-quality SC populations from heterogeneous cultures. To enable fluorescence-activated cell sorting (FACS)-based purification of hPSC-SCs, we screened a library of 242 antibodies for human surface antigens that specifically mark GFAP+ SCs (Figures S4A and S4B). We identified 11 surface antibodies that stained more than 20% of GFAP+ SCs, with CD44, CD49e, CD81, and CD98 marking the majority of the target population (Figure S4B). Analysis of surface marker expression in our LP and HP scRNA-seq datasets revealed transcripts that were specifically enriched in each cell type (Figure S4C). Interestingly, this list included 9 of the 11 surface marker hits identified by antibody screening. Among these, CD46, CD146, CD147, and CD166 were enriched in mature SCs in both datasets, whereas CD9, CD49e, and CD171 were enriched only in HP mature SCs. CD44 was highly expressed in SCPD in both LP and HP cultures, whereas CD81 enrichment was specific to the LP population (Figure S4C). Further validation revealed that CD98 was the only marker specifically expressed in SCs, but not in NCs or SCPs (Figure S4D). These populations expressed CD49D, a marker previously shown to mark the early SOX10+ NC lineage (Fattahi et al., 2016).

[0202] Taken together, these data demonstrate that our hPSC differentiation system generates scalable, proliferative human SC cultures that can be further enriched using FACS.

[0203] hPSC-SCs promote neuronal maturation and myelination in vitro and are transplanted into injured sciatic nerves in rats. SCs play a fundamental role in maintaining and protecting the structure and function of peripheral nerves. Myelinating SCs are specialized glial cells that form lipid-rich myelin sheaths around axons and enable fast neuronal signal propagation in the PNS. Because our hPSC-derived mature SCs express high levels of genes involved in myelination and lipid metabolism (Figures 1F and 1G, Figure S3H), we set out to identify and characterize myelinating SCs (mySCs) in our LP and HP mature SCs. We modularly scored the top differentially expressed primary mouse mySCs and a curated list of defined myelinogenic genes (Calder et al., 2015) in our LP and HP mature SCs (Figure 2A, Table S3). We identified more than 25% of mature SCs in each dataset as myelinating (Figure 2A). LP and HP mySCs were specifically enriched for multiple neurotrophic factors, as well as neurotransmitter and postsynaptic signaling genes (Figures S5A and S5B). For example, members of the TGF and FGF protein families were highly enriched in mySCs compared with other mature SCs in both datasets (Figure S5A). BDNF expression was specific to HP mySCs (Figure S5A). To define the unique functional properties of these cells, we performed pathway enrichment analysis using GO BP, KEGG, and Reactome gene sets for genes significantly upregulated in LP and HP mySCs (Figure 2B). Interestingly, among the top 50 significantly enriched pathways, we identified multiple pathways related to axon development, myelination, neuron development, synapse assembly, cell adhesion, and cell motility. These are well-established physiological characteristics of myelinating SCs. HP mySCs specifically upregulated pathways related to extracellular matrix organization, cell adhesion, and cell motility. It is interesting to note that SCs are known to contribute to the deposition and organization of ECM components, the formation of lamellipodia and cytoplasmic processes, and the formation of contacts between radial sorting and myelinating and axonal recognition, suggesting that our LP and HP mySCs possess molecular programs that enable myelinating SCs to perform their functions.

[0204] (Table S3) TIFF2025531671000304.tif118128

[0205] Because cell adhesion molecules (CAMs) play an important role in SC association with axons, neural components, and the ECM, and cell adhesion was the most significantly enriched GO term in both LP and HP mySCs (Figure 2B), we sought to determine the specific CAMs enriched in mySC populations. We modularly scored a list of cell adhesion molecules combining cell-cell and cell-matrix gene sets in our LP and HP mature SCs and identified many CAMs that were specifically depleted and enriched in LP and HP mySCs compared with other mature SCs (Figure S5C). For example, HLA-DR, which was also a hit in our SC antibody screen (Figures S4B and S5C), was enriched in both LP and HP mySCs. The proinflammatory interleukin IL18 was depleted, whereas MAG and KIT were enriched in both mySC populations (Figure S5C). Members of the contactin protein family (CNTN1, 4, and 6), which are axon-associated CAMs and play a role in the formation of axonal connections in the developing nervous system, were specifically enriched in HP mySCs (fig. S5C). Similarly, HP mySCs were highly enriched for PLXNB3, which is important for axon guidance and cell migration (fig. S5C).

[0206] To assess the ability of hPSC-derived SCs to functionally interact with neurons, we established cocultures with hPSC-derived sensory neurons (Chambers et al., 2012) and motor neurons (Calder and Tchieu, 2015) (Figure 2C). RFP-labeled SCs (day 60) were mixed with GFP-labeled sensory neurons (day 50) and analyzed at 72 h of coculture. SCs closely associated with sensory neurons by aligning along their processes (Figure 2D). Similarly, cocultures of SCs and hPSC-derived motor neurons (day 25) showed strong interactions along neuronal fibers (Figure 2E). The long-term process of human cell maturation in hPSC-derived lineages remains a major obstacle in the field. Glial cells, such as astrocytes, have been shown to promote the functional maturation of hPSC-derived CNS neurons (Tang et al., 2013). To assess the effect on SC maturation, we performed calcium imaging in hPSC-derived motor neurons at days 40 and 70 of differentiation (15 and 55 days of coculture). Interestingly, there was a significant increase in the calcium response of stage-matched motor neurons cocultured with SCs (Figure S6A). At day 70, responsiveness to glutamate and KCl stimulation was further improved and remained distinct from cultures containing motor neurons alone (Figure S6B). Our findings demonstrate the ability of hPSC-SCs to regulate neuronal function in vitro. To determine whether hPSC-SCs are functional and capable of producing myelin in vivo, we asked whether they could survive and engraft in a rat model of sciatic nerve injury. We depleted endogenous SCs via mechanical squeezing of the nerve at the injury site and injected RFP-labeled hPSC-SCs (Figure 2F). Transplanted SCs were easily detected 8 weeks after neural injection using the human-specific nuclear marker SC101 (Figure 2G). Transplanted hPSC-SCs made close contact with host neurons (Figure 2H) and expressed the myelin markers MAG and PO (Figure 2I). In mature myelinated fibers, sodium channels are located at the nodes of Ranvier, the site of action potential electrical generation.This region is adjacent to the CASPR-expressing domain (paranodal region), where the axonal membrane is in close contact with the myelin membrane. An axonal membrane domain characterized by potassium channel expression is adjacent to the CASPR+ region. Notably, we observed proper localization of both sodium and potassium channels in axons wrapped by RFP-labeled hPSC-SCs (Figure 2J-L). These studies demonstrate the ability of hPSC-SCs to engraft and produce myelin that properly associates with nerve fibers and nodes of Ranvier in injured adult peripheral nerves.

[0207] These results demonstrate that our hPSC-derived cultures of functional SCs provide a framework for modeling pathologies in which SCs play a central role in disease initiation and progression. For example, a large subset of Charcot-Marie-Tooth disease (CMT) patients suffer from debilitating myelin defects caused by genetic mutations. Importantly, genes associated with CMT, including demyelinating CMT1, axonal CMT2, and intermediate CMT, were expressed by our SC cultures, confirming their applicability for modeling CMT pathophysiology in future studies (Figure S7).

[0208] hPSC-derived SCs enable modeling, mechanistic understanding, and treatment of diabetic peripheral neuropathy In addition to rare genetic defects such as CMT, SCs are associated with a wide range of other neurological disorders. The most prominent form of acquired neuropathy is diabetic peripheral neuropathy (DPN), which results from progressive degeneration of peripheral nerves (Simmons and Feldman, 2002). Although symptoms result from neuronal dysfunction, it is unclear whether sensory neuron damage is the primary event in DPN, and there is evidence that SC degeneration and peripheral demyelination may be contributing factors (Eckersley, 2002). As proof of concept, we describe the use of our human hPSC differentiation system to model DPN by investigating the effects of high glucose on sensory neurons and SCs (Figure 3A).

[0209] Sensory neurons showed no obvious toxicity at glucose levels up to 45 mM. In contrast, hPSC-derived SC cultures were highly sensitive to even moderately elevated glucose levels (Figure 3B). High glucose treatment induced oxidative stress in SC cultures as measured by MitoSOX staining (Figure 3C).

[0210] Given the sophisticated glucotoxicity in SCs, strategies to prevent glucose-mediated cell damage in SCs may represent novel therapeutic opportunities for treating DPN. We established a high-throughput screening (HTS) assay to measure the viability of hPSCs-SCs in the presence of 30 mM glucose, and the Prestwick library containing 1,120 small molecules approved by the FDA, EMA, or other regulatory agencies (Figure 3D, Figure S8A). We identified several hit compounds that significantly increased SC viability under high-glucose conditions (Figure 3E, Table S4). Gaining mechanistic insight into the protective effects of these hits could elucidate the mechanisms of glucotoxicity in SCs. Given that the library compounds target many different cellular pathways, we sought to determine shared pathways among the candidate drugs that improved SC viability under high-glucose conditions using our previously established analytical approach (Samuel et al., 2020).

[0211] (Table S4) TIFF2025531671000305.tif204161TIFF2025531671000306.tif214161TIFF2025531671000307.tif209161TIFF2025531671000308.tif209161TIFF2025531671000309.tif214161TIFF2025531671000310.tif219161TIFF2025531671000311.tif214161TIFF2025531671000312.tif209161TIFF2025531671000313.tif209161TIFF2025531671000314.tif219161TIFF2025531671000315.tif219161TIFF2025531671000316.tif214161TIFF2025531671000317.tif209161TIFF2025531671000318.tif219161TIFF2025531671000319.tif214161TIFF2025531671000320.tif209161TIFF2025531671000321.tif213161TIFF2025531671000322.tif219161TIFF2025531671000323.tif214161TIFF2025531671000324.tif209161TIFF2025531671000325.tif209161TIFF2025531671000326.tif209161TIFF2025531671000327.tif209161TIFF2025531671000328.tif209161TIFF2025531671000329.tif209161TIFF2025531671000330.tif214161TIFF2025531671000331.tif209161TIFF2025531671000332.tif209161TIFF2025531671000333.tif209161TIFF2025531671000334.tif209161TIFF2025531671000335.tif214161TIFF2025531671000336.tif209161TIFF2025531671000337.tif209161TIFF2025531671000338.tif209161TIFF2025531671000339.tif209161TIFF2025 531671000340.tif209161TIFF2025531671000341.tif219161TIFF2025531671000342.tif2 09161TIFF2025531671000343.tif219161TIFF2025531671000344.tif209161TIFF20255316 71000345.tif219161TIFF2025531671000346.tif219161TIFF2025531671000347.tif85128.

[0212] We first predicted drug-protein interactions across the entire Prestwick library using a similarity ensemble approach (SEA) (Keiser et al., 2007). Next, we calculated a weighted total z-score for each protein by adding the normalized z-scores across all compounds targeting each protein, which we used in iPAGE GO analysis (Goodarzi et al., 2009). Among the GO terms associated with SC-protected drug candidates, we identified oxidative phosphorylation (OXPHOS), nitrogen metabolism, and metallopeptidases (Figure 3F). We then validated the expression of these candidate pathways in our HP cultures using module scoring analysis (Figure S8B). To determine the degree to which positive z-scores were enriched among drugs targeting each protein, we performed a Fisher's exact test. From this analysis, we identified 33 proteins as significant drug targets, filtered based on average sum z-score >0, false discovery rate (FDR) <0.25, and Fisher's p <0.1 (Figure 3G-I).

[0213] We performed protein-protein interaction network analysis using the STRING database to identify interactions among our 33 significant drug targets (Szklarczyk et al., 2019). In the resulting network, IL6, NR3C1, PGR, and PTGS2 had the highest degree centrality (Figure 3J). Furthermore, for more comprehensive target prediction, we generated a list of potential targets of the top hits derived from the HTS dataset (Figure S8C) or computational predictions using network- and similarity-based algorithms (Figure 3K, Table S5). Surprisingly, many of the potential target proteins were shared among multiple hits, including potassium channels (KCN), estrogen and progesterone receptors (ESR and PGR), prostaglandin synthases (PTGS), and prostaglandin receptors (PTGER). Notably, the predicted target pattern of tolbutamide was similar to that of our top hit, bupropion (BP) (Figure 3K).

[0214] (Table S5) TIFF2025531671000348.tif235167TIFF2025531671000349.tif214167TIFF20255316710 00350.tif219167TIFF2025531671000351.tif225167TIFF2025531671000352.tif225167

[0215] (Table S5 (continued)) TIFF2025531671000353.tif224167TIFF2025531671000354.tif230167TIFF2025531671000355.tif230167TIFF2025531671 000356.tif230167TIFF2025531671000357.tif231167TIFF2025531671000358.tif227167TIFF2025531671000359.tif62167

[0216] Our top hit compound, BP, is a widely used antidepressant commercially available as Wellbutrin®. BP demonstrated a dose-dependent effect in rescuing the viability of high-glucose-treated SC cultures (Figure S8D). In accordance with previous reports of glucose-mediated activation of the oxidative stress response (Correa-Silva et al., 2018; Kowluru et al., 2003), we observed activation of a cellular inflammatory response via nuclear NF-κB p65 localization in SCs exposed to high glucose, a phenotype that was neutralized by BP treatment (Figure S8E).

[0217] To understand the mechanism of glucotoxicity in SC cultures and its rescue with BP, we performed unbiased transcriptional and metabolite profiling (Fig. 3L). We compared the gene expression profiles of SCs treated with low and high glucose, regardless of BP treatment, and then performed pathway enrichment analysis of the differentially expressed genes. Of the 1,559 SC transcripts enriched or depleted in response to high glucose, 66 showed opposite expression patterns in cells treated with BP (Fig. S9A). Among these, PTGER4 was the only gene shared with the list of predicted BP targets (Fig. 3I and K, Table S5). Affected processes in cells exposed to high glucose included several metabolic and cell cycle signaling pathways, as well as BP treatment-regulated pathways related to DNA replication and transcription, cell cycle, and stress response.

[0218] To better understand the metabolic consequences of high glucose and BP treatment, we performed metabolomics and integrated the data with our bulk RNA sequencing results. The detected primary metabolites were classified into seven categories based on their abundance patterns in response to glucose and BP (Fig. S10A and B). Many of the metabolites accumulated or were depleted in SCs exposed to high glucose. BP treatment reversed the response in a subset of these metabolites, specifically in groups 4 and 7. Metabolic pathway enrichment analysis suggested modulation in the citric acid cycle, urea cycle, amino acid metabolism, glycolysis, and gluconeogenesis (Fig. S10A and B). In parallel, BP treatment increased the detected levels of the TCA cycle metabolites succinate, fumarate, citrate, α-ketoglutarate, and malate (Fig. S10, Fig. S11A). Citrate concentrations decreased in response to high glucose and were reversed by BP treatment. Expression of the citrate transporter SLC13A2 followed the same trend (Figures S10 and S11A). Exposure to high glucose caused an increase in cellular urea accompanied by an increase in the transcript level of the plasma membrane urea transporter SLC14A2 (Figures S10 and S11A). Both urea concentration and its transporter mRNA levels decreased in the presence of BP (Figures S10 and S11A). We observed an increase in cellular pyruvate in response to high glucose, which was reduced by BP treatment (Figures S10 and S11A). Cellular lactate levels decreased after BP treatment accompanied by downregulation of the membrane monocarboxylate transporter SLC16A3, SLC16A14, and SLC5A2 RNA levels (Figures S10 and S11A). It has previously been reported that elevated glucose levels can activate the polyol pathway in several cell types (Oates, 2002). The polyol pathway metabolizes excess intracellular glucose to sorbitol and subsequently to fructose through two enzymatic steps catalyzed by aldose reductase (AR) and sorbitol dehydrogenase (SDH), respectively. Osmotic and oxidative stress caused by the polyol pathway has been proposed as a mediator of tissue damage in response to high glucose, based on studies in the lens more than 50 years ago (Van Heyningen, 1959).Sorbitol accumulation is involved in peripheral nerve damage in multiple animal models of diabetes (Mizisin, 2014; Oates, 2002). We asked whether hPSC-SCs exhibit increased sorbitol levels in response to high glucose as a potential mechanism for their selective vulnerability. Consistent with studies in mice (Maekawa et al., 2001; Mizisin and Powell, 1993), we observed a higher AR-to-SDH ratio in hPSC-derived SCs compared with sensory neurons (Figure S11B). Furthermore, SCs, but not sensory neurons, showed increased sorbitol levels when exposed to high glucose (Figure S11C). BP treatment dose-dependently reduced sorbitol accumulation in SCs (Figure S11D). Similarly, we observed increased cellular levels of fructose in SCs treated with high glucose, and BP treatment countered this effect (Figure S11E). This is consistent with the protective effect of aldose reductase inhibitors in diabetic SCs (Hao et al., 2015). Collectively, these results point to a global metabolic shift in high-glucose-treated SCs that is reversed by BP treatment.

[0219] Pathway enrichment analysis of transcripts upregulated in response to high glucose and reversed in response to BP revealed the glycerolipid metabolism and ErbB signaling pathways (Figure 3M). Transcriptional changes in the glycerolipid metabolism pathway (Figure 3N) were accompanied by corresponding changes in pathway metabolites measured in the metabolomics dataset (Figure 3O). Increased triacylglycerol breakdown and diacylglycerol breakdown to free fatty acids and glycerol were suggested by the upregulation of LIPG and PNLIPRP3 transcripts in response to high glucose treatment (Figure 3N). This is intriguing given the important role of lipid metabolism in myelin production and SC physiology (Figure S3H). Furthermore, through the action of prostaglandin synthase, the glycerolipid metabolism pathway is directly linked to prostaglandin metabolism. This piqued our interest for several additional reasons. For example, prostaglandin E2 receptor (PTGER4) and prostaglandin-endoperoxide synthase 2 (PTGS2) were among the top significant protein targets identified by our high-throughput drug screening (Figure 3I). Notably, they were two parts of a protein-protein interaction network, with PTGS2 exhibiting high network centrality (Figure 3J), and PTGS and PTGER were part of a protein family shared among our top drug hits (Figure 3K). Furthermore, PTGER4 transcripts had an inverted pattern of expression in response to high glucose in the presence and absence of BP (Figure S9A). We aimed to investigate the effect of PTGER4 in SC glucotoxicity using a genetic approach. We used CRISPR-Cas9 ribonucleoprotein (RNP) to knock out PTGER4 in Schwann cells and observed a significant decrease in cleaved caspase-3 in response to high glucose treatment, indicating less susceptibility to glucotoxicity in the absence of PTGER4 (Figure 3P).

[0220] Bupropion rescues disease phenotypes in mouse models of DPN Given the remarkable ability of BP to rescue hPSC-SC viability in vitro, we next evaluated the therapeutic potential of BP in a mouse model of DPN. We treated wild-type C57BL6 mice with the pancreatic beta cell-specific toxin streptozotocin (STZ), which resulted in beta cell death, impaired insulin production, and hyperglycemia in the mice (Wu and Huan, 2001). In DPN, sensory nerve damage generally results in loss of sensation in the limbs. We evaluated the effects of BP treatment in STZ-treated mice by measuring heat sensation as a readout of sensory nerve function and by histological analysis of the sciatic nerve to assess structural damage (Figure 4A). STZ-treated mice showed a dramatic increase in blood glucose levels, independent of BP treatment, compared with nondiabetic control animals, indicating that BP treatment did not affect glucose levels. Hyperglycemic mice maintained without BP treatment showed a delayed response to heat stimulation at 7 and 8 weeks after STZ treatment. Notably, BP-treated diabetic mice showed no significant difference in response time compared with normal, nondiabetic animals (Figure 4B). Histological analysis revealed a significant increase in the proportion of TUNEL+ apoptotic cells in the sciatic nerves of STZ mice. BP + STZ-treated animals showed significantly fewer apoptotic cells than vehicle + STZ-treated animals (Figures 4C and 4D). Finally, we evaluated the effects of STZ and BP treatment on peripheral myelin using transmission electron microscopy. We observed a greater proportion of fibers with damaged myelin in the sciatic nerves of STZ-treated animals, which was significantly reduced in BP + STZ-treated animals (Figures 4E and 4F). These data indicate that BP can partially prevent DPN in STZ-treated mice. To evaluate whether BP can reverse sensory impairment and has therapeutic potential in more advanced disease states, we initiated BP treatment 8 weeks after STZ in a separate cohort of mice. These mice already showed delayed reaction times to heat stimuli before treatment with BP, but showed no significant differences 4–8 weeks after BP treatment compared with normal mice.These studies demonstrate the potent therapeutic effect of BP in the STZ model of DPN.

[0221] Taken together, these results highlight the promise of hPSC-derived cultures for modeling SC disorders, understanding disease mechanisms, and identifying potential therapeutic strategies.

[0222] Discussion Our study reports highly efficient derivation of SCs from hPSCs, overcoming limitations of previous studies, such as low yield, delayed differentiation, limited SC maturation, and lack of myelination data (Huang et al., 2017; Kim et al., 2017; Lee et al., 2007; Liu et al., 2012, 2012; Ziegler et al., 2011). Key features of our hPSC-based model are the scalability and purity of the resulting SCs and the ability to culture the cells for long periods without losing SC characteristics. In contrast, primary SCs tend to rapidly lose their characteristics during long-term culture, leading to increased contamination of SC cultures with fibroblast-like cells. Important developmental questions now accessible using this novel differentiation technique include the mechanisms controlling the transition from multipotent NC stem cells to committed SCs, and studies of human SC plasticity, which have provided data in mice suggesting that both melanocytes and parasympathetic neurons can originate from early SC lineages (Adameyko et al., 2009; Bonnamour et al., 2021; Espinosa-Medina et al., 2014; Nitzan et al., 2013). Our stepwise SC differentiation protocol generates SCs following the developmental process thought to occur in vivo. Therefore, it provides a powerful framework for lineage tracing studies that reveal how NC and SC fate specification mechanisms lead to the emergence of SCs and other SCPDs, such as melanocytes.

[0223] Our high-resolution transcriptomic profiling of hPSC-derived SCs is the first study to provide a comprehensive molecular characterization of human SCs at two differentiation stages. In addition to confirming the authenticity of the differentiated Schwann cell type, it allows us to identify specific transcription factors, SC functional markers, and surface molecules. Combining these data with high-throughput antibody screening, we identify novel markers for the prospective isolation of mature SCs. The identification of CD98 as a surface marker for the prospective isolation of committed SCs represents a powerful tool for such studies. Based on the proof-of-concept data presented herein, research on SC-mediated neuronal maturation and myelination should be another area of ​​focus. Modeling PNS pathologies and developing drug screening platforms for compounds that modulate peripheral myelination may be of particular interest. A striking feature of cultured hPSC-derived SCs is their gene expression pattern, which not only confirms their SC identity but also suggests that pluripotent-derived cells match the expression pattern of adult SCs. This is in contrast to most other in vitro-derived hPSC lines, such as neurons, which express fetal stage markers (Studer et al., 2015). Autologous SCs are currently being tested for applications in regenerative medicine targeting both PNS and CNS disorders (Lavdas et al., 2008; Rodrigues et al., 2012). Our transplantation data demonstrate robust engraftment of hPSC-SCs in models of traumatic nerve injury. Although future studies are needed to evaluate their long-term engraftment and therapeutic potential in models of PNS and CNS injury, our results lay a solid foundation for the application of hPSC-SCs in regenerative medicine, including spinal cord injury.

[0224] We present an hPSC-based DPN model that revealed selective vulnerability of SCs to diabetes-associated hyperglycemia. While the majority of our data were obtained in response to high glucose exposure, we observed a decrease in SC viability even with more moderate increases in glucose. Through our high-throughput screening, we identified candidate drugs that counteract glucotoxicity in SCs. Interestingly, many proteins are shared among the predicted targets of these drugs. For validation studies, we focused on our top candidate, bupropion. Transcriptional and metabolic profiling of stressed and rescued SCs provided intriguing insights into the cellular consequences of SC glucotoxicity and the mechanism of BP rescue. By comparing the list of predicted BP targets with 66 transcripts whose expression patterns were reversed under glucose and BP treatment, we identified PTGER4 as a potential target mediating SC glucotoxicity. These proteins convert arachidonate to PGH2, a precursor of other prostaglandins, including PGD2, PGF2, and PGE2, and PGE2 is a ligand for PTGER4. Furthermore, PTGER4 is one of the key targets for our HTS library compound analysis for hits that improve SC survival under high glucose conditions. Furthermore, prostaglandin analogs have shown some evidence of modest efficacy in randomized clinical trials for DPN (Boulton et al., 2005).

[0225] The ability of BP to counter glucose-mediated SC toxicity correlated with a decrease in intracellular glucose and sucrose levels. BP treatment resulted in a decrease in cellular lactate concentration and its membrane transporters, which may be potentially important given the importance of lactate as a fuel in neuron-SC metabolic coupling (Babetto et al., 2020; Domenech-Estevez et al., 2015). Our transcriptional and metabolomic profiling provided evidence for an upregulation of glycolysis and a downregulation of mitochondrial respiration in SCs in response to high glucose, which was rescued by BP treatment. iPAGE gene set enrichment analysis also identified oxidative phosphorylation as a pathway that may mediate the HTS hit's protective effect. Interestingly, BP appears to be the only antidepressant commonly associated with modest weight loss in patients, rather than weight gain (Arterburn et al., 2016). It is tempting to speculate whether BP-mediated changes in glucose metabolism may be related to their systemic effects. By performing pathway enrichment analysis, we identified significant changes in glycerolipid metabolism under high glucose conditions that were reversed upon BP treatment, which has important functional relevance given the role of lipid metabolism and myelin synthesis in SC function.

[0226] Importantly, our in vivo studies demonstrate that BP treatment can rescue DPN-related behavioral disorders and neuronal damage. The fact that BP is a widely used drug should greatly facilitate any future testing in DPN patients. Interestingly, BP has shown some benefit in treating patients suffering from neuropathic pain (Semenchuk et al., 2001), raising the question of whether the effects of drugs on alternative indications may also be mediated by their effects on SC vulnerability. In addition to BP, we have identified several additional compounds that can rescue Schwann cell vulnerability. It will be interesting to determine whether these compounds act via a common or distinct mechanism and whether they exhibit in vivo activity in STZ mice comparable to BP.

[0227] In conclusion, our findings facilitate human SC-based research for applications in regenerative medicine and human disease modeling. This study further implicates SC defects in the pathogenesis of DPN and presents BP as an FDA-approved drug capable of treating DPN-related damage in vitro and in vivo.

[0228] Materials and Methods Human pluripotent stem cell (hPSC) culture The hPSC line H9 (WA-09) and derivatives (SOX10::GFP, SYN::ChR2-YFP, SYN::YFP, PHOX2B:GFP, EF1::RFP EDNRB- / -) were maintained on mouse embryonic fibroblasts (MEFs, Global Stem, Rockville, MD) in KSR (Life Technologies, 10828-028) containing hPSC medium as previously described (Chambers et al., 2009) or seeded on Geltrex™-coated plates and maintained in chemically defined medium (E8) as previously described (Barber et al., 2019). Cells were subjected to mycoplasma testing at monthly intervals and STR profiled to confirm cell identity at the beginning of the study.

[0229] Neural crest induction hPSC differentiation toward NC was performed using knockout serum (KSR) medium or chemically defined Essential 6 (E6) medium according to previously established methods (Fattahi et al., 2016; Tchieu et al., 2017). Briefly, when monolayer cultures of hPSCs reached approximately 70% confluency, the neural crest induction protocol was initiated (D0) by aspirating maintenance medium (E8) and replacing it with neural crest induction medium A [BMP4 (1 ng ml-1), SB431542 (10 μM), and CHIR 99021 (600 nM) in Essential 6 medium]. Subsequently, cultures were fed with D2 neural crest induction medium B [SB431542 (10 μM) and CHIR 99021 (1.5 μM) in Essential 6 medium] until D12. Next, developmental precursors were formed between D12 and D30 to facilitate the selection of glial precursor lineages. At that time, medium B on D12 was removed, and the NC monolayer was dissociated using Accutase (30 min, 37°C, 5% CO). After centrifuging the sample at 290 × g for 1 min, the cells were resuspended in NC-C medium [neurobasal medium, FGF2 (10 ng ml-1), CHIR 99021 (3 μM), N2 supplement (10 μl ml-1), B27 supplement (20 μl ml-1), glutagro (10 μl ml-1), and MEM NEAA (10 μl ml-1)] and transferred to ultra-low attachment plates to form free-floating 3D developmental precursors. After 2 days, when free-floating developmental precursors could be observed, they were gently gathered in the center of each well using a vortex motion. The old medium was then carefully aspirated from the periphery of each well without removing the developmental precursors. After adding fresh NC-C medium, the cultures were incubated for 48 hours (37°C and 5% CO2) before passage with Accutase. Similarly, the cultures were fed with fresh medium every other day and passaged every 4 days until D30.

[0230] Induction and expansion of Schwann cells from hPSCs On day 1, NC cells were aggregated into 3D spheroids (5 million cells / well) in Ultra Low Attachment 6-well culture plates (Fisher Scientific, 3471) and cultured in Neurobasal (NB) medium supplemented with L-glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156), and B27 (Life Technologies, 17504044), containing CHIR (3 μM, Tocris Bioscience, 4423), FGF2 (10 ng / ml, R&D Systems, 233-FB-001MG / CF), and NRG1 (10 ng / ml, R&D 378-SM-025). After 14 days of suspension culture, spheroids were seeded onto polyornithine / laminin / fibronectin (PO / LM / FN)-coated dishes (prepared as previously (17)) in Neurobasal (NB) medium supplemented with NRG1 (20 ng / ml, R&D378-SM-025), FGF2 (10 ng / ml, R&D Systems, 233-FB-001MG / CF), and cAMP (100 mM, Sigma, D0260), supplemented with L-glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156), and B27 (Life Technologies, 17504044). SC precursors migrate out of the seeded spheroids and differentiate into SCs within 10 days. For long-term expansion, cells were cultured on PO / LM / FN-coated dishes in Schwann cell medium (Sciencell, 1701). Cells were fixed for immunostaining or harvested for gene expression analysis at days 25, 35, 50, and 100 of differentiation.

[0231] FACS and immunofluorescence (IF) analysis For IF, cells were fixed with 4% paraformaldehyde (PFA, Affymetrix-USB, 19943) for 20 minutes, then blocked and permeabilized with 1% bovine serum albumin (BSA, Thermo Scientific, 23209) and 0.3% Triton X-100 (Sigma, T8787). Cells were then incubated overnight at 4°C in primary antibody solution and stained with fluorophore-conjugated secondary antibodies for 1 hour at room temperature. The stained cells were then incubated with DAPI (1 ng / ml, Sigma, D9542-5MG) and washed several times before imaging. For flow cytometry analysis, cells were dissociated with Accutase (Innovative Cell Technologies, AT104), fixed and permeabilized using BD Cytofix / Cytoperm (BD Bioscience, 554722) solution, washed, blocked, and permeabilized using BD Perm / Wash buffer (BD Bioscience, 554723) according to the manufacturer's instructions. Cells were then stained with primary (overnight at 4°C) and secondary (30 minutes at room temperature) antibodies and analyzed using a flow cytometer (FlowJo software). A list of primary antibodies and dilutions is provided in Table S6.

[0232] (Table S6) TIFF2025531671000360.tif150167

[0233] Single-cell RNA sequencing (scRNA-seq) data analysis ScRNA-seq processing FASTq files were aligned to the human GRCh38 reference transcriptome using the 10X Genomics CellRanger 6.0.0 pipeline ( Zheng et al., 2017 ) to generate a gene expression count matrix using the “include introns” option.

[0234] Quality Control and Cell Filtering Datasets were analyzed using Seurat v4 in R v4.1.0 (Hao et al., 2021). The number of reads mapped to mitochondrial and ribosomal transcripts per cell was derived using the "PercentageFeatureSet" function. We identified and then removed low-quality cells independently for each dataset based on the number of unique features captured per cell, the number of unique molecular identifiers (UMIs) captured per cell, and the percentage of mitochondrial gene transcripts per cell. Datasets were filtered based on the following quality control metrics: nFeatures > 200, nFeatures < 7000, nCounts < 40000, and mitochondrial reads < 20%.

[0235] Dimensionality Reduction, Clustering, and Annotation The transcript count matrix was log-normalized using a scaling factor of 10,000 with 2,000 variable features identified using the "vst" method. Cell cycle duration distribution was predicted using the "CellCycleScoring" function with the S and G2M features from Seurat available in "cc.genes." The 2,000 most variable feature set was scaled and centered, and the following data variables were regressed: nFeatures, nCounts, mitochondrial gene percentage, ribosomal gene percentage, S score, and G2M score. Principal component analysis (PCA) was performed using default settings. PCA reduction was used to perform uniform manifold approximation and projection (UMAP) dimensionality reduction. Shared nearest neighbor (SNN) graphs were calculated using default settings, followed by cell clustering, which was achieved using the default Louvain algorithm. Quality control metrics were visualized for each cluster to identify and remove low-quality cell clusters (see Quality Control and Cell Filtering). After removing low-quality cell clusters, the above pipeline was re-run for each dataset. The number of principal components used in UMAP reduction and SNN calculations was determined by the principal component standard deviations specific to each dataset. The resolution used for clustering each dataset can be found in Table S7. Cluster markers were derived using the Wilcoxon rank-sum test. Cluster annotation was based on the expression of known cell-type marker genes. Following cell-type annotation, gene dropout values ​​were imputed using adaptive thresholded low-rank approximation (ALRA) (Linderman et al., 2018). Rank k approximation was automatically selected for each dataset with default values ​​selected for all other parameters. Imputed gene expression is shown in all plots and is used as the default in all downstream analyses unless otherwise noted.

[0236] (Table S7) TIFF2025531671000361.tif112167

[0237] Analysis of publicly available datasets Primary tissue-derived Schwann cell type markers for Schwann cell precursors, myelinating, and non-myelinating Schwann cells were obtained from Tasdemir-Yilmaz et al. (2021) using the interactive webpage Pagoda2 by performing differential feature expression analysis of cell type clusters of interest across the entire dataset of cells. Differentially expressed (DE) genes were sorted by Z-score and converted to human gene names with the "biomaRt" (Durinck et al., 2009) package using the human and mouse genome databases available in Ensemble.

[0238] Characterization of gene cluster expression Gene lists were compiled for genes belonging to transcription factors, surface markers, cell adhesion, neurotransmitter receptors, and neurotrophic factor functional groups from the Molecular Signatures Database (MSigDB) (Liberzon et al., 2011). For each dataset, gene lists were filtered to remove low-abundance genes (detected in fewer than 25% of cells in each cell-type cluster). Genes were then determined to be exclusively expressed by a cluster if more than 25% of cells within that cluster expressed only that gene.

[0239] To further selectively filter the transcription factor and surface marker gene sets, we derived genes shared by the transcription factor and surface marker gene sets and a cell type-specific differentially expressed (DE) gene list.

[0240] Cell-type transcriptional signature scoring To find transcriptionally similar cell populations between the two datasets, first, differentially expressed (DE) genes in the reference dataset were calculated from the imputed gene counts using the "FindAllMarkers" function, using the Wilcoxon rank-sum test to return only genes with a fold change (FC) greater than 0.25. The reference DE gene list was then filtered to remove genes not present in the query dataset. Next, for each cell cluster in the reference dataset, a transcriptional signature gene list was created from the top 100 DE genes, filtered for a p-value less than 0.05 and sorted by fold change (FC). The query dataset was then scored for the transcriptional signature gene list of each reference dataset cell cluster using the "AddModuleScore" function, based on the imputed feature counts of the query dataset.

[0241] Identification and specification of myelin-forming Schwann cells A myelinating Schwann cell (mySC)-specific marker gene set was curated by combining publicly available dataset-derived markers (Jessen and Mirsky, 2005) with standard myelinogenesis-related markers for a total of 21 marker genes (Table S3). LP- and HP-specific mature Schwann cell clusters were subsets from the entire LP and HP datasets and scored for the mySC gene set using the "AddModuleScore" function. Cells with a positive mySC score were isolated and identified as mySCs for further analysis.

[0242] Gene ontology analysis Cell type-specific DE genes of interest with positive fold change (FC) were calculated from imputed gene counts using the "FindAllMarkers" function. Each gene set was filtered to include genes with p-values ​​<0.05 and sorted by decreasing fold change. Up to 250 genes from each cell type-specific dataset were used for gene function profiling analysis by selecting pathways from the GO Biological Processes, KEGG, and Reactome databases using the g:Profiler (Raudvere et al., 2019) online tool. Enrichment terms were ranked by decreasing negative log10-transformed p-values.

[0243] Surface marker screening Screening for specific surface antigens was performed on hPSC-SCs at day 80 of differentiation using the BD Lyoplate library® (BD, 560747). Cells were seeded into 96-well plates (10,000 cells / well) and stained with primary and secondary antibodies according to the manufacturer's instructions. Stained wells were fixed for whole-plate imaging and quantification. The percentage of total GFAP double-positive cells was quantified for each antibody. Top hits (>60% double-positive) were further validated using flow cytometry.

[0244] Gene expression analysis For RNA sequencing, total RNA was extracted using the RNeasy RNA Purification Kit (Qiagen, 74106). For qRT-PCR assays, total RNA samples were reverse transcribed into cDNA using Superscript II Reverse Transcriptase (Life Technologies, 18064-014). qRT-PCR reactions were set up using QuantiTect SYBR Green PCR Mix (Qiagen, 204148). Each data point represents three independent biological replicates. RNA-seq reads were mapped to the human reference genome (hg19) using TopHat v2.0. TopHat was run with default parameters except for coverage search. Alignments were then quantified using HTSeq, and differential gene expression was calculated using DESeq normalized to the cranial neural crest samples.

[0245] Viability assay To monitor SC viability, cells were assayed for LDH activity using the CytoTox96 Cytotoxicity Assay Kit (Promega, G1780). Briefly, cells were seeded at 30,000 cells / cm2 in 96-well plates. Supernatants and cell lysates were harvested after 24 hours and assayed for LDH activity using a plate reader (absorbance at 490 nm). Cytotoxicity was calculated by dividing the LDH signal in the supernatant by the total LDH signal (from the lysate + supernatant). Cells were cultured in Schwann cell medium (Sciencell, 1701) on PO / LM / FN-coated dishes during the assay.

[0246] Calcium imaging MN-only cultures and MN-SC cocultures were subjected to calcium imaging on days 40 and 70 after coculture, as previously described (Barreto-Chang and Dolmetsch, 2009). Briefly, cells were loaded with 2 μmol / L Fluo-4 AM dissolved in a 1:1 (v / v) ratio of 20% Pluronic®-F127 and DMSO at a stock concentration of 1 mmol / L for 45 min at room temperature in a Tyrode's solution consisting of (mmol / L): 140 NaCl, 5.4 KCl, 1 MgCl, 1.8 CaCl, 10 glucose, and 10 HEPES at pH 7.4. For activation, cells were spiked with a solution containing glutamate (50 mM) or KCl (300 mM). Time-lapse images were acquired using an Axiovert Inverted Microscope (Zeiss) on a heated stage. Ratiometric analysis was performed using Metamorph Software (Molecular Devices).

[0247] Transplantation of hPSC-SCs into the rat sciatic nerve and histological evaluation All procedures were performed in accordance with NIH guidelines and approved by the local Institutional Animal Care and Use Committee (IACUC). Rats were anesthetized with isoflurane gas, and both sciatic nerves were exposed below the sciatic notch and crushed twice at the same position for 30 seconds using Dumont #5 forceps. Immediately afterwards, 3 x 10 4A cell suspension of 100 hPSCs / µl Schwann cells was transplanted by injecting approximately 3-4 µl into the proximal and distal locations of the crush site using a glass micropipette. Survival times ranged from 2 to 8 weeks. For immunohistochemistry, tissues were fixed by intracardiac perfusion of 4% PFA in 0.1 M PBS. Sciatic nerves were dissected from rats 2, 3, 4, and 8 weeks after crush lesion and transplantation. After dissection, the sciatic nerves were prepared by placing them in 30% sucrose in 0.1 M PBS overnight and embedding them in OCT blocks for cryosectioning, or by removing the perineurium and teasing them in cold 0.1 M phosphate buffer (pH 7.4). Several nerves were dissected after perfusion and immunostained to examine individual axons. Regenerating axons distal to the crush site were analyzed.

[0248] Metabolite measurements High-glucose, low-glucose, and drug-treated SCs and sensory neurons were subjected to biochemical assays of sorbitol (Abcam, ab118968), glucose (Abcam, ab65333), pyruvate (Abcam, ab65342), and 2DG uptake (Abcam, ab136955). Measurements were performed according to the manufacturer's instructions. Data were normalized according to cell number and averaged across three to six biological replicates.

[0249] High-throughput screening assay for drugs that reverse glucose-mediated SC cytotoxicity Chemical compound screening was performed using the Prestwick Chemical Library®. RFP-labeled hPSC-SCs were seeded into 384-well plates (1,000 cells / well) and treated with 30 mM glucose immediately before compound addition. Compounds were added at 1 μM concentrations. After 72 h, plates were treated with DAPI for 10 min, washed twice, and fixed for whole-plate imaging. The number of viable cells was quantified for each well by counting the number of DAPI-negative, RFP-positive cells. For validation of selected hit compounds (bupropion HCl, Sigma, B102), cells were treated with various concentrations of the compound for dose-response analysis. The highest non-toxic dose (0.7 μM, based on sorbitol reduction and viability) was used for follow-up experiments.

[0250] Drug target prediction The Z-scores of primary hit compounds were calculated as Z = (x - μ) / σ. X is the number of viable cells. μ is the average number of viable cells, and σ is the standard deviation of all compounds and the DMSO control. Normalized z-score values ​​reported for all compounds were first transformed to N(0,1) using the bestNormalize package (v1.4.0) in R (v3.5.1). Treatments with transformed z-scores greater than 2 were selected, resulting in 16 hit compounds.

[0251] To identify proteins most likely to be targeted by SC protective drugs against glucotoxicity, we used two independent tests. First, we calculated a combined z-score, which combined our normalized z-scores from all treatments associated with a particular protein. Second, we performed a Fisher's exact test to determine whether the target protein was enriched among targets of treatments with positive z-scores. We report the correlation between the p-values ​​calculated by these two independent tests. For all compounds, potential target proteins were identified as described above. Next, a weighted sum z-score was calculated for each protein by combining the normalized z-scores across all treatments (Zaykin, 2011). Next, p-values ​​were calculated based on the combined z-scores and adjusted using p.adjust (method = FDR). As an orthogonal approach, for each protein, we recorded the number of treatments with a positive normalized z-score and the total number of compounds predicted to target that protein. Using the sum of counts for all other proteins and drugs, we performed a Fisher exact test to assess the extent to which positive z-scores were enriched among treatments likely to affect the protein of interest. As expected, the two p-values, the combined z-score and Fisher, generally correlate.

[0252] Protein-protein interaction network construction. Protein-protein interaction network analysis was performed using the Search Tool for the Search of Interacting Genes (STRING) database. The minimum required interaction score was set to 0.4, corresponding to a medium level of confidence. Edge thickness indicates the degree of data support from active interaction sources: text mining, experiments, databases, co-expression, neighborhood, gene fusion, and co-occurrence.

[0253] iPAGE pathway enrichment analysis The iPAGE algorithm was used for gene set and pathway enrichment analysis (Goodarzi et al., 2009). iPAGE first quantizes continuous input data into evenly distributed bins and then calculates the mutual information (MI) between the vector of gene values ​​within each class bin and the binary vector of gene set membership. The significance of the calculated MI values ​​is then assessed by a randomization-based statistical test. Finally, it uses a high-order geometric distribution to determine the level at which significantly informative pathways are over-represented (red) or under-represented (blue) in each class bin. The resulting p-values ​​are used to plot a heatmap visualization, in which rows represent significant pathways and columns correspond to class bins. We first ordered target genes identified from FDA-approved drug library screening with positive z-scores (see Drug Target Prediction above) based on their combined z-scores from left to right, and then divided them into seven evenly distributed bins. Next, we assessed the enrichment (red boxes) and depletion (blue boxes) of various gene sets across the spectrum (MSigDB v6.0). Gene sets provided were from the Molecular Signatures Database (MSigDB v6.0) database (Liberzon et al., 2015). Here, we report enriched gene sets from Cluster 2 (C2, curated gene sets) and Cluster 5 (C5, ontology gene sets).

[0254] NF-kB staining Image analysis was performed using FIJI (Schindelin et al., 2012). Watershedding was applied to separate nuclei grouped together. Nuclear ROIs were overlaid on the NF-KB images to calculate nuclear colocalization. Several measurements were performed to analyze NF-KB in each condition. Because the cytoplasm could not be isolated to represent the cell to which it belongs, and the average value of the mean intensity of the cytoplasmic ROI does not account for the distribution of intensity per ROI area, the integrated densities of the cytoplasmic ROIs were summed and divided by the sum of the cytoplasmic ROI areas to obtain the mean intensity of NF-KB across all cytoplasm in each image. The mean intensity of each nucleus was divided by the mean intensity of the cytoplasm to obtain the ratio of nuclear to cytoplasmic NF-KB expression.

[0255] Metabolomics hPSC-Sc were treated with 5 mM and 30 mM glucose for 72 hours and harvested for metabolomic analysis. Frozen whole-cell pellets from at least three biological replicates were submitted to the West Coast Metabolomics Center at the University of California, Davis, for untargeted primary metabolomic analysis using an Agilent 6890 gas chromatograph and a Pegasus III Time-of-Flight mass spectrometer. Heat maps were generated and pathway analysis was performed using MetaboAnalyst (Xia et al., 2009).

[0256] Fructose, lactate and sorbitol measurement Cellular levels of fructose, lactate, and sorbitol were measured by colorimetric assays according to the instructions provided by the manufacturers. The following kits were used: fructose (Abcam, ab83380), lactate (Abcam, ab65331), and sorbitol (Abcam, ab118968).

[0257] CRISPR knockout of PTGER4 Schwann PTGER4 ribonucleoprotein (RNP) complexes were constructed by mixing 180 pmol of multi-guide sgRNA (Synthego, USA) and 20 pmol of Cas9 2NLS (Berkeley QB3) per reaction in Lonza electroporation buffer P3 (Lonza, Switzerland). hPSC-derived Schwann cells were detached using trypsin and washed twice with PBS. Immediately prior to electroporation, 250,000 cells per reaction were resuspended in Lonza electroporation buffer P3. Cells were mixed with RNP and electroporated using a Lonza 4D 96-well electroporation system with pulse code DS-137. Ten minutes after nucleofection, cells were diluted with warm medium and plated. The medium was changed the following day, and cells were passaged as needed until the day of assay. For quantification, images were analyzed with NIH ImageJ software by measuring the fluorescence intensity of individual cells via manual region-of-interest selection.

[0258] Drug treatment of diabetic mice All procedures were performed in accordance with NIH guidelines and approved by the local Institutional Animal Care and Use Committee (IACUC). Male C57BL6 mice aged 3–8 weeks were treated with a single IP injection of STZ (180 mg / kg, Sigma, 85882) to induce pancreatic beta cell death. Blood glucose levels were measured at weekly intervals by collecting a drop of blood from the tip of the tail using a standard glucometer (Freestyle Lite), starting 1 week after treatment. BP treatment was initiated 1 week after STZ treatment. BP was mixed with 1.63 mg / g of standard chow for oral administration at approximately 300 mg / kg daily. The dose was calculated based on average daily food intake (5.5 g / day) and initial body weight (30 g).

[0259] Mouse thermosensitivity test Thermal nociception was assessed using the hot plate test. The hot plate (Ugo Basile 35100) consisted of a metal surface (55°C) with a transparent Plexiglas cylinder to house the mouse. The subject was placed on the constant-temperature hot plate, and the latency required to show discomfort, assessed by either licking / shaking the hind paw or jumping, was determined. Typical baseline latencies were 5-10 seconds, with a maximum latency of 30 seconds. All animals that did not show discomfort were removed after the maximum latency of 30 seconds to avoid tissue damage.

[0260] statistical analysis Data are presented as mean ± SEM and were obtained from at least three independent experiments. Data for replicates (n) are shown in the figures. Statistical analysis was performed using ANOVA with Student's t-test (comparing two groups) or Dunnett's test (comparing multiple groups to control). The distribution of raw data approximated a normal distribution for data with a sufficient number of replicates to test for normality (Kolmogorov-Smirnov normality test).

[0261] References Adameyko, I., Lallemend, F., Aquino, JB, Pereira, JA, Topilko, P., Muller, T., Fritz, N., Beljajeva, A., Mochii, M., Liste, I., et al. (2009). Schwann Cell Precursors from Nerve Innervation Are a Cellular Origin of Melanocytes in Skin.Cell 139,366-379. Arterburn, D., Sofer, T., Boudreau, DM, Bogart, A., Westbrook, EO, Theis, MK, Simon, G., and Haneuse, S. (2016). Long-Term Weight Change after Initiating Second-Generation Antidepressants. J. Clin. Med. 5, E48. Babetto,E.,Wong,K.M.,and Beirowski,B.(2020).A glycolytic shift in Schwann cells supports injured axons.Nat.Neurosci.23,1215-1228. Bajpai,R.,Chen,D.A.,Rada-Iglesias,A.,Zhang,J.,Xiong,Y.,Helms,J.,Chang,C.-P.,Zhao,Y.,Swigut,T.,and Wysocka,J.(2010).CHD7 cooperates with PBAF to control multipotent neural crest formation.Nature 463,958-962. Barber,K.,Studer,L.,and Fattahi,F.(2019).Derivation of enteric neuron lineages from human pluripotent stem cells.Nat.Protoc.14,1261-1279. Barreto-Chang,O.L.,and Dolmetsch,R.E.(2009).Calcium imaging of cortical neurons using Fura-2 AM.J.Vis.Exp.JoVE 1067. Bonnamour,G.,Soret,R.,and Pilon,N.(2021).Dhh-expressing Schwann cell precursors contribute to skin and cochlear melanocytes,but not to vestibular melanocytes.Pigment Cell Melanoma Res.34,648-654. Boulton,A.J.M.,Vinik,A.I.,Arezzo,J.C.,Bril,V.,Feldman,E.L.,Freeman,R.,Malik,R.A.,Maser,R.E.,Sosenko,J.M.,Ziegler,D.,et al.(2005).Diabetic neuropathies:a statement by the American Diabetes Association.Diabetes Care 28,956-962. Calder,E.L.,Tchieu,J.,Steinbeck,J.A.,Tu,E.,Keros,S.,Ying,S.-W.,Jaiswal,M.K.,Cornacchia,D.,Goldstein,P.A.,Tabar,V.,et al.(2015).Retinoic Acid-Mediated Regulation of GLI3 Enables Efficient Motoneuron Derivation from Human ESCs in the Absence of Extrinsic SHH Activation.J.Neurosci.Off.J.Soc.Neurosci.35,11462-11481. Callaghan,B.C.,Cheng,H.T.,Stables,C.L.,Smith,A.L.,and Feldman,E.L.(2012).Diabetic neuropathy:clinical manifestations and current treatments.Lancet Neurol.11,521-534. Chambers,S.M.,Fasano,C.A.,Papapetrou,E.P.,Tomishima,M.,Sadelain,M.,and Studer,L.(2009).Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling.Nat.Biotechnol.27,275-280. Chambers,S.M.,Qi,Y.,Mica,Y.,Lee,G.,Zhang,X.-J.,Niu,L.,Bilsland,J.,Cao,L.,Stevens,E.,Whiting,P.,et al.(2012).Combined small-molecule inhibition accelerates developmental timing and converts human pluripotent stem cells into nociceptors.Nat.Biotechnol.30,715-720. Correa-Silva,S.,Alencar,A.P.,Moreli,J.B.,Borbely,A.U.,de S Lima,L.,Scavone,C.,Damasceno,D.C.,Rudge,M.V.C.,Bevilacqua,E.,and Calderon,I.M.P.(2018).Hyperglycemia induces inflammatory mediators in the human chorionic villous.Cytokine 111,41-48. Domenech-Estevez,E.,Baloui,H.,Repond,C.,Rosafio,K.,Medard,J.-J.,Tricaud,N.,Pellerin,L.,and Chrast,R.(2015).Distribution of monocarboxylate transporters in the peripheral nervous system suggests putative roles in lactate shuttling and myelination.J.Neurosci.Off.J.Soc.Neurosci.35,4151-4156. Durinck,S.,Spellman,P.T.,Birney,E.,and Huber,W.(2009).Mapping identifiers for the integration of genomic datasets with the R / Bioconductor package biomaRt.Nat.Protoc.4,1184-1191. Eckersley,L.(2002).Role of the Schwann cell in diabetic neuropathy.Int.Rev.Neurobiol.50,293-321. Espinosa-Medina,I.,Outin,E.,Picard,C.A.,Chettouh,Z.,Dymecki,S.,Consalez,G.G.,Coppola,E.,and Brunet,J.-F.(2014).Neurodevelopment.Parasympathetic ganglia derive from Schwann cell precursors.Science 345,87-90. Fattahi,F.,Steinbeck,J.A.,Kriks,S.,Tchieu,J.,Zimmer,B.,Kishinevsky,S.,Zeltner,N.,Mica,Y.,El-Nachef,W.,Zhao,H.,et al.(2016).Deriving human ENS lineages for cell therapy and drug discovery in Hirschsprung disease.Nature 531,105-109. Finzsch,M.,Schreiner,S.,Kichko,T.,Reeh,P.,Tamm,E.R.,Bosl,M.R.,Meijer,D.,and Wegner,M.(2010).Sox10 is required for Schwann cell identity and progression beyond the immature Schwann cell stage.J.Cell Biol.189,701-712. Gfeller,D.,Grosdidier,A.,Wirth,M.,Daina,A.,Michielin,O.,and Zoete,V.(2014).SwissTargetPrediction:a web server for target prediction of bioactive small molecules.Nucleic Acids Res.42,W32-38. Goodarzi,H.,Elemento,O.,and Tavazoie,S.(2009).Revealing global regulatory perturbations across human cancers.Mol.Cell 36,900-911. Hao,W.,Tashiro,S.,Hasegawa,T.,Sato,Y.,Kobayashi,T.,Tando,T.,Katsuyama,E.,Fujie,A.,Watanabe,R.,Morita,M.,et al.(2015).Hyperglycemia Promotes Schwann Cell Dedifferentiation and De-myelination via Sorbitol Accumulation and Igf1 Protein Down-regulation.J.Biol.Chem.290,17106-17115. Hao,Y.,Hao,S.,Andersen-Nissen,E.,Mauck,W.M.,Zheng,S.,Butler,A.,Lee,M.J.,Wilk,A.J.,Darby,C.,Zager,M.,et al.(2021).Integrated analysis of multimodal single-cell data.Cell 184,3573-3587.e29. Huang,C.-W.,Huang,W.-C.,Qiu,X.,Fernandes Ferreira da Silva,F.,Wang,A.,Patel,S.,Nesti,L.J.,Poo,M.-M.,and Li,S.(2017).The Differentiation Stage of Transplanted Stem Cells Modulates Nerve Regeneration.Sci.Rep.7,17401. Huang,D.W.,Sherman,B.T.,and Lempicki,R.A.(2009a).Bioinformatics enrichment tools:paths toward the comprehensive functional analysis of large gene lists.Nucleic Acids Res.37,1-13. Huang,D.W.,Sherman,B.T.,and Lempicki,R.A.(2009b).Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.Nat.Protoc.4,44-57. Jessen,K.R.,and Mirsky,R.(2005).The origin and development of glial cells in peripheral nerves.Nat.Rev.Neurosci.6,671-682. Jessen,K.R.,Mirsky,R.,and Lloyd,A.C.(2015).Schwann Cells:Development and Role in Nerve Repair.Cold Spring Harb.Perspect.Biol.7,a020487. Keiser,M.J.,Roth,B.L.,Armbruster,B.N.,Ernsberger,P.,Irwin,J.J.,and Shoichet,B.K.(2007).Relating protein pharmacology by ligand chemistry.Nat.Biotechnol.25,197-206. Kim,H.-S.,Lee,J.,Lee,D.Y.,Kim,Y.-D.,Kim,J.Y.,Lim,H.J.,Lim,S.,and Cho,Y.S.(2017).Schwann Cell Precursors from Human Pluripotent Stem Cells as a Potential Therapeutic Target for Myelin Repair.Stem Cell Rep.8,1714-1726. Kim,S.,Chen,J.,Cheng,T.,Gindulyte,A.,He,J.,He,S.,Li,Q.,Shoemaker,B.A.,Thiessen,P.A.,Yu,B.,et al.(2019).PubChem 2019 update:improved access to chemical data.Nucleic Acids Res.47,D1102-D1109. Kowluru,R.A.,Koppolu,P.,Chakrabarti,S.,and Chen,S.(2003).Diabetes-induced activation of nuclear transcriptional factor in the retina,and its inhibition by antioxidants.Free Radic.Res.37,1169-1180. Lavdas,A.A.,Papastefanaki,F.,Thomaidou,D.,and Matsas,R.(2008).Schwann cell transplantation for CNS repair.Curr.Med.Chem.15,151-160. Lee,G.,Kim,H.,Elkabetz,Y.,Al Shamy,G.,Panagiotakos,G.,Barberi,T.,Tabar,V.,and Studer,L.(2007).Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells.Nat.Biotechnol.25,1468-1475. Liberzon,A.,Subramanian,A.,Pinchback,R.,Thorvaldsdottir,H.,Tamayo,P.,and Mesirov,J.P.(2011).Molecular signatures database(MSigDB) 3.0.Bioinforma.Oxf.Engl.27,1739-1740. Liberzon,A.,Birger,C.,Thorvaldsdottir,H.,Ghandi,M.,Mesirov,J.P.,and Tamayo,P.(2015).The Molecular Signatures Database(MSigDB) hallmark gene set collection.Cell Syst.1,417-425. Linderman,G.C.,Zhao,J.,and Kluger,Y.(2018).Zero-preserving imputation of scRNA-seq data using low-rank approximation. Liu,Q.,Spusta,S.C.,Mi,R.,Lassiter,R.N.T.,Stark,M.R.,Hoke,A.,Rao,M.S.,and Zeng,X.(2012).Human neural crest stem cells derived from human ESCs and induced pluripotent stem cells:induction,maintenance,and differentiation into functional schwann cells.Stem Cells Transl.Med.1,266-278. Liu,T.,Lin,Y.,Wen,X.,Jorissen,R.N.,and Gilson,M.K.(2007).BindingDB:a web-accessible database of experimentally determined protein-ligand binding affinities.Nucleic Acids Res.35,D198-201. Maekawa,K.,Tanimoto,T.,Okada,S.,Suzuki,T.,Suzuki,T.,and Yabe-Nishimura,C.(2001).Expression of aldose reductase and sorbitol dehydrogenase genes in Schwann cells isolated from rat:effects of high glucose and osmotic stress.Brain Res.Mol.Brain Res.87,251-256. Mathias,S.L.,Hines-Kay,J.,Yang,J.J.,Zahoransky-Kohalmi,G.,Bologa,C.G.,Ursu,O.,and Oprea,T.I.(2013).The CARLSBAD database:a confederated database of chemical bioactivities.Database J.Biol.Databases Curation 2013,bat044. Menendez,L.,Yatskievych,T.A.,Antin,P.B.,and Dalton,S.(2011).Wnt signaling and a Smad pathway blockade direct the differentiation of human pluripotent stem cells to multipotent neural crest cells.Proc.Natl.Acad.Sci.108,19240-19245. Mica,Y.,Lee,G.,Chambers,S.M.,Tomishima,M.J.,and Studer,L.(2013).Modeling neural crest induction,melanocyte specification,and disease-related pigmentation defects in hESCs and patient-specific iPSCs.Cell Rep.3,1140-1152. Mizisin,A.P.(2014).Mechanisms of diabetic neuropathy:Schwann cells.Handb.Clin.Neurol.126,401-428. Mizisin,A.P.,and Powell,H.C.(1993).Schwann cell injury is attenuated by aldose reductase inhibition in galactose intoxication.J.Neuropathol.Exp.Neurol.52,78-86. Newbern,J.,and Birchmeier,C.(2010).Nrg1 / ErbB signaling networks in Schwann cell development and myelination.Semin.Cell Dev.Biol.21,922-928. Nitzan,E.,Pfaltzgraff,E.R.,Labosky,P.A.,and Kalcheim,C.(2013).Neural crest and Schwann cell progenitor-derived melanocytes are two spatially segregated populations similarly regulated by Foxd3.Proc.Natl.Acad.Sci.U.S.A.110,12709-12714. Oates,P.J.(2002).Polyol pathway and diabetic peripheral neuropathy.Int.Rev.Neurobiol.50,325-392. Raudvere,U.,Kolberg,L.,Kuzmin,I.,Arak,T.,Adler,P.,Peterson,H.,and Vilo,J.(2019).g:Profiler:a web server for functional enrichment analysis and conversions of gene lists(2019 update).Nucleic Acids Res.47,W191-W198. Rodrigues,M.C.O.,Rodrigues,A.A.,Glover,L.E.,Voltarelli,J.,and Borlongan,C.V.(2012).Peripheral Nerve Repair with Cultured Schwann Cells:Getting Closer to the Clinics.Sci.World J.2012,e413091. Samuel,R.M.,Majd,H.,Richter,M.N.,Ghazizadeh,Z.,Zekavat,S.M.,Navickas,A.,Ramirez,J.T.,Asgharian,H.,Simoneau,C.R.,Bonser,L.R.,et al.(2020).Androgen Signaling Regulates SARS-CoV-2 Receptor Levels and Is Associated with Severe COVID-19 Symptoms in Men.Cell Stem Cell 27,876-889.e12. Schindelin,J.,Arganda-Carreras,I.,Frise,E.,Kaynig,V.,Longair,M.,Pietzsch,T.,Preibisch,S.,Rueden,C.,Saalfeld,S.,Schmid,B.,et al.(2012).Fiji:an open-source platform for biological-image analysis.Nat.Methods 9,676-682. Semenchuk,M.R.,Sherman,S.,and Davis,B.(2001).Double-blind,randomized trial of bupropion SR for the treatment of neuropathic pain.Neurology 57,1583-1588. Simmons,Z.,and Feldman,E.L.(2002).Update on diabetic neuropathy.Curr.Opin.Neurol.15,595-603. Siramshetty,V.B.,Eckert,O.A.,Gohlke,B.-O.,Goede,A.,Chen,Q.,Devarakonda,P.,Preissner,S.,and Preissner,R.(2018).SuperDRUG2:a one stop resource for approved / marketed drugs.Nucleic Acids Res.46,D1137-D1143. Studer,L.,Vera,E.,and Cornacchia,D.(2015).Programming and Reprogramming Cellular Age in the Era of Induced Pluripotency.Cell Stem Cell 16,591-600. Szklarczyk,D.,Gable,A.L.,Lyon,D.,Junge,A.,Wyder,S.,Huerta-Cepas,J.,Simonovic,M.,Doncheva,N.T.,Morris,J.H.,Bork,P.,et al.(2019).STRING v11:protein-protein association networks with increased coverage,supporting functional discovery in genome-wide experimental datasets.Nucleic Acids Res.47,D607-D613. Tang,X.,Zhou,L.,Wagner,A.M.,Marchetto,M.C.N.,Muotri,A.R.,Gage,F.H.,and Chen,G.(2013).Astroglial cells regulate the developmental timeline of human neurons differentiated from induced pluripotent stem cells.Stem Cell Res.11,743-757. Tasdemir-Yilmaz,O.E.,Druckenbrod,N.R.,Olukoya,O.O.,Dong,W.,Yung,A.R.,Bastille,I.,Pazyra-Murphy,M.F.,Sitko,A.A.,Hale,E.B.,Vigneau,S.,et al.(2021).Diversity of developing peripheral glia revealed by single-cell RNA sequencing.Dev.Cell 56,2516-2535.e8. Tchieu,J.,Zimmer,B.,Fattahi,F.,Amin,S.,Zeltner,N.,Chen,S.,and Studer,L.(2017).A Modular Platform for Differentiation of Human PSCs into All Major Ectodermal Lineages.Cell Stem Cell 21,399-410.e7. Wu,K.K.,and Huan,Y.(2001).Streptozotocin-Induced Diabetic Models in Mice and Rats.In Current Protocols in Pharmacology,(John Wiley & Sons,Inc.),p. Xia,J.,Psychogios,N.,Young,N.,and Wishart,D.S.(2009).MetaboAnalyst:a web server for metabolomic data analysis and interpretation.Nucleic Acids Res.37,W652-660. Yamanishi,Y.,Kotera,M.,Moriya,Y.,Sawada,R.,Kanehisa,M.,and Goto,S.(2014).DINIES:drug-target interaction network inference engine based on supervised analysis.Nucleic Acids Res.42,W39-45. Zaykin,D.V.(2011).Optimally weighted Z-test is a powerful method for combining probabilities in meta-analysis.J.Evol.Biol.24,1836-1841. Zheng,GXY,Terry,JM,Belgrader,P.,Ryvkin,P.,Bent,ZW,Wilson,R.,Ziraldo,SB,Wheeler,TD,McDermott,GP,Zhu,J.,et al.(2017).Massively parallel digital transcriptional profiling of single cells.Nat.Commun.8,14049. Ziegler, L., Grigoryan, S., Yang, IH, Thakor, NV, and Goldstein, RS (2011). Efficient generation of schwann cells from human embryonic stem cell-derived neurospheres. Stem Cell Rev.7, 394-403.

[0262] Example 2 Culture methods for differentiation and culture of hPSCs into neural crest cells and Schwann cells. Human pluripotent stem cell (hPSC) culture The hPSC line H9 (WA-09) and derivatives (SOX10::GFP; EF1::RFP) were maintained on mouse embryonic fibroblasts (MEFs, Global Stem, Rockville, MD) in KSR (Life Technologies, 10828-028) containing hPSC medium as previously described, or seeded on Gel-trex™-coated (Thermo Fisher Scientific, A1413302) plates as previously described and maintained in chemically defined Essential 8 (E8) medium. 10 WTC11 cells were also maintained in E8 medium.

[0263] Neural crest (NC) and Schwann cell (SC) differentiation To induce neural crest-derived Schwann cells, we used two different methods. In option 1, we performed neural crest induction using Knockout Serum Replacement (KSR, Life Technologies, 10828028) containing medium and SC induction medium supplemented with NRG1 (R&D 378-SM-025). In option 2, we used Essential 6 (E6, Life Technologies, A1516401) medium for neural crest induction and SC induction medium without additional NRG1 supplements.

[0264] NC Induction Option 1 hESCs were seeded on Matrigel (BD Biosciences, 354234)-coated dishes (10 cells / cm) in hESC medium containing 10 ng / ml FGF2 (R&D Systems, 233-FB-001MG / CF). NC differentiation was initiated in knockout serum replacement (KSR) medium (KO DMEM + 15% KSR, L-glutamine (Life Technologies, 25030-081), and NEAA (Life Technologies, 11140-050) containing LDN193189 (100 nM, Stemgent, Cambridge, MA) and SB431542 (10 mM, Tocris, Ellisville, MI). For NC induction, from days 2 to 11, LDN and SB were added plus 3 mM CHIR99021 (Tocris). Cells were treated with KSR medium (Human Embryonic Stem Cells, 4423). KSR medium was gradually replaced with increasing amounts of N2 medium from days 4 to 10, as previously described. Differentiated cells were sorted for CD49D on day 11. CNS progenitor control cells were generated by treatment with LDN and SB from days 0 to 11, as previously described [58—Chambers, S.M., Fasano, C.A., Papapetrou, E.P., Tomishima, M., Sadelain, M., and Studer, L. (2009). Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat. Biotechnol. 27, 275-280, incorporated by reference in its entirety]. Throughout this disclosure, day 0 refers to the day the medium was switched from hESC medium to differentiation medium. Differentiation days in the text and figures refer to the number of days from the pluripotent stage (day 0).

[0265] Schwann Cell Induction and Expansion Option 1 On day 11, NC cells were aggregated into 3D spheroids (5 million cells / well) in Ultra Low Attachment 6-well culture plates (Fisher Scientific, 3471) and cultured in Neurobasal (NB) medium supplemented with L-glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156), and B27 (Life Technologies, 17504044), containing CHIR (3 mM, Tocris Bioscience, 4423), FGF2 (10 ng / ml, R&D Systems, 233-FB-001MG / CF), and NRG1 (10 ng / ml, R&D 378-SM-025). After 14 days of suspension culture, spheroids were seeded onto polyornithine / laminin / fibronectin (PO / LM / FN)-coated dishes (prepared as previously (17)) in Neurobasal (NB) medium supplemented with NRG1 (20 ng / ml, R&D 378-SM-025), FGF2 (10 ng / ml, RD Systems, 233-FB-001MG / CF), and cAMP (100 mM, Sigma, D0260), supplemented with L-glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156), and B27 (Life Technologies, 17504044). SC precursors migrate out of the seeded spheroids and differentiate into SCs within 10 days. For long-term expansion, cells were cultured in Schwann cell medium (Sciencell, 1701, without FBS) on PO / LM / FN-coated dishes. Cells were fixed for immunostaining or harvested for gene expression analysis at days 25, 35, 50, and 100 of differentiation.

[0266] NC Induction Option 2 hPSC differentiation into NCs was performed according to previously established methods using chemically defined Essential 6 (E6, Life Technologies, A1516401) medium. Briefly, when monolayer cultures of hPSCs reached approximately 70% confluency, the NC induction protocol was initiated (Day 0) by aspirating the maintenance medium (E8, Life Technologies, A2858501) and replacing it with NC induction medium A [BMP4 (1 ng ml-1, R&D Systems, 314-BP), SB431542 (10 mM, R&D Systems, 1614), and CHIR 99021 (600 nM, Tocris Bioscience, 4423)] in Essential 6 medium (Life Technologies, A1516401). D2-D12 cultures were subsequently fed with NC induction medium B [SB431542 (10 mM) and CHIR 99021 (1.5 mM) in Essential 6 medium].

[0267] Schwann Cell Induction and Expansion Option 2 On D12, NCs were dissociated to form 3D spheroids and maintained until D30 to promote the emergence of glial progenitor cells. To do so, we removed Medium B on D12 and dissociated the NC monolayer using Accutase (Innovative Cell Technologies, AT104) at 37°C and 5% CO for 30 min. After centrifuging the sample at 290 × g for 1 min, we repopulated the cells with NC-C medium [FGF2 (10 ng ml-1, R&D Systems, 233-FB-001MG / CF), CHIR 99021 (3 mM), N2 supplement (10 ml ml-1, CTS, A1370701), B27 supplement (20 ml ml-1, Life The cells were resuspended in MEM NEAAs (10 ml ml, Corning, 25-025-CI) in 10% ethanol (Life Technologies, 17504044), Glutagro (10 ml ml, Corning, 25-015-CI), and Neurobasal Medium (Life Technologies, 21103049) and transferred to ultra-low attachment plates (Fisher Scientific, 3471) to form free-floating 3D developmental precursors. After 2 days, when free-floating developmental precursors could be observed, they were gently gathered in the center of each well using a vortexing motion. The old medium was then carefully aspirated from the periphery of each well without removing the developmental precursors. After adding fresh NC-C medium, the cultures were incubated for 48 hours (37°C and 5% CO2) before passaging using Accutase. Similarly, the cultures were fed with fresh medium every other day and passaged every four days until D30. On D30, free-floating developmental precursors were dissociated using Accutase, cultured in FBS-free Schwann cell medium (Sciencell, 1701), and plated onto dishes coated with polyornithine / laminin / fibronectin (PO / LM / FN, Sigma, P3655, Cultrex 3400-10, and Corning, 356008, 15 mg / ml, 2 mg / ml, and 2 mg / ml, respectively). Typically, starting from one 6-well plate on D0, three 10 cm PO / LM / FN-coated dishes should be obtained by D30. To improve induction efficiency and purity, cells were treated with FBS-free Schwann cell medium containing 10 mM SB431542 from D32 to D42 and 100 mM cAMP (Sigma, D0260) from D32 to D39. Cultures were fed with fresh medium daily and passaged using 0.05% trypsin (Fisher Scientific, MT25052CI) when the cultures expanded and became confluent (>80%). Long-term maintenance and expansion of cells was in Schwann cell medium without FBS.

[0268] FACS and immunofluorescence (IF) analysis For IF, cells were fixed with 4% paraformaldehyde (PFA, SCBT, sc-281692) for 20 min, then blocked and permeabilized with 1% bovine serum albumin (BSA, Thermo Scientific, 23209) and 0.3% Triton X-100 (Sigma, T8787). Cells were then incubated overnight at 4°C in primary antibody solution and stained with fluorophore-conjugated secondary antibodies for 1 h at RT. The stained cells were then incubated with DAPI (1 ng / ml, Sigma, D9542-5MG) and washed several times before imaging. For flow cytometry analysis, cells were dissociated with Accutase (Innovative Cell Technologies, AT104), fixed and permeabilized using BD Cytofix / Cytoperm (BD Bioscience, 554722) solution, and then washed, blocked, and permeabilized using BD Perm / Wash buffer (BD Bioscience, 554723) according to the manufacturer's instructions. Cells were then stained with primary (overnight at 4°C) and secondary (30 min at room temperature) antibodies and analyzed using a flow cytometer (FlowJo software). The antibodies and diluents used were CD49D (Biolegend, 304301, 1:800), CHAT (Proteintech, 20747-1-AP, 1:1000), CHAT (Sigma, AB144P, 1:1000), GFAP (Abcam, ab4674, 1:1000), MAG (Millipore, LS-C279052-200, 1:200), MBP (Millipore, MAB386, 1:200), MPZ (Abcam, ab39375, 1:500), NFH (Encor, RPCA-NF-H, 1:1000), NFkB p65 (Invitrogen, 710048, 1:500), and PMP22 (Novus The following were used: S100 (Thermo Scientific, RB-9018-P0, 1:500), S101 (Takara, Y40400, 1:1000), TUBB3 (Millipore Sigma, ab9354, 1:350), and TUBB3 (Biolegend, 801202, 1:1500).

Claims

1. A composition comprising one or more Schwann cells, said Schwann cells comprising CD98 or a functional fragment thereof comprising at least about 70% of the sequence relative to CD98.

2. The composition of claim 1 , wherein the cells are derived from neural crest (NC) cells.

3. 3. The composition of claim 1 or claim 2, wherein the cells are cultured for more than about 35 days.

4. The composition of any one of claims 1 to 3, wherein the cells are cultured for more than about 58 days.

5. 10. The composition of claim 1, further comprising one or a combination of S100, myelin binding protein (MBP), and GFAP.

6. The cells SOX10, POU3F2, GAP43, or SOX10, POU3F2, and functional fragments thereof containing at least about 70% sequence identity to GAP43. The composition of any one of claims 1 to 5, further comprising one or a combination of:

7. The cells PMP22, SOX10, POU3F2, GAP43, NGFR, MP2, CD46, CD146, CD147, CD166, ERBB3, GDNF, or PMP22, SOX10, POU3F2, GAP43, NGFR, MP2, CD46, CD146, CD147, CD166, ERBB3, and functional fragments thereof comprising at least about 70% sequence identity to GDNF. The composition of any one of claims 1 to 6, further comprising one or a combination of:

8. The cells FOX01, TBX19, MATN2, PLAT, or FOX01, TBX19, MATN2 and functional fragments thereof containing at least about 70% sequence identity to PLAT.

8. The composition of claim 7, further comprising one or a combination of:

9. The cells PMP22, POU3F2, GAP43, NGFR, MP2, CD46, CD146, CD147, CD166, ERBB3, GDNF, CD9, CD49e, CD171, or PMP22, POU3F2, GAP43, NGFR, MP2, CD46, CD146, CD147, CD166, ERBB3, GDNF, CD9, CD49e, and functional fragments thereof comprising at least about 70% sequence identity to one of CD171. The composition of any one of claims 1 to 4, further comprising one or a combination of:

10. 1. A composition comprising one or more Schwann cells, said Schwann cells comprising: MPZ, MAG, PMPP22, PLLP, or Functional fragments thereof containing at least about 70% of the sequence of MPZ, MAG, PMPP22, or PLLP. A composition comprising:

11. 1. A composition comprising one or more Schwann cells, said Schwann cells comprising: POU6F2, CD44, CD81, or Functional fragments thereof containing at least about 70% of the sequence of MPZ, MAG, PMPP22, and PLLP. A composition comprising:

12. The composition of any one of claims 1 to 11, comprising greater than about 70% Schwann cells.

13. The composition of any one of claims 1 to 11, comprising greater than about 80% Schwann cells.

14. The composition of any one of claims 1 to 11, comprising greater than about 90% Schwann cells.

15. The composition of any one of claims 1 to 11, wherein the cells are derived from human pluripotent stem cells.

16. The composition of any one of claims 1 to 11, wherein the cells are cultured for at least about 2 weeks.

17. (i) a composition according to any one of claims 1 to 16, comprising a therapeutically effective amount of Schwann cells; (ii) a pharmaceutically acceptable carrier; and A pharmaceutical composition comprising:

18. A tissue culture system comprising the composition of any one of claims 1 to 16 and a tissue culture medium.

19. 20. The system of claim 18, wherein the system further comprises a solid substrate on which the cells are disposed.

20. A method for differentiating pluripotent stem cells into Schwann cells, comprising exposing neural crest cells to an effective amount of FGF2 for a time sufficient to differentiate the neural crest into Schwann cells.

21. A method for enriching Schwann cells in a cell culture, comprising exposing a composition of pluripotent stem cells to FGF2 for a period of time sufficient for the neural crest cells to express SOX10 or a functional fragment thereof.

22. 22. The method of claim 21, further comprising exposing the composition of neural crest cells to a WNT pathway activator for a time sufficient for the neural crest cells to express SOX10 or a functional fragment thereof.

23. The method of claims 21 and 22, further comprising exposing the composition of neural crest cells to SB431542 and / or dbcAMP for a time sufficient for the neural crest cells to express one or a combination of POU3F1, PMP22, MBP, MPZ, AQP4, or functional fragments thereof.

24. 24. The method of claim 23, wherein the step of exposing the composition of neural crest cells to SB431542 and / or dbcAMP comprises exposure for a time sufficient to differentiate the neural crest cells into Schwann cells.

25. 25. The method of any one of claims 21 to 24, wherein the one or more exposing steps are cumulatively carried out for more than about 19 days.

26. A method for culturing one or more Schwann cells, the method comprising exposing one or more neural crest cells to tissue culture medium comprising FGF2, SB431542 and / or dbcAMP, or derivatives or functional fragments thereof.

27. 27. The method of claim 26, wherein the one or more exposing steps are cumulatively carried out for more than about 19 days.

28. The method of claims 26 and 27, further comprising differentiating human pluripotent stem cells into neural crest cells prior to the exposing step.

29. 1. A method for screening one or more agents for neuromodulatory activity, comprising: i) culturing a composition according to any one of claims 1 to 17 in a tissue culture system containing one or more healthy or dysfunctional neuronal cells; ii) exposing said composition to one or more agents; iii) monitoring the composition for neuromodulatory activity; and iv) identifying the one or more agents as toxic to cells of the nervous system if the neuromodulatory activity inhibits, prevents or reduces the growth or health of the cells compared to the neuromodulatory activity of healthy nerve cells in the absence of the one or more agents; or identifying the one or more agents as inducing neuronal repair if the neuromodulatory activity improves or restores the function of the dysfunctional neuron compared to the neuromodulatory activity of the dysfunctional neuron in the absence of the one or more agents; A method comprising:

30. 20. A method of transplanting a Schwann cell population into a subject in need thereof by administering to the subject the pharmaceutical composition of claim 17.

31. A method of treating spinal cord injury in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of Schwann cells and a pharmaceutically effective carrier.

32. 32. The method of claim 31, wherein the composition is administered intravenously.

33. A method for treating diabetic peripheral neuropathy in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of Schwann cells and a pharmaceutically effective carrier.

34. 34. The method of claim 33, wherein the composition is administered intravenously.

35. 1. A method of treating diabetic peripheral neuropathy in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of bupropion or a derivative or salt thereof and a pharmaceutically effective carrier.

36. 36. The method of claim 35, wherein the subject is a human.

37. 36. The method of claim 35, wherein the pharmaceutical composition is administered via suppository, topical contact, oral, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intracranial, intranasal, or subcutaneous administration.

38. 38. The method of any one of claims 35 to 37, further comprising co-administering a therapeutically effective amount of a second active agent.

39. 1. A method of treating glucose-mediated SC toxicity in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of bupropion or a derivative or salt thereof and a pharmaceutically effective carrier.

40. 40. The method of claim 39, wherein the subject is a human.

41. 40. The method of claim 39, wherein the pharmaceutical composition is administered via suppository, topical contact, oral, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intracranial, intranasal, or subcutaneous administration.

Images