Methods for making and using differentiated nerve cells

Abstract

The present disclosure provides methods for differentiating stem and progenitor cells into nerve cells by a regimen that does not use a BMP4 pathway inhibitor that results in SMAD inhibition. Some aspects of the present disclosure relate to methods for differentiating stem cells or progenitor cells into nerve cells, the methods comprising (i) contacting stem cells or progenitor cells with a differentiation composition; and (ii) culturing the cells in the micropores to form spheroids. Other aspects relate to methods for differentiating stem cells or progenitor cells into nerve cells, the methods comprising: (i) contacting the stem cells or progenitor cells with a differentiation composition wherein the differentiation composition comprises an ALK inhibitor: one or more of DMH1, DMH2, K02288, A83-01, or a combination thereof; and (ii) culturing the cells in the micropores to form spheroids. Also described are nerve cells, spheroids, populations of cells or populations of spheroids produced by the methods according to the claims.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 329,713, filed on April 11, 2022, which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application includes a sequence listing submitted in XML format and incorporated herein by reference in its entirety. The XML copy created on April 11, 2023 is named "CLFRP0404WO_SL" and is 12 kilobytes in size. Background Art

[0005] Ⅰ. Technical Field

[0006] The present invention relates to the fields of cell biology and disease treatment.

[0007] II. Background

[0008] Cell populations that retain the ability to differentiate into many specialized cell types can be used to develop large numbers of lineage-specific differentiated cell populations. It is expected that these lineage-specific differentiated cell populations can be used for cell replacement therapy of patients suffering from diseases that result in loss of function of a defined cell population. In addition to their direct therapeutic value, lineage-specific differentiated cells are also valuable research tools for a variety of purposes, including in vitro screening assays for identifying, determining and testing functional specificity, or for testing the delivery of therapeutic molecules to treat cell lineage-specific diseases. For example, in the case of Parkinson's disease, it is the loss of midbrain dopaminergic (DA) neurons that leads to the appearance of disease symptoms. Therefore, methods for generating DA neuronal cells from multipotent cells are needed, as such cells can be used both in therapy and in disease models, for example, for identifying new treatments for treating neurodegenerative diseases. Summary of the invention

[0009] The present disclosure provides a method for differentiating stem cells and progenitor cells into neural cells by not using a BMP4 pathway inhibitor that causes SMAD inhibition. The method includes a method for differentiating stem cells or progenitor cells into neural cells, the method including: (i) contacting stem cells or progenitor cells with a differentiation composition; and (ii) culturing cells in microwells to form spheroids and / or neurospheres. A method for differentiating stem cells or progenitor cells into neural cells is also described, the method including (i) contacting stem cells or progenitor cells with a differentiation composition, wherein the differentiation composition comprises an ALK inhibitor: one or more of DMH1, DMH2, K02288, A83-01; and (ii) culturing cells in microwells to form spheroids and / or neurospheres. Neural cells, spheroids, cell groups, or spheroid groups produced by the methods described in the claims are also described. The method also includes a method for treating a disease in a mammalian subject, comprising administering to the subject a therapeutically effective amount of a neural cell, spheroid, or neurosphere of the present disclosure, a population of cells, spheroids, or neurospheres. Also described are methods of screening a test compound comprising: (a) contacting a test compound with a cell, spheroid, or neurosphere of the disclosure; and (b) measuring the function, physiology, or viability of the cell.

[0010] The term "spheroid" refers to cells in a three-dimensional spherical structure. The spheroid may be composed of or substantially composed of progenitor cells, such as or for example, at least

[0011] 40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85.86,87,88,89,90,91,92,93,94,95,96,97,98,99, or 100%

[0012] The cells of the invention may be progenitor cells. Neurospheres are composed of further differentiated cells, such as cells belonging to the neural cell lineage. Neurospheres may include or contain at least

[0013] 20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99, or 100%

[0014] (or any range derivable therein) are cells that are neural progenitor cells.

[0015] Compounds DMH1, DMH2, K02288 and A83-01 are also referred to as ALK inhibitors. The ALK inhibitor may consist of DMH1, DMH2, K02288 or A83-01. The ALK inhibitor may contain DMH2 or consist of DMH2. The ALK inhibitor may consist of DMH1 and DMH2. The ALK inhibitor may consist of K02288 and DMH2. The ALK inhibitor may consist of A8301 and DMH2.

[0016] Stem cells or progenitor cells may include induced pluripotent stem cells (induced pluripotent stem cells, iPSC) or embryonic stem (embryonic stem, ES) cells. Stem cells or progenitor cells may include hematopoietic stem cells or progenitor cells. Stem cells or progenitor cells may be further limited to totipotent stem cells, pluripotent stem cells or multipotent stem cells. Cells may include embryonic stem (ES) cells. Cells may be human cells or derived from human cells. Cells may be human ES cells. Human ES cells may include or be further limited to HS420 cells. Stem cells or progenitor cells may not include iPSC, ES cells, hematopoietic stem cells or progenitor cells, totipotent cells, pluripotent cells, multipotent stem cells, human cells, cells derived from human cells or HS420 cells.

[0017] Contacting the cells can include contacting the cells for a period of substantially continuous contact of about 1 to 7 days. The term "substantially continuous contact" refers to contact for at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the time during a period of time, but does not exclude brief periods of non-contact, such as periods in which the cells may undergo washing, replated, trypsinization, or changes in cell culture medium. A period of time, such as a period of time during which a compound or ALK inhibitor is in contact with a cell, can include at least, at most, about, or exactly

[0018] 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,5 100 days (or any range derivable therein). Contacting, e.g., contacting cells with a compound or ALK inhibitor as described herein, can be defined from a certain time period, e.g., from the addition of differentiation medium ( FIG. 12 ), where day 0 is the day that the cells are first contacted with the differentiation medium. Contacting can be defined from day 100 to day 101 (or any range derivable therein).

[0019] -14,-13,-12,-11,-10,-9,-8,-7,-6,-5,-4,-3,-2,-1,0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,3 0,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,or 72

[0020] starts on a certain day and ends on a specific day, such as

[0021] 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52 ,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,or 100

[0022] days, or any range derivable therein.

[0023] The cell can be contacted with 0.01 to 5 μM of a compound described herein and / or an ALK inhibitor. The cell can be contacted with at least, at most, about, or exactly

[0024] 0.01,0.02,0.03,0.04,0.05,0.06,0.07,0.08,0.09,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1,1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9,2,2.1,2.2,2.3,2.4,2.5,2.6,2.7,2.8,2.9,3,3.1,3.2,3.3, 3.4,3.5,3.6,3.7,3.8,3.9,4,4.1,4.2,4.3,4.4,4.5,4.6,4.7,4.8,4.9,5,5.5,6,6.5,7,7.5,8,8.5,9,9.5,10,10.5,11,11.5,12,12.5,13,13.5,14,14.5,15,15.5,16,16.5,17,17.5,18,18.5,19, or 20 μM

[0025] The cell may be contacted with 0.2 μM of a compound or ALK inhibitor described herein (or any range derivable therein). The cell may be contacted with 0.8 μM of a compound or ALK inhibitor described herein.

[0026] The method may also include contacting the cell with a Rho Kinase (ROCK) inhibitor. The method may not include contacting the cell with a ROCK inhibitor. The ROCK inhibitor may include Y27632. Other ROCK inhibitors that can be used in the methods of the present disclosure include Fasudil, Ripasudil, Netarsudil, RKI-1447, GSK429286A, and Y30141. The method may not include contacting the cell with Y27632, Fasudil, Ripasudil, Netarsudil, RKI-1447, GSK429286A, and / or Y30141. The cell may be contacted with 5 to 15 μM of a ROCK inhibitor. The cell may be contacted with at least, at most, about, or exactly

[0027] 1,1.5,2,2.5,3,3.5,4,4.5,5,5.5,6,6.5,7,7.5,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,30,35,40,45,50,55,60,65,70,75, or 80 μM, nM, or mM ROCK inhibitor (or any range derivable therein). The cell may be contacted with the ROCK inhibitor before contacting with the ALK inhibitor. The cell may be contacted with the ROCK inhibitor immediately before contacting with the ALK inhibitor. The contact with the ROCK inhibitor and the contact with the ALK inhibitor compound may overlap for a period of time. The contact with the ROCK inhibitor and the contact with the ALK inhibitor may be non-overlapping and include a time period between when the cell is in contact with the ROCK inhibitor and when the cell is in contact with the ALK inhibitor. The cell may be contacted with the ROCK inhibitor after the ALK inhibitor. The contact with the ROCK inhibitor and the contact with the ALK inhibitor may be non-overlapping and include the time period between when the cell is in contact with the ALK inhibitor and when the cell is in contact with the ROCK inhibitor. The time period may be at least, at most or about 1, 2, 3, 4, 5, 10, 15, 30 or 45 minutes or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 18 or 24 hours, or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days (or any range derivable therefrom). The cell may be contacted with the ROCK inhibitor for a period of 1 to 48 hours. The cell may be contacted with the ROCK inhibitor

[0028] 14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47, or 48 hours, or 2.5,3,3.5,4,4.5,5,5.5,6,6.5, or 7 days, or 1, 2, 3, 4, or 5 weeks (or any range derivable therein). Contact with a ROCK inhibitor can be defined as starting from a certain time period, for example, starting from the addition of differentiation medium (Figure 12), where day 0 is the day when the cells are first contacted with the differentiation medium. Contact can be defined as starting from a certain time period, for example, starting from the addition of differentiation medium (Figure 12), where day 0 is the day when the cells are first contacted with the differentiation medium. Contact can be defined as starting from a certain time period, for example, starting from the addition of differentiation medium (Figure 12), where day 0 is the day when the cells are first contacted with the differentiation medium.

[0029] -14,-13,-12,-11,-10,-9,-8,-7,-6,-5,-4,-3,-2,-1,0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,3 0,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,or72

[0030] starts on a certain day and ends on a specific day, such as

[0031] 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52 ,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,or 100

[0032] days, or any range derivable therein.

[0033] The method may not include contacting the cell, spheroid or neurosphere with one or more Smad inhibitors. The method may not include contacting the cell, spheroid or neurosphere with LDN193189 and / or SB431542. The method may not include contacting the cell, spheroid or neurosphere with LDN193189 and SB431542. The method may not include dual Smad inhibition or single Smad inhibition. The method may not include contacting the cell with Noggin protein or a Noggin regulator (e.g., an activator or repressor of Noggin or a direct activator or repressor of Noggin). The method may not include contacting the cell with a BMP4 inhibitor. The method may not include contacting the cell with Noggin and / or Chordin or its activator.

[0034] The nerve cell can be further defined as a dopaminergic neuron, a glutamatergic neuron, a serotonergic neuron, a cholinergic neuron, a GABAergic neuron, a motor neuron, an astrocyte or an oligodendrocyte. The nerve cell can be further defined as the nerve cell described herein. The term "dopaminergic neuron" or "DA neuron" refers to a neuron having the ability to produce dopamine (3,4-dihydroxyphenylethylamine). Dopaminergic neurons do not need to produce dopamine all the time, but only need to have the ability to produce dopamine. DA neurons can be DA neurons of the A8 group, A9 group, A10 group, A11 group, A12 group, A13 group, A14 group, A15 group, A16 group, Aaq group or telencephalon group. The nerve cell may not include dopaminergic neurons, glutamatergic neurons, serotonergic neurons, cholinergic neurons, GABAergic neurons, motor neurons, astrocytes or oligodendrocytes. The DA neurons may not include DA neurons of the A8 group, A9 group, A10 group, A11 group, A12 group, A13 group, A14 group, A15 group, A16 group, Aaq group, or telencephalic group.

[0035] Contacting cells with a compound or ALK inhibitor described herein may include culturing cells in a cell culture medium comprising the compound or ALK inhibitor. The cell culture medium may include one or more of: DMEM culture medium, neurobasal culture medium, GSK inhibitor, cAMP, GDNF, BDNF, amino acids, X-VIVO culture medium, antimicrobial agents, B-27 supplements, N-2 supplements and L-glutamine, sonic hedgehog protein (SHH), purmorphamin, FGF-8 protein (fibroblast growth factor 8), FGF-20, TGF-B3 and gamma secretase inhibitors. Gamma secretase inhibitors include, for example, DAPT (ref CAS 208255-80-5) and compound E (ref CAS209986-17-4). The method may not include contacting the cells with one or more of: DMEM medium, neurobasal medium, GSK inhibitors, cAMP, GDNF, BDNF, amino acids, X-VIVO medium, antimicrobial agents, B-27 supplements, N-2 supplements and L-glutamine, sonic hedgehog protein (SHH), purine amines, FGF-8 protein (fibroblast growth factor 8), FGF-20, TGF-B3 and gamma secretase inhibitors, DAPT (ref CAS208255-80-5), compound E (ref CAS 209986-17-4), GSK3 inhibitors or CHIR99021. The cells may also be contacted with a GSK3 (glycogen synthase kinase 3) inhibitor for a period of time. The GSK3 inhibitor may include CHIR99021. The GSK3 inhibitor may be added after 3 days after contact with the differentiation medium. The GSK3 inhibitor may be added 2, 3, 4, 5, 6, 7 or 8 days (or any range derivable therein) after contact with the differentiation medium. The time period may be 5 to 15 days. The time period may be 10 days. Contact with the GSK3 inhibitor, cAMP, GDNF, BDNF, amino acids, X-VIVO medium, antimicrobial agents, B-27 supplement, N-2 supplement, L-glutamine, sonic hedgehog protein (SHH), purine amines, FGF-8 protein (fibroblast growth factor 8), FGF-20, TGF-B3, gamma secretase inhibitors, DAPT (ref CAS208255-80-5), compound E (ref CAS209986-17-4) and / or CHIR99021 ...

[0036] -14,-13,-12,-11,-10,-9,-8,-7,-6,-5,4,-3,-2,-1,0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32, 33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71, or 72 days and ends on a particular day, e.g.

[0037] 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52 ,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69.70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,or 100

[0038] days, or any range derivable therein.

[0039] The method may include or further include contacting the cells with one or more of FHF8, SHH and purine amine for a period of time. The cells may be contacted with FHF8, SHH and / or purine amine 1 day after contact with the differentiation medium. The period of time may be 3 to 10 days. The period of time may be 7 days.

[0040] The method may include or further include contacting the cells with one or more of cAMP, GDNF, BDNF, TGFB3, FGF20 and a gamma secretase inhibitor for a period of time. The cells may be contacted with cAMP, GDNF, BDNF, TGFB3, FGF20 and / or a gamma secretase inhibitor 8 days after contact with the differentiation medium. The period of time may be 15 to 40 days.

[0041] Contact with GSK3 inhibitors, cAMP, GDNF, BDNF, amino acids, X-VIVO medium, antimicrobial agents, B-27 supplement, N-2 supplement, L-glutamine, sonic hedgehog protein (SHH), purine amines, FGF-8 protein (fibroblast growth factor 8), FGF-0, TGFB3, gamma secretase inhibitors, DAPT (ref CAS208255-80-5), compound E (ref CAS209986-17-4) and / or CHIR99021 can be performed in the first

[0042] -14,-13,-12,-11,-10,-9,-8,-7,-6,-5,-4,-3,-2,-1,0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32, 33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71, or 72 days and ends on a particular day, e.g.

[0043] 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52 ,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69.70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,or 100

[0044] days, or any range derivable therein.

[0045] The method may include or further include contacting the cell with ascorbic acid. The ascorbic acid may be in a concentration of 100 to 400 μM. The concentration of the ascorbic acid may be, is at least, or is at most

[0046] 20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,105,110,115,120,125,130,135,140,145,150,155,160,165,170,175,180,185,190,195,200,2 05,210,215,220,225,230,235,240,245,250,255,260,265,270,275,280,285,290,295,300,305,310,315,320,325,330,335,340,345,350,355,360,365,370 ,375,380,385,390,395,400,405,410,415,420,425,430,435,440,445,450,455,460,465,470,475,480,485,490,495,500,505,510,515,520,525,530,535, 540,545,550,555.560,565,570,575,580,585,590,595,600,605,610,615,620,625,630,635,640,645,650.655,660,665,670,675,680,685,690,695, or 700 μM

[0047] (or any range derivable therein). The concentration of ascorbic acid when contacted with the cells may be 200 μM. The cells may be contacted with ascorbic acid within a period of 8 to 15 days after contact with the differentiation medium. The cells may be contacted with ascorbic acid for the following, or at least the following, or at most the following time period after contact with the differentiation medium:

[0048] 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52 ,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99, or 100 days

[0049] (or any range derivable therein). The method may not include contacting the cells with ascorbic acid. The cells may be contacted with ascorbic acid for a period of 13 days after contact with the differentiation medium. The cells may be contacted with ascorbic acid for a period of 10 to 40 days. The cells may be contacted with ascorbic acid for the following, at least the following, or at most the following time periods:

[0050] 8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,5 5,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99, or 100 days,

[0051] or any range derivable therein.

[0052] -14,-13,-12,-11,-10,-9,-8,-7,-6,-5,-4,-3,-2,-1,0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,3 0,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,or72

[0053] starts on a certain day and ends on a specific day, such as

[0054] 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52 ,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,or 100

[0055] days, or any range derivable therein.

[0056] The differentiation composition may comprise an extracellular matrix or the method may further comprise contacting the cells, spheroids or neurospheres with an extracellular matrix for a period of time. The extracellular matrix may be laminin and / or Geltrex. The method may not comprise contacting the cells, spheroids or neurospheres with an extracellular matrix. The differentiation composition may not comprise an extracellular matrix. Other extracellular matrices that may or may not be included in the methods and compositions include collagen, elastin, fibronectin, hyaluronic acid, tenascin, hyaluoronan and proteoglycans. The differentiation composition may comprise an RGD peptide or the method may further comprise contacting the cells, spheroids or neurospheres with an RGD peptide for a period of time. The tripeptide Arg-Gly-Asp (RGD) is composed of arginine, glycine and aspartic acid. It was originally identified as an amino acid sequence within the extracellular matrix protein fibronectin that mediates cell attachment.

[0057] The cells, spheroids and / or neurospheres may be cultured under hypoxic conditions for a period of time. The period of time may be 1 to 60 days. The hypoxic conditions may comprise 0 to 10% oxygen. The hypoxic conditions may be 2 to 5% oxygen or may be 3% oxygen. The period of time may be 1 to 60 days. The hypoxic conditions may include, include at least, or include at most

[0058] 0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29, or 30%

[0059] Oxygen, or any range derivable therein. The time period can be

[0060] 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or any range derivable therein.

[0061] -14,-13,-12,-11,-10.-9,-8,-7,-6,-5,-4,-3,-2,-1,O,1,2,3,4,5,6,7,8 ,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,3 0,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69.70,71,or72

[0062] starts on a certain day and ends on a specific day, such as

[0063] 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52 ,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,or 100

[0064] days, or any range derivable therein.

[0065] The method may include or further include contacting the cell with a HIF-1α stabilizer. The HIF-1α stabilizer may include one or more of the following: dimethyloxalylglycine, FG4592, CoCl2, deferoxamine mesylate, cyclometallated iridium (III) metal complex 1a, 1-(imidazole-1-ylmethyl)-3,5-diphenylpyrazole, 3,5-diphenyl-1-(pyrazol-1-ylmethyl)pyrazole, N-[(3,5-diphenylpyrazol-1-yl)methyl]-N-phenylaniline, (3,5-diphenylpyrazol-1-yl)methyl]diethylamine and (3,5-diphenylpyrazol-1-yl)methyl]diisopropylamine. The method may not include contacting the cell with a HIF-1α stabilizer. The method may not include contacting the cells with dimethyloxalylglycine, FG4592, CoCl2, deferoxamine mesylate, cyclometallated iridium (III) metal complex 1a, 1-(imidazol-1-ylmethyl)-3,5-diphenylpyrazole, 3,5-diphenyl-1-(pyrazol-1-ylmethyl)pyrazole, N-[(3,5-diphenylpyrazol-1-yl)methyl]-N-phenylaniline, (3,5-diphenylpyrazol-1-yl)methyl]diethylamine and / or (3,5-diphenylpyrazol-1-yl)methyl]diisopropylamine. The HIF-1α stabilizer may be contacted with the cells for a period of time. The period of time may be

[0066] 1, 2, 3, 4, 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 days, or any range derivable therein. The HIF-1α stabilizer may be contacted with the cell for at least 1 day.

[0067] -14,-13,-12,-11,-10,-9,-8,-7,-6,-5,-4,-3,-2,-1,0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,3 0,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,or72

[0068] starts on a certain day and ends on a specific day, such as

[0069] 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52 ,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,or 100

[0070] days, or any range derivable therein.

[0071] The ratio of the number of spheroids or neurospheres to the volume of the cell culture medium may be about 1000 spheroids or neurospheres to 200 to 1000 μl of cell culture medium. The ratio of the number of spheroids or neurospheres to the volume of the cell culture medium may be about 1000 spheroids or neurospheres to 200 to 500 μl of cell culture medium. The ratio of the number of spheroids or neurospheres to the volume of the cell culture medium may be about, may be at most, or may be at least

[0072] 500,505,510,515,520,525,530,535,540,545,550,555,560,565,570,575,580,585,590,595,600,605,610,615,620,625,630,635,640,645,650,655,660,665,670, 675,680,685,690,695,700,705,710,715,720,725,730,735,740,745,750,755,760,765,770,775,780,785,790,795,800,805,810,815,820,825,830,835,840,845, 850,855,860,865,870,875,880,885,890,895,900,905,910,915,920,925,930.935,940,945,950,955,960,965,970,975,980,985,990,995,1000,1000,1100,1200, 1300,1400,1500,1600,1700,1800,1900,2000,2100,2200,2300,2400,2500,2600,2700,2800,2900,3000,3100,3200,3300,3400,3500,3600,3700,3800,3900,or 4000

[0073] spheroids or neurospheres than about, at least, or at most

[0074] 100,105,110,115,120,125,130,135,140,145,150,155,160,165,170,175,180,185,190,195,200,205,210,215,220,225,230,235,240,245,250,255,260,265,270,275,280,285,290,295,300,305,310,315,320,3 25,330,335,340,345,350,355,360,365,370,375,380,385,390,395,400,405,410,415,420,425,430,435,440,445,450,455,460,465,470,475,480,485,490,495,500,505,510,515,520,525,530,535,540,545,550 ,555,560,565,570,575,580,585,590,595,600,605,610,615,620,625,630,635,640.645,650,655,660,665,670,675,680,685,690,695,700,705,710,715,720,725,730,735.740,745,750,755,760,765,770,775,7 80,785,790,795,800,805,810,815,820,825,830,835,840,845,850,855,860,865,870,875,880,885,890,895,900,905,910,915,920,925,930,935,940,945,950,955,960,965,970,975,980,985,990,995, or 1000 μl

[0075] Cell culture medium, or any range derivable therein.

[0076] The ratio can be maintained for a period of at least day 0 to day 42, wherein day 0 is the day the cells are first contacted with the differentiation medium. The ratio can be maintained for a period of, at least, or at most:

[0077] 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49, or 50 days,

[0078] or any range derivable therein.

[0079] The stem cell or progenitor cell may comprise exogenously expressed Nurr-1 and / or Pitx3. The stem cell or progenitor cell may comprise a heterologous nucleic acid encoding Nurr-1 and / or Pitx3 protein or a functional fragment thereof. The neural cell may be further defined as Nestin+, Pax-6+ and Sox-1+ cells. The method may not include cells comprising Nurr-1 and / or Pitx3, or the cell may not comprise exogenous expression of Nurr-1 and / or pitx3 and / or a heterologous nucleic acid encoding Nurr-1 and / or Pitx3.

[0080] Culturing cells in microwells may include culturing cells on a substrate material comprising a hydrophilic and porous material layer having a plurality of persistent well-shaped indentations on its outer surface supported by a semipermeable membrane on its inner surface, wherein the well-shaped indentations have a diameter of 100 μm. 2 To about 1mm 2 The hole and about 100pm 2 To about 1mm 2 The bottom surface of the semipermeable membrane, wherein the hydrophilic and porous material layer has a porosity that allows oxygen and cell nutrients to pass through. The semipermeable membrane may have pores with a diameter of about 1 μm to about 200 nm, such as about 2 μm to about 40 nm. The semipermeable membrane may have a diameter of, at least, or at most

[0081] 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40，41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65.66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,13 2,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173,174,175,176,177,178,179,180,181,182,183,184,185,186,187,188,189,19 0,191,192,193,194,195,196,197,198,199,200,201,202,203,204,205,206,207,208,209,210,211,212,213,214,215,216,217,218,219,220,221,222,223,224,225,226,227,228,229,230,231,232,233,234,235,236,237,238,239,240,241,242,243,244,245,246,247,248 8, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, or 300 (or any range derivable therein) pm or nm pores.

[0082] After a period of contact with a compound or ALK inhibitor described herein, the percentage of non-neuronal cells in the cell culture, spheroid or neurosphere can be less than 30% of the cell population of the present disclosure. After a period of contact with a compound or ALK inhibitor described herein, the percentage of non-neuronal cells in the cell culture can be less than 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% (or any range derivable therein) of the cell population. The time period can be 4 to 8 days. The time period can be at least, at most, or about

[0083] 0.5, 1, 2, 3, 4, 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, or 30 days or any range derivable therein.

[0084] The term "3D cell culture" as used herein refers to cell culture in which cells are allowed to grow or interact with their surroundings in all three dimensions. This can be achieved by culturing cells on low adhesion plates, bioreactors, or small capsules. Cells grown in 3D culture can take the shape of spheroids or neurospheres as they grow.

[0085] Unless otherwise specified, the term "cell" as used herein may include spheroids and neurospheres since they are composed of cells.

[0086] The method may not include exposing the cell to the following

[0087] -14,-13,-12,-11,-10,-9,-8,-7,-6,-5,-4,-3,-2,-1,0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,4 4,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,or 100

[0088] day, or any range derivable therein, of exposure to: ALK inhibitors, DMH1, DMH2, K02288, A83-01, ROCK inhibitors, Y27632, fasudil, rosudil, nesadil, RKI-1447, GSK429286A, Y30141, LDN193189, SB431542, DMEM medium, neurobasal medium, GSK inhibitors, cAMP, GDNF, BDNF, amino acids, X-VIVO medium, antimicrobials, B-27 supplement, N-2 supplement and L-glutamine, SHH, purine amines, FGF-8 protein, FGF-20, TGF-B3 and gamma secretase inhibitors, DAPT (ref CAS208255-80-5), compound E (refCAS209986-17-4), GSK3 inhibitor, CHIR99021, dimethyloxalylglycine, FG4592, CoCl2, deferoxamine mesylate, cyclometallated iridium (III) metal complex 1a, 1-(imidazol-1-ylmethyl)-3,5-diphenylpyrazole, 3,5-diphenyl-1-(pyrazol-1-ylmethyl)pyrazole, N-[(3,5-diphenylpyrazol-1-yl)methyl]-N-phenylaniline, (3,5-diphenylpyrazol-1-yl)methyl]diethylamine and / or (3,5-diphenylpyrazol-1-yl)methyl]diisopropylamine.

[0089] The disease may include a neurodegenerative disease. The subject may be a human subject. The subject may be a mammal. The subject may include a laboratory animal, a pig, a rat, a goat, a rabbit, a cat, a dog, a horse, or a mouse. The spheroid or neurosphere may be dissociated prior to administration. The method may not include dissociating the spheroid or neurosphere prior to administration.

[0090] Throughout this application, the term "about" is used according to its plain and ordinary meaning in the arts of cellular and molecular biology to indicate that a value includes the standard deviation of error for the device or method employed to determine the value.

[0091] When used in conjunction with the term "comprising", the use of a noun without a quantifier may mean "one", but it also complies with the meaning of "one or more", "at least one", and "one or more than one".

[0092] As used herein, the terms "or / or" and "and / or / or" are used to describe multiple components that are combined or exclusive of each other. For example, "x, y and / or z" can refer to "x" alone, "y" alone, "z" alone, "x, y and z", "(x and y) or z", "x or (y and z)", or "x or y or z". It is specifically contemplated that x, y, or z can be specifically excluded from an embodiment or aspect.

[0093] The words "comprising" (and any variations thereof), "having" (and any variations thereof), "including" (and any variations thereof), "characterized by" (and any variations thereof), or "containing" (and any variations thereof) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0094] Compositions and methods of use thereof may "comprise", "consist essentially of" or "consist of" any ingredient or step disclosed throughout this specification. The phrase "consisting of" excludes any unspecified element, step, or ingredient. The phrase "consisting essentially of" limits the scope of the subject matter to the specified substances or steps and those that do not materially affect its basic and novel characteristics. It is contemplated that some embodiments and aspects described in the context of the term "comprising", may also be implemented in the context of the term "consisting of" or "consisting essentially of".

[0095] It is particularly contemplated that any limitation discussed about one embodiment or aspect of the present invention may be applied to any other embodiment or aspect of the present invention. In addition, any composition of the present invention may be used for any method of the present invention, and any method of the present invention may be used to produce or utilize any composition of the present invention. Some aspects of the embodiments set forth in the examples are also embodiments that may be implemented in the context of some embodiments discussed elsewhere in different embodiments or elsewhere in the application (e.g., in summary of the invention, specific embodiments, claims, and accompanying drawings).

[0096] Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that although the detailed description and specific examples indicate some specific embodiments of the present invention, they are given by way of example only, because from this detailed description, various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] The following drawings form part of this specification and are included to further illustrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in conjunction with the detailed description of specific embodiments presented herein.

[0098] Figure 1A to 1B. Dopaminergic differentiation assay. Shown are culture methods including 3D-immersion (A) and 3D-AirLiwell (B).

[0099] Figure 2 .Dopaminergic neurospheres are more homogenous and standardized.

[0100] Figure 3 Expression of neural precursor and neuronal markers by qPCR (top) and expression of midbrain and dopaminergic-specific markers by qPCR (bottom). Figure 3 Better neural induction and specification of some markers for 3D-ALi neurospheres were shown.

[0101] Figure 4 . Gene expression fold changes of 3D-ALi / 3D-i after 6 weeks of differentiation by RNA-seq. Figure 4 Higher expression of dopaminergic precursor markers but lack of neuronal maturation were shown by 3D-ALi.

[0102] Figure 5 . Gene expression fold changes of 3D-ALi / 3D-i after 6 weeks of differentiation by RNA-seq. Neurospheres cultured by 3D-ALi showed downregulation of some cell proliferation markers after 6 weeks of culture.

[0103] Figure 6 . Expression of tyrosine hydroxylase by western blotting. 3D-ALi has the ability to produce dopamine but exhibits a lack of maturation, which was determined by western blotting and HPLC.

[0104] Figure 7 . Dopamine Dosage by HPLC. 3D-ALi has the ability to produce dopamine but exhibits a lack of maturation as determined by western blot and HPLC.

[0105] Figure 8 .3D-Ali neurospheres are electrophysiologically functional and have more stable and homogenous electrical signals.

[0106] Fig. 9 .3D-Ali dopaminergic neurospheres cultured on extracellular matrix have the ability to mature.

[0107] Fig.10 .3D-Ali dopaminergic neurons observed by electron microscopy were more homogeneous and pure.

[0108] Fig.11 The chemical structures of DMH1, DMH2, A83-01 and K02288 are shown.

[0109] Fig. 12A B to B illustrate some methodological aspects for differentiating stem cells into neural cells using 3D culture.

[0110] Fig.13Ato C. Single-cell RNA-seq analysis of dopaminergic neurospheres at 6 weeks.

[0111] Fig.14 . Quantitative PCR analysis of dopaminergic neurospheres at 6 weeks.

[0112] Fig.15 .3D-Immersed neurospheres produced more dopamine than 3D-Ali neurospheres, as measured by HPLC.

[0113] Fig.16 The electrical signals of neurons contained in 3D-Ali neurospheres are more similar to physiology and are highly standardized.

[0114] Fig.17 .Experimental design and modification of mature solutions.

[0115] Fig.18 .Effects of hypoxia and laminin on dopaminergic maturation.

[0116] Fig.19 . Testing of volume:neurosphere ratio and its effect on dopaminergic maturation.

[0117] Fig. 20 .Design of a plan for adding antioxidants.

[0118] Fig.21A to B. Effect of antioxidant addition on DA maturation. Fig.21A Shown is the gross morphology of neurospheres with added antioxidants. Fig.21B Shown are the effects of certain markers when neurospheres were cultured with antioxidants in the 3D-Ali method.

[0119] Fig. 22 Dopaminergic differentiation with the addition of Alk-I compounds in the 3D-Ali protocol. The concentrations of the compounds tested were 0.2 μM and 0.8 μM.

[0120] Fig.23A to D. Quantitative PCR results: dopaminergic differentiation with the addition of Alk-I compounds in the 3D-Ali protocol. Shown are qPCR results for (A) Ki-67 and Nestin; (B) Pax6 and B3-Tub; (C) Lmx1a and Nurr-1; and (D) Map-2 and TH at weeks 2 and 4. Fig.23AThe data of each group of 12 bars shown in D represent from left to right: 1: control day 0, 2: LDN193189 / SB431542, 3: DMSO 0.2 μM, 4: DMSO 0.8 μM, 5: DMH1 0.2 μM, 6: DMH2 0.2 μM, 7: A-8301 0.2 μM, 8: K02288 0.2 μM, 9: DMH1 0.8 μM, 10: DMH2 0.8 μM, 11: A-8301 0.8 μM, 12: K02288 0.8 μM. Fig.23D The 11 bars in the middle bottom bar graph represent data from left to right: 1: control day 0, 2: LDN193189 / SB431542, 3: DMSO 0.2 μM, 4: DMSO 0.8 μM, 5: DMH1 0.2 μM, 6: DMH2 0.2 μM, 7: A-8301 0.2 μM, 8: K022880.2 μM, 9: DMH1 0.8 μM, 10: DMH2 0.8 μM, and 11: A-8301 0.8 μM. DETAILED DESCRIPTION OF THE INVENTION

[0122] The formation of neural ectoderm is a key step in the differentiation of pluripotent stem cells into neural cells and tissues. The present disclosure provides an effective alternative to the method using the Smad inhibition scheme (various combinations of Noggin, LDN193189 and SB431542). The inventors hypothesize that differentiating progenitor cells into mature neurons in three-dimensional cell culture will provide advantages over differentiation methods in two-dimensional cell culture. These advantages include: 1) no cell separation (which can induce chromosomal changes and increase the risk of canceration of cells); 2) reduction in cell mortality due to enzymatic cell separation; 3) simplification of cell culture protocols; 4) improved standardization and homogeneity of transplanted cells; 5) cells are more representative of physiological neural tissue; and 6) increased yield of neuronal differentiated cells.

[0123] I. Definition

[0124] The term "differentiation" used with respect to cells in a differentiated cell system refers to a process in which a cell differentiates from one cell type (e.g., a pluripotent, totipotent, or multipotent differentiable cell) into another cell type (e.g., a fully differentiated cell). More generally, the term "differentiation" refers to a process in which an unspecialized stem cell or precursor cell acquires the characteristics of a specialized or fully differentiated cell (e.g., a brain, heart, liver, or muscle cell). Differentiation is controlled by the interaction of the cell's genes with extracellular physical and chemical conditions, typically through signal transduction pathways involving proteins embedded in the cell surface.

[0125] Progenitor cells in the present disclosure are included in somatic cells. Progenitor cells are multipotent cells. Totipotent cells, multipotent cells and pluripotent cells can be "stem cells", which can differentiate into one or more different cell types. The terms "stem cell", "embryonic stem cell", "induced pluripotent stem cell" have been described above.

[0126] In this specification, "stem cells" refer to cells that can be cultured in vitro and differentiated into cells of various lineages constituting the body. Specifically, they include ES cells, pluripotent stem cells derived from fetal primordial germ cells (EG cells: Proc Natl Acad Sci USA. 1998, 95: 13726-31), pluripotent stem cells derived from testes (GS cells: Nature, 2008, 456: 344-9), induced pluripotent stem cells derived from somatic cells (induced pluripotent stem cells; iPS cells), and human pluripotent somatic stem cells (neural stem cells), preferably iPS cells and ES cells, more preferably iPS cells.

[0127] II. ALK Inhibitors

[0128] Pluripotent stem cells (PSC) derived from embryos or induced by somatic cells have the ability to differentiate into a wide variety of cell types for tissue modeling and cell therapy. In vitro simulation of embryonic development provides the best method for producing differentiated cells with defined characteristics. The first natural specialization of embryonic tissue occurs during the gastrulation stage, with the differentiation of the three germ layers ectoderm, mesoderm and endoderm, from which all adult tissues will be derived.

[0129] Various combinations between Noggin, LDN 193189 and SB 431542 are commonly used and are often referred to as "dual Smad inhibition". It represents the most widely used method to date for inducing early neural specialization of PSCs in vitro. Herein, a report of a differentiation method is described that does not include "dual Smad inhibition" but rather achieves differentiation by inducing neural specialization of pluripotent stem cells in vitro using 4 chemicals that inhibit ALK: DMH1, DMH2, K02288 and A83-01 (Table 1).

[0130] Table 1: Compounds

[0131]

[0132] III. Cell Source

[0133] The present disclosure relates to the differentiation of the starting population of stem cells or progenitor cells to neural cells. Stem cells or progenitor cells can be cells described herein and / or cells derived from sources described herein. Stem cells or progenitor cells can be ES cells. It is contemplated that ES cells derived from any warm-blooded animal, preferably mammals, can be used. Some examples of mammals include mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cattle, horses, goats, monkeys and humans. Some preferred examples of ES cells include ES cells derived from humans. Stem cells or progenitor cells may not include cells derived from or taken from human fetuses.

[0134] Some specific examples of ES cells include ES cells of mammals etc. established by culturing early embryos before implantation, ES cells established by culturing early embryos prepared by nuclear transplantation of somatic cell nuclei, and ES cells obtained by changing genes on chromosomes of these ES cells by genetic modification methods. Each ES cell can be prepared according to a method generally performed in the relevant field or known literature.

[0135] Mouse ES cells were established in 1981 by Evans et al. (1981, Nature 292: 154-6) and Martin GR et al. (1981, Proc Natl Acad Sci 78: 7634-8), and can be purchased from, for example, Sumitomo Dainippon Pharma Co., Ltd (Osaka, Japan) and the like.

[0136] Human ES cells were established in 1998 by Thomson et al. (Science, 1998, 282: 1145-7), and are available from the WiCell Institute (available on the World Wide Web at wisell.org / , Madison, Wis., USA), the National Institutes of Health, Kyoto University, etc., and can be purchased from, for example, Cellartis (available on the World Wide Web at cellartis.com / , Sweden), etc.

[0137] The stem cell or progenitor cell can be an iPSC (also referred to as an iPS cell). As an iPS cell, an iPS cell derived from any warm-blooded animal, preferably a mammal, can be used. Some examples of mammals include mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cattle, horses, goats, monkeys and humans. Some preferred examples of iPS cells include iPS cells derived from humans.

[0138] Some specific examples of iPS cells include cells that have acquired pluripotency in ES cells, which can be obtained by introducing multiple genes into somatic cells such as skin cells, etc. For example, iPS cells obtained by introducing Oct3 / 4 gene, Klf4 gene, c-Myc gene and Sox2 gene, and iPS cells obtained by introducing Oct3 / 4 gene, Klf4 gene and Sox2 gene (Nat Biotechnol 2008; 26: 101-106). In addition to these, methods for further reducing transgenes (Nature. 2008 Jul. 31; 454 (7204): 646-50), methods using low molecular weight compounds (Cell Stem Cell. 2009 Jan. 9; 4 (1): 16-9, Cell Stem Cell. 2009 Nov. 6; 5 (5): 491-503), methods using transcription factor proteins instead of genes (Cell Stem Cell. 2009 May 8; 4 (5): 381-4), etc. The generated iPS cells can be used in the present invention regardless of the method of their generation.

[0139] Some examples of human iPS cell lines specifically include the 253G1 strain (an iPS cell line prepared by expressing OCT4 / SOX2 / KLF4 in skin fibroblasts of a 36-year-old woman), the 201B7 strain (an iPS cell line prepared by expressing OCT4 / SOX2 / KLF4 / c-MYC in skin fibroblasts of a 36-year-old woman), the 1503-iPS (297A1) (an iPS cell line prepared by expressing OCT4 / SOX2 / KLF4 / c-MYC in skin fibroblasts of a 73-year-old woman), the 1392-iPS (297F1) (an iPS cell line prepared by expressing OCT4 / SOX2 / KLF4 / c-MYC in skin fibroblasts of a 56-year-old man), the NHDF-iPS (297L1) (an iPS cell line prepared by expressing OCT4 / SOX2 / KLF4 / c-MYC in skin fibroblasts of a newborn boy), and the like.

[0140] IV. Cell Culture Methods

[0141] Included herein are methods for differentiating stem cells or progenitor cells or the starting population of stem cells and / or progenitor cells into neural cells. The methods of the present disclosure may include or also include culturing cells in a culture medium as defined herein and / or include or also include supplements and components as described herein. These methods will be discussed in more detail below. It is also specifically contemplated that the methods of the present disclosure may not include culturing cells in a culture medium as described herein or with supplements as described herein.

[0142] Cell culture can be 3-dimensional (3D cell culture). The 3D environment provides a balance between the accumulation of paracrine factors and the renewal of nutrients in the extracellular matrix. The derivation of neurons in 3D also has the following advantages: i) the use of low volumes of culture medium, and therefore the amount of new neuroactive substances used for determination is small; and ii) the choice of multiplexing (96-well plate format), which is essential for automated screening activities.

[0143] In order to realize the three-dimensional characteristics of cell culture, cells can be cultivated or differentiated in matrix, support or micropore. In principle, suitable matrix or support that can be used for three-dimensional cell culture are known to professionals. Therefore, such matrix or support can be any matrix or support. For example, matrix or support can be an extracellular matrix comprising natural molecules or synthetic polymers, biological and synthetic mixtures (hybrid), metals, ceramics and bioactive glass or carbon nanotubes.

[0144] Some exemplary natural extracellular matrix molecules include collagen, basement membranes (e.g., laminin or fibrin), alginate, chitosan, hyaluronic acid, silk fibroin, cellulose acetate, casein, chitin, fibrinogen, gelatin, elastin, or poly(hydroxyalkanoate). Synthetic extracellular matrix polymers include modified forms of hyaluronic acid (HA), modified forms of poly(ethylene glycol, PEG), self-assembling protein hydrogels, poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), polyurethane, or PGS. Biological and synthetic mixtures may include, for example, polycaprolactone-chitosan, PLLA-hydroxyapatite, hydroxyapatite-bioglass-ceramic, poly(hydroxyalkanoate)-bioglass, hydroxyapatite-collagen, PCL-gelatin, or PCL-collagen. Exemplary metals include tantalum, magnesium and its alloys, titanium and its alloys or nitinol (nickel and titanium alloys). Some examples of ceramic and bioactive glass matrix / scaffold include titanium and tricalcium phosphate, hydroxyapatite and tricalcium phosphate, bioactive silicate glass (SiO2--Na2O--CaO--P2O5), hydroxyapatite and bioglass, calcium phosphate glass or phosphate glass. Carbon nanotubes can be constructed using graphite ranging from 0.4 to 2nm. Carbon nanotubes may include CNT-polycaprolactone, CNT-ceramic matrix, 45S5 bioglass-CNT, CNT inlaid with gelatin hydrogel, CNT-TiO2, CNT-laminin, CNT or CNT-TGF-β grafted with polyacrylic acid.

[0145] Matrix or support can also be hydrogel, for example matrix gel (matrigel), fibrin gel or alginate gel. Optionally, growth factors and other molecules can be added to matrix gel. Matrix gel can also be mixed with culture medium. For example, matrix gel can be diluted with the medium as described herein or in the embodiments. Matrix gel can also be BD Matrigel.

[0146] The three-dimensional cell culture used in the present invention may not include organoid culture. Organoids are three-dimensional tissue structures, usually generated from pluripotent stem cells (PSCs), but also, for example, from neuroepithelial stem cells, which self-organize and reproduce complex aspects of their organ counterparts, ranging from physiological processes to regeneration and disease.

[0147] The method may include contacting the cells or spheroids with an activator of the Hedgehog signaling pathway, or the differentiation medium may include an activator of the Hedgehog signaling pathway. The "Hedgehog signaling pathway" or "SHH pathway" is well known in the art and has been described, for example, in Choudhry et al. (2014) "Sonic hedgehog signaling pathway: a complex network." Ann Neurosci. 21(1): 28-31. Hedgehog ligands, including, for example, Sonic Hedgehog, Indian hedgehog, and / or Desert hedgehog, bind to receptors, including, for example, patched or patched-smoothed receptor complexes, which induce downstream signaling cascades. Downstream target genes of SHH signaling include GLI1, GL12, and / or GL13. Therefore, the term "activator of the Hedgehog signaling pathway" also refers to an activator of any of the above molecules that form part of the signaling pathway.

[0148] The method may include contacting the cells or spheroids with an activator of SHH, or the differentiation medium may include an activator of SHH. Exemplary activators of sonic hedgehog (SHH) signaling include purmorphamine (PMA; 2-(1-naphthyloxy)-6-(4-morpholinoanilino)-9-cyclohexylpurine 9-cyclohexyl-N-[4-(4-morpholino)phenyl]-2-(1-naphthyloxy), CAS No. 483367-10-8), SHH, smoothened agonist (SAG; 3-chloro-N-[trans-4(methylamino)cyclohexyl]-N-[[3-(4-pyridyl)phenyl]methyl]-benzo[b]thiophene-2-carboxamide, CAS No. 912545-86-9) and Hh-Ag 1.5 (3-chloro-4,7-difluoro-N-(4-(methylamino)cyclohexyl)-N-(3-(pyridin-4-yl)benzyl)benzo[b]thiophene-2-carboxamide, CAS No.: 612542-14-0). Therefore, the SHH pathway activator can be purine amine. The SHH pathway activator can also be a recombinant or truncated form of SHH that retains the SHH pathway activation function, such as SHHC24II.

[0149] The method may include contacting the cell or spheroid with a neurotrophic factor, or the differentiation medium may include a neurotrophic factor. As used herein, the term "neurotrophic factor" relates to a family of proteins that regulate the survival, development and function of neurons. Exemplary neurotrophic factors include insulin-like growth factor 1 (Insulin-like growth factor, IGF), fibroblast growth factor (Fibroblast growth factor, FGF), transforming growth factor β (Transforming growth factor, TGF), leukemia inhibitory factor (Leukemia inhibitory factor, LIF), nerve growth factor (nerve growth factor, NGF), brain-derived neurotrophic factor (brain-derivedneurotrophic factor, BDNF), neurotrophin-3 (neurotrophin-3, NT-3), neurotrophin-4 (neurotrophin-4, NT-4) and GDNF ligand family and ciliary neurotrophic factor (ciliary neurotrophicfactor, CNTF). The GDNF ligand family includes glial cellline-derived neurotrophic factor (GDNF), neurturin (NRTN), artemin (ARTN) and persephin (PSPN).

[0150] The method may include contacting the cells or spheroids with an antioxidant, or the differentiation medium may include an antioxidant. Antioxidants are molecules that inhibit the oxidation of other molecules. The terms "oxidation" and "antioxidants" are well known in the art and have been described in, for example, Nordberg J, Arner ES. (2001) "Reactive oxygen species, antioxidants, and the mammalian thioredoxin system." Free Radic Biol Med. 31 (11): 1287-312. In short, oxidation is a chemical reaction involving the loss of electrons or the increase of oxidation states. Oxidation reactions can produce free radicals. These free radicals can then start chain reactions. When a chain reaction occurs in a cell, it can cause damage or death to the cell. Antioxidants terminate these chain reactions by removing free radical intermediates and inhibit other oxidation reactions. Therefore, antioxidants refer to inhibitors of molecules involved in cellular oxidation processes.

[0151] Some exemplary antioxidants include ascorbic acid, superoxide dismutase 1, superoxide dismutase 2, superoxide dismutase 3, glutathione, lipoic acid, epigallocatechin gallate, curcumin, melatonin, hydroxytyrosol, ubiquinone, catalase, vitamin E or uric acid. Thus, the antioxidant may be ascorbic acid.

[0152] The method may include contacting the cells or spheroids with an activator of the activin / transforming growth factor-β (TGF-β) signaling pathway, or the differentiation medium may include an activator of the activin / transforming growth factor-β signaling pathway. The activin / TGF-β signaling pathway is known in the art and includes, for example, receptor ligands, including, for example, TGFB1, TGFB2, TGFB3, activin A, activin B, activin AB, and / or NODAL, binding to a heterotetrameric receptor complex consisting of two type I receptor kinases (including, for example, TGFBR2, ACVR2A, and / or ACVR2B) and two type II receptor kinases (including, for example, TGFBR1, ACVR1 B, and / or ACVR1C). Exemplary activators of the activin / TGF-β3 signaling pathway include TGF-β1, TGF-β2, TGF-β3, activin A, activin B, activin AB, or nodal. Thus, an activator of the activin / TGF-β signaling pathway may be TGF-β3.

[0153] The method may include contacting the cells or spheroids with a cAMP analog, or the differentiation medium may include a cAMP analog. Such cAMP analogs are compounds having physical, chemical, biochemical or pharmacological properties similar to cyclic adenosine monophosphate (cAMP). Exemplary cAMP analogs include forskolin, 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8CPT-2Me-cAMP), 8-chloro-cAMP (8-Cl-cAMP), Bucladesine, Rp-adenosine.3.,5.,-cyclic monothiophosphate sodium salt (Rp-cAMPS), Sp-8-hydroxyadenosine.3.,5.,-cyclic monothiophosphate sodium salt (Sp-80H-cAMPS) and Rp8-hydroxyadenosine.3.,5.,-cyclic monothiophosphate sodium salt (Rp-80H-cAMPS) or dbcAMP. Thus, the cAMP analog may be dbcAMP.

[0154] The method may comprise contacting the cells or spheroids with N2B27 medium (into which the different compounds are diluted), or the differentiation medium may comprise N2B27 medium (into which the different compounds are diluted). This means that the medium comprises an N2 supplement and a B27 supplement. Both of these supplements are well known to those skilled in the art and are freely available. The B27 supplement may be a B27 supplement without vitamin A.

[0155] Differentiation medium may include neurobasal medium and / or DMEM-F12 medium. For example, both mediums are available from Gibco. For example, N2B27 medium may include equal amounts of neurobasal medium and DMEM / F12 medium.

[0156] Cell culture medium can be a culture medium known in the art and used to culture stem cells, such as STEMFLEX cell culture medium. Culture medium can include a culture medium for culturing neurons, such as neural basal culture medium, neural basal-A culture medium or neural progenitor cell basal culture medium. Culture medium can include NS-A culture medium, BME culture medium, BGJb culture medium, CMRL 1066 culture medium, Glasgow MEM culture medium, improved MEM zinc selection culture medium, IMDM culture medium, culture medium 199 culture medium, EagleMEM culture medium, αMEM culture medium, DMEM culture medium, DMEM / F12 culture medium, ham culture medium, RPMI 1640 culture medium, Fischer's culture medium and mixed culture medium thereof, etc. Cell culture medium can usually be purchased from Invitrogen, SIGMA, Wako PureChemical Industries, Ltd., Sumitomo Dainippon Pharma Co., Ltd., etc.

[0157] The culture medium to be used in the differentiation method can be a serum-containing culture medium or a serum-free culture medium (e.g., knockout culture medium). As used herein, serum-free culture medium means a culture medium that does not contain unregulated or unpurified serum, and a culture medium containing purified blood-derived components and animal tissue-derived components (e.g., growth factors) corresponds to a serum-free culture medium. When the culture medium to be used in the differentiation method is a serum-containing culture medium, and mammalian serum, such as fetal bovine serum, etc. can be used as serum. The concentration of serum in the culture medium is generally 0.01 to 20% by weight or 0.1 to 10% by weight.

[0158] The culture medium to be used in the differentiation method may also contain a serum substitute. Some examples of serum substitutes include albumin (e.g., lipid-rich albumin), transferrin, fatty acids, collagen precursors, trace elements (e.g., zinc, selenium), B-27 supplements, N2 supplements, alternative knockout serum substitutes, 2-mercaptoethanol, 3'thioglycerol, and their equivalents. These concentrations in the culture medium are the same as the concentrations of the aforementioned serum in the culture medium.

[0159] The culture medium to be used in the method of the present disclosure may also contain lipids, amino acids (e.g., non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, inorganic salts, antibiotics (e.g., penicillin and streptomycin) or antibacterial agents (e.g., amphotericin B), etc. The concentrations of these in the culture medium are the same as those of the aforementioned serum in the culture medium.

[0160] Other culture conditions such as culture temperature, CO2 concentration, etc. can be appropriately determined. Although the culture temperature is not particularly limited, it is, for example, about 30 to 40° C., preferably about 37° C. The CO2 concentration is, for example, about 1 to 10%, preferably about 5%.

[0161] The method may include evaluating the cells, for example, by evaluating protein expression using antigen-antibody reaction, by evaluating gene expression using quantitative RT-PCR, etc. The method may also include evaluating the cells for expression of cell markers, such as Pax6, Otx2, FoxA2, ​​Lmx1a, and Msx1.

[0162] Cell culture conditions can be provided for the culture of neural cells or progenitor cells as provided herein. The starting cells of the selected population can comprise at least or about 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 The seeding density of the starting cell population can be at least or about 10, 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 cells / ml, or any range derivable therein.

[0163] The culture container for culturing the cells of the present disclosure or their progeny cells may include, but is not specifically limited to, bottles, bottles for tissue culture, dishes, culture dishes, dishes for tissue culture, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, microslides, chamber slides, tubes, trays, The cells can be cultured in any volume, for example, in a chamber, culture bag, and roller bottle, as long as they can culture cells therein. The cells can be cultured in the following volume: at least or about 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350 ml, 400 ml, 450 ml, 500 ml, 550 ml, 600 ml, 800 ml, 1000 ml, 1500 ml, or any range that can be derived therein, depending on the demand for cultivation. The culture vessel can be a bioreactor, which can refer to any device or system that supports a biologically active environment. The volume of a bioreactor can be at least or about 2, 4, 5, 6, 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 500 liters, 1, 2, 4, 6, 8, 10, 15 cubic meters, or any range that can be derived therein.

[0164] The culture container can be cell-adhesive or non-adhesive and is selected according to the purpose. The cell-adhesive culture container can be coated with any substance for cell adhesion, such as extracellular matrix (ECM) to improve the adhesion of the blood vessel surface to the cells. The substance for cell adhesion can be any material intended to attach stem cells or feeder cells (if used). Substances for cell adhesion include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, laminin 521, fibronectin, and mixtures thereof, such as Matrigel TM , and lysed cell membrane preparations.

[0165] The culture method may include culturing human stem cells, such as HS420, in Stemflex medium (ThermoFisher) on laminin 521 coated tissue culture flasks (ThermoFisher). On "day 0" of differentiation, HS420 cells may be plated at 70% confluence and passaged using Accutase (ThermoFisher). The cells may be suspended in X-VIVO medium (LONZA, ref BE04-380Q) supplemented with 1% penicillin / streptomycin, 10 μM ROCK inhibitor (Y27632, Abcam) and neural inducer (ALK inhibitor). 2200 human pluripotent stem cells per microwell in the supplemented X-VIVO medium may be deposited in a microwell plate. For example, on a substrate material contained in molded microwells on an insert in an air-liquid interface (720 microwells per well). Supplemented X-VIVO medium may be added under the insert. In order to properly distribute the cells in each microwell, the substrate material can be gently stirred and placed on a stable support for 15 minutes to allow the cells to settle to the bottom of the wells. After these 15 minutes, the medium from the cell suspension can be removed by aspiration with a 0.5×16 mm needle, and the spheroids can be just covered by a thin film of the remaining medium, and the plate is then incubated at 37°C for 24 hours.

[0166] On "Day 1" of differentiation, 24 hours after spheroid formation, the culture medium under the insert can be replaced with fresh culture medium made from the following combination: half X-VIVO medium (Lonza) and half Neurobasal medium (ThermoFisher), which does not contain ROCK inhibitors and is supplemented with 1% penicillin / streptomycin, 1% non-essential amino acids, 1% B-27 supplement (ThermoFisher), L-glutamine, 100 ng / ml SHH, 2 μM purine amine and 100 ng / ml FGF-8 and one or more of neural inducers (ALK inhibitors).

[0167] On "Day 3" of differentiation, half of the medium can be changed to gradually replace the X-VIVO medium with the Neurobasal medium. The Neurobasal medium can contain 1% penicillin / streptomycin, 1% non-essential amino acids, 1% B-27 supplement (ThermoFisher), L-glutamine, 100ng / ml SHH, 2μM purine amine, 100ng / ml FGF-8, 3μM CHIR99021 and neural inducer (ALK inhibitor).

[0168] On "Day 8" of differentiation, half of the culture medium can be replaced with neurobasal medium supplemented with one or more of the following: 1% penicillin / streptomycin, 1% non-essential amino acids, 1% B-27 supplement, 3 μM CHIR99021, 0.5 mM cAMP, 20 ng / mL GDNF, 20 ng / mL BDNF, 5 ng / mL FGF20, 1 ng / mL TGF-B3 and 1 μM Compound E.

[0169] On "Day 13" of differentiation, half of the medium can be changed. CHIR99021 can be removed. The medium can be replaced with a neurobasal medium containing one or more of the following: 1% penicillin / streptomycin, 1% non-essential amino acids, 1% B-27 supplement, 0.5 mM cAMP, 20 ng / mL GDNF, 20 ng / mL BDNF, 5 ng / mL FGF20, 1 ng / mL TGF-B3, and 1 μM Compound E. FIG. 12 illustrates a 3-dimensional culture method.

[0170] V. Cell Culture Substrate

[0171] The method includes culturing cells in a microwell. The microwell may be part of a substrate. The substrate may include a hydrophilic and porous material layer having a plurality of persistent well-shaped indentations on its outer surface supported by a semipermeable membrane on its inner surface, wherein the well-shaped indentations have a diameter of 1 μm. 2 Up to 1mm 2 The hole and about 1pm 2 Up to 1mm 2 bottom surface.

[0172] The expression "semipermeable membrane" refers to a membrane that allows the passage of solvents and a portion of solutes. It allows certain substances present in the solution to pass preferentially over other substances, depending on the size of the pores. It encompasses hydrophilic membranes, filtration membranes and porous membranes.

[0173] The hydrophilic and porous material can be a hydrophilic gel that allows the aqueous cell culture medium to diffuse and does not adhere to the cells. Generally speaking, the corresponding hydrophilic gel can be made from a polymer selected from the group consisting of agarose, polyvinyl alcohol (PVA), agar-agar, alginate, hyaluronic acid, pectin or starch or any equivalent synthetic gel.

[0174] The corresponding hydrophilic gels can be made of polymers of materials from cytocompatible natural (or modified) sources, such as carbohydrates (e.g., agarose, agar-agar, alginate, starch, pectin, chitosan, dextran, polysaccharides), proteins or ECM components (e.g., laminin, collagen, hyaluronic acid, fibrin), peptides (e.g., gelatin), and mixtures thereof; or gels derived from natural ECM, preferably Matrigel (a recombinant basement membrane preparation extracted from mouse sarcoma (a tumor rich in extracellular matrix proteins) (this material, once isolated, contains about 60% laminin, 30% type IV collagen, and 8% nestin. Nestin is a bridging molecule that interacts with laminin and type IV collagen and contributes to the structural organization of these extracellular matrix molecules. The Coming Matrigel matrix also contains heparan sulfate proteoglycans (beaded proteoglycans), transforming growth factor (TGF-β), epidermal growth factor (epidermal growth factor) and β-actin. factor, EGF), insulin-like growth factor (IGF-1), fibroblast growth factor (bFGF), tissue plasminogen activator and other growth factors naturally present in EHS tumors. There are also residual matrix metalloproteinases derived from tumor cells), Myogel (extracted from human benign tumor tissue "leiomyomas" based on human extracellular matrix Myogel, as described in: Abberton et al., 2008, Cells Tissues Organs, 188 (4): 347-58. doi: 10.1159 / 000121575. Epub 2008 Mar 20. PMID: 18354248, 'Salo, et al., 2015, BMC Cancer, 15, 981 https: doi.or 10.1186 / sl 2885-015-1944-z, or Cartigel (extracellular matrix extract of cartilage) or a synthetic source material selected from the group consisting of polyethylene glycol), polyaliphatic polyurethane, polyether polyurethane, polyethylene copolymer, polyamide, polyvinyl alcohol, poly(ethylene oxide), polypropylene oxide, polyethylene glycol, polypropylene glycol, polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide, poly(hydroxyethyl acrylate), poly(hydroxyethyl methacrylate), and mixtures thereof.

[0175] Hydrophilic gels can be prepared by a number of methods known in the art and involve covalent crosslinking or non-covalent assembly, or a combination of both. Covalent crosslinking can also be performed as part of the synthesis process, for example by using difunctional monomers during polymerization, or alternatively by crosslinking of preformed macromolecules of hydrophilic polymers, where the polymers are linear or branched, using natural or synthetic macromolecules in the process.

[0176] The ductile layer of hydrophilic and porous material can be a hydrophilic gel layer formed at a temperature higher than the gelling temperature of the gel (e.g., 20°C to 100°C higher than the gelling temperature). Generally speaking, a ductile layer of agarose is formed by depositing agarose on a semipermeable membrane at a temperature of 75°C to 100°C to form a composite matrix. When higher than the gelling temperature, the ductile layer of the hydrophilic gel can be about 5mm to 10cm large and about 2mm to 10mm thick. Once formed, the hydrophilic gel can prevent the adhesion of cells and spheroids, thereby avoiding the use of anti-adhesion solutions before centrifugation during cell culture. The ductile layer of hydrophilic and porous material can have a porosity that allows oxygen and cell nutrients to pass through. The porosity can range from about 10nm to about 500pm. A ductile layer of a hydrophilic and porous material (eg, agarose) may have a porosity ranging from about 100 nm to about 500 mm, which may be occupied primarily by water molecules, allowing the passage of nutrients, small molecules, chemical compounds, and even proteins (eg, albumin).

[0177] The substrate may have 4 to 1,536 wells or may have, have at least or have at most 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260 0,265,270,275,280,285,290,295,300,305,310,315,320,325,330,335,340,345,350,355,360,365,370,375,380,385,390,395,400,405,410,415,420,425,430,435,440,445,450,455,460,465,470,475,480,485,490,495,500,505,510,515,520,525,530 ,535,540,545,550,555,560,565,570,575,580,585,590,595,600,605,610,615,620,625,630,635,640,645,650,655,660,665,670,675,680,685,690,695,700,705,710,715,720,725,730,735,740,745,750,755,760,765,770,775,780,785,790,795,800 ,805,810,815,820,825,830,835,840,845,850,855,860,865,870,875,880,885,890,895,900,905,910,915,920,925,930,935,940,945,950,955,960,965,970,975,980,985,990,995,1000,1000,1100,1200,1300,1400,1500,1600,1700,1800,1900,or 2000

[0178] (or any range derivable therein) well-shaped indentations.

[0179] Spheroid formation can be achieved over time by gravity for about 2 to 5 hours, or by subjecting a cell culture vessel containing a floating substrate material seeded with cells to centrifugation to induce cell aggregation. The cell culture vessel can be centrifuged at 100 x g for a period of 5 to 10 minutes.

[0180] VI. Nerve cells

[0181] The method includes making the cell differentiate into nerve cells. The method can be used to produce one or more types of nerve cells, including motor neurons, sensory neurons and interneurons. Typical neurons are composed of cell bodies (referred to as cell bodies), dendrites and axons. The method can be used to produce cholinergic neurons, GABAergic neurons, motor neurons, astrocytes, oligodendrocytes, glutamatergic neurons, dopaminergic neurons and / or serotonergic neurons. The method can be used to produce dopaminergic neurons. The method can include making nerve cells differentiate into neurons, astrocytes, oligodendrocytes, dopamine neurons or motor neurons, pyramidal neurons, motor neurons, spinal ventral horn motor neurons, ventral midbrain neurons, interneurons, glial cells, radial glial cells, retinal pigment epithelium, oligodendrocytes, dopamine neurons, GABA neurons, glutamatergic neurons, catecholamine neurons, serotonergic neurons and cholinergic neurons.

[0182] The method can also include the evaluation of progenitor cells and / or neural cells. For example, the generation or differentiation of neural cells in the cell population can be determined by the presence of certain cell markers. These markers can vary according to the species or organism used to start the population. Some examples of neural cell markers in organisms such as humans include transcription factors or structural proteins. Some examples of transcription factors include MYT1L, BRN2, SOX1, PAX6, NKX6.1, OLIG2, NGN2, LHX3, ISL1 / 2 and HB9. Other neural markers include tubulin (e.g., Tubb2a and Tubb2b), Map2, synaptic proteins (e.g., Syn1 and Syn2), synaptophysin, synaptotagmin (e.g., Syt1, Syt4, Syt13, Syt16), neurogenic differentiation factor (NeuroD), cholineacetyltransferase (ChAT) (e.g., ChAT), neurofilaments, neuromelanin, Tuj1, Thy1, Chat, GluR (kainite 1), neurogenic differentiation factor 1 (Neurod 1), etc. The expression of excitatory and inhibitory neurotransmitter receptors can also be used to assess the number and quality of the generated neural cells. In addition, gross cell morphology can be used to identify neural cells in a non-neural cell population. The neural cells of the present disclosure may also not include one or more of the markers listed herein, such as MYT1L, BRN2, SOX1, PAX6, NKX6.1, OLIG2, NGN2, LHX3, ISL1 / 2 and HB9, Tubb2a, Tubb2b, Map2, synapsin, Syn1, Syn2, synaptophysin, synaptotagmin, Syt1, Syt4, Syt13, Syt 16, neurogenic differentiation factor, choline acetyltransferase (ChAT), capsule ChAT, neurofilament, neuromelanin, Tuj1, Thy1, Chat, GluR (kainite 1) and neurogenic differentiation factor 1.

[0183] The presence of the neural cell can also be assessed functionally. For example, the cell can be assessed based on electrophysiological characteristics. These assessments can be performed using patch-clamp recordings. Other functional characteristics include the ability to fire action potentials, to generate outward currents in response to glycine, GABA, or kainite, and to generate inward currents in response to glutamate.

[0184] Neural cells can be assessed, and therefore identified, by the presence of one or more (including 2, 3, 4, 5 or more) of any of the foregoing characteristics and / or markers.

[0185] The neural cells or cell populations can also be evaluated for expression of markers characteristic of non-neural initiating cell populations. In some cases, reprogramming can be evaluated by an increase in the expression of neural markers and a decrease in the expression of non-neural initiating cell markers.

[0186] Nerve cells may also be defined as dopaminergic (DA) neurons. DA neurons may be determined by evaluating changes in expression of proteins and genes specifically expressed by dopaminergic neurons (in this specification, the above proteins and genes are sometimes referred to as dopaminergic neuron markers). Evaluation of changes in expression of the above dopaminergic neuron cell markers may be performed, for example, by a protein expression evaluation method utilizing antigen-antibody reaction, by a gene expression evaluation method utilizing quantitative RT-PCR, and the like. Some examples of the above dopaminergic neuron cell markers present in the midbrain include tyrosine hydroxylase (TH), OTX2, FOXA2, LMX1A, LMX1B, PITX3, EN1, and NURR1 genes / proteins.

[0187] In addition, whether the dopaminergic neurons obtained by the production method of the present invention have functions equivalent to those of in vivo dopaminergic neurons can be determined by evaluating dopamine release, and responsiveness to oxidative stress and drug stimulation.

[0188] The cells obtained during the process of the production method of the present invention and the dopaminergic neurons of the present invention can be cryopreserved and thawed. The freezing and thawing methods of the cells are known in the relevant art and are not particularly limited as long as they do not affect the differentiation potential, viability, dopamine production ability, etc. of the cells. For example, the dopaminergic neurons of the present invention can be stored at -80°C by washing the cells with PBS, separating them from the culture dish with a cell dispersion solution (e.g., Accutase (registered trademark) Innovative Cell Technologies), removing the cell dispersion solution, and suspending the cells in a cryopreservation solution (e.g., cell banker 2 (LSIMedience Corporation)). Some examples of thawing methods include a method that includes thawing in a constant temperature tank at 37°C, washing the cryopreservation solution by centrifugation, and suspending in a culture medium for use, etc. When freezing and thawing the cells obtained during the process of the production method of the present invention, Nurr1-positive dopaminergic neurons can also be induced from the cells after thawing.

[0189] VII. Methods of using nerve cells

[0190] The methods of the present disclosure relate to the generation of neural progenitor cells that can be used to treat a subject. For example, the cells generated by the methods of the present disclosure can be used to treat neurodegenerative diseases. Some non-limiting examples of neurodegenerative diseases include Alzheimer's disease; epilepsy; Huntington's Disease; Parkinson's disease; stroke; spinal cord injury; traumatic brain injury; Lewy body dementia; Pick's disease; Niewmann-Pick disease; amyloid angiopathy; cerebral amyloid angiopathy; systemic amyloidosis; hereditary cerebral hemorrhage with amyloidosis of the Dutch type; inclusion body myositis; mild cognitive impairment; Down's syndrome; and neuromuscular disorders, including amyotrophic lateral sclerosis (ALS), multiple sclerosis, and muscular dystrophy, including Duchennedystrophy, Becker muscular dystrophy, Landouzy-Dejerine muscular dystrophy, and limb-girdle muscular dystrophy. The invention also includes neurodegenerative diseases caused by stroke, head trauma, spinal cord injury, or other damage to the brain, peripheral nerves, central nervous system or neuromuscular system. The methods described herein may relate to methods for preventing a disease or health-related condition in a subject.

[0191] The present disclosure provides a medicament comprising neurons produced by the method of the present disclosure. As used herein, neurons are not particularly limited as long as they are cells obtained by the above-mentioned production method of the present disclosure.

[0192] In this drug, neurons can be used as they are, or as cell aggregates such as precipitates obtained by passing through a filter through concentration, etc. In addition, the drug can also be added with a protective agent such as DMSO (dimethyl sulfoxide) and stored frozen. In order to use the drug more safely, the drug can be treated under such conditions as heat treatment, radiation treatment, etc. while retaining neuronal function and denaturing pathogenic proteins. In addition, in order to prevent the growth of neurons in an amount exceeding that necessary, the drug is subjected to a combination of the above-mentioned treatments, growth is inhibited by pretreatment with mitomycin C, etc., and treatment is performed by a method including introducing genes for metabolic enzymes that do not naturally exist in mammals into neurons, administering an inactivated form of the agent as needed to allow the agent to be converted into a toxin only in neurons into which genes for metabolic enzymes that do not naturally exist in mammals have been introduced, thereby causing cell elimination (suicide gene therapy), etc.

[0193] Since the drug of the present disclosure is safe and has low toxicity, it can be administered to mammals (eg, humans, mice, rats, guinea pigs, pigs, monkeys).

[0194] The patient's own cells can be used to prepare neural cells, or the cells of donors with tissue compatibility types in the tolerable range can be used for the medicine of the present disclosure. When sufficient cells cannot be obtained due to age, constitution, etc., cells embedded with polyethylene glycol or silicon capsules, porous containers, etc. can also be transplanted to avoid rejection. The dosage (amount to be transplanted) and the frequency of administration (number of times to be transplanted) of the medicine of the present disclosure can be appropriately determined according to the age, weight, symptoms, etc. of the patient receiving the administration.

[0195] By administering (transplanting) a drug containing neurons of the present disclosure, it can be effectively implanted into the patient, which in turn enables effective production (release) of dopamine in the patient. Therefore, the drug of the present disclosure can be used to treat diseases caused by reduced dopamine production (release), for example, neurodegenerative diseases such as Parkinson's disease, Huntington's disease, Alzheimer's disease, epilepsy and schizophrenia, etc.

[0196] The neural cells of the present disclosure can be used in methods for screening drug compounds, such as compounds for treating neurodegenerative diseases. For example, the test compound can be contacted with the neurons of the present disclosure alone or in combination with other drugs, and the morphological or functional changes of the neurons can be measured to evaluate whether the test compound can be used as a drug. Some examples of methods for measuring functional changes include measuring the amount of dopamine produced or released from neurons. Dopaminergic neurons can be cells that exhibit the same phenotype as the disease to be treated, and particularly preferred are dopaminergic neurons produced by inducing differentiation of stem cells produced by somatic cells derived from the disease.

[0197] Some examples of test compounds include peptides, proteins, antibodies, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, etc. As used herein, test compounds can form salts. As salts, physiologically acceptable acids (e.g., inorganic acids, organic acids), salts of alkalis (e.g., alkali metal salts, alkaline earth metal salts, aluminum salts), etc. are used, and some examples of such salts include salts of inorganic acids (e.g., hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), salts of organic acids (e.g., acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid), sodium salts, potassium salts, calcium salts, magnesium salts, barium salts and aluminum salts can be used.

[0198] The drugs obtained using the above screening can be formulated using physiologically acceptable additives and according to known methods.

[0199] VIII. Other Medications

[0200] It is contemplated that the methods of the present disclosure include administration of additional agents. Additional agents may include one or more BMP inhibitors, such as noggin, tenascin, dorsomorphin, LDN-193189 (4-(6-(4-(piperazine-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline hydrochloride), dorsomorphin (6-[4-(2-piperidin-1-ylethoxy)phenyl]-3-pyridin-4-ylpyrazolo[1,5-a]pyrimidine), etc.; TGFβ family inhibitors, such as SB431542 (4-[4(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide), A-83-01 (3-(6-methylpyridin-2-yl)-1-phenylthiocarbamoyl

[0013] The invention relates to the invention of the invention to a group of drugs which are directed against GSK3β, such as pyridinecarbonitrile (pyridinecarbonitrile), pyrimidinecarbonitrile (pyrimidinecarbonitrile), pyridinecarbonitrile (pyridinecarbonitrile), pyridinecarbonitrile (pyridinecarbonitrile), pyridinecarbonitrile (pyridinecarbonitrile), pyridinecarbonitrile (pyridinecarbonitrile), pyridinecarbonitrile (pyridinecarbonitrile), pyridinecarbonitrile (pyridinecarbonitrile), pyridinecarbonitrile (pyridinecarbonitrile), pyridinecarbonitrile (pyridinecarbonitrile), pyridinecarbonitrile (pyridinecarbonitrile), pyridinecarbonitrile (pyridinecarbonitrile), pyridinecarbonitrile (pyridinecarbonitrile), pyridinecarbonitrile (pyridinecarbonitrile), pyridinecarbonitrile (pyridinecarbonitrile), pyridinecarbonitrile (pyridinecarbonitrile), pyridinecarbonitrile (pyridinecarbonitrile), pyridinecarbonitrile (pyridinecarbonitrile), pyridinecarbonitrile ( The method may not include contacting the cell with one or more additional agents described herein.The method may not include contacting the cell with one or more BMP inhibitors, such as tenascin, doxorubicin, noggin, LDN193189 (4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline hydrochloride), doxorubicin (6-[4-(2-piperidin-1-ylethoxy)phenyl]-3-pyridin-4-ylpyrazolo[1,5-a]pyrimidine), etc.; TGFβ family inhibitors, such as SB431542 (4-[4(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide); GSK3β inhibitors, such as Such as CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile) and BIO (6-bromo-indirubin-3'-oxime); smoothened agonists, such as purinamine (N-(4-morpholinophenyl)-2-(1-naphthyloxy)-9-cyclohexyl-9H-purin-6-amine) and SAG (N-methyl-N'-(3-pyridylbenzyl)-N'-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane); and growth factors, such as sonic hedgehog (SHH) and fibroblast growth factor-8 (FGF8).

[0201] The additional agent may include one or more of an activator of the phosphatidylinositol 3-kinase signaling pathway and an activator of the MAPK signaling pathway. The additional agent may not include one or more of an activator of the phosphatidylinositol 3-kinase signaling pathway and an activator of the MAPK signaling pathway. Additional agents may include one or more of the following: midkine, pleiotrophin, insulin-like growth factor-1, inhibitors of TGF-β superfamily signaling pathways, A83-01, SB431542, doxormorphine, inhibitors of Wnt signaling pathways, PNU-74654, Dickkopf, activators of Notch signaling pathways (e.g., delta-1, delta-2, delta-3, delta-4, Jagged-1, Jagged-2), activators of protein kinase signaling pathways (e.g., forskolin or dibutyryl-cAMP), activators of the tyrosine kinase anaplastic lymphoma kinase (ALK), and activators of insulin-like growth factor (IGF) receptors, and inhibitors of SMAD2, SMAD3, SMAD4, SMAD1, SMAD5, SMAD8, inhibitors of Wnt or LRP binding to Frizzled, or inhibitors of β-catenin stabilization. The method may not include contacting the cells described herein with one or more of an activator of the phosphatidylinositol 3-kinase signaling pathway and an activator of the MAPK signaling pathway. The additional agent may not include one or more of an activator of the phosphatidylinositol 3-kinase signaling pathway and an activator of the MAPK signaling pathway. Additional agents may include one or more of the following: midkine, pleiotrophin, insulin-like growth factor-1, inhibitors of TGF-β superfamily signaling pathways, SB431542, inhibitors of Wnt signaling pathways, PNU-74654, Dickkopf, activators of Notch signaling pathways (e.g., Delta-1, Delta-2, Delta-3, Delta-4, Jagged-1, Jagged-2), activators of protein kinase signaling pathways (e.g., forskolin or dibutyryl-cAMP), activators of the tyrosine kinase anaplastic lymphoma kinase (ALK), and activators of the insulin-like growth factor (IGF) receptor, and inhibitors of SMAD2, SMAD3, SMAD4, SMAD1, SMAD5, SMAD8, inhibitors of Wnt or LRP binding to Frizzled, or inhibitors of β-catenin stabilization.

[0202] IX. Examples

[0203] The following examples are included to illustrate some preferred aspects of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in the practice of the present invention, and therefore can be considered to constitute preferred modes for its practice. However, based on this disclosure, it will be appreciated by those skilled in the art that many changes may be made to some of the disclosed specific embodiments without departing from the spirit and scope of the present invention and still obtaining the same or similar results.

[0204] Example 1: Three-dimensional cell culture for differentiating stem cells or progenitor cells into neural cells in vitro.

[0205] A. Materials and Methods

[0206] 1. Chemicals, proteins and antibodies

[0207] LDN193189 was provided by Axonmedchem (ref 1509) and stored at 5mM in DMSO at -20°C. SB431542 was provided by Abcam (ref ab120163) and stored at 50mM in DMSO at -20°C. FGF-8 (fibroblast growth factor 8, ref GFH176-5) was provided by Peprotech and stored at 100μg / mL in pure water at -20°C. SHH (sonic hedgehog, ref GFH168-5) was provided by Cell Guidance and stored at 100μg / mL in pure water at -20°C. Purineamine (ref SML0868) was provided by Calbiochem and stored at 10mM in pure DMSO at -20°C. CHIR99021 (ref ct99021) was provided by Axon Medchem and stored at a concentration of 7.5 mM in DMSO at -20°C. cAMP (cyclic adenosine monophosphate, ref D0627) was provided by Sigma and stored at a concentration of 0.5 M in DMSO at -20°C. BDNF (brain-derived neurotrophic factor, ref GFH1-2) was provided by Cell Guidance and stored at a concentration of 100 μg / mL in purified water at -20°C. GDNF (glial-derived neurotrophic factor, ref GFH2-2) was provided by Cell Guidance and stored at a concentration of 100 μg / mL in purified water at -20°C. TGFB3 (transforming growth factor β3, ref GFH109-2) was provided by CellGuidance and stored at a concentration of 100 μg / mL in purified water at -20°C. FGF20 (fibroblast growth factor 20, ref 100-41) was provided by Peprotech and stored at a concentration of 100 μg / mL in purified water at -20°C. Compound E, a γ-secretase inhibitor (ref CAS209986-17-4), was provided by Calbiochem and stored at a concentration of 5 mM in pure DMSO at -20°C. For immunostaining, the following antibodies were used: mouse anti-βIII-tubulin (Sigma), rabbit polyclonal anti-TH (Merck), goat anti-mouse IgG-Alexa 555 (Life technologies), goat anti-rabbit IgG-Alexa 488 (Lifetechnologies).

[0208] 2. Embryonic Stem Cell Culture and Differentiation

[0209] The human embryonic stem cell (hESC) line HS420 (gift from Dr. Outi Hovatta, Karolinska institute, Sweden) was cultured in Stemflex medium (Thermofisher) on laminin 521-coated tissue culture flasks (Thermofisher) according to the manufacturer's instructions. HS420 cells at 70% confluence were passaged in X-VIVO medium (LONZA, ref BE4-380Q) supplemented with 1% penicillin / streptomycin, 10 μM ROCK inhibitor (Y27632, abcam), 0.5 μM LDN193189 and 10 μM SB431542 in 3D cell culture plates.

[0210] Dopaminergic neurospheres are made by using the following two different 3D cell culture techniques. The first 3D cell culture technique is the standard method "3D-immersion", which first forces spheroids to gather in plastic microwells, then takes out and transfers to the standard culture plate maintained under stirring (Figure 1A). In the second technique used, called the "3D-AirLiwell" method, spheroids are forced to gather in non-adhesive microwells molded in medium permeability agarose. Compared with 3D-immersion, they are kept in microwells alone without any transfer, immersion and stirring. The semipermeable membrane at the bottom of the molded microwell allows their long-term stability, and also establishes air / liquid interface conditions (Figure 1B) that are conducive to gas and air exchange.

[0211] a. Fabrication of dopaminergic neurospheres by forced aggregation followed by immersion (3Di)

[0212] HS420 cells were deposited in Aggrewell-800TM (Stemcell technologies, 6-well plate, 2 ml per well) at a ratio of 2'200 cells per microwell (the microwell plate used here contained 1'800 microwells per well) in supplemented X-VIVO medium. In order to properly distribute the cells in each microwell, the plate was gently shaken and placed on a stable support. After 15 minutes, the plate was incubated at 37°C for 24 hours to generate spheroids. 24 hours after spheroid formation, spheroids were collected and subsequently transferred to standard 6-well plates in the following combination: half X-VIVO and half Neurobasal medium, which does not contain ROCK inhibitors, supplemented with 1% penicillin / streptomycin, 1% non-essential amino acids, 1% B-27 supplement (Thermofisher), L-glutamine, 0.5 μM SB431542, 10 μM LDN193189 (double SMAD inhibition cocktail), 100 ng / ml SHH, 2 μM purine amine and 100 ng / ml FGF8. Spheroids were then cultured in 3 ml of medium per well under constant agitation (60 rpm, orbital shaker) at 37°C and 5% CO2. On day 3, 3 μM CHIR99021 was added. Neurobasal medium gradually replaced X-VIVO medium. On day 8, replace the medium with neurobasal medium supplemented with 1% penicillin / streptomycin, 1% non-essential amino acids, 1% B-27 supplement, 3 μM CHIR99021, 0.5 mM cAMP, 20 ng / mL GDNF, 20 ng / mL BDNF, 5 ng / mL FGF20, 1 ng / mL TGF-B3, and 1 μM Compound E. On day 13, remove CHIR99021. Note: For each medium change, refresh half of the medium and use 1.5 to 2 ml per well.

[0213] b. Fabrication of dopaminergic neurospheres by using 3D-AirLiwell technology (3D-ALi)

[0214] For neural differentiation, 2200 human pluripotent stem cells per microwell in supplemented X-VIVO medium were deposited on a substrate material contained in molded microwells on an insert in an air-liquid interface (720 microwells per well). In order to correctly distribute the cells in each microwell, the substrate material was gently shaken and placed on a stable support for 15 minutes. Supplemented X-VIVO medium was added under the insert. The medium from the cell suspension added on the microwells was then removed by aspiration with a 0.5×16 mm needle and the spheroids were just covered by a thin film of residual medium (10), and the plate was then incubated at 37° C. for 24 hours.

[0215] 24 hours after spheroid formation, the medium under the insert was replaced with fresh medium made from a combination of half X-VIVO and half Neurobasal medium, which does not contain ROCK inhibitors, supplemented with 1% penicillin / streptomycin, 1% non-essential amino acids, 1% B-27 supplement (Thermofisher), L-glutamine, 0.5 μM SB431542, 10 μM LDN193189 (double SMAD inhibition cocktail), 100 ng / ml SHH, 2 μM purine amine, and 100 ng / ml FGF8. Spheroids were then cultured in their respective microwells in a conventional incubator at 37°C and 5% CO2. On day 3, 3 μM CHIR99021 was added. Neurobasal medium gradually replaced X-VIVO medium. On day 8, replace the medium with neurobasal medium supplemented with 1% penicillin / streptomycin, 1% non-essential amino acids, 1% B-27 supplement, 3 μM CHIR99021, 0.5 mM cAMP, 20 ng / mL GDNF, 20 ng / mL GDNF, 5 ng / mL FGF20, 1 ng / mL TGF-B3, and 1 μM Compound E. On day 13, remove CHIR99021. Note: For each medium change, refresh half of the medium and use 0.5 ml per well.

[0216] 3. Quantitative RT-PCR and RNA Sequencing

[0217] For qRT-PCR, RNA was extracted from dopaminergic neurospheres cultured in immersion (3D-i) and air-liquid culture interface (3D-ALi) from 3 different cell batches of HS420 cells. The time points for RNA extraction were: 3rd, 8th, 15th, 22nd, 29th, 43rd, 57th days. Total RNA was isolated using the Qiagen RNeasy kit according to the manufacturer's instructions. RNA concentration was determined by a spectrometer (Thermo Scientific NanoDrop 2000), and RNA quality was verified by a 2100 bioanalyzer (Agilent). Quantitative PCR was performed with Tecan Freedom Evo using SYBR green fluorescence. cDNA was generated from RNA using a prime scriptRT kit (Takara). All primers were diluted at a concentration of 0.86 μM and a master mix (Power SYBR Green Master Mix, Thermo Fisher Scientific) was used. For RNA sequencing, the same RNA used in qPCR was used to analyze gene expression at week 6 of differentiation (= day 43).

[0218]

[0219] 4. Dopamine Dosage by HPLC

[0220] Dopamine production was analyzed by high-pressure liquid chromatography (HPLC) after extraction from dopaminergic neurospheres cultured submerged in 0.1N perchloric acid (HClO4) (3D-i) or in an air-liquid culture interface (3D-ALi). Cells were lysed in a small volume (250 μL) at 4°C for 15 minutes with vigorous vortexing every 5 minutes. The supernatant was recovered after centrifugation and used immediately for dopamine dosing or stored at -20°C. Catecholamines were measured by HPLC with electrochemical detection in coulometric mode. Separation of analytes was performed on a reverse phase column, Symmetry C-18, 5 mm (4.6·150 mm 2 ) (Waters Corporation) in isocratic mode at a flow rate of 1 mL / min. Coulometric detector parameters, Coulochem III (Thermo scientific), were used to adjust the cell at a potential of +200 mV. For the analytical cell, electrode 1 at a potential of +100 mV and electrode 2 at a potential of -100 mV were used.

[0221] 5. Western Blot

[0222] Total protein was extracted from dopaminergic neurospheres differentiated for 1 month by homogenizing the cells in RIPA buffer (1×) and incubating on ice for one hour. The lysate was centrifuged at 13 rpm for 10 minutes and the supernatant was used for western blot analysis. For each condition, 10, 20, 30 and 40 μg of protein were analyzed by western blot to evaluate the expression of protein tyrosine hydroxylase.

[0223] 6. Electrophysiology via MEA Arrays

[0224] Dopaminergic neurospheres were transferred to the center of the multi-porous MEA device using a dissecting microscope. Eight to four recording electrodes were selected for each given condition in triplicate. Then, an amplifier and data acquisition system for electrophysiological recording were used. The signal-to-noise ratio (SNR) was measured as the standard deviation of the voltage during the 30-minute recording, using the signal as the average peak-to-peak voltage of the spikes recorded in the same 30-minute time.

[0225] 7. Plating of Neurospheres on Extracellular Matrix, Immunocytochemistry and Electron Microscopy

[0226] On day 29, neurospheres were plated on poly-ornithine / laminin coated tissue culture plates in mature neural basal medium supplemented with 1% penicillin / streptomycin, 1% non-essential amino acids, 1% B-27 supplement, 3 μM CHIR99021, 0.5 mM cAMP, 20 ng / mL GDNF, 20 ng / mL GDNF, 5 ng / mL FGF20, 1 ng / mL TGF-B3, and 1 μM compound E. After 1 week of culture on coverslips, cells on glass coverslips were fixed with 1 mL of 4% paraformaldehyde in PBS for 30 minutes at room temperature. Cells were then washed three times with 2 mL of PBS. Cells were permeabilized with PBS containing 0.3% Triton X-100 (Sigma) and incubated with primary antibodies overnight in PBS containing 5% bovine serum albumin (Sigma).

[0227] The cells were washed three times with 2 mL of PBS and then incubated with secondary antibodies in PBS containing 5% bovine serum albumin for 1.5 hours. The cells were washed three times with PBS and then exposed to 300 nM DAPI in PBS (Sigma) for 15 minutes at room temperature. After washing three times in PBS, the cells were rinsed with water and mounted on glass slides using Fluorsave reagent (Merck Millipore). In addition, dopaminergic neurospheres plated on glass coverslips were dehydrated and then covered with a gold nanolayer (20 nm) and visualized by electron microscopy.

[0228] B. Results

[0229] First, the homogeneity of dopaminergic neurospheres was studied over 60 days using the previously described dopaminergic specialization protocol. Dopaminergic neurospheres cultured with 3D conventional "3D-immersion" were compared with 3D-AirLiwell neurospheres. As a practical reminder, in the standard method "3D-immersion", spheroids were first forced to aggregate in plastic microwells, then removed and transferred to standard culture plates maintained under stirring. In contrast, using the "3D-AirLiwell" method, spheroids were forced to aggregate in non-adhesive microwells molded in medium permeability agarose. Compared to 3D-immersion, they were kept alone in the microwells without any transfer, immersion, and stirring. The semipermeable membrane at the bottom of the molded microwells allows their long-term stability, and also establishes air / liquid interface conditions that are conducive to gas and air exchange. To more precisely compare and characterize the morphology of dopaminergic spheroids in 3D-Immersed vs. 3D-AirLiwell, dopaminergic differentiation was performed during 2 months and the evolution of their size and shape was followed by light microscopy. Figure 2 As shown in , at week 0, there were no significant differences in both circular shape and size between the two methods. However, after 2 weeks of culture, dopaminergic neurospheres cultured with 3D-AirLiwell were more homogeneous than those cultured on 3D-Immersion. Software-based quantification of the distribution range of area, roundness, perimeter, circularity, and diameter ( Figure 2 ) showed that the variability was significantly lower in 3D-AirLiwell. In addition, spheroids that initially had a round shape maintained their round morphology in 3D-AirLiwell, while those in 3D-Immersion evolved into more heterogeneous and irregular shapes.

[0230] Then, dopaminergic specification was monitored more quantitatively at different time points by using Q-RT-PCR. In addition to dopaminergic specification, the expression of some markers involved in early or late neural induction was analyzed, as well as cell proliferation at different time points during the 2-month differentiation period ( Figure 3 ). First, for the expression of the cell proliferation marker Ki67, no differences were observed between the two 3D cell culture methods. Nestin (also an early NPC marker) and βIII-tubulin (a neuronal marker) did not show any significant regulation. These proteins are not normally upregulated in cells during the neural induction process, but rather reassemble more in the cytosol to form denser cytoskeletal fibers.

[0231] In contrast and as expected, the transcription factor Pax-6, which is upregulated during the early neural induction process, was increased in the "3D-AirLiwell" spheroids from day 15 until the end of differentiation. In summary, these observations enhance the ability of 3D-AirLiwell to support the switch between hESCs and NPCs. Regarding specific markers, Lmx1a, an early midbrain marker upregulated in dopaminergic precursors, was also upregulated in 3D-AirLiwell spheroids from week 2 until the end of differentiation. Finally, tyrosine hydroxylase, a marker of mature dopaminergic neurons, showed upregulation in 3D-immersion spheroids. Therefore, these Q-RT-PCR results indicate that the 3D-AirLiwell method allows for better midbrain regionalization of NPCs, but clearly determines the lack of maturity.

[0232] In order to have a global perspective on gene expression, RNA-seq analysis was performed to compare spheroids from three different cell batches of the HS420 cell line cultured by both methods at week 6 (= day 43). Figure 4 In the graph of fold change between 3D-AirLiwell and 3D-Immersion, positive bars mean upregulation in 3D-AirLiwell, and conversely negative bars mean upregulation in 3D-Immersion. The combination of Lmx1a, Otx-2, and FoxA2 is the best correlate for estimating the early regional state of NPCs, because dopaminergic NPCs are derived from ventral midbrain cells, i.e., triple-positive cells. In the left panel ( Figure 4 ), these 3 markers were upregulated in spheroids cultured in "3D-AirLiwell". In addition, other transcription factors involved in the development of dopaminergic neurons (e.g., Nurr-1, MSX1, FoxA2) were also upregulated in 3D-ALi, confirming the suggestion of better dopaminergic regionalization and specificity of NPCs. In contrast, late dopaminergic markers (e.g., TH, EN-1 (engrailed-1), DRD2 (dopamine receptor), VMAT2 (dopamine transporter)) were upregulated in spheroids cultured with the standard 3D immersion method ( Figure 4 In addition, the expression of other specific maturation markers was also analyzed. For example, all maturation markers (NeuN, Neurofilament heavy chain (Neurofilament heavy)..., Figure 4 ), confirming the lack of maturation in spheroids cultured using the 3D-AirLiwell method.

[0233] In addition, the expression of cell proliferation markers was analyzed. Figure 5As shown in Figure 2, cell proliferation markers were all downregulated in neurospheres cultured by the 3D-ALi method, suggesting a huge advantage in terms of tumor risk, which may be lower for cells cultured by this technology.

[0234] However, a lack of maturation of dopaminergic neurospheres cultured by "3D-AirLiwell" was also determined at the protein level. Dopaminergic differentiation was performed using the two 3D methods "3D-Immersion" and "3D-AirLiwell", and western blot analysis of spheroids was performed on day 33 of differentiation ( Figure 6 ). As expected, we observed lower amounts of tyrosine hydroxylase protein in samples from spheroids cultured in “3D-AirLiwell”, indicating a lack of maturation by this new approach.

[0235] In addition, dopamine production was analyzed by high-pressure liquid chromatography (HPLC) after extraction from dopaminergic neurospheres cultured in immersion (3D-i) and air-liquid culture interface (3D-Ali). As expected, the amount of dopamine produced by 3D-ALi dopaminergic neurospheres was lower than that by 3D-i neurospheres ( Figure 7 ).

[0236] Regarding the electrophysiological aspects of dopaminergic neurospheres, spontaneous electrical activity generated by neurons differentiated by both techniques was measured. Both neurospheres cultured in 3D-immersion and 3D-AirLiwell were alive and electrophysiologically active. Figure 8 As shown in the left part, the waveform of the 3D-AirLiwell neurosphere signal is more homogenous and grouped into 2 similar shapes, while the shapes are more heterogeneous for 3D immersion and grouped into more than 4 different shapes. Dopaminergic neurospheres cultured in 3D immersion showed significantly higher frequencies, amplitudes of spikes and bursts, and higher numbers of spikes, the number of bursts indicating more neuronal network activity. However, neurospheres cultured in 3D-AirLiwell showed calm and stable activity over time, while those in 3D-immersion had more epileptic activity ( Figure 8 ).

[0237] At week 4 of differentiation, neuronal maturation of dopaminergic neurospheres was induced by plating them on extracellular matrix (ECM)-coated plates. After 1 week of maturation, immunofluorescence staining with neuronal markers β-III tubulin and tyrosine hydroxylase (TH), a specific marker for dopaminergic neurons, was performed ( Fig. 9). Both methods can obtain abundant neurons around the plate-seeded spheroids, confirming that 3D-AirLiwell spheroids maintain the characteristics of neuronal maturation. Mature neurons on the ECM-coated plates were also observed by electron microscopy ( Fig.10 ). This allows the shape of the neuronal network and cells to be analyzed with better resolution. Using the immersion method, the presence of invasive large flat cells (2) was observed (by DAPI staining and electron microscopy) under the neuronal network (1), suspected to be proliferative NPCs (because they appeared to be nestin+, Pax-6+, data not shown). In contrast and significantly, 3D-AirLiwell did not induce this cell population, indicating a higher level of homogeneity of the cell preparation.

[0238] To further characterize the neurospheres generated by the 3D-Ali method and the 3D-i method, single-cell RNA sequencing was performed to determine the phenotypes of different cells in the neurospheres ( Fig.13A ). The majority of cells in 3D-Ali neurospheres were located in neuronal clusters (86.5%), whereas only half of the cells in 3D-i neurospheres were located in neuronal cell type clusters. 3D-I neurospheres had a higher percentage of myeloid progenitor cells (23.2% in 3D-i vs. 0.3% in 3D-Ali) and fibroblasts (15.9% in 3D-i vs. 0% in 3D-Ali) ( Fig. 13B To C). Therefore, 3D dopaminergic differentiation under 3D-Ali conditions produced a more homogeneous cell population than under 3D-I conditions. It was also found that tyrosine hydroxylase (TH) mRNA was increased under 3D-Ali conditions ( Fig.14 ), but no differences in dopamine content were detected ( Fig.15 ). The 3D-Ali condition also provided electrical signals that were more indicative of stable neurons ( Fig.16 ).

[0239] To improve the maturation step, 2.5% or 10% of laminin 511 (Takkara) or (growth factor reduced extracellular matrix, ThermoFisher) was added to the cell culture medium to promote neuronal maturation of spheroids from day 1 to the end of differentiation and from day 13 to the end. RGD peptides can also be tested to promote maturation by adding them to the culture medium similar to laminin or geltrex.

[0240] The second strategy will involve culturing dopaminergic spheroids under hypoxic conditions from day 1 to the end of differentiation and from day 13 to the end. In addition, the combination of hypoxia and the addition of extracellular matrix (laminin or Geltrex) or RGD peptide to the culture medium will also be tested. These combinations will be applied from day 1 to the end of differentiation and from day 13 to the end.

[0241] The inventors tested whether the addition of extracellular matrix and / or hypoxia could improve the development of mature neurons. 2.5% and 10% laminin and normoxia (20% oxygen) or hypoxia (3% oxygen) ( Fig.17 ).like Fig.18 As shown in, hypoxia was shown to enhance and accelerate the maturation of dopaminergic neurons at day 29, and laminin did not provide any significant effect in either normoxic or hypoxic conditions. It is further hypothesized that the effects demonstrated by hypoxia can be reproduced by adding a HIF-1α stabilizer to the cell culture medium. It is further hypothesized that other changes to cell culture, such as changes in the volume of cell culture medium, can provide improvements in the maturation of neurons. The inventors have found that the ratio between the number of neurospheres and the volume of cell culture medium can provide improvements in the maturation of cells. In AirLiwell (6-well plates), 1.5 million cells were seeded per well (6-well AirLiwell plates) to produce 2350 neurospheres. Under these conditions, 750 μl of cell culture medium provides a more effective method for differentiating neurons ( Fig.19 ).

[0242] The present inventors also tested the effects of different antioxidants on three-dimensional dopaminergic neuron differentiation. Fig. 20 The scheme design of adding antioxidants is shown. Fig.21A As shown in Figures 4 and 5, antioxidants had no effect on overall neuronal differentiation. Comparable gene expression of the neuronal markers b3-tubulin (TUBB3) and MAP2 was observed, and a similar downregulation of the proliferation marker ki67 was also observed. Most of the antioxidants tested attenuated the expression of TH during dopaminergic neuron differentiation. However, ascorbic acid (vitamin C) was found to result in a significant enhancement in DA neuron differentiation ( Fig.21B ).

[0243] It is also expected that overexpression of factors that promote the maturation of dopaminergic neurons can be stably transduced in hESC (HS420 cell line) to improve dopaminergic maturation. The factors selected may include Nurr-1 and Pitx3. In fact, these two transcription factors are very important for the maturation and maintenance of dopaminergic neurons in vivo. To strengthen this selection, these two transcription factors are weakly expressed by spheroids. In addition, many studies emphasize that Nurr1 and Pitx3 are essential for the maturation of dopaminergic neurons. It is worth noting that neuron-specific promoters will be used to overexpress these two transcription factors. For example, the Tα1α-tubulin promoter verified in previous studies will be tested to specifically target these factors in neurons. [Suter DM, Cartier L, Bettiol E, Tirefort D, Jaconi ME, Dubois-Dauphin M, Krause KH Stem Cells.2006Mar;24(3):615-23.doi:10.1634 / stemcells.2005-0226.Epub 2005Nov 17.PMID:16293575].

[0244] Example 2: Neuronal cell differentiation in three-dimensional cell culture

[0245] To investigate the effects of other ALK inhibitors (ALKi) described previously, and to potentially replace dual-SMAD inhibition in the protocol using 3D-AirLiwell technology, the human embryonic stem cell line HS420 was differentiated into dopaminergic neurospheres as described previously in Example 1, and the effects of DMH1, DMH2, K02288 and A8301 alone or in combination on hESC neuronal induction were compared with the combination of LDN193189 and SB431542 compounds. The results of differentiation were analyzed by q-PCR after 2 and 4 weeks of dopaminergic differentiation ( Fig.23A Cells were exposed to compounds during one week at day 0 at test concentrations of 0.2 μM and 0.8 μM in minimal medium suitable for early neural induction. In addition to the ALKi compounds, compounds and proteins mentioned in the protocol and described above for the first week of induction were added to the culture medium, such as purine amines, SHH, and FGF-8 ( Fig. 22). DMH1, DMH2, A-8301, and K02288 were found to have comparable activity to LDN / SB (dual smad inhibition) in general neural differentiation. In contrast, different ALK inhibitors had different effects on dopaminergic differentiation: DMH1 and DMH2 were roughly comparable with LDN / SB and A-8301 and K02288 had no major effect on dopaminergic differentiation. The combination of DMH2 and A-8301 provided synergistic effects in 2D cell culture.

[0246] ***

[0247] According to the present disclosure, all methods disclosed and claimed herein can be carried out and implemented without excessive experimentation. Although the compositions and methods of the present invention have been described according to some preferred embodiments, it is obvious to those skilled in the art that the steps or sequence of steps of the methods described herein and the methods described herein can be changed without departing from the concept, spirit and scope of the present invention. More specifically, it will be obvious that certain medicaments related in both chemical and physiological aspects can be used to replace the medicaments described herein, and the same or similar results will be achieved at the same time. All such similar substitutions and changes that are obvious to those skilled in the art are considered to be within the spirit, scope and concept of the present invention defined by the appended claims.

Claims

1. A method for differentiating stem cells or progenitor cells into neural cells, the method comprising (i) contacting the stem cells or progenitor cells with a differentiation composition, wherein the differentiation composition comprises one or more of the ALK inhibitors: DMH1, DMH2, K02288 and A83-01; and (ii) culturing the cells in microwells to form spheroids and / or neurospheres; wherein the ALK inhibitor does not comprise LDN193189 and SB431542; wherein the cells, spheroids and / or neurospheres are cultured under hypoxic conditions; and wherein the ratio of the number of spheroids or neurospheres to the volume of cell culture medium is about 1000 spheroids or neurospheres to 200 to 500 μl of cell culture medium.

2. A method for differentiating stem cells or progenitor cells into neural cells, the method comprising (i) contacting the stem cells or progenitor cells with a differentiation composition; and (ii) culturing the cells in microwells to form spheroids and / or neurospheres.

3. A method for differentiating stem cells or progenitor cells into neural cells, the method comprising (i) contacting the stem cells or progenitor cells with a differentiation composition, wherein the differentiation composition comprises an ALK inhibitor: one or more of DMH1, DMH2, K02288 and A83-01; and (ii) culturing the cells in microwells to form spheroids and / or neurospheres.

4. The method of claim 3, wherein the ALK inhibitor consists of DMH1, DMH2, K02288 or A83-01.

5. The method of claim 3 or 4, wherein the ALK inhibitor comprises or consists of DMH2.

6. The method of claim 3, wherein the ALK inhibitor consists of DMH1 and DMH2.

7. The method of claim 3, wherein the ALK inhibitor consists of K02288 and DMH2.

8. The method of claim 3, wherein the ALK inhibitor consists of A8301 and DMH2.

9. The method of any one of claim 8, wherein the ALK inhibitor does not comprise LDN193189 and / or SB431542.

10. The method of any one of claims 3 to 9, wherein the concentration of the ALK inhibitor in the differentiation composition is 0.1 to 1 μM.

11. The method of any one of claims 2 to 10, wherein the stem or progenitor cell comprises an induced pluripotent stem cell (iPSC) or an embryonic stem (ES) cell.

12. The method of claim 11, wherein the stem or progenitor cells comprise embryonic stem (ES) cells.

13. The method of claim 12, wherein the ES cells are human ES cells.

14. The method of claim 13, wherein the human ES cells comprise HS420 cells.

15. The method of any one of claims 2 to 8, wherein the stem or progenitor cell comprises a totipotent stem cell, a pluripotent stem cell, or a multipotent stem cell.

16. The method of any one of claims 3 to 13, wherein contacting the cells comprises contacting the cells for a period of substantially continuous contact of about 1 to 7 days.

17. The method of any one of claims 3 to 16, wherein the cell is contacted with 0.01 to 5 μM of the ALK inhibitor.

18. The method of claim 17, wherein the cells are contacted with 10 μM ALK inhibitor.

19. The method of any one of claims 2 to 18, wherein the method further comprises contacting the stem or progenitor cell with a Rho kinase (ROCK) inhibitor.

20. The method of claim 19, wherein the ROCK inhibitor comprises Y27632.

21. The method of claim 19 or 20, wherein the cells are contacted with 5 to 15 μM ROCK inhibitor.

22. The method of any one of claims 2 to 21, wherein the cells are contacted with the ROCK inhibitor prior to contacting with the differentiation composition.

23. The method of any one of claims 2 to 21, wherein the period of time during which the cells are contacted with the ROCK inhibitor and with the differentiation composition overlaps.

24. The method of claim 22 or 23, wherein the cells are contacted with the ROCK inhibitor for a period of 1 to 48 hours.

25. The method of any one of claims 2 to 24, wherein the method does not comprise contacting the cell with a Smad inhibitor and / or a BMP4 inhibitor.

26. The method of claim 25, wherein the method does not comprise contacting the cell with LDN193189 and / or SB431542.

27. The method of any one of claims 2 to 26, wherein the method does not comprise dual Smad inhibition or single Smad inhibition.

28. The method of any one of claims 2 to 27, wherein the method does not comprise contacting the cell with noggin.

29. The method of any one of claims 2 to 28, wherein the neural cell is further defined as a dopaminergic neuron, a glutamatergic neuron, a serotonergic neuron, a cholinergic neuron, a GABAergic neuron, a motor neuron, an astrocyte, or an oligodendrocyte.

30. The method of claim 29, wherein the neural cells are defined as dopaminergic neurons.

31. The method of any one of claims 2 to 30, wherein contacting the cell with a compound, an ALK inhibitor, or a composition comprises culturing the cell in a cell culture medium comprising the compound, an ALK inhibitor, or a composition.

32. The method of claim 31, wherein the cell culture medium comprises one or more of: DMEM medium, neurobasal medium, GSK inhibitor, cAMP, GDNF, BDNF, amino acids, X-VIVO medium, antimicrobial agents, B-27 supplement, N-2 supplement and L-glutamine, sonic hedgehog protein (SHH), purine amine, FGF-8 protein (fibroblast growth factor 8), FGF-20, TGF-B3 and a gamma secretase inhibitor.

33. The method of any one of claims 24 to 32, wherein the cell is further contacted with a GSK3 (glycogen synthase kinase 3) inhibitor for a period of time.

34. The method of claim 33, wherein the GSK3 inhibitor comprises CHIR99021.

35. The method of claim 33 or 34, wherein the GSK3 inhibitor is added after 3 days after contacting with the differentiation medium.

36. The method of any one of claims 33 to 35, wherein the period of time is 5 to 15 days.

37. The method of claim 36, wherein the period of time is 10 days.

38. The method of any one of claims 32 to 37, wherein the method comprises or further comprises contacting the cell with one or more of FHF8, SHH and purine amine for a period of time.

39. The method of claim 38, wherein the cells are contacted with FHF8, SHH and / or purine amines one day after contacting with the differentiation medium.

40. The method of claim 38 or 39, wherein the period of time is 3 to 10 days.

41. The method of claim 40, wherein the period of time is 7 days.

42. The method of any one of claims 32 to 41, wherein the method comprises or further comprises contacting the cell with one or more of cAMP, GDNF, BDNF, TGFB3, FGF20, and a gamma secretase inhibitor for a period of time.

43. The method of claim 42, wherein the cells are contacted with cAMP, GDNF, BDNF, TGFB3, FGF20 and / or a gamma secretase inhibitor eight days after contacting with the differentiation medium.

44. The method of claim 42 or 43, wherein the period of time is from 15 to 40 days.

45. The method of any one of claims 2 to 44, wherein the method comprises or further comprises contacting the cell with ascorbic acid.

46. ​​The method of claim 45, wherein the concentration of ascorbic acid contacted with the cells is 100 to 400 μM.

47. The method of claim 46, wherein the concentration of ascorbic acid contacted with the cells is 200 μM.

48. The method of any one of claims 45 to 47, wherein the cells are contacted with ascorbic acid over a period of 8 to 15 days after contact with the differentiation medium.

49. The method of claim 48, wherein the cells are contacted with ascorbic acid over a period of 13 days after contacting with the differentiation medium.

50. The method of any one of claims 2 to 49, wherein the cells are contacted with ascorbic acid for a period of 10 to 40 days.

51. The method of any one of claims 2 to 50, wherein the differentiation composition comprises an extracellular matrix, or the method further comprises contacting the cells, spheroids or neurospheres with an extracellular matrix for a period of time.

52. The method of claim 51, wherein the extracellular matrix comprises laminin and / or Geltrex.

53. The method of any one of claims 2 to 44, wherein the differentiation composition does not comprise an extracellular matrix, or the method does not comprise contacting the cells, spheroids or neurospheres with an extracellular matrix.

54. The method of any one of claims 2 to 53, wherein the differentiation composition comprises an RGD peptide, or the method further comprises contacting the cell, spheroid or neurosphere with an RGD peptide for a period of time.

55. The method of any one of claims 2 to 54, wherein the cells, spheroids and / or neurospheres are cultured under hypoxic conditions for a period of time.

56. The method of any one of claims 42 to 55, wherein the period of time is 1 to 60 days.

57. The method of claim 55 or 56, wherein the hypoxic conditions comprise 0 to 10% oxygen.

58. The method of claim 57, wherein the hypoxic conditions comprise 3% oxygen.

59. The method of any one of claims 2 to 58, wherein the method further comprises contacting the cell with a HIF-1 alpha stabilizer.

60. The method of claim 59, wherein the HIF-1α stabilizer comprises one or more of the following: dimethyloxalylglycine, FG4592, CoCl2, deferoxamine mesylate, cyclometallated iridium (III) metal complex 1a, 1-(imidazol-1-ylmethyl)-3,5-diphenylpyrazole, 3,5-diphenyl-1-(pyrazol-1-ylmethyl)pyrazole, N-[(3,5-diphenylpyrazol-1-yl)methyl]-N-phenylaniline, (3,5-diphenylpyrazol-1-yl)methyl]diethylamine and (3,5-diphenylpyrazol-1-yl)methyl]diisopropylamine.

61. The method of any one of claims 2 to 60, wherein the ratio of the number of spheroids or neurospheres to the volume of cell culture medium is about 1000 spheroids or neurospheres to 200 to 1000 μl of cell culture medium.

62. The method of any one of claims 2 to 60, wherein the ratio of the number of spheroids or neurospheres to the volume of cell culture medium is about 1000 spheroids or neurospheres to 200 to 500 μl of cell culture medium.

63. The method of claim 61 or 62, wherein the ratio is maintained for a period of at least day 0 to day 42, wherein day 0 is the day the cells are first contacted with the differentiation medium.

64. The method of any one of claims 3 to 63, wherein the stem or progenitor cell comprises exogenously expressed Nurr-1 and / or Pitx3.

65. The method of any one of claims 2 to 64, wherein the stem or progenitor cell comprises a heterologous nucleic acid encoding a Nurr-1 and / or Pitx3 protein or a functional fragment thereof.

66. The method of any one of claims 2 to 65, wherein the neural cells are further defined as Nestin+, Pax-6+, and Sox-1+ cells.

67. The method of any one of claims 2 to 66, wherein culturing the cells in microwells comprises culturing the cells on a substrate material comprising a hydrophilic and porous material layer having a plurality of persistent well-shaped indentations on its outer surface supported by a semipermeable membrane on its inner surface, wherein the well-shaped indentations have a diameter of 100 μm. 2 To about 1mm 2 The hole and about 100pm 2 To about 1mm 2 The bottom surface, wherein the hydrophilic and porous material layer has a porosity that allows oxygen and cell nutrients to pass through.

68. The method of claim 67, wherein the semipermeable membrane has pores with a diameter of about 1 nm to about 200 pm, such as about 2 nm to about 40 pm.

69. A spheroid or neurosphere produced by the method of any one of claims 3 to 68.

70. A population of cells, spheroids or neurospheres produced by the method of any one of claims 3 to 68.

71. The cell population, spheroid population or neurosphere population of claim 70, wherein the percentage of non-neural cells in the cell culture or spheroid after a period of contact with the differentiation composition is less than 30%.

72. The cell population of claim 71, wherein the time period is 4 to 8 days.

73. A method for treating a disease in a mammalian subject, comprising administering to the subject a therapeutically effective amount of the neural cell population, spheroid population, or neurosphere population of claim 70.

74. The method of claim 73, wherein the disease comprises a neurodegenerative disease.

75. The method of claim 73 or 74, wherein the subject is a human subject.

76. The method of any one of claims 73 to 75, wherein the spheroids are dissociated prior to administration.

77. The method of any one of claims 73 to 75, wherein the method does not comprise dissociation of the spheroids prior to administration.

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