Thermoreversible polymers with improved stability and methods and uses thereof

Abstract

Provided herein are thermoreversible polymers with improved stability, hydrogel compositions comprising the thermoreversible polymers, and in vitro methods for generating an enriched cell population for inhibitory GABAergic cortical interneurons from human pluripotent cells using the hydrogel compositions. In some embodiments, the pluripotent cells are human pluripotent stem cells (hPSCs) (e.g., human embryonic stem cells or human induced pluripotent stem cells (iPSCs)).

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 595,847, filed November 3, 2023, and U.S. Provisional Patent Application No. 63 / 595,841, filed November 3, 2023, the entire contents of each of which are incorporated herein by reference. [Background technology]

[0002] BACKGROUND OF THE INVENTION Human pluripotent stem cells (hPSCs) have the ability to differentiate into every cell type in the body and therefore have broad applications for regenerative medicine. However, large-scale expansion and differentiation of hPSCs remains a major challenge in the field, preventing these transformative therapies from reaching the large patient population in need. Herein, we describe the creation of a well-defined, synthetic, thermoreversible hydrogel that enables large-scale growth and differentiation of hPSCs into sensitive cell types. This improved hydrogel formulation allows for a lower viscosity gel, enabling the encapsulation of sensitive cell types using a diverse range of liquid handling systems, thanks to the unique properties of the polymer blend and molecular weight (MW) that provide a low-viscosity hydrogel ideal for the culture and differentiation of hPSCs into all three germ layers. This minimizes shear during the cell encapsulation process for large-scale bioreactor systems. The blend and MW also allow for greater flow rates during encapsulation and greater control over bead size / shape. The polymer MW and PEG length are optimized to provide a stable hydrogel that maintains structure during the long differentiation process and offers a range of stiffness that supports hPSC expansion and differentiation into cells of all three germ layers. This formulation is also compatible with functionalization, allowing for controlled protein presentation and release. Using this thermoreversible hydrogel, we were able to expand hPSCs on a large scale and differentiate them into several cell fates, including midbrain dopaminergic neurons, cortical interneurons, pancreatic endodermal progenitor cells, and hematopoietic stem cells. Summary of the Invention [Means for solving the problem]

[0003] (Summary of the Invention) Provided herein is a scalable in vitro method for generating postmitotic inhibitory GABAergic cortical interneurons (cINs) from human stem cells (e.g., human pluripotent or multipotent cells), comprising encapsulating the stem cells in a three-dimensional synthetic hydrogel under conditions that generate a population of cells comprising cINs. In some embodiments, the pluripotent cells are human pluripotent stem cells (hPSCs) (e.g., human embryonic stem cells or human induced pluripotent stem cells (iPSCs)). Preferably, the method is xeno-free, and the hydrogel does not contain extracellular matrix (ECM) proteins.

[0004] In a preferred embodiment, the three-dimensional hydrogel for use in the method is a thermoresponsive (e.g., thermoreversible) hydrogel. In some embodiments, the three-dimensional hydrogel is a polyethylene glycol-poly(N-isopropylacrylamide) (PEG-PNIPAAM)-based hydrogel. In some embodiments, the thermoresponsive hydrogel has a lower critical solution temperature (LCST) of less than about 30°C, less than about 29°C, less than about 28°C, less than about 27°C, or less than about 26°C. In related embodiments, the thermoresponsive hydrogel has one or more of the following properties: (a) an LCST of about 12°C to about 32°C, preferably between about 20°C and 24°C, more preferably about 22°C; (b) a stiffness of about 100 Pascals (Pa) to about 8000 Pascals (Pa), preferably about 800 Pa to about 1000 Pa; (c) a liquidus viscosity of about 200 cP to about 4000 cP, preferably about 200 cP to about 1000 cP.

[0005] Also provided herein are novel thermoreversible hydrogels useful in the methods described herein.

[0006] Also provided herein is a composition comprising a population of cells produced by the methods described herein, wherein the population of cells is enriched for postmitotic inhibitory GABAergic cortical interneurons. Preferably, the composition does not contain ECM and is xeno-free.

[0007] Also provided herein is the use of a composition comprising cIN produced by the methods described herein in the treatment of a neurological disorder. In some preferred embodiments, the neurological disorder is epilepsy. [Brief explanation of the drawings]

[0008] [Figure 1] Figure 1 is a schematic diagram showing the two-step synthesis of thermoreversible poly(NIPAAm-co-Bam)-b-PEG graft copolymer of formula (III). Molar ratios are indicated by lowercase letters between the intermediate reactants. AIBN refers to azobisisobutyronitrile. Butylamine is shown; however, other lower alkylamines can be substituted in this reaction.

[0009] [Figure 2] Figure 2 shows a comparison of the properties of acrylate-based versus acrylamide-based thermoreversible polymers. LCST = lower critical solution temperature.

[0010] [Figure 3] Figure 3 shows a comparison of the properties of low MW acrylate-based thermoreversible polymers versus high MW acrylate-based thermoreversible polymers. LCST = lower critical solution temperature.

[0011] [Figure 4] FIG. 4 shows a comparison of the effect of PEG molecular weight on the properties of acrylate-based thermoreversible polymers.

[0012] [Figure 5A-B]Figures 5A-5D show a comparison of the effect of PEG:PNIPAAm polymer wt / wt% on the properties of acrylate-based thermoreversible polymers. Figures 5C and 5D show hPSC viability throughout a scalable encapsulation process using various hydrogel formulations. Figure 5C: Varying the PEG:PNIPAAm ratio allowed for an increase in flow rate to meet the minimum 2 mL / min required for scale-up. Furthermore, the lower shear of the 1:3 formulation maximizes cell viability during encapsulation (second plot, measured 24 hours after encapsulation). The highest yield was also obtained with this 1:3 formulation. Figure 5D: 3D hydrogel core / shell design consisting of a core of hydrogel + cells and an outer layer of an acellular shell. A representative image of encapsulated hPSC aggregates in the 3D hydrogel of Formula III is shown on the right. [Figure 5C-D] Figures 5A-5D show a comparison of the effect of PEG:PNIPAAm polymer wt / wt% on the properties of acrylate-based thermoreversible polymers. Figures 5C and 5D show hPSC viability throughout a scalable encapsulation process using various hydrogel formulations. Figure 5C: Varying the PEG:PNIPAAm ratio allowed for an increase in flow rate to meet the minimum 2 mL / min required for scale-up. Furthermore, the lower shear of the 1:3 formulation maximizes cell viability during encapsulation (second plot, measured 24 hours after encapsulation). The highest yield was also obtained with this 1:3 formulation. Figure 5D: 3D hydrogel core / shell design consisting of a core of hydrogel + cells and an outer layer of an acellular shell. A representative image of encapsulated hPSC aggregates in the 3D hydrogel of Formula III is shown on the right.

[0013] [Figure 6A] FIG. 6 shows the effect of isobutyl versus n-butyl at position R2 of Formula III on the properties of acrylate-based thermoreversible polymers.

[0014] [Figure 6B] Figures 6.5A-6.5C show the effect of -H (hydrogel) versus -CH3 (methyl) at position R4 of Formula III on the properties of acrylate-based thermoreversible polymers. 6.5A shows the effect on material properties. 6.5B shows the difference in temperature-based gelation (green = methyl / CH3, gray = hydrogen / H). 6.5C shows the effect on hydrogel encapsulation. [Figure 6C] Figures 6.5A-6.5C show the effect of -H (hydrogel) versus -CH3 (methyl) at position R4 of Formula III on the properties of acrylate-based thermoreversible polymers. 6.5A shows the effect on material properties. 6.5B shows the difference in temperature-based gelation (green = methyl / CH3, gray = hydrogen / H). 6.5C shows the effect on hydrogel encapsulation. [Figure 6D] Figures 6.5A-6.5C show the effect of -H (hydrogel) versus -CH3 (methyl) at position R4 of Formula III on the properties of acrylate-based thermoreversible polymers. 6.5A shows the effect on material properties. 6.5B shows the difference in temperature-based gelation (green = methyl / CH3, gray = hydrogen / H). 6.5C shows the effect on hydrogel encapsulation.

[0015] [Figure 7] FIG. 7 shows the pendant functionalization of PEG groups (R1 in Formula III) with inert structures (methoxy and hydroxyl) and functional structures (acrylate, biotin, and DBCO).

[0016] [Figure 8] FIG. 8 shows the backbone functionalization of the polymer (R3 in Formula III) with methacrylate, maleimide, and DBCO.

[0017] [Figure 9] FIG. 9 shows the presentation and release of proteins (FGF and heparin) from the polymer.

[0018] [Figure 10A-B] 10A-10B show the effect of various groups (-NH2, -OCH3, and -AC) at the pendant PEG position R1 of Formula III and various solvents on the polymer synthesis reaction parameters.

[0019] [Figure 11A] Figures 11A-11E show a comparison of the generation of MGE progenitor cells from human pluripotent stem cells (hPSCs) using various neural induction protocols, performed in 2D cell culture and in 3D thermoreversible hydrogels, as assessed by FOXG1 and DLX1 expression (qPCR, Figures 11B and 11C) (fold change compared to undifferentiated hPSCs) and FOXG1 and NKX2-1 expression (flow cytometry, Figures 11D and 11E). [Figure 11B-C] Figures 11A-11E show a comparison of the generation of MGE progenitor cells from human pluripotent stem cells (hPSCs) using various neural induction protocols, performed in 2D cell culture and in 3D thermoreversible hydrogels, as assessed by FOXG1 and DLX1 expression (qPCR, Figures 11B and 11C) (fold change compared to undifferentiated hPSCs) and FOXG1 and NKX2-1 expression (flow cytometry, Figures 11D and 11E). [Figure 11D-E] Figures 11A-11E show a comparison of the generation of MGE progenitor cells from human pluripotent stem cells (hPSCs) using various neural induction protocols, performed in 2D cell culture and in 3D thermoreversible hydrogels, as assessed by FOXG1 and DLX1 expression (qPCR, Figures 11B and 11C) (fold change compared to undifferentiated hPSCs) and FOXG1 and NKX2-1 expression (flow cytometry, Figures 11D and 11E).

[0020] [Figure 12A]Figures 12A-12F show a comparison of the generation of cortical interneurons from human pluripotent stem cells (hPSCs) using various neural induction protocols, performed in 2D cell culture and in 3D thermoreversible hydrogels, as assessed by the expression of FOXG1, CALB1, and GAD1 (qPCR, Figures 12B-12D) (fold change compared to undifferentiated hPSCs) and FOXG1 and NKX2-1 (flow cytometry, Figures 12E-12F). [Figure 12B-C] Figures 12A-12F show a comparison of the generation of cortical interneurons from human pluripotent stem cells (hPSCs) using various neural induction protocols, performed in 2D cell culture and in 3D thermoreversible hydrogels, as assessed by the expression of FOXG1, CALB1, and GAD1 (qPCR, Figures 12B-12D) (fold change compared to undifferentiated hPSCs) and FOXG1 and NKX2-1 (flow cytometry, Figures 12E-12F). [Figure 12D-E] Figures 12A-12F show a comparison of the generation of cortical interneurons from human pluripotent stem cells (hPSCs) using various neural induction protocols, performed in 2D cell culture and in 3D thermoreversible hydrogels, as assessed by the expression of FOXG1, CALB1, and GAD1 (qPCR, Figures 12B-12D) (fold change compared to undifferentiated hPSCs) and FOXG1 and NKX2-1 (flow cytometry, Figures 12E-12F). [Figure 12F] Figures 12A-12F show a comparison of the generation of cortical interneurons from human pluripotent stem cells (hPSCs) using various neural induction protocols, performed in 2D cell culture and in 3D thermoreversible hydrogels, as assessed by the expression of FOXG1, CALB1, and GAD1 (qPCR, Figures 12B-12D) (fold change compared to undifferentiated hPSCs) and FOXG1 and NKX2-1 (flow cytometry, Figures 12E-12F).

[0021] [Figure 13]Figure 13. Thermoreversible hydrogels for generating hPSC-derived cortical interneurons in a 3D environment. a) A well-defined PNIPAM-based synthetic hydrogel (liquid at low temperatures and rapidly gels when heated to 37 °C; b, c). By controlling the temperature of the system, hPSCs are mixed with this hydrogel at low temperatures and extruded into warm culture medium, resulting in the encapsulation of these cells in porous gel beads. These can be used in well-plate assays (b) or easily scaled to perfusion bioreactor systems (c). Through the use of dedicated media formulations, cells can be differentiated into any cell type of interest within these beads. d) Differentiation paradigms and factors used to generate hPSC-derived cINs in 3D hydrogels. e) Progression of marker expression throughout the hPSC differentiation process into cINs and assays used to assess differentiation efficiency.

[0022] [Figure 14A] Figure 14. Expression of lineage-specific markers of cortical interneuron differentiation. a) At day 10 of differentiation in thermoreversible hydrogel beads, cells begin to express the ventral telencephalon marker FoxG1. By day 18, cells express FoxG1 and NKX2-1, markers of MGE progenitors. After 35 days of differentiation in hydrogel beads, cells significantly down-regulate NKX2-1 expression while maintaining high FoxG1 expression, consistent with a mature cIN ​​phenotype. b) Representative flow cytometry plots corresponding to the quantification shown in a. Data are shown as the mean + standard deviation of two biological replicates. [Figure 14B]Figure 14. Expression of lineage-specific markers of cortical interneuron differentiation. a) At day 10 of differentiation in thermoreversible hydrogel beads, cells begin to express the ventral telencephalon marker FoxG1. By day 18, cells express FoxG1 and NKX2-1, markers of MGE progenitors. After 35 days of differentiation in hydrogel beads, cells significantly down-regulate NKX2-1 expression while maintaining high FoxG1 expression, consistent with a mature cIN ​​phenotype. b) Representative flow cytometry plots corresponding to the quantification shown in a. Data are shown as the mean + standard deviation of two biological replicates.

[0023] [Figure 15A] Figure 15. Gene expression analysis of hPSC differentiation into cortical interneurons. a) Transcript expression analysis by qPCR in cells collected on days 18 and 35. Fold change (2^(-ΔΔCt)) compared to gene expression in hPSCs is expressed as fold change compared to hPSCs. CALB1 = calbindin; PV = parvalbumin; SST = somatostatin. b) Immunocytochemical analysis of gene expression in day 35 cells, showing high expression of FoxG1 and NCAM, as well as SST. The absence of ki67-positive cells indicates postmitotic cells. c) Quantification of b. Data are shown as the mean + standard deviation of two biological replicates. [Figure 15B] Figure 15. Gene expression analysis of hPSC differentiation into cortical interneurons. a) Transcript expression analysis by qPCR in cells collected on days 18 and 35. Fold change (2^(-ΔΔCt)) compared to gene expression in hPSCs is expressed as fold change compared to hPSCs. CALB1 = calbindin; PV = parvalbumin; SST = somatostatin. b) Immunocytochemical analysis of gene expression in day 35 cells, showing high expression of FoxG1 and NCAM, as well as SST. The absence of ki67-positive cells indicates postmitotic cells. c) Quantification of b. Data are shown as the mean + standard deviation of two biological replicates. [Figure 15C]Figure 15. Gene expression analysis of hPSC differentiation into cortical interneurons. a) Transcript expression analysis by qPCR in cells collected on days 18 and 35. Fold change (2^(-ΔΔCt)) compared to gene expression in hPSCs is expressed as fold change compared to hPSCs. CALB1 = calbindin; PV = parvalbumin; SST = somatostatin. b) Immunocytochemical analysis of gene expression in day 35 cells, showing high expression of FoxG1 and NCAM, as well as SST. The absence of ki67-positive cells indicates postmitotic cells. c) Quantification of b. Data are shown as the mean + standard deviation of two biological replicates.

[0024] [Figure 16A-B] Figure 16. Viability of hPSCs encapsulated in thermoreversible hydrogel beads and their differentiation efficiency into cortical interneurons compared to standard 2D processes. a) Post-harvest viability of hPSC-derived cells after 35 days of differentiation in standard 2D culture or 3D culture in the novel hydrogel of formula (III) described herein. b) Quantification of a). c) Marker expression analyzed by flow cytometry in hPSC-derived cells at various harvest time points shows higher differentiation efficiency in the novel hydrogel of formula (III) compared to standard 2D methods (% FoxG1+ cells (fold change) at days 10, 18, and 35, and % Nkx2-1+ cells (fold change) at day 18, for 2D versus 3D culture). Data are presented as the mean + standard deviation of two biological replicates. [Figure 16B]Figure 16. Viability of hPSCs encapsulated in thermoreversible hydrogel beads and their differentiation efficiency into cortical interneurons compared to standard 2D processes. a) Post-harvest viability of hPSC-derived cells after 35 days of differentiation in standard 2D culture or 3D culture in the novel hydrogel of formula (III) described herein. b) Quantification of a). c) Marker expression analyzed by flow cytometry in hPSC-derived cells at various harvest time points shows higher differentiation efficiency in the novel hydrogel of formula (III) compared to standard 2D methods (% FoxG1+ cells (fold change) at days 10, 18, and 35, and % Nkx2-1+ cells (fold change) at day 18, for 2D versus 3D culture). Data are presented as the mean + standard deviation of two biological replicates. [Figure 16C] Figure 16. Viability of hPSCs encapsulated in thermoreversible hydrogel beads and their differentiation efficiency into cortical interneurons compared to standard 2D processes. a) Post-harvest viability of hPSC-derived cells after 35 days of differentiation in standard 2D culture or 3D culture in the novel hydrogel of formula (III) described herein. b) Quantification of a). c) Marker expression analyzed by flow cytometry in hPSC-derived cells at various harvest time points shows higher differentiation efficiency in the novel hydrogel of formula (III) compared to standard 2D methods (% FoxG1+ cells (fold change) at days 10, 18, and 35, and % Nkx2-1+ cells (fold change) at day 18, for 2D versus 3D culture). Data are presented as the mean + standard deviation of two biological replicates.

[0025] [Figures 17A-C]Figures 17A-17C show the expansion of hESCs and hiPSCs in 3D hydrogels containing the thermoreversible polymer of Formula III. Figure 17A: Three culture scales compatible with the 3D hydrogel. Positive displacement pipette (PDP) droplets consist of only the core 3D hydrogel, allowing for rapid screening of multiple culture conditions at a small scale. Core / shell beads (C / S beads)-static consist of cells encapsulated in 3D hydrogel beads fabricated with a hydrogel + cellular core and an outer acellular shell, and are cultured statically in well plates. C / S beads-spinner are cells encapsulated in 3D hydrogel core / shell beads grown in a rotating environment using spinner flasks. Figure 17B: An exemplary human embryonic stem cell line grown in 3D hydrogels for 8 consecutive days achieves an 80-fold fold change at all three culture scales, achieving a yield of up to 20e6 cells / mL of 3D hydrogel at harvest with high viability. Figure 17C: Example of a human induced pluripotent stem cell line grown in 3D hydrogel for 8 consecutive days, achieving a fold change of over 100-fold when grown in encapsulated C / S beads, with a yield of up to 25e6 cells / mL of 3D hydrogel at harvest with high viability.

[0026] [Figure 18A] Figures 18A-18C. Reproducible hPSC expansion in bioreactors at various scales utilizing 3D hydrogels containing the polymer of Formula III. Figure 18A: Schematic of the scalable bioreactor system containing 3D hydrogel beads. Figure 18B: Pluripotency marker expression determined by flow cytometry for each lot. Figure 18C: Gene expression analysis performed by qPCR, showing strong correlation in gene expression for all four lots of hPSC expansion in 3D hydrogels. Pearson correlation index is shown for each plot. [Figure 18B]Figures 18A-18C. Reproducible hPSC expansion in bioreactors at various scales utilizing 3D hydrogels containing the polymer of Formula III. Figure 18A: Schematic of the scalable bioreactor system containing 3D hydrogel beads. Figure 18B: Pluripotency marker expression determined by flow cytometry for each lot. Figure 18C: Gene expression analysis performed by qPCR, showing strong correlation in gene expression for all four lots of hPSC expansion in 3D hydrogels. Pearson correlation index is shown for each plot. [Figure 18C] Figures 18A-18C. Reproducible hPSC expansion in bioreactors at various scales utilizing 3D hydrogels containing the polymer of Formula III. Figure 18A: Schematic of the scalable bioreactor system containing 3D hydrogel beads. Figure 18B: Pluripotency marker expression determined by flow cytometry for each lot. Figure 18C: Gene expression analysis performed by qPCR, showing strong correlation in gene expression for all four lots of hPSC expansion in 3D hydrogels. Pearson correlation index is shown for each plot.

[0027] [Figure 19] Figure 19. 3D hydrogels containing polymers of Formula III enable efficient hPSC differentiation into pancreatic endoderm cells (PE). A 100 mL spinner flask contains hPSCs encapsulated in core-shell 3D hydrogel beads. Cells were expanded and differentiated in the 3D hydrogel platform. Performance is compared to standard PE differentiation in suspension culture.

[0028] [Figure 20A-C]Figures 20A-20D. 3D hydrogels containing the polymer of Formula III enable efficient hPSC differentiation into pancreatic endoderm (PE) cells. Figure 20A: Stages of hPSC differentiation into pancreatic endoderm (PE) cells. Figure 20B: Differentiation efficiency was assessed by expression of the PE marker PDX-1 (pancreatic and duodenal homeobox-1). Figure 20C: Use of 3D hydrogel technology enables 45-fold higher PE cell generation compared to standard suspension methods. Figure 20D: 3D hydrogel technology utilizing improved gel formulations allows for greater control over PE aggregate size compared to standard suspension methods, minimizing undesired effects of nutrient limitation (e.g., cell death (necrotic core) and suboptimal differentiation). [Figure 20D] Figures 20A-20D. 3D hydrogels containing the polymer of Formula III enable efficient hPSC differentiation into pancreatic endoderm (PE) cells. Figure 20A: Stages of hPSC differentiation into pancreatic endoderm (PE) cells. Figure 20B: Differentiation efficiency was assessed by expression of the PE marker PDX-1 (pancreatic and duodenal homeobox-1). Figure 20C: Use of 3D hydrogel technology enables 45-fold higher PE cell generation compared to standard suspension methods. Figure 20D: 3D hydrogel technology utilizing improved gel formulations allows for greater control over PE aggregate size compared to standard suspension methods, minimizing undesired effects of nutrient limitation (e.g., cell death (necrotic core) and suboptimal differentiation).

[0029] [Figure 21A]Figures 21A-21C. 3D hydrogels containing polymers of Formula III enable efficient hPSC differentiation into midbrain dopaminergic cells (mDA). Figure 21A: A 100 mL spinner flask contains hPSCs encapsulated in core-shell 3D hydrogel beads. Cells were expanded and differentiated in the 3D hydrogel platform. Figure 21B: During a 16-day differentiation process using a positive displacement pipette (PDP) droplet format, collected mDA cells showed high viability. Differentiation efficacy was assessed by the presence of FoxA2, ​​a marker for mDA progenitor cells. Figure 21C. Cells encapsulated in 3D hydrogel C / S beads were expanded and differentiated for 16 days in a 100 mL spinner flask format. Collected mDA cells showed high viability. Differentiation efficacy was assessed by the presence of FoxA2, ​​a marker for mDA progenitor cells. [Figure 21B-C] Figures 21A-21C. 3D hydrogels containing polymers of Formula III enable efficient hPSC differentiation into midbrain dopaminergic cells (mDA). Figure 21A: A 100 mL spinner flask contains hPSCs encapsulated in core-shell 3D hydrogel beads. Cells were expanded and differentiated in the 3D hydrogel platform. Figure 21B: During a 16-day differentiation process using a positive displacement pipette (PDP) droplet format, collected mDA cells showed high viability. Differentiation efficacy was assessed by the presence of FoxA2, ​​a marker for mDA progenitor cells. Figure 21C. Cells encapsulated in 3D hydrogel C / S beads were expanded and differentiated for 16 days in a 100 mL spinner flask format. Collected mDA cells showed high viability. Differentiation efficacy was assessed by the presence of FoxA2, ​​a marker for mDA progenitor cells.

[0030] [Figure 22A-B]Figures 22A-C. 3D hydrogels containing the polymer of Formula III enable efficient hPSC differentiation into hematopoietic stem cells (HSCs). Figure 22A) hPSC differentiation into HSCs following a ready-to-use media formulation (StemDiff Hematopoietic Kit). Figure 22B) Representative images of hPSCs cultured in hydrogels on the day of induction (stage 1) and on the day of harvest (end of stage 2). Figure 22C) Harvest analysis of hPSC-derived HSCs differentiated in the improved hydrogel system. [Figure 22C] Figures 22A-C. 3D hydrogels containing the polymer of Formula III enable efficient hPSC differentiation into hematopoietic stem cells (HSCs). Figure 22A) hPSC differentiation into HSCs following a ready-to-use media formulation (StemDiff Hematopoietic Kit). Figure 22B) Representative images of hPSCs cultured in hydrogels on the day of induction (stage 1) and on the day of harvest (end of stage 2). Figure 22C) Harvest analysis of hPSC-derived HSCs differentiated in the improved hydrogel system.

[0031] [Figure 23A-B] Figures 23A-C. 3D hydrogels containing polymers of Formula III enable efficient HSC expansion. Figure 23A) Thawing and expansion of human umbilical cord blood-derived CD34+ HSCs in 3D hydrogels. Figure 23B) Representative images of HSCs expanded in hydrogels in SFEM II medium supplemented with StemSpan CD34+ Expansion Supplement (StemDiff Hematopoietic Kit). Figure 23C) Collection analysis of HSCs expanded for 8 days in the improved hydrogel system. [Figure 23C] Figures 23A-C. 3D hydrogels containing polymers of Formula III enable efficient HSC expansion. Figure 23A) Thawing and expansion of human umbilical cord blood-derived CD34+ HSCs in 3D hydrogels. Figure 23B) Representative images of HSCs expanded in hydrogels in SFEM II medium supplemented with StemSpan CD34+ Expansion Supplement (StemDiff Hematopoietic Kit). Figure 23C) Collection analysis of HSCs expanded for 8 days in the improved hydrogel system. DETAILED DESCRIPTION OF THE INVENTION

[0032] (Detailed Description of the Invention) (definition)

[0033] As used herein, "activator" refers to a compound that increases, induces, stimulates, activates, promotes, or enhances the activation of the signaling function of a molecule or pathway (e.g., Wnt signaling, SHH signaling, etc.).

[0034] As used herein, the term "cell population" or "cell population" refers to a group of at least two cells.In a non-limiting example, the cell population can comprise at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, or at least about 1000 cells.The population can be a pure population that contains one type of cell (for example, a population of dopaminergic neurons or a population of undifferentiated stem cells).Alternatively, the population can contain more than one type of cell (for example, a mixed cell population).

[0035] As used herein, the term "stem cell" refers to a cell that has the capacity to divide in culture for an indefinite period of time to give rise to specialized cells.

[0036] As used herein, the terms "embryonic stem cells" and "ESCs" refer to primitive (undifferentiated) cells derived from a preimplantation embryo, capable of dividing for extended periods in culture without differentiation, and known to develop into cells and tissues of the three primary germ layers. Human embryonic stem cells refer to embryonic stem cells derived from a human embryo. As used herein, the terms "human embryonic stem cells" or "hESCs" refer to a type of pluripotent stem cell derived from an early human embryo (up to the blastocyst stage) that is known to be capable of dividing for extended periods in culture without differentiation, and known to develop into cells and tissues of the three primary germ layers.

[0037] As used herein, the term "embryonic stem cell line" refers to a population of embryonic stem cells that have been cultured for days, months, or even years without differentiation under in vitro conditions that allow proliferation.

[0038] As used herein, "pluripotent" refers to the ability to develop into an organism's three developmental germ layers, including endoderm, mesoderm, and ectoderm.

[0039] As used herein, the term "induced pluripotent stem cell" or "iPSC" refers to a type of pluripotent stem cell formed by the introduction of certain embryonic genes (e.g., but not limited to, the OCT4 transgene, the SOX2 transgene, and the KLF4 transgene) into somatic cells (see, e.g., Takahashi and Yamanaka, Cell, 126, 663-676 (2006) (incorporated herein by reference)).

[0040] As used herein, the term "neuron" refers to a nerve cell, which is the main functional unit of the nervous system. A neuron consists of a cell body and its processes (axon and one or more dendrites). Neurons transmit information to other neurons or cells by releasing neurotransmitters at synapses.

[0041] As used herein, the term "undifferentiated" refers to cells that have not yet developed into specialized cell types.

[0042] As used herein, the term "differentiation" refers to the process by which an unspecialized embryonic cell acquires the characteristics of a specialized cell (e.g., a neuronal, cardiac, hepatic, or muscle cell). Differentiation is controlled by the interaction of the cell's genes with the physical and chemical conditions outside the cell, usually through signaling pathways involving proteins embedded in the cell surface.

[0043] As used herein, the term "inducing differentiation" in reference to a cell refers to changing from its default cell type (genotype and / or phenotype) to a non-default cell type (genotype and / or phenotype). Thus, "inducing differentiation in a stem cell" refers to inducing the stem cell (e.g., a human stem cell) to divide into progeny cells with characteristics (e.g., genotype (e.g., changes in gene expression as determined by genetic analysis (e.g., microarray)) and / or phenotype (e.g., changes in expression of one or more protein markers)) that differ from the stem cell.

[0044] As used herein, the term "marker" or "cell marker" or "biomarker" refers to a gene or protein that identifies a particular cell or cell type. A marker for a cell may not be limited to one marker, and may refer to a "pattern" of markers such that a specified group of markers may identify one cell or cell type from another.

[0045] As used herein, the term "linker" or "linkage" refers to a linking moiety that connects two groups and has a backbone length of 100 atoms or less. The linker or linkage can be a covalent bond connecting two groups, or a chain between 1 atom and 100 atoms in length (e.g., 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, 8 carbon atoms, 10 carbon atoms, 12 carbon atoms, 14 carbon atoms, 16 carbon atoms, 18 carbon atoms, or 20 carbon atoms in length), and the linker can be linear, branched, cyclic, or single-atom. In certain cases, one, two, three, four, five, or more carbon atoms in the linker backbone can be optionally replaced with a sulfur heteroatom, a nitrogen heteroatom, or an oxygen heteroatom. The bond between backbone atoms can be saturated or unsaturated, and usually there are one, two, or three or fewer unsaturated bonds in the linker backbone. The linker may contain one or more substituents, for example, an alkyl group, an aryl group, or an alkenyl group. Linkers may include, but are not limited to, poly(ethylene glycol); ethers, thioethers, tertiary amines, alkyls (which may be linear or branched (e.g., methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl))), and the like. The linker backbone may include a cyclic group (e.g., an aryl, heterocycle, or cycloalkyl group), with two or more atoms (e.g., two atoms, three atoms, or four atoms) of the cyclic group contained in the backbone. The linker may or may not be cleavable.

[0046] "Alkyl" refers to a monovalent saturated aliphatic hydrocarbyl group having 1 to 10 carbon atoms (e.g., 1 to 6 carbon atoms, or 1 to 5, or 1 to 4, or 1 to 3 carbon atoms). In some instances, a "lower alkyl" is an alkyl group having 1 to 6 carbon atoms. This term includes, by way of example, straight-chain and branched-chain hydrocarbyl groups, such as methyl (CH-), ethyl (CHCH-), n-propyl (CHCHCH-), isopropyl ((CH)CH-), n-butyl (CHCHCHCH-), isobutyl ((CH)CHCH-), sec-butyl ((CH)(CHCH)CH-), t-butyl ((CH)C-), n-pentyl (CHCHCHCHCHCH-), and neopentyl ((CH)CCH-).

[0047] The term "substituted alkyl" refers to an alkyl group in which one or more carbon atoms in the alkyl chain have been replaced with a heteroatom (e.g., -O-, -N-, -S-, -S(O) n -NR- (R is hydrogen or alkyl)), and having from 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, -SO2-heteroaryl, and -NR a R bwherein R and R may be the same or different and are selected from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, and heterocycle.

[0048] As used herein, the terms "chemoselective functional group" and "chemoselective tag" are used interchangeably, and refer to chemoselective reactive groups that selectively react with each other to form a covalent bond. Chemoselective functional groups of interest include, but are not limited to, two thiol groups, a thiol and a maleimide, or a thiol and an iodoacetamide, and groups that can react with each other by click chemistry (e.g., an azide group and an alkyne group (e.g., a cyclooctyne group)). Chemoselective functional groups of interest include, but are not limited to, thiol, alkyne, cyclooctyne, azide, phosphine, maleimide, alkoxyamine, aldehyde, and their protected versions and precursors. In certain embodiments, the chemoselective functional group is a thiol.

[0049] In some embodiments, the term "end group" refers to the group(s) (e.g., H, alkyl or substituted alkyl, and / or residual components of the initiator used during polymerization) generated as a result of any convenient polymerization method of the subject comonomers described herein.

[0050] As used herein, the term "modifying agent" refers to any convenient agent that provides a desired property of interest (e.g., a desired physical and / or biological property) and can be conjugated to a thermoreversible polymer, for example, via a chemoselective functional group on a side-chain linker or terminus of the polymer. Such agents may belong to the categories of small molecules, proteins, peptides, sugars, polynucleotides, etc. Modifiers of interest include, but are not limited to, ligands, substrates, enzymes, pharmaceuticals (e.g., chemotherapeutic drugs), plasmids, polynucleotides, bioactive peptides, antibodies, biomarkers, biosensors, catalysts, elements, cell targeting agents, small molecule drugs, fluorescent / radioactive / optical imaging agents, peptides / proteins / enzymes, nucleic acids (e.g., siRNA / RNA / DNA), metal-based compounds / catalysts, site-specific cell targeting agents (e.g., compounds / ligands / antibodies), smart adjuvants, and gene therapy vectors. In certain embodiments, the modulator is selected from heparin, hyaluronic acid, a specific binding member, a peptide, a nucleic acid, gelatin, fibronectin, collagen, laminin, bFGF, EGF, insulin, progesterone, glucose, thymosin beta-4, SHH, noggin, activin, TGFb3, FGF8, BDNF, GDNF, NT3, PDGF-AA, and IGF- 1. In certain cases, the modulator is a cytokine, a member of the BMP family (e.g., TGFβ or activin), a neutrophin (e.g., NT3 or BDNF), or a hedgehog protein (e.g., SHH).

[0051] Any convenient method can be used to conjugate the modifier to the thermoreversible polymer. Conjugation methods and chemistries of interest include, but are not limited to, those described by Greg Hermanson in Bioconjugate Techniques (3rd ed.), 2013, Academic Press. In certain embodiments, the modifier is a protein. In certain embodiments, the modifier is a peptide. In certain embodiments, the modifier is peptidic and can be conjugated to the thermoreversible polymer by covalent bonding to the N-terminus or C-terminus, or to the peptidic material, or by covalent bonding to an amino acid side chain (e.g., an amino-, thiol-, hydroxyl-, carboxylic acid-, or phenol-containing amino acid side group, or derivatives thereof) (e.g., via a terminal functional group and / or a side chain functional group). In certain embodiments, the modifier is heparin. In certain embodiments, the heparin modifier is linked by a thiol bond. In certain cases, the heparin can be linked to a subject polymer by conjugation to the carboxylic acid group of the heparin. For example, Figure 9 shows an exemplary method for linking thiol-heparin to the acrylate group of a polymer by Michael addition. In certain embodiments, two or more modifiers (e.g., heparin and hyaluronic acid) can be linked to each other in addition to the thermoreversible polymer.

[0052] As used herein, lower critical solution temperature (LCST) refers to the critical temperature below which the components of a mixture are miscible in all compositions. The term "lower" indicates that the LCST is the lower boundary for a temperature interval of partial miscibility or miscibility for only certain compositions.

[0053] The inventors have discovered that the methods described herein allow for at least a 10-fold increase in the generation of MGE progenitor cells, with at least four-fold higher cell viability at the time of harvest, compared to standard 2D culture formats.

[0054] In some embodiments, an in vitro method for differentiating human stem cells is provided, the method comprising: encapsulating human stem cells in a three-dimensional synthetic hydrogel; and contacting the encapsulated human stem cells with at least one inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling and at least one Wingless (Wnt) antagonist for a predetermined period of time; and contacting the cells with at least one activator of Sonic Hedgehog (SHH) signaling for a predetermined period of time to obtain a cell population comprising MGE progenitor cells. In some embodiments, the cell population comprising the MGE progenitor cells exhibits the following characteristics: (a) at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 86% or at least 87% of the cell population are FoxG1 positive; and (b) at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, or at least 77% of the cell population are NKX2-1 positive. In some embodiments, the FOXG1-positive and NKX2-1-positive MGE progenitor cells comprise at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% of the cell population, as measured, for example, by flow cytometry, immunocytochemistry, and / or qPCR.

[0055] In a preferred embodiment, the method further comprises contacting the generated encapsulated MGE progenitor cells with at least one neurotrophic factor and, optionally, a Notch inhibitor for a predetermined period of time to obtain a population of cells comprising differentiated inhibitory GABAergic cortical interneurons (cINs) that are FOXG1-positive. In some embodiments, the cIN-containing cell population exhibits the following characteristics: (a) at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, or at least 77% of the cell population are FoxG1-positive, as measured, for example, by flow cytometry, immunocytochemistry, and / or qPCR; (b) at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82% of the cell population are FoxG1-positive; , at least 83%, or at least 84% are GABA positive; (c) at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, or at least 83% of the cell population are PV positive; (d) less than 30%, less than 25%, less than 24%, or less than 23% of the cell population are NKX2-1 positive; and / or (e) less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the cell population are ki67 positive.

[0056] (synthetic hydrogel)

[0057] In some embodiments, synthetic hydrogels for use in the methods described herein are polyethylene glycol-poly(N-isopropylacrylamide) (PEG-PNIPAAM)-based hydrogels that are solid at 37° C. Preferred PEG-PNIPAAM hydrogels include those described in U.S. Pat. No. 10,982,055 and WIPO Publication No. 2022 / 251137A1, the entire contents of each of which are incorporated herein by reference.

[0058] In some preferred embodiments, the hydrogel comprises a thermoreversible polymer comprising an N-isopropylacrylamide (NIPAAM) comonomer, a lower alkylamine comonomer, and a poly(ethylene glycol) (PEG) comonomer, wherein the terminal PEG monomer is substituted with alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl. Preferably, the lower alkylamine comonomer comprises n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, or isopentyl; and the terminal PEG monomer is substituted with an alkoxy group.

[0059] In some embodiments, the thermoreversible polymer has the formula (I): [ka] Including, wherein a, b, and c represent the mole fractions of the comonomers, and a, b, and c are each greater than 0, preferably a>0.8; 0.2>b>0; and 0.1>c>0; and PEG n is a polyethylene glycol polymer, and n is an integer from 1 to 2500; R 1 is alkyl or substituted alkyl, preferably C1-C6 alkyl, more preferably butyl; R 2 is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl, preferably alkoxy, more preferably methoxy; G 1 and G 2are each independently selected from a polymer segment, an end group, a linker, and an attached modifying substance.

[0060] In a related embodiment, the thermoreversible polymer has the formula (II): [ka] wherein n is 1 to 2500; G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifying substance.

[0061] Other synthetic hydrogels may be utilized in the above methods, including, but not limited to, those described in U.S. Pat. Nos. 6,897,064 and 10,982,055 and U.S. Patent Application Publication No. 2024 / 0294713, the contents of each of which are incorporated herein by reference in their entirety.

[0062] Formula III: [ka] Also provided herein are novel thermoreversible polymers comprising: where a, b, c, and d represent the mole fractions of comonomers in the polymer, and a, b, and c are each greater than 0; PEG n is a polyethylene glycol polymer, R 1 is any terminal or functional group, if present, not including a primary amine; R 2 is a lower alkyl group, R 3 is an end group or functional group or linked modifier, if present; R 4 is hydrogen or a lower alkyl group, G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and a linked modifying substance; The molecular weight of the polymer is greater than 50 kDa.

[0063] The present inventors have discovered that the thermoreversible polymer of Formula III has several advantages over the thermoreversible polymers disclosed, for example, in U.S. Pat. No. 10,982,055 and U.S. Patent Application Publication No. 2024 / 0294713. Briefly, as exemplified herein, the thermoreversible polymers according to the present disclosure exhibit a combination of properties that make them uniquely suitable for three-dimensional cell culture. By way of example, three-dimensional hydrogels comprising the thermoreversible polymer of Formula III exhibit, among other things: (1) long-term stability (which allows a long differentiation process to occur during cell culture), (2) reduced viscosity compared to prior art polymers (which allows efficient cell encapsulation), and (3) adequate rigidity to support cell growth. These characteristics result in significant improvements in cell viability and cell yield.

[0064] In preferred embodiments, the thermoreversible polymer of Formula III has one or more, preferably all, of the following properties: (a) an LCST of 12°C to 32°C, (b) a stiffness of 100 Pa to 8000 Pa, (c) a viscosity of 100 cP to 2000 cP, and (d) a molecular weight of 50 kDa to 500 kDa.

[0065] In some embodiments, the R of the thermoreversible polymer of Formula III 1 is absent. In other embodiments, R of the thermoreversible polymer of Formula III is 1is a functional group. In some embodiments, the functional group is a chemoselective functional group, non-limiting examples of which include two thiol groups, a thiol and a maleimide, or a thiol and an iodoacetamide, and groups that can react with each other via click chemistry (e.g., an azide group and an alkyne group (e.g., a cycloalkyne group, e.g., dibenzocyclooctyne (DBCO))). Functional groups include, but are not limited to, acrylate, thiol, hydroxyl, alkoxy (e.g., methoxy), alkyne, cycloalkyne, azide, hydrazide, phosphine, maleimide, carboxylic acid, alkoxyamine, aldehyde, biotin, silane, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), NHS ester, toluenesulfonyl (Tos), and protected versions and precursors thereof.

[0066] In some embodiments, the R of the thermoreversible polymer of Formula III 1 is a terminal group, non-limiting examples of which include C 1~ Examples of C alkoxy include, but are not limited to, C alkoxy (e.g., methoxy, ethoxy, n-propoxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, or isopentoxy), alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl. 1 is not alkyl (e.g., other than n-butyl) or substituted alkyl.

[0067] In some embodiments, the R of the thermoreversible polymer of Formula III 2is lower alkyl, optionally selected from methyl, ethyl, propyl, n-butyl, pentyl, isopropyl, isobutyl, isopentyl, tert-butyl, cyclopropyl, and cyclobutyl. 2 is n-butyl. The inventors have discovered that n-butyl at this position exhibits higher stiffness, higher gel stability, and a lower gel LCST at similar viscosity compared to other conformations (e.g., isobutyl). In some embodiments, R 2 is lower alkyl other than isobutyl.

[0068] In some embodiments, the R of the thermoreversible polymer of Formula III 3 is absent. In other embodiments, R of the thermoreversible polymer of Formula III is 3 is a modifier, optionally selected from heparin, hyaluronic acid, a specific binding member, a peptide, a nucleic acid, gelatin, fibronectin, collagen, laminin, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin, progesterone, glucose, stromal cell-derived factor 1 (SDF-1), thymosin beta-4, sonic hedgehog (SHH), noggin, activin, transforming growth factor beta (TGF-β), FGF8, brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), neurotrophic factor 3 (NT3), platelet-derived growth factor (PDGF), IL-16, IL-2, and insulin-like growth factor 1 (IGF-1).

[0069] In some preferred embodiments, R 4 is a lower alkyl group, especially R 4 is methyl.

[0070] In some cases, G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifier. 1 and G 2are each independently selected from heparin, hyaluronic acid, a member of a specific binding pair, a polypeptide, a nucleic acid, and a carboxyl group. 1 and G 2 are each independently a modified substance selected from gelatin, elastin, fibronectin, collagen, and laminin. 1 and G 2 are each independently selected from a chemokine, a peptide hormone, and a growth factor. 1 and G 2 are each independently selected from fibroblast growth factor, epidermal growth factor, hepatic growth factor, insulin, stromal cell-derived factor 1, thymosin beta-4, sonic hedgehog, noggin, activin, transforming growth factor, bone morphogenetic factor, brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, neurotrophin 3, platelet-derived growth factor, FGF-2, FGF-8, keratinocyte growth factor, or insulin-like growth factor. 1 and G 2 are each independently selected from chain transfer agents, non-limiting examples of which include dithioesters, dithiocarbamates, trithiocarbonates, or xanthates. 1 and G 2 are each independently selected from a chain transfer agent containing a thiolcarbonylthio group, and a thermal initiator (e.g., azobisisobutyronitrile (AIBN)).

[0071] In some cases, a>0.8; 0.1>b>0; 0.2>c>0, and 0.1>d>0.

[0072] In some embodiments, the thermoreversible polymer of Formula III has a molecular weight (MW) of between 50 kDa and 500 kDa, or between 50 kDa and 250 kDa. In certain embodiments, the thermoreversible polymer has a MW of at least 50 kDa, at least 60 kDa, at least 70 kDa, at least 80 kDa, or at least 90 kDa. In certain embodiments, the thermoreversible polymer has a MW in the range of about 50 kDa to about 250 kDa, e.g., about 50 kDa to about 200 kDa, about 50 kDa to about 150 kDa, about 50 kDa to about 100 kDa, or about 50 kDa to about 75 kDa. In certain embodiments, the thermoreversible polymer has a MW of about 60 kDa to about 140 kDa, about 70 kDa to about 130 kDa, about 80 kDa to about 120 kDa, or about 90 kDa to about 110 kDa. In some embodiments, the MW is about 80 kDa, about 85 kDa, about 90 kDa, about 95 kDa, about 100 kDa, about 105 kDa, about 110 kDa, about 115 kDa, or about 120 kDa. In certain preferred embodiments, the thermoreversible polymer has a MW of about 50 kDa to about 250 kDa, or about 50 kDa to about 150 kDa.

[0073] Any convenient poly(ethyl glycol) (PEG) polymer group can be utilized as a side chain in the thermoreversible polymer of Formula (III). In some embodiments of Formula (III), PEG n is a polyethyl glycol polymer having a MW of about 2 kDa to about 20 kDa. In a related embodiment, PEG n has a MW of about 2 kDa or greater, e.g., 2 kDa to 20 kDa, or 2 kDa to 10 kDa, or 3 kDa to 20 kDa, or 3 kDa to 10 kDa. n has a MW of about 2 kDa to about 9 kDa, about 3 kDa to about 8 kDa, about 4 kDa to about 7 kDa, or about 4 kDa to about 6 kDa. n The PEG has a MW of about 2 kDa, about 3 kDa, about 4 kDa, about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, or about 9 kDa.n The groups may be modified with any convenient group (including terminal modifications).

[0074] In certain embodiments, the thermoreversible polymer of Formula III has a weight:weight (w / w) ratio of PEG:PNIPAAm copolymer greater than 1:2. In some embodiments, the w / w ratio of PEG:PNIPAAm copolymer is about 1:2.5, about 1:2.75, about 1:3.0, about 1:3.5, about 1:3.75, about 1:4.0, about 1:4.25, or about 1:4.5. In some embodiments, the w / w ratio of PEG:PNIPAAm copolymer is about 1:3 to about 1:4.5, or about 1:4. In some embodiments, the w / w ratio of PEG:PNIPAAm copolymer is measured according to relative molecular ratios by nuclear magnetic resonance (NMR) and total polymer molecular weight by gel permeation chromatography (GPC) (GPC-NMR analysis).

[0075] In some embodiments, the weight of the PNIPAAm copolymer is defined as the weight of the copolymer backbone after polymerization of the comonomer (i.e., before addition of PEG). For example, in some embodiments, the weight of the PNIPAAm copolymer is [ka] of the following copolymers: [ka] In a related embodiment, the weight of PEG is defined as the weight after polymerization of PEG before reaction with its copolymer backbone. [ka] is defined as the weight of

[0076] In particularly preferred embodiments, the thermoreversible polymer of Formula II comprises a PEG:PNIPAAM weight ratio of greater than 1:2 and a MW of between 50 kDa and 250 kDa, including any range therebetween.

[0077] The inventors have discovered that polymers of Formula III having a PEG:PNIPAAM weight ratio greater than 1:2 (e.g., a 1:3 ratio) and a low MW (e.g., 50 kDa to 250 kDa) offer several surprising advantages over the polymers disclosed in, for example, U.S. Pat. No. 10,982,055 and U.S. Patent Application Publication No. 2024 / 0294713. These advantages include, but are not limited to, (1) hydrogels with lower viscosity (which minimize shear during the cell encapsulation process for large-scale bioreactor cell culture systems), (2) higher flow rates (which reach 2 mL / min), and (3) greater control of bead size and shape (higher circularity and lower tail formation frequency), (4) optimal stiffness range for maintaining three-dimensional structure during the long cell differentiation process and supporting stem cell expansion and differentiation into all three germ layers, and (5) improved functionalization capabilities for controlled protein presentation and release. Thus, the thermoreversible polymers of Formula III provide a scalable three-dimensional cell culture system, for example, for time periods relevant to the generation of functional neurons.

[0078] In certain embodiments, the thermoreversible polymer of Formula III has an LCST within the range of 12° C. to 32° C., e.g., 12° C. to 30° C., 15° C. to 30° C., 15° C. to 25° C., or 10° C. to 20° C. In preferred embodiments, the thermoreversible polymer of Formula III has an LCST of about 20° C. to about 22° C.

[0079] Also provided is a composition comprising: a) a three-dimensional hydrogel comprising a thermoreversible polymer of Formula III; and b) cells encapsulated within the hydrogel. The three-dimensional hydrogel-cell composition is useful for generating a desired number of cells by culturing the three-dimensional hydrogel-cell composition under conditions and for a period of time sufficient to generate the desired number of cells. In some embodiments, the period of time sufficient to generate the desired number of cells is at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 26 days, at least 27 days, at least 28 days, at least 29 days, at least 30 days, at least 31 days, at least 32 days, at least 33 days, at least 34 days, at least 35 days, at least 40 days, at least 45 days, at least 50 days, at least 55 days, at least 60 days, at least 3 months, at least 4 months, at least 6 months, or longer. Such cells may include stem cells, differentiated cells, etc. The thermoreversible polymer-cell composition of Formula III is particularly useful for differentiating cells, for example, to generate a desired number of differentiated cells over a relatively long period of time. The three-dimensional thermoreversible polymer-cell composition of the present disclosure may be implanted into an individual in need thereof, where cells proliferate and / or differentiate within the implanted thermoreversible polymer-cell composition and migrate out of the implanted thermoreversible polymer-cell composition.

[0080] In some aspects, the present disclosure provides a composition comprising a plurality of hydrogel capsules, wherein at least 90%, at least 95%, at least 98%, or at least 99% of the hydrogel capsules in the composition comprise cells and a hydrogel encapsulating the cells, the hydrogel encapsulating the cells comprising a thermoreversible polymer of Formula III. In some embodiments, at least 90% of the hydrogel capsules each comprise a plurality of cells, e.g., at least 100, at least 200, at least 500, at least 700, at least 800, at least 900, or more cells. As shown in the examples of the present disclosure, the thermoreversible polymer of Formula III provides improvements, including but not limited to, bead (i.e., capsule) uniformity, bead circularity, bead sphericity, bead volume and diameter reduction, flow rate for producing beads, and bead shape resulting from gravity drip extrusion.

[0081] The present disclosure provides a method for producing differentiated cells from stem or progenitor cells, the method comprising culturing stem or progenitor cells in a three-dimensional hydrogel composition comprising a thermoreversible polymer of Formula III for a time suitable for inducing differentiation of the stem or progenitor cells under conditions suitable for inducing differentiation of the stem or progenitor cells. The conditions for inducing differentiation of stem or progenitor cells depend in part on the differentiated cells desired. The conditions may include including one or more factors in the hydrogel that induce differentiation. In some embodiments, the conditions suitable for differentiation of the stem cells include culturing the stem or progenitor cells in the hydrogel composition for at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 26 days, at least 27 days, at least 28 days, at least 29 days, at least 30 days, at least 31 days, at least 32 days, at least 33 days, at least 34 days, at least 35 days, at least 40 days, at least 45 days, at least 50 days, at least 55 days, at least 60 days, at least 3 months, at least 4 months, at least 6 months, or longer to generate a differentiated cell population.

[0082] In some embodiments, the present disclosure provides methods for expanding stem cells, the methods comprising encapsulating single cells or multicellular clusters in a three-dimensional hydrogel comprising a thermoreversible polymer of formula III, and culturing the cells under suitable stem cell expansion conditions.

[0083] The thermoreversible polymer can be prepared using any convenient method. A variety of polymerization methods can be used to prepare the base polymer material, including, for example, polyacrylate, polyacrylamide, and mixtures thereof. A variety of derivatization methods can be used to introduce any convenient functionality into the base polymer material of interest. A variety of chemoselective conjugation chemicals, linkers, functional groups, and modifiers can be used to prepare further derivatives and conjugates of the base polymer material of interest and their derivatives.

[0084] In some embodiments, a method for preparing a thermoreversible polymer of Formula III is provided, comprising: (i) polymerizing a comonomer population comprising N-isopropylacrylamide (NIPAAm), an alkyl methacrylate (preferably butyl methacrylate (BMA)), and N-acryloxysuccinimide (NASI) in a single, ideal solvent that maximizes monomer solubility to form a PNIPAAM-co-PBMA-co-PNASI copolymer backbone; (ii) reacting the copolymer with a mono-PEG amine (e.g., methoxy-PEG amine) to form a [PNIPAAM-co-PBMA-co-PNASI]-b-[PEG] copolymer; and (iii) reacting the [PNIPAAM-co-PBMA-co-PNASI]-b-[PEG] with isopropylamine to form a thermoreversible polymer of Formula III. Optimally, the initiator concentration in step (i) controls the backbone molecular weight and, therefore, the total polymer molecular weight. In preferred embodiments, the w / w ratio of PEG amine to PNIPAAM-co-PBMA-co-PNASI copolymer backbone (controlled by initiator concentration) is greater than 1:2, preferably greater than 1:3. In some embodiments, the w / w ratio of PEG amine to PNIPAAM-co-PBMA-co-PNASI copolymer is about 1:2.5, about 1:2.75, about 1:3.0, about 1:3.5, about 1:3.75, about 1:4.0, about 1:4.25, or about 1:4.5. In some embodiments, the w / w ratio of PEG amine to PNIPAAm copolymer is about 1:3 to about 1:4.5, or about 1:4. In some embodiments, a thermoreversible polymer of Formula III is provided, produced by such a process.

[0085] In some embodiments, the single solvent is selected from acetone, acetonitrile, benzene, chloroform, dichloromethane, dimethylformamide, dimethylsulfoxide, dioxane, ethyl acetate, pyridine, ethanol, methanol, tetrahydrofuran, toluene, and water.

[0086] (stem cells)

[0087] The term "stem cell" refers to a cell that is totipotent, pluripotent, or multipotent and can differentiate into one or more different cell types. This term includes, but is not limited to, embryonic stem cells, stem cells isolated from organs (e.g., skin stem cells), and induced pluripotent stem cells (iPSCs). The term "totipotency" refers to the ability of a cell to differentiate into any type of cell in a differentiated organism as well as cells of extraembryonic matter (e.g., placenta). As used herein, the term "induced pluripotent stem cell" or "iPSC" refers to a type of pluripotent stem cell that is similar to an embryonic stem cell but is generated when somatic (e.g., adult) cells are reprogrammed to enter an embryonic stem cell-like state by forcing them to express factors critical for maintaining the "sternness" of embryonic stem cells (ESCs) (i.e., the ability of embryonic stem cells to be directed to commit to different differentiation pathways). As used herein, the term "progenitor" in reference to a cell refers to an intermediate cellular stage where the cell is no longer a pluripotent stem cell, nor is it a fully committed cell. Progenitor cells in this disclosure are included within somatic cells.

[0088] The term "pluripotent" refers to a cell line that is capable of differentiating into any terminally differentiated cell type.

[0089] The term "multipotent" refers to a cell line that is capable of differentiating into at least two terminally differentiated cell types.

[0090] The term "embryonic stem cell" refers to a primitive (undifferentiated) cell derived from a preimplantation embryo or early embryo (for example, including up to the blastocyst stage), which can divide for a long period of time in culture without differentiation, and can develop into the cells and tissues of the three primary germ layers. Embryonic stem cells can also be isolated from an embryo, placenta, or umbilical cord.

[0091] The term "embryonic stem cell line" refers to a population of embryonic stem cells (e.g., human embryonic stem cell lines SA01, VUB01, HUES 24, H1, H9, WT3, HUES1) that have been cultured for days, months, or even years under in vitro conditions that allow proliferation without differentiation.

[0092] The term "induced pluripotent stem cells" or "iPSCs" refers to a type of pluripotent stem cell similar to embryonic stem cells (see, e.g., Takahashi and Yamanaka, Cell, 126, 663-676 (2006) (incorporated herein by reference)) formed by the introduction of certain embryonic genes (e.g., OCT4 transgene, SOX2 transgene, and KLF4 transgene) into somatic cells. Examples of somatic cells include, but are not limited to, bone marrow cells, epithelial cells, fibroblast cells, hematopoietic cells, hepatocytes, intestinal cells, mesenchymal cells, myeloid progenitor cells, and spleen cells. Alternatively, iPSCs can be generated by reprogramming somatic cells to enter an embryonic stem cell-like state by forcing them to express factors critical for maintaining embryonic stem cell (ESC) "rigor" (i.e., the ability of embryonic stem cells to be directed to commit to distinct differentiation pathways).

[0093] Methods for culturing stem cells, particularly human embryonic stem cells, are known in the art and are described in WO2006 / 029297, WO2006 / 019366 and WO2006 / 029198 (all to Thomson and Ludwig), and WO2008 / 089351 (Bergendahl and Thomson), which are incorporated herein by reference in their entireties.

[0094] (Inhibitor of "Small Mothers Against Decapentaplegic" (SMAD))

[0095] Small Mothers Against Decapentaplegic (SMAD) is a general term for a class of signaling molecules that can regulate the directed differentiation of stem cells. SMADs are intracellular proteins that transmit extracellular signals from transforming growth factor-β ligands to the nucleus, where the extracellular signal activates the transcription of downstream genes. SMADs are members of a class of signaling molecules that can regulate the directed differentiation of stem cells.

[0096] In the context of the disclosed methods, inhibitors of SMAD signaling include compounds that interact with SMADs and / or molecules associated with or other components of SMAD signaling, reducing or blocking their activity. Inhibitors may directly bind to SMAD signaling and cause a conformational change, reduce or prevent expression of SMAD-encoding genes or SMAD target genes, reduce SMAD protein levels, and / or interfere with the interaction of SMADs with one or more signaling partners.

[0097] Inhibitors also include molecules that indirectly regulate the biological activity of SMADs by interfering with upstream signaling molecules (e.g., located within the extracellular domain); example signaling molecules and effects include Noggin, which sequesters bone morphogenetic proteins and inhibits the activation of ALK receptors 1, 2, 3, and 6, thereby preventing downstream SMAD activation. Similarly, Chordin, Cerberus, and Follistatin similarly sequester extracellular activators of SMAD signaling. The transmembrane protein Bambi also acts as a pseudoreceptor to sequester extracellular TGFβ signaling molecules.

[0098] Antibodies that block activin, nodal, TGFβ, and BMP are contemplated for use to neutralize extracellular activators of SMAD signaling, etc. Thus, in one embodiment, an inhibitor of the present disclosure induces (alters) or shifts differentiation from an early state cell type to a non-early state cell type (e.g., one of the methods of the present disclosure comprising at least three inhibitors generated non-early state neural progenitor cells).

[0099] The inhibitors disclosed herein "alter" or "reduce" or "block" early-state signaling, for example, to direct cell differentiation toward non-early-state cell types as described herein to generate the cortical interneurons disclosed herein. Thus, the inhibitors of the present disclosure can be biological compounds (natural or synthetic) or small molecules, for example, to increase or decrease signal molecule activity that supports generating the cortical interneurons of the present disclosure.

[0100] Inhibitors have been described in terms of competitive inhibition (binding to the active site in a manner that precludes or reduces binding of another known binding compound) and allosteric inhibition (binding to a protein in a manner that alters the conformation of the protein in a manner that interferes with binding of a compound to the active site of the protein), in addition to inhibition induced by binding to and affecting a molecule upstream from a named signaling molecule, which in turn causes inhibition of the named molecule.

[0101] SMAD inhibitors that can be advantageously used in the methods disclosed herein include those well known and readily available to those skilled in the art. SMAD inhibitors that have been used for neural conversion of human ESCs and iPSCs are described in Chambers et al., Nat Biotechnol, 27:275-280 (2009).

[0102] Exemplary SMAD inhibitors that can be used in the methods and compositions disclosed herein include SB431542, LDN-193189, noggin PD169316, SB203580, LY364947, A77-01, A-83-01, BMP4, GW788388, GW6604, SB-505124, lerdelimumab, metelimumab, GC-I008, AP-12009, AP-11OI4, LY550410, LY580276, LY364947, LY2109761, SB-505124, E-616452 (RepSox ALK inhibitor), SD-208, SMI6, NPC-30345, Ki26894, SB-203580, SD-093, activin-M108A, P144, soluble TBR2-Fc, DMH-1, a compound called dorsomorphin dihydrochloride, and derivatives and / or variants thereof, each of which derivatives and / or variants has one or more SMAD inhibitory activity.

[0103] (a) TGFβ / activin / nodal pathway inhibitor

[0104] SMAD signaling pathway inhibition includes inhibition of the TGFβ / activin / nodal pathway and the BMP pathway. Exemplary TGFβ / activin pathway inhibitors include, but are not limited to, TGFβ receptor inhibitors, inhibitors of SMAD 2 / 3 phosphorylation, inhibitors of the interaction between SMAD 2 / 3 and SMAD 4, and activators / agonists of SMAD 6 and SMAD 7. Furthermore, the following classifications are for organizational purposes only, and one skilled in the art will recognize that a compound may affect one or more points in the pathway and therefore function in more than one of the defined classifications.

[0105] TGFβ receptor (e.g., ALK5) inhibitors may include antibodies against TGFβ receptors (e.g., ALK5), dominant-negative variants of TGFβ receptors (e.g., ALK5), and antisense nucleic acids that suppress the expression of TGFβ receptors (e.g., ALK5). Exemplary TGFβ receptor / ALK5 inhibitors include SB431542 (see, e.g., Inman et al., Molecular Pharmacology, 62(1):65-74(2002)), A-83-01 (also known as 3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide (see, e.g., Tojo et al., Cancer Science, 96(11):791-800(2005) and commercially available, e.g., from Toicris Bioscience);2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine, Wnt3a / BIO (see, e.g., Dalton et al., WO 2008 / 094597, incorporated herein by reference), BMP4 (see Dalton, supra), GW788388 (-{4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridin-2-yl}-N(tetrahydro-2H-pyran-4-yl)benzamide) (see, e.g., Gellibert et al., Journal of Medicinal Chemistry, 49(7):2210-2221 (2006)), SM16 (see, e.g., Suzuki et al., Cancer Research, 67(5):2351-2359 (2007)), IN-I 130 (3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-IH-imidazol-2-yl)methyl)benzamide) (see, e.g., Kim et al., Xenobiotica, 38(3):325-339 (2008)), GW6604 (2-phenyl-4-(3-pyridin-2-yl-IH-pyrazol-4-yl)pyridine) (see, e.g., de Gouville et al., Drug News Perspective, 19(2):85-90 (2006)), SB-505124 (2-(5-benzo[1,3]dioxol-5-yl-2-tertbutyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride) (see, e.g., DaCosta et al., Molecular Pharmacology, 65(3):744-752 (2004)) and pyrimidine derivatives (see, for example, those listed in WO2008 / 006583, incorporated herein by reference). In some embodiments, the method includes use of the SMAD inhibitor SB431542 at a concentration of about 5 μM to 15 μM, preferably about 10 μM, for a period of about 5 days to about 10 days, preferably about 7 days to about 8 days.

[0106] Furthermore, while the term "ALK5 inhibitor" is not intended to include nonspecific kinase inhibitors, the term "ALK5 inhibitor" should be understood to include inhibitors that inhibit ALK4 and / or ALK7 in addition to ALK5 (e.g., SB-431542, etc.). See, for example, Inman et al., J Mol Pharmacol, 62(1):65-74 (2002). Without intending to limit the scope of the present invention, it is believed that ALK5 inhibitors affect the mesenchymal-epithelial transition / transition (MET) process. The TGFβ / activin pathway is a driver of epithelial-mesenchymal transition (EMT). Therefore, inhibiting the TGFβ / activin pathway can promote the MET (i.e., reprogramming) process.

[0107] Specific examples of inhibitors include SU5416; 2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride (SB-505124); lerdelimumab (CAT-152); methelimumab (CAT-192); GC-I008; ID11; AP-12009; AP-11OI4; LY550410; LY580276; LY364947; LY21097 61; SB-505124; SB-431542; SD-208; SMI6; NPC-30345; Ki26894; SB-203580; SD-093; Gleevec; 3,5,7,2',4'-pentahydroxyflavone (morin); activin-M108A; P144; soluble TBR2-Fc; and tumor cells transfected with antisense targeting the TGFβ receptor. See, e.g., Wrzesinski et al., Clinical Cancer Research, 13(18):5262-5270 (2007); Kaminska et al., Acta Biochimica Polonica, 52(2):329-337 (2005); and Chang et al., Frontiers in Bioscience, 12:4393-4401 (2007).

[0108] The inhibitor of SMAD2 / 3 phosphorylation can include antibodies against SMAD2 or SMAD3, dominant 5 negative variants of SMAD2 or SMAD3, and antisense nucleic acids that target SMAD2 or SMAD3.Specific examples of inhibitors include PD169316; SB203580; SB-431542; LY364947; A77-01; and 3,5,7,2',4'-pentahydroxyflavone (morin).(See, for example, Wrzesinski (supra); Kaminska (supra); Shimanuki et al., Oncogene, 26:3311-3320 (2007); and Kataoka et al., EP1992360 (incorporated herein by reference)).

[0109] SB-431542 (i.e., CAS 301836-41-9; IUPAC 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide) is a commercially available small molecule inhibitor of SMADs, which can reduce or block transforming growth factor β (TGFβ) / activin-nodal signaling.

[0110] Inhibitors of the interaction between SMAD2 / 3 and SMAD4 may include antibodies against SMAD2, SMAD3, and / or smad4, dominant-negative variants of SMAD2, SMAD3, and / or smad4, as well as antisense nucleic acids targeting SMAD2, SMAD3, and / or smad4. Specific examples of inhibitors of the interaction between SMAD2 / 3 and SMAD4 include, but are not limited to, Trx-SARA, Trx-xFoxH1b, and Trx-Lef1. (See, for example, Cui et al., Oncogene, 24:3864-3874 (2005) and Zhao et al., Molecular Biology of the Cell, 17:3819-15 3831 (2006)).

[0111] (b) BMP inhibitors

[0112] Exemplary BMP pathway inhibitors include, but are not limited to, noggin, BMP receptor inhibitors, inhibitors of SMAD 1 / 5 / 8 phosphorylation, inhibitors of the interaction of SMAD 1 / 5 / 8 with SMAD 4, and activators / agonists of SMAD 6 and SMAD 7. The classifications below are for organizational purposes only, and one of skill in the art will recognize that a compound may affect one or more points in the pathway and therefore function in more than one of the defined classifications.

[0113] Inhibitors of SMAD 1 / 5 / 8 phosphorylation include, but are not limited to, antibodies against SMAD 1, SMAD 5, or SMAD 8, dominant-negative variants of SMAD 1, SMAD 5, or SMAD 8, antisense nucleic acids targeting SMAD 1, SMAD 5, or SMAD 8, and small molecules targeting SMAD 1, SMAD 5, or SMAD 8. Specific examples of inhibitors include LDN-193189 and dorsomorphin (commercially available, e.g., from Stemgent).

[0114] BMP receptor inhibitor includes but is not limited to antibody to BMP receptor, dominant negative variant of BMP receptor, siRNA that targets BMP receptor or antisense nucleic acid that targets BMP receptor, or small molecule that targets BMP receptor.Specific examples of inhibitor include but are not limited to DMH-1, dorsomorphin dihydrochloride and LDN-193189 (for example, commercially available from Tocris Biosciences).

[0115] LDN193189 (i.e., DM-3189, IUPAC 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinolone) is a commercially available small molecule inhibitor of SMAD signaling. LDN193189 is also a highly potent small molecule inhibitor of the protein tyrosine kinases (PTKs) ALK2, ALK3, and ALK6, and inhibits signaling of members of the ALK1 and ALK3 families of type I TGFβ receptors, resulting in inhibition of the transmission of multiple biological signals, including bone morphogenetic protein (BMP) signals BMP2, BMP4, BMP6, BMP7, and activin cytokine signals, and subsequent inhibition of SMAD phosphorylation of Smad1, Smad5, and Smad8. Yu et al., Nat Med 14:1363-1369 (2008) and Ctmy et al., Bioorg Med Chem Lett 18:4388-4392 (2008). In some embodiments, the method comprises use of the BMP inhibitor LDN-193189 at a concentration of about 50 nM to about 150 nM, preferably about 100 nM, for a period of about 10 days to about 20 days, preferably about 14 days to about 16 days.

[0116] (c) Dual SMAD inhibitors

[0117] The SMAD signaling inhibitor can include the dual SMAD inhibitors SB431542 and LDN-193189, or their functional derivatives and / or variants. As used herein, the terms "LSB" and "XLSB" are previously described in the definitions section. The dual SMAD inhibitor substantially increases the efficiency of differentiation.

[0118] According to the methods of the present disclosure, SB431542 can be contacted with pluripotent and / or multipotent cells in in vitro culture at a final concentration of about 0.1 μM to about 1 mM. LDN-193189 can be contacted with pluripotent and / or multipotent cells in in vitro culture at a final concentration of about 1 nM to about 10 μM.

[0119] (Wingless (Wnt) signaling antagonist)

[0120] "Wingless" or "Wnt" refers to a signaling pathway composed of Wnt family ligands and Wnt family receptors (e.g., Frizzled receptors and LRPDerailed / RYK receptors) that is mediated with or without β-catenin. Wnt proteins have been implicated in oncogenesis and several developmental processes, including the regulation of cell fate and patterning during embryonic development.

[0121] The Wnt pathway includes any of the proteins downstream or upstream of Wnt protein activity. For example, it can include LRPS, LRP6, Dkk, GSK-3, Wnt10B, Wnt6, Wnt3 (e.g., Wnt3A), Wnt1, or any of the other proteins discussed herein, and the genes encoding these proteins.

[0122] The Wnt pathway also includes pathways downstream of Wnt, such as the LRPS pathway or HBM pathway, the Dkk pathway, the p-catenin pathway, the MAPKAPK2 pathway, the OPG / RANK pathway, etc. By "LRP5 pathway" and "IBM pathway" is meant any protein / gene, including an LRP5 mutant or HBM mutant, and a protein downstream of an LRPS mutant or HBM mutant. By "β-catenin pathway" is meant any protein / gene, including β-catenin and a protein downstream of β-catenin. By "MAPKAPK2 pathway" is meant any protein / gene, including MAPKAPK2 and a protein downstream of MAPKAPK2. By "OPG / RANKL pathway" is meant any protein / gene, including OPG / RANKL and a protein downstream of OPG and RANKL. By "Dkk pathway" is meant any protein / gene involved in the interaction of Dkk-1, which is part of the Wnt pathway, with LRP5 and / or LRP6. Dkk-I inhibits LRP5 activity.

[0123] The term "Wnt antagonist" used herein refers not only to any agent that can act by directly inhibiting the normal function of Wnt protein, but also to any agent that inhibits Wnt signaling pathway, thereby reproducing the function of Wnt.Examples of Wnt signaling antagonists include XAV939 (Hauang et al., Nature, 461:614-620 (2009)), vitamin A (retinoic acid), lithium, flavonoids, Dickkopf1 (Dkk1), insulin-like growth factor binding protein (IGFBP) (WO2009 / 131166) and siRNA against β-catenin.Exemplary Wnt antagonists include, but are not limited to, XAV939, IWP-2, DKK1 (Dickkopf protein 1) and IWR1. Additional Wnt inhibitors include, but are not limited to, IWR compounds, IWP compounds, and other Wnt inhibitors described in WO09155001 and Chen et al., Nat Chem Biol, 5:100-7 (2009). In some embodiments, the method comprises use of the Wnt antagonist IWP-2 at a concentration of about 1 μM to about 10 μM, preferably about 5 μM, for a period of about 5 days to about 10 days, preferably about 7 days or about 8 days.

[0124] XAV939 is a potent small molecule inhibitor of tankyrase (TNKS)1 and tankyrase (TNKS)2, with IC 50The values ​​are 11 nM and 4 nM, respectively. Huang et al., Nature, 461:614-620 (2009). By inhibiting TNKS activity, XAV939 increases the protein levels of the axin-GSK3β complex and promotes the degradation of β-catenin in SW480 cells. Known antagonists of Wnt signaling also include Dickkopf proteins, secreted Frizzled-related proteins (sFRPs), Wnt inhibitory factor 1 (WIF-1), and Soggy. Members of the Dickkopf-related protein family (Dkk-1 to Dkk-4) are secreted proteins with two cysteine-rich domains separated by a linker region. Dkk-3 and Dkk-4 also contain a single prokineticin domain. Dkk-1, Dkk-2, Dkk-3, and Dkk-4 function as antagonists of canonical Wnt signaling by binding to LRP5 / 6 and preventing its interaction with the Wnt-Frizzled complex. Dkk-1, Dkk-2, Dkk-3, and Dkk-4 also bind to cell surface Kremen-1 or Kremen-2 and promote the internalization of LRP5 / 6. The antagonist activity of Dkk-3 has not been demonstrated. Dkk proteins have distinct expression patterns in adult and embryonic tissues and have a wide range of effects on tissue development and morphogenesis.

[0125] The Dkk family also includes Soggy, which is homologous to Dkk-3 but not to other family members. sFRPs are a family of five Wnt-binding glycoproteins similar to membrane-bound Frizzled. They are the largest family of Wnt inhibitors and comprise two groups: sFRP1, sFRP2, and sFRP5, and sFRP3 and sFRP4. All are secreted, originate from unique genes, and are not alternatively spliced ​​forms of the Frizzled family. Each sFRP contains an N-terminal cysteine-rich domain (CRO). Other antagonists of Wnt signaling include WIF-1 (Wnt inhibitory factor 1), a secreted protein that binds to Wnt proteins and inhibits their activity.

[0126] In some embodiments, the present disclosure relates to inhibitors and / or antagonists of the SMAD signaling pathway and the Wnt signaling pathway. SMAD inhibitors include SB431542, LDN-193189, noggin PD169316, SB203580, LY364947, A77-01, A-83-01, BMP4, GW788388, GW6604, SB-505124, lerdelimumab, meterimumab, GC-I008, AP-12009, AP-110I4, LY550410, LY580276, and LY364947. Wnt antagonists include, but are not limited to, LY2109761, SB-505124, SB-431542, SD-208, SMI6, NPC-30345, Ki26894, SB-203580, SD-093, activin-M108A, P144, soluble TBR2-Fc, DMH-1, dorsomorphin dihydrochloride, and their derivatives. Wnt antagonists include, but are not limited to, XAV939, DKK1, SFRP-1, SFRP-2, SFRP-5, SFRP-3, SFRP-4, WIF-1, Soggy, IWP-2, IWR1, and their derivatives.

[0127] In some embodiments of these methods, SB431542 and LDN193189 can be used in combination to inhibit the SMAD signaling pathway. In other embodiments, XAV939 can be used to antagonize the Wnt signaling pathway.

[0128] In other embodiments of these methods, the concentration of XAV939 in the cell culture can be about 0.2 μM to about 20 μM; the concentration of LDN193189 in the cell culture can be about 10 nM to about 1000 nM, and the concentration of SB431542 in the cell culture can be about 1 μM to about 100 μM. For example, the concentration of XAV939 can be about 2 μM, the concentration of LDN193189 can be about 100 nM, and the concentration of SB431542 can be about 10 μM.

[0129] In further embodiments of these methods, the forebrain progenitor cells are generated by contacting the stem cells with XAV939, LDN193189, and / or SB431542 for a duration of about 5 days to about 40 days. In related embodiments, the forebrain progenitor cells are generated by contacting the stem cells with XAV939, LDN193189, and / or SB431542 for a duration of about 10 days to about 25 days.

[0130] In a further aspect of the method according to any of these three embodiments, the Wnt signaling antagonist may be selected from the group consisting of XAV939, DKK1, DKK-2, DKK-3, Dkk-4, SFRP-1, SFRP-2, SFRP-5, SFRP-3, SFRP-4, WIF-1, Soggy, IWP-2, IWR1, ICG-001, KY0211, Wnt-059, LGK974, IWP-L6, and derivatives and / or variants thereof, each of which has one or more Wnt signaling antagonist activities. For example, the Wnt signaling antagonist may include XAV939 or a functional derivative and / or variant thereof. XAV939 may be contacted with pluripotent and / or multipotent cells in in vitro culture at a final concentration of about 10 nM to about 500 μM.

[0131] Various cell culture media and supplements can be used to differentiate stem cells into forebrain progenitor cells, including KSR medium, N2 medium (NaHCO, DMEM / F12 with N2B supplement (Stem Cell Technologies)), and Neurobasal medium with B27 supplement (Gibco) and N2 supplement (Invitrogen). Kriks et al., Nature, 480:547-551 (2011). In some embodiments, the cells are maintained on mouse embryonic fibroblasts (MEFs) as previously described, dissociated with Accutase (Innovative Cell Technologies) for differentiation, or dissociated with Dispase for passaging (Chambers et al., Nat Biotechnol, 27:275-280 (2009)).

[0132] (Sonic Hedgehog (SHH) activator)

[0133] NKX2.1, a transcription factor marker for the ventral forebrain progenitor population, can be used to monitor differentiation. Sussel et al., Development, 126:3359-3370 (1999) and Xu et al., J Neurosci, 24:2612-2622 (2004). Inhibition of Wnt signaling promotes ventralization by enhancing the production of FOXG1 and subsequently inducing the regulated SHH-mediated differentiation of multipotent and pluripotent cells toward an NKX2.1+ forebrain progenitor fate.

[0134] The generation of such neuronal cell lineages and populations can be achieved by contacting multipotent and / or pluripotent cells with one or more inhibitors of SMAD signaling and one or more antagonists of Wnt signaling for a duration sufficient to induce the generation of one or more markers of cortical interneurons or their precursors, followed by contacting neuronal progenitor cells (e.g., neuronal progenitor cells generated as disclosed herein) with one or more activators of SHH signaling for a predetermined period of time.

[0135] As used herein, the term "activator" in the context of SHH refers to a compound that promotes and / or enhances SHH signaling, thereby inducing neuronal progenitor cells to differentiate into cortical interneurons or their precursors, hypothalamic neurons or their precursors, and / or pre-optic chorionic neurons or their precursors. Examples of SHH signaling pathway activators useful in the present disclosure include proteins belonging to the Hedgehog family (e.g., SHH), inhibitors of the interaction of Pte with Smo, Smo receptor activators, Shh receptor activators (e.g., Hg-Ag, purmorphamine, etc.), substances that increase Ci / Gli family levels, inhibitors of intracellular degradation of Ci / Gli factors, and SHH overexpression constructs or Ci / Gli overexpression constructs resulting from transfection.

[0136] In some aspects of the method, an SHH signaling pathway activator (e.g., SHH plus purmorphamine) is added to the cell culture for the entire duration or a portion of the duration of the culture. In some embodiments, the concentration of the SHH activator in the cell culture is about 10 ng / mL to about 5000 ng / mL for SHH (or recombinant SHH) and about 0.1 μM to about 20 μM for purmorphamine. In some preferred embodiments, the concentration of the SHH activator in the cell culture is about 50 ng / mL to about 500 ng / mL for SHH (or recombinant SHH) and about 0.5 μM to about 4 μM for purmorphamine.

[0137] Purmorphamine has the name 9-cyclohexyl-N-[4-(morpholinyl)phenyl]-2-(1-naphthalenyloxy)-9H-purin-6-amine and the chemical formula C 31 H 32 Purmorphamine is a commercially available small molecule with NO. The structure of purmorphamine is listed below. Purmorphamine binds to and activates the seven-transmembrane Smo receptor in the Hedgehog signaling pathway.

[0138] In yet further aspects of the method according to any of these three embodiments, the SHH signaling activator may be selected from the group consisting of smoothened agonist (SAG), SAG analogs, SHH, C25-SHH, C24-SHH, purmorphamine, Hg-Ag, and derivatives and / or variants thereof, each of which has one or more SMAD inhibitory activities. For example, the SHH signaling activator may include recombinant SHH and purmorphamine, or functional derivatives and / or variants thereof. The recombinant SHH may be contacted with pluripotent and / or multipotent cells in in vitro culture at a final concentration of about 5 ng / mL to about 5 μg / mL. The purmorphamine may be contacted with pluripotent and / or multipotent cells in in vitro culture at a final concentration of about 0.1 μM to about 20 μM. In some embodiments, the method comprises use of the SSH activator SAG at a concentration of about 0.05 μM to about 5 μM, preferably about 0.1 μM, for a period of about 15 days to about 25 days, preferably about 21 days or about 22 days.

[0139] The differential timing of SHH differentiation activation is crucial for generating distinct ventral progenitor cells with distinct anterior-posterior identities. Early activation of SHH signaling in the induction of hESC-derived progenitor cells expressing markers of the hypothalamic anlage requires the presence of FGF-8. Kriks et al., Nature, 480:547-551 (2011). Neuronal progenitor cells can be contacted with one or more activators of SHH signaling after a predetermined period of passaging following generation of neuronal progenitor cells and / or following contact of pluripotent and / or multipotent cells with one or more inhibitors of SMADs and / or one or more antagonists of Wnt signaling.

[0140] For example, contacting the neuronal progenitor cells with one or more activators of SHH signaling can be initiated about 4 to about 20 days, or about 8 to about 18 days, after contacting the pluripotent and / or multipotent cells with one or more inhibitors of SMAD and one or more antagonists of Wnt signaling. Contacting the neuronal progenitor cells with one or more activators of SHH signaling can be for a period of about 5 to about 30 days, or about 8 to about 16 days.

[0141] As used herein, "FGF receptor (FGFR) agonist" refers to a molecule that can activate FGFR (e.g., a molecule that binds to FGFR, induces receptor dimerization, and activates the P13K and Ras / ERK signaling pathways). Non-limiting examples of FGFR agonists include FGF2, FGF8, and SUN11602. In a preferred embodiment, the FGFR agonist is FGF8 (e.g., recombinantly produced FGF8). According to the methods described herein, cells are contacted with an FGFR agonist (e.g., FGF8) to shift the balance toward rostralization. In some embodiments, the cells are contacted with FGF8 for a period of about 10 to about 20 days, preferably for a period of about 12 to about 16 days, and more preferably for about 13, 14, 15, or 16 days. Preferably, contact of the cells with an FGFR agonist (e.g., FGF8) is initiated about 5 to about 10 days, preferably about 6, 7, or 8 days, after initial contact of the cells with one or more SMAD inhibitors and / or the WNT inhibitor and / or the SHH activator.

[0142] In some preferred embodiments, the method includes (i) LDN193189 (LDN) to inhibit BMP signaling, (ii) SB-431542 (SB) to inhibit TGFβ signaling, (iii) recombinant FGF8, (iv) smoothened agonist (SAG; 3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-[3-(pyridin-4-yl)benzyl]-1-benzothiophene-2-carboxamide) to activate sonic hedgehog signaling, (iv) IWP2 to inhibit WNT signaling, and (v) FGF8 to shift the balance toward rostralization.

[0143] (Further maturity)

[0144] MGE progenitor cells can be differentiated into cINs, preferably postmitotic cINs, by contacting the cells with a neurotrophic factor (e.g., but not limited to, glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF)) for a predetermined amount of time according to the methods described herein. Preferably, the cells are simultaneously contacted with a Notch inhibitor (e.g., DAPT). In some embodiments, the predetermined amount of time is at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 26 days, at least 27 days, at least 28 days, at least 29 days, or at least 30 days.

[0145] (a marker for cortical interneurons)

[0146] As used herein, the term "marker" or "cell marker" refers to a gene or protein that identifies a particular cell or cell type. The marker for a cell may not be limited to one marker. Marker may refer to a "pattern" of markers, such that a specified group of markers can identify one cell or cell type from another cell or cell type.

[0147] Markers of cortical interneurons and / or cortical interneuron progenitor cells have been described and are readily available to those skilled in the art, including, for example, SST, PV, GABA, calbindin, LHX6, RAX, FOXA2, FOXG1, OLIG2, MASH1, NKX6.2, VGLUT1, MAP2, CTIP2, SATB2, TBR1, DLX2, ASCL1, and ChAT.

[0148] In some embodiments, differentiation into postmitotic cIN progenitor cells is identified by expression of one or more markers selected from FOXG1, PV, SST, calbindin, DCX, ASCL1, TUJ1, GABA, GAD1, VGAT, VGLUT1, and GAD67. In related embodiments, postmitotic cIN progenitor cells are identified by lack of expression of one or more markers selected from NKX2-1 and OLIG2, and optionally Ki67.

[0149] In some embodiments, differentiation into telencephalic cells is identified by the expression of FoxG1 and / or the lack of expression of RAX.

[0150] In some embodiments, differentiation into ventral telencephalic cells is specified by the expression of FoxG1 and DLX2 and the absence of EMX1 expression.

[0151] In some embodiments, differentiation into MGE progenitor cells is identified by expression of one or more markers selected from FOXG1, NKX2-1, NKX2-2, ASCL1, SIX6, OLIG2, NKX6.2, DLX1 / 2 and LHX6.

[0152] In a related embodiment, postmitotic cIN progenitor cells are distinguished from MGE progenitor cells by detecting expression of at least NKX2-1 and OLIG2, wherein MGE progenitor cells are identified by expression of NKX2-1 and lack of expression of OLIG2, and postmitotic cIN progenitor cells are identified by expression of OLIG2 and lack of expression of NKX2-1.

[0153] It is understood that the pluripotent and / or multipotent cells can be human or mouse cells, which can be selected from the group consisting of embryonic stem cells, adult stem cells, neural stem cells, induced pluripotent cells, engineered pluripotent cells, primary progenitor cells, induced progenitor cells, and engineered progenitor cells.

[0154] The contacting with the SMAD inhibitor and / or the contacting with the Wnt signaling antagonist can be performed simultaneously or sequentially. The contacting can be for a duration of about 5 days to about 30 days.

[0155] The method according to the present disclosure can be used to generate cortical interneurons and their precursors in quantities and purity that cannot be obtained by the state of the art. In some embodiments, the large number of pure functional cortical interneurons obtained by using the method of the present disclosure can be used to study seizures, schizophrenia or autism, and other neurological disorders. Similarly, these cells can also be used in cell therapy.

[0156] (composition)

[0157] In a further embodiment, the present disclosure provides a composition comprising one or more in vitro differentiated neuronal cells that produce one or more markers of cortical interneuron cells and / or one or more markers of cortical interneuron progenitor cells, wherein the in vitro differentiated neuronal cells are produced by (a) contacting a multipotent or pluripotent cell encapsulated in a synthetic hydrogel with two or more inhibitors of SMAD signaling, (b) contacting the multipotent or pluripotent cell with one or more inhibitors of Wnt signaling, and (c) contacting the multipotent or pluripotent cell with one or more activators of SHH signaling.

[0158] The compositions disclosed herein may comprise a mixture of two or more types of cells, in which cortical interneurons constitute at least about 30% of the total number of cells, or at least about 40% of the total number of cells, or at least about 50% of the total number of cells, or at least about 60% of the total number of cells, or at least about 70% of the total number of cells, or at least about 80% of the total number of cells, or at least about 90% of the total number of cells, or at least about 95% of the total number of cells.

[0159] The composition may comprise a mixture of two or more types of cells, in which NKX2.1+ / PV+ cortical interneurons constitute at least about 5%, or at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80% of the total number of cells in the composition.

[0160] The composition may comprise a mixture of two or more types of cells, in which gamma-aminobutyric acid (GABA)ergic inhibitory interneurons constitute at least about 5%, or at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80% of the total number of cortical interneurons in the composition.

[0161] Cortical interneurons or interneuron progenitor cells can be modified with a transgene that expresses a detectable marker, such as CT-2 or green fluorescent protein (GFP). It is understood that these detectable markers can be replaced with other detectable markers without departing from this aspect of the disclosure.

[0162] The cortical interneuron progenitor cells of the present invention give rise to functional interneurons, which exhibit the morphological, neurochemical, and electrophysiological properties of mature interneurons. The immature interneuron precursor preparations can be matured in vitro under controlled culture conditions that mimic their in vivo neuronal environment. Alternatively, the immature interneuron progenitor cells mature after transplantation and migration within the cerebral cortex of a mammalian subject (e.g., a human subject). When transplanted into the cerebral cortex, the immature interneuron progenitor cells of the present invention can migrate extensively in a non-radial (i.e., tangential) manner. Upon migration, the cortical interneuron progenitor cells can mature into interneurons that express parvalbumin and Kv3.1, which exhibit a rapidly spiking action potential discharge pattern. Alternatively, the cortical interneuron progenitor cells of the present invention can mature into somatostatin-expressing interneurons, which exhibit the rebound, adaptive, non-rapid-spiking firing pattern characteristic of this interneuron subgroup. These somatostatin-expressing interneurons can further express neuropeptide Y.

[0163] The cortical interneuron progenitor cells can mature into interneurons with a mean resting membrane potential of about -40 mV to about -70 mV. Over time, the mean resting membrane potential becomes more hyperpolarized, ranging from about -55 mV to about -70 mV.

[0164] Methods for the Treatment of Disorders and Diseases

[0165] In vitro derivation of neuronal cells from stem or progenitor cells has important clinical implications and is also important for disease modeling and drug screening. Conditions suitable for treatment according to this method of the present invention include, but are not limited to, seizure disorders (e.g., epilepsy or infantile spasms); neuropsychiatric disorders (e.g., autism, schizophrenia, anxiety, and eating disorders); neurodevelopmental disorders (e.g., holoprosencephaly or microcephaly); and Parkinson's disease. Initial studies using hPSCs have primarily targeted neurodegenerative disorders known to affect specific neuronal types, such as midbrain dopamine neurons in Parkinson's disease (PD) (Kriks et al., Nature, 480:547-551 (2011); Soldner et al., Cell, 136:964-977 (2009); and Soldner et al., Cell, 146:318-331 (2011)) or motor neurons in amyotrophic lateral sclerosis (ALS) (Dimos et al., Science, 321:1218-1221 (2008)) and spinal muscular atrophy (SMA) (Ebert et al., Nature, 457:277-280 (2009)). More recent studies suggest the potential for addressing complex neurological disorders such as schizophrenia (Brennand et al., Nature, 473:221-225 (2011)) or autism-related syndromes (Marchetto et al., Cell, 143:527-539 (2010) and Pasca et al., Nat Med, 17:1657-1662 (2011)).

[0166] The cells of the present disclosure can be delivered by intraparenchymal or intraventricular transplantation, as described in U.S. Patent Nos. 5,082,670 and 5,650,148 (Gage et al.) and U.S. Patent Application Publication No. 20060141622 (Johe et al.), which are incorporated herein by reference in their entireties. Intraparenchymal transplantation can be achieved by injecting immature interneuron progenitor cells into the host brain parenchyma, or by surgically creating a cavity to expose the host brain parenchyma and then placing the cell graft into the cavity. Both methods provide parenchymal apposition between the transplanted cells and the host brain tissue at the time of transplantation, and both promote anatomical integration between the graft and the host brain tissue. Alternatively, the graft can be placed in a ventricle (e.g., a cerebral ventricle) or subdurally (e.g., on the surface of the host brain where the graft is separated from the host brain parenchyma by the intervening pia mater, or the intervening arachnoid and pia mater). Transplantation into the ventricle can be achieved by injection of donor cells, or by growing the cells in a matrix (e.g., 30% collagen) to form a solid tissue plug that can then be implanted into the ventricle to prevent displacement of the graft. For subdural transplantation, the cells can be injected near the brain surface after making a slit in the dura. This is important if the graft is required to become an integral part of the host brain and survive for the life of the host.

[0167] Regardless of this survival issue, the transplantation of neuronal cells and neuronal precursors described in this disclosure results in successful engraftment of large numbers of cells that can be studied following their maturation in vivo.

[0168] Methods for the Treatment of Neurodegenerative Disorders and Diseases

[0169] In certain embodiments, the present disclosure provides methods for the treatment of disorders and diseases associated with neurodegeneration, including, for example, seizure disorders, Parkinson's disease (PD) and Alzheimer's disease (AD), comprising the in vivo administration of cortical interneurons to a patient suffering from a seizure disorder, PD or AD, wherein the cortical interneurons are generated by the methods disclosed herein.

[0170] Methods for the Treatment of Mental Disorders and Illnesses

[0171] In other embodiments, the present disclosure provides methods for the treatment of psychiatric disorders and illnesses, including, for example, schizophrenia and autism-related disorders.

[0172] Unlike PD, ALS, or SMA, the neuronal types important for modeling schizophrenia or autism are less well defined, and no attempts have been made to target neuronal subtype identities in their studies. Excellent work has recently been done to establish protocols for the induction of human ESC-derived cortical projection neurons. Espuny-Camacho et al., Neuron, 77:440-456 (2013) and Shi et al., Nat Neurosci, 15:477-486, S471 (2012).

[0173] However, inhibitory neurons (e.g., cortical interneurons) may have a particularly important role in schizophrenia or autism. Insel, Nature, 468:187-193 (2010) and Lewis et al., Nat Rev Neurosci, 6:312-324 (2005).

[0174] Current paradigms for modeling and treating human psychiatric disorders use patient-specific iPSC-derived neurons. Brennand et al., Nature, 473:221-225 (2011); Cheung et al., Hum Mol Genet, 20:2103-2115 (2011); Chiang et al., Mol Psychiatry, 16:358-360 (2011); Marchetto et al., Cell, 143:527-539 (2010); and Pasca et al., Nat Med, 17:1657-1662 (2011). However, these published studies were performed in mixed neuronal cultures of unclear neuronal subtype identity, and characterization of subtype-specific synaptic and functional properties was limited. Aggregation of postmortem findings has been utilized in attempts to link genetic defects to psychiatric disorders (e.g., interneuron-associated Erbb4 receptor in schizophrenia). Fazzari et al., Nature, 464:1376–1380 (2010).

[0175] As disclosed herein, the present disclosure provides purified populations of mature cortical interneurons that can be used as models of human psychiatric disorders and diseases, and in therapeutic regimens for the treatment of such psychiatric disorders and diseases. Furthermore, the data presented herein demonstrate that highly efficient induction of cortical interneurons is possible after timed exposure to developmental cues.

[0176] Without wishing to be bound by theory, it is believed that putative hESC-derived GABAergic interneurons receive synaptic input from other human interneurons and from mouse excitatory projection neurons. Cells exhibiting neurochemical properties of cortical interneurons adopt highly mature physiological characteristics within 30 days of plating in mouse cortical cultures. The mechanism of accelerated in vitro maturation of NKX2.1:GFP+ neurons in mouse cortical cultures is currently unknown, but the involvement of species-specific timing factors is inferred from the data presented herein. The data of the present disclosure further demonstrate that synaptically active cortical interneurons can be induced in vitro and may be useful for modeling and treating cortical interneuron pathologies in psychiatric disorders, including, but not limited to, schizophrenia and autism.

[0177] The generation of hESC-derived PV-expressing neurons and the presence of relatively fast-spiking, non-adaptive neurons in these cultures is particularly interesting given the implication of PV interneuron dysfunction in schizophrenia (Beasley and Reynolds, Schizophr Res, 24:349-355 (1997) and Woo et al., Am J Psychiatry, 154:1013-1015 (1997)). Rapidly spiking PV+ cortical interneurons are observed late during prenatal development in primates and continue to mature into early adulthood (Anderson et al., Neuroscience, 67:7-22 (1995) and Insel, Nature, 468:187-193 (2010)). Considering the role of PV+ neurons in various pathological conditions, the data presented herein support the modeling of such dysfunctional neuronal conditions and their treatment by administering cortical interneurons and / or their precursors.

[0178] Thus, in certain aspects of these embodiments, the present disclosure provides for the generation of enriched cortical interneuron subgroups (e.g., somatostatin+ cells and PV+ cells). MGE progenitor cells can be under the control of SHH signaling, with high SHH signaling levels promoting the generation of somatostatin+ cells and lower SHH signaling levels promoting the generation of PV+ neurons. Xu et al., Neuron, 65:328-340 (2010).

[0179] Any method known in the art for measuring gene expression can be used, particularly quantitative methods (e.g., real-time quantitative PCR or microarrays, or methods using gene reporter expression), or qualitative methods (e.g., immunostaining or cell sorting methods that identify cells that display specific biomarkers, including cell surface markers).

[0180] Some embodiments of the present invention are illustrated in the following sections.

[0181] Item 1. A thermoreversible polymer having improved stability over time, Formula (III): [ka] Including, In the formula, a, b, c, and d represent the mole fractions of the polymer; PEG n is a polyethylene glycol polymer, and n is an integer from 1 to 2500; R 1 is any end group, if present, other than a primary amine; R 2 is a lower alkyl group; R 3 is a functional group or linked modifier, if present; G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and a linked modifying substance; Thermoreversible polymer.

[0182] Item 2. The thermoreversible polymer according to Item 1, wherein the molecular weight (MW) of the polymer is about 0 kilodaltons (kDa) to about 250 kilodaltons (kDa) or about 50 kDa to about 200 kDa.

[0183] Section 3.R 1 is selected from methoxy, ethoxy, n-propoxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy and isopentoxy 1~ Item 1 or 2. The thermoreversible polymer according to item 1 or 2, which is a C6 alkoxy.

[0184] Section 4.R 1 Item 4. The thermoreversible polymer according to item 3, wherein is methoxy.

[0185] Section 5.R 2 is selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, isopropyl, isobutyl, isopentyl, tert-butyl, cyclopropyl, and cyclobutyl.

[0186] Section 6.R 2 Item 6. The thermoreversible polymer according to item 5, wherein is butyl.

[0187] Section 7.R 3 Item 7. The thermoreversible polymer according to any one of items 1 to 6, wherein no

[0188] Section 8.R 1 is methoxy and R 2 is butyl and R 3 Item 2. The thermoreversible polymer according to Item 1, wherein the polymer has a MW of about 0 kDa to about 250 kDa.

[0189] Section 9. PEG n Item 9. The thermoreversible polymer according to any one of Items 1 to 8, having a MW of about 1 kilodalton (kDa) to about 50 kilodaltons (kDa).

[0190] Section 10.R 3

[0023] Item 10. The thermoreversible polymer according to any one of items 1 to 9, wherein is a chemoselective functional group selected from thiol, alkyne, cyclooctyne, azide, phosphine, maleimide, alkoxyamine, aldehyde, and protected versions or precursors thereof.

[0191] Section 11.R 3 Item 10. The thermoreversible polymer according to any one of Items 1 to 9, wherein the modifying substance is selected from heparin, hyaluronic acid, a specific binding member, a peptide, a nucleic acid, gelatin, fibronectin, collagen, laminin, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin, progesterone, glucose, stromal cell-derived factor 1 (SDF-1), thymosin beta-4, sonic hedgehog (SHH), noggin, activin, transforming growth factor beta (TGF-β), FGF8, brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), neurotrophic factor 3 (NT3), platelet-derived growth factor (PDGF), IL-16, IL-2, and insulin-like growth factor 1 (IGF-1).

[0192] Item 12. The thermoreversible polymer according to any one of Items 1 to 11, wherein the thermoreversible polymer has one or more of the following properties: (a) an LCST of about 12°C to about 32°C, preferably about 19°C to about 23°C; (b) a stiffness of about 100 Pa to about 8000 Pa, preferably about 100 Pa to about 3000 Pa; and (c) a viscosity of about 100 cP to about 1500 cP.

[0193] Item 13. A three-dimensional hydrogel comprising the thermoreversible polymer according to any one of Items 1 to 12 and an aqueous buffer solution.

[0194] Item 14. An in vitro method for generating a cell population enriched for MGE progenitor cells from an initial population of human stem cells, the method comprising: (a) encapsulating an initial population of human stem cells in the three-dimensional hydrogel of paragraph 13; and (b) contacting the encapsulated human stem cells with at least one inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling and at least one Wingless (Wnt) antagonist; and contacting the cells with at least one activator of Sonic Hedgehog (SHH) signaling and an FGFR agonist, thereby obtaining a cell population enriched for MGE progenitor cells that express FOXG1 and at least one additional marker indicative of MGE progenitor cells. A method comprising:

[0195] Item 15. The method according to Item 14, wherein the human stem cells are selected from the group consisting of human embryonic stem cells, human adult stem cells, human neural stem cells, human induced pluripotent cells, human primary progenitor cells, and human induced progenitor cells.

[0196] Item 16. The method of Item 14, wherein the contacting with the at least one inhibitor of SMAD signaling and the contacting with the at least one Wnt antagonist are carried out simultaneously or sequentially, each having a duration of between about 5 days and about 30 days.

[0197] Clause 17. The method of clause 16, wherein contacting the cells with the at least one Wnt antagonist is initiated within 5 days, preferably within 4 days, within 3 days, within 2 days, or within 1 day of initial contacting the cells with the at least one inhibitor of SMAD signaling, and preferably contacting the cells with the at least one Wnt antagonist is initiated simultaneously with initial contacting the cells with the at least one inhibitor of SMAD signaling.

[0198] Item 18. The at least one inhibitor of SMAD signaling is selected from the group consisting of SB431542, LDN-193189, noggin PD169316, SB203580, LY364947, A77-01, A-83-01, BMP4, GW788388, GW6604, SB-505124, lerdelimumab, meterimumab, GC-I008, AP-12009, AP-110I4, LY550410, LY580276, LY364947, LY2109761, SB-505124, E-616452 (RepSox ALK inhibitor), SD-208, SMI6, NPC-30345, [ka] Item 15. The method of item 14, wherein the antibody is selected from the group consisting of SB-203580, SD-093, activin-M108A, P144, soluble TBR2-Fc, DMH-1, dorsomorphin dihydrochloride, derivatives thereof, and combinations thereof.

[0199] Clause 19. The method of clause 18, wherein said at least one inhibitor of SMAD signaling comprises SB431542 and LDN-193189.

[0200] Clause 20. The method of clause 14, wherein the at least one Wnt antagonist is selected from the group consisting of XAV939, DKK1, DKK-2, DKK-3, Dkk-4, SFRP-1, SFRP-2, SFRP-5, SFRP-3, SFRP-4, WIF-1, Soggy, IWP-2, IWR1, ICG-001, KY0211, Wnt-059, LGK974, IWP-L6, derivatives thereof, and combinations thereof, and preferably the at least one Wnt antagonist comprises IWP-2.

[0201] Item 21. The method of Item 14, wherein the at least one activator of SHH signaling is selected from the group consisting of smoothened agonist (SAG), SAG analogs, SHH, C25-SHH, C24-SHH, purmorphamine, Hg-Ag, derivatives thereof, and combinations thereof.

[0202] Item 22. (i) the contact of the cells with the at least one activator of SHH signaling is completed within about 5 to about 30 days, preferably about 18 to 23 days, more preferably about 19 to 22 days, and even more preferably about 20 or 21 days from the start of the contact; (ii) the initial contact of the cells with the at least one activator of SHH signaling is completed within about 0 days from the initial contact of the cells with the at least one inhibitor of SMAD signaling and from the initial contact of the cells with the at least one inhibitor of WNT signaling. (iii) the initial contact of the cells with the at least one inhibitor of SMAD signaling is between 0 and 4 days from the start of the initial contact of the cells with the at least one Wnt antagonist; (iv) the contact of the cells with the at least one inhibitor of SMAD signaling is terminated between 6 and 14 days from the start of the contact, and / or (v) the contact of the cells with the at least one Wnt antagonist is terminated between 6 and 8 days from the start of the contact, preferably about 7 days from the start of the contact.

[0203] Clause 23. The method of Clause 14, wherein the at least one additional marker is selected from the group consisting of NKX2-1, NKX2-2, ASCL1, SIX6, OLIG2, NKX6.2, DLX1 / 2 and LXH6.

[0204] Item 24. The method of Item 14, wherein at least about 60%, at least 65%, at least 70%, at least 75%, or at least 80% of the obtained cell population expresses FOXG1 and NKX2-1.

[0205] Item 25. The method of Item 14, wherein at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% of the obtained cell population comprises MGE progenitor cells.

[0206] Clause 26. The method of clause 14, further comprising (c) contacting the cells after a predetermined amount of time with at least one neurotrophic factor (e.g., GDNF, BDNF) and optionally a Notch inhibitor (e.g., DAPT) to produce a cell population enriched for differentiated inhibitory GABAergic cortical interneurons (cINs) that express FOXG1 and at least one additional marker indicative of cortical interneuron cells.

[0207] Item 27. The method according to Item 26, comprising the step of contacting the cells with at least one neurotrophic factor and a Notch inhibitor.

[0208] Item 28. The method according to Item 27, comprising the step of contacting the cells with GDNF, BDNF and DAPT.

[0209] Item 29. The method of any one of Items 26 to 28, wherein the step of contacting the cells with at least one neurotrophic factor and, optionally, a Notch inhibitor is performed after the step of contacting the cells with the at least one inhibitor of SMAD signaling, at least one Wnt antagonist, and at least one activator of SHH signaling has been completed.

[0210] Item 30. The method of any one of Items 26 to 29, wherein the step of contacting the cells with at least one neurotrophic factor and optionally a Notch inhibitor is terminated between 7 and 30 days after initiation, and / or the step of contacting the cells with at least one neurotrophic factor and optionally a Notch inhibitor is terminated at least about 10 days, at least about 12 days, or at least about 14 days after initiation.

[0211] Clause 31. The method of Clause 26, wherein the at least one additional marker is selected from the group consisting of PV, SST, calbindin, DCX, ASCL1, TUJ1, GABA, GAD1, VGAT, vGLUT1, and GAD67.

[0212] Item 32. The method of any one of Items 26 to 31, wherein at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% of the obtained cIN cell population expresses parvalbumin (PV).

[0213] Item 33. The method according to any one of Items 26 to 32, wherein less than about 5% of the obtained cIN cell population expresses Ki67.

[0214] Paragraph 34. A composition comprising a population of cells produced by the method of any one of paragraphs 14 to 26, wherein at least 50% of the cells, or at least 60% of the cells, or at least 70% of the cells, or at least 80% of the cells, or at least 90% of the cells, or at least 95% of the cells are MGE progenitor cells, preferably the method does not include a step for purifying or further enriching the MGE progenitor cells after step (b).

[0215] Paragraph 35. A composition comprising a population of cells produced by the method of any one of paragraphs 17 to 33, wherein at least 50% of the cells, or at least 60% of the cells, or at least 70% of the cells, or at least 80% of the cells, or at least 90% of the cells, or at least 95% of the cells are cIN, and preferably the method does not include a step for purifying or further enriching the cIN ​​cells after step (c).

[0216] Item 36. At least 50% of the cells, or at least 60% of the cells, or at least 70% of the cells, or at least 80% of the cells, or at least 90% of the cells, or at least 95% of the cells are NKX2.1 - / PV + Item 36. The composition according to Item 35, wherein

[0217] Item 37. Use of the composition according to any one of Items 34 to 36 in the treatment of a neurological disorder.

[0218] Item 38. The use according to Item 37, wherein the neurological disorder is a seizure disorder.

[0219] 39. An in vitro method for generating a cell population enriched for MGE progenitor cells from an initial population of human stem cells, the method comprising: (a) encapsulating an initial population of human stem cells in a three-dimensional hydrogel; and (b) contacting the encapsulated human stem cells with at least one inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling and at least one Wingless (Wnt) antagonist; and contacting the cells with at least one activator of Sonic Hedgehog (SHH) signaling and an FGFR agonist, thereby obtaining a cell population enriched for MGE progenitor cells that express FOXG1 and at least one additional marker indicative of MGE progenitor cells. A method comprising:

[0220] Clause 40: The method according to Clause 39, wherein the human stem cells are selected from the group consisting of human embryonic stem cells, human adult stem cells, human neural stem cells, human induced pluripotent cells, human primary progenitor cells, and human induced progenitor cells.

[0221] Paragraph 41. The method of Paragraph 39, wherein the contacting with the at least one inhibitor of SMAD signaling and the contacting with the at least one Wnt antagonist are carried out simultaneously or sequentially, each having a duration of between about 5 days and about 30 days.

[0222] Clause 42. The method of clause 41, wherein contacting the cells with the at least one Wnt antagonist is initiated within 5 days, preferably within 4 days, within 3 days, within 2 days, or within 1 day of initial contacting the cells with the at least one inhibitor of SMAD signaling, and preferably contacting the cells with the at least one Wnt antagonist is initiated simultaneously with initial contacting the cells with the at least one inhibitor of SMAD signaling.

[0223] Item 43. The at least one inhibitor of SMAD signaling is selected from the group consisting of SB431542, LDN-193189, noggin PD169316, SB203580, LY364947, A77-01, A-83-01, BMP4, GW788388, GW6604, SB-505124, lerdelimumab, meterimumab, GC-I008, AP-12009, AP-110I4, LY550410, LY580276, LY364947, LY2109761, SB-505124, E-616452 (RepSox ALK inhibitor), SD-208, SMI6, NPC-30345, [ka] 40. The method of claim 39, wherein the antibody is selected from the group consisting of SB-203580, SD-093, activin-M108A, P144, soluble TBR2-Fc, DMH-1, dorsomorphin dihydrochloride, derivatives thereof, and combinations thereof.

[0224] Clause 44. The method of Clause 43, wherein said at least one inhibitor of SMAD signaling comprises SB431542 and LDN-193189.

[0225] Clause 45. The method of Clause 39, wherein the at least one Wnt antagonist is selected from the group consisting of XAV939, DKK1, DKK-2, DKK-3, Dkk-4, SFRP-1, SFRP-2, SFRP-5, SFRP-3, SFRP-4, WIF-1, Soggy, IWP-2, IWR1, ICG-001, KY0211, Wnt-059, LGK974, IWP-L6, derivatives thereof, and combinations thereof, preferably wherein the at least one Wnt antagonist comprises IWP-2.

[0226] Clause 46. The method of clause 39, wherein the at least one activator of SHH signaling is selected from the group consisting of smoothened agonist (SAG), SAG analogs, SHH, C25-SHH, C24-SHH, purmorphamine, Hg-Ag, derivatives thereof, and combinations thereof.

[0227] Item 47. (i) the contact of the cells with the at least one activator of SHH signaling is terminated within about 5 to about 30 days, preferably about 18 to 23 days, more preferably about 19 to 22 days, and even more preferably about 20 or 21 days from the start of the contact; (ii) the initial contact of the cells with the at least one activator of SHH signaling is terminated within about 0 days from the initial contact of the cells with the at least one inhibitor of SMAD signaling and from the initial contact of the cells with the at least one inhibitor of WNT signaling. (iii) the initial contact of the cells with the at least one inhibitor of SMAD signaling is between 0 and 4 days from the start of the initial contact of the cells with the at least one Wnt antagonist; (iv) the contact of the cells with the at least one inhibitor of SMAD signaling is terminated between 6 and 14 days from the start of the contact, and / or (v) the contact of the cells with the at least one Wnt antagonist is terminated between 6 and 8 days from the start of the contact, preferably about 7 days from the start of the contact.

[0228] Clause 48. The method of Clause 39, wherein the at least one additional marker is selected from the group consisting of NKX2-1, NKX2-2, ASCL1, SIX6, OLIG2, NKX6.2, DLX1 / 2 and LXH6.

[0229] Clause 49. The method of clause 39, wherein at least about 60%, at least 65%, at least 70%, at least 75%, or at least 80% of the resulting cell population expresses FOXG1 and NKX2-1.

[0230] Clause 50. The method of Clause 39, wherein at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% of the obtained cell population comprises MGE progenitor cells.

[0231] Clause 51. The method of clause 39, further comprising (c) contacting the cells after a predetermined amount of time with at least one neurotrophic factor (e.g., GDNF, BDNF) and optionally a Notch inhibitor (e.g., DAPT) to produce a cell population enriched for differentiated inhibitory GABAergic cortical interneurons (cINs) that express FOXG1 and at least one additional marker indicative of cortical interneuron cells.

[0232] Clause 52. The method of Clause 51, comprising the step of contacting the cells with at least one neurotrophic factor and a Notch inhibitor.

[0233] Item 53. The method according to Item 52, comprising the step of contacting the cells with GDNF, BDNF and DAPT.

[0234] Paragraph 54. The method of any one of paragraphs 51 to 53, wherein the step of contacting the cells with at least one neurotrophic factor and, optionally, a Notch inhibitor, is performed after the steps of contacting the cells with the at least one inhibitor of SMAD signaling and at least one Wnt antagonist and at least one activator of SHH signaling have been completed.

[0235] Paragraph 55. The method of any one of paragraphs 51 to 54, wherein the step of contacting the cells with at least one neurotrophic factor and optionally a Notch inhibitor is terminated between 7 and 30 days after initiation, and / or the step of contacting the cells with at least one neurotrophic factor and optionally a Notch inhibitor is terminated at least about 10 days, at least about 12 days, or at least about 14 days after initiation.

[0236] Clause 56. The method of Clause 51, wherein the at least one additional marker is selected from the group consisting of PV, SST, calbindin, DCX, ASCL1, TUJ1, GABA, GAD1, VGAT, vGLUT1, and GAD67.

[0237] Paragraph 57. The method of any one of paragraphs 51 to 56, wherein at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% of the obtained cIN cell population expresses parvalbumin (PV).

[0238] Paragraph 58. The method of any one of paragraphs 51 to 57, wherein less than about 5% of the obtained cIN cell population expresses KI67.

[0239] Paragraph 59. A composition comprising a population of cells produced by the method of any one of paragraphs 39 to 50, wherein at least 50% of the cells, or at least 60% of the cells, or at least 70% of the cells, or at least 80% of the cells, or at least 90% of the cells, or at least 95% of the cells are MGE progenitor cells, preferably the method does not include a step for purifying or further enriching the MGE progenitor cells after step (b).

[0240] Paragraph 60. A composition comprising a population of cells produced by the method of any one of paragraphs 51 to 58, wherein at least 50% of the cells, or at least 60% of the cells, or at least 70% of the cells, or at least 80% of the cells, or at least 90% of the cells, or at least 95% of the cells are cIN, and preferably the method does not include a step for purifying or further enriching the cIN ​​cells after step (c).

[0241] Item 61. At least 50% of the cells, or at least 60% of the cells, or at least 70% of the cells, or at least 80% of the cells, or at least 90% of the cells, or at least 95% of the cells are NKX2.1 - / PV + Item 61. The composition according to item 60, wherein

[0242] Item 62. Use of the composition according to any one of Items 59 to 61 in the treatment of a neurological disorder.

[0243] Item 63. The use according to Item 62, wherein the neurological disorder is a seizure disorder. [Example]

[0244] (Example) The following examples illustrate preferred embodiments of the present invention and are not intended to limit the scope of the invention in any way. Although the invention has been described in relation to its preferred embodiments, various modifications thereof will become apparent to those skilled in the art upon reading the present application.

[0245] Example 1 The generation of cINs from human stem cells requires multiple factors, careful timing, and a long maturation time frame, and therefore the 3D hydrogel must remain stable over these relatively long periods.

[0246] The following structure is disclosed in U.S. Patent Application Publication No. 2024 / 0294713: [ka] Hydrogels containing the acrylamide polymer were first tested specifically for their stability during the duration of cIN generation from human stem cells.

[0247] Generation of cINs from human stem cells was not possible using hydrogels containing such acrylamide polymers. This was because the hydrogels were not stable over the entire 35-day differentiation period at any of the molecular weights tested (0 kDa to 500 kDa). These hydrogels also did not have the appropriate peak stiffness range for stability (<1200 Pa), exhibited high viscosities (>2000 cP), had low stability / high swell ratios (>1.5) in aqueous environments, and high LCSTs (>25). These gels did not retain their structure over time in aqueous environments or under the shear stress of mixing in spinner flasks or bioreactors. Finally, these gels did not adequately extrude and encapsulate cells, and they had low sphericity (<0.7), large bead diameters (>4 mm), and large tail formation ratios (>0.5).

[0248] Therefore, structural improvements to increase stability were examined.

[0249] The following structure corresponds to Formula III: [ka] It was first found that utilizing acrylates in polymers with (see process in Figure 1) resulted in a decreased LCST, increased gel stiffness, reduced viscosity and increased gel stability compared to acrylamide.

[0250] In the formula, R 1 is methoxy and R 2 is butyl and R 4 is methyl, (d) is absent, and the MW of this polymer is 250 kDa to 500 kDa. The results are shown in Table 1 below and in Figure 2: [Table 1]

[0251] Next, the properties of the acrylate polymer of the above structure were modified to further improve the stability of the hydrogel over time. Polymer backbones of various molecular weights were created by varying the initiator concentration. It was found that decreasing the overall polymer molecular weight (which is a function of backbone length, which directly controls the length of the polymer; decreasing backbone length corresponds to decreasing the molecular weight of the polymer) increased stiffness, maintained the LCST, decreased liquidus viscosity, and increased stability (all functional groups remained as defined above); the results are shown in Table 2 below and Figure 3: [Table 2]

[0252] Thus, 3D hydrogels containing low molecular weight acrylate-based polymers have been found to offer significant improvements over prior art thermoreversible polymers for 3D culture of stem cells, for example, to generate cINs. In particular, 3D hydrogels containing low molecular weight acrylate-based polymers exhibit increased stability (which allows for the long differentiation process required to generate cINs), reduced liquid phase viscosity (which allows for efficient cell encapsulation), and appropriate stiffness to support neural growth while maintaining a favorable LCST.

[0253] The effect of varying the molecular weight of PEG in the polymer of Formula III on the properties of three-dimensional hydrogels was examined. It was surprisingly found that PEG lengths of 1k or shorter resulted in poor gel performance at the matched wt% (15 wt%), while PEG lengths of 10k or longer hindered gel flexibility and node stability. Therefore, a PEG weight of approximately 5k was found to be optimal. See Figure 4.

[0254] Next, the effect of varying the weight ratio of PEG to polymer backbone in the polymer of Formula III was examined. It was discovered that increasing the PEG:PNIPAAm ratio beyond 1:2 allowed for improvements in synthesis and performance in encapsulation and cell growth. See Table 3 below and Figure 5A. [Table 3]

[0255] Increasing the PEG:PNIPAAm weight ratio beyond 1:2 increases gel stiffness, decreases gel viscosity, increases gel stability, and decreases gel LCT (see Figure 5A). Without being bound by theory, these benefits may stem from increased participation of PNIPAAm in gel node formation as well as stronger associations that support temperature-based gel formation.

[0256] Increasing the PEG:PNIPAAm copolymer ratio beyond 1:2 also allows for improvements to cell encapsulation, as the encapsulated beads exhibit decreased bead diameter, decreased bead volume, increased bead sphericity, and a reduced proportion of beads containing tails after gravity drop encapsulation (see Figure 5B).

[0257] Further improvements at PEG:PNIPAAm weight ratios greater than 1:2 include improvements to encapsulation and cell performance (e.g., greater flow rates at lower shear stress, higher immediate cell viability, higher 7-day cell viability, and higher cell yields). See Figure 5C.

[0258] Next, position R of formula III 2 The effect of alkyl groups in the polymers was investigated. Briefly, the polymers of Formula III (R 1 = methoxy, R 4 = methyl, (d) = 0, and R 2 is n-butyl or isobutyl): [ka] was prepared and tested.

[0259] The steric structure of the pendant alkyl group (n-butyl vs. isobutyl) was found to be important, with n-butyl surprisingly exhibiting higher stiffness and similar viscosity, higher gel stability, and a lower gel LCST compared to isobutyl (see Figure 6).

[0260] The steric structure of the backbone acrylate group was found to be important because methyl (from butyl methacrylate) at position R4 of Formula III exhibits higher stiffness and similar viscosity, higher gel stability, and a lower gel LCST compared to hydrogen at position R4. See Figures 6.5A-6.5B. Furthermore, polymers of Formula III with methyl at position R4 exhibit smaller bead diameters and bead volumes, higher bead sphericity, and similar bead-tail ratio formation. See Figure 6.5C.

[0261] The polymer of Formula III described herein contains a combination of features that make it uniquely useful for encapsulating and suspending cells in three-dimensional hydrogel bioreactors for in-tank scale-up production for long-term cell culture / differentiation protocols (e.g., generation of functional neurons from stem cells). The hydrogels, including encapsulated differentiated cells (e.g., neurons), can be used for therapeutic purposes in humans. The system maintains cell (e.g., stem cell) viability and avoids harsh cell recovery methods from the system.

[0262] The polymer of Formula III may be functionalized at multiple positions, including pendant and backbone positions. For example, functionalization may be achieved by adding monoamine-PEGs terminating in individual functional groups such as: 1 A polymer of formula III was prepared having the formula: R 1 = methoxy; R 1 = hydroxyl; R 1 = acrylate; R 1 = biotin; R 1 =DBCO. See Figure 7.

[0263] Skeletal position R 3 A polymer of formula III was prepared having the following groups in R 3 = methacrylate; R 3 = maleimide; R 3= DBCO. Functionalization was achieved by reacting free NASI groups with amine-conjugated functional groups prior to isopropylamine saturation, see Figure 8.

[0264] To demonstrate the ability of the polymer of formula III to bind and release proteins in the context of stem cell differentiation, position R 1 The polymer of formula III bearing an acrylate at was reacted with thiol-proteins (FGF and heparin) via thiol-Michael addition. See Figure 9.

[0265] Surprisingly, the pendant PEG groups (positions R 1 We found that the position R 1 Groups other than primary amines at R allow for higher reaction v / v %, higher yield per volume of solvent, and more reproducible synthesis. See Figure 10A. Amine-terminated R 1 Making PEG requires the use of bifunctional diamino-PEG monomers in its synthesis, which has the potential for unintended covalent crosslinking. This can significantly impact the polymer's performance, reproducibility, and viscosity, and can prevent the polymer from reliquefying. To attempt to avoid this, syntheses using diamino-PEG require very dilute reactants (<2 w / v%) during synthesis, limiting the yield per unit volume of the polymer upon scale-up and exhibiting batch-to-batch variability. Syntheses using monoamine-PEG eliminate the potential for unintended crosslinking, allowing for a more reproducible synthesis and enabling scale-up of the synthesis.

[0266] Furthermore, we found that solvent selection for Reaction 1 is crucial for successful polymer synthesis and the incorporation of each monomer in the intended molar ratio, as well as for precise control of its molecular weight. Certain solvents with ideal solubility properties for all monomers exhibit high reaction efficiency with complete incorporation (A). Meanwhile, some solvents polymerize only those monomers in which they are soluble, but not all monomers (B). Some solvents inhibit the complete polymerization of the reaction (C-D). Some polymers are not suitable for any reaction (E-F). Solvents tested ranged from polar protic solvents, polar aprotic solvents, and nonpolar solvents in standard and anhydrous forms of various conformations. Solvents tested included acetone, acetonitrile, benzene, chloroform, dichloromethane, dimethylformamide, dimethyl sulfoxide, dioxane, ethyl acetate, pyridine, ethanol, methanol, tetrahydrofuran, toluene, and water. See Figure 10B.

[0267] Example 2 Several alternative neural induction protocols were evaluated for the generation of postmitotic cIN progenitor cells from human stem cells in 3D hydrogels, varying the media formulation and timing of addition of various components.

[0268] In particular, the culture protocols described in the following references were evaluated for a 35-day differentiation process: (1) Maroof et al., Cell Stem Cell, 12(5):559-572 (2013); (2) Nicholas et al., Cell Stem Cell, 12(5):573-586 (2013); and (3) Kim et al., Stem Cells, 32(7):1789-1804 (2014) (the entire contents of each of which are incorporated herein by reference). Alternative methods that do not include wnt inhibition were tested for each protocol. An alternative method that uses SB instead of AZD (an ERK inhibitor) was tested for Studer's protocol.

[0269] Dual SMAD inhibition and wnt inhibition for 9 days, SHH activation for 8 days, and no added final specification / maturation factors according to Maroof et al.:

number

[0270] Dual SMAD inhibition and wnt inhibition for 14 days, SHH activation for 35 days, and BDNF / DAPT for 11 days according to Nicholas et al.:

number

[0271] According to Kim et al., dual SMAD inhibition and wnt inhibition for 7 days, FGF8 added for 14 days, SHH activation for 21 days, and BDNF / GDNF / DAPT added for 14 days:

number

[0272] The results of using these neural induction protocols were evaluated by assessing the generation of MGE on day 18 (Figure 11A). As shown in Figures 11B-11E, by day 18, MGE progenitor cells, characterized by expression of FoxG1, NKX2-1, and DLK1 / 2, were efficiently generated exclusively in 3D hydrogels using the Kim et al. protocol.

[0273] The results of using these neural induction protocols were evaluated by assessing the generation of cortical interneurons on day 35 (Figure 12A). As shown in Figures 12B-12F, by day 35, MGE-derived cortical interneurons or cortical interneuron progenitor cells, characterized by FoxG1 expression, calbindin expression, Gad1 expression, and downregulation of Nkx2-1, were efficiently generated using Kim et al.'s protocol. Notably, although not as efficient as Kim et al.'s protocol, Maroof et al.'s protocol tested in 3D hydrogels was significantly superior to the original protocol performed in 2D cell culture (see Figures 12A-12E). Furthermore, all media substitution methods that did not include Wnt inhibition or by substituting SB with AZD were less efficient at generating MGE progenitor cells.

[0274] Example 3 Proof-of-concept studies were performed utilizing low molecular weight acrylate-backbone 3D hydrogels to demonstrate robust induction of encapsulated hPSCs into postmitotic cINs with therapeutic potential to treat neurological disorders (e.g., epilepsy).

[0275] (method)

[0276] Preparation of 3D PEG-PNIPAAM Hydrogels. A thermoreversible graft copolymer was fabricated using a two-step synthesis process (Figure 1). In this process, PEG represents the hydrophilic block, PNIPAAm represents the hydrophobic block, and the alkyl pendant groups (described here as butyl chains but can include any alkyl chain) act as temperature-shifting moieties. To fabricate this thermoreversible graft copolymer, a mixture of NIPAAm, N-acryloxysuccinimide (NASI), and an alkyl-chain methacrylate was first copolymerized by standard radical polymerization. After reprecipitation and drying, the resulting functionalizable copolymer was then mixed with a monoamine-terminated PEG block. The amine-terminated group was attached to the PNIPAAm-co-PNIPAAm-co-MA backbone via an amidation reaction between the amine and N-hydroxysuccinimide (NHS). Finally, the remaining NHS groups were converted to PNIPAAm by the addition of isopropylamine, and the resulting polymer was dried, dialyzed, and lyophilized.

[0277] (hPSC expansion)

[0278] Human PSC cells (H9 human embryonic stem cells (WA09, WiCell, Madison, WI, passages 53–58)) were maintained in E8 medium (Gibco, Billings, MT) on Matrigel (BD, San Jose, CA) according to the manufacturer's recommendations and passaged using Versene (Thermo Fisher, Waltham, MA).

[0279] (Cell encapsulation, differentiation and collection)

[0280] For differentiation, hPSCs were dissociated with Accutase (Stem Cell Technologies, Vancouver, BC, Canada) and encapsulated using PEG-PNIPAAM hydrogels at a final 10 wt / v% concentration using a concentration of 250,000 cells / ml of gel. The hydrogel and cells were mixed on ice, then 50 μl droplets were created and extruded into well plates as shown in Figure 13b for a total of five gel droplets per well. The plates were incubated at 37°C for 15 minutes to allow gelation to occur, forming dome-shaped beads. Complete E8 medium heated to 37°C and supplemented with Rock inhibitor Y-27632 (Selleck Chemicals, Houston, TX) was then added (final 5% v / v (medium / gel)), and the cells were incubated at 37°C with 5% CO2. Fifty percent of the medium was replaced daily to ensure the plates remained above 33°C. After 48 hours in expansion conditions, the culture medium was replaced with differentiation medium containing DMEM, knockout serum replacement (KSR, 20%), 2 mM L-glutamine, and 10 μM β-mercaptoethanol (all from Thermo Fisher Scientific). For neural induction, cells were treated with LDN193189 (100 nM, Stemgent, Cambridge, MA) from day 0 (D0) to day 14 (D14) and SB431542 (10 μM, Tocris, Minneapolis, MN) from day 0 (D0) to day 7 (D7). For MGE induction, cells were treated with IWP2 (5 μM, Selleck Chem) from day 0 (D0) to day 7 (D7), with SAG (0.1 μM, XcessBio, Chicago, IL) from day 0 (D0) to day 21 (D21), and with FGF8 (100 ng / ml, Peprotech) from day 8 (D8) to day 21 (D21).On day D22, the medium was replaced with DMEM F / 12 (Stem Cell Technologies) containing 10 ng / ml GDNF (R&D Systems Minneapolis, MN), 10 ng / ml BDNF (R&D), and 2.5 μM DAPT (Tocris) for further differentiation and maturation. At the indicated time points, aggregates were dissociated into single cells on a rotating platform in the presence of Accumax (Innovative Cell Technologies, San Diego, CA) and TrypLE (Thermo Fisher Scientific).

[0281] (Cell counting and viability analysis)

[0282] Cells were stained with AOPI and counted using K2 image capture equipment and Matrix software (PerkinElmer, Waltham, Mass.) to obtain total cell number and percentage viability.

[0283] (Flow cytometry)

[0284] Differentiated cells were dissociated and fixed in CytoFix / CytoPerm solution (BD) for 20 minutes and washed with Perm / Wash (BD). For staining, cells were incubated with primary antibodies for 30 minutes. After washing with Perm / Wash, Alexa 647-conjugated secondary antibodies (Thermo Fisher) were added and incubated for an additional 30 minutes. After washing with Perm / Wash, cells were resuspended in PBS and analyzed using an Attune NxT Flow Cytometer (Thermo Fisher). Raw data were analyzed using NovoExpress (Agilent, Santa Clara, CA) software. Ten thousand events were used per analysis.

[0285] (immunocytochemistry)

[0286] For immunofluorescence staining, fixed cells were incubated with Intercept Blocking Buffer (LI-COR Biosciences, Lincoln, NE) for 30 minutes and permeabilized with 0.25% Triton®-x for 10 minutes. Cells were then incubated with primary antibodies diluted in blocking buffer overnight at 4°C. After rinsing with PBS, samples were incubated with fluorochrome-conjugated secondary antibodies (Alexa 488- or Alexa 647-conjugated IgG; Thermo Fisher Scientific) and Hoechst 33342 (4 mg / ml) in blocking buffer at room temperature for 1 hour. After rinsing with PBS, imaging was performed using a Cytation 5 imaging system (Agilent), and images were analyzed using FIJI image analysis software (Schindelin, J. et al., Fiji: An open-source platform for biological image analysis, Nature Methods, Vol. 9, preprint (2012) at https: / / doi.org / 10.1038 / nmeth.2019).

[0287] (Quantitative PCR)

[0288] Total RNA was prepared using the RNeasy kit (Qiagen, Germantown, MD), and cDNA derived from total RNA was generated using the RT2 First Strand kit (Qiagen). For quantitative analysis of transcript expression, real-time PCR analysis was performed using the RT2 SYBR Green qPCR Mastermixes (Qiagen) and the AriaMX Real Time PCR System (Agilent). Primers were designed using the Integrated DNA Technologies PrimerQuest tool (Coralville, IA). The mRNA expression level for each gene was normalized to that of the ACTB gene. Their relative values ​​were calculated by setting the value of the normalized control as 1.

[0289] (result)

[0290] Encapsulation of human pluripotent stem cells in thermoreversible hydrogels for differentiation into cortical interneurons

[0291] The copolymer hydrogel utilized herein is based on hydrophilic poly(ethylene glycol) (PEG) and temperature-sensitive poly(N-isopropylacrylamide) (PNIPAAm). When heated above its lower critical solution temperature (LCST), the PNIPAAm component becomes increasingly hydrophobic, allowing micelle formation and essentially physical "crosslinking" the PEG-PNIPAAm polymer (Figure 13a). This hydrogel can be used to encapsulate and differentiate hPSCs in scaled-down (well plate) models and stirred cultures (including perfusion stirred-tank bioreactors) by simply extruding hydrogel droplets containing single cells or small cell clusters into warm culture medium, resulting in the formation of gel capsules containing these cells (Figures 13b and 13c). This encapsulation method enables a well-defined, synthetic, xeno-free, and scalable platform for the production of clinically relevant cell types.

[0292] The feasibility of differentiating human pluripotent stem cells (hPSCs) into GABAergic cortical interneuron progenitor cells encapsulated in the hydrogel was tested using a well-established culture medium (Kim, T.G. et al., Efficient specification of interneurons from human pluripotent stem cells by dorsoventral and rostrocaudal modulation, Stem Cells, 32, (2014)) (Figure 13d). Briefly, human embryonic stem cells (WA09) were seeded into the hydrogel beads and expanded for 48 hours in minimal growth medium (Gibco-E8) supplemented with ROCK inhibitor (Y-27632) to maximize cell viability, as previously described (Watanabe, K. et al., A ROCK inhibitor permits survival of dissociated human embryonic stem cells, Nat Biotechnol, 25, (2007)). To promote differentiation of hPSCs into the neuroectoderm lineage, we used the ALK2 / 3 inhibitor LDN193189 and the ALK5 / 7 inhibitor SB431542, as previously described (Kim, TG et al., Efficient specification of interneurons from human pluripotent stem cells by dorsoventral and rostrocaudal modulation, Stem Cells, 32, (2014)). Furthermore, we used IWP2, a chemical inhibitor of the Wnt pathway, to promote rostralization of early neuroectoderm and subsequently inhibit dorsalization of neuroectoderm (Kim, TG, 2014). We used activator of the sonic hedgehog (SHH) pathway (SAG) to define MGE identity over LGE. To counteract the caudalizing effect of SHH activation, FGF8 was added to shift the balance towards rostralization, efficiently generating MGE progenitors (Kim, TG, 2014).Finally, glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF) and the γ-secretase inhibitor DAPT were added to promote further differentiation of MGE progenitor cells into cINs and further neural maturation.

[0293] To monitor the patterning efficiency of the ventral telencephalon, MGE progenitor, and cIN progenitor cells, previously described markers characterizing these stages were used for quantitative PCR (qPCR), flow cytometry (FC), and immunocytochemistry (ICC) (Figure 13e) (Marin, O., Human cortical interneurons take their time, Cell Stem Cell, Vol. 12, preprint at https: / / doi.org / 10.1016 / j.stem.2013.04.017 (2013); Kim, T. G. et al., Efficient specification of interneurons from human pluripotent stem cell by dorsoventral and rostrocaudal modulation, Stem Cells, 32, (2014); Nicholas, C. R. et al., Functional maturation of hPSC-derived forebrain interneurons requires an extended timeline and mimics human neural development, Cell Stem Cell, 12, (2013); Cunningham, M. et al., hPSC-derived maturing GABAergic interneurons ameliorate seizures and abnormal behavior in epileptic mice, Cell Stem Cell, 15, (2014); and Maroof, AM et al., Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells, Cell Stem Cell, 12, (2013)).

[0294] Efficient differentiation of hPSCs into GABAergic cortical interneurons in a three-dimensional hydrogel platform

[0295] Flow cytometry analysis enabled us to determine cell identity and purity at various time points throughout differentiation. By day 10 of differentiation of hPSCs encapsulated in 3D hydrogels, these cells began to express FoxG1, an early marker of ventral telencephalic progenitors (Figures 14a and 14b). By day 18, an average of 88.44% of the cells expressed FoxG1, and 78.4% expressed the MGE marker NKX2-1, indicating strong patterning toward telencephalic MGE progenitors (Figures 14a and 14b). Upon further maturation, by day 35, cells showed high expression of FoxG1 (78.14%) and significant downregulation of NXK2-1 to 22.52%, consistent with a mature postmitotic cIN phenotype (Figures 14a and 14b). To confirm the MGE progenitor and cIN phenotypes, qPCR and ICC analyses were performed (Figure 51). qPCR analysis demonstrated elevated expression of ventral telencephalic and MGE progenitor markers, FoxG1 and DLX2, by day 18 compared to undifferentiated hPSCs, as well as upregulation of SST, suggesting early induction of genes that characterize MGE-derived mature cIN ​​populations (Figure 15a). By day 35 of differentiation, cells showed upregulation of cIN-like genes (e.g., GAD1 (glutamic acid decarboxylase, involved in GABA biosynthesis)), calbindin (CALB1), and further upregulation of SST (Figure 15a). Consistent with this qPCR analysis, ICC analysis at day 35 showed a high percentage of cells expressing FoxG1, the neural cell adhesion protein NCAM, high GABA synthesis (85.1% GABA+), and mature cIN ​​markers PV (84.5% PV+) and SST (42.3% SST+), consistent with the MGE origin of these cells. Furthermore, ki67 expression was less than 1%, indicating that the majority of these cells were indeed postmitotic (Figures 15b and 15c).

[0296] The thermoreversible hydrogel culture platform provides high hPSC viability and high differentiation efficiency into GABAergic cortical interneurons compared to standard 2D culture methods.

[0297] Next, we sought to compare the efficiency of our synthetic 3D hydrogel platform with that of cIN generation using standard 2D methods (Maroof, AM et al., Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells, Cell Stem Cell, 12, (2013)). We first compared post-harvest viability. After 35 days of differentiation, cells harvested from 3D hydrogels grown as cell aggregates exhibited high (>80%) viability, whereas cells harvested from 2D cultures exhibited low viability (approximately 20%) (Figures 16a and 16b). This indicated that our gentler harvesting and detachment method from the thermoreversible hydrogel provided substantial improvements in cell viability upon harvest compared to standard 2D culture. Furthermore, we evaluated the differentiation efficiency of cells encapsulated in the novel hydrogel of formula (III) compared to cells produced in 2D. We found that the percentage of cells expressing FoxG1 was approximately 800-fold higher in 3D compared to 2D by day 10 (Figure 16c). By day 18, the percentage of FoxG1+ cells was approximately 100-fold higher in 3D compared to 2D, and NK2X-1 expression was approximately 10-fold higher in 3D compared to 2D (Figure 15c). By day 35, we obtained approximately 100-fold higher FoxG1 expression in 3D compared to 2D (Figure 16c). Together, these findings demonstrate higher differentiation efficiency into cINs when hPSCs are encapsulated in 3D synthetic hydrogels and grown as aggregates compared to standard 2D methods.

[0298] (Consideration)

[0299] We demonstrated that hPSCs, when encapsulated in 3D hydrogels, can be robustly differentiated into MGE progenitors and cINs, with over 80% expression of the MGE progenitor marker FoxG1 and over 75% expression of Nkx2-1 by day 18, followed by over 75% FoxG1 expression by day 35, with a high percentage of cells expressing the cIN ​​markers PV, SST, GAD1, and GABA, along with a strong downregulation of Nkx2-1 expression to only approximately 22%. This, combined with the nearly absent ki67 expression, indicates that the 3D thermoreversible hydrogel platform enables the efficient generation of mature postmitotic cINs.

[0300] Furthermore, a side-by-side comparison between standard 2D cell generation methods and the synthetic 3D hydrogel process using the same media profile demonstrated the impressive advantages of this synthetic 3D hydrogel culture platform, with an approximately 800-fold higher FoxG1+ cell percentage by day 18 and an approximately 100-fold higher FoxG1+ cell percentage by day 35. Combined with this, the gentle collection process resulted in approximately four-fold higher viability compared to cell detachment from the 2D platform. Functional differentiation and maturation of hPSCs into cINs in vitro has been suggested to occur over an extended period, resembling their long maturation process during human brain development (Marin, O., Human cortical interneurons take their time, Cell Stem Cell, Vol. 12). Interestingly, marker expression analysis of cells collected at day 35 from the novel hydrogel of formula (III) suggests an accelerated maturation timeframe compared to that previously reported using conventional culture methods.Downregulation of Nkx2-1 was observed after 5 weeks of in vivo transplantation of 5-week-old cells (Kim, TG et al., Efficient specification of interneurons from human pluripotent stem cell by dorsoventral and rostrocaudal modulation, Stem Cells, 32, (2014)), after 30 weeks of differentiation (from 98% to 66% expression (Nicholas, CR et al., Functional maturation of hPSC-derived forebrain interneurons requires an extended timeline and mimics human neural development, Cell Stem Cell, 12, (2013)), or 4 months after transplantation of 3-week-old cells (showing downregulation of NKX2 to approximately 29%) (Zhu, Q. et al., Human cortical interneurons optimized for grafting specifically integrate, abort seizures, and display prolonged efficacy without Over-inhibition, Neuron (2023), doi:10.1016 / j.neuron.2022.12.014). In addition, SST expression was only detected after 20–30 weeks of differentiation (approximately 12%–40%) (Nicholas CR et al., supra) or 5 months after transplantation (Kim TG et al., supra).

[0301] Regarding PV expression, only 10% of cells were reported to express this protein at 15 weeks of differentiation (Nicholas CR et al., supra) or 4–5 months after in vivo transplantation (8–10%) (Kim TG et al., supra; Zhu, Q. et al., supra). In contrast, using the present method, 84.5% of PV+ cINs were detected by day 35. Culturing NKX2-1+ MGE progenitor cells in mouse cortical extracts has previously been reported to provide an accelerated timeframe for cIN maturation, allowing detection of PV expression by day 30 of in vitro differentiation. This suggests that specific cell-cell interactions and / or signaling molecules are key to accelerating this maturation process, although the mechanisms involved have not been identified (Maroof, AM et al., Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells, Cell Stem Cell, 12, (2013)). Without being bound by theory, the methods described herein may promote cell-cell interactions and boost autocrine and paracrine signaling by capturing within the hydrogel environment key signaling molecules secreted by these cells that are otherwise washed away in conventional 2D culture or 3D conventional suspension platforms. Collectively, the data presented herein demonstrate robust differentiation of cINs from hPSCs in 3D synthetic hydrogels (e.g., the novel hydrogels of Formula (III)), with substantially higher viability and differentiation efficiency compared to standard 2D culture, and accelerated differentiation periods compared to maturation timeframes reported using standard 2D culture or hybrid 2D-suspension systems.

[0302] Example 4 A proof-of-concept study was performed utilizing low molecular weight (Formula III) acrylate-backbone 3D hydrogels to demonstrate robust expansion of encapsulated hPSCs.

[0303] (method)

[0304] Preparation of 3D PEG-PNIPAAM Hydrogels. A thermoreversible graft copolymer was fabricated using a two-step synthesis process (Figure 1). In this process, PEG represents the hydrophilic block, PNIPAAm represents the hydrophobic block, and the alkyl pendant groups (described here as butyl chains but can include any alkyl chain) act as temperature-shifting moieties. To fabricate this thermoreversible graft copolymer, a mixture of NIPAAm, N-acryloxysuccinimide (NASI), and an alkyl-chain methacrylate was first copolymerized by standard radical polymerization. After reprecipitation and drying, the resulting functionalizable copolymer was then mixed with a monoamine-terminated PEG block. The amine-terminated group was attached to the PNIPAAm-co-PNIPAAm-co-MA backbone via an amidation reaction between the amine and N-hydroxysuccinimide (NHS). Finally, the remaining NHS groups were converted to NIPAAm by the addition of isopropylamine, and the resulting polymer was dried, dialyzed, and lyophilized.

[0305] (hPSC expansion)

[0306] Human PSC cells (H9 human embryonic stem cells (WA09, WiCell, Madison, WI, passages 53–58)) were maintained in E8 medium (Gibco, Billings, MT) on Matrigel (BD, San Jose, CA) according to the manufacturer's recommendations and passaged using Versene (Thermo Fisher, Waltham, MA) or ReleSR (Stem Cell Technologies, Vancouver, BC, Canada).

[0307] (Cell encapsulation, differentiation and collection)

[0308] For expansion, hPSCs were dissociated with Accutase (Stem Cell Technologies, Vancouver, BC, Canada) and encapsulated in PEG-NIPAAM hydrogels at a final concentration of 2.5% to 10% wt / v, using a concentration of 250,000 cells / ml of gel. The hydrogel and cells were mixed, encapsulated, and extruded into vessels of various sizes (100 mL to 1 L). Complete E8 medium, heated to 37°C and supplemented with Rock of Ice inhibitor Y-27632 (Selleck Chemicals, Houston, TX), was then added, and the cells were incubated at 37°C with 5% CO2. At the end of the expansion process (day 7), aggregates were dissociated into single cells on a rotating platform in the presence of Accumax (Innovative Cell Technologies, San Diego, CA) and TrypLE (Thermo Fisher Scientific).

[0309] (Cell counting and viability analysis)

[0310] Cells were stained with AOPI and counted using a K2 image capture device and Matrix software (PerkinElmer, Waltham, MA) to obtain total cell number and percentage viability.

[0311] (Flow cytometry)

[0312] Cells were detached, fixed in CytoFix / CytoPerm solution (BD) for 20 minutes, and washed with Perm / Wash (BD). For staining, cells were incubated with primary antibodies for 30 minutes. After washing with Perm / Wash, cells were resuspended in PBS and analyzed using an Attune NxT Flow Cytometer (Thermo Fisher). Raw data were analyzed using NovoExpress (Agilent, Santa Clara, CA) software. Ten thousand events were used per analysis.

[0313] (Quantitative PCR)

[0314] Total RNA was prepared using the RNeasy kit (Qiagen, Germantown, MD), and cDNA derived from total RNA was generated using the RT2 First Strand kit (Qiagen). For quantitative analysis of transcript expression, real-time PCR analysis was performed using the RT2 SYBR Green qPCR Mastermixes (Qiagen) and the AriaMX Real Time PCR System (Agilent). Primers were designed using the Integrated DNA Technologies PrimerQuest tool (Coralville, IA). The mRNA expression level for each gene was normalized to that of the ACTB gene. Their relative values ​​were calculated by setting the value of the normalized control as 1.

[0315] (Consideration)

[0316] This gel formulation enabled the creation of beads composed of core and shell shapes. These beads allowed for maximum cell retention during scale-up (Figure 5D). Three different culture scales were used to rapidly iterate various conditions in a scale-down model (positive displacement pipette (PDP), static, and spinner, Figure 17A) that closely predicts performance in a bioreactor. Using this improved formulation, we demonstrated high viability during encapsulation. High viability throughout the encapsulation process enabled scalable hPSC expansion, with encapsulation flow rates as low as 2 ml gel / min. An exemplary expansion process using this formulation is shown in Figure 18B, demonstrating reproducible, high-quality hPSC expansion across four different bioreactor scales (Figure 18A), ranging from 100 mL to 1 L of vessel volume, as confirmed by flow cytometry (Figure 18B) and qPCR (Figure 18C). Furthermore, as shown in 17B, 17C, we demonstrated robust expansion in this hydrogel using both human embryonic stem cell (hESC) and human induced pluripotent stem cell (hiPSC) lines. Example 5 A proof-of-concept study was performed utilizing low molecular weight acrylate-backbone 3D hydrogels to demonstrate robust induction of encapsulated hPSCs into pancreatic endodermal progenitor (PE) cells.

[0317] (method)

[0318] Preparation of 3-Dimensional PEG-PNIPAAM Hydrogels A two-step synthetic process was used to fabricate thermoreversible graft copolymers as described for Example 4 (FIG. 1).

[0319] (hPSC expansion)

[0320] Human PSC cells (H9 human embryonic stem cells (WA09, WiCell, Madison, WI, passages 53–58)) were maintained in E8 medium (Gibco, Billings, MT) on Matrigel (BD, San Jose, CA) according to the manufacturer's recommendations and passaged using Versene (Thermo Fisher, Waltham, MA) or ReleSR (Stem Cell Technologies, Vancouver, BC, Canada).

[0321] (Cell encapsulation, differentiation and collection)

[0322] For differentiation, hPSCs were dissociated with Accutase (Stem Cell Technologies, Vancouver, BC, Canada) and encapsulated using PEG-PNIPAAM hydrogels at a final concentration of 2.5 wt / v% to 10 wt / v% using a concentration of 250,000 cells / ml of gel. The hydrogel and cells were mixed, encapsulated, and extruded into a 100 mL spinner. The spinner was incubated at 37°C for 15 minutes to allow gelation. Complete E8 medium, heated to 37°C and supplemented with Rock inhibitor Y-27632 (Selleck Chemicals, Houston, TX), was then added, and the cells were incubated at 37°C with 5% CO2. Fifty percent of the medium was replaced daily to ensure the plates remained above 33°C. After 48 hours in expansion conditions, the culture medium was replaced with differentiation medium according to the manufacturer's recommendations (STEMdiff, Definitive Endoderm, Stem Cell Technologies, Vancouver, BC, Canada) (FIG. 20A). At the indicated time points (FIG. 19), aggregates were dissociated into single cell populations on a rotating platform in the presence of Accumax (Innovative Cell Technologies, San Diego, CA) and TrypLE (Thermo Fisher).

[0323] (Cell counting and viability analysis)

[0324] Cells were stained with AOPI and counted using a K2 image capture device and Matrix software (PerkinElmer, Waltham, MA) to obtain total cell number and percentage viability.

[0325] (Flow cytometry)

[0326] Cells were detached, fixed in CytoFix / CytoPerm solution (BD) for 20 minutes, and washed with Perm / Wash (BD). For staining, cells were incubated with primary antibodies for 30 minutes. After washing with Perm / Wash, cells were resuspended in PBS and analyzed using an Attune NxT Flow Cytometer (Thermo Fisher). Raw data were analyzed using NovoExpress (Agilent, Santa Clara, CA) software. Ten thousand events were used per analysis.

[0327] (Consideration)

[0328] Increased gel stability allowed for a longer differentiation process and complete vessel exchange volume without compromising gel integrity, both of which were key to ensuring high differentiation efficiency during scale-up. As shown in Figure 19, PE cells were generated in this improved 3D hydrogel system using a pre-made media formulation and compared to the standard process of suspending cells in the hydrogel. As a result, we obtained 45-fold higher PE production compared to 3D suspension (standard culture), greater control over aggregate size, high post-seeding viability (>90%), and expression of the PE progenitor marker PDX-1 (Figures 20B-20D). Notably, the greater control over aggregate size revealed by this 3D culture method allows for the generation of aggregates smaller than 500 μm. This indicates that hydrogels containing the thermoreversible polymer of Formula III have broad implications for clinical manufacturing. This is because aggregates above this size may exhibit a necrotic core due to nutrient limitation and / or suboptimal differentiation efficiency due to heterogeneous responses to differentiation cues throughout the aggregate.

[0329] Example 6 A proof-of-concept study was performed utilizing low molecular weight acrylate-backbone 3D hydrogels to demonstrate robust induction of encapsulated hPSCs into midbrain dopaminergic cells (mDAs).

[0330] (method)

[0331] Preparation of 3-Dimensional PEG-PNIPAAM Hydrogels A two-step synthetic process was used to fabricate thermoreversible graft copolymers as described for Example 4 (FIG. 1).

[0332] (hPSC expansion)

[0333] Human PSC cells (H9 human embryonic stem cells (WA09, WiCell, Madison, WI, passages 53–58)) were maintained in E8 medium (Gibco, Billings, MT) on Matrigel (BD, San Jose, CA) according to the manufacturer's recommendations and passaged using Versene (Thermo Fisher, Waltham, MA) or ReleSR (Stem Cell Technologies, Vancouver, BC, Canada).

[0334] (Cell encapsulation, differentiation and collection)

[0335] For differentiation, hPSCs were dissociated with Accutase (Stem Cell Technologies, Vancouver, BC, Canada) and encapsulated using PEG-PNIPAAM hydrogels at a final concentration of 2.5 wt / v% to 10 wt / v% using a concentration of 500,000 cells / ml of gel. The hydrogel and cells were mixed and plated into plates using a positive displacement pipette (PDP) or encapsulated and extruded into a 100 mL spinner. The plates were incubated at 37°C for 15 minutes to allow gelation and form dome-shaped beads. Complete E8 medium, heated to 37°C and supplemented with Rock inhibitor Y-27632 (Selleck Chemicals, Houston, TX), was then added, and the cells were incubated at 37°C with 5% CO2. Fifty percent of the medium was replaced daily to ensure the plates remained above 33°C. After 48 hours in expansion conditions, the culture medium was replaced with differentiation medium (Figure 21A) as previously described (Adil et al., Sci Rep 7, 40573 (2017)). On day 16 of differentiation (Figure 21A), aggregates were dissociated into single cell populations on a rotating platform in the presence of Accumax (Innovative Cell Technologies, San Diego, CA) and TrypLE (Thermo Fisher).

[0336] (Cell counting and viability analysis)

[0337] Cells were stained with AOPI and counted using a K2 image capture device and Matrix software (PerkinElmer, Waltham, MA) to obtain total cell number and percentage viability.

[0338] (Flow cytometry)

[0339] Cells were detached, fixed in CytoFix / CytoPerm solution (BD) for 20 minutes, and washed with Perm / Wash (BD). For staining, cells were incubated with primary antibodies for 30 minutes. After washing with Perm / Wash, cells were resuspended in PBS and analyzed using an Attune NxT Flow Cytometer (Thermo Fisher). Raw data were analyzed using NovoExpress (Agilent, Santa Clara, CA) software. Ten thousand events were used per analysis.

[0340] (Consideration)

[0341] Improved gel stability allowed for a longer differentiation process and complete vessel volume exchange without compromising gel integrity, both of which were key to ensuring high differentiation efficiency during scale-up. As shown in Figures 21B and 21C, mDAs were generated in this improved 3D hydrogel system. At various scales (PDP and 100 mL spinner), high differentiation efficiency was achieved, as measured by FoxA2 expression (70%–90%), a marker for ventral midbrain progenitor cells, with high viability (>80%) upon harvest (Figures 21B and 21C). This data demonstrates that this improved hydrogel formulation is compatible with scalable culture systems, possesses high gel stability over the long differentiation process, and protects cells from shear stress generated by bioreactor mixing.

[0342] Example 7 Proof-of-concept studies were performed utilizing low molecular weight acrylate-backbone 3D hydrogels to demonstrate robust induction of encapsulated hPSCs into hematopoietic stem cells (HSCs), as well as expansion of primary HSC cells.

[0343] (method)

[0344] Preparation of 3-Dimensional PEG-PNIPAAM Hydrogels A two-step synthetic process was used to fabricate thermoreversible graft copolymers as described for Example 4 (FIG. 1).

[0345] (hPSC expansion)

[0346] Human PSC cells (H9 human embryonic stem cells (WA09, WiCell, Madison, WI, passages 53–58)) were maintained in E8 medium (Gibco, Billings, MT) on Matrigel (BD, San Jose, CA) according to the manufacturer's recommendations and passaged using Versene (Thermo Fisher, Waltham, MA) or ReleSR (Stem Cell Technologies, Vancouver, BC, Canada).

[0347] (Cell encapsulation, differentiation and collection)

[0348] For HSC differentiation, hPSCs were dissociated with Accutase (Stem Cell Technologies, Vancouver, BC, Canada) and encapsulated in PEG-PNIPAAM hydrogels at a final concentration of 2.5 wt / v% to 10 wt / v% using a concentration of 500,000 cells / ml of gel. The hydrogel and cells were mixed and plated into plates using a positive displacement pipette (PDP). The plates were incubated at 37°C for 15 minutes to allow gelation and form dome-shaped beads. Complete E8 medium, heated to 37°C and supplemented with Rock inhibitor Y-27632 (Selleck Chemicals, Houston, TX), was then added, and the cells were incubated at 37°C with 5% CO2. Fifty percent of the medium was replaced daily to ensure the plates remained above 33°C. After 48 hours in expansion conditions, the culture medium was replaced with differentiation medium according to the manufacturer's recommendations for the StemDiff Hematopoietic Kit (Stem Cell Technologies, Vancouver, BC, Canada) (Figure 22A). On day 12 of differentiation (Figure 22A), cells were harvested by cooling the hydrogel to 4°C. For HSC expansion, primary human umbilical cord blood CD34+ HSCs (Stem Cell Technologies, Vancouver, BC, Canada) were thawed, encapsulated in the hydrogel as described above, and seeded using the PDP method. Cells were expanded for 8 days in SFEM II medium supplemented with StemSpan CD34+ Expansion Supplement (Figure 23A). Cells were then harvested by cooling the hydrogel to 4°C.

[0349] (Cell counting and viability analysis)

[0350] Cells were stained with AOPI and counted using a K2 image capture device and Matrix software (PerkinElmer, Waltham, MA) to obtain total cell number and percentage viability.

[0351] (Consideration)

[0352] The improved gel stability allowed for a longer differentiation process and complete vessel exchange volume without compromising gel integrity, both of which were key to ensuring high differentiation efficiency during scale-up. As shown in Figures 22B and 22C, the improved 3D hydrogel system described above was used to differentiate hPSCs into HSCs. Furthermore, human umbilical cord blood CD34+ HSCs were also successfully expanded in this 3D hydrogel system (Figures 22B and 22C). This data demonstrates that the improved hydrogel formulation is compatible with HSC expansion and differentiation from hPSCs, has high gel stability throughout the long differentiation process, and protects cells from shear stress caused by bioreactor mixing during scale-up. HSCs are promising cell therapy candidates with a wide range of therapeutic applications, from sickle cell disease and β-thalassemia to blood cancers.

[0353] Example 8 Comparison of hydrogels containing thermoreversible polymers of Formula III with hydrogels containing thermoreversible polymers disclosed in US Pat. No. 10,982,055.

[0354] The synthesis of the thermoreversible polymer disclosed in US 10,982,055 is shown in Figure 3 of US 10,982,055. The synthesis of the thermoreversible polymer of Formula III of the present disclosure is shown in Figure 1. The following table summarizes the key differences in the synthesis of each polymer: [Table 4]

[0355] While the materials and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that modifications may be made to the methods described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention.

Claims

1. 1. A thermoreversible polymer with improved stability over time, comprising: Formula (III): 【Chemistry 13】 Including, wherein (a), (b), (c), and (d) represent the mole fractions of comonomers in the polymer, and (a), (b), and (c) are each greater than 0; PEG ｎ is a polyethylene glycol polymer, and n is an integer; R １ is any terminal group or functional group, if present, other than a primary amine; R ２ is a lower alkyl group; R ３ is an end group or functional group or linked modifier, if present; R ４ is hydrogen or a lower alkyl group; G １ and G ２ are each independently selected from a polymer segment, an end group, a linker, and a linked modifying substance; The molecular weight of the polymer is greater than 50 kDa. Thermoreversible polymer.

2. 10. The thermoreversible polymer of claim 1, wherein the molecular weight (MW) of the polymer is from about 50 kDa to about 250 kDa.

3. The PEG ｎ 3. The thermoreversible polymer of claim 1 or 2, wherein is a polyethyl glycol polymer having a MW of about 1 kDa to about 50 kDa or about 2 kDa to about 20 kDa.

4. 4. The thermoreversible polymer of any one of claims 1 to 3, having a weight:weight (w / w) ratio of PEG:PNIPAAm copolymer of greater than about 1:2, preferably having a weight:weight (w / w) ratio of PEG:PNIPAAm copolymer of about 1:2.5 to about 1:4.

5.

5. R １ The thermoreversible polymer according to any one of claims 1 to 4, wherein is absent or is any terminal group or functional group other than alkyl or substituted alkyl.

6. R １ is selected from methoxy, ethoxy, n-propoxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy and isopentoxy １ ~C ６ The thermoreversible polymer according to any one of claims 1 to 5, which is alkoxy or hydroxy.

7. R １ is methoxy, or R １ The thermoreversible polymer of claim 6 , wherein is hydroxyl, biotin, or DBCO.

8. R ２ The thermoreversible polymer according to any one of claims 1 to 7, wherein is other than isobutyl.

9. R ２ The thermoreversible polymer of any one of claims 1 to 7, wherein is selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, isopropyl, isobutyl, isopentyl, tert-butyl, cyclopropyl, and cyclobutyl.

10. R ２ is n-butyl, isobutyl, or tera-butyl, and preferably R ２ The thermoreversible polymer of claim 9, wherein is n-butyl.

11. R ３ The thermoreversible polymer according to any one of claims 1 to 10, wherein

12. R １ is methoxy, and R ２ is n-butyl, and preferably R ４ The thermoreversible polymer according to any one of claims 1 to 4, wherein is methyl.

13. 13. The thermoreversible polymer of claim 12, wherein (d) is absent.

14. R ３ The thermoreversible polymer according to any one of claims 1 to 13, wherein

15. R １ and / or R ３ is a chemoselective functional group selected from acrylate, methacrylate, biotin, streptavidin, thiol, alkyne, cyclooctyne, azide, phosphine, maleimide, alkoxyamine, aldehyde, and protected versions or precursors thereof.

16. R １ and / or R ３ is a modifying substance selected from heparin, hyaluronic acid, a specific binding member, a peptide, a nucleic acid, gelatin, fibronectin, collagen, laminin, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin, progesterone, glucose, stromal cell-derived factor 1 (SDF-1), thymosin beta-4, sonic hedgehog (SHH), noggin, activin, transforming growth factor beta (TGF-β), FGF8, brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), neurotrophic factor 3 (NT3), platelet-derived growth factor (PDGF), IL-16, IL-2, and insulin-like growth factor 1 (IGF-1).

17. A thermoreversible polymer according to any one of claims 1 to 16, having one or more, preferably all, of the following properties: (a) an LCST of about 12°C to about 32°C, preferably about 19°C to about 23°C; (b) a stiffness of about 100 Pa to about 8000 Pa; and (c) a viscosity of about 100 cP to about 2000 cP.

18. A three-dimensional hydrogel comprising the thermoreversible polymer of any one of claims 1 to 17 and an aqueous buffer solution.

19. 19. The three-dimensional hydrogel of claim 18, which is stable in a buffered environment at 37°C for a period of at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, or at least 8 weeks, or at least 3 months.

20. 18. A composition comprising a plurality of hydrogel capsules, wherein at least 90%, preferably at least 95%, of the hydrogel capsules in the composition comprise at least one type of cell and a hydrogel encapsulating the cells, the hydrogel encapsulating the cells being a hydrogel comprising the thermoreversible polymer of any one of claims 1 to 17 and an aqueous buffer solution.

21. 21. The composition of claim 20, wherein at least 90% of the hydrogel capsules in the composition each contain at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, or at least 9000 cells, preferably stem cells.

22. 1. A method for expanding or generating a population of differentiated cells from stem or progenitor cells, comprising: Culturing the stem or progenitor cells in the three-dimensional hydrogel of claim 18 or 19 under conditions suitable for inducing differentiation of the stem or progenitor cells. A method comprising:

23. 23. The method of claim 22, wherein the conditions suitable for expansion or differentiation of the stem or progenitor cells comprise culturing the stem or progenitor cells in the hydrogel composition for a period of at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, or at least 8 weeks, or at least 3 months.

24. 1. An in vitro method for generating a cell population enriched for MGE progenitor cells from an initial population of human stem cells, comprising: (a) encapsulating an initial population of human stem cells in a three-dimensional hydrogel according to claim 18 or 19; and (b) contacting the encapsulated human stem cells with at least one inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling and at least one Wingless (Wnt) antagonist; and contacting the cells with at least one activator of Sonic Hedgehog (SHH) signaling and an FGFR agonist, thereby obtaining a cell population enriched for MGE progenitor cells that express FOXG1 and at least one additional marker indicative of MGE progenitor cells. A method comprising:

25. 25. The method of claim 24, wherein the human stem cells are selected from the group consisting of human embryonic stem cells, human adult stem cells, human neural stem cells, human induced pluripotent cells, human primary progenitor cells, and human induced progenitor cells.

26. 25. The method of claim 24, wherein said contacting with at least one inhibitor of SMAD signaling and said contacting with at least one Wnt antagonist are performed simultaneously or sequentially, each having a duration of between about 5 days and about 30 days.

27. 27. The method of claim 26, wherein contacting the cells with the at least one Wnt antagonist is initiated within 5 days, preferably within 4 days, within 3 days, within 2 days, or within 1 day of initial contacting the cells with the at least one inhibitor of SMAD signaling, and preferably contacting the cells with the at least one Wnt antagonist is initiated simultaneously with initial contacting the cells with the at least one inhibitor of SMAD signaling.

28. the at least one inhibitor of SMAD signaling is selected from the group consisting of SB431542, LDN-193189, Noggin PD169316, SB203580, LY364947, A77-01, A-83-01, BMP4, GW788388, GW6604, SB-505124, lerdelimumab, meterimumab, GC-I008, AP-12009, AP-110I4, LY550410, LY580276, LY364947, LY2109761, SB-505124, E-616452 (RepSox ALK inhibitor), SD-208, SMI6, NPC-30345, 【Chemistry 14】 , SB-203580, SD-093, activin-M108A, P144, soluble TBR2-Fc, DMH-1, dorsomorphin dihydrochloride, derivatives thereof, and combinations thereof.

29. 29. The method of claim 28, wherein said at least one inhibitor of SMAD signaling comprises SB431542 and LDN-193189.

30. 25. The method of claim 24, wherein the at least one Wnt antagonist is selected from the group consisting of XAV939, DKK1, DKK-2, DKK-3, Dkk-4, SFRP-1, SFRP-2, SFRP-5, SFRP-3, SFRP-4, WIF-1, Soggy, IWP-2, IWR1, ICG-001, KY0211, Wnt-059, LGK974, IWP-L6, derivatives thereof, and combinations thereof, preferably wherein the at least one Wnt antagonist comprises IWP-2.

31. 25. The method of claim 24, wherein the at least one activator of SHH signaling is selected from the group consisting of smoothened agonist (SAG), SAG analogues, SHH, C25-SHH, C24-SHH, purmorphamine, Hg-Ag, derivatives thereof, and combinations thereof.

32. (i) contacting the cells with the at least one activator of SHH signaling is terminated between about 5 days and about 30 days from the start of the contacting, preferably between about 18 days and 23 days from the start of the contacting, more preferably between about 19 days and 22 days from the start of the contacting, and even more preferably between about 20 days or 21 days from the start of the contacting; (ii) the initial contact of the cell with the at least one activator of SHH signaling is between about 0 and about 10 days, preferably about 0 days, from the initial contact of the cell with the at least one inhibitor of SMAD signaling and from the initial contact of the cell with the at least one inhibitor of WNT signaling; (iii) initial contact of the cell with the at least one inhibitor of SMAD signaling is between 0 and 4 days after initial contact of the cell with the at least one Wnt antagonist; (iv) contacting the cells with the at least one inhibitor of SMAD signaling is terminated between 6 and 14 days from the start thereof, and / or (v) contacting the cells with the at least one Wnt antagonist is terminated between 6 and 8 days from the initiation thereof, preferably about 7 days from the initiation thereof; 32. The method of claim 31 .

33. 25. The method of claim 24, wherein the at least one additional marker is selected from the group consisting of NKX2-1, NKX2-2, ASCL1, SIX6, OLIG2, NKX6.2, DLX1 / 2 and LXH6.

34. 25. The method of claim 24, wherein at least about 60%, at least 65%, at least 70%, at least 75%, or at least 80% of the resulting cell population expresses FOXG1 and NKX2-1.

35. 25. The method of claim 24, wherein at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% of the resulting cell population comprises MGE progenitor cells.

36. (c) after a predetermined amount of time, contacting the cells with at least one neurotrophic factor (e.g., GDNF, BDNF) and optionally a Notch inhibitor (e.g., DAPT) to produce a cell population enriched for differentiated inhibitory GABAergic cortical interneurons (cINs) that express FOXG1 and at least one additional marker indicative of cortical interneuron cells.

25. The method of claim 24, further comprising:

37. 37. The method of claim 36, comprising contacting the cells with at least one neurotrophic factor and a Notch inhibitor.

38. 38. The method of claim 37, comprising contacting the cells with GDNF, BDNF and DAPT.

39. 39. The method of any one of claims 36 to 38, wherein the step of contacting the cells with at least one neurotrophic factor and optionally a Notch inhibitor is performed after the steps of contacting the cells with at least one inhibitor of SMAD signaling and at least one Wnt antagonist and at least one activator of SHH signaling have been completed.

40. 40. The method of any one of claims 36-39, wherein the step of contacting the cells with at least one neurotrophic factor and optionally a Notch inhibitor is terminated between 7 and 30 days after initiation, and / or the step of contacting the cells with at least one neurotrophic factor and optionally a Notch inhibitor is terminated at least about 10 days, at least about 12 days, or at least about 14 days after initiation.

41. 37. The method of claim 36, wherein the at least one additional marker is selected from the group consisting of PV, SST, calbindin, DCX, ASCL1, TUJ1, GABA, GAD1, VGAT, vGLUT1, and GAD67.

42. 42. The method of any one of claims 36 to 41, wherein at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% of the resulting cIN cell population expresses parvalbumin (PV).

43. 43. The method of any one of claims 36 to 42, wherein less than about 5% of the obtained cIN cell population expresses Ki67.

44. 37. A composition comprising a population of cells produced by the method of any one of claims 24 to 36, wherein at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95% of the cells are MGE progenitor cells, preferably the method does not include a step for purifying or further enriching the MGE progenitor cells after step (b).

45. 44. A composition comprising a population of cells produced by the method of any one of claims 27 to 43, wherein at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95% of the cells are cIN, preferably the method does not include a step for purifying or further enriching the cIN ​​cells after step (c).

46. At least 50% of the cells, or at least 60% of the cells, or at least 70% of the cells, or at least 80% of the cells, or at least 90% of the cells, or at least 95% of the cells are NKX2.1 － / PV ＋ 46. ​​The composition of claim 45, wherein:

47. Use of a composition according to any one of claims 44 to 46 in the treatment of neurological disorders.

48. 48. The use of claim 47, wherein the neurological disorder is a seizure disorder.

49. A method for preparing a thermoreversible polymer of formula III, comprising: (i) polymerizing a comonomer population comprising N-isopropylacrylamide (NIPAAm), an alkyl methacrylate, and N-acryloxysuccinimide (NASI) in a single solvent in the presence of an initiator concentration effective to form a PNIPAAM-co-PBMA-co-PNASI copolymer backbone having a molecular weight (MW) of at least 50 kDa; (ii) reacting the copolymer with monoPEG amine to form a [PNIPAAM-co-PBMA-co-PNASI]-b-[PEG] copolymer; and (iii) reacting the [PNIPAAM-co-PBMA-co-PNASI]-b-[PEG] with isopropylamine to form a thermoreversible polymer of formula III A method comprising:

50. 50. The method of claim 49, wherein the single solvent is selected from acetone, acetonitrile, benzene, chloroform, dichloromethane, dimethylformamide, dimethylsulfoxide, dioxane, ethyl acetate, pyridine, ethanol, methanol, tetrahydrofuran, toluene, and water.

51. 51. A thermoreversible polymer produced by the method of claim 49 or 50.

52. 52. A three-dimensional hydrogel comprising the thermoreversible polymer of claim 51.

53. The thermoreversible polymer of claims 1 to 17, further comprising expansion of human hematopoietic stem cells for at least one week.

54. The thermoreversible polymer according to claims 1 to 17, further comprising pluripotent stem cells, and further comprising a step of differentiating them into midbrain dopaminergic cells, pancreatic endoderm cells and hematopoietic stem cells.

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