IPSC-derived astrocytes and methods of use thereof

Abstract

The present disclosure provides methods of generating neural precursor cells having glial preference from induced pluripotent cells and further differentiating the neural precursor cells into astrocytes. Further provided herein are methods of screening assays using astrocytes, models that mimic the human brain, and cell therapies.

Description

[0001] [Priority claim]

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 356,787, filed on June 29, 2022, the entire contents of which are hereby incorporated by reference. [Background of the invention]

[0004] [1. Field]

[0005] The present invention relates generally to the fields of molecular biology and medicine. More specifically, it relates to a method for differentiating induced pluripotent stem cells to produce astrocytes.

[0006] [2. Description of related technologies]

[0007] Astrocytes play a central role in brain development and play important roles in the central nervous system (CNS) by maintaining brain homeostasis, providing metabolic support to neurons, regulating connectivity of neural circuits, and controlling blood flow as a component of the blood-brain barrier. Astrocytes undergo transformation after injury or develop disease after injury (e.g., reactive astrogliosis). These reactive astrocytes play a role in the development and progression of many neurological diseases. [Summary of the Invention]

[0008] Certain embodiments of the present disclosure provide an in vitro method for generating astrocytes from induced pluripotent stem cells (iPSCs), comprising: (a) obtaining a starting population of neural precursor cells (NPCs) derived from iPSCs; (b) culturing the NPCs in the presence of at least one leukemia inhibitory factor (LIF) receptor ligand for a time sufficient to generate astrocyte progenitor cells (APCs); and (c) further culturing the APCs in the presence of at least one LIF receptor ligand and a lipid concentrate for a time sufficient to generate a population of astrocytes.

[0009] In some aspects, iPSC is cultured in a serum-free medium with defined components. In some aspects, the method complies with Good Manufacturing Practice (GMP). In some aspects, one or more of steps (a) to (c) are carried out under xeno-free conditions, feeder-free conditions and / or unconditioned medium conditions. In some aspects, each of steps (a) to (d) is carried out under xeno-free conditions, feeder-free conditions and / or unconditioned medium conditions. In some aspects, each of steps (a) to (d) is performed under determined conditions. In some aspects, iPSC is human iPSC.

[0010] In some aspects, 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 the iPSCs in the absence of a ROCK inhibitor or bubitastatin; (c) pretreating the iPSCs in the presence of a GSK3 inhibitor; (d) differentiating the iPSCs into a population of NPCs. In some aspects, the ECM protein is: laminin, fibronectin, vitronectin, matrigel TM , tenascin, entactin, thrombospondin, elastin, gelatin and / or collagen. In some aspects, the ECM protein is basement membrane extract (BME) purified from mouse Engelbreth-Holm-Swarm tumor. In some aspects, the ECM protein is matrigel TM , laminin or vitronectin. In certain aspects, the extracellular matrix protein is matrigel TM In certain aspects, the methods do not comprise inhibition of SMAD signaling.

[0011] In a specific aspect, steps (a) to (b) are carried out under hypoxic conditions. In some aspects, the cultivation of steps (a) to (b) is further defined as adherent 2-dimensional culture. In some aspects, step (a) lasts for about 24 hours. In a specific aspect, step (b) lasts for about 48 hours. In some aspects, the ROCK inhibitor is H1152. In some aspects, step (c) is carried out under normoxic conditions. In some aspects, step (c) performs about 72 hours. In a specific aspect, the GSK3 inhibitor is CHIR99021, BIO or SB-216763. In a specific aspect, the GSK3 inhibitor is CHIR99021.

[0012] In some aspects, step (d) includes forming aggregates in the presence of a ROCK inhibitor. In some aspects, cell culture is three-dimensional (3D) culture. In some aspects, step (d) includes culturing on an ultra-low attachment plate, a rotator or a bioreactor. In some aspects, step (d) lasts for about 8 days (e.g., 5, 6, 7, 8, 9 or 10 days).

[0013] In some aspects, NPC expresses CD24, CD184 and CD271. In some aspects, the method also includes detecting the expression of CD56, CD15, Sox1, Nestin, β3-tubulin, microglobulin and / or Pax-6 in the NPC group. In some aspects, at least 70% (e.g., 75%, 80%, 85% or 90%) of the NPC group is positive for CD24 and Nestin. In some aspects, NPC expresses Pax6 and Nestin. In some aspects, after step (b), compared with iPSC, SSEA-4 and TRA-1-60 expression of APC is reduced. In some aspects, NPC is cryopreserved.

[0014] In some aspects, iPSC is derived from a healthy donor. In some aspects, iPSC is from a donor with a disease. In some aspects, the disease is Alexander disease or leukodystrophy. In some aspects, iPSC includes destruction of TREM2, APOE, methyl-CpG binding protein 2 (MeCP2) and / or alpha-synuclein (SCNA). In some aspects, astrocytes are terminal astrocytes positive for CD44, S100b, NFIX, GLAST and / or GFAP. In some aspects, astrocytes are SSEA4 and CD44 positive. In some aspects, at least 30% (e.g., 35%, 40%, 45% or 50%) of astrocyte populations are SSEA4 and CD44 positive. In some aspects, astrocytes have functional glutamate uptake and / or the development of neural networks.

[0015] In some aspects, at least one LIF receptor ligand is a leukemia inhibitory factor protein (LIF), a cilia-derived neurotrophic factor protein (CNTF), an oncostatin-M protein (OSM) and / or a cardiotrophic factor 1 (CT-1). In some aspects, step (b) further comprises culturing in the presence of a lipid concentrate, EGF, JAGG1 and / or DLL1. In some aspects, step (b) comprises culturing in the presence of LIF, CNTF, OSM, JAGG1, a lipid concentrate and EGF. In some aspects, step (b) comprises culturing in the presence of LIF, CNTF, OSM, DLL1, a lipid concentrate and EGF. In some aspects, step (b) comprises culturing in the presence of LIF, CNTF, OSM, JAGG1, DLL1, a lipid concentrate and EGF. In some aspects, step (b) comprises culturing in the presence of LIF, CNTF, OSM, JAGG1, DLL1, a lipid concentrate and EGF. In some aspects, step (b) comprises culturing in the presence of LIF, CNTF, OSM, JAGG1, DLL1, a lipid concentrate and EGF. In certain aspects, step (b) comprises culturing in the presence of LIF, CNTF, OSM, DLL1, CT1, a lipid concentrate, and EGF.

[0016] In some aspects, the APCs are cultured in the presence of LIF, CNTF, Oncostatin-M and / or CT-1. In some aspects, the APCs are cultured in the presence of LIF and CNTF. In some aspects, the concentration of LIF, CNTF, Oncostatin-M and / or CT-1 is about 1 to 20 ng / mL (e.g., 1, 5, 10, 15 or 20 ng / mL). In specific aspects, the concentration of LIF, CNTF, Oncostatin-M and / or CT-1 is about 10 ng / mL.

[0017] In some aspects, step (b) comprises culturing NPC on a Geltrex-coated surface. In some aspects, step (b) lasts 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).

[0018] In certain aspects, step (c) comprises culturing the cells on a vitronectin-coated surface.

[0019] In some aspects, the lipid concentrate is a lipid concentrate determined by chemical composition. In some aspects, the lipid concentrate determined by chemical composition comprises saturated fatty acids and unsaturated fatty acids. In specific aspects, the lipid concentrate determined by chemical composition comprises arachidonic acid, cholesterol, DL-α-tocopheryl acetate, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid and / or stearic acid.

[0020] In some aspects, step (c) lasts for about 4 to 7 weeks (e.g., 4, 5, 6 or 7 weeks). In some aspects, astrocytes express CD44, NFIX and / or GFAP. In some aspects, astrocytes express CD56, S100B, CD44, GFAP, NFIX and / or GLAST. In some aspects, at least 80% (e.g., 80%, 85%, 90% or 95%) of astrocyte populations are positive for S100B, CD44 and / or NFIX. In some aspects, at least 30% (e.g., 30%, 35%, 40%, 45% or 50%) of astrocyte populations are positive for CD56 and / or GFAP. In particular aspects, astrocytes maintain network activity and absorb excess glutamate. In certain aspects, astrocytes secrete IL-1ra, IL-6, IL-8 (CXCL8), IL-10, CCL5 (RANTES), CCL7, CCL20, CXCL1, CXCL2 and / or CXCL5 upon stimulation with IL-1α and / or TNFα.

[0021] Further provided herein is a pharmaceutical composition comprising a population of astrocytes produced according to the present embodiment or aspects thereof and a pharmaceutically acceptable carrier. In certain aspects, at least 30% (e.g., 30%, 35%, 40%, 45% or 50%) of the astrocyte population are positive for SSEA4 and CD44. In particular, in certain aspects, at least 45% of the astrocyte population are positive for SSEA4 and CD44.

[0022] Another embodiment provides a composition comprising at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95% or 99%) astrocyte groups that are positive for S100B, CD44 and / or NFIX, wherein the astrocyte group is differentiated from iPSC. In some aspects, at least 80% of the astrocyte group is positive for S100B, CD44 and / or NFIX. In some aspects, at least 30% of the astrocyte group is positive for SSEA and CD44. In some aspects, at least 45% of the astrocyte group is positive for SSEA and CD44. In some aspects, the composition also includes neurons.

[0023] A further embodiment provides a method for screening a test compound, comprising introducing the test compound into the astrocyte population of this embodiment or aspects thereof. In certain aspects, the method further comprises measuring the viability and / or function of the astrocytes.

[0024] Another embodiment provides use of a composition of this embodiment or aspects thereof as a model of neurodegenerative disease or injury.

[0025] In yet another embodiment, a co-culture is provided, comprising astrocytes and / or neural precursor cells, endothelial cells and pericytes produced by the present embodiment or aspects thereof. Also provided herein is the use of the co-culture of the present embodiment or aspects thereof for simulating human brain development or neurodegeneration.

[0026] Another embodiment provides a kit comprising astrocytes produced by the method of the present embodiment or aspects thereof. In some aspects, the kit further comprises endothelial cells and / or pericytes. Further provided herein is a neurodegeneration model comprising the co-culture of the present embodiment or aspects thereof.

[0027] Also provided herein is a method for treating a neurodegenerative disease, comprising administering to a subject an effective amount of an astrocyte composition of the present embodiment or aspects thereof. In certain aspects, the disease is Alexander disease or leukodystrophy.

[0028] Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific embodiments, although indicating the preferred embodiments of the present invention, are given only by way of illustration, because various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description.

Brief Description of the Figures

[0030] The following drawings constitute part of this specification and are included to further demonstrate certain aspects of the present invention. The present invention may be better understood by referring to one or more of these drawings in combination with the detailed description of specific embodiments given herein.

[0031] Figure 1 : Schematic diagram depicting the method of generating neural progenitor cells (NPCs).

[0032] Figures 2A to 2D: Characterization of NPCs by flow cytometry. Cell surface (Figures 2A and 2C) and intracellular antigens (Figures 2B to 2D) were stained for intracellular antigens. Pluripotency markers SSEA-4 and TRA-1-60 decreased significantly during the 14-day differentiation process, while neural progenitor cell (NPC) markers such as CD24, CD184, and CD271 began to appear throughout the differentiation process and increased over time. NPCs from all 5 tested lines showed high expression of neural progenitor markers Pax6 and Nestin. Expression of neural progenitor markers SOX1, doublecortin (DCX), and pan-neuronal marker β3-tubulin (Tuj) was observed in donors 1279 and 1279MeCP2, but expression was low or absent in 11995, 21527, 1434, and 1434APOE lines.

[0033] Figures 3A-3F: Activation of the LIF receptor is sufficient to distinguish astrocytes from NPCs. (Figure 3A) A simplified schematic diagram of the differentiation procedure for generating astrocytes from NPCs, showing the amount and length of time of application of LIF receptor agonists. (Figure 3B) Phase contrast images of cells seeded on a PLO / laminin matrix (B top) at day 60, and ICC staining of astrocyte markers CD44, NFIX, and GFAP proteins at days 35 and 80 after differentiation. It can be seen that a relatively small proportion of cells express CD44 and GFAP at day 35 of the process, while at day 80, CD44 expression appears to be completely uniform across the population, and GFAP expression increases dramatically across the population. (Figure 3C) Quantification of the percentage of cells positive for several astrocyte markers at day 70 using flow cytometry. High expression of astrocyte and astrocyte progenitor markers S100B, NFIX, and CD44 demonstrates the ability of this process to generate highly pure astrocytes. GFAP and GLAST expression indicated that many of these cells expressed functional markers of mature astrocytes. Low expression of CD15 and SSEA4 indicated that multipotent cells were no longer observed in the culture. (Figure 3D) The theoretical cumulative yield during differentiation showed that the cell growth was significantly more than 10,000-fold, making this protocol cost-effective. (Figure 3E) Time course glutamate uptake efficiency. Glutamate uptake was performed 7 days after seeding 96-well plates on Matrigel. 20 μM glutamate was added for 60 minutes 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 to the corresponding +TBOA sample. (Figure 3F) Microelectrode analysis (MEA) raster plots of different cultures before (baseline) and after glutamate application. 120000iCell Gluta neurons were cultured alone or co-cultured with 20000iCell astrocytes or NPC astrocytes in BrainPhys medium for 23 days. Synchronous bursting activity was seen in all conditions, indicating the formation of mature synaptic networks. The number of action potentials observed in network bursts observed in co-cultures containing either iCell Astrocytes or NPC Astrocytes was higher compared to iCellGlutaNeuron monocultures, showing an effect on astrocyte co-culture-induced network formation. Additional application of exogenous glutamate was sufficient to block all synchronous bursting in iCell GlutaNeuron monocultures, but not in co-culture samples, indicating increased robustness of the cultures to external perturbations.

[0034] Figures 4A to 4C :31 kinds of culture medium matrix experiments. ( Figure 4A) Fold expansion analysis was performed on 31 culture media to determine the potential of each culture media to induce NPC proliferation over the 14-day differentiation process. Culture media 5, 6, 7, 8, 13, 21, 22, 23, 30, and 31 showed an almost complete lack of cells at the end of day 14 and were therefore excluded from further analysis. Figure 4B ) Purity analysis on day 14 was performed by flow cytometry for expression of astrocyte lineage-related markers. Samples were collected on day 14 for 31 media tested, but Figure 4A The exception was a sample that showed a complete loss of cells in . Samples from all tested cultures showed a lack of the pluripotency marker SSEA4 and nearly uniform expression of the astrocyte lineage marker NFIX. Although co-expression of astrocyte progenitor markers was higher in cultures 10, 14, 16, 24, and 29, neural progenitor markers remained largely unchanged between samples. Figure 4C ) Of the 31 media compared, 11 media components had the best performance based on expansion rate and purity and were selected for further experiments. These were media 2, 3, 4, 10, 12, 14, 16, 18, 24, 26, and 29.

[0035] Figures 5A to 5E :11 and 5 culture medium matrix experiments. ( Figure 5A ) Flow cytometry analysis of 11 media was performed to determine the best combination of proliferation and astrocyte progenitor marker expression at D35 in this process, as shown in Figure 4. Almost uniform expression of the astrocyte lineage marker NFIX can be observed in all conditions (>90% of cells expressed NFIX in all media conditions except media 18), as well as the lack of pluripotency marker SSEA4 (<10% SSEA4 expression in all media conditions, except media 14, 16, 24 and 29, where expression was less than 20%) and NPC marker CD15 (CD15 expression was less than 5% in all conditions). It can be clearly seen that certain conditions upregulated the expression of astrocyte markers CD44, S100B and GLAST (media conditions 14, 16, 24 and 29 showed >75% CD44 / S100B co-expressing cells, and media 14, 16 and 24 showed >60% GLAST expression). These conditions use media 14, 16, 24, and 29 and are therefore most effective in specifying the astrocyte lineage of NPC cells. Figure 5B ) Cumulative fold expansion analysis of 11 media matrices over 35 days of differentiation. It can be seen that media 14, 16, 24, and 29 have significantly higher proliferation through D35 compared to the other media conditions, with more than 25-fold expansion during this period. This is consistent with Figure 5AThis, combined with the higher expression of astrocyte markers in the culture medium, suggests that these media are clearly optimal for inducing robust differentiation of astrocytes from NPC cells. Figure 5C ) Immunocytochemistry of D49 cells from a 5-medium matrix experiment. Astrocyte progenitor and astrocyte markers NFIX and CD44 can be seen to be increased in cells differentiated using medium 14, 16, 24, and 29 compared to the control medium 3. Expression of the mature astrocyte marker GFAP was robustly expressed in cells differentiated using medium 14, 16, 24, and 29, while very little GFAP was observed in cells differentiated using medium 3. ( Figure 5D )Glutamate uptake assay of D49 cells from a 5-medium comparison study. Glutamate uptake is a common and well-recognized indicator of astrocyte function in vitro. The glutamate transporter inhibitor TBOA was used as a control to confirm that the removal of glutamate from the culture 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 show a decrease in glutamate measured in the culture medium after 1 hour of incubation. Cells differentiated using media 3 were unable to take up glutamate significantly. ( Figure 5E ) Overview of the top five media out of a comparison of 11 for further investigation.

[0036] Fig. 6A ~6J: Reemergence of SSEA4+ as a marker of astrocyte progenitor cells. ( Fig. 6A ) Representative flow cytometry plots of surface CD56 / SSEA4 co-staining at D28 and intracellular CD44 / SSEA4 co-staining at D28 and D35 in medium 14 condition. The emergence of cells co-expressing the astrocyte markers CD44 and SSEA4, a classical marker of multipotent cells, is demonstrated. This marker combination on astrocytes has not been previously demonstrated. Time course of surface SSEA4 expression and intracellular CD44 expression from D7 to D35, (Figure 6B) condition 14, (Figure 6D) condition 16, (Figure 6E) condition 24 and (Figure 6F) condition 29. After D35, the emergence of a CD44 and SSEA4 co-positive population can be observed in astrocytes differentiated from medium conditions 14, 16 and 24, where expression increases from less than 20% on D35, but increases to more than 90% by day 42. This was not observed in condition 29 or control cells differentiated using medium 3. Media conditions 14, 16 and 24 were continued to D42. Differential expression of SSEA4 and CD44 by surface and intracellular staining at D28 and D35. (FIG. 6G) Summary of expression profiles for condition 14, (FIG. 6H) condition 16, (FIG. 6I) condition 24 and (FIG. 6J) condition 29.

[0037] Figures 7A-7C: Post-thaw optimization and functional analysis. (Figure 7A) Immunocytochemistry of astrocytes cultured 7 days post-thaw in Astro3 medium, AMM, or BrainPhys complete medium. All media used post-thaw were equally able to support the expression of astrocyte marker genes GFAP, CD44, and NFIX. (Figure 7B) Glutamate uptake assay of astrocytes cultured 7 days post-thaw in Astro3 medium, AMM, or BrainPhys complete medium. All media were equally able to support astrocyte function, and astrocytes cultured in all media showed robust glutamate uptake function. (Figure 7C) Quantitative results of MEA data analysis. iCell GlutaNeurons were cultured alone or co-cultured with NPC astrocytes. An increase in network burst frequency can be seen in neurons co-cultured with NPC astrocytes (Gluta+C1).

[0038] Figures 8A to 8J : Astrocytes from four batches were thawed and cultured in Astro 3 medium for 7 days and then stimulated in basal medium for 24 hours. Supernatants were collected and assayed using R&D's custom Luminex assay on a FLEXMAP 3D instrument according to the manufacturer's instructions. Each condition was run in duplicate and the mean of all four batches was compared to the control (unstimulated) condition to calculate the fold change. Error bars are ±1SEM. ( Fig. 8A )Astrocytes are capable of robust secretion of IL-1ra following stimulation with IL-1α, IFN-γ, TNF-α, or a combination thereof. Stimulation with IL-1α or TNF-α resulted in a greater than 3-fold increase in the induction of IL-1ra secretion. Combining TNF-α with IL-1α or IL-1β resulted in an approximately 14-fold increase over unstimulated controls. The combination of IFN-γ with TNF-α produced the highest fold change over control, with greater than 84-fold increase in IL-1ra secretion compared to control. IL-1ra binds to the cell surface IL-1 receptor, blocking the binding of proinflammatory IL-1α and IL-1β. ( Figure 8B )Astrocytes are capable of robust secretion of IL-6 following stimulation with IL-1α, TNF-α, or a combination thereof. IFN-γ alone or in combination with TNF-α resulted in a 32-fold and 39-fold increase in IL-6 secretion, respectively, compared to unstimulated controls. IL-α stimulation resulted in a greater than 400-fold increase in IL-6 secretion compared to controls. TNF-α in combination 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 are capable of robust secretion of IL-8 / CXCL8 following stimulation with IL-1α, TNF-α, or a combination thereof. TNF-α alone or in combination with IFN-γ resulted in an 898-fold and 665-fold increase in IL-8 secretion, respectively, compared to unstimulated controls. IL-α stimulation resulted in a greater than 3,300-fold increase in IL-8 secretion compared to controls. TNF-α in combination 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 to attract proinflammatory immune cells following injury. The analyte also plays a role in the recruitment and differentiation of oligodendrocyte precursor cells (OPCs) to promote remyelination. ( Fig.8D )Astrocytes are capable of robust IL-10 secretion following stimulation with IL-1α, IFN-γ, TNF-α, or a combination thereof. IFN-γ stimulation resulted in a 161-fold increase in IL-10 secretion over unstimulated controls. IL-α stimulation resulted in a greater than 415-fold increase in IL-10 secretion 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 is secreted by astrocytes to reduce iNos activity and decrease astrogliosis. ( Fig. 8E ) Astrocytes are able to robustly secrete CCL5 / RANTES after stimulation with IL-1α, TNF-α, or their combination. IL-1α stimulation resulted in a 156-fold increase in CCL5 / RANTES secretion over 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 controls the motility of peripheral immune cells. ( Figure 8F) Astrocytes are able to robustly secrete CCL7 after stimulation with IL-1α, TNF-α, or a combination thereof. TNF-α stimulation resulted in a 1184-fold increase in CCL7 secretion compared to controls. IL-1α stimulation resulted in a 2177-fold increase in CCL7 secretion compared to unstimulated controls. IFN-γ and TNF-α stimulation 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 are able to robustly secrete CCL20 after stimulation with IL-1α, TNF-α, or a combination thereof. Stimulation with IL-1α or TNF-α alone increased CCL20 secretion by more than 18-fold compared to unstimulated controls. Compared with the control group, the combination of TNF-α with IL-1α, IL-1β, or IFN-γ increased CCL20 secretion by more than 100-fold. CCL20 is secreted by astrocytes in response to proinflammatory stimuli and attracts T cells, B cells, and DCs. ( Figure 8H )Astrocytes are capable of robust secretion of CXCL1 following stimulation with IL-1α, TNF-α, or a combination thereof. TNF-α alone or in combination with IFN-γ resulted in >9-fold greater CXCL1 secretion compared to controls. Stimulation with IL-1α increased CXCL1 secretion 84-fold compared to unstimulated controls. TNF-α in combination with IL-1α or IL-1β increased CXCL1 secretion >450-fold compared to controls. CXCL1 is secreted by astrocytes, recruits neutrophils to sites of infection, and increases BBB permeability. ( Figure 8I )Astrocytes are capable of robust secretion of CXCL2 following stimulation with IL-1α, TNF-α, or a combination thereof. TNF-α alone or in combination with IFN-γ resulted in over 51-fold increase in CXCL2 secretion compared to controls. IL-1α stimulation increased CXCL2 secretion 379-fold compared to unstimulated controls. TNF-α in combination with IL-1α or IL-1β increased CXCL2 secretion over 1585-fold compared to controls. CXCL2 activates CXCR2 found on oligodendrocytes and OPCs, thereby increasing OPC proliferation and differentiation. ( Figure 8J) Astrocytes are able to robustly secrete CXCL5 after stimulation with IL-1α, TNF-α, or their combination. TNF-α alone or in combination with IFN-γ resulted in over 47-fold more CXCL5 secretion than controls. Stimulation with IL-1α increased CXCL5 secretion 161-fold compared to unstimulated controls. TNF-α in combination with IL-1α or IL-1β increased CXCL5 secretion over 872-fold compared to controls. CXCL5 is secreted by astrocytes in response to injury and activates microglia, resulting in reduced microglial phagocytosis and inhibition. [Specific implementation method]

[0039] In certain embodiments, the present disclosure provides methods for producing neural precursor cells (NPCs) with a glial cell preference, also referred to herein as iPSC-derived glial cell progenitor cells, for producing astrocytes. In particular aspects, the method is a defined serum-free method for generating astrocytes, e.g., for preclinical and / or clinical applications.

[0040] In this study, NPCs with a glial bias were generated from multiple iPSC lines and placed in a chemically defined astrocyte differentiation medium to efficiently generate terminal astrocytes. Terminal astrocytes express key astrocyte markers such as CD44, NFIX, and GFAP, and demonstrate glutamate uptake and promote the development of neuronal networks. In process marker analysis, the reappearance of SSEA4 (a pluripotent stem cell marker) was identified, which was co-expressed with the astrocyte progenitor marker CD44. The current study further demonstrates that terminal astrocytes can be successfully cryopreserved and used for multi-cell detection-specific applications, preclinical applications, or clinical applications before or after cryopreservation. Further embodiments provide methods for identifying agents that regulate astrocyte viability or function, and a tri-culture kit for simulating neurodegeneration. Astrocytes produced 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 the present method can be used for disease modeling, drug discovery and / or regenerative medicine.

[0041] I. Definition

[0042] As used in the specification herein, "a" or "an" may mean one or more. As used in the claims herein, when used in conjunction with the word "comprising", the word "a" or "an" may mean one or more.

[0043] The term "or" is used in the claims to mean "and / or" unless explicitly stated to refer only to alternatives or the alternatives are mutually exclusive, although the present disclosure supports a definition referring only to alternatives and "and / or." As used herein, "another" may mean at least one second or longer.

[0044] The term "essentially" should be understood to mean that the methods or compositions include only the specified steps or materials, as well as those that do not materially affect the basic and novel characteristics of such methods and compositions.

[0045] As used herein, a composition or culture medium that is "substantially free" of a particular substance or material contains ≤30%, ≤20%, ≤15%, more preferably ≤10%, even more preferably ≤5%, or most preferably ≤1% of the substance or material.

[0046] As used herein, the terms "substantially" or "approximately" may be applied to modify any quantitative comparison, value, measurement, or other representation that may permissibly vary without resulting in a change in the basic function to which it is related.

[0047] The term "about" generally means within the standard deviation of the stated value as determined using standard analytical techniques for measuring the stated value. These terms may also be used by citing ± 5% of the stated value.

[0048] As used herein, "substantially free," with respect to a particular component, is used herein to mean that none of the specified component is purposely formulated into the composition and / or is present only as a contaminant or in trace amounts. Thus, the total amount of the specified component resulting from any accidental contamination of the composition is much less than 0.05%, and preferably less than 0.01%. The most preferred composition is one in which standard analytical methods cannot detect any amount of the specified component.

[0049] As used herein, "feeder-free" or "feeder-independent" refers to cultures supplemented with cytokines and growth factors (e.g., TGFβ, bFGF, LIF) as a substitute for 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 animal-based matrices (e.g., Matrigel). TM ) or grown on matrices such as fibronectin, collagen, or vitronectin. These methods allow human stem cells to remain in a largely undifferentiated state without the need for a “feeder layer” of mouse fibroblasts.

[0050] A "feeder layer" is defined herein as a coating of cells, such as on the bottom of a culture dish. Feeder cells can release nutrients into the culture medium and provide a surface to which other cells (such as pluripotent stem cells) can attach.

[0051] The terms "defined" or "completely defined," when used in relation to a culture medium, extracellular matrix, or culture condition, refer to a culture medium, extracellular matrix, or culture condition in which the chemical composition and amounts of substantially all components are known. For example, a defined medium does not contain factors of unknown composition, such as fetal bovine serum, bovine serum albumin, or human serum albumin. Typically, a defined medium comprises a basal medium (e.g., Dulbecco's Modified Eagle Medium (DMEM), F12, or Roswell Park Memorial Institute Medium (RPMI) 1640, which contains amino acids, vitamins, inorganic salts, buffers, antioxidants, and an energy source) supplemented with recombinant albumin, chemically defined lipids, and recombinant insulin. An example of a completely defined medium is Essential 8 TM Culture medium.

[0052] For culture media, extracellular matrices or culture systems used for human cells, the term "xeno-free (XF)" refers to the condition that the materials used are not of non-human animal origin.

[0053] "Treatment" or "treatment" includes (1) inhibiting a subject or patient from experiencing or displaying pathology or symptoms of a disease (e.g., arresting further development of the pathology and / or symptomology), (2) ameliorating a disease in a subject or patient who is experiencing or displaying pathology or symptoms of a disease (e.g., reversing the pathology and / or symptoms), and / or (3) achieving any measurable reduction in a disease in a subject or patient who is experiencing or displaying pathology or symptoms of a disease.

[0054] "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 susceptible to the disease but does not yet experience or display any or all of the pathology or symptoms of the disease, and / or (2) slowing the onset of the pathology or symptoms of a disease in a subject or patient who may be at risk and / or susceptible but does not yet experience or display any or all of the pathology or symptoms of the disease.

[0055] As used herein, the term "patient" or "subject" refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or a transgenic species thereof. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human patients are adults, adolescents, infants, and fetuses.

[0056] The term "effective", as that term is used in the specification and / or claims, means sufficient to achieve a desired, expected or intended result. "Effective amount", "therapeutically effective amount" or "pharmaceutically effective amount" in the context of treating a patient or subject with a compound, refers to an amount of a compound that, when administered to a subject or patient for treatment or prevention of a disease, is sufficient to affect such treatment or prevention.

[0057] As generally used herein, "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues, organs and / or body fluids of humans and animals without excessive toxicity, irritation, allergic response or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0058] "Induced pluripotent stem cells (iPSC)" are cells produced by reprogramming somatic cells by expressing or inducing the expression of a combination of multiple factors (referred to herein as reprogramming factors). iPSC can be produced using fetal, postnatal, neonatal, infant 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 Oct3 / 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 somatic cells into pluripotent stem cells.

[0059] The term "extracellular matrix proteins" refers to molecules that provide structural and biochemical support to surrounding cells. Extracellular matrix proteins can be recombinant or refer to fragments or peptides. Examples include collagen and heparan sulfate.

[0060] "Three-dimensional (3-D) culture" refers to an artificially created environment in which biological cells are allowed to grow or interact with their surroundings in all three dimensions. 3-D cultures can be grown in a variety of cell culture vessels, such as bioreactors, small capsules in which cells can grow as spheroids, or non-adherent culture plates. In some aspects, 3-D cultures are scaffold-free. In contrast, "two-dimensional (2-D)" culture refers to cell cultures, such as a monolayer of cells on an adherent surface.

[0061] As used herein, the "disruption" of a gene refers to the elimination or reduction of the expression of one or more gene products encoded by the subject gene in a cell compared to the expression level of the gene product in the absence of the disruption. Example gene products include mRNA and protein products encoded by the gene. In some cases, the disruption is transient or reversible, and in other cases permanent. In some cases, the disruption is a functional or full-length protein or mRNA, although truncated or non-functional products may be produced. In some embodiments herein, gene activity or function, rather than expression, is disrupted. Gene disruption is usually induced by artificial methods, i.e., by adding or introducing compounds, molecules, complexes or compositions, and / or by disrupting genes or nucleic acids associated with genes, such as at the DNA level. Example methods of gene disruption include gene silencing, knockout, knockout and / or gene disruption techniques, such as gene editing. Examples include antisense techniques, such as RNAi, siRNA, shRNA and / or ribozymes, which typically result in a transient reduction in expression, and gene editing techniques that result in the inactivation or disruption of targeted genes, for example, by inducing breakage and / or homologous recombination. Examples include insertions, mutations and deletions. The disruption typically results in suppression and / or complete lack of expression of the normal or "wild-type" product encoded by the gene. Examples of such gene disruptions are insertions, frameshifts and missense mutations, deletions, knock-ins and knockouts of genes or gene portions, including deletions of entire genes. Such disruptions may occur in the coding region, for example, in one or more exons, resulting in the inability to produce a full-length product, a functional product, or any product, for example by inserting a stop codon. Such disruptions may also occur due to disruption of promoters or enhancers or other regions that affect transcriptional activation, thereby preventing transcription of the gene. Gene disruptions include gene targeting, including targeted gene inactivation by homologous recombination.

[0062]

II. Induced pluripotent stem cells

[0063] In some embodiments, the method involves differentiating iPSCs. The induction of pluripotency was first demonstrated in 2006 using mouse cells (Yamanaka et al., 2006) and in 2007 using human cells (Yu et al., 2007; Takahashi et al., 2007) by reprogramming somatic cells through the introduction of transcription factors associated with pluripotency. Pluripotent stem cells can be maintained in an undifferentiated state and can be differentiated into any adult cell type.

[0064] Except germ cells, any somatic cell can be used as the starting point of iPSC. For example, the cell type can be a keratinocyte, a fibroblast, a hematopoietic cell, a mesenchymal cell, a hepatocyte or a gastric cell. T cells can also be used as a somatic cell source for reprogramming (U.S. Patent No. 8,741,648). There is no limit to the degree of cell differentiation or the age of the animal from which the cells are collected; even undifferentiated progenitor cells (including somatic stem cells) and finally differentiated mature cells can also be used as the source of somatic cells in the methods disclosed herein. iPSC can be grown under conditions known to differentiate human ES cells into specific cell types and express human ES cell markers, including: SSEA-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81.

[0065] A.HLA matching

[0066] The major histocompatibility complex (MHC) is the main cause of immune rejection in allogeneic organ transplantation. There are three major MHC class I haplotypes (A, B, and C) and three major MHC class II haplotypes (DR, DP, and DQ).

[0067] If the donor cells are HLA homozygous, i.e. contain identical alleles for each antigen presenting protein, then the MHC compatibility between the donor and recipient increases significantly. Most individuals are heterozygous for MHC class I and class II genes, but some individuals are homozygous for these genes. These homozygous individuals can serve as super donors, and grafts generated from their cells can be transplanted into all individuals homozygous or heterozygous for that haplotype. Furthermore, if homozygous donor cells have a haplotype found at high frequency in the population, these cells may be suitable for transplantation therapy in a large number of individuals.

[0068] Therefore, iPSC can be produced by the somatic cells of the treated subject, or another subject with the same or substantially the same HLA type as the patient. In one case, the major HLA (e.g., three major loci of HLA-A, HLA-B and HLA-DR) of the donor is the same as the major HLA of the recipient. In some cases, the somatic cell donor may be a super donor; therefore, iPSC derived from MHC homozygous super donors can be used to produce differentiated cells. Therefore, iPSC from super donors can be transplanted into homozygous or heterozygous subjects of the haplotype. For example, iPSC can be homozygous at two HLA alleles (such as HLA-A and HLA-B). Therefore, the iPSC produced by super donors can be used in the method disclosed herein to produce differentiated cells that may "match" a large number of potential receptors.

[0069]

B. Reprogramming Factors

[0070] Somatic cells can be reprogrammed to produce induced pluripotent stem cells (iPSC) using any method known to those skilled in the art. Any person skilled in the art can easily produce induced pluripotent stem cells; for example, see published U.S. Patent Application No. 20090246875, published U.S. Patent Application No. 2010 / 0210014; published U.S. Patent Application No. 20120276636; U.S. Patent No. 8,058,065; U.S. Patent No. 8,129,187; U.S. Patent No. 8,278,620; PCT Publication No. WO 2007 / 069666 A1 and U.S. Patent No. 8,268,620, incorporated herein by reference. Typically, nuclear reprogramming factors are used to produce 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 used. In other embodiments, Oct3 / 4, Sox2, c-Myc, and Klf4 are used. In certain aspects, 5, 6, 7, or 8 reprogramming factors are used.

[0071] Cells are treated with nuclear reprogramming substances, which are generally factors capable of inducing iPSCs from somatic cells or nucleic acids encoding these substances (including forms integrated in vectors). Nuclear reprogramming substances generally include at least Oct3 / 4, Klf4 and Sox2 or nucleic acids encoding these molecules. Functional inhibitors of p53, L-myc or nucleic acids encoding L-myc, and Lin28 or Lin28b or nucleic acids encoding Lin28 or Lin28b can be used as additional nuclear reprogramming substances. Nanog can also be used for nuclear reprogramming.As disclosed in published U.S. patent application No. 20120196360, exemplary reprogramming factors for producing 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) Oct3 / 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, HPV16 E6; (15) Oct3 / 4, Klf4, Sox2, TERT, HPV16E7; (16) Oct3 / 4, Klf4, Sox2, TERT, HPV16E6, 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 by Esrrg). In one non-limiting example, Oct3 / 4, Klf4, Sox2, and c-Myc are used.In other embodiments, Oct4, Nanog and Sox2 are used; for example, see U.S. Patent No. 7,682,828, which is 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 used. In other non-limiting examples, Oct3 / 4, Klf4, Sox2 and Sal 4 are used. Factors such as Nanog, Lin28, Klf4 or c-Myc can improve reprogramming efficiency and can be expressed from several different expression vectors. For example, an integration vector can be used, such as a system based on EBV elements (U.S. Patent No. 8,546,140). In a further aspect, the reprogramming protein 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 inhibitors, leukemia inhibitory factor (LIF), p53 inhibitors, NF-κB inhibitors, or combinations thereof. These regulatory factors may include small molecules, inhibitory nucleotides, expression cassettes, or protein factors. It is expected that almost any iPS cell or cell line can be used.

[0072] Mouse and human cDNA sequences of these nuclear reprogramming materials can be obtained with reference to the NCBI accession numbers mentioned in WO 2007 / 069666, which is incorporated herein by reference. Methods for introducing one or more reprogramming materials or nucleic acids encoding these reprogramming materials are known in the art, such as disclosed in published U.S. Patent Application No. 2012 / 0196360 and U.S. Patent No. 8,071,369, both of which are incorporated herein by reference.

[0073] Once derived, iPSCs can be cultured in a medium sufficient to maintain pluripotency. iPSCs can be used with various culture media and techniques developed for culturing pluripotent stem cells (more specifically, embryonic stem cells), as described in U.S. Patent No. 7,442,548 and U.S. Patent Publication No. 2003 / 0211603. For mouse cells, leukemia inhibitory factor (LIF) is added to ordinary culture medium as a differentiation inhibitor for culture. For human cells, basic fibroblast growth factor (bFGF) is preferably added instead of LIF. Other iPSC culture and maintenance methods known to those skilled in the art can be used.

[0074] In certain embodiments, undefined conditions can be used; for example, pluripotent cells can be cultured on fibroblast feeder cells or culture medium exposed to fibroblast feeder cells to maintain the stem cells in an undifferentiated state. In some embodiments, cells are co-cultured with mouse embryonic fibroblasts (as feeder cells) that have been treated with radiation or antibiotics to terminate cell division. Alternatively, defined, feeder-independent culture systems (e.g., TESR TM culture medium (Ludwig et al., 2006a; Ludwig et al., 2006b) or E8 TM culture medium (Chen et al., 2011)) to culture and maintain pluripotent cells in a largely undifferentiated state.

[0075]

C.Plasmid

[0076] In some embodiments, iPSC can be modified to express exogenous nucleic acid, for example, including an enhancer operably connected to a promoter and a nucleic acid sequence encoding a first marker. The construct may also include other elements, such as a ribosome binding site (internal ribosome binding sequence) for translation initiation and a transcription / translation terminator. Generally, it is 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.

[0077] In some embodiments, the plasmid encoding the marker consists 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, including a tyrosinase enhancer and (4) a multiple cloning site for incorporation of various nucleic acid cassettes; (5) a nucleic acid sequence encoding a marker operably linked to a tyrosinase promoter. There are many known plasmid vectors in the art for inducing protein-encoding nucleic acids. These include, but are not limited to, the vectors disclosed in U.S. Pat. No. 6,103,470; U.S. Pat. No. 7,598,364; U.S. Pat. No. 7,989,425; and U.S. Pat. No. 6,416,998, which are incorporated herein by reference. In certain aspects, the plasmid contains a "suicide gene" that, upon administration of the prodrug, affects the conversion of the gene product into a compound that kills its 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.

[0078] Viral gene delivery systems can be RNA-based or DNA-based viral vectors. Episomal gene delivery systems can be plasmids, episomal vectors based on Epstein-Barr virus (EBV), vectors based on yeast, vectors based on adenovirus, episomal vectors based on simian virus 40 (SV40), vectors based on bovine papilloma virus (BPV), or lentiviral vectors.

[0079] 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; synthesized or absorbed by cells); nucleic acids (e.g., mRNA, or enzymatically active nucleic acid molecules) or polysaccharides. Includes determinants of any such cellular components that can be detected by antibodies, lectins, probes, or nucleic acid amplification reactions, which are specific to markers of target cell types. These markers can also be identified by biochemical or enzyme assays or biological reactions, depending on the function of the gene product. The nucleic acid sequences encoding these markers can be operably linked to a tyrosinase enhancer. In addition, other genes can also be included, such as genes that may affect stem cell differentiation, cell function, physiology, or pathology.

[0080]

D.Conveying system

[0081] To introduce nucleic acids, such as DNA or RNA, into the presently disclosed engineered cell lines, any suitable method for delivering nucleic acids for transformation of cells may be used, as described herein or otherwise known to those of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA, such as by ex vivo transfection (Wilson et al., 1989, Nabel et al., 1989), injection (U.S. Pat. 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, all of which are incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S. Pat. No. 5,789,215, incorporated herein by reference); by electroporation (U.S. Pat. No. 5,384,253, incorporated herein by reference; Tur-Kaspa et al., 1986; Potter et al., 1984); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by using DEAE-dextran followed by polyethylene glycol (Gopal, 1985); by direct sonication (Fechheimer et al., 1987); by 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); by microprojectile bombardment (PCT Application No. WO 94 / 09699 and 95 / 06128; U.S. Pat. Nos. 5,610,042; 5,322,783, 5,563,055, 5,550,318, 5,538,877 and 5,538,880, all of which are incorporated herein by reference); by silicon carbide fiber agitation (Kaeppler et al., 1990; U.S. Pat. Nos. 5,302,523 and 5,464,765, all of which are incorporated herein by reference); by Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,591,616 and 5,563,055, all of which are incorporated herein by reference); by desiccation / inhibition-mediated DNA uptake (Potrykus et al., 1985), and any combination of such methods. By applying such techniques, organelles, cells, tissues or organisms can be stably or transiently transformed.

[0082] 1. Viral vector

[0083] Viral vectors can be provided in certain aspects of the present disclosure. When generating recombinant viral vectors, non-essential genes are usually replaced by genes or coding sequences of heterologous (or non-natural) proteins. Viral vectors are an expression construct that utilizes viral sequences to introduce nucleic acids and possible proteins into cells. Certain viruses infect cells or enter cells through receptor-mediated endocytosis, and the ability to integrate into the host cell genome and stably and effectively express viral genes makes them attractive candidates for transferring exogenous nucleic acids to cells (e.g., mammalian cells). Non-limiting examples of viral vectors that can be used to deliver nucleic acids in certain aspects of the present disclosure are described below.

[0084] Retroviruses hold promise as gene delivery vectors because they are able to integrate their genes into the host genome, transfer large amounts of foreign genetic material, infect a wide range of species and cell types, and be packaged in specialized cell lines (Miller, 1992).

[0085] In order to construct a retroviral vector, nucleic acid is inserted into the viral genome to replace certain viral sequences to produce a virus with a replication defect. In order to produce virions, a packaging cell line containing gag, pol and env genes (but without LTR and packaging components) was constructed (Mann et al., 1983). When a recombinant plasmid containing cDNA is introduced into a special cell line together with the retroviral LTR and packaging sequence (for example, by calcium phosphate precipitation), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles and then secreted into the culture medium (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The culture medium containing the recombinant retrovirus is then collected, selectively concentrated, and used for gene transfer. Retroviral vectors can infect a variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al., 1975).

[0086] Lentivirus is a complex retrovirus that contains, in addition to the common retroviral genes gag, pol and env, other genes with regulatory or structural functions. Lentivirus vectors are well known in the art (e.g., see Naldini et al., 1996; Zufferey et al., 1997; Blomer et al., 1997; U.S. Patents 6,013,516 and 5,994,136).

[0087] Recombinant lentiviral vectors are capable of infecting non-dividing cells and can be used for in vivo and ex vivo gene transfer and expression of nucleic acid sequences. For example, recombinant lentiviruses capable of infecting non-dividing cells (wherein suitable host cells are transfected with two or more vectors carrying packaging functions, i.e., gag, pol and env, as well as rev and tat) are described in U.S. Pat. No. 5,994,136, which is incorporated herein by reference.

[0088] 【2. Free carrier】

[0089] The use of extrachromosomal (i.e., episomal) vectors based on plasmids or liposomes may also be provided in certain aspects of the present disclosure. For example, such episomal vectors may include oriP-based vectors and / or vectors encoding EBNA-1 derivatives. These vectors may allow large fragments of DNA to be introduced into cells and maintained extrachromosomally, replicated once per cell cycle, efficiently distributed to daughter cells, and substantially not evoke an immune response.

[0090] In particular, EBNA-1 is the only viral protein required for the replication of oriP-based expression vectors, and it does not trigger a cellular immune response because it has developed an efficient mechanism to bypass the processing required to present its antigens on MHC class I molecules (Levitskaya et al., 1997). In addition, EBNA-1 can enhance the expression of cloned genes in trans, inducing the expression of cloned genes up to 100-fold in some cell lines (Langle-Rouault et al., 1998; Evans et al., 1997). Finally, such oriP-based expression vectors are inexpensive to manufacture.

[0091] In some aspects, the reprogramming factor is expressed from an expression cassette comprising one or more exogenous episomal genetic elements (see U.S. Patent Publication 2010 / 0003757, incorporated herein by reference). Therefore, iPSCs may be substantially free of exogenous genetic elements, such as retroviral or lentiviral vector elements. These iPSCs are prepared by using extrachromosomal replication vectors (i.e., episomal vectors), which are capable of episomal replication so that iPSCs are substantially free of exogenous vectors or viral elements (see U.S. Patent No. 8,546,140, ​​incorporated herein by reference; Yu et al., 2009). Many DNA viruses, such as adenovirus, simian vacuolating virus 40 (SV40) or bovine papilloma virus (BPV), or plasmids containing budding yeast ARS (autonomous replication sequence), replicate extrachromosomally or episomally in mammalian cells. These episomal plasmids are essentially free of all these disadvantages associated with integration vectors (Bode et al., 2001). For example, as described above, lymphotrophic herpes virus-based or Epstein-Barr virus (EBV) may replicate extrachromosomally and help deliver reprogramming genes to somatic cells. Useful EBV elements are OriP and EBNA-1, or variants or functional equivalents thereof. Another advantage of episomal vectors is that exogenous elements are lost over time after being introduced into cells, resulting in self-sustaining iPSCs that are essentially free of these elements.

[0092] Other extrachromosomal vectors include other vectors based on lymphotrophic herpesviruses. Lymphotrophic herpesvirus is a herpesvirus that can replicate in lymphoblasts (e.g., human B lymphoblasts) and become a plasmid during a part of its natural life cycle. Herpes simplex virus (HSV) is not a "lymphotrophic" herpesvirus. Typical lymphotrophic herpesviruses include, but are not limited to, EBV, Kaposi's sarcoma herpesvirus (KSHV); herpesvirus saimiri (HS) and Marek's disease virus (MDV). In addition, other episomal-based vector sources, such as yeast ARS, adenovirus, SV40 or BPV, are also contemplated.

[0093] Any skilled artisan will be well able to construct 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).

[0094] The vector may also contain other components or functions that further regulate gene delivery and / or gene expression, or otherwise provide components or functions that provide beneficial properties to the target cell. For example, such other components include components that affect binding or targeting to cells (including components that mediate cell type or tissue specific binding); components that affect cellular uptake of vector nucleic acid; components that affect the localization of polynucleotides in cells after uptake (e.g., agents that mediate nuclear localization); and components that affect the expression of polynucleotides.

[0095] Such components can also include markers, such as detectable and / or selective markers, which can be used to detect or select cells that have been ingested and are expressing the nucleic acid delivered by the vector. Such components can be provided as natural features of the vector (e.g., using certain viral vectors with components or functions that mediate binding and ingestion), or the vector can be modified to provide such functions. Such vectors are known in the art in a wide variety and are generally available. When the vector is maintained in a host cell, the vector can be stably replicated as an autonomous structure by the cell during mitosis, incorporated into the genome of the host cell, or maintained in the nucleus or cytoplasm of the host cell.

[0096] 【3. Regulatory elements】

[0097] The expression cassette contained in the reprogramming vectors useful in the present disclosure preferably comprises (in the 5' to 3' direction) a eukaryotic transcriptional promoter operably linked to a protein coding sequence, splicing signals including intervening sequences, and a transcription termination / polyadenylation sequence.

[0098]

a. Promoter / enhancer

[0099] The expression construct provided herein comprises a promoter that drives programmed gene expression. The promoter generally includes a sequence that serves to locate the start site of RNA synthesis. The most famous example is the TATA box, but in some promoters lacking the TATA box, for example, the promoter of the mammalian terminal deoxynucleotidyl transferase gene and the promoter of the SV40 late gene, discrete elements covering the start site itself help to fix the start position. Additional promoter elements regulate the frequency of transcription initiation. Typically, these promoters are located in the region 30 to 110 bp upstream of the start site, although it has been demonstrated that many promoters also include functional elements downstream of the start site. In order to place the coding sequence under the "control" of the promoter, the 5' end of the transcription start site of the transcription reading frame of the selected promoter is positioned "downstream" (i.e., 3') of the selected promoter. The "upstream" promoter stimulates the transcription of DNA and promotes the expression of the encoded RNA.

[0100] The spacing between promoter elements is often flexible so that promoter function is retained when elements are inverted or moved relative to each other. In the tk promoter, the spacing between promoter elements can be increased to 50 bp before activity begins to decline. Depending on the promoter, individual elements appear to function either cooperatively or independently to activate transcription. Promoters may or may not be associated with "enhancers," which refer to cis-acting regulatory sequences involved in the activation of transcription of a nucleic acid sequence.

[0101] The promoter can be a promoter naturally associated with a nucleic acid sequence, such as can be obtained by isolating a 5' non-coding sequence located upstream of a coding segment and / or an exon. Such a promoter can be referred to as "endogenous". Similarly, an enhancer can be an enhancer naturally associated with a nucleic acid sequence, located downstream or upstream of the sequence. Alternatively, certain advantages can be obtained by placing the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not usually associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer that is not usually associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, as well as promoters or enhancers isolated from any other virus, prokaryote or eukaryotic cell, and promoters or enhancers that are not "naturally present", i.e., different elements containing different transcriptional regulatory regions, and / or mutations that change expression. For example, the most commonly used promoters in recombinant DNA construction include β-lactamase (penicillinase), lactose, and tryptophan (trp) promoter systems. In addition to synthesizing promoter and enhancer nucleic acid sequences, recombinant cloning and / or nucleic acid amplification techniques (including PCR) can also be used to generate promoter and enhancer nucleic acid sequences. TM ) are used in conjunction with the compositions disclosed herein to generate sequences (see U.S. Pat. Nos. 4,683,202 and 5,928,906, both of which are incorporated herein by reference). In addition, control sequences that direct transcription and / or expression of sequences within non-nuclear organelles (e.g., mitochondria, chloroplasts, etc.) can be used.

[0102] Naturally, it will be important to employ promoters and / or enhancers that effectively direct the expression of the DNA fragment in the organelle, cell type, tissue, organ, or organism selected for expression. Those skilled in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression, (e.g., see Sambrook et al., 1989, incorporated herein by reference). The promoter employed may be constitutive, tissue-specific, inducible, and / or useful under appropriate conditions to direct high-level expression of the introduced DNA fragment, such as in large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0103] In addition, any promoter / enhancer combination (e.g., according to the Eukaryotic Promoter Database EPDB) can also be used to drive expression. Use of T3, T7 or SP6 cytoplasmic expression systems is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters, either as part of a delivery complex or as an additional gene expression construct, if the appropriate bacterial polymerase is provided.

[0104] Non-limiting example promoters include early or late viral promoters, such as the SV40 early or late promoter, the cytomegalovirus (CMV) immediate early promoter, 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), the metallothionein promoter (Karin et al., 1989; Richards et al., 1984); and tandem response element promoters, such as the cyclic AMP response element promoter (cre), the serum response element promoter (sre), the phorbol ester promoter (TPA) and the response element promoter (tre), close to the minimal TATA box. The human growth hormone promoter sequence (eg, the human growth hormone minimal promoter described in Genbank, Accession No. X05244, nucleotides 283-341) or the mouse mammary tumor promoter (available from ATCC, Catalog No. ATCC 45007) may also be used.

[0105] Tissue-specific transgenic expression, particularly for reporter gene expression in hematopoietic cells and expression derived from programmed hematopoietic cell precursors, may be desirable as a means of identifying derived hematopoietic cells and precursors. In order to increase specificity and activity, the use of cis-acting regulatory elements has been considered. For example, hematopoietic cell-specific promoters may be used. Many such hematopoietic cell-specific promoters are known in the art.

[0106] In certain aspects, the methods of the present disclosure also relate to enhancer sequences, i.e., nucleic acid sequences that increase promoter activity, and regardless of their orientation, even at relatively long distances (up to several kilobases from the target promoter). However, enhancer function is not necessarily limited to such long distances, as they may also function in the vicinity of a given promoter.

[0107] Many hematopoietic cell promoter and enhancer sequences have been identified and may be useful in the current method. For example, see U.S. Patent No. 5,556,954; U.S. Patent Application No. 20020055144; U.S. Patent Application No. 20090148425. [b. Initiation Signal and Linked Expression]

[0108] Specific start signals can also be used in the expression constructs provided in the present disclosure for efficient translation of coding sequences. These signals include ATG start codons or adjacent sequences. It may be necessary to provide exogenous translation control signals, including ATG start codons. One of ordinary skill in the art will be able to easily determine this and provide the necessary signals. As is well known, the start codon must be "in frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. Exogenous translation control signals and start codons can be natural or synthetic. Expression efficiency can be improved by including appropriate transcription enhancer elements.

[0109] In certain embodiments, internal ribosome entry site (IRES) elements are used to create polygenic or polycistronic information. IRES elements can bypass the ribosome scanning model of 5' methylated Cap-dependent translation and start translation at internal sites (Pelletier and Sonenberg, 1988). IRES elements from two members of the picornavirus family (poliomyelitis and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well as IRES elements from mammalian information (Macejak and Sarnow, 1991). IRES elements can be connected to heterologous open reading frames. Multiple open reading frames can be transcribed together, and each reading frame is separated by IRES, thereby producing polycistronic information. With IRES elements, ribosomes can access each open reading frame for efficient translation. Multiple genes can be effectively expressed using a single promoter / enhancer to transcribe a single message (see U.S. Patent Nos. 5,925,565 and 5,935,819, all incorporated herein by reference).

[0110] In addition, certain 2A sequence elements can be used to create linkage or co-expression of programmed genes in constructs provided herein. For example, a cleavage sequence can be used to co-express genes by connecting open reading frames to form a single cisson. A typical cleavage sequence is F2A (foot-and-mouth disease virus 2A) or a "2A-like" sequence (e.g., Thosea asigna virus 2A; T2A) (Minskaia and Ryan, 2013). In a specific embodiment, the F2A cleavage peptide is used to link the expression of genes in a multi-lineage construct.

[0111]

c. Copy starting point

[0112] In order to propagate the vector in the host cell, it may contain one or more origins of replication sites (commonly referred to as "ori"), for example, a nucleic acid sequence corresponding to oriP of EBV as described above or a genetically engineered oriP that has a similar or enhanced function in programming, which is a specific nucleic acid sequence for initiating replication. Alternatively, the replication origins or autonomous replication sequences (ARS) of other extrachromosomal replicating viruses as described above may be used.

[0113] [d. Selection and screenable markers]

[0114] In certain embodiments, cells containing nucleic acid constructs can be identified in vitro or in vivo by including a marker in the expression vector. Such markers will confer recognizable changes to the cells, making it easy to identify cells containing the expression vector. Typically, a selectable marker is a marker that confers a property that allows selection. The presence of a positive selectable marker allows selection, while the presence of a negative selectable marker prevents selection. An example of a positive selectable marker is a drug resistance marker.

[0115] Usually, comprising drug selection markers helps the cloning and identification of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, ZEOCIN and histidine are useful selection markers. In addition to conferring the markers that allow the phenotype of the transformants to be distinguished according to the conditions, other types of markers are also considered, including screenable markers, such as GFP, which is based on colorimetric analysis. Alternatively, screenable enzymes can be used as negative selection markers, such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT). Those skilled in the art will also know how to use immune markers, which may be combined with FACS analysis. As long as it can be expressed simultaneously with the nucleic acid encoding the gene product, the marker used is considered to be unimportant. More examples of selection and screenable markers are well known to those skilled in the art.

[0116]

E.Gene disruption

[0117] In certain aspects, the expression, activity or function of the TREM2, APOE, MeCP2 and / or SCNA genes is disrupted in a cell, such as a PSC (e.g., ESCs or iPSC). In some embodiments, gene disruption is achieved by creating a disruption in a gene, such as a knockout, insertion, missense or frameshift mutation, such as a biallelic frameshift mutation, a deletion of all or part of a gene, such as one or more exons or portions thereof, and / or knock-in. For example, disruption can be 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), specifically designed to target gene sequences or portions thereof.

[0118] In some embodiments, the expression, activity and / or function of a gene is disrupted by disrupting the gene. In some aspects, the gene is disrupted so that its expression is reduced by at least 20%, 30% or 40%, typically by at least 50%, 60%, 70%, 80%, 90% or 95%, compared to expression without the gene disruption or without the introduction of components that affect the disruption.

[0119] In some embodiments, the disruption is transient or reversible, such that expression of the gene is restored at a later time. In other embodiments, the disruption is irreversible or temporary, e.g., permanent.

[0120] In some embodiments, gene disruption is performed by inducing one or more double-strand breaks and / or one or more single-strand breaks in a gene, typically in a targeted manner. In some embodiments, the double-strand or single-strand breaks are made by a nuclease (e.g., an endonuclease), such as a gene-targeted nuclease. In some aspects, the break is induced in the coding region of the gene, e.g., in an exon. For example, in some embodiments, the induction occurs near the N-terminal portion of the coding region, e.g., in the first exon, in the second exon, or in a subsequent exon.

[0121] In some aspects, double-strand or single-strand breaks are repaired by a cell repair process, such as by non-homologous end joining (NHEJ) or homology directed repair (HDR). In some aspects, the repair process is prone to error and causes gene disruption, such as frameshift mutations, such as biallelic frameshift mutations, which may cause gene knockout completely. For example, in some aspects, interruption includes induced deletions, mutations and / or insertions. In some embodiments, destruction causes the presence of early termination codons. In some aspects, the presence of insertions, deletions, translocations, frameshift mutations and / or premature termination codons can cause the expression, activity and / or function of genes to be interrupted.

[0122] In some embodiments, gene disruption is achieved using antisense technology, such as by RNA interference (RNAi), short interfering RNA (siRNA), short hairpin (shRNA) and / or ribozymes are used to selectively inhibit or suppress the expression of a gene. siRNA technology is RNAi, which uses double-stranded RNA molecules, whose sequences are homologous to the mRNA nucleotide sequences transcribed from the gene, and sequences complementary to the nucleotide sequences. siRNA is usually homologous / complementary to a region of the mRNA transcribed from the gene, or may be siRNA comprising multiple RNA molecules, which are homologous / complementary to different regions. In some aspects, siRNA is composed of a polycistronic construct. In specific aspects, siRNA inhibits translation of wild-type and mutant proteins from endogenous mRNA.

[0123] In some embodiments, the disruption is achieved using a DNA targeting molecule, such as a DNA binding protein or a DNA binding nucleic acid, or a complex, compound or composition comprising the same, which specifically binds or hybridizes to a gene. In some embodiments, the DNA targeting molecule includes a DNA binding domain, such as a zinc finger protein (ZFP) DNA binding domain, a transcription activator-like protein (TAL) or a TAL effector (TALE) DNA binding domain, a clustered regularly interspaced short palindromic repeat (CRISPR) DNA binding domain, or a DNA binding domain from a giant nuclease. Zinc fingers, TALEs and CRISPR system binding domains can be engineered to bind to a predetermined nucleotide sequence, such as by engineering (changing one or more amino acids) the recognition helical region of a naturally occurring zinc finger or TALE protein. Engineered DNA binding proteins (zinc fingers or TALEs) are non-naturally occurring proteins. Rational design criteria include the application of substitution rules and computerized algorithms for processing information in a database, storing information and binding data for existing ZFP and / or TALE designs. See, for example, 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. Publication No. 2011 / 0301073.

[0124] 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, to promote the inhibition or destruction of a gene. For example, in some embodiments, gene disruption is performed by a fusion protein comprising a DNA binding protein and a heterologous regulatory domain or functional fragment thereof. In some aspects, the domain comprises a transcription factor domain, such as an activator, a repressor, a coactivator, a co-inhibitor, a silencing agent, an oncogene, a DNA repair enzyme and its associated factors and modifiers, a DNA rearrangement enzyme and its associated factors and modifiers, a chromatin-associated protein and its modifiers, such as a kinase, an acetylase and a deacetylase, and a DNA modifying enzyme, such as a methyltransferase, a topoisomerase, a helicase, a ligase, a kinase, a phosphatase, a polymerase, an endonuclease and its associated factors and modifiers. For example, see U.S. Patent Application Publication Nos. 2005 / 0064474; 2006 / 0188987 and 2007 / 0218528, which are incorporated herein by reference in their entirety for detailed information on the fusion of a DNA binding domain and a nuclease cleavage domain. In some aspects, 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 complexes or fusion proteins containing nucleases that consist of a sequence-specific DNA binding domain fused or complexed with a non-specific DNA cleavage molecule such as a nuclease.

[0125] In certain aspects, these targeted chimeric nucleases or complexes containing nucleases perform precise genetic modification by inducing targeted double-strand breaks or single-strand breaks, stimulating cellular DNA repair mechanisms, including error-prone non-homologous end joining (NHEJ) and homology-directed repair (HDR). In some embodiments, the nuclease is an endonuclease, such as a zinc finger nuclease (ZFN), a TALE nuclease (TALEN), and an RNA-guided endonuclease (RGEN), such as a CRISPR-associated (Cas) protein or a giant nuclease.

[0126] In some embodiments, a donor nucleic acid is provided, for example, a donor plasmid or nucleic acid encoding a genetically engineered antigen receptor, and a gene editing site is inserted by HDR after the introduction of DSBs. Therefore, in some embodiments, the disruption of the gene and the introduction of the antigen receptor (eg, CAR) are performed simultaneously, so that the gene is partially disrupted by knocking in or inserting a nucleic acid encoding CAR.

[0127] In some embodiments, no donor nucleic acid is provided. In certain aspects, NHEJ-mediated repair following introduction of a DSB results in insertion or deletion mutations, thereby resulting in gene disruption, for example, by generating a missense mutation or a frameshift.

[0128] [1. ZFP and ZFN]

[0129] 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.

[0130] 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 thereof is a protein or domain within a larger protein that binds to DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized by the coordination of zinc ions. Zinc finger DNA binding proteins are often abbreviated as zinc finger proteins or ZFPs. ZFPs include artificial ZFP domains that target specific DNA sequences, typically 9 to 18 nucleotides, produced by the assembly of a single finger.

[0131] ZFPs include those with a single finger domain of about 30 amino acids in length and comprising an alpha helix containing two invariant histidine residues coordinated by zinc to two single beta-turned cysteines and having two, three, four, five, or six fingers. Typically, the sequence specificity of a ZFP can be altered by amino acid substitutions at four helical positions (-1, 2, 3, and 6) on the zinc finger recognition helix. Thus, in some embodiments, a ZFP or a molecule containing a ZFP is non-naturally occurring, e.g., designed to bind to a selected target site.

[0132] In certain aspects, disruption of MeCP2 is performed by contacting a first target site in a gene with a first ZFP, thereby disrupting the gene. In some embodiments, a target site in a gene is contacted with a fusion ZFP comprising six fingers and a regulatory domain, thereby inhibiting expression of the gene.

[0133] In some embodiments, the step of further contacting comprises contacting a second target site in the gene with a second ZFP. In some aspects, the first and second target sites are adjacent. In some embodiments, the first and second ZFPs are covalently linked. In some aspects, the first ZFP is a fusion protein comprising a regulatory domain or at least two regulatory domains.

[0134] In some embodiments, the first and second ZFPs are fusion proteins, each ZFP comprises a regulatory domain or each comprises at least two regulatory domains. In some embodiments, the regulatory domain is a transcriptional repressor, a transcriptional activator, an endonuclease, a methyltransferase, a histone acetyltransferase, or a histone deacetylase.

[0135] In some embodiments, ZFP is by the ZFP nucleic acid encoding that is operably connected to a promoter. In some aspects, the method also comprises the step of first applying nucleic acid to the cell in the form of lipid: nucleic acid complex or naked nucleic acid. In some embodiments, ZFP is by expression vector encoding, and this expression vector comprises the ZFP nucleic acid that is operably connected to a promoter. In some embodiments, ZFP is by the nucleic acid encoding that is operably connected to an inducible promoter. In some aspects, ZFP is by the nucleic acid encoding that is operably connected to a weak promoter.

[0136] In some embodiments, the target site is the upstream of the gene transcription start site. In some aspects, the target site is adjacent to the transcription start site of the gene. In some aspects, the target site is adjacent to the RNA polymerase pause site downstream of the gene transcription start site.

[0137] 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 liS-type restriction endonuclease and one or more zinc finger binding domains, which may be engineered or unengineered. In some embodiments, the cleavage domain is from the liS-type restriction endonuclease Fok I. Fok I typically catalyzes double-stranded cleavage of DNA, 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other strand.

[0138] In some embodiments, ZFN targets genes present in engineered cells. In certain aspects, ZFN effectively generates double-strand breaks (DSBs), such as at predetermined sites in the coding region of a gene. Typical targeting regions include exons, regions encoding N-terminal regions, first exons, second exons, and promoter or enhancer regions. In some embodiments, transient expression of ZFN promotes efficient and permanent destruction of target genes in engineered cells. In particular, in some embodiments, delivery of ZFN results in permanent destruction of genes with an efficiency of more than 50%.

[0139] Many gene-specific engineered zinc fingers are available on the market. For example, Sangamo Biosciences (Richmond, California, USA) has collaborated with Sigma-Aldrich (St. Louis, Missouri, USA) to develop a platform (CompoZr) for zinc finger construction that allows researchers to completely bypass zinc finger construction and validation and provide specifically targeted zinc fingers for thousands of proteins (Gaj et al., Trends in Biotechnology, 2013, 31(7), 397-405). In some embodiments, commercially available zinc fingers or custom designed zinc fingers are used.

[0140] TAL, TALE and TALEN

[0141] 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 in a transcription activator-like protein effector (TALE) protein, see, e.g., U.S. Patent Publication No. 2011 / 0301073, incorporated herein by reference in its entirety.

[0142] TALE DNA binding domains or TALEs are polypeptides comprising one or more TALE repeat domains / units. The repeat domains are involved in the binding of TALEs to their cognate target DNA sequences. A single "repeat unit" (also referred to as a "repeat") is typically 33 to 35 amino acids in length and exhibits at least some sequence homology with other TALE repeat sequences in naturally occurring TALE proteins. Each TALE repeat unit includes 1 or 2 DNA binding residues, which constitute the repeat variable diresidue (RVD), which is typically located at position 12 and / or 13 of the repeat sequence. The natural (canonical) code for DNA recognition of these TALEs has been determined, so the HD sequences at positions 12 and 13 result in binding to cytosine (C), NG to T, NI to A, NN to G or A, NO to T, and non-classical (atypical) RVDs are also known. See U.S. Patent Publication No. 2011 / 0301073. In some embodiments, TALEs can target any gene by designing a TAL array specific for a target DNA sequence. The target sequence typically begins with thymidine.

[0143] In some embodiments, the molecule is a DNA-binding endonuclease, such as a TALE nuclease (TALEN). In certain aspects, TALEN is a fusion protein comprising a DNA binding domain derived from TALE and a nucleic acid catalytic domain for cutting a nucleic acid target sequence.

[0144] In some embodiments, TALEN recognizes and cuts the target sequence in the gene. In some aspects, the cutting of DNA can cause double-strand breaks. In some aspects, the break stimulates the rate of homologous recombination or non-homologous end joining (NHEJ). Generally, NHEJ is an imperfect repair process, which usually causes the DNA sequence of the cutting site to change. In some aspects, the repair mechanism involves the residues of reconnecting the two DNA ends by directly reconnecting (Critchlow and Jackson, 1998) or by the end connection mediated by so-called microhomology. In some embodiments, repair by NHEJ causes small insertions or deletions, and can be used to destroy and thus inhibit genes. In some embodiments, modification can be the replacement, deletion or addition of at least one nucleotide. In some aspects, cells in which cleavage-induced mutagenesis events have occurred, i.e., mutagenesis events that are continuous with NHEJ events, can be identified and / or selected by methods well known in the art.

[0145] In some embodiments, TALE repeats are assembled to specifically target genes. A TALEN library targeting 18,740 human protein-coding genes has been constructed. Custom-designed TALE arrays are commercially available through Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, Kentucky, USA), and Life Technologies (Grand Island, New York, USA).

[0146] In some embodiments, TALEN is introduced as a trans gene encoded by one or more plasmid vectors. In some aspects, the plasmid vector can contain a selection marker for identifying and / or selecting cells that receive the vector. [3. RGEN (CRISPR / Cas system)]

[0147] In some embodiments, one or more DNA binding nucleic acids are used for destruction, such as by RNA-guided endonucleases (RGENs). For example, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-related (Cas) proteins can be used for destruction. In general, "CRISPR systems" collectively refer to transcripts and other elements that participate in CRISPR-related ("Cas") gene expression or guide its activity, including sequences encoding Cas genes, tracr (trans-activating 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 CRISPR loci.

[0148] CRISPR / Cas nucleases or CRISPR / Cas nuclease systems may include a non-coding RNA molecule (guide) RNA that binds to DNA with sequence specificity, and a Cas protein (e.g., Cas9) having nuclease function (e.g., two nuclease domains). One or more elements of a CRISPR system may be derived from a type I, type II, or type III CRISPR system, e.g., from a specific organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes.

[0149] In some aspects, Cas nuclease and gRNA (including crRNA specific to the target sequence and fixed tracrRNA fusion) are introduced into the cell. Typically, the target site at the 5' end of gRNA uses complementary base pairing to target the Cas nuclease to the target site, such as a gene. The target site can be selected according to the position of the 5' adjacent motif (PAM) sequence of the protospacer region, for example, usually NGG or NAG. In this regard, the gRNA is targeted to the 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. In general, the feature of the CRISPR system is to promote the formation of CRISPR complex elements at the target sequence site. Typically, "target sequence" generally refers to a sequence designed as a guide sequence with complementarity, wherein the hybridization between the target sequence and the guide sequence promotes the formation of the CRISPR complex. It is not necessarily necessary to be fully complementary, as long as there is enough complementarity to cause hybridization and promote the formation of the CRISPR complex.

[0150] The CRISPR system can induce a double-strand break (DSB) at a target site, which is then broken down as described herein. In other embodiments, Cas9 variants considered "nickases" are used to cut single strands at a target site. For example, pairs of nickases can be used to increase specificity, each nickase being guided by a pair of different gRNA targeting sequences so that when the nicks are introduced simultaneously, 5' overhangs are introduced. In other embodiments, a catalytically inactive Cas9 is fused to a heterologous effector domain (e.g., a transcriptional repressor or activator) to affect gene expression.

[0151] 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 the cell, such as within an organelle. Typically, a sequence or template that can be used to reorganize a target locus comprising a target sequence is referred to as an "editing template" or "editing polynucleotide" or "editing sequence". In some aspects, an exogenous template polynucleotide may be referred to as an editing template. In some aspects, recombination is homologous recombination.

[0152] Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (including a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs from) the target sequence. The tracr sequence can comprise or consist of all or a portion of a wild-type tracr sequence (e.g., about or more than 20, 26, 32, 45, 48, 54, 63, 67, 85 or more nucleotides of a wild-type tracr sequence), and can also form part of a CRISPR complex, such as by hybridizing to all or a portion of a tracr partner sequence (operably linked to a guide sequence) along at least a portion of the tracr sequence. The tracr sequence has sufficient complementarity to the tracr mate sequence to hybridize and participate in the formation of the CRISPR complex, such as at least 50%, 60%, 70%, 80%, 90%, 95% or 99% sequence complementarity along the length of the tracr mate sequence when optimally aligned.

[0153] One or more vectors driving the expression of one or more elements of the CRISPR system can be introduced into a cell so that the expression of the elements of the CRISPR system directly forms a CRISPR complex at one or more target sites. Components can also be delivered to cells as proteins and / or RNA. For example, the Cas enzyme, the guide sequence connected to the tracr-mate sequence, and the tracr sequence can all be operably connected to separate regulatory elements on different vectors. Alternatively, two or more elements expressed from the same or different regulatory elements can be combined into a single vector, and one or more additional vectors provide any components of the CRISPR system that are not included in the first vector. The vector may contain one or more insertion sites, such as restriction endonuclease recognition sequences (also called "cloning sites"). In some embodiments, 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 in the cell.

[0154] The vector may comprise a regulatory element operably linked to an enzyme-coding sequence encoding 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, Cse1, 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, 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, accession number Q99ZW2.

[0155] The CRISPR enzyme can be Cas9 (e.g., from S. pyogenes or S. pneumonia). The CRISPR enzyme can directly cut one or both strands at the location of the target sequence, for example, 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 so that the mutated CRISPR enzyme lacks the ability to cut one or both strands of a target polynucleotide comprising a target sequence. For example, an aspartate-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cuts both strands to a nickase (cutting a single strand). In some embodiments, the Cas9 nickase can be used in combination with a guide sequence, for example, two guide sequences that target the sense strand and the antisense strand of a DNA target, respectively. This combination allows the two strands to be nicked and used to induce NHEJ or HDR.

[0156] In some embodiments, the enzyme coding sequence encoding the CRISPR enzyme is optimized for codons optimized for expression in a specific cell (e.g., a eukaryotic cell). The eukaryotic cell can be a cell from a specific organism (e.g., a mammal) or a cell derived from a specific organism, including but not limited to humans, mice, rats, rabbits, dogs, or non-human primates. In general, 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 of the native sequence with a more frequent or most commonly used codon in the host cell gene while maintaining the native amino acid sequence. Various species show particular biases for certain codons for specific amino acids. Codon bias (differences in codon usage between organisms) is generally associated with the translation efficiency of messenger RNA (mRNA), which in turn is believed to depend on factors such as the properties of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell generally reflects the most commonly used codons in peptide synthesis. Therefore, genes can be customized based on codon optimization to achieve optimal gene expression in a given organism.

[0157] Typically, a guide sequence is any polynucleotide sequence that has sufficient complementarity to a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more when optimally aligned using a suitable alignment algorithm.

[0158] Any suitable algorithm can be used to determine the optimal alignment of sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transform (e.g., BurrowsWheeler 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).

[0159] The CRISPR enzyme can be part of a fusion protein comprising one or more heterologous protein domains. The CRISPR enzyme fusion protein can contain any other protein sequence, as well as an optional linker sequence between any two domains. Examples of protein domains that can be fused to the CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, and protein domains having 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), β-galactosidase, β-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins, including blue fluorescent protein (BFP). CRISPR enzymes can be fused to gene sequences encoding proteins or protein fragments that bind to DNA molecules or bind to other cellular molecules, including, but not limited to, maltose binding protein (MBP), S tags, Lex A DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions.

[0160]

III. Astrocyte differentiation method

[0161] In some embodiments, a method for generating differentiated cells from a substantially single cell suspension of pluripotent stem cells (PSCs) (e.g., human iPSCs) is provided. In some embodiments, the PSCs are cultured to pre-confluence to prevent any cell aggregation. In some aspects, the PSCs are cultured to a ... TM or TRYPLE TM For example, PSCs can be dissociated by incubation with 5% paraformaldehyde (e.g., 4% paraformaldehyde ...

[0162] Once a single cell suspension of PSCs is obtained at a known cell density, the cells are typically seeded in an appropriate culture vessel, such as a tissue culture plate, such as a culture flask, a 6-well, 24-well, or 96-well plate. The culture vessel used to culture cells may include, but is not limited to, a culture flask, a tissue culture flask, a dish, a culture dish, a tissue culture dish, a multi-dish, a microplate, a multi-well plate, a microslide, a chamber slide, a test tube, a tray, Chambers, culture bags and roller bottles, as long as it is capable of culturing stem cells therein. Cells can be cultured in 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 derived therefrom, depending on the needs of the culture. In certain embodiments, the culture vessel can be a bioreactor, which can refer to any in vitro device or system that supports a biologically active environment so that cells can proliferate. The volume of the bioreactor is at least or about 2, 4, 5, 6, 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 500 L, 1, 2, 4, 6, 8, 10, 15 cubic meters, or any range derivable therein.

[0163] In certain aspects, PSCs, such as iPSCs, are plated at a cell density suitable for efficient differentiation. Typically, the 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, cells can be seeded at a cell density of about 50,000 to about 400,000 cells per well. In exemplary methods, cells are seeded at a cell 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).

[0164] PSC (e.g., iPSC) is usually cultured on a culture plate coated with one or more cell adhesion proteins to promote cell adhesion 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 the culture surface as a means of providing solid support for the growth of pluripotent cells. The term "extracellular matrix" is recognized in the art. Its components include one or more of the following proteins: fibronectin, laminin, vitronectin, tenascin, Entactin, thrombospondin, elastin, gelatin, collagen, fibrillin, merosin, ankyrin, cartilage junction protein, link protein, bone sialoprotein, osteocalcin, osteopontin, epinectin, hyaluronic adhesion protein, crude fiber modulin, epidermal integrin and galin protein. In an exemplary method, PSC grows on a culture plate coated with vitronectin or fibronectin. In some embodiments, the cell adhesion protein is a human protein.

[0165] Extracellular matrix (ECM) proteins can be naturally derived and purified from human or animal tissues, or, alternatively, ECM proteins can be genetically engineered recombinant proteins or proteins synthesized in nature. ECM proteins can be whole proteins or peptide fragments in the form of natural or engineered peptide fragments. Examples of ECM proteins that may be useful in cell culture matrices include laminin, type I collagen, type IV collagen, fibronectin, and vitronectin. In some embodiments, the matrix composition includes peptide fragments of synthetically generated fibronectin or recombinant fibronectin. In some embodiments, the matrix composition is xeno-free. For example, in a xeno-free matrix for culturing human cells, matrix components of human origin can be used, wherein any non-human animal components can be excluded.

[0166] In some aspects, the total protein concentration in the matrix composition may 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 a more preferred embodiment, the total protein concentration in the matrix composition is about 5 μg / mL to about 200 μg / mL.

[0167] Cells can be cultured with the nutrients necessary to support the growth of each specific cell population. Typically, cells are cultured in a growth medium that includes a carbon source, a nitrogen source, and a buffer to maintain pH. The medium may also include 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 TM (E8 TM ) medium and supplemented with various nutrients, such as non-essential amino acids and vitamins, to promote stem cell growth. Examples of minimum essential media include, but are not limited to, minimum essential medium Eagle (MEM) alpha medium, Dulbecco's modified Eagle medium (DMEM), RPMI-1640 medium, 199 medium, and F12 medium. In addition, the minimum essential medium may be supplemented with additives, such as horse, calf, or fetal bovine serum. Alternatively, the culture medium may be serum-free. In other cases, the growth medium may contain "knockout serum replacement," referred to herein as a serum-free formulation, which is optimized to grow and maintain undifferentiated cells, such as stem cells, in culture. For example, KNOCKOUT TM Serum replacements are disclosed in US Patent Application No. 2002 / 0076747, which is incorporated herein by reference. Preferably, PSCs are cultured in a fully defined feeder-free medium.

[0168] Therefore, PSCs are typically cultured in a fully defined medium after plating. In certain aspects, about 18 to 24 hours after plating, the medium is aspirated and replaced with fresh medium (e.g., E8 TM In some aspects, the single-cell PSC is cultured in a completely defined medium for about 1, 2, or 3 days after plating. Preferably, the single-cell PSC is cultured in a completely defined medium for about 2 days before undergoing a differentiation process.

[0169] In some embodiments, the culture medium may or may not contain any substitute for serum. Serum substitutes may include materials that appropriately contain albumin (for example, but not limited to, lipid-rich albumin, albumin substitutes such as recombinant albumin, plant starch, dextran and protein hydrolysate), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol or its equivalent. Serum substitutes may be prepared by methods disclosed in, for example, International Publication No. WO 98 / 30679. Alternatively, any commercially available material may be used to obtain more convenience. Commercially available materials include KNOCKOUT TMSerum Replacement (KSR), Chemically Defined Lipid Concentrate (Gibco), and GLUTAMAX TM (Gibco).

[0170] Other culture conditions can be defined appropriately. For example, the culture temperature is about 30-40° C., for example, at least or about 31, 32, 33, 34, 35, 36, 37, 38, 39° C., but is not particularly limited thereto. In one embodiment, the cells are cultured at 37° C. The CO2 concentration can be about 1% to 10%, for example, about 2-5%, or any range derived therefrom. The oxygen tension can be at least, up to or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20%, or any range derivable therefrom.

[0171] iPSCs, NPCs and / or astrocytes can be cultured in a defined medium including, but not limited to, DMEM-F12, E8, E6, Neurobasal medium, Minimum Essential Medium (MEM), and / or BrainPhys Neuronal Medium.

[0172] A. Neural progenitor cells (NPC)

[0173] iPSCs can be differentiated into NPCs with a glial bias, also known as glial progenitor cells. iPSCs can be harvested with EDTA and plated on an extracellular matrix (e.g., Matrigel) in Essential8 medium containing a ROCK inhibitor (e.g., at a concentration of 0.1-10 μM, such as 1 μM H1152). The cells are then fed with Essential 8 medium for 1 to 3 days, for example, 2 days.

[0174] The cells may then be pretreated with a medium comprising DMEM-F12 and a GSK3 inhibitor (eg CHIR99021, eg at a concentration of about 1-5 μM, particularly about 3 μM) under normoxic conditions for about 2-4 days, eg 3 days.

[0175] The cells can then be dissociated with TrypLE (Gibco) and aggregates can be formed using a medium containing essential 6 (Gibco), N2 supplement (Gibco) and a ROCK inhibitor (e.g., H1152, e.g., at a concentration of 0.1 to 10 μM, e.g., 1 μM). Aggregates can be formed in ultra-low adsorption bottles, rotators (e.g., 500 mL), or bioreactors (e.g., PBS microbioreactors). Aggregates can be cultured for 5 to 10 days, e.g., 8 days, and supplemented with NPC differentiation medium without ROCK inhibitor every other day. Aggregates can be dissociated with TrypLE in a 37°C water bath for 10 to 15 minutes. Dissociated cells can be filtered (e.g., 100 μM filter) and cryopreserved (e.g., using CryoStorCS10 (BioLifeSolutions)). NPCs may be from apparently healthy normal or diseased donors.

[0176] Cells can be stained for the pluripotency markers SSEA-4 and TRA-1-60 as well as NPC markers such as CD24, CD184, and CD271, which appear initially and increase over time. Other markers may include Nestin and PAX6.

[0177] In certain aspects, the current methods do not form neurospheres. In certain aspects, the current differentiation methods include culturing in the absence of a SMAD inhibitor, a TGFβ inhibitor, and / or a γ-secretase inhibitor.

[0178]

B. Differentiation of NPCs into astrocytes

[0179] An exemplary differentiation procedure for generating astrocytes from NPCs is shown in Figure 3A. NPCs can be grown at 20 k / cm 2The cells are seeded at a density of 100 μg / mL in a container coated with an extracellular matrix protein (e.g., Geltrex) in a medium containing DMEM / F12 medium supplemented with N2 and B27 (+vitA). The culture can be carried out under normoxic conditions, such as 20% oxygen. The stage 1 astrocyte culture medium may contain a chemically defined lipid concentrate (Gibco Product No. 11905031, such as 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) with delta-like canonical Notch ligand 1 (DLL1) and / or human Jagged 1Fc chimeric protein (JAGG1) (e.g., at a concentration of 1 to 25 ng / mL, especially about 10 ng / mL. The first stage astrocyte differentiation medium may further contain one or more LIF receptor ligands, such as leukemia inhibitory factor protein (LIF), ciliary neurotrophic factor protein (CNTF), oncostatin M protein and / or cardiotrophic factor 1 (CT-1) (e.g., each at a concentration of 1 to 25 ng / mL, especially about 10 ng / ml). Specifically, the first stage astrocyte culture medium includes CNTF, oncostatin-M, LIF and CT-1. The culture may be continued for about 1 to 3 weeks, for example, about 2 weeks, and passaged when confluent.

[0180] The lipid concentrate can be a concentrated lipid emulsion comprising saturated fatty acids and unsaturated fatty acids. For example, the lipid concentrate may include arachidonic acid (e.g., at a concentration of 2 mg / L), cholesterol (e.g., at a concentration of 220 mg / L), DL-α-tocopheryl acetate (e.g., at a concentration of 70 mg / L), linoleic acid (e.g., at a concentration of 10 mg / L), linolenic acid (e.g., at a concentration of 10 mg / L), myristic acid (e.g., at a concentration of 10 mg / L), oleic acid (e.g., at a concentration of 10 mg / L), palmitic acid (e.g., at a concentration of 10 mg / L), palmitoleic acid (e.g., at a concentration of 10 mg / L), and stearic acid (e.g., at a concentration of 10 mg / L). The lipid concentrate may also include Tween Pluronic F-68 and ethanol.

[0181] Next, for stage 2, the culture medium can be changed to an astrocyte culture medium containing one or more LIF receptor ligands, such as LIF, CNTF, oncostatin M and / or CT-1, in particular LIF and CNTF. The stage 2 astrocyte differentiation medium may also contain a lipid concentrate. The culture may last for about 3 to 8 weeks, such as 4 to 7 weeks, in particular 4 weeks, 5 weeks, 6 weeks or 7 weeks. The cells can then be stained for astrocyte markers CD44, NFIX and GFAP.

[0182] Astrocyte differentiation medium (e.g., stage 1 or stage 2) may further comprise one or more activators of the Notch pathway. One or more activators of the Notch pathway may 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).

[0183] In a specific aspect, the basal medium may include DMEM / F12, N2 supplement, B27 and retinoic acid supplement (e.g., at a concentration of 1%), GlutaMAX and penicillin / streptomycin. Astrocyte culture medium may also include OSM, CNTF and LIF (e.g., at a concentration of 5 to 25 ng / mL, particularly about 10 ng / mL). Astrocyte culture medium may also include DLL1 (e.g., at a concentration of 5 to 50 ng / mL, particularly about 10 ng / mL), JAGG1 (e.g., at a concentration of 5 to 50 ng / mL, particularly about 10 ng / mL), lipid concentrate (e.g., at a concentration of 1 to 5%, particularly about 2%), CT-1 (e.g., at a concentration of 5 to 50 ng / mL, particularly about 10 ng / mL) and / or EGF (e.g., at a concentration of 5 to 50 ng / mL, particularly about 20 ng / mL).

[0184] Astrocytes express several proteins that can be used as markers for detection using methods such as immunocytochemistry, Western blot analysis, flow cytometry, or enzyme-linked immunosorbent assay (ELISA). Astrocytes are stained for the surface markers, CD44 and glutamate aspartate transporter (GLAST), as well as the 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). Cell markers can be detected at the mRNA level, for example, by reverse transcriptase polymerase chain reaction (RT-PCR), Northern blot analysis, or using publicly available sequence data. Dot blot analysis is performed using sequence-specific primers in a standard amplification method. Expression of tissue-specific markers is considered positive if the expression detected at the protein or mRNA level is at least or about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold or 9-fold, more specifically 10-fold, 20-fold, 30-fold, 40-fold, 50-fold or more than that of control cells (e.g., undifferentiated pluripotent stem cells or other unrelated cell types).

[0185]

C. Differentiated culture medium

[0186] Cells can be cultured with the nutrients needed to support the growth of each specific cell population. Typically, cells are cultured in a growth medium that includes 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, pH indicators, and inorganic salts. Exemplary growth media include minimal essential media, such as Dulbecco's Modified Eagle's Medium (DMEM) or ESSENTIAL 8 TM (E8 TM ) medium and supplemented with various nutrients, such as non-essential amino acids and vitamins, to promote stem cell growth. Examples of minimum essential media include, but are not limited to, minimum essential medium Eagle (MEM) alpha medium, Dulbecco's modified Eagle medium (DMEM), RPMI-1640 medium, 199 medium, and F12 medium. In addition, the minimum essential medium may be supplemented with additives, such as horse, calf, or fetal bovine serum. Alternatively, the culture medium may be serum-free. In other cases, the growth medium may contain "knockout serum replacement," referred to herein as a serum-free formulation, which is optimized to grow and maintain undifferentiated cells, such as stem cells, in culture. For example, KNOCKOUT TM Serum replacements are disclosed in US Patent Application No. 2002 / 0076747, which is incorporated herein by reference. Preferably, PSCs are cultured in a defined, feeder-free medium.

[0187] In some embodiments, the culture medium may or may not contain any substitute for serum. Serum substitutes may include materials that appropriately contain albumin (such as lipid-rich albumin, albumin substitutes, such as recombinant albumin, plant starch, dextran and protein hydrolysate), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol or its equivalent. Serum substitutes can be prepared by methods disclosed in, for example, International Publication No. WO 98 / 30679. Alternatively, any commercially available material can be used to obtain more convenience. Commercially available materials include KNOCKOUT TM Serum Replacement (KSR), Chemically Defined Lipid Concentrate (Gibco), and GLUTAMAX TM (Gibco).

[0188] Other culture conditions can be defined appropriately. For example, the culture temperature is about 30-40° C., for example, at least or about 31, 32, 33, 34, 35, 36, 37, 38, 39° C., but is not particularly limited thereto. In one embodiment, the cells are cultured at 37° C. The CO2 concentration can be about 1% to 10%, for example, about 2-5%, or any range derived therefrom. The oxygen tension can be at least, up to or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20%, or any range derivable therefrom.

[0189]

D. Cryopreservation

[0190] Cells produced by the methods disclosed herein can be cryopreserved, for example, see PCT Publication No. 2012 / 149484A2, which is incorporated herein by reference, at any stage of the process, such as NPC, stage I astrocytes, or stage II astrocytes. Cells can be cryopreserved with or without a matrix. In several embodiments, the storage temperature range is 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, 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 several embodiments, liquid nitrogen (or other similar liquid coolants) are used to store cells. In further embodiments, the cells are stored for more than about 6 hours. In additional embodiments, the cells are stored for about 72 hours. In several embodiments, the cells are stored for 48 hours to about 1 week. In 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. The cells may also be stored for longer periods of time. The cells may be cryopreserved alone or on a substrate, such as any substrate disclosed herein.

[0191] In some embodiments, additional cryoprotectants can be used. For example, cells can be cryopreserved in a cryopreservation solution comprising one or more cryoprotectants (such as DM80), serum albumin (such as human or bovine serum albumin). In certain embodiments, the solution comprises 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 comprises 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 a specific embodiment, the solution comprises 2.5% DMSO. In another specific embodiment, the solution comprises 10% DMSO.

[0192] For example, during cryopreservation, the cells can be cooled at a rate of about 1°C / min. 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, the cells are cooled to 4°C and then cooled at a rate of about 1°C / min. The frozen cells can be transferred to the gas phase of liquid nitrogen before thawing. For example, in some embodiments, once the cells reach about -80°C, they are transferred to a liquid nitrogen storage area. A controlled rate refrigerator can also be used for freezing. The frozen cells can be thawed at a temperature of about 25°C to about 40°C, usually at a temperature of about 37°C.

[0193]

IV. How to use

[0194] The present disclosure provides a method for generating NPCs and astrocytes. These cell populations can be used for many important research, development and commercial purposes. These include, but are not limited to, transplantation or implantation of cells in vivo; in vitro screening of growth / regulatory factors, drug compounds, etc.; elucidation of the mechanisms of disease and infection; study of the mechanisms of action of drugs and / or growth factors; diagnosis and monitoring of disease in patients; gene therapy; and production of biologically active products, to name a few.

[0195] A. Test compound screening

[0196] The cell lines produced by the methods disclosed herein can be used for any methods and applications of iPSCs or differentiated cells known to date. For example, a method for evaluating a compound can be provided, comprising determining the pharmacological or toxicological properties of the compound on a cell line. A method for evaluating the effect of a compound on a cell culture can also be provided, comprising: (a) contacting a cell culture provided herein with a compound; (b) determining the effect of the compound on the cell culture.

[0197] The cell culture can be used commercially to screen factors (e.g., solvents, small molecule drugs, peptides, oligonucleotides) or environmental conditions (e.g., culture conditions or manipulations) that affect the properties of such cells and their various progeny. For example, the test compound can be a chemical compound, a small molecule, a polypeptide, a growth factor, a cytokine, or other biological agent.

[0198] In one embodiment, a method includes contacting a cell culture with a test agent and determining whether the test agent regulates the activity or function of cells in a cell population. In some applications, screening analysis is used to identify agents that regulate cell proliferation, change cell differentiation, or affect cell viability. Screening tests can be performed in vitro or in vivo. The method for screening and determining a candidate drug includes a method suitable for high-throughput screening. For example, a cell culture can be positioned or placed on a culture dish, a culture bottle, a roller bottle, or a culture dish (for example, a single multi-well culture dish or a culture dish, such as 8, 16, 32, 64, 96, 384, and 1536 multi-well plates or culture dishes), and can be selected to be placed in a specified position to identify potential therapeutic molecules. For example, libraries that can be screened include small molecule libraries, siRNA libraries, and adenovirus transfection vector libraries.

[0199] Other screening applications involve the testing of pharmaceutical compounds for their effects on retinal tissue maintenance or repair. Screening may be done because the compound is intended to have a pharmacological effect on the cells or because a compound intended to have an effect in other ways may have unexpected side effects on cells of this tissue type.

[0200]

B. Treatment and transplantation

[0201] Other embodiments may also provide for using the cell lines to treat a disease or condition. In another aspect, the present disclosure provides a method of treating an individual in need thereof, comprising administering to the individual a composition comprising an engineered cell.

[0202] In order to determine the suitability of the cell composition for therapeutic administration, cells can first be tested in a suitable animal model. On the one hand, the ability of the cell line to survive and maintain its phenotype in vivo is assessed. The composition is transplanted into an immunodeficient animal (e.g., a nude mouse or an immunodeficient animal made by chemical or irradiation). After a period of growth, tissue is harvested and the cells derived from the pluripotent stem cells are assessed to be still present.

[0203] Applicable diseases 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, depression, alcoholism, insomnia, mania, Alzheimer's disease, epilepsy and diabetic neuropathy. In specific aspects, the disease is Alexander disease or leukodystrophy.

[0204] [C. Pharmaceutical composition]

[0205] Also provided herein are pharmaceutical compositions and formulations comprising the present cells and a pharmaceutically acceptable carrier. In certain aspects, the present compositions provide a population of astrocytes that express at least SSEA4 and CD44.

[0206] Therefore, according to the present invention, the cell composition for administration to a subject can be formulated in any conventional manner, using one or more physiologically acceptable carriers, including excipients and adjuvants, which facilitate processing of the compound into a pharmaceutically acceptable preparation. The correct formulation depends on the selected route of administration.

[0207] The pharmaceutical compositions and formulations described herein can be prepared by mixing the active ingredients (eg, cells) having the desired degree of purity in the form of a lyophilized formulation or an aqueous solution with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 22 ndPharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to: buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethylammonium chloride; benzalkonium chloride; benzalkonium 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; 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 dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; counterions to form salts, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein also include internal drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX, International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases (such as chondroitinase).

[0208] D. Distribution for Commercial, Therapeutic and Research Purposes

[0209] In some embodiments, a reagent system is provided that includes cells present at any time during manufacture, distribution, or use. The kit may contain any combination of cells described in the present disclosure and undifferentiated pluripotent stem cells or other differentiated cell types, typically sharing the same genome. Each cell type may be packaged together in the same facility or at different locations, at the same or different times, or in different containers, controlled by the same entity or different entities sharing a business relationship. The pharmaceutical composition may be optionally packaged in a suitable container with written instructions for the desired purpose, such as mechanical toxicology.

[0210] In some embodiments, a kit is provided, which may include, for example, one or more culture media and components for producing cells. The reagent system may be packaged in an aqueous medium or, where appropriate, in a lyophilized form. The container device of the kit generally includes at least one vial, test tube, flask, bottle, syringe or other container device, in which the components can be placed, preferably in appropriate equal portions. If there are multiple components in the kit, the kit generally also includes a second, third or other additional container, in which the other components can be placed separately. However, a vial may contain various combinations of components. The components of the kit may be provided in the form of dry powders. When the reagents and / or components are provided in the form of dry powders, the powder can be reconstituted by adding a suitable solvent. It is conceivable that the solvent may also be provided in another container. The kit of the present disclosure will generally also include a device for tightly sealing the kit components for commercial sale. Such containers may include injection or blow-molded plastic containers in which the required vials are retained. The kit may also include instructions for use, such as printed or electronic versions, such as digital versions.

[0211] [V. Embodiment]

[0212] The following examples are included to demonstrate preferred embodiments of the present invention. It should be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in the practice of the present invention and therefore can be considered to constitute preferred modes for its implementation. However, it should be appreciated by those skilled in the art based on this disclosure that many changes can be made in the disclosed specific embodiments and that similar or similar results can still be obtained without departing from the spirit and scope of the present invention.

[0213] [Example 1: Generation of astrocytes]

[0214] Human induced pluripotent stem cells (hiPSCs) were harvested with EDTA and cultured at 15,000 c / cm in Essential 8 medium (Gibco) containing 1 μM ROCK inhibitor H1152 (Sigma). 2 The cells were plated on tissue culture (TC) treated plates coated with Matrigel (Corning) 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. Starting from day 3, the cells were preactivated 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 microbioreactors. Aggregates were cultured for 8 days and fed every other day with NPC differentiation medium without H1152. On day 14, aggregates were dissociated using TrypLE in a 37°C water bath for 10-15 minutes. Dissociated cells were filtered 100 μM and cryopreserved using CryoStor CS10 (BioLifeSolutions). NPCs were generated from 5 different hPSC cell lines from apparently healthy normal donors and diseased donors.

[0215] Cell surface (Figures 2A and 2C) and intracellular antigens (Figures 2B to 2D) were stained. The pluripotency markers SSEA-4 and TRA-1-60 decreased significantly during the 14-day differentiation process, while NPC markers such as CD24, CD184, and CD271 began to appear and increased over time. NPCs from all five tested lines showed high Pax6 and Nestin expression, 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 minutes and then quenched with medium containing B27 supplement (Gibco). For cell surface antigens, 100,000 cells were stained with antibody diluent for 30 minutes at room temperature. Propidium iodide (Sigma) was used to exclude dead cells. For intracellular antigens, 100,000 cells were first stained for viability for 10 minutes using fixable viability stain 620 (BD Biosciences), 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 dilutions. Cells were analyzed using an Accuri C6 flow cytometer (BD Biosciences).

[0216] A schematic diagram of the differentiation procedure for generating astrocytes from NPCs is shown in Figure 3A. NPCs were cultured at 20 k / cm 2The density of 100 μg / ml was seeded in a container coated with Geltrex, and DMEM / F12 medium supplemented with N2 and B27 (+vitA) and LIF receptor ligands (LIF, CNTF, oncostatin-M and CT-1, all 10 ng / ml) was used for two weeks and passaged at confluence. After two weeks, the culture medium was replaced with LIF+CNTF and lipid concentrate for the rest of the differentiation. The cells were fixed in 4% paraformaldehyde (Sigma) for 60 minutes and stored in DPBS (Gibco) for immunocytochemistry. The primary antibody was diluted in ICC permeation buffer (DPBS+2% FBS+0.2% Triton X-100) and incubated overnight at 4°C. After the primary antibody incubation, the antibody was removed and the cells were washed 3 times in 100 μL DPBS per well for 10 minutes. The secondary antibody was then applied for 1 hour at RT. After the secondary incubation, Hoescht33342 (Invitrogen Cat#H3570) was added for 10 minutes. The cells were then washed 3 times with 100 μL DPBS per well for 10 minutes and imaged on a Molecular Devices Image Xpress automated microscope. Phase contrast images of cells seeded on a PLO / laminin matrix (B top) on day 60 and ICC staining of astrocyte markers CD44, NFIX, and GFAP proteins on days 35 and 80 after differentiation are shown in Figure 3B. The percentage of positive cells for several astrocyte markers on day 70 was quantified using flow cytometry as shown in Figure 3C.

[0217] The theoretical cumulative yield during differentiation showed significant cell growth (Figure 3D). The time course glutamate uptake efficiency (the percentage of glutamate concentration in -TBOA samples to the corresponding +TBOA samples) is shown in Figure 3E. NPC-derived astrocytes can take up glutamate from culture medium comparable to iCell astrocytes. For glutamate uptake assays, cells were plated in triplicate under control and +TBOA (Tocris Cat#1223) conditions at a density of 30,000 cells per well on vitronectin-coated 96-well plates. The cells were then cultured for 7 or 14 days before the start of the assay. On the day of the assay, the cells were washed once with HBSS (Gibco) and incubated in HBSS or HBSS+300μM TBOA for 1 hour at 37°C 5% CO2. After incubation, 20 μM glutamate was added to the wells incubated with HBSS, and 20 μM glutamate + 300 μM TBOA was added to the wells containing HBSS + 300 μM TBOA and placed at 37 ° C 5% CO2 for 1 hour. After incubation, the samples were moved to a separate 96-well plate and centrifuged at 400 × G for 10 minutes. After centrifugation, the remaining glutamate concentration in the samples was analyzed using the Glutamate-Glo detection kit (Promega Cat# J7021) according to the manufacturer's instructions.

[0218] Microelectrode assay (MEA) raster plots of different cultures before (baseline) and after glutamate administration are shown in Figure 3F. The results show that both iCell astrocytes and NPC-astrocytes are able to maintain network activity and take up excess glutamate and protect neurons. To further evaluate the function of the derived astrocytes, the electrical activity of astrocyte-neuron co-cultures was compared with neuronal monocultures on MEA. Briefly, 120K iCell Gluta neurons were dotted with 30K iCell astrocytes or NPC-astrocytes on 48 classic MEA plates (Axion Biosystems). Cells were replenished and recorded using BrainPhys complete medium. On the day of recording, 50% of the spent medium was replaced with complete BrainPhys medium approximately 2 to 4 hours before data acquisition, and recorded for 5 to 10 minutes (300 to 600 seconds) to fully capture network bursting behavior. On day 23, neuronal activity was recorded as baseline, then 20 μM l -glutamate was added to four wells of three groups (iCell Gluta neurons alone, iCell Gluta neurons co-cultured with iCell astrocytes, and iCell Gluta neurons co-cultured with NPC-derived astrocytes) and recorded again 1 hour later.

[0219] Next, 31 media combinations were evaluated. Figure 4A Fold expansion analysis of 31 culture media over a 14-day differentiation time course is shown. Purity analysis at day 14 was performed by flow cytometry for expression of astrocyte lineage-related markers. Figure 4B shown. Figure 4C The compositions of 11 media with the best performance in terms of expansion rate and purity in a comparison of 31 media are provided and these media were selected for further experiments.

[0220] Figure 5A Marker expression at day 35 of differentiation is shown. The cells expressed less than 1% CD15, about 90-100% (e.g., greater than 95%, 96%, 97%, 98%, or 99%) CD56, less than 20% (e.g., less than 15%, 10%, 5%, or 1%) SSEA4, about 90-100% (e.g., greater than 95%, 96%, 97%, 98%, or 99%) NFIX, and less than 10% (e.g., less than 5% or 1%) GFAP. Expression of CD44 / S100b varied from about 15% to greater than 80%, and expression of GLAST varied from about 2% to greater than 70%. Flow cytometric analysis comparing 11 media as shown. Figure 5B The cumulative fold expansion analysis of 11 culture media during 35 days of differentiation is shown in Figure 5C Condition 14 showed about 28-fold expansion, condition 16 showed about 36-fold expansion, and condition 29 showed about 34-fold expansion. The five culture media were further evaluated by immunocytochemistry of D49 cells ( Figure 5E )( Figure 5E ). Then, glutamate uptake assay was performed on D49 cells from the 5 media comparison study ( Figure 5D ).

[0221] Further studies showed the re-emergence of SSEA4+ as a marker for astrocyte progenitors. Representative flow cytometry images of surface CD56 / SSEA4 co-staining on D28 and intracellular CD44 / SSEA4 co-staining on D28 and D35 under medium 14 conditions are shown in Figure 2. Fig. 6AAs shown. With condition 14, CD44 / SSEA4 cells increased from 15.8% on day 28 to 31.3% on day 35. The time course of surface SSEA4 expression and intracellular CD44 expression from D7 to D35 is shown for (Fig. 6B) condition 3, (Fig. 6C) condition 14, (Fig. 6D) condition 16, (Fig. 6E) condition 24, and (Fig. 6F) condition 29. Medium conditions 14, 16, and 24 were continued until D42. Conditions 14, 16, and 24 resulted in an increase in SSEA4 from less than 20% to more than 95% after day 35, and a gradual increase in CD44 from approximately 40% on day 14 to more than 80% on day 35. The differential expression of SSEA4 and CD44 by surface and intracellular staining on D28 and D35 was measured. A summary of the expression profiles is provided for (FIG 6G) condition 14, (FIG 6H) condition 16, (FIG 6I) condition 24, and (FIG 6J) condition 29. For conditions 14, 16, and 24, more than 60% of SSEA4-positive cells were present on day 28, and more than 80% of SSEA4-positive cells were present on day 35. CD44 / SSEA4-positive cells were about 20% to 40%, such as about 25% to 30%.

[0222] Immunocytochemistry of astrocytes cultured in Astro3 medium, AMM, or BrainPhys complete medium 7 days after thawing is shown in Figure 7A. Glutamate uptake assay of astrocytes cultured in Astro3 medium, AMM, or BrainPhys complete medium 7 days after thawing is shown in Figure 7C. Quantitative results of MEA data analysis are shown in Figure 7C.

[0223] To further evaluate the function of the derived astrocytes, the electrical activity of astrocyte-neuron co-cultures was compared with that of neuron monocultures on MEA. Luminex analysis of the concentrations of common astrocyte secretory proteins is shown in Figure 7D. Luminex analysis of the concentrations of cytokine- or LPS-induced reactive astrocyte secretory proteins is shown in Figure 7E, and RNA-Seq analysis of common astrocyte markers is shown in Figure 7F.

[0224]

Table 1: Antibody list

[0225] Antibody supplier Part Number SSEA4 BDBioSciences 560796 TRA160 Biolegend 330614 CD56 BDBioSciences 555516 CD15 Biolegend 301908 CD44 BDBioSciences 555478 CD44 BDBioSciences 559942 CD271 Miltenyi Biotec 130-113-421 CD24 Biolegend 311110 CD184 Biolegend 306506 TUJ BDBioSciences 560381 Nestin BDBioSciences 560393 Pax6 Miltenyi Biotec 130-123-267 Pax6 Biolegend 901301 SOX1 BDBioSciences 562224 DCX BDBioSciences 561505 NFIX ThermoFisher PA5-64917 GFAP Dako Z0334

[0226] Astrocytes from four batches were thawed and cultured in Astro 3 medium for 7 days and then stimulated in basal medium for 24 hours. Supernatants were collected and assayed using R&D's custom Luminex assay on a FLEXMAP 3D instrument according to the manufacturer's instructions. Astrocytes were able to robustly secrete IL-1ra ( Fig. 8A ). Stimulation with IL-1α or TNF-α resulted in a greater than 3-fold increase in the induction of IL-1ra secretion. Combining TNF-α with IL-1α or IL-1β resulted in an approximately 14-fold increase over unstimulated controls. The combination of IFN-γ with TNF-α produced the highest fold change over control, with more than 84-fold increase in IL-1ra secretion compared to control. IL-1ra binds to the cell surface IL-1 receptor, blocking the binding of pro-inflammatory IL-1α and IL-1β.

[0227] Astrocytes can robustly secrete IL-6 ( Figure 8B ). IFN-γ alone or in combination with TNF-α resulted in a 32-fold and 39-fold increase in IL-6 secretion, respectively, compared to unstimulated controls. IL-α stimulation resulted in a more than 400-fold increase in IL-6 secretion compared to controls. TNF-α in combination 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.

[0228] Astrocytes can 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 resulted in an 898-fold and 665-fold increase in IL-8 secretion, respectively, compared to unstimulated controls. IL-α stimulation resulted in a greater than 3,300-fold increase in IL-8 secretion compared to controls. TNF-α in combination 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 pro-inflammatory immune cells following injury. The analyte also plays a role in the recruitment and differentiation of oligodendrocyte precursor cells (OPCs) to promote remyelination.

[0229] Astrocytes can robustly secrete IL-10 ( Fig.8D ). IFN-γ stimulation resulted in a 161-fold increase in IL-10 secretion compared to unstimulated controls. IL-α stimulation resulted in a more than 415-fold increase in IL-10 secretion 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 is secreted by astrocytes to reduce iNos activity and decrease astrogliosis.

[0230] Astrocytes can robustly secrete CCL5 / RANTES after stimulation with IL-1α, TNF-α, or their combination. Fig. 8EIL-1α stimulation resulted in a 156-fold increase in CCL5 / RANTES secretion 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 controls the motility of peripheral immune cells.

[0231] Astrocytes can robustly secrete CCL7 after stimulation with IL-1α, TNF-α, or their combination. Figure 8F ). TNF-α stimulation resulted in a 1184-fold increase in CCL7 secretion compared to the control group. IL-1α stimulation resulted in a 2177-fold increase in CCL7 secretion compared to the unstimulated control. IFN-γ and TNF-α stimulation increased CCL7 secretion 3745-fold compared to the unstimulated control. TNF-α in combination with IL-1α or IL-1β increased CCL7 secretion by more than 15,000-fold compared to the control group. CCL7 is important for astrocyte-microglia interactions and induces microglial activation.

[0232] Astrocytes can 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. Combination of TNF-α 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.

[0233] Astrocytes can robustly secrete CXCL1 after stimulation with IL-1α, TNF-α, or their combination. Figure 8H ). TNF-α alone or in combination with IFN-γ resulted in more than 9-fold increase in CXCL1 secretion compared to controls. Stimulation with IL-1α increased CXCL1 secretion 84-fold compared to unstimulated controls. TNF-α in combination with IL-1α or IL-1β increased CXCL1 secretion more than 450-fold compared to controls. CXCL1 is secreted by astrocytes, recruits neutrophils to sites of infection, and increases BBB permeability.

[0234] Astrocytes can robustly secrete CXCL2 after stimulation with IL-1α, TNF-α, or their combination. Figure 8I). TNF-α alone or in combination with IFN-γ resulted in CXCL2 secretion greater than 51-fold compared to controls. IL-1α stimulation increased CXCL2 secretion 379-fold compared to unstimulated controls. TNF-α in combination with IL-1α or IL-1β increased CXCL2 secretion greater than 1585-fold compared to controls. CXCL2 activates CXCR2 found on oligodendrocytes and OPCs, thereby increasing OPC proliferation and differentiation.

[0235] Astrocytes can robustly secrete CXCL5 after stimulation with IL-1α, TNF-α, or their combination. Figure 8J ). TNF-α alone or in combination with IFN-γ resulted in >47-fold increase in CXCL5 secretion compared to controls. Stimulation with IL-1α increased CXCL5 secretion 161-fold compared to unstimulated controls. TNF-α in combination with IL-1α or IL-1β increased CXCL5 secretion >872-fold compared to controls. CXCL5 is secreted by astrocytes in response to injury and activates microglia, resulting in decreased microglial phagocytosis and inhibition.

[0236]

Table 2: Culture medium composition

[0237]

Culture medium 1 to 8

[0238] Medium 1 Medium 2 Medium 3 Medium 4 Medium 5 Medium 6 Medium 7 Medium 8 Basic M Basic M Basic M Basic M Basic M Basic M Basic M Basic M Lipid concentrate Lipid concentrate Lipid concentrate Lipid concentrate CT1 CT1 CT1 CT1 EGF EGF EGF EGF

[0239]

Culture medium 9-16

[0240]

[0241]

Culture medium 17-24

[0242]

[0243]

Culture medium 25-31

[0244] Medium 25 Medium 26 Medium 27 Medium 28 Medium 29 Medium 30 Medium 31 Basic M Basic M Basic M Basic M Basic M Basic M Basic M DLL1 DLL1 DLL1 DLL1 DLL1 DLL1 DLL1 JAGG1 JAGG1 JAGG1 JAGG1 JAGG1 JAGG1 JAGG1 Lipid concentrate Lipid concentrate Lipid concentrate CT1 CT1 CT1 CT1 EGF EGF EGF EGF

[0245]

Basic culture medium

[0246] Basic culture medium Final concentration DMEM / F12 N2 1％ B27+RA 2％ GlutaMAX 1％ Pen / Strep 1％ OSM 10 ng / mL CNTF 10 ng / mL LIF 10 ng / mL

[0247]

Variable ingredients

[0248] Variable components Final concentration DLL1 10 ng / mL JAGG1 10 ng / mL Lipid concentrate 2％ CT-1 Series 10 ng / mL EGF 20 ng / mL

[0249]

***

[0250] All methods disclosed and claimed herein can be made and executed without undue experimentation in view of the present disclosure. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes may be applied to the methods and steps or order of steps described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain chemically and physiologically related agents may be substituted for the agents described herein while the same or similar results will be obtained. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be in accordance with the spirit, scope, and concept of the invention as defined by the appended claims.

[0251]

References

[0252] The following references are specifically incorporated herein by reference to the extent they provide exemplary procedural or other details supplementary to those described herein.

[0253] Alexander et al., Proc. Nat. Acad. Sci. USA, 85:5092-5096, 1988.

[0254] Ausubel et al., Current Protocols in Molecular Biology, Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., MA, 1996.

[0255] Blomer et al., 1997.

[0256] Chen et al., Nature Methods 8:424-429, 2011.

[0257] Ercolani et al., J. Biol. Chem., 263:15335-15341, 1988.

[0258] Evans, et al., In: Cancer Principles and Practice of Oncology, Devita et al. (Eds.), Lippincot-Raven, NY, 1054-1087, 1997.

[0259] Fechheimer et al., Proc Natl. Acad. Sci. USA, 84:8463-8467, 1987.

[0260] International Publication No.WO 2012 / 149484

[0261] International Publication No.WO 02 / 016536

[0262] International Publication No.WO 03 / 016496

[0263] International Publication No.WO 2007 / 069666

[0264] International Publication No.WO 2007 / 069666

[0265] International Publication No.WO 98 / 30679

[0266] International Publication No.WO 98 / 30679

[0267] International Publication No.WO 98 / 53058

[0268] International Publication No.WO 98 / 53059

[0269] International Publication No.WO 98 / 53060

[0270] Karin et al.Cell,36:371-379,1989.

[0271] Langle-Rouault et al.,J.Virol.,72(7):6181-6185,1998.

[0272] Levitskaya et al.,Proc.Natl.Acad.Sci.USA,94(23):12616-12621,1997.

[0273] Ludwig et al.,Nat.Biotechnol.,24:185-187,2006b.

[0274] Ludwig et al., Nat. Methods, 3:637-646, 2006a.

[0275] Maniatis, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1988.

[0276] Ng,Nuc.Acid Res.,17:601-615,1989.

[0277] Pelletier and Sonenberg,Nature,334(6180):320-325,1988.

[0278] Quitsche et al., J. Biol. Chem., 264:9539-9545, 1989.

[0279] Richards et al., Cell, 37:263-272, 1984.

[0280] Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd Ed. Cold Spring Harbor 1997.

[0281] Takahashi et al., Cell, 131:861-872, 2007.

[0282] U.S. Patent No. 4,683,202

[0283] U.S. Patent No. 5,556,954

[0284] U.S. Patent No. 5,925,565

[0285] U.S. Patent No. 5,928,906

[0286] U.S. Patent No. 5,935,819

[0287] U.S. Patent No. 6,103,470

[0288] U.S. Patent No. 6,140,081

[0289] U.S. Patent No. 6,416,998

[0290] U.S. Patent No. 6,453,242

[0291] U.S. Patent No. 6,534,261

[0292] U.S. Patent No. 7,442,548

[0293] U.S. Patent No. 7,598,364

[0294] U.S. Patent No. 7,682,828

[0295] U.S. Patent No. 7,989,425

[0296] U.S. Patent No. 8,058,065

[0297] U.S. Patent No. 8,071,369

[0298] U.S. Patent No. 8,129,187

[0299] U.S. Patent No. 8,268,620

[0300] U.S. Patent No. 8,278,620

[0301] U.S. Patent No. 8,546,140

[0302] U.S. Patent No. 8,546,140

[0303] U.S. Patent No. 8,741,648

[0304] U.S. Patent Publication No. 2002 / 0076747

[0305] U.S. Patent Publication No. 2002 / 0076747

[0306] U.S. Patent Publication No. 20020055144

[0307] U.S. Patent Publication No. 2003 / 0211603

[0308] U.S. Patent Publication No. 2005 / 0064474

[0309] U.S. Patent Publication No. 2005 / 0260186

[0310] U.S. Patent Publication No. 2006 / 0104968

[0311] U.S. Patent Publication No. 2006 / 0188987

[0312] U.S. Patent Publication No. 2007 / 0218528

[0313] U.S. Patent Publication No. 20090148425

[0314] U.S. Patent Publication No. 20090246875

[0315] U.S. Patent Publication No. 2010 / 0210014

[0316] U.S. Patent Publication No. 2011 / 0301073

[0317] U.S. Patent Publication No. 2011 / 0301073

[0318] U.S. Patent Publication No. 2011 / 0301073

[0319] U.S. Patent Publication No. 2012 / 0196360

[0320] U.S. Patent Publication No. 20120276636

[0321] Yamanaka et al., Cell, 131(5):861-72, 2007. Yu et al., Science, 318:1917, 2007.

Claims

1. An in vitro method for generating astrocytes from induced pluripotent stem cells (iPSCs), comprising: (a) Obtaining a starting population of iPSC-derived neural progenitor cells (NPCs); (b) culturing the NPCs in the presence of at least one leukemia inhibitory factor (LIF) receptor ligand for a period of time sufficient to generate astrocyte progenitor cells (APCs); and (c) further culturing the APCs in the presence of at least one LIF receptor ligand and the lipid concentrate for a period of time sufficient to generate a population of astrocytes.

2. The method according to claim 1, wherein: iPSCs were cultured in serum-free defined medium.

3. The method according to claim 1 or 2, wherein: The method complies with the Good Manufacturing Practice (GMP) for pharmaceutical production.

4. The method according to any one of claims 1 to 3, wherein: One or more of steps (a) to (d) are performed under xeno-free conditions, feeder-free conditions and / or conditioned medium-free conditions.

5. The method according to 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 according to any one of claims 1 to 3, wherein: Each of steps (a) to (d) is performed under certain conditions.

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

8. The method according to any one of claims 1 to 7, wherein: The starting group of NPCs to obtain include: (a) iPSCs were cultured on a surface coated with extracellular matrix (ECM) proteins in the presence of ROCK inhibitor. (b) iPSCs were further cultured in the absence of ROCK inhibitor or bubistatin; (c) iPSCs were pretreated in the presence of GSK3 inhibitors; (d) Differentiation of iPSCs into NPC populations.

9. The method according to claim 8, wherein: ECM proteins are: laminin, fibronectin, vitronectin, matrigel TM , tenascin, entactin, thrombospondin, elastin, gelatin and / or collagen.

10. The method according to claim 8, wherein: ECM proteins were purified from basement membrane extract (BME) from mouse Engelbreth-Holm-Swarm tumors.

11. The method according to claim 8, wherein: The ECM protein is Matrigel TM , laminin, or vitronectin.

12. The method according to claim 8, wherein: The extracellular matrix protein is matrigel TM .

13. The method according to any one of claims 8 to 12, wherein: The method does not comprise inhibiting SMAD signaling.

14. The method according to any one of claims 8 to 13, wherein: Steps (a) to (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) to (b) is further defined as adherent two-dimensional culture.

16. The method according to any one of claims 8 to 15, wherein: Step (a) lasts for about 24 hours.

17. The method according to any one of claims 8 to 16, wherein: Step (b) lasts 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 according to any one of claims 8 to 18, wherein: Step (c) is carried out under normoxic conditions.

20. The method according to any one of claims 8 to 19, wherein: Step (c) is performed for about 72 hours.

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

22. The method according to any one of claims 8 to 20, wherein: GSK3 inhibitor CHIR99021.

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

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

25. The method according to any one of claims 8 to 24, wherein: Step (d) comprises culturing on ultra-low attachment plates, spinners or bioreactors.

26. The method according to any one of claims 8 to 25, wherein: Step (d) lasts for about 8 days.

27. The method according to any one of claims 8 to 26, wherein: NPCs express CD24, CD184 and CD271.

28. The method according to 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 according to any one of claims 8 to 28, wherein: At least 70% of the NPC population was positive for CD24 and Nestin.

30. The method according to any one of claims 8 to 29, wherein: NPCs express Pax6 and Nestin.

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

32. The method according to any one of claims 8 to 31, wherein: Keep NPCs frozen.

33. The method according to any one of claims 1 to 32, wherein: iPSCs are derived from healthy donors.

34. The method according to any one of claims 1 to 33, wherein: iPSCs are derived from donors with the disease.

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

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

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

38. The method according to any one of claims 1 to 35, wherein: Astrocytes were positive for SSEA4 and CD44.

39. The method according to any one of claims 1 to 35, wherein: At least 30% of the astrocyte population was positive for SSEA4 and CD44.

40. The method of claim 37, wherein: Astrocytes have functional glutamate uptake and / or development of neural networks.

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

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

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

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

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

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

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

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

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

51. The method according to any one of claims 41 to 49, wherein: The concentration of LIF, CNTF, oncostatin-M and / or CT-1 is approximately 10 ng / mL.

52. The method according to any one of claims 1 to 51, wherein: Step (b) involves culturing NPCs on a Geltrex-coated surface.

53. The method according to any one of claims 1 to 52, wherein: Step (b) lasts for about 2 weeks.

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

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

56. The method of claim 55, wherein: Chemically defined lipid concentrates include saturated and unsaturated fatty acids.

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

58. The method according to any one of claims 1 to 55, wherein: Step (c) lasts for about 4 to 7 weeks.

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

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

61. The method according to any one of claims 1 to 60, wherein: At least 80% of the astrocyte population was positive for S100B, CD44 and / or NFIX.

62. The method according to any one of claims 1 to 61, wherein: At least 30% of the astrocyte population was positive for CD56 and / or GFAP.

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

64. The method according to any one of claims 1 to 63, wherein: 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. The method according to any one of claims 1 to 63, wherein: 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 the astrocyte population produced according to any one of claims 1 to 63 and a pharmaceutically acceptable carrier.

67. The composition according to claim 66, wherein At least 30% of the astrocyte population was positive for SSEA4 and CD44.

68. The composition according to claim 66, wherein At least 45% of the astrocyte population was positive for SSEA4 and CD44.

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

70. The composition according to claim 69, wherein At least 80% of the astrocyte population was positive for S100B, CD44 and / or NFIX.

71. The composition of claim 69, wherein At least 30% of the astrocyte population was positive for SSEA4 and CD44.

72. The composition of claim 69, wherein At least 45% of the astrocyte population was positive for SSEA4 and CD44.

73. The composition of claim 69, further comprising neurons.

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

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

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

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

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

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

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

81. A neurodegeneration model comprising the co-culture according to claim 77.

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

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

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