Compositions comprising neurons and methods of using the same

Abstract

The disclosure includes compositions and pharmaceutical compositions and kits comprising novel brain progenitor cells. In some embodiments, the disclosure relates to methods of making the cell lines as well as methods of using the cell lines to perform functional assays while being manipulated in culture.

Description

COMPOSITIONS COMPRISING NEURONSAND METHODS OF USING THE SAMESTATEMENT REGARDING FEDERALLY SPONSORED RESEARCH0001 This invention was made with government support under grant number DP5OD024558 awarded by the National Institutes of Health. The government has certain rights in this invention.RELATED APPLICATIONS0002 This application is an International Application filed on August 30, 2024, which claims priority to U.S. Provisional Application No. 63 / 579,830, filed August 31, 2023, which is hereby incorporated herein by reference in its entirety.SUBMISSION OF SEQUENCE LISTING0003 The Sequence Listing associated with this application is filed in electronic format via Patent Center and hereby incorporated by reference into the specification in its entirety. The name of the text file containing the Sequence Listing is STFD-001-PCT_SL.xml. The size of the text file is 87,031 bytes and the .xml file was created on August 30, 2024.FIELD0004 The disclosure relates to cell lines comprising one or a plurality of brain progenitor cells, including, in some embodiments, hindbrain neuronal cell lines. The disclosure also relates to methods of making the same, methods of differentiating stem cells to arrive at the cell lines and systems configured to grow and perform functional assays using the compositions comprising the cell lines.BACKGROUND0005 The brain is arguably the most complex organ within the vertebrate body. Defining the origins, diversity, and lineage hierarchy of the earliest brain progenitors is imperative to understand brain development and evolution [1-5], It will also delineate a developmental roadmap to differentiate human pluripotent stem cells (hPSCs) into various brain cell-types in vitro for regenerative medicine, disease modeling and other applications [6-10],0006 During the process of gastrulation, pluripotent cells differentiate into the three germ layers: endoderm, mesoderm, and ectoderm. In the developing mouse embryo, the ectoderm arises at ~E6.75-7.0 (6.75-7.0 days)

[0011] , Ectoderm is subsequently subdivided at -E7.5 into neural ectoderm, border ectoderm (neural plate border), and surface ectoderm; these respectively generate the brain, neural crest, and skin [11,12], The major subdivisions of the brain — forebrain, midbrain, and hindbrain — arise by -E8.5 [13,14],0007 Whereas the forebrain executes higher-level cognitive processes, the hindbrain constitutes most of the brainstem and is involved in life-sustaining processes such as breathing, eating, sleep, and wakefulness [15-17], Indeed, hindbrain injury is often deadly, and can impair consciousness, sensation, reflexes and breathing

[0018] , As a case in point, diffuse intrinsic pontine glioma, a childhood hindbrain cancer, is invariably fatal (with a 99% 5-year mortality rate), underscoring the vital importance of the hindbrain

[0019] , Consequently, the ability to produce human hindbrain cells in vitro could broadly accelerate studies of hindbrain biology and disease. In turn, such an enterprise is predicated on an understanding of early hindbrain development [15- 17].0008 There has been considerable interest in the extracellular signals that specify neural fate. Pioneering studies revealed that organizer cells in the embryo [20,21] produce diffusible TGFbeta and BMP inhibitors to induce neural fate in vivo [22-26], Accordingly, inhibiting TGFbeta and BMP (“dual SMAD inhibition”)

[0027] , often alongside WNT inhibition [28-31], became the principal strategy to differentiate hPSCs into neural cell-types in vitro. This breakthrough has enabled many successes in generating human forebrain and midbrain cells from hPSCs [28-51], In contrast, it has generally been more challenging to produce hindbrain cells in vitro, with some exceptions [37,40,52-57], This intimates an incomplete understanding of the earliest hindbrain progenitors and the extracellular signals that induce them.0009 Neural ectoderm represents the earliest brain-restricted progenitor. Classical studies by Nieuwkoop in 1952 [58,59] led to the idea that neural ectoderm harbors the broad potential to generate the forebrain, midbrain, and hindbrain [2,6,7,60], Indeed, the forebrain, midbrain, and hindbrain are anatomically contiguous, consistent with a scenario in which they originate from a common neural precursor. Current methods to differentiate hPSCs into brain cell-types implicitly assume the existence of a common neural progenitor that can generate the forebrain, midbrain, and hindbrain [6,7], Nevertheless, classical fate maps of zebrafish

[0061] , frog

[0062] , chicken

[0063] ,and mouse

[0064] embryos have instead suggested that during gastrulation, neural ectoderm cells may have already become fated to give rise to certain parts of the brain.SUMMARY OF EMBODIMENTS00010 The disclosure provides the ability to produce human hindbrain, human forebrain, and human midbrain cells in vitro as well as forebrain, midbrain and hindbrain progenitor cells. This ability can broadly accelerate studies of hindbrain biology and disease. Compositions comprising cells of the disclosure may also be used for therapies in the event that cell transplantation into subjects is needed to replace aberrant or dysfunctional cell types in the brains of the subjects.00011 The disclosure relates to a composition comprising a cell or plurality of cells, wherein the cells comprise SOX2 or a functional variant thereof and are deficient in NANOG. In some embodiments, the cell or plurality of cells are from about 12 to about 48 hours in culture. In some embodiments, the cell or plurality of cells are frozen. In some embodiments, the cell or plurality of cells are frozen at about 0 degrees Celsius or about -200 degrees Celsius. In some embodiments, the cell or plurality of cells comprise a cell membrane that is columnar or cuboidal in shape and wherein the composition further comprises an extracellular matrix peptide. In some embodiments, the cell or plurality of cells comprises one or a combination of GBX2, H0XA1, HOXB1, or one or a combination of functional variants thereof that comprise at least about 75% sequence identity to GBX2, H0XA1, HOXB1. In some embodiments, the cell or plurality of cells comprise OTX2 or a functional variant thereof. In some embodiments, the cell or plurality of cells comprise one or a combination of FOXG1, LHX2, SIX3, PAX6, or one or a combination of functional variants thereof that comprise at least about 75% sequence identity to FOXG1, LHX2, SIX3, PAX6. In some embodiments, the cell or plurality of cells comprise one or a combination of LHX2, EMX2, PAX6, or one or a combination of variants thereof that comprise at least about 75% sequence identity to LHX2, EMX2, PAX6. In some embodiments, the cell or plurality of cells comprises one or a combination of HNF1B, PAX6, PAX3, PAX7, NGN2, OLIG3, GDF7, GSX2, MSX1, or one or a combination of functional variants thereof that comprise at least about 75% sequence identity to HNF1B, PAX6, PAX3, PAX7, NGN2, OLIG3, GDF7, GSX2, MSX1.00012 In some embodiments, the cell or plurality of cells are at least about 48 hours in culture, the composition further comprises a WNT activator or derivative thereof. In some embodiments,the composition is free of a WNT inhibitor. In some embodiments, the composition further comprises one or a combination of an HH inhibitor, an FGF / ERK inhibitor, or a derivative thereof. In some embodiments, the cell or plurality of cells comprise one or a combination of SOX2, LHX2, NKX2.1, GLI1, F0XG1 or one or a combination of functional variants thereof that comprise at least about 75% sequence identity to SOX2, LHX2, NKX2.1, GLI1, F0XG1. In some embodiments, the cell or plurality of cells further comprise one or a combination of LHX2 and F0XG1, or one or a combination of functional variants thereof that comprise at least about 75% sequence identity to LHX2 and F0XG1.00013 In some embodiments, the cell or plurality of cells comprise one or a combination of EN1, EN2, PAX2, PAX5 or one or a combination of functional variants thereof that comprise at least about 75% sequence identity to EN1, EN2, PAX2, PAX5.00014 In some embodiments, the cells are at least about 36 hours in culture. In some embodiments, the composition further comprises a Rho Kinase inhibitor. In some embodiments, the cell or plurality of cells comprises one or a combination of MAFB, HNF1B, KROX20, MEIS1, MEIS2, HOXA2, HOXA3, HOXB2, HOXB3, or one or a combination of functional variants thereof that comprise at least about 75% sequence identity to MAFB, HNF1B, KROX20, MEIS1, MEIS2, HOXA2, HOXA3, HOXB2, HOXB3. In some embodiments, the cells are at least about 48 hours in culture. In some embodiments, the composition further comprises an FGF activator, RA activator, TGFBR inhibitor, BMPR inhibitor and WNT inhibitor.00015 In some embodiments, the cell or plurality of cells comprises one or a combination of HNF1B, PAX6, PAX3, PAX7, NGN2, OLIG3, GDF7, GSX2, MSX1, or one or a combination of functional variants thereof that comprise at least about 75% sequence identity to HNF1B, PAX6, PAX3, PAX7, NGN2, OLIG3, GDF7, GSX2, MSX1.00016 In some embodiments, the composition comprises a cell or plurality of cells, wherein the cell or plurality of cells comprise MAPT and SNAP25 or functional variants thereof. In some embodiments, the cell or plurality of cells are deficient in NANOG and SOX2. In some embodiments the cell or plurality of cells are at least about 9 days in culture. In some embodiments the cell or plurality of cells further comprise one or a plurality of neurites extending from their cellular body. In some embodiments, the cell or plurality of cells further comprise TBR1, RELN, EBF2, LHX1, LHX5, VGLUT2, or one or a combination of functionalvariants thereof that comprise at least about 75% sequence identity to TBR1, RELN, EBF2, LHX1, LHX5, VGLUT2. In some embodiments, the cell or plurality of cells further comprise DLX1, SP8, LHX1, LHX5, LHX6, DLX5, VGAT, NKX2.1, PR0X1, ISL1, POMC, or one or a combination of functional variants thereof that comprise at least about 75% sequence identity to DLX1, SP8, LHX1, LHX5, LHX6, DLX5, VGAT, NKX2.1, PR0X1, ISL1, POMC In some embodiments, the cell or plurality of cells further comprise TLX3, ASCL1, LBX1, LMX1B, LHX1, LHX5, PAX2, or one or a combination of functional variants thereof that comprise at least about 75% sequence identity to TLX3, ASCL1, LBX1, LMX1B, LHX1, LHX5, PAX2. In some embodiments the cell or plurality of cells further comprise ISL1, CHAT, VACHT, ACHE, CHT, LHX3, SOX14, SIM1, EN1, GAD1, EVX1, PH0X2A, PH0X2B, H0XA1, H0XA3, or one or a combination of functional variants thereof that comprise at least about 75% sequence identity to ISL1, CHAT, VACHT, ACHE, CHT, LHX3, SOX14, SIM1, EN1, GAD1, EVX1, PH0X2A, PH0X2B, H0XA1, H0XA3.00017 In some embodiments, the disclosure provides a composition comprising a cell or plurality of cells, wherein the cell or plurality of cells comprise PAX3, PAX7, and TFAP2A, or functional variants thereof. In some embodiments, the cell or plurality of cells are deficient in NANOG. In some embodiments, the cell or plurality of cells are at least about 36 hours in culture. In some embodiments, the composition further comprises a BMP activator and WNT activator, or a derivative thereof. In some embodiments, the composition further comprises a TGFP inhibitor and an FGF / ERK inhibitor, or derivative thereof.00018 In some embodiments, the disclosure provides a composition comprising a cell or plurality of cells, wherein the cell or plurality of cells comprise GATA3 and TFAP2A, or functional variants thereof. In some embodiments, the cell or plurality of cells are deficient in NANOG. In some embodiments, the cell or plurality of cells are at least about 36 hours in culture. In some embodiments, the composition further comprises a BMP activator and WNT inhibitor, or derivative thereof. In some embodiments, the composition further comprises a TGFp inhibitor and an FGF / ERK inhibitor, or derivative thereof.00019 In some embodiments, the disclosure provides a composition comprising a cell or plurality of cells, wherein the cell or plurality of cells are from a stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell. In some embodiments, the cell or plurality of cells are human.00020 In some embodiments, the disclosure provides a pharmaceutical composition comprising a therapeutically affective amount of any of the embodiments above and a pharmaceutically acceptable carrier.00021 In some embodiments, the disclosure provides a kit comprising:(a) the composition or pharmaceutical compositions of any of claims 1 through 47; and(b) tissue culture medium and / or tubing configured for transfer of cells. In some embodiments, the kit further comprises instructions to culture or transfer the cells. In some embodiments, the kit is frozen.00022 In some embodiments, the disclosure provides a composition comprising a cell or plurality of cells. In some embodiments, cells have a phenotype comprising SOX2 or a functional variant thereof and are deficient in NANOG. In some embodiments, the cell or plurality of cell are from about 12 to about 48 hours in culture. In some embodiments, the cell or plurality of cells are frozen. In some embodiments, the cell or plurality of cells are frozen at -200 degrees Celsius. In some embodiments, the cell or plurality of cells comprise a cell membrane that is columnar or cuboidal in shape and wherein the composition further comprises an extracellular matrix peptide. In some embodiments, the cell or plurality of cells comprise OTX2 or a functional variant thereof. In some embodiments, the cell or plurality of cells are at least about 48 hours in culture.00023 In some embodiments, the cell or plurality of cells have a phenotype further comprising one or a combination of FOXG1, LHX2, SIX3, PAX6, or one or a combination of functional variants thereof that comprise at least about 75% sequence identity to FOXG1, LHX2, SIX3, PAX6. In some embodiments, the cell or plurality of cells are at least about 48 hours in culture. In some embodiments, the cell or plurality of cells have a phenotype comprising one or a combination of LHX2, EMX2, PAX6, or one or a combination of variants thereof that comprise at least about 75% sequence identity to LHX2, EMX2, PAX6. In some embodiments, the cell or plurality of cells are at least about 48 hours in culture.00024 In some embodiments, the composition further comprises a WNT activator or derivative thereof. In some embodiments, the composition is free of a WNT inhibitor. In some embodiments, the composition further comprises one or a combination of an HH inhibitor, an FGF / ERK inhibitor, or a derivative thereof.00025 In some embodiments, the cell or plurality of cells has a phenotype further comprising one or a combination of NKX2.1, GLI1 or one or a combination of functional variants thereof that comprise at least about 75% sequence identity to NKX2.1, GLI1.00026 In some embodiments, the cell or plurality of cells has a phenotype further comprising one or a combination of LHX2 and FOXG1, or one or a combination of functional variants thereof that comprise at least about 75% sequence identity to LHX2 and FOXG1.00027 In some embodiments, the cell or plurality of cells have a phenotype further comprising one or a combination of EN1, EN2, PAX2, PAX5 or one or a combination of functional variants thereof that comprise at least about 75% sequence identity to EN1, EN2, PAX2, PAX5.00028 In some embodiments, the cell or plurality of cells has a phenotype comprising one or a combination of GBX2, HOXA1, HOXB1, or one or a combination of functional variants thereof that comprise at least about 75% sequence identity to GBX2, HOXA1, HOXB1. In some embodiments, the cells are at least about 36 hours in culture. In some embodiments, the composition further comprises a Rho Kinase inhibitor. In some embodiments, the cell or plurality of cells has a phenotype further comprising one or a combination of MAFB, HNF1B, KROX20, MEIS1, MEIS2, HOXA2, HOXA3, HOXB2, HOXB3, or one or a combination of functional variants thereof that comprise at least about 75% sequence identity to MAFB, HNF1B, KROX20, MEIS1, MEIS2, HOXA2, HOXA3, HOXB2, HOXB3. In some embodiments, the cells are at least about 48 hours in culture. In some embodiments, the composition further comprising an FGF activator. In some embodiments, the composition further comprises one or a combination of TGF0 pathway inhibitor, a BMP pathway inhibitor, a WNT inhibitor, an RA activator, or a derivative thereof.00029 In some embodiments, the cell or plurality of cells has a phenotype comprising one or a combination of HNF1B, PAX6, PAX3, PAX7, NGN2, OLIG3, GDF7, GSX2, MSX1, or one or a combination of functional variants thereof that comprise at least about 75% sequence identity to HNF1B, PAX6, PAX3, PAX7, NGN2, OLIG3, GDF7, GSX2, MSX1.00030 In some embodiments, the disclosure provides a composition comprising a cell or plurality of cells. The cell or plurality of cells have a phenotype comprising MAPT and SNAP25 or functional variants thereof. In some embodiments, the cell or plurality of cells are deficient in NANOG and SOX2. In some embodiments, the cell or plurality of cells are at least about 9 daysin culture. In some embodiments, the cell or plurality of cells further comprise one or a plurality of neurites extending from their cellular body.00031 In some embodiments, the cell or plurality of cells have a phenotype further comprising TBR1, RELN, EBF2, LHX1, LHX5, VGLUT2, or one or a combination of functional variants thereof that comprise at least about 75% sequence identity to TBR1, RELN, EBF2, LHX1, LHX5, VGLUT2.00032 In some embodiments, the cell or plurality of cells have a phenotype further comprising DLX1, SP8, LHX1, LHX5, LHX6, DLX5, VGAT, NKX2.1, PROXI, ISL1, POMC, or one or a combination of functional variants thereof that comprise at least about 75% sequence identity to DLX1, SP8, LHX1, LHX5, LHX6, DLX5, VGAT, NKX2.1, PROXI, ISL1, POMC.00033 In some embodiments, the cell or plurality of cells have a phenotype further comprising TLX3, ASCL1, LBX1, LMX1B, LHX1, LHX5, PAX2, or one or a combination of functional variants thereof that comprise at least about 75% sequence identity to TLX3, ASCL1, LBX1, LMX1B, LHX1, LHX5, PAX2.00034 In some embodiments, the cell or plurality of cells have a phenotype further comprising ISL1, CHAT, VACHT, ACHE, CHT, LHX3, SOX14, SIM1, EN1, GAD1, EVX1, PHOX2A, PHOX2B, HOXA1, HOXA3, or one or a combination of functional variants thereof that comprise at least about 75% sequence identity to ISL1, CHAT, VACHT, ACHE, CHT, LHX3, SOX14, SIM1, EN1, GAD1, EVX1, PHOX2A, PHOX2B, H0XA1, H0XA3.00035 In some embodiments, the disclosure provides a composition comprising a cell or plurality of cells. The cell or plurality of cells have a phenotype comprising PAX3, PAX7, and TFAP2A, or functional variants thereof. In some embodiments, the cell or plurality of cells are deficient in NANOG. In some embodiments, the cell or plurality of cells are at least about 36 hours in culture. In some embodiments, the composition further comprises a BMP activator and WNT activator, or a derivative thereof. In some embodiments, the composition further comprises a TGFp inhibitor and an FGF / ERK inhibitor, or derivative thereof.00036 In some embodiments, the disclosure provides a composition comprising a cell or plurality of cells. The cell or plurality of cells has a phenotype comprising GATA3 and TFAP2A, or functional variants thereof. In some embodiments, the cell or plurality of cells are deficient in NANOG. In some embodiments, the cell or plurality of cells are at least about 36 hours in culture. In some embodiments, the composition further comprises a BMP activator andWNT inhibitor, or derivative thereof. In some embodiments, further comprises a TGFp inhibitor and an FGF / ERK inhibitor, or derivative thereof. In some embodiments, the cell or plurality of cells are from a stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell. In some embodiments, the cell or plurality of cells are human.00037 In some embodiments, the disclosure provides a pharmaceutical composition comprising a therapeutically affective amount of a composition comprising a cell or plurality of cells comprising a phenotype disclosed herein; and a pharmaceutically acceptable carrier.00038 In some embodiments, the disclosure provides a kit. The kit comprises (a) (i) a composition comprising a cell or plurality of cells comprising a phenotype disclosed herein and / or (ii) a pharmaceutical compositions comprising a composition comprising a cell or plurality of cells comprising a phenotype disclosed herein and a pharmaceutically acceptable carrier; and (b) tissue culture medium and / or tubing configured for transfer of cells.In some embodiments, the disclosure provides a method of inducing differentiation of a cell into a mammalian ectodermal progenitor cell comprising: (i) exposing one or a combination of inhibitors chosen from: a TGFp inhibitor, a BMP inhibitor, an FGF / ERK inhibitor, and a WNT inhibitor to the cell for a time period sufficient to differentiate the cell into an ectodermal progenitor cell. In some embodiments, the cell is a stem cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a human pluripotent stem cell. In some embodiments, the time period for the method is from about 12 hours to about 24 hours. In some embodiments, the time period is from about 12 hours to about 48 hours. In some embodiments, the ectodermal progenitor cell comprises SOX2; and is deficient in expression of one or a combination of: NANOG, H0XA1, HOXB1, GBX2, PAX3, GATA3, and TFAP2A.In some embodiments, the disclosure provides a method of differentiating a cell into an anterior neural ectodermal cell comprising (i) exposing the cell to one or a combination of inhibitors chosen from: a TGFP inhibitor, a BMP inhibitor, and a WNT inhibitor for a time period sufficient to differentiate the cell into an anterior neural ectodermal cell. In some embodiments, the cell is an ectodermal progenitor cell comprising SOX2; and is deficient in expression of one or a combination of: NANOG, HOXA1, HOXB 1, GBX2, PAX3, GATA3, and TFAP2A. In some embodiments, the time period for the method is from about 12 hours to about 24 hours. In some embodiments, the method further comprises exposing the cell to a Rho Kinase inhibitor contemporaneously with or in sequence with step (i). In some embodiments, the methodfurther comprises seeding from about 35,000 to about 75,000 cells prior to exposing the cells to one or a combination of inhibitors. In some embodiments, the disclosure provides a method of differentiating a cell into a forebrain progenitor cell comprising: (i) exposing the cell to one or a combination of inhibitors chosen from: a BMP inhibitor and a WNT inhibitor for a time period sufficient to differentiate the cell into a forebrain progenitor cell. In some embodiments, the time period for the method is from about 12 hours to about 48 hours. In some embodiments, the method further comprises exposing the cell to an FGF2 activator contemporaneously with or in sequence with step (i). In some embodiments, the FGF2 activator is exposed to the cell for no more than about 24 hours in duration. In some embodiments, the cell is an anterior ectodermal progenitor cell comprising SOX2 and OTX2; and that is deficient in expression of one or a combination of: NANOG, H0XA1, HOXB1, GBX2, PAX3, GAT A3 and TFAP2A.In some embodiments, the disclosure provides a method of differentiating a cell into adorsal forebrain progenitor cell comprising (i) exposing the cell to one or a combination of modulators chosen from: a FGF / ERK pathway inhibitor, a Hedgehog inhibitor, and a WNT activator for a time period sufficient to differentiate the cell into a dorsal forebrain cell. In some embodiments, the cell is an anterior ectodermal progenitor cell comprising SOX2 and OTX2; and that is deficient in expression of one or a combination of: NANOG, H0XA1, HOXB1, GBX2, PAX3, GATA3 and TFAP2A. In some embodiments, the dorsal forebrain cell comprises SOX2, LHX2, EMX2, and PAX6; and is deficient in NANOG, H0XA1 and HOXB1. In some embodiments, the step of exposing is performed in from about 48 hours to about 72 hours.In some embodiments, the disclosure provides a method of differentiating a cell into a midbrain progenitor cell comprising (i) exposing the cell to one or a combination of modulators chosen from: an FGF / ERK pathway activator, and a WNT activator for a time period sufficient to differentiate the cell into a dorsal hindbrain cell. In some embodiments, the cell is an ectodermal progenitor cell comprising SOX2 and OTX2; and that is deficient in expression of one or a combination of: NANOG, H0XA1, HOXB1, GBX2, PAX3, GATA3 and TFAP2A. In some embodiments, the midbrain progenitor cell comprises SOX2, OTX2, EN1, EN2, PAX2, PAX5; and is deficient in NANOG, HOXA1 and HOXB1. In some embodiments, the step of exposing is performed in from about 36 hours to about 48 hours.00039 In some embodiments, the disclosure provides a method of inducing differentiation of acell into a posterior neural progenitor cell comprising: (i) exposing the cell to one or a combination of modulators chosen from: a TGFp inhibitor, a BMP inhibitor, a WNT inhibitor, a FGF pathway activator, and a retinoic acid pathway activator for a time period sufficient to differentiate the cell into a posterior neural progenitor. In some embodiments, the cell comprises SOX2 and is deficient in NANOG. In some embodiments, the cell is a human cell. In some embodiments, the time period for the method is from about 12 hours to about 24 hours. In some embodiments, the method further comprises exposing the cell to a Rho Kinase inhibitor contemporaneously with or in sequence with step (i). In some embodiments, the method further seeding from about 35,000 to about 75,000 cells prior to exposing the cell to one or a combination of modulators. In some embodiments, the posterior neural progenitor cell comprises SOX2, GBX2, HOXA1, HOXB1; and is deficient in expression of one or a combination of: NANOG, PAX3, GAT A3 and TFAP2A.00040 In some embodiments, the disclosure provides a method of inducing differentiation of a cell into a hindbrain neural progenitor cell comprising: (i) exposing the cell to one or a combination of modulators chosen from a WNT activator and an FGF pathway activator for a time period sufficient to differentiate the cell into a hindbrain neural progenitor cell. In some embodiments, the hindbrain neural progenitor cell comprises one or a combination of SOX2, GBX2, MAFB, HNF1B, KROX20, MEIS1, MEIS2, H0XA1, H0XA2, H0XA3, HOXB1, HOXB2, HOXB3 and is deficient in NANOG. In some embodiments, the step of exposing is performed from about 24 hours to about 48 hours. In some embodiments, the cell comprises SOX2, GBX2, H0XA1, HOXB1; and is deficient in expression of one or a combination of: NANOG, PAX3, GAT A3 and TFAP2A.00041 In some embodiments, the disclosure provides a method of inducing differentiation of a cell into a hindbrain dorsal cell comprising: (i) exposing the cell to one or a combination of modulators chosen from a WNT activator, a BMP inhibitor, and a Hedgehog inhibitor for a time period sufficient to differentiate the cell into a dorsal hindbrain cell. In some embodiments, the time period is from about 48 to about 72 hours. In some embodiments, the cell comprises one or a combination of SOX2, GBX2, MAFB, HNF1B, KROX20, MEIS1, MEIS2, HOXA1, HOXA2, H0XA3, HOXB1, HOXB2, HOXB3 and is deficient in NANOG. In some embodimentsthe dorsal hindbrain cell comprises one or a combination of: SOX2, HNF1B, PAX6, PAX3, PAX7, NGN2, 0LIG3, GDF7, GSX2, MSX1; and wherein the dorsal hindbrain cell is deficient in NANOG.00042 In some embodiments, the disclosure provides a method of inducing differentiation of a cell into a hindbrain ventral cell comprising: (i) exposing the cell to one or a combination of modulators chosen from a WNT inhibitor and a Hedgehog activator for a time period sufficient to differentiate the cell into a ventral hindbrain cell. In some embodiments, the time period is from about 48 to about 72 hours. In some embodiments, the cell comprises one or a combination of: SOX2, GBX2, MAFB, HNF1B, KROX20, MEIS1, MEIS2, HOXA1, HOXA2, H0XA3, HOXB1, HOXB2, HOXB3 and is deficient in NANOG. In some embodiments, the ventral hindbrain cell comprises one or a combination of: SOX2, HNF1B, NKX2.2, NKX6.1, NKX6.2, OLIG2, PHOX2B; and wherein the dorsal hindbrain cell is deficient in NANOG.00043 In some embodiments, the disclosure provides a method of inducing differentiation of a cell into a neuron comprising: (i) exposing the cell to one or a combination of modulators chosen from a neurotrophic growth factor for a time period sufficient to differentiate the cell into a neural cell. In some embodiments, the step of exposing further comprises exposure to one or a combination of: a NOTCH pathway inhibitor, ascorbic acid and an adenyl cyclase activator. In some embodiments, the neural cell comprises:(a) one or a combination of MAPT, SNAP25, TBR1, RELN, EBF2, LHX1, LHX5, VGLUT2; and is deficient in NANOG; or(b) one or a combination of MAPT, SNAP25, DLX1, SP8, LHX1, LHX5, LHX6, DLX5, VGAT, NKX2.1, PROXI, ISL1, POMC; and is deficient in NANOG; or(c) one or a combination of MAPT, SNAP25, TLX3, ASCL1, LBX1, LMX1B, LHX1, LHX5, PAX2; and is deficient in NANOG; or(d) one or a combination of MAPT, SNAP25, ISL1, CHAT, YACHT, ACHE, CHT, LHX3, SOX14, SIM1, EN1, GAD1, EVX1, PHOX2A, PHOX2B, HOXA1, HOXA3; and is deficient in NANOG. In some embodiments, the cell is chosen from: a dorsal forebrain progenitor cell, a ventral forebrain progenitor cell, a dorsal hindbrain progenitor cell, and a ventral hindbrain progenitor cell. In some embodiments, the step of exposing is from about 5 to about 7 days.00044 In some embodiments, the disclosure provides a method of differentiating a cell into anon-neural ectodermal progenitor cell comprising; (i) exposing one or a combination of modulators chosen from: a TGFp inhibitor, a BMP activator, an FGF / ERK inhibitor, and a WNT activator to the cell for a time period sufficient to differentiate the cell into an ectodermal progenitor cell for a time period sufficient to differentiate the cell into a neural cell. In some embodiments, the time period is from about 12 hours to about 24 hours. In some embodiments, the method further comprises exposing the cell to a Rho Kinase inhibitor contemporaneously with or in sequence with step (i). In some embodiments, the method further comprises seeding from about 35,000 to about 75,000 cells prior to exposing the cell to one or a combination of inhibitors. In some embodiments, the non-neural ectodermal progenitor cell comprises one of a combination of PAX3, PAX7, TFAP2A.00045 In some embodiments, the disclosure provides a method of differentiating a cell into a non-neural ectodermal cell comprising; (i) exposing one or a combination of modulators chosen from: a TGFp inhibitor, a BMP activator, an FGF / ERK inhibitor, and a WNT inhibitor to the cell for a time period sufficient to differentiate the cell into an ectodermal progenitor cell for a time period sufficient to differentiate the cell into a non-neural ectodermal cell. In some embodiments, the time period is from about 12 hours to about 24 hours. In some embodiments, the method further comprises exposing the cell to a Rho Kinase inhibitor contemporaneously with or in sequence with step (i). In some embodiments, the method further comprises seeding from about 35,000 to about 75,000 cells prior to exposing the cell to one or a combination of inhibitors. In some embodiments, the non-neural ectodermal cell comprises one of a combination of GATA3 and TFAP2A. In some embodiments, the non-neural ectodermal cell is deficient in SOX2.00046 In some embodiments, the disclosure provides a method of culturing a cell comprising exposing the cell to one or more tissue culture mediums in Table 1. In some embodiments, the disclosure provides a method of culturing border ectoderm (Dl-2, 24 hours): DI definitive ectoderm cells comprising adding ROCK inhibitor to cell culture media to promote cell survival during seeding. In some embodiments, the ROCK inhibitor is thiazovivin. In some embodiments, the disclosure provides a method of differentiating pluripotent cells, for example hPSCs or iPSCs, comprising enzymatically dissociating the pluripotent cells (e.g., hPSCs or iPSCs) and plating them as single cells in a culture medium comprising ROCK inhibitor to cell culture media to promote cell survival during seeding. In some embodiments, the ROCK inhibitor is thiazovivin.00047 In some embodiments, the disclosure provides a method of testing the effect of a test compound on differentiation of D2 posterior neural ectoderm cells into hindbrain cells comprising culturing the D2 posterior neural ectoderm cells in hindbrain media comprising the test compound and observing the fate of the cultured cells by one or more of quantitative PCR, immunocytochemistry, flow cytometry, single-cell RNA sequencing and OMniATAC-seq for hindbrain cell markers. In some embodiments, the D2 posterior neural ectoderm cells are cultured for 72 hours. In some embodiments, the hindbrain media is replaced every 24 hours. 00048 In some embodiments, the disclosure provides a method of testing the effect of a test compound on differentiation of D4 hindbrain progenitor cells into dorsal hindbrain cells comprising culturing the D4 hindbrain progenitor cells in dorsal hindbrain media comprising the test compound and observing the fate of the cultured cells by assessing the presence of dorsal hindbrain cell markers by any means, for example, by one or more of quantitative PCR, immunocytochemistry, flow cytometry, single-cell RNA sequencing and OMniATAC-seq. In some embodiments, the D4 hindbrain progenitor cells are cultured for 72 hours. In some embodiments, the dorsal hindbrain media is replaced every 24 hours.00049 In some embodiments, the disclosure provides a method of testing the effect of a test compound on differentiation of D4 hindbrain progenitor cells into ventral hindbrain cells comprising culturing the D4 hindbrain progenitor cells in ventral hindbrain media comprising the test compound and observing the fate of the cultured cells by assessing the presence of ventral hindbrain cell markers by any means, for example, one or more of quantitative PCR, immunocytochemistry, flow cytometry, single-cell RNA sequencing and OMniATAC-seq. In some embodiments, the D4 hindbrain progenitor cells are cultured for 72 hours. In some embodiments, the ventral hindbrain media is replaced every 24 hours.00050 In some embodiments, the disclosure provides a method of testing the effect of a test compound on differentiation of pluripotent cell-derived D7 cells, for example, hPSC or iPSC- derived D7 cells into neurons comprising culturing the hPSC or iPSC-derived D7 cells in neuronal differentiation media comprising BDNF, GDNF, Forskolin, 2-Phospho-L-ascorbic acid, and DAPT and the test compound and observing the fate of the cultured cells by assessing the presence of neuronal cell markers by any means, for example, by one or more of quantitative PCR, immunocytochemistry, flow cytometry, single-cell RNA sequencing and OMniATAC-seq. In some embodiments, the hPSC or iPSC-derived D7 cells are cultured for 7 days. In someembodiments, the basal media is gradually changed from primarily CDM2 media to primarily neurobasal media over the 7 days.00051 In some embodiments, the disclosure provides a method of differentiating day 2 posterior neural ectoderm into hindbrain progenitor cells corresponding to specific rhombomere identities comprising activating FGF and WNT in the day 2 posterior neural ectoderm. In some embodiments, the day 2 posterior neural ectoderm is differentiated for two days.00052 In some embodiments, the disclosure provides a method of generating hindbrain-specific motor neurons comprising culturing day-7 pluripotent cell-derived, for example, hPSC or iPSC- derived, dorsal hindbrain and or ventral hindbrain neural progenitor cells with BDNF, GDNF, Forskolin and Vitamin C, while blocking NOTCH. In some embodiments, the neural progenitor cells are cultured for 7 days.00053 In some embodiments, the disclosure provides a method of generating posterior neural ectoderm comprising culturing HPSCs or iPSCs under conditions that simultaneously activate FGF and RA alongside neural induction signals BMP, TGFP and WNT. In some embodiments, the culturing is carried out over two days.00054 In some embodiments, the disclosure provides a method for testing the effect of a test compound on posterior neural ectoderm, comprising culturing the posterior neural ectoderm in a media containing the test compound and observing the fate or response of the cultured cells, for example, by one or more of quantitative PCR, immunocytochemistry, flow cytometry, singlecell RNA sequencing and OMniATAC-seq.00055 In some embodiments, the disclosure provides an in vitro cell culture comprising hindbrain motor neurons and a culture media. In some embodiments, the hindbrain motor neurons are dorsal hindbrain motor neurons. In some embodiments, the hindbrain motor neurons are ventral hindbrain motor neurons.00056 In some embodiments, the disclosure provides an in vitro cell culture comprising posterior neural ectoderm and a culture media. The disclosure also relates to a method of culturing cells in vitro wherein one or more of the cells are taken from a composition disclosed herein and the method comprises exposing the cells to one of the disclosed culture mediums disclosed in the specification. In some embodiments, the cells are cultured for about the duration of time identified in Table 1 of the disclosure. In some embodiments, the cells are cultured for about 12, 24, 48, 36, or about 48 hours or more in the culture medium disclosed herein. In someembodiments, the cells are cultured for about 1, 2, 3, 4, 5, 6, or about 7 days or more in one of the culture mediums disclosed herein.BRIEF DESCRIPTION OF DRAWINGS00057 FIGS. 1 A-1J show in vivo identification, and in vitro generation of, anterior and posterior neural ectoderm. FIG.1A shows a summary of hPSC differentiation into ectodermal cell-types. FIG. IB shows in situ staining of E6.75 mouse embryo. Scale bar = 50 pm. FIG. 1C shows Singlecell RNA-sequencing of E6.75 mouse embryos; analysis performed on a previously published resource. Embryonic cells are shown, with extraembryonic cells excluded (Elf5 and Rhox5 log2 normalized expression = 0). FIG. ID shows Flow cytometry of Hl hPSCs prior to, and after, differentiation into definitive ectoderm for 24 hours. FIG. IE shows in situ staining of E7.5 mouse embryo. Scale bar = 50 pm. FIG. IF shows Single-cell RNA-sequencing ofE7.5 mouse embryos; analysis performed on a previously published resource. Neural ectoderm cells (identified by log2 normalized expression of Sox2 > 0.5, and Nanog, Tfap2a, Rhox5, Foxjl, and Elf5 = 0) are shown. FIG. 1G shows qPCR of Hl hPSCs differentiated into definitive ectoderm for 24 hours, and then subsequently treated with neural-inducing signals (A-83-01 [1 pM], LDN193189 [250 nM] and XAV939 [1 pM]), in the presence or absence of FGF2 (20 ng / mL) and / or retinoid pathway agonist TTNPB (50 nM) for 24 hours. qPCR data normalized to undifferentiated hPSCs. FIG. 1H shows qPCR of Hl hPSCs differentiated into definitive ectoderm for 24 hours, followed by differentiation into either aNE, pNE, border ectoderm or surface ectoderm for 24 hours. qPCR data normalized to undifferentiated hPSCs. FIG. II shows Immunostaining of Hl hPSCs differentiated into definitive ectoderm for 24 hours, followed by differentiation into aNE or pNE for 24 hours. Scale bar = 100 pm. FIG. 1 J shows immunostaining of Hl hPSCs differentiated into definitive ectoderm for 24 hours, followed by differentiation into border or surface ectoderm for 24 hours. Scale bar = 100 pm.00058 FIGS. 2A and 2B show genetic lineage-tracing of neural ectoderm reveals two parallel brain progenitors in the E7.5 mouse embryo Genetic lineage-tracing of neural ectoderm reveals two parallel brain progenitors in the E7.5 mouse embryo. FIG. 2A shows E7.5 Sox2-CreER, Confetti mouse embryos exposed to 0.5 mg 4OHT (intraperitoneally delivered into pregnant females) to label single E7.5 Sox2+neural ectoderm progenitors with one of three detectable fluorescent colors (red, cyan, or yellow). Subsequently, E9.5 embryos were immunostained tovisualize neural ectoderm -derived clones and where they resided in relation to the forebrain, midbrain, and hindbrain. The forebrain / midbrain domain was molecularly determined by Otx2 immunostaining (left). Quantification of the locations of all neural ectoderm-derived clones analyzed in this study (right). Scale bar = 100 m. FIG. 2B shows E7.5 Gbx2-CreER ROSA26- LoxP-STOP-LoxP-tdTomato mouse embryos exposed to 1 mg 4OHT (intraperitoneally delivered into pregnant females) to label E7.5 Gbx2+pNE with tdTomato. Subsequently, staining was performed on E8.5-E11.5 embryos to visualize pNE-derived progeny in relation to the forebrain, midbrain, and hindbrain. The forebrain / midbrain domain was molecularly determined by Otx2 in situ staining (for E8.5 and E9.5) and Otx2 immunostaining (for El 1.5). Additionally, immunostaining was performed on El 8.5 embryos to visualize pNE-derived progeny, and the hindbrain domain was molecularly determined by the presence of serotoninergic neurons, which are present in the hindbrain, but not the midbrain. Scale bar = 100 pm (for E8.5 and E9.5) and 500 pm (for El 1.5 and El 8.5).00059 FIGS. 3A-3F show that hPSC-derived anterior and posterior neural ectoderm are restricted to respectively differentiate into forebrain / midbrain vs. hindbrain progenitors in vitro. FIG. 3A shows the strategy to differentiate hPSCs into forebrain, midbrain, and hindbrain progenitors within 4 days in vitro. FIG. 3B shows qPCR (left) and immunostaining (right) of Hl hPSC-derived day -2 anterior neural ectoderm or posterior neural ectoderm that was treated with forebrain-inducing signals for 48 hours. Scale bar = 120 pm. FIG. 3C shows qPCR (left) and immunostaining (right) of Hl hPSC-derived day-2 anterior neural ectoderm or posterior neural ectoderm that was treated with midbrain-inducing signals for 48 hours. Scale bar = 120 pm. FIG. 3D shows a summary of rhombomere marker expression in the early hindbrain, with the proviso that certain markers shift over time (top). Immunostaining of Hl hPSCs differentiated into definitive ectoderm for 24 hours, followed by posterior neural ectoderm for 24 hours, and finally, hindbrain progenitors after an additional 48 hours (bottom). FIG. 3E shows qPCR (left) and immunostaining (right) of Hl hPSC-derived day-2 anterior neural ectoderm or posterior neural ectoderm that was treated with hindbrain-inducing signals for 48 hours. Scale bar = 120 pm. FIG. 3F shows that Hl hPSCs were differentiated into either aNE or pNE within 2 days; pNE was fluorescently labeled with ViaFluor 488 SE dye, and then aNE (uncolored) and pNE (dye-labeled) were mixed, and treated with either forebrain-,midbrain-, or hindbraininducing signals for 2 additional days, prior to fixation and immunostaining. Scale bar = 100 pm.00060 FIGS. 4A-H show that different accessible chromatin landscapes distinguish hPSC-derived anterior vs. posterior neural ectoderm. FIG. 4A shows differentially accessible chromatin regions in hPSC-derived aNE or pNE, as detected by OmniATACseq. Each dot indicates a single chromatin region, which was assigned to its nearest gene. FIG. 4B shows transcription factor motifs that were respectively enriched in either aNE- or pNE-specific accessible chromatin regions, with statistical significance shown. FIG. 4C shows OmniATACseq of the hPSC-derived aNE and pNE treated with 48 hours of forebraininducing signals. FIG. 4D shows OmniATACseq of the hPSC- derived aNE and pNE treated with 48 hours of hindbraininducing signals. FIG. 4E shows Hl hPSCs differentiated into either aNE or pNE within 2 days; pNE was fluorescently labeled with ViaFluor 488 SE dye, and then aNE (uncolored) and pNE (dye-labeled) were mixed, and treated with either forebrain-, midbrain-, or hindbrain-inducing signals for 2 additional days, prior to fixation and immunostaining. Scale bar = 100 pm. FIG. 4F shows Differentially accessible chromatin regions in hPSC-derived aNE or pNE, as detected by OmniATACseq. Each dot indicates a single chromatin region, which was assigned to its nearest gene. FIG. 4G shows transcription factor motifs that were respectively enriched in either aNE- or pNE-specific accessible chromatin regions, with statistical significance shown. FIG. 4H shows OmniATACseq of the indicated hPSC- derived cell-types.00061 FIGS. 5A-5C show generation of dorsal forebrain, ventral forebrain, dorsal hindbrain, and ventral hindbrain progenitors from hPSCs in vitro. FIG. 5A shows the strategy to differentiate hPSCs into dorsal forebrain, ventral forebrain, dorsal hindbrain and ventral hindbrain progenitors within 7 days in vitro. FIG. 5B shows immunostaining of Hl hPSC- derived day-4 forebrain or hindbrain progenitors that were treated with dorsalizing or ventralizing signals (see Fig. 4a) for 72 hours. Scale bar = 120 pm. FIG. 5C shows scRNAseq of H7 hPSC-derived day-4 forebrain or hindbrain progenitors, and day-7 dorsal forebrain, ventral forebrain, dorsal hindbrain, or ventral hindbrain progenitors. Cells were colored by the duration and type of differentiation they were subjected to.00062 FIGS. 6A-6F show generation of hindbrain motor neurons from hPSCs in vitro. FIG. 6A shows immunostaining of Hl hPSC-derived day-7 dorsal forebrain, ventral forebrain, dorsal hindbrain, or ventral hindbrain progenitors that were exposed to the same neuron-inducing signals for 7 additional days in vitro. Scale bar = 120 pm. FIG. 6B shows immunostaining of Hl hPSC-derived day-7 dorsal forebrain, ventral forebrain, dorsal hindbrain, or ventral hindbrain progenitors that were exposed to the same neuron-inducing signals for 7 additional days in vitro. Scale bar = 120 pm. FIG. 6C shows immunostaining of Hl hPSC-derived day-7 dorsal forebrain, ventral forebrain, dorsal hindbrain, or ventral hindbrain progenitors that were exposed to the same neuron-inducing signals for 7 additional days in vitro. Scale bar = 120 pm. FIG. 6D shows scRNAseq of H7 hPSC-derived day-14 ventral hindbrain population. FIG. 6E shows live Ca2+imaging of WTC AAVSl-CAG-GCaMP f^ Cs differentiated into ventral hindbrain neurons for 30 days, which exhibited spontaneous Ca2+transients as assessed within individual cells (fe Z) and across the culture (right). Scale bar = 50 pm. FIG. 6F shows electrophysiological activity of Hl hPSC-derived ventral hindbrain neurons on day 27 of differentiation, showing voltagedependent K+or Na+channel currents elicited by depolarization of the holding potential from -90 mV to 50 mV in voltage-clamp mode (top) and action potentials elicited by injection of pulsed (300 pA, 5 ms, 10 Hz) or prolonged (50 pA, 1.5 seconds) currents in current-clamp mode (bottom).00063 FIGS. 7A-7E shows evolutionary conservation of anterior and posterior ectoderm. FIG. 7A shows a summary of evolutionary relationships. FIG. 7B shows single-cell RNA-sequencing of gastrulating macaque embryo (Carnegie Stage [CS9]; embryonic day 23 [E23]); analysis performed on a previously published resource. Neural ectoderm cells (identified by log2 normalized expression of SOX2 > 1 and, TFAP2A, HAND1, and APLNR = 0) are shown. FIG. 7C shows single-cell RNA-sequencing of gastrulating chicken embryo (Hamburger-Hamilton stage 5 [HH5]); analysis performed on a previously published resource. Neural ectoderm cells are shown and were defined as cells within the annotated “ectoderm” cluster with log2 normalized expression of Sox2 > 0 (top). In situ staining of HH5 stage chicken embryo, scale bar = 500 pm (bottom). FIG. 7D shows single-cell RNA-sequencing of gastrulating zebrafish embryo (8 hours post-fertilization [h.p.f ], 75% epiboly); analysis performed on a previously published resource. Neural ectoderm cells (identified by log2 normalized expression of sox2 > 0.5 and tfap2a < 0.5) are shown (top). In situ staining of 8 h.p.f. zebrafish embryo, scale bar = 50 pm (bottom). FIG.7E shows in situ staining of gastrulating acorn worm embryo (at the flat-plate stage). Scale bar = 100 pm.00064 FIGS. 8A-8I show results of differentiation of hPSCs into definitive ectoderm within 24 hours, and in vivo analysis of anterior and posterior neural ectoderm. FIG. 8A shows qPCR of H7 hPSCs differentiated for 24 hours with “dual SMAD inhibitors” (TGF0 inhibitor A-83-01 [1 pM] and BMP inhibitor LDN-193189 [250 nM]), in the presence or absence of WNT inhibitor (C59, 1 pM) and ERK inhibitor (Trametinib, 250 nM). “DO” = day 0 hPSCs. qPCR data normalized to undifferentiated hPSCs. FIG. 8B shows qPCR of H7 hPSCs differentiated for 24 hours with “dual SMAD inhibitors” (TGF0 inhibitor A-83-01 [1 pM] and BMP inhibitor LDN- 193189 [250 nM]), in the presence or absence of individual ERK inhibitors (PD0325901, Trametinib, AZD6244, GDC0623 or Pimasertib) or FGFR inhibitors (AZD4547, PD173074 or BGJ398). qPCR data normalized to undifferentiated hPSCs. FIG. 8C shows qPCR of Hl hPSCs seeded at different densities, and then differentiated into definitive ectoderm with BMP, ERK, TGF0 and WNT inhibitors (LDN-193189, Trametinib, A-83-01 and C59, respectively) for 24 hours. qPCR data normalized to undifferentiated hPSCs. Open circle indicates a seeding density of 520 cells / mm2, which was the baseline used throughout this study. FIG. 8D shows qPCR of Hl, H7, SUN004.1 and SUN004.2 hPSCs prior to, or after, differentiation into definitive ectoderm with BMP, ERK, TGF0 and WNT inhibitors (LDN-193189, Trametinib, A-83-01 and C59, respectively) for 24 hours. qPCR data normalized to undifferentiated hPSCs. FIG. 8E shows flow cytometry of Hl hPSCs differentiated into definitive ectoderm for 24 hours. The percentage of SOX2+ NANOG- definitive ectoderm cells is shown. Individual datapoints depict the outcomes of independent experiments. FIG. 8F shows immunostaining ofH7 hPSCs prior to, or after, differentiation into definitive ectoderm for 24 hours. Scale bar = 100 pm. FIG. 8G shows flow cytometry of HES3 MIXL1-GFP hPSCs differentiated into definitive ectoderm (day 1), anterior neural ectoderm (day 2), posterior neural ectoderm (day 2), border ectoderm (day 2), surface ectoderm (day 1), or mid primitive streak (day 1, as a positive control to induce MIXL1 expression). Negligible numbers of MIXL1+ primitive streak cells emerged after ectoderm differentiation, reaffirming the suppression of spurious primitive streak differentiation. FIG. 8H shows in situ staining of E7.5 mouse embryo, showing overlap of Sox2 and Gbx2 expression in pNE. Scale bar = 25 pm. FIG. 81 shows single-cell RNA-sequencing of E7.5 mouse embryos; analysis performed on a previously-published resource. Neural ectoderm cells (identified by log2normalized expression of Sox2 > 0.5, and Nanog, Tfap2ct, Rhox5, Foxjl, and Elf5 = 0) are shown.00065 FIGS. 9A-9G show differentiation of hPSCs into anterior or posterior neural ectoderm within 48 hours. FIG. 9A shows qPCR of Hl hPSCs differentiated into definitive ectoderm for 24 hours, followed by BMP, TGFp and WNT inhibitors (LDN-193189, A-83-01 and C59, respectively) for 24 hours to generate anterior neural ectoderm. To assess the importance of each anterior neural ectoderm-inducing signal, each factor was individually withheld. qPCR data normalized to undifferentiated hPSCs. (“DO” = day 0 hPSCs.) FIG. 9B shows qPCR of Hl hPSCs differentiated into definitive ectoderm for 24 hours, followed by a combination of FGF and RA agonists (FGF2 and TTNPB, respectively) together with BMP, TGF0 and WNT inhibitors (LDN-193189, A-83-01 and C59, respectively) for 24 hours to generate posterior neural ectoderm. To assess the importance of each posterior neural ectoderm-inducing signal, each factor was individually withheld. qPCR data normalized to undifferentiated hPSCs. FIG. 9C shows results of qPCR of Hl hPSCs differentiated into definitive ectoderm for 24 hours, followed by differentiation into posterior neural ectoderm for 24 hours. During posterior neural ectoderm induction, different doses of FGF agonist (FGF2) or RA agonist (TTNPB) were added to assess dose-dependent effects. Open circles indicate the optimal doses of FGF2 (20 ng / mL) and TTNPB (50 nM) for posterior neural ectoderm induction used as a baseline throughout this study. qPCR data normalized to undifferentiated hPSCs. FIG. 9D shows in situ staining of E7.5 mouse embryo, showing that Raldh2 (the rate-limiting enzyme in retinoic acid [RA] synthesis) is expressed adjacent to pNE. Scale bar = 50 pm. FIG. 9E shows qPCR of Hl hPSCs differentiated into definitive ectoderm for 24 hours, followed by differentiation into anterior neural ectoderm for 24 hours (with A-83-01, LDN-193189, and C59). Alternatively, to test the effect of WNT signaling on anterior neural ectoderm induction, WNT inhibitor (C59) was withheld, and WNT agonist (CHIR99021) was added instead. qPCR data normalized to undifferentiated hPSCs. FIG. 9F shows results of qPCR of Hl hPSCs differentiated into definitive ectoderm for 24 hours, followed by differentiation into posterior neural ectoderm for 24 hours (with A-83-01, LDN- 193189, C59, FGF2 and TTNPB). Alternatively, to test the effect of WNT signaling on posterior neural ectoderm induction, WNT inhibitor (C59) was withheld, and WNT agonist (CHIR99021) was added instead. qPCR data normalized to undifferentiated hPSCs. FIG. 9G shows qPCR of Hl hPSCs differentiated for 48 hours, using two separate strategies. First, hPSCs weredifferentiated into definitive ectoderm for 24 hours, followed by either anterior or posterior neural ectoderm induction for 24 hours (as described in this study). Alternatively, hPSCs were differentiated with BMP and TGFp inhibitors (LDN-193189 and A-83-01, respectively; “2i”) together with WNT agonist (CHIR99021, 2 pM) for 48 hours, similarly to a previous report. qPCR data normalized to undifferentiated hPSCs.00066 FIGS. 10A-10F show results of differentiation of hPSCs into border or surface ectoderm within 48 hours. FIG. 10A shows qPCR of Hl hPSCs differentiated into definitive ectoderm for 24 hours, followed by treatment with non-neural signals (A-83-01 and PD0325901), in combination with different levels of BMP signaling (BMP agonist BMP4 [0.05-30 ng / mL] or alternatively, BMP inhibitor LDN-193189), in the presence or absence of WNT agonist (CHIR99021), for 24 hours. White circles indicate optimal BMP doses for border ectoderm induction (0.5 ng / mL BMP4 + CHIR99021) or surface ectoderm induction (30 ng / mL BMP4). qPCR data normalized to undifferentiated hPSCs. FIG. 10B shows qPCR of Hl hPSCs differentiated into definitive ectoderm for 24 hours, followed by border ectoderm-inducing signals (A-83-01, BMP4 [0.5 ng / mL], and PD0325901), in combination with different levels of WNT signaling (WNT agonist CHIR99021 [3-8 pM] or WNT inhibitor C59), for 24 hours. qPCR data normalized to undifferentiated hPSCs. FIG. 10C shows qPCR of Hl hPSCs differentiated into definitive ectoderm for 24 hours, followed by border ectoderm-inducing signals (A-83-01, CHIR99021 [3 pM], and PD0325901), in combination with different levels of BMP signaling (BMP agonist BMP4 [0.05-30 ng / mL] or alternatively, BMP inhibitor LDN- 193189), for 24 hours. qPCR data normalized to undifferentiated hPSCs. FIG. 10D shows qPCR of Hl hPSCs differentiated into definitive ectoderm for 24 hours, followed by border ectoderminducing signals (A-83-01, BMP4, CHIR99021, and PD0325901) for 24 hours to generate border ectoderm. To assess the importance of each border ectoderm-inducing signal, each factor was individually withheld. qPCR data normalized to undifferentiated hPSCs. FIG. 10E shows qPCR of Hl hPSCs differentiated into definitive ectoderm for 24 hours, followed by surface ectoderminducing signals (A-83-01, C59, and PD0325901), in the presence or absence of BMP4 (0-30 ng / mL), for 24 hours. qPCR data normalized to undifferentiated hPSCs. FIG. 10F shows qPCR of Hl hPSCs differentiated into definitive ectoderm for 24 hours, followed by surface ectoderminducing signals (A-83-01, BMP4, C59, and PD0325901) for 24 hours. To assess the importanceof each border ectoderm-inducing signal, each factor was individually withheld. qPCR data normalized to undifferentiated hPSCs.00067 FIGS. 11A-E show results of differentiation of hPSCs into anterior neural, posterior neural, border or surface ectoderm within 48 hours. FIG. 11A shows qPCR of Hl hPSCs differentiated into definitive ectoderm for 24 hours, followed by surface ectoderm-inducing signals (A-83-01, BMP4, C59, and PD0325901), in the presence or absence of retinoid agonist (TTNPB, 0.5-500 nM), for 24 hours. qPCR data normalized to undifferentiated hPSCs. FIG. 1 IB shows a summary of hPSC differentiation strategy. FIG. 11C shows qPCR of H7 hPSCs differentiated into definitive ectoderm for 24 hours, followed by either anterior neural ectoderm, posterior neural ectoderm, border ectoderm or surface ectoderm, for 24 hours. qPCR data normalized to undifferentiated hPSCs. FIG. 1 ID shows qPCR of SUN004.1 hPSCs differentiated into definitive ectoderm for 24 hours, followed by either anterior neural ectoderm, posterior neural ectoderm, border ectoderm or surface ectoderm, for 24 hours. qPCR data normalized to undifferentiated hPSCs. FIG. 1 IE shows qPCR of SUN004.2 hPSCs differentiated into definitive ectoderm for 24 hours, followed by either anterior neural ectoderm, posterior neural ectoderm, border ectoderm or surface ectoderm, for 24 hours. qPCR data normalized to undifferentiated hPSCs.00068 FIGS. 12A-12D show lineage tracing of neural ectoderm cells within mouse embryos. FIG. 12 A shows in situ staining (i) and immunostaining (ii) of E9.5 and El 8.5 Gbx2-Cre R ROSA26-LoxP-STOP-LoxP-tdTomato mouse embryos that were not exposed to 4OHT. This negative control revealed minimal Gbx2-CreER-dnven recombination in the absence of 4OHT, although a minor degree of recombination was observed in the E18.5 diencephalon in the absence of 4OHT. Scale bar = 500 pm (E9.5) and 1 mm (E18.5). FIG. 12B shows results of E7.0 Gbx2-CreER, ROSA26-LoxP-STOP-LoxP-tdTomato mouse embryos exposed to 1 mg 4OHT (intraperitoneally delivered into pregnant females) to label Gbx2+pNE with tdTomato. Given the 12-hour labeling window of 4OHT92(-E7.0-E7.5), this would be expected to label incipiently- emerging Gbx2+pNE (which arises at -E7.5) with tdTomato. Subsequently, in situ staining was performed on E8.5 embryos to visualize pNE-derived progeny in relation to the expression of Otx2 (which marks the forebrain / midbrain). Scale bar = 100 pm. FIG. 12C shows results of E7.0 Gbx2-CreER ROSA26-LoxP-STOP-LoxP-tdTomato mouse embryos exposed to 1 mg 4OHT (intraperitoneally delivered into pregnant females) to label Gbx2+pNE with tdTomato.Subsequently, in situ staining was performed on E8.5 embryos to visualize pNE-derived progeny in relation to the expression of Wntl (which marks the midbrain / hindbrain boundary) and Gbx2 (which marks the hindbrain). Scale bar = 100 pm. FIG. 12D shows results of E7.0 Gbx2-CreER, ROSA26-LoxP-STOP-LoxP-tdTomato mouse embryos were exposed to 1 mg 4OHT (intraperitoneally delivered into pregnant females) to label Gbx2+pNE with tdTomato.Subsequently, in situ staining was performed on E8.5 embryos to visualize pNE-derived progeny in relation to the expression of Krox20 (Egr 2), which is first turned on in rhombomere 3 (r3), and shortly afterwards, in r5134. Scale bar = 100 pm.00069 FIGS. 13A-13F show results of differentiation of hPSCs into forebrain progenitors within 4 days. FIG. 13A shows qPCR of Hl hPSCs differentiated into definitive ectoderm for 24 hours, followed by anterior neural ectoderm for 24 hours, and then subsequently treated with either basal medium (CDM2) for 48 hours, or supplemented with WNT inhibitor DKK1 (LRP5 / 6 inhibitor, 300 ng / mL), LGK974 (Porcupine inhibitor, 100 nM), C59 (Porcupine inhibitor, 1 pM), or XAV939 (Tankyrase inhibitor, 1 pM). This revealed that XAV939 was most effective at upregulating forebrain marker F0XG1. qPCR data normalized to undifferentiated hPSCs. FIG. 13B shows qPCR of Hl hPSCs differentiated into definitive ectoderm for 24 hours, followed by anterior neural ectoderm for 24 hours, and then subsequently treated with XAV939 for 48 hours to generate forebrain progenitors. During this final 48 hour interval, FGF2 (20 ng / mL) was added for the first 24 hour interval (“day 3”), the second 24 hour interval (“day 4”), or the entirety of the 48 hour interval (“days 3-4”), in the presence or absence of BMP receptor inhibitor LDN193189 (100 nM). This revealed that day 3 FGF2 treatment, together with BMP inhibition on days 3-4, was most effective at upregulating forebrain markers FOXGl and SIX3. qPCR data normalized to undifferentiated hPSCs. FIG. 13C shows qPCR of H7 hPSCs differentiated into definitive ectoderm for 24 hours, followed by anterior neural ectoderm for 24 hours, and then subsequently treated with XAV939 + LDN193189 + FGF2 (2-100 ng / mL) for 24 hours, and finally XAV939 + LDN193189 for 24 hours to generate forebrain progenitors. On the third day of differentiation (“D3”), different doses of FGF2 (2-100 ng / mL) were tested, or alternatively, exogenous FGF2 was withheld (no notation), in the presence or absence of FGFR inhibitor PD173074 (100 ng / mL). This revealed that 20 ng / mL of FGF2 on day 3 of differentiation was most effective at upregulating forebrain markers SIX3 and FOXGE qPCR data normalized to undifferentiated hPSCs. FIG. 13D shows immunostaining of Hl hPSCs differentiated intodefinitive ectoderm for 24 hours, followed by anterior neural ectoderm for 24 hours, and then subsequently into SIX3+forebrain progenitors for an additional 48 hours. Scale bar = 120 pm. This is the same experiment and field of view shown in Fig. 3b, except immunostaining for a different protein was performed. FIG. 13E shows qPCR of Hl hPSCs differentiated into definitive ectoderm for 24 hours, followed by anterior neural ectoderm for 24 hours, and then subsequently treated with XAV939 + LDN193189 + FGF2 for 24 hours, and finally XAV939 + LDN193189 for 24 hours to generate forebrain progenitors. To assess the importance of each forebrain-inducing signal (XAV939, LDN193189 or FGF2), each individual signal was respectively withheld during the 48 hour interval of forebrain induction. qPCR data normalized to undifferentiated hPSCs. FIG. 13F shows qPCR of Hl hPSCs differentiated into definitive ectoderm for 24 hours, followed by anterior neural ectoderm for 24 hours, and then subsequently treated with XAV939 + LDN193189 + FGF2 for 24 hours, and finally XAV939 + LDN193189 for 24 hours to generate forebrain progenitors. Alternatively, TGF0 agonist (Activin, 5 or 50 ng / mL) or TGF0 inhibitors (either A-83-01 [1 pM] or SB-505124 [2 pM]) were added during the 48 hour interval of forebrain induction. This revealed that TGFP activation or inhibition did not enhance forebrain progenitor specification. qPCR data normalized to undifferentiated hPSCs. 00070 FIGS. 14A-14G show results of differentiation of hPSCs into midbrain progenitors within 4 days. FIG. 14A shows qPCR of Hl hPSCs differentiated into definitive ectoderm for 24 hours, followed by anterior neural ectoderm for 24 hours, and then subsequently treated with the following WNT pathway modulators for 48 hours: WNT inhibitor (XAV939, 1 pM), RSPO2 (25 nM), RSPO2 + WNT3A (increasing concentrations of 10, 100 or 1000 ng / mL), RSPO2 + NGS WNT -FC (increasing concentrations of 0.03, 0.3 or 3 nM), or CHIR99021 (increasing concentrations of 0.75, 1.5 or 3 pM). For WNT3A or NGS WNT -FC treatment conditions, RSPO2 was added to increase cell -surface levels of FRIZZLED receptors, thereby enhancing cellular responsiveness to these WNT pathway agonists. qPCR data normalized to undifferentiated hPSCs. FIG. 14B shows qPCR of Hl hPSCs differentiated into definitive ectoderm for 24 hours, followed by anterior neural ectoderm for an additional 24 hours, and then subsequently treated with either WNT inhibitor (XAV939, 1 pM) or WNT agonist (CHIR99021, 1.5-4 pM) for an additional 48 hours. This showed that WNT induced midbrain progenitors, whereas WNT inhibitor specified forebrain progenitors. qPCR data normalized to the sample that displayed the highest expression in this experiment. FIG. 14C shows in situ staining of E8.5Tcf / Lef:H2B:Venus reporter mouse embryo for Foxgl (forebrain marker) and Verms (indicating WNT transcriptional response). There is a posteriorizing gradient of WNT signaling, with minimal WNT signaling in Foxgl+forebrain, but high WNT signaling in the midbrain. FIG. 14D shows qPCR of Hl hPSCs differentiated into definitive ectoderm for 24 hours, followed by anterior neural ectoderm for 24 hours, and then subsequently differentiated towards midbrain with WNT agonist (CHIR99021, 3 pM) for 48 hours, in the presence or absence of FGF2 (20 ng / mL) on the first 24 hour interval, the second 24 hour interval, or the entire 48 interval of midbrain induction. qPCR data normalized to undifferentiated hPSCs. FIG. 14E shows qPCR of Hl hPSCs differentiated into definitive ectoderm for 24 hours, followed by anterior neural ectoderm for 24 hours, and then subsequently differentiated towards midbrain with WNT agonist (CHIR99021, 3 pM) for 48 hours, in the presence or absence of FGF2 (2-200 ng / mL) on last 24 hours of midbrain induction. qPCR data normalized to undifferentiated hPSCs. FIG. 14F shows qPCR of Hl hPSCs differentiated into definitive ectoderm for 24 hours, followed by anterior neural ectoderm for 24 hours, and then subsequently differentiated towards midbrain with WNT agonist (CHIR99021, 3 pM) for 48 hours. Alternatively, TGF0 agonist (Activin, 5 or 50 ng / mL) or TGFP inhibitor (A-83-01, 1 pM) was added during the 48 hour interval of midbrain induction. qPCR data normalized to undifferentiated hPSCs. FIG. 14G shows qPCR of Hl hPSCs differentiated into definitive ectoderm for 24 hours, followed by anterior neural ectoderm for 24 hours, and then subsequently differentiated towards midbrain with WNT agonist (CHIR99021, 3 pM) for 48 hours. Alternatively, BMP agonist (BMP4, 5 or 50 ng / mL) or BMP inhibitor (LDN- 193189, 100 nM) was added during the 48 hour interval of midbrain induction. qPCR data normalized to undifferentiated hPSCs. FIG. 14H shows qPCR of Hl hPSCs differentiated into definitive ectoderm for 24 hours, followed by anterior neural ectoderm for 24 hours, and then subsequently differentiated towards midbrain for 48 hours (CHIR99021 on days 3-4, and FGF2 on day 4). To assess the importance of each midbrain-inducing signal (CHIR99021 or FGF2), each individual signal was respectively withheld during the 48 hour interval of midbrain induction. qPCR data normalized to undifferentiated hPSCs.00071 FIGS. 15A-15G show results of differentiation of hPSCs into hindbrain progenitors within 4 days. FIG. 15A shows qPCR of Hl hPSCs differentiated into definitive ectoderm for 24 hours, followed by posterior neural ectoderm for 24 hours, and then further differentiated for 48 hours in the presence or absence of FGF2 (10-200 ng / mL) or FGFR inhibitor (PD173074, 100nM). qPCR data normalized to undifferentiated hPSCs. FIG. 15B shows qPCR of Hl hPSCs differentiated into definitive ectoderm for 24 hours, followed by posterior neural ectoderm for 24 hours, and then further differentiated for 48 hours in FGF2 (20 ng / mL), in the presence or absence of WNT agonist (CHIR99021, 0.5-1.5 pM) or WNT inhibitor (XAV939, 1 pM). qPCR data normalized to undifferentiated hPSCs. FIG. 15C shows qPCR of Hl hPSCs differentiated into definitive ectoderm for 24 hours, followed by posterior neural ectoderm for 24 hours, and then further differentiated into hindbrain for 48 hours by FGF2 + CHIR99021. Alternatively, during the 48 hour interval of hindbrain induction, either FGF2 or CHIR99021 were individually withheld. This revealed that both FGF and WNT signaling are required for maximal MAFB expression. qPCR data normalized to levels found in hindbrain progenitors (e.g., cells treated with FGF2 + CHIR99021). FIG. 15D shows qPCR of Hl hPSCs differentiated into definitive ectoderm for 24 hours, followed by posterior neural ectoderm for 24 hours, and then further differentiated into hindbrain for 48 hours by FGF2 + CHIR99021. Alternatively, during the 48 hour interval of hindbrain induction, either FGF2 or CHIR99021 were individually withheld for 24 intervals (“day 3” = the first 24 hour interval, whereas “day 4” = the second 24 hour interval). This revealed that both FGF and WNT signaling are continuously required for 48 hours to achieve maximal MAFB expression. qPCR data normalized to undifferentiated hPSCs. FIG. 15E shows qPCR of Hl hPSCs differentiated into definitive ectoderm for 24 hours, followed by posterior neural ectoderm for 24 hours, and then further differentiated into hindbrain by FGF2 + CHIR99021 for 24, 48 or 72 hours. This revealed that FGF and WNT treatment for 48 hours was optimal for hindbrain specification (as denoted by a white circle). qPCR data normalized to undifferentiated hPSCs. FIG. 15F shows scRNAseq of H7 hPSC-derived day-4 hindbrain progenitors, and day-7 dorsal hindbrain or ventral hindbrain progenitors. In the legend, cells were colored by the duration and type of differentiation they were subjected to. An asterisk (*) indicates that the legend is identical to that shown in FIG. 4C and is reproduced here to aid the interpretation of other images shown in this subpanel. FIG. 15G shows results of Hl hPSCs differentiated into definitive ectoderm for 24 hours, and then subsequently differentiated into either anterior neural ectoderm (aNE) or posterior neural ectoderm (pNE) within an additional 24 hours. hPSC-derived aNE and pNE cells were then dissociated and then re-plated in either forebrain-, midbrain-, or hindbrain-induced signals for 48 hours, and then were fixed for immunostaining.00072 FIGS. 16A-16I Hl show differentiation of hPSCs into dorsal forebrain and ventral forebrain progenitors within 7 days FIG. 16A shows Hl hPSCs were differentiated into definitive ectoderm within 24 hours, and then anterior neural ectoderm within 24 hours, followed by forebrain progenitors within 48 hours, which were then treated with Vismodegib + PD173074 for 72 hours, in the presence or absence of WNT agonist (CHIR99021, 1-6 pM) or WNT inhibitor (XAV939, 1 pM). This revealed that 2 pM of CHIR99021 (indicated by white circle) was optimal for inducing dorsal forebrain marker EMX2. qPCR data normalized to undifferentiated hPSCs. FIG. 16B shows Hl hPSCs were differentiated into definitive ectoderm within 24 hours, and then anterior neural ectoderm within 24 hours, followed by forebrain progenitors within 48 hours, which were then treated with CHIR99021 (2 pM) + PD 173074 (100 nM) for 72 hours, in the presence or absence of FGF2 (20 ng / mL) or FGFR inhibitor (PD173074, 100 nM). This revealed that FGF inhibition enhanced dorsal forebrain markers EMX2 and PAX6, while preventing erroneous WNT-induced expression of midbrain marker EN1. qPCR data normalized to undifferentiated hPSCs. FIG. 16C shows Hl hPSCs were differentiated into definitive ectoderm within 24 hours, and then anterior neural ectoderm within 24 hours, followed by forebrain progenitors within 48 hours, which were then treated with CHIR99021 (2 pM) + PD 173074 (100 nM) for 72 hours, in the presence or absence of BMP4 (10 ng / mL) or BMPR inhibitors (DMH1 [250 nM] or LDN193189 [100 nM]). FIG. 16D shows Hl hPSCs were differentiated into definitive ectoderm within 24 hours, and then anterior neural ectoderm within 24 hours, followed by forebrain progenitors within 48 hours, which were then treated with XAV939 (1 pM) in the presence or absence of increasing doses of HEDGEHOG pathway agonist 21K (0.1-5 nM) for 72 hours to generate ventral forebrain. This showed that high HEDGEHOG pathway activation, together with WNT inhibition, led to maximum expression of ventral forebrain markers. qPCR data normalized to the sample that displayed the highest expression in this experiment. FIG. 16E shows Hl hPSCs were differentiated into definitive ectoderm within 24 hours, and then posterior neural ectoderm within 24 hours, followed by hindbrain progenitors within 48 hours, which were then treated with HEDGEHOG inhibitor (Vismodegib, 150 nM) and WNT agonist (CHIR99021, 2 pM) for 72 hours to generate dorsal forebrain, in the presence or absence of BMP agonist (BMP4,10 ng / rnL) or BMPR inhibitor (LDN193189, 100 nM). This showed that BMP inhibition promoted expression of dorsal forebrain markers GSX2 and PAX3. FIG. 16F shows Hl hPSCs were differentiated into definitive ectoderm within 24 hours, and then posterior neural ectoderm within 24 hours, followed by hindbrain progenitors within 48 hours, which were then treated with HEDGEHOG pathway inhibitor Vismodegib (150 nM) in the presence or absence of increasing doses of WNT pathway agonist CHIR99021 (1-4 pM) for 72 hours to generate dorsal forebrain. This showed that moderate WNT pathway activation, together with HH inhibition, led to maximum expression of dorsal hindbrain markers GSX2 and OLIG3. FIG. 16G shows scRNAseq of H7 hPSC-derived day-4 hindbrain progenitors, and day-7 dorsal hindbrain or ventral hindbrain progenitors. In the legend, cells were colored by the duration and type of differentiation they were subjected to. An asterisk (*) indicates that the legend is identical to that shown in Fig. 4c, and is reproduced here to aid the interpretation of other images shown in this subpanel. FIG. 16H shows Hl hPSCs were differentiated into definitive ectoderm within 24 hours, and then posterior neural ectoderm within 24 hours, followed by hindbrain progenitors within 48 hours, which were then treated with either Vismodegib (150 nM) + CHIR99021 (2 pM) + DMH1 (250 nM) for 72 hours to generate dorsal forebrain, or alternatively, 2 IK (5 nM) + XAV939 (1 pM) for 72 hours to generate ventral forebrain. qPCR data normalized to the sample that displayed the highest expression in this experiment. FIG. 161 shows Hl hPSCs were differentiated into definitive ectoderm within 24 hours, and then posterior neural ectoderm within 24 hours, followed by hindbrain progenitors within 48 hours, which were then treated with WNT pathway inhibitor XAV939 (1 pM) in the presence or absence of increasing doses of HEDGEHOG pathway agonist 21K (50 pM- 5 nM) for 72 hours to generate ventral hindbrain. This showed that high doses of HEDGEHOG pathway activation, together with WNT inhibition, were required to maximally induce ventral hindbrain markers. qPCR data normalized to the sample that displayed the highest expression in this experiment.00073 FIGS. 17A-17H show differentiation of hPSCs into dorsal and ventral forebrain and dorsal and ventral hindbrain neurons within 14 days. FIG. 17A shows scRNAseq of H7 hPSC-derived dorsal forebrain, ventral forebrain, dorsal hindbrain and ventral hindbrain populations on day 14 of differentiation. An asterisk (*) indicates imagesidentical to those shown in FIG. 5D and reproduced here to aid the interpretation of other images shown in this subpanel. FIG. 17B shows scRNAseq of H7 hPSC-derived dorsal forebrain populations on day 14 of differentiation reveals expression of Cajal-Retzius neuron markers. FIG. 17C shows scRNAseq of H7 hPSC-derived ventral forebrain populations on day 14 of differentiation, which reveals expression of cortical interneuron markers. FIG. 17D shows scRNAseq of H7 hPSC-derived ventral forebrain populations on day 14 of differentiation, which reveals expression of hypothalamus-associated markers. hPSC-derived groups I and II express PROXI, LHX5 and ADCYAP 1 , which are expressed in vivo by various regions of the hypothalamus, including the preoptic area, paraventricular nucleus, and supraoptic nucleus. hPSC-derived group I is additionally defined by OTP, the archetypic transcription factor required for the development of paraventricular and supraoptic nuclei. Instead, hPSC-derived group II is additionally defined by POU4F1 (BRN3A) and EBF1, and may correspond to an early Pou4fl+ Ebfl+ Pome- hypothalamic lineage in vivo. hPSC-derived group II also expresses ADCYAP 1RI (the ADYCAP1 receptor), which is expressed by multiple brain regions, including the preoptic area, paraventricular nucleus, and supraoptic nucleus. Finally, hPSC-derived group III is demarcated by ISL1 and POMC, which are expressed within the arcuate nucleus of the hypothalamus. FIG. 17E shows qPCR of Hl hPSC-derived day-7 ventral hindbrain progenitors differentiated towards neurons for 7 additional days. qPCR data normalized to the sample that displayed the highest expression in this experiment. FIG. 17F shows scRNAseq of H7 hPSC-derived ventral hindbrain populations on day 14 of differentiation, which reveals expression of multiple ventral hindbrain neuron subtype markers (left), whose identities were extrapolated from ventral spinal cord neuron subtype markers. Cartoon of progenitor domains within the developing ventral hindbrain, based on embryological studies. FIG. 17G shows scRNAseq of H7 hPSC-derived dorsal hindbrain populations on day 14 of differentiation, which reveals expression of multiple dorsal hindbrain neuron subtype markers (left). Cartoon of progenitor domains within the developing ventral hindbrain, based on embryological studies. FIG. 17H shows quantification of intracellular acetylcholine levels in Hl hPSC-derived ventral hindbrain neurons on day 48 of differentiation.00074 FIGS. 18A-18F show a functional assessment of hPSC-derived dorsal and ventral hindbrain neurons. FIG. 18A shows live Ca2+imaging of WTC AAVSl-CAG-GCaMP6f\^ Cs differentiated into dorsal hindbrain neurons for 30 days, which exhibited spontaneous Ca2+transients as assessed within individual cells (left) and across the culture (right). Scale bar = 50 pm. FIG. 18B shows electrophysiological activity of Hl hPSC-derived dorsal hindbrain neurons on day 30 of differentiation, showing voltage-dependent K+(left) or Na+(right) channel currents elicited by depolarization of the holding potential from -90 mV to 50 mV in voltage-clamp mode. FIG. 18C shows current-clamp characterization of Hl hPSC-derived dorsal hindbrain neurons on day 30 of differentiation, showing action potentials elicited by injection of pulsed (500 pA, 5 ms, 10 Hz, left) or prolonged (50 pA, 1.5 seconds, right) currents in current-clamp mode. FIG. 18D shows a summary of the current-voltage relationship of voltage-dependent K+- currents from dorsal and ventral hindbrain neurons. Currents were measured from -90 mV to +50 mV holding potentials, n (number of cells) = 4. Data are mean ± S.E.M. FIG. 18E shows a summary of the current-voltage relationship of voltage-dependent Na+-currents from dorsal and ventral hindbrain neurons. Currents were measured from -90 mV to +50 mV holding potentials, n (number of cells) = 4. Data are mean ± S.E.M. FIG. 18F shows a Summary of the number of spikes elicited by the prolonged current injection, as shown in panel C, left.00075 FIGS. 19A-19C show genome-wide ATACseq reveals differences in accessible chromatin landscape between aNE and pNE, as well as their differentiation into forebrain vs. hindbrain. FIG. 19A shows Z-score heatmaps of all differentially accessible regions (DARs) with FDR<0.05 between Day 2 hPSC-derived aNE vs. pNE. FIG. 19B shows Z-score heatmaps of all differentially accessible regions (DARs) with FDR<0.05 between Day 4 hPSC-derived aNE — Forebrain vs. pNE — Forebrain. FIG. 19C shows Z-score heatmaps of all differentially accessible regions (DARs) with FDR<0.05 between Day 4 hPSC-derived aNE — Hindbrain vs. pNE — Hindbrain.DETAILED DESCRIPTION OF EMBODIMENTS00076 The disclosure relates to compositions comprising ectodermal cells and differentiated cells arising or derived therefrom. The disclosure also relates to a culture of cells comprising one or a plurality of ectodermal cells or brain progenitor cells. In some embodiments, the brain progenitor cells are forebrain, midbrain or hindbrain cells. The disclosure relates to compositionscomprising cell lines comprising any one or combination of the foregoing cells. The disclosure further relates to methods of differentiating pluripotent stem cells into any two dimensional or three-dimensional cultures comprising ectodermal cells and brain progenitor cells disclosed herein. The disclosure further relates to pharmaceutical compositions comprising the ectodermal progenitor cells, the brain progenitor cells, the forebrain cells, the midbrain cells, the hindbrain cells or any combination thereof; and a pharmaceutically acceptable carrier. In some embodiments, the ectodermal progenitor cells display a phenotype that is deficient in NANOG but SOX2 positive. In some embodiments, the disclosure relates to cell compositions comprising either one or a plurality of cells disclosed herein or a cell line comprising one or a plurality of cells disclosed herein. The disclosure relates to a method of making one or more of the foregoing cells by exposing the one or more cells to tissue culture medium. In some embodiments, the tissue culture medium is any tissue culture medium disclosed in Table 1. In some embodiments, the methods of making or differentiating the cells is from about 12 hours to about 48 hours. In some embodiments, a method of making a hindbrain, midbrain or forebrain cell is accomplished in no more than 10, 9, 8, or 7 days.Definitions00077 Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994), provide one skilled in the art with a general guide to many of the terms used in the present application. Additionally, the practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Such techniques are explained fully in the literature, such as, "Molecular Cloning: A Laboratory Manual", 2nd edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (MJ. Gait, ed., 1984); "Animal Cell Culture" (R.I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology", 4th edition (D.M. Weir & C.C. Blackwell, eds., Blackwell Science Inc., 1987); "Gene Transfer Vectors for Mammalian Cells" (J.M. Miller & M.P. Calos, eds., 1987); "Current Protocols in Molecular Biology" (F.M. Ausubel et al., eds., 1987); and "PCR: The Polymerase Chain Reaction", (Mullis et al., eds., 1994).00078 As used in the present disclosure and claims, the singular forms “a”, “an” and “the” include plural forms unless the context clearly dictates otherwise.00079 It is understood that wherever embodiments are described herein with the language “comprising” otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are also provided. It is also understood that wherever embodiments are described herein with the language “consisting essentially of’ otherwise analogous embodiments described in terms of “consisting of’ are also provided.00080 The term "about" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%„ ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. For recitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6,9, and 7.0 are explicitly contemplated.00081 The term “and / or” as used in a phrase such as “A and / or B” herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).00082 The term “substantially free of’ as used herein refers to a composition that only has trace or negligible amounts of the substance to which it refers. In some embodiments, substantially free means that the composition comprises only about 0.1%, 0.2%, 0.3% 0.4% or 0.5% of the substance to which it refers. In some embodiments, substantially free means that the composition comprises less than about 1.0% of the substance to which it refers relative to the number or mass of substances in the compositions and confers no biological effect to the compositions.00083 The term "culture vessel" as used herein is defined as any vessel suitable for growing, culturing, cultivating, proliferating, propagating, or otherwise similarly manipulating cells. A culture vessel may also be referred to herein as a "culture insert". In some embodiments, the culture vessel is made out of biocompatible plastic and / or glass. In some embodiments, the plastic is a thin layer of plastic comprising one or a plurality of pores that allow diffusion of protein, nucleic acid, nutrients (such as heavy metals and hormones) antibiotics, and other cellculture medium components through the pores. Tn some embodiments, the pores are not more than about 0.1, 0.5 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 microns wide. In some embodiments, the culture vessel comprises a hydrogel matrix and free of a base or any other structure. In some embodiments, the culture vessel is designed to contain a hydrogel or hydrogel matrix and various culture mediums. In some embodiments, the culture vessel consists of or consists essentially of a hydrogel or hydrogel matrix. In some embodiments, the only plastic component of the culture vessel is the components of the culture vessel that make up the side walls and / or bottom of the culture vessel that separate the volume of a well or zone of cellular growth from a point exterior to the culture vessel. In some embodiments, the culture vessel comprises a hydrogel and one or a plurality of isolated ectodermal cells disclosed herein or brain progenitor cells disclosed herein or hindbrain cells disclosed herein. In some embodiments, the culture vessel comprises a hydrogel and one or a plurality of isolated cells disclosed herein, to which one or a plurality of neuronal cells are seeded.00084 The term “exposing” as used herein refers to bringing a disclosed compound and a cell, target receptor, or other biological entity together in direct or indirect contact, in such a manner that the compound can affect the activity of the cell (e.g., receptor, cell, etc ). Directly this can occur by physical contact between the disclosed compound and the cell, receptor or other entity; i.e., by interacting with the target or cell itself, or indirectly this can occur by interacting with another molecule, co-factor, factor, or protein on which the activity of the cell is dependent. In some embodiments, the activity of the cell in response to the compound or molecule is differentiation. In some embodiments, the compound is one or more differentiation factors. In some embodiments, the differentiation factor or factors are a component of the cell culture medium.00085 “Derivatives” of the compounds disclosed herein are pharmaceutically acceptable salts, prodrugs, deuterated forms, radio-actively labeled forms, isomers, solvates and combinations thereof. The “combinations” mentioned in this context are refer to derivatives falling within at least two of the groups: pharmaceutically acceptable salts, prodrugs, deuterated forms, radio- actively labeled forms, isomers, and solvates. Examples of radio-actively labeled forms include compounds labeled with tritium, phosphorous-32, iodine-129, carbon-11, fluorine-18, and the like. The compounds described herein may be present in the form of pharmaceutically acceptable salts. For use in medicines, the salts of the compounds described herein refer to non-toxic“pharmaceutically acceptable salts.” Pharmaceutically acceptable salt forms include pharmaceutically acceptable acidic / anionic or basic / cationic salts. Suitable pharmaceutically acceptable acid addition salts of the compounds described herein include e.g., salts of inorganic acids (such as hydrochloric acid, hydrobromic, phosphoric, nitric, and sulfuric acids) and of organic acids (such as, acetic acid, benzenesulfonic, benzoic, methanesulfonic, and p- toluenesulfonic acids). Examples of pharmaceutically acceptable base addition salts include e.g., sodium, potassium, calcium, ammonium, organic amino, or magnesium salt. As used herein, the term “salt” refers to acid or base salts of the compounds used in the methods of the present disclosure. Illustrative examples of acceptable salts are mineral acid (hydrochloric acid, hydrobromic acid, phosphoric acid, and the like) salts, organic acid (acetic acid, propionic acid, glutamic acid, citric acid and the like) salts, quaternary ammonium (methyl iodide, ethyl iodide, and the like) salts.00086 The term “progenitor cell” as used herein is defined as a cell that is pluripotent cell exposed to cell medium that comprises differentiation factors but remains pluripotent, multipotent, orat least partially undifferentiated. In some embodiments, a progenitor cell comprises SOX2 or variatns thereof. In some embodiments, a progenitor cell is deficient in NANOG or variants thereof.00087 The term “pluripotent stem cell” as used herein is defined as a cell that is self-replicating capable of developing into cells and tissues of the three primary germ layers. Pluripotent stem cells include embryonic and induced pluripotent cells as defined herein. Contemplated pluripotent stem cells originate from mammals, e.g., human, mouse, rat, monkey, horse, goat, sheep, dog, cat etc.00088 The term “induced pluripotent stem cell” (iPSC) means a type of pluripotent cell made by reprogramming a somatic cell to have the same properties as embryonic stem cells, namely, the ability to self-renew and differentiate into the three primary germ layers. In some embodiments, iPSCs include mammalian cells, e.g., human, mouse, rat, monkey, horse, goat, sheep, dog, cat etc., reprogrammed to express SOX2 but are deficient in NANOG. In some embodiments, iPSCs comprise reprogrammed primary cell lines. In some embodiments, iPSCs are obtained from a repository, such as the Coriell Institute for Medical Research (e.g., Catalog ID GM25256 (WTC- 11), GM25430, GM23392, GM23396, GM24666, GM27177, GM24683), California Institute for Regenerative Medicine: California’s Stem Cell Agency (e.g., CW60261, CW60354, CW60359,CW60480, CW60335, CW60280, CW60594, CW60083, CW60086, CW60087, CW60167, CW60186), and the American Type Culture Collection (ATCC®) (e.g., ATCC-DYR0530 Human Induced Pluripotent Stem (IPS) Cells (ATCC® ACS-1012™, ATCC® ACS-1011™, ATCC® Number: ACS-1024™, ATCC® Number: ACS-1028™, ATCC® Number: ACS- 1031™, ATCC® Number: ACS-1004™, ATCC® Number: ACS-1029™, ATCC® Number: ACS-1020™, ATCC® Number: ACS-1007™, ATCC® Number: ACS-1030™ ). Induced pluripotent stem cells may be derived from cell types such as fibroblasts taken from the skin, lung, or vein of subjects that are apparently healthy or diseased.00089 As defined herein, the term “inhibition,” “inhibit,” “inhibiting,” and the like in reference to a protein-inhibitor (e.g., antagonist) interaction means negatively affecting (e.g., decreasing) the activity or function of the protein relative to the activity or function of the protein in the absence of the inhibitor. In embodiments inhibition refers to reduction of expression of a certain protein within a certain pathway. In some embodiments, inhibition refers to a reduction in the activity of a signal transduction pathway or signaling pathway. Thus, inhibition includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signal transduction or enzymatic activity or the amount of a protein in a disclosed culture, system or cell.00090 The term “embryonic stem cell line” as used herein is defined as a cell derived from the inner cell mass of the pre-implantation blastocyst capable of self-renewal and differentiation into the three primary germ layers. In some embodiments, embryonic stem cell lines listed in the NIH Human Embryonic Stem Cell Registry, e.g., CHB-1, CHB-2, CHB-3, CHB-4, CHB-5, CHB-6, CHB-8, CHB-9, CHB-10, CHB-11, CHB-12, RUES1, RUES2, HUES 1, HUES 2, HUES 3, HUES 4, HUES 5, HUES 6, HUES 7, HUES 8, HUES 9, HUES 10, HUES 11, HUES 12, HUES 13, HUES 14, HUES 15, HUES 16, HUES 17, HUES 18, HUES 19, HUES 20, HUES 21, HUES 22, HUES 23, HUES 24, HUES 26, HUES 27, HUES 28, CyT49, RUES3, WA01 (HI), UCSF4, NYUES1, NYUES2, NYUES3, NYUES4, NYUES5, NYUES6, NYUES7, MFS5, HUES 48, HUES 49, HUES 53, HUES 65, HUES 66, UCLA 1, UCLA 2, UCLA 3, WA07 (H7), WA09 (H9), WA13 (H13), WA14 (H14), HUES 62, HUES 63, HUES 64, CT1, CT2, CT3, CT4, MA135, Endeavour-2, WIBR1, WIBR2, HUES 45, Shef 3, Shef 6, WIBR3, WIBR4, WIBR5, WIBR6, BJNheml9, BJNhem20, SA001, SA002, UCLA 4, UCLA 5, UCLA 6, HUES PGD 13, HUES PGD 3, ESI-014, ESL017, HUES PGD 11, HUES PGD 12, WAI 5, WA16, WA17,WAI 8, WAI 9, etc. In some embodiments, embryonic stem cells comprise gene(s) associated with diseases or disorders.00091 The term “rho kinase inhibitor” means a compound that decreases the activity of rho kinase. In some embodiments, the rho kinase inhibitor is N-[(3-Hydroxyphenyl)methyl]-N'-[4- (4-pyridinyl)-2-thiazolyl]urea dihydrochloride (RKI-1447), (+)-(R)-trans-4-(l-aminoethyl)-N-(4- pyridyl)cyclohexanecarboxamide dihydrochloride (Y-27632), Fasudil (HA-1077), Hydroxyfasudil (HA 1100 hydrochloride), Thiazovivin, GSK429286A, Narciclasine, and / or (+)- (R)-trans4-(l-aminoethyl)-N-(lH-pyrrolo[2,3-b]pyridin-4-yl)cyclohexanecarboxamide dihydrochloride (Y-30141).00092 The term "hydrogel" as used herein is defined as any water-insoluble, crosslinked, three- dimensional network of polymer chains with the voids between polymer chains filled with or capable of being filled with water. The term "hydrogel matrix" as used herein is defined as any three-dimensional hydrogel construct, system, device, or similar structure. In some embodiments, the hydrogel or hydrogel matrix comprises one or more proteins and / or glycoproteins. In some embodiments, the hydrogel or hydrogel matrix comprises one or more of the following proteins: collagen, gelatin, elastin, titin, laminin, fibronectin, fibrin, keratin, silk fibroin, and any derivatives or combinations thereof. In some embodiments, the hydrogel or hydrogel matrix comprises Matrigel® or vitronectin. In some embodiments, the hydrogel or hydrogel matrix can be solidified into various shapes, for example, a bifurcating shape designed to mimic a neuronal tract. In some embodiments, the hydrogel or hydrogel matrix comprises poly (ethylene glycol) dimethacrylate (PEG). In some embodiments, the hydrogel or hydrogel matrix comprises Puramatrix. In some embodiments, the hydrogel or hydrogel matrix comprises glycidyl methacrylate-dextran (MeDex). In some embodiments, two or more hydrogels or hydrogel matrixes are used simultaneously cell culture vessel. In some embodiments, two or more hydrogels or hydrogel matrixes are used simultaneously in the same cell culture vessel but the hydrogels are separated by a wall that create independently addressable microenvironments in the tissue culture vessel such as wells. In a multiplexed tissue culture vessel it is possible for some embodiments to include any number of aforementioned wells or independently addressable location within the cell culture vessel such that a hydrogel matrix in one well or location is different or the same as the hydrogel matrix in another well or location of the cell culture vessel.00093 The term “two-dimensional culture” as used herein is defined as cultures of cells that lie flat on hydrogels, including Matrigel® and vitronectin, disposed in culture vessels with only a one to four cell height. In some embodiments, two-dimensional culture is not more than 3 cells high. In some embodiments, two-dimensional culture is not more than 2 cells high. In some embodiments, two-dimensional culture is not more than 1 cell high.00094 As used herein, a “three-dimensional culture” is defined as a culture of cells that take a three dimensional shape while in culture. In some embodiments, the three-dimensional cultures are more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cells in height. In some embodiments, the three- dimensional culture has an organized shape self-assembled by way of simple culturing methods. In some embodiments, the three-dimensional culture comprises one or a plurality of cell types disclosed herein.00095 "Variant" used herein with respect to a nucleic acid means a nucleic acid sequence comprising (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid sequence that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid sequence that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequences substantially identical thereto. "Variant" with respect to a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, conservative substitution of amino acids, or addition of at least one amino acid as compared to a reference sequence, but the prptide or polypeptide retains at least one biological activity of the reference sequence upon which it is based. Variant may also mean a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity. A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157: 105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result inproteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101, incorporated fully herein by reference. Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity, as is understood in the art. Substitutions may be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hyrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties. Nucleic acid molecules or nucleic acid sequences of the disclosure include those that encode amino acid sequences comprising one or more of: SOX2, OTX2, FOXG1, LHX2, SIX3, EN1, EN2, PAX2, PAX3, PAX5, PAX6, PAX7, EMX2, NKX2.1, GLI1, GBX2, H0XA1, HOXB1, MAFB, HNF1B, KROX20, MEIS1, MEIS2, HOXA2, H0XA3, HOXB2, HOXB3, NGN2, OLIG3, GDF7, GSX2, MSX1, MAPT, SNAP25, TBR1, RELN, EBF2, LHX1, LHX5, VGLUT2, DLX1, SP8, LHX6, DLX5, VGAT, NKX2.1, PROXI, ISL1, POMC, TLX3, ASCL1, LBX1, LMX1B, CHAT, VACHT, ACHE, CHT, LHX3, SOX14, SIM1, GAD1, EVX1, PHOX2A, PHOX2B, TFAP2A GATA3 and variants or functional fragments thereof that possess no less than about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the coding sequences of the foregoing.00096 The "percent identity" or "percent homology" of two polynucleotide or two polypeptide sequences is determined by comparing the sequences using the GAP computer program (a part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) using its default parameters. "Identical" or "identity" as used herein in the context of two or more nucleic acids or amino acid sequences, may mean that the sequences have a specified percentage of residues that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions inthe specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) may be considered equivalent. Identity may he performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0. Briefly, the BLAST algorithm, which stands for Basic Local Alignment Search Tool is suitable for determining sequence similarity. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length Win the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension for the word hits in each direction are halted when: 1) the cumulative alignment score falls off by the quantity X from its maximum achieved value; 2) the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or 3) the end of either sequence is reached. The Blast algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The Blast program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 10915- 10919, which is incorporated herein by reference in its entirety) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands. The BLAST algorithm (Karlin et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 5873-5787, which is incorporated herein by reference in its entirety) and Gapped BLAST perform a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide sequences would occur by chance. For example, a nucleic acid is considered similar to another if the smallest sum probability in comparison of the test nucleic acid to the other nucleic acid is less than about 1, less than about 0.1, less than about 0.01, andless than about 0.001 . Two single-stranded polynucleotides are "the complement" of each other if their sequences can be aligned in an anti-parallel orientation such that every nucleotide in one polynucleotide is opposite its complementary nucleotide in the other polynucleotide, without the introduction of gaps, and without unpaired nucleotides at the 5' or the 3' end of either sequence. A polynucleotide is "complementary" to another polynucleotide if the two polynucleotides can hybridize to one another under moderately stringent conditions. Thus, a polynucleotide can be complementary to another polynucleotide without being its complement.00097 The terms "functional fragment" means any portion of a polypeptide or nucleic acid sequence from which the respective full-length polypeptide or nucleic acid relates that is of a sufficient length and has a sufficient structure to confer a biological affect that is at least similar or substantially similar to the full-length polypeptide or nucleic acid upon which the fragment is based. In some embodiments, a functional fragment is a portion of a full-length or wild-type nucleic acid sequence that encodes any one of the nucleic acid sequences disclosed herein, and said portion encodes a polypeptide of a certain length and / or structure that is less than full-length but encodes a domain that still biologically functional as compared to the full-length or wild-type protein. In some embodiments, the functional fragment may have a reduced biological activity, about equivalent biological activity, or an enhanced biological activity as compared to the wildtype or full-length polypeptide sequence upon which the fragment is based.00098 The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a subject, such as a human. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. 00099 The phrase "pharmaceutically acceptable carrier" is art recognized and includes a pharmaceutically acceptable material, composition or vehicle, suitable for administering compounds of the present invention to mammals. The carriers include liquid or solid fdler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin, which is incorporated herein by reference in its entirety.000100 The term "salt" refers to acidic salts formed with inorganic and / or organic acids, as well as basic salts formed with inorganic and / or organic bases. Examples of these acids and bases are well known to those of ordinary skill in the art. Such acid addition salts will normally be pharmaceutically acceptable although salts of non-pharmaceutically acceptable acids may be of utility in the preparation and purification of the compound in question. Acid addition salts of the compounds of the invention are most suitably formed from pharmaceutically acceptable acids, and include for example those formed with inorganic acids e.g. hydrochloric, hydrobromic, sulphuric or phosphoric acids and organic acids e.g. succinic, malaeic, acetic or fumaric acid. Other non-pharmaceutically acceptable salts e.g. oxalates can be used for example in the isolation of the compounds of the invention, for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt. Also included within the scope of the invention are solvates and hydrates of the invention.000101 The conversion of a given compound salt to a desired compound salt is achieved by applying standard techniques, in which an aqueous solution of the given salt is treated with a solution of base e.g. sodium carbonate or potassium hydroxide, to liberate the free base which is then extracted into an appropriate solvent, such as ether. The free base is then separated from the aqueous portion, dried, and treated with the requisite acid to give the desired salt.000102 In vivo hydrolyzable esters or amides of certain compounds of the invention can be formed by treating those compounds having a free hydroxy or amino functionality with the acidchloride of the desired ester in the presence of a base in an inert solvent such as methylene chloride or chloroform. Suitable bases include triethylamine or pyridine. Conversely, compounds of the invention having a free carboxy group can be esterified using standard conditions which can include activation followed by treatment with the desired alcohol in the presence of a suitable base.000103 Examples of pharmaceutically acceptable addition salts include, without limitation, the non-toxic inorganic and organic acid addition salts such as the hydrochloride derived from hydrochloric acid, the hydrobromide derived from hydrobromic acid, the nitrate derived from nitric acid, the perchlorate derived from perchloric acid, the phosphate derived from phosphoric acid, the sulphate derived from sulphuric acid, the formate derived from formic acid, the acetate derived from acetic acid, the aconate derived from aconitic acid, the ascorbate derived from ascorbic acid, the benzene sulphonate derived from benzensulphonic acid, the benzoate derived from benzoic acid, the cinnamate derived from cinnamic acid, the citrate derived from citric acid, the embonate derived from embonic acid, the enantate derived from enanthic acid, the fumarate derived from fumaric acid, the glutamate derived from glutamic acid, the glycolate derived from glycolic acid, the lactate derived from lactic acid, the maleate derived from maleic acid, the mal onate derived from malonic acid, the mandelate derived from mandelic acid, the methanesulphonate derived from methane sulphonic acid, the naphthalene-2-sulphonate derived from naphtalene-2-sulphonic acid, the phthalate derived from phthalic acid, the salicylate derived from salicylic acid, the sorbate derived from sorbic acid, the stearate derived from stearic acid, the succinate derived from succinic acid, the tartrate derived from tartaric acid, the toluene-p- sulphonate derived from p-toluene sulphonic acid, and the like. Particularly preferred salts are sodium, lysine and arginine salts of the compounds of the invention. Such salts can be formed by procedures well known and described in the art.000104 Other acids such as oxalic acid, which cannot be considered pharmaceutically acceptable, can be useful in the preparation of salts useful as intermediates in obtaining a chemical compound of the invention and its pharmaceutically acceptable acid addition salt. Metal salts of a chemical compound of the invention include alkali metal salts, such as the sodium salt of a chemical compound of the invention containing a carboxy group. Mixtures of isomers obtainable according to the invention can be separated in a manner known per se into the individual isomers; diastereoisomers can be separated, for example, by partitioning betweenpolyphasic solvent mixtures, recrystallization and / or chromatographic separation, for example over silica gel or by, e.g., medium pressure liquid chromatography over a reversed phase column, and racemates can be separated, for example, by the formation of salts with optically pure salt-forming reagents and separation of the mixture of diastereoisomers so obtainable, for example by means of fractional crystallization, or by chromatography over optically active column materials.000105 Media. In some implementations, the methods and compositions of the invention utilize or comprise medium, also known as growth medium, cell culture medium, or cell medium. The medium will comprise factors and components necessary to support the growth, proliferation, and maintenance of cells in culture, for example amino acids, sugars, and ions such as calcium, magnesium, potassium, sodium, and phosphate required for cell survival, growth, and maintenance. In a primary implementation, the growth medium is a basal medium. In a primary embodiment, the medium is a defined medium comprising, e g. comprising non-animal derived factors. Exemplary defined basal media known in the art include, for example, CDM1, CDM2, CDM3, DMEM, and RPMI.000106 Other components in the culture medium may be those usually used in animal cell culture media, and examples thereof include amino acids, vitamins, lipid factors, energy sources, osmo-regulators, iron sources, and pH buffers. In addition to these components, for example, a trace metal element, a surfactant, a proliferation cofactor, and a nucleoside may be contained. Such culture medium components, including the peptide composed of specific kinds of amino acids, may be separately added to a cell culture medium. Specifically, for example, a high concentration of peptide and culture medium components may be added to the cell culture at the same time or at different times.000107 Specific examples of the other components in the culture medium embodiments include amino acids, such as L-alanine, L-arginine, L-asparagine, L-asparaginic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L- methionine, L-ornithine, L-phenylalanine, L-proline, L-threonine, L-triptophane, and L-valine, preferably, L-alanine, L-arginine, L-asparagine, L-asparaginic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L- proline, L-threonine, L-triptophane, and L-valine; vitamins, such as i-inositol, biotin, folic acid, lipoic acid, nicotine amide, nicotinic acid, p-aminobenzoic acid, calcium pantothenate, pyridoxalhydrochloride, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin Bl 2, and ascorbic acid, preferably, biotin, folic acid, lipoic acid, nicotine amide, calcium pantothenate, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, and ascorbic acid; lipid factors, such as choline chloride, choline tartrate, linoleic acid, oleic acid, and cholesterol, preferably, choline chloride; energy sources, such as glucose, galactose, mannose, and fructose, preferably, glucose; osmo-regulators, such as sodium chloride, potassium chloride, and potassium nitrate, preferably, sodium chloride; iron sources, such as EDTA iron, iron citrate, ferrous chloride, ferric chloride, ferrous sulfate, ferric sulfate, and ferric nitrate, preferably, ferric chloride, EDTA iron, and iron citrate; and pH buffers, such as sodium hydrogen carbonate, calcium chloride, sodium dihydrogen phosphate, HEPES, and MOPS, preferably, sodium hydrogen carbonate.000108 In addition to these components, the culture medium may further contain, for example, trace amounts of metal elements, such as copper sulfate, manganese sulfate, zinc sulfate, magnesium sulfate, nickel chloride, tin chloride, magnesium chloride, and / or sodium silicite, preferably, copper sulfate, zinc sulfate, and / or magnesium sulfate; a surfactant, such as Tween 80 or Pluronic F68; a proliferation cofactor, such as recombinant insulin, recombinant IGF, recombinant EGF, recombinant FGF, recombinant PDGF, recombinant TGF-a, ethanolamine hydrochloride, sodium selenite, retinoic acid, or putrescine hydrochloride, preferably, sodium selenite, ethanolamine hydrochloride, recombinant IGF, or putrescine hydrochloride; and a nucleoside, such as deoxyadenosine, deoxycytidine, deoxyguanosine, adenosine, cytidine, guanosine, or uridine. In the above-described preferred embodiments of the present invention, the culture medium may contain an antibiotic, such as streptomycin, penicillin G potassium, or gentamicin; and a pH indicator, such as phenol red.000109 The contents of the other components in the culture medium may be appropriately determined depending on the kind of cells to be cultured and the kind of a desired protein, and, preferably, the amino acid is in the range of from about 0.05 to about 1500 mg / L, the vitamin is in the range of 0.001 to 10 mg / L, the lipid factor is in the range of 0 to 200 mg / L, the energy source is in the range of 1 to 20 g / L, the osmo-regulator is in the range of 0.1 to 10000 mg / L, the iron source is in the range of 0.1 to 500 mg / L, the pH buffer is in the range of 1 to 10000 mg / L, the trace metal element is in the range of 0.00001 to 200 mg / L, the surfactant isin the range of 0 to 5000 mg / L, the proliferation cofactor is in the range of 0.05 to 10000 pg / L, and the nucleoside is in the range of 0.001 to 50 mg / L.000110 The pH of the culture medium varies depending on cells to be cultured, but is usually from about 6.8 to to about 7.6, and is from about 7.0 to about 7.4 in many cases. The cell culture medium according to the present invention, preferably, does not contain serum derived from mammals.Cells000111 The disclosure relates to cells and, specifically ectodermal progenitor cells and well as differentiated forebrain, midbrain, and hindbrain cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human. The disclosure also relates to transformed cells lines that are optionally exposed to cell culture medium. In some embodiments, cells of the disclosure comprise or express an amino acid sequence comprising one or a combination of two or more biomarkers disclosed in Table 1.000112 In some embodiments, cells of the disclosure comprise or express an amino acid sequence comprising one or a combination of variants or functional fragments of the biomarkers disclosed in Table 1. In such embodiments, the functional fragment may retain 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity to the wild-type human sequence upon which the sequence is derived. In some embodiments, the functional fragment may retain 85%, 80%, 75%, 70%, 65%, or 60% sequence identity to the wild-type sequence upon which the sequence is derived. In some embodiments, the functional fragment may retain about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, or about 60% sequence identity to the amino acid sequence of Table 1.000113 Compositions of the disclosure include embodiments that comprise cells comprising SOX2 or variants or functional fragments thereof that comprise no less than about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SOX2.000114 In some embodiments cells of the disclosure comprise OTX2 or variants or functional fragments thereof that possess no less than about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity, to OTX2.000115 In some embodiments cells of the disclosure comprise one or a combination of: FOXG1, LHX2, SIX3, EN1, EN2, PAX2, PAX3, PAX5, PAX6, PAX7, EMX2, NKX2.1, GLI1, GBX2, HOXA1, HOXB1, MAFB, HNF1B, KROX20, MEIS1, MEIS2, HOXA2, HOXA3, HOXB2, HOXB3, NGN2, OLIG3, GDF7, GSX2, MSX1, MAPT, SNAP25, TBR1, RELN, EBF2, LHX1, LHX5, VGLUT2, DLX1, SP8, LHX6, DLX5, VGAT, NKX2.1, PROXI, ISL1, POMC, TLX3, ASCL1, LBX1, LMX1B, CHAT, VACHT, ACHE, CHT, LHX3, SOX14, SIM1, GAD1, EVX1, PHOX2A, PHOX2B, TFAP2A GAT A3; or one or a combination of variants or functional fragments of FOXG1, LHX2, SIX3, EN1, EN2, PAX2, PAX3, PAX5, PAX6, PAX7, EMX2, NKX2.1, GLI1, GBX2, HOXA1, HOXB1, MAFB, HNF1B, KROX20, MEIS1, MEIS2, HOXA2, HOXA3, HOXB2, HOXB3, NGN2, OLIG3, GDF7, GSX2, MSX1, MAPT, SNAP25, TBR1, RELN, EBF2, LHX1, LHX5, VGLUT2, DLX1, SP8, LHX6, DLX5, VGAT, NKX2.1, PROXI, ISL1, POMC, TLX3, ASCL1, LBX1, LMX1B, CHAT, VACHT, ACHE, CHT, LHX3, SOX14, SIM1, GAD1, EVX1, PHOX2A, PHOX2B, TFAP2A GATA3 that possess no less than about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity of, respectively of FOXG1, LHX2, SIX3, EN1, EN2, PAX2, PAX3, PAX5, PAX6, PAX7, EMX2, NKX2.1, GLI1, GBX2, HOXA1, HOXB1, MAFB, HNF1B, KROX20, MEIS1, MEIS2, HOXA2, HOXA3, HOXB2, HOXB3, NGN2, OLIG3, GDF7, GSX2, MSX1, MAPT, SNAP25, TBR1, RELN, EBF2, LHX1, LHX5, VGLUT2, DLX1, SP8, LHX6, DLX5, VGAT, NKX2.1, PROXI, ISL1, POMC, TLX3, ASCL1, LBX1, LMX1B, CHAT, VACHT, ACHE, CHT, LHX3, SOX14, SIM1, GAD1, EVX1, PHOX2A, PHOX2B, TFAP2A, GAT A3. In some embodiments cells of the disclosure have a phenotype comprising OTX2 or variants or functional fragments thereof that possess no less than about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity, to OTX2. In some embodiments cells of the disclosure have a phenotype comprising one or a combination of: FOXG1, LHX2, SIX3, EN1, EN2, PAX2, PAX3, PAX5, PAX6, PAX7, EMX2, NKX2.1, GLI1, GBX2, HOXA1, HOXB1, MAFB, HNF1B, KROX20, MEIS1, MEIS2, HOXA2, HOXA3, HOXB2, HOXB3, NGN2, OLIG3, GDF7, GSX2, MSX1, MAPT, SNAP25, TBR1, RELN, EBF2, LHX1, LHX5, VGLUT2, DLX1, SP8, LHX6, DLX5, VGAT, NKX2.1, PROXI, ISL1, POMC, TLX3, ASCL1, LBX1, LMX1B, CHAT, VACHT, ACHE, CHT, LHX3, SOX14, SIM1, GAD1, EVX1, PHOX2A, PHOX2B, TFAP2A GATA3; or one or a combination of variants or functional fragments of FOXG1,LHX2, SIX3, EN1, EN2, PAX2, PAX3, PAX5, PAX6, PAX7, EMX2, NKX2.1, GLI1, GBX2, H0XA1, HOXB1, MAFB, HNF1B, KROX20, MEIS1, MEIS2, HOXA2, HOXA3, HOXB2, HOXB3, NGN2, OLIG3, GDF7, GSX2, MSX1, MAPT, SNAP25, TBR1, RELN, EBF2, LHX1, LHX5, VGLUT2, DLX1, SP8, LHX6, DLX5, VGAT, NKX2.1, PROXI, ISL1, POMC, TLX3, ASCL1, LBX1, LMX1B, CHAT, VACHT, ACHE, CHT, LHX3, SOX14, SIM1, GAD1, EVX1, PHOX2A, PHOX2B, TFAP2A GATA3 that possess no less than about 70%, about 75%, about 80%>, about 85%, about 90%>, about 95%, about 96%, about 97%>, about 98%, or about 99% sequence identity of, respectively of FOXG1, LHX2, SIX3, EN1, EN2, PAX2, PAX3, PAX5, PAX6, PAX7, EMX2, NKX2.1, GLI1, GBX2, HOXA1, HOXB1, MAFB, HNF1B, KROX20, MEIS1, MEIS2, HOXA2, HOXA3, HOXB2, HOXB3, NGN2, OLIG3, GDF7, GSX2, MSX1, MAPT, SNAP25, TBR1, RELN, EBF2, LHX1, LHX5, VGLUT2, DLX1, SP8, LHX6, DLX5, VGAT, NKX2.1, PROXI, ISL1, POMC, TLX3, ASCL1, LBX1, LMX1B, CHAT, VACHT, ACHE, CHT, LHX3, SOX14, SIM1, GAD1, EVX1, PHOX2A, PHOX2B, TFAP2A, GATA3.System000116 The present disclosure also relates to a system comprising: (i) a cell culture vessel optionally comprising a hydrogel; (ii) one or a plurality of cells disclosed herein either in suspension or as a component of an adherent culture; and (iii) one or plurality of differentiation factors or reagents disclosed as a component of cell culture medium of Table 1.000117 In some embodiments, the system further comprises one or combination of culture mediums disclosed herein. The disclosure also relates to a method of culturing ectodermal progenitor cells or brain cells in a system, the system comprising: (i) a cell culture vessel optionally comprising a hydrogel; (ii) one or a plurality of stem cells either in suspension or as a component of a spheroid; and (iii) one or plurality of differentiation factors. In some embodiments, the system further comprises one or combination of culture mediums disclosed herein. In some embodiments, the methods relate to replacing medium during a culture time of form about 0.5 to about 15 days at least one time to (i) expose one or a plurality of stem cells to a first cell medium for a time period sufficient to differentiate the one or plurality of stem cells into ectodermal progenitor cells and the sequentially replacing the medium to (ii) expose one or plurality of ectodermal progenitor cells to a second cell medium for a time period sufficient todifferentiate the one or plurality of ectodermal progenitor cells into neuronal or non-neuronal cells.000118 In some embodiments, the disclosure relates to a system comprising a cell culture unit comprising one or a plurality of cell reactor surfaces housed in at least a first compartment, the one or plurality of cell reactor surfaces in fluid connection with a first and second media line, the first media line in fluid communication with a first media inlet, the second media line in fluid communication to a first media outlet; a gas transfer module in operable connection to the one or plurality of cell reactor surfaces; and a first gas inlet in operable connection to the gas transfer module.000119 In some embodiments, the system comprises one or plurality of cell reactor surfaces that have a surface area from about 0.2 meters squared to about 100 meters squared. In some embodiments, the one or plurality of cell reactor surfaces are configured in a cylindrical form with a hollow volume fixed within a cylindrical first compartment; wherein the first media line and the second media line are positioned on opposite faces of the cylinder. In one embodiment of the above aspects and embodiments, the first media line is attached to a first sealable aperture configured for sterile attachment of a cell culture media source. In some embodiments, the first gas inlet is attached to a second sealable aperture configured for sterile attachment of a gas source. In some embodiments, the system further comprises an apheresis unit in fluid communication with the cell culture unit. In one embodiment of the above aspects and embodiments, the system further comprises a harvesting compartment in fluid communication with the cell culture unit. In some embodiments, the system further comprises a pump and a fluid regulator in operable contact with the first media line, wherein the pump is capable of generating pressure in the first media line and wherein the fluid regulator is capable of regulating the speed of fluid from the pump through the first compartment and into the second media line. In some embodiments, the gas module comprises a gas pump and a gas regulator connected to the first compartment by a first gas line; wherein the first compartment comprises at least one gas outlet; wherein the gas pump is configured to generate air pressure from the pump to the first compartment through the first gas line, wherein the at least one gas outlet is a vent or in configured for sterile connection to a vent; and wherein the gas regulator is configured to regulate the speed of gas from the pump through the first compartment. In some embodiments, the system further comprises one or a plurality of ectodermal cells disclosed herein comprisingone or a combination of biomarkers disclosed herein. In some embodiments, the system further comprises one or a plurality of forebrain, midbrain or hindbrain cells. In some embodiments, the one or plurality of forebrain, midbrain or hindbrain cells comprise one or a combination of two or more of the biomarkers (or have a phenotype comprising the biomarkers) from Table 1. In some embodiments, the forebrain, midbrain or hindbrain cells are derived from the ectodermal progenitor cells disclosed herein.000120 In some embodiments, a system comprising a cell culture unit is utilized to culture and expand a T-cell population described herein. In some embodiments, the cell culture unit comprises one or a plurality of cell reactor surfaces housed in at least a first compartment, the one or plurality of cell reactor surfaces in fluid connection with a first and second media line, the first media line in fluid communication with a first media inlet, the second media line in fluid communication to a first media outlet. In some embodiments, the one or plurality of cell reactor surfaces are configured in a cylindrical form with a hollow volume fixed within a cylindrical first compartment; wherein the first media line and the second media line are positioned on opposite faces of the cylindrical first compartment. The first media line can be attached to a first sealable aperture configured for sterile attachment of a cell culture media source. In some embodiments, the system further comprises a pump and a fluid regulator in operable contact with the first media line, wherein the pump is capable of generating pressure in the first media line and wherein the fluid regulator is capable of regulating the speed of fluid from the pump through the first compartment and into the second media line.000121 The one or plurality of cell reactor surfaces can have a surface area from about 0.5 m2 to about 100.0 m2, including any value therein, such as about 3 m2, about 4 m2, about 5 m2, about 6 m2, about 7 m2, about 8 m2, about 9 m2, about 10 m2, about 11 m2, about 12 m2, about 13 m2, about 14 m2, about 15 m2, about 16 m2, about 17 m2, about 18 m2, about 19 m2, about 20 m2, about 21 m2, about 22 m2, about 23 m2, about 24 m2, about 25 m2, about 30 m2, about 40 m2, about 50 m2, about 60 m2, about 70 m2, about 80 m2, about 90 m2, or about 100 m2 , or about 105 m2.000122 In some embodiments, the system further comprises a gas transfer module in operable connection to the one or plurality of cell reactor surfaces. In some embodiments, the gas module comprises a gas pump and a gas regulator connected to the first compartment by a first gas line. In such embodiments, the first compartment comprises at least one gas outlet. The gas pump isconfigured to generate air pressure from the pump to the first compartment through the first gas line. The gas outlet can be one or more vents or the gas outlet can be configured for sterile connection to one or more vents. The gas regulator is capable of regulating the speed of gas from the pump through the first compartment.000123 Some embodiments further comprise a first gas inlet in operable connection to the gas transfer module. In some embodiments, the first gas inlet is attached to a second sealable aperture configured for sterile attachment of a gas source. The gas source can be any known gas storage and / or delivery system, such as for example a container or a tank.000124 A cell culture system as described herein can be used to expand ectodermal progenitor cells through culturing and / or differentiate one or a plurality of cells in the system and allowing the progenitor cells to grow in the first compartment for a time period sufficient to proliferate and / or to differentiate in response to exposure to cell culture medium disclosed herein. Cells of the disclosure can be introduced into the system through the system’s first compartment. In some embodiments, the cells are hindbrain, midbrain or forebrain cells comprising the biomarkers disclosed in Table 1. In some embodiments, the time period sufficient to proliferate and / or to differentiate in response to exposure of certain cell culture medium is from about 12 hours to about 48 hours.000125 The disclosure also relates to a system comprising a cell culture unit comprising one or a plurality of cell reactor surfaces housed in a plurality of compartments, each compartment separated by a removable partition first compartment comprising at least one cell reactor surface, at least one cell reactor surface in fluid connection with a first and second media line, the first media line in fluid communication with a first media inlet, the second media line in fluid communication to a first media outlet. In some embodiments, the cell culture unit comprises a single cell culture chamber comprising multiple partitions, each partition independently removable and independently in fluid connection with the first and the second media line and each partition or set of partitions defining a distinct compartment. In some embodiments, the cell culture unity comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more compartments, each compartment separated by and / or defined by one or more partitions. In some embodiments, the compartments are configured in a grid or linear pattern. In some embodiments, each partition separating one compartment from another compartment may be removed such that the cell reactor surface of a first compartment is or becomes contiguous with a cell reactor surface of a second compartment.The removal of one or more partitions allows for an increased surface area onto which cells from one compartment (such as the first compartment) may proliferate and / or grow into another compartment (such as the second compartment) during a method of culturing. In some embodiments, the cell culture unit comprises a set of side walls defining a single surface area divided among 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more compartments each compartment with at least one or a plurality of cell reactor surfaces. In some embodiments, each compartment has at least a first cell reactor surface. The disclosure relates to a method of growing disclosed cell populations on a tissue culture system disclosed herein, wherein primary sets of human stem cells are plated at about a concentration of from about 0.001 to about 10 million cells per milliliter into one or more compartments of the cell culture unit and then allowed to grow to a confluent layer on surface area of from about 1 to about 200 squared centimeters. In some embodiments, the method further comprises removing one or more partitions to allow the cells to grow in a second compartment until confluence, when again, optionally, another partition may successively be removed to allow for more surface are for expanded culture. In some embodiments the method of culturing further comprises repeating the step of removing a partition for each of the compartments into which cells should grow. In some embodiments, the cell culture unit comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more partitions each of which corresponding to the physical barrier between a second and third compartment, between a third and fourth compartment, between a fourth and fifth compartment, between a fifth and sixth compartment, between a sixth and seventh compartment, between a seventh and eighth compartment, between an eighth and ninth compartment, between a ninth and tenth compartment, between a tenth and eleventh compartment, and / or between an eleventh and twelfth compartment, respectively.000126 In some embodiments, iPSCs of human or non-human origin are seeded into the cell culture unit and exposed to one or a plurality of cell culture mediums in sequential steps of Table 1.000127 In some embodiments, one or more of the partitions comprise an interior portion, a frame portion and an exterior portion. The interior portion of the partition is positioned in the closed portion of the system; the frame portion spans a wall of the culture system separating the interior of the culture system to the exterior of the system; and the exterior portion is positioned outside of the system. In some embodiments, a seal operably fits around the frame portion of oneor more of the partitions such that removal of the partition does not introduce pathogens to and / or does not expose the environment outside of the tissue culture system to the interior of the tissue culture system.000128 In some embodiments, the cell density of each compartment is from about 0.1 to about 10 million cells per mb of cell culture media. In some embodiments, the cell density of each compartment is from about 0.1 to about 10 million cells per mL of cell culture media. In some embodiments, the cell density of each compartment is from about 0.5 to about 10 million cells per mL of cell culture media. In some embodiments, the cell density of each compartment is from about 1.0 to about 10 million cells per mL of cell culture media. In some embodiments, the cell density of each compartment is from about 2 million to about 10 million cells per mL of cell culture media. In some embodiments, the cell density of each compartment is from about 3 million to about 10 million cells per mL of cell culture media. In some embodiments, the cell density of each compartment is from about 4 million to about 10 million cells per mL of cell culture media. In some embodiments, the cell density of each compartment is from about 5 million to about 10 million cells per mL of cell culture media. In some embodiments, the cell density of each compartment is from about 6 million to about 10 million cells per mL of cell culture media. In some embodiments, the cell density of each compartment is from about 7 million to about 10 million cells per mL of cell culture media. In some embodiments, the cell density of each compartment is from about 8 million to about 10 million cells per mL of cell culture media. In some embodiments, the cell density of each compartment is from about 9 million to about 10 million cells per mL of cell culture media. In some embodiments, the cell density of each compartment is from about 0.1 million to about 20 million cells per mL of cell culture media. In some embodiments, the cell density of each compartment is from about 0.1 million to about 50 million cells per mL of cell culture media.000129 In some embodiments, cells of the disclosure are seeded on a cell reactor surface with from about 20,000 to about 80,000 cells. In some embodiments, methods of the disclosure comprise exposing the seeded cells to cell culture medium disclosed herein in sequential steps for a time period disclosed on Table 1.Compositions000130 In some embodiments, the composition comprises no less than about 1,000, about5,000, about 10,000, about 15,000, about 20,000, about 25,000, about 30,000, about 35,000, about 40,000, about 45,000, about 50,000, or about 60,000 cells. In some embodiments, the composition comprises from about 25,000 to about 100,000 cells. In some embodiments, the composition comprises from about 35,000 to about 100,000 cells. In some embodiments, the composition comprises from about 45,000 to about 100,000 cells. In some embodiments, the composition comprises from about 55,000 to about 100,000 cells. In some embodiments, the composition comprises from about 75,000 to about 100,000 cells.000131 In some embodiments, the compositions of the disclosure comprise a hydrogel. The hydrogel or hydrogel matrixes can have various thicknesses. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 10 pm to about 3000 pm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 150 pm to about 3000 pm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 200 pm to about 3000 pm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 250 pm to about 3000 pm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 300 pm to about 3000 pm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 350 pm to about 3000 pm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 400 pm to about 3000 pm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 450 pm to about 3000 pm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 500 pm to about 3000 pm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 550 pm to about 3000 pm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 600 pm to about 3000 pm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 650 pm to about 3000 pm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 700 pm to about 3000 pm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 750 pm to about 3000 pm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 800 pm to about 3000 pm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 850 pm to about 3000 pm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 900 pm to about 3000 pm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about950 gm to about 3000 gm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 1000 gm to about 3000 gm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 1500 gm to about 3000 gm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 2000 gm to about 3000 gm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 2500 gm to about 3000 gm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 gm to about 2500 gm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 gm to about 2000 gm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 gm to about 1500 gm.000132 In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 gm to about 1000 gm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 gm to about 950 gm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 gm to about 900 gm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 gm to about 850 gm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 gm to about 800 gm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 gm to about 750 gm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 gm to about 700 gm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 gm to about 650 gm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 gm to about 600 gm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 gm to about 550 gm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 gm to about 500 gm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 gm to about 450 gm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 gm to about 400 gm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 gm to about 350 gm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 gm to about 300 gm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 gm to about 250 gm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 gm to about 200 gm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 gm to about 150 gm. In some embodiments, thethickness of the hydrogel or hydrogel matrix is from about 300 gm to about 600 gm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 400 gm to about 500 gm.000133 In some embodiments, the hydrogel or hydrogel matrix comprises one or more synthetic polymers. In some embodiments, the hydrogel or hydrogel matrix comprises one or more of the following synthetic polymers: polyethylene glycol (polyethylene oxide), polyvinyl alcohol, poly- 2-hydroxyethyl methacrylate, polyacrylamide, silicones, and any derivatives or combinations thereof. In some embodiments, the hydrogel or hydrogel matrix comprises one or more synthetic and / or natural polysaccharides. In some embodiments, the hydrogel or hydrogel matrix comprises one or more of the following polysaccharides: hyaluronic acid, heparin sulfate, heparin, dextran, agarose, chitosan, alginate, and any derivatives or combinations thereof.000134 Depending on the particular embodiment, groups of cells may be placed according to any suitable shape, geometry, and / or pattern. For example, independent groups of cells may be deposited as compositions, and the compositions may be arranged within a three dimensional grid, or any other suitable three dimensional pattern. The compositions may all comprise approximately the same number of disclosed cells and be approximately the same size, or alternatively, different compositions may have different numbers of cells and different sizes. In some embodiments, multiple compositions may be arranged in shapes such as an L or T shape, radially from a single point or multiple points, sequential compositions in a single line or parallel lines, tubes, cylinders, toroids, hierarchically branched vessel networks, high aspect ratio objects, thin closed shells, organoids, or other complex shapes which may correspond to geometries of tissues, vessels or other biological structures. In some embodiments, the composition comprises a combination of at least about 2 different cell types disclosed herein. In some embodiments, the composition comprises over about 50% of one type of cell relative to the total number of cells in the composition, over about 60% of one type of cell relative to the total number of cells in the composition, over about 70% of one type of cell relative to the total number of cells in the composition, over about 80% of one type of cell relative to the total number of cells in the composition, over about 90% of one type of cell relative to the total number of cells in the composition, over about 95% of one type of cell relative to the total number of cells in the composition, over about 30% of the cells, over about 40% of the cells relative to the total number of cells in the composition, over about 10% of the cells relative to the total number of cells in thecomposition, over about 20% of the cells relative to the total number of cells in the composition, over about 25% of the cells relative to the total number of cells in the composition, over about 30% of the cells relative to the total number of cells in the composition, over about 35% of the cells relative to the total number of cells in the composition, over about 45% of the cells relative to the total number of cells in the composition, over about 55% of the cells relative to the total number of cells in the composition.000135 In some embodiments, the composition is a pharmaceutical composition, wherein the pharmaceutical composition comprises a desired dosage, which in some aspects includes a desired dose or number of cells and / or a desired ratio of neuronal cell subpopulations. Thus, the dosage of cells, in some embodiments is based on a total number of cells (or number per m2 body surface area or per kg body weight) and a desired ratio of the individual populations or subtypes. In some embodiments, the dosage of cells is based on a desired total number (or number per m2 body surface area or per kg of body weight) of cells in the individual populations or of individual cell types. In some embodiments, the dosage is based on a combination of such features, such as a desired number of total cells, desired ratio, and desired total number of cells in the individual populations.000136 In some embodiments, the composition comprises a desired dose of total cells, such as a desired dose of subtypes of neuronal or non-neuronal cells, e.g., ectodermal cells, forebrain, midbrain, or hindbrain cells. In some aspects, the desired dose is a desired number of cells, a desired number of cells per unit of body surface area or a desired number of cells per unit of body weight of the subject to whom the cells are administered, e.g., cells / m2 or cells / kg. In some aspects, the desired dose is at or above a minimum number of cells or minimum number of cells per unit of body surface area or body weight. In some aspects, among the total cells, administered at the desired dose, the individual populations or sub-types are present at or near a desired output ratio as described herein, e.g., within a certain tolerated difference or error of such a ratio.000137 Thus, in some embodiments, the dosage is based on a desired fixed dose of total cells and a desired ratio, and / or based on a desired fixed dose of two or more, e.g., each, of the individual neuronal subpopulations. Thus, in some embodiments, the dosage is based on a desired fixed or minimum dose of neuronal subpopulations and a desired ratio thereof.000138 Tn certain embodiments, composition is administered to the subject at a range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges.000139 In some embodiments, the dose of total cells and / or dose of individual neuronal subpopulations of cells is within a range of between at or about 104 and at or about 109 cells / meter2 (m2) body surface area, such as between 105 and 106 cells / m2 body surface area, for example, at or about 1 x 105 cells / m2, 1.5x 105 cells / m2, 2x 105 cells / m2, or 1 x 106 cells / m2 body surface area. For example, in some embodiments, the cells are administered at, or within a certain range of error of, between at or about 104 and at or about 109 neuronal cells / meter2 (m2) body surface area, such as between 105 and 106 neuronal cells / m2 body surface area, for example, at or about 1x 105 neuronal / m2, 1.5x105 neuronal / m2, 2x 105 neuronal cells / m2, or 1 x 106 neuronal cells / m2 body surface area.000140 In some embodiments, the cells are administered at or within a certain range of error of between at or about 104 and at or about 109 cells / meter2 (m2) body weight, such as between 105 and 106 cells / m2body weight, for example, at or about 1x105 cells / m2, 1.5 x 105 cells / m2, 2x 105 cells / kg, or 1 x 106 cells / m2body surface area.000141 In some embodiments, the cells are in culture and quiescent for about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40 45, 50, 55, or about 60 days old in culture.000142 The disclosure furthers relates to a pharmaceutical composition comprising a therapeutically effective amount of one or a plurality of any of the enteric neurons disclosedherein and a pharmaceutically acceptable carrier. The pharmaceutical compositions of the disclosure can further include one or more compatible active ingredients which are aimed at providing the composition with another pharmaceutical effect in addition to that provided by the disclosed enteric neurons. “Compatible” as used herein means that the active ingredients of such a composition are capable of being combined with each other in such a manner so that there is no interaction that would substantially reduce the efficacy of each active ingredient or the composition under ordinary use conditions. Such one or more compatible active ingredients may include, but not limited to, any of the chemotherapeutic agents, neuroprotectants and / or neuromodulators, or immunosuppressive agents disclosed elsewhere herein. In some embodiments, the agents are administered in the form of a salt, e.g., a pharmaceutically acceptable salt. “Pharmaceutically acceptable salts” means physiologically and pharmaceutically acceptable salts of compounds, such as oligomeric compounds, i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. Suitable pharmaceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, and sulphuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic acids, for example, p-toluenesulphonic acid. 000143 The choice of carrier in the pharmaceutical composition may be determined in part by the particular method used to administer the composition. Accordingly, there is a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some embodiments, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition.000144 In addition, buffering agents in some aspects are included in the composition. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some embodiments, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, LippincottWilliams & Wilkins 21 st ed. (May 1 , 2005) or Remington’s Pharmaceutical Sciences, 18th or 19th ed. published by the Mack Publishing Company of Easton, Pa., both are incorporated herein by reference.000145 In some embodiments, the pharmaceutical composition comprises the disclosed composition in an amount that is effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Thus, in some embodiments, the methods of administration include administration of the disclosed composition at effective amounts. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.000146 Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions, which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Pharmaceutical compositions may be prepared / formulated, packaged, or sold in formulations suitable for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, intra- lesional, buccal, ophthalmic, intravenous, intra-organ or another route of administration.000147 The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonicsodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations may include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt. For parenteral application, suitable vehicles consist of solutions, e.g., oily or aqueous solutions, as well as suspensions, emulsions, or implants. Aqueous suspensions may contain substances, which increase the viscosity of the suspension and include, for example, sodium carboxymethyl cellulose, sorbitol and / or dextran.Methods000148 The disclosure relates to methods of culturing cells disclosed herein. In some embodiments, the disclosure relates to a method of culturing one or a plurality of progenitor cells comprising: (a) exposing the one or plurality of cells to a basal medium, for example, CDM2 media from about 12 hours to about 36 hours. In some embodiments, the method comprises a step of seeding from about 30,000 cells to about 100,000 cells prior to performing step (a). In some embodiments, the disclosure relates to a method of culturing one or a plurality of ectodermal progenitor cells comprising: (a) exposing the one or plurality of cells to a basal medium, for example, CDM2 media for a time period sufficient to differentiate the one or plurality of cells into ectodermal progenitor cells. In some embodiments, the ectodermal progenitor cells are express SOX2 protein or a variant thereof and are deficient in NANOG. In some embodiments, the time period sufficient to differentiate the one or plurality of cells into ectodermal progenitor cells is about 24 hours. In some embodiments, the method further comprises (b) exposing the cell or plurality of cells to FGF / ERK pathway inhibitor. In some embodiments, the cells are stem cells. In some embodiments, the cells are mammalian. In some embodiments, the cells are human. In some embodiments, the cells are human stem cells. In some embodiments, the cells are human pluripotent stem cells. In some embodiments, the disclosure relates to a method of differentiating one or a plurality of human stem cells into a ectodermal progenitor cell comprising: (a) exposing the one or plurality of cells to a basal medium, for example, CDM2 media from about 12 hours to about 36 hours. In some embodiments, the method comprises a step of seeding from about 30,000 cells to about 100,000cells prior to performing step (a). Tn some embodiments, the disclosure relates to a method of culturing one or a plurality of ectodermal progenitor cells comprising: (a) exposing the one or plurality of cells to a basal medium, for example, CDM2 media.000149 The disclosure relates to methods of culturing ectodermal progenitor cells disclosed herein. In some embodiments, the disclosure relates to a method of culturing one or a plurality of ectodermal progenitor cells comprising: (a) exposing the one or plurality of cells to neurobasal media from about 12 hours to about 36 hours. In some embodiments, the method comprises a step of seeding the cells at a density of from about 30,000 cells / cm2 to about 100,000 cells / cm2 prior to performing step (a). In some embodiments, the disclosure relates to a method of culturing one or a plurality of ectodermal progenitor cells comprising: (a) exposing the one or plurality of cells to neurobasal media for a time period sufficient to differentiate the one or plurality of cells into ectodermal progenitor cells. In some embodiments, the ectodermal progenitor cells are express SOX2 protein or a variant thereof and OTX2 protein or variant thereof. In some embodiments, the time period sufficient to differentiate the one or plurality of cells into ectodermal progenitor cells is about 24 hours. In some embodiments, the time period sufficient to differentiate the one or plurality of cells into an anterior neural ectodermal progenitor. In some embodiments, the method further comprises exposing the ectodermal progenitor cells to one or a combination of Rho Kinase Inhibitors for a time period sufficient to induce differentiation of the cells into anterior neural ectodermal cells. In some embodiments, the culture media comprises a TGFP inhibitor, BMP inhibitor and WNT inhibitor. The disclosure relates to methods of differentiating stem cells disclosed herein into one or a plurality anterior neural ectoderm cells. In some embodiments, the plurality of anterior neural ectoderm cells are about 36 hours in culture. In some embodiments, the method of differentiating comprises exposing the cells to a basal medium, for example, CDM2 media followed by neurobasal media for a total duration of from about 24 hours to about 48 hours.000150 The disclosure relates to a method of culturing an anterior ectodermal cell or cells disclosed herein. In some embodiments, method of culturing comprises (a) exposing the one or plurality of anterior ectodermal cells to a basal medium, for example, CDM2 media from about 24 hours to about 48 hours. In some embodiments, the method comprises culturing the anterior ectodermal cells disclosed herein are culture for a time sufficient to differentiate the cell or cells into forebrain progenitor cells. In some embodiments, the forebrain progenitor cells compriseS0X2, 0TX2, F0XG1, LHX2, SIX3, PAX6 or any functional variants of the foregoing. In some embodiments, the methods of the disclosure comprise a method of differentiating a stem cell into a forebrain progenitor cell comprising SOX2, 0TX2, F0XG1, LHX2, SIX3, PAX6 or any functional variant thereof. In some embodiments, the methods of culturing and differentiating the cell or cells are exposed to a basal medium, for example, CDM2 media for a first time period and second sequential time period, wherein the first time period is from about 18 to about 30 hours; and wherein the second time period is from about 18 hours to about 30 hours. In some embodiments, the first time period comprises exposing the cell or cells to a basal medium, for example, CDM2 media comprising a WNT inhibitor and / or a BMP inhibitor.000151 In some embodiments, the method further comprises exposing the cell or cells to an FGF activator for about 18 hours, 19 hours, 20, hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hour or 28 hours of the first time period or the second time period, but not both time periods. In some embodiments, the method comprises differentiating a stem cell into a forebrain progenitor cell comprising (a) exposing the cell or cell to a basal medium, for example, CDM2 media for from about 36 to about 52 hours, wherein the basal medium, for example, CDM2 culture media comprises a WNT inhibitor and / or a BMP inhibitor for from about 36 hours to about 52 hours; and (b) exposing the cell or cells to an FGF pathway activator (optionally, an FGF agonist) for from about 18 to about 30 hours of the total 30 to 52 hours. In some embodiments, step (a) is performed for about 48 hours and step (b) is performed for about 24 hours of the 48 hour period of step (a). In some embodiments, prior to steps (a) and (b), the method further comprises differentiating the stem cell into an ectodermal progenitor cell and differentiating the ectodermal progenitor cell into an anterior neural progenitor cell.000152 In some embodiments, the disclosure also relates to a method of culturing comprises (a) exposing the one or plurality of anterior ectodermal cells to a basal medium, for example, CDM2 media from about 24 hours to about 48 hours. In some embodiments, the method comprises culturing the anterior ectodermal cells disclosed herein are culture for a time sufficient to differentiate the cell or cells into midbrain progenitor cells. In some embodiments, the forebrain progenitor cells comprise one or a combination of: SOX2, OTX2, EN1, EN2, PAX2, PAX5 or any functional variants of the foregoing. In some embodiments, the methods of the disclosure comprise a method of differentiating a stem cell into a forebrain progenitor cell comprising one or a combination of: SOX2, OTX2, EN1, EN2, PAX2, PAX5 or any functional variant thereof.In some embodiments, the methods of culturing and differentiating the cell or cells are exposed to a basal media, for example, CDM2 media for a first time period and second sequential time period, wherein the first time period is from about 18 to about 30 hours; and wherein the second time period is from about 18 hours to about 30 hours. In some embodiments, the first time period comprises exposing the cell or cells to a basal medium, for example, CDM2 media comprising a WNT pathway activator. In some embodiments, the method further comprises exposing the cell or cells to an FGF activator for about 18 hours, 19 hours, 20, hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hour or 28 hours of the first time period or the second time period, but not both time periods. In some embodiments, the method comprises differentiating a stem cell into a midbrain progenitor cell comprising (a) exposing the cell or cell to a basal medium, for example, CDM2 media for from about 36 to about 52 hours, wherein the basal medium, for example, CDM2 culture media comprises a WNT pathway activator for from about 36 hours to about 52 hours; and (b) exposing the cell or cells to an FGF pathway activator (optionally, an FGF agonist) for from about 18 to about 30 hours of the total 30 to 52 hours. In some embodiments, step (a) is performed for about 48 hours and step (b) is performed for about 24 hours of the 48 hour period of step (a). In some embodiments, prior to steps (a) and (b), the method further comprises differentiating the stem cell into an ectodermal progenitor cell and differentiating the ectodermal progenitor cell into an anterior neural progenitor cell. 000153 Some embodiments of the disclosure include methods of culturing an ectodermal progenitor cell comprising SOX2 or a variant thereof and deficient in NANOG or a variant thereof for a time period sufficient to differentiate the cell into a posterior ectodermal progenitor cell. The disclosure relates to methods of culturing posterior ectodermal cells disclosed herein. In some embodiments, the disclosure relates to a method of culturing one or a plurality of ectodermal progenitor cells comprises: (a) exposing the one or plurality of cells to a basal medium, for example, CDM2 media from about 12 hours to about 36 hours. In some embodiments, the method comprises a step of seeding the cells at a density of from about 30,000 cells / cm2 to about 100,000 cells / cm2 prior to performing step (a). In some embodiments, the disclosure relates to a method of culturing one or a plurality of ectodermal progenitor cells comprising: (a) exposing the one or plurality of cells to a basal medium, for example, CDM2 media for a time period sufficient to differentiate the one or plurality of cells into posterior ectodermal progenitor cells. In some embodiments, the posterior ectodermal progenitor cells areexpress one or a combination of SOX2 or a functional variant thereof; GBX2 or a functional variant thereof; H0XA1 or a functional variant thereof; and H0XB1 or a functional variant thereof. In some embodiments, the time period sufficient to differentiate the one or plurality of cells into ectodermal progenitor cells is about 24 hours. In some embodiments, the time period sufficient to differentiate the one or plurality of cells into a posterior neural ectodermal progenitor is from about 12 to about 24 hours. In some embodiments, the basal medium, for example, CDM2 media comprises a TGFp pathway inhibitor. In some embodiments, the basal medium, for example, CDM2 media comprises a BMP pathway inhibitor. In some embodiments, the basal medium, for example, CDM2 media comprises an Retinoic acid pathway agonist. In some embodiments, the basal medium, for example, CDM2 media comprises an FGF pathway activator. In some embodiments, the basal medium, for example, CDM2 media comprises a WNT pathway inhibitor. In some embodiments, the basal medium, for example, CDM2 media comprises one or a combination of Rho Kinase Inhibitors. In some embodiments, the time period sufficient to induce differentiation of the cells into posterior neural ectodermal cells. In some embodiments, the culture media comprises: a TGFp inhibitor, BMP inhibitor, a retinoic acid agonist, a WNT inhibitor and an FGF pathway activator. In some embodiments, the culture media comprises: a TGFP inhibitor, BMP inhibitor, a retinoic acid agonist, a WNT inhibitor and an FGF pathway activator., and a Rho Kinase inhibitor. The disclosure relates to methods of differentiating stem cells disclosed herein into one or a plurality posterior neural ectoderm cells. In some embodiments, the plurality of posterior neural ectoderm cells are about 24 hours in culture.000154 The disclosure relates to a method of culturing a hindbrain progenitor cell. In some embodiments, the hindbrain progenitor cells comprise one or a combination of: SOX2, GBX2, MAFB, HNF1B, KROX20, MEIS1, MEIS2, H0XA1, H0XA2, H0XA3, HOXB1, HOXB2, HOXB3, or a functional variant of any of the foregoing. In some embodiments, the methods of the disclosure include a method of differentiating a posterior ectodermal progenitor cell into a hindbrain progenitor cell. In some embodiments of culturing or differentiating, the method comprises (a) exposing one or a plurality of a posterior ectodermal progenitor cell to a basal media, for example, CDM2 media comprising a WNT pathway activator. In some embodiments the basal medium, for example, CDM2 media further comprises an FGF pathway activator. In some embodiments, the methods of culturing or differentiating the one or plurality posteriorectodermal progenitor cells comprises exposing the one or plurality of cells to a basal medium, for example, CDM2 media to the cells for from about 24 to about 48 hours. The disclosure relates to a method of culturing a stem cell or differentiating a stem cell into a hindbrain progenitor cell, wherein the hindbrain progenitor cells comprise one or a combination of: SOX2, GBX2, MAFB, HNF1B, KROX20, MEIS1, MEIS2, H0XA1, H0XA2, H0XA3, H0XB1, H0XB2, H0XB3, or a functional variant of any of the foregoing.000155 The disclosure relates to a method of culturing a ventral or a dorsal hindbrain progenitor cell. In some embodiments, the hindbrain cells comprise: one or a combination of: SOX2, HNF1B, PAX6, PAX3, PAX7, NGN2, OLIG3, GDF7, GSX2, MSX1, or a functional variant of any of the foregoing, if the hindbrain cell is a dorsal hindbrain progenitor cell; or one or a combination of: SOX2, HNF1B, NKX2.2, NKX6.1, NKX6.2, OLIG2, PHOX2B, or a functional variant of any of the foregoing, if the hindbrain cell is a ventral hindbrain progenitor cell.000156 In some embodiments, the methods of the disclosure include a method of differentiating a hindbrain progenitor cell into a dorsal hindbrain progenitor cell or a ventral hindbrain progenitor cell. In some embodiments of culturing or differentiating one or a plurality of cells into a dorsal hindbrain progenitor cell, the method comprises (a) exposing one or a plurality of a hindbrain progenitor cells to a basal medium, for example, CDM2 media comprising a WNT pathway activator, a Hedgehog inhibitor and a BMP inhibitor. In some embodiments, the step of exposing is from about 2 to about 3 days.000157 In some embodiments, the methods of the disclosure include a method of culturing or differentiating a hindbrain progenitor cell into a ventral hindbrain progenitor cell, the method comprising (a) exposing one or a plurality of a hindbrain progenitor cells to a basal media, for example, CDM2 media comprising a WNT inhibitor and a Hedgehog activator. In some embodiments, the step of exposing is from about 2 to about 3 days.000158 In some embodiments, the disclosure relates to methods of culturing or differentiating a stem cell into a dorsal hindbrain progenitor cell or ventral hindbrain progenitor cell by culturing the stem cell for about 6 to about 7 days. In such embodiments, the stem cell is differentiated into an ectodermal progenitor cell; the ectodermal progenitor cell is differentiated into a hindbrain progenitor cell; and then subsequently the hindbrain progenitor cell is differentiated into a dorsal or ventral hindbrain progenitor cell. In some embodiments, the stem cell is a human pluripotent stem cell.000159 Further embodiments relate to a method of differentiating or culturing dorsal hindbrain progenitor cells or ventral hindbrain progenitor cells into dorsal hindbrain neural cells or ventral hindbrain neural cells, respectively. The aforementioned method comprises (a) exposing the dorsal hindbrain progenitor cells to a neuronal differentiation media for from about 4 days to about 7 days, wherein the neural differentiation media comprises one or more neurotrophic growth factors, such as GDNF and / or BDNF or functional variants thereof. In some embodiments, the neural differentiation media further comprises one or a combination of: an adenylate cyclase agonist, ascorbic acid or a derivative thereof, and a NOTCH pathway inhibitor or a functional variant thereof. In some embodiments, the step (a) is performed for at least about 5, 6, or about 7 days, wherein neural differentiation media slowly transitioned over about 5, 6, or about 7 days from basal media, for example, CDM2 media. The change of media differentiates the dorsal hindbrain progenitor to dorsal hindbrain neuronal cells. The aforementioned method in respect to ventral hindbrain neural cells comprises (a) exposing the ventral hindbrain progenitor cells to a neuronal differentiation media for from about 4 days to about 7 days, wherein the neural differentiation media comprises one or more neurotrophic growth factors, such as GDNF and / or BDNF or functional variants thereof. In some embodiments, the neural differentiation media further comprises one or a combination of: an adenylate cyclase agonist, ascorbic acid or a derivative thereof, and a NOTCH pathway inhibitor or a functional variant thereof. In some embodiments, the step (a) is performed for at least about 5, 6, or about 7 days, wherein neural differentiation media slowly transitioned over about 5, 6, or about 7 days from a basal media, for example, CDM2 media. The change of media differentiates the ventral hindbrain progenitor to ventral hindbrain neuronal cells.000160 The disclosure relates to a method of culturing a stem cell or differentiating a stem cell into a dorsal or ventral hindbrain neuronal cell, wherein the ventral hindbrain neuronal cells comprise one or a combination of: MAPT, SNAP25, ISL1, CHAT, VACHT, ACHE, CHT, LHX3, SOX14, SIM1, EN1, GAD1, EVX1, PHOX2A, PHOX2B, H0XA1, H0XA3, or a functional variant of any of the foregoing; or wherein the dorsal hindbrain neuronal cells comprise one or a combination of: MAPT, SNAP25, TLX3, ASCL1, LBX1, LMX1B, LHX1, LHX5, PAX2, or a functional variant of the foregoing. In some embodiments, the method of culturing or differentiating is from about 12 to about 14 days.000161 Further embodiments relate to a method of culturing or a method of differentiating forebrain progenitor cells into dorsal and ventral forebrain progenitor cells. Some embodiments of the disclosure relate to forebrain progenitor cells comprising one or a combination of: SOX2, OTX2, FOXG1, LHX2, SIX3, PAX6, or functional variants of any of the foregoing; and differentiating the forebrain progenitor cells to dorsal forebrain progenitor cells, wherein the dorsal forebrain progenitor cells comprise one or a combination of: SOX2, LHX2, EMX2, PAX6, or variants of any of the foregoing. Some embodiments of the disclosure relate to forebrain progenitor cells comprising one or a combination of: SOX2, OTX2, FOXG1, LHX2, SIX3, PAX6, or functional variants of any of the foregoing; and differentiating the forebrain progenitor cells to ventral forebrain progenitor cells, wherein the ventral forebrain progenitor cells comprise one or a combination of: SOX2, LHX2, NKX2.1, GLI1, FOXG1, or variants of any of the foregoing. The methods of differentiating or culturing comprise a step of exposing the forebrain progenitor cells to a cell culture medium comprising one or a combination of: a Hedgehog inhibitor, a FGF pathway inhibitor, and a WNT activator if the method is for preparation or differentiation of dorsal forebrain progenitor cells. The methods of differentiating or culturing comprise a step of exposing the forebrain progenitor cells to a cell culture medium comprising one or a combination of: a Hedgehog activator, a FGF pathway inhibitor, and a WNT inhibitor if the method is for preparation or differentiation of ventral forebrain progenitor cells. The aforementioned steps of exposing can be performed for from about 2 to about 3 days.000162 Any of the resultant ventral or dorsal forebrain progenitor cells can be further differentiated into ventral or dorsal forebrain neural cells by exposing such progenitor cells to neural differentiation medium. Similar to hindbrain neural cell production, some embodiments of the disclosure relate to methods of manufacturing forebrain neural cells, of either dorsal or ventral phenotypes, by exposing the ventral or dorsal forebrain progenitor cells to the neural differentiation medium for from about 5 to about 7 days, wherein the neural differentiation medium is slowly and progressively added to a basal medium, for example, CDM2 medium. In some embodiments, the step of exposing results in production of ventral forebrain neural cells comprising one or a combination of: MAPT, SNAP25, TBR1, RELN, EBF2, LHX1, LHX5, VGLUT2, or functional variants thereof. In some embodiments, the step of exposing results in production of ventral forebrain neural cells comprising one or a combination of: MAPT,SNAP25, DLX1 , SP8, LHX1 , LHX5, LHX6, DLX5, VGAT, NKX2.1, PROXI, ISL1 , POMC, or functional variants thereof.000163 The disclosure also relates to a method of making, culturing or differentiating stem cells or ectodermal progenitor cells into surface ectoderm or border ectodermal cells. The method comprise the steps identified in Table 1. In some embodiment one or a plurality of stem cells are exposed to successive rounds of culturing whereby cell culture medium of Table 1 is exposed to the cells to arrive at non-neural progenitor cells.000164 Some embodiments of a basal media, for example, CDM2 media include the following: 000165 50% IMDM + GlutaMAX (Thermo Fisher, 31980-097) + 50% F12 + GlutaMAX (Thermo Fisher, 31765-092) + 1 mg / mL polyvinyl alcohol (Sigma, P8136-250G) + 1% v / v chemically defined lipid concentrate (Thermo Fisher, 11905-031) + 450 pM 1 -thioglycerol (Sigma, M6145-100ML) + 0.7 pg / mL recombinant human insulin (Sigma, 11376497001) + 15 pg / mL human transferrin (Sigma, 10652202001) + 1% v / v penicillin / streptomycin (Thermo Fisher, 15070-063). Ang et al., 2022; Loh et al., 2014; Loh et al., 2016):000166 Some embodiments of neurobasal medium comprise: Neurobasal Neurobasal media (Thermo Fisher) supplemented with 1% v / v N2 (Thermo Fisher), 2% v / v B27 w / o Vitamin A (Thermo Fisher), and 1% v / v penicillin / streptomycin (Thermo Fisher).

[0058] FGF / ERK pathway inhibitors000167 Cell culture media of the disclosure may comprise a FGF / ERK pathway inhibitor. In some embodiments, the FGF / ERK pathway inhibitor is chosen from one or a combination of: 000168 Trametinib, PD173074, AZD4547, BGJ398 (FGF receptor inhibitor),000169 PD0325901 (ERK inhibitor), or a derivative thereof. In some embodiments, the FGF pathway inhibitor or ERK pathway inhibitor is exposed to one or a plurality of cells at a concentration of from about 0.001 pM to about 1000 pM. In some embodiments, the concentration of the FGF pathway inhibitor is about 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, or about 400 nM in media.000170000171 BMP pathway inhibitors000172 Cell culture media of the disclosure may comprise a BMP pathway inhibitor. In some embodiments the BMP pathway inhibitor is chosen from one or a combination of:000173 LDN193189 or a derivative thereof, DMH1 or derivative thereof, Dorsomorphin / Compound C or derivative thereof, NOGGIN or a functional variant thereof, CHORDIN or a functional variant thereof. In some embodiments, the BMP pathway inhibitor is exposed to one or a plurality of cells at a concentration of from about 0.001 pM to about 1000 pM. In some embodiments, the concentration of the FGF pathway inhibitor is about 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, or about 400 nM in media.000174000175 WNT pathway inhibitor000176 Cell culture media of the disclosure may comprise a BMP pathway inhibitor. In some embodiments the WNT pathway inhibitor is chosen from one or a combination of:000177 C59, XAV939, IWR1, LGK974 or derivatives thereof, or DKK1 or a functional variant thereof. In some embodiments, the WNT pathway inhibitor is exposed to one or a plurality of cells at a concentration of from about 0.001 pM to about 1000 pM. In some embodiments, the concentration of the WNT pathway inhibitor is about 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, or about 400 nM in a volume of media.000178000179 TGFp pathway inhibitor000180 Cell culture media of the disclosure may comprise a TGFp pathway inhibitor. In some embodiments the TGFp pathway inhibitor is chosen from one or a combination of:000181 A8301, SB-431542, LY 364947, SB-505124, RepSox or derivatives thereof; or Follistatin, Leftyl, or Lefty2 or a functional variant thereof. In some embodiments, the TGFp pathway inhibitor is exposed to one or a plurality of cells at a concentration of from about 0.001 pM to about 1000 pM. In some embodiments, the concentration of the TGFp pathway inhibitor is about 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 500 nM, 600 nM, 700 nM 800 nM, 900 nM, or about 1 pM in a volume of media.000182000183 RHO Kinase inhibitor000184 Cell culture media of the disclosure may comprise a RHO Kinase inhibitor. In some embodiments the RHO Kinase inhibitor is chosen from one or a combination of:000185 Thiazovivin, Y27632, TC-S 7001, Chroman 1 or a derivative thereof. In some embodiments, the RHO Kinase inhibitor is exposed to one or a plurality of cells at aconcentration of from about 0.001 pM to about 1000 pM. In some embodiments, the concentration of the RHO Kinase inhibitor is about 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, or about 1 pM in a volume of media.000186000187 WNT pathway activator000188 Cell culture media of the disclosure may comprise a WNT pathway activator. In some embodiments, the WNT pathway activator is chosen from one or a combination of: CHIR99021 or functional variant thereof, WNT3A or functional variant thereof; RSPO1 or functional variant thereof, RSPO2 or functional variant thereof, RSPO3 or functional variant thereof, RSPO4 or functional variant thereof, lithium chloride, BIO or derivative thereof, BlO-acetoxime or derivative thereof. In some embodiments, the WNT pathway activator is exposed to one or a plurality of cells at a concentration of from about 0.001 pM to about 1000 pM. In some embodiments, the concentration of the WNT pathway activator is about 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 pM, 2 pM or 3 pM in a volume of media.000189000190 FGF pathway activator000191 Cell culture media of the disclosure may comprise a FGF pathway activator. In some embodiments, the FGF pathway activator is chosen from one or a combination of: FGF1 or a functional variant thereof; FGF2 or a functional variant thereof; FGF8 or a functional variant thereof; FGF10 or a functional variant thereof. In some embodiments, the FGF pathway activator is exposed to one or a plurality of cells at a concentration of from about 0.001 ng / mL of medium to about 1000 ng / mL of medium. In some embodiments, the concentration of the FGF pathway activator is about 1 ng / mL of medium, 5 ng / mL of medium, 10 ng / mL of medium, 20 ng / mL of medium, 30 ng / mL of medium, 40 ng / mL of medium, 50 ng / mL of medium, 60 ng / mL of medium, 70 ng / mL of medium, 80 ng / mL of medium, 90 ng / mL of medium, or about 100 ng / mL of medium in a volume of media.000192000193 Hedgehog inhibitor000194 Cell culture media of the disclosure may comprise a Hedgehog inhibitor. In some embodiments, the Hedgehog inhibitor is chosen from one or a combination of: Vismodegib or a functional derivative thereof; or XL-139 or a functional derivative thereof. In some embodiments, the Hedgehog inhibitor is exposed to one or a plurality of cells at a concentration of from about 0.001 pM to about 1000 pM. In some embodiments, the concentration of the WNT pathway activator is about 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 pM, 2 pM or 3 pM in a volume of media.000195000196 Hedgehog activator000197 Cell culture media of the disclosure may comprise a Hedgehog inhibitor. In some embodiments, the Hedgehog activator is chosen from one or a combination of: SHH or a functional variant thereof; or SAG 21Kor a functional derivative thereof. In some embodiments, the Hedgehog inhibitor is exposed to one or a plurality of cells at a concentration of from about 0.001 pM to about 1000 pM. In some embodiments, the concentration of the WNT pathway activator is about 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 pM, 2 pM or 3 pM in a volume of media.000198000199 In any of the methods or systems disclosed herein, differentiation factors may be used may be disclosed in Table 1 with at least about 70% sequence identity to the above sequences. Alternatively, and in some embodiments, the differentiation factors identified in Table 1 or their derivatives are used in the methods of culturing or the methods of differentiating a cell. In any of the methods or systems disclosed herein, the differentiation factors used may be functional fragments or variants of the polypeptides disclosed above with at least about 80% sequence identity to the above sequences. In any of the methods or systems disclosed herein, the differentiation factors used may be functional fragments or variants of the polypeptides disclosed above with at least about 85% sequence identity to the above sequences. In any of the methods or systems disclosed herein, the differentiation factors used may be functional fragments or variants of the polypeptides disclosed above with at least about 90% sequence identity to the above sequences. In any of the methods or systems disclosed herein, the differentiation factors used may be functional fragments or variants of the polypeptides disclosed above with at least about 95% sequence identity to the above sequences. In any of the methods or systems disclosedherein, the differentiation factors used may be functional derivatives of the small molecules disclosed above. The methods of the disclosure relate to the sequential exposure of a culture of cells to two or more different tissue culture mediums. In some embodiments, the methods relate to the sequential exposure of cells of the present disclosure or the tissue culture medium described herein.000200 Methods for characterizing cell subtypes are well known in the art, for example flow cytometry, which is described in Pockley et ah, Curr Protoc Toxicol. 2015 Nov 2; 66: 18.8.1-34, which is incorporated herein by reference.000201 The time period sufficient for the disclosed cells to proliferate and / or differentiate varies depending on, e.g., the features of the system and the type of cell population.000202 In some embodiments, the cells can be co-cultured with one or a plurality of brain cell subtypes or agents. In such embodiments, the one or plurality of cells can present with a neuromodulator agent or suspected neuromodulatory agent for a period of time sufficient to stimulate a response from the cell. The agents can be disclosed or have unknown function. If the agent is bested tested for neurotoxicity, the agents may be exposed to the cells for a time period sufficient to test the neurotoxicity of the agents. In some embodiments, agents are exposed to the cells for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more hours at a dosage of from about 0.1 mg / mL of cell culture medium to about 100 mg / mL of cell culture medium.000203 The present disclosure provides devices, methods, and systems involving production, maintenance, and physiological interrogation of neural cells in microengineered configurations designed to mimic native nerve tissue anatomy. It is another object of the disclosure to provide a medium to high-throughput assay of neurological function for the screening of pharmacological and / or toxicological properties of chemical and biological agents. In some embodiments, the agents are cells, such as any type of cell disclosed herein, or antibodies, such as antibodies that are used to treat clinical disease. In some embodiments, the agents are any drugs or agents that are used to treat human disease such that toxi cities, effects or neuromodulation can be compared among a new agent which is a proposed mammalian treatment and existing treatments from human disease. In some embodiments, new agents for treatment of human disease are treatments for neurodegenerative disease and are compared to existing treatments for neurodegenerative disease.000204 Similarly, information gathered from imaging can determine quantitative metrics for the degree of cell toxicology and lends further insight into toxic and neuroprotective mechanisms of various agents or compounds of interest. In some embodiments, the at least one agent comprises a small chemical compound. In some embodiments, the at least one agent comprises at least one environmental or industrial pollutant. In some embodiments, the at least one agent comprises one or a combination of small chemical compounds chosen from: chemotherapeutics, analgesics, cardiovascular modulators, cholesterol, neuroprotectants, neuromodulators, immunomodulators, anti-inflammatories, and anti-microbial drugs.Screening Methods000205 The present disclosure also relates to a method of evaluating the toxicity of an agent comprising: (a) culturing one or more neuronal cells on any of the devices disclosed herein; (b) exposing at least one agent to the one or more neuronal cells; (c) measuring and / or observing one or more metrics of the neuronal cell health; and (d) correlating one or more metrics of the one or more neuronal cells with the toxicity of the agent, such that, if the metrics are indicative of decreased cell viability, the agent is characterized as toxic and, if the metrics are indicative of unchanged or increased cell viability, the agent is characterized as non-toxic; wherein step (c) optionally comprises and / or observing one or more morphometric changes of the one or more neuronal cells; and wherein step (d) optionally comprises correlating one or more morphometric changes of the one or more neuronal cells with the toxicity of the agent, such that, if the changes are indicative of decreased cell viability, the agent is characterized as toxic and, if the changes are indicative of unchanged or increased cell viability, the agent is characterized as non-toxic. In some embodiments, the neuronal cells are any of the cells disclosed herein.000206 In some embodiments, the at least one agent comprises a small chemical compound. In some embodiments, the at least one agent comprises at least one environmental or industrial pollutant. In some embodiments, the at least one agent comprises one or a combination of small chemical compounds chosen from: chemotherapeutics, analgesics, cardiovascular modulators, cholesterol level modulators, neuroprotectants, neuromodulators, immunomodulators, antiinflammatories, and anti-microbial drugs such as bacterial antibiotics. In some embodiments, the at least one agent comprises a therapeutically effective amount of an antibody, such as a clinically relevant monoclonal antibody such as Tysabri.000207 The present disclosure also relates to method of measuring the amount or degree of myelination or demyelination of one or more axons of one or a plurality of neuronal cells, said method comprising: (a) culturing one or more neuronal cells explants on any of the devices disclosed herein for a time and under conditions sufficient to grow at least one axon; (b) measuring and / or observing one or more morphometric changes of the one or more neuronal cells; and (c) correlating one or more morphometric changes of the one or more neuronal cells with a quantitative or qualitative change of myelination of the neuronal cells.000208 The present disclosure also relates to a method of measuring myelination or demyelination of one or more axons of one or a plurality of neuronal cells, said method comprising: (a) culturing one or more neuronal cells on any of the devices disclosed herein for a time and under conditions sufficient to grow at least one axon; (b) measuring and / or observing one or more physiological metrics of the one or more neuronal cells; and (c) correlating one or more metrics of the one or more neuronal with a quantitative or qualitative change of myelination of the neuronal cells; wherein step (b) optionally comprises and / or observing one or more morphometric changes of the one or more neuronal cells; and wherein step (c) optionally comprises correlating one or more morphometric changes of the one or more neuronal cells with the quantitative or qualitative change of myelination of the neuronal cells.000209 The present disclosure also relates to a method of measuring myelination or demyelination of one or more axons of one or a plurality of neuronal cells, said method comprising: (a) culturing one or more neuronal cells on any of the devices disclosed herein for a time and under conditions sufficient to grow at least one axon; and (b) detecting the amount of myelination on one or a plurality of axons of the one or more neuronal cells and / or one or more tissue explants. In each of the above embodiments, the step should comprise one or a plurality of neural cells in culture with one or a combination of agents with known or unknow toxicity. 000210 In some embodiments, the step of detecting the amount of myelination on one or a plurality of axons of the one or more neuronal cells comprises exposing the cells to an antibody that binds to myelin.000211 In some embodiments, the method further comprises (i) exposing one or a plurality of neuronal cells and / or one or a plurality of tissue explants in the presence of SCs to at least one agent after steps (a) and (b); (ii) measuring and / or observing one or more expression patterns of the cells, measuring and / or observing one or more morphometric changes and / or detecting thequantitative amount of myelin from the one or a plurality of neuronal cells; (iii) calculating a change of measurements, observations and / or quantitative amount of myelin from the one or a plurality of neuronal cells in the presence and absence of the agent; and (iv) correlating the change of measurements, observations and / or quantitative amount of myelin from the one or a plurality of neuronal cells to the presence or absence of the agent.000212 In some embodiments, the at least one agent comprises at least one environmental or industrial pollutant. In some embodiments, the at least one agent comprises one or a combination of small chemical compounds chosen from: chemotherapeutics, analgesics, cardiovascular modulators, cholesterol level modulators, neuroprotectants, neuromodulators, immunomodulators, anti-inflammatories, and anti-microbial drugs.000213 The present disclosure also relates to a method of measuring myelination or demyelination of one or more axons of one or a plurality of neuronal cells, said method comprising: (a) culturing one or more neuronal cells on any of the devices disclosed herein for a time and under conditions sufficient to grow at least one axon; and (b) quantifying the levels of myelination of such one or more neuronal cells based on the presence of the number and / or density of axons in the culture. In some embodiments, the method further comprises exposing the one or more neuronal cells to an agent. In some embodiments, the at least one agent comprises at least one environmental or industrial pollutant.000214 In some embodiments, the at least one agent comprises one or a combination of small chemical compounds chosen from: chemotherapeutics, analgesics, cardiovascular modulators, cholesterol level modulators, neuroprotectants, neuromodulators, immunomodulators, antiinflammatories, and anti-microbial drugs.000215 In some embodiments, the at least one agent comprises a small chemical compound. In some embodiments, the at least one agent comprises at least one environmental or industrial pollutant. In some embodiments, the at least one agent comprises one or a combination of small chemical compounds chosen from: chemotherapeutics, analgesics, cardiovascular modulators, cholesterol, neuroprotectants, neuromodulators, immunomodulators, anti-inflammatories, and anti-microbial drugs.000216 In some embodiments, the at least one agent comprises one or a combination of chemotherapeutics chosen from: Actinomycin, Alitretinoin, All-trans retinoic acid, Azacitidine, Azathioprine, Bexarotene, Bleomycin, Bortezomib, Capecitabine, Carboplatin, Chlorambucil,Cisplatin, Cyclophosphamide, Cytarabine, Dacarbazine(DTIC), Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Erlotinib, Etoposide, Fluorouracil, Gefitinib, Gemcitabine, Hydroxyurea, Idarubicin, Imatinib, Irinotecan, Mechlorethamine, Melphalan, Mercaptopurine, Methotrexate, Mitoxantrone, Nitrosoureas, Oxaliplatin, Paclitaxel, Pemetrexed, Romidepsin, Tafluposide, Temozolomide (Oral dacarbazine), Teniposide, Tioguanine (formerly Thioguanine), Topotecan, Tretinoin, Valrubicin, Vemurafenib, Vinblastine Vincristine, Vindesine, Vinorelbine, Vismodegib, and Vorinostat.000217 In some embodiments, the at least one agent comprises one or a combination of analgesics chosen from: Paracetoamol, Non-steroidal anti-inflammatory drugs (NSAIDs), COX- 2 inhibitors, opioids, flupirtine, tricyclic antidepressants, carbamaxepine, gabapentin, and pregabalin.000218 In some embodiments, the at least one agent comprises one or a combination of cardiovascular modulators chosen from: nepicastat, cholesterol, niacin, Scutellaria, prenylamine, dehydroepiandrosterone, monatepil, esketamine, niguldipine, asenapine, atomoxetine, flunarizine, milnacipran, mexiletine, amphetamine, sodium thiopental, flavonoid, bretylium, oxazepam, and honokiol.000219 In some embodiments, the at least one agent comprises one or a combination of neuroprotectants and / or neuromodulators chosen from: tryptamine, galanin receptor 2, phenylalanine, phenethylamine, N-methylphenethylamine, adenosine, kyptorphin, substance P, 3-methoxytyramine, catecholamine, dopamine, GAB A, calcium, acetylcholine, epinephrine, norepinephrine, and serotonin.000220 In some embodiments, the at least one agent comprises one or a combination of immunomodulators chosen from: clenolizimab, enoticumab, ligelizumab, simtuzumab, vatelizumab, parsatuzumab, Imgatuzumab, tregalizaumb, pateclizumab, namulumab, perakizumab, faralimomab, patritumab, atinumab, ublituximab, futuximab, and duligotumab. 000221 In some embodiments, the at least one agent comprises one or a combination of antiinflammatories chosen from: ibuprofen, aspirin, ketoprofen, sulindac, naproxen, etodolac, fenoprofen, diclofenac, flurbiprofen, ketorolac, piroxicam, indomethacin, mefenamic acid, meloxicam, nabumetone, oxaprozin, ketoprofen, famotidine, meclofenamate, tolmetin, and sal sal ate.000222 In some embodiments, the at least one agent comprises one or a combination of antimicrobials chosen from: antibacterials, antifungals, antivirals, antiparasitics, heat, radiation, and ozone.000223 The present disclosure also relates to a method of detecting and / or quantifying neuronal cell growth viability comprising: (a) quantifying one or a plurality of neuronal cells; (b) culturing the one or more neuronal cells on any of the systems disclosed herein; and (c) calculating the number of neuronal cells in the composition after culturing for a time period sufficient to allow growth of the one or plurality of cells.000224 Methods of differentiating or generating cell subtypes disclosed herein can be accomplished by following any one or multiple successive steps comprising exposure to the disclosed cell culture medium:Step 1 of differentiation: Day 1 (definitive ectoderm induction, 24 hours)000225 Differentiation of pluripotent cells, e.g., hPSCs into definitive ectoderm represents the very first step of ectoderm differentiation. Definitive ectoderm can be subsequently used to generate anterior neural ectoderm, posterior neural ectoderm, border ectoderm, and surface ectoderm. The disclosure relates to a cell or cell line comprising or expressing SOX2 and deficient in NANOG. In some embodiments, compositions comprising the cells also comprise a cell culture media comprising one or a combination of components disclosed below. In some embodiments, the cell culture media comprises a FGF pathway inhibitor. In some embodiments, the aforementioned cells are characterized as definitive ectodermal cells. In some embodiments, the cells are differentiated into definitive ectodermal cells after human stem cells are exposed to the below culture medium for from about 12 to about 24 hours.Step 2 of differentiation: Day 2 (anterior neural ectoderm induction, 24 hours)000226 hPSC-derived definitive ectoderm (step 1) was dissociated, and re-seeded at 37,500 cells / cm2-75,000 cells / cm2 in the below media for 24 hours to generate anterior neural ectoderm (step 2). Anterior neural ectoderm is the starting point for subsequent forebrain or midbrain differentiation. As mentioned above, dissociation and re-seeding of cells at this stage improves differentiation efficiencies, but is not absolutely required for differentiation. ROCK inhibitor (see below) enhances cell survival during dissociation and re-seeding, but is not critical for differentiation.000227 The disclosure relates to a cell or cell line comprising or expressing SOX2 and OTX2. In some embodiments, compositions comprising the cells also comprise a cell culture media comprising one or a combination of components disclosed below. In some embodiments, the cell culture media comprises a TGFp inhibitor, a BMP and WNT inhibitors. In some embodiments, the aforementioned cells are characterized as anterior neural ectodermal cells. In som embodiments, the cells are differentiated into anterior neural ectodermal cells after exposure to the below culture medium for from about 12 to about 24 hours.Step 3 of differentiation: Day 3-4 (forebrain progenitors, 48 hours)000228 Anterior neural ectoderm (generated in step 2) was further differentiated into forebrain progenitors. Please see the special note below that FGF2 is only added for a 24 hour period of this 48 hour step. The disclosure relates to a cell or cell line comprising or expressing SOX2, OTX2, FOXG1, LHX2, SIX3, PAX6. In some embodiments, compositions comprising the cells also comprise a cell culture media comprising one or a combination of components disclosed below. In some embodiments, the cell culture media comprises a FGF agonist. In some embodiments, the aforementioned cells are characterized as forebrain progenitor cells.Step 4 of differentiation: Day 4-7 (dorsal forebrain, 72 hours)000229 Forebrain progenitors (generated in step 3) were further differentiated into dorsal forebrain progenitors. The disclosure relates to a cell or cell line comprising or expressing SOX2,LHX2, EMX2, PAX6. In some embodiments, compositions comprising the cells also comprise a cell culture media comprising one or a combination of components disclosed below. In some embodiments, the cell culture media comprises a WNT pathway activator. In some embodiments, the aforementioned cells are characterized as dorsal forebrain progenitor cells.Step 4 of differentiation: Day 4-7 (ventral forebrain, 72 hours)000230 Forebrain progenitors (generated in step 3) were further differentiated into ventral forebrain progenitors.000231 The disclosure relates to a cell or cell line comprising or expressing SOX2, LHX2, NKX2.1, GLI1, FOXG1. In some embodiments, compositions comprising the cells also comprise a cell culture media comprising one or a combination of components disclosed below. In some embodiments, the cell culture media comprises a WNT inhibitors and FGF / ERK inhibitor. In some embodiments, the aforementioned cells are characterized as ventral forebrain progenitor cells.Step 3 of differentiation: Day 3-4 (midbrain, 48 hours)000232 Anterior neural ectoderm (generated in step 2) was further differentiated into midbrain progenitors. Please see the special note below that FGF2 is only added for a 24 hour period of this 48 hour step. The disclosure relates to a cell or cell line comprising or expressing SOX2, OTX2, EN1, EN2, PAX2, PAX5. In some embodiments, compositions comprising the cells also comprise a cell culture media comprising one or a combination of components disclosed below. In some embodiments, the cell culture media comprises and FGF activator. In some embodiments, the aforementioned cells are characterized as midbrain progenitor cells.Step 2 of differentiation: Day 2 (posterior neural ectoderm, 24 hours)000233 hPSC-derived definitive ectoderm (step 1) was dissociated, and re-seeded at 37,500 cells / cm2-75,000 cells / cm2 in the below media for 24 hours to generate posterior neural ectoderm (step 2). Posterior neural ectoderm is the starting point for subsequent hindbrain differentiation. As mentioned above, dissociation and re-seeding of cells at this stage improves differentiation efficiencies, but is not absolutely required for differentiation. ROCK inhibitor (see below) enhances cell survival during dissociation and re-seeding, but is not critical for differentiation.000234 The disclosure relates to a cell or cell line comprising or expressing The disclosure relates to a cell or cell line comprising or expressing SOX2, GBX2, H0XA1, H0XB1. In some embodiments, compositions comprising the cells also comprise a cell culture media comprising one or a combination of components disclosed below. In some embodiments, the cell culture media comprises and FGF activator. In some embodiments, the aforementioned cells are characterized as posterior neural ectoderm cells.Step 3 of differentiation: Day 3-4 (hindbrain, 48 hours)000235 Posterior neural ectoderm (generated in step 2) was further differentiated into hindbrain progenitors.000236 The disclosure relates to a cell or cell line comprising or expressing The disclosure relates to a cell or cell line comprising or expressing SOX2, GBX2, MAFB, HNF1B, KROX20, MEIS1, MEIS2, H0XA1, H0XA2, H0XA3, HOXB1, H0XB2, H0XB3. In someembodiments, compositions comprising the cells also comprise a cell culture media comprising one or a combination of components disclosed below with basal medium for at least about 2 days. In some embodiments, the cell culture media comprises and FGF activator. In some embodiments, the aforementioned cells are characterized as hindbrain progenitor cells.Step 4 of differentiation: Day 4-7 (dorsal hindbrain, 72 hours)000237 Hindbrain progenitors (generated in step 3) were further differentiated into dorsal hindbrain progenitors.000238 The disclosure relates to a cell or cell line comprising or expressing The disclosure relates to a cell or cell line comprising or expressing SOX2, HNF1B, NKX2.2, NKX6.1, NKX6.2, OLIG2, PHOX2B. In some embodiments, compositions comprising the cells also comprise a cell culture media comprising one or a combination of components disclosed below with basal medium for at least about 2 days. In some embodiments, the aforementioned cells are characterized as dorsal hindbrain progenitor cells.Step 4 of differentiation: Day 4-7 (ventral hindbrain, 72 hours)000239 Hindbrain progenitors (generated in step 3) were further differentiated into ventral hindbrain progenitors.000240 The disclosure relates to a cell or cell line comprising or expressing SOX2, HNF1B, NKX2.2, NKX6.1, NKX6.2, OLIG2, PHOX2B. In some embodiments, compositions comprising the cells also comprise a cell culture media comprising one or a combination of components disclosed below in the table.Step 5 of differentiation: Day 7-14 (all neuronal types, one week)000241 Day 7 hP SC -derived dorsal forebrain, ventral forebrain, dorsal hindbrain, or ventral hindbrain progenitors (see above, generated in Step 4) were further differentiated for 7 days in neuronal differentiation media (below). This neuronal differentiation media was modified from a previously reported method (Qi et al., 2017). Exposure of hPSC-derived dorsal forebrain progenitors to this neuronal differentiation media generated dorsal forebrain-specific neurons, and so on for the other types of neural progenitors and neurons.000242 This differentiation step is initially started in CDM2 basal medium, and the cells are progressively transitioned into Neurobasal basal medium.000243 The disclosure relates to a cell or cell line comprising or expressing MAPT, SNAP25, TBR1, RELN, EBF2, LHX1, LHX5, VGLUT2. The disclosure relates to a cell or cell line comprising or expressing MAPT, SNAP25, DLX1, SP8, LHX1, LHX5, LHX6, DLX5, VGAT, NKX2.1, PROXI, ISL1, POMC. The disclosure relates to a cell or cell line comprising or expressing MAPT, SNAP25, TLX3, ASCL1, LBX1, LMX1B, LHX1, LHX5, PAX2. The disclosure relates to a cell or cell line comprising or expressing MAPT, SNAP25, ISL1, CHAT, VACHT, ACHE, CHT, LHX3, SOX 14, SIM1, EN1, GAD1, EVX1, PHOX2A, PHOX2B, H0XA1, HOXA3. In some embodiments, compositions comprising the cells also comprise a cell culture media comprising one or a combination of components disclosed below in the table.Step 2 of differentiation: Day 2 (border ectoderm, 24 hours)000244 hPSC-derived definitive ectoderm (step 1) was dissociated, and re-seeded at 37,500 cells / cm2 in the below media for 24 hours to generate border ectoderm (step 2). As mentionedabove, dissociation and re-seeding of cells at this stage improves differentiation efficiencies, but is not absolutely required for differentiation. ROCK inhibitor (see below) enhances cell survival during dissociation and re-seeding, but is not critical for differentiation.000245 The disclosure relates to a cell or cell line comprising or expressing PAX3, PAX7, TFAP2A. In some embodiments, compositions comprising the cells also comprise a cell culture media comprising BMP activator, WNT activator, TGF0 inhibitor, and FGF / ERK inhibitor. In some embodiments, the cell or cell line is characterized as a definitive ectoderm.Step 2 of differentiation: Day 2 (surface ectoderm, 24 hours)000246 hPSC-derived definitive ectoderm (step 1) was dissociated, and re-seeded at 37,500 cells / cm2 in the below media for 24 hours to generate surface ectoderm (step 2). As mentioned above, dissociation and re-seeding of cells at this stage improves differentiation efficiencies, but is not absolutely required for differentiation. ROCK inhibitor (see below) enhances cell survival during dissociation and re-seeding, but is not critical for differentiation. The disclosure relates to a cell or cell line comprising or expressing GATA3 and TFAP2A. In some embodiments, compositions comprising the cells also comprise a cell culture media comprising a BMP activator, a WNT inhibitor, a TGF inhibitor, and an FGF / ERK inhibitor.TABLE Z: Protein Biomarkers000247 Cells of the disclosure include embodiments that comprise or express (have a phenotype comprising) any one or combination of two or more of the following protein biomarker or functional variants of any of the following comprising at least about 75% sequence identity to SEQ ID NO: 1 through SEQ ID NO: 65.PAX5 5079 NP_057953.1 (SEQ ID NO:1)MDLEKNYPTPRTSRTGHGGVNQLGGVFVNGRPLPDWRQRIVELAHQGVRPCDISRQ LRVSHGCVSKILGRYYETGSIKPGVIGGSKPKVATPKWEKIAEYKRQNPTMFAWEIRDRLLAE RVCDNDTVPSVSSINRIIRTKVQQPPNQPVPASSHSIVSTGSVTQVSSVSTDSAGSSYSISGILGI TSPSADTNKRKRDEGIQESPVPNGHSLPGRDFLRKQMRGDLFTQQQLEVLDRVFERQHYSDI FTTTEPIKPEQTTEYSAMASLAGGLDDMKANLASPTPADIGSSVPGPQSYPIVTGRDLASTTLP GYPPHVPPAGQGSYSAPTLTGMVPGSEFSGSPYSHPQYSSYNDSWRFPNPGLLGSPYYYSA AARGAAPPAAATAYDRHPAX6 5080 NP_001355839.1 (SEQ ID NO:2)M CEAFYCESGQTSEISEPH I RAPWNPAAPSQSQH A EQGGRSSAAEI KRAVAGAN RLP ALSLTCGGRTCLWLKPSPGSDSHVCTGGHSGVNQLGGVFVNGRPLPDSTRQKIVELAHSGA RPCDISRILQVSNGCVSKILGRYYETGSIRPRAIGGSKPRVATPEWSKIAQYKRECPSIFAWEIR DRLLSEGVCTNDNIPSVSSINRVLRNLASEKQQMGADGMYDKLRMLNGQTGSWGTRPGWY PGTSVPGQPTQDGCQQQEGGGENTNSISSNGEDSDEAQMRLQLKRKLQRNRTSFTQEQIEA LEKEFERTHYPDVFARERLAAKIDLPEARIQVWFSNRRAKWRREEKLRNQRRQASNTPSHIPI SSSFSTSVYQPIPQPTTPVSSFTSGSMLGRTDTALTNTYSALPPMPSFTMANNLPMQPPVPSQ TSSYSCMLPTSPSVNGRSYDTYTPPHMQTHMNSQPMGTSGTTSTGLISPGVSVPVQVPGSEP DMSQYWPRLQPAX7 5081 NP_002575.1 (SEQ ID NO:3)MAALPGTVPRMMRPAPGQNYPRTGFPLEVSTPLGQGRVNQLGGVFINGRPLPNHIRH KIVEMAHHGIRPCVISRQLRVSHGCVSKILCRYQETGSIRPGAIGGSKPRQVATPDVEKKIEEY KRENPGMFSWEIRDRLLKDGHCDRSTVPSGLVSSISRVLRIKFGKKEEEDEADKKEDDGEKK AKHSIDGILGDKGNRLDEGSDVESEPDLPLKRKQRRSRTTFTAEQLEELEKAFERTHYPDIYT REELAQRTKLTEARVQVWFSNRRARWRKQAGANQLAAFNHLLPGGFPPTGMPTLPPYQLP DSTYPTTTISQDGGSTVHRPQPLPPSTMHQGGLAAAAAAADTSSAYGARHSFSSYSDSFMNP AAPSNHMNPVSNGLSPQVMSILGNPSAVPPQPQADFSISPLHGGLDSATSISASCSQRADSIKPGDSLPTSQAYCPPTYSTTGYSVDPVAGYQYGQYGQSECLVPWASPVPIPSPTPRASCLFMESYKWSGWGMSISQMEKLKSSQMEQFTPH0X2A 401 NP_005160.2 (SEQ ID NO:4)MDYSYLNSYDSCVAAMEASAYGDFGACSQPGGFQYSPLRPAFPAAGPPCPALGSSN CALGALRDHQPAPYSAVPYKFFPEPSGLHEKRKQRRIRTTFTSAQLKELERVFAETHYPDIYT REELALKIDLTEARVQVWFQNRRAKFRKQERAASAKGAAGAAGAKKGEARCSSEDDDSKE STCSPTPDSTASLPPPPAPGLASPRLSPSPLPVALGSGPGPGPGPQPLKGALWAGVAGGGG GGPGAGAAELLKAWQPAESGPGPFSGVLSSFHRKPGPALKTNLFPHOX2B 8929 NP_003915.2 (SEQ ID NO:5)MYKMEYSYLNSSAYESCMAGMDTSSLASAYADFSSCSQASGFQYNPIRTTFGATSGC PSLTPGSCSLGTLRDHQSSPYAAVPYKLFTDHGGLNEKRKQRRIRTTFTSAQLKELERVFAET HYPDIYTREELALKIDLTEARVQVWFQNRRAKFRKQERAAAAAAAAAKNGSSGKKSDSSRD DESKEAKSTDPDSTGGPGPNPNPTPSCGANGGGGGGPSPAGAPGAAGPGGPGGEPGKGG AAAAAAAAAAAAAAAAAAAAGGLAAAGGPGQGWAPGPGPITSIPDSLGGPFASVLSSLQR PNGAKAALVKSSMFPOMC 5443 NP_001306133.1 (SEQ ID NO:6)MPRSCCSRSGALLLALLLQASMEVRGWCLESSQCQDLTTESNLLECIRACKPDLSAE TPMFPGNGDEQPLTENPRKYVMGHFRWDRFGRRNSSSSGSSGAGQKREDVSAGEDCGPLP EGGPEPRSDGAKPGPREGKRSYSMEHFRWGKPVGKKRRPVKVYPNGAEDESAEAFPLEFK RELTGQRLREGDGPDGPADDGAGAQADLEHSLLVAAEKKDEGPYRMEHFRWGSPPKDKRY GGFMTSEKSQTPLVTLFKNAIIKNAYKKGEPROX1 5629 NP_001257545.1 (SEQ ID NO:7)MPDHDSTALLSRQTKRRRVDIGVKRTVGTASAFFAKARATFFSAMNPQGSEQDVEYS WQHADGEKSNVLRKLLKRANSYEDAMMPFPGATIISQLLKNNMNKNGGTEPSFQASGLSST GSEVHQEDICSNSSRDSPPECLSPFGRPTMSQFDMDRLCDEHLRAKRARVENIIRGMSHSPS VALRGNENEREMAPQSVSPRESYRENKRKQKLPQQQQQSFQQLVSARKEQKREERRQLKQ QLEDMQKQLRQLQEKFYQIYDSTDSENDEDGNLSEDSMRSEILDARAQDSVGRSDNEMCEL DPGQFIDRARALIREQEMAENKPKREGNNKERDHGPNSLQPEGKHLAETLKQELNTAMSQV VDTWKVFSAKPSRQVPQVFPPLQIPQARFAVNGENHNFHTANQRLQCFGDVIIPNPLDTFGN VQMASSTDQTEALPLWRKNSSDQSASGPAAGGHHQPLHQSPLSATTGFTTSTFRHPFPLPL MAYPFQSPLGAPSGSFSGKDRASPESLDLTRDTTSLRTKMSSHHLSHHPCSPAHPPSTAEGL SLSLIKSECGDLQDMSEISPYSGSAMQEGLSPNHLKKAKLMFFYTRYPSSNMLKTYFSDVKF NRCITSQLIKWFSNFREFYYIQMEKYARQAINDGVTSTEELSITRDCELYRALNMHYNKANDFE VPERFLEVAQITLREFFNAIIAGKDVDPSWKKAIYKVICKLDSEVPEIFKSPNCLQELLHERELN 5649 NP_005036.2 (SEQ ID NO:8)MERSGWARQTFLLALLLGATLRARAAAGYYPRFSPFFFLCTHHGELEGDGEQGEVLI SLHIAGNPTYYVPGQEYHVTISTSTFFDGLLVTGLYTSTSVQASQSIGGSSAFGFGIMSDHQFG NQFMCSWASHVSHLPTTNLSFIWIAPPAGTGCVNFMATATHRGQVIFKDALAQQLCEQGAPT DVTVHPHLAEIHSDSIILRDDFDSYHQLQLNPNIWVECNNCETGEQCGAIMHGNAVTFCEPYG PRELITTGLNTTTASVLQFSIGSGSCRFSYSDPSIIVLYAKNNSADWIQLEKIRAPSNVSTIIHILYL PEDAKGENVQFQWKQENLRVGEVYEACWALDNILIINSAHRQWLEDSLDPVDTGNWLFFPG ATVKHSCQSDGNSIYFHGNEGSEFNFATTRDVDLSTEDIQEQWSEEFESQPTGWDVLGAVIGT ECGTIESGLSMVFLKDGERKLCTPSMDTTGYGNLRFYFVMGGICDPGNSHENDIILYAKIEGRK EH ITLDTLSYSSYKVPSLVSWI N PELQTPATKFCLRQKN H QGHNRN VWAVDFFH VLPVLPSTMSHMIQFSINLGCGTHQPGNSVSLEFSTNHGRSWSLLHTECLPEICAGPHLPHSTVYSSENYS GWNRITIPLPNAALTRNTRIRWRQTGPILGNMWAIDNVYIGPSCLKFCSGRGQCTRHGCKCDP GFSGPACEMASQTFPMFISESFGSSRLSSYHNFYSIRGAEVSFGCGVLASGKALVFNKDGRR QLITSFLDSSQSRFLQFTLRLGSKSVLSTCRAPDQPGEGVLLHYSYDNGITWKLLEHYSYLSY HEPRIISVELPGDAKQFGIQFRWWQPYHSSQREDVWAIDEIIMTSVLFNSISLDFTNLVEVTQSL GFYLGNVQPYCGHDWTLCFTGDSKLASSMRYVETQSMQIGASYMIQFSLVMGCGQKYTPHM DNQVKLEYSTNHGLTWHLVQEECLPSMPSCQEFTSASIYHASEFTQWRRVIVLLPQKTWSSA TRFRWSQSYYTAQDEWALDSIYIGQQCPNMCSGHGSCDHGICRCDQGYQGTECHPEAALPS TIMSDFENQNGWESDWQEVIGGEIVKPEQGCGVISSGSSLYFSKAGKRQLVSWDLDTSWVDF VQFYIQIGGESASCNKPDSREEGVLLQYSNNGGIQWHLLAEMYFSDFSKPRFVYLELPAAAKT PCTRFRWWQPVFSGEDYDQWAVDDIIILSEKQKQIIPVINPTLPQNFYEKPAFDYPMNQMSVW LMLANEGMVKNETFCAATPSAMIFGKSDGDRFAVTRDLTLKPGYVLQFKLNIGCANQFSSTAP VLLQYSHDAGMSWFLVKEGCYPASAGKGCEGNSRELSEPTMYHTGDFEEWTRITIVIPRSLA SSKTRFRWIQESSSQKNVPPFGLDGVYISEPCPSYCSGHGDCISGVCFCDLGYTAAQGTCVS NVPNHNEMFDRFEGKLSPLWYKITGAQVGTGCGTLNDGKSLYFNGPGKREARTVPLDTRNIR LVQFYIQIGSKTSGITCIKPRTRNEGLIVQYSNDNGILWHLLRELDFMSFLEPQIISIDLPQDAKTP ATAFRWWQPQHGKHSAQWALDDVLIGMNDSSQTGFQDKFDGSIDLQANWYRIQGGQVDIDC LSMDTALIFTENIGKPRYAETWDFHVSASTFLQFEMSMGCSKPFSNSHSVQLQYSLNNGKDW HLVTEECVPPTIGCLHYTESSIYTSERFQNWKRITVYLPLSTISPRTRFRWIQANYTVGADSWAI DNWLASGCPWMCSGRGICDAGRCVCDRGFGGPYCVPWPLPSILKDDFNGNLHPDLWPEV YGAERGNLNGETIKSGTSLIFKGEGLRMLISRDLDCTNTMYVQFSLRFIAKSTPERSHSILLQF SISGGITWHLMDEFYFPQTTNILFINVPLPYTAQTNATRFRLWQPYNNGKKEEIWIVDDFIIDGN NVNNPVMLLDTFDFGPREDNWFFYPGGNIGLYCPYSSKGAPEEDSAMVFVSNEVGEHSITTR DLNVNENTIIQFEINVGCSTDSSSADPVRLEFSRDFGATWHLLLPLCYHSSSHVSSLCSTEHHP SSTYYAGTMQGWRREWHFGKLHLCGSVRFRWYQGFYPAGSQPVTWAIDNVYIGPQCEEMC NGQGSCINGTKCICDPGYSGPTCKISTKNPDFLKDDFEGQLESDRFLLMSGGKPSRKCGILSS GNNLFFNEDGLRMLMTRDLDLSHARFVQFFMRLGCGKGVPDPRSQPVLLQYSLNGGLSWS LLQEFLFSNSSNVGRYIALEIPLKARSGSTRLRWWQPSENGHFYSPWVIDQILIGGNISGNTVL EDDFTTLDSRKWLLHPGGTKMPVCGSTGDALVFIEKASTRYWSTDVAVNEDSFLQIDFAASC SVTDSCYAIELEYSVDLGLSWHPLVRDCLPTNVECSRYHLQRILVSDTFNKWTRITLPLPPYTR SQATRFRWHQPAPFDKQQTWAIDNVYIGDGCIDMCSGHGRCIQGNCVCDEQWGGLYCDDPE TSLPTQLKDNFNRAPSSQNWLTVNGGKLSTVCGAVASGMALHFSGGCSRLLVTVDLNLTNAE FIQFYFMYGCLITPNNRNQGVLLEYSVNGGITWNLLMEIFYDQYSKPGFVNILLPPDAKEIATRF RWWQPRHDGLDQNDWAIDNVLISGSADQRTVMLDTFSSAPVPQHERSPADAGPVGRIAFDM FMEDKTSVNEHWLFHDDCTVERFCDSPDGVMLCGSHDGREVYAVTHDLTPTEGWIMQFKISV GCKVSEKIAQNQIHVQYSTDFGVSWNYLVPQCLPADPKCSGSVSQPSVFFPTKGWKRITYPL PESLVGNPVRFRFYQKYSDMQWAIDNFYLGPGCLDNCRGHGDCLREQCICDPGYSGPNCYL THTLKTFLKERFDSEEIKPDLWMSLEGGSTCTECGILAEDTALYFGGSTVRQAVTQDLDLRGA KFLQYWGRIGSENNMTSCHRPICRKEGVLLDYSTDGGITWTLLHEMDYQKYISVRHDYILLPE DALTNTTRLRWWQPFVISNGIWSGVERAQWALDNILIGGAEINPSQLVDTFDDEGTSHEENW SFYPNAVRTAGFCGNPSFHLYWPNKKKDKTHNALSSRELIIQPGYMMQFKIWGCEATSCGDL HSVMLEYTKDARSDSWQLVQTQCLPSSSNSIGCSPFQFHEATIYNSVNSSSWKRITIQLPDHV SSSATQFRWIQKGEETEKQSWAIDHVYIGEACPKLCSGHGYCTTGAICICDESFQGDDCSVFS HDLPSYIKDNFESARVTEANWETIQGGVIGSGCGQLAPYAHGDSLYFNGCQIRQAATKPLDLT RASKIMFVLQIGSMSQTDSCNSDLSGPHAVDKAVLLQYSVNNGITWHVIAQHQPKDFTQAQR VSYNVPLEARMKGVLLRWWQPRHNGTGHDQWALDHVEWLVSTRKQNYMMNFSRQHGLR HFYNRRRRSLRRYPSIM1 6492 NP_001361698.1 (SEQ ID N0:9)MKEKSKNAARTRREKENSEFYELAKLLPLPSAITSQLDKASIIRLTTSYLKMRWFPEG LGEAWGHSSRTSPLDNVGRELGSHLLQTLDGFIFWAPDGKIMYISETASVHLGLSQVELTGN SIYEYIHPADHDEMTAVLTAHQPYHSHFVQEYEIERSFFLRMKCVLAKRNAGLTCGGYKVIHCS GYLKIRQYSLDMSPFDGCYQNVGLVAVGHSLPPSAVTEIKLHSNMFMFRASLDMKLIFLDSRV AELTGYEPQDLIEKTLYHHVHGCDTFHLRCAHHLLLVKGQVTTKYYRFLAKHGGWVWVQSYA TIVHNSRSSRPHCIVSVNYVLTDTEYKGLQLSLDQISASKPAFSYTSSSTPTMTDNRKGAKSRL SSSKSKSRTSPYPQYSGFHTERSESDHDSQWGGSPLTDTASPQLLDPADRPGSQHDASCAY RQFSDRSSLCYGFALDHSRLVEERHFHTQACEGGRCEAGRYFLGTPQAGREPWWGSRAAL PLTKASPESREAYENSMPHIASVHRIHGRGHWDEDSWSSPDPGSASESGDRYRTEQYQSSP HEPSKIETLIRATQQMIKEEENRLQLRKAPSDQLASINGAGKKHSLCFANYQQPPPTGEVCHG SALANTSPCDHIQQREGKMLSPHENDYDNSPTALSRISSPNSDRISKSSLILAKDYLHSDISPH QTAGDHPTVSPNCFGSHRQYFDKHAYTLTGYALEHLYDSETIRNYSLGCNGSHFDVTSHLRM QPDPAQGHKGTSVIITNGSSIX3 6496 NP_005404.1 (SEQ ID NO:10)MVFRSPLDLYSSHFLLPNFADSHHRSILLASSGGGNGAGGGGGAGGGSGGGNGAG GGGAGGAGGGGGGGSRAPPEELSMFQLPTLNFSPEQVASVCETLEETGDIERLGRFLWSLP VAPGACEAINKHESILRARAWAFHTGNFRDLYHILENHKFTKESHGKLQAMWLEAHYQEAEK LRGRPLGPVDKYRVRKKFPLPRTIWDGEQKTHCFKERTRSLLREWYLQDPYPNPSKKRELA QATGLTPTQVGNWFKNRRQRDRAAAAKNRLQHQAIGPSGMRSLAEPGCPTHGSAESPSTA ASPTTSVSSLTERADTGTSILSVTSSDSECDVSNAP25 6616 NP_001309836.1 (SEQ ID NO:11)MAEDADMRNELEEMQRRADQLADESLESTRRMLQLVEESKDAGIRTLVMLDEQGEQ LERIEEGMDQINKDMKEAEKNLTDLGKFCGLCVCPCNKLKSSDAYKKAWGNNQDGWASQP ARWDEREQMAISGGFIRRVTNDARENEMDENLEQVSGIIGNLRHMALDMGNEIDTQNRQIDR IMEKADSNKTRIDEANQRATKMLGSGSOX1 48403 NP_004180.1 (SEQ ID NO:12)MSKPSDHIKRPMNAFMVWSRGQRRKMAQENPKMHNSEISKRLGAEWKLLSEAEKRPYIDEAKRLRAQHMKEHPDYKYRPRRKPKNLLKKDRYVFPLPYLGDTDPLKAAGLPVGASDGLLSAPEKARAFLPPASAPYSLLDPAQFSSSAIQKMGEVPHTLATGALPYASTLGYQNGAFGSLSCPSQHTHTHPSPTNPGYWPCNCTAWSASTLQPPVAYILFPGMTKTGIDPYSSAHATAMSOX2 6657 NP_003097.1 (SEQ ID NO:13)MYNMMETELKPPGPQQTSGGGGGNSTAAAAGGNQKNSPDRVKRPMNAFMVWSRG QRRKMAQENPKMHNSEISKRLGAEWKLLSETEKRPFIDEAKRLRALHMKEHPDYKYRPRRK TKTLMKKDKYTLPGGLLAPGGNSMASGVGVGAGLGAGVNQRMDSYAHMNGWSNGSYSMM QDQLGYPQHPGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTP GMALGSMGSWKSEASSSPPWTSSSHSRAPCQAGDLRDMISMYLPGAEVPEPAAPSRLHM SQHYQSGPVPGTAINGTLPLSHMSP8 221833 NP_874359.2 (SEQ ID N0:14)MATSLLGEEPRLGSTPLAMLAATCNKIGSPSPSPSSLSDSSSSFGKGFHPWKRSSSS SSASCNWGSSLSSFGVSGASRNGGSSSAAAAAAAAAAAAAALVSDSFSCGGSPGSSAFSL TSSSAAAAAAAAAAAASSSPFANDYSVFQAPGVSGGSGGGGGGGGGGSSAHSQDGSHQP VFISKVHTSVDGLQGIYPRVGMAHPYESWFKPSHPGLGAAGEVGSAGASSWWDVGAGWIDV QNPNSAAALPGSLHPAAGGLQTSLHSPLGGYNSDYSGLSHSAFSSGASSHLLSPAGQHLMD GFKPVLPGSYPDSAPSPLAGAGGSMLSAGPSAPLGGSPRSSARRYSGRATCDCPNCQEAE RLGPAGASLRRKGLHSCHIPGCGKVYGKTSHLKAHLRWHTGERPFVCNWLFCGKRFTRSDE LQRHLRTHTGEKRFACPVCNKRFMRSDHLSKHVKTHSGGGGGGGSAGSGSGGKKGSDTDS EHSAAGSPPCHSPELLQPPEPGHRNGLETBR1 10716 NP_006584.1 (SEQ ID NO:15)MQLEHCLSPSIMLSKKFLNVSSSYPHSGGSELVLHDHPIISTTDNLERSSPLKKITRGM TNQSDTDNFPDSKDSPGDVQRSKLSPVLDGVSELRHSFDGSAADRYLLSQSSQPQSAATAP SAMFPYPGQHGPAHPAFSIGSPSRYMAHHPVITNGAYNSLLSNSSPQGYPTAGYPYPQQYGH SYQGAPFYQFSSTQPGLVPGKAQVYLCNRPLWLKFHRHQTEMIITKQGRRMFPFLSFNISGLD PTAHYNIFVDVILADPNHWRFQGGKWVPCGKADTNVQGNRVYMHPDSPNTGAHWMRQEISF GKLKLTNNKGASNNNGQMWLQSLHKYQPRLHWEVNEDGTEDTSQPGRVQTFTFPETQFIA VTAYQNTDITQLKIDHNPFAKGFRDNYDTIYTGCDMDRLTPSPNDSPRSQIVPGARYAMAGSFL QDQFVSNYAKARFHPGAGAGPGPGTDRSVPHTNGLLSPQQAEDPGAPSPQRWFVTPANNR LDFAASAYDTATDFAGNAATLLSYAAAGVKALPLQAAGCTGRPLGYYADPSGWGARSPPQY CGTKSGSVLPCWPNSAAAAARMAGANPYLGEEAEGLAAERSPLPPGAAEDAKPKDLSDSS WIETPSSIKSIDSSDSGIYEQAKRRRISPADTPVSESSSPLKSEVLAQRDCEKNCAKDISGYYG FYSHSTFAP2A 7020 NP_001358995.1 (SEQ ID NO: 16)MKMLWKLTDNIKYEDCEDRHDGTSNGTARLPQLGTVGQSPYTSAPPLSHTPNADFQPPYFPPPYQPIYPQSQDPYSHVNDPYSLNPLHAQPQPQHPGWPGQRQSQESGLLHTHRGLPHQLSGLDPRRDYRRHEDLLHGPHALSSGLGDLSIHSLPHAIEEVPHVEDPGINIPDQTVIKKGPVSLSKSNSNAVSAIPINKDNLFGGWNPNEVFCSVPGRLSLLSSTSKYKVTVAEVQRRLSPPECLNASLLGGVLRRAKSKNGGRSLREKLDKIGLNLPAGRRKAANVTLLTSLVEGEAVHLARDFGYVCETEFPAKAVAEFLNRQHSDPNEQVTRKNMLLATKQICKEFTDLLAQDRSPLGNSRPNPIL EPGIQSCLTHFNLISHGFGSPAVCAAVTALQNYLTEALKAMDKMYLSNNPNSHTDNNAKSSDK EEKHRKTLX3 30012 NP_066305.2 (SEQ ID NO:17)MEAPASAQTPHPHEPISFGIDQILNSPDQDSAPAPRGPDGASYLGGPPGGRPGATYP SLPASFAGLGAPFEDAGSYSVNLSLAPAGVIRVPAHRPLPGAVPPPLPSALPAMPSVPTVSSL GGLNFPWMESSRRFVKDRFTAAAAL.TPFTVTRRIGHPYQNRTPPKRKKPRTSFSRVQICELE KRFHRQKYLASAERAALAKSLKMTDAQVKTWFQNRRTKWRRQTAEEREAERQQASRLMLQ LQHDAFQKSLNDSIQPDPLCLHNSSLFALQNLQPWEEDSSKVPAVTSLVVACHT 6572 NP_003046.2 (SEQ ID N0:18)MESAEPAGQARAAATKLSEAVGAALQEPRRQRRLVLVIVCVALLLDNMLYMVIVPIVP DYIAHMRGGGEGPTRTPEVWEPTLPLPTPANASAYTANTSASPTAAWPAGSALRPRYPTESE DVKIGVLFASKAILQLLVNPLSGPFIDRMSYDVPLLIGLGVMFASTVLFAFAEDYATLFAARSLQ GLGSAFADTSGIAMIADKYPEEPERSRALGVALAFISFGSLVAPPFGGILYEFAGKRVPFLVLAA VSLFDALLLLAVAKPFSAAARARANLPVGTPIHRLMLDPYIAWAGALTTCNIPLAFLEPTIATW MKHTMAASEWEMGMAWLPAFVPHVLGVYLTVRLAARYPHLQWLYGALGLAVIGASSCIVPA CRSFAPLWSLCGLCFGIALVDTALLPTLAFLVDVRHVSVYGSVYAIADISYSVAYALGPIVAGHI VHSLGFEQLSLGMGLANLLYAPVLLLLRNVGLLTRSRSERDVLLDEPPQGLYDAVRLRERPVS GQDGEPRSPPGPFDACEDDYNYYYTRSVGAT 140679 NP_542119.1 (SEQ ID NO:19)MATLLRSKLSNVATSVSNKSQAKMSGMFARMGFQAATDEEAVGFAHCDDLDFEHRQ GLQMDILKAEGEPCGDEGAEAPVEGDIHYQRGSGAPLPPSGSKDQVGGGGEFGGHDKPKIT AWEAGWNVTNAIQGMFVLGLPYAILHGGYLGLFLIIFAAWCCYTGKILIACLYEENEDGEWR VRDSYVAIANACCAPRFPTLGGRWNVAQIIELVMTCILYVWSGNLMYNSFPGLPVSQKSWSII ATAVLLPCAFLKNLKAVSKFSLLCTLAHFVINILVIAYCLSRARDWAWEKVKFYIDVKKFPISIGII VFSYTSQIFLPSLEGNMQQPSEFHCMMNWTHIAACVLKGLFALVAYLTWADETKEVITDNLPG SIRAWNIFLVAKALLSYPLPFFAAVEVLEKSLFQEGSRAFFPACYSGDGRLKSWGLTLRCALV VFTLLMAIYVPHFALLMGLTGSLTGAGLCFLLPSLFHLRLLWRKLLWHQVFFDVAIFVIGGICSV SGFVHSLEGLIEAYRTNAEDVGLUT2 57084 NP_065079.1 (SEQ ID NQ:20)MESVKQRILAPGKEGLKNFAGKSLGQIYRVLEKKQDTGETIELTEDGKPLEVPERKAP LCDCTCFGLPRRYIIAIMSGLGFCISFGIRCNLGVAIVDMVNNSTIHRGGKVIKEKAKFNWDPET VGMIHGSFFWGYIITQIPGGYIASRLAANRVFGAAILLTSTLNMLIPSAARVHYGCVIFVRILQGL VEGVTYPACHGIWSKWAPPLERSRLATTSFCGSYAGAVIAMPLAGILVQYTGWSSVFYVYGSF GMVWYMFWLLVSYESPAKHPTITDEERRYIEESIGESANLLGAMEKFKTPWRKFFTSMPVYAII VANFCRSWTFYLLLISQPAYFEEVFGFEISKVGMLSAVPHLVMTIIVPIGGQIADFLRSKQILSTT TVRKIMNCGGFGMEATLLLWGYSHTRGVAISFLVLAVGFSGFAISGFNVNHLDIAPRYASILMG ISNGVGTLSGMVCPIIVGAMTKNKSREEWQYVFLIAALVHYGGVIFYAIFASGEKQPWADPEET SEEKCGFIHEDELDEETGDITQNYINYGTTKSYGATTQANGGWPSGWEKKEEFVQGEVQDSH SYKDRVDYSACHE 43 NP_000656.1 (SEQ ID NO:21)MRPPQCLLHTPSLASPLLLLLLWLLGGGVGAEGREDAELLVTVRGGRLRGIRLKTPG GPVSAFLGIPFAEPPMGPRRFLPPEPKQPWSGWDATTFQSVCYQYVDTLYPGFEGTEMWNP NRELSEDCLYLNVWTPYPRPTSPTPVLVWIYGGGFYSGASSLDVYDGRFLVQAERTVLVSMN YRVGAFGFLALPGSREAPGNVGLLDQRLALQWVQENVAAFGGDPTSVTLFGESAGAASVG MHLLSPPSRGLFHRAVLQSGAPNGPWATVGMGEARRRATQLAHLVGCPPGGTGGNDTELVA CLRTRPAQVLVNHEWHVLPQESVFRFSFVPWDGDFLSDTPEALINAGDFHGLQVLVGWKD EGSYFLVYGAPGFSKDNESLISRAEFLAGVRVGVPQVSDLAAEAWLHYTDWLHPEDPARLR EALSD WG DH N WCPVAQLAG RLAAQGARVYAYVFEH RASTLSWPLWM G VPH G YEI EFI FG I PLDPSRNYTAEEKIFAQRLMRYWANFARTGDPNEPRDPKAPQWPPYTAGAQQYVSLDLRPLEVRRGLRAQACAFWNRFLPKLLSATDTLDEAERQWKAEFHRWSSYMVHWKNQFDHYSKQDRCSDLASCL1 429 NP_004307.2 (SEQ ID NO: 22)M ESSAKM ESGGAGQQPQPQPQQPFLPPAACFFATAAAAAAAAAA AAAQSAQQQQ QQQQQQQQAPQLRPAADGQPSGGGHKSAPKQVKRQRSSSPELMRCKRRLNFSGFGYSLP QQQPAAVARRNERERNRVKLVNLGFATLREHVPNGAANKKMSKVETLRSAVEYIRALQQLLD EHDAVSAAFQAGVLSPTISPNYSNDLNSMAGSPVSSYSSDEGSYDPLSPEEQELLDFTNWFCHAT 1103 NP_066264.4 (SEQ ID NO: 23)MAAKTPSSEESGLPKLPVPPLQQTLATYLQCMRHLVSEEQFRKSQAIVQQFGAPGGL GETLQQKLLERQEKTANWVSEYWLNDMYLNNRLALPVNSSPAVIFARQHFPGTDDQLRFAAS LISGVLSYKALLDSHSIPTDCAKGQLSGQPLCMKQYYGLFSSYRLPGHTQDTLVAQNSSIMPE PEHVIVACCNQFFVLDWINFRRLSEGDLFTQLRKIVKMASNEDERLPPIGLLTSDGRSEWAEA RTVLVKDSTNRDSLDMIERCICLVCLDAPGGVELSDTHRALQLLHGGGYSKNGANRWYDKSL QFWGRDGTCGWCEHSPFDGIVLVQCTEHLLKHVTQSSRKLIRADSVSELPAPRRLRWKCS PEIQGHLASSAEKLQRIVKNLDFIVYKFDNYGKTFIKKQKCSPDAFIQVALQLAFYRLHRRLVP TYESASIRRFQEGRVDNIRSATPEALAFVRAVTDHKAAVPASEKLLLLKDAIRAQTAYTVMAIT GMAIDNHLLALRELARAMCKELPEMFMDETYLMSNRFVLSTSQVPTTTEMFCCYGPWPNGY GACYNPQPETILFCISSFHSCKETSSSKFAKAVEESLIDMRDLCSLLPPTESKPLATKEKATRP SQGHQPCHT 60482 NP_001291934.1 (SEQ ID NO: 24)MAFHVEGLIAIIVFYLLILLVGIWAAWRTKNSGSAEERSEAIIVGGRDIGLLVGGFTMTAT WVGGGYINGTAEAVYVPGYGLAWAQAPIGYSLSLILGGLFFAKPMRSKGYVTMLDPFQQIYG KRMGGLLFIPALMGEMFWAAAIFSALGATISVIIDVDMHISVIISALIATLYTLVGGLYSVAYTDW QLFCIFVGLWISVPFALSHPAVADIGFTAVHAKYQKPWLGTVDSSEVYSWLDSFLLLMLGGIPW QAYFQRVLSSSSATYAQVLSFLAAFGCLVMAIPAILIGAIGASTDWNQTAYGLPDPKTTEEADMI LPIVLQYLCPVYISFFGLGAVSAAVMSSADSSILSASSMFARNIYQLSFRQNASDKEIVWVMRIT VFVFGASATAMALLTKTVYGLWYLSSDLVYIVIFPQLLCVLFVKGTNTYGAVAGYVSGLFLRITG GEPYLYLQPLIFYPGYYPDDNGIYNQKFPFKTLAMVTSFLTNICISYLAKYLFESGTLPPKLDVF DAWARHSEENMDKTILVKNENIKLDELALVKPRQSMTLSSTFTNKEAFLDVDSSPEGSGTED NLQDLX1 1745 NP_835221.2 (SEQ ID NO: 25)MTMTTMPESLNSPVSGKAVFMEFGPPNQQMSPSPMSHGHYSMHCLHSAGHSQPDGAYSSASSFSRPLGYPYVNSVSSHASSPYISSVQSYPGSASLAQSRLEDPGADSEKSTWEGGEVRFNGKGKKIRKPRTIYSSLQLQALNRRFQQTQYLALPERAELAASLGLTQTQVKIWFQNKRSKFKKLMKQGGAALEGSALANGRALSAGSPPVPPGWNPNSSSGKGSGGNAGSYIPSYTSWYPSAHQEAMQQPQLMDLX5 1749 NP_005212.1 (SEQ ID NO: 26)MTGVFDRRVPSIRSGDFQAPFQTSAAMHHPSQESPTLPESSATDSDYYSPTGGAPHGYCSPTSASYGKALNPYQYQYHGVNGSAGSYPAKAYADYSYASSYHQYGGAYNRVPSATNQPEKEVTEPEVRMVNGKPKKVRKPRTIYSSFQLAALQRRFQKTQYLALPERAELAASLGLTQTQVKIWFQNKRSKIKKIMKNGEMPPEHSPSSSDPMACNSPQSPAVWEPQGSSRSLSHHPHAHPPTSNQSPASSYLENSASWYTSAASSINSHLPPPGSLQHPLALASGTLYEBF2 64641 NP_073150.2 (SEQ ID NO: 27)MFGIQDTLGRGPTLKEKSLGAEMDSVRSWVRNVGWDANVAAQSGVALSRAHFEKQ PPSNLRKSNFFHFVLALYDRQGQPVEIERTAFVDFVENDKEQGNEKTNNGTHYKLQLLYSNG VRTEQDLYVRLI DSVTKQPI AYEGQNKN PEM CRVLLTH EVMCSRCCEKKSCGN RN ETPSDPVI IDRFFLKFFLKCNQNCLKTAGNPRDMRRFQWLSTTVNVDGHVLAVSDNMFVHNNSKHGRRA RRLDPSEATPCIKAISPSEGWTTGGAMVIIIGDNFFDGLQWFGTMLVWSELITPHAIRVQTPPR HIPGWEVTLSYKSKQFCKGAPGRFIYTALNEPTIDYGFQRLQKVIPRHPGDPERLAKEMLLKR AADLVEALYGTPHNNQDIILKRAADIAEALYSVPRNPSQLPALSSSPAHSGMMGINSYGSQLG VSISESTQGNNQGYIRNTSSISPRGYSSSSTPQQSNYSTSSNSMNGYSNVPMANLGVPGSPG FLNGSPTGSPYGIMSSSPTVGSSSTSSILPFSSSVFPAVKQKSAFAPVIRPQGSPSPACSSGNG NGFRAMTGLWPPMEMX2 2018 NP_004089.1 (SEQ ID NO: 28)MFQPAPKRCFTIESLVAKDSPLPASRSEDPIRPAALSYANSSPINPFLNGFHSAAAAAA GRGVYSNPDLVFAEAVSHPPNPAVPVHPVPPPHALAAHPLPSSHSPHPLFASQQRDPSTFYP WLIHRYRYLGHRFQGNDTSPESFLLHNALARKPKRIRTAFSPSQLLRLEHAFEKNHYWGAER KQLAHSLSLTETQVKVWFQNRRTKFKRQKLEEEGSDSQQKKKGTHHINRWRIATKQASPEEI DVTSDDEN1 2019 NP_001417.3 (SEQ ID NO: 29)MEEQQPEPKSQRDSALGAAAAATPGGLSLSLSPGASGSSGSGSDGDSVPVSPQPAP PSPPAAPCLPPLAHHPHLPPHPPPPPPQHLAAPAHQPQPAAQLHRTTNFFIDNILRPDFGCKK EQPPPQLLVAAAARGGAGGGGRVERDRGQTAAGRDPVHPLGTRAPGAASLLCAPDANCGP PDGSQPAAAGAGASKAGNPAAAAAAAAAAVAAAAAAAAAKPSDTGGGGSGGGAGSPGAQ GTKYPEHGNPAILLMGSANGGPWKTDSQQPLVWPAWVYCTRYSDRPSSGPRTRKLKKKKN EKEDKRPRTAFTAEQLQRLKAEFQANRYITEQRRQTLAQELSLNESQIKIWFQNKRAKIKKAT GIKNGLALHLMAQGLYNHSTTTVQDKDESEEN2 2020 NP_001418.2 (SEQ ID NO: 30)MEENDPKPGEAAAAVEGQRQPESSPGGGSGGGGGSSPGEADTGRRRALMLPAVLQ APGNHQHPHRITNFFIDNILRPEFGRRKDAGTCCAGAGGGRGGGAGGEGGASGAEGGGGA GGSEQLLGSGSREPRQNPPCAPGAGGPLPAAGSDSPGDGEGGSKTLSLHGGAKKGGDPGGPLDGSLKARGLGGGDLSVSSDSDSSQAGANLGAQPMLWPAWVYCTRYSDRPSSGPRSRKPKKKNPNKEDKRPRTAFTAEQLQRLKAEFQTNRYLTEQRRQSLAQELSLNESQIKIWFQNKRAKIKKATGNKNTLAVHLMAQGLYNHSTTAKEGKSDSEEVX1 2128 NP_001980.1 (SEQ ID N0: 31)MESRKDMWFLDGGQLGTLVGKRVSNLSEAVGSPLPEPPEKMVPRGCLSPRAVPPAT RERGGGGPEEEPVDGLAGSAAGPGAEPQVAGAAMLGPGPPAPSVDSLSGQGQPSSSDTES DFYEEIEVSCTPDCATGNAEYQHSKGSGSEALVGSPNGGSETPKSNGGSGGGGSQGTLACS ASDQMRRYRTAFTREQIARLEKEFYRENYVSRPRRCELAAALNLPETTIKVWFQNRRMKDKR QRLAMTWPHPADPAFYTYMMSHAAAAGGLPYPFPSHLPLPYYSPVGLGAASAASAAASPFS GSLRPLDTFRVLSQPYPRPELLCAFRHPPLYPGPAHGLGASAGGPCSCLACHSGPANGLAP RAAAASDFTCASTSRSDSFLTFAPSVLSKASSVALDQREEVPLTRF0XG1 2290 NP_005240.3 (SEQ ID NO: 32)MLDMGDRKEVKMIPKSSFSINSLVPEAVQNDNHHASHGHHNSHHPQHHHHHHHHHH HPPPPAPQPPPPPQQQQPPPPPPPAPQPPQTRGAPAADDDKGPQQLLLPPPPPPPPAAALD GAKADGLGGKGEPGGGPGELAPVGPDEKEKGAGAGGEEKKGAGEGGKDGEGGKEGEKK NGKYEKPPFSYNALIMMAIRQSPEKRLTLNGIYEFIMKNFPYYRENKQGWQNSIRHNLSLNKC FVKVPRHYDDPGKGNYWMLDPSSDDVFIGGTTGKLRRRSTTSRAKLAFKRGARLTSTGL.TFM DRAGSLYWPMSPFLSLHHPRASSTLSYNGTTSAYPSHPMPYSSVLTQNSLGNNHSFSTANGL SVDRLVNGEIPYATHHLTAAALAASVPCGLSVPCSGTYSLNPCSVNLLAGQTSYFFPHVPHPS MTSQSSTSMSARAASSSTSPQAPSTLPCESLRPSLPSFTTGLSGGLSDYFTHQNQGSSSNPL IHGAD1 2571 NP_000808.2 (SEQ ID NO: 33)MASSTPSSSATSSNAGADPNTTNLRPTTYDTWCGVAHGCTRKLGLKICGFLQRTNSL EEKSRLVSAFKERQSSKNLLSCENSDRDARFRRTETDFSNLFARDLLPAKNGEEQTVQFLLE WDILLNYVRKTFDRSTKVLDFHHPHQLLEGMEGFNLELSDHPESLEQILVDCRDTLKYGVRT GHPRFFNQLSTGLDIIGLAGEWLTSTANTNMFTYEIAPVFVLMEQITLKKMREIVGWSSKDGD GIFSPGGAISNMYSIMAARYKYFPEVKTKGMAAVPKLVLFTSEQSHYSIKKAGAALGFGTDNVI LIKCNERGKIIPADFEAKILEAKQKGYVPFYVNATAGTTVYGAFDPIQEIADICEKYNLWLHVDA AWGGGLLMSRKHRHKLNGIERANSVTWNPHKMMGVLLQCSAILVKEKGILQGCNQMCAGYL FQPDKQYDVSYDTGDKAIQCGRHVDIFKFWLMWKAKGTVGFENQINKCLELAEYLYAKIKNR EEFEMVFNGEPEHTNVCFWYIPQSLRGVPDSPQRREKLHKVAPKIKALMMESGTTMVGYQP QGDKANFFRMVISNPAATQSDIDFLIEEIERLGQDLGATA3 2625 NP_001002295.1 (SEQ ID NO: 34)MEVTADQPRWVSHHHPAVLNGQHPDTHHPGLSHSYMDAAQYPLPEEVDVLFNIDGQGNHVPPYYGNSVRATVQRYPPTHHGSQVCRPPLLHGSLPWLDGGKALGSHHTASPWNLSPFSKTSIHHGSPGPLSVYPPASSSSLSGGHASPHLFTFPPTPPKDVSPDPSLSTPGSAGSARQDEKECLKYQVPLPDSMKLESSHSRGSMTALGGASSSTHHPITTYPPYVPEYSSGLFPPSSLLGGSPTGFGCKSRPKARSSTEGRECVNCGATSTPLWRRDGTGHYLCNACGLYHKMNGQNRPLIKPKRRLSAARRAGTSCANCQTTTTTLWRRNANGDPVCNACGLYYKLHNINRPLTMKKEGIQTRNRKMSSKSKKCKKVHDSLEDFPKNSSFNPAALSRHMSSLSHISPFSHSSHMLTTPTPMHPPS SLSFGPHHPSSMVTAMGGBX2 2637 NP_001476.2 (SEQ ID NO: 35)MSAAFPPSLMMMQRPLGSSTAFSIDSLIGSPPQPSPGHFVYTGYPMFMPYRPWLPP PPPPPPALPQAALQPALPPAHPHHQIPSLPTGFCSSLAQGMALTSTLMATLPGGFSASPQHQ EAAAARKFAPQPLPGGGNFDKAEALQADAEDGKGFLAKEGSLLAFSAAETVQASLVGAVRG QGKDESKVEDDPKGKEESFSLESDVDYSSDDNLTGQAAHKEEDPGHALEETPPSSGAAGST TSTGKNRRRRTAFTSEQLLELEKEFHCKKYLSLTERSQIAHALKLSEVQVKIWFQNRRAKWK RVKAGNANSKTGEPSRNPKIWPIPVHVSRFAIRSQHQQLEQARPGDF7 151449 NP_878248.2 (SEQ ID NO: 36)MDLSAAAALCLWLLSACRPRDGLEAAAVLRAAGAGPVRSPGGGGGGGGGGRTLAQ AAGAAAVPAAAVPRARAARRAAGSGFRNGSWPHHFMMSLYRSLAGRAPAGAAAVSASGH GRADTITGFTDQATQDESAAETGQSFLFDVSSLNDADEWGAELRVLRRGSPESGPGSWTSP PLLLLSTCPGAARAPRLLYSRAAEPLVGQRWEAFDVADAMRRHRREPRPPRAFCLLLRAVA GPVPSPLALRRLGFGWPGGGGSAAEERAVLWSSRTQRKESLFREIRAQARALGAALASEP LPDPGTGTASPRAVIGGRRRRRTALAGTRTAQGSGGGAGRGHGRRGRSRCSRKPLHVDFKE LGWDDWIIAPLDYEAYHCEGLCDFPLRSHLEPTNHAIIQTLLNSMAPDAAPASCCVPARLSPISI LYIDAANNWYKQYEDMWEACGCRGLI1 2735 NP_005260.1 (SEQ ID NO: 37)MFNSMTPPPISSYGEPCCLRPLPSQGAPSVGTEGLSGPPFCHQANLMSGPHSYGPAR ETNSCTEGPLFSSPRSAVKLTKKRALSISPLSDASLDLQTVIRTSPSSLVAFINSRCTSPGGSYG HLSIGTMSPSLGFPAQMNHQKGPSPSFGVQPCGPHDSARGGMIPHPQSRGPFPTCQLKSEL DMLVGKCREEPLEGDMSSPNSTGIQDPLLGMLDGREDLEREEKREPESVYETDCRWDGCSQ EFDSQEQLVHHINSEHIHGERKEFVCHWGGCSRELRPFKAQYMLWHMRRHTGEKPHKCTF EGCRKSYSRLENLKTHLRSHTGEKPYMCEHEGCSKAFSNASDRAKHQNRTHSNEKPYVCKL PGCTKRYTDPSSLRKHVKTVHGPDAHVTKRHRGDGPLPRAPSISTVEPKREREGGPIREESR LTVPEGAMKPQPSPGAQSSCSSDHSPAGSAANTDSGVEMTGNAGGSTEDLSSLDEGPCIAG TGLSTLRRLENLRLDQLHQLRPIGTRGLKLPSLSHTGTTVSRRVGPPVSLERRSSSSSSISSAY TVSRRSSLASPFPPGSPPENGASSLPGLMPAQHYLLRARYASARGGGTSPTAASSLDRIGGL PMPPWRSRAEYPGYNPNAGVTRRASDPAQAADRPAPARVQRFKSLGCVHTPPTVAGGGQN FDPYLPTSVYSPQPPSITENAAMDARGLQEEPEVGTSMVGSGLNPYMDFPPTDTLGYGGPEG AAAEPYGARGPGSLPLGPGPPTNYGPNPCPQQASYPDPTQETWGEFPSHSGLYPGPKALG GTYSQCPRLEHYGQVQVKPEQGCPVGSDSTGLAPCLNAHPSEGPPHPQPLFSHYPQPSPPQ YLQSGPYTQPPPDYLPSEPRPCLDFDSPTHSTGQLKAQLVCNYVQSQQELLWEGGGREDAP AQEPSYQSPKFLGGSQVSPSRAKAPVNTYGPGFGPNLPNHKSGSYPTPSPCHENFWGANR ASHRAAAPPRLLPPLPTCYGPLKVGGTNPSCGHPEVGRLGGGPALYPPPEGQVCNPLDSLDLDNTQLDFVAILDEPQGLSPPPSHDQRGSSGHTPPPSGPPNMAVGNMSVLLRSLPGETEFLNSSAGSX2 170825 NP_573574.2 (SEQ ID NO: 38)MSRSFYVDSLIIKDTSRPAPSLPEPHPGPDFFIPLGMPPPLVMSVSGPGCPSRKSGAFCVCPLCVTSHLHSSRGSVGAGSGGAGAGVTGAGGSGVAGAAGALPLLKGQFSSAPGDAQFCPRVNHAHHHHHPPQHHHHHHQPQQPGSAAAAAAAAAAAAAAAALGHPQHHAPVCTATTYNVADPRRFHCLTMGGSDASQVPNGKRMRTAFTSTQLLELEREFSSNMYLSRLRRIEIATYLNLSEKQVKIWFQNRRVKHKKEGKGTQRNSHAGCKCVGSQVHYARSEDEDSLSPASANDDKEISPLHNF1B 6928 NP_000449.1 (SEQ ID NO: 39)MVSKLTSLQQELLSALLSSGVTKEVLVQALEELLPSPNFGVKLETLPLSPGSGAEPDT KPVFHTLTNGHAKGRLSGDEGSEDGDDYDTPPILKELQALNTEEAAEQRAEVDRMLSEDPW RAAKM I KGYM QQH N I PQREWDVTGLNQSH LSQH LN KGTPM KTQKRAALYTWYVRKQREI L RQFNQTVQSSGNMTDKSSQDQLLFLFPEFSQQSHGPGQSDDACSEPTNKKMRRNRFKWGP ASQQILYQAYDRQKNPSKEEREALVEECNRAECLQRGVSPSKAHGLGSNLVTEVRVYNWFA NRRKEEAFRQKLAMDAYSSNQTHSLNPLLSHGSPHHQPSSSPPNKLSGVRYSQQGNNEITS SSTISHHGNSAMVTSQSVLQQVSPASLDPGHNLLSPDGKMISVSGGGLPPVSTLTNIHSLSHH NPQQSQNLIMTPLSGVMAIAQSLNTSQAQSVPVINSVAGSLAALQPVQFSQQLHSPHQQPLM QQSPGSHMAQQPFMAAVTQLQNSHMYAHKQEPPQYSHTSRFPSAMWTDTSSISTLTNMSS SKQCPLQAWHOXA1 3198 NP_005513.2 (SEQ ID NO: 40)MDNARMNSFLEYPILSSGDSGTCSARAYPSDHRITTFQSCAVSANSCGGDDRFLVGR GVQIGSPHHHHHHHHRHPQPATYQTSGNLGVSYSHSSCGPSYGSQNFSAPYSPYALNQEAD VSGGYPQCAPAVYSGNLSSPMVQHHHHHQGYAGGAVGSPQYIHHSYGQEHQSLALATYNNS LSPLHASHQEACRSPASETSSPAQTFDWMKVKRNPPKTGKVGEYGYLGQPNAVRTNFTTKQ LTELEKEFHFNKYLTRARRVEIAASLQLNETQVKIWFQNRRMKQKKREKEGLLPISPATPPGN DEKAEESSEKSSSSPCVPSPGSSTSDTLTTSHHOXA2 3199 NP_006726.1 (SEQ ID NO: 41)MNYEFEREIGFINSQPSLAECLTSFPPVADTFQSSSIKTSTLSHSTLIPPPFEQTIPSLNPGSHPRHGAGGRPKPSPAGSRGSPVPAGALQPPEYPWMKEKKAAKKTALLPAAAAAATAAATGPACLSHKESLEIADGSGGGSRRLRTAYTNTQLLELEKEFHFNKYLCRPRRVEIAALLDLTERQVKVWFQNRRMKHKRQTQCKENQNSEGKCKSLEDSEKVEEDEEEKTLFEQALSVSGALLEREGYTFQQNALSQQQAPNGHNGDSQSFPVSPLTSNEKNLKHFQHQSPTVPNCLSTMGQNCGAGLNNDSPEALEVPSLQDFSVFSTDSCLQLSDAVSPSLPGSLDSPVDISADSLDFFTDTLTTIDLQHLNYH0XA3 3200 NP_705895.1 (SEQ ID NO: 42)MQKATYYDSSAIYGGYPYQAANGFAYNANQQPYPASAALGADGEYHRPACSLQSPS SAGGHPKAHELSEACLRTLSAPPSQPPSLGEPPLHPPPPQAAPPAPQPPQPAPQPPAPTPAA PPPPSSASPPQNASNNPTPANAAKSPLLNSPTVAKQIFPWMKESRQNTKQKTSSSSSGESCA GDKSPPGQASSKRARTAYTSAQLVELEKEFHFNRYLCRPRRVEMANLLNLTERQIKIWFQNR RM KYKKDQKGKGM LTSSGGQSPSRSPVPPGAGGYLNSM H SLVNSVPYEPQSPPPFSKPPQ GTYGLPPASYPASLPSCAPPPPPQKRYTAAGAGAGGTPDYDPHAHGLQGNGSYGTPHIQGS PVFVGGSYVEPMSNSGPALFGLTHLPHAASGAMDYGGAGPLGSGHHHGPGPGEPHPTYTDL TGHHPSQGRIQEAPKLTHLHOXB1 3211 NP_002135.2 (SEQ ID NO: 43)MDYNRMNSFLEYPLCNRGPSAYSAHSAPTSFPPSSAQAVDSYASEGRYGGGLSSPAFQQNSGYPAQQPPSTLGVPFPSSAPSGYAPAACSPSYGPSQYYPLGQSEGDGGYFHPSSYGAQLGGLSDGYGAGGAGPGPYPPQHPPYGNEQTASFAPAYADLLSEDKETPCPSEPNTPTARTFDWMKVKRNPPKTAKVSEPGLGSPSGLRTNFTTRQLTELEKEFHFNKYLSRARRVEIAATLELNETQVKIWFQNRRMKQKKREREEGRVPPAPPGCPKEAAGDASDQSTCTSPEASPSSVTSHOXB2 3212 NP_002136.1 (SEQ ID NO: 44)MNFEFEREIGFINSQPSLAECLTSFPAVLETFQTSSIKESTLIPPPPPFEQTFPSLQPGASTLQRPRSQKRAEDGPALPPPPPPPLPAAPPAPEFPWMKEKKSAKKPSQSATSPSPAASAVPASGVGSPADGLGLPEAGGGGARRLRTAYTNTQLLELEKEFHFNKYLCRPRRVEIAALLDLTERQVKVWFQNRRMKHKRQTQHREPPDGEPACPGALEDICDPAEEPAASPGGPSASRAAWEACCHPPEWPGALSADPRPLAVRLEGAGASSPGCALRGAGGLEPGPLPEDVFSGRQDSPFLPDLNFFAADSCLQLSGGLSPSLQGSLDSPVPFSEEELDFFTSTLCAIDLQFPHOXB3 3213 NP_001371676.1 (SEQ ID NO: 45)MQKATYYDNAAAALFGGYSSYPGSNGFGFDVPPQPPFQAATHLEGDYQRSACSLQS LGNAAPHAKSKELNGSCMRPGLAPEPLSAPPGSPPPSAAPTSATSNSSNGGGPSKSGPPKC GPGTNSTLTKQIFPWMKESRQTSKLKNNSPGTAEGCGGGGGGGGGGGSGGSGGGGGGGG GGDKSPPGSAASKRARTAYTSAQLVELEKEFHFNRYLCRPRRVEMANLLNLSERQIKIWFQN RRMKYKKDQKAKGLASSSGGPSPAGSPPQPMQSTAGFMNALHSMTPSYESPSPPAFGKAH QNAYALPSNYQPPLKGCGAPQKYPPTPAPEYEPHVLQANGGAYGTPTMQGSPVYVGGGGYA DPLPPPAGPSLYGLNHLSHHPSGNLDYNGAPPMAPSQHHGPCEPHPTYTDLSSHHAPPPQG RIQEAPKLTHLISL1 3670 NP_002193.2 (SEQ ID NO: 46)MGDMGDPPKKKRLISLCVGCGNQIHDQYILRVSPDLEWHAACLKCAECNQYLDESCT CFVRDGKTYCKRDYIRLYGIKCAKCSIGFSKNDFVMRARSKVYHIECFRCVACSRQLIPGDEFA LREDGLFCRADHDWERASLGAGDPLSPLHPARPLQMAAEPISARQPALRPHVHKQPEKTTRVRTVLNEKQLHTLRTCYAANPRPDALMKEQLVEMTGLSPRVIRVWFQNKRCKDKKRSIMMK QLQQQQPNDKTNIQGMTGTPMVAASPERHDGGLQANPVEVQSYQPPWKVLSDFALQSDIDQ PAFQQLVNFSEGGPGSNSTGSEVASMSSQLPDTPNSMVASPIEAKROX20 1959 NP_000390.2 (SEQ ID NO: 47)MMTAKAVDKIPVTLSGFVHQLSDNIYPVEDLAATSVTIFPNAELGGPFDQMNGVAGDG MINIDMTGEKRSLDLPYPSSFAPVSAPRNQTFTYMGKFSIDPQYPGASCYPEGIINIVSAGILQG VTSPASTTASSSVTSASPNPLATGPLGVCTMSQTQPDLDHLYSPPPPPPPYSGCAGDLYQDP SAFLSAATTSTSSSLAYPPPPSYPSPKPATDPGLFPMIPDYPGFFPSQCQRDLHGTAGPDRKP FPCPLDTLRVPPPLTPLSTIRNFTLGGPSAGVTGPGASGGSEGPRLPGSSSAAAAAAAAAAY NPHHLPLRPILRPRKYPNRPSKTPVHERPYPCPAEGCDRRFSRSDELTRHIRIHTGHKPFQCRI CMRNFSRSDHLTTHIRTHTGEKPFACDYCGRKFARSDERKRHTKIHLRQKERKSSAPSASVP APSTASCSGGVQPGGTLCSSNSSSLGGGPLAPCSSRTRTPLBX1 10660 NP_006553.2 (SEQ ID NO: 48)MTSKEDGKAAPGEERRRSPLDHLPPPANSNKPLTPFSIEDILNKPSVRRSYSLCGAAH LLAAADKHAQGGLPLAGRALLSQTSPLCALEELASKTFKGLEVSVLQAAEGRDGMTIFGQR QTPKKRRKSRTAFTNHQIYELEKRFLYQKYLSPADRDQIAQQLGLTNAQVITWFQNRRAKLKR DLEEMKADVESAKKLGPSGQMDIVALAELEQNSEATAGGGGGCGRAKSRPGSPVLPPGAPK APGAGALQLSPASPLTDQPASSQDCSEDEEDEEIDVDDLHX1 3975 NP_005559.2 (SEQ ID NO: 49)MVHCAGCKRPILDRFLLNVLDRAWHVKCVQCCECKCNLTEKCFSREGKLYCKNDFFR CFGTKCAGCAQGISPSDLVRRARSKVFHLNCFTCMMCNKQLSTGEELYIIDENKFVCKEDYLS NSSVAKENSLHSATTGSDPSLSPDSQDPSQDDAKDSESANVSDKEAGSNENDDQNLGAKRR GPRTTIKAKQLETLKAAFAATPKPTRHIREQLAQETGLNMRVIQVWFQNRRSKERRMKQLSA LGARRHAFFRSPRRMRPLVDRLEPGELIPNGPFSFYGDYQSEYYGPGGNYDFFPQGPPSSQ AQTPVDLPFVPSSGPSGTPLGGLEHPLPGHHPSSEAQRFTDILAHPPGDSPSPEPSLPGPLH SMSAEVFGPSPPFSSLSVNGGASYGNHLSHPPEMNEAAVWLHX2 9355 NP_004780.3 (SEQ ID NO: 50)MLFHSLSGPEVHGVIDEMDRRAKSEAPAISSAIDRGDTETTMPSISSDRAALCAGCGG KISDRYYLLAVDKQWHMRCLKCCECKLNLESELTCFSKDGSIYCKEDYYRRFSVQRCARCHL GISASEMVMRARDLVYHLNCFTCTTCNKMLTTGDHFGMKDSLVYCRLHFEALLQGEYPAHFN HADVAAAAAAAAAAKSAGLGAAGANPLGLPYYNGVGTVQKGRPRKRKSPGPGADLAAYNA ALSCNENDAEHLDRDQPYPSSQKTKRMRTSFKHHQLRTMKSYFAINHNPDAKDLKQLAQKT GLTKRVLQVWFQNARAKFRRNLLRQENTGVDKSTDAALQTGTPSGPASELSNASLSPSSTP TTLTDLTSPTLPTVTSVLTSVPGNLEGHEPHSPSQTTLTNLFLHX3 8022 NP_001350675.1 (SEQ ID NO: 51)MTLNMKETRWGAPAPEIPLCAGCDQHILDRFILKALDRHWHSKCLKCSDCHTPLAER CFSRGESVYCKDDFFKRFGTKCAACQLGIPPTQWRRAQDFVYHLHCFACWCKRQLATGDE FYLMEDSRLVCKADYETAKQREAEATAKRPRTTITAKQLETLKSAYNTSPKPARHVREQLSSE TGLDM RWQVWFQN RRAKEKRLKKDAGRQRWGQYFRN M KRSRGGSKSDKDSVQEGQDS DAEVSFPDEPSLAEMGPANGLYGSLGEPTQALGRPSGALGNFSLEHGGLAGPEQYRELRPG SPYGVPPSPAAPQSLPGPQPLLSSLVYPDTSLGLVPSGAPGGPPPMRVLAGNGPSSDLSTGS SGGYPDFPASPASWLDEVDHAQFLHX5 64211 NP_071758.1 (SEQ ID NO: 52)MMVHCAGCERPILDRFLLNVLDRAWHIKCVQCCECKTNLSEKCFSREGKLYCKNDFF RRFGTKCAGCAQGISPSDLVRKARSKVFHLNCFTCMVCNKQLSTGEELYVIDENKFVCKDDY LSSSSLKEGSLNSVSSCTDRSLSPDLQDALQDDPKETDNSTSSDKETANNENEEQNSGTKRR GPRTTIKAKQLETLKAAFAATPKPTRHIREQLAQETGLNMRVIQVWFQNRRSKERRMKQLSA LGARRHAFFRSPRRMRPLGGRLDESEMLGSTPYTYYGDYQGDYYAPGSNYDFFAHGPPSQA QSPADSSFLAASGPGSTPLGALEPPLAGPHAADNPRFTDMISHPDTPSPEPGLPGTLHPMPG EVFSGGPSPPFPMSGTSGYSGPLSHPNPELNEAAVWLHX6 26468 NP_055183.2 (SEQ ID NO: 53)MYWKHENAAPALPEGCRLPAEGGPATDQVMAQPGSGCKATTRCLEGTAPPAMAQS DAEALAGALDKDEGQASPCTPSTPSVCSPPSAASSVPSAGKNICSSCGLEILDRYLLKVNNLI WHVRCLECSVCRTSLRQQNSCYIKNKEIFCKMDYFSRFGTKCARCGRQIYASDWVRRARGN AYHLACFACFSCKRQLSTGEEFGLVEEKVLCRIHYDTMIENLKRAAENGNGLTLEGAVPSEQD SQPKPAKRARTSFTAEQLQVMQAQFAQDNNPDAQTLQKLADMTGLSRRVIQVWFQNCRAR HKKHTPQHPVPPSGAPPSRLPSALSDDIHYTPFSSPERARMVTLHGYIESQVQCGQVHCRLP YTAPPVHLKADMDGPLSNRGEKVILFQYLMX1B 4010 NP_001167617.1 (SEQ ID NO: 54)MDIATGPESLERCFPRGQTDCAKMLDGIKMEEHALRPGPATLGVLLGSDCPHPAVCE GCQRPISDRFLMRVNESSWHEECLQCAACQQALTTSCYFRDRKLYCKQDYQQLFAAKCSGC MEKIAPTEFVMRALECVYHLGCFCCCVCERQLRKGDEFVLKEGQLLCKGDYEKEKDLLSSVS PDESDSVKSEDEDGDMKPAKGQGSQSKGSGDDGKDPRRPKRPRTILTTQQRRAFKASFEVS SKPCRKVRETLAAETGLSVRWQVWFQNQRAKMKKLARRHQQQQEQQNSQRLGQGEPGP GQGLGQEVLSSRMEGMMASYTPLAPPQQQIVAMEQSPYGSSDPFQQGLTPPQMPGNDSIFH DIDSDTSLTSLSDCFLGSSDVGSLQARVGNPIDRLYSMQSSYFASMAFB 9935 NP_005452.2 (SEQ ID NO: 55)MAAELSMGPELPTSPLAMEYVNDFDLLKFDVKKEPLGRAERPGRPCTRLQPAGSVS STPLSTPCSSVPSSPSFSPTEQKTHLEDLYWMASNYQQMNPEALNLTPEDAVEALIGSHPVP QPLQSFDSFRGAHHHHHHHHPHPHHAYPGAGVAHDELGPHAHPHHHHHHQASPPPSSAAS PAQQLPTSHPGPGPHATASATAAGGNGSVEDRFSDDQLVSMSVRELNRHLRGFTKDEVIRLKQKRRTLKNRGYAQSCRYKRVQQKHHLENEKTQLIQQVEQLKQEVSRLARERDAYKVKCEKLANSGFREAGSTSDSPSSPEFFLMAPT 4137 NP_001116538.2 (SEQ ID NO: 56)MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDG SEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEA AGHVTQEPESGKWQEGFLREPGPPGLSHQLMSGMPGAPLLPEGPREATRQPSGTGPEDTE GGRHAPELLKHQLLGDLHQEGPPLKGAGGKERPGSKEEVDEDRDVDESSPQDSPPSKASP AQDGRPPQTAAREATSIPGFPAEGAIPLPVDFLSKVSTEIPASEPDGPSVGRAKGQDAPLEFT FHVEITPNVQKEQAHSEEHLGRAAFPGAPGEGPEARGPSLGEDTKEADLPEPSEKQPAAAP RGKPVSRVPQLKARMVSKSKDGTGSDDKKAKTSTRSSAKTLKNRPCLSPKHPTPGSSDPLI QPSSPAVCPEPPSSPKYVSSVTSRTGSSGAKEMKLKGADGKTKIATPRGAAPPGQKGQANA TRIPAKTPPAPKTPPSSATKQVQRRPPPAGPRSERGEPPKSGDRSGYSSPGSPGTPGSRSRT PSLPTPPTREPKKVAWRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGK VQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQV EVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPWS GDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGLMEIS1 4211 NP_002389.1 (SEQ ID NO: 57)MAQRYDDLPHYGGMDGVGIPSTMYGDPHAARSMQPVHHLNHGPPLHSHQYPHTAH TNAMAPSMGSSVNDALKRDKDAIYGHPLFPLLALIFEKCELATCTPREPGVAGGDVCSSESF NEDIAVFAKQIRAEKPLFSSNPELDNLMIQAIQVLRFHLLELEKVHELCDNFCHRYISCLKGKM PIDLVIDDREGGSKSDSEDITRSANLTDQPSWNRDHDDTASTRSGGTPGPSSGGHTSHSGDN SSEQGDGLDNSVASPSTGDDDDPDKDKKRHKKRGIFPKVATNIMRAWLFQHLTHPYPSEEQK KQLAQDTGLTILQVNNWFINARRRIVQPMIDQSNRAVSQGTPYNPDGQPMGGFVMDGQQHM GIRAPGPMSGMGMNMGMEGQWHYMMEIS2 4212 NP_733777.1 (SEQ ID NO: 58)MAQRYDELPHYGGMDGVGVPASMYGDPHAPRPIPPVHHLNHGPPLHATQHYGAHAP HPNVMPASMGSAVNDALKRDKDAIYGHPLFPLLALVFEKCELATCTPREPGVAGGDVCSSDS FNEDIAVFAKQVRAEKPLFSSNPELDNLMIQAIQVLRFHLLELEKVHELCDNFCHRYISCLKGK MPIDLVIDERDGSSKSDHEELSGSSTNLADHNPSSWRDHDDATSTHSAGTPGPSSGGHASQS GDNSSEQGDGLDNSVASPGTGDDDDPDKDKKRQKKRGIFPKVATNIMRAWLFQHLTHPYPS EEQKKQLAQDTGLTILQVNNWFINARRRIVQPMIDQSNRAGFLLDPSVSQGAAYSPEGQPMG SFVLDGQQHMGIRPAGPMSGMGMNMGMDGQWHYMMSX1 4487 NP_002439.2 (SEQ ID NO: 59)MAPAADMTSLPLGVKVEDSAFGKPAGGGAGQAPSAAAATAAAMGADEEGAKPKVSPSLLPFSVEALMADHRKPGAKESALAPSEGVQAAGGSAQPLGVPPGSLGAPDAPSSPRPLGHFSVGGLLKLPEDALVKAESPEKPERTPWMQSPRFSPPPARRLSPPACTLRKHKTNRKPRTPFTTAQLLALERKFRQKQYLSIAERAEFSSSLSLTETQVKIWFQNRRAKAKRLQEAELEKLKMAAKPMLPPAAFGLSFPLGGPAAVAAAAGASLYGASGPFQRAALPVAPVGLYTAHVGYSMYHLTNGN2 63973 NP_076924.1 (SEQ ID NO: 60)MFVKSETLELKEEEDVLVLLGSASPALAALTPLSSSADEEEEEEPGASGGARRQRGA EAGQGARGGVAAGAEGCRPARLLGLVHDCKRRPSRARAVSRGAKTAETVQRIKKTRRLKAN NRERNRMHNLNAALDALREVLPTFPEDAKLTKIETLRFAHNYIWALTETLRLADHCGGGGGG LPGALFSEAVLLSPGGASAALSSSGDSPSPASTWSCTNSPAPSSSVSSNSTSPYSCTLSPASP AGSDMDYWQPPPPDKHRYAPHLPIARDCINKX2.1 7080 NP_001073136.1 (SEQ ID NO: 61)MWSGGSGKARGWEAAAGGRSSPGRLSRRRIMSMSPKHTTPFSVSDILSPLEESYKK VGMEGGGLGAPLAAYRQGQAAPPTAAMQQHAVGHHGAVTAAYHMTAAGVPQLSHSAVGGY CNGNLGNMSELPPYQDTMRNSASGPGWYGANPDPRFPAISRFMGPASGMNMSGMGGLGS LGDVSKNMAPLPSAPRRKRRVLFSQAQVYELERRFKQQKYLSAPEREHLASMIHLTPTQVKI WFQNHRYKMKRQAKDKAAQQQLQQDSGGGGGGGGTGCPQQQQAQQQSPRRVAVPVLVK DGKPCQAGAPAPGAASLQGHAQQQAQHQAQAAQAAAAAISVGSGGAGLGAHPGHQPGSA GQSPDLAHHAASPAALQGQVSSLSHLNSSGSDYGTMSCSTLLYGRTWOLIG3 167826 NP_786923.1 (SEQ ID NO: 62)MNSDSSSVSSRASSPDMDEMYLRDHHHRHHHHQESRLNSVSSTQGDMMQKMPGESLSRAGAKAAGESSKYKIKKQLSEQDLQQLRLKINGRERKRMHDLNLAMDGLREVMPYAHGPSVRKLSKIATLLLARNYILMLTSSLEEMKRLVGEIYGGHHSAFHCGTVGHSAGHPAHAANSVHPVHPILGGALSSGNASSPLSAASLPAIGTIRPPHSLLKAPSTPPALQLGSGFQHWAGLPCPCTICQMPPPPHLSALSTANMARLSAESKDLLKOTX2 5015 NP_001257452.1 (SEQ ID NO: 63)MMSYLKQPPYAVNGLSLTTSGMDLLHPSVGYPATPRKQRRERTTFTRAQLDVLEALF AKTRYPDIFMREEVALKINLPESRVQVWFKNRRAKCRQQQQQQQNGGQNKVRPAKKKTSPA REVSSESGTSGQFTPPSSTSVPTIASSSAPVSIWSPASISPLSDPLSTSSSCMQRSYPMTYTQA SGYSQGYAGSTSYFGGMDCGSYLTPMHHQLPGPGATLSPMGTNAVTSHLNQSPASLSTQGY GASSLGFNSTTDCLDYKDQTASWKLNFNADCLDYKDQTSSWKFQVLPAX2 5076 NP_001291498.1 (SEQ ID NO: 64)MEERAGPAVLKLSLRGDTAAAAALLPLLCLPMDMHCKADPFSAMHPGHGGVNQLG GVFVNGRPLPDWRQRIVELAHQGVRPCDISRQLRVSHGCVSKILGRYYETGSIKPGVIGGSK PKVATPKWDKIAEYKRQNPTMFAWEIRDRLLAEGICDNDTVPSVSSINRIIRTKVQQPFHPTPD GAGTGVTAPGHTIVPSTASPPVSSASNDPVGSYSINGILGIPRSNGEKRKRDEDVSEGSVPNGDSQSGVDSLRKHLRADTFTQQQLEALDRVFERPSYPDVFQASEHIKSEQGNEYSLPALTPGLDEVKSSLSASTNPELGSNVSGTQTYPWTGRDMASTTLPGYPPHVPPTGQGSYPTSTLAGMVPGSEFSGNPYSHPQYTAYNEAWRFSNPALLSSPYYYSAAPRGSAPAAAAAAYDRHPAX3 5077 NP_852122.1 (SEQ ID NO: 65)MTTLAGAVPRMMRPGPGQNYPRSGFPLEVSTPLGQGRVNQLGGVFINGRPLPNHIRH KIVEMAHHGIRPCVISRQLRVSHGCVSKILCRYQETGSIRPGAIGGSKPKQVTTPDVEKKIEEY KRENPGMFSWEIRDKLLKDAVCDRNTVPSVSSISRILRSKFGKGEEEEADLERKEAEESEKKA KHSIDGILSERASAPQSDEGSDIDSEPDLPLKRKQRRSRTTFTAEQLEELERAFERTHYPDIYT REELAQRAKLTEARVQVWFSNRRARWRKQAGANQLMAFNHLIPGGFPPTAMPTLPTYQLSE TSYQPTSIPQAVSDPSSTVHRPQPLPPSTVHQSTIPSNPDSSSAYCLPSTRHGFSSYTDSFVPP SGPSNPMNPTIGNGLSPQVMGLLTNHGGVPHQPQTDYALSPLTGGLEPTTTVSASCSQRLDH MKSLDSLPTSQSYCPPTYSTTGYSMDPVTGYQYGQYGQSKPWTF000248 In some embodiments, the present disclosure uses human neural and non-neural cells to grow nerve tissue in a three dimensional environment in which cells mimick native nerve architecture and expression patterns, allowing more biologically relevant measurement of morphometric data. In some embodiments, the present disclosure uses neuronal and nonneuronal cells derived from primary human tissue or human stem cells. In other embodiments, neuronal ccells may be derived from human stem cells, including induced pluripotent stem cells. 000249 It is another object of this disclosure to provide neural cells and non-neural cells that mimic native anatomical and physiological features and that is susceptible to evaluation using high-throughput electrophysiological stimulation and recording methods.000250 It is another object of the disclosure to provide a low, medium or high-throughput assay of neurological function for the screening of pharmacological and / or toxicological properties of chemical and biological agents. In some embodiments, the agents are cells, such as any type of cell disclosed herein, or antibodies, such as antibodies that are used to treat clinical disease, in some embodiments, the agents are any drugs or agents that are used to treat human disease such that toxicities, effects or neuromodulation can be compared among a new agent which is a proposed mammalian treatment and existing treatments from human disease. In some embodiments, new agents for treatment of human disease are treatments for neurodegenerative disease and are compared to existing treatments for neurodegenerative disease.000251 It is another object of the present disclosure to provide methods of replicating, manipulating, modifying, and evaluating mechanisms underlying myelin-compromising diseases and peripheral neuropathies.000252 It is another object of the present disclosure to allow medium to high-throughput assay of neuromodulation in human neural cells for the screening of pharmacological and / or toxicological activities of chemical and biological agents.000253 It is another object of the present disclosure to employ unique assembly of technologies such as 2D and 3D microengineered neural bundles in conjunction with optical and electrochemical stimulation and recording of human neural cell populations.000254 It is another object of the present disclosure to quantify evoked post-synaptic potentials in a biomimetic, engineered thalamocortical circuits. Our observation of antidromically- generated population spike in neural tracts suggest that they are capable of population-level physiology, such as the conduction of compound action potentials and postsynaptic potentials. 000255 It is another object of the present disclosure to utilize optogenetic methods, hardware and software control of illumination, and fluorescent imaging to allow for noninvasive stimulation and recording of multi-unit physiological responses to evoked potentials in neural circuits.ReferencesEach of the following references is incorporated by reference in its entirety:1 Hemmati-Brivanlou, A. & Melton, D. Vertebrate embryonic cells will become nerve cells unless told otherwise. Ce / / 88, 13-17, doi: 10.1016 / s0092-8674(00)81853-x (1997).2 Lumsden, A. & Krumlauf, R. Patterning the vertebrate neuraxis. Science 274, 1109-1115 (1996).3 Stern, C. D. Neural induction: old problem, new findings, yet more questions. Development 132, 2007-2021, doi: 10.1242 / dev.01794 (2005).4 Rubenstein, J. L., Shimamura, K., Martinez, S. & Puelles, L. Regionalization of the prosencephalic neural plate. Annu Rev Neurosci 21, 445-477, doi : 10.1146 / annurev. neuro .21.1.445 (1998).5 Molyneaux, B. J., Arlotta, P, Menezes, J. R. L. & Macklis, J. D. Neuronal subtype specification in the cerebral cortex. Nature Reviews Neuroscience 8, 427-437, doi:10.1038 / nrn2151 (2007).6 Tao, Y. & Zhang, S.-C. Neural Subtype Specification from Human Pluripotent Stem Cells. Cell Stem Cell 19, 573-586, doi:10.1016 / j.stem.2016.10.015 (2016).7 Muguruma, K. & Sasai, Y. In vitro recapitulation of neural development using embryonic stem cells: from neurogenesis to histogenesis. Development, Growth & Differentiation 54, 349-357, doi: 10.11 ll / j,1440-169X.2012.01329.x (2012).8 Pa§ca, S. P. The rise of three-dimensional human brain cultures. Nature 553, 437-445, doi: 10.1038 / nature25032 (2018).9 Velasco, S., Paulsen, B. & Arlotta, P. 3D Brain Organoids: Studying Brain Development and Disease Outside the Embryo. Annu Rev Neurosci 43, 375-389, doi: 10.1146 / annurev-neuro- 070918-050154 (2020).10 Hansen, D. V, Rubenstein, J. L. R. & Kriegstein, A. R. Deriving excitatory neurons of the neocortex from pluripotent stem cells. Neuron 70, 645-660, doi: 10.1016 / j. neuron.2011.05.006 (2011).11 Li, L. et al. Location of transient ectodermal progenitor potential in mouse development. Development 140, 4533-4543 (2013).12 Groves, A. K. & LaBonne, C. Setting appropriate boundaries: fate, patterning and competence at the neural plate border. Developmental Biology 389, 2-12, doi: 10.1016 / j.ydbio.2013.11.027 (2014).13 Shimamura, K., Hartigan, D. J., Martinez, S., Puelles, L. & Rubenstein, J. L. Longitudinal organization of the anterior neural plate and neural tube. Development 121, 3923- 3933, doi:10.1242 / dev,121.12.3923 (1995).14 Joyner, A. L., Liu, A. & Millet, S. Otx2, Gbx2 and Fgf8 interact to position and maintain a mid-hindbrain organizer. Current Opinion in Cell Biology 12, 736-741 (2000).15 Moens, C. B. & Prince, V. E. Constructing the hindbrain: insights from the zebrafish. Dev Dyn 224, 1-17, doi: 10.1002 / dvdy.10086 (2002).16 Krumlauf, R. & Wilkinson, D. G. Segmentation and patterning of the vertebrate hindbrain. Development 148, doi: 10.1242 / dev, 186460 (2021).17 Philippidou, P. & Dasen, J. S. Hox genes: choreographers in neural development, architects of circuit organization. Neuron 80, 12-34, doi : 10.1016 / j . neuron.2013.09.020 (2013).18 Benghanem, S. et al. Brainstem dysfunction in critically ill patients. Crit Care 24, 5, doi : 10.1186 / s 13054-019-2718-9 (2020).19 Aziz-Bose, R. & Monje, M. Diffuse intrinsic pontine glioma: molecular landscape and emerging therapeutic targets. Curr Opin Oncol 31, 522-530, doi : 10.1097 / CCG.0000000000000577 (2019).20 Spemann, H. & Mangold, H. Induction of embryonic primordia by implantation of organizers from a different species. Archiv fur Mikroskopische Anatomie und Entwicklungsmechanik 45, 13-38-38 (1924).21 Beddington, R. S. Induction of a second neural axis by the mouse node. Development (Cambridge, England) 120, 613-620-620 (1994).22 Hemmati-Brivanlou, A. & Melton, D. A. A truncated activin receptor inhibits mesoderm induction and formation of axial structures in Xenopus embryos. Nature 359, 359609a359600, doi: 10.1038 / 359609a0 (1992).23 Hemmati-Brivanlou, A. & Melton, D. A. Inhibition of activin receptor signaling promotes neuralization in Xenopus. Cell 77, 273-281-281, doi: 10.1016 / 0092-8674(94)90319-0 (1994).24 Piccolo, S., Sasai, Y, Lu, B. & De Robertis, E. M. Dorsoventral patterning in Xenopus: inhibition of ventral signals by direct binding of chordin to BMP-4. Cell 86, 589-598, doi : 10.1016 / s0092-8674(00)80132-4 (1996).25 Zimmerman, L. B., De Jesus-Escobar, J. M. & Harland, R. M. The Spemann organizer signal noggin binds and inactivates bone morphogenetic protein 4. Cell 86, 599-606, doi : 10.1016 / s0092-8674(00)80133-6 (1996).26 Fainsod, A. et al. The dorsalizing and neural inducing gene folli statin is an antagonist of BMP-4. Meeh Dev 63, 39-50, doi:10.1016 / s0925-4773(97)00673-4 (1997).27 Chambers, S. M. et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nature biotechnology 27, 275-280, doi: 10.1038 / nbt. 1529 (2009).28 Maroof, A. M. et al. Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells. Cell Stem Cell 12, 559-572, doi: 10.1016 / j.stem.2013.04.008 (2013).29 Tchieu, J. et al. A Modular Platform for Differentiation of Human PSCs into All Major Ectodermal Lineages. Cell Stem Cell 2\, 399-410.e397, doi: 10.1016 / j.stem.2017.08.015 (2017).30 Kadoshima, T. et al. Self-organization of axial polarity, inside-out layer pattern, and species-specific progenitor dynamics in human ES cell-derived neocortex. Proceedings of the National Academy of Sciences of the United States of America 110, 20284-20289, doi: 10.1073 / pnas.1315710110 (2013).31 Nicholas, C. R. et al. Functional Maturation of hPSC-Derived Forebrain Interneurons Requires an Extended Timeline and Mimics Human Neural Development. Cell Stem Cell 12, 573-586, doi:10.1016 / j.stem.2013.04.005 (2013).32 Cederquist, G. Y. et al. Specification of positional identity in forebrain organoids. Nat Biotechnol 37, 436-444, doi: 10.1038 / s41587-019-0085-3 (2019).33 Qi, Y. et al. Combined small -molecule inhibition accelerates the derivation of functional cortical neurons from human pluripotent stem cells. Nature Biotechnology 35, 154-163, doi: 10.1038 / nbt.3777 (2017).34 Kriks, S. et al. Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease. Nature (2011).35 Fasano, C. A., Chambers, S. M., Lee, G., Tomishima, M. J. & Studer, L. Efficient derivation of functional floor plate tissue from human embryonic stem cells. Cell Stem Cell 6, 336-347, doi: 10.1016 / j. stem.2010.03.001 (2010).36 De Santis, R., Etoc, F., Rosado-Olivieri, E. A. & Brivanlou, A. H. Self-organization of human dorsal -ventral forebrain structures by light induced SHH. Nat Commun 12, 6768, doi : 10.1038 / s41467-021 -26881 -w (2021 ).37 Rifes, P et al. Modeling neural tube development by differentiation of human embryonic stem cells in a microfluidic WNT gradient. Nat Biotechnoi ' 38, 1265-1273, doi:10.1038 / s41587- 020-0525-0 (2020).38 Ozair, M. Z. et al. hPSC Modeling Reveals that Fate Selection of Cortical Deep Projection Neurons Occurs in the Subplate. Cell Stem Cell 23, 60-73 e66, doi : 10.1016 / j . stem.2018.05.024 (2018).39 Grealish, S. et al. Human ESC-derived dopamine neurons show similar preclinical efficacy and potency to fetal neurons when grafted in a rat model of Parkinson's disease. Cell Stem Cell 15, 653-665, doi: 10.1016 / j. stem.2014.09.017 (2014).40 Kirkeby, A. et al. Generation of regionally specified neural progenitors and functional neurons from human embryonic stem cells under defined conditions. Cell Reports 1, 703-714, doi: 10.1016 / j. celrep.2012.04.009 (2012).41 Kirkeby, A. et al. Predictive Markers Guide Differentiation to Improve Graft Outcome in Clinical Translation of hESC-Based Therapy for Parkinson's Disease. Cell Stem Cell 20, 135-148, doi: 10.1016 / j. stem.2016.09.004 (2017).42 Bhaduri, A. et al. Cell stress in cortical organoids impairs molecular subtype specification. Nature 110, 20284-20233, doi: 10.1038 / s41586-020-1962-0 (2020).43 Pollen, A. A. et al. Establishing Cerebral Organoids as Models of Human-Specific Brain Evolution. Cell 176, 743-756.e717, doi: 10.1016 / j cell.2019.01.017 (2019).44 Quadrato, G. et al. Cell diversity and network dynamics in photosensitive human brain organoids. Nature 18, 736-716, doi: 10.1038 / nature22047 (2017).45 Velasco, S. et al. Individual brain organoids reproducibly form cell diversity of the human cerebral cortex. Nature, 1-18, doi: 10.1038 / s41586-019-1289-x (2019).46 Camp, J. G. et al. Human cerebral organoids recapitulate gene expression programs of fetal neocortex development. Proceedings of the National Academy of Sciences of the United States of America 112, 15672-15677, doi: 10.1073 / pnas.1520760112 (2015).47 Kanton, S. et al. Organoid single-cell genomic atlas uncovers human-specific features of brain development. Nature 574, 418-422, doi: 10.1038 / s41586-019-1654-9 (2019).48 He, Z. et al. Lineage recording in human cerebral organoids. Nat Methods 19, 90-99, doi : 10.1038 / s41592-021 -01344-8 (2022).49 Fleck, J. S. et al. Inferring and perturbing cell fate regulomes in human brain organoids. Nature, doi: 10.1038 / s41586-022-05279-8 (2022).50 Birey, F. et al. Assembly of functionally integrated human forebrain compositions. Nature 545, 54-59, doi: 10.1038 / nature22330 (2017).51 Kikuchi, T. et al. Human iPS cell-derived dopaminergic neurons function in a primate Parkinson's disease model. Nature 548, 592-596, doi: 10.1038 / nature23664 (2017).52 Lu, J. et al. Generation of serotonin neurons from human pluripotent stem cells. Nature Biotechnology 34, 89-94, doi:10.1038 / nbt.3435 (2016).53 Lippmann, E. S. et al. Deterministic HOX patterning in human pluripotent stem cell- derived neuroectoderm. Stem Cell Reports 4, 632-644, doi: 10.1016 / j.stemcr.2015.02.018 (2015).54 Iyer, N. R. et al. Modular derivation of diverse, regionally discrete human posterior CNS neurons enables discovery of transcriptomic patterns. Sci Adv 8, eabn7430, doi: 10.1126 / sciadv.abn7430 (2022).55 Valiulahi, P. etal. Generation of caudal -type serotonin neurons and hindbrain-fate organoids from hPSCs. Stem Cell Reports 16, 1938-1952, doi: 10.1016 / j.stemcr.2021.06.006 (2021).56 Maury, Y. et al. Combinatorial analysis of developmental cues efficiently converts human pluripotent stem cells into multiple neuronal subtypes. Nature Biotechnology 33, 89-96, doi: 10.1038 / nbt.3049 (2015).57 Funato, K., Smith, R. C., Saito, Y. & Tabar, V. Dissecting the impact of regional identity and the oncogenic role of human-specific NOTCH2NL in an hESC model of H3.3G34R-mutant glioma. Cell Stem Cell 28, 894-905 e897, doi: 10.1016 / j.stem.2021.02.003 (2021).58 Nieuwkoop, P. D. Activation and organization of the central nervous system in amphibians. Part II. Differentiation and organization. Journal of Experimental Zoology 120, 33- 81, doi:httDs: / / doi.org / 10.1002 / iez.1401200103 (1952).59 Nieuwkoop, P. D. Activation and organization of the central nervous system in amphibians. Part I. Induction and activation. Journal of Experimental Zoology 120, 1-31, doi : https : / / onlinelibrary. wiley. com / doi / 10.1002 / j ez, 1401200102 ( 1952).60 Al Anber, A. & Martin, B. L Transformation of a neural activation and patterning model. EMBO Rep 20, e48060, doi: 10.15252 / embr.201948060 (2019).61 Woo, K. & Fraser, S. E. Order and coherence in the fate map of the zebrafish nervous system. Development 121, 2595-2609, doi:10.1242 / dev.l21.8.2595 (1995).62 Keller, R., Shih, J. & Sater, A. The cellular basis of the convergence and extension of the Xenopus neural plate. Dev Dyn 193, 199-217, doi: 10.1002 / aja.1001930302 (1992).63 Fernandez-Garre, P, Rodriguez-Gallardo, L., Gallego-Diaz, V, Alvarez, I. S. & Puelles, L. Fate map of the chicken neural plate at stage 4. Development 129, 2807-2822, doi: 10.1242 / dev.129.12.2807 (2002).64 Tam, P. P. Regionalisation of the mouse embryonic ectoderm: allocation of prospective ectodermal tissues during gastrulation. Development 107, 55-67 (1989).65 Pijuan Sala, B. et al. A single-cell molecular map of mouse gastrulation and early organogenesis. Nature 1-25, doi:10.1038 / s41586-019-0933-9 (2019).66 Loh, K. M. et al. Efficient Endoderm Induction from Human Pluripotent Stem Cells by Logically Directing Signals Controlling Lineage Bifurcations. Cell Stem Cell 14, doi: 10.1016 / j.stem.2013.12.007 (2014).67 Loh, K. M. et al. Mapping the Pairwise Choices Leading from Pluripotency to Human Bone, Heart, and Other Mesoderm Cell Types. Cell 166, doi: 10.1016 / j. cell.2016.06.011 (2016).68 Greber, B. et al. FGF signalling inhibits neural induction in human embryonic stem cells. £M5(9 J(2011).69 Ang, S. L., Conlon, R. A., Jin, O. & Rossant, J. Positive and negative signals from mesoderm regulate the expression of mouse Otx2 in ectoderm explants. Development 120, 2979- 2989 (1994).70 Bouillet, P, Chazaud, C., Oulad-Abdelghani, M., Dolle, P. & Chambon, P. Sequence and expression pattern of the Stra7 (Gbx-2) homeobox-containing gene induced by retinoic acid in P19 embryonal carcinoma cells. Dev Dyn 204, 372-382, doi: 10.1002 / aja.1002040404 (1995).71 Wassarman, K. M. et al. Specification of the anterior hindbrain and establishment of a normal mid / hindbrain organizer is dependent on Gbx2 gene function. Development 124, 2923- 2934, doi: 10.1242 / dev, 124.15.2923 (1997).72 Wood, H. B. & Episkopou, V. Comparative expression of the mouse Soxl, Sox2 and Sox3 genes from pre-gastrulation to early somite stages. Meeh Dev 86, 197-201 (1999).73 Zhang, X. et al. Pax6 is a human neuroectoderm cell fate determinant. Cell Stem Cell 7, 90-100 (2010).74 Murphy, P. & Hill, R. E. Expression of the mouse labial-like homeobox-containing genes, Hox 2.9 and Hox 1.6, during segmentation of the hindbrain. Development 111, 61-74, doi: 10.1242 / dev.lll.1.61 (1991).75 Sun, X., Meyers, E. N., Lewandoski, M. & Martin, G. R. Targeted disruption of Fgf8 causes failure of cell migration in the gastrulating mouse embryo. Genes & Development 13, 1834-1846 (1999).76 Rhinn, M. etal. Cloning, expression and relationship of zebrafish gbxl and gbx2 genes to Fgf signaling. Meeh Dev 120, 919-936, doi:10.1016 / s0925-4773(03)00135-7 (2003).77 Liu, A., Losos, K. & Joyner, A. L. FGF8 can activate Gbx2 and transform regions of the rostral mouse brain into a hindbrain fate. Development 126, 4827-4838, doi: 10.1242 / dev.126.21.4827 (1999).78 Li, J. Y. & Joyner, A. L. Otx2 and Gbx2 are required for refinement and not induction of mid-hindbrain gene expression. Development 128, 4979-4991, doi: 10.1242 / dev.128.24.4979 (2001).79 Niswander, L. & Martin, G. R. Fgf-4 expression during gastrulation, myogenesis, limb and tooth development in the mouse. Development 114, 755-768 (1992).80 Ribes, V, Le Roux, I., Rhinn, M., Schuhbaur, B. & Dolle, P. Early mouse caudal development relies on crosstalk between retinoic acid, Shh and Fgf signalling pathways. Development 136, 665-676, doi: 10.1242 / dev.016204 (2009).81 Crossley, P. H. & Martin, G. R. The mouse Fgf8 gene encodes a family of polypeptides and is expressed in regions that direct outgrowth and patterning in the developing embryo. Development 121, 439-451 (1995).82 Nordstrom, U., Jessell, T. M. & Edlund, T. Progressive induction of caudal neural character by graded Wnt signaling. Nat Neurosci 5, 525-532 (2002).83 Wilson, P. A., Lagna, G., Suzuki, A. & Hemmati-Brivanlou, A. Concentration-dependent patterning of the Xenopus ectoderm by BMP4 and its signal transducer Smadl. Development 124, 3177-3184, doi: 10.1242 / dev,124.16.3177 (1997).84 Faure, S., de Santa Barbara, P, Roberts, D. J. & Whitman, M. Endogenous patterns of BMP signaling during early chick development. Developmental Biology 244, 44-65 (2002).85 Arnold, K. et al. Sox2+ Adult Stem and Progenitor Cells Are Important for Tissue Regeneration and Survival ofMice. Cell Stem Cell 9, 317-329, doi: 10.1016 / j. stem.2011.09.001 (2011).86 Snippert, H. J. et al. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell 143, 134-144, doi: 10.1016 / j. cell.2010.09.016 (2010).87 Chen, L., Guo, Q. & Li, J. Y. Transcription factor Gbx2 acts cell-nonautonomously to regulate the formation of lineage-restriction boundaries of the thalamus. Development 136, 1317- 1326, doi: 10.1242 / dev.030510 (2009).88 Madisen, L. et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nature Neuroscience 13, 133-140, doi: 10.1038 / nn.2467 (2010).89 Sunmonu, N. A., Li, K., Guo, Q. & Li, J. Y. H. Gbx2 and Fgf8 are sequentially required for formation of the midbrain-hindbrain compartment boundary. Development 138, 725-734, doi: 10.1242 / dev.055665 (2011).90 Jahn, H. M. et al. Refined protocols of tamoxifen injection for inducible DNA recombination in mouse astroglia. Set Rep-uk 8, 5913, doi : 10.1038 / s41598-018-24085-9 PMID - 29651133 (2018).91 Zovein, A. C. et al. Fate tracing reveals the endothelial origin of hematopoietic stem cells. Cell Stem Cell 3, 625-636 (2008).92 Ganat, Y. M. et al. Early Postnatal Astroglial Cells Produce Multilineage Precursors and Neural Stem Cells In Vivo. The Journal of Neuroscience 26, 8609-8621, doi:10.1523 / jneurosci.2532-06.2006 PMID - 16914687 (2006).93 Stent, G. S. The role of cell lineage in development. Philos Trans R Soc Lond B Biol Sci 312, 3-19, doi: 10.1098 / rstb.1985.0174 (1985).94 Waddington, C. H. Organisers and Genes. (Cambridge University Press, 1940).95 Ferrer- Vaquer, A. et al. A sensitive and bright single-cell resolution live imaging reporter of Wnt / p-catenin signaling in the mouse. BMC Dev Biol 10, 121, doi: 10.1186 / 1471-213X-10-121 (2010).96 Cordes, S. P. & Barsh, G. S. The mouse segmentation gene kr encodes a novel basic domain-leucine zipper transcription factor. Cell 9, 1025-1034, doi: 10.1016 / 0092- 8674(94)90033-7 (1994).97 Wiellette, E. L. & Sive, H. vhnfl and Fgf signals synergize to specify rhombomere identity in the zebrafish hindbrain. Development 130, 3821-3829, doi:10.1242 / dev.00572 (2003).98 Maves, L., Jackman, W. & Kimmel, C. B. FGF3 and FGF8 mediate a rhombomere 4 signaling activity in the zebrafish hindbrain. Development 129, 3825-3837, doi : 10.1242 / dev.129.16.3825 (2002).99 Marin, F. & Charnay, P. Hindbrain patterning: FGFs regulate Krox20 and mafB / kr expression in the otic / preotic region. Development 127, 4925-4935, doi: 10.1242 / dev.127.22.4925 (2000).100 Ye, W., Shimamura, K., Rubenstein, J. L., Hynes, M. A. & Rosenthal, A. FGF and Shh signals control dopaminergic and serotonergic cell fate in the anterior neural plate. Cell 93, 755- 766, doi: 10.1016 / s0092-8674(00)81437-3 (1998).101 Ribisi, S., Jr. et al. Ras-mediated FGF signaling is required for the formation of posterior but not anterior neural tissue in Xenopus laevis. Dev Biol 227, 183-196, doi: 10.1006 / dbio.2000.9889 (2000).102 Cox, W. G. & Hemmati-Brivanlou, A. Caudalization of neural fate by tissue recombination and bFGF. Development 121, 4349-4358 (1995).103 Corces, M. R. et al. An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nature Methods 528, 142, doi: 10.1038 / nmeth.4396 (2017).104 Murcia-Ramon, R. et al. Neuronal tangential migration from Nkx2.1 -positive hypothalamus. Brain Struct Funct 225, 2857-2869, doi: 10.1007 / s00429-020-02163-x (2020).105 Xu, Q., Tam, M. & Anderson, S. A. Fate mapping Nkx2.1 -lineage cells in the mouse telencephalon. J Comp Neurol 506, 16-29, doi: 10.1002 / cne.21529 (2008).106 Hernandez -Miranda, L. R., Muller, T. & Birchmeier, C. The dorsal spinal cord and hindbrain: From developmental mechanisms to functional circuits. Dev Biol 432, 34-42, doi: 10.1016 / j.ydbio.2016.10.008 (2017).107 Pattyn, A. et al. Coordinated temporal and spatial control of motor neuron and serotonergic neuron generation from a common pool of CNS progenitors. Genes Dev 17, 729- 737, doi: 10.1101 / gad.255803 (2003).108 Ericson, J. et al. Sonic hedgehog induces the differentiation of ventral forebrain neurons: A common signal for ventral patterning within the neural tube. Cell 81, 747-756, doi: 10.1016 / 0092-8674(95)90536-7 PMID - 7774016 (1995).109 Rash, B. G. & Grove, E. A. Patterning the Dorsal Telencephalon: ARole for Sonic Hedgehog? The Journal of Neuroscience 27, 11595-11603, doi: 10.1523 / jneurosci.3204-07.2007 PMID - 17959802 (2007).110 Backman, M. et al. Effects of canonical Wnt signaling on dorso-ventral specification of the mouse telencephalon. Developmental Biology 279, 155-168, doi: 10.1016Zj.ydbio.2004.12.010 PMID - 15708565 (2005).111 Gunhaga, L. et al. Specification of dorsal telencephalic character by sequential Wnt and FGF signaling. Nat Neurosci 6, 701-707, doi: 10.1038 / nnl068 (2003).112 Gray, P. A. Transcription factors and the genetic organization of brain stem respiratory neurons. J Appl Physiol (1985) 104, 1513-1521, doi: 10.1152 / japplphysiol.01383.2007 (2008).113 Qi, Y. et al. Combined small-molecule inhibition accelerates the derivation of functional cortical neurons from human pluripotent stem cells. Nature Biotechnology 35, 154, doi: 10.1038 / nbt.3777 (2017).114 Hannibal, J., Mikkelsen, J. D., Fahrenkrug, J. & Larsen, P. J. Pituitary adenylate cyclase- activating peptide gene expression in corticotropin-releasing factor-containing parvicellular neurons of the rat hypothalamic paraventricular nucleus is induced by colchicine, but not byadrenalectomy, acute osmotic, ether, or restraint stress. Endocrinology 136, 4116-4124, doi: 10.1210 / endo.136.9.7649120 (1995).115 Palkovits, M., Somogyvari-Vigh, A. & Arimura, A. Concentrations of pituitary adenylate cyclase activating polypeptide (PACAP) in human brain nuclei. Brain Res 699, 116-120, doi: 10.1016 / 0006-8993(95)00869-r (1995).116 Nasif, S. et al. Islet 1 specifies the identity of hypothalamic melanocortin neurons and is critical for normal food intake and adiposity in adulthood. Proc Natl Acad Sci USA 112, El 861- 1870, doi: 10.1073 / pnas.1500672112 (2015).117 Zhuang, S. et al. Expression of Isll during mouse development. Gene Expr Patterns 13, 407-412, doi:10.1016 / j.gep.2013.07.001 (2013).118 Pattyn, A., Morin, X., Cremer, H., Goridis, C. & Brunet, J. F. Expression and interactions of the two closely related homeobox genes Phox2a and Phox2b during neurogenesis.Development 124, 4065-4075, doi: 10.1242 / dev.124.20.4065 (1997).119 Sieber, M. A. et al. Lbxl acts as a selector gene in the fate determination of somatosensory and viscerosensory relay neurons in the hindbrain. JNenrosci 27, 4902-4909, doi : 10.1523 / JNEUROSCI.0717-07.2007 (2007).120 Chen, T. W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295-300, doi:10.1038 / naturel2354 (2013).121 Maddah, M. et al. A non-invasive platform for functional characterization of stem-cell- derived cardiomyocytes with applications in cardiotoxicity testing. Stem Cell Reports 4, 621-631, doi: 10.1016 / j.stemcr.2015.02.007 (2015).122 Zhai, J. et al. Primate gastrulation and early organogenesis at single-cell resolution. Nature, 1-7, doi: 10.1038 / s41586-022-05526-y (2022).123 Williams, R. M., Lukoseviciute, M., Sauka-Spengler, T. & Bronner, M. E. Single-cell atlas of early chick development reveals gradual segregation of neural crest lineage from the neural plate border during neurulation. Elife 11, doi: 10.7554 / eLife.74464 (2022).124 Wagner, D. E. et al. Single-cell mapping of gene expression landscapes and lineage in the zebrafish embryo. Science 360, eaar4362, doi:10.1126 / science.aar4362 PMID - 29700229 (2018).125 Paxton, C. N., Bleyl, S. B., Chapman, S. C. & Schoenwolf, G. C. Identification of differentially expressed genes in early inner ear development. Gene Expr Patterns 10, 31-43, doi: 10.1016 / j.gep.2009.11.002 (2010).126 Li, Y, Allende, M. L., Finkelstein, R. & Weinberg, E. S. Expression of two zebrafish orthodenticle-related genes in the embryonic brain. Meeh Dev 48, 229-244, doi: 10.1016 / 0925- 4773(94)90062-0 (1994).127 Pani, A. M. et al. Ancient deuterostome origins of vertebrate brain signalling centres. Nature 483, 289, doi:10.1038 / naturel0838 PMID - 22422262 (2012).128 Lowe, C. J. Molecular insights into deuterostome evolution from hemichordate developmental biology. Curr Top Dev Biol 141, 75-117, doi: 10.1016 / bs.ctdb.2020.12.002 (2021).129 Metzis, V. et al. Nervous System Regionalization Entails Axial Allocation before Neural Differentiation. Cell 175, 1105-1118.el 117, doi:10.1016 / j.cell.2018.09.040 (2018).130 Simon, C. S. & Hadjantonakis, A. K. Top to Tail: Anterior-Posterior Patterning Precedes Regional Nervous System Identity. Cell 175, 905-907, doi : 10.1016 / j . cell.2018.10.040 (2018).131 Henrique, D., Abranches, E., Verrier, L. & Storey, K. G. Neuromesodermal progenitors and the making of the spinal cord. Development 142, 2864-2875, doi: 10.1242 / dev.119768 (2015).132 Pani, A. M. et al. Ancient deuterostome origins of vertebrate brain signalling centres. Nature 483, 289-294, doi: 10.1038 / naturel0838 (2012).133 Loh, K. M. et al. Mapping the Pairwise Choices Leading from Pluripotency to Human Bone, Heart, and Other Mesoderm Cell Types. Cell 166, 451-467, doi: 10.1016 / j. cell.2016.06.011 (2016).134 Schneider-Maunoury, S. et al. Disruption of Krox-20 results in alteration of rhombomeres 3 and 5 in the developing hindbrain. Cell 75, 1199-1214, doi: 10.1016 / 0092-8674(93)90329-0 (1993).135 Miao, Y. et al. Next-Generation Surrogate Wnts Support Organoid Growth and Deconvolute Frizzled Pleiotropy In Vivo. Cell Stem Cell 27, 840-851 e846, doi: 10.1016 / j.stem.2020.07.020 (2020).136 Moreau, M. X., Saillour, Y, Cwetsch, A. W., Pierani, A. & Causeret, F. Single-cell transcriptomics of the early developing mouse cerebral cortex disentangle the spatial and temporal components of neuronal fate acquisition. Development 148, doi:10.1242 / dev,197962 (2021).137 Lavado, A. & Oliver, G. Proxl expression patterns in the developing and adult murine brain. Dev Dyn 236, 518-524, doi : 10.1002 / dvdy.21024 (2007).138 Abelian, A., Vernier, B., Retaux, S. & Medina, L. Similarities and differences in the forebrain expression of Lhxl and Lhx5 between chicken and mouse: Insights for understanding telencephalic development and evolution. J Comp Neurol 518, 3512-3528, doi: 10.1002 / cne.22410 (2010).139 Acampora, D. et al. Progressive impairment of developing neuroendocrine cell lineages in the hypothalamus of mice lacking the Orthopedia gene. Genes Dev 13, 2787-2800, doi: 10.1101 / gad.13.21.2787 (1999).140 Yu, H., Rubinstein, M. & Low, M J. Developmental single-cell transcriptomics of hypothalamic POMC neurons reveal the genetic trajectories of multiple neuropeptidergic phenotypes. Elife 11, doi: 10.7554 / eLife.72883 (2022).141 Hashimoto, H. et al. Distribution of the mRNA for a pituitary adenylate cyclase- activating polypeptide receptor in the rat brain: an in situ hybridization study. J Comp Neurol 371, 567-577, doi:10.1002 / (SICI)1096-9861(19960805)371:4<567::AID-CNE6>3.0.CO;2-2 (1996).142 Alaynick, W. A., Jessell, T. M. & Pfaff, S. L. Snapshot: spinal cord development. Cell 146, 178-178. el71 (2011).143 Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 9, 676-682, doi: 10.1038 / nmeth.2019 (2012).144 Andrews, S. FastQC: A Quality Control Tool for High Throughput Sequence Data, <http: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / > (2010).145 Krueger, F. Trim Galore, <https: / / www.bioinformatics.babraham.ac.uk / projects / trim galore / > (2012).146 Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nature Methods 9, 357-359, doi: 10.1038 / nmeth, 1923 (2012).147 Zhang, Y. et al. Model-based analysis of ChlP-Seq (MACS). Genome Biol 9, R137 (2008).148 Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology 15, 550, doi: 10.1186 / sl3059-014-0550-8 (2014).149 Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol Cell 38, 576-589 (2010).150 Thomson, J. A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145-1147 (1998).151 Ang, L. T. et al. Generating human artery and vein cells from pluripotent stem cells highlights the arterial tropism of Nipah and Hendra viruses. Cell 185, 2523-2541 e2530, doi : 10.1016 / j . cell .2022.05.024 (2022) .152 Davis, R. P. et al. Targeting a GFP reporter gene to the MIXL1 locus of human embryonic stem cells identifies human primitive streak-like cells and enables isolation of primitive hematopoietic precursors. Blood 111, 1876-1884 (2008).153 Chen, L., Guo, Q. & Li, J. Y. H. Transcription factor Gbx2 acts cell-nonautonomously to regulate the formation of lineage-restriction boundaries of the thalamus. Development 136, 1317- 1326, doi: 10.1242 / dev.030510 PMID - 19279136 (2009).154 Madisen, L. et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nature Neuroscience 13, 133-140, doi: 10.1038 / nn.2467 PMID - 20023653 (2009).155 Snippert, H. J. et al. Intestinal Crypt Homeostasis Results from Neutral Competition between Symmetrically Dividing Lgr5 Stem Cells. Cell 143, 134-144, doi: 10.1016 / j. cell.2010.09.016 PMID - 20887898 (2010).156 Choi, H. M. T. et al. Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust. Development 145, devl65753-165122, doi: 10.1242 / dev.165753 (2018).157 Kim, Y. S. etal. Crystal structure of the natural ani on-conducting channelrhodopsin GtACRl. Nature 561, 343-348, doi : 10.1038 / s41586-018-0511 -6 (2018).158 Pijuan-Sala, B. et al. A single-cell molecular map of mouse gastrulation and early organogenesis. Nature, 1-6, doi: 10.1038 / s41586-019-0933-9 (2019).159 Williams, R. M., Lukoseviciute, M., Sauka-Spengler, T. & Bronner, M. E. Single-cell atlas of early chick development reveals gradual segregation of neural crest lineage from the neural plate border during neurulation. eLife 11, e74464, doi: 10.7554 / elife.74464 PMID - 35088714 (2022).EXAMPLESEXPERIMENTAL MODEL AND SUBJECT DETAILSCell culture000256 All cells in this study were cultured in standard incubator conditions (20% 02, 5% CO2 and 37 °C).Human pluripotent stem cell lines000257 Multiple hPSC lines, including both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) were used in this study: wild-type Hl, H7, and H9 hESCsl50 (WiCell), wild-type SUN004.1 and SUN004.2 hiPSCsl51 (provided by Hiromitsu Nakauchi’s laboratory), HES3 MIXL1-GFP hPSCsl52 (provided by Edouard Stanley’s laboratory), and WTC AAVSl-CAG-GCaMP6f hiPSCs!21 (provided by Bruce Conklin’s laboratory). Of note, SUN004.1 and SUN004.2 represent two different, clonally-derived hiPSClines reprogrammed from peripheral blood mononuclear cells obtained from the same healthy adult volunteer (SUN004) 151.Mouse models000258 Gbx2CreER / + micel53 were originally developed by, and generously provided by, James Li’s laboratory (JAX #022135) and maintained on a CD1 background, heterozygous for the targeted allele. Mice carrying the Gbx2CreER allele were identified by PCR as described by Jackson Laboratory. Sox2CreER / + mice85 were originally developed by Konrad Hochedlinger’ s laboratory, were obtained from Jackson Laboratory (JAX #017593), and maintained on a C57BL / 6 background, heterozygous for the targeted allele. Mice carrying the Sox2CreER allele were identified by PCR as described by Jackson Laboratory. R26-CAG- LoxP-STOP-LoxP-tdTomato (“Ail4”) micel54 were originally developed by the Allen Brain Institute, were obtained from Jackson Laboratory (JAX #007914), and were maintained homozygous for the targeted allele. R26-Confetti mice 155 were originally developed by Hans Clevers’s laboratory, were obtained from Jackson Laboratory (JAX #017492), and were respectively maintained as homozygous for the targeted allele. Tcf / Lef:H2B-GFP mice95 were originally developed by Anna-Katerina Hadjantonakis’s laboratory, were obtained from the Jackson Laboratory (#013752), and maintained on a CD1 background, heterozygous for the targeted allele.Mouse husbandry and lineage tracing000259 Mice of the desired genetic backgrounds were mated, leading to timed pregnancies. Noon on the day a vaginal plug was detected was designated as E0.5. For Gbx2CreER-mediated lineage tracing, 1 mg of (Z)-4-Hydroxytamoxifen (Sigma) in corn oil was delivered via intraperitoneal injection to pregnant female mice at the relevant labeling timepoint. For Sox2CreER-mediated lineage tracing, 0.5 mg of (Z)-4-Hydroxytamoxifen was administered as described above.In situ hybridization of whole mount mouse embryos000260 Hybridization chain reaction v3.0 (HCR3)156 was used to perform whole-mount fluorescent in situ hybridization (FISH) of mouse embryos. Embryos were dissected in ice-cold4% paraformaldehyde and subsequently fixed either for 1 hour (E8.0 and earlier), or alternatively overnight (E8.25 onward), prior to methanol dehydration. Hybridization mRNA probes, amplifiers, and buffers were obtained from Molecular Instruments, and the protocol for whole mount mouse embryos were followed as per the manufacturer’s instructions with the following modifications: Proteinase K digestions varied with embryonic stage; E6.5: 5 min; E7.5: 7.5 min; E8.5: 10 min; E9.5: 15 min. Embryos were incubated in SSCT + DAPI prior to mounting.Immunostaining of whole mount mouse embryos000261 Embryos were dissected in ice-cold PBS and fixed overnight on a rocker at 4C in 1% paraformaldehyde. They were washed 3 x 10 min in PBS at room temperature before being blocked overnight in embryo blocking buffer (3% BSA, 0.2% Tween-20 in PBS). Embryos were then incubated with primary antibodies (diluted in embryo blocking buffer) for 48 hours at 4C with gentle rocking. Subsequently, the embryos were washed for 4 hours in PBST (0.2% Tween- 20 in PBS) before being transferred to incubation with secondary antibodies for 48 hours at 4C with gentle rocking. Embryos were cleared overnight in Rapiclear 1.49 (Sunjin labs) before being mounted, also in Rapiclear 1.49.Imaging of mouse embryos000262 Images were collected at room temperature on Olympus FV3000, Leica SP8, or Zeiss LSM980 confocal microscopes using 405, 488, 514, 561, and 640 nm wavelengths and 4X / 0.16, 10X / 0.4, and 20X / 0.75 dry objectives, and 40X / 1.3 oil objective. Data was displayed with FIJI ImageJ software.Computational analysis of confetti images000263 Confetti images were imported into Imaris 10 for quantification. Cells were identified in CFP, YFP, and RFP channels separately using the Spots function. Clones were identified as spots within 50 pm of each other in the same channel using the Imaris SplitSpots plugin function. A surface feature of the forebrain and midbrain were generated from the Otx2 immunofluorescent signal in far-red. Clones were defined relative to the forebrain / midbrain surface element. Clones with less than 2 cells across the midbrain-hindbrain boundary were assigned to the region in which the majority of cells resided.Basement membrane matrix for cell culture experiments000264 hPSCs were maintained and differentiated on cell culture plates that been pre-coated with Geltrex basement membrane matrix, as described previouslyl51. Geltrex (Thermo Fisher) was diluted 1 : 100 in DMEM / F 12 (Thermo Fisher) and was kept at 4°C. To coat cell culture plates, Geltrex solution was added to cell culture plastics for at least 1 hour at 37°C. The volume of Geltrex solution added was roughly equivalent to half the working volume of the well or dish (e g., 1 mL or 0.5 mL of Geltrex solution was added per well of a 6-well or 12-well plate, respectively). After coating with Geltrex, the basement membrane solution was aspirated, leaving behind a thin film; subsequently, cells were plated on the Geltrex-coated cell culture plastics.Human pluripotent stem cell culture000265 Undifferentiated hPSCs were maintained as described previously 151 in either mTeSRl or mTeSR Plus media (StemCell Technologies) supplemented with 1% penicillin / streptomycin (Thermo Fisher), on Geltrex-coated cell culture plates. When the undifferentiated hPSC cultures became partially confluent, undifferentiated hPSCs were passaged by treating them with EDTA (Versene, Thermo Fisher) for 7 minutes at room temperature. After removal of EDTA, mTeSR was re-added and the plate was manually scraped to generate clumps, which were reseeded onto new Geltrex-coated plates. hPSC differentiation into ectodermal derivatives000266 The day before beginning differentiation (“day -1” [D-l ]), hPSCs were enzymatically dissociated using Accutase (Thermo Fisher) and plated as single cells from 30,000-100,000 cells / cm2 on Geltrex-coated cell culture plates in mTeSR or mTeSR Plus + 1% penicillin / streptomycin + ROCK inhibitor thiazovivin (1 pM, Tocris). Inclusion of ROCK inhibitor was critical to ensure survival of single cells during seeding. After 24 hours, cells were ready for subsequent differentiation. All differentiations up to 7 days were conducted in Chemically Defined Medium 2 (CDM2), described previously66,67. Briefly, CDM2 is comprised of 50% IMDM + GlutaMAX (Thermo Fisher, 31980-097), 50% F12 + GlutaMAX (Thermo Fisher), polyvinyl alcohol (1 mg / mL, Sigma) + chemically defined lipid concentrate(1% v / v, Thermo Fisher), 1 -thioglycerol (450 gM, Sigma), recombinant human insulin (0.7 gg / mL, Sigma), human transferrin (15 gg / mL, Sigma), penicillin / streptomycin (1% v / v, Thermo Fisher). To create CDM2, polyvinyl alcohol was brought into suspension by gentle warming and magnetic stirring, and the media was filtered through a 0.22 pm filter prior to use. Definitive ectoderm (DO-1, 24 hours): DO hPSCs were briefly washed before differentiation with DMEM / F12 to remove any traces of mTeSR Plus. They were then differentiated toward definitive ectoderm in CDM2 media supplemented with A8301 (1 gM, Tocris), Trametinib (250 nM, Cellagen Technology), C59 (1 gM, Tocris), and LDN193189 (100 nM, Stemgent) for 24 hours. Subsequently, DI definitive ectoderm was used for either anterior neural ectoderm, posterior neural ectoderm, border ectoderm, or surface ectoderm induction. Anterior neural ectoderm (DI -2, 24 hours): DI definitive ectoderm cells were resplit using Accutase (Thermo Fisher) and plated as single cells on Geltrex-coated cell culture plates in CDM2 supplemented with A8301 (1 gM, Tocris), LDN193189 (100 nM, Stemgent), C59 (1 gM, Tocris), and Thiazovivin (1 gM, Tocris) for 24 hours. Thiazovivin was added to promote cell survival after enzymatic passaging. For experiments ending after two days of differentiation, cells were plated at 37,500 cells / cm2, and for experiments ending after more than 2 days of differentiation (i.e., towards a forebrain fate), 75,000 cells / cm2 were plated. After 24 hours of anterior neural ectoderm media, cells were used for either forebrain or midbrain induction. Posterior neural ectoderm (DI -2, 24 hours): DI definitive ectoderm cells were resplit using Accutase (Thermo Fisher) and plated as single cells on Geltrex-coated cell culture plates in CDM2 supplemented with A8301 (1 gM, Tocris), LDN193189 (100 nM, Stemgent), TTNPB (50 nM, Tocris), FGF2 (20 ng / mL, R&D Systems), XAV939 (1 gM, Tocris), and Thiazovivin (1 gM, Tocris) for 24 hours. Thiazovivin was added to promote cell survival after enzymatic passaging. For experiments ending after two days of differentiation, cells were plated at 37,500 cells / cm2, and for experiments ending after more than 2 days of differentiation (i.e., toward a hindbrain fate), 150,000 cells / cm2 were plated. After 24 hours of posterior neural ectoderm media, cells were used for hindbrain induction. Border ectoderm (Dl-2, 24 hours): DI definitive ectoderm cells were resplit using Accutase (Thermo Fisher) and plated as single cells at 37,500 cells / cm2 on Geltrex-coated cell culture plates in CDM2 supplemented with A8301 (1 gM, Tocris), PD0325901 (100 nM, Tocris), CHIR99012 (3 gM, Tocris), BMP4 (0.5 ng / mL, R&D Systems), and Thiazovivin (1 gM, Tocris) for 24 hours. Thiazovivin was added to promote cell survivalafter enzymatic passaging. Surface ectoderm (DI -2, 24 hours): DI definitive ectoderm cells were resplit using Accutase (Thermo Fisher) and plated as single cells at 37,500 cells / cm2 on Geltrex-coated cell culture plates in CDM2 supplemented with A8301 (1 pM, Tocris), PD0325901 (100 nM, Tocris), BMP4 (30 ng / mL, R&D Systems), C59 (1 pM, Tocris), and Thiazovivin (1 pM, Tocris) for 24 hours. Thiazovivin was added to promote cell survival after enzymatic passaging. Forebrain (D2-4, 48 hours): D2 anterior neural ectoderm cells were briefly washed with DMEM / F12 prior to addition of forebrain media. D3 forebrain media consisted of CDM2 supplemented with XAV939 (1 pM, Tocris), LDN193189 (100 nM, Stemgent), and FGF2 (20 ng / mL, R&D Systems), and was changed after 24 hours into D4 forebrain media, which consisted of CDM2 supplemented with XAV939 (1 pM) and LDN192189 (100 nM). Subsequently, forebrain cells could be further differentiated toward either dorsal forebrain or ventral forebrain fates. Midbrain (D2-4, 48 hours): D2 anterior neural ectoderm cells were briefly washed with DMEM / F12 prior to addition of midbrain media. D3 midbrain media consisted of CDM2 supplemented with CHIR99021 (3 pM, Tocris) and was changed after 24 hours into D4 midbrain media, which consisted of CDM2 supplemented with CHIR99021 (3 pM) and FGF2 (20 ng / mL, R&D Systems).000267 Hindbrain (D2-4, 48 hours): D2 posterior neural ectoderm cells were briefly washed with DMEM / F12 prior to addition of hindbrain media. D3 and D4 hindbrain media consisted of CDM2 supplemented with CHIR99021 (0.75 pM, Tocris) and FGF2 (20 ng / mL, R&D Systems). Hindbrain media was replaced after 24 hours. Subsequently, hindbrain cells could be further differentiated toward either dorsal hindbrain or ventral hindbrain fates.000268 Dorsal Forebrain (D4-7, 72 hours): D4 forebrain progenitors were briefly washed with DMEM / F12 prior to addition of dorsal forebrain media. D5, D6, and D7 dorsal forebrain media consisted of CDM2 supplemented with CHIR99021 (2 pM, Tocris), Vismodegib (150 nM, Selleck Chemicals), and PD173074 (100 nM, Tocris). Dorsal forebrain differentiation was conducted for 72 hours, and dorsal forebrain media was replaced every 24 hours.000269 Ventral Forebrain (D4-7, 72 hours): D4 forebrain progenitors were briefly washed with DMEM / F12 prior to addition of ventral forebrain media. D5, D6, and D7 dorsal forebrain media consisted of CDM2 supplemented with XAV939 (1 pM, Tocris) and SAG 21K (5 nM, Tocris). Ventral forebrain differentiation was conducted for 72 hours, and ventral forebrain media was replaced every 24 hours.000270 Dorsal Hindbrain (D4-7, 72 hours): D4 hindbrain progenitors were briefly washed with DMEM / F12 prior to addition of dorsal hindbrain media. D5, D6, and D7 dorsal hindbrain media consisted of CDM2 supplemented with CHIR99021 (2 pM, Tocris), Vismodegib (150 nM, Selleck Chemicals), and LDN193189 (100 nM, Tocris). Dorsal hindbrain differentiation was conducted for 72 hours, and dorsal hindbrain media was replaced every 24 hours.000271 Ventral Hindbrain (D4-7, 72 hours): D4 hindbrain progenitors were briefly washed with DMEM / F12 prior to addition of ventral hindbrain media. D5, D6, and D7 ventral hindbrain media consisted of CDM2 supplemented with XAV939 (1 pM, Tocris) and SAG 21K (5 nM, Tocris). Ventral hindbrain differentiation was conducted for 72 hours, and ventral hindbrain media was replaced every 24 hours.000272 Neuronal differentiation (D7-14, 7 days): hPSC-derived day 7 dorsal forebrain, day 7 ventral forebrain, day 7 dorsal hindbrain, and day 7 ventral forebrain progenitors were subsequently differentiated into neurons for 7 days. Neuronal differentiation media was modified from a previously-reported methodi 13. From day 8-14, cells were differentiated in BDNF (20 ng / ml, PeproTech), GDNF (20 ng / ml, PeproTech), Forskolin (10 pM, Tocris), 2-Phospho-L- ascorbic acid (200 pg / ml, Sigma- Aldrich), DAPT (lOpM, R&D Systems) with different basal media used on each day of differentiation. The general approach was to gradually transition differentiating cells from CDM2 to Neurobasal differentiation media. Media composition was as follows: 1) D7-8 media (i.e., first 24 hours of neuronal differentiation) contained: 25% Neurobasal media (see below) + 75% CDM2 media was added; 2) D8-9 media (i.e., second 24 hours of neuronal differentiation): 50% Neurobasal media + 50% CDM2 media was added; 3) D9-10 (i.e., third 24 hours of neuronal differentiation), 75% Neurobasal media + 25% CDM2 media was added; 4) DI 0-14 (i.e., remaining 96 hours of neuronal differentiation), 100% Neurobasal media. In other words, throughout the entire neuronal differentiation phase, cells were exposed to the same concentration of neuronal differentiation signals (BDNF, GDNF, Forskolin, 2-Phospho-L-ascorbic acid, and DAPT) while a gradual change in basal media occurred. “Neurobasal media” is comprised of Neurobasal media (Thermo Fisher) supplemented with 1% v / v N2 (Thermo Fisher), 2% v / v B27 w / o Vitamin A (Thermo Fisher), and 1% v / v penicillin / streptomycin (Thermo Fisher). The media was passed through a sterilizing 0.22 pM filter before use.000273 Finally, as a negative control, hPSCs were differentiated into mid primitive streak as described previously 133, with Activin A (30 ng / mL) + BMP4 (40 ng / mL) + CHIR99021 (6 pM) + FGF2 (20 ng / mL) in CDM2 basal media for 24 hours.Fluorescent labeling of hPSC-derived cells for coculture experiments000274 Day 2 anterior and posterior neural ectoderm (aNE and pNE, respectively) were generated as described. On day 2, aNE and pNE cells were lifted using Accutase (Thermo Fisher). aNE cells sat on ice while pNE cells were resuspended at 106 cells / mL in prewarmed PBS containing 1 pM ViaFluor 488 Cell Proliferation dye (Biotium) for 10 minutes at 37DC. Dye was prepared on the day of the labeling. Cells were washed with DMEM / F12 (Thermo Fisher) prior to pelleting. For mixed cultures, aNE and dye-labeled pNE were mixed at a 1 : 1 ratio prior to plating and subsequent culture in either forebrain, midbrain, or hindbrain media, as described. On day 4, cells were fixed and stained as described prior to imaging.Quantitative PCR000275 Cells were lysed with RNeasy Lysis Buffer (Qiagen) and RNA was subsequently extracted using the RNeasy Plus Kit (Qiagen) as per the manufacturer’s instructions. RNA was reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qPCR was subsequently performed on the cDNA with the SensiFAST SYBR Lo-ROX Kit (Bioline) on a QuantStudio 5 qPCR machine (Thermo Fisher). qPCR primer sequences are provided in Table SI. The cycling parameters were as follows: initial dissociation (95 °C, 2 mins) followed by 40 cycles of amplification and SYBR signal detection (95 °C dissociation, 5 seconds; 60 °C annealing, 10 seconds; followed by 72 °C extension, 30 seconds), with a final series of steps to generate a dissociation curve at the end of each run. During qPCR data analysis, the fluorescence threshold to determine Ct values was set at the linear phase of amplification. Expression of all genes was normalized to the levels of the reference gene YWHAZ.Immunocytochemistry of hPSC-derived cell-types000276 Adherent cells were washed once with PBS, fixed for 10 minutes in 4% paraformaldehyde (Fisher Scientific) at room temperature, and then washed three more times with PBS. They were subsequently permeabilized with permeabilization / blocking solution (0.1%Triton-XlOO + 5% donkey serum + PBS) for 1 hour at 4 °C. Subsequently, primary antibodies were added in permeabilization / blocking solution overnight at 4 °C. Cells were then washed three times with PBS, and secondary antibodies were added in permeabilization / blocking solution for 1 hour at 4 °C. Cells were then...

Claims

CLAIMS1. A composition comprising a cell or plurality of cells, wherein the cells comprise SOX2 or a functional variant thereof and are deficient in NANOG.

2. The composition of claim 1, wherein the cell or plurality of cell are from about 12 to about 48 hours in culture.

3. The composition of either of claims 1 or 2 wherein the cell or plurality of cells are frozen.

4. The composition of claim 3, wherein the cell or plurality of cells are frozen at -200 degrees Celsius.

5. The composition of cany of claims 1 through 4, wherein the cell or plurality of cells comprise a cell membrane that is columnar or cuboidal in shape and wherein the composition further comprises an extracellular matrix peptide.

6. The composition of any of claims 1 through 5, wherein the cell or plurality of cells comprise OTX2 or a functional variant thereof.

7. The composition of claim 6, wherein the cell or plurality of cells are at least about 48 hours in culture.

8. The composition of any of claims 1 through 7, wherein the cell or plurality of cells comprise one or a combination of FOXG1, LHX2, SIX3, PAX6, or one or a combination of functional variants thereof that comprise at least about 75% sequence identity to FOXG1, LHX2, SIX3, PAX6.

9. The composition of claim 8, wherein the cell or plurality of cells are at least about 48 hours in culture.

10. The composition of any of claims 1 through 7, wherein the cell or plurality of cells comprise one or a combination of LHX2, EMX2, PAX6, or one or a combination of variants thereof that comprise at least about 75% sequence identity to LHX2, EMX2, PAX6.

11. The composition of claim 10, wherein the cell or plurality of cells are at least about 48 hours in culture.

12. The composition of either of claims 10 or 11, wherein the composition further comprises a WNT activator or derivative thereof.

13. The composition of claim 10, wherein the composition is free of a WNT inhibitor.

14. The composition of claim 13 further comprising one or a combination of an HH inhibitor, an FGF / ERK inhibitor, or a derivative thereof.

15. The composition of any of claims 1 through 14, wherein the cell or plurality of cells comprise one or a combination of NK.X2.1, GLI1 or one or a combination of functional variants thereof that comprise at least about 75% sequence identity to NK.X2.1, GLI1.

16. The composition of claim 15, wherein the cell or plurality of cells further comprise one or a combination of LHX2 and FOXG1, or one or a combination of functional variants thereof that comprise at least about 75% sequence identity to LHX2 and FOXG1.

17. The composition of any of claims 1 through 7, wherein the cell or plurality of cells comprise one or a combination of EN1, EN2, PAX2, PAX5 or one or a combination of functional variants thereof that comprise at least about 75% sequence identity to EN1, EN2, PAX2, PAX5.

18. The composition of any of claims 1 through 5, wherein the cell or plurality of cells comprises one or a combination of GBX2, HOXA1, HOXB1, or one or a combination offunctional variants thereof that comprise at least about 75% sequence identity to GBX2, H0XA1, EI0XB1.

19. The composition of claim 18, wherein the cells are at least about 36 hours in culture.

20. The composition of any of claims 17 through 19 further comprising a Rho Kinase inhibitor.

21. The composition of any of claims 18 through 20, wherein the cell or plurality of cells comprises one or a combination of MAFB, HNF1B, KROX20, MEIS1, MEIS2, HOXA2, H0XA3, HOXB2, HOXB3, or one or a combination of functional variants thereof that comprise at least about 75% sequence identity to MAFB, HNF1B, KROX20, MEIS1, MEIS2, HOXA2, H0XA3, HOXB2, HOXB3.

22. The composition of claim 18, wherein the cells are at least about 48 hours in culture.

23. The composition of any of claims 16 through 17 further comprising an FGF activator.

24. The composition of claim 18 or claim 23 further comprising one or a combination of TGFP pathway inhibitor, a BMP pathway inhibitor, a WNT inhibitor, an RA activator, or a derivative thereof.

25. The composition of any of claims 1 through 5 or 21 through 24, wherein the cell or plurality of cells comprises one or a combination of HNF1B, PAX6, PAX3, PAX7, NGN2, OLIG3, GDF7, GSX2, MSX1, or one or a combination of functional variants thereof that comprise at least about 75% sequence identity to HNF1B, PAX6, PAX3, PAX7, NGN2, OLIG3, GDF7, GSX2, MSX1.

26. A composition comprising a cell or plurality of cells, wherein the cell or plurality of cells comprise MAPT and SNAP25 or functional variants thereof.

27. The composition of claim 26, wherein the cell or plurality of cells are deficient in NANOG and SOX2.

28. The composition of either of claims 26 or 27, wherein the cell or plurality of cells are at least about 9 days in culture.

29. The composition of any of claims 26 through 28, wherein the cell or plurality of cells further comprise one or a plurality of neurites extending from their cellular body.

30. The composition of any of claims 26 through 29, wherein the cell or plurality of cells further comprise TBR1, RELN, EBF2, LHX1, LHX5, VGLUT2, or one or a combination of functional variants thereof that comprise at least about 75% sequence identity to TBR1, RELN, EBF2, LHX1, LHX5, VGLUT2.

31. The composition of any of claims 26 through 29, wherein the cell or plurality of cells further comprise DLX1, SP8, LHX1, LHX5, LHX6, DLX5, VGAT, NKX2.1, PROXI, ISL1, POMC, or one or a combination of functional variants thereof that comprise at least about 75% sequence identity to DLX1, SP8, LHX1, LHX5, LHX6, DLX5, VGAT, NKX2.1, PROXI, ISL1, POMC.

32. The composition of any of claims 26 through 29, wherein the cell or plurality of cells further comprise TLX3, ASCL1, LBX1, LMX1B, LHX1, LHX5, PAX2, or one or a combination of functional variants thereof that comprise at least about 75% sequence identity to TLX3, ASCL1, LBX1, LMX1B, LHX1, LHX5, PAX2.

33. The composition of any of claims 26 through 29, wherein the cell or plurality of cells further comprise ISL1, CHAT, VACHT, ACHE, CHT, LHX3, SOX14, SIM1, EN1, GAD1, EVX1, PHOX2A, PHOX2B, HOXA1, HOXA3, or one or a combination of functional variants thereof that comprise at least about 75% sequence identity to ISL1, CHAT, VACHT, ACHE, CHT, LHX3, SOX 14, SIM1, EN1, GAD1, EVX1, PHOX2A, PHOX2B, H0XA1, HOXA3.

34. A composition comprising a cell or plurality of cells, wherein the cell or plurality of cells comprise PAX3, PAX7, and TFAP2A, or functional variants thereof.

35. The composition of claim 34, wherein the cell or plurality of cells are deficient in NANOG.

36. The composition of either of claims 34 or 35, wherein the cell or plurality of cells are at least about 36 hours in culture.

37. The composition of any of claims 34 through 36 further comprising a BMP activator and WNT activator, or a derivative thereof.

38. The composition of claim 37 further comprising a TGFp inhibitor and an FGF / ERK inhibitor, or derivative thereof.

39. A composition comprising a cell or plurality of cells, wherein the cell or plurality of cells comprise GATA3 and TFAP2A, or functional variants thereof.

40. The composition of claim 39, wherein the cell or plurality of cells are deficient in NANOG41. The composition of either of claims 39 or 40, wherein the cell or plurality of cells are at least about 36 hours in culture.

42. The composition of any of claims 39 through 41 further comprising a BMP activator and WNT inhibitor, or derivative thereof.

43. The composition of claim 42 further comprising a TGFp inhibitor and an FGF / ERK inhibitor, or derivative thereof.

44. The composition of any of claims 1 through 43, wherein the cell or plurality of cells are from a stem cell.

45. The composition of claim 44, wherein the stem cell is an induced pluripotent stem cell.

46. The composition of any of claims 1 through 45, wherein the cell or plurality of cells are human.

47. A pharmaceutical composition comprising a therapeutically affective amount of the composition of any of claims 1 through 46; and a pharmaceutically acceptable carrier.

48. A kit compri si ng :(a) the composition or pharmaceutical compositions of any of claims 1 through 47; and(b) tissue culture medium and / or tubing configured for transfer of cells.

49. The kit of claim 48 further comprising instructions to culture or transfer the cells.

50. The kit of either of claims 48 or 49, wherein the kit is frozen.

51. A method of inducing differentiation of a cell into a mammalian ectodermal progenitor cell comprising: (i) exposing one or a combination of inhibitors chosen from: a TGFp inhibitor, a BMP inhibitor, an FGF / ERK inhibitor, and a WNT inhibitor to the cell for a time period sufficient to differentiate the cell into an ectodermal progenitor cell.

52. The method of claim 51, wherein the cell is a stem cell.

53. The method of any of claims 51 or 52, wherein the cell is a human cell.

54. The method of any of claims 51 through 53, wherein the cell is a human pluripotent stem cell.

55. The method of any of claims 51 through 54, wherein the time period is from about 12 hours to about 24 hours.

56. The method of any of claims 51 through 55, wherein the ectodermal progenitor cell comprises SOX2; and is deficient in expression of one or a combination of: NANOG, H0XA1, H0XB1, GBX2, PAX3, GATA3, and TFAP2A.

57. A method of differentiating a cell into an anterior neural ectodermal cell comprising exposing the cell to one or a combination of inhibitors chosen from: a TGFP inhibitor, a BMP inhibitor, and a WNT inhibitor for a time period sufficient to differentiate the cell into an anterior neural ectodermal cell.

58. The method of claim 57, wherein the cell is an ectodermal progenitor cell comprising SOX2; and is deficient in expression of one or a combination of: NANOG, H0XA1, H0XB1, GBX2, PAX3, GATA3, and TFAP2A.

59. The method of any of claims 57 through 58, wherein the time period is from about 12 hours to about 24 hours.

60. The method of any of claims 57 through 59 further comprising exposing the cell to a Rho Kinase inhibitor contemporaneously with or in sequence with step (i).

61. The method of any of claims 57 through 60 further comprising seeding from about 35,000 cells / cm2of cell culture surface area to about 75,000 cells / cm2of cell culture surface area prior to exposing the cell to one or a combination of inhibitors.

62. A method of differentiating a cell into a forebrain progenitor cell comprising: (i) exposing the cell to one or a combination of inhibitors chosen from: a BMP inhibitor and a WNT inhibitor for a time period sufficient to differentiate the cell into a forebrain progenitor cell.

63. The method of claim 62, wherein the time period is from about 12 hours to about 48 hours.

64. The method of any of claims 62 through 63 further comprising exposing the cell to an FGF2 activator contemporaneously with or in sequence with step (i).

65. The method of claim 64, wherein the FGF2 activator is exposed to the cell for no more than about 24 hours in duration.

66. The method of any of claims 62 through 65, wherein the cell is an anterior ectodermal progenitor cell comprising SOX2 and 0TX2; and that is deficient in expression of one or a combination of: NANOG, H0XA1, H0XB1, GBX2, PAX3, GATA3 and TFAP2A.

67. A method of differentiating a cell into a dorsal forebrain progenitor cell comprising exposing the cell to one or a combination of modulators chosen from: a FGF / ERK pathway inhibitor, a Hedgehog inhibitor, and a WNT activator for a time period sufficient to differentiate the cell into a dorsal forebrain cell.

68. The method of claim 67, wherein the cell is an anterior ectodermal progenitor cell comprising SOX2 and 0TX2; and that is deficient in expression of one or a combination of: NANOG, H0XA1, H0XB1, GBX2, PAX3, GATA3 and TFAP2A.

69. The method of any of claim 67 or 68, wherein the dorsal forebrain cell comprises SOX2, LHX2, EMX2, and PAX6; and is deficient in NANOG, H0XA1 and H0XB1.

70. The method of any of claims 67 through 69, wherein the step of exposing is performed in from about 48 hours to about 72 hours.

71. A method of differentiating a cell into a midbrain progenitor cell comprising (i) exposing the cell to one or a combination of modulators chosen from: an FGF / ERK pathway activator, and a WNT activator for a time period sufficient to differentiate the cell into a dorsal hindbrain cell.

72. The method of claim 71, wherein the cell is an ectodermal progenitor cell comprising SOX2 and 0TX2; and that is deficient in expression of one or a combination of: NANOG, H0XA1, H0XB1, GBX2, PAX3, GATA3 and TFAP2A.

73. The method of any of claim 71 or 72, wherein the midbrain progenitor cell comprises SOX2, 0TX2, EN1, EN2, PAX2, PAX5; and is deficient in NANOG, H0XA1 and H0XB1.

74. The method of any of claims 71 through 73, wherein the step of exposing is performed in from about 36 hours to about 48 hours.

75. A method of inducing differentiation of a cell into a posterior neural progenitor cell comprising: (i) exposing the cell to one or a combination of modulators chosen from: a TGFp inhibitor, a BMP inhibitor, a WNT inhibitor, a FGF pathway activator, and a retinoic acid pathway activator for a time period sufficient to differentiate the cell into a posterior neural progenitor.

76. The method of claim 75, wherein the cell comprises SOX2 and is deficient in NANOG.

77. The method of any of claims 75 or 76, wherein the cell is a human cell.

78. The method of any of claims 75 through 77, wherein the time period is from about 12 hours to about 24 hours.

79. The method of any of claims 75 through 78 further comprising exposing the cell to a Rho Kinase inhibitor contemporaneously with or in sequence with step (i).

80. The method of any of claims 75 through 79 further comprising seeding from about 35,000 cell / cm2 of cell culture surface area to about 75,000 cells / cm2cell culture surface area prior to exposing the cell to one or a combination of modulators.

81. The method of any of claims 75 through 79, wherein the posterior neural progenitor cell comprises SOX2, GBX2, H0XA1, H0XB1; and is deficient in expression of one or a combination of: NANOG, PAX3, GATA3 and TFAP2A.

82. A method of inducing differentiation of a cell into a hindbrain neural progenitor cell comprising: (i) exposing the cell to one or a combination of modulators chosen from a WNT activator and an FGF pathway activator for a time period sufficient to differentiate the cell into a hindbrain neural progenitor cell.

83. The method of claim 82, wherein the hindbrain neural progenitor cell comprises one or a combination of SOX2, GBX2, MAFB, HNF1B, KROX20, MEIS1, MEIS2, H0XA1, H0XA2, H0XA3, H0XB1, H0XB2, H0XB3 and is deficient in NANOG.

84. The method of claims 82 or 83, wherein the step of exposing is performed from about 24 hours to about 48 hours.

85. The method of any of claims 82 through 84, wherein the cell comprises SOX2, GBX2, H0XA1, H0XB1; and is deficient in expression of one or a combination of: NANOG, PAX3, GATA3 and TFAP2A.

86. A method of inducing differentiation of a cell into a dorsal hindbrain cell comprising: (i) exposing the cell to one or a combination of modulators chosen from a WNT activator, a BMP inhibitor, and a Hedgehog inhibitor for a time period sufficient to differentiate the cell into a dorsal hindbrain cell.

87. The method of claim 86, wherein the time period is from about 48 to about 72 hours.

88. The method of any of claims 86 or 87, wherein the cell comprises one or a combination of SOX2, GBX2, MAFB, HNF1B, KROX20, MEIS1, MEIS2, H0XA1, H0XA2, H0XA3, H0XB1, H0XB2, H0XB3 and is deficient in NANOG.

89. The method of any of claims 86 through 88, wherein the dorsal hindbrain cell comprises one or a combination of: SOX2, HNF1B, PAX6, PAX3, PAX7, NGN2, OLIG3, GDF7, GSX2, MSX1; and wherein the dorsal hindbrain cell is deficient in NANOG.

90. A method of inducing differentiation of a cell into a hindbrain ventral cell comprising: (i) exposing the cell to one or a combination of modulators chosen from a WNT inhibitor and a Hedgehog activator for a time period sufficient to differentiate the cell into a ventral hindbrain cell.

91. The method of claim 90, wherein the time period is from about 48 to about 72 hours.

92. The method of any of claims 90 or 91, wherein the cell comprises one or a combination of: SOX2, GBX2, MAFB, HNF1B, KROX20, MEIS1, MEIS2, H0XA1, HOXA2, H0XA3, HOXB1, HOXB2, HOXB3 and is deficient in NANOG.

93. The method of any of claims 90 through 92, wherein the ventral hindbrain cell comprises one or a combination of: SOX2, HNF1B, NKX2.2, NKX6.1, NKX6.2, OLIG2, PHOX2B; and wherein the dorsal hindbrain cell is deficient in NANOG.

94. A method of inducing differentiation of a cell into a neuron comprising: (i) exposing the cell to one or a combination of modulators chosen from a neurotrophic growth factor for a time period sufficient to differentiate the cell into a neural cell.

95. The method of claim 94, wherein the step of exposing further comprises exposure to one or a combination of: a NOTCH pathway inhibitor, ascorbic acid and an adenylyl cyclase activator.

96. The method of any of claims 94 or 95, wherein the neural cell comprises:(a) one or a combination of MAPT, SNAP25, TBR1, RELN, EBF2, LHX1, LHX5, VGLUT2; and is deficient in NANOG; or(b) one or a combination of MAPT, SNAP25, DLX1, SP8, LHX1 , LHX5, LHX6, DLX5, VGAT, NKX2.1, PR0X1, ISL1, POMC; and is deficient in NANOG; or(c) one or a combination of MAPT, SNAP25, TLX3, ASCL1, LBX1, LMX1B, LHX1, LHX5, PAX2; and is deficient in NANOG; or(d) one or a combination of MAPT, SNAP25, ISL1, CHAT, VACHT, ACHE, CHT, LHX3, SOX14, SIM1, EN1, GAD1, EVX1, PH0X2A, PH0X2B, H0XA1, H0XA3; and is deficient in NANOG.

97. The method of any of claims 94 through 96, wherein the cell is chosen from: a dorsal forebrain progenitor cell, a ventral forebrain progenitor cell, a dorsal hindbrain progenitor cell, and a ventral hindbrain progenitor cell.

98. The method of any of claims 94 through 96, wherein the step of exposing is from about 5 to about 7 days.

99. A method of differentiating a cell into a non-neural ectodermal progenitor cell comprising; (i) exposing one or a combination of modulators chosen from: a TGF inhibitor, a BMP activator, an FGF / ERK inhibitor, and a WNT activator to the cell for a time period sufficient to differentiate the cell into an ectodermal progenitor cell for a time period sufficient to differentiate the cell into a neural cell.

100. The method of claim 99, wherein the time period is from about 12 hours to about 24 hours.

101. The method of any of claims 99 or 100 further comprising exposing the cell to a Rho Kinase inhibitor contemporaneously with or in sequence with step (i).

102. The method of any of claims 99 through 101 further comprising seeding from about 35,000 cells / cm2 of cell culture surface area to about 75,000 cells / cm2 of cell culture surface area prior to exposing the cell to one or a combination of inhibitors.

103. The method wherein the non-neural ectodermal progenitor cell comprises one of a combination of PAX3, PAX7, TFAP2A, or a functional variant thereof.

104. A method of differentiating a cell into a non-neural ectodermal cell comprising; (i) exposing one or a combination of modulators chosen from: a TGFp inhibitor, a BMP activator, an FGF / ERK inhibitor, and a WNT inhibitor to the cell for a time period sufficient to differentiate the cell into an ectodermal progenitor cell for a time period sufficient to differentiate the cell into a non-neural ectodermal cell.

105. The method of claim 104, wherein the time period is from about 12 hours to about 24 hours.

106. The method of any of claims 104 or 105 further comprising exposing the cell to a Rho Kinase inhibitor contemporaneously with or in sequence with step (i).

107. The method of any of claims 104 through 106 further comprising seeding from about 35,000 cells / cm2of cell culture surface area to about 75,000 cells / cm2of cell culture surface area prior to exposing the cell to one or a combination of inhibitors.

108. The method wherein the non-neural ectodermal cell comprises one of a combination of GATA3 and TFAP2A.

109. The method of claim 108, wherein the non-neural ectodermal cell is deficient in SOX2.

110. A method of culturing a cell comprising exposing the cell to one or more tissue culture mediums in Table 1.

111. A method of differentiating border ectoderm cells comprising culturing the border ectoderm in a culture media comprising ROCK inhibitor during seeding.

112. The method according to claim 11, wherein the ROCK inhibitor is thiazovivin.

113. A method of differentiating hPSCs or iPSCs comprising enzymatically dissociating hPSCs or iPSCs from their solid support and re-plating them as single cells in a culture medium comprising one or more ROCK inhibitors.

114. The method according to claim 113, wherein the ROCK inhibitor is thiazovivin.

115. A method of testing the effect of an agent on growth or viability of cells comprising exposing the cells to the agent and observing the cells for morphological changes and / or by measuring expression of mRNA or protein by performing one or a combination of: quantitative PCR, immunocytochemistry, flow cytometry, single-cell RNA sequencing and OMniATAC-seq for hindbrain cell markers.

116. The method according to claim 115, wherein the cells are cultured for about 72 hours.

117. The method according to claim 116, wherein the media is replaced about every 24 hours.

118. A method of testing the effect of an agenton hindbrain progenitor cells or dorsal hindbrain cells or ventral hindbrain cells comprising culturing the hindbrain progenitor cells in cell culture media comprising the agent and measuring expression of mRNA or protein in the cells by one or more of quantitative PCR, immunocytochemistry, flow cytometry, single-cell RNA sequencing and OMniATAC-seq for dorsal hindbrain cell markers.

119. The method according to claim 118, wherein the cells are cultured for about 72 hours.

120. The method according to claim 119, wherein the media is replaced about every 24 hours.

121. A method of testing the effect of an agent on differentiation of cells comprising culturing the cells in media comprising the test compound and detecting expression of mRNA or protein in the cultured cells by one or more of: quantitative PCR, immunocytochemistry, flow cytometry, single-cell RNA sequencing and OmniATAC-seq .

122. The method according to claim 121, wherein the cells are hindbrain progenitor cells cultured for about 72 hours.

123. The method according to claim 122, wherein the media is replaced about every 24 hours.

124. A method of testing the effect of an agent on cells comprising culturing the cells in cell culture media comprising the agent and detecting expression of mRNA or protein in the cultured cells by one or more of: quantitative PCR, immunocytochemistry, flow cytometry, single-cell RNA sequencing and OmniATAC-seq; and / or detecting or observing morphological changes to the cells after exposure to the agent125. The method according to claim 124, wherein the cells are cultured for a time period of about 7 days.

126. The method according to claim 125, wherein the basal media is gradually changed from CDM2 media to neurobasal media over the time period.

127. A method of differentiating posterior neural ectoderm into hindbrain progenitor cells comprising exposing the posterior neural ectoderm with activators of FGF and WNT.

128. The method according to claim 127, wherein the posterior neural ectoderm is cultured for about two days.

129. A method of generating hindbrain-specific motor neurons comprising culturing day-7 hPSC or iPSC-derived dorsal hindbrain and or ventral hindbrain neural progenitor cells with BDNF, GDNF, Forskolin and Vitamin C, while blocking NOTCH.

130. The method according to claim 129, wherein the neural progenitor cells are cultured for 7 days.

131. A method of generating posterior neural ectoderm comprising culturing human pluripotent stem cells or iPSCs under conditions that simultaneously activate FGF and RA alongside neural induction signals BMP, TGFP and WNT.

132. The method according to claim 131, wherein the culturing is performed in about two days.

133. A method for testing the effect of an agent on cells comprising culturing cells disclosed herein the agent with the cells; and observing the cultured cells for morphometric changes and / or performing by one or more of quantitative PCR, immunocytochemistry, flow cytometry, single-cell RNA sequencing and OmniATAC-seq.

134. An in vitro cell culture system comprising hindbrain motor neurons and a cell culture media.

135. The in vitro cell culture system according to claim 134, wherein the hindbrain motor neurons are dorsal hindbrain motor neurons or ventral hindbrain neurons.

136. An in vitro cell culture system comprising posterior neural ectoderm and a cell culture media.

Images