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WO2020219696A1 - Dispositifs et procédés de génération de cellules progénitrices d'oligodendrocytes - Google Patents

Dispositifs et procédés de génération de cellules progénitrices d'oligodendrocytes Download PDF

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WO2020219696A1
WO2020219696A1 PCT/US2020/029553 US2020029553W WO2020219696A1 WO 2020219696 A1 WO2020219696 A1 WO 2020219696A1 US 2020029553 W US2020029553 W US 2020029553W WO 2020219696 A1 WO2020219696 A1 WO 2020219696A1
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days
cells
cell
inhibitor
signaling
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David V. Schaffer
Riya MUCKOM
Douglas S. Clark
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University of California Berkeley
University of California San Diego UCSD
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University of California Berkeley
University of California San Diego UCSD
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0623—Stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/415—Wnt; Frizzeled
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/42—Notch; Delta; Jagged; Serrate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers

Definitions

  • hPSCs Human pluripotent stem cells
  • OPCs oligodendrocyte progenitor cells
  • hPSC-OPCs have recently advanced to a Phase II clinical trial and are even being considered for additional myelin-associated diseases in the central nervous system (CNS), such as adrenoleukodystrophy, multiple sclerosis (MS), or injury from radiation.
  • CNS central nervous system
  • MS multiple sclerosis
  • mDA midbrain dopaminergic neurons
  • the present disclosure provides a device for screening multiple cell culture conditions, the device comprising a multi-pillar plate and a multi- well plate, and providing multiple cell culture conditions.
  • the present disclosure further provides methods of screening multiple cell culture conditions for neural cell differentiation culture conditions for generating neural cells using a device of the present disclosure. Also provided are methods for generating neural cells under the cell culture conditions identified in the method of screening using the device of the present disclosure. Further provided are methods for generating an OPC by culturing an hPSC under specific conditions that promote optimal differentiation of the hPSC to OPC.
  • methods are provided for treating a myelin-associate disorder in the central nervous system by administering to a subject in need therefore an OPC produced from an hPSC cultured according to the methods disclosed herein.
  • FIG. 1 shows a high-level schematic of upstream screening and downstream scale-up for robust production hPSC-derived CRTs.
  • Thousands of culture conditions are screened for optimal marker expression profile of cell type in micro-scale systems and the optimal protocol is translated first to bench-scale systems and ultimately to large-scale culture systems to produce enough quantity to meet patient needs.
  • FIG. 2 shows a microscale High-Throughput Culture System for Advanced Screening.
  • a micropillar chip with cells suspended in a 3D hydrogel is stamped to a complementary micro well chip containing isolated media conditions to generate 532 independent
  • FIG. 3 shows an overall workflow developed for large scale fully-defined production of pure OPCs for therapeutic application.
  • Stirred tank bioreactor culture (1), FACS enrichment (2), and downstream applications such as cryopreservation, further scale-up, and functional assays (3) are subcomponents of the overall workflow.
  • FIG. 4 shows early culture parameters play a large role in OPC differentiation
  • FIG. 5 shows the strategic addition of signaling antagonists to modulate OPC specification.
  • FIG. 6 shows the fine tuning of temporal profiles of RA and SAG to influence OPC specification.
  • FIG. 7 shows a factorial ANOVA model of individual and combinatorial effects of 12 culture parameters on expression of key markers in OPC specification.
  • FIG. 8 shows a generalizable platform for screening parameters for optimization of upstream CRT production.
  • A) Timeline of small molecule addition for differentiation of midbrain dopaminergic neurons from human pluripotent stem cells.
  • FIG. 9 shows a comparison of stirred and static culture environment for
  • thermoresponsive gel-encapsulated OPC differentiation A) Schematic of OPCs encapsulated in thermoresponsive gel within spinner flask bioreactor. B) Quantitative modeling in COMSOL of physicochemical environment at cross section of bioreactor i. glucose concentration with and without agitation (50 RPM) ii. Shear stress with 50 RPM agitation. C) i. Bright field image of encapsulated neurospheres in bioreactor after 10 days of growth ii. Viability assay iii.
  • thermoresponsive gels Distribution of growth in thermoresponsive gels in stirred and static culture environments measured by neurosphere diameter at Day 10; scale bar represents 100 microns iv.
  • FIG. 10 shows the expansion of hESCs in thermoreversible gel in a bioreactor.
  • FIG. 11 shows the in-process immunocytochemical analysis of early germ line markers during first 15 days of bioreactor culture.
  • FIG. 12 shows the in-process immunocytochemical analysis of early OPC and non-OPC markers between Day 15-25 of bioreactor culture.
  • FIG. 13 shows the development of an aggregate dissociation process to achieve viable single cell suspension before and after FACS sorting.
  • B) Viable dissociation of neural aggregates involves: i) plating on laminin coated dishes; ii) Allowing cells to migrate from the aggregate; and iii) Applying accutase enzymatic dissociation reagent and straining through 40 micron mesh.
  • C) Staining for propidium iodide reveals high (-80%) viable cells.
  • D) Microscopy of sorted PDGFRa-i- cells reveals highly viable cells with processes morphologically resembling OPCs.
  • FIG. 14A-14C shows later stage assessment of OPC differentiation protocol built from screening data.
  • A) Flow cytometry indicating H9- and TCTF- derived OPCs express PDGFRa after 60 days of culture.
  • FIG. 15 shows an immunocytochemical comparison of OPC and non-OPC markers before and after sorting.
  • FIG. 16 shows passaging and cryopreservation viability of purified hESC-derived
  • FIG. 17 shows small molecule agonists affect maturation and myelination ability of
  • OPCs A) MBP+ cells after 100 days of differentiation.
  • FIG. 18 shows a comparison of Integrated Expansion/Differentiation Bioreactor
  • FIG. 19A-19D shows H9 seeding and viability across the micropillar culture chip.
  • Controlled modulation of seeding density of cells onto micropillars shown by fluorescence microscopy of Hoechst stained cells seeded at four different densities consistent across the micropillar chip; scale bar represents 100 microns ii.
  • FIG. 20A-20E shows a longitudinal study of proliferation and differentiation on
  • FIG. 21 shows an image analysis pipeline for quantification of nuclear and cytoplasmic cellular markers and co-expression.
  • FIG. 22 shows controlled modulation of seeding density of cells onto micropillars.
  • FIG. 23 shows the duration of Neural Induction affects OPC differentiation efficiency.
  • FIG. 24A-24C Shows combinatorial liquid dispensing and DMSO and SAG dose
  • A) Validation of liquid dispensing layout for full factorial combinatorial study. Full factorial design with n 4 factors at a range of doses dispensed into the microwell chip validated by using fluorescent beads representing the identity and concentration of each factor. One chip contains 90 unique culture combinations with four technical replicates each, totaling 360 independent microsites. Scale bar represents 1 mm.
  • FIG. 25 shows temporal profiles of SAG influence OPC specification.
  • FIG. 26A-26C shows temporal profiles of RA influence OPC specification.
  • FIG. 27 shows temporal profiles of RA influence co-expression of OPC and/or motor neuron markers.
  • FIG. 28 shows an iterative process to improve Factorial ANOVA model.
  • FIG. 29 shows a comparison of aggregate distribution in Suspension culture
  • FIG. 30 shows replication of biphasic Wnt effect on 01ig2 expression across hPSC lines.
  • FIG. 31 show OPCs implanted in a HD mouse model.
  • A“three-dimensional culture system,” as used herein, refers to an environment that is created to allow biological cells to grow or interact with its surroundings in all three dimensions.
  • biocompatible refers to a polymer that does not cause substantial toxic or injurious effects to: i) a mammalian cell in in vitro cell culture; and/or ii) an individual upon implantation.
  • hydrogel refers to a network of polymer chains that are hydrophilic, sometimes found as a colloidal gel in which water is the dispersion medium.
  • thermoresponsive refers to a polymer that responds to temperature.
  • a thermoresponsive hydrogel is a certain state (e.g., solid) above a certain temperature, but is maintained as a liquid at a lower temperature.
  • a thermoresponsive polymer is liquid at 4°C, and is a gel or semi-solid at 37°C.
  • factor refers to a biologically active factor.
  • a factor as used in the present disclosure may be a small molecule or a peptide.
  • a“combination of factors” refers to a combination of biologically active factors that function together to achieve a result (e.g., differentiation of oligodendrocyte precursor cells).
  • an "effective amount" of a factor or combination of factors is an amount that, when in contact with target suitable cells, provides a functional effect that results in a desired outcome, e.g., differentiation of pluripotent stem cells into oligodendrocyte precursor cells.
  • An effective amount should be readily scalable depending on the number of target suitable cells that are subject to a factor or combination of factors to obtain the desired outcome.
  • progenitor cell refers to an undifferentiated cell that is
  • progenitor cells can produce progeny that are capable of differentiating into more than one cell type.
  • oligodendrocyte precursor cell “oligodendrocyte progenitor cell”
  • OPC oligodendrocyte progenitor cell
  • pre-oligodendrocyte precursor cell or“pre-OPC” refers to a cell that becomes an oligodendrocyte precursor cell upon differentiation.
  • pre-OPC as used herein may refer to, e.g., a neural committed pluripotent stem cell, a neural committed human embryonic stem cell, and the like.
  • treatment refers to obtaining a desired
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease or at risk of acquiring the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; (c) relieving the disease, i.e., causing regression of the disease; and (d) replacing a lost function that results from the disease.
  • the terms“individual,”“host,”“subject,” and“patient” are used interchangeably herein, and refer to a mammal, including, but not limited to, human and non-human primates, including simians and humans; mammalian sport animals (e.g., horses, camels, etc.); mammalian farm animals (e.g., sheep, goats, cows, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.). In some cases, the individual is a human.
  • mammalian sport animals e.g., horses, camels, etc.
  • mammalian farm animals e.g., sheep, goats, cows, etc.
  • mammalian pets dogs, cats, etc.
  • rodents e.g., mice, rats, etc.
  • the present disclosure provides a device for screening multiple cell culture conditions, the device comprising a multi-pillar plate and a multi- well plate, the device providing multiple cell culture conditions.
  • the present disclosure further provides methods of screening multiple cell culture conditions for neural cell differentiation culture conditions for generating neural cells using a device of the present disclosure. Also provided are methods for generating neural cells under the cell culture conditions identified in the method of screening using the device of the present disclosure.
  • the present disclosure provides a device for screening multiple cell culture conditions.
  • the device may comprise a multi-pillar plate comprising one or more pillars, wherein each pillar comprises a gel composition deposited thereon, wherein the gel composition comprises one or more stem cells.
  • the device may further comprise a multi- well plate comprising one or more cell culture mediums contained in one or more wells where each of the one or more culture mediums or wells are subjected to one or more culture conditions.
  • the multi-pillar plate may be mated or joined with or contacted with the multi- well plate such that each of the one or more pillars is inserted into a well of the multi-well plate.
  • each of the one or more pillars is contacted with one or more cell culture mediums in the one or more wells of the multi- well plate under one or more cell culture conditions.
  • the contacting of the one or more pillars with the one or more cell culture mediums in the one or more wells under the one or more cell culture conditions generates a medium-contacted stem cell.
  • the medium-contacted stem cell differentiates into to a neural cell, e.g., a neural progenitor cell such as an OPC or a midbrain dopaminergic neuron.
  • a device of the present disclosure may be used to screen for neural cell differentiation culture conditions.
  • An aspect of the device is a multi-pillar plate.
  • a“multi-pillar plate” refers to any support having one or more pillars or posts positioned on the surface of the support.
  • Each of the one or more pillars may have a surface on which an amount of gel comprising at least one stem cell is deposited.
  • the multi-pillar plate may be configured to mate with a multi well plate such that the one or more pillars may be inserted into one or more wells of the multi well plate.
  • each of the one or more pillars are dimensioned to lie within the volume of each of the one or more wells of the multi-well plate.
  • the multi-pillar plate may include one or more pillars arranged in any configuration, e.g., a rectangular grid, a circular configuration, a linear configuration, etc., on a support.
  • the one or more pillars may be regularly arranged at a substantially constant spacing.
  • the one or more pillars are irregularly arranged such that the spacing between each pillar varies.
  • the multi-pillar plate includes a two-dimensional array of pillars.
  • the two- dimensional array of pillars may include 2 to 10,000 pillars, such as, e.g., 8 to 5,000 pillars, 10 to 1,000 pillars, e.g., 10 to 900 pillars, 20 to 800 pillars, 30 to 700 pillars, 40 to 600 pillars, or 50 to 100 pillars, e.g., 12, 24, 48, 96, 384, 1096 and 1536 pillars.
  • the density of the pillars in the two-dimensional array may range from 1 pillar/cm 2 to 600 pillars/cm 2 , such as 2 pillars/cm 2 to 200 pillars/cm 2 .
  • the pillars are arranged in rows and columns.
  • the pillars are arranged in an array having two or more rows.
  • the pillars are arranged in an array having two or more columns.
  • the one or more pillars of the multi-pillar plate may comprise any suitable dimensions.
  • the dimensions of the one or more pillars are such that each of the one or more pillars is dimensioned to lie within the volume of one or more wells of a multi-well plate. In some cases, the dimensions of the one or more pillars are such that each of the one or more pillars comprises a surface that receives a gel composition such that a gel composition may be stably deposited thereon. In some cases, the surface upon which the gel composition is deposited is the top surface of the pillar.
  • the one or more pillars may have any suitable shape such as, but not limited to, cylinders, cones, spheres, etc.
  • the sectional shape of each pillar may be, e.g., circular, oval, hexagonal, rectangular, square or other shapes.
  • a cross-section of the one or more pillars may have any suitable diameter that ranges from 0.1 pm to 1000 pm such as, e.g., 0.5 pm to 500 pm, 1 pm to 100 pm, or 10 pm to 50 pm.
  • the one or more pillars may have a substantially constant diameter or irregular diameters.
  • the pillars may have any suitable height such as, but not limited to, 0.1 pm to 1000 pm such as, e.g., 0.5 pm to 500 pm, 1 pm to 100 pm, or 10 pm to 50 pm.
  • the one or more pillars may be of a substantially constant height or irregular diameters.
  • the multi-pillar plate support and one or more pillars may be constructed of any suitable material.
  • the multi-pillar plate is constructed of a material that can provide sufficient rigidity for insertion of the one or more pillars into one or more wells of a multi-well plate.
  • the multi-pillar plate is constructed of a material that is compatible, e.g., biocompatible, with a gel composition comprising at least one stem cell.
  • the material of the multi-pillar may be transparent or opaque, as desired.
  • the multi-pillar plate is fabricated from a polymeric material. Suitable materials include polymers (e.g. polycarbonate, silicone, etc.), polystyrene, glass, polyacrylate, ceramics, and metals.
  • the one or more pillars may comprise a gel composition deposited
  • the gel composition may comprise a hydrogel cell matrix.
  • the hydrogel cell matrix may include a plurality of hydrogel polymers. Any suitable hydrogel polymers can be used in the hydrogel cell matrices provided herein.
  • Hydro gel polymers may include the following monomers: lactic acid, glycolic acid, acrylic acid, 1 -hydroxy ethyl methacrylate (HEM A), ethyl methacrylate (EM A), propylene glycol methacrylate (PEMA), acrylamide (A AM), N- vinylpyrrolidone, methyl methacrylate (MMA), glycidyl methacrylate (GDMA), glycol methacrylate (GMA), ethylene glycol, fumaric acid, and the like.
  • Common cross linking agents include tetraethylene glycol dimethacrylate (TEGDMA) and N,N'-methylenebisacrylamide.
  • TEGDMA tetraethylene glycol dimethacrylate
  • N,N'-methylenebisacrylamide N,N'-methylenebisacrylamide.
  • the hydrogel can be homopoly meric, or can comprise co-polymers of two or more of the aforementioned polymers.
  • the hydrogel polymer that encapsulates the stem cell or progenitor cell is generally hydrophilic.
  • Suitable hydrogel polymers include, but are not limited to, poly(N-isopropylacrylamide) (pNIPAAm); poly(N-isopropylacrylamide-co-acrylic acid); hyaluronic acid or hyaluronate; crossl inked hyaluronic acid or hyaluronate; pHEMA; or copolymers of p(NIPAAm)-based sIPNs and other hydrogel sIPNs (semi-interpenetrating networks).
  • the hydrogel polymer is a hyaluronic acid (HyA) polymer.
  • the hydrogel polymer is an acrylated hyaluronic acid (HyA) polymer.
  • the hydrogel is a temperature-sensitive hydrogel.
  • a temperature- sensitive hydrogel is a polyacrylic acid or derivative thereof, e.g., poly (N-isopropylacrylamide) gel, and the increase in temperature causes the hydrogel to contract, thereby forcing an agent out of the hydrogel.
  • the temperature-sensitive hydrogel is an interpenetrating hydrogel network of poly(acrylamide) and poly( acrylic acid), and the increase in temperature causes the hydrogel to swell, thereby allowing an agent to diffuse out of the gel.
  • the temperature required for triggering release of an agent from the hydrogel is generally about normal body temperature, e.g., about 37°C.
  • the stiffness module of a subject hydrogel matrix can be in the range of from about 15 Pascals (Pa) to about 850 Pa, e.g., from about 15 Pa to about 20 Pa, from about 20 Pa to about 50 Pa, from about 50 Pa to about 100 Pa, from about 100 Pa to about 150 Pa, from about 150 Pa to about 200 Pa, from about 200 Pa to about 250 Pa, from about 250 Pa to about 300 Pa, from about 300 Pa to about 350 Pa, from about 350 Pa to about 400 Pa, from about 400 Pa to about 500 Pa, from about 500 Pa to about 600 Pa, from about 600 Pa to about 700 Pa, from about 700 Pa to about 800 Pa, or from about 800 Pa to about 850 Pa.
  • Pa Pascals
  • the gel composition comprises a biocompatible polymer. In some cases, the
  • biocompatible polymer is thermoresponsive and forms a hydrogel at certain temperatures. Some thermoresponsive polymers can undergo reversible phase transition from a liquid state at lower temperatures to a solid state at higher temperatures. In some cases, a biocompatible
  • thermoresponsive polymer can be a liquid at around 4°C.
  • a biocompatible thermoresponsive polymer can be a liquid at a temperature that is below about 25°C, below about 22°C, below about 20°C, below about 18°C, below about 16°C, below about 14°C, below about 12°C, below about 10°C, below about 8°C, below about 6°C, below about 4°C, below about 2°C, or around CPC.
  • a biocompatible thermoresponsive polymer can be a liquid at a temperature in the range of from about 2°C to 4°C, from 4°C to 6°C, from 2°C to 6°C, from 6°C to 10°C, or from 2°C to 10°C.
  • a biocompatible thermoresponsive polymer of the present disclosure is a solid, semi-solid, or a gel at around 37°C.
  • a biocompatible thermoresponsive polymer can be a solid, semi-solid, or a gel at a temperature of at least 30°C, at least 32°C, at least 34°C, at least 35°C, at least 36°C, at least 37°C, at least 38°C, at least 40°C, at least 42°C.
  • a biocompatible thermoresponsive polymer can be a gel at a temperature in the range of from about 34°C to 35°C, from 35°C to 38°C, or from 34°C to 37°C.
  • a biocompatible thermoresponsive polymer can be a liquid at a temperature in the range of from about 2°C to 4°C, from 4°C to 6°C, from 2°C to 6°C, from 6°C to 10°C, or from 2°C to 10°C; and is a gel at a temperature in the range of from about 34°C to 35°C, from 35°C to 38°C, or from 34°C to 37°C.
  • a biocompatible thermoresponsive polymer of the present disclosure is a liquid at 4°C and becomes a gel when warmed to 37°C.
  • a gel composition comprising any one of the hydrogel cell matrices provided herein and a stem cell or progenitor cell encapsulated in the matrix.
  • the density of cells in a hydrogel matrix can range from about 10 2 cells/cubic centimeter (cc) hydrogel matrix to about 10 9 cells/cc.
  • the density of cells in a hydrogel matrix can range from about 10 2 cells/cc to about 10 4 cells/cc, from about 10 4 cells/cc to about 10 s cells/cc, from about 10 s cells/cc to about 10 6 cells/cc, from about 10 6 cells/cc to about 10 7 cells/cc, from about 10 7 cells/cc to about 10 s cells/cc, or from about 10 s cells/cc to about 10 9 cells/cc.
  • the weight percent of the cells suspended in the hydrogel matrix can range from 0.01 weight% to about 1 weight%, e.g., 0.01 weight%, 0.02 weight%, 0.03 weight%, 0.04 weight%, 0.05 weight%, from 0.05 weight% to about 0.1 weight%, from about 0.1 weight% to about 0.25 weight%, from about 0.25 weight% to about 0.5 weight%, from about 0.5 weight% to about 0.75 weight%, or from about 0.75 weight% to 1 weight%.
  • Any suitable stem cell can be used in the hydrogel cell matrix system provided herein.
  • stem cell refers to an undifferentiated cell that can be induced to proliferate.
  • the stem cell is capable of self-maintenance, meaning that with each cell division, one daughter cell will also be a stem cell.
  • Stem cells can be obtained from embryonic, post natal, juvenile or adult tissue.
  • Stem cells include totipotent stem cells, pluripotent stem cells, and multipotent stem cells.
  • Suitable stem cells include induced pluripotent stem cells.
  • Stem cells can be human stem cells, non-human primate stem cells, rodent (e.g., mouse; rat) stem cells, ungulate stem cells, ovine stem cells, equine stem cells, feline stem cells, canine stem cells, and the like.
  • Stem cells include, e.g., hematopoietic stem cells, embryonic stem cells (e.g., pre
  • mesenchymal stem cells include, but are not limited to, keratinocytes, adipocytes, cardiomyocytes, neurons, osteoblasts, pancreatic islet cells, retinal cells, and the like.
  • Suitable human embryonic stem (ES) cells include, but are not limited to, any of a variety of available human ES lines, e.g., BGOl(hESBGN-Ol), BG02 (hESBGN-02), BG03 (hESBGN-03) (BresaGen, Inc.; Athens, GA); SA01 (Sahlgrenska 1), SA02 (Sahlgrenska 2) (Cellartis AB; Goeteborg, Sweden); ES01 (HES-1), ES01 (HES-2), ES03 (HES-3), ES04 (HES- 4), ES05 (HES-5), ES06 (HES-6) (ES Cell International; Singapore); UC01 (HSF-1), UC06 (HSF-6) (University of California, San Francisco; San Francisco, CA); WA01 (HI), WA07 (H7), WA09 (H9), WA09/O
  • Hadassah lines are given as the National Institutes of Health (NIH) code, followed in parentheses by the provider code. See, e.g., U.S. Patent No. 6,875,607.
  • the Hadassah lines (HADC-100, HADC-102, and HADC-106) are also suitable for use; the Hadassah cell lines are available from, e.g., Kadimastem and Fineage Therapeutics.
  • Suitable human ES cell lines can be positive for one, two, three, four, five, six, or all seven of the following markers: stage-specific embryonic antigen-3 (SSEA-3); SSEA-4; TRA 1- 60; TRA 1-81; Oct-4; GCTM-2; and alkaline phosphatase.
  • SSEA-3 stage-specific embryonic antigen-3
  • SSEA-4 SSEA-4
  • TRA 1- 60 TRA 1-81
  • Oct-4 Oct-4
  • GCTM-2 alkaline phosphatase
  • Embryonic stem cells can be isolated from primate tissue (U.S. Pat. No. 5,843,780;
  • hESCs Human embryonic stem cells
  • Equivalent cell types to hESCs include their pluripotent derivatives, such as primitive ectoderm-like (EPL) cells, as outlined in WO 01/51610 (Bresagen).
  • the zona pellucida is removed from developed blastocysts by brief exposure to pronase (Sigma).
  • the inner cell masses are isolated by immunosurgery, in which blastocysts are exposed to a 1:50 dilution of rabbit anti-human spleen cell antiserum for 30 min, then washed for 5 min three times in DMEM, and exposed to a 1:5 dilution of Guinea pig complement (Gibco) for 3 min (Solter et al., Proc. Natl. Acad. Sci. U.S.A. 1975, 72:5099).
  • lysed trophectoderm cells are removed from the intact inner cell mass (ICM) by gentle pipetting, and the ICM plated on mEF feeder layers.
  • inner cell mass-derived outgrowths are dissociated into clumps, either by exposure to calcium and magnesium-free phosphate-buffered saline (PBS) with 1 mM EDTA, by exposure to dispase or trypsin, or by mechanical dissociation with a micropipette; and then replated on mEF in fresh medium.
  • PBS calcium and magnesium-free phosphate-buffered saline
  • ESC-like morphology is characterized as compact colonies with apparently high nucleus to cytoplasm ratio and prominent nucleoli. Resulting ESCs are then routinely split every 1-2 weeks by brief trypsinization, exposure to Dulbecco's PBS (containing 2 mM EDTA), exposure to type IV collagenase (about 200 U/mL; Gibco) or by selection of individual colonies by micropipette.
  • hEGCs Human Embryonic Germ cells
  • hEGCs Human Embryonic Germ cells
  • isotonic buffer 0.1 mL 0.05% trypsin/0.53 mM sodium EDTA solution (BRL) and cut into ⁇ 1 mm 3 chunks.
  • the cells are incubated 1 h or overnight at 37°C in about 3.5 mL EG growth medium (DMEM containing D-glucose, NaHCOq 15% ES qualified fetal calf serum; 2 mM glutamine; 1 mM sodium pyruvate; 1000-2000 U/mL human recombinant leukemia inhibitory factor; 1-2 ng/mL human recombinant bFGF; and 10 mM forskolin (in 10% DMSO).
  • the cells are then resuspended in 1-3 mL of EG growth medium, and plated onto a feeder layer (e.g., STO cells, ATCC No. CRL 1503, inactivated with 5000 rad g-irradiation).
  • the first passage is done after 7-10 days, and then cultured with daily replacement of medium until cell morphology consistent with EG cells is observed, typically after 7-30 days or 14 passages.
  • hESCs can be obtained from established lines obtainable from public depositories (for example, the WiCell Research Institute, Madison Wis. U.S.A., or the American Type Culture Collection, Manassas Va., U.S.A.).
  • U.S. Patent Publication 2003-0113910 Al reports pluripotent stem cells derived without the use of embryos or fetal tissue. It may also be-possible to reprogram cord blood or other progenitor cells into PSCs by using a factor that induces the pluripotent phenotype (Chambers et al., Cell. 2003, 113:643; Mitsui et al., Cell. 2003, 113:631). Under appropriate conditions, any cell that otherwise meets the definitions for PSCs or ESCs can be used.
  • the stem cell is a hematopoietic stem cell (HSC).
  • HSCs are mesoderm- derived cells that can be isolated from bone marrow, blood, cord blood, fetal liver and yolk sac. HSCs are characterized as CD34 + and CD3 . HSCs can repopulate the erythroid, neutrophil- macrophage, megakaryocyte and lymphoid hematopoietic cell lineages in vivo. In vitro, HSCs can be induced to undergo at least some self-renewing cell divisions and can be induced to differentiate to the same lineages as is seen in vivo. As such, HSCs can be induced to differentiate into one or more of erythroid cells, megakaryocytes, neutrophils, macrophages, and lymphoid cells.
  • the stem cell is a neural stem cell (NSC).
  • NSCs neural stem cells
  • a neural stem cell is a multipotent stem cell which is capable of multiple divisions, and under specific conditions can produce daughter cells which are neural stem cells, or neural progenitor cells that can be neuroblasts or glioblasts, e.g., cells committed to become one or more types of neurons and glial cells respectively.
  • Methods of obtaining NSCs are known in the art.
  • the stem cell is a mesenchymal stem cell (MSC).
  • MSCs originally
  • MSC derived from the embryonal mesoderm and isolated from adult bone marrow, can differentiate to form muscle, bone, cartilage, fat, marrow stroma, and tendon.
  • Methods of isolating MSC are known in the art; and any known method can be used to obtain MSC. See, e.g., U.S. Pat. No. 5,736,396, which describes isolation of human MSC.
  • the stem cell is an induced pluripotent stem (iPS) cell.
  • iPS induced pluripotent stem
  • induced pluripotent stem (iPS) cell is a pluripotent stem cell induced from a somatic cell, e.g., a differentiated somatic cell. iPS cells are capable of self-renewal and differentiation into cell fate- committed stem cells, including neural stem cells, as well as various types of mature cells.
  • iPS cells can be generated from somatic cells, including skin fibroblasts, using, e.g., known methods. iPS cells produce and express on their cell surface one or more of the following cell surface antigens: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, and Nanog. In some cases, iPS cells produce and express on their cell surface SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, and Nanog. iPS cells express one or more of the following genes: Oct- 3/4, Sox2, Nanog, GDF3, REX1, FGF4, ESG1, DPPA2, DPPA4, and hTERT.
  • an iPS cell expresses Oct-3/4, Sox2, Nanog, GDF3, REX1, FGF4, ESG1, DPPA2, DPPA4, and hTERT.
  • Methods of generating iPS are known in the art, and any such method can be used to generate iPS. See, e.g., Takahashi and Yamanaka (2006) Cell 126:663-676; Yamanaka et. al. (2007) Nature 448:313-7; Wernig et. al. (2007) Nature 448:318-24; Maherali (2007) Cell Stem Cell 1:55-70; Nakagawa et al. (2008) Nat. Biotechnol. 26:101; Takahashi et al. (2007) Cell 131:861; Takahashi et al. (2007) Nat. Protoc. 2:3081; and Okita et al. (2007 Nature 448:313.
  • iPS cells can be generated from somatic cells (e.g., skin fibroblasts) by genetically modifying the somatic cells with one or more expression constructs encoding Oct-3/4 and Sox2.
  • somatic cells are genetically modified with one or more expression constructs comprising nucleotide sequences encoding Oct-3/4, Sox2, c-myc, and Klf4.
  • somatic cells are genetically modified with one or more expression constructs comprising nucleotide sequences encoding Oct-4, Sox2, Nanog, and LIN28.
  • the present disclosure provides a multi-well plate comprising one or more wells.
  • the term“multi-well plate” refers to any support having a plurality of wells for holding an amount of cell culture media.
  • the one or more wells of the multi-well plate may receive and hold a culture medium from a liquid handling system.
  • the multi-well plate may be compatible with a multi-pillar plate of the present disclosure.
  • each of the one or more wells of the multi-well plate may receive or mate with a pillar of the multi-pillar plate.
  • Each well comprises one or more cell culture conditions.
  • each well may comprise a cell culture medium subjected to one or more cell culture conditions.
  • the multi-well plate may include a plurality of wells arranged in any configuration, e.g., a rectangular grid, a circular configuration, a linear configuration, etc.
  • the one or more wells may be regularly arranged at a substantially constant spacing. In some cases, the one or more wells are irregularly arranged such that the spacing between each well varies. In some instances, the multi- well plate includes a two-dimensional array of wells.
  • the two-dimensional array of wells may include 2 to 10,000 wells, such as, e.g., 8 to 5,000 wells, 10 to 1,000 wells, e.g., 10 to 900 wells, 20 to 800 wells, 30 to 700 wells, 40 to 600 wells, or 50 to 100 wells, e.g., 12, 24, 48, 96, 384, 1096 and 1536 wells.
  • the density of the wells in the two-dimensional array may range from 1 well/cm 2 to 300 wells/cm 2 , such as 2 wells/cm 2 to 200 wells/cm 2 .
  • the wells are arranged in rows and columns. In some instances, the wells are arranged in an array having two or more rows. In some instances, the wells are arranged in an array having two or more columns. In some instances, the sample wells are configured in a multi-well format.
  • Each of the one or more wells may have any suitable size and shape.
  • the well may receive and hold a volume of cell culture medium, e.g., 50 nL to 1000 nL, such as, e.g., 60 nL to 900 nL, 70 nL to 800 nL, 80 nL to 700 nL, 90 nL to 600 nL, 100 nL to 500 nL.
  • the well may include different configurations, e.g., diameters, shapes, as desired.
  • the one or more wells may be sized to receive one or more pillars of the multi-pillar plate.
  • the height of a well ranges from 0.1 pm to 1000 pm such as, e.g., 0.5 pm to 500 pm, 1 pm to 100 pm, or 10 pm to 50 pm.
  • the one or more wells may have any convenient volume.
  • the well has a volume ranging from, e.g., 50 nL to 1000 nL, such as, e.g., 60 nL to 900 nL, 70 nL to 800 nL, 80 nL to 700 nL, 90 nL to 600 nL, 100 nL to 500 nL.
  • the well has a volume of lOpL or less, such as 5pL or less, 2pL or less, 1.5pL or less, l.OpL or less, 0.7 pL or less, 0.5 pL or less, 0.2pL or less.
  • Each of the one or more wells of the multi-well plate may contain an amount of cell culture medium under one or more cell culture conditions.
  • the one or more cell culture conditions may support the growth and/or differentiation of a stem cell contained within the gel composition, e.g., to an oligodendrocyte progenitor cell or midbrain dopaminergic neuron.
  • the one or more cell culture conditions may comprise one or more seeding densities.
  • the device of the present disclosure provides for the culturing and differentiating of cells (e.g., differentiation of stem cells to neural cells, e.g., OPCs or midbrain dopaminergic neurons) with densities as high as about lxlO 9 cells/mL of hydrogel.
  • the device provides for the culturing and differentiating of cells (e.g., differentiation of pluripotent stem cells to OPCs or to oligodendrocytes) with densities as high as about lxlO 9 cells/mL of hydrogel, as high as about lxlO 7 cells/mL of hydrogel, as high as about lxlO 6 cells/mL of hydrogel, as high as about 1x10 s cells/mL of hydrogel, as high as about lxlO 4 cells/mL of hydrogel, or as high as about lxlO 3 cells/mL of hydrogel.
  • cells e.g., differentiation of pluripotent stem cells to OPCs or to oligodendrocytes
  • densities as high as about lxlO 9 cells/mL of hydrogel, as high as about lxlO 7 cells/mL of hydrogel, as high as about lxlO 6 cells/mL of hydrogel, as high as about 1x10
  • the device of the present disclosure provides for the culturing and differentiating of subject cells with a cell density in the range of from about l x lO 3 cells/mL to about 5 x 10 3 cells/mL, from about 5 x 10 3 cells/mL to about l x lO 4 cells/mL, from about l x lO 4 cells/mL to about 5 x 10 4 cells/mL, from about 10 s cells/mL to about 5 x 10 s cells/mL, from about 5 x 10 s cells/mL to about 10 6 cells/mL, from about 10 6 cells/mL to about 5 x 10 6 cells/mL, from about 5 x 10 6 cells/mL to about 10 7 cells/mL, from about 10 7 cells/mL to about 5 x 10 7 cells/mL, from about 5 x 10 7 cells/mL to about 10 s cells/mL, from about 10 s cells/mL to about 5 x 10 s cells/mL, from
  • the device of the present disclosure provides for the culturing and differentiating of subject cells with densities as high as about lxlO 9 cells/cc (lxlO 9 cells/cubic centimeter) of hydrogel.
  • a device of the present disclosure provides for the culturing and differentiating of subject cells with densities as high as about lxlO 9 cells/cc of hydrogel, as high as about lxlO 7 cells/cc of hydrogel, as high as about lxlO 6 cells/cc of hydrogel, as high as about 1x10 s cells/cc of hydrogel, as high as about lxlO 4 cells/cc of hydrogel, as high as about lxlO 3 cells/cc of hydrogel.
  • the one or more culture conditions may comprise one or more factors that support
  • the one or more culture conditions comprises a combination of two or more factors that support growth and/or differentiation of a cell contained within the gel composition.
  • the two or more factors drive the differentiation of oligodendrocyte precursor cells (OPCs) from pluripotent stem cells (PSCs).
  • OPCs oligodendrocyte precursor cells
  • PSCs pluripotent stem cells
  • Suitable factors for inclusion in the one or more cell culture mediums of the present disclosure include, but are not limited to, a Sonic hedgehog (Shh) signaling pathway agonist, a Wnt signaling pathway agonist, retinoic acid, and a dual-SMAD inhibitor.
  • suitable factors may include an antagonist of the SHH pathway, an antagonist of the Wnt pathway, and an antagonist of the Notch pathway.
  • one or more cell culture mediums or wells comprises a combination of factors comprising an Shh signaling pathway agonist (e.g., SAG), a Wnt signaling pathway agonist (e.g., CHIR99021), and retinoic acid.
  • one or more cell culture mediums or wells comprises a combination of factors comprising an Shh signaling pathway antagonist (e.g., GANTT61), a Wnt signaling pathway antagonist (e.g., IWP-2), and a Notch signaling pathway antagonist (DAPT).
  • an Shh signaling pathway antagonist e.g., GANTT61
  • a Wnt signaling pathway antagonist e.g., IWP-2
  • DAPT Notch signaling pathway antagonist
  • one or more cell culture mediums or wells comprises a combination of factors comprising an Shh signaling pathway agonist, a Wnt signaling pathway agonist, retinoic acid, a dual-SMAD inhibitor, an antagonist of the SHH pathway, an antagonist of the Wnt pathway, and an antagonist of the Notch pathway.
  • one or more cell culture mediums or wells may include a combination of factors comprising SAG, CHIR99021, retinoic acid, a dual-SMAD inhibitor such as SB431542 and/or LDN189193 (or LDN193189), GANTT61, IWP-2, and DAPT.
  • the combination of factors is present in the device of the present disclosure in an amount effective to induce differentiation of neural cells, e.g., OPCs or midbrain dopaminergic neurons, from stem cells, e.g., hESCs, PSCs, etc.
  • the at least one stem cell of the gel composition may be exposed to the one or more factors, e.g., a combination of the factors described above, for any suitable amount of time, e.g., 5 minutes to 2 weeks, e.g., 5-30 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 30 minutes to 1 hour, 1 hour, 1-2 hours, 2 hours, 1-3 hours, 3 hours, 2-4 hours, 4 hours, , 3-5 hours, 5 hours, 4-6 hours, 6 hours, 8 hours, 10 hours, 12-24 hours, 12 hours, 18 hours, 24 hours, 24-36 hours, 36 hours, 36-48, 48 hours, 2 days-1 week, 2 days-90 days, 2 days-80 days, 2 days-70 days, 2 days- 60 days, 2 days- 50 days, 2 days- 40 days, 2 days- 30 days, 2 days-20 days, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14
  • the amount of the one or more factors may be modulated over a period of time.
  • the amount of the one or more factors may be constant, increased, or decreased over a period of time, e.g., 5 minutes to 2 weeks, e.g., 5-30 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 30 minutes to 1 hour, 1 hour, 1-2 hours, 2 hours, 1-3 hours, 3 hours, 2-4 hours, 4 hours, , 3-5 hours, 5 hours, 4-6 hours, 6 hours, 8 hours, 10 hours, 12-24 hours, 12 hours, 18 hours, 24 hours, 24-36 hours, 36 hours, 36-48, 48 hours, 2 days-1 week, 2 days-90 days, 2 days-80 days, 2 days-70 days, 2 days- 60 days, 2 days- 50 days, 2 days- 40 days, 2 days- 30 days, 2 days-20 days, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14
  • one or more factors may be increased or decreased by at least 20 fold, at least 15 fold, at least 10 fold, at least 9 fold, at least 8 fold, at least 7 fold, at least 6 fold, at least 5 fold, at least 4 fold, at least 3 fold, at least 2 fold, or at least 1 fold.
  • one or more initial factors are introduced to one or more cell culture mediums before or after one or more additional factors are introduced to one or more cell culture mediums.
  • An Shh signaling pathway agonist used in combination with one or more other factors in a three- dimensional biocompatible thermoresponsive polymer of the present disclosure can be SAG, where SAG is a compound of the formula:
  • Shh signaling pathway can be used.
  • other Shh signaling pathway agonists can be used, including, but not limited to, the SAG analog 3,4-dichloro-/V-(cA-4-(methylamino)cyclohexyl)- /V-(3-pyridin-4-ylbenzyl)benzo[Z?]thiophene-2-carboxamide, SAG derivatives, an Shh polypeptide and/or variant thereof, an Shh protein-polymer conjugate, purmorphamine, purmorphamine derivatives, the synthetic non-peptidyl small molecule Hh-Ag (Frank- Kamenetsky et a , J. Biol.
  • Shh signaling pathway agonists that may find use in the present disclosure include agonists that are derived from several sources of hedgehog protein.
  • a suitable Shh signaling pathway agonist can be a Shh polypeptide that has a hydrophobic palmitoyl group appended to the alpha- amine of the N-terminal cysteine.
  • the Shh signaling pathway agonist is SAG.
  • a suitable Shh signaling pathway agonist includes an Shh polypeptide.
  • a suitable Shh polypeptide can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100%, amino acid sequence identity to amino acids 24-462 of the Shh amino acid set forth in SEQ ID NO:l.
  • the Shh signaling pathway agonist is present in the device of the present
  • the Shh signaling pathway agonist can be present in the device of the present disclosure in a concentration of from about 0.5 mM to about 2 mM.
  • the Shh signaling pathway agonist can be present in the device of the present disclosure in a
  • concentration of from about 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, or 0.8 mM, to about 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2 mM, 2.1 mM, 2.2 mM, 2.3 mM, 2.4 mM, or 2.5 mM.
  • the agonist is present in a concentration of from about 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, or 0.8 mM, to about 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2 mM, 2.1 mM, 2.2 mM, 2.3 mM, 2.4 mM, or 2.5 mM.
  • the agonist can be present in a concentration from about 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, or 1.5 mM, to about 8 mM, 8.5 mM, 9 mM, 9.5 mM, 9.6 mM, 9.7 mM, 9.8 mM, 9.9 mM, 10 mM, 10.1 mM, 10.2 mM, 10.3 mM, 10.4 mM, 10.5 mM, 11 mM, 11.5 mM, or 12 mM.
  • the agonist can be present in a concentration from about 10 nM to about 1000 nM such as, e.g., from about 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, to about 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM.
  • a Wnt signaling pathway agonist included in a three-dimensional biocompatible polymer of the present disclosure can be CHIR99021, where CHIR99021 is a compound of the formula:
  • Wnt signaling pathway can be used.
  • other Wnt signaling pathway agonists can be used, including, but not limited to, CHIR-99021 analogs and derivatives, WAY-316606 (Bodine et al., Bone. 2009, 44(6): 1063-1068), (hetero)arylpyrimidines (Gilbert et al., Bioorg. Med. Chem. Lett. 2010, 20(l):366-370), IQ-1 (Miyabashi et al., Proc. Natl. Acad. Sci. U.S.A. 2007,
  • the Wnt signaling pathway agonist (e.g., CHIR-99021) is present in the device of the present disclosure in a concentration of from about 1 mM to about 10 mM.
  • the Wnt signaling pathway agonist can be present in the device of the present disclosure in a concentration of from about 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, or 1.5 mM, to about 8 mM, 8.5 mM, 9 mM, 9.5 mM, 9.6 mM, 9.7 mM, 9.8 mM, 9.9 mM, 10 mM, 10.1 mM, 10.2 mM, 10.3 mM, 10.4 mM, 10.5 mM, 11 mM, 11.5 mM, or 12 mM.
  • the agonist is present in a concentration of from about 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, or 0.8 mM, to about 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2 mM, 2.1 mM, 2.2 mM, 2.3 mM, 2.4 mM, or 2.5 mM.
  • the agonist can be present in a concentration from about 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, or 1.5 mM, to about 8 mM, 8.5 mM, 9 mM, 9.5 mM, 9.6 mM, 9.7 mM, 9.8 mM, 9.9 mM, 10 mM, 10.1 mM, 10.2 mM, 10.3 mM, 10.4 mM, 10.5 mM, 11 mM, 11.5 mM, or 12 mM.
  • the agonist can be present in a concentration from about 10 nM to about 1000 nM such as, e.g., from about 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, to about 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM.
  • biocompatible polymer of the present disclosure to drive differentiation of OPCs comprises a Sonic hedgehog (Shh) signaling pathway agonist, a Wnt signaling pathway agonist, and retinoic acid.
  • Retinoic acid or a retinoic acid analog, can be included in a device of the present disclosure.
  • Suitable retinoic acid analogs include, e.g., retinoic acid isomers [all-trans-retinoic acid (ATRA), 9-cis-retinoic acid (9CRA) and 13-cis-retinoic acid (13CRA)] and their oxidized derivatives [19- hydroxy and 19-oxo derivatives of ATRA (19-hydroxy- ATRA and 19-oxo-ATRA), 19-oxo derivative of 9CRA (19-oxo-9CRA), and 19-hydroxy derivative of 13CRA (19-hydroxy- 13CRA)].
  • ATRA all-trans-retinoic acid
  • 9CRA 9-cis-retinoic acid
  • 13CRA 13-cis-retinoic acid
  • retinoic acid (or a retinoic acid analog) is present in the device of the present disclosure in a concentration of from about 10 nM to about 1000 nM.
  • retinoic acid can be present in the device of the present disclosure in a concentration of from about 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, or 80 nM, to about 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, or 200 nM.
  • retinoic acid is present in a concentration of from about 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, or 0.8 mM, to about 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2 mM, 2.1 mM, 2.2 mM, 2.3 mM, 2.4 mM, or 2.5 mM.
  • retinoic acid can be present in a concentration from about 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, or 1.5 mM, to about 8 mM, 8.5 mM, 9 mM, 9.5 mM, 9.6 mM, 9.7 mM, 9.8 mM, 9.9 mM, 10 mM, 10.1 mM, 10.2 mM, 10.3 mM, 10.4 mM, 10.5 mM, 11 mM, 11.5 mM, or 12 mM.
  • retinoic acid can be present in a concentration from about 10 nM to about 1000 nM such as, e.g., from about 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, to about 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM.
  • Dual-SMAD inhibitors Dual-SMAD inhibitors
  • a device of the present disclosure can include a factor or factors that lead to dual-SMAD inhibition.
  • Dual-SMAD inhibition can be achieved by inhibition of type I activin receptor-like kinase receptors and type I bone morphogenetic protein (BMP) receptors, i.e., inhibition of activin and BMP signaling.
  • BMP bone morphogenetic protein
  • Smad2 and Smad3 are effectors in the TGFbeta/activin pathway; Smadl/5/9 are effectors in the TGFbeta/activin pathway; Smadl/5/9 are effectors in the TGFbeta/activin pathway; Smadl/5/9 are effectors in the TGFbeta/activin pathway; Smadl/5/9 are effectors in the TGFbeta/activin pathway; Smadl/5/9 are effectors in the TGFbeta/activin pathway
  • TGFbeta/activin and BMP signaling pathways can regulate the differentiation of hPSCs into non-neuroectodermal lineages.
  • two inhibitors - one for TGFbeta/activin and one for BMP signaling - are used.
  • Use of two inhibitors can induce differentiation of a stem cell into a neuroectodermal lineage, which is an early step towards getting an OPC (or a neuron).
  • Use of two inhibitors - one for TGFbeta/activin and one for BMP signaling - is termed“dual SMAD inhibition.” In some cases, the inhibitors do not inhibit the Smad proteins themselves but instead inhibit other members in the pathways.
  • SB431542 inhibits TGFbeta/activin receptors (which lie upstream of Smad2/3), and LDN189193 inhibits BMP receptors (which lie upstream of Smadl/5/9).
  • a “dual-Smad inhibitor” refers to an agent that inhibits the TGFbeta/activin pathway or that inhibits BMP signaling.
  • dual-SMAD inhibition is achieved by administering SB431542 and
  • dual-SMAD inhibition is achieved by administering SB431542 and
  • suitable dual-SMAD inhibitors of the present disclosure can be a combination of factors such as SB431542 and LDN 189193, where SB431542 is a compound of the formula:
  • LDN193189 is a compound of the formula:
  • a suitable dual-SMAD inhibitor of the present disclosure is a single factor such as compound C (Zhou et al., Stem Cells. 2012, 28(10): 1741-1750).
  • Dual-SMAD inhibitors of the present disclosure that are a combination of factors inhibit both activin signaling (factors that may include, but are not limited to, SB431542 and derivatives, SB505124, activin-M108, inhibin, betaglycan, follistatin, follistatin-related gene (FLRG), Cripto, BAMBI (for review, see, Harrison et al., Trends Endocrin. Metabol.
  • BMP signaling factors that may include, but are not limited to, LDN189193 and derivatives, Noggin (Chambers et al., Nat. Biotechnol. 2009, 27(3):275-280), and the like.
  • Other dual-SMAD inhibitors may include, but are not limited to, Dorsomorphin and Dorsomorphin analogs DMH1, DMH2 and LDN (Langenfeld et al., PLoS One. 2013, 8(4):e61256).
  • a combination of factors that function together with a three-dimensional biocompatible polymer of the present disclosure to drive differentiation of OPCs comprises a Sonic hedgehog (Shh) signaling pathway agonist, a Wnt signaling pathway agonist, and retinoic acid.
  • the combination of factors further comprises a dual-SMAD inhibitor (e.g., SB431542 and Noggin; or SB431542 and LDN189193) that is present in a device of the present disclosure, where SB431542 is present at a concentration of about from about 1 mM to about 20 mM (e.g., about 1 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, 20 mM, etc.), and wherein Noggin is present at a concentration of about 500 ng/mL (e.g., about 400 ng/mL, about 450 ng/mL, about 470 ng/mL, about 480 ng/mL, about 490 ng/mL, about 510 ng/mL, about 520 ng/mL, about 530 ng/m
  • the combination of factors comprises SB431542 and LDN189193, where SB431542 is present at a concentration of about from about 1 mM to about 20 mM (e.g., about 1 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, 20 mM, etc.); and where LDN189193 is present at a concentration of from about 10 nM to about 250 nM (e.g., about 10 nM, about 20 nM, about 50 nM, about 75 nM, about 100 nM, about 125 nM, about 200 nM, or about 250 nM).
  • SB431542 is present at a concentration of about from about 1 mM to about 20 mM (e.g., about 1 mM, about 5 mM, about 6 mM, about 7
  • the inhibitor is present in a concentration of from about 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, or 0.8 mM, to about 1.5 mM, 1.6 mM,
  • the inhibitor can be present in a concentration from about 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM,
  • the inhibitor can be present in a concentration from about 10 nM to about 1000 nM such as, e.g., from about 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, to about 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM,
  • Shh signaling pathway antagonists that have the same effect on the Shh signaling pathway can be used.
  • the Shh signaling pathway antagonist is present in the device of the
  • the Shh signaling pathway antagonist can be present in the device of the present disclosure in a concentration of from about 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, or 0.8 mM, to about 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2 mM, 2.1 mM, 2.2 mM, 2.3 mM, 2.4 mM, or 2.5 mM.
  • the antagonist is present in a concentration of from about 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, or 0.8 mM, to about 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2 mM, 2.1 mM, 2.2 mM, 2.3 mM, 2.4 mM, or 2.5 mM.
  • the antagonist can be present in a concentration from about 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM,
  • the antagonist can be present in a concentration from about 10 nM to about 1000 nM such as, e.g., from about 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, to about 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM.
  • a Wnt signaling pathway antagonist included in a three-dimensional biocompatible polymer of the present disclosure can be IWP-2 is a compound of the formula:
  • Wnt signaling pathway can be used.
  • the Wnt signaling pathway antagonist (e.g., IWP-2) is present in the device of the present disclosure in a concentration of from about 1 mM to about 10 mM.
  • the Wnt signaling pathway antagonist can be present in the device of the present disclosure in a concentration of from about 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, or 1.5 mM, to about 8 mM, 8.5 mM, 9 mM, 9.5 mM, 9.6 mM, 9.7 mM, 9.8 mM, 9.9 mM, 10 mM, 10.1 mM, 10.2 mM, 10.3 mM, 10.4 mM, 10.5 mM, 11 mM, 11.5 mM, or 12 mM.
  • the antagonist is present in a concentration of from about 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, or 0.8 mM, to about 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2 mM, 2.1 mM, 2.2 mM, 2.3 mM, 2.4 mM, or 2.5 mM.
  • the antagonist can be present in a concentration from about 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, or 1.5 mM, to about 8 mM, 8.5 mM, 9 mM, 9.5 mM, 9.6 mM, 9.7 mM, 9.8 mM, 9.9 mM, 10 mM, 10.1 mM, 10.2 mM, 10.3 mM, 10.4 mM, 10.5 mM, 11 mM, 11.5 mM, or 12 mM.
  • the antagonist can be present in a concentration from about 10 nM to about 1000 nM such as, e.g., from about 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, to about 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM.
  • a Notch signaling pathway antagonist included in a three-dimensional biocompatible polymer of the present disclosure can be DAPT is a compound of the formula:
  • Notch signaling pathway antagonists that have the same effect on the Notch signaling pathway can be used.
  • the Notch signaling pathway antagonist (e.g., DAPT) is present in the device of the present disclosure in a concentration of from about 1 mM to about 10 mM.
  • the Notch signaling pathway antagonist can be present in the device of the present disclosure in a concentration of from about 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, or 1.5 mM, to about 8 mM, 8.5 mM, 9 mM, 9.5 mM, 9.6 mM, 9.7 mM, 9.8 mM, 9.9 mM, 10 mM, 10.1 mM, 10.2 mM, 10.3 mM, 10.4 mM, 10.5 mM, 11 mM, 11.5 mM, or 12 mM.
  • the antagonist is present in a concentration of from about 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, or 0.8 mM, to about 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2 mM, 2.1 mM, 2.2 mM, 2.3 mM, 2.4 mM, or 2.5 mM.
  • the antagonist can be present in a concentration from about 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, or 1.5 mM, to about 8 mM, 8.5 mM, 9 mM, 9.5 mM, 9.6 mM, 9.7 mM, 9.8 mM, 9.9 mM, 10 mM, 10.1 mM, 10.2 mM, 10.3 mM, 10.4 mM, 10.5 mM, 11 mM, 11.5 mM, or 12 mM.
  • the antagonist can be present in a concentration from about 10 nM to about 1000 nM such as, e.g., from about 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, to about 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM.
  • a combination of factors that function together with a three-dimensional biocompatible polymer of the present disclosure to drive differentiation of OPCs comprises a Sonic hedgehog (Shh) signaling pathway antagonist, a Wnt signaling pathway agonist, and DAPT.
  • a device of the present disclosure can be part of a system that provides for analysis and/or purification of cells generated using the device.
  • a system of the present disclosure comprises: a) a device of the present disclosure; and b) one or more of: i) a wide field fluorescent microscope; ii) a confocal fluorescent microscope; iii) a flow cytometry device; iv) computer hardware and software for data analysis (for analysis of data generated using a device of the present disclosure); v) image processing hardware; and software; vi) image analysis hardware and software.
  • the present disclosure provides a method of screening multiple cell culture conditions.
  • the method screens for neural cell differentiation culture conditions.
  • Such methods at least comprise, for example, depositing one or more gel compositions comprising at least one stem cell onto one or more pillars of a multi-pillar plate; contacting each of the one or more pillars with one or more cell culture mediums in one or more wells of a multi- well plate, under one or more cell culture conditions, generating a medium-contacted stem cell; and detecting at least one neural cell marker in the medium-contacted stem cell, wherein an increase in expression of the at least one marker in the medium-contacted stem cell, compared to a control, indicates that the one or more cell culture conditions modulate stem cell differentiation to neural cells.
  • the screening method may use the device of the present disclosure comprising a multi-pillar plate and a multi-well plate, as described in the detail above.
  • a screening method of the present disclosure allows a person of skill in the art to identify one or more culture conditions that drives the generation of OPCs from suitable cells as previously described (e.g., a pluripotent stem cell).
  • one or more culture conditions identified by screening methods of the present disclosure increases the rate of generation of OPCs from suitable cells, e.g., by 10%, by 20%, by 30%, by 40%, by 50%, by 60%, by 70%, by 80%, by 90%, by 100% or more (e.g., by 120%, by 150%, by 200% or more) in about 80 days or less, 50 days or less, 40 days or less, 30 days or less, 20 days or less, 15 days or less, 10 days or less, or in about 5 days or less, etc.
  • suitable cells e.g., by 10%, by 20%, by 30%, by 40%, by 50%, by 60%, by 70%, by 80%, by 90%, by 100% or more (e.g., by 120%, by 150%, by 200% or more) in about 80 days or less, 50 days or less, 40 days or less, 30 days or less, 20 days or less, 15 days or less, 10 days or less, or in about 5 days or less, etc.
  • the method comprises modulating any suitable amount or concentration of one or more factors over a period of time.
  • the amount of the one or more factors may be constant, increased, or decreased over a period of time, e.g., 5 minutes to 2 weeks, e.g., 5-30 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 30 minutes to 1 hour, 1 hour, 1-2 hours, 2 hours, 1-3 hours, 3 hours, 2-4 hours, 4 hours, , 3-5 hours,
  • one or more factors may be increased or decreased by at least 20 fold, at least 15 fold, at least 10 fold, at least 9 fold, at least 8 fold, at least 7 fold, at least 6 fold, at least 5 fold, at least 4 fold, at least 3 fold, at least 2 fold, or at least 1 fold.
  • one or more initial factors are introduced to one or more cell culture mediums before or after one or more additional factors are introduced to one or more cell culture mediums.
  • Suitable factors for inclusion in the one or more cell culture mediums of the present disclosure include, but are not limited to, a Sonic hedgehog (Shh) signaling pathway agonist, a Wnt signaling pathway agonist, retinoic acid, and a dual-SMAD inhibitor.
  • suitable factors may include an antagonist of the SHH pathway, an antagonist of the Wnt pathway, and an antagonist of the Notch pathway.
  • one or more cell culture mediums or wells comprises a combination of factors comprising an Shh signaling pathway agonist (e.g., SAG), a Wnt signaling pathway agonist (e.g., CHIR99021), and retinoic acid.
  • one or more cell culture mediums or wells comprises a combination of factors comprising an Shh signaling pathway antagonist (e.g., GANTT61), a Wnt signaling pathway antagonist (e.g., IWP-2), and a Notch signaling pathway antagonist (DAPT).
  • an Shh signaling pathway antagonist e.g., GANTT61
  • a Wnt signaling pathway antagonist e.g., IWP-2
  • DAPT Notch signaling pathway antagonist
  • one or more cell culture mediums or wells comprises a combination of factors comprising an Shh signaling pathway agonist, a Wnt signaling pathway agonist, retinoic acid, a dual-SMAD inhibitor, an antagonist of the SHH pathway, an antagonist of the Wnt pathway, and an antagonist of the Notch pathway.
  • one or more cell culture mediums or wells may include a combination of factors comprising SAG, CHIR99021, retinoic acid, a dual-SMAD inhibitor such as SB431542 and/or LDN189193 (or LDN193189), GANTT61, IWP-2, and DAPT.
  • the combination of factors is present in the device of the present disclosure in an amount effective to induce differentiation of neural cells, e.g., OPCs or midbrain dopaminergic neurons, from stem cells, e.g., hESCs, PSCs, etc.
  • the subject methods comprise depositing one or more gel compositions comprising at least one stem cell onto one or more pillars of a multi-pillar plate by any suitable liquid handling system.
  • the one or more gel compositions is deposited onto a surface of the one or more pillars, e.g., a top surface of one or more pillars.
  • any suitable amount of gel composition may be deposited or dispensed onto each of the one ore more micropillars such as, e.g., from 50 nL to 1000 nL, such as, e.g., 60 nL to 900 nL, 70 nL to 800 nL, 80 nL to 700 nL, 90 nL to 600 nL, 100 nL to 500 nL.
  • the concentration of cells in a deposited gel composition can range from about 50 mM to about 500 mM, e.g., from about 50 mM to about 75 mM, from about 75 mM to about 100 mM, from about 100 mM to about 125 mM, from about 125 mM to about 150 mM, from about 150 mM to about 200 mM, from about 200 mM to about 250 mM, from about 250 mM to about 300 mM, from about 300 mM to about 350 mM, from about 350 mM to about 400 mM, from about 400 mM to about 450 mM, or from about 450 mM to about 500 mM.
  • the weight percent of the cells suspended in the deposited gel composition can range from 0.01 weight% to about 1 weight%, e.g., 0.01 weight%, 0.02 weight%, 0.03 weight%, 0.04 weight%, 0.05 weight%, from 0.05 weight% to about 0.1 weight%, from about 0.1 weight% to about 0.25 weight%, from about 0.25 weight% to about 0.5 weight%, from about 0.5 weight% to about 0.75 weight%, or from about 0.75 weight% to 1 weight%.
  • the methods of the present disclosure comprise contacting each of the one or more pillars with one or more cell culture mediums in one or more wells of a multiwell plate, under one or more cell culture conditions.
  • the multi-pillar plate is mated with the multi- well plate such that the one or more pillars are inserted into the one or more wells.
  • a pillar may be inserted into a well such that the pillar fills the volume or a portion of the volume of the well.
  • the one or more gel compositions comprising the at least one stem cell may contact, e.g., may be submerged in, the one or more cell culture mediums.
  • the contacting of the one or more pillars with the one or more culture mediums under one or more culture conditions may generate a medium-contacted stem cell.
  • the term“medium- contacted stem cell” refers to a stem cell encapsulated in the gel composition that has been contacted with a cell culture medium contained in a well of the multi- well plate, under one or more cell culture conditions.
  • the differentiated cell is a neural cell.
  • the contacting generates a neural progenitor cell, e.g., an oligodendrocyte progenitor cell, expressing one or more markers including, e.g., 01ig2, Nkx2.2, or Tujl.
  • the contacting generates a neural cell, e.g., midbrain dopaminergic (mDA) neuron expressing one or more markers including, e.g., tyrosine hydroxylase (TH) and Tuj 1.
  • mDA midbrain dopaminergic
  • the method comprises detecting at least one phenotypic marker, e.g., neural cell marker, in the medium-contacted stem cell, wherein an increase in expression of the at least one marker in the medium-contacted stem cell, compared to a control, indicates that the one or more cell culture conditions modulate stem cell differentiation to neural cells.
  • a control of the subject methods may include, e.g., a stem cell that has not been contacted with any cell culture medium under the one or more cell culture conditions of the methods.
  • the control includes a stem cell that has been contacted with cell culture medium under conventional culture conditions to induce differentiation to a neural cell.
  • Suitable neural cell markers include, but are not limited to, 01ig2, Nkx2.2, TH, and Tujl.
  • suitable neural progenitor cell e.g., OPC
  • markers comprise 01ig2, Nkx2.2, and Tujl.
  • suitable midbrain dopaminergic (mDA) neuron markers comprise TH and Tujl.
  • the method comprises detecting a combination of neural markers, e.g., at least 2 or at least 3 neural markers.
  • the at least one neural cell marker may be labeled with a detectable label.
  • the neural cell markers may be directly or indirectly labelled using a variety of available methods known in the art. Labelled markers offer the convenience of automatic and high-throughput detection.
  • the detectable label is a protein that can generate a detectable signal.
  • the protein may include, but is not limited to, a protein enzyme capable of catalyzing conversion of a substrate to a detectable reaction product, either directly or indirectly, which have been used, for example, in cell-based screening assays.
  • enzymes such as b-galactosidase, b- glucuronidase (GUS), b-lactamase, alkaline phosphatase, peroxidase (e.g., horse radish peroxidase), chloramphenicol acetyltransferase (CAT) and luciferase. Any of a range of enzymes capable of producing a detectable product either directly or indirectly may be so modified or may occur naturally.
  • detectable proteins include green fluorescent proteins, which have characteristic detectable emission spectra, and have been modified to alter their emission spectra, as described in PCT WO 96/23810, the disclosure of which is incorporated herein, and fluorescent protein from an Anthozoa species (see, e.g., Matz et al., Nat. Biotechnol. 1999, 17:969-973); and the like. Fusions of fluorescent proteins with other proteins, and DNA sequences encoding the fusion proteins which are expressed in cells are described in PCT WO 95/07463, the disclosure of which is incorporated herein.
  • Suitable fluorescent proteins include, but are not limited to, green fluorescent protein
  • GFP blue fluorescent variant of GFP
  • BFP blue fluorescent variant of GFP
  • CFP yellow fluorescent variant of GFP
  • YFP yellow fluorescent variant of GFP
  • EGFP enhanced GFP
  • ECFP enhanced CFP
  • EYFP enhanced YFP
  • GFPS65T Emerald, Topaz (TYFP)
  • Citrine mCitrine
  • GFPuv destabilised EGFP
  • dECFP destabilised ECFP
  • EYFP destabilised EYFP
  • mCFPm Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed- monomer, J-Red, dimer2, t-dimer2(12), mRFPl, pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein and kindling protein, Phycobiliproteins and Phy
  • fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrapel, mRaspberry, mGrape2, mPlum (Shaner et al. (2005) Nat. Methods 2:905-909), and the like. Any of a variety of fluorescent and colored proteins from Anthozoan species, as described in, e.g., Matz et al. (1999) Nature Biotechnol. 17:969-973, is suitable for use.
  • the present disclosure provides methods of generating a plurality of neural cells, e.g., oligodendrocyte precursor cells (OPCs), oligodendrocytes, or midbrain neurons, using a three- dimensional culture system.
  • the method of generating neural cells comprises culturing suitable cells, e.g., pluripotent stem cells, using a three-dimensional culture system, e.g., under one or more cell culture conditions identified in the screen using the device of the present disclosure.
  • the method of generating neural cells comprises differentiating suitable cells by culturing the cells using a three-dimensional culture system, e.g., under one or more cell culture conditions identified in the screen using the device of the present disclosure.
  • the method of generating neural cells comprises differentiating suitable cells (e.g., pluripotent stem cells) by culturing the cells using a three-dimensional culture system that comprises a biocompatible polymer, e.g., a thermoresponsive biocompatible polymer, and one factor, or a combination of factors, which are present in the system in an effective concentration to drive the differentiation of the cells into neural cells.
  • suitable cells e.g., pluripotent stem cells
  • a three-dimensional culture system that comprises a biocompatible polymer, e.g., a thermoresponsive biocompatible polymer, and one factor, or a combination of factors, which are present in the system in an effective concentration to drive the differentiation of the cells into neural cells.
  • methods of the present disclosure include methods for the generation and/or differentiation of neural cells in a large- scale manner.
  • the methods comprise generating and/or differentiating neural cells in any suitable container having a volume ranging from lmL to 100 mL, such as, e.g., from lOmL to 90 mL, from 20 mL to 80 mL, from 30 mL to 70 mL, and from 40 mL to 50 mL.
  • the three-dimensional culture system comprises a biocompatible polymer.
  • the gel composition may comprise a hydrogel cell matrix.
  • the hydrogel cell matrix may include a plurality of hydrogel polymers. Any suitable hydrogel polymers can be used in the hydrogel cell matrices provided herein.
  • Hydro gel polymers may include the following monomers: lactic acid, glycolic acid, acrylic acid, 1-hydroxyethyl methacrylate (HEM A), ethyl methacrylate (EM A), propylene glycol methacrylate (PEMA), acrylamide (A AM), N-vinylpyrrolidone, methyl methacrylate (MMA), glycidyl methacrylate (GDMA), glycol methacrylate (GMA), ethylene glycol, fumaric acid, and the like.
  • Common cross linking agents include tetraethylene glycol dimethacrylate (TEGDMA) and N,N'-methylenebisacrylamide.
  • TEGDMA tetraethylene glycol dimethacrylate
  • N,N'-methylenebisacrylamide N,N'-methylenebisacrylamide.
  • the hydrogel can be homopolymeric, or can comprise co-polymers of two or more of the aforementioned polymers.
  • hydrogel polymer that encapsulates the stem cell or progenitor cell e.g., an
  • oligodendrocyte progenitor cell is generally hydrophilic.
  • Suitable hydrogel polymers include, but are not limited to, poly(N-isopropylacrylamide) (pNIPAAm); poly(N- isopropylacrylamide-co-acrylic acid); hyaluronic acid or hyaluronate; cross! inked hyaluronic acid or hyaluronate; pHEMA; or copolymers of p(NIPAAm)-based sIPNs and other hydrogel sIPNs (semi-interpenetrating networks).
  • the hydrogel polymer is a hyaluronic acid (HyA) polymer.
  • the hydrogel polymer is an acrylated hyaluronic acid (HyA) polymer.
  • the hydrogel is a temperature-sensitive hydrogel. In some cases, a
  • temperature-sensitive hydrogel is a polyacrylic acid or derivative thereof, e.g., poly (N- isopropylacrylamide) gel, and the increase in temperature causes the hydrogel to contract, thereby forcing an agent out of the hydrogel.
  • the temperature-sensitive hydrogel is an interpenetrating hydrogel network of poly (acrylamide) and poly (acrylic acid), and the increase in temperature causes the hydrogel to swell, thereby allowing an agent to diffuse out of the gel.
  • the temperature required for triggering release of an agent from the hydrogel is generally about normal body temperature, e.g., about 37°C.
  • the stiffness module of a subject hydrogel matrix can be in the range of from about 15
  • Pascals (Pa) to about 850 Pa e.g., from about 15 Pa to about 20 Pa, from about 20 Pa to about 50 Pa, from about 50 Pa to about 100 Pa, from about 100 Pa to about 150 Pa, from about 150 Pa to about 200 Pa, from about 200 Pa to about 250 Pa, from about 250 Pa to about 300 Pa, from about 300 Pa to about 350 Pa, from about 350 Pa to about 400 Pa, from about 400 Pa to about 500 Pa, from about 500 Pa to about 600 Pa, from about 600 Pa to about 700 Pa, from about 700 Pa to about 800 Pa, or from about 800 Pa to about 850 Pa.
  • the gel composition comprises a biocompatible polymer.
  • the biocompatible polymer is thermoresponsive and forms a hydrogel at certain temperatures. Some thermoresponsive polymers can undergo reversible phase transition from a liquid state at lower temperatures to a solid state at higher temperatures.
  • a biocompatible thermoresponsive polymer of the present disclosure is a liquid at around 4°C.
  • a biocompatible thermoresponsive polymer of the present disclosure is a liquid at a temperature that is below about 25°C, below about 22°C, below about 20°C, below about 18°C, below about 16°C, below about 14°C, below about 12°C, below about 10°C, below about 8°C, below about 6°C, below about 4°C, below about 2°C, or around 0°C.
  • a biocompatible thermoresponsive polymer of the present disclosure is liquid at a temperature in the range of from about 2°C to 4°C, from 4°C to 6°C, from 2°C to 6°C, from 6°C to 10°C, or from 2°C to 10°C.
  • a biocompatible thermoresponsive polymer of the present disclosure is a solid, semi-solid, or a gel at around 37°C.
  • a biocompatible thermoresponsive polymer of the present disclosure is a solid, semi-solid, or a gel at a temperature of at least 30°C, at least 32°C, at least 34°C, at least 35°C, at least 36°C, at least 37°C, at least 38°C, at least 40°C, at least 42°C.
  • a biocompatible thermoresponsive polymer of the present disclosure is a gel at a temperature in the range of from about 34°C to 35°C, from 35°C to 38°C, or from 34°C to 37°C.
  • a biocompatible thermoresponsive polymer of the present disclosure is a gel at a temperature in the range of from about 34°C to 35°C, from 35°C to 38°C, or from 34°C to 37°C.
  • a biocompatible thermoresponsive polymer of the present disclosure is a gel at a temperature in the range of from
  • thermoresponsive polymer of the present disclosure is a liquid at a temperature in the range of from about 2°C to 4°C, from 4°C to 6°C, from 2°C to 6°C, from 6°C to 10°C, or from 2°C to 10°C; and is a gel at a temperature in the range of from about 34°C to 35°C, from 35°C to 38°C, or from 34°C to 37°C.
  • a biocompatible thermoresponsive polymer of the present disclosure is liquid at 4°C and becomes a gel when warmed to 37°C.
  • Suitable thermoresponsive and/or thermoreversible biopolymers may further include those described in WO 2017/062374, the disclosure of which is incorporated by reference herein.
  • a gel composition comprising any one of the
  • hydrogel cell matrices provided herein and a stem cell or progenitor cell encapsulated in the matrix.
  • the concentration of cells in a subject hydrogel matrix can range from about 50 mM to about 500 mM, e.g., from about 50 mM to about 75 mM, from about 75 mM to about 100 mM, from about 100 mM to about 125 mM, from about 125 mM to about 150 mM, from about 150 mM to about 200 mM, from about 200 mM to about 250 mM, from about 250 mM to about 300 mM, from about 300 mM to about 350 mM, from about 350 mM to about 400 mM, from about 400 mM to about 450 mM, or from about 450 mM to about 500 mM.
  • the weight percent of the cells suspended in the hydrogel matrix can range from 0.01 weight% to about 1 weight%, e.g., 0.01 weight%, 0.02 weight%, 0.03 weight%, 0.04 weight%, 0.05 weight%, from 0.05 weight% to about 0.1 weight%, from about 0.1 weight% to about 0.25 weight%, from about 0.25 weight% to about 0.5 weight%, from about 0.5 weight% to about 0.75 weight%, or from about 0.75 weight% to 1 weight%.
  • methods using a three-dimensional culture system of the present disclosure allows for rapid generation of neural cells, e.g., OPCs or midbrain neurons.
  • a subject method of generating neural cells using a three-dimensional culture system of the present disclosure require substantially less than 110-150 days, e.g., substantially less than 110 days, substantially less than 100 days, to generate at least 10 3 neural cells.
  • a subject method of generating neural cells using a three-dimensional culture system of the present disclosure requires less than 50 days, e.g., less than 40 days, less than 30 days, less than 20 days, less than 10 days, etc., to generate at least 10 3 neural cells.
  • a subject method of generating neural cells using a three-dimensional culture system of the present disclosure requires from about 18 days to about 20 days, e.g., about 18-20 days, about 15-20 days, about 15 days, about 20 days, etc., to generate at least 10 3 neural cells.
  • a method of the present disclosure for generating neural cells uses a three-dimensional culture system of the present disclosure, generates from about 10 3 neural cells to about 10 9 neural cells (e.g., from about 10 3 to about 10 4 , from about 10 4 to about 10 s , from about 10 s to about 10 6 , from about 10 6 to about 10 7 , from about 10 7 to about 10 s , or from about 10 s to 10 9 , or more than 10 9 , neural cells) in a period of time of from about 5 days to about 30 days, e.g., from about 5 days to about 7 days, from about 7 days to about 10 days, from about 10 days to about 15 days, from about 15 days to about 20 days, from about 20 days to about 25 days, or from about 25 days to about 30 days.
  • neural cells e.g., from about 10 3 to about 10 4 , from about 10 4 to about 10 s , from about 10 s to about 10 6 , from about 10 6 to about 10 7 , from about 10 7 to about
  • a method of the present disclosure for generating neural cells uses a three- dimensional culture system of the present disclosure, generates from about 10 3 neural cells to about 10 9 neural cells (e.g., from about 10 3 to about 10 4 , from about 10 4 to about 10 s , from about 10 s to about 10 6 , from about 10 6 to about 10 7 , from about 10 7 to about 10 s , or from about 10 s to 10 9 , or more than 10 9 , neural cells) in a period of time of from about 5 days to about 30 days, e.g., from about 5 days to about 7 days, from about 7 days to about 10 days, from about 10 days to about 15 days, from about 15 days to about 20 days, from about 20 days to about 25 days, or from about 25 days to about 30 days, starting with from 10 to 100 pluripotent cell cells.
  • neural cells e.g., from about 10 3 to about 10 4 , from about 10 4 to about 10 s , from about 10 s to about 10 6 , from about
  • the present disclosure provides methods of generating a high percentage of viable neural cells that can be characterized by the detection of a single, or combination, of any phenotypic marker(s) that may include any of the above-mentioned phenotypic markers.
  • the present disclosure provides methods of generating at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% viable neural cells that can be characterized by the detection of a single, or combination, of any phenotypic marker(s) that may include any of the above mentioned phenotypic markers (e.g., 01ig2 and NKX2.2) relative to the total amount of cells that result from using the present methods.
  • any phenotypic marker(s) that may include any of the above mentioned phenotypic markers (e.g., 01ig2 and NKX2.2) relative to the total amount of cells that result from using the present methods.
  • the disclosure provides that interactions of the cultured hPSCs with different signal modulators as well as the interactions of the signal modulators among each other impacts the differentiation of hPSCs to OPCs.
  • One or more of the following conditions can promote differentiation of hPSCs to OPCs: 1) inhibition of BMP signaling; 2) presence in the culture medium of RA; 3) activation of WNT signaling early during culture; 4) inhibition of WNT signaling later during culture; 5) inhibition of SHH early during culture; 6) activation of SHH later during culture; and 7) inhibition of Notch pathway later during culture.
  • inhibition of BMP signaling is performed at least up to the beginning of the later phase of culture, e.g., at least up to the 15 th day of culture.
  • hPSC seeding density in the hydrogel used to culture the hPSCs for example, density between 10 and 500 cells/pillar (i.e., about 100 nl of hydrogel), increases OPC differentiation. Accordingly, in certain embodiments, the hPSCs are cultured in a hydrogel at a density of between 10 and 500 cells per 100 nl of hydrogel.
  • hPSCs are cultured in a hydrogel at a density of from 10 hPSCs per 100 nl hydrogel to about 15 hPSCs per 100 nl hydrogel, from about 15 hPSCs per 100 nl hydrogel to about 20 hPSCs per 100 nl hydrogel, from about 20 hPSCs per 100 nl hydrogel to about 25 hPSCs per 100 nl hydrogel, from about 25 hPSCs per 100 nl hydrogel to about 30 hPSCs per 100 nl hydrogel, from about 30 hPSCs per 100 nl hydrogel to about 35 hPSCs per 100 nl hydrogel, from about 35 hPSCs per 100 nl hydrogel to about 40 hPSCs per 100 nl hydrogel, from about 40 hPSCs per 100 nl hydrogel to about 45 hPSCs per 100 nl hydrogel, or from about 45 hPSCs per 100 nl
  • the hPSCs are cultured in a hydrogel at a density of from 25 cells per 100 nl hydrogel to about 500 cells per 100 nl hydrogel; e.g., at a density of from about 25 to about 50 cells per 100 nl hydrogel, from about 50 to about 100 cells per 100 nl hydrogel, from about 100 to about 200 cells per 100 nl hydrogel, from about 200 to about 300 cells per 100 nl hydrogel, from about 300 to about 400 cells per 100 nl hydrogel, or from about 400 to about 500 cells per 100 nl hydrogel.
  • the inhibitor of BMP signaling is LDN193189, SB431542, or a combination thereof. Additional examples of inhibitors of BMP signaling are known in the art and such embodiments are within the purview of the invention. Moreover, appropriate concentration of a selected BMP signaling inhibitor can be readily determined by a person of ordinary skill in the art.
  • Activators and inhibitors of WNT signaling as well as their appropriate concentrations can be readily determined by a person of ordinary skill in the art. Non-limiting examples of WNT inhibitors are described in the United States Application Publication Numbers
  • the WNT inhibitor is IWP-2. Additional examples of WNT inhibitors are known in the art and can be used in a method of the present disclosure. Non-limiting examples of WNT activators are described in the United States Application Publication Numbers 20200113913, 20200109371, 20190241615, and 20190144828, the contents of which are incorporated herein by reference in their entireties. In some cases, the WNT activator is CHIR. Additional examples of WNT activators are known in the art and can be used in a method of the present disclosure.
  • Activators and inhibitors of SHH signaling as well as their appropriate concentrations can be readily determined by a person of ordinary skill in the art. Non-limiting examples of SHH inhibitors are described in the United States Application Publication Numbers
  • the SHH inhibitor is GANTT61. Additional examples of SHH inhibitors are known in the art and can be used in a method of the present disclosure. Non limiting examples of SHH activators are described in the United States Application Publication Numbers 20100317699, 20120245184, 20020198236, and 20060287385, the contents of which are incorporated herein by reference in their entireties. In some cases, the SHH activator is smoothened agonist (SAG). Additional examples of SHH activators are known in the art and can be used in a method of the present disclosure.
  • SAG smoothened agonist
  • the inhibitor of Notch signaling is DAPT. Additional examples of inhibitors of Notch signaling are known in the art and can be used in a method of the present disclosure. Moreover, appropriate concentration of a selected Notch inhibitor can be readily determined by a person of ordinary skill in the art.
  • the present disclosure further provides a method for inducing
  • differentiation of an hPSC into an OPC comprising culturing the hPSC in a medium comprising one or more of the following: 1) an inhibitor of BMP signaling; 2) RA; 3) an activator of WNT signaling early during culture; 4) an inhibitor of WNT signaling later during culture; 5) an inhibitor of SHH early during culture; 6) an activator of SHH later during culture (e.g., starting on day 4); and 7) an inhibitor of Notch pathway later during culture.
  • the hPSC is cultured for about 21 days in a medium comprising one or more of the following: 1) an inhibitor of BMP signaling at least up to 15 days (e.g., from day 1 to day 15 of culture; or from day 1 to day 21 of culture); 2) RA throughout the entire 21 days of culture; 3) an activator of WNT signaling during days 1 to 4 of culture (e.g., during days 1-3, during days 1-4, etc.; but not during days 5-21 of culture); 4) an inhibitor of WNT signaling during days 5 to 21 of culture (but not during days 1-4 of culture); 5) an inhibitor of SHH during days 1 to 4 of culture (but not during days 5-21 of culture); 6) an activator of SHH during days 4 to 21 of culture (but not during days 1-3 of culture); and 7) an inhibitor of Notch pathway during days 15 to 21 of culture (but not during days 1-14 of culture).
  • the hPSC is cultured for about 21 days in a medium comprising all of (1) to
  • the hPSCs are cultured in a hydrogel at a density of between 10 and 500 cells per 100 nl of hydrogel.
  • the present disclosure further provides an OPC produced from an hPSC according to the methods disclosed herein.
  • OPCs produced using a method of the present disclosure can be purified, e.g., a population of cells is generated in which at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%, of the cells in the population are OPCs.
  • Pharmaceutical compositions are also provided comprising an OPC produced from an hPSC according to the methods disclosed herein and a pharmaceutically acceptable carrier.
  • the present disclosure further provides methods of treating a neurological disorder by administering to a subject in need thereof an OPC produced from an hPSC according to the methods disclosed herein.
  • the present disclosure provides a method comprising: a) generating a composition comprising OPCs, as described above; and b) administering the composition to an individual in need thereof.
  • the composition to be administered can comprise from 10 to 10 7 OPCs (where the OPCs are generated using a method of the present disclosure; e.g., from about 10 OPCs to about 10 2 OPCs, from about 10 2 OPCs to about 10 3 OPCs, from about 10 3 OPCs to about 10 4 OPCs, from about 10 4 OPCs to about 10 s OPCs, from about 10 s OPCs to about 10 6 OPCs, or from about 10 6 OPCs to about 10 7 OPCs, are administered.
  • the composition comprising the OPCs further includes a small molecule that increases myelination, where such small molecules include, e.g., DAPT and ketoconazole.
  • the diseases treated according to the methods disclosed herein include myelin-associated disorder in the CNS, such as radiation therapy-induced injury, Huntington’s Disease, Acute disseminated encephalomyelitis (ADEM); Acute hemorrhagic leukoencephalitis; Acute optic neuritis; Acute transverse myelitis; Adrenoleukodystrophy;
  • myelin-associated disorder in the CNS such as radiation therapy-induced injury, Huntington’s Disease, Acute disseminated encephalomyelitis (ADEM); Acute hemorrhagic leukoencephalitis; Acute optic neuritis; Acute transverse myelitis; Adrenoleukodystrophy;
  • Adrenomyeloneuropathy Alexander Disease; Alzheimer’s Disease; aminoacidurias;
  • myelinoclastic sclerosis extrapontine myelinolysis; Gaucher disease; Guillain-Barre syndrome; Hereditary neuropathy; hereditary neuropathy with liability to pressure palsy; HTLV-1- associated myelopathy; Hurler syndrome; Hypomyelination; hypoxic brain injury; Krabbe Disease; Leber hereditary optic atrophy and related mitochondrial disorders; leukodystrophic disorders; Marburg multiple sclerosis; Marchiafava-Bignami disease; Metachromatic leukodystrophy; multiple sclerosis; multiple system atrophy; myelinoclastic disorders;
  • myelopathy nerve injury; neuromyelitis optica; Neuromyelitis optica (NMO); Niemann-Pick disease; optic neuropathy; optic-spinal multiple sclerosis; Osmotic Demyelination Syndrome; Parkinsons; Pelizaeus-Merzbacher Disease; peripheral neuropathy; Phenylketonuria; primary progressive multiple sclerosis (PPMS); progressive inflammatory neuropathy; progressive multifocal leukoencephalopathy; Progressive subcortical ischemic demyelination; progressive- onset multiple sclerosis; relapsing-onset multiple sclerosis; relapsing-remitting multiple sclerosis (RRMS); reperfusion injury; Schilder disease; secondary progressive multiple sclerosis (SPMS); Solitary sclerosis; Spinal Cord Injury; Subacute sclerosing panencephalitis; Tabes dorsalis; Tay- Sachs disease; Traumatic Brain Injury; Tropical spastic paraparesis; Tumefactive multiple sclerosis; or Vitamin B12
  • the OPCs are used for treating a myelin associated disorder in the CNS.
  • the OPCs can be administered within the brain or the spinal cord of the subject at a specific site, for example, at the site of demyelination and/or injury.
  • the methods can comprise administering an OPC in the subject via the epidural or intracerebral route.
  • aspects of the methods further include cryopreserving the neural cells generated by the subject methods.
  • the neural cells may be cryopreserved from 0 hr to 30 hr after the contacting has occurred; for example, the neural cells may be cryopreserved from 0 hr to 24 hr, from 0 hr to 12 hr, or from 0 hr to 6 hr after the contacting has occurred. Any known method used to successfully cryopreserve neural cells, e.g., OPCs, may be applied.
  • the neural cells may be preserved in any standard cryopreservation solution.
  • the cells can be maintained such that once it is determined that the cells may be utilized in a downstream application, e.g., transplantation, the cells can be thawed and, e.g., be injected, transplanted, expanded, etc.
  • methods of the present disclosure may include flow cytometrically assaying one or more cells generated by the subject methods, e.g., by fluorescence activated cell sorting. See, e.g., Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No.
  • flow cytometrically assaying the sample involves using a flow cytometer capable of simultaneous excitation and detection of multiple fluorophores.
  • Methods of the present disclosure may involve image cytometry, such as is described in Holden et al. (2005) Nature Methods 2:773 and Valet, et al. 2004 Cytometry 59:167-171, the disclosures of which are incorporated herein by reference.
  • methods of the present disclosure may include assaying one or more cells generated by the subject methods with fluorescence microscopy.
  • Fluorescence microscopy methods include irradiating light onto a labeled object, executing an excitation and fluorescence emission process on the object using the irradiated light, capturing emitted fluorescence, and observing information, such as the image of the object. Any suitable fluorescence microscopy methods for detecting fluorescently labeled specific binding members may be used. Fluorescence microscopy methods are well known in the art. See, e.g., Lichtman et al. Nature Methods (2005) 2: 910-919; Combs et al.
  • the sample may be assayed by wide field fluorescence microscopy, laser scanning confocal microscopy, two photon laser scanning fluorescence microscopy, and the like.
  • a device for screening multiple culture conditions comprising:
  • a multi-pillar plate comprising one or more pillars, wherein each pillar comprises a gel composition deposited thereon, wherein the gel composition comprises a stem cell, and
  • a multi-well plate comprising one or more cell culture mediums in one or more wells, each well comprising one or more cell culture conditions.
  • conditions comprises one or more seeding densities.
  • conditions comprises one or more amounts of one or more factors.
  • IWP-2 IWP-2
  • DAPT DAPT
  • a method for screening multiple cell culture conditions comprising:
  • each of the one or more pillars with one or more cell culture mediums in one or more wells of a multi-well plate (e.g., of the device of any one of aspects 1-11), under one or more cell culture conditions, generating a medium-contacted stem cell; and
  • detecting at least one neural cell marker in the medium-contacted stem cell wherein an increase in expression of the at least one marker in the medium-contacted stem cell, compared to a control, indicates that the one or more cell culture conditions modulate stem cell differentiation to neural cells.
  • oligodendrocyte progenitor cells oligodendrocyte progenitor cells.
  • conditions comprises one or more seeding densities.
  • conditions comprises one or more amounts or concentrations of one or more factors.
  • [00182] 22 The method of Aspect 21, wherein the one or more factors comprises a SHH signaling pathway agonist, a Wnt signaling pathway agonist, and a Retinoic acid signaling pathway agonist.
  • IWP-2 IWP-2
  • DAPT DAPT
  • a method for inducing differentiation of an hPSC into an OPC comprising culturing the hPSC in a medium comprising one or more of the following: 1) an inhibitor of BMP signaling; 2) retinoic acid (RA); 3) an activator of WNT signaling early during culture; 4) an inhibitor of WNT signaling later during culture; 5) an inhibitor of SHH early during culture; 6) an activator of SHH later during culture; and 7) an inhibitor of Notch pathway later during culture.
  • [00193] 33 The method according to Aspect 31 or 32, comprising culturing the hPSC in a medium comprising all of the following: 1) an inhibitor of BMP signaling for at least up to 15 days; 2) RA throughout the 21 days; 3) an activator of WNT signaling during days 0 to 4; 4) an inhibitor of WNT signaling during days 5 to 21; 5) an inhibitor of SHH during days 0 to 4, 6) an activator of SHH during days 5 to 21, and 7) an inhibitor of Notch pathway during days 15 to 21. [00194] 34.
  • the inhibitor of BMP signaling is LDN193189, SB431542, or a combination thereof; 2) the activator of WNT signaling is CHIR99021, 3) the inhibitor of WNT signaling is IWP-2; 4) the inhibitor of SHH is GANTT61; 5) the activator of SHH is smoothened agonist, and 6) the inhibitor of Notch pathway is DAPT.
  • medium comprises a hydrogel comprising the cells immersed in a liquid medium.
  • a pharmaceutical composition comprising: a) an OPC according to claim 38; and b) a pharmaceutically acceptable carrier.
  • a method for treating a neurological disorder comprising
  • a method for treating a neurological disorder comprising: a)
  • Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pi, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like.
  • oligodendrocyte progenitor cells OPCs
  • mDA neurons midbrain dopaminergic neurons
  • the optimized culture conditions from the screen are then applied toward a scaled up process for large-scale production of purified hESC-derived OPCs that leverages the strengths of a synthetic thermoreversible hydrogel to encapsulate cells in 3D during the differentiation process.
  • 3D microencapsulation of cells within synthetic thermoresponsive hydrogels is advantageous for several reasons including xeno-free composition, scalable production, and ease of cell retrieval without harsh chemical or mechanical gel degradation throughout and at the end of the production process, enabling feasible in-process sampling and therefore a higher degree of within-process knowledge.
  • a single reactor was capable of producing on the order of 10 million OPC precursors (defined by expression of OPC marker 01ig2) in 2 weeks.
  • OPCs that can be applied to neural aggregates after harvesting from 3D thermoreversible hydrogel cultures.
  • Purification strategies for therapeutic cell types may be advantageous compared to heterogeneous cell mixtures due to the higher product composition knowledge and decreased risk of contaminating cell types and that may cause unwanted and potentially dangerous side effects, such as cyst formation or teratomas in the patient.
  • Purified OPCs can be cryopreserved and/or applied toward several downstream applications, such as preclinical safety and efficacy studies.
  • the 3D screening and analysis strategy presented in this disclosure is relevant for numerous emerging cell replacement therapy candidates for which conversion of a stem or progenitor cell, such as a human pluripotent stem cells, to a therapeutically relevant cell type requires searching through a large in vitro design space of doses, durations, dynamics, and combinations of signaling cues over several weeks of culture.
  • the 3D context of the screening platform disclosed herein enables high- throughput investigation into neurodevelopment that can offer unique perspectives beyond what is capable in 2D screening platforms, for example, by recapitulating cell-to-cell interactions, cytoskeletal arrangement, and multicellular patterning in 3D.
  • the lumen structures that were observed during the neural induction period (FIG. 20B) in response to caudalizing conditions (high Wnt and RA) could be the basis of future organoid screening strategies to probe early multicellular arrangement and the effect of lumen size and shape on cell fate determination at various positions along the rostro-caudal and dorso-ventral axes.
  • Wnt signaling pathway A potential case of biphasic activity was observed for the Wnt signaling pathway as both activation and inhibition appeared to increase expression of OPC markers Nkx2.2 and Oiig2. This effect was seen with initial Wnt activation by CHIR during Days 0-3 of OPC differentiation followed by inhibition by IWP-2 during days 4-21 of OPC differentiation.
  • the Wnt pathway has shown stage-specific activity in cardiac and hematopoietic development, which may thus be a conserved feature across several developmental systems. Wnt signals play an important role in the gastrulation of the embryo to form the primitive streak, yet in the subsequent stages of spinal cord development, Wnt signals induce a dorsalizing effect whereas oligodendrocytes originate from the motor neuron domain on the ventral side.
  • oligodendrocytes created through this protocol having expressed OTX2 at Day 10 (FIG. 20C) are thought to resemble forebrain OPCs, rather than spinal cord OPCs, perhaps due to the exposure to Wnt antagonist, IWP-2, inducing a more rostral position during the differentiation window.
  • Wnt antagonist IWP-2
  • the fluorescence microscopy readout used in this methodology is in the form of image data, which is commonly quantified by segmenting cells to score as positive or negative for marker expression. Dense cell populations can confound scoring methodology, which is further complicated if cellular markers are localized in the cytoplasm because of overlapping cell bodies. Therefore, it is advantageous to choose nuclear localized cell specific markers in the design of the screening experiment, such as 01ig2 and Nkx2.2 expression for OPC
  • thermoresponsive hydrogels are advantageous because of the ease of cell retrieval without harsh chemical or mechanical gel degradation throughout and at the end of the production process, (Lei et al. (2013) supra; Lin et al. (2017) Sci. Rep. 7:40191) enabling feasible in-process sampling and therefore a higher degree of within-process knowledge.
  • a pilot study to assess a stirred bioreactor configuration for the upscaling of 3D thermoreversible gel- encapsulated OPC production from hPSCs is described.
  • modular purification strategies can be applied to enrich for cell types of interest, such as PDGFRa+ OPCs from neural aggregates.
  • Pure populations of cells may be advantageous compared to heterogeneous cell mixtures for applications in cell therapy due to the decreased risk of contaminating cell types that may cause unwanted and potentially dangerous side effects, such as cyst formation or teratomas in the patient (DeFrancesco (2009) Nat.
  • a custom-made Nkx2.2-Cre H9 reporter line that constitutively expresses DsRed protein but switches to GFP expression upon exposure to Cre recombinase was then employed to longitudinally monitor proliferation and differentiation of hPSCs to Nkx2.2+ oligodendrocyte progenitors in 3D on the microchip platform.
  • a small range of culture conditions from previously published protocols of OPC differentiation was selected for an initial, pilot differentiation experiment, and the GFP expression was quantified at later stages of differentiation. Cellular morphological changes due to neural lineage commitment and maturation were clearly observed at later stages in the 3D differentiation (FIG. 20) as cultures were maintained and monitored for more than 80 days on the microchip.
  • Fluorescence image analysis pipelines for quantification of nuclear and cytoplasmic marker expression via immunocytochemistry for endpoint analyses were then built (FIG. 21). Together, these results support the robust and long-term culture potential and cellular marker expression readout of this miniaturization methodology for hPSC differentiation screening.
  • Oiig2 expression is substantially modulated by tuning early 3D culture parameters
  • hPSCs differentiation of hPSCs was induced via inhibition of BMP signaling using the dual SMAD inhibition approach, (Chambers et al. (2009) Nat. Biotechnol. 27:275) with LDN193189 and SB431542, and a range of neural induction durations were tested before introducing agonists for Wnt, retinoic acid (RA), and SHH developmental signaling pathways.
  • RA retinoic acid
  • SHH developmental signaling pathways A strong inverse correlation between neural induction time and OPC specification was observed such that shorter neural induction duration resulted in up to 6-fold higher 01ig2 expression in some cases (FIG. 23).
  • GANTT61 is an antagonist of the SHH pathway
  • IWP-2 is an antagonist of the Wnt pathway
  • DAPT is an antagonist of the Notch pathway (FIG. 5 A).
  • 01ig2 expression were measured and the proportion of cells co-expressing both OPC markers was quantified. Most notably, a significant decrease in %01ig2 was observed in response to DAPT across all conditions tested (FIG. 5 Bi). This could point to a role for Notch signaling in maintaining or promoting specification of 01ig2+ progenitors - a hypothesis not previously examined to our knowledge - and serves as preliminary evidence to test Notch agonists such as DLL-4 in follow-up studies of OPC optimization. This effect may be mediated by an interaction with the SHH pathway (Kong et al. (2015) Dev Cell 33:373). Interestingly, the same trend was not observed with respect to %Nkx2.2.
  • the micropillar/microwell chip was applied to screen through numerous temporal profiles of SAG, as well as RA due to its analogous role along the rostro-caudal axis during spinal cord development, by dividing the signal window into“Early” and“Late” stages that were dosed independently to form constant, increasing, and decreasing dose profiles over time (FIG. 6 A).
  • Tujl expression was measured and the proportion of 01ig2+ cells that co-expressed Tuj 1 was calculated to potentially identify any modulators of the balance between 01ig2+ cells that proceed down a motor neuron fate (also Tujl+) versus an oligodendrocyte fate (also Nkx2.2+).
  • Category 2 composed of phenotypes ranking low on OPC progenitor fate (low 01ig2 and/or Nkx2.2 expression), all shared the low dosing of retinoic acid at 0.1 mM throughout the duration of the experiment, further emphasizing the high impact of RA on OPC yield.
  • Category 3 composed of the highest 01ig2 and Nkx2.2 expression as well as
  • the parameter coefficients were analyzed as a measure of relative influence on the expression of a certain endpoint phenotype, such as 01ig2+Nkx2.2+ cells, and could be interpreted as a sensitivity analysis of key parameters on the OPC specification process and the most significant parameters were sorted by their effect magnitude (FIG. 7 B).
  • RA The dosage of RA was among the most impactful parameters in this study for expression of 01ig2 and Nkx2.2 markers (FIG. 7 Bi,ii).
  • a high dose of RA (1 mM) early in the differentiation (Day 0-1) was ranked to be the top most influential culture parameter in the acquisition of OPC fate (co-expression of 01ig2 and Nkx2.2) (FIG. 7 Bi-iii).
  • the presence of SAG in the early portion of differentiation was markedly more significant to OPC fate induction than in the later portion, in line with the previous analysis (FIG. 4).
  • Novel additions IWP-2 and GANT were observed to correlate positively with co-expression of 01ig2 and Nkx2.2 as well.
  • this analysis identified two cases of culture variables interacting in a synergistic manner to promote OPC differentiation - between RA and SAG doses in the early differentiation period to promote Nkx2.2 expression, and between CF1IR duration and GANT dose to promote co-expression of Nkx2.2 and 01ig2.
  • this micro-scale screening strategy can enable robust sensitivity analysis that simplifies the parameter space to only the most influential culture components that can then be prioritized in future optimizations when culture configurations undergo a major change, such as during scale up processes, to save time and resources at large scale.
  • a new differentiation protocol is provided based on the parameters isolated in this screen to have the most influence in specifying 01ig2+ Nkx2.2+ progenitors (FIG. 7Biii) and carried out the differentiation into the later stages of OPC maturation in a larger scale format to assess the ability of this optimized protocol to create mature oligodendrocytes.
  • the protocol was able to produce PDGFRa expressing cells by Day 60 across multiple hPSC lines, as well as 04 expressing cells by Day 75 and MBP expressing cells and myelination ability at Day 100 ( Figure S10).
  • Microculture chip can be applied to multiple types of 3D hPSC differentiation screens
  • the screening and analysis methodology presented here is relevant to several up and coming cell replacement therapy candidates for which production from a precursor cell type requires searching through a large in vitro design space of doses, durations, dynamics, and combinations of signaling cues over several weeks of culture, such as those derived from human pluripotent stem cells (Trounson et al. (2016) Nat. Rev. Mol. Cell Biol. 17:194).
  • hESCs differentiated to OPCs based on scoring across 5 dimensions: ease of scalability, precedence for cGMP compliance, macroenvironmental homogeneity, ease of implementation, and capital cost.
  • FACS purification process developed here as the second subcomponent of the production process. Purified cells can be applied toward functional analysis immediately, further expanded, or cryopreserved until needed (FIG. 3).
  • FIG. 9 A a pilot scale experiment was implemented in order to assess the effects of a stirred culture environment on the viability, growth, and differentiation of hPSCs to OPCs because of the need for agitation in large scale bioreactors to mitigate chemical gradients that form from cell metabolism.
  • COMSOL was used to create a dynamic quantitative model of the culture environment and provide estimates of the magnitude of chemical and shear force gradients within a stirred setting (FIG. 9 B). The model showed significant differences in the chemical environment across the bioreactor with (50 RPM) and without agitation.
  • hPSCs were encapsulated in thermoreversible gel in single cell form in the pilot scale bioreactor and were placed in OPC differentiation media starting Day 0. On Day 4, stirring at 0 or 50 RPM was introduced. No observable effects on gel integrity were observed during the course of agitation. On Day 10, measurements of viability and neurosphere growth were recorded (FIG. 9 C).
  • thermoreversible gel encapsulated differentiation process was then scaled up further by implementing the thermoreversible gel encapsulated differentiation process in 50mL spinner flasks.
  • H9 hESCs were seeded into thermoreversible gel at a density of 1E5 cells/mL gel and the cell encapsulated gel was extruded through a syringe to create continuous fibers in a 50 mL volume of cell culture media in a spinner flask bioreactor.
  • Cells were expanded in E8 maintenance medium for 2 days before beginning the OPC differentiation process. Cell aggregates remained viable and continued to proliferate over the course of multiple weeks in the bioreactor (FIG. 10).
  • Immunocytochemical analysis of the cell aggregates over the differentiation in the bioreactor revealed pluripotent cells on Day 0 and large proportions (-100%) of mitotic cells throughout the first 6 days of culture. Additionally, neuroectodermal differentiation measured by Pax6 expression peaked around Day 6 and began to decline. Mesodermal and endodermal differentiation measured by Brachyury and Gata4 was not detected (FIG. 11). Later timepoints of the bioreactor culture revealed detectable OPC differentiation, measured by 01ig2 and Nkx2.2 expression, by Day 15. Neuronal marker Tujl was also detected at Day 25 (FIG. 12).
  • the sorted PDGFRa+ population was then expanded and viability after passaging as well as cryopreservation in two preservatives: 10% DMSO and cGMP-compliant CryoStore solution was checked and high levels of viability (>90%) of the cells was found (FIG. 16).
  • continued culturing of OPCs in maturation media was performed and MBP+ mature oligodendrocytes were observed after 150 days.
  • the purified OPCs were applied to preliminary functional assay to determine myelination ability, measured by co-localization of MBP and Tujl (FIG. 17).
  • the progenitor PDGFRa+ cells are multipotent, and the media formulation may be adjusted to promote higher ratios of oligodendrocytes to astrocytes or neurons.
  • H9s Fluman embryonic stem cells (H9s, NIH Stem Cell Registry #0062) were subcultured in monolayer format on a layer of 1 % Matrigel and maintained in Essential 8 medium during expansion. At 80% confluency, H9s were passaged using Versene solution and replated at a 1:8 split.
  • H9s were dissociated into single cells using Accutase solution and resuspended in
  • H9s were counted and resuspended at defined densities in 50% Matrigel solution on ice. While chilled, 100 nL of, H9s in 50% Matrigel solution were deposited onto the micropillars using a custom robotic liquid handling program and then incubated at 37C for 20 minutes to promote gelation of 3D cultures. The micropillar chip was then inverted and placed into a fresh microwell chip containing cell culture media (Table 1). All liquid dispensing into the microculture platform was performed with a DIGILAB Omnigrid Micro liquid handler with customized programs for deposition patterns. Media were changed daily by transferring the micropillar chip into a microwell chip containing fresh media every other day using a custom made mechanical“Chip Swapper” for consistent transfer. Technical replicates included two different dispensing patterns to average out positional effects across the microchip.
  • IWP-2 varied Tocris, 3533
  • GANTT61 varied Enzo, ALX-270-482-M001
  • the micropillar chip was carefully removed from the wellchip and placed in new wellchip containing Calcein AM, Ethidium Homodimer, and Hoechst diluted in sterile PBS (dilution details in Table 2). The chip was incubated for 20 minutes and then transferred to a new wellchip containing PBS and individual
  • the micropillar chip was carefully removed from the wellchip and placed into a bath of 4% parafomaldehyde for 15 minutes to fix cell cultures. Then, the micropillar chip was washed twice in PBS for 5 minutes each and placed into a bath of 0.25% Triton-X + 5% donkey serum in PBS for 10 minutes to permeabilize cells. After permeabilization, the micropillar chip was washed 5 times in 5% donkey serum for 5 minutes each, dried, transferred to a wellchip containing primary antibodies of interest diluted in PBS+donkey serum (dilution details in Table 2), and stored overnight at 4C.
  • the micropillar chip was washed twice in PBS for 5 minutes each, dried, placed into a micro well chip containing the corresponding secondary antibodies (dilution details in Table 2), and incubated at 37C for 2 hours. After secondary staining, the micropillar chip was washed twice in PBS for 5 minutes each, dried, and placed into a wellchip containing PBS; individual microenvironments were imaged using fluorescent confocal microscopy.
  • Mouse anti-Oct4 1 100 SCBT, sc-5279 Mouse anti-Brachyury 1 : 100 SCBT, sc-379321 Rabbit anti-Gata4 1 : 100 SCBT, sc-9053 Mouse anti-PDGFRa Alexa647 1 : 100 SCBT, sc-398206
  • Rabbit anti-GFAP 1 1000 Abeam, ab7260 Donkey anti-Rabbit Cy3 1:250 Jackson, 711-165-152
  • Stained micropillar chips were sealed with a polypropylene film (GeneMate T-2452-1) and imaged with a 20x objective using a Perkin Elmer Opera Phenix automated confocal fluorescence microscope available in the High-Throughput Screening Facility at UC Berkeley. Laser exposure time and power was kept constant for a fluorescence channel within an imaging set.
  • H9s were counted and resuspended at defined densities in 10% Mebiol Gel and kept chilled on ice. While chilled, H9s in liquefied gel were aspirated into a syringe and then extruded through a 16-gauge blunt end needle (StemCell Technologies) into warm Essential 8 medium containing lOuM Y- 27632. Media with extruded gel fibers was transferred to a 125mL Spinner Flask bioreactors and placed on a cell culture stir plate in a cell culture incubator and maintained at 37°C and 5% CO2.
  • Neural aggregates were collected from the 3D gel at a range of timepoints and plated on laminin -coated dishes for 20 days to dissociate aggregates into single cells with Accutase enzymatic reagent.
  • Single cell suspensions are stained with anti-PDGFRa antibody conjugated to a phycoerythrin (PE) fluorophore and/or anti-04 antibody conjugated to an Alexa 488 fluorophore, and propidium iodide (PI) for lh at 4°C wrapped in aluminum foil.
  • PE phycoerythrin
  • PI propidium iodide
  • Appropriate fluorescence-minus-one (FMO) controls were prepared as well.
  • Stained suspensions were transported on ice to the sorting facility at UC Berkeley and sorted using an Aria Fusion Cell Sorter Cells are gated using FMO controls and selected for high fluorescence in the 561 laser line for PDGFRa expression, and low fluorescence in the 640 laser line for viability. Sorted population is replated on laminin -coated dishes and cultured in OPC maturation media.
  • Single cell OPC suspensions were made by Versene or Accutase based dissociation from culture plates and centrifuged at 200G for 2 minutes. Pellets were resuspended in either 10% DMSO or 100% CyroStore reagent and transferred to a cryovial. Cry o vials were stored in an IPA filled Mr. Frosty in a -80°C freezer overnight and then transferred to a liquid nitrogen tank for long term storage.
  • a mouse model of HD, R6/2 strain was used to test the ability of the OPCs cultured according to the methods disclosed herein for the ability to survive implantation, migrate and engraft into the host brain tissue, and alleviate symptoms of HD that are caused by dysmyelinated neurons.
  • OPCs were produced in a bioreactor in a 3D
  • thermoresponsive polymer gel and prepared for implantation into the striatum of R6/2 mice (FIG. 31 A).
  • OPCs were implanted in mice at 5 weeks of age.
  • Male mice injected with OPCs displayed an increased lifespan compared to mice injected with a control vehicle (FIG. 3 IB).
  • male R6/2 mice displayed an increase in weight, a metric representing general health, following the OPC implantation compared to control mice, whereas female R6/2 mice did not display any differences in weight over the duration of the study compared to the control mice (FIG. 31C).
  • immunohistochemisty images of the cell graft 4 weeks post injection revealed OPCs (positive for 01ig2 marker) that survived the implantation. Analysis of the cells relative to the injection tract suggests that the cells began migrating along the corpus callosum of the brain. (FIG. 3 ID).

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Abstract

La présente invention concerne un dispositif de criblage de multiples conditions de culture cellulaire, le dispositif comprenant une plaque multi-piliers et une plaque multi-puits, le dispositif fournissant de multiples conditions de culture cellulaire. La présente invention concerne en outre des procédés de criblage de multiples conditions de culture cellulaire pour des conditions de culture de différenciation de cellules neurales pour générer des cellules neurales à l'aide d'un dispositif de la présente invention. L'invention concerne également des procédés de génération de cellules neurales dans les conditions de culture cellulaire identifiées dans le procédé de criblage à l'aide d'un dispositif de la présente invention. De plus, l'invention concerne des procédés de mise en culture de hPSC pour produire des cellules progénitrices d'oligodendrocytes (OPC). L'invention concerne également des procédés de traitement d'une maladie associée à la myéline du système nerveux central par l'administration des cellules progénitrices d'oligodendrocytes produites selon les procédés de l'invention.
PCT/US2020/029553 2019-04-26 2020-04-23 Dispositifs et procédés de génération de cellules progénitrices d'oligodendrocytes Ceased WO2020219696A1 (fr)

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WO2022211893A1 (fr) * 2021-03-30 2022-10-06 Trailhead Biosystems Inc. Procédés et compositions pour générer des cellules progénitrices d'oligodendrocytes
WO2023229819A1 (fr) * 2022-05-24 2023-11-30 Bluerock Therapeutics Lp Procédés de fabrication de cellules progénitrices d'oligodendrocytes
WO2025097051A1 (fr) * 2023-11-03 2025-05-08 Axent Biosciences Inc. Polymères thermoréversibles à stabilité améliorée et procédés et utilisations associés

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WO2006105278A2 (fr) * 2005-03-29 2006-10-05 The Regents Of The University Of California Regulation de la destinee de cellules souches par reseau reglable
WO2009040423A1 (fr) * 2007-09-26 2009-04-02 INSERM (Institut National de la Santé et de la Recherche Médicale) Utilisation de dérivés de tocophérol comme inhibiteurs de la voie de signalisation notch
US8492147B2 (en) * 2004-06-18 2013-07-23 Riken Method of inducing the differentiation of embryonic stem cells into nerve by serum-free suspension culture
US20160175800A1 (en) * 2014-12-18 2016-06-23 Wisconsin Alumni Research Foundation Covalently-immobilized hydrogel arrays in multi-well plates
WO2017062374A1 (fr) * 2015-10-05 2017-04-13 The Regents Of The University Of California Compositions et méthodes pour générer des précurseurs d'oligodendrocytes
WO2019213366A1 (fr) * 2018-05-02 2019-11-07 The Trustees Of Columbia University In The City Of New York Procédés pour induire une différenciation terminale dans des cellules souches par interférence avec la réplication d'adn, procédés pour induire une différenciation pancréatique, et cellules différenciées obtenues à partir de ceux-ci
US20200087623A1 (en) * 2018-08-03 2020-03-19 The Regents Of The University Of California Generation of Human Spinal Cord Neural Stem Cells

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US8492147B2 (en) * 2004-06-18 2013-07-23 Riken Method of inducing the differentiation of embryonic stem cells into nerve by serum-free suspension culture
WO2006105278A2 (fr) * 2005-03-29 2006-10-05 The Regents Of The University Of California Regulation de la destinee de cellules souches par reseau reglable
WO2009040423A1 (fr) * 2007-09-26 2009-04-02 INSERM (Institut National de la Santé et de la Recherche Médicale) Utilisation de dérivés de tocophérol comme inhibiteurs de la voie de signalisation notch
US20160175800A1 (en) * 2014-12-18 2016-06-23 Wisconsin Alumni Research Foundation Covalently-immobilized hydrogel arrays in multi-well plates
WO2017062374A1 (fr) * 2015-10-05 2017-04-13 The Regents Of The University Of California Compositions et méthodes pour générer des précurseurs d'oligodendrocytes
WO2019213366A1 (fr) * 2018-05-02 2019-11-07 The Trustees Of Columbia University In The City Of New York Procédés pour induire une différenciation terminale dans des cellules souches par interférence avec la réplication d'adn, procédés pour induire une différenciation pancréatique, et cellules différenciées obtenues à partir de ceux-ci
US20200087623A1 (en) * 2018-08-03 2020-03-19 The Regents Of The University Of California Generation of Human Spinal Cord Neural Stem Cells

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Publication number Priority date Publication date Assignee Title
WO2022211893A1 (fr) * 2021-03-30 2022-10-06 Trailhead Biosystems Inc. Procédés et compositions pour générer des cellules progénitrices d'oligodendrocytes
US12385009B2 (en) 2021-03-30 2025-08-12 Trailhead Biosystems Inc. Methods and compositions for generating oligodendrocyte progenitor cells
WO2023229819A1 (fr) * 2022-05-24 2023-11-30 Bluerock Therapeutics Lp Procédés de fabrication de cellules progénitrices d'oligodendrocytes
WO2025097051A1 (fr) * 2023-11-03 2025-05-08 Axent Biosciences Inc. Polymères thermoréversibles à stabilité améliorée et procédés et utilisations associés

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