Thermoreversible polymers with improved stability and methods and uses thereof

Abstract

This article provides a thermally reversible polymer with improved stability, a hydrogel composition comprising the thermally reversible polymer, and an in vitro method for generating a cell population rich in inhibitory GABAergic cortical interneurons from human pluripotent stem cells using the hydrogel composition.

Description

Cross-references to related applications

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 595,847, filed November 3, 2023, and U.S. Provisional Patent Application Serial No. 63 / 595,841, filed November 3, 2023, the entire contents of each of which are incorporated herein by reference. Background Technology

[0002] Human pluripotent stem cells (hPSCs) possess the potential to differentiate into any cell type in vivo, and therefore have broad applications in regenerative medicine. However, large-scale expansion and differentiation of hPSCs remains a major challenge in this field, hindering these transformative therapies from reaching a wide range of patients in need. Here we describe the production of a synthetic, fully defined, thermally reversible hydrogel capable of facilitating the large-scale growth and differentiation of hPSCs into sensitive cell types. An improved hydrogel formulation yields a low-viscosity gel, enabling the encapsulation of sensitive cell types using a variety of liquid handling systems, thanks to these unique properties: the polymer formulation and molecular weight (MW) make the low-viscosity hydrogel ideal for hPSC culture and differentiation into all three germ layers. This minimizes shear forces during cell encapsulation in large-scale bioreactor systems. This formulation and MW also allow for faster flow rates during encapsulation and better control over bead size / geometry. The optimal MW and PEG length of the polymer yield a stable hydrogel that maintains its structure during the lengthy differentiation process and has a range of stiffness to support hPSC expansion and differentiation into all three germ layer cells. This formulation is also suitable for functionalization, enabling controlled protein presentation and release. Using this thermally reversible hydrogel, we can massively expand hPSCs and differentiate them into several cell types, including midbrain dopaminergic neurons, cortical interneurons, pancreatic endoderm progenitor cells, and hematopoietic stem cells. Summary of the Invention

[0003] This article provides an in vitro scalable method for generating postmitotic inhibitory GABAergic cortical interneurons (cINs) from human stem cells (e.g., human pluripotent stem cells or multipotent stem cells), the method comprising encapsulating the stem cells in a three-dimensional synthetic hydrogel under conditions that generate a cell population containing cINs. In some aspects, the pluripotent cells are human pluripotent stem cells (hPSCs), such as human embryonic stem cells or human induced pluripotent stem cells (iPSCs). Preferably, the method is free of xenogeneic components, and the hydrogel does not contain extracellular matrix (ECM) proteins.

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

[0005] This paper also provides novel thermally reversible hydrogels that can be used in the methods described herein.

[0006] This document also provides a composition comprising a cell population generated by the method described herein, wherein the cell population is rich in postmitotic inhibitory GABAergic cortical interneurons. Preferably, the composition is free of ECM and xenogeneic components.

[0007] This document also provides the use of compositions comprising cIN produced by the methods described herein in the treatment of neurological disorders. In some preferred embodiments, the neurological disorder is epilepsy. Attached Figure Description

[0008] Figure 1 This is a schematic diagram illustrating the two-step synthesis of the thermally reversible poly(NIPAAm-co-Bam)-b-PEG graft copolymer of formula (III). The molar ratios between intermediate reactions are indicated by lowercase letters. AIBN refers to azobisisobutyronitrile. Butylamine is illustrated; however, other lower alkylamines can also be substituted in the reaction.

[0009] Figure 2 This paper presents a comparison of the properties of acrylate-based thermally reversible polymers and acrylamide-based thermally reversible polymers. LCST = Lower Critical Dissolution Temperature.

[0010] Figure 3 This study presents a comparison of the properties of low-MW acrylate-based thermally reversible polymers with high-MW acrylate-based thermally reversible polymers. LCST = Lower Critical Dissolution Temperature.

[0011] Figure 4 This study compares the effects of PEG molecular weight on the properties of acrylate-based thermally reversible polymers.

[0012] Figures 5A to 5D This study demonstrates a comparison of the effects of PEG:PNIPAAM polymer weight / weight% on the properties of acrylate-based thermally reversible polymers. Figure 5C and Figure 5D The hPSC activity is shown in the scalable encapsulation process using different hydrogel formulations. Figure 5C Changing the PEG:PNIPAAm ratio increased the flow rate to meet the minimum requirement of 2 mL / min for scale-up. Furthermore, the lower shear force of the 1:3 formulation maximized cell viability after encapsulation (second figure, measured 24 hours post-encapsulation). Maximum yield was also achieved using the 1:3 formulation. Figure 5D The three-dimensional hydrogel core / shell design consists of a hydrogel, a cell nucleus, and a cell-free outer shell. The right side shows a representative image of hPSC aggregates encapsulated in a three-dimensional hydrogel of Formula III.

[0013] Figure 6 illustrates R in Equation III 2 The effect of isobutyl and n-butyl groups at different positions on the properties of acrylate-based thermally reversible polymers.

[0014] Figures 6.5A to 6.5C illustrate R in Equation III. 4 The effect of -H (hydrogel) and -CH3 (methyl) at the position on the properties of acrylate-based thermally reversible polymers. 6.5A shows the effect on material properties. 6.5B shows the temperature-based differences in gelation (green = methyl / CH3, gray = hydrogen / H). 6.5C shows the effect on hydrogel encapsulation.

[0015] Figure 7 It shows the PEG group (R of Formula III) 1 Functionalization of side chains with inert structures (methoxy and hydroxy) and functional structures (acrylate, biotin, and DBCO).

[0016] Figure 8 Demonstrates main-chain functionalization of polymers with methacrylates, maleimide, and DBCO (R of Formula III) 3 ).

[0017] Figure 9 The presentation of proteins (FGF and heparin) and their release from polymers were demonstrated.

[0018] Figures 10A to 10B The position R of the PEG side chain in Formula III is shown. 1 The effects of various functional groups (-NH2, -OCH3 and -AC) and different solvents on the polymer synthesis reaction parameters.

[0019] Figures 11A to 11EThis study demonstrates a comparison of MGE progenitor cells generated from human pluripotent stem cells (hPSCs) using various neural induction protocols in both two-dimensional cell cultures and three-dimensional thermally reversible hydrogels, such as via FOXG1 and DLX1 expression (qPCR). Figure 11B and Figure 11C (Fold change relative to undifferentiated hPSCs) and expression of FOXG1 and NKX2-1 (FC, flow cytometry, Figure 11D and Figure 11E As assessed.

[0020] Figures 12A to 12F This study demonstrates a comparison of cortical interneurons generated from human pluripotent stem cells (hPSCs) using various neural induction protocols performed in two-dimensional cell cultures and three-dimensional thermally reversible hydrogels, such as via expression of FOXG1, CALB1, and GAD1 (qPCR). Figures 12B to 12D (Fold change relative to undifferentiated hPSCs) and expression of FOXG1 and NKX2-1 (FC, flow cytometry, Figures 12E to 12F As assessed.

[0021] Figure 13 Thermally reversible hydrogels for generating hPSC-derived cortical interneurons in a three-dimensional environment: a) Fully defined synthetic PNIPAM-based hydrogels (liquid at low temperatures, rapidly gelling upon heating to 37°C. b, c). hPSCs were mixed with the hydrogel at low temperatures by controlling the system temperature and extruded into warm culture media, thereby encapsulating these cells in porous gel beads. These can be used for well plate assays (b) or easily extended to perfusion bioreactor systems (c). Cells can be differentiated into any cell type of interest within these beads using specialized culture medium formulations. d) Differentiation paradigms and factors for generating hPSC-derived cINs in three-dimensional hydrogels. e) Evolution of marker expression during hPSC differentiation into cINs, and assays for assessing differentiation potency.

[0022] Figure 14. Expression of lineage-specific markers of cortical interneuron differentiation. a) On day 10 of differentiation in thermoreversible hydrogel beads, cells began to express the ventral telebrain marker FoxG1. By day 18, cells expressed FoxG1 and NKX2-1, markers of MGE progenitor cells. After 35 days of differentiation in hydrogel beads, NKX2-1 expression was significantly downregulated while maintaining high FoxG1 expression, consistent with the mature cIN ​​phenotype. b) Representative flow cytometry plots corresponding to the quantifications shown in a. Data are expressed as mean + sd of two biological replicates.

[0023] Figure 15. Gene expression analysis of hPSCs differentiated into cortical interneurons. a) Transcriptional expression analysis of cells harvested on days 18 and 35 by qPCR. Fold change relative to gene expression in hPSCs (expressed as 2^(-ΔΔCt)). CALB1 = calcium-binding protein; PV = albumin; SST = somatostatin. b) Immunocytochemical analysis of gene expression in cells on day 35 showed high expression of FoxG1, NCAM, and SST. The absence of Ki67-positive cells indicates post-mitotic cells. c) b. Quantitative data are expressed as mean + sd of two biological replicates.

[0024] Figure 16. Viability and differentiation potency of hPSCs differentiating into cortical interneurons encapsulated in thermally reversible hydrogel beads compared to the standard two-dimensional process. a) Post-harvest viability of hPSC-derived cells after 35 days of differentiation in standard two-dimensional cultures or three-dimensional cultures in novel formula (III) hydrogels as described herein. b) Quantification of a). c) Flow cytometry analysis of biomarker expression in hPSC-derived cells at different harvest time points, showing that the novel formula (III) hydrogel has higher differentiation potency (%FoxG1+ cells, fold change at days 10, 18, and 35 in two-dimensional and three-dimensional cultures, and %Nkx2-1+ cells, fold change at day 18) compared to the standard two-dimensional method. Data are expressed as mean + sd of two biological replicates.

[0025] Figures 17A to 17C The amplification of hESC and hiPSC in a three-dimensional hydrogel containing a thermoreversible polymer of formula III is demonstrated. Figure 17A Three culture scales compatible with 3D hydrogels: Positive phase displacement pipette (PDP) droplets consist only of a core 3D hydrogel and allow for rapid screening of small-scale, multi-condition culture. Core / shell beads (C / S beads) – Static structures consisting of cells encapsulated in 3D hydrogel beads, which are composed of a hydrogel core and a cell-free outer shell, and are cultured under static conditions in well plates. C / S beads – Rotators encapsulate cells in 3D hydrogel core / shell beads, allowing for cell growth in a rotating environment using a spinneret flask. Figure 17B An exemplary human embryonic stem cell line was continuously grown in a three-dimensional hydrogel for 8 days, achieving an 80-fold fold change across all three culture scales, with a yield of up to 20e. 6 A three-dimensional hydrogel with cells per mL, exhibiting high viability at harvest. Figure 17C Examples of human induced pluripotent stem cell lines were continuously grown in a three-dimensional hydrogel for 8 days. When grown in encapsulated C / S beads, the fold change exceeded 100-fold, and the yield reached 25e. 6A three-dimensional hydrogel with cells per mL, exhibiting high viability at harvest.

[0026] Figures 18A to 18C Reproducible hPSC amplification in bioreactors of different sizes using a three-dimensional hydrogel containing a polymer of formula III. Figure 18A Schematic diagram of a scalable bioreactor system containing three-dimensional hydrogel beads. Figure 18B The expression of pluripotency markers in each batch was determined by flow cytometry. Figure 18C Gene expression analysis using qPCR showed strong correlations in gene expression among all four batches of hPSC amplified in the three-dimensional hydrogel. Each figure displays Pearson's correlation indexes.

[0027] Figure 19 A three-dimensional hydrogel containing a polymer of formula III effectively differentiated hPSCs into pancreatic endoderm cells (PE). A 100-mL spin bottle contained hPSCs encapsulated in core-shell three-dimensional hydrogel beads. Cells expanded and differentiated on the three-dimensional hydrogel platform. Performance was compared with standard PE differentiation in suspension culture.

[0028] Figures 20A to 20D Three-dimensional hydrogels containing polymers of formula III can effectively differentiate hPSCs into pancreatic endodermal cells (PE). Figure 20A The various stages at which hPSCs differentiate into pancreatic endodermal (PE) cells. Figure 20B Differentiation power was assessed by the expression of the PE marker PDX-1 (pancreatic and duodenal homeobox-1). Figure 20C Compared with standard suspension methods, the use of three-dimensional hydrogel technology can increase the yield of PE cells by 45 times. Figure 20D Compared to standard suspension methods, three-dimensional hydrogel technology using improved gel formulations allows for better control over the size of PE aggregates, minimizing the adverse effects of nutrient limitation (such as cell death, necrotic core, and underdifferentiation).

[0029] Figures 21A to 21C Three-dimensional hydrogels containing polymers of formula III can effectively differentiate hPSCs into midbrain dopaminergic cells (mDA). Figure 21A The 100-mL rotary flask contains hPSCs encapsulated in core-shell three-dimensional hydrogel beads. Cells expand and differentiate on the three-dimensional hydrogel platform. Figure 21B After a 16-day differentiation process using positive phase displacement pipette (PDP) droplets, harvested mDA cells exhibited high viability. Differentiation efficacy was assessed by the presence of FoxA2 (a marker of mDA progenitor cells). Figure 21CCells encapsulated in three-dimensional hydrogel C / S beads were expanded and differentiated for 16 days in a 100 ml rotary flask. The harvested mDA cells exhibited high viability. Differentiation efficacy was assessed by the presence of FoxA2 (a marker of mDA progenitor cells).

[0030] Figures 22A to 22C Three-dimensional hydrogels containing polymers of formula III can effectively differentiate hPSCs into hematopoietic stem cells (HSCs). Figure 22A hPSCs were differentiated into HSCs using a readily available culture medium formulation (StemDiff hematopoietic kit). Figure 22B Representative images of hPSCs on the day of induction (stage 1), during culture in hydrogel, and on the day of harvest (end of stage 2). Figure 22C Harvest analysis of hPSC-derived HSCs differentiated in an improved hydrogel system.

[0031] Figures 23A to 23C Three-dimensional hydrogels containing polymers of formula III can achieve efficient HSC amplification. Figure 23A Thawing and amplification of CD34+ HSCs derived from human umbilical cord blood in a three-dimensional hydrogel. Figure 23B Representative image of HSCs amplified in a hydrogel in SFEM II medium (StemDiff Hematopoietic Kit) supplemented with StemSpanCD34+ amplification supplement. Figure 23C Harvest analysis of HSCs amplified for 8 days in an improved hydrogel system. Detailed Implementation

[0032] definition As used in this article, "activator" refers to a compound that increases, induces, stimulates, activates, promotes, or enhances the activation of molecular or pathway signal transduction functions, such as Wnt signal transduction and SHH signal transduction.

[0033] As used herein, the term "cell population" or "cell group" refers to a group of at least two cells. In non-limiting examples, a cell population may contain 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 may be a pure population containing one cell type, such as a population of dopaminergic neurons, or it may be a population of undifferentiated stem cells. Alternatively, the population may contain more than one cell type, such as a mixed cell population.

[0034] As used in this article, the term "stem cell" refers to a cell that can divide indefinitely and produce specialized cells in culture.

[0035] As used herein, the terms "embryonic stem cells" and "ESCs" refer to primitive (undifferentiated) cells derived from preimplantation embryos that are capable of prolonged division without differentiation in culture and are known to develop into cells and tissues of the three major germ layers. Human embryonic stem cells refer to embryonic stem cells derived from human embryos. As used herein, the terms "human embryonic stem cells" or "hESCs" refer to a class of pluripotent stem cells derived from early human embryos (up to and including the blastocyst stage) that are capable of prolonged division without differentiation in culture and are known to develop into cells and tissues of the three major germ layers.

[0036] As used in this article, the term "embryonic stem cell line" refers to a population of embryonic stem cells that can proliferate undifferentiated under in vitro culture conditions for days, months, or even years.

[0037] As used in this article, “pluripotency” refers to the ability of an organism to develop into one of the three developmental germ layers (endoderm, mesoderm, and ectoderm).

[0038] As used herein, the term “induced pluripotent stem cell” or “iPSC” refers to a class of pluripotent stem cells formed by introducing certain embryonic genes (such as, but not limited to, transgenes of OCT4, SOX2, and KLF4) (see, for example, Takahashi and Yamanaka Cell 126, 663-676 (2006), which are incorporated herein by reference).

[0039] As used in this article, the term "neuron" refers to a nerve cell, which is the primary 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.

[0040] As used in this article, the term "undifferentiated" refers to cells that have not yet developed into specialized cell types.

[0041] As used in this article, “differentiation” refers to the process by which undifferentiated embryonic cells acquire the characteristics of specialized cells, such as neurons, heart, liver, or muscle cells. Differentiation is controlled by the interaction of cellular genes with extracellular physical and chemical conditions, typically through signaling pathways involving proteins embedded on the cell surface.

[0042] As used herein, the term "induced differentiation" refers to changing a default cell type (genotype and / or phenotype) to a non-default cell type (genotype and / or phenotype). Therefore, "induced stem cell differentiation" refers to inducing stem cells (e.g., human stem cells) to divide into daughter cells with characteristics different from those of the stem cells, such as genotype (e.g., changes in gene expression as determined by genetic analysis such as microarrays) and / or phenotype (e.g., changes in the expression of one or more protein markers).

[0043] As used herein, the terms “marker,” “cell marker,” or “biomarker” refer to a gene or protein that identifies a particular cell or cell type. A cell marker may not be limited to a single marker; a marker can refer to a “pattern” of markers that allows a specified set of markers to distinguish one cell or cell type from another.

[0044] As used herein, the term "linker" or "linkage" refers to a linking portion that connects two groups, with a main chain length of 100 atoms or less. A linker or linkage can be a covalent bond connecting two groups or a chain of 1 to 100 atoms in length, such as chains of 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, or 20 carbon atoms. Linkers can be linear, branched, cyclic, or monoatomic. In some cases, one, two, three, four, or five or more carbon atoms in the linker main chain may optionally be substituted with sulfur, nitrogen, or oxygen heteroatoms. The bonds between the main chain atoms can be saturated or unsaturated, and typically no more than one, two, or three unsaturated bonds exist in the linker main chain. Linkers may contain one or more substituent groups, such as alkyl, aryl, or alkenyl groups. Linkers may include, but are not limited to, poly(ethylene glycol); ethers, thioethers, tertiary amines, and alkyl groups, and may be straight-chain or branched, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), etc. The linker backbone may contain cyclic groups, such as aryl groups, heterocyclic groups, or cycloalkyl groups, wherein two or more atoms (e.g., two, three, or four atoms) of the cyclic group are contained in the backbone. Linkers may be cleavable or non-cleavable.

[0045] "Alkyl" refers to a monovalent saturated aliphatic hydrocarbon group having 1 to 10 carbon atoms, or 1 to 6 carbon atoms, 1 to 5 carbon atoms, 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In some cases, "lower alkyl" is an alkyl group having 1 to 6 carbon atoms. This term includes, for example, straight-chain and branched hydrocarbon groups, such as methyl (CH3—), ethyl (CH3CH2—), n-propyl (CH3CH2CH2—), isopropyl ((CH3)2CH—), n-butyl (CH3CH2CH2CH2—), isobutyl ((CH3)2CHCH2—), sec-butyl ((CH3)(CH3CH2)CH—), tert-butyl ((CH3)3C—), n-pentyl (CH3CH2CH2CH2CH2—), and neopentyl ((CH3)3CCH2—).

[0046] The term "substituted alkyl" refers to an alkyl group as defined herein, wherein one or more carbon atoms in the alkyl chain have optionally been replaced by heteroatoms such as —O—, —N—, —S—, —S(O). n —(where n is 0 to 2), —NR— (where R is hydrogen or alkyl), and having 1 to 5 substituents selected from the group consisting of: alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, amide, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxy, oxo, thionyl, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclic, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy. Heterocyclic, heterocyclic, hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-aryl, —SO-heteroaryl, —SO2-alkyl, —SO2-aryl, —SO2-heteroaryl and —NR a R b R and R can be the same or different, and are selected from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic.

[0047] 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 covalent bonds. Chemoselective functional groups of interest include, but are not limited to, two thiol groups, thiols and maleimide or iodoacetamide, and groups that can react with each other via click chemistry, such as azide and alkyne groups (e.g., cyclooctyne groups). Chemoselective functional groups of interest include, but are not limited to, thiols, alkynes, cyclooctynes, azides, phosphine, maleimide, alkoxyamine, aldehydes and their protected versions, and their precursors. In some embodiments, the chemoselective functional group is a thiol.

[0048] In some embodiments, the term "terminal group" refers to a group produced by any convenient subject comonomer polymerization method described herein, such as H, alkyl or substituted alkyl and / or residual components of the initiator used during polymerization.

[0049] As used herein, the term "modifier" refers to any convenient agent that provides desired properties of interest (e.g., desired physical and / or biological properties) and is capable of conjugating to a thermally reversible polymer (e.g., via polymer side-chain linkers or chemically selective functional groups at the ends). 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 agents), 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 (siRNA / RNA / DNA, etc.), metal-based compounds / catalysts, site-specific cell-targeting agents (compounds / ligands / antibodies, etc.), and smart adjuvants and gene therapy vectors. In some embodiments, the modifier is selected from heparin, hyaluronic acid, specific binding members, peptides, nucleic acids, gelatin, fibronectin, collagen, laminin, bFGF, EGF, insulin, progesterone, glucose, thymosin β-4, SHH, noggin, activin, TGFβ3, FGF8, BDNF, GDNF, NT3, PDGF-AA, and IGF-1. In other cases, the modifier is a cytokine, a member of the BMP family (e.g., TGFβ or activin), a neurotrophic factor (e.g., NT3 or BDNF), or hedgehog protein (e.g., SHH).

[0050] Any convenient method can be used to conjugate the modifier to the thermally reversible polymer. Conjugation methods and chemical approaches of interest include, but are not limited to, those described by Greg Hermanson in *Bioconjugate Techniques* (3rd edition), 2013, Academic Press. In some embodiments, the modifier is a protein. In some embodiments, the modifier is a peptide. In some embodiments, the modifier is a peptide and can be conjugated to the thermally reversible polymer (e.g., via terminal and / or side chain functional groups) by covalent attachment to the N-terminus or C-terminus of the peptide agent, or by covalent attachment to an amino acid side chain (e.g., an amino acid side chain group containing an amino, thiol, hydroxyl, carboxylic acid, or phenol, or a derivative thereof). In some embodiments, the modifier is heparin. In some embodiments, the heparin modifier is linked via a thiol bond. In some cases, heparin can be linked to the subject polymer via conjugation to the carboxylic acid group of heparin. For example, Figure 9An exemplary method for attaching heparin thiol to the acrylate groups of a polymer via Michael addition is illustrated. In some embodiments, two or more modifiers (e.g., heparin and hyaluronic acid) may be interconnected in addition to the thermally reversible polymer.

[0051] As used herein, the lower critical solution temperature (LCST) is the critical temperature at which all components of a mixture are miscible with all other components. The term "lower" in the terminology indicates that the LCST is the lower limit of the temperature range in which components are partially miscible or miscible only with certain components.

[0052] The inventors have discovered that, compared to standard two-dimensional culture methods, the method described herein can increase the yield of MGE progenitor cells by at least 10 times and increase cell viability at harvest by at least 4 times.

[0053] In some aspects, 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 Small Mothers Against Decapentaplegic (SMAD) signaling inhibitor and at least one Wingless (Wnt) antagonist for a predetermined amount of time; and contacting the cells with at least one Sonic Hedgehog (SHH) signaling activator for a predetermined amount of time to obtain a cell population containing MGE progenitor cells. In some aspects, the cell population containing 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%, at least 86%, or at least 87% of the cell population is 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 is NKX2-1 positive. In some respects, FOXG1-positive NKX2-1-positive MGE progenitor cells constitute at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% of the cell population, for example, as measured by flow cytometry, immunocytochemistry, and / or qPCR.

[0054] In a preferred aspect, the method further includes contacting the resulting encapsulated MGE progenitor cells with at least one neurotrophic factor and optionally a notch inhibitor for a predetermined time period to obtain a cell population comprising FOXG1-positive differentiation-inhibiting GABAergic cortical interneurons (cINs). In some respects, the cell population containing cIN 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 is FoxG1 positive; (b) at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% of the cell population is 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 is PV positive; (d) less than 30%, less than 25%, less than 24%, or less than 23% of the cell population is 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 is Ki67 positive, as measured by, for example, flow cytometry, immunocytochemistry, and / or qPCR.

[0055] Synthetic hydrogels In some embodiments, the synthetic hydrogels used in the methods described herein are based on polyethylene glycol-poly(N-isopropylacrylamide) (PEG-PNIPAAM) hydrogels that are solid at 37°C. Preferred PEG-PNIPAAM hydrogels include those described in U.S. Patent No. 10,982,055 and WIPO Publication No. 2022 / 251137A1, the entire contents of each of which are incorporated herein by reference.

[0056] In some preferred embodiments, the hydrogel comprises a thermally reversible 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, aralkyl, substituted aralkyl, heteroaryl, substituted heteroaryl, heteroaryl, and substituted heteroaryl. 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.

[0057] In some implementations, the thermally reversible polymer includes formula (I): Where: a, b, and c are the mole fractions of the comonomer, wherein a, b, and c are each greater than zero, preferably a > 0.8; 0.2 > b > 0; and 0.1 > c > 0; PEG n It is a polyethylene glycol polymer, and n is an integer from 1 to 2500; R 1 It is an alkyl or substituted alkyl group, preferably a C1-C6 alkyl group, more preferably a deltabutyl group; R 2 It is an alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, aralkyl, substituted aralkyl, heteroaryl, substituted heteroaryl, heteroaryl, and substituted heteroaryl, preferably alkoxy, more preferably methoxy; and G 1 and G 2 Each component is independently selected from polymer segments, terminal groups, linkers, and linking modifiers.

[0058] In the relevant implementation scheme, the thermally reversible polymer includes formula (II): Where n is from 1 to 2500; and G 1 and G 2 Each component is independently selected from polymer segments, terminal groups, linkers, and linking modifiers.

[0059] Other synthetic hydrogels may be used in the method, including but not limited to those described in U.S. Patent Nos. 6,897,064 and 10,982,055 and U.S. Patent Publication No. 2024 / 0294713, the entire contents of each of which are incorporated herein by reference.

[0060] This article also provides novel thermally reversible polymers comprising Formula III, and three-dimensional hydrogels comprising said novel thermally reversible polymers and their use in the methods described herein (e.g., culturing and / or differentiating stem cells): (III) Where a, b, c, and d represent the mole fractions of the copolymer monomers in the polymer, with a, b, and c each being greater than 0.

[0061] PEG n It is a polyethylene glycol polymer R 1 (If it exists) is remove Any terminal group or functional group other than primary amine R 2 It is a lower alkyl group R 3(If present) is a terminal group, functional group, or linker. R 4 It is a hydrogen or lower alkyl group G 1 and G 2 Each component is independently selected from polymer segments, terminal groups, linkers, and linking modifiers. And the molecular weight of the polymer is greater than 50 kDa.

[0062] The inventors have discovered that the thermally reversible polymer of Formula III has several advantages over, for example, the thermally reversible polymers disclosed in U.S. Patent No. 10,982,055 and U.S. Patent Publication No. 2024 / 0294713. In short, as illustrated herein, the thermally reversible polymers according to this disclosure exhibit a combination of properties that make them particularly suitable for three-dimensional cell culture. For example, a three-dimensional hydrogel comprising the thermally reversible polymer of Formula III. especially It exhibits: (1) long-term stability, enabling long-term differentiation processes during cell culture; (2) lower viscosity compared to existing polymers, thus enabling efficient cell encapsulation; and (3) appropriate stiffness to support cell growth. These features significantly improve cell viability and cell yield.

[0063] In a preferred aspect, the thermally reversible polymer of Formula III has one or more (preferably all) of the following properties: (a) LCST of 12-32°C; (b) stiffness of 100-8000 Pa; (c) viscosity of 100-2000 cP; and (d) molecular weight of 50-500 kDa.

[0064] In some respects, the R of the thermally reversible polymer of formula III 1 It does not exist. In other respects, the R of the thermally reversible polymer of formula III... 1 It is a functional group. In some aspects, the functional group is a chemoselective functional group, non-limiting examples of which include two thiol groups, thiols and maleimide or iodoacetamide, and groups that can react with each other via click chemistry, such as azides and alkyne groups (e.g., cycloalkyne groups, such as dibenzocyclooctyne (DBCO)). Functional groups include, but are not limited to, acrylates, thiols, hydroxyl groups, alkoxy groups (e.g., methoxy groups), alkynes, cycloalkynes, azides, hydrazides, phosphine, maleimide, carboxylic acids, alkoxyamines, aldehydes, biotin, silanes, 1,2-distearate-sn-glycerol-3-phosphoethanolamine (DSPE), NHS esters, toluenesulfonyl (Tos) and their protected versions and precursors.

[0065] In some respects, the R of the thermally reversible polymer of formula III 1It is a terminal group, and non-limiting examples include, but are not limited to, C1-C6 alkoxy groups (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, aralkyl, substituted aralkyl, heteroaryl, substituted heteroaryl, heteroaryl, and substituted heteroaryl. In some aspects, R 1 It is not an alkyl group (e.g., not n-butyl) or a substituted alkyl group.

[0066] In some respects, the R of the thermally reversible polymer of formula III 2 It is a lower alkyl group, optionally selected from methyl, ethyl, propyl, n-butyl, pentyl, isopropyl, isobutyl, isopentyl, tert-butyl, cyclopropyl, and cyclobutyl. In some embodiments, R 2 It is n-butyl. The inventors have discovered that, compared to other conformations such as isobutyl, the n-butyl at this position exhibits higher stiffness, similar viscosity, higher gel stability, and a lower gel LCST. In some embodiments, R... 2 It is a lower alkyl group other than isobutyl.

[0067] In some respects, the R of the thermally reversible polymer of formula III 3 It does not exist. In other respects, the R of the thermally reversible polymer of formula III... 3 It is a modifier, optionally selected from heparin, hyaluronic acid, specific binding members, peptides, nucleic acids, gelatin, fibronectin, collagen, laminin, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin, progesterone, glucose, stromal cell-derived factor-1 (SDF-1), thymosin β-4, sound hedgehog factor (SHH), noggin, activin, transforming growth factor-β (TGF-β), FGF8, brain-derived neurotrophic factor (BDNF), glial cell-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).

[0068] In some preferred embodiments, R 4 It is a lower alkyl group, especially when R 4 When it is methyl.

[0069] In some cases, G 1 and G 2 Each component is independently selected from polymer segments, terminal groups, linkers, and linking modifiers. In related aspects, G... 1 and G 2Each is independently selected from heparin, hyaluronic acid, members of specific binding pairs, polypeptides, nucleic acids, and carboxyl groups. In related aspects, G... 1 and G 2 Each is an independent modifier selected from gelatin, elastin, fibronectin, collagen, and laminin. In related aspects, G... 1 and G 2 Each is independently selected from chemokines, peptide hormones, and growth factors. In some implementations, G... 1 and G 2 Each of these is independently selected from fibroblast growth factor, epidermal growth factor, liver growth factor, insulin, stromal cell-derived factor-1, thymosin β-4, sound hedgehog factor, noggin, activin, transforming growth factor, bone morphogenetic protein, brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, neurotrophic factor-3, platelet-derived growth factor, FGF-2, FGF-8, keratinocyte growth factor, or insulin-like growth factor. In some aspects, G... 1 and G 2 Each is independently selected from chain transfer agents, and non-limiting examples include dithioesters, dithiocarbamates, trithiocarbonates, or xanthate esters. In some aspects, G 1 and G 2 Each is independently selected from chain transfer agents and thermal initiators containing thiol carbonyl thio groups (such as azobisisobutyronitrile (AIBN)).

[0070] In some cases, a>0.8; 0.1>b>0; 0.2>c>0, and 0.1>d>0.

[0071] In some aspects, the molecular weight (MW) of the thermally reversible polymer of Formula III is between 50 and 500 kDa or between 50 and 250 kDa. In some embodiments, the MW of the thermally reversible polymer is at least 50 kDa, at least 60 kDa, at least 70 kDa, at least 80 kDa, or at least 90 kDa. In some embodiments, the MW of the thermally reversible polymer is in the range of about 50 kDa to about 250 kDa, such as 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 some embodiments, the MW of the thermally reversible polymer is 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 some preferred embodiments, the MW of the thermally reversible polymer is about 50 kDa to about 250 kDa or about 50 to about 150 kDa.

[0072] Any suitable poly(ethylene glycol) (PEG) polymer group can be used as a side chain of the thermally reversible polymer of formula (III). In some embodiments of formula (III), PEG n It is a polyethylene glycol polymer with a molecular weight (MW) of approximately 2-20 kDa. In the relevant embodiments, PEG... n The MW is about 2 kDa or greater, such as 2 kDa to 20 kDa, or 2 kDa to 10 kDa, or 3 kDa to 20 kDa, or 3 kDa to 10 kDa. In some implementations, PEG n The MW is approximately 2 kDa to approximately 9 kDa, approximately 3 kDa to approximately 8 kDa, approximately 4 kDa to approximately 7 kDa, and approximately 4 kDa to approximately 6 kDa. In some implementations, PEG n The MW values ​​are approximately 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, or 9 kDa. PEG n The group can be modified with any suitable group, including terminal modifications.

[0073] In some embodiments, the weight-to-weight (weight / weight) ratio of the thermally reversible polymer of Formula III, PEG, to PNIPAAm copolymer is greater than 1:2. In some embodiments, the weight / weight ratio of PEG to 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 weight / weight ratio of PEG to PNIPAAm copolymer is about 1:3 to about 1:4.5, or about 1:4. In some embodiments, the weight / weight ratio of PEG to PNIPAAm copolymer is measured using nuclear magnetic resonance (NMR) based on the relative molecular ratio and using gel permeation chromatography (GPC) based on the total polymer molecular weight (GPC-NMR analysis).

[0074] In some aspects, the weight of the PNIPAAm copolymer is defined as the weight of the copolymer backbone after the comonomer polymerization (i.e., before the addition of PEG). For example, in some aspects, the weight of the PNIPAAm copolymer is defined as the following weight: After polymerization of comonomers: In related aspects, the weight of PEG is defined as before reaction with the copolymer backbone. The weight.

[0075] In a particularly preferred embodiment, the PEG:PNIPAAM weight ratio of the thermally reversible polymer of Formula II is greater than 1:2 and the MW is between 50 kDa and 250 kDa (inclusive).

[0076] The inventors have discovered that polymers of Formula III with a PEG:PNIPAAM weight ratio greater than 1:2 (e.g., 1:3) and a lower MW (e.g., 50 kDa-250 kDa) have several surprising advantages over polymers disclosed, for example, in U.S. Patent No. 10,982,055 and U.S. Patent Publication No. 2024 / 0294713, including, but not limited to: (1) a lower viscosity hydrogel, minimizing shear forces during cell encapsulation in large-scale bioreactor cell culture systems; (2) faster flow rates, reaching 2 mL / min; and (3) greater control over bead size and geometry (higher roundness and lower tail formation frequency); (4) maintenance of a three-dimensional structure during prolonged cell differentiation processes and an optimal range of stiffness to support stem cell proliferation and differentiation into all three germ layers; and (5) improved functionalization capabilities for controlling protein presentation and release. Therefore, the thermally reversible polymers of Formula III provide scalable three-dimensional cell culture systems, for example, for generating functional neurons, over relevant time periods.

[0077] In some embodiments, the LCST of the thermally reversible polymer of Formula III is in the range of 12-32°C, such as 12-30°C, 15-30°C, 15-25°C, or 10-20°C. In a preferred embodiment, the LCST of the thermally reversible polymer of Formula III is about 20°C to about 22°C.

[0078] A composition is also provided comprising: a) a three-dimensional hydrogel comprising a thermally reversible polymer of formula III; and b) cells encapsulated within the hydrogel. The three-dimensional hydrogel-cell composition can be used to produce a desired number of cells by culturing the composition under conditions and for a time period sufficient to produce the desired number of cells. In some aspects, the time period sufficient to produce 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 suitable for differentiating cells, for example, generating a desired number of differentiated cells over a relatively long period of time. The three-dimensional thermoreversible polymer-cell composition of this disclosure can be implanted into an individual in need, wherein cells proliferate and / or differentiate within the implanted thermoreversible polymer-cell composition and migrate out of the implanted thermoreversible polymer-cell composition.

[0079] In some aspects, this 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 comprise cells and a hydrogel encapsulating the cells, wherein the hydrogel encapsulating the cells comprises a thermally reversible polymer of Formula III. In some embodiments, at least 90% of the hydrogel capsules each comprise a plurality of cells, for example, 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 working examples of this disclosure, the thermally reversible polymer of Formula III provides improvements, including but not limited to, bead (i.e., capsule) uniformity, bead roundness, bead sphericity, reduction in bead volume and diameter, bead production flow rate, and bead shape produced by gravity drop extrusion.

[0080] This disclosure provides a method for generating differentiated cells from stem cells or precursor cells, the method comprising culturing stem cells or precursor cells for a period of time in a three-dimensional hydrogel composition comprising a thermally reversible polymer of formula III under conditions suitable for inducing differentiation of stem cells or precursor cells. The conditions for inducing differentiation of stem cells or precursor cells depend in part on the desired differentiated cells. The conditions may include the inclusion of one or more differentiation-inducing factors in the hydrogel. In some aspects, suitable conditions for stem cell differentiation include culturing stem cells or precursor cells in a hydrogel composition for a period of 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 produce a differentiated cell population.

[0081] In several aspects, this disclosure provides a method for expanding stem cells, the method comprising encapsulating a single cell or multi-cell cluster in a three-dimensional hydrogel containing a thermally reversible polymer of formula III, and culturing the cells under suitable stem cell expansion conditions.

[0082] The subject thermally reversible polymer can be prepared using any convenient method. A variety of polymerization methods can be used to prepare the basic polymer material, including polyacrylates, polyacrylamides, and mixtures thereof. Various derivatization methods can be used to introduce any convenient functionality into the subject basic polymer material. A variety of chemically selective conjugation chemistry, linkers, functional groups, and modifiers can be used in the preparation of further derivatives and conjugates of the subject basic polymer material and its derivatives.

[0083] In some aspects, a method for preparing a thermally reversible polymer of formula III is provided, the method comprising the steps of: (i) polymerizing a group of comonomers comprising N-isopropylacrylamide (NIPAAm), alkyl methacrylate (preferably butyl methacrylate (BMA)), and N-acryloyloxysuccinimide (NASI) in a single ideal solvent that maximizes monomer solubility to form a PNIPAAM-co-PBMA-co-PNASI copolymer backbone; and (ii) reacting the copolymer with a mono-PEG amine (e.g., methoxyPEG amine) to form a [PNIPAAM-co-PBMA-co-PNASI]-b-[PEG] copolymer; and (iii) reacting [PNIPAAM-co-PBMA-co-PNASI]-b-[PEG] with isopropylamine to form a thermally reversible polymer of formula III. Preferably, the concentration of the initiator in step (i) controls the molecular weight of the backbone, thereby controlling the total polymer molecular weight. In a preferred embodiment, the weight / weight ratio (controlled by the initiator concentration) of the PEG amine to the PNIPAAM-co-PBMA-co-PNASI copolymer backbone is greater than 1:2, and preferably greater than 1:3. In some embodiments, the weight / weight ratio of the PEG amine to the 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 weight / weight ratio of the PEG amine to the PNIPAAM copolymer is about 1:3 to about 1:4.5, or about 1:4. In some embodiments, a thermally reversible polymer of Formula III produced by this method is provided.

[0084] In some respects, the single solvent is selected from acetone, acetonitrile, benzene, chloroform, dichloromethane, dimethylformamide, dimethyl sulfoxide, dioxane, ethyl acetate, pyridine, ethanol, methanol, tetrahydrofuran, toluene, and water.

[0085] stem cells The term "stem cell" refers to a pluripotent, totipotent, or multipotent cell capable of differentiating 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 "pluripotent" refers to the cell's ability to differentiate into any type of cell in a different organism, as well as the ability to differentiate into extraembryonic cells (such as placental cells). As used herein, the terms "induced pluripotent stem cells" or "iPSCs" refer to a class of pluripotent stem cells that resemble embryonic stem cells but are produced when somatic cells (e.g., adult cells) are reprogrammed into an embryonic stem cell-like state. This reprogramming is achieved by forcibly expressing factors essential for maintaining the "stemness" of embryonic stem cells (ESCs) (i.e., their ability to be guided into different differentiation pathways). As used herein, the term "progenitor cell" refers to an intermediate cell stage in which the cell is no longer a pluripotent stem cell and has not yet fully differentiated into a cell. Progenitor cells in this disclosure include somatic cells.

[0086] The term "pluripotent" refers to a cell line that is capable of differentiating into any terminally differentiated cell type.

[0087] The term "multipotent" refers to a cell line that is capable of differentiating into at least two terminally differentiated cell types.

[0088] The term "embryonic stem cells" refers to primitive (undifferentiated) cells derived from preimplantation or early embryos (e.g., up to and including the blastocyst stage), capable of prolonged division without differentiation in culture, and able to develop into cells and tissues of the three major germ layers. Embryonic stem cells can also be isolated from the embryo, placenta, or umbilical cord.

[0089] The term "embryonic stem cell line" refers to a population of embryonic stem cells that can proliferate undifferentiated for days, months or even years under in vitro conditions (e.g., human embryonic stem cell lines SA01, VUB01, HUES 24, H1, H9, WT3, HUES1).

[0090] The term "induced pluripotent stem cells" or "iPSCs" refers to a class of pluripotent stem cells, similar to embryonic stem cells, created by introducing certain embryonic genes (such as transgenic OCT4, SOX2, and KLF4) into somatic cells (see, for example, Takahashi and Yamanaka Cell 126, 663-676 (2006), incorporated herein by reference). Examples of somatic cells include, but are not limited to, bone marrow cells, epithelial cells, fibroblasts, hematopoietic cells, hepatocytes, intestinal cells, mesenchymal cells, myeloid progenitor cells, and spleen cells. Alternatively, iPSCs can also be generated by reprogramming somatic cells into an embryonic stem cell-like state by forcing somatic cells to express factors essential for maintaining the "stemness" of embryonic stem cells (ESCs)—that is, their ability to be guided into different differentiation pathways.

[0091] Methods for culturing stem cells, particularly human embryonic stem cells, are known in the art and are described in Thomson and Ludwig WO2006 / 029297, WO2006 / 019366 and WO2006 / 029198 and Bergendahl and Thomson WO2008 / 089351, which are hereby incorporated by reference in their entirety.

[0092] Inhibitors of "Small Mothers Against Decapentaplegic" (SMAD) Small Mothers against Decapentaplegic (SMAD) generally refers to a class of signaling molecules that regulate the directed differentiation of stem cells. SMADs are intracellular proteins that transduce extracellular signals from transforming growth factor β ligands to the cell nucleus, where they activate downstream gene transcription, and are members of a signaling molecule class that regulates the directed differentiation of stem cells.

[0093] As currently disclosed, SMAD signaling inhibitors include compounds that interact with and reduce or block the activity of SMAD and / or SMAD-related molecules or other components of SMAD signaling. Inhibitors can bind directly to SMAD signaling and cause conformational changes, reduce or prevent the expression of genes encoding SMAD or SMAD target genes, decrease SMAD protein levels, and / or interfere with the interaction of SMAD with one or more signaling chaperones.

[0094] Inhibitors also include molecules that indirectly regulate SMAD biological activity by blocking upstream signaling molecules (e.g., within extracellular domains). Examples of such signaling molecules and their functions include: Noggin, which blocks 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 also block extracellular activators of SMAD signaling in a similar manner. Bambi, a transmembrane protein, can also act as a pseudoreceptor to block extracellular TGFβ signaling molecules.

[0095] Consider using antibodies that block activin, nodal, TGFβ, and BMP to neutralize extracellular activators of SMAD signaling. Therefore, in one embodiment, the inhibitors of this disclosure induce (alter) or alter differentiation from the default cell type to a non-default cell type; for example, one method of this disclosure comprises at least three inhibitors that generate non-default neural progenitor cells.

[0096] The inhibitors disclosed herein "alter," "reduce," or "block" default signaling to guide cell differentiation into non-default cell types, as described herein, for the generation of cortical interneurons. Therefore, the inhibitors of this disclosure can be biological compounds (natural or synthetic) or small molecules that enhance or attenuate the activity of signaling molecules that contribute to the generation of, for example, the cortical interneurons of this disclosure.

[0097] Inhibitors can be described in various ways, including competitive inhibition (binding to the active site by excluding or reducing the binding of another known binding compound) and allosteric inhibition (binding to a protein by altering its conformation, thereby interfering with the binding of the compound to the active site of the protein). In addition, they can also include inhibition caused by binding to and affecting a molecule upstream of a specified signaling molecule, thereby leading to the inhibition of the specified molecule.

[0098] SMAD inhibitors that can be advantageously utilized in the methods disclosed herein include those well-known and readily available to those skilled in the art. (Chambers et al.) Nat Biotechnol SMAD inhibitors for neural conversion of human ESC and iPSC are described in 27:275-280 (2009).

[0099] Exemplary SMAD inhibitors that can be used in the methods and compositions disclosed herein include the specified compounds 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, and E-616452 (RepSox). ALK inhibitors), SD-208, SMI6, NPC-30345, Ki26894, SB-203580, SD-093, activin-M108A, P144, soluble TBR2-Fc, DMH-1, doxorphine dihydrochloride and their derivatives and / or variants, wherein each of their derivatives and / or variants has one or more SMAD inhibitory activities.

[0100] a) Inhibitors of the TGFβ / activin / Nodal pathway Inhibition of SMAD signaling pathways 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 classification is for tissue purposes only, and those skilled in the art will understand that compounds can affect one or more aspects of the pathway, and therefore may act in more than one defined category.

[0101] TGF β receptor (e.g., ALK5) inhibitors may include antibodies against TGF β receptor (e.g., ALK5), its dominant and negative variants, and antisense nucleic acids that inhibit its expression. Exemplary TGFβ receptor / ALK5 inhibitors include, but are not limited to, SB431542 (see, for example, 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-pyrazol-1-thiocarboxamide (see, for example, Tojo, et al., Cancer Science 96(11):791-800 (2005), and commercially available from, for example, ToicrisBioscience); 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthylidine, Wnt3a / BIO (see, for example, Dalton, et al., WO2008 / 094597, incorporated herein by reference), BMP4 (see, Dalton, ibid.), GW788388 (-{4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridin-2-yl}-N(tetrahydro-2H-pyran-4-yl)benzamide) (see, for example, Gellibert, et al., Journal of Medicinal Chemistry 49(7):2210-2221 (2006)), SM16 (see, for example, Suzuki, et al., Cancer Research 67(5):2351-2359 (2007)), IN-I 130 (3-((5-(6-methylpyridin-2-yl)-4-(quinoxaloline-6-yl)-1H-imidazol-2-yl)methyl)benzamide) (see, for example, Kim, et al., Xenobiotica 38(3):325-339 (2008)), GW6604 (2-phenyl-4-(3-pyridin-2-yl-1H-pyrazol-4-yl)pyridine) (see, for example, de Gouville, et al., Drug NewsPerspective 19(2):85-90 (2006)), SB-505124 (2-(5-benzo[1,3]dioxacyclopenten-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride) (see, for example, DaCosta et al., Molecular Pharmacology65(3):744-752 (2004)) and pyrimidine derivatives (see, for example, those listed in WO2008 / 006583, which are incorporated herein by reference). In some aspects, the method includes using the SMAD inhibitor SB431542 at a concentration of about 5-15 μM, preferably about 10 μM, for a period of about 5 to about 10 days, preferably about 7 to 8 days.

[0102] Furthermore, while "ALK5 inhibitors" is not intended to encompass non-specific kinase inhibitors, "ALK5 10 inhibitors" should be understood to include inhibitors that, in addition to ALK5, also inhibit ALK4 and / or ALK7, such as SB-431542. See, for example, Inman et al. J Mol Phamacol 62(1):65-74 (2002). Without intending to limit the scope of this disclosure, ALK5 inhibitors are believed to affect the mesenteric-epithelial transition (MET) process. The TGFβ / activin pathway is a driver of epithelial-mesenchymal transition (EMT). Therefore, inhibition of the TGFβ / activin pathway may promote the MET (i.e., reprogramming) process.

[0103] Specific examples of inhibitors include, but are not limited to, SU5416; 2-(5-benzo[1,3]dioxacyclopenten-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride (SB-505124); and lerdelimumb. (CAT-152); metilumab (CAT-192); GC-I008; ID11; AP-12009; AP-11OI4; LY550410; LY580276; LY364947; LY2109761; 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 antisense transfection of tumor cells targeting the TGFβ receptor. See, for example, 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).

[0104] Inhibitors of SMAD 2 / 3 phosphorylation can include antibodies against SMAD2 or SMAD3, their dominant 5-negative variants, and antisense nucleic acids targeting them. Specific examples of inhibitors include PD169316; SB203580; SB-431542; LY364947; A77-01; and 3,5,7,2′,4′-pentahydroxyflavone (Morin). (See, for example, Wrzesinski, ibid.; Kaminska, ibid.; Shimanuki, et al., Oncogene 26:3311-3320 (2007); and Kataoka, et al., EP 1992360, which are incorporated herein by reference.) SB-431542 (i.e., CAS 301836-41-9; IUPAC 4-[4-(1,3-benzodioxane-5-yl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide) is a commercially available small molecule SMAD inhibitor that can reduce or block transforming growth factor β (TGFβ) / activin-Nodal signaling.

[0105] Inhibitors of the SMAD 2 / 3 and SMAD 4 interaction include antibodies against SMAD2, SMAD3, and / or smad4, their dominant and negative variants, and antisense nucleic acids targeting them. Specific examples of SMAD 2 / 3 and SMAD4 interaction inhibitors 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).) (b) BMP inhibitors 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 between SMAD 1 / 5 / 8 and SMAD 4, and activators / agonists of SMAD 6 and SMAD 7. The classification described below is for organizational purposes only, and those skilled in the art will understand that compounds can affect one or more steps in the pathway, and therefore compounds can function in more than one defined category.

[0106] Inhibitors of SMAD 1 / 5 / 8 phosphorylation include, but are not limited to, antibodies against SMAD 1, SMAD 5, or SMAD 8, their dominant and negative variants, antisense nucleic acids targeting them, and small molecules. Specific examples of inhibitors include LDN-193189 and doxorphine (e.g., commercially available from Stemgent, for example).

[0107] BMP receptor inhibitors include, but are not limited to, antibodies against the BMP receptor, its dominant and negative variants, siRNAs or antisense nucleic acids targeting it, or small molecules. Specific examples of inhibitors include, but are not limited to, DMH-1, doxorphine dihydrochloride, and LDN-193189 (commercially available from, for example, Tocris Biosciences).

[0108] 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 SMAD signaling inhibitor. LDN193189 is also a highly potent small-molecule inhibitor of ALK2, ALK3, and ALK6 protein tyrosine kinases (PTKs). It inhibits the signaling of ALK1 and ALK3 family members of the type I TGFβ receptor, thereby inhibiting the transmission of multiple biological signals, including bone morphogenetic proteins (BMPs) BMP2, BMP4, BMP6, BMP7, and activin cytokine signaling, and subsequently inhibits 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 aspects, the method includes using the BMP inhibitor LDN-193189 at a concentration of about 50-150 nM (preferably about 100 nM) for a period of about 10 days to about 20 days (preferably about 14-16 days).

[0109] (c) Dual SMAD inhibitors SMAD signaling inhibitors may include dual SMAD inhibitors SB431542 and LDN-193189, or their functional derivatives and / or variants. The terms “LSB” and “XLSB” as used herein have previously been described in the definitions section. Dual SMAD inhibitors significantly improve differentiation efficiency.

[0110] According to the method of this disclosure, SB431542 can be contacted with pluripotent cells and / or pluripotent stem cells in vitro to a final concentration of about 0.1 μM to about 1 mM. LDN-193189 can be contacted with pluripotent stem cells and / or pluripotent stem cells in vitro to a final concentration of about 1 nM to about 10 μM.

[0111] Wingless (Wnt) signal transduction antagonist "Wingless" or "Wnt" refers to a signaling pathway composed of Wnt family ligands and Wnt family receptors (such as Frizzled and LRPterailed / RYK receptors), mediated or not mediated by β-catenin. Wnt proteins are involved in tumorigenesis and various developmental processes, including regulating cell fate and patterns during embryogenesis.

[0112] The Wnt pathway includes any protein downstream or upstream of Wnt protein activity. For example, this may include LRPS, LRP6, Dkk, GSK-3, Wnt10B, Wnt6, Wnt3 (e.g., Wnt 3A), Wnt1, or any other protein discussed herein, as well as the genes encoding these proteins.

[0113] The Wnt pathway also includes downstream pathways of Wnt, such as the LRPS or HBM pathway, the Dkk pathway, the β-catenin pathway, the MAPKAPK2 pathway, and the OPG / RANK pathway. The "LRP5 pathway" and "IBM pathway" refer to any protein / gene including LRP5 or HBM mutants, as well as proteins downstream of LRPS or HBM mutants. The "β-catenin pathway" refers to any protein / gene including β-catenin and its downstream proteins. The "MAPKAPK2 pathway" refers to any protein / gene including MAPKAPK2 and proteins downstream of MAPKAPK2. The "OPG / RANKL pathway" refers to any protein / gene including OPG / RANKL and proteins downstream of OPG and RANKL. The "Dkk pathway" refers to any protein / gene involved in the interaction between Dkk-1 and LRP5 and / or LRP6, which are part of the Wnt pathway. Dkk-I inhibits LRP5 activity.

[0114] As used herein, the term "Wnt antagonist" refers not only to any agent that can directly inhibit the normal function of the Wnt protein, but also to any agent that can inhibit the Wnt signaling pathway and thus restore Wnt function. Examples of Wnt signaling antagonists include XAV939 (Hauang et al.). Nature461:614-620 (2009)), vitamin A (retinoic acid), lithium, flavonoids, Dickkopf1 (Dkk1), insulin-like growth factor binding protein (IGFBP) (WO2009 / 131166), and siRNA targeting β-catenin. Exemplary Wnt antagonists include, but are not limited to, XAV939, IWP-2, DKK1 (Dickkopf protein 1), and IWR1. Other Wnt inhibitors include, but are not limited to, IWR compounds, IWP compounds, and WO09155001 and Chen et al. Nat Chem Biol Other Wnt inhibitors described in 5:100-7 (2009). In some aspects, the method includes using a Wnt antagonist IWP-2 at a concentration of about 1-10 μM, preferably about 5 μM, for a period of about 5 to about 10 days, preferably about 7 or 8 days.

[0115] XAV939 is a potent small-molecule telomere anchoring enzyme (TNKS) 1 and 2 inhibitor with an IC50 value of 100%. 50 The values ​​are 11 nM and 4 nM, respectively. Huang et al., Nature 461:614-620 (2009). XAV939 increases the protein level of the axin-GSK3β complex by inhibiting TNKS activity and promotes the degradation of β-catenin in SW480 cells. Known Wnt signaling antagonists also include Dickkopf proteins, secretory coil-associated protein (sFRP), Wnt repressor factor 1 (WIF-1), and Soggy. Members of the Dickkopf-associated protein family (Dkk-1 to Dkk-4) are secretory proteins with two cysteine-rich domains separated by a linker region. Dkk-3 and Dkk-4 also have a prodynein domain. Dkk-1, Dkk-2, Dkk-3, and Dkk-4 act as classic Wnt signaling antagonists by binding to LRP5 / 6, preventing LRP5 / 6 from interacting with the Wnt coil-associated 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. Antagonistic activity of Dkk-3 has not yet been confirmed. Dkk proteins exhibit different expression patterns in adult and embryonic tissues and have a wide range of effects on tissue development and morphogenesis.

[0116] The Dkk family also includes Soggy, which is homologous to Dkk-3 but dissimilar to other family members. sFRP is a family of five Wnt-binding glycoproteins, which are similar to membrane-bound Frizzled glycoproteins. The largest family of Wnt inhibitors comprises two groups: the first group consists of sFRP1, sFRP2, and sFRP5, and the second group includes sFRP3 and sFRP4. They are all secreted and derived from unique genes, and are not alternative splicing 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.

[0117] In some embodiments, this disclosure relates to inhibitors and / or antagonists of the SMAD and Wnt signaling pathways. SMAD inhibitors include, but are not limited to, SB431542, LDN-193189, and Noggin. PD169316, SB203580, LY364947, A77-01, A-83-01, BMP4, GW788388, GW6604, SB-505124, Dilimumab, Metemumab, GC-I008, AP-12009, AP-110I4, LY550410, LY580276, LY364947, LY2109761, SB-505124, SB-431542, SD-208, SMI6, NPC-30345, Ki26894, SB-203580, SD-093, Activin-M108A, P144, Soluble TBR2-Fc, DMH-1, Doxorphine dihydrochloride and its 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.

[0118] In some applications, SB431542 and LDN193189 can be used in combination to suppress the SMAD signaling pathway. In other applications, XAV939 can be used to antagonize the Wnt signaling pathway.

[0119] In other aspects of these methods, the concentration of XAV939 in the cell culture can be from about 0.2 μM to 20 μM; the concentration of LDN193189 in the cell culture can be from about 10 nM to about 1000 nM; and the concentration of SB431542 in the cell culture can be from 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.

[0120] In a further aspect of these methods, prebrain progenitor cells are generated by exposing stem cells to XAV939, LDN193189, and / or SB431542 for a duration of approximately 5 days to approximately 40 days. In a related aspect, prebrain progenitor cells are generated by exposing stem cells to XAV939, LDN193189, and / or SB431542 for a duration of approximately 10 days to approximately 25 days.

[0121] In a further aspect of the method according to any of these three embodiments, the Wnt signaling 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, and their derivatives and / or variants, wherein each of these derivatives and / or variants has one or more Wnt signaling antagonist activities. For example, the Wnt signaling antagonist may comprise XAV939 or its functional derivatives and / or variants. XAV939 may be contacted with pluripotent stem cells and / or pluripotent stem cells in vitro at a final concentration of about 10 nM to about 500 μM.

[0122] Stem cells can be differentiated into prebrain progenitor cells using a variety of cell culture media and supplements, including KSR medium, N2 medium (DMEM / F12 with NaHCO3 and N2B supplement (Stem Cell Technologies)), and Neurobasal medium with B27 (Gibco) and N2 supplement (Invitrogen). Kriks et al., Nature 480:547-551 (2011). In some embodiments, cells are maintained on mouse embryonic fibroblasts (MEF), as previously described, and dissociated with Accutase (Innovative Cell Technologies) for differentiation, or with dispersing enzymes for passage (Chambers et al., Nat Biotechnol27:275-280 (2009)).

[0123] Sound Hedgehog (SHH) Activator The transcription factor marker NKX2.1 of the ventral forebrain progenitor cell 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 enhances FOXG1 production, which in turn promotes ventralization by inducing controlled SHH-mediated differentiation of pluripotent and multipotent cells into NKX2.1+ prebrain progenitor cells.

[0124] Such neuronal cell lineages and populations can be generated by contacting neuronal precursor cells (such as those generated herein) with one or more SHH signaling activators, said contact taking place at a predetermined time after pluripotent and / or pluripotent cells have been contacted with one or more SMAD signaling inhibitors and one or more Wnt signaling antagonists, and for a duration sufficient to induce the generation of one or more markers of cortical interneurons or their precursors.

[0125] As used herein, the term "activator" refers to a compound that, in the context of SHH, promotes and / or enhances SHH signaling, thereby inducing the differentiation of neuronal precursor cells into cortical interneurons or their precursors, hypothalamic neurons or their precursors, and / or preoptic chorionic neurons or their precursors. Examples of SHH signaling pathway activators available in this disclosure include: proteins belonging to the hedgehog family (e.g., SHH), inhibitors of Pte-Smo interaction, Smo receptor activators, Shh receptor activators (e.g., Hg-Ag, purinemorphine, etc.), substances that increase Ci / Gli family levels, inhibitors of intracellular degradation of Ci / Gli factors, and transfected SHH overexpression constructs or Ci / Gli overexpression constructs.

[0126] In some aspects of this method, an SHH signaling pathway activator (e.g., SHH + purinemorphine) is added to the cell culture for the entire or part of the culture duration. In some embodiments, the concentration of the SHH activator in the cell culture is from about 10 ng / mL to about 5000 ng / mL for SHH (or recombinant SHH) and from about 0.1 μM to about 20 μM for purinemorphine. In some preferred embodiments, the concentration of the SHH activator in the cell culture is from about 50 ng / mL to about 500 ng / mL for SHH (or recombinant SHH) and from about 0.5 μM to about 4 μM for purinemorphine.

[0127] Purinemorphamine is a commercially available small molecule with the name 9-cyclohexyl-N-[4-(morpholinyl)phenyl]-2-(1-naphthoxy)-9H-purine-6-amine and the chemical formula C. 31 H 32 N6O2. The structure of purinemorphine is listed below. Purinemorphine binds to and activates the 7-transmembrane Smo receptor in the hedgehog signaling pathway.

[0128] In a further aspect of the method according to any of these three embodiments, the SHH signaling activator is selected from the group consisting of smoothed agonists (SAG), SAG analogs, SHH, C25-SHH, C24-SHH, purinemorphine, Hg-Ag, and their derivatives and / or variants, wherein each of these derivatives and / or variants has one or more SMAD inhibitory activities. For example, the SHH signaling activator may comprise recombinant SHH and purinemorphine, or functional derivatives and / or variants thereof. The recombinant SHH may be contacted in vitro with pluripotent stem cells and / or multipotent stem cells at a final concentration of about 5 ng / mL to about 5 μg / mL. Purinemorphine may be contacted in vitro with pluripotent stem cells and / or multipotent stem cells at a final concentration of about 0.1 μM to about 20 μM. In some aspects, the method includes using an SSH activator SAG at a concentration of about 0.05-5 μM, preferably about 0.1 μM, for a period of about 15 days to about 25 days, preferably about 21 or 22 days.

[0129] The timing of SHH differentiation activation is crucial for triggering the generation of different ventral progenitor cells with distinct anterior and posterior identities. Early activation of SHH signaling in hESC-derived progenitor cells expressing hypothalamic primordium markers requires the presence of FGF-8. (Kriks et al.,) Nature 480:547-551 (2011). After neuronal precursor cells are generated and / or pluripotent cells and / or multipotent cells are contacted with one or more SMAD inhibitors and / or one or more Wnt signaling antagonists, neuronal precursor cells may be contacted with one or more SHH signaling activators after a predetermined time.

[0130] For example, after exposing pluripotent cells and / or pluripotent cells to one or more SMAD inhibitors and one or more Wnt signaling antagonists, neuronal precursor cells may be exposed to one or more SHH signaling activators within approximately 4 to 20 days or approximately 8 to 18 days. The exposure time for neuronal precursor cells to one or more SHH signaling activators may be approximately 5 to 30 days or approximately 8 to 16 days.

[0131] As used herein, "FGF receptor (FGFR) agonist" means a molecule capable of activating FGFR (e.g., a molecule that binds to FGFR and induces receptor dimerization, and activates the PI3K 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., recombinant FGF8). According to the methods described herein, cells are contacted with an FGFR agonist (such as FGF8) to shift the equilibrium toward cephalic lateralization. In some aspects, cells are contacted with FGF8 for a period of about 10 to about 20 days, preferably about 12 to about 16 days, more preferably about 13, 14, 15, or 16 days. Preferably, cells are first exposed to an FGFR agonist (such as FGF8) about 5 to 10 days, preferably about 6, 7 or 8 days, after the cells have been first exposed to one or more SMAD inhibitors and / or after the cells have been first exposed to a WNT inhibitor and / or after the cells have been first exposed to an SHH activator.

[0132] In some preferred aspects, the method includes: (i) LDN193189 (LDN) to suppress BMP signaling; (ii) SB-431542 (SB) to suppress TGFβ signaling; (iii) recombinant FGF8; (iv) a smoothed agonist (SAG; 3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-[3-(pyridin-4-yl)benzyl]-1-benzothiophene-2-carboxamide) to activate acoustic hedgehog signaling; (iv) IWP2 to suppress WNT signaling; and (v) FGF8 to shift the balance toward head lateralization.

[0133] Further maturation According to the method described herein, MGE progenitor cells can differentiate into cIN, preferably into postmitotic cIN, by exposing cells to neurotrophic factors (such as, but not limited to, glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF)) for a predetermined time. Preferably, the cells are simultaneously exposed to a notch inhibitor (such as DAPT). In some aspects, the predetermined 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.

[0134] Markers of cortical interneurons As used herein, the term "marker" or "cell marker" refers to a gene or protein that identifies a specific cell or cell type. A cell may have more than one marker. A marker can also refer to a "pattern" of markers that allows a specific set of markers to identify different cells or cell types.

[0135] Markers for cortical interneurons and / or cortical interneuron precursor cells have been described and are readily available to those skilled in the art, including, for example, SST, PV, GABA, calcium-binding protein, LHX6, RAX, FOXA2, FOXG1, OLIG2, MASH1, NKX6.2, VGLUT1, MAP2, CTIP2, SATB2, TBR1, DLX2, ASCL1, and ChAT.

[0136] In some respects, postmitotic cIN precursor cells are identified by the expression of one or more markers selected from FOXG1, PV, SST, calcium-binding protein, DCX, ASCL1, TUJ1, GABA, GAD1, VGAT, VGLUT1, and GAD67. In other respects, postmitotic cIN precursor cells are identified by the absence of expression of one or more markers selected from NKX2-1 and OLIG2, and optionally Ki67.

[0137] In some respects, differentiation into telencephalon cells is identified by the lack of expression of FoxG1 and / or RAX.

[0138] In some respects, differentiation into ventral terminal brain cells is identified by the expression of FoxG1 and DLX2 and / or the lack of EMX1 expression.

[0139] In some respects, differentiation into MGE progenitor cells is identified by the expression of one or more markers selected from FOXG1, NKX2-1, NKX2-2, ASCL1, SIX6, OLIG2, NKX6.2, DLX1 / 2, and LHX6.

[0140] In related aspects, postmitotic cIN precursor cells can be distinguished from MGE progenitor cells by detecting the expression of at least NKX2-1 and OLIG2. MGE progenitor cells are identified by the expression of NKX2-1 and the lack of expression of OLIG2, while postmitotic cIN precursor cells are identified by the expression of OLIG2 and the lack of expression of NKX2-1.

[0141] It is understandable that pluripotent cells and / or pluripotent cells can be human cells or mouse cells, and they can be selected from a group consisting of free embryonic stem cells, adult stem cells, neural stem cells, induced pluripotent stem cells, engineered pluripotent stem cells, primary progenitor cells, induced progenitor cells, and engineered progenitor cells.

[0142] Contact with SMAD inhibitors and / or Wnt signaling antagonists can be carried out simultaneously or sequentially. Contact may last from approximately 5 to approximately 30 days.

[0143] The methods disclosed herein can be used to generate cortical interneurons and their precursors in quantities and purity unattainable by existing technologies. In some embodiments, the large quantities of pure, functional cortical interneurons obtained using the methods of this disclosure can be used to study epilepsy, schizophrenia, autism, and other neurological disorders. Similarly, these cells can also be used in cell therapy.

[0144] Composition In a further embodiment, this disclosure provides a composition comprising one or more in vitro differentiated neuronal cells that generate one or more markers of cortical interneurons and / or cortical interneuron precursor cells, wherein the in vitro differentiated neuronal cells are generated by: (a) contacting pluripotent cells or pluripotent stem cells encapsulated in a synthetic hydrogel with two or more SMAD signaling inhibitors; (b) contacting the pluripotent cells or pluripotent stem cells with one or more Wnt signaling inhibitors; and (c) contacting the pluripotent cells or pluripotent stem cells with one or more SHH signaling activators.

[0145] The compositions disclosed herein may comprise a mixture of two or more cell types, wherein cortical interneurons comprise at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95% of the total number of cells.

[0146] The composition may comprise a mixture of two or more cell types, wherein NKX2.1+ / PV+ cortical interneurons comprise 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.

[0147] The composition may comprise a mixture of two or more cell types, wherein γ-aminobutyric acid (GABA) inhibits interneurons comprising 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.

[0148] Cortical interneurons or interneuron precursor cells can be modified with transgenes that express detectable biomarkers, such as, for example, CT-2 or green fluorescent protein (GFP). It should be understood that these detectable biomarkers can be interchanged with other detectable biomarkers without departing from this aspect of the disclosure.

[0149] The cortical interneuron precursor cells of this invention produce functional interneurons that exhibit the morphological, neurochemical, and electrophysiological properties of mature interneurons. Immature interneuron precursor preparations can... exist Matured under controlled in vitro culture conditions that mimic the in vivo neuronal environment. Alternatively, immature interneuron precursor cells mature after transplantation and migration into the cerebral cortex of a mammalian subject (e.g., a human subject). The immature interneuron precursor cells of the present invention, after transplantation into the cerebral cortex, can migrate extensively in a non-radial (i.e., tangential) manner. After migration, cortical interneuron precursor cells can mature into interneurons expressing albumin and Kv3.1, which exhibit a rapid spike action potential firing pattern. Alternatively, the cortical interneuron precursor cells of the present invention can mature into interneurons expressing somatostatin, exhibiting the characteristic rebound, adaptive, non-rapid spike firing pattern of this subgroup of interneurons. These somatostatin-expressing interneurons can further express neuropeptide Y.

[0150] Cortical interneuron precursor cells can mature into interneurons with a mean resting membrane potential of approximately -40 mV to approximately -70 mV. Over time, the mean resting membrane potential becomes more hyperpolarized, ranging from approximately -55 mV to approximately -70 mV.

[0151] Methods for treating symptoms and diseases The in vitro derivation of neurons from stem cells or progenitor cells has significant clinical implications and is crucial for disease modeling and drug screening. Disorders for which the method according to the invention is applicable include, but are not limited to, epileptic conditions such as epilepsy or infantile spasms; neuropsychiatric disorders such as autism, schizophrenia, anxiety disorders, and eating disorders; neurodevelopmental disorders such as holoencephaly or microcephaly; and Parkinson's disease. Early research using hPSCs primarily focused on neurodegenerative disorders known to affect specific types of neurons, 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., Cell146:318-331 (2011)) or motor neurons in amyotrophic lateral sclerosis (ALS) (Dimos et al., Science 321:1218-1221 (2008)) and motor neurons in spinal muscular atrophy (SMA) (Ebert et al., Nature 457:277-280 (2009)). Recent research suggests the potential to address complex neuronal 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)).

[0152] The cells disclosed herein can be delivered via intraparenchymal or intraventricular transplantation, as described in U.S. Patent Nos. 5,082,670 and 5,650,148 to Gage et al. and U.S. Patent Publication No. 20060141622 to Johe et al., all of which are hereby incorporated by reference in their entirety. Intraparenchymal transplantation can be achieved by injecting immature interneuronal precursor cells into the host brain parenchyma, or by surgically creating a cavity to expose the host brain parenchyma and then placing the cell graft within the cavity. Both methods can achieve parenchymal adhesion between the transplanted cells and the host brain tissue at transplantation and both contribute to anatomical integration between the transplanted tissue and the host brain tissue. Alternatively, the graft can be placed in the ventricle, such as the cerebral ventricle, or subdurally, for example on the surface of the host brain, separated from the host brain parenchyma by the intervening pia mater or arachnoid mater and pia mater. Transplantation to the ventricles is accomplished by injecting donor cells or by growing cells in a matrix such as 30% collagen to form a solid tissue block, which is then implanted into the ventricles to prevent graft displacement. For subdural transplantation, cells can be injected around the surface of the brain after a small incision is made in the dura mater. This is crucial if the graft is to become an integral part of the host's brain and survive throughout the host's life.

[0153] Leaving aside the issue of survival, the neuronal and progenitor cell transplantation described in this disclosure can successfully transplant a large number of cells, which can... exist The samples will be studied after maturing in vivo.

[0154] Treatment methods for neurodegenerative diseases In some embodiments, this disclosure provides methods for treating conditions and diseases associated with neurodegeneration, including, for example, epilepsy, Parkinson's disease (PD), and Alzheimer's disease (AD), methods comprising in vivo administration of cortical interneurons to a patient suffering from epilepsy, PD, or AD, these cortical interneurons being generated by the methods disclosed herein.

[0155] Treatment methods for mental illnesses and diseases In other embodiments, this disclosure provides methods for treating mental illnesses and disorders, including, for example, schizophrenia and autism-related conditions.

[0156] Unlike PD, ALS, or SMA, the key neuronal types for modeling schizophrenia or autism are not well-defined, and these studies have not attempted to directly identify neuronal subtypes. Recent progress has been made in establishing derivation schemes for 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).

[0157] However, inhibitory neurons, such as cortical interneurons, may play a particularly important role in schizophrenia or autism. Nature 468:187-193 (2010) and Lewis et al., Nat Rev Neurosci 6:312-324 (2005).

[0158] Current paradigms for modeling and treating mental illnesses employ 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 conducted in mixed neural cultures with unidentified neuronal subtypes and limited characterization of subtype-specific synapses and functional properties. Using the convergence of autopsy findings, attempts have been made to link genetic defects to mental illnesses, such as interneuron-associated Erbb4 receptors in schizophrenia. Fazzari et al., Nature464:1376-1380 (2010).

[0159] As disclosed herein, this disclosure provides a purified population of mature cortical interneurons that can be used as a model of human mental illnesses and diseases, and can be used in therapeutic regimens for treating such mental illnesses and diseases. Furthermore, the data presented herein demonstrate that cortical interneurons can be efficiently induced upon timed exposure to developmental signals.

[0160] While not wanting to be bound by theory, it is believed that hypothesized hESC-derived GABAergic interneurons receive synaptic input from other interneurons and excitatory mouse projection neurons. Cells exhibiting the neurochemical properties of cortical interneurons acquired fairly mature physiological characteristics 30 days after seeding on mouse cortical cultures. Although the mechanism of accelerated in vitro maturation of NKX2.1:GFP+ neurons in mouse cortical cultures is currently unclear, the data presented in this paper suggest that species-specific time factors are involved. Currently available data further indicate that... exist In vitro acquisition of synaptically active cortical interneurons can be used for modeling and treatment of cortical interneuron pathology in mental disorders, including but not limited to schizophrenia and autism.

[0161] Given the impact of PV interneuron dysfunction on schizophrenia, the generation of hESC-derived PV-expressing neurons and the presence of relatively fast-firing, maladaptive neurons in these cultures are of particular interest. (Beasley and Reynolds) Schizophr Res 24:349-355 (1997) and Woo et al., Am J Psychiatry 154:1013-1015 (1997). Rapidly firing PV+ cortical interneurons can be observed in the late prenatal development of primates, and they continue to mature into early adulthood. Anderson et al. Neuroscience 67:7-22 (1995) and Insel, Nature 468:187-193 (2010). Given the role of PV+ neurons under various pathological conditions, the data presented in this paper support modeling this dysfunctional neuronal state and treating these dysfunctional neuronal states by applying cortical interneurons and / or their precursors.

[0162] Therefore, in some aspects of these embodiments, this disclosure provides for the enrichment of cortical interneuron subpopulations, such as somatostatin+ and PV+ cells. MGE progenitor cells may be controlled by SHH signaling, with high SHH signaling levels promoting the generation of somatostatin+ cells and low SHH signaling levels promoting the generation of PV+ neurons. (Xu et al.) Neuron 65:328-340 (2010).

[0163] Any method known in the art for measuring gene expression can be used, particularly quantitative methods such as real-time quantitative PCR or microarrays, or methods using gene reporter expression, or qualitative methods such as immunostaining or cell sorting methods, to identify cells exhibiting specific biomarkers (including cell surface markers).

[0164] The following items list several embodiments of the present invention: Project 1. A thermally reversible polymer with improved stability over time, comprising formula (III): Where a, b, c, and d represent the mole fraction of the polymer; PEG n It is a polyethylene glycol polymer, and n is an integer from 1 to 2500; R 1 (If present) is any terminal group other than a primary amine; R 2 It is a lower alkyl group; R 3 (If present) is a functional group or a linker; and G 1 and G 2 Each component is independently selected from polymer segments, terminal groups, linkers, and linking modifiers.

[0165] Project 2. The thermally reversible polymer as described in Project 1, wherein the molecular weight (MW) of the polymer is about 0 to about 250 kilodaltons (kDa) or about 50 to about 200 kDa.

[0166] Project 3. A thermally reversible polymer as described in Project 1 or Project 2, wherein R 1 It is a C1-C6 alkoxy group selected from methoxy, ethoxy, n-propoxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy.

[0167] Project 4. A thermally reversible polymer as described in Project 3, wherein R 1 It is a methoxy group.

[0168] Item 5. A thermally reversible polymer as described in any one of Items 1 to 4, wherein R 2 The group consisting of methyl, ethyl, propyl, butyl, pentyl, isopropyl, isobutyl, isopentyl, tert-butyl, cyclopropyl, and cyclobutyl is selected.

[0169] Project 6. A thermally reversible polymer as described in Project 5, wherein R 2 It is butyl.

[0170] Item 7. The thermally reversible polymer as described in any one of Items 1 to 6, wherein R 3 It does not exist.

[0171] Project 8. A thermally reversible polymer as described in Project 1, wherein R 1 It is a methoxy group, R 2 It is butyl, and R 3 It does not exist, and the MW of the polymer is about 0 to about 250 kDa.

[0172] Item 9. The thermally reversible polymer as described in any one of Items 1 to 8, wherein PEG n Its molecular weight is approximately 1 to approximately 50 kilodaltons (kDa).

[0173] Item 10. The thermally reversible polymer as described in any one of Items 1 to 9, wherein R 3 It is a chemically selective functional group selected from thiols, alkynes, cyclooctynes, azides, phosphine, maleimide, alkoxyamine, aldehydes and their protected versions or precursors.

[0174] Item 11. A thermally reversible polymer as described in any one of Items 1 to 9, wherein R 3 It is selected from the following modifiers: heparin, hyaluronic acid, specific binding members, peptides, nucleic acids, gelatin, fibronectin, collagen, laminin, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin, progesterone, glucose, stromal cell-derived factor-1 (SDF-1), thymosin β-4, sound hedgehog factor (SHH), noggin, activin, transforming growth factor-β (TGF-β), FGF8, brain-derived neurotrophic factor (BDNF), glial cell-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).

[0175] Item 12. A thermally reversible polymer as described in any one of Items 1 to 11, wherein the thermally reversible polymer has one or more of the following properties: (a) an LCST of about 12°C to 32°C, preferably about 19°C to 23°C; (b) a stiffness of about 100 Pa to 8000 Pa, preferably about 100 Pa to 3000 Pa; and (c) a viscosity of about 100 cP to 1500 cP.

[0176] Item 13. A three-dimensional hydrogel comprising any one of items 1 to 12, a thermally reversible polymer, and a buffered aqueous solution.

[0177] Project 14. An in vitro method for generating a cell population rich in MGE progenitor cells from an initial human stem cell population, the method comprising: (a) Encapsulating the initial human stem cell population in the three-dimensional hydrogel described in Project 13; and (b) Contact the encapsulated human stem cells with at least one Small Mothers Against Decapentaplegic (SMAD) signaling inhibitor and at least one Wingless (Wnt) antagonist; and contact the cells with at least one Sound Hedgehog (SHH) signaling activator and an FGFR agonist to obtain a cell population rich in MGE progenitor cells expressing FOXG1 and at least one additional marker indicating MGE progenitor cells.

[0178] Item 15. The method as described in 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.

[0179] Item 16. The method of Item 14, wherein the contact with the at least one SMAD signaling inhibitor and the contact with the at least one Wnt antagonist are performed simultaneously or sequentially, and the duration of each contact is between about 5 days and about 30 days.

[0180] Item 17. The method of Item 16, wherein the contact of the cell with the at least one Wnt antagonist begins within 5 days, preferably 4 days, 3 days, 2 days or 1 day after the first contact of the cell with the at least one SMAD signaling inhibitor, preferably wherein the contact of the cell with the at least one Wnt antagonist begins simultaneously with the first contact of the cell with the at least one SMAD signaling inhibitor.

[0181] Item 18. The method of Item 14, wherein the at least one SMAD signaling inhibitor is selected from the group consisting of: SB431542, LDN-193189, Noggin PD169316, SB203580, LY364947, A77-01, A-83-01, BMP4, GW788388, GW6604, SB-505124, ledelimumab, metilumab, GC-I008, AP-12009, AP-110I4, LY550410, LY580276, LY364947, LY2109761, SB-505124, E-616452 (RepSox) ALK inhibitors), SD-208, SMI6, NPC-30345, KÏ26894, SB-203580, SD-093, activin-M108A, P144, soluble TBR2-Fc, DMH-1, doxorphine dihydrochloride, their derivatives and combinations thereof.

[0182] Item 19. The method of Item 18, wherein the at least one SMAD signaling inhibitor comprises SB431542 and LDN-193189.

[0183] Item 20. The method of Item 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, preferably wherein the at least one Wnt antagonist includes IWP-2.

[0184] Item 21. The method of Item 14, wherein the at least one SHH signaling activator is selected from the group consisting of Smoothened agonists (SAG), SAG analogs, SHH, C25-SHH, C24-SHH, purinemorphine, Hg-Ag, derivatives thereof, and combinations thereof.

[0185] Project 22. The method of Item 21, wherein (i) the contact of the cells with the at least one SHH signaling activator is completed within approximately 5 to approximately 30 days after the start of the process, preferably within approximately 18 to 23 days after the start of the process, more preferably within approximately 19 to 22 days after the start of the process, and even more preferably within approximately 20 or 21 days after the start of the process; (ii) the first contact of the cells with the at least one SHH signaling activator is spaced approximately 0 to approximately 10 days after the first contact of the cells with the at least one SMAD signaling inhibitor and from the first contact of the cells with the at least one WNT signaling inhibitor, preferably within approximately 0 days; (iii) the first contact of the cells with the at least one SMAD signaling inhibitor is spaced approximately 0 to 4 days after the first contact of the cells with the at least one Wnt antagonist; (iv) the contact of the cells with the at least one SMAD signaling inhibitor is completed within 6 to 14 days after the start of the process; and / or (v) the contact of the cells with the at least one Wnt antagonist is completed within 6 to 8 days after the start of the process, preferably within approximately 7 days after the start of the process.

[0186] Item 23. The method of Item 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.

[0187] Item 24. The method as described in 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.

[0188] Item 25. The method as described in 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 contains MGE progenitor cells.

[0189] Item 26. The method of Item 14, the method further comprising (c) after a predetermined time, contacting the cells with at least one neurotrophic factor (e.g., GDNF, BDNF) and an optional notch inhibitor (e.g., DAPT) to generate a cell population rich in differentiation-inhibiting GABAergic cortical interneurons (cINs) expressing FOXG1 and at least one additional marker indicating cIN cells.

[0190] Item 27. The method of Item 26, wherein the method comprises contacting the cells with at least one neurotrophic factor and a notch inhibitor.

[0191] Item 28. The method of Item 27, wherein the method includes contacting the cells with GDNF, BDNF and DAPT.

[0192] Item 29. The method of any one of Items 26 to 28, wherein after contacting the cells with the at least one SMAD signaling inhibitor, the at least one Wnt antagonist and the at least one SHH signaling activator, the cells are contacted with at least one neurotrophic factor and optionally a notch inhibitor.

[0193] Item 30. The method of any one of items 26 to 29, wherein contacting the cells with at least one neurotrophic factor and optionally a notch inhibitor is completed within 7 to 30 days after initiation, and / or contacting the cells with at least one neurotrophic factor and optionally a notch inhibitor is completed at least about 10 days, at least about 12 days, or at least about 14 days after initiation.

[0194] Item 31. The method of Item 26, wherein the at least one additional biomarker is selected from the group consisting of PV, SST, calcium-binding protein, DCX, ASCL1, TUJ1, GABA, GAD1, VGAT, vGLUT1, and GAD67.

[0195] 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 albumin (PV).

[0196] Item 33. The method of any one of items 26 to 32, wherein less than about 5% of the obtained cIN cell population expresses Ki67.

[0197] Item 34. A composition comprising a cell population produced by the method of any one of Items 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 wherein the method does not include a step of purifying or further enriching the MGE progenitor cells after step (b).

[0198] Item 35. A composition comprising a cell population produced by the method of any one of Items 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 ​​cells, preferably wherein the method does not include a step of purifying or further enriching cIN cells after step (c).

[0199] Item 36. The composition of Item 35, 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 NKX2.1 - / PV + .

[0200] Item 37. Use of the composition described in any one of items 34 to 36 in the treatment of neurological disorders.

[0201] Item 38. Use as described in Item 37, wherein the neurological condition is epilepsy.

[0202] Project 39. An in vitro method for generating a cell population rich in MGE progenitor cells from an initial human stem cell population, the method comprising: (a) Encapsulating an initial human stem cell population in a three-dimensional hydrogel; and (b) Contact the encapsulated human stem cells with at least one Small Mothers Against Decapentaplegic (SMAD) signaling inhibitor and at least one Wingless (Wnt) antagonist; and contact the cells with at least one Sound Hedgehog (SHH) signaling activator and an FGFR agonist to obtain a cell population rich in MGE progenitor cells expressing FOXG1 and at least one additional marker indicating MGE progenitor cells.

[0203] Item 40. The method as described in Item 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.

[0204] Item 41. The method of Item 39, wherein the contact with the at least one SMAD signaling inhibitor and the contact with the at least one Wnt antagonist are performed simultaneously or sequentially, and the duration of each contact is between about 5 days and about 30 days.

[0205] Item 42. The method of Item 41, wherein the contact of the cell with the at least one Wnt antagonist begins within 5 days, preferably 4 days, 3 days, 2 days or 1 day after the first contact of the cell with the at least one SMAD signaling inhibitor, preferably wherein the contact of the cell with the at least one Wnt antagonist begins simultaneously with the first contact of the cell with the at least one SMAD signaling inhibitor.

[0206] Item 43. The method of Item 39, wherein the at least one SMAD signaling inhibitor is selected from the group consisting of: SB431542, LDN-193189, Noggin PD169316, SB203580, LY364947, A77-01, A-83-01, BMP4, GW788388, GW6604, SB-505124, ledelimumab, metilumab, GC-I008, AP-12009, AP-110I4, LY550410, LY580276, LY364947, LY2109761, SB-505124, E-616452 (RepSox) ALK inhibitors), SD-208, SMI6, NPC-30345, KÏ26894, SB-203580, SD-093, activin-M108A, P144, soluble TBR2-Fc, DMH-1, doxorphine dihydrochloride, their derivatives and combinations thereof.

[0207] Item 44. The method of Item 43, wherein the at least one SMAD signaling inhibitor comprises SB431542 and LDN-193189.

[0208] Item 45. The method of Item 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 includes IWP-2.

[0209] Item 46. The method of Item 39, wherein the at least one SHH signaling activator is selected from the group consisting of Smoothened agonists (SAG), SAG analogs, SHH, C25-SHH, C24-SHH, purinemorphine, Hg-Ag, derivatives thereof, and combinations thereof.

[0210] Project 47. The method of Item 46, wherein (i) the contact of the cells with the at least one SHH signaling activator is completed within approximately 5 to approximately 30 days after the start of the process, preferably approximately 18 to 23 days after the start of the process, more preferably approximately 19 to 22 days after the start of the process, and even more preferably within approximately 20 or 21 days after the start of the process; (ii) the first contact of the cells with the at least one SHH signaling activator is spaced approximately 0 to approximately 10 days after the first contact of the cells with the at least one SMAD signaling inhibitor and from the first contact of the cells with the at least one WNT signaling inhibitor, preferably approximately 0 days; (iii) the first contact of the cells with the at least one SMAD signaling inhibitor is spaced approximately 0 to 4 days after the first contact of the cells with the at least one Wnt antagonist; (iv) the contact of the cells with the at least one SMAD signaling inhibitor is completed within 6 to 14 days after the start of the process; and / or (v) the contact of the cells with the at least one Wnt antagonist is completed within 6 to 8 days after the start of the process, preferably within approximately 7 days after the start of the process.

[0211] Item 48. The method of Item 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.

[0212] Item 49. The method as described in Item 39, 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.

[0213] Item 50. The method as described in Item 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 contains MGE progenitor cells.

[0214] Item 51. The method of Item 39, the method further comprising (c) after a predetermined time, contacting the cells with at least one neurotrophic factor (e.g., GDNF, BDNF) and optionally a notch inhibitor (e.g., DAPT) to generate a cell population rich in differentiation-inhibiting GABAergic cortical interneurons (cINs) expressing FOXG1 and at least one additional marker indicating cIN cells.

[0215] Item 52. The method of Item 51, wherein the method comprises contacting the cells with at least one neurotrophic factor and a notch inhibitor.

[0216] Item 53. The method of Item 52, wherein the method includes contacting the cells with GDNF, BDNF and DAPT.

[0217] Item 54. The method of any one of items 51 to 53, wherein after contacting the cells with the at least one SMAD signaling inhibitor, at least one Wnt antagonist and at least one SHH signaling activator, the cells are contacted with at least one neurotrophic factor and optionally a notch inhibitor.

[0218] Item 55. The method of any one of items 51 to 54, wherein contacting the cells with at least one neurotrophic factor and optionally a notch inhibitor is completed within 7 to 30 days after initiation, and / or contacting the cells with at least one neurotrophic factor and optionally a notch inhibitor is completed at least about 10 days, at least about 12 days, or at least about 14 days after initiation.

[0219] Item 56. The method of Item 51, wherein the at least one additional biomarker is selected from the group consisting of PV, SST, calcium-binding protein, DCX, ASCL1, TUJ1, GABA, GAD1, VGAT, vGLUT1, and GAD67.

[0220] Item 57. The method of any one of Items 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 albumin (PV).

[0221] Item 58. The method of any one of items 51 to 57, wherein less than about 5% of the obtained cIN cell population expresses KI67.

[0222] Item 59. A composition comprising a cell population produced by the method of any one of Items 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 wherein the method does not include a step of purifying or further enriching the MGE progenitor cells after step (b).

[0223] Item 60. A composition comprising a cell population produced by the method of any one of Items 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 ​​cells, preferably wherein the method does not include a step of purifying or further enriching cIN cells after step (c).

[0224] Item 61. The composition of Item 60, 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 NKX2.1 - / PV + .

[0225] Item 62. Use of the composition described in any one of Items 59 to 61 in the treatment of neurological disorders.

[0226] Item 63. The use as described in Item 62, wherein the neurological disorder is epilepsy.

[0227] Example The following examples illustrate preferred embodiments of the invention and are not intended to limit the scope of the invention in any way. While the invention has been described in conjunction with its preferred embodiments, those skilled in the art should be able to understand various modifications thereto upon reading this application.

[0228] Example 1 The generation of cIN from human stem cells requires multiple factors, precise time control, and a relatively long maturation period. Therefore, three-dimensional hydrogels should remain stable during these relatively long periods.

[0229] The hydrogel of an acrylamide polymer comprising the following structure disclosed in U.S. Patent Publication No. 2024 / 0294713 was initially... especially Its stability was tested over the duration of cIN generation from human stem cells: Hydrogels containing such acrylamide polymers are unstable throughout the 35-day differentiation period and therefore cannot generate cINs from human stem cells, regardless of the tested molecular weight (0 kDa to 500 kDa). These hydrogels also exhibit unsuitable peak stiffness ranges (stability) (<1200 Pa), high viscosity (>2000 cP), low stability / high swelling ratio in aqueous environments (>1.5), and high LCST (>25). These gels fail to maintain their structure for extended periods in aqueous environments or under the shear stresses of mixing in rotating flasks or bioreactors. Finally, these gels are poorly extruded and encapsulated for cells, exhibiting low sphericity (<0.7), large bead diameters (>4 mm), and a high tail formation ratio (>0.5).

[0230] Therefore, structural improvement schemes to enhance stability were tested.

[0231] First, determine which acrylate with the following structure will be used in the polymer (see [link to polymer description]). Figure 1 (The process in the middle): Corresponding to equation III, where R 1 It is a methoxy group, R 2 It is butyl, R 4 It is methyl, and (d) is absent, and the polymer has a MW of 250-500 kDa, which, relative to acrylamide, leads to a decrease in LCST, an increase in gel stiffness, a decrease in viscosity, and an improvement in gel stability.

[0232] The results are shown in Table 1 below. Figure 2 As shown in the image.

[0233] Table 1

[0234] Next, the properties of the acrylate polymer with the above structure were modified to further improve the stability of the hydrogel over time. By changing the initiator concentration, polymer backbones of various molecular weights were generated. Studies showed that reducing the overall molecular weight of the polymer (molecular weight is a function of backbone length, which directly controls the polymer length; shortening the backbone length corresponds to a decrease in polymer molecular weight) increases stiffness, maintains LCST, reduces liquid phase viscosity, and improves stability (all functional groups are defined as described above); the results are shown in Table 2 below. Figure 3 The text shows: Table 2

[0235] Therefore, this study determined that three-dimensional hydrogels containing low molecular weight acrylate-based polymers offer significant improvements over existing thermally reversible polymers, for example, in the three-dimensional culture of stem cells to generate cINs. In particular, the three-dimensional hydrogels containing low molecular weight acrylate-based polymers exhibit greater stability, enabling the long-duration differentiation process required for cIN generation, reducing liquid phase viscosity for efficient cell encapsulation, and possessing appropriate stiffness to support neural growth while maintaining favorable LCST.

[0236] The effect of the molecular weight of PEG in modified Form III polymers on the properties of three-dimensional hydrogels was investigated. Surprisingly, the study found that PEG with a length of 1 kJ or less resulted in poor gel performance at a matched weight percentage (15 wt%), while PEG with a length of 10 kJ or more hindered gel flexibility and nodal stability. Therefore, a PEG molecular weight of approximately 5 kJ was determined to be optimal. See also Figure 4 .

[0237] Next, the effect of the weight ratio of PEG to the polymer backbone in the modified III polymer was tested. The study found that increasing the PEG to PNIPAAm ratio above 1:2 improved synthesis and encapsulation performance, as well as cell growth. See Table 3 below. Figure 5A , Table 3

[0238] Increasing the weight ratio of PEG to PNIPAAm to greater than 1:2 increases gel stiffness, decreases gel viscosity, increases gel stability, and decreases gel LCT. See also Figure 5A Unrestricted by theory, these advantages may stem from the increased involvement of PNIPAMAm in gel node formation and stronger association to support temperature-based gel formation.

[0239] Increasing the PEG to PNIPAAm copolymer ratio to greater than 1:2 also improves cell encapsulation, as the encapsulated beads exhibit reduced bead diameter, reduced bead volume, increased bead sphericity, and a lower fraction of tail-containing beads after gravity-drip encapsulation. See also Figure 5B .

[0240] When the weight ratio of PEG to PNIPAAm is greater than 1:2, other improvements include enhanced encapsulation and cell performance, such as faster flow rates and lower shear stress, higher immediate cell viability, higher 7-day cell viability, and higher cell yield. See also Figure 5C .

[0241] Next, the position R of Equation III was studied. 2The effect of the alkyl group at the position. In short, polymers of formula III (R...) were prepared and tested. 1 = Methoxy, R 4 = Methyl, (d) = 0, and R 2 (It is n-butyl or isobutyl): The conformations of the side-chain alkyl groups (n-butyl and isobutyl) were determined to be significant. Surprisingly, n-butyl exhibited higher stiffness, similar viscosity, higher gel stability, and lower gel LCST compared to isobutyl. See Figure 6.

[0242] The conformation of the main-chain acrylate group was identified as significant because the methyl group at position R4 (from butyl methacrylate) in Formula III exhibits higher stiffness, similar viscosity, higher gel stability, and lower gel LCST compared to the hydrogen at position R4. See Figures 6.5A to 6.5B. Furthermore, the polymer of Formula III with a methyl group at position R4 exhibits smaller bead diameter and volume, higher bead sphericity, and similar bead tail number formation. See Figure 6.5C.

[0243] Polymers of Formula III, as described herein, possess several characteristics that make them uniquely suited for encapsulating and suspending cells in three-dimensional hydrogel bioreactors for large-scale production in tanks for long-term cell culture / differentiation protocols, such as generating functional neurons from stem cells. Current hydrogels (containing encapsulated differentiated cells, such as neurons) can be used for therapeutic purposes in humans. Current systems maintain cell (e.g., stem cells) viability and avoid the need for harsh methods of cell recovery from the system.

[0244] The polymer of Formula III can be functionalized at multiple positions, including both side-chain and main-chain positions. For example, functionalization can be achieved by adding a monoamine-PEG with a corresponding functional group at the end, as shown below. Creating R at the side-chain position 1 Polymers of Formula III containing the following groups: R 1 = Methoxy group; R 1 = Hydroxyl group; R 1 = Acrylate; R 1 = Biotin; R 1 =DBCO. See also Figure 7 .

[0245] Created in main chain location R 3 Polymers of Formula III containing the following groups: R 3 = Methacrylate; R 3 = Maleimide; R 3 =DBCO. See also Figure 8 Functionalization is achieved by reacting free NASSI groups with amine-conjugated functional groups before isopropylamine saturation.

[0246] To demonstrate the ability of the polymer of formula III to bind and release proteins in a stem cell differentiation environment, position R will be... 1 Polymers of formula III containing acrylate react with thiol-proteins (FGF and heparin) via thiol-Michael addition. See also Figure 9 .

[0247] Surprisingly, it was discovered (location R) 1 The PEG group at position R has a significant impact on polymer synthesis. Specifically, the PEG group at position R... 1 The group at the [specific location] is not a primary amine, but another group, which allows for higher reaction volume / volume%, higher solvent yield per unit volume, and more reproducible synthesis. See [link to documentation]. Figure 10A The R-terminus generates amine-terminated compounds. 1 The synthesis requires the use of bifunctional diaminoPEG monomers, which carries the risk of unintended covalent crosslinking. This can significantly impact polymer properties, reproducibility, and viscosity, and may prevent polymer reliquefaction. To avoid this, synthesis using diaminoPEG requires very dilute reactions (<2 wt% / volume), limiting polymer yield per unit volume at scale-up and resulting in batch-to-batch variability. Using monoamine-PEG eliminates the possibility of unintended crosslinking, enabling more reproducible synthesis and scale-up.

[0248] Furthermore, it was found that solvent selection for reaction 1 was crucial for the successful synthesis of the polymer, the introduction of each monomer at the desired molar ratio, and precise control of the molecular weight. Some solvents exhibited ideal solubility for all monomers, resulting in high reaction efficiency and complete monomer polymerization (A); while some solvents could only polymerize soluble monomers and not all monomers (B); and some solvents inhibited complete polymerization (CD); and some polymers were unsuitable for any reaction (EF). Solvents tested included standard and anhydrous polar protic solvents, polar aprotic solvents, and nonpolar solvents of various structural conformations. Solvents tested included acetone, acetonitrile, benzene, chloroform, dichloromethane, dimethylformamide, dimethyl sulfoxide, dioxane, ethyl acetate, pyridine, ethanol, methanol, tetrahydrofuran, toluene, and water. See also Figure 10B Example 2 Several alternative neural induction protocols were evaluated for the generation of postmitotic cIN precursor cells from human stem cells in a three-dimensional hydrogel, using different culture medium formulations and different times of component addition.

[0249] Specifically, 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), all of which are incorporated herein by reference in their entirety. For each protocol, alternatives that do not inhibit Wnt were tested. In the Studer protocol, an alternative to SB was tested by replacing AZD (an ERK inhibitor).

[0250] According to Maroof et al., dual inhibition of SMAD and Wnt lasted for 9 days, SHH activation lasted for 8 days, and no final specialization / maturation factor was added. According to Nicholas et al. of Dual inhibition of SMAD and Wnt lasted for 14 days, SHH activation lasted for 35 days, and BDNF / DAPT was added for 11 days: According to Kim et al., dual inhibition of SMAD and Wnt lasted for 7 days, FGF8 addition lasted for 14 days, SHH activation lasted for 21 days, and BDNF / GDNF / DAPT addition lasted for 14 days. On day 18, the outcome of the neural induction protocol was evaluated by assessing the production of MGE. Figure 11A ).like Figures 11B to 11E As shown, by day 18, using only Kim et al.'s method, MGE progenitor cells characterized by the expression of FoxG1, NKX2-1, and DLK1 / 2 could be effectively generated in a three-dimensional hydrogel.

[0251] On day 35, the outcome of the neural induction protocol was assessed by evaluating the generation of cortical interneurons. Figure 12A ).like Figures 12B to 12F As shown, by day 35, the protocol used by Kim et al. effectively generated MGE-derived cortical interneurons or cortical interneuron precursor cells characterized by expression of FoxG1, Calbindin, Gad1, and downregulation of Nkx2-1. Notably, while not as effective as Kim et al.'s protocol, the protocol tested by Maroof et al. in a three-dimensional hydrogel was significantly superior to the original protocol performed in two-dimensional cell cultures (see [link to original protocol]). Figures 12A to 12EFurthermore, all culture medium alternatives that do not inhibit Wnt or replace SB with AZD are less efficient in generating MGE progenitor cells.

[0252] Example 3 A proof-of-concept study was conducted using a low molecular weight acrylate backbone three-dimensional hydrogel to demonstrate that encapsulated hPSCs can be stably derived into postmitotic cINs with therapeutic potential for treating neurological disorders such as epilepsy.

[0253] method Preparation of three-dimensional PEG-PNIPAAM hydrogel A two-step synthesis process is used to produce thermally reversible graft copolymers. Figure 1 The copolymer is named PNIPAAm-co-PNASI-co-MA, where PEG represents the hydrophilic block, PNIPAAm represents the hydrophobic block, and alkyl side groups (described here as butyl chains, but may include any alkyl chain) act as temperature-transition moieties. To produce the thermally reversible graft copolymer, a mixture of NIPAAm, N-acryloyloxysuccinimide (NASI), and alkyl-chain methacrylates was first copolymerized via standard free radical polymerization. After reprecipitation and drying, the resulting functionalized copolymer was then mixed with monoamine-terminated PEG blocks. The amine-terminated groups were attached to the PNIPAAm-co-PNASI-co-MA backbone via an amidation reaction of amine and N-hydroxysuccinimide (NHS). Finally, the remaining NHS groups were converted to PNIPAAm by adding isopropylamine, and the resulting polymer was dried, dialyzed, and lyophilized.

[0254] hPSC amplification As recommended by the manufacturer, human PSC cells (H9 human embryonic stem cells (WA09, WiCell, Madison, WI, passaged 53-58 times)) were maintained on Matrigel (BD, San Jose, CA) in E8 medium (Gibco, Billings, MT) and passaged using Versene (Thermo Fisher, Waltham, MA).

[0255] Cell encapsulation, differentiation, and harvesting For differentiation, hPSCs were dissociated using Accutase (Stem Cell Technologies, Vancouver, BC, Canada) and encapsulated in PEG-PNIPAAM hydrogel at a final weight / volume concentration of 250,000 cells / ml. The hydrogel and cells were mixed on ice, and then 50 μL droplets were produced and extruded into well plates, as shown. Figure 13As shown in b, there are a total of 5 gel droplets in each well. The plate was incubated at 37°C for 15 min to gel and form dome-shaped beads. Complete E8 medium supplemented with Rock Inhibitor Y-27632 (Selleck Chemicals, Houston, TX) was heated to 37°C, followed by the addition of (final 5% V / Volume medium / gel), and the cells were incubated at 37°C and 5% CO2. Fifty percent of the medium was replaced daily to ensure the plate remained above 33°C. After culturing under amplification conditions for 48 hours, the medium was replaced with differentiation medium containing DMEM, knockout serum substitute (KSR, 20%), 2 mM L-glutamine, and 10 μM β-mercaptoethanol (all from Thermo Fisher). For neural induction, cells were treated with LDN193189 (100 nM, Stemgent, Cambridge, MA) from D0 to D14 and with SB431542 (10 μM, Tocris, Minneapolis, MN) from D0 to D7. For MGE induction, cells were treated with IWP2 (5 μM, SelleckChem) from D0 to D7, with SAG (0.1 μM, XcessBio, Chicago, IL) from D0 to D21, and with FGF8 (100 ng / ml, Peprotech) from D8 to D21. On D22, the medium was changed to 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 a specified time point, on a rotating platform, in the presence of Accumax (Innovative Cell Technologies, San Diego, CA) and TrypLE (Thermo Fisher), aggregates were dissociated into individual cells.

[0256] Cell counting and viability analysis Cells were stained using AOPI and counted using a K2 image capture device and Matrix software (PerkinElmer, Waltham, MA) to obtain total cell count and viability percentage.

[0257] Flow cytometry Differentiated cells were dissociated and fixed in CytoFix / CytoPerm solution (BD) for 20 min, followed by washing with Perm / Wash (BD). For staining, cells were incubated with the primary antibody for 30 min. After washing with Perm / Wash, Alexa647-conjugated secondary antibody (Thermo Fisher) was added, and the cells were incubated for another 30 min. 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.

[0258] Immunocytochemistry For immunofluorescence staining, fixed cells were incubated with Intercept blocking buffer (LI-CORBiosciences, Lincoln, NE) for 30 min and permeabilized with 0.25% Triton-x for 10 min. Cells were then incubated overnight at 4°C with primary antibody diluted in blocking buffer. After rinsing with PBS, samples were incubated with fluorescently labeled secondary antibody (Alexa 488 or Alexa 647 labeled IgG; Thermo Fisher) and Hoechst 33342 (4 mg / ml) in blocking buffer at room temperature for 1 hour. After rinsing with PBS, images were created using a Cytation 5 imaging system (Agilent) and analyzed using FIJI image analysis software (Schindelin, J. et al. Fiji: An open-source platform for biological-image analysis). Nature Methods Volume 9 Preprint at https: / / doi.org / 10.1038 / nmeth.2019 (2012)) Analyze the image.

[0259] Quantitative PCR Total RNA was prepared using the RNeasy kit (Qiagen, Germantown, MD), and cDNA was generated from the total RNA using the RT2 first-strand kit (Qiagen). For quantitative analysis of transcript expression, real-time PCR was performed using the RT2 SYBR GreenqPCR Mastermixes (Qiagen) and the AriaMX real-time PCR system (Agilent). Primers were designed using the PrimerQuest tool (Coralville, IA) for integrative DNA technology. mRNA expression levels for each gene were normalized to ACTB gene expression levels. Relative values ​​were calculated by setting the normalization value for the control group to 1.

[0260] result Human pluripotent stem cells were encapsulated in a thermoreversible hydrogel for differentiation into cortical interneurons. The copolymer hydrogel used in this paper is based on hydrophilic poly(ethylene glycol) (PEG) and temperature-sensitive poly(N-isopropylacrylamide) (PNIPAAm). When heated above the lowest critical solution temperature (LCST), the hydrophobicity of the PNIPAMAm component increases, leading to micelle formation, essentially a physical "crosslinking" of the PEG-PNIPAAm polymer. Figure 13 a). This hydrogel can be used to encapsulate and differentiate hPSCs in scaled-down (well plate) models and stirred cultures (including perfusion stirred tank bioreactors). Simply extruding a droplet of hydrogel containing a single cell or small cell cluster into a warm culture medium forms a gel capsule containing these cells. Figure 13 b and Figure 13 c). This encapsulation method can establish a fully defined, synthetic, xenogeneic, and scalable platform for manufacturing clinically relevant cell types.

[0261] The feasibility of differentiating human pluripotent stem cells (hPSCs) into GABAergic cortical interneuron precursor cells encapsulated in hydrogels was tested using well-established culture media (Kim, TG, et al.). Interneurons were efficiently selected from human pluripotent stem cells through dorsoventral and cephalocaudal modulation. Stem Cells 32, (2014) Figure 13d). In short, human embryonic stem cells (WA09) were seeded into hydrogel beads and expanded for 48 hours in minimal proliferation medium (Gibco-E8) supplemented with a 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 hPSC differentiation into the neuroectodermal lineage, the ALK2 / 3 inhibitor LDN193189 and the ALK5 / 7 inhibitor SB431542 were used, 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, the Wnt pathway inhibitor IWP2 was used to promote cephaladization of early neuroectoderm, followed by inhibition of dorsalization (Kim, TG, 2014). Sound hedgehog (SHH) pathway activator (SAG) was used to define the distinction between MGE and LGE. To counteract the caudalizing effect of SHH activation, FGF8 was added to shift the balance towards cephaladization, thereby effectively generating MGE progenitor cells (Kim, TG, 2014). Finally, to promote further differentiation of MGE progenitor cells into cIN and further promote neural maturation, glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), and the γ-secretase inhibitor DAPT were added.

[0262] To monitor the pattern-forming potency of ventral telencephalon, MGE progenitor cells, and cIN progenitor cells, quantitative PCR (qPCR), flow cytometry (FC), and immunocytochemistry (ICC) were performed using previously described markers characterizing these stages. Figure 13 e) (Marín, O. Human cortical interneurons take their time. Cell Stem CellVolume 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 cells 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, A. M. et al. Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells. Cell Stem Cell 12, (2013)).

[0263] Efficiently differentiating hPSCs into GABAergic cortical interneurons on a three-dimensional hydrogel platform Flow cytometry analysis allowed us to determine cell identity and purity at different time points during differentiation. Ten days after differentiation, hPSCs encapsulated in a 3D hydrogel began expressing FoxG1, an early marker of ventral telencephalon progenitors (Fig. 14a and 14b). By day 18, an average of 88.44% of cells expressed FoxG1, and 78.4% expressed the MGE marker NKX2-1, indicating strong pattern formation of telencephalon-derived MGE progenitors (Fig. 14a and 14b). With further maturation, by day 35, cells exhibited high expression of FoxG1 (78.14%) and a significant downregulation of NKX2-1 to 22.52%, consistent with the post-mitotic cIN phenotype (Fig. 14a and 14b). qPCR and ICC analyses were performed to confirm the MGE progenitor and cIN phenotype (Fig. 51). qPCR analysis showed that, compared with undifferentiated hPSCs, by day 18, the expression of FoxG1 and DLX2, markers of ventral telencephalon and MGE progenitor cells, was higher, and SST was also upregulated, indicating early induction of genes characterizing the mature cIN ​​cell population derived from MGE (Fig. 15a). At day 35 of differentiation, cells exhibited upregulation of cIN-like genes (such as GAD1 (glutamate decarboxylase, involved in GABA biosynthesis) and calcium-binding protein (CALB1)) and further upregulation of SST (Fig. 15a). Consistent with this qPCR analysis, ICC analysis at day 35 showed a high percentage of cells expressing FoxG1, consistent with the MGE origin of these cells, high synthesis of the neural cell adhesion protein NCAM, high levels of GABA (85.1% GABA+), and mature cIN ​​markers PV (84.5% PV+) and SST (42.3% SST+). Furthermore, ki67 was less than 1%, indicating that most of these cells were indeed post-mitotic (Fig. 15b and Fig. 15c).

[0264] Compared with standard two-dimensional culture methods, the thermoreversible hydrogel culture platform can significantly improve the conversion of hPSCs to GABA-functionalized cells. The vitality and efficacy of interneuron differentiation in the layer.

[0265] Next, we attempted to compare the effectiveness of the synthetic three-dimensional hydrogel platform for cIN generation with that of standard two-dimensional methods (Maroof, AM et al., Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells). Cell Stem Cell12, (2013)). We first compared the viability after harvest. After 35 days of differentiation, cells harvested from the three-dimensional hydrogel, which grew as cell aggregates, showed higher (>80%) viability, while cells harvested from the two-dimensional culture showed lower viability (approximately 20%) (Figs. 16a and 16b). This indicates that the method of gentle harvesting and dissociation from the thermally reversible hydrogel significantly improves cell viability at harvest compared to standard two-dimensional culture. Furthermore, we evaluated the differentiation potency of cells encapsulated in the novel formula (III) hydrogel compared to cells produced in two dimensions. We found that by day 10, the percentage of cells expressing FoxG1 in the three-dimensional culture was approximately 800-fold higher than in the two-dimensional culture (Fig. 16c). By day 18, the percentage of FoxG1+ cells in the three-dimensional culture was approximately 100-fold higher than in the two-dimensional culture, and NK2X-1 expression was approximately 10-fold higher (Fig. 15c). By day 35, we obtained approximately 100-fold higher FoxG1 expression in three dimensions compared to two dimensions (Figure 16c). Overall, these results demonstrate that when hPSCs are encapsulated and grown as aggregates in three-dimensional synthetic hydrogels, they exhibit greater efficacy in differentiating into cINs compared to standard two-dimensional methods.

[0266] discuss We have demonstrated that when hPSCs are encapsulated in a three-dimensional hydrogel, they can robustly differentiate into MGE progenitor cells and cINs. By day 18, expression of the MGE progenitor marker FoxG1 exceeded 80%, and Nkx2-1 expression exceeded 75%. By day 35, FoxG1 expression exceeded 75%, while cells expressed high percentages of cIN markers PV, SST, GAD1, and GABA, whereas Nkx2-1 expression was strongly downregulated to only about 22%. This, along with the near absence of Ki67 expression, indicates that a three-dimensional thermoreversible hydrogel platform can effectively generate mature postmitotic cINs.

[0267] Furthermore, using the same culture medium composition, a side-by-side comparison was made between standard two-dimensional cell generation methods and the synthetic three-dimensional hydrogel process. The results showed the remarkable superiority of the synthetic three-dimensional hydrogel culture platform, with approximately 800-fold higher percentages of FoxG1+ cells by day 18 and approximately 100-fold higher by day 35. In addition, the gentler harvesting process resulted in approximately 4-fold higher cell viability compared to cells isolated from a two-dimensional platform. Studies have shown that hPSCs… exist The process of functional differentiation and maturation into cIN in vitro is a long period of time, similar to the long maturation process of cIN during human brain development (Marín, O. Human cortical interneurons take their time). Cell Stem CellVolume 12). Interestingly, biomarker expression analysis of cells harvested from the novel type (III) hydrogel on day 35 showed an accelerated maturation timeframe compared to results currently reported using conventional culture methods. Nkx2-1 was reportedly downregulated under the following conditions: exist Five weeks after transplantation of 5-week-old cells in vivo (Kim, TG et al. Efficient specification of interneurons from human pluripotent stem cells by dorsoventral and rostrocaudal modulation). Stem Cells 32, (2014)), after 30 weeks of differentiation (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, NKX2 was downregulated to about 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). Furthermore, SST expression was only detected 20–30 weeks after differentiation (approximately 12–40%) (Nicholas CR et al., ibid.) or 5 months post-transplantation (Kim T.G. et al., ibid.).

[0268] Regarding PV expression, it has been reported that only 10% of cells express this protein after 15 weeks of differentiation (Nicholas CR et al., ibid.), or existOnly 8-10% of cells expressed this protein 4-5 months after in vivo transplantation (Kim TG et al., ibid.; Zhu, Q. et al., ibid.). In contrast, using the current method, 84.5% of PV+ cINs were detected at day 35. Previously, it was reported that culturing NKX2-1+ MGE progenitor cells on mouse cortical extracts accelerated their maturation to cIN timeframe, and PV expression was detectable 30 days after in vitro differentiation. This suggests that the presence of certain cell-cell interactions and / or signaling molecules is key to accelerating this maturation process, although the mechanisms involved are not yet fully understood (Maroof, AM et al., Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells). Cell Stem Cell 12, (2013)). Without being theoretically limited, the method described herein can capture these key signaling molecules secreted by cells in a hydrogel. environment In this process, cell-cell interactions are promoted, and autocrine and paracrine signaling is enhanced, while these signaling molecules would otherwise be washed away in conventional two-dimensional cultures or three-dimensional suspension platforms. In summary, the data presented in this paper demonstrate that robust differentiation from hPSCs to cINs can be achieved in three-dimensional synthetic hydrogels, such as the novel formula (III), with significantly higher viability and differentiation potency compared to standard two-dimensional cultures, and an accelerated differentiation cycle compared to the maturation timeframe reported using standard two-dimensional cultures or hybrid two-dimensional suspension systems.

[0269] Example 4 A proof-of-concept study was conducted using a low molecular weight acrylate backbone three-dimensional hydrogel (Formula III) to demonstrate robust amplification of encapsulated hPSCs.

[0270] method Preparation of three-dimensional PEG-PNIPAAM hydrogel A two-step synthesis process is used to produce thermally reversible graft copolymers. Figure 1The copolymer is named NIPAAm, where PEG represents the hydrophilic block, PNIPAAm represents the hydrophobic block, and alkyl side groups (described here as butyl chains, but may include any alkyl chain) act as temperature-transition moieties. To produce the thermally reversible graft copolymer, a mixture of NIPAAm, N-acryloyloxysuccinimide (NASI), and alkyl-chain methacrylates was first copolymerized via standard free radical polymerization. After reprecipitation and drying, the resulting functionalized copolymer was then mixed with monoamine-terminated PEG blocks. The amine-terminated groups were attached to the PNIPAAm-co-PNASI-co-MA backbone via an amidation reaction of amine and N-hydroxysuccinimide (NHS). Finally, the remaining NHS groups were converted to NIPAAm by adding isopropylamine, and the resulting polymer was dried, dialyzed, and lyophilized.

[0271] hPSC amplification As recommended by the manufacturer, human PSC cells (H9 human embryonic stem cells (WA09, WiCell, Madison, WI, passaged 53-58 times)) were maintained on Matrigel (BD, San Jose, CA) in E8 medium (Gibco, Billings, MT) and passaged using Versene (Thermo Fisher, Waltham, MA) or ReleSR (Stem Cell Technologies, Vancouver, BC, Canada).

[0272] Cell encapsulation, differentiation, and harvesting For expansion, hPSCs were dissociated using Accutase (Stem Cell Technologies, Vancouver, BC, Canada) and encapsulated in PEG-NIPAAM hydrogels at a final concentration of 250,000 cells / ml (2.5%–10% by weight / volume). The hydrogel and cells were mixed, encapsulated, and extruded into containers of varying sizes (100 mL to 1 L). Complete E8 medium supplemented with Rock Inhibitor Y-27632 (Selleck Chemicals, Houston, TX) was heated to 37°C, then added, and the cells were incubated at 37°C with 5% CO2. At the end of the expansion process (7 days), the 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).

[0273] Cell counting and viability analysis Cells were stained using AOPI and counted using a K2 image capture device and Matrix software (PerkinElmer, Waltham, MA) to obtain total cell count and viability percentage.

[0274] Flow cytometry Cells were dissociated and fixed in CytoFix / CytoPerm solution (BD) for 20 min, followed by washing with Perm / Wash (BD). For staining, cells were incubated with the primary antibody for 30 min. 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.

[0275] Quantitative PCR Total RNA was prepared using the RNeasy kit (Qiagen, Germantown, MD), and cDNA was generated from the total RNA using the RT2 first-strand kit (Qiagen). For quantitative analysis of transcript expression, real-time PCR was performed using the RT2 SYBR GreenqPCR Mastermixes (Qiagen) and the AriaMX real-time PCR system (Agilent). Primers were designed using the PrimerQuest tool (Coralville, IA) for integrative DNA technology. mRNA expression levels for each gene were normalized to ACTB gene expression levels. Relative values ​​were calculated by setting the normalization value for the control group to 1.

[0276] discuss This gel formulation produces beads composed of core-shell geometry, thus achieving maximum cell retention at scale-up. Figure 5D Three different culture scales were used to rapidly iterate different conditions in a scaled-down model, thereby more accurately predicting performance in bioreactors (positive displacement pipette - PDP - static and rotary). Figure 17A Using this improved formulation, we demonstrated its high viability after encapsulation. This high viability throughout the encapsulation process enabled scalable amplification of hPSCs, with encapsulation flow rates as low as 2 ml gel / min. Figure 18B An exemplary amplification process using this formulation is demonstrated, showing results at four different bioreactor scales ( Figure 18AHigh-quality, reproducible amplification of hPSCs can be achieved in various container volumes (from 100 mL to 1 L), such as by flow cytometry. Figure 18B The results were verified by qPCR (18C). Furthermore, as shown in 17B and 17C, both human embryonic stem cell lines (hESC) and human induced pluripotent stem cell lines (hiPSC) exhibited robust expansion in this hydrogel.

[0277] Example 5 A proof-of-concept study was conducted using a low molecular weight acrylate backbone three-dimensional hydrogel to demonstrate that the encapsulated hPSCs robustly derive into pancreatic endodermal progenitor cells (PE).

[0278] method Preparation of three-dimensional PEG-PNIPAAM hydrogel A two-step synthesis process is used to produce thermally reversible graft copolymers. Figure 1 As described in Example 4.

[0279] hPSC amplification As recommended by the manufacturer, human PSC cells (H9 human embryonic stem cells (WA09, WiCell, Madison, WI, passaged 53-58 times)) were maintained on Matrigel (BD, San Jose, CA) in E8 medium (Gibco, Billings, MT) and passaged using Versene (Thermo Fisher, Waltham, MA) or ReleSR (Stem Cell Technologies, Vancouver, BC, Canada).

[0280] Cell encapsulation, differentiation, and harvesting For differentiation, hPSCs were dissociated using Accutase (Stem Cell Technologies, Vancouver, BC, Canada) and encapsulated in PEG-PNIPAAM hydrogel at a final concentration of 250,000 cells / ml (2.5%–10% by weight / volume). The hydrogel and cells were mixed, encapsulated, and extruded into 100 mL spinnerets. The spinnerets were incubated at 37°C for 15 min to allow gelation. Complete E8 medium supplemented with Rock Inhibitor Y-27632 (Selleck Chemicals, Houston, TX) was heated to 37°C and then added, and the cells were incubated at 37°C with 5% CO2. The medium was changed 50% daily, ensuring the plate remained above 33°C. After 48 hours of expansion under amplification conditions, the medium was replaced with differentiation medium (STEMdiff, Definitive Endoderm, Stem Cell Technologies, Vancouver, BC, Canada) as recommended by the manufacturer. Figure 20A At a specified time point ( Figure 19 On a rotating platform, in the presence of Accumax (Innovative Cell Technologies, San Diego, CA) and TrypLE (ThermoFisher), aggregates are dissociated into individual cells.

[0281] Cell counting and viability analysis Cells were stained using AOPI and counted using a K2 image capture device and Matrix software (PerkinElmer, Waltham, MA) to obtain total cell count and viability percentage.

[0282] Flow cytometry Cells were dissociated and fixed in CytoFix / CytoPerm solution (BD) for 20 min, followed by washing with Perm / Wash (BD). For staining, cells were incubated with the primary antibody for 30 min. 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.

[0283] discuss Improved gel stability allows for a longer differentiation process and enables complete vascular exchange without compromising gel integrity; both of these factors are crucial for ensuring high differentiation efficiency during large-scale scaling. Figure 19 As shown, in the improved three-dimensional hydrogel system, PE cells were generated in this hydrogel using a readily available culture medium formulation, and compared with the standard process in suspension. Therefore, compared with three-dimensional suspension (standard culture), PE yield was increased by 45-fold, aggregate size control was better, post-inoculation viability was higher (>90%), and the PE progenitor cell marker PDX-1 was expressed. Figures 20B to 20D Notably, this three-dimensional culture method allows for better control of aggregate size, resulting in aggregates smaller than 500 μm. This suggests that hydrogels containing the thermally reversible polymer of Formula III have broad implications for clinical manufacturing, as aggregates larger than this size may result in necrotic cores due to nutrient limitations and / or poor differentiation efficiency due to uneven responses of the aggregates to differentiation signals.

[0284] Example 6 A proof-of-concept study was conducted using a low molecular weight acrylate backbone three-dimensional hydrogel to demonstrate robust derivatization of encapsulated hPSCs into midbrain dopaminergic cells (mDA).

[0285] method Preparation of three-dimensional PEG-PNIPAAM hydrogel A two-step synthesis process is used to produce thermally reversible graft copolymers. Figure 1 As described in Example 4.

[0286] hPSC amplification As recommended by the manufacturer, human PSC cells (H9 human embryonic stem cells (WA09, WiCell, Madison, WI, passaged 53-58 times)) were maintained on Matrigel (BD, San Jose, CA) in E8 medium (Gibco, Billings, MT) and passaged using Versene (Thermo Fisher, Waltham, MA) or ReleSR (Stem Cell Technologies, Vancouver, BC, Canada).

[0287] Cell encapsulation, differentiation, and harvesting For differentiation, hPSCs were dissociated using Accutase (Stem Cell Technologies, Vancouver, BC, Canada) and encapsulated in PEG-PNIPAAM hydrogel at a final concentration of 2.5%–10% by weight / volume, resulting in a gel concentration of 500,000 cells / ml. The hydrogel and cells were mixed and seeded into plates using positive displacement pipettes (“PDPs”), or encapsulated and extruded into 100 mL spinnerets. The plates were incubated at 37°C for 15 min to allow gelation and the formation of dome-shaped beads. Complete E8 medium supplemented with Rock Inhibitor Y-27632 (Selleck Chemicals, Houston, TX) was heated to 37°C and subsequently added, and the cells were incubated at 37°C with 5% CO2. Fifty percent of the medium was replaced daily, ensuring the plates remained above 33°C. After 48 hours of culture under amplification conditions, the medium was replaced with differentiation medium as previously described (Adil et al.). Sci Rep 7, 40573 (2017)) ( Figure 21A On the 16th day of differentiation ( Figure 21A On a rotating platform, in the presence of Accumax (Innovative Cell Technologies, San Diego, CA) and TrypLE (Thermo Fisher), aggregates are dissociated into individual cells.

[0288] Cell counting and viability analysis Cells were stained using AOPI and counted using a K2 image capture device and Matrix software (PerkinElmer, Waltham, MA) to obtain total cell count and viability percentage.

[0289] Flow cytometry Cells were dissociated and fixed in CytoFix / CytoPerm solution (BD) for 20 min, followed by washing with Perm / Wash (BD). For staining, cells were incubated with the primary antibody for 30 min. 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.

[0290] discuss Improved gel stability allows for a longer differentiation process and enables complete vascular exchange without compromising gel integrity; both of these are crucial for ensuring high differentiation efficiency at scale-up. Figure 21B and Figure 21C As shown, mDA was generated in the improved three-dimensional hydrogel system. High differentiation efficacy was achieved at different scales (PDP and 100 mL spinneret), as measured by FoxA2 expression (70%-90%). FoxA2 is a marker of ventral midbrain progenitor cells, and the cells exhibited high viability (>80%) at harvest. Figure 21B and Figure 21C This data demonstrates that the improved hydrogel formulation is compatible with scalable culture systems, exhibits high gel stability during the lengthy differentiation process, and protects cells from the shear stress generated by bioreactor mixing.

[0291] Example 7 A proof-of-concept study was conducted using a low molecular weight acrylate backbone three-dimensional hydrogel to demonstrate robust derivatization of encapsulated hPSCs into hematopoietic stem cells (HSCs) and expansion of primary HSCs.

[0292] method Preparation of three-dimensional PEG-PNIPAAM hydrogel A two-step synthesis process is used to produce thermally reversible graft copolymers. Figure 1 As described in Example 4.

[0293] hPSC amplification As recommended by the manufacturer, human PSC cells (H9 human embryonic stem cells (WA09, WiCell, Madison, WI, passaged 53-58 times)) were maintained on Matrigel (BD, San Jose, CA) in E8 medium (Gibco, Billings, MT) and passaged using Versene (Thermo Fisher, Waltham, MA) or ReleSR (Stem Cell Technologies, Vancouver, BC, Canada).

[0294] Cell encapsulation, differentiation, and harvesting For HSC differentiation, hPSCs were dissociated using Accutase (Stem Cell Technologies, Vancouver, BC, Canada) and encapsulated in PEG-PNIPAAM hydrogel at a final concentration of 2.5%–10% by weight / volume, resulting in a gel concentration of 500,000 cells / ml. The hydrogel and cells were mixed and seeded into plates using positive displacement pipettes (“PDP”). The plates were incubated at 37°C for 15 min to allow gelation and the formation of dome-shaped beads. Complete E8 medium supplemented with Rock Inhibitor Y-27632 (Selleck Chemicals, Houston, TX) was heated to 37°C and subsequently added, and the cells were incubated at 37°C with 5% CO2. The medium was changed 50% daily, ensuring the plates remained above 33°C. After 48 hours of amplification, the medium was replaced with differentiation medium as recommended by the manufacturer of the StemDiff hematopoietic kit (Stem Cell Technologies, Vancouver, BC, Canada). Figure 22A On the 12th day of differentiation ( Figure 22A The hydrogel was cooled to 4°C to harvest cells. For HSC amplification, primary human umbilical cord blood CD34+ HSCs (Stem Cell Technologies, Vancouver, BC, Canada) were thawed and encapsulated in hydrogels as described above, and seeded using the PDP method. Cells were amplified for 8 days in SFEM II medium supplemented with StemSpan CD34+ amplification supplement. Figure 23A Cells were then harvested by cooling the hydrogel to 4°C.

[0295] Cell counting and viability analysis Cells were stained using AOPI and counted using a K2 image capture device and Matrix software (PerkinElmer, Waltham, MA) to obtain total cell count and viability percentage.

[0296] discuss Improved gel stability allows for a longer differentiation process and enables complete vascular exchange without compromising gel integrity; both of these are crucial for ensuring high differentiation efficiency at scale-up. Figure 22B and Figure 22C As shown, an improved three-dimensional hydrogel system was used to differentiate hPSCs into HSCs. Furthermore, human umbilical cord blood CD34+ HSCs were also successfully amplified in this three-dimensional hydrogel system. Figure 23B and Figure 23CThis data demonstrates that the improved hydrogel formulation is compatible with HSC amplification and hPSC differentiation, exhibiting high gel stability during the lengthy differentiation process and protecting cells from shear stresses generated during bioreactor mixing and scale-up. HSCs are a promising cell therapy candidate with broad therapeutic applications, ranging from sickle cell disease and β-thalassemia to blood cancers.

[0297] Example 8 The hydrogel containing the thermally reversible polymer of Formula III was compared with the hydrogel containing the thermally reversible polymer disclosed in U.S. Patent No. 10,982,055.

[0298] The synthesis of thermally reversible polymers, as disclosed in US Patent No. 10,982,055, is described in US 10,982,055. Figure 3 The synthesis of the thermally reversible polymer of Formula III disclosed herein is shown. Figure 1 As shown. The following table summarizes the main differences in the synthesis of the corresponding polymers: Table 4

[0299] While the materials and methods of the invention have been described by way of preferred embodiments, it will be apparent to those skilled in the art that variations may be applied to the methods described herein without departing from the spirit and scope of the invention. All such similar substitutions and modifications that will be apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the invention.

Claims

1. A thermally reversible polymer with stability that improves over time, comprising formula (III): Wherein (a), (b), (c) and (d) represent the mole fractions of the copolymer monomers in the polymer, wherein (a), (b) and (c) are each greater than 0; PEG n It is a polyethylene glycol polymer, and n is an integer; R 1 (If present) is any terminal group or functional group other than a primary amine; R 2 It is a lower alkyl group; R 3 (If present) is a terminal group, functional group, or linker; R 4 It is hydrogen or a lower alkyl group; G 1 and G 2 Each component is independently selected from polymer segments, terminal groups, linkers, and linking modifiers. The polymer has a molecular weight greater than 50 kDa.

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

3. The thermally reversible polymer of claim 1 or 2, wherein the PEG n It is a polyethylene glycol polymer with a molecular weight ratio (MW) of about 1 kDa to about 50 kDa or about 2 kDa to about 20 kDa.

4. The thermally reversible polymer according to any one of claims 1 to 3, wherein the weight:w / w ratio of the PEG to PNIPAAm copolymer of the thermally reversible polymer is greater than about 1:2, preferably wherein the weight:w / w ratio of the PEG to PNIPAAm copolymer of the thermally reversible polymer is about 1:2.5 to about 1:4.

5.

5. The thermally reversible polymer according to any one of claims 1 to 4, wherein R 1 It does not contain any terminal groups or functional groups other than alkyl or substituted alkyl groups.

6. The thermally reversible polymer according to any one of claims 1 to 5, wherein R 1 It is a C1-C6 alkoxy group selected from methoxy, ethoxy, n-propoxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy, or a hydroxyl group.

7. The thermally reversible polymer of claim 6, wherein R 1 It is a methoxy group, or R in it. 1 It is hydroxyl, biotin, or DBCO.

8. The thermally reversible polymer according to any one of claims 1 to 7, wherein R 2 It is not isobutyl.

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

10. The thermally reversible polymer of claim 9, wherein R 2 It is n-butyl, isobutyl, or tert-butyl, preferably wherein R 2 It is n-butyl.

11. The thermally reversible polymer according to any one of claims 1 to 10, wherein R 3 It does not exist.

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

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

14. The thermally reversible polymer according to any one of claims 1 to 13, wherein R 3 It does not exist.

15. The thermally reversible polymer according to any one of claims 1 to 13, wherein R 1 and / or R 3 It is a chemically selective functional group selected from acrylates, methacrylates, biotin, streptavidin, thiols, alkynes, cyclooctyne, azides, phosphine, maleimide, alkoxyamine, aldehydes, and their protected versions or precursors.

16. The thermally reversible polymer according to any one of claims 1 to 13, wherein R 1 and / or R 3 It is selected from the following modifiers: heparin, hyaluronic acid, specific binding members, peptides, nucleic acids, gelatin, fibronectin, collagen, laminin, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin, progesterone, glucose, stromal cell-derived factor-1 (SDF-1), thymosin β-4, sound hedgehog factor (SHH), noggin, activin, transforming growth factor-β (TGF-β), FGF8, brain-derived neurotrophic factor (BDNF), glial cell-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. The thermally reversible polymer according to any one of claims 1 to 16, wherein the thermally reversible polymer has one or more, and preferably all, of the following properties: (a) an LCST of about 12°C to 32°C, preferably about 19°C to 23°C; (b) a stiffness of about 100 Pa to 8000 Pa; and (c) a viscosity of about 100 cP to 2000 cP.

18. A three-dimensional hydrogel comprising the thermally reversible polymer of any one of claims 1 to 17 and a buffered aqueous solution.

19. The three-dimensional hydrogel of claim 18, wherein the hydrogel remains stable at 37°C in a buffered environment for a period of at least one week, at least two weeks, at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, or at least eight weeks or at least three months.

20. A composition comprising a plurality of hydrogel capsules, wherein at least 90%, preferably at least 95%, of the hydrogel capsules comprises at least one cell and a hydrogel encapsulating the cell, wherein the hydrogel encapsulating the cell is a hydrogel comprising the thermally reversible polymer of any one of claims 1 to 17 and a buffered aqueous solution.

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 wherein the cells are stem cells.

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

23. The method of claim 22, wherein the conditions suitable for the expansion or differentiation of the stem cells or precursor cells include culturing the stem cells or precursor cells in the hydrogel composition for a period of at least one week, at least two weeks, at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, or at least eight weeks or at least three months.

24. An in vitro method for generating a cell population rich in MGE progenitor cells from an initial human stem cell population, the method comprising: (a) Encapsulating an initial human stem cell population in the three-dimensional hydrogel of claim 18 or 19; as well as (b) Contact the encapsulated human stem cells with at least one Small Mothers Against Decapentaplegic (SMAD) signaling inhibitor and at least one Wingless (Wnt) antagonist; and contact the cells with at least one Sound Hedgehog (SHH) signaling activator and an FGFR agonist to obtain a cell population rich in MGE progenitor cells expressing FOXG1 and at least one additional marker indicating MGE progenitor cells.

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. The method of claim 24, wherein the contact with the at least one SMAD signaling inhibitor and the contact with the at least one Wnt antagonist are performed simultaneously or sequentially, and the duration of each contact is between about 5 days and about 30 days.

27. The method of claim 26, wherein the contact between the cell and the at least one Wnt antagonist begins within 5 days, preferably 4 days, 3 days, 2 days or 1 day after the first contact between the cell and the at least one SMAD signaling inhibitor, preferably wherein the contact between the cell and the at least one Wnt antagonist begins simultaneously with the first contact between the cell and the at least one SMAD signaling inhibitor.

28. The method of claim 24, wherein the at least one SMAD signaling inhibitor is selected from the group consisting of: SB431542, LDN-193189, Noggin PD169316, SB203580, LY364947, A77-01, A-83-01, BMP4, GW788388, GW6604, SB-505124, ledelimumab, metilumab, GC-I008, AP-12009, AP-110I4, LY550410, LY580276, LY364947, LY2109761, SB-505124, E-616452 (RepSox) ALK inhibitors), SD-208, SMI6, NPC-30345, KÏ26894, SB-203580, SD-093, activin-M108A, P144, soluble TBR2-Fc, DMH-1, doxorphine dihydrochloride, their derivatives and combinations thereof.

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

30. 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 includes IWP-2.

31. The method of claim 24, wherein the at least one SHH signaling activator is selected from the group consisting of Smoothened agonists (SAG), SAG analogs, SHH, C25-SHH, C24-SHH, purinemorphine, Hg-Ag, their derivatives, and combinations thereof.

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

33. 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. 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 obtained cell population expresses FOXG1 and NKX2-1.

35. 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 obtained cell population contains MGE progenitor cells.

36. The method of claim 24, further comprising (c) after a predetermined time, contacting the cells with at least one neurotrophic factor (e.g., GDNF, BDNF) and optionally a notch inhibitor (e.g., DAPT) to generate a cell population rich in differentiation-inhibiting GABAergic cortical interneurons (cINs) expressing FOXG1 and at least one additional marker indicating cIN cells.

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

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

39. The method of any one of claims 36 to 38, wherein after contacting the cells with the at least one SMAD signaling inhibitor, the at least one Wnt antagonist and the at least one SHH signaling activator, the cells are then contacted with at least one neurotrophic factor and optionally a notch inhibitor.

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

41. The method of claim 36, wherein the at least one additional biomarker is selected from the group consisting of PV, SST, calcium-binding protein, DCX, ASCL1, TUJ1, GABA, GAD1, VGAT, vGLUT1, and GAD67.

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 obtained cIN cell population expresses albumin (PV).

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. A composition comprising a cell population produced by the method of any one of claims 24 to 36, 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 wherein the method does not include a step of purifying or further enriching the MGE progenitor cells after step (b).

45. A composition comprising a cell population produced by the method of any one of claims 27 to 43, 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 ​​cells, preferably wherein the method does not include a step of purifying or further enriching cIN cells after step (c).

46. ​​The composition of claim 45, 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 NKX2.

1. - / PV + .

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

48. The use as described in claim 47, wherein the neurological disorder is epilepsy.

49. A method for preparing a thermally reversible polymer of formula III, the method comprising the following steps: (i) In a single solvent, under conditions where the initiator concentration is sufficient to form a PNIPAAM-co-PBMA-co-PNASI copolymer backbone with a MW of at least 50 kDa, a group of comonomers comprising N-isopropylacrylamide (NIPAAm), alkyl methacrylate, and N-acryloyloxysuccinimide (NASI) is polymerized; and (ii) the copolymer is reacted with a monoPEG amine to form a [PNIPAAM-co-PBMA-co-PNASI]-b-[PEG] copolymer; and (iii) the [PNIPAAM-co-PBMA-co-PNASI]-b-[PEG] is reacted with isopropylamine to form a thermally reversible polymer of Formula III.

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

51. A thermally reversible polymer, produced by the method of claim 49 or 50.

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

53. The thermally reversible polymer of claims 1 to 17, further comprising expanding artificial hematopoietic stem cells for at least one week.

54. The thermally reversible polymer of claims 1 to 17, further comprising pluripotent stem cells and differentiating these pluripotent stem cells into midbrain dopaminergic cells, pancreatic endoderm cells, and hematopoietic stem cells.

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