IPSC-derived astrocytes and methods for their use

Abstract

The present disclosure provides methods for generating glial-biased neural progenitor cells from induced pluripotent cells and further differentiating the neural progenitor cells into astrocytes. Further provided herein are methods for using astrocytes in screening assays, models that mimic the human brain, and cell therapy.

Description

[Technical Field]

[0001] Priority claims This application claims priority to U.S. Provisional Patent Application No. 63 / 356,787, filed June 29, 2022, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] 1. Field The present invention relates generally to the fields of molecular biology and medicine, and more particularly to a method for differentiating induced pluripotent stem cells to generate astrocytes.

[0003] 2. Description of Related Technology Astrocytes play a vital role in the central nervous system (CNS) by having a central role in brain development, maintaining brain homeostasis, providing metabolic support to neurons, regulating neural circuit connectivity, and controlling blood flow as an integral part of the blood-brain barrier. Astrocytes undergo transformation after injury or post-injury conditions, such as reactive astrogliosis. These reactive astrocytes are involved in the development and progression of many neurological disorders. Summary of the Invention [Means for solving the problem]

[0004] Certain embodiments of the present disclosure provide an in vitro method for generating astrocytes from induced pluripotent stem cells (iPSCs), the method comprising: (a) obtaining a starting population of neural progenitor cells (NPCs) derived from the iPSCs; (b) culturing the NPCs in the presence of at least one leukemia inhibitory factor (LIF) receptor ligand for a period sufficient to produce astrocyte precursor cells (APCs); and (c) further culturing the APCs in the presence of at least one LIF receptor ligand and a lipid concentrate for a period sufficient to produce a population of astrocytes.

[0005] In some embodiments, the iPSCs are cultured in serum-free defined medium. In certain embodiments, the method is compliant with Good Manufacturing Practice (GMP). In some embodiments, one or more of steps (a)-(c) are performed under xeno-free, feeder-free, and / or conditioned medium-free conditions. In certain embodiments, each of steps (a)-(c) is performed under xeno-free, feeder-free, and / or conditioned medium-free conditions. In some embodiments, each of steps (a)-(c) is performed under defined conditions. In some embodiments, the iPSCs are human iPSCs.

[0006] In some embodiments, obtaining a starting population of NPCs comprises: (a) culturing iPSCs on a surface coated with an extracellular matrix (ECM) protein in the presence of a ROCK inhibitor; (b) further culturing iPSCs in the absence of a ROCK inhibitor or blebbistatin; (c) pretreating iPSCs in the presence of a GSK3 inhibitor; and (d) differentiating the iPSCs into a population of NPCs. In some embodiments, the ECM protein is laminin, fibronectin, vitronectin, MATRIGEL™, tenascin, entactin, thrombospondin, elastin, gelatin, and / or collagen. In certain embodiments, the ECM protein is basement membrane extract (BME) purified from mouse EHS (Engelbreth-Holm-Swarm) tumor. In some embodiments, the ECM protein is MATRIGEL™, laminin, or vitronectin. In some embodiments, the extracellular matrix protein is MATRIGEL™. In certain embodiments, the method does not involve inhibition of SMAD signaling.

[0007] In certain embodiments, steps (a) and (b) are performed under hypoxic conditions. In some embodiments, the culture in steps (a) and (b) is further defined as an adherent two-dimensional culture. In some embodiments, step (a) is for about 24 hours. In certain embodiments, step (b) is for about 48 hours. In certain embodiments, the ROCK inhibitor is H1152. In some embodiments, step (c) is performed under normoxic conditions. In some embodiments, step (c) is performed for about 72 hours. In certain embodiments, the GSK3 inhibitor is CHIR99021, BIO, or SB-216763. In certain embodiments, the GSK3 inhibitor is CHIR99021.

[0008] In some embodiments, step (d) comprises forming aggregates in the presence of a ROCK inhibitor. In certain embodiments, the cell culture is a three-dimensional (3D) culture. In some embodiments, step (d) comprises culturing on an ultra-low attachment plate, spinner, or bioreactor. In some embodiments, step (d) is for about 8 days (e.g., 5, 6, 7, 8, 9, or 10 days).

[0009] In some embodiments, the NPCs express CD24, CD184, and CD271. In some embodiments, the method further comprises detecting expression of CD56, CD15, Sox1, nestin, β3-tubulin, microglobulin, and / or Pax-6 in the NPC population. In some embodiments, the NPC population is at least 70% (e.g., 75%, 80%, 85%, or 90%) positive for CD24 and nestin. In some embodiments, the NPCs express Pax6 and nestin. In certain embodiments, the APCs after step (b) have reduced expression of SSEA-4 and TRA-1-60 compared to iPSCs. In certain embodiments, the NPCs are cryopreserved.

[0010] In some embodiments, the iPSCs are derived from a healthy donor. In certain embodiments, the iPSCs are derived from a donor with a disease. In some embodiments, the disease is Alexander disease or leukodystrophy. In some embodiments, the iPSCs contain disruptions in TREM2, APOE, methyl-CpG binding protein 2 (MeCP2), and / or alpha-synuclein (SCNA). In certain embodiments, the astrocytes are terminal astrocytes positive for CD44, S100b, NFIX, GLAST, and / or GFAP. In some embodiments, the astrocytes are positive for SSEA4 and CD44. In some embodiments, at least 30% (e.g., 35%, 40%, 45%, or 50%) of the astrocyte population are positive for SSEA4 and CD44. In certain embodiments, the astrocytes exhibit functions in glutamate uptake and / or neural network development.

[0011] In certain embodiments, the at least one LIF receptor ligand is leukemia inhibitory factor protein (LIF), ciliary-derived neurotrophic factor protein (CNTF), oncostatin-M protein (OSM), and / or cardiotrophin 1 (CT-1). In some embodiments, step (b) further comprises culturing in the presence of a lipid concentrate, EGF, JAGG1, and / or DLL1. In certain embodiments, step (b) comprises culturing in the presence of LIF, CNTF, OSM, JAGG1, a lipid concentrate, and EGF. In some embodiments, step (b) comprises culturing in the presence of LIF, CNTF, OSM, DLL1, a lipid concentrate, and EGF. In certain embodiments, step (b) comprises culturing in the presence of LIF, CNTF, OSM, JAGG1, DLL1, a lipid concentrate, and EGF. In some embodiments, step (b) comprises culturing in the presence of LIF, CNTF, OSM, JAGG1, DLL1, a lipid concentrate, and EGF. In certain embodiments, step (b) comprises culturing in the presence of LIF, CNTF, OSM, DLL1, CT1, a lipid concentrate, and EGF.

[0012] In some embodiments, APCs are cultured in the presence of LIF, CNTF, oncostatin-M, and / or CT-1. In certain embodiments, APCs are cultured in the presence of LIF and CNTF. In some embodiments, LIF, CNTF, oncostatin-M, and / or CT-1 are present at a concentration of about 1-20 ng / mL (e.g., 1, 5, 10, 15, or 20 ng / mL). In certain embodiments, LIF, CNTF, oncostatin-M, and / or CT-1 are present at a concentration of about 10 ng / mL.

[0013] In some embodiments, step (b) comprises culturing NPCs on a Geltrex-coated surface. In certain embodiments, step (b) is for about 2 weeks (e.g., 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or 20 days).

[0014] In certain embodiments, step (c) comprises culturing the cells on a surface coated with vitronectin.

[0015] In some embodiments, the lipid concentrate is a lipid concentrate of known composition. In some embodiments, the lipid concentrate of known composition comprises saturated fatty acids and unsaturated fatty acids. In certain embodiments, the lipid concentrate of known composition comprises arachidonic acid, cholesterol, DL-α-tocopherol acetate, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid, and / or stearic acid.

[0016] In some embodiments, step (c) is performed for about 4 weeks to 7 weeks (e.g., 4 weeks, 5 weeks, 6 weeks, or 7 weeks). In some embodiments, the astrocytes express CD44, NFIX, and / or GFAP. In particular embodiments, the astrocytes express CD56, S100B, CD44, GFAP, NFIX, and / or GLAST. In some embodiments, the population of astrocytes is at least 80% (e.g., 80%, 85%, 90%, or 95%) positive for S100B, CD44, and / or NFIX. In some embodiments, the population of astrocytes is at least 30% (e.g., 30%, 35%, 40%, 45%, or 50%) positive for CD56 and / or GFAP. In particular embodiments, the astrocytes maintain network activity and take up excess glutamate. In some embodiments, astrocytes secrete IL-1ra, IL-6, IL-8 (CXCL8), IL-10, CCL5 (RANTES), CCL7, CCL20, CXCL1, CXCL2, and / or CXCL5 after stimulation with IL-1α and / or TNFα.

[0017] Further provided herein is a pharmaceutical composition comprising a population of astrocytes produced according to this embodiment or aspects thereof and a pharmaceutically acceptable carrier. In some embodiments, the population of astrocytes is at least 30%, e.g., 30%, 35%, 40%, 45%, or 50%, positive for SSEA4 and CD44. In certain embodiments, the population of astrocytes is at least 45% positive for SSEA4 and CD44.

[0018] Another embodiment provides a composition comprising a population of astrocytes that are at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, or 99%) positive for S100B, CD44, and / or NFIX, where the population of astrocytes is differentiated from iPSCs. In some embodiments, the population of astrocytes is at least 80% positive for S100B, CD44, and / or NFIX. In certain embodiments, the population of astrocytes is at least 30% positive for SSEA and CD44. In some embodiments, the population of astrocytes is at least 45% positive for SSEA and CD44. In some embodiments, the composition further comprises neurons.

[0019] Further embodiments provide methods of screening test compounds comprising introducing the test compound into a population of astrocytes of this embodiment or aspect thereof, hi some aspects, the method further comprises measuring astrocyte viability and / or function.

[0020] Another embodiment provides for the use of a composition of this embodiment or aspect thereof as a model for neurodegenerative disease or injury.

[0021] In yet another embodiment, there is provided a co-culture comprising astrocytes and / or neural progenitor cells, endothelial cells, and pericytes produced according to this embodiment or aspect thereof. Also provided herein is the use of the co-culture of this embodiment or aspect thereof to mimic human brain development or neurodegeneration.

[0022] Another embodiment provides a kit comprising astrocytes produced by the method of this embodiment or any aspect thereof. In some aspects, the kit further comprises endothelial cells and / or pericytes. Further provided herein is a model of neurodegeneration comprising the co-culture of this embodiment or any aspect thereof.

[0023] Also provided herein are methods for treating a neurodegenerative disease comprising administering to a subject an effective amount of an astrocyte composition of this embodiment or aspect thereof, hi some aspects, the disease is Alexander disease or leukodystrophy.

[0024] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0025] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief explanation of the drawings]

[0026] [Figure 1] Schematic diagram showing how neural progenitor cells (NPCs) are generated.

[0027] [Figures 2A-2D] Characterization of NPCs by flow cytometry. Cells were stained for both cell surface (Figures 2A and 2C) and intracellular antigens (Figures 2B-2D). Pluripotency markers SSEA-4 and TRA-1-60 significantly decreased during the 14-day differentiation process, whereas neural progenitor cell (NPC) markers such as CD24, CD184, and CD271 began to appear during differentiation and increased over time. NPCs from all five lines tested showed high expression of the neural progenitor markers Pax6 and nestin. Expression of the neural progenitor markers SOX1, doublecortin (DCX), and the pan-neuronal marker β3-tubulin (Tuj) was observed in donors 1279 and 1279 MeCP2, but low or absent expression was observed in lines 11995, 21527, 1434, and 1434 APOE.

[0028] [Figures 3A-3F]Activation of LIF receptors is sufficient to differentiate astrocytes from NPCs. (Figure 3A) A simplified schematic of the differentiation procedure for generating astrocytes from NPCs, showing the amount and length of time LIF receptor agonist was applied. (Figure 3B) Phase-contrast images of cells plated on a PLO / Laminin substrate at day 60 (top of B), as well as ICC staining for the astrocyte markers CD44, NFIX, and GFAP at days 35 and 80 after differentiation. At day 35 of the process, relatively few cells appeared to express CD44 and GFAP. However, by day 80, CD44 expression appeared completely uniform throughout the population, and GFAP expression dramatically increased throughout the population. (Figure 3C) Quantification of the percentage of cells positive for several astrocyte markers at day 70 using flow cytometry. High expression of the astrocyte and astrocyte progenitor markers S100B, NFIX, and CD44 demonstrates the ability of this process to generate highly pure astrocytes. The expression of GFAP and GLAST indicates that many of these cells express functional markers of mature astrocytes. Low expression of CD15 and SSEA4 indicates that pluripotent cells are no longer observed in the cultures. (Figure 3D) The theoretical cumulative yield during the differentiation process represents a significant cell expansion of over 10,000-fold, enhancing the cost-effectiveness of this protocol. (Figure 3E) Time course of glutamate uptake efficiency. Glutamate uptake was measured after 7 days of plating in 96-well plates with Matrigel. 20 μM glutamate was added for 60 min in the presence or absence of the glutamate transporter inhibitor DL-TBOA (TBOA). The measurements shown represent the percentage of glutamate concentration in the -TBOA sample relative to the corresponding +TBOA sample. (Figure 3F) Microelectrode assay (MEA) roster plot of different cultures before (baseline) and after glutamate application. 120,000 iCell GlutaNeurons were cultured alone or co-cultured with 20,000 iCell astrocytes or NPC astrocytes in BrainPhys medium for 23 days.Synchronized bursting activity was visible in all conditions, indicating the formation of mature synaptic networks. The number of action potentials observed in network bursts was greater in cocultures containing either iCell astrocytes or NPC astrocytes compared to iCell GlutaNeuron monocultures, indicating an effect on network formation induced by astrocyte coculture. Additional application of exogenous glutamate was sufficient to abolish all synchronized bursts in iCell GlutaNeuron monocultures, but not in coculture samples, indicating increased robustness of the cultures against outside perturbations.

[0029] [Figures 4A-4C] Matrix experiment of 31 media. (Figure 4A) Fold expansion analysis of 31 media tested to determine their respective potential to induce NPC proliferation over a 14-day differentiation time course. Media 5, 6, 7, 8, 13, 21, 22, 23, 30, and 31 showed a near-complete lack of cells at the end of day 14 and were excluded from further analysis. (Figure 4B) Purity analysis of astrocyte lineage-associated marker expression by flow cytometry on day 14. Samples were collected on day 14 of the 31 media test, except for the sample showing a complete loss of cells in Figure 4A. Samples from all media tested show a lack of the pluripotency marker SSEA4 and near-uniform expression of the astrocyte lineage marker NFIX. Neural progenitor markers varied little between samples, but co-expression of astrocyte progenitor markers was higher in media 10, 14, 16, 24, and 29. (Figure 4C) In a comparison of 31 media for further experiments, the compositions of the 11 media that performed best based on growth and purity were identified. The 11 media are media 2, 3, 4, 10, 12, 14, 16, 18, 24, 26, and 29.

[0030] [Figures 5A-5E]Eleven media and five media matrix experiments. (Figure 5A) Flow cytometry analysis of the eleven media determined from Figure 4 to have the best combination of proliferation and astrocyte progenitor marker expression at D35 of the process. Nearly uniform expression of the astrocyte lineage marker NFIX (>90% of cells express NFIX in all media conditions except media 18) can be observed in all conditions, as well as a lack of the pluripotency marker SSEA4 (<10% SSEA4 expression in all media conditions except media 14, 16, 24, and 29, where expression was less than 20%) and the NPC marker CD15 (CD15 expression was less than 5% in all conditions). Certain conditions clearly upregulate the expression of the astrocyte markers CD44, S100B, and GLAST (media conditions 14, 16, 24, and 29 show >75% CD44 / S100B co-expression, while media 14, 16, and 29 show >60% GLAT expression). These conditions, using media 14, 16, 24, and 29, are the most effective for identifying the astrocyte lineage from NPC cells. (Figure 5B) Cumulative fold expansion analysis of 11 media matrices over 35 days of differentiation. Media 14, 16, 24, and 29 demonstrate significantly higher proliferation by D35 than the other media conditions, with over 25-fold expansion over this period. This, combined with the higher expression of astrocyte markers from Figure 5A, indicates that these media are clearly optimal for inducing robust astrocyte differentiation from NPC cells. (Figure 5C) Immunocytochemistry of D49 cells from a five-medium matrix experiment. Astrocyte progenitor and astrocyte markers NFIX and CD44 were significantly increased in cells differentiated using media 14, 16, 24, and 29 compared to the control medium 3. Expression of the mature astrocyte marker GFAP was robustly observed in cells differentiated using media 14, 16, 24, and 29, whereas GFAP was barely observed in cells differentiated using medium 3. (Figure 5D) Glutamate uptake assay of D49 cells from a five-medium comparison study. Glutamate uptake is a commonly accepted indicator of astrocyte function in vitro.The glutamate transporter inhibitor TBOA was used as a control to confirm that all removal of glutamate from the medium was due to the presence and activity of these astrocyte-specific transporters. Cells differentiated using media 14, 16, 24, and 29 were able to demonstrate a decrease in measured glutamate in the medium after 1 hour of incubation. Cells differentiated using medium 3 were unable to significantly uptake glutamate. (Figure 5E) A summary of the five media that performed best in the comparison of 11 media is provided for further study.

[0031] [Figures 6A-6J] Reemergence of SSEA4+ as an astrocyte progenitor marker. (Figure 6A) Representative flow cytometry plots of surface CD56 / SSEA4 co-staining at D28 and intracellular CD44 / SSEA4 co-staining at both D28 and D35 from medium 14. These plots demonstrate the emergence of cells co-expressing the astrocyte marker CD44 and SSEA4, a classic marker of pluripotent cells. This combination of markers on astrocytes has not been previously demonstrated. (Figure 6B) Time course of surface SSEA4 expression and intracellular CD44 expression from D7 to D35 in medium 3, (Figure 6C) condition 14, (Figure 6D) condition 16, (Figure 6E) condition 24, and (Figure 6F) condition 29. The emergence of a CD44 and SSEA4 co-positive population can be observed after D35 in astrocytes differentiated in medium conditions 14, 16, and 24, where expression increased from less than 20% by D35 to over 90% by day 42. This occurrence was not observed in control cells differentiated with condition 29 or medium 3. Medium conditions 14, 16, and 24 were continued until D42. Differential expression of SSEA4 and CD44 by surface and intracellular staining at D28 and D35. Summary of the expression profiles of (Figure 6G) condition 14, (Figure 6H) condition 16, (Figure 6I) condition 24, and (Figure 6J) condition 29.

[0032] [Figure 7A-7C]Post-thaw optimization and functional assays. (Figure 7A) Immunocytochemistry of astrocytes cultured in either Astro3 medium, AMM medium, or BrainPhys Complete medium 7 days after thawing. All media used after thawing were equally capable of supporting the expression of the astrocyte marker genes GFAP, CD44, and NFIX. (Figure 7B) Glutamate uptake assay of astrocytes cultured in either Astro3 medium, AMM medium, or BrainPhys Complete medium 7 days after thawing. All media were equally capable of supporting astrocyte function, and astrocytes cultured in all media demonstrated robust glutamate uptake function. (Figure 7C) Quantified results from MEA data analysis. iCell GlutaNeurons were cultured alone or in co-culture with NPC astrocytes. Increased network burst frequency was observed in neurons co-cultured with NPC astrocytes (Gluta+C1).

[0033] [Figures 8A-8J]Four lots of astrocytes were thawed and cultured in Astro3 medium for 7 days, followed by stimulation with basal medium for 24 hours. Supernatants were collected and analyzed using an R&D custom Luminex assay on a FLEXMAP 3D instrument according to the manufacturer's instructions. Each condition was performed in duplicate, and fold changes were calculated by comparing the average of all four lots with the control (unstimulated) condition. Error bars are + / - 1 SEM. (Figure 8A) Astrocytes were able to robustly secrete IL-1ra after stimulation with IL-1α, IFN-γ, TNF-α, or their combinations. Stimulation with IL-1α or TNF-α induced IL-1ra secretion more than threefold. TNF-α combined with IL-1α or IL-1β increased IL-1ra secretion approximately 14-fold over unstimulated controls. The combination of IFN-γ and TNF-α resulted in the highest fold change over controls, increasing IL-1ra secretion more than 84-fold compared to controls. IL-1ra binds to the cell surface IL-1 receptor and blocks the binding of proinflammatory IL-1α and IL-1β. (Figure 8B) Astrocytes were able to robustly secrete IL-6 after stimulation with IL-1α, TNF-α, or their combination. IFN-γ alone or in combination with TNF-α increased IL-6 secretion 32-fold and 39-fold, respectively, compared to unstimulated controls. IL-α stimulation increased IL-6 secretion by more than 400-fold compared to controls. TNF-α combined with IL-1α or IL-1β increased IL-6 secretion by more than 3,300-fold compared to controls. IL-6 attracts proinflammatory T cells and promotes demyelination. (Figure 8C) Astrocytes were able to robustly secrete IL-8 / CXCL8 after stimulation with IL-1α, TNF-α, or their combination. TNF-α alone or in combination with IFN-γ increased IL-8 secretion 898-fold and 665-fold, respectively, over unstimulated controls. IL-α stimulation increased IL-8 secretion more than 3,300-fold compared to controls. TNF-α combined with IL-1α or IL-1β increased IL-8 secretion more than 12,000-fold compared to controls. IL-8 / CXCL8 is secreted by astrocytes and attracts pro-inflammatory immune cells after injury.This analyte also plays a role in the recruitment and differentiation of oligodendrocyte progenitor cells (OPCs) and promotes remyelination. (Figure 8D) Astrocytes were able to robustly secrete IL-10 after stimulation with IL-1α, IFN-γ, TNF-α, or their combinations. IFN-γ stimulation increased IL-10 secretion 161-fold compared to unstimulated controls. IL-α stimulation increased IL-10 secretion by more than 415-fold compared to controls. TNF-α alone or TNF-α combined with IL-1α, IL-1β, or IFN-γ increased IL-10 secretion by more than 1000-fold compared to controls. IL-10 is secreted by astrocytes, reduces iNos activity, and alleviates astrogliosis. (Figure 8E) Astrocytes were able to robustly secrete CCL5 / RANTES after stimulation with IL-1α, TNF-α, or their combinations. IL-1α stimulation increased CCL5 / RANTES secretion 156-fold compared to unstimulated controls. TNF-α alone or in combination with IL-1α, IL-1β, or IFN-γ increased CCL5 / RANTES secretion by more than 2500-fold compared to controls. CCL5 / RANTES regulates peripheral immune cell activity (Figure 8F). Astrocytes were able to robustly secrete CCL7 after stimulation with IL-1α, TNF-α, or their combination. TNF-α stimulation increased CCL7 secretion 1184-fold compared to controls. IL-1α stimulation increased CCL7 secretion 2177-fold compared to unstimulated controls. TNF-α-stimulated IFN-γ increased CCL7 secretion 3745-fold compared to unstimulated controls. TNF-α combined with IL-1α or IL-1β increased CCL7 secretion by more than 15,000-fold compared to controls. CCL7 is important for astrocyte-microglia interactions and induces microglial activation. (G) Astrocytes were able to robustly secrete CCL20 after stimulation with IL-1α, TNF-α, or their combination. Stimulation with IL-1α or TNF-α alone increased CCL20 secretion by more than 18-fold compared to unstimulated controls.TNF-α combined with IL-1α, IL-1β, or IFN-γ increased CCL20 secretion by more than 100-fold compared to controls. CCL20 is secreted by astrocytes in response to proinflammatory stimuli and attracts T cells, B cells, and DCs (Figure 8H). Astrocytes were able to robustly secrete CXCL1 after stimulation with IL-1α, TNF-α, or their combinations. TNF-α alone or combined with IFN-γ increased CXCL1 secretion by more than 9-fold compared to controls. Stimulation with IL-1α increased CXCL1 secretion by 84-fold compared to unstimulated controls. TNF-α combined with IL-1α or IL-1β increased CXCL1 secretion by more than 450-fold compared to controls. CXCL1 is secreted by astrocytes and recruits neutrophils to sites of infection and increases BBB permeability. (Figure 8I) Astrocytes were able to robustly secrete CXCL2 after stimulation with IL-1α, TNF-α, or their combination. TNF-α alone or in combination with IFN-γ increased CXCL2 secretion by more than 51-fold compared to controls. Stimulation with IL-1α increased CXCL2 secretion by 379-fold compared to unstimulated controls. TNF-α in combination with IL-1α or IL-1β increased CXCL2 secretion by more than 1585-fold compared to controls. CXCL2 activates CXCR2, which is found on oligodendrocytes or OPCs, thereby promoting OPC proliferation and differentiation. (Figure 8J) Astrocytes were able to robustly secrete CXCL5 after stimulation with IL-1α, TNF-α, or their combination. TNF-α alone or in combination with IFN-γ increased CXCL5 secretion by more than 47-fold compared to controls. Stimulation with IL-1α increased CXCL5 secretion 161-fold over unstimulated controls. TNF-α combined with IL-1α or IL-1β increased CXCL5 secretion more than 872-fold compared to controls. CXCL5 is secreted by astrocytes in response to injury, activating microglia and reducing microglial phagocytosis and inhibition. DETAILED DESCRIPTION OF THE INVENTION

[0034] Description of exemplary embodiments In certain embodiments, the present disclosure provides methods for generating neural progenitor cells (NPCs) with a glial bias, also referred to herein as iPSC-derived glial progenitor cells, for the generation of astrocytes. In certain aspects, the methods are defined, serum-free methods for generating astrocytes, such as for preclinical and / or clinical applications.

[0035] In this study, glial-biased NPCs were generated from multiple iPSC lines and placed in astrocyte differentiation medium with a defined composition to efficiently generate end-stage astrocytes. End-stage astrocytes expressed key astrocyte markers, such as CD44, NFIX, and GFAP, exhibited glutamate uptake, and promoted neuronal network development. During this process, marker analysis identified re-expression of SSEA4 (a pluripotent stem cell marker) co-expressed with the astrocyte progenitor marker CD44. This study further demonstrated successful cryopreservation of end-stage astrocytes and their use in multiple cell assay-specific, preclinical, or clinical applications before and after cryopreservation. Further embodiments provide methods for identifying drugs that modulate astrocyte viability or function, as well as a triple culture kit for mimicking neurodegeneration. Astrocytes generated by this method can also be used in cell therapy applications, including therapeutic applications for treating neurological and brain-related diseases or conditions. Astrocytes generated by this method can be used in disease modeling, drug discovery, and / or regenerative medicine.

[0036] I. Definition As used herein, "a" or "an" may mean one or more. As used herein in the claims, when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more.

[0037] Use of the term "or" in the claims is used to mean "and / or," unless expressly indicated to refer to alternatives only or where the alternatives are mutually exclusive, but the present disclosure supports a definition that refers to alternatives only and "and / or." As used herein, "another" can mean at least a second or more.

[0038] The term "essentially" should be understood to mean that the method or composition includes only certain steps or materials that do not materially affect the basic and novel characteristics of those methods and compositions.

[0039] As used herein, a composition or medium that is "substantially free" of a particular substance or material contains 30% or less, 20% or less, 15% or less, more preferably 10% or less, even more preferably 5% or less, or most preferably 1% or less of the substance or material.

[0040] As used herein, the terms "substantially" or "approximately" may be applied to modify quantitative comparisons, values, measurements, or other expressions that may vary to an acceptable degree without resulting in a change in the basic function to which they relate.

[0041] The term "about" generally means within the standard deviation of the stated value, as determined using standard analytical techniques to measure the stated value. The term may also be used to refer to plus or minus 5% of the stated value.

[0042] As used herein, "essentially free" with respect to a particular component is used herein to mean that none of the particular component is intentionally incorporated into the composition and / or is present only as a contaminant or in trace amounts. Thus, the total amount of the particular component resulting from any unintentional contamination of the composition is well below 0.05%, preferably below 0.01%. Most preferred are compositions in which the amount of the particular component is undetectable using standard analytical methods.

[0043] "Feeder-free" or "feeder-independent" is used herein to refer to cultures supplemented with cytokines and growth factors (e.g., TGFβ, bFGF, LIF) in lieu of a feeder cell layer. Thus, "feeder-free" or feeder-independent culture systems and media can be used to culture and maintain pluripotent cells in an undifferentiated and proliferative state. In some cases, feeder-free cultures utilize an animal-based matrix (e.g., MATRIGEL™) or are cultured on substrates such as fibronectin, collagen, or vitronectin. These approaches allow human stem cells to be maintained in an essentially undifferentiated state without the need for a "feeder layer" of mouse fibroblasts.

[0044] A "feeder layer" is defined herein as a coating layer of cells, such as the bottom of a culture dish. Feeder cells can release nutrients into the medium and provide a surface to which other cells, such as pluripotent stem cells, can attach.

[0045] The terms "defined" or "completely defined," when used in reference to a medium, extracellular matrix, or culture condition, refer to a medium, extracellular matrix, or culture condition in which the chemical composition and amount of nearly all components are known. For example, a defined medium does not contain undefined factors such as fetal bovine serum, bovine serum albumin, or human serum albumin. Typically, a defined medium contains a basal medium (e.g., Dulbecco's Modified Eagle's Medium (DMEM), F12, or Roswell Park Memorial Institute Medium (RPMI) 1640, containing amino acids, vitamins, inorganic salts, buffers, antioxidants, and an energy source) supplemented with recombinant albumin, lipids of known composition, and recombinant insulin. An example of a completely defined medium is Essential 8™ medium.

[0046] For media, extracellular matrices, or culture systems used with human cells, the term "xeno-free (XF)" refers to the condition in which the materials used are not derived from non-human animals.

[0047] "Treatment" or "treating" includes (1) inhibiting the disease in a subject or patient experiencing or exhibiting the symptoms or symptomology of the disease (e.g., arresting further development of the symptoms and / or symptomology), (2) ameliorating the disease in a subject or patient experiencing or exhibiting the symptoms or symptomology of the disease (e.g., reversing the symptoms and / or symptomology), and / or (3) causing a measurable reduction in the disease in a subject or patient experiencing or exhibiting the symptoms or symptomology of the disease.

[0048] "Prophylactic treatment" includes (1) reducing or alleviating the risk of developing a disease in a subject or patient who may be at risk and / or predisposed to the disease, but who has not yet experienced or displayed any or all of the symptoms or symptomology of the disease, and / or (2) delaying the onset of symptoms or symptomology of the disease in a subject or patient who may be at risk and / or predisposed to the disease, but who has not yet experienced or displayed any or all of the symptoms or symptomology of the disease.

[0049] As used herein, the term "patient" or "subject" refers to a living mammal, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human patients are adults, juveniles, infants, and fetuses.

[0050] The term "effective," as that term is used in the specification and / or claims, means adequate to achieve a desired, expected, or intended result. An "effective amount," "therapeutically effective amount," or "pharmaceutically effective amount," when used in connection with treating a patient or subject with a compound, means the amount of a compound that, when administered to a subject or patient for treating or preventing a disease, is sufficient to affect the treatment or prevention of such disease.

[0051] Generally, as used herein, "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs and / or body fluids of human beings and animals without undue toxicity, irritation, allergic response or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0052] "Induced pluripotent stem cells (iPSCs)" are cells generated by reprogramming somatic cells through the expression or induction of expression of a combination of factors (referred to herein as reprogramming factors). iPSCs can be generated using fetal, postnatal, neonatal, juvenile, or adult somatic cells. In certain embodiments, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, Oct4 (sometimes referred to as Oct 3 / 4), Sox2, c-Myc, Klf4, Nanog, and Lin28. In some embodiments, somatic cells are reprogrammed by expressing at least two reprogramming factors, at least three reprogramming factors, or four reprogramming factors to reprogram the somatic cells into pluripotent stem cells.

[0053] The term "extracellular matrix protein" refers to molecules that provide structural and biochemical support to surrounding cells. Extracellular matrix proteins can be recombinant and also refer to fragments or peptides thereof. Examples include collagen and heparin sulfate.

[0054] "Three-dimensional (3D) culture" refers to an artificially created environment in which living cells can grow or interact with their surroundings in all three dimensions. 3D cultures can be grown in a variety of cell culture vessels, such as bioreactors, small capsules in which cells can grow into spheroids, or non-adherent culture plates. In certain embodiments, the 3D culture is scaffold-free. In contrast, "two-dimensional (2-D)" culture refers to cell culture, such as a monolayer on an adhesive surface.

[0055] As used herein, "disruption" of a gene refers to the elimination or reduction of expression of one or more gene products encoded by a gene of interest in a cell compared to the expression level of the gene product in the absence of the disruption. Exemplary gene products include mRNA and protein products encoded by the gene. The disruption may be temporary or reversible, or permanent. Despite the fact that truncated or non-functional products may be produced, in some cases, the disruption is of a functional or full-length protein or mRNA. In some embodiments herein, the activity or function of a gene is disrupted, as opposed to expression. Gene disruption is generally induced by artificial methods, i.e., by the addition or introduction of a compound, molecule, complex, or composition, and / or by disruption of the nucleic acid of the gene or nucleic acid associated with the gene, such as at the DNA level. Exemplary methods of gene disruption include gene disruption techniques such as gene silencing, knockdown, knockout, and / or gene editing. Examples include antisense technologies such as RNAi, siRNA, shRNA, and / or ribozymes, which generally result in gene editing techniques that result in transient reduction of expression and inactivation or disruption of targeted genes, for example, by inducing cleavage and / or homologous recombination. Examples include insertion, mutation, and deletion. Disruption typically results in the suppression and / or complete absence of expression of the normal or "wild-type" product encoded by the gene. Examples of such gene disruptions include insertions, frameshift and missense mutations, deletions, knock-ins, and knock-outs of genes or portions of genes, including deletion of the entire gene. Such disruptions can occur in coding regions, such as one or more exons, resulting in the inability to produce a full-length product, a functional product, or any product, such as by inserting a stop codon. Such disruptions can also occur by disrupting promoters or enhancers or other regions that affect transcriptional activation to prevent gene transcription. Gene disruption includes gene targeting, which involves inactivating a targeted gene by homologous recombination.

[0056] II. Induced pluripotent stem cells In some embodiments, the method relates to differentiation of iPSCs. Induction of pluripotency was first achieved using mouse cells in 2006 (Yamanaka et al. 2006) and human cells in 2007 (Yu et al. 2007; Takahashi et al. 2007) by reprogramming somatic cells with the introduction of pluripotency-associated transcription factors. Pluripotent stem cells can be maintained in an undifferentiated state and differentiated into any mature cell type.

[0057] All somatic cells, with the exception of germ cells, can be used as the starting point for iPSCs. For example, cell types can be keratinocytes, fibroblasts, hematopoietic cells, mesenchymal cells, hepatocytes, or gastric cells. T cells can also be used as a source of somatic cells for reprogramming (U.S. Patent No. 8,741,648). There are no limitations on the degree of differentiation of the cells or the age of the animal from which the cells were collected; undifferentiated progenitor cells (including somatic stem cells) and even terminally differentiated mature cells can be used as a source of somatic cells in the methods disclosed herein. iPSCs can be grown under conditions known to differentiate human ES cells into specific cell types and can express human ES cell markers, including SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81.

[0058] A.HLA compatibility The major histocompatibility complex (MHC) is the primary cause of immune rejection of allogeneic organ transplants. There are three major class I MHC haplotypes (A, B, C) and three major MHC class II haplotypes (DR, DP, DQ).

[0059] The MHC compatibility between the donor and the recipient is significantly increased when the donor cells are HLA homozygous, i.e., contain identical alleles for each antigen-presenting protein. Most individuals are heterozygous for MHC class I and II genes, but certain individuals are homozygous for these genes. These homozygous individuals can serve as super donors, and grafts made from their cells can be transplanted into all individuals who are homozygous or heterozygous for that haplotype. Furthermore, if the homozygous donor cells have a haplotype that is frequently found in the population, these cells can be applied to transplantation therapy for a large number of individuals.

[0060] Thus, iPSCs can be generated from the somatic cells of the subject to be treated or from another subject with the same or substantially the same HLA type as the patient. In some cases, the donor's major HLA (e.g., the three major loci: HLA-A, HLA-B, and HLA-DR) is identical to the recipient's major HLA. In some cases, the somatic cell donor can be a super donor; thus, iPSCs derived from an MHC homozygous super donor can be used to generate differentiated cells. Thus, iPSCs derived from a super donor can be transplanted into a subject who is either homozygous or heterozygous for that haplotype. For example, iPSCs can be homozygous for two HLA alleles, such as HLA-A and HLA-B. Thus, iPSCs generated from a super donor can be used in the methods disclosed herein to generate differentiated cells that can potentially "match" a large number of potential recipients.

[0061] B. Reprogramming Factors Somatic cells can be reprogrammed to generate induced pluripotent stem cells (iPSCs) by using methods known to those skilled in the art.Those skilled in the art can easily generate induced pluripotent stem cells: for example, see US Patent Application Publication No. 20090246875, US Patent Application Publication No. 2010 / 0210014; US Patent Application Publication No. 20120276636; US Patent No. 8,058,065; US Patent No. 8,129,187; US Patent No. 8,278,620; PCT Publication No. WO 2007 / 069666A1 and US Patent No. 8,268,620, which are incorporated herein by reference.Generally, nuclear reprogramming factors are used to generate pluripotent stem cells from somatic cells. In some embodiments, at least two, at least three, or at least four of Klf4, c-Myc, Oct3 / 4, Sox2, Nanog, and Lin28 are utilized. In other embodiments, Oct3 / 4, Sox2, c-Myc, and Klf4 are utilized. In some aspects, five, six, seven, or eight reprogramming factors are used.

[0062] Cells are generally treated with nuclear reprogramming agents, which are one or more factors capable of inducing iPSCs from somatic cells, or nucleic acids encoding these agents (including those incorporated into vectors). Nuclear reprogramming agents generally include at least Oct3 / 4, Klf4, Sox2, or nucleic acids encoding these molecules. Additional nuclear reprogramming agents can be used, including p53, L-myc, or nucleic acids encoding L-myc, and Lin28 or Lin28b, or functional inhibitors of nucleic acids encoding Lin28 or Lin28b. Nanog can also be used for nuclear reprogramming.As disclosed in U.S. Patent Application Publication No. 20120196360, exemplary reprogramming factors for generating iPSCs include: (1) Oct3 / 4, Klf4, Sox2, L-Myc (Sox2 can be replaced with Sox1, Sox3, Sox15, Sox17, or Sox18; Klf4 can be replaced with Klf1, Klf2, or Klf5); (2) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, SV40 large T antigen (SV40LT); (3) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, human papillomavirus (HPV) 16 E6; (4) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, HPV16 E7. (5)Oct3 / 4, Klf4, Sox2, L-Myc, TERT, HPV16 E6, HPV16 E7;(6)Oct3 / 4, Klf4, Sox2, L-Myc, TERT, Bmil;(7)Oct3 / 4, Klf4, Sox2, L-Myc, Lin28;(8)Oct3 / 4 , Klf4, Sox2, L-Myc, Lin28, SV40LT;(9)Oct3 / 4, Klf4, Sox2, L-Myc, Lin28, TERT, SV40LT;(10)Oc t3 / 4, Klf4, Sox2, L-Myc, SV40LT; (11) Oct3 / 4, Esrrb, Sox2, L-Myc (Esrrb can be replaced by Esrrg); (12) Oct3 / 4, Klf4, Sox2; (13) Oct3 / 4, Klf4, Sox2, TERT, SV40LT; (14) Oct3 / 4, Klf4, Sox2, TERT, HP (15) Oct3 / 4, Klf4, Sox2, TERT, HPV16 E7; (16) Oct3 / 4, Klf4, Sox2, TERT, HPV16 E6, HPV16 E7; (17) Oct3 / 4, Klf4, Sox2, TERT, Bmil; (18) Oct3 / 4, Klf4, Sox2, Lin28 (19) Oct3 / 4, Klf4, Sox2, Lin28, SV40LT; (20) Oct3 / 4, Klf4, Sox2, Lin28, TERT, SV40LT; (21) Oct3 / 4, Klf4, Sox2, SV40LT; or (22) Oct3 / 4, Esrrb, Sox2 (Esrrb can be replaced with Esrrg).In one non-limiting example, Oct3 / 4, Klf4, Sox2, and c-Myc are utilized. In other embodiments, Oct4, Nanog, and Sox2 are utilized; see, for example, U.S. Patent No. 7,682,828, incorporated herein by reference. These factors include, but are not limited to, Oct3 / 4, Klf4, and Sox2. In other examples, factors include, but are not limited to, Oct3 / 4, Klf4, and Myc. In some non-limiting examples, Oct3 / 4, Klf4, c-Myc, and Sox2 are utilized. In other non-limiting examples, Oct3 / 4, Klf4, Sox2, and Sal4 are utilized. Factors such as Nanog, Lin28, Klf4, or c-Myc can improve reprogramming efficiency and can be expressed from several different expression vectors. For example, integrating vectors such as the EBV element-based system can be used (U.S. Patent No. 8,546,140). In further embodiments, protein reprogramming can be directly introduced into somatic cells by protein transduction. Reprogramming can further include contacting the cells with one or more signaling receptors, including glycogen synthase kinase 3 (GSK-3) inhibitors, mitogen-activated protein kinase kinase (MEK) inhibitors, transforming growth factor beta (TGF-β) receptor inhibitors or signal transduction inhibitors, leukemia inhibitory factor (LIF), p53 inhibitors, NF-κB inhibitors, or combinations thereof. These modulators can include small molecules, inhibitory nucleotides, expression cassettes, or protein factors. It is expected that virtually any iPS cell or cell line can be used.

[0063] The mouse and human cDNA sequences for these nuclear reprogramming agents are available by reference to the NCBI accession numbers set forth in WO 2007 / 069666, which is incorporated herein by reference. Methods for introducing one or more reprogramming agents or nucleic acids encoding these reprogramming agents are known in the art and are disclosed, for example, in U.S. Patent Application Publication No. 2012 / 0196360 and U.S. Patent No. 8,071,369, both of which are incorporated herein by reference.

[0064] Once obtained, iPSCs can be cultured in a medium sufficient to maintain pluripotency. iPSCs can be used with a variety of media and techniques developed for culturing pluripotent stem cells, more specifically embryonic stem cells, as described in U.S. Pat. No. 7,442,548 and U.S. Patent Application Publication No. 2003 / 0211603. For mouse cells, culture is performed by adding leukemia inhibitory factor (LIF) as a differentiation inhibitor to standard culture medium. For human cells, it is desirable to add basic fibroblast growth factor (bFGF) instead of LIF. Other methods for culturing and maintaining iPSCs that would be known to those skilled in the art can be used.

[0065] In certain embodiments, non-limiting conditions can be used; for example, pluripotent cells can be cultured with fibroblast feeder cells or in medium exposed to fibroblast feeder cells to maintain the stem cells in an undifferentiated state. In some embodiments, cells are cultured in the presence of mouse embryonic fibroblasts, which have been treated with radiation or antibiotics to arrest cell division, as feeder cells. Alternatively, pluripotent cells can be cultured and maintained in a substantially undifferentiated state using a defined feeder-independent culture system, such as TESR™ medium (Ludwig et al., 2006a; Ludwig et al., 2006b) or E8™ medium (Chen et al., 2011).

[0066] C. Plasmid In some embodiments, iPSCs can be engineered to express an exogenous nucleic acid, such as a nucleic acid sequence encoding a first marker and an enhancer operably linked to a promoter. This construct may also include other elements, such as a ribosome binding site (internal ribosome binding sequence) for translation initiation and a transcription / translation terminator. It is generally advantageous to transfect cells with the construct. Suitable vectors for stable transfection include, but are not limited to, retroviral vectors, lentiviral vectors, and Sendai virus.

[0067] In some embodiments, the marker-encoding plasmid is composed of: (1) a high-copy-number origin of replication, (2) a selectable marker, such as, but not limited to, the neo gene for antibiotic selection with kanamycin, (3) a transcription termination sequence containing a tyrosinase enhancer, and (4) a multiple cloning site for incorporating various nucleic acid cassettes; and (5) a nucleic acid sequence encoding the marker operably linked to the tyrosinase promoter. Numerous plasmid vectors are known in the art for inducing protein-encoding nucleic acids. These include, but are not limited to, the vectors disclosed in U.S. Pat. Nos. 6,103,470; 7,598,364; 7,989,425; and 6,416,998, which are incorporated herein by reference. In some embodiments, the plasmid contains a "suicide gene" that, upon administration of a prodrug, transfers the gene product to a compound that kills the host cell. Examples of suicide gene / prodrug combinations that can be used are truncated EGFR and cetuximab; herpes simplex virus-thymidine kinase (HSV-tk) and ganciclovir, acyclovir, or FIAU; oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinase thymidylate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside.

[0068] Viral gene delivery systems can be RNA-based or DNA-based viral vectors. Episomal gene delivery systems can be plasmids, Epstein-Barr virus (EBV)-based episomal vectors, yeast-based vectors, adenovirus-based vectors, Simian virus 40 (SV40)-based episomal vectors, bovine papillomavirus (BPV)-based vectors, or lentiviral vectors.

[0069] Markers include, but are not limited to, fluorescent proteins (e.g., green fluorescent protein or red fluorescent protein), enzymes (e.g., horseradish peroxidase or alkaline phosphatase or firefly / renilla luciferase or nanoluc), or other proteins. Markers can be proteins (including secreted proteins, cell surface proteins, or internal proteins; proteins synthesized or taken up by the cell); nucleic acids (such as mRNA or enzymatically active nucleic acid molecules), or polysaccharides. This includes determinants of such cellular components that are detectable by antibodies, lectins, probes, or nucleic acid amplification reactions specific for the marker of the cell type of interest. Markers can also be identified by biochemical or enzymatic assays, or biological responses that depend on the function of the gene product. Nucleic acid sequences encoding these markers can be operably linked to a tyrosinase enhancer. In addition, other genes, such as genes that can affect stem cell differentiation, cell function, physiology, or pathology, can be included.

[0070] D. Delivery Systems Introduction of nucleic acids, such as DNA or RNA, into the engineered cell lines of the present disclosure can use any suitable method for nucleic acid delivery for cell transformation, as described herein or as would be known to one of skill in the art. Such methods include, but are not limited to, ex vivo transfection (Wilson et al., 1989; Nabel et al., 1989), injection (U.S. Patent Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466, and 5,580,859, each of which is incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S. Patent No. 5,789,215, incorporated herein by reference); electroporation (U.S. Patent No. 5,384,253, incorporated herein by reference; Tur-Kaspa et al., 1989; al., 1986; Potter et al., 1984); calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); the use of DEAE-dextran followed by polyethylene glycol (Gopal, 1985); direct sonication (Fechheimer et al., 1987); liposome-mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991) and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988); particle bombardment (WO 94 / 09699 and WO 95 / 06128, each of which is incorporated herein by reference; U.S. Pat. Nos. 5,610,042; 5,322,783; 5,563,055; 5,550,318; 5,538,877; and 5,538,880); agitation with silicon carbide fibers (Kaeppler et al. al., 1990; U.S. Pat. Nos. 5,302,523 and 5,464,765, each of which is incorporated herein by reference; Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,591,616 and 5,563,055, each of which is incorporated herein by reference); desiccation / inhibition-mediated DNA uptake (Potrykus et al., 1985), and direct delivery of DNA by any combination of such methods. By applying techniques such as these, organelles, cells, tissues, or organisms can be stably or transiently transformed.

[0071] 1. Viral Vectors Viral vectors can be provided in certain embodiments of the present disclosure. In creating recombinant viral vectors, non-essential genes are typically replaced with genes or coding sequences for heterologous (or non-native) proteins. Viral vectors are a type of expression construct that utilizes viral sequences to introduce nucleic acids and, in some cases, proteins into cells. Certain viruses are attractive candidates for introducing foreign nucleic acids into cells (e.g., mammalian cells) due to their ability to infect or enter cells via receptor-mediated endocytosis, integrate into the host cell genome, and stably and efficiently express viral genes. Non-limiting examples of viral vectors that can be used to deliver nucleic acids in certain embodiments of the present disclosure are described below.

[0072] Retroviruses are promising gene delivery vectors due to their ability to integrate their genes into the host genome, introduce large amounts of foreign genetic material, infect a wide range of species and cell types, and be packaged in specialized cell lines (Miller, 1992).

[0073] To construct retroviral vectors, nucleic acids are inserted into the viral genome in place of specific viral sequences, generating viruses that are replication-defective. To produce virions, packaging cell lines containing the gag, pol, and env genes but lacking the long-term repeat (LTR) and packaging components are constructed (Mann et al., 1983). When a recombinant plasmid containing a cDNA, along with retroviral long-term repeat (LTR) and packaging sequences, is introduced into specialized cell lines (e.g., by calcium phosphate precipitation), the packaging sequences enable the RNA transcripts of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture medium (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The medium containing the recombinant retrovirus is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors can infect a wide range of cell types. However, integration and stable expression require host cell division (Paskind et al., 1975).

[0074] Lentiviruses are complex retroviruses that contain, in addition to the common retroviral genes gag, pol, and env, other genes with regulatory or structural functions. Lentiviral vectors are well known in the art (see, e.g., Naldini et al., 1996; Zufferey et al., 1997; Blomer et al., 1997; U.S. Patent Nos. 6,013,516 and 5,994,136).

[0075] Recombinant lentiviral vectors are capable of infecting non-dividing cells and can be used for gene transfer and expression of nucleic acid sequences both in vivo and ex vivo. For example, recombinant lentiviruses capable of infecting non-dividing cells (suitable host cells are transfected with two or more vectors having packaging functions, i.e., gag, pol, and env, and rev and tat) are described in U.S. Patent No. 5,994,136, incorporated herein by reference.

[0076] 2. Episomal Vectors The use of plasmid- or liposome-based extrachromosomal (i.e., episomal) vectors can also be provided in certain embodiments of the present disclosure. Such episomal vectors can include, for example, oriP-based vectors and / or vectors encoding derivatives of EBNA-1. These vectors can allow large fragments of DNA to be introduced into cells, maintained extrachromosomally, replicate once per cell cycle, divide efficiently into daughter cells, and not provoke an immune response.

[0077] Notably, EBNA-1, the only viral protein required for replication of oriP-based expression vectors, has developed an efficient mechanism to bypass the processing required for antigen presentation on MHC class I molecules and thus does not elicit a cellular immune response (Levitskaya et al., 1997). Furthermore, EBNA-1 acts in trans to promote cloned gene expression, and can induce cloned gene expression up to 100-fold in some cell lines (Langle-Rouault et al., 1998; Evans et al., 1997). Finally, production of such oriP-based expression vectors is inexpensive.

[0078] In certain embodiments, reprogramming factors are expressed from expression cassettes contained in one or more exogenous episomal genetic elements (see U.S. Patent Application Publication No. 2010 / 0003757, incorporated herein by reference). Thus, iPSCs can be essentially free of exogenous genetic elements, such as retroviral or lentiviral vector elements. These iPSCs are prepared using extrachromosomally replicating vectors (i.e., episomal vectors), which are vectors that can replicate episomally so that iPSCs are essentially free of exogenous vectors or viral elements (see U.S. Patent No. 8,546,140; Yu et al., 2009, incorporated herein by reference). Plasmids containing some DNA viruses, such as adenovirus, simian vacuolar virus 40 (SV40), bovine papillomavirus (BPV), or budding yeast ARS (Autonomously Replicating Sequences), replicate extrachromosomally or episomally in mammalian cells. These episomal plasmids essentially avoid all of the drawbacks associated with integrating vectors (Bode et al., 2001). For example, lymphotrophic herpesvirus-based vectors, including the Epstein-Barr virus (EBV) defined above, replicate extrachromosomally and can aid in the delivery of reprogramming genes to somatic cells. Useful EBV elements include OriP and EBNA-1, or their mutants or functional equivalents. An additional advantage of episomal vectors is that after introduction into cells, exogenous elements are lost over time, resulting in self-sustaining iPSCs essentially free of these elements.

[0079] Other extrachromosomal vectors include other lymphotrophic herpesvirus-based vectors. Lymphotrophic herpesviruses are herpesviruses that replicate in lymphoblasts (e.g., human B lymphoblasts) and become plasmids as part of their natural life cycle. Herpes simplex virus (HSV) is not a "lymphotrophic" herpesvirus. Exemplary lymphotrophic herpesviruses include, but are not limited to, EBV, Kaposi's sarcoma herpesvirus (KSHV); herpesvirus saimiri (HS), and Marek's disease virus (MDV). Other sources of episome-based vectors, such as yeast ARS, adenovirus, SV40, or BPV, are also contemplated.

[0080] Those skilled in the art would be well-versed in the art of constructing vectors by standard recombinant techniques (see, for example, Maniatis et al., 1988 and Ausubel et al., 1994, both of which are incorporated herein by reference).

[0081] Vectors may also contain other components or functionalities that further regulate gene delivery and / or gene expression, or that otherwise provide beneficial properties to target cells. Such other components include, for example, components that affect cell binding or targeting (including components that mediate cell-type or tissue-specific binding), components that affect uptake of vector nucleic acid by cells, components that affect localization of polynucleotides within cells after uptake (such as agents that mediate nuclear localization), and components that affect expression of polynucleotides.

[0082] Such components may also include markers, such as detectable and / or selectable markers, that can be used to detect or select cells that have taken up and are expressing the nucleic acid delivered by the vector. Such components may be provided as natural features of the vector (e.g., the use of certain viral vectors that have components or functions that mediate binding and uptake), or the vector may be modified to provide such functions. A large variety of such vectors are known in the art and are generally available. When a vector is maintained in a host cell, it may be stably replicated by the cell as an autonomous structure during mitosis, integrated into the genome of the host cell, or maintained in the nucleus or cytoplasm of the host cell.

[0083] 3. Regulatory Elements The expression cassette contained in the reprogramming vectors useful in the present disclosure preferably comprises (5' to 3') a eukaryotic transcriptional promoter operably linked to a protein coding sequence, splice signals including intervening sequences, and transcription termination / polyadenylation sequences.

[0084] a. Promoter / enhancer The expression constructs provided herein include a promoter that drives the expression of a programming gene. Promoters generally contain sequences that function to position the start site for RNA synthesis. The most well-known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late gene, discrete elements located above the start site itself help to anchor the start position. Additional promoter elements regulate the frequency of transcription initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although some promoters have been shown to contain functional elements downstream of the start site as well. To place a coding sequence "under the control" of a promoter, the 5' end of the transcription start site of the transcriptional reading frame is positioned "downstream" (i.e., 3') of the selected promoter. The "upstream" promoter stimulates DNA transcription and promotes expression of the encoded RNA.

[0085] Spacing between promoter elements is often flexible, so that promoter function is maintained when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased by up to 50 bp before activity begins to decline. Depending on the promoter, individual elements appear to function cooperatively or independently to activate transcription. Promoters may or may not be used in combination with "enhancers," which refer to cis-acting regulatory sequences involved in the transcriptional activation of a nucleic acid sequence.

[0086] A promoter may be one naturally associated with a nucleic acid sequence, as can be obtained by isolating the 5' non-coding sequences located upstream of a coding segment and / or exon. Such a promoter may be referred to as "endogenous." Similarly, an enhancer may be one naturally associated with a nucleic acid sequence located downstream or upstream of that sequence. Alternatively, certain advantages may be obtained by placing a coding nucleic acid segment under the control of a recombinant or heterologous promoter (which refers to a promoter not normally associated with a nucleic acid sequence in its natural environment). A recombinant or heterologous enhancer also refers to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, promoters or enhancers isolated from any other virus or prokaryotic or eukaryotic cell, and promoters or enhancers that are "not naturally occurring," i.e., promoters or enhancers containing different elements of different transcriptional regulatory regions and / or mutations that alter expression. For example, promoters most commonly used in recombinant DNA construction include the β-lactamase (penicillinase), lactose, and tryptophan (trp) promoter systems. In addition to producing promoter and enhancer nucleic acid sequences synthetically, sequences can be produced in connection with the compositions disclosed herein using nucleic acid amplification techniques, including recombinant cloning and / or PCR™ (see U.S. Pat. Nos. 4,683,202 and 5,928,906, each of which is incorporated herein by reference). Furthermore, it is contemplated that control sequences that direct transcription and / or expression of sequences in non-nuclear organelles, such as mitochondria and chloroplasts, can be used as well.

[0087] Naturally, it will be important to use a promoter and / or enhancer that effectively directs expression of the DNA segment in the organelle, cell type, tissue, organ, or organism selected for expression. Those skilled in the art of molecular biology are generally familiar with the use of promoter, enhancer, and cell type combinations for protein expression (see, e.g., Sambrook et al. 1989, incorporated herein by reference). The promoter used may be constitutive, tissue-specific, inducible, and / or useful under appropriate conditions for inducing high-level expression of the introduced DNA segment, which is advantageous in large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0088] Additionally, any promoter / enhancer combination (e.g., as in the Eukaryotic Promoter Database EPDB) can be used to drive expression. Use of the T3, T7, or SP6 cytoplasmic expression systems is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional gene expression construct.

[0089] Non-limiting examples of promoters include early or late viral promoters, such as the SV40 early or late promoter, the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus (RSV) early promoter; eukaryotic promoters, such as the β-actin promoter (Ng, 1989; Quitsche et al., 1989), the GADPH promoter (Alexander et al., 1988; Ercolani et al., 1988), and the metallothionein promoter (Karin et al., 1989; Richards et al., 1984); and tethered response element promoters, such as the cyclic AMP response element promoter (cre), serum response element promoter (sre), phorbol ester promoter (TPA), and minimal TATA box proximal response element promoter (tre). It is also possible to use the human growth hormone promoter sequence (e.g., the human growth hormone minimal promoter described in Genbank, accession number X05244, nucleotides 283-341) or the mouse mammary tumor promoter (available from ATCC, catalog number ATCC 45007).

[0090] Tissue-specific expression of a transgene, particularly a reporter gene expression in hematopoietic cells and precursors of hematopoietic cells derived from programming, would be desirable as a method of identifying the derived hematopoietic cells and precursors. To enhance both specificity and activity, the use of cis-acting regulatory elements is contemplated. For example, a hematopoietic cell-specific promoter can be used. Many such hematopoietic cell-specific promoters are known in the art.

[0091] In certain embodiments, the methods of the present disclosure also relate to enhancer sequences, i.e., nucleic acid sequences that enhance promoter activity and have the potential to act in cis over relatively long distances (up to several kilobases away from the target promoter), regardless of their orientation. However, enhancer function is not necessarily limited to such long distances and can also function in the immediate vicinity of a given promoter.

[0092] Many hematopoietic cell promoter and enhancer sequences have been identified and may be useful in the present methods. See, e.g., U.S. Patent No. 5,556,954; U.S. Patent Application Publication No. 20020055144; U.S. Patent Application Publication No. 20090148425.

[0093] b. Initiation signal and linked expression Specific initiation signals can also be used in the expression constructs provided herein for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. It may be necessary to provide exogenous translational control signals, including the ATG initiation codon. One of ordinary skill in the art would be able to readily determine this and provide the necessary signals. It is well known that the initiation codon must be "in frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. Exogenous translational control signals and initiation codons can be either natural or synthetic. The efficiency of expression can be improved by including appropriate transcriptional enhancer elements.

[0094] In certain embodiments, internal ribosome entry site (IRES) elements are used to generate multigene or polycistronic messages. IRES elements can bypass the ribosome scanning model of 5'C methylated cap-dependent translation and initiate translation at internal sites (Pelletier and Sonenberg, 1988). IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), and IRESs from mammalian messages have been described (Macejak and Sarnow, 1991). IRES elements can link heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, generating polycistronic messages. IRES elements allow each open reading frame access to ribosomes for efficient translation. A single promoter / enhancer can be used to efficiently express multiple genes to transcribe a single message (see U.S. Pat. Nos. 5,925,565 and 5,935,819, each of which is incorporated herein by reference).

[0095] Additionally, certain 2A sequence elements can be used to link or co-express programming genes in the constructs provided herein. For example, cleavage sequences can be used to link open reading frames to form a single cistron, thereby co-expressing genes. Exemplary cleavage sequences are F2A (foot-and-mouth disease virus 2A) or "2A-like" sequences (e.g., Thosea asigna virus 2A; T2A) (Minskaia and Ryan, 2013). In certain embodiments, F2A-cleavage peptides are used to link the expression of genes in multi-lineage constructs.

[0096] c. Origin of replication To propagate a vector in a host cell, the vector may contain one or more origin of replication sites (often referred to as "ori"), such as a nucleic acid sequence corresponding to the EBV oriP described above or a genetically engineered oriP with a similar or enhanced function in programming, where such oriP is a specific nucleic acid sequence from which replication is initiated. Alternatively, the origin of replication of other extrachromosomally replicating viruses, or an autonomously replicating sequence (ARS), can be used, as described above.

[0097] d. Selectable and Screenable Markers In certain embodiments, cells containing the nucleic acid construct can be identified in vitro or in vivo by including a marker in the expression vector. Such a marker confers a distinguishable change to the cell, allowing for easy identification of cells containing the expression vector. Generally, a selectable marker is a marker that confers a property that allows for selection. A positive selectable marker is a marker whose presence allows for its selection, while a negative selectable marker is a marker whose presence inhibits its selection. An example of a positive selectable marker is a drug resistance marker.

[0098] Typically, the inclusion of a drug selection marker aids in the cloning and identification of transformants; for example, genes conferring resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selection markers. In addition to markers that confer phenotypes that allow for the identification of transformants based on the implementation of conditions, other types of markers are contemplated, including colorimetrically based screenable markers such as GFP. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) can be utilized as negative selection markers. Those skilled in the art will also know how to use immunological markers, possibly in combination with FACS analysis. The marker used is not believed to be critical, so long as it can be expressed simultaneously with the nucleic acid encoding the gene product. Further examples of selectable and screenable markers are well known to those skilled in the art.

[0099] E. Gene disruption In certain embodiments, the expression, activity, or function of the TREM2, APOE, MeCP2, and / or SCNA genes is disrupted in cells such as PSCs (e.g., ESCs or iPSCs). In some embodiments, gene disruption is carried out by disrupting the gene, such as by knockout, insertion, frameshift mutation, such as a missense or biallelic frameshift mutation, deletion and / or knock-in of all or part of the gene, for example, one or more exons or portions thereof. For example, disruption can be carried out by sequence-specific or targeted nucleases, including DNA-binding targeted nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), and RNA-guided nucleases such as CRISPR-associated nucleases (Cas), which are specifically designed to target the sequence of a gene or a portion thereof.

[0100] In some embodiments, disruption of gene expression, activity and / or function is carried out by disrupting the gene, hi some aspects, the gene is disrupted such that its expression is reduced by at least or about 20, 30, or 40%, generally at least or about 50, 60, 70, 80, 90, or 95%, compared to expression in the absence of the gene disruption or in the absence of components introduced to effect the disruption.

[0101] In some embodiments, the disruption is transient or reversible, such that expression of the gene is subsequently restored. In other embodiments, the disruption is not reversible or transient, e.g., permanent.

[0102] In some embodiments, gene disruption is typically carried out by inducing one or more double-strand breaks and / or one or more single-strand breaks in a gene in a targeted manner. In some embodiments, double-strand or single-strand breaks are carried out by a nuclease, for example, an endonuclease, such as a gene-targeting nuclease. In some embodiments, the break is induced in the coding region of a gene, for example, in an exon. For example, in some embodiments, the break occurs near the N-terminal end of the coding region, for example, in the first exon, the second exon, or a subsequent exon.

[0103] In some embodiments, double-stranded or single-stranded breaks undergo repair through cellular repair processes, such as non-homologous end joining (NHEJ) or homology-directed repair (HDR). In some embodiments, the repair process is error-prone and results in disruption of the gene, such as a frameshift mutation, e.g., a biallelic frameshift mutation, which can result in a complete knockout of the gene. For example, in some embodiments, the disruption includes inducing a deletion, mutation, and / or insertion. In some embodiments, the disruption results in the presence of a premature stop codon. In some embodiments, the presence of an insertion, deletion, translocation, frameshift mutation, and / or premature stop codon results in disruption of gene expression, activity, and / or function.

[0104] In some embodiments, gene disruption is achieved using antisense technology, such as RNA interference (RNAi), short interfering RNA (siRNA), short hairpin (shRNA), and / or ribozymes, to selectively suppress or prevent gene expression. siRNA technology is RNAi using double-stranded RNA molecules that have a sequence homologous to and a sequence complementary to the nucleotide sequence of mRNA transcribed from a gene. siRNAs generally contain multiple RNA molecules that are homologous / complementary to one region of mRNA transcribed from a gene, or homologous / complementary to different regions. In some embodiments, siRNAs are included in polycistronic constructs. In certain embodiments, siRNAs suppress both wild-type and mutant protein translation from endogenous mRNA.

[0105] In some embodiments, disruption is achieved using a DNA-targeting molecule, such as a DNA-binding protein or DNA-binding nucleic acid, or a complex, compound, or composition comprising the same, that specifically binds to or hybridizes with the gene. In some embodiments, the DNA-targeting molecule comprises a DNA-binding domain, such as a zinc finger protein (ZFP) DNA-binding domain, a transcription activator-like protein (TAL) or TAL effector (TALE) DNA-binding domain, a clustered regularly interspaced short palindromic repeats (CRISPR) DNA-binding domain, or a DNA-binding domain from a meganuclease. Zinc finger, TALE, and CRISPR system binding domains can be engineered to bind to a predetermined nucleotide sequence, for example, through manipulation (changing one or more amino acids) of the recognition helix region of a naturally occurring zinc finger or TALE protein. Engineered DNA-binding proteins (zinc finger or TALE) are non-naturally occurring proteins. Rational criteria for design include the application of substitution rules and computerized algorithms to process information in databases storing information on existing ZFP and / or TALE designs and binding data. See, e.g., U.S. Patent Nos. 6,140,081; 6,453,242; and 6,534,261. See also WO 98 / 53058; WO 98 / 53059; WO 98 / 53060; WO 02 / 016536; and WO 03 / 016496, and U.S. Patent Application Publication No. 2011 / 0301073.

[0106] In some embodiments, the DNA targeting molecule, complex, or combination comprises a DNA binding molecule and one or more additional domains, such as an effector domain, for promoting gene suppression or disruption.For example, in some embodiments, gene disruption is carried out by a fusion protein comprising a DNA binding protein and a heterologous regulatory domain or a functional fragment thereof.In some embodiments, the domain includes, for example, transcription factor domains such as activators, repressors, coactivators, corepressors, silencers, oncogenes, DNA repair enzymes and their associated factors and modifiers, DNA rearrangement enzymes and their associated factors and modifiers, chromatin-associated proteins and their modifiers, such as kinases, acetylases and deacetylases, and DNA modifying enzymes, such as methyltransferases, topoisomerases, helicases, ligases, kinases, phosphatases, polymerases, endonucleases, and their associated factors and modifiers. For details about the fusion of DNA binding domains and nuclease cleavage domains, see, for example, U.S. Patent Application Publication Nos. 2005 / 0064474; 2006 / 0188987 and 2007 / 0218528, which are incorporated herein by reference in their entirety. In some embodiments, the additional domain is a nuclease domain. Thus, in some embodiments, gene disruption is facilitated by gene or genome editing using engineered proteins, such as nucleases and nuclease-containing complexes or fusion proteins, which are composed of sequence-specific DNA binding domains fused or complexed with non-specific DNA cleavage molecules, such as nucleases.

[0107] In some embodiments, these targeted chimeric nucleases or nuclease-containing complexes induce targeted double-strand or single-strand breaks and stimulate cellular DNA repair mechanisms, including error-prone non-homologous end joining (NHEJ) and homology-directed repair (HDR), thereby achieving precise gene modification. In some embodiments, the nuclease is an endonucleases such as zinc finger nucleases (ZFNs), TALE nucleases (TALENs), and RNA-guided endonucleases (RGENs) or meganucleases, such as CRISPR-associated (Cas) proteins.

[0108] In some embodiments, a donor nucleic acid, e.g., a donor plasmid or a nucleic acid encoding an engineered antigen receptor, is provided and inserted by HDR at the site of gene editing following the introduction of a DSB. Thus, in some embodiments, gene disruption and introduction of an antigen receptor, e.g., a CAR, are performed simultaneously, whereby the gene is partially disrupted by knock-in or insertion of a nucleic acid encoding a CAR.

[0109] In some embodiments, no donor nucleic acid is provided. In some aspects, NHEJ-mediated repair after introduction of a DSB results in an insertion or deletion mutation that can cause gene disruption, for example, by creating a missense mutation or a frameshift.

[0110] 1. ZFPs and ZFNs In some embodiments, the DNA targeting molecule comprises a DNA binding protein, such as one or more zinc finger proteins (ZFPs) or transcription activator-like proteins (TALs), fused to an effector protein, such as an endonuclease. Examples include ZFNs, TALEs, and TALENs.

[0111] In some embodiments, the DNA-targeting molecule comprises one or more zinc finger proteins (ZFPs) or domains thereof that bind to DNA in a sequence-specific manner. A ZFP or domain is a protein or domain within a larger protein that binds to DNA in a sequence-specific manner via one or more zinc fingers, a region of amino acid sequence within the binding domain whose structure is stabilized by the coordination of zinc ions. The term zinc finger DNA-binding protein is often abbreviated as zinc finger protein or ZFP. Among ZFPs are artificial ZFP domains, typically 9-18 nucleotides in length, that are generated by assembly of individual fingers to target specific DNA sequences.

[0112] ZFPs include those in which the single finger domain is approximately 30 amino acids long, contains two invariant histidine residues coordinated through zinc with two cysteines in a single beta turn, and contains an alpha helix with two, three, four, five, or six fingers. Generally, the sequence specificity of ZFPs can be altered by making amino acid substitutions at the four helical positions (-1, 2, 3, and 6) of the zinc finger recognition helix. Thus, in some embodiments, ZFPs or ZFP-containing molecules are engineered to bind to non-native, e.g., selected, target sites.

[0113] In some embodiments, disruption of MeCP2 is carried out by contacting a first target site of the gene with a first ZFP, thereby disrupting the gene. In some embodiments, the target site of the gene is contacted with a fusion ZFP comprising six fingers and a regulatory domain, thereby inhibiting expression of the gene.

[0114] In some embodiments, the contacting step further comprises contacting a second target site within the gene with a second ZFP. In some embodiments, the first and second target sites are adjacent. In some embodiments, the first and second ZFPs are covalently linked. In some embodiments, the first ZFP is a fusion protein comprising a regulatory domain or at least two regulatory domains.

[0115] In some embodiments, the first and second ZFPs each comprise a regulatory domain or are fusion proteins each comprising at least two regulatory domains, hi some embodiments, the regulatory domains are transcriptional repressors, transcriptional activators, endonucleases, methyltransferases, histone acetyltransferases, or histone deacetylases.

[0116] In some embodiments, the ZFP is encoded by a ZFP nucleic acid operably linked to a promoter. In some aspects, the method further comprises first administering the nucleic acid to the cell in a lipid:nucleic acid complex or as naked nucleic acid. In some embodiments, the ZFP is encoded by an expression vector comprising a ZFP nucleic acid operably linked to a promoter. In some embodiments, the ZFP is encoded by a nucleic acid operably linked to an inducible promoter. In some aspects, the ZFP is encoded by a nucleic acid operably linked to a weak promoter.

[0117] In some embodiments, the target site is upstream of the transcription start site of the gene. In some embodiments, the target site is adjacent to the transcription start site of the gene. In some embodiments, the target site is adjacent to an RNA polymerase pause site downstream of the transcription start site of the gene.

[0118] In some embodiments, the DNA targeting molecule is or comprises a zinc finger DNA binding domain fused to a DNA cleavage domain to form a zinc finger nuclease (ZFN). In some embodiments, the fusion protein comprises a cleavage domain (or cleavage half-domain) from at least one type 1iS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered. In some embodiments, the cleavage domain is derived from the type 1iS restriction endonuclease Fok I. Fok I catalyzes double-stranded cleavage of DNA, typically 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other strand.

[0119] In some embodiments, ZFNs target genes present in engineered cells. In some embodiments, ZFNs efficiently generate double-strand breaks (DSBs) at predetermined sites in the coding region of a gene, for example. Typical targeted regions include exons, regions encoding the N-terminal region, the first exon, the second exon, and promoter or enhancer regions. In some embodiments, transient expression of ZFNs promotes highly efficient and permanent disruption of target genes in engineered cells. Notably, in some embodiments, delivery of ZFNs results in permanent gene disruption with an efficiency of over 50%.

[0120] Many gene-specific engineered zinc fingers are commercially available. For example, Sangamo Biosciences (Richmond, CA, USA) has developed a platform for zinc finger construction (CompoZr) in collaboration with Sigma-Aldrich (St. Louis, MO, USA), allowing researchers to completely bypass the construction and validation of zinc fingers and providing zinc fingers specifically targeted to thousands of proteins (Gaj et al., Trends in Biotechnology, 2013, 31(7), 397-405). In some embodiments, commercially available zinc fingers are used or custom-designed.

[0121] 2. TAL, TALE and TALEN In some embodiments, the DNA targeting molecule comprises a naturally occurring or engineered (non-naturally occurring) transcription activator-like protein (TAL) DNA binding domain, such as a transcription activator-like protein effector (TALE) protein. See, e.g., U.S. Patent Application Publication No. 2011 / 0301073, which is incorporated herein by reference in its entirety.

[0122] A TALE DNA-binding domain, or TALE, is a polypeptide containing one or more TALE repeat domains / units. The repeat domain is responsible for binding of the TALE to its cognate target DNA sequence. A single "repeat unit" (also called a "repeat") is typically 33-35 amino acids long and exhibits at least some sequence homology to other TALE repeat sequences in naturally occurring TALE proteins. Each TALE repeat unit typically contains one or two DNA-binding residues at positions 12 and / or 13 of the repeat, constituting a repeat variable dimer (RVD). The natural (canonical) code for DNA recognition of these TALEs has been determined such that the HD sequence at positions 12 and 13 binds to cytosine (C), NG binds to T, NI binds to A, NN binds to G or A, and NO binds to T; non-canonical (atypical) RVDs are also known. See US Patent Application Publication No. 2011 / 0301073. In some embodiments, TALEs can target any gene by designing TAL arrays with specificity for the target DNA sequence, which generally begins with a thymidine.

[0123] In some embodiments, the molecule is a DNA-binding endonuclease, such as a TALE nuclease (TALEN). In some embodiments, a TALEN is a fusion protein comprising a DNA-binding domain derived from a TALE and a nuclease catalytic domain for cleaving a nucleic acid target sequence.

[0124] In some embodiments, TALENs recognize and cleave target sequences in genes. In some embodiments, DNA cleavage results in double-strand breaks. In some embodiments, cleavage stimulates the rate of homologous recombination or non-homologous end joining (NHEJ). Generally, NHEJ is an imperfect repair process that often leads to changes in the DNA sequence at the cleavage site. In some embodiments, the repair mechanism involves rejoining the remains of the two DNA ends via direct religation (Critchlow and Jackson, 1998) or so-called microhomology-mediated end joining. In some embodiments, NHEJ-mediated repair results in small insertions or deletions that can be used to disrupt and thereby silence genes. In some embodiments, the modification can be a substitution, deletion, or addition of at least one nucleotide. In some embodiments, cells in which a cleavage-induced mutagenesis event, i.e., a mutagenesis event subsequent to an NHEJ event, has occurred can be identified and / or selected by methods well known in the art.

[0125] In some embodiments, TALE repeats are assembled to specifically target genes. A library of TALENs targeting 18,740 human protein-coding genes has been constructed. Custom-designed TALE arrays are commercially available from Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, KY, USA), and Life Technologies (Grand Island, NY, USA).

[0126] In some embodiments, the TALENs are introduced as transgenes encoded by one or more plasmid vectors. In some embodiments, the plasmid vectors may contain selectable markers that provide for identification and / or selection of cells that have received the vector.

[0127] 3. RGEN (CRISPR / Cas system) In some embodiments, the disruption is carried out using one or more DNA-binding nucleic acids, such as RNA-guided endonuclease (RGEN)-mediated disruption. For example, the disruption can be carried out using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins. Generally, the term "CRISPR system" refers collectively to the transcripts and other elements involved in the expression or directing the activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, tracr (transactivating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr-mate sequences (including "direct repeats" and tracrRNA-processed partial direct repeats in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), and / or other sequences and transcripts from the CRISPR locus.

[0128] A CRISPR / Cas nuclease or CRISPR / Cas nuclease system can include a non-coding RNA molecule (guide) RNA that binds to DNA in a sequence-specific manner and a Cas protein (e.g., Cas9) with nuclease function (e.g., two nuclease domains). One or more elements of the CRISPR system can be derived from a Type I, Type II, or Type III CRISPR system, and can be derived from a particular organism that contains an endogenous CRISPR system, such as Streptococcus pyogenes.

[0129] In some embodiments, a Cas nuclease and a gRNA (comprising a fusion of a target sequence-specific crRNA and an immobilized tracrRNA) are introduced into a cell. Generally, a target site at the 5' end of the gRNA allows the Cas nuclease to target a target site, e.g., a gene, using complementary base pairing. Target sites can typically be selected based on their location directly 5' of a protospacer adjacent motif (PAM) sequence, such as NGG or NAG. In this regard, the gRNA targets a desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. Generally, CRISPR systems are characterized by elements that promote the formation of a CRISPR complex at the site of the target sequence. Typically, the term "target sequence" generally refers to a sequence to which the guide sequence is designed to be complementary, and hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. Absolute complementarity is not necessary, provided there is sufficient complementarity to allow hybridization to occur and promote formation of a CRISPR complex.

[0130] The CRISPR system can induce a double-strand break (DSB) at the target site, followed by disruption as described herein. In another embodiment, a Cas9 variant considered a "nickase" is used to nick a single strand at the target site. Paired nickases can be used, for example, to improve specificity, each directed by a pair of different gRNAs targeting sequences such that simultaneous introduction of a nick introduces a 5' overhang. In another embodiment, catalytically inactive Cas9 is fused to a heterologous effector domain, such as a transcriptional repressor or activator, to affect gene expression.

[0131] The target sequence may comprise any polynucleotide, such as a DNA or RNA polynucleotide. The target sequence may be located in the nucleus or cytoplasm of a cell, such as within a cellular organelle. Generally, a sequence or template that can be used for recombination into a target locus that comprises a target sequence is referred to as an "editing template," or an "editing polynucleotide," or an "editing sequence." In some embodiments, an exogenous template polynucleotide may be referred to as an editing template. In some embodiments, the recombination is homologous recombination.

[0132] Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands within or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs therefrom). A tracr sequence that may comprise or consist of all or a portion of a wild-type tracr sequence (e.g., about 20 or more, about 26 or more, about 32 or more, about 45 or more, about 48 or more, about 54 or more, about 63 or more, about 67 or more, about 85 or more nucleotides of the wild-type tracr sequence) can form part of a CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence operably linked to a guide sequence. The tracr sequence has sufficient complementarity to the tracr mate sequence to hybridize and participate in the formation of a CRISPR complex (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity along the length of the tracr mate sequence when optimally aligned).

[0133] One or more vectors driving the expression of one or more elements of the CRISPR system can be introduced into a cell so that expression of the elements of the CRISPR system directs the formation of a CRISPR complex at one or more target sites. Components can also be delivered to a cell as protein and / or RNA. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence can each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more elements expressed from the same or different regulatory elements can be combined into a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. A vector can contain one or more insertion sites, such as restriction endonuclease recognition sequences (also called "cloning sites"). In some embodiments, the one or more insertion sites are located upstream and / or downstream of one or more sequence elements of one or more vectors. When multiple different guide sequences are used, a single expression construct can be used to target CRISPR activity to multiple different corresponding target sequences within a cell.

[0134] The vector can include regulatory elements operably linked to an enzyme coding sequence that encodes a CRISPR enzyme, such as a Cas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Csel, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. These enzymes are known; for example, the amino acid sequence of the S. pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2.

[0135] The CRISPR enzyme can be Cas9 (e.g., from S. pyogenes or S. pneumoniae). CRISPR enzymes can direct cleavage of one or both strands at the location of a target sequence, such as within the target sequence and / or within the complement of the target sequence. The vector can encode a CRISPR enzyme that is mutated relative to the corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence. For example, an aspartic acid to alanine substitution (D10A) in the RuvC I catalytic domain of S. pyogenes Cas9 converts Cas9 from a nuclease that cleaves both strands to a nickase (single-strand cleavage). In some embodiments, Cas9 nickase can be used in combination with guide sequences, e.g., two guide sequences that target the sense and antisense strands of a DNA target, respectively. This combination nicks both strands and can be used to induce NHEJ or HDR.

[0136] In some embodiments, the enzyme coding sequence encoding the CRISPR enzyme is codon-optimized for expression in a particular cell, such as a eukaryotic cell. The eukaryotic cell may be of or derived from a particular organism, such as a mammal, including, but not limited to, a human, mouse, rat, rabbit, dog, or non-human primate. Generally, codon optimization refers to the process of modifying a nucleic acid sequence to enhance expression in a target host cell by replacing at least one codon in the native sequence with a codon more frequently or most frequently used in the host cell's genes while maintaining the native amino acid sequence. Various species exhibit specific biases toward specific codons for specific amino acids. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of messenger RNA (mRNA) translation, which is thought to depend, among other things, on the properties of the codon being translated and the availability of specific transfer RNA (tRNA) molecules. The dominance of selected tRNAs within a cell generally reflects the codons most frequently used in peptide synthesis. Therefore, genes can be tailored for optimal gene expression in a given organism based on codon optimization.

[0137] Generally, a guide sequence is any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about 50% or more, about 60% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97.5% or more, about 99% or more, or more.

[0138] Optimal alignment can be determined using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transformation (e.g., Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0139] CRISPR enzymes can be part of fusion proteins containing one or more heterologous protein domains. CRISPR enzyme fusion proteins can contain any additional protein sequences and optionally linker sequences between any two domains. Examples of protein domains that can be fused to CRISPR enzymes include, but are not limited to, epitope tags, reporter gene sequences, and protein domains with one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), autofluorescent proteins including HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and blue fluorescent protein (BFP). CRISPR enzymes can be fused to genetic sequences encoding proteins or protein fragments that bind to DNA molecules or other cellular molecules, including, but not limited to, maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions.

[0140] III. Astrocyte differentiation method In some embodiments, methods are provided for generating differentiated cells from essentially single-cell suspensions of pluripotent stem cells (PSCs), such as human iPSCs. In some embodiments, PSCs are cultured to confluence beforehand to prevent any cell aggregation. In certain embodiments, PSCs are dissociated by incubation with a cell dissociation enzyme, such as TRYPSIN™ or TRYPLE™. PSCs can also be dissociated into an essentially single-cell suspension by pipetting. Additionally, blebbistatin (e.g., about 2.5 μM) can be added to the culture medium to prevent cells from adhering to the culture vessel and prolong PSC viability after dissociation into single cells. Alternatively, a ROCK inhibitor can be used instead of blebbistatin to prolong PSC viability after dissociation into single cells.

[0141] Once a single cell suspension of PSCs is obtained at a known cell density, the cells are typically seeded into an appropriate culture vessel, such as a flask, tissue culture plate, such as a 6-well, 24-well, or 96-well plate. Culture vessels used to culture the cells can include, but are not limited to: flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multiwell plates, microslides, chamber slides, tubes, trays, CELLSTACK® chambers, culture bags, and roller bottles, provided that stem cells can be cultured therein. Cells can be cultured in volumes of at least or about 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350 ml, 400 ml, 450 ml, 500 ml, 550 ml, 600 ml, 800 ml, 1000 ml, 1500 ml, or any range of volumes derivable therein depending on the needs of the culture. In certain embodiments, the culture vessel can be a bioreactor, which can refer to any ex vivo device or system that supports a biologically active environment in which cells can grow. The bioreactor can have a volume of at least or about 2, 4, 5, 6, 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 500 liters, 1, 2, 4, 6, 8, 10, 15 cubic meters, or any range derivable therein.

[0142] In certain embodiments, PSCs, such as iPSCs, are plated at a cell density suitable for efficient differentiation. Generally, cells are plated at a density of about 10,000 to about 75,000 cells / cm. 2 For example, about 15,000 to about 40,000 cells / cm 2In a 6-well plate, the cells can be plated at a cell density of about 50,000 to about 400,000 cells per well. In an exemplary method, the cells are plated at a density of about 100,000, about 150,000, about 200,000, about 250,000, about 300,000, or about 350,000 cells per well, e.g., about 200,000 cells per well.

[0143] PSCs, such as iPSCs, are typically cultured on culture plates coated with one or more cell adhesion proteins to promote cell attachment while maintaining cell viability. For example, preferred cell adhesion proteins include extracellular matrix proteins, such as vitronectin, laminin, collagen, and / or fibronectin, which can be used to coat culture surfaces as a means of providing solid support for pluripotent cell growth. The term "extracellular matrix" is art-recognized. Its components include one or more of the following proteins: fibronectin, laminin, vitronectin, tenascin, entactin, thrombospondin, elastin, gelatin, collagen, fibrillin, merosin, anchorin, chondronectin, link protein, bone sialoprotein, osteocalcin, osteopontin, epinectin, hyaluronectin, undulin, epiligrin, and kalinin. In an exemplary method, PSCs are grown on culture plates coated with vitronectin or fibronectin. In some embodiments, the cell adhesion protein is a human protein.

[0144] Extracellular matrix (ECM) proteins can be naturally occurring and purified from human or animal tissues. Alternatively, ECM proteins can be genetically engineered recombinant proteins or synthetic in nature. ECM proteins can be whole proteins or in the form of natural or engineered peptide fragments. Examples of ECM proteins that may be useful in matrices for cell culture include laminin, collagen I, collagen IV, fibronectin, and vitronectin. In some embodiments, the matrix composition comprises synthetically produced peptide fragments of fibronectin or recombinant fibronectin. In some embodiments, the matrix composition is xeno-free. For example, xeno-free matrices for culturing human cells can use human-derived matrix components, in which case any non-human animal components can be omitted.

[0145] In some embodiments, the total protein concentration in the matrix composition can be about 1 ng / mL to about 1 mg / mL. In some preferred embodiments, the total protein concentration in the matrix composition is about 1 μg / mL to about 300 μg / mL. In more preferred embodiments, the total protein concentration in the matrix composition is about 5 μg / mL to about 200 μg / mL.

[0146] Cells can be cultured with nutrients necessary to support the growth of each specific cell population. Generally, cells are cultured in a growth medium containing a carbon source, a nitrogen source, and a buffer to maintain pH. The medium may also contain fatty acids or lipids, amino acids (such as non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, pyruvate, buffers, and inorganic salts. Exemplary growth media include minimal essential media, such as Dulbecco's Modified Eagle's Medium (DMEM) or ESSENTIAL 8™ (E8™) medium, supplemented with various nutrients, such as non-essential amino acids and vitamins, to promote stem cell proliferation. Examples of minimal essential media include, but are not limited to, Minimum Essential Medium Eagle's (MEM) Alpha Medium, Dulbecco's Modified Eagle's Medium (DMEM), RPMI-1640 Medium, 199 Medium, and F12 Medium. Additionally, minimal essential media can be supplemented with additives such as horse, calf, or fetal bovine serum. Alternatively, the medium can be serum-free. In other cases, the growth medium may contain a "Knockout serum replacement," referred to herein as a serum-free formulation optimized for the growth and maintenance of undifferentiated cells, such as stem cells, in culture. KNOCKOUT™ serum replacement is disclosed, for example, in U.S. Patent Application Publication No. 2002 / 0076747, which is incorporated herein by reference. Preferably, PSCs are cultured in a feeder-free, completely defined medium.

[0147] Thus, PSCs are generally cultured in a complete defined culture medium after plating. In certain embodiments, approximately 18-24 hours after plating, the medium is aspirated and fresh medium, such as E8™ medium, is added to the culture. In certain embodiments, single-cell PSCs are cultured in a complete defined culture medium for approximately 1, 2, or 3 days after plating. Preferably, single-cell PSCs are cultured in a complete defined culture medium for approximately 2 days before proceeding with the differentiation process.

[0148] In some embodiments, the medium may or may not contain any serum substitute. Serum substitutes may include materials that suitably contain albumin (e.g., but not limited to, lipid-rich albumin, albumin substitutes such as recombinant albumin, plant starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, or equivalents thereof. Serum substitutes can be prepared, for example, by methods disclosed in WO 98 / 30679. Alternatively, any commercially available material can be used for greater convenience. Commercially available materials include KNOCKOUT™ Serum Replacer (KSR), lipid concentrate with known composition (Gibco), and GLUTAMAX™ (Gibco).

[0149] Other culture conditions can be defined as appropriate. For example, the culture temperature can be about 30 to 40°C, for example, at least about 31, 32, 33, 34, 35, 36, 37, 38, or 39°C, but is not limited thereto. In one embodiment, cells are cultured at 37°C. The CO2 concentration can be about 1 to 10%, for example, about 2 to 5%, or any range derivable therein. The oxygen partial pressure can be at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20%, or any range derivable therein.

[0150] iPSCs, NPCs, and / or astrocytes can be cultured in defined media including, but not limited to, DMEM-F12, E8, E6, Neurobasal medium, Minimum Essential Medium (MEM), and / or BrainPhys Neuronal Medium.

[0151] A. Neural progenitor cells (NPCs) iPSCs can differentiate into glial-biased NPCs, also known as glial progenitor cells. iPSCs can be harvested with EDTA and plated on an extracellular matrix (e.g., MATRIGEL®)-coated surface in Essential 8 medium containing a ROCK inhibitor (e.g., H1152 at a concentration of 0.1-10 μM, e.g., 1 μM). Culture can be performed under hypoxic conditions, e.g., 5% oxygen. The ROCK inhibitor can then be removed after 12-48 hours, e.g., 24 hours. The cells can then be fed with Essential 8 medium for 1-3 days, e.g., 2 days.

[0152] The cells can then be pretreated with a medium containing DMEM-F12 and a GSK3 inhibitor (e.g., CHIR99021 at a concentration of about 1-5 μM, particularly about 3 μM) under normoxic conditions for about 2-4 days, e.g., 3 days.

[0153] The cells can then be dissociated using TrypLE (Gibco) and aggregates formed using NPC differentiation medium containing Essential 6 (Gibco), N2 supplement (Gibco), and a ROCK inhibitor (e.g., H1152 at a concentration of 0.1-10 μM, e.g., 1 μM). Aggregates can be formed in ultra-low attachment flasks, spinners (e.g., 500 mL), or bioreactors (e.g., PBS mini-bioreactors). Aggregates can be cultured for 5-10 days, e.g., 8 days, and fed every other day with NPC differentiation medium without ROCK inhibitor. Aggregates can be dissociated using TrypLE in a 37°C water bath for 10-15 minutes. Dissociated cells can be filtered (e.g., through a 100 μM filter) and cryopreserved (e.g., using CryoStorCS10 (BioLifeSolutions)). NPCs can be generated from apparently healthy or diseased donors.

[0154] Cells can be stained for pluripotency markers SSEA-4 and TRA-1-60, which begin to appear and increase over time, as well as NPC markers such as CD24, CD184, and CD271. Additional markers can include nestin and PAX6.

[0155] In certain embodiments, the method does not form neurospheres. In some embodiments, the differentiation method comprises culturing in the absence of a SMAD inhibitor, a TGFβ inhibitor, and / or a gamma secretase inhibitor.

[0156] B. Differentiation of NPCs into Astrocytes A schematic diagram of an exemplary differentiation procedure for generating astrocytes from NPCs is shown in Figure 3A. NPCs are cultured at 20 kJ / cm in extracellular matrix protein (e.g., Geltrex)-coated vessels in a medium containing DMEM / F12 medium supplemented with N2 and B27 (+vitA). 2 The cells can be seeded at a density of 1000 μg / well. Culture can be performed in normoxia, such as 20% oxygen. Stage 1 astrocyte medium can include a lipid concentrate of known composition (Gibco, Cat. No. 11905031, e.g., at a concentration of 1-5%, particularly about 2%, 3%, or 4%) and epidermal growth factor (e.g., at a concentration of 5-50 ng / mL, particularly about 10, 20, or 30 ng / mL) in combination with Delta-Like Canonical Notch Ligand 1 (DLL1) and / or human Jagged 1 Fc chimeric protein (JAGG1) (e.g., at a concentration of 1-25 ng / mL, particularly about 10 ng / mL). Stage 1 astrocyte differentiation medium may further comprise one or more LIF receptor ligands, e.g., leukemia inhibitory factor protein (LIF), ciliary-derived neurotrophic factor protein (CNTF), oncostatin M protein, and / or cardiotrophin 1 (CT-1) (e.g., each at a concentration of 1-25 ng / mL, particularly about 10 ng / mL). In certain embodiments, stage 1 astrocyte medium comprises CNTF, oncostatin M, LIF, and CT-1. Cultures may be cultured for about 1-3 weeks, e.g., about 2 weeks, and can be passaged upon confluence.

[0157] Lipid concentrate can be a concentrated lipid emulsion containing saturated fatty acid and unsaturated fatty acid.For example, lipid concentrate can contain arachidonic acid (for example, 2 mg / L concentration), cholesterol (for example, 220 mg / L concentration), DL-α-tocopherol acetate (for example, 70 mg / L concentration), linoleic acid (for example, 10 mg / L concentration), linolenic acid (for example, 10 mg / L concentration), myristic acid (for example, 10 mg / L concentration), oleic acid (for example, 10 mg / L concentration), palmitic acid (for example, 10 mg / L concentration), palmitoleic acid (for example, 10 mg / L concentration), and stearic acid (for example, 10 mg / L concentration).Lipid concentrate can further contain Tween 80 (registered trademark), Pluronic F-68, and ethyl alcohol.

[0158] Next, in Stage 2, the medium can be changed to an astrocyte medium containing one or more LIF receptor ligands, such as LIF, CNTF, oncostatin M, and / or CT-1, particularly LIF and CNTF. The astrocyte differentiation medium in Stage 2 can further contain a lipid concentrate. Culture can be for approximately 3 to 8 weeks, e.g., 4 to 7 weeks, particularly 4, 5, 6, or 7 weeks. Cells can then be stained for the astrocyte markers CD44, NFIX, and GFAP.

[0159] The astrocyte differentiation medium (e.g., stage 1 or stage 2) can further include one or more activators of the Notch pathway. The one or more activators of the Notch pathway can be Jagged 1 protein, Jagged 2 protein, and / or Delta-like protein 1 (DLL1), Delta-like protein 2 (DLL2), or Delta-like protein 3 (DLL3).

[0160] In certain embodiments, the basal medium may contain DMEM / F12, N2 supplement, B27 and retinoic acid supplement (e.g., at a concentration of 1%), GlutaMAX, and penicillin / streptomycin. The astrocyte medium may further contain OSM, CNTF, and LIF (e.g., at a concentration of 5-25 ng / mL, particularly about 10 ng / mL). The astrocyte medium may further contain DLL1 (e.g., at a concentration of 5-50 ng / mL, particularly about 10 ng / mL), JAGG1 (e.g., at a concentration of 5-50 ng / mL, particularly about 10 ng / mL), lipid concentrate (e.g., at a concentration of 1-5%, particularly about 2%), CT-1 (e.g., at a concentration of 5-50 ng / mL, particularly about 10 ng / mL), and / or EGF (e.g., at a concentration of 5-50 ng / mL, particularly about 20 ng / mL).

[0161] Astrocytes express several proteins that can serve as markers for detection by methods such as immunocytochemistry, Western blot analysis, flow cytometry, or enzyme-linked immunosorbent assay (ELISA). Astrocytes can be stained for surface markers, CD44 and glutamate-aspartate transporter (GLAST), as well as intracellular markers, glial fibrillary acidic protein (GFAP), excitatory amino acid transporter 1 (EAAT1), glutamine synthetase (GS), aquaporin 4 (AQP4), and S100 calcium-binding protein B (S100β). Cellular markers can be detected at the mRNA level by, for example, reverse transcriptase polymerase chain reaction (RT-PCR), Northern blot analysis, or dot blot hybridization analysis using sequence-specific primers in standard amplification methods using published sequence data (GENBANK®). Expression of a tissue-specific marker, detected at the protein or mRNA level, is considered positive if the level is at least or about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or 9-fold, more particularly 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or more, than the level of a control cell, e.g., an undifferentiated pluripotent stem cell or other unrelated cell type.

[0162] C. Differentiation medium Cells can be cultured with nutrients necessary to support the growth of each specific population of cells. Generally, cells are cultured in a growth medium containing a carbon source, a nitrogen source, and a buffer to maintain pH. The medium can also contain fatty acids or lipids, amino acids (such as non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, pyruvate, buffers, pH indicators, and inorganic salts. Exemplary growth media include minimal essential media, such as Dulbecco's Modified Eagle's Medium (DMEM) or ESSENTIAL 8™ (E8™) medium, supplemented with various nutrients, such as non-essential amino acids and vitamins, to enhance stem cell growth. Examples of minimal essential media include, but are not limited to, Minimal Essential Medium Eagle's (MEM) Alpha Medium, Dulbecco's Modified Eagle's Medium (DMEM), RPMI-1640 Medium, 199 Medium, and F12 Medium. Additionally, minimal essential media can be supplemented with additives such as horse, calf, or fetal bovine serum. Alternatively, the medium can be serum-free. In other cases, the growth medium may comprise a "knockout serum replacement," referred to herein as a serum-free formulation optimized for growing and maintaining undifferentiated cells, such as stem cells, in culture. KNOCKOUT™ serum replacement is disclosed, for example, in U.S. Patent Application Publication No. 2002 / 0076747, which is incorporated herein by reference. Preferably, PSCs are cultured in a feeder-free, completely defined medium.

[0163] In some embodiments, the medium may or may not contain any serum substitute. Serum substitutes may include materials that appropriately contain albumin (lipid-rich albumin, albumin substitutes such as recombinant albumin, vegetable starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, or equivalents thereof. Serum substitutes can be prepared, for example, by methods disclosed in WO 98 / 30679. Alternatively, and more conveniently, commercially available materials can be used. Commercially available materials include KNOCKOUT™ Serum Replacer (KSR), lipid concentrates of known composition (Gibco), and GLUTAMAX™ (Gibco).

[0164] Other culture conditions can be defined as appropriate. For example, the culture temperature can be about 30 to 40°C, for example, at least about 31, 32, 33, 34, 35, 36, 37, 38, or 39°C, but is not particularly limited thereto. In one embodiment, cells are cultured at 37°C. The CO2 concentration can be about 1 to 10%, for example, about 2 to 5%, or any range derivable therein. The oxygen partial pressure can be at least up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20%, or any range derivable therein.

[0165] D. Cryopreservation Cells produced by the methods disclosed herein can be cryopreserved at any stage of the process, such as NPCs, stage I astrocytes, or stage II astrocytes; see, for example, PCT Publication WO 2012 / 149484 A2, incorporated herein by reference. Cells can be cryopreserved with or without a substrate. In some embodiments, storage temperatures range from about -50°C to about -60°C, about -60°C to about -70°C, about -70°C to about -80°C, about -80°C to about -90°C, or about -90°C to about -100°C, and overlapping ranges thereof. In some embodiments, lower temperatures are used for storage (e.g., maintenance) of cryopreserved cells. In some embodiments, liquid nitrogen (or other similar liquid coolant) is used to preserve the cells. In further embodiments, the cells are preserved for more than about 6 hours. In further embodiments, the cells are preserved for about 72 hours. In some embodiments, the cells are preserved for 48 hours to about 1 week. In still other embodiments, the cells are stored for about 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In further embodiments, the cells are stored for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Cells can also be stored for longer periods of time. Cells can be cryopreserved separately or on a substrate, such as any of the substrates disclosed herein.

[0166] In some embodiments, an additional cryoprotectant can be used. For example, cells can be cryopreserved in a cryopreservation solution containing one or more cryoprotectants, such as DM80, and serum albumin, such as human or bovine serum albumin. In certain embodiments, the solution contains about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% DMSO. In other embodiments, the solution contains about 1% to about 3%, about 2% to about 4%, about 3% to about 5%, about 4% to about 6%, about 5% to about 7%, about 6% to about 8%, about 7% to about 9%, or about 8% to about 10% dimethyl sulfoxide (DMSO) or albumin. In certain embodiments, the solution contains 2.5% DMSO. In another specific embodiment, the solution contains 10% DMSO.

[0167] Cells can be cooled, for example, at about 1°C / min during cryopreservation. In some embodiments, the cryopreservation temperature is about -80°C to about -180°C or about -125°C to about -140°C. In some embodiments, cells are cooled to 4°C before cooling at about 1°C / min. Cryopreserved cells can be transferred to the vapor phase of liquid nitrogen before thawing for use. In some embodiments, for example, once the cells reach about -80°C, they are transferred to a liquid nitrogen storage area. Cryopreservation can also be performed using a controlled rate freezer. Cryopreserved cells can be thawed, for example, at a temperature of about 25°C to about 40°C, typically about 37°C.

[0168] IV.How to use Methods are provided by which NPCs and astrocytes can be produced. These cell populations can be used for many important research, development, and commercial purposes. These include, but are not limited to, in vivo cell transplantation or implantation; in vitro screening of growth / regulatory factors, pharmaceutical compounds, etc.; elucidating mechanisms of disease and infection; studying mechanisms by which drugs and / or growth factors act; diagnosing and monitoring disease in patients; gene therapy; and producing biologically active products, to name just a few.

[0169] A. Screening of Test Compounds The cell lines generated by the methods disclosed herein can be used in any method and application currently known in the art for iPSCs or differentiated cells. For example, methods for evaluating compounds can be provided, including assaying the pharmacological or toxicological properties of the compounds on cell lines. Methods for evaluating the effects of compounds on cell cultures can also be provided, comprising: a) contacting a cell culture provided herein with a compound; and b) assaying the effects of the compound on the cell culture.

[0170] Cell cultures can be used commercially to screen factors (solvents, small molecule drugs, peptides, oligonucleotides, etc.) or environmental conditions (e.g., culture conditions or manipulations) that affect the properties of such cells and their various progeny. For example, test compounds can be chemical compounds, small molecules, polypeptides, growth factors, cytokines, or other biological agents.

[0171] In one embodiment, the method includes contacting a cell culture with a test agent and determining whether the test agent modulates the activity or function of cells in the population. In some applications, the screening assay is used to identify agents that modulate cell proliferation, alter cell differentiation, or affect cell viability. The screening assay can be performed in vitro or in vivo. Methods for screening and identifying candidate agents include methods suitable for high-throughput screening. For example, cell cultures can be placed or arranged in appropriate locations in culture dishes, flasks, roller bottles, or plates (e.g., single multiwell dishes, or dishes such as 8-, 16-, 32-, 64-, 96-, 384-, and 1536-multiwell plates or dishes), optionally in defined locations, for the identification of potential therapeutic molecules. Libraries that can be screened include, for example, small molecule libraries, siRNA libraries, and adenoviral transfection vector libraries.

[0172] Another screening application involves testing the effect of pharmaceutical compounds on the maintenance or repair of retinal tissue. Screening can be done because the compounds are designed to have a pharmacological effect on cells, or because compounds designed to have an effect elsewhere may have unintended side effects on cells of this tissue type.

[0173] B. Treatment and Transplantation Other embodiments may also provide for the use of the cell lines for the treatment of a disease or disorder. In another aspect, the present disclosure provides a method of treating an individual in need thereof, the method comprising administering to the individual a composition comprising engineered cells.

[0174] To determine the suitability of a cell composition for therapeutic administration, the cells can first be tested in an appropriate animal model. In one embodiment, the ability of the cell line to survive and maintain its phenotype in vivo is evaluated. The composition is transplanted into an immunodeficient animal (e.g., nude mice or animals rendered immunodeficient chemically or by irradiation). Tissue is harvested after a growth period and evaluated for the presence or absence of pluripotent stem cell-derived cells.

[0175] Indications for use include, but are not limited to, autism, RETT syndrome, schizophrenia, fragile X syndrome, Angelman syndrome, Timothy syndrome, Parkinson's disease, amyotrophic lateral sclerosis, Alzheimer's disease, progressive supranuclear palsy, multiple sclerosis, Huntington's disease, multiple system atrophy, spinocerebellar degeneration, traumatic nerve injury, spinal cord injury, stroke, cerebral hemorrhage, cerebral thrombosis, cerebral embolism, macular degeneration, tremor, tardive dyskinesia, panic disorder, anxiety disorder, depression, alcoholism, insomnia, mania, Alzheimer's disease, epilepsy, and diabetic neuropathy. In certain embodiments, the disease is Alexander disease or leukodystrophy.

[0176] C. Pharmaceutical Compositions Also provided herein are pharmaceutical compositions and formulations comprising the cells and a pharmaceutically acceptable carrier. In some embodiments, the compositions provide a population of astrocytes that express at least SSEA4 and CD44.

[0177] Thus, the cell compositions for administration to a subject according to the present invention can be formulated in any conventional manner using one or more physiologically acceptable carriers containing excipients and auxiliary agents that facilitate processing of the compound into a pharmaceutically usable preparation. The appropriate formulation will vary depending on the selected route of administration.

[0178] The pharmaceutical compositions and formulations described herein comprise an active ingredient (e.g., cells) having a desired purity in the form of a lyophilized formulation or an aqueous solution, optionally in one or more pharmaceutically acceptable carriers (see Remington's Pharmaceutical Sciences 22 ndPharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; Examples of suitable pharmaceutically acceptable carriers include, but are not limited to, proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersing agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, a sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0179] D. Distribution for Commercial, Therapeutic, and Research Purposes In some embodiments, reagent systems are provided that contain cells present at any time during manufacture, distribution, or use. Kits can include any combination of cells described in this disclosure in combination with undifferentiated pluripotent stem cells or other differentiated cell types, which often share the same genome. Each cell type can be packaged together, in separate containers within the same facility, or in different locations at the same or different times, under the control of the same or different entities sharing a business relationship. Pharmaceutical compositions can optionally be packaged in appropriate containers with written instructions for the desired purpose, such as mechanistic toxicity.

[0180] In some embodiments, kits are provided that may include, for example, one or more media and components for producing cells. Reagent systems can be packaged either in aqueous media or in lyophilized form, as needed. The container means of the kits generally includes at least one vial, test tube, flask, bottle, syringe, or other container means into which the components may be placed, preferably appropriately divided. Where multiple components are included in the kit, the kit will generally also include a second, third, or other additional container into which the additional components may be separately disposed. However, various combinations of components may be included in vials. The components of the kits may be provided as dry powders. Where reagents and / or components are provided as dry powders, the powders can be reconstituted by the addition of a suitable solvent. It is contemplated that the solvent may also be provided in a separate container means. The kits of the present disclosure also typically include means for containing the components of the kit in close confinement for commercial sale. Such containers may include injection- or blow-molded plastic containers into which the desired vials are retained. The kits may also include instructions for use, such as in printed or electronic, e.g., digital, format. [Example]

[0181] The following examples are included to demonstrate preferred embodiments of the invention. Those of skill in the art should understand that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and as such can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, understand that many modifications to the specific embodiments which are disclosed can be made and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0182] Example 1 - Preparation of astrocytes Human induced pluripotent stem cells (hiPSCs) were harvested with EDTA and plated at 15,000 c / cm onto tissue culture (TC)-treated plates coated with Matrigel (Corning) in Essential 8 medium (Gibco) containing 1 μM ROCK inhibitor H1152 (Sigma). 2 The cells were plated in 1000 ml of PBS and cultured under hypoxic conditions. The ROCK inhibitor was removed on day 1, and the cells were fed with Essential 8 medium for 2 days. From day 3, the cells were pretreated with medium consisting of DMEM-F12 (Gibco) and 3 μM CHIR99021 (Stemgent) and cultured under normoxic conditions. After 3 days, the cells were dissociated with TrypLE (Gibco) and aggregates were formed using NPC differentiation medium consisting of Essential 6 (Gibco), N2 supplement (Gibco), and H1152 (1 μM) (Figure 1). Aggregates were formed in ultra-low attachment flasks, 500 mL spinners, or PBS mini-bioreactors. The aggregates were cultured for 8 days and fed with NPC differentiation medium without H1152 every other day. On day 14, the aggregates were dissociated with TrypLE for 10–15 min in a 37°C water bath. Dissociated cells were filtered at 100 μM and cryopreserved using CryoStorCS10 (BioLifeSolutions). NPCs were generated from five different hPSC lines from both apparently healthy and diseased donors.

[0183] Cells were stained for both cell surface (Figures 2A and 2C) and intracellular antigens (Figures 2B-2D). Pluripotency markers SSEA-4 and TRA-1-60 significantly decreased during the 14-day differentiation process, whereas NPC markers such as CD24, CD184, and CD271 began to appear and increased over time. NPCs from all five lines tested showed high expression of Pax6 and nestin, while the highest expression of SOX1, doublecortin (DCX), and β3-tubulin (Tuj) was observed in donor 1279. Cells were dissociated with Accutase (Gibco) for 5 min and quenched with medium containing B27 supplement (Gibco). For cell surface antigens, 100,000 cells were stained with antibody dilutions for 30 min at room temperature. Dead cells were excluded using propidium iodide (Sigma). For intracellular antigens, 100,000 cells were first stained for viability using Fixable Viability Stain 620 (BD Biosciences) for 10 minutes, followed by fixation with 4% formaldehyde (Sigma) for 15 minutes. Cells were permeabilized with 0.1% Triton (Sigma) or 0.1% saponin (MP Biomedicals) and stained overnight with antibody diluent. Cells were analyzed using an Accuri C6 flow cytometer (BD Biosciences).

[0184] A schematic diagram of the differentiation procedure for generating astrocytes from NPCs is shown in Figure 3A. NPCs were cultured in Geltrex-coated vessels at a density of 20 kJ / cm. 2Cells were seeded at a density of 100 μL per well and cultured for 2 weeks in DMEM / F12 medium supplemented with N2 and B27 (+vitA) and LIF receptor ligands (LIF, CNTF, oncostatin M, and CT-1, all at 10 ng / ml). They were passaged upon reaching confluence. After 2 weeks, the medium was changed to LIF + CNTF and lipid concentrate for the remainder of differentiation. Cells were fixed in 4% paraformaldehyde (Sigma) for 60 minutes and stored in DPBS (Gibco) for immunocytochemistry. Primary antibodies were diluted in ICC Permeation Buffer (DPBS + 2% FBS + 0.2% Triton X-100) and incubated overnight at 4°C. After primary antibody incubation, the antibodies were removed and the cells were washed three times with 100 μL DPBS per well for 10 minutes. Secondary antibodies were then applied for 1 hour at RT. After secondary antibody incubation, Hoescht 33342 (Invitrogen, Catalog No. H3570) was added for 10 minutes. Cells were then washed three times with 100 μL of DPBS per well for 10 minutes and imaged using a Molecular Devices Image Xpress automated microscope. Phase-contrast images of cells plated on PLO / Laminin substrate at day 60 (top of Figure 3B) and ICC staining for astrocyte markers CD44, NFIX, and GFAP protein at 35 and 80 days of differentiation are shown in Figure 3B. Quantification of the percentage of positive cells for several astrocyte markers at day 70 using flow cytometry is shown in Figure 3C.

[0185] The theoretical cumulative yield during the differentiation process indicated significant cell proliferation (Figure 3D). The glutamate uptake efficiency (percentage of glutamate concentration in the -TBOA sample relative to the corresponding +TBOA sample) over time is shown in Figure 3E. NPC-derived astrocytes were able to uptake glutamate from the culture medium equivalently to iCell astrocytes. For the glutamate uptake assay, cells were plated in triplicate at a density of 30,000 cells per well on vitronectin-coated 96-well plates in both control and +TBOA (Tocris, catalog no. 1223) conditions. Cells were then cultured for 7 or 14 days before starting the assay. On the day of the assay, cells were washed once with HBSS (Gibco) and incubated in either HBSS or HBSS + 300 μM TBOA at 37°C and 5% CO2 for 1 hour. After incubation, 20 μM glutamate was added to wells incubated with HBSS, and 20 μM glutamate + 300 μM TBOA was added to wells containing HBSS + 300 μM TBOA for 1 h at 37°C, 5% CO2. After incubation, samples were transferred to another 96-well plate and centrifuged at 400 × G for 10 min. After centrifugation, the remaining glutamate concentration in the samples was analyzed using the Glutamate-Glo Assay kit (Promega, catalog number J7021) according to the manufacturer's instructions.

[0186] Microelectrode assay (MEA) roster plots of the different cultures before (baseline) and after glutamate application are shown in Figure 3F. These results indicate that both iCell astrocytes and NPC astrocytes maintained network activity, uptaked excess glutamate, and protected neurons. To further evaluate the resulting astrocyte function, the electrical activity of astrocyte-neuron cocultures was compared with that of neuron monocultures in MEAs. Briefly, 120K iCell Gluta Neurons were interspersed with 30K iCell astrocytes or NPC astrocytes in 48 classic MEA plates (Axion Biosystems). Cells were fed and recorded using BrainPhys complete medium. On the day of recording, approximately 2–4 h before data acquisition, 50% of the spent medium was replaced with BrainPhys complete medium, and recording was performed for 5–10 min (300–600 s) to adequately capture network burst behavior. On day 23, neuronal activity was recorded as baseline, and then 20 μM l -glutamate was added to four wells for each of the three groups (iCell Gluta Neuron alone, iCell Gluta Neuron cocultured with iCell astrocytes, and iCell Gluta Neuron cocultured with NPC-derived astrocytes), and recordings were made again after 1 h.

[0187] Next, 31 media compositions were evaluated. Figure 4A shows the fold growth analysis of the 31 media matrices over a 14-day differentiation time course. Figure 4B shows the purity analysis at day 14 for astrocyte lineage-associated marker expression by flow cytometry. Figure 4C shows the compositions of the 11 media that performed best based on growth and purity in the comparison of the 31 media selected for further experimentation.

[0188] Figure 5A shows marker expression at day 35 of differentiation. Cells showed less than 1% CD15 expression, approximately 90-100% (e.g., greater than 95%, 96%, 97%, 98%, or 99%) CD56 expression, less than 20% (e.g., less than 15%, 10%, 5%, or 1%) SSEA4 expression, approximately 90-100% (e.g., greater than 95%, 96%, 97%, 98%, or 99%) NFIX expression, and less than 10% (e.g., less than 5% or 1%) GFAP expression. CD44 / S100b expression varied from approximately 15% to greater than 80%, and GLAST expression varied from approximately 2% to greater than 70%. Flow cytometry analysis of the comparison of the 11 media matrices is shown in Figure 5B, and cumulative fold expansion analysis of the 11 media matrices over 35 days of differentiation is shown in Figure 5C. Condition 14 showed approximately 28-fold growth, condition 16 showed approximately 36-fold growth, and condition 29 showed approximately 34-fold growth. The five media matrices (Figure 5E) were further evaluated by immunocytochemistry of D49 cells (Figure 5E). Glutamate uptake assays of D49 cells from the five media comparison studies were then performed (Figure 5D).

[0189] Further studies demonstrated the re-emergence of SSEA4+ as an astrocyte progenitor cell marker. Representative flow cytometry plots of surface CD56 / SSEA4 co-staining at D28 and intracellular CD44 / SSEA4 co-staining at both D28 and D35 in medium condition 14 are shown in Figure 6A. CD44 / SSEA4 cells increased from 15.8% at D28 to 31.3% at D35 in condition 14. The time course of surface SSEA4 expression and intracellular CD44 expression from D7 to D35 is shown in (Figure 6B) condition 3, (Figure 6C) condition 14, (Figure 6D) condition 16, (Figure 6E) condition 24, and (Figure 6F) condition 29. Medium conditions 14, 16, and 24 were continued until D42. In conditions 14, 16, and 24, SSEA4 expression increased from less than 20% to over 95% after day 35, while CD44 expression gradually increased from approximately 40% on day 14 to over 80% on day 35. Differential expression of SSEA4 and CD44 was measured by surface and intracellular staining at day 28 and day 35. (Figure 6G) Condition 14, (Figure 6H) Condition 16, (Figure 6I) Condition 24, and (Figure 6J) Condition 29 expression profiles are summarized. SSEA4-positive cells were over 60% on day 28 and over 80% on day 35 in conditions 14, 16, and 24. CD44 / SSEA4-positive cells were approximately 20-40%, e.g., approximately 25-30%.

[0190] Immunocytochemistry of astrocytes cultured in either Astro3 medium, AMM medium, or BrainPhys Complete medium 7 days after thawing is shown in Figure 7A. Glutamate uptake assay of astrocytes cultured in either Astro3 medium, AMM medium, or BrainPhys Complete medium 7 days after thawing is shown in Figure 7C. Quantified results from MEA data analysis are shown in Figure 7C.

[0191] To further evaluate the functionality of the resulting astrocytes, we compared the electrical activity of the astrocyte-neuron cocultures with that of neuron monocultures in MEAs. Luminex analysis of common astrocyte-secreted protein concentrations is shown in Figure 7D. Luminex analysis of cytokine- or LPS-induced responsive astrocyte-secreted protein concentrations is shown in Figure 7E. RNA-Seq analysis of common astrocyte markers is shown in Figure 7F.

[0192] [Table 1]

[0193] Four lots of astrocytes were thawed and cultured in Astro 3 medium for 7 days, followed by stimulation with basal medium for 24 hours. Supernatants were collected and analyzed on a FLEXMAP 3D instrument using a custom R&D Luminex assay according to the manufacturer's instructions. Astrocytes were able to robustly secrete IL-1ra after stimulation with IL-1α, IFN-γ, TNF-α, or their combinations (Figure 8A). Stimulation with IL-1α or TNF-α induced IL-1ra secretion by more than threefold. The combination of TNF-α with IL-1α or IL-1β resulted in an approximately 14-fold increase compared to unstimulated controls. The combination of IFN-γ and TNF-α resulted in the highest fold change compared to controls, with IL-1ra secretion exceeding 84-fold compared to controls. IL-1ra binds to the IL-1 receptor on the cell surface and blocks the binding of proinflammatory IL-1α and IL-1β.

[0194] Astrocytes were able to robustly secrete IL-6 after stimulation with IL-1α, TNF-α, or their combination (Figure 8B). IFN-γ alone or in combination with TNF-α, respectively, increased IL-6 secretion 32- and 39-fold compared to unstimulated controls. IL-α stimulation increased IL-6 secretion by more than 400-fold compared to controls. TNF-α combined with IL-1α or IL-1β increased IL-6 secretion by more than 3,300-fold compared to controls. IL-6 attracts proinflammatory T cells and promotes demyelination.

[0195] Astrocytes were able to robustly secrete IL-8 / CXCL8 after stimulation with IL-1α, TNF-α, or their combination (Figure 8C). TNF-α alone or in combination with IFN-γ, respectively, increased IL-8 secretion 898-fold and 665-fold compared to unstimulated controls. IL-α stimulation increased IL-8 secretion by more than 3,300-fold compared to controls. TNF-α combined with IL-1α or IL-1β increased IL-8 secretion by more than 12,000-fold compared to controls. IL-8 / CXCL8 is secreted by astrocytes and attracts proinflammatory immune cells after injury. This analyte also plays a role in the recruitment and differentiation of oligodendrocyte progenitor cells (OPCs) and promotes remyelination.

[0196] Astrocytes were able to robustly secrete IL-10 after stimulation with IL-1α, IFN-γ, TNF-α, or their combinations (Figure 8D). IFN-γ stimulation increased IL-10 secretion 161-fold compared to unstimulated controls. IL-α stimulation increased IL-10 secretion by more than 415-fold compared to controls. TNF-α alone or in combination with IL-1α, IL-1β, or IFN-γ increased IL-10 secretion by more than 1000-fold compared to controls. IL-10, secreted by astrocytes, reduces iNos activity and attenuates astrogliosis.

[0197] Astrocytes were able to robustly secrete CCL5 / RANTES after stimulation with IL-1α, TNF-α, or their combination (Figure 8E). IL-1α stimulation increased CCL5 / RANTES secretion 156-fold over unstimulated controls. TNF-α alone or in combination with IL-1α, IL-1β, or IFN-γ increased CCL5 / RANTES secretion more than 2500-fold compared to controls. CCL5 / RANTES regulates the movement of peripheral immune cells.

[0198] Astrocytes were able to robustly secrete CCL7 after stimulation with IL-1α, TNF-α, or their combination (Figure 8F). TNF-α stimulation increased CCL7 secretion 1184-fold over controls. IL-1α stimulation increased CCL7 secretion 2177-fold over unstimulated controls. TNF-α-stimulated IFN-γ increased CCL7 secretion 3745-fold over unstimulated controls. TNF-α combined with IL-1α or IL-1β increased CCL7 secretion more than 15,000-fold compared to controls. CCL7 is important for astrocyte-microglia interactions and induces microglial activation.

[0199] Astrocytes were able to robustly secrete CCL20 after stimulation with IL-1α, TNF-α, or their combination (Figure 8G). Stimulation with IL-1α or TNF-α alone increased CCL20 secretion by more than 18-fold compared to unstimulated controls. TNF-α combined with IL-1α, IL-1β, or IFN-γ increased CCL20 secretion by more than 100-fold compared to controls. CCL20 is secreted by astrocytes in response to proinflammatory stimuli and attracts T cells, B cells, and DCs.

[0200] Astrocytes were able to robustly secrete CXCL1 after stimulation with IL-1α, TNF-α, or their combination (Figure 8H). TNF-α alone or in combination with IFN-γ increased CXCL1 secretion by more than 9-fold compared to controls. Stimulation with IL-1α increased CXCL1 secretion by 84-fold compared to unstimulated controls. TNF-α combined with IL-1α or IL-1β increased CXCL1 secretion by more than 450-fold compared to controls. CXCL1 is secreted by astrocytes and recruits neutrophils to sites of infection and increases BBB permeability.

[0201] Astrocytes were able to robustly secrete CXCL2 after stimulation with IL-1α, TNF-α, or their combination (Figure 8I). TNF-α alone or in combination with IFN-γ increased CXCL2 secretion by more than 51-fold compared to controls. Stimulation with IL-1α increased CXCL2 secretion by 379-fold compared to unstimulated controls. TNF-α combined with IL-1α or IL-1β increased CXCL2 secretion by more than 1585-fold compared to controls. CXCL2 activates CXCR2, which is found on oligodendrocytes and OPCs, promoting OPC proliferation and differentiation.

[0202] Astrocytes were able to robustly secrete CXCL5 after stimulation with IL-1α, TNF-α, or their combination (Figure 8J). TNF-α alone or in combination with IFN-γ increased CXCL5 secretion by more than 47-fold compared to controls. Stimulation with IL-1α increased CXCL5 secretion by 161-fold compared to unstimulated controls. TNF-α combined with IL-1α or IL-1β increased CXCL5 secretion by more than 872-fold compared to controls. CXCL5 is secreted by astrocytes in response to injury and activates microglia, resulting in decreased microglial phagocytosis and inhibition.

[0203] [Table 2] JPEG2025525436000003.jpg206170

[0204] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes can be made in the methods and in the steps or series of steps of the methods described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents that are chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims. References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. Alexander et al., Proc. Nat. Acad. Sci. USA, 85:5092-5096, 1988. Ausubel et al.,Current Protocols in Molecular Biology,Greene Publ.Assoc.Inc.& John Wiley & Sons,Inc.,MA,1996.Blomer et al.,1997 Chen et al.,Nature Methods 8:424-429,2011. Ercolani et al., J. Biol. Chem., 263:15335-15341, 1988. Evans, et al., In: Cancer Principles and Practice of Oncology, Devita et al. (Eds.), Lippincott-Raven, NY, 1054-1087, 1997. Fechheimer et al., Proc Natl. Acad. Sci. USA, 84:8463-8467, 1987. International Publication No. 2012 / 149484 Brochure International Publication No. 02 / 016536 Brochure International Publication No. 03 / 016496 Brochure International Publication No. 2007 / 069666 Pamphlet International Publication No. 2007 / 069666 Pamphlet International Publication No. 98 / 30679 Brochure International Publication No. 98 / 30679 Brochure International Publication No. 98 / 53058 Brochure International Publication No. 98 / 53059 Brochure International Publication No. 98 / 53060 Brochure Karin et al. Cell, 36:371-379, 1989. Langle-Rouault et al., J. Virol., 72(7):6181-6185, 1998. Levitskaya et al., Proc. Natl. Acad. Sci. USA, 94(23):12616-12621, 1997. Ludwig et al., Nat. Biotechnol., 24:185-187, 2006b. Ludwig et al., Nat. Methods, 3:637-646, 2006a. Maniatis, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1988. Ng,Nuc.Acid Res.,17:601-615,1989. Pelletier and Sonenberg, Nature, 334(6180):320-325, 1988. Quitsche et al., J. Biol. Chem., 264:9539-9545, 1989. Richards et al., Cell, 37:263-272, 1984. Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd Ed. Cold Spring Harbor 1997. Takahashi et al., Cell, 131:861-872, 2007. U.S. Patent No. 4,683,202 U.S. Patent No. 5,556,954 U.S. Patent No. 5,925,565 U.S. Patent No. 5,928,906 U.S. Patent No. 5,935,819 U.S. Patent No. 6,103,470 U.S. Patent No. 6,140,081 U.S. Patent No. 6,416,998 U.S. Patent No. 6,453,242 U.S. Patent No. 6,534,261 U.S. Patent No. 7,442,548 U.S. Patent No. 7,598,364 U.S. Patent No. 7,682,828 U.S. Patent No. 7,989,425 U.S. Patent No. 8,058,065 U.S. Patent No. 8,071,369 U.S. Patent No. 8,129,187 U.S. Patent No. 8,268,620 U.S. Patent No. 8,278,620 U.S. Patent No. 8,546,140 U.S. Patent No. 8,546,140 U.S. Patent No. 8,741,648 US Patent Application Publication No. 2002 / 0076747 US Patent Application Publication No. 2002 / 0076747 US Patent Application Publication No. 20020055144 US Patent Application Publication No. 2003 / 0211603 US Patent Application Publication No. 2005 / 0064474 US Patent Application Publication No. 2005 / 0260186 US Patent Application Publication No. 2006 / 0104968 US Patent Application Publication No. 2006 / 0188987 US Patent Application Publication No. 2007 / 0218528 US Patent Application Publication No. 20090148425 US Patent Application Publication No. 20090246875 US Patent Application Publication No. 2010 / 0210014 US Patent Application Publication No. 2011 / 0301073 US Patent Application Publication No. 2011 / 0301073 US Patent Application Publication No. 2011 / 0301073 US Patent Application Publication No. 2012 / 0196360 US Patent Application Publication No. 20120276636 Yamanaka et al.,Cell,131(5):861-72,2007. Yu et al.,Science,318:1917,2007.

Claims

1. (a) obtaining a starting population of neural progenitor cells (NPCs) derived from iPSCs; (b) culturing the NPCs in the presence of at least one leukemia inhibitory factor (LIF) receptor ligand for a period of time sufficient to produce astrocyte precursor cells (APCs); and (c) further culturing the APCs in the presence of at least one LIF receptor ligand and a lipid concentrate for a period of time sufficient to produce a population of astrocytes.

1. An in vitro method for generating astrocytes from induced pluripotent stem cells (iPSCs), comprising:

2. 10. The method of claim 1, wherein the iPSCs are cultured in a serum-free defined medium.

3. 3. The method of claim 1 or 2, which is compliant with Good Manufacturing Practices (GMP).

4. 4. The method of claim 1, wherein one or more of steps (a) to (d) are performed under xeno-free, feeder-free, and / or conditioned medium-free conditions.

5. The method of any one of claims 1 to 3, wherein each of steps (a) to (d) is performed under xeno-free conditions, feeder-free conditions, and / or conditioned medium-free conditions.

6. The method of any one of claims 1 to 3, wherein each of steps (a) to (d) is carried out under limited conditions.

7. The method of any one of claims 1 to 6, wherein the iPSCs are human iPSCs.

8. Obtaining the starting population of NPCs includes: (a) culturing iPSCs on an extracellular matrix (ECM) protein-coated surface in the presence of a ROCK inhibitor; (b) further culturing the iPSCs in the absence of a ROCK inhibitor or blebbistatin; (c) pretreating the iPSCs in the presence of a GSK3 inhibitor; The method of any one of claims 1 to 7, comprising (d) differentiating the iPSCs into a population of NPCs.

9. 9. The method of claim 8, wherein the ECM protein is laminin, fibronectin, vitronectin, MATRIGEL™, tenascin, entactin, thrombospondin, elastin, gelatin, and / or collagen.

10. 9. The method of claim 8, wherein the ECM protein is a basement membrane extract (BME) purified from a mouse Engelbreth-Holm-Swarm tumor.

11. 9. The method of claim 8, wherein the ECM protein is MATRIGEL™, laminin, or vitronectin.

12. 9. The method of claim 8, wherein the extracellular matrix protein is MATRIGEL™.

13. The method of any one of claims 8 to 12, which does not involve inhibition of SMAD signaling.

14. The method according to any one of claims 8 to 13, wherein steps (a) and (b) are carried out under hypoxic conditions.

15. The method according to any one of claims 8 to 14, wherein the culture in steps (a) and (b) is further defined as an adherent two-dimensional culture.

16. 16. The method of any one of claims 8 to 15, wherein step (a) is for about 24 hours.

17. 17. The method of any one of claims 8 to 16, wherein step (b) is for about 48 hours.

18. The method according to any one of claims 8 to 17, wherein the ROCK inhibitor is H1152.

19. The method of any one of claims 8 to 18, wherein step (c) is carried out under normoxic conditions.

20. 20. The method of any one of claims 8 to 19, wherein step (c) is carried out for about 72 hours.

21. 21. The method of any one of claims 8 to 20, wherein the GSK3 inhibitor is CHIR99021, BIO, or SB-216763.

22. 21. The method of any one of claims 8 to 20, wherein the GSK3 inhibitor is CHIR99021.

23. 23. The method of any one of claims 8 to 22, wherein step (d) comprises forming aggregates in the presence of a ROCK inhibitor.

24. The method of any one of claims 8 to 23, wherein the cell culture is a three-dimensional (3D) culture.

25. 25. The method of any one of claims 8 to 24, wherein step (d) comprises culturing on an ultra-low attachment plate, a spinner, or a bioreactor.

26. 26. The method of any one of claims 8 to 25, wherein step (d) is for about 8 days.

27. The method of any one of claims 8 to 26, wherein the NPC expresses CD24, CD184, and CD271.

28. The method of any one of claims 8 to 27, further comprising detecting the expression of CD56, CD15, Sox1, nestin, β3-tubulin, microglobulin, and / or Pax-6 in the NPC population.

29. The method of any one of claims 8 to 28, wherein the population of NPCs is at least 70% positive for CD24 and nestin.

30. The method of any one of claims 8 to 29, wherein the NPC expresses Pax6 and nestin.

31. The method of any one of claims 8 to 30, wherein after step (b), the APCs have reduced expression of SSEA-4 and TRA-1-60 compared to the iPSCs.

32. The method of any one of claims 8 to 31, wherein the NPCs are cryopreserved.

33. The method of any one of claims 1 to 32, wherein the iPSCs are derived from a healthy donor.

34. 34. The method of any one of claims 1 to 33, wherein the iPSCs are derived from a diseased donor.

35. 35. The method of claim 34, wherein the disease is Alexander disease or leukodystrophy.

36. 36. The method of any one of claims 1 to 35, wherein the iPSCs comprise disruptions in TREM2, APOE, methyl-CpG binding protein 2 (MeCP2), and / or alpha-synuclein (SCNA).

37. 36. The method of any one of claims 1 to 35, wherein the astrocytes are terminal astrocytes positive for CD44, S100b, NFIX, GLAST, and / or GFAP.

38. The method of any one of claims 1 to 35, wherein the astrocytes are positive for SSEA4 and CD44.

39. 36. The method of any one of claims 1 to 35, wherein at least 30% of the astrocyte population is positive for SSEA4 and CD44.

40. 38. The method of claim 37, wherein the astrocytes exhibit functional glutamate uptake and / or neural network development.

41. 41. The method of any one of claims 1 to 40, wherein the at least one LIF receptor ligand is leukemia inhibitory factor protein (LIF), ciliary derived neurotrophic factor protein (CNTF), oncostatin-M protein (OSM), and / or cardiotrophin 1 (CT-1).

42. 42. The method of any one of claims 1 to 41, wherein step (b) further comprises culturing in the presence of a lipid concentrate, EGF, JAGG1, and / or DLL1.

43. 43. The method of any one of claims 1 to 42, wherein step (b) comprises culturing in the presence of LIF, CNTF, OSM, JAGG1, a lipid concentrate, and EGF.

44. 43. The method of any one of claims 1 to 42, wherein step (b) comprises culturing in the presence of LIF, CNTF, OSM, DLL1, a lipid concentrate, and EGF.

45. 43. The method of any one of claims 1 to 42, wherein step (b) comprises culturing in the presence of LIF, CNTF, OSM, JAGG1, DLL1, a lipid concentrate, and EGF.

46. 43. The method of any one of claims 1 to 42, wherein step (b) comprises culturing in the presence of LIF, CNTF, OSM, JAGG1, CT1, a lipid concentrate, and EGF.

47. 43. The method of any one of claims 1 to 42, wherein step (b) comprises culturing in the presence of LIF, CNTF, OSM, DLL1, CT1, a lipid concentrate, and EGF.

48. The method of any one of claims 1 to 47, wherein the APCs are cultured in the presence of LIF, CNTF, oncostatin-M, and / or CT-1.

49. The method of any one of claims 1 to 48, wherein the APCs are cultured in the presence of LIF and CNTF.

50. 50. The method of any one of claims 41 to 49, wherein LIF, CNTF, oncostatin-M, and / or CT-1 are present at a concentration of about 1 to 20 ng / mL.

51. 50. The method of any one of claims 41 to 49, wherein LIF, CNTF, oncostatin-M, and / or CT-1 are present at a concentration of about 10 ng / mL.

52. 52. The method of any one of claims 1 to 51, wherein step (b) comprises culturing NPCs on a surface coated with Geltrex.

53. 53. The method of any one of claims 1 to 52, wherein step (b) is for about 2 weeks.

54. 54. The method of any one of claims 1 to 53, wherein step (c) comprises culturing the cells on a surface coated with vitronectin.

55. 55. The method of any one of claims 1 to 54, wherein the lipid concentrate is a chemically defined lipid concentrate.

56. 56. The method of claim 55, wherein the chemically defined lipid concentrate comprises saturated and unsaturated fatty acids.

57. 56. The method of claim 55, wherein the chemically defined lipid concentrate comprises arachidonic acid, cholesterol, DL-α-tocopherol acetate, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid, and stearic acid.

58. 56. The method of any one of claims 1 to 55, wherein step (c) is for about 4 to 7 weeks.

59. 59. The method of any one of claims 1 to 58, wherein the astrocytes express CD44, NFIX, and / or GFAP.

60. 60. The method of any one of claims 1 to 59, wherein the astrocytes express CD56, S100B, CD44, GFAP, NFIX, and / or GLAST.

61. 61. The method of any one of claims 1 to 60, wherein the population of astrocytes is at least 80% positive for S100B, CD44, and / or NFIX.

62. 62. The method of any one of claims 1 to 61, wherein the population of astrocytes is at least 30% positive for CD56 and / or GFAP.

63. 63. The method of any one of claims 1 to 62, wherein the astrocytes maintain network activity and take up excess glutamate.

64. 64. The method of any one of claims 1 to 63, wherein the astrocytes secrete IL-1ra, IL-6, IL-8 (CXCL8), IL-10, CCL5 (RANTES), CCL7, CCL20, CXCL1, CXCL2, and / or CXCL5 after stimulation with IL-1α and / or TNFα.

65. 64. The method of any one of claims 1 to 63, wherein the astrocytes secrete IL-1ra, IL-6, IL-8 (CXCL8), IL-10, CCL5 (RANTES), CCL7, CCL20, CXCL1, CXCL2, and CXCL5 after stimulation with IL-1α and / or TNFα.

66. A pharmaceutical composition comprising a population of astrocytes produced according to any one of claims 1 to 63 and a pharmaceutically acceptable carrier.

67. 67. The composition of claim 66, wherein the astrocyte cell population is at least 30% positive for SSEA4 and CD44.

68. 67. The composition of claim 66, wherein the astrocyte cell population is at least 45% positive for SSEA4 and CD44.

69. A composition comprising a population of astrocytes that are at least 70% positive for S100B, CD44, and / or NFIX, wherein the population of astrocytes is differentiated from iPSCs.

70. 70. The composition of claim 69, wherein the astrocyte cell population is at least 80% positive for S100B, CD44, and / or NFIX.

71. 70. The composition of claim 69, wherein the astrocyte cell population is at least 30% positive for SSEA4 and CD44.

72. 70. The composition of claim 69, wherein the astrocyte cell population is at least 45% positive for SSEA4 and CD44.

73. 70. The composition of claim 69, further comprising a neuron.

74. A method for screening a test compound, comprising introducing the test compound into a population of astrocytes according to any one of claims 66 to 73.

75. 75. The method of claim 74, further comprising measuring astrocyte viability and / or function.

76. 74. Use of a composition according to any one of claims 66 to 73 as a model for a neurodegenerative disease or injury.

77. A co-culture comprising astrocytes and / or neural progenitor cells, endothelial cells, and pericytes produced by the method of any one of claims 1 to 63.

78. 78. Use of the co-culture of claim 77 to mimic human brain development or neurodegeneration.

79. A kit comprising astrocytes produced by the method of any one of claims 1 to 63.

80. 80. The kit of claim 79, further comprising endothelial cells and / or pericytes.

81. 78. A model of neurodegeneration comprising the co-culture of claim 77.

82. A method for treating a neurodegenerative disease, comprising administering to a subject an effective amount of an astrocyte composition according to any one of claims 66 to 73.

83. 83. The method of claim 82, wherein the disease is Alexander disease or leukodystrophy.

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