Methods for producing and using differentiated neural cells - Patents.com

Abstract

The present disclosure provides a method for differentiating stem cells and progenitor cells into neural cells by a method that does not use an inhibitor of the BMP4 pathway that causes SMAD inhibition.An aspect of the present disclosure relates to a method for differentiating stem cells or progenitor cells into neural cells, comprising: (i) contacting stem cells or progenitor cells with a differentiation composition; and (ii) culturing the cells in a microwell to form spheroids.An additional aspect relates to a method for differentiating stem cells or progenitor cells into neural cells, comprising: (i) contacting stem cells or progenitor cells with a differentiation composition that comprises one or more of ALK inhibitors: DMH1, DMH2, K02288, A83-01 or combinations thereof; and (ii) culturing the cells in a microwell to form spheroids.Also described are neural cells, spheroids, populations of cells or populations of spheroids produced by the methods of the claims.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 329,713, filed April 11, 2022, which is incorporated by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted in XML format, and is incorporated by reference in its entirety. Said XML copy, created on April 11, 2023, is entitled "CLFRP0404WO_SL" and is 12 kilobytes in size.

[0003] I. FIELD OF THE INVETION The present invention relates to the fields of cell biology and disease treatment. [Background technology]

[0004] II. Background Cell populations that retain the ability to differentiate into multiple specialized cell types are useful for developing multiple lineage-specific differentiated cell populations. These lineage-specific differentiated cell populations may find use in cell replacement therapy for patients with diseases that result in the loss of function of a particular cell population. In addition to their direct therapeutic value, lineage-specific differentiated cells are also valuable research tools for a variety of purposes, such as in vitro screening assays to identify, confirm and test 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. Thus, there is a need for methods to generate DA neuronal cells from pluripotent cells, since such cells can be used therapeutically and in disease models, for example, to identify new therapeutic substances for the treatment of neurodegenerative diseases. Summary of the Invention

[0005] The present disclosure provides a method for differentiating stem cells and progenitor cells into neural cells by a method that does not use an inhibitor of the BMP4 pathway that results in SMAD inhibition. The method includes a method for differentiating stem cells or progenitor cells into neural cells, comprising: (i) contacting stem cells or progenitor cells with a differentiation composition; and (ii) culturing the cells in a microwell to form spheroids and / or neurospheres. Also described is a method for differentiating stem cells or progenitor cells into neural cells, comprising: (i) contacting stem cells or progenitor cells with a differentiation composition that includes one or more of the following ALK inhibitors: DMH1, DMH2, K02288, and A83-01; and (ii) culturing the cells in a microwell to form spheroids and / or neurospheres. Also described is a neural cell, spheroid, population of cells, or population of spheroids produced by the method of the claims. The methods also include a method of treating a disease in a mammalian subject comprising administering to the subject a therapeutically effective number of a neural cell, spheroid or neurosphere of the present disclosure, a population of cells, spheroids or neurospheres. Also described is a method of screening a test compound comprising: (a) contacting the test compound with a cell, spheroid or neurosphere of the present disclosure; and (b) measuring the function, physiology or viability of the cell.

[0006] The term spheroid refers to cells that are three-dimensional spherical in shape. Spheroids can be cultured in a manner that allows for the formation of cells that are 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% of the cells in the spheroid. (or any extent derivable therein) may be precursors or may consist of or consist essentially of progenitor cells, such that at least 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% (or any extent derivable therein) of the cells in the spheroid may be precursors. Neurospheres are composed of more differentiated cells, such as cells committed to the neuronal lineage.Neurospheres are a type of cell that expresses neural precursors. 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% (or any range derivable therein), or at least 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 ...10, 110, 111, 112, 113, 114, 115, 116, 117, 118, 11 3, 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% (or any range derivable therein) of the cells.

[0007] The 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 include 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.

[0008] The stem or progenitor cells may include induced pluripotent stem cells (iPSCs) or embryonic stem (ES) cells. The stem or progenitor cells may include hematopoietic stem or progenitor cells. The stem or progenitor cells may be further defined as totipotent, pluripotent or multipotent stem cells. The cells may include embryonic stem (ES) cells. The cells may be human cells or derived from human cells. The cells may be human ES cells. The human ES cells may include HS420 cells or may be further defined as HS420 cells. The stem or progenitor cells may exclude iPSCs, ES cells, hematopoietic stem or progenitor cells, totipotent cells, pluripotent cells, multipotent stem cells, human cells, cells derived from human cells, or HS420 cells.

[0009] The step of contacting the cells may include contacting the cells for a substantially continuous contact period of about 1-7 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 particular period, but does not exclude shorter non-contact periods, such as periods during which the cells may be washed, re-plated, trypsinized, or have cell culture medium changed. The period of time, e.g., the period during which the compound or ALK inhibitor is contacted with the cells, is at least 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, 109, 109, 109, 1000, 1010, 1020, 1030, 104 , 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 (or any range derivable therein), at most 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 (or any range derivable therein), about 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 (or any range derivable therein), or exactly 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 5, 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 (or any range derivable therein). Contact, such as contacting cells with a compound described herein or an ALK inhibitor, may be defined from a particular time period, for example from the time when differentiation medium is added (FIG. 12), where day 0 is the day the cells are first contacted with differentiation medium. Contact may be from -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, 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, 10 9, 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, orThe period may end on a particular day, such as day 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, or any range derivable therein.

[0010] The cells may be contacted with 0.01 to 5 μM of a compound described herein and / or an ALK inhibitor. The cells may be contacted with at least 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 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, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 3.10, 3.11, 3.12, 3.13, 3.14, 3.15, 3.16, 3.17, 3.18, 3.19, 3.20, 3.21, 3.22, 3.23, 3.24, 3.25, 3.26, 3.27, 3.28, 3.29, 3.30, 3.31, 3.32, 3.33, 3.34, 3.35, 3.36, 3.37, 3.38, 3.39, 3.40, 3.41, 3.42, 3.43, 3.44, 3.45, 3.46, 3.47, 3.48, 3.49, 3.50, 3.51, 3.52, 3.53, 3 .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 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, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 3.10, 3.11, 3.12, 3.13, 3.14, 3.15, 3.16, 3.17, 3.18, 3.19, 3.20, 3.21, 3.22, 3.23, 3.24, 3.25, 3.26, 3.27, 3.28, 3.29, 3.30, 3.31, 3.32, 3.33, 3.34, 3.35, 3.36, 3.37, 3.38, 3.39, 3.40, 3.41, 3.42, 3.43, 3.44, 3.45, 3.46, 3.47, 3.48, 3.49, 3.50, 3.51, 3.52, 3.53, 37, 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 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, The cells may be contacted with 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 (or any range derivable therein) of a compound described herein or an ALK inhibitor. The cells may be contacted with 0.2 μM of a compound described herein or an ALK inhibitor. The cells may be contacted with 0.8 μM of a compound described herein or an ALK inhibitor.

[0011] The method may further include contacting the cell with a Rho kinase (ROCK) inhibitor. The method may exclude contacting the cell with a ROCK inhibitor. The ROCK inhibitor may include Y27632. Other ROCK inhibitors useful in the methods of the present disclosure include Fasudil, Ripasudil, Netarsudil, RKI-1447, GSK429286A and Y30141. The method may exclude contacting the cell with Y27632, Fasudil, Ripasudil, Netarsudil, RKI-1447, GSK429286A and / or Y30141. The cell may be contacted with 5-15 μM of the ROCK inhibitor.The cells are at least 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 of ROCK inhibitor (or any range derivable therein), at most 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 about 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 of ROCK inhibitor (or any range derivable therein); The cells may be contacted with μM, nM or mM of the ROCK inhibitor (or any range derivable therein), or with exactly 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 of the ROCK inhibitor (or any range derivable therein). The cells may be contacted with the ROCK inhibitor before contact with the ALK inhibitor. The cells may be contacted with the ROCK inhibitor immediately before contact 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 not overlap, including a period of time between when the cells are contacted with the ROCK inhibitor and when the cells are contacted with the ALK inhibitor. The cells 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 not overlap, including a period of time between when the cells are contacted with the ALK inhibitor and when the cells are contacted with the ROCK inhibitor.This period of time shall be at least 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 therein), and at most 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. The cells may be contacted with the ROCK inhibitor for a period of 1 to 48 hours. The cells may be contacted with the ROCK inhibitor for a period of 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, 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 may be defined as from a particular time period, for example from the time when differentiation medium is added (Figure 12), where day 0 is the day the cells are first contacted with differentiation medium.Contacts are -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, or may begin on day 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, It may end on a particular day, such as day 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, or any range derivable therein.

[0012] The method may exclude contacting the cells, spheroids or neurospheres with one or more Smad inhibitors. The method may exclude contacting the cells, spheroids or neurospheres with LDN193189 and / or SB431542. The method may exclude contacting the cells, spheroids or neurospheres with LDN193189 and SB431542. The method may exclude dual or mono Smad inhibition. The method may exclude contacting the cells with Noggin protein or Noggin regulators, such as activators or inhibitors of Noggin or direct activators or inhibitors of Noggin. The method may exclude contacting the cells with a BMP4 inhibitor. The method may exclude contacting the cells with Noggin and / or Chordin or activators thereof.

[0013] The neuron may 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 neuron may be further defined as a neuron as described herein. The term "dopaminergic neuron" or "DA neuron" refers to a neuron that has the ability to produce dopamine (3,4-dihydroxyphenylethylamine). A dopaminergic neuron does not always have to produce dopamine, but only has to have the ability to produce dopamine. The DA neuron may be a DA neuron of group A8, group A9, group A10, group A11, group A12, group A13, group A14, group A15, group A16, group Aaq, or group telencephalic DA neuron. The neural cells may be excluding dopaminergic neurons, glutamatergic neurons, serotonergic neurons, cholinergic neurons, GABAergic neurons, motor neurons, astrocytes or oligodendrocytes. The DA neurons may be excluding A8, A9, A10, A11, A12, A13, A14, A15, A16, Aaq or telencephalic DA neurons.

[0014] Contacting cells with the compound or ALK inhibitor described herein may include culturing cells in a cell culture medium that includes the compound or ALK inhibitor.Cell culture medium may include one or more of DMEM medium, Neurobasal medium, GSK inhibitor, cAMP, GDNF, BDNF, amino acid, X-VIVO medium, antibacterial agent, B-27 supplement, N-2 supplement and L-glutamine, sonic hedgehog protein (SHH), purmorphamine, FGF-8 protein (fibroblast growth factor 8), FGF-20, TGF-B3 and gamma secretase inhibitor.Gamma secretase inhibitors include, for example, DAPT (reference number CAS 208255-80-5) and compound E (reference number CAS 209986-17-4). The method may exclude contacting the cells with 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), purmorphamine, FGF-8 protein (fibroblast growth factor 8), FGF-20, TGF-B3 and gamma secretase inhibitor, DAPT (reference number CAS 208255-80-5), Compound E (reference number CAS 209986-17-4), GSK3 inhibitor or CHIR99021. The cells may further 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 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 period may be 5-15 days. The period may be 10 days.GSK3 inhibitors, cAMP, GDNF, BDNF, amino acids, X-VIVO medium, antibacterial agents, B-27 supplement, N-2 supplement, L-glutamine, sonic hedgehog protein (SHH), purmorphamine, FGF-8 protein (fibroblast growth factor 8), FGF-20, TGF-B3, gamma secretase inhibitors, DAPT (reference number CAS 208255-80-5), Compound E (reference number CAS 209986-17-4) and / or contact with CHIR99021, -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, May begin on day 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, or may begin on day 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, It may end on a particular day, such as day 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, or any range derivable therein.

[0015] The method may include or further include contacting the cells with one or more of FHF8, SHH and purmorphamine for a period of time. The cells may be contacted with FHF8, SHH and / or purmorphamine one day after contact with the differentiation medium. The period may be 3-10 days. The period may be 7 days.

[0016] 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 may be 15 to 40 days.

[0017] GSK3 inhibitors, cAMP, GDNF, BDNF, amino acids, X-VIVO medium, antibacterial agents, B-27 supplement, N-2 supplement, L-glutamine, sonic hedgehog protein (SHH), purmorphamine, FGF-8 protein (fibroblast growth factor 8), FGF-0, TGFB3, gamma secretase inhibitors, DAPT (reference number CAS 208255-80-5), compound E (reference number CAS 209986-17-4) and / or contact with CHIR99021, -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, May begin on day 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, or may begin on day 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, It may end on a particular day, such as day 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, or any range derivable therein.

[0018] The method may include or further include contacting the cells with ascorbic acid. The ascorbic acid may be at a concentration of 100-400 μM. The concentration of ascorbic acid may be 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, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 3 65, 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, 5 35, 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 (or any range derivable therein), and may be at least 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, 265, 270, 275, 280, 285, 290, 300, 310, 320, 330, 340, 350, 360, 370, 385, 390, 400, 410, 420, 430, 440, 450, 465, 470, 485, 490, 500, 510, 520, 530, 540, 550, 560, 570, 585, 590, 600, 610, 620, 630, 640, 650, 665, 670, 685, 690, 700, 710, 720, 730, 740, 750, 765, 7 5, 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 (or any range derivable therein), or at most 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, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 3 50, 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 (or any range derivable therein). The ascorbic acid may be at a concentration of 200 μM upon contact with the cells. The cells may be contacted with the ascorbic acid for a period of 8 to 15 days after contact with the differentiation medium. After contact with differentiation medium, the cells were incubated for 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 (or any range derivable therein), or at least 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, 8 0, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 days (or any range derivable therein), or at most 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 (or any range derivable therein), or at most 1, 2, The cells may be contacted with ascorbic acid for a period of 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 days (or any range derivable therein). The method may exclude the step of 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.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, 9 5, 96, 97, 98, 99, or 100 days, or any range derivable therein, or at least 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, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 120, 121, 122, 123, 124, 1 8, 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 or any range derivable therein, or at most 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 The composition may be contacted with ascorbic acid for 0, 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, or any range derivable therein. Contact with ascorbic acid may reduce the following: -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,It may start on day 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, The period may end on a particular day, such as day 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, or any range derivable therein.

[0019] The differentiation composition may include an extracellular matrix, or the method may further include 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 exclude contacting the cells, spheroids, or neurospheres with an extracellular matrix. The differentiation composition may exclude an extracellular matrix. Other extracellular matrices that may be included or excluded in the methods and compositions include collagen, elastin, fibronectin, hyaluronic acid, tenascin, hyaluronan, and proteoglycans. The differentiation composition may include an RGD peptide, or the method may further include contacting the cells, spheroids, or neurospheres with an RGD peptide for a period of time. The tripeptide Arg-Gly-Asp (RGD) consists of arginine, glycine, and aspartic acid. It was originally identified as an amino acid sequence within the extracellular matrix protein fibronectin that mediates cell adhesion.

[0020] The cells, spheroids and / or neurospheres may be cultured under hypoxic conditions for a period of time. The period may be from 1 to 60 days. The hypoxic conditions may include 0-10% oxygen. The hypoxic conditions may be 2-5% oxygen, or may be 3% oxygen. The period may be from 1 to 60 days. The hypoxic conditions may include 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% oxygen, or any range derivable therein, including at least 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% oxygen, or any range derivable therein, or at most 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% oxygen, or any range derivable therein. The period of time may be 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.Hypoxic conditions were -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, 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, or any range derivable therein.

[0021] The method may include or further include contacting cells with HIF-1α stabilizer.The HIF-1α stabilizer may include one or more of dimethyloxalylglycine, FG4592, CoCl2, deferoxamine mesylate, cyclometalated 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.The method may exclude contacting cells with HIF-1α stabilizer. The method may exclude 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 time period may be 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.Contact of HIF-1α stabilizers with cells results in -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, 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, May begin on day 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, or on day 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, 1, 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, or any range derivable therein.

[0022] The ratio of the number of spheroids or neurospheres to the amount of cell culture medium may be about 1000 spheroids or neurospheres for 200-1000 μl of cell culture medium.The ratio of the number of spheroids or neurospheres to the amount of cell culture medium may be about 1000 spheroids or neurospheres for 200-500 μl of cell culture medium. The ratio of the number of spheroids or neurospheres to the amount of cell culture medium is approximately 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, 300, 305, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 8 0, 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, 5 30, 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 or 1000 μl of cell culture medium or any range derivable therein,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, 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 or 1000 μl of cell culture medium or any range derivable therein, or at most 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, 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, 7 15, 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 or 1000 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, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 885, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1200, 1210, 1220, 1230, 1240, 1250, 1260, 1270, 1280, 1290, 1300, 1310, 1320, 13 25, 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, 79 0, 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 spheroids or neurospheres, at most 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, 8 60, 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 spheroids or neurospheres or at least 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 885, 890, 900, 9100, 9200, 9300, 940, 950, 960, 970, 985, 990, 1000, 10100, 10200, 10300 90, 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, 74 5, 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,The cell may be 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 spheroids or neurospheres. ,

[0023] The ratio may be maintained for a period of at least from day 0 to day 42, where day 0 is the day the cells are first contacted with the differentiation medium. The ratio is at least 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, or any range derivable therein. 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 days, or any range derivable therein, or at most 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, or any range derivable therein.

[0024] The stem or progenitor cells may contain exogenously expressed Nurr-1 and / or Pitx3. The stem or progenitor cells may contain heterologous nucleic acids encoding Nurr-1 and / or Pitx3 proteins or functional fragments thereof. The neural cells may be further defined as Nestin+, Pax-6+ and Sox-1+ cells. The method may eliminate cells that contain exogenous expression of heterologous nucleic acids encoding Nurr-1 and / or Pitx3 and / or heterologous nucleic acids encoding Nurr-1 and / or Pitx3, or the cells may eliminate exogenous expression of heterologous nucleic acids encoding Nurr-1 and / or Pitx3 and / or heterologous nucleic acids encoding Nurr-1 and / or Pitx3.

[0025] The step of culturing cells in the microwells may include culturing the cells on a substrate material comprising a layer of hydrophilic porous material having a plurality of continuous well-shaped depressions on its outer surface supported by a semipermeable membrane on its inner surface, wherein the well-shaped depressions are 100 pm or less in diameter. 2 From about 1 mm 2 of opening and about 100 pm 2 From about 1 mm 2wherein the hydrophilic porous material layer has a porosity that allows the passage of oxygen and cellular nutrients. The semi-permeable membrane may have pores having a diameter of about 1 pm to about 200 nm, for example about 2 pm to about 40 nm. Semipermeable membranes are 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, 132, 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, 190, 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, 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, at least 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, 132, 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, 1 70, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 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, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 2 60, 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, or at most 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, 5 2, 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, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 1 45, 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, 190, 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, 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) may have pores with diameters of pm or nm.

[0026] The percentage of non-neuronal cells in the cell culture, spheroid or neurosphere after contact with a compound described herein or an ALK inhibitor for a period of time may be less than 30% in the cell population of the present disclosure. The percentage of non-neuronal cells in the cell culture after contact with a compound described herein or an ALK inhibitor for a period of time may 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) in the cell population. The period of time may be 4 to 8 days. The period shall be at least 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, and at most 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, The range may be 0, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or any range derivable therein, or about 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.

[0027] As used herein, the term "3D cell culture" refers to a cell culture in which cells are made to grow or interact with their surroundings all in three dimensions. This can be achieved by growing cells on low-attachment plates, bioreactors, or small capsules. Cells grown in 3D culture may take on the shape of spheroids or neurospheres as they grow.

[0028] The term "cells" as used herein, unless otherwise specified, may include spheroids and neurospheres, since these are composed of cells.

[0029] The method includes -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, 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, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, At days 1, 92, 93, 94, 95, 96, 97, 98, 99 or 100, or any range derivable therein, the cells were treated with ALK inhibitors, DMH1, DMH2, K02288, A83-01, ROCK inhibitors, Y27632, fasudil, ripasudil, netarsudil, 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, purmorphamine, FGF-8 protein, FGF-20, TGF-B3 and gamma secretase inhibitors, DAPT (Reference No. CAS 208255-80-5), compound E (Reference No. CAS 209986-17-4), GSK3 inhibitors, 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 may be omitted.

[0030] 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 spheroids or neurospheres may be dissociated prior to administration. The method may exclude dissociation of the spheroids or neurospheres prior to administration.

[0031] Throughout this application, the term "about" is used in accordance with its plain and ordinary meaning within the field of cellular and molecular biology to indicate that a value includes the standard deviation of error for the device or method being utilized to determine the value.

[0032] When used in conjunction with the term "comprising," the use of the words "a" or "an" can mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more."

[0033] As used herein, the terms "or" and "and / or" are utilized to describe multiple elements in combination or mutually exclusive. For example, "x, y, and / or z" can refer to "x" only, "y" only, "z" only, "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.

[0034] The words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), "characterized by" (and any form of including, such as "characterized as") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0035] The compositions and methods for their use may "comprise," "consist essentially of," or "consist of" any of the components or steps disclosed throughout this application. The phrase "consisting of" excludes any elements, steps, or components not specified. The phrase "consisting essentially of" limits the scope of the described subject matter to the specified materials or steps and those that do not materially affect its basic and novel characteristics. It is contemplated that 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."

[0036] It is specifically contemplated that any limitation discussed with respect to one embodiment or aspect of the present invention may be applied to any other embodiment or aspect of the present invention.Furthermore, any composition of the present invention may be used in any method of the present invention, and any method of the present invention may be used to make or utilize any composition of the present invention.Aspects of the embodiments described in the examples are also embodiments that may be implemented in the context of embodiments discussed elsewhere in different examples or elsewhere in this application, for example in the Summary of the Invention, Detailed Description of the Embodiments, Claims, and Figure Legend Descriptions.

[0037] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating particular embodiments of the present invention, are given by way of illustration only, since various modifications and changes within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0038] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0039] [Figure 1] Dopaminergic differentiation experiment. Culture methods including 3D-immersion (A) and 3D-AirLiwell (B) are shown. [Diagram 2] Dopaminergic neurospheres are more homogeneous and standardized. [Diagram 3] Expression of neural precursor and neuronal markers by qPCR (top) and midbrain and dopaminergic specific markers by qPCR (bottom), showing superior neural induction and specification for several markers in 3D-ALi neurospheres. [Figure 4]3D-ALi / 3D-i fold change in gene expression after 6 weeks of differentiation by RNA-seq, showing higher expression of dopaminergic precursor markers but a lack of neuronal maturation with 3D-ALi. [Diagram 5] Fold change in gene expression after 6 weeks of differentiation in 3D-ALi / 3D-i by RNA-seq. After 6 weeks in culture, neurospheres cultured in 3D-ALi showed downregulation of several cell proliferation markers. [Figure 6] Tyrosine hydroxylase expression by Western blot. Although 3D-ALi have the ability to produce dopamine, the lack of maturation was confirmed by Western blot and HPLC. [Figure 7] Dopamine content by HPLC. Although 3D-ALi have the capacity to produce dopamine, the lack of maturation was confirmed by Western blot and HPLC. [Figure 8] The 3D-Ali neurospheres are electrophysiologically functional, and the electrical signals are more stable and homogeneous. [Figure 9] 3D-Ali dopaminergic neurospheres cultured on extracellular matrix have the capacity to mature. [Figure 10] 3D-Ali dopaminergic neurons observed by electron microscopy are more homogenous and pure. [Figure 11] The chemical structures of DMH1, DMH2, A83-01 and K02288 are shown. [Figure 12A] 1 illustrates aspects of a method for differentiating stem cells into neural cells using 3D culture. [Figure 12B] 1 illustrates aspects of a method for differentiating stem cells into neural cells using 3D culture. [Figure 13A] Single-cell RNA-seq analysis of dopaminergic neurospheres at 6 weeks. [Figure 13B] Single-cell RNA-seq analysis of dopaminergic neurospheres at 6 weeks. [Figure 13C]Single-cell RNA-seq analysis of dopaminergic neurospheres at 6 weeks. [Figure 14] Quantitative PCR analysis of dopaminergic neurospheres at 6 weeks. [Figure 15] 3D-submerged neurospheres produce more dopamine than 3D-Ali neurospheres as measured by HPLC. [Figure 16] The electrical signals of neurons contained in 3D-Ali neurospheres are more similar to physiological functions and are highly standardized. [Figure 17] Experimental design and operation of the maturation protocol. [Figure 18] Effects of hypoxia and laminin on dopaminergic maturation. [Figure 19] Examination of the volume:neurosphere ratio and its effect on dopaminergic maturation. [Figure 20] Protocol design with added antioxidants. [Figure 21A] Effect of antioxidant addition on DA maturation. Overall morphology of neurospheres after antioxidant addition is shown. [Figure 21B] Effect of antioxidant addition on DA maturation. The effect of specific markers when neurospheres were cultured with antioxidants in the 3D-Ali method is shown. [Figure 22] Dopaminergic differentiation with the addition of Alk-I compounds in the 3D-Ali protocol. The concentrations of compounds tested were 0.2 μM and 0.8 μM. [Figure 23A]Quantitative PCR results: dopaminergic differentiation with the addition of Alk-I compound in the 3D-Ali protocol. qPCR results at weeks 2 and 4 are shown for (A) Ki-67 and Nestin; (B) Pax6 and B3-Tub; (C) Lmx1a and Nurr-1; and (D) Map-2 and TH. Data shown in each group of 12 bars in Figures 23A-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. The 11 bars in the bottom bar graph of FIG. 23D 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 and 11: A-8301 0.8 μM. [Figure 23B] See legend to Figure 23A. [Figure 23C] See legend to Figure 23A. [Figure 23D] See legend to Figure 23A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] Detailed Description of the Invention The formation of neuroectoderm is a crucial step in the differentiation of pluripotent stem cells into neural cells and tissues. This disclosure provides an effective alternative to methods using Smad inhibition protocols (various combinations of Noggin, LDN193189 and SB431542). We hypothesized that differentiating progenitor cells into mature neurons in three-dimensional cell cultures would have advantages over differentiation methods in two-dimensional cell cultures. These advantages include: 1) no cell detachment (which can induce chromosomal changes and increase the risk of cancerization of cells), 2) reduced cell mortality due to enzymatic cell detachment, 3) simplified cell culture protocols, 4) improved standardization and homogeneity of transplanted cells, 5) cells more representative of physiological neural tissue, and 6) improved yield of neurally differentiated cells.

[0041] I. Definition The term "differentiation" as used with respect to cells in a differentiated cell line refers to the process by which a cell differentiates from one cell type (e.g., a multipotent, totipotent, or pluripotent differentiable cell) to another cell type, such as a fully differentiated cell. More generally, the term "differentiation" refers to the process by which an unspecialized stem or progenitor cell acquires the characteristics of a specialized or fully differentiated cell, such as a brain, heart, liver, or muscle cell. Differentiation is usually controlled by the interaction of the cell's genes with the physical and chemical conditions outside the cell through signaling pathways involving proteins embedded in the cell surface.

[0042] The precursor cells in this disclosure are included in somatic cells. The precursor cells are multipotent cells. Totipotent cells, pluripotent cells, and multipotent cells can be "stem cells" that can differentiate into one or more different cell types. The terms "stem cell", "embryonic stem cell", and "induced pluripotent stem cell" have been described above.

[0043] In this specification, the term "stem cell" refers to a cell that can be cultured in vitro and differentiate into cells of multiple lineages that constitute a living body. Specifically, it includes 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.

[0044] II. ALK inhibitors Pluripotent stem cells (PSCs) derived from embryos or from somatic cells have the capacity to differentiate into a wide variety of cell types of interest for tissue modeling and cell therapy. Mimicking embryonic development in vitro represents the best approach to generate differentiated cells with well-defined properties. The first natural specification of embryonic tissues occurs at the gastrulation stage and involves the differentiation of the three germ layers: ectoderm, mesoderm and endoderm, from which all adult tissues are derived.

[0045] Multiple combinations of Noggin, LDN 193189 and SB 431542 are commonly used and are commonly referred to as "dual SMAD inhibition". This is the most widely used method to date to induce early neural specification of PSCs in vitro. Described herein is a report of a differentiation method that does not involve "dual Smad inhibition" but instead uses four chemicals that inhibit ALK: DMH1, DMH2, K02288 and A83-01 (Table 1) to achieve differentiation and induce neural specification of pluripotent stem cells in vitro.

[0046] (Table 1) Compounds TIFF2025512359000001.tif83160

[0047] III. Cell sources The present disclosure relates to the differentiation of a starting population of stem or progenitor cells into neural cells. The stem or progenitor cells may be those described herein and / or derived from the sources described herein. The stem or progenitor cells may be ES cells. It is contemplated that ES cells derived from any warm-blooded animal, preferably a mammal, may be used. Examples of mammals include mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cows, horses, goats, monkeys, and humans. Preferred examples of ES cells include ES cells derived from humans. The stem or progenitor cells may exclude cells derived from or taken from a human fetus.

[0048] Specific examples of ES cells include ES cells of mammals and the like established by culturing early pre-implantation embryos, ES cells established by culturing early embryos prepared by nuclear transfer of somatic cell nuclei, and ES cells obtained by modifying genes on the chromosomes of these ES cells by genetic engineering methods. Each ES cell can be prepared according to methods commonly used in the relevant field or known literature.

[0049] Mouse ES cells were established in 1981 by Evans et al. (1981, Nature 292: 154-6) and Martin G R. et al. (1981, Proc Natl Acad Sci 78: 7634-8), and can be purchased from, for example, Sumitomo Dainippon Pharma Co., Ltd. (Osaka, Japan).

[0050] Human ES cells were established in 1998 by Thomson et al. (Science, 1998, 282: 1145-7) and are available from WiCell Research Institute (available on the World Wide Web at wicell.org / , Madison, Wis., USA), the US National Institute of Health, Kyoto University, and the like, and can be purchased from, for example, Cellartis (available on the World Wide Web at cellartis.com / , Sweden).

[0051] The stem or progenitor cells may be iPSCs (also known as iPS cells). The iPS cells may be derived from any warm-blooded animal, preferably a mammal. Examples of mammals include mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cows, horses, goats, monkeys, and humans. A preferred example of the iPS cells is a human-derived iPS cell.

[0052] Specific examples of iPS cells include cells that have acquired pluripotency like 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). Other methods include further reducing the amount of introduced genes (Nature. 2008 Jul. 31; 454 (7204): 646-50), 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), and using transcription factor proteins instead of genes (Cell Stem Cell. 2009 May 8; 4(5): 381-4). The produced iPS cells can be used for the present invention, regardless of the production method.

[0053] Examples of human iPS cell lines include the 253G1 line (an iPS cell line prepared by expressing OCT4 / SOX2 / KLF4 in skin fibroblasts of a 36-year-old woman), the 201B7 line (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), and the NHDF-iPS (297 L1). (An iPS cell line prepared by expressing OCT4 / SOX2 / KLF4 / c-MYC in skin fibroblasts from a newborn boy).

[0054] IV. Cell culture method Included herein is a method for differentiating stem cells or progenitor cells, or a starting population of stem cells and / or progenitor cells, into neural cells.The disclosed method may comprise or further comprise the step of culturing cells in the medium defined herein and / or comprising or further comprising the supplements and ingredients described herein.These methods will be discussed in more detail below.It is further specifically intended that the disclosed method may exclude the step of culturing cells in the medium or medium with supplements described herein.

[0055] Cell culture may be three-dimensional (3D cell culture). The 3D environment provides a balance between the accumulation of paracrine factors in the extracellular matrix and the regeneration of nutrients. Derivation of neurons in 3D also has the advantages of i) using less medium and thus less amount of novel neuroactive substances used in the assay, ii) having the option of multiplexing (96-well plate format), essential for automated screening activities.

[0056] To achieve the three-dimensional characteristics of cell culture, cells can be grown or differentiated in a matrix, scaffold or microwell. In principle, suitable matrices or scaffolds that can be used in three-dimensional cell culture are known to those skilled in the art. Thus, such matrices or scaffolds can be any matrix or scaffold. For example, matrices or scaffolds can be extracellular matrices, including any of natural molecules or synthetic polymers, bio- and synthetic hybrids, metals, ceramics, and bioactive glasses or carbon nanotubes.

[0057] Exemplary natural extracellular matrix molecules include collagen, basement membranes such as laminin or fibrin, alginate, chitosan, hyaluronic acid, silk fibroin, cellulose acetate, casein, chitin, fibrinogen, gelatin, elastin, or poly-(hydroxyalkanoates). Synthetic extracellular matrix polymers include hyaluronic acid (HA)-modified, polyethylene glycol (PEG)-modified, self-assembling protein hydrogels, poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), polyurethane, or PGS. Biological and synthetic hybrids can include, for example, polycaprolactone-chitosan, PLLA-hydroxyapatite, hydroxyapatite-bioglass-ceramic, poly-(hydroxylalkanoates)-bioglass, hydroxyapatite-collagen, PCL-gelatin, or PCL-collagen. Exemplary metals include tantalum, magnesium and its alloys, titanium and its alloys, or nitinol (an alloy of nickel and titanium). Examples of ceramic and bioactive glass matrices / scaffolds 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 0.4-2 nm graphite. Carbon nanotubes can include CNT-polycaprolactone, CNT-ceramic matrix, 45S5 bioglass-CNT, CNT studded with gelatin hydrogel, CNT-TiO2, CNT-laminin, CNT grafted with polyacrylic acid or CNT-TGF-β.

[0058] The matrix or scaffold may be a hydrogel, such as Matrigel, fibrin gel, or alginate gel. Optionally, growth factors and other molecules may be added to the Matrigel. The Matrigel may also be mixed with a culture medium. For example, the Matrigel may be diluted with a culture medium as described herein or in the Examples. The Matrigel may be BD Matrigel™.

[0059] The three-dimensional cell culture used in the present invention may exclude organoid culture.Organoid is a three-dimensional tissue structure, which is often generated from pluripotent stem cells (PSCs), but also from, for example, neuroepithelial stem cells, self-organizes, and reproduces the complex aspects of corresponding organs, from physiological processes to regeneration and disease.

[0060] The method may include contacting the cells or spheroids with a hedgehog signaling pathway activator, or the differentiation medium may include a hedgehog signaling pathway activator. "Hedgehog signaling pathway" or "SHH pathway" is well known in the art and 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, and induce downstream signaling cascades. Downstream target genes of SHH signaling include GLI1, GL12, and / or GL13. Thus, the term "activator of hedgehog signaling pathway" also refers to an activator of any one of the above molecules that form part of this signaling pathway.

[0061] 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-naphthoxy)-6-(4-morpholinoanilino)-9-cyclohexylpurine 9-cyclohexyl-N-[4-(4-morpholinyl)phenyl]-2-(1-naphthalenyloxy), CAS number: 483367-10-8), SHH, smoothened agonist (SAG; 3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-[[3-(4-pyridinyl)phenyl]methyl]-benzo[b]thiophene-2-carboxamide, CAS number: 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 number: 612542-14-0). Thus, the SHH pathway activator can be purmorphamine. The SHH pathway activator can also be a recombinant or truncated form of SHH that retains SHH pathway activation function, such as SHH C24II.

[0062] The method may include contacting the cells or spheroids with a neurotrophin, or the differentiation medium may include a neurotrophin. The term "neurotrophin" as used herein refers to a family of proteins that regulate the survival, development and function of neurons. Exemplary neurotrophins include insulin-like growth factor 1 (IGF), fibroblast growth factor (FGF), transforming growth factor beta (TGF), leukemia inhibitory factor (LIF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), and ligands of the GDNF family and ciliary neurotrophic factor (CNTF). Ligands of the GDNF family include glial cell line-derived neurotrophic factor (GDNF), neurturin (NRTN), artemin (ARTN) and persephin (PSPN).

[0063] The method may include contacting the cells or spheroids with an antioxidant, or the differentiation medium may include an antioxidant. An antioxidant is a molecule that inhibits the oxidation of other molecules. The terms "oxidation" and "antioxidant" are well known in the art and are described, for example, in Nordberg J, Arner E S. (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 that involves the loss of electrons or an increase in oxidation state. Oxidation reactions can generate free radicals. These radicals can then initiate chain reactions. If a chain reaction occurs within 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. Thus, antioxidants refer to inhibitors of molecules involved in the oxidation process of cells.

[0064] 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 can be ascorbic acid.

[0065] The method may include contacting the cell or spheroid with an activator of activin / transforming growth factor-β (TGF-β) signaling pathway, or the differentiation medium may include an activator of activin / transforming growth factor-β (TGF-β) signaling pathway. Activin / TGF-β signaling pathway is known in the art and includes receptor ligands, including, for example, TGFB1, TGFB2, TGFB3, activin A, activin B, activin AB and / or NODAL, and binds 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 activin / TGF-β3 signaling pathway include TGFβ1, TGFβ2, TGFβ3, activin A, activin B, activin AB or nodal. Thus, an activator of the activin / TGF-β signaling pathway can be TGFβ3.

[0066] The method may include contacting the cells or spheroids with a cAMP analog, or the differentiation medium may include a cAMP analog, such a cAMP analog being a compound that has similar physical, chemical, biochemical or pharmacological properties as 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 monophosphorothioate sodium salt (Rp-cAMPS), Sp-8-hydroxyadenosine 3.,5.,-cyclic monophosphorothioate sodium salt (Sp-80H-cAMPS) and Rp8-hydroxyadenosine 3.,5.,-cyclic monophosphorothioate sodium salt (Rp-80H-cAMPS) or dbcAMP. Thus, the cAMP analog can be dbcAMP.

[0067] The method may include contacting the cell or spheroid with N2B27 medium (in which various compounds are diluted), or the differentiation medium may include N2B27 medium (in which various compounds are diluted). This means that the medium includes N2 supplement and B27 supplement. Both supplements are well known to those skilled in the art and are freely available. The B27 supplement can be B27 supplement without vitamin A.

[0068] The differentiation medium may include Neurobasal medium and / or DMEM-F12 medium. Both media can be obtained, for example, from Gibco. The N2B27 medium can include, for example, equal amounts of Neurobasal medium and DMEM / F12 medium.

[0069] The cell culture medium may be one known and used in the art for culturing stem cells, such as STEMFLEX cell culture medium. The medium may include a medium for culturing neurons, such as NEUROBASAL medium, NEUROBASAL-A medium, or Neural Progenitor Basal medium. The medium may include NS-A medium, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM zinc option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, DMEM / F12 medium, Ham's medium, RPMI 1640 medium, Fisher's medium, and mixtures thereof. Cell culture media can generally be purchased from Invitrogen, SIGMA, Wako Pure Chemical Industries, Ltd., Sumitomo Dainippon Pharma Co., Ltd., and the like.

[0070] The medium used in the present differentiation method may be a serum-containing medium or a serum-free medium (such as KNOCKOUT medium). As used herein, serum-free medium means a medium that does not contain unconditioned or unpurified serum, and a medium containing purified blood-derived components and animal tissue-derived components (e.g., growth factors) corresponds to a serum-free medium. When the medium used in the present differentiation method is a serum-containing medium, the serum may be a mammalian serum such as fetal bovine serum. The serum concentration in the medium is generally 0.01 to 20 wt % or 0.1 to 10 wt %.

[0071] The medium used in this differentiation method may contain serum substitute.Examples of serum substitute include albumin (e.g., lipid-rich albumin), transferrin, fatty acid, collagen precursor, trace elements (e.g., zinc, selenium), B-27 supplement, N2 supplement, Replacement KnockOut serum substitute, 2-mercaptoethanol, 3'thiolglycerol, and their equivalents.The concentration of these in the medium is the same as the concentration of the serum in the medium.

[0072] The medium used in the method of the present disclosure may 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 medium are the same as the concentrations of the serum in the medium.

[0073] Other culture conditions such as culture temperature, CO2 concentration, etc. can be appropriately determined. The culture temperature is not particularly limited, but 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%.

[0074] Methods can include evaluating cells, for example, evaluating protein expression by using antigen-antibody reactions, evaluating gene expression by using quantitative RT-PCR, etc. Methods can also include evaluating cells for expression of cell markers, such as Pax6, Otx2, FoxA2, ​​Lmx1a, and Msx1.

[0075] Cell culture conditions can be provided for culturing the neural cells or progenitor cells provided herein. The starting cells of the selected population are cultured at least 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 or any range of cells that can be derived therefrom, or about 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13The starting cell population may contain at least 10, 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 cells / ml or any range derivable therein or about 10, 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 The seeding density may be 100 cells / ml or any range derivable therein.

[0076] Culture vessels used to culture the cells of the present disclosure or their progeny cells can include, but are not limited to, flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, microslides, chamber slides, tubes, trays, CellSTACK (registered trademark) chambers, culture bags, and roller bottles, so long as the cells can be cultured therein. The cells may be cultured in a volume of at least 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 derivable therein 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 derivable therein depending on the needs of the culture. The culture vessel may be a bioreactor, which may refer to any device or system that supports a biologically active environment. The bioreactor may have a volume of at least 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 derivable therein 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 derivable therein.

[0077] The culture vessel can be cell-adhesive or non-adhesive, and can be selected depending on the purpose. The cell-adhesive culture vessel can be coated with any cell-adhesive substrate, such as extracellular matrix (ECM), to improve the adhesion of the vessel surface to cells. The cell-adhesive substrate can be any material intended to attach stem cells or feeder cells (if used). The cell-adhesive substrate includes collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, laminin 521, fibronectin and mixtures thereof, such as Matrigel™, as well as dissolved cell membrane preparations.

[0078] The culture method may include culturing human stem cells, such as HS420, in Stemflex medium (ThermoFisher) on laminin 521-coated tissue culture flasks (ThermoFisher). At differentiation "day 0", HS420 cells may be plated at 70% confluency and passaged using Accutase™ (ThermoFisher). 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 supplemented X-VIVO medium may be deposited in a microwell plate, for example, on a substrate material in molded microwells on an insert (720 microwells per well), at the air-liquid interface. Supplemented X-VIVO medium may be added underneath the insert. To accurately distribute the cells into each microwell, the substrate material can be gently agitated and placed on a stable support for 15 minutes to allow the cells to settle to the bottom of the wells, after which the medium from the cell suspension can be removed by aspiration with a 0.5×16 mm needle, leaving the spheroids only covered with a thin film of residual medium (10), and the plate can then be incubated at 37° C. for 24 hours.

[0079] On differentiation "day 1", 24 hours after spheroid formation, the medium underneath the inserts can be replaced with fresh medium made by combining half X-VIVO medium (Lonza) without ROCK inhibitor and half Neurobasal medium (ThermoFisher) supplemented with 1% penicillin / streptomycin, 1% non-essential amino acids, 1% B-27 supplement (ThermoFisher), L-glutamine, 100 ng / ml SHH, 2 μM purmorphamine and 100 ng / ml FGF-8 and one or more of the following neural inducers (ALK inhibitors).

[0080] On differentiation "day 3", X-VIVO medium may be gradually replaced with Neurobasal medium by exchanging half of the medium. Neurobasal medium may contain 1% penicillin / streptomycin, 1% non-essential amino acids, 1% B-27 supplement (ThermoFisher), L-glutamine, 100 ng / ml SHH, 2 μM purmorphamine, 100 ng / ml FGF-8, 3 μM CHIR99021 and neural inducer (ALK inhibitor).

[0081] On differentiation "day 8", half of the medium may 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.

[0082] On differentiation "day 13", half of the medium may be changed. CHIR99021 may be removed. The medium may be replaced with 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. Figure 12 illustrates the 3D culture method.

[0083] V. Cell culture substrate The method includes culturing cells in microwells. The microwells may be part of a substrate. The substrate may include a layer of hydrophilic porous material having a plurality of continuous well-shaped depressions on its outer surface supported by a semipermeable membrane on its inner surface, the well-shaped depressions being less than 1 pm in diameter. 2 to 1 mm 2 and approximately 1 pm 2 to 1 mm 2 The base surface is

[0084] The term "semipermeable membrane" refers to a membrane that allows the passage of solvents and some solutes. Semipermeable membranes allow certain substances present in a solution to pass preferentially over other substances, depending on the size of the pores. Semipermeable membranes include hydrophilic membranes, filter membranes, and porous membranes.

[0085] The hydrophilic porous material may be a hydrophilic gel that allows the diffusion of aqueous cell culture medium and does not adhere to the cells. Typically, the hydrophilic gel may therefore be made from a polymer selected from agarose, polyvinyl(alcohol) (PVA), agar-agar, alginate, hyaluronic acid, pectin or starch or any equivalent synthetic gel.

[0086] Hydrophilic gels can therefore be made from polymers of cytocompatible naturally derived (or modified) materials such as carbohydrates (e.g. agarose, agar, alginate, starch, pectin, chitosan, dextran, polysachrides), proteins or ECM components (e.g. laminin, collagen, hyaluronan, fibrin), peptides (e.g. gelatin) and mixtures thereof, or gels derived from natural ECM, preferably Matrigel™ (a reconstituted basement membrane preparation extracted from a mouse sarcoma, a tumor rich in extracellular matrix proteins (this material, when isolated, contains approximately 60% laminin, 30% collagen IV and 8% entactin). Entactin is a bridging molecule that interacts with laminin and collagen IV and contributes to the structural organization of these extracellular matrix molecules. Coming Matrigel™ Matrix also contains heparan sulfate proteoglycan (perlecan), transforming growth factor (TGF-β), epidermal growth factor (EGF), insulin-like growth factor (IGF-1), fibroblast growth factor (bFGF), tissue plasminogen activator, and other growth factors that occur naturally in EHS tumors.Residual matrix metalloproteinases derived from tumor cells, Myogel (a human-based extracellular matrix, Myogel, is extracted from human benign tumor tissue "leiomyoma" 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 There are also synthetically derived materials selected from poly(ethylene glycol), polyaliphatic polyurethanes, polyether polyurethanes, polyethylene copolymers, polyamides, polyvinyl alcohols, poly(ethylene oxide), polypropylene oxide, polyethylene glycol, polypropylene glycol, polytetramethylene oxide, polyvinylpyrrolidone, polyacrylamide, poly(hydroxyethyl acrylate), poly(hydroxyethyl methacrylate) and mixtures thereof.

[0087] Hydrophilic gels can be prepared by many methods known in the art that involve covalent cross-linking or non-covalent assembly or a combination of both. Covalent cross-linking can further be carried out as part of the synthesis process, for example by using bifunctional monomers during polymerization, or alternatively by cross-linking preformed macromolecules of hydrophilic polymers, which can be linear or branched, using natural or synthetic polymers during the process.

[0088] The malleable layer of hydrophilic porous material may be a layer of hydrophilic gel formed at a temperature above the gelling temperature of the gel (e.g., 20°C to 100°C above said gelling temperature). Typically, the malleable 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. The malleable layer of hydrophilic gel may be about 5 mm to 10 cm in size and about 2 mm to 10 mm in thickness once above the gelling temperature. Once formed, the hydrophilic gel inhibits adhesion of cells and spheroids, avoiding the use of anti-adhesion solutions prior to centrifugation during cell culture. The malleable layer of hydrophilic porous material may have a porosity that allows the passage of oxygen and cell nutrients. The porosity may range from about 10 nm to about 500 pm. A malleable layer of hydrophilic porous material such as agarose may have a porosity ranging from about 100 nm to about 500 mm, most of which is occupied by water molecules, allowing the passage of nutrients, small molecules, compounds, and even proteins such as albumin.

[0089] The substrate may have 4 to 1,536 well-shaped depressions, or may have 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, 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, 53 0, 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(or any range derivable therein) of well-shaped depressions, 0, 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, 52 5, 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, 80 0, 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(or any range derivable therein), or 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, 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, 5 25, 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, 8 00, 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 (or any range derivable therein).

[0090] The formation of spheroids can be achieved by gravity over a period of about 2 to 5 hours, or by subjecting the cell culture vessel containing the cell-seeded suspended substrate material to centrifugation to induce cell aggregation. The cell culture vessel may be centrifuged at 100 x g for 5 to 10 minutes.

[0091] VI. Neurons The method includes differentiation of the cells into neural cells. The method may be used to generate one or more types of neural cells, including motor neurons, sensory neurons, and interneurons. A typical neuron consists of a cell body (referred to as a neuronal cell body), dendrites, and an axon. The method may be used to generate cholinergic neurons, GABAergic neurons, motor neurons, astrocytes, oligodendrocytes, glutamatergic neurons, dopaminergic neurons, and / or serotonergic neurons. The method may be used to generate dopaminergic neurons. The method may include differentiating the neural cells into neurons, astrocytes, oligodendrocytes, dopamine neurons, or motor neurons, pyramidal neurons, motor neurons, spinal cord anterior horn motor neurons, neurons of the ventral midbrain, interneurons, glial cells, radial glial cells, retinal pigment epithelium, oligodendrocytes, dopamine neurons, GABA neurons, glutamatergic neurons, catecholinergic neurons, serotonergic neurons, and cholinergic neurons.

[0092] The method may also include the evaluation of progenitor cells and / or neural cells. For example, the production or differentiation of neural cells in a cell population may be determined by the presence of certain cell markers. These markers may vary depending on the species or organism used for the starting population. Examples of neural cell markers in organisms such as humans include transcription factors or structural proteins. 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, synapsin (e.g., Syn1 and Syn2), synaptophysin, synaptotagmin (e.g., Syt1, Syt4, Syt13, Syt16), NeuroD, choline acetyltransferase (ChAT) (e.g., vesicular ChAT), neurofilament, neuromelanin, Tuj1, Thy1, Chat, GluR (Kainite 1), Neurod 1, and the like. Expression of excitatory and inhibitory neurotransmitter receptors can also be used to assess the number and quality of the generated neural cells. Additionally, gross cell morphology may be used to identify neural cells in a population of non-neuronal cells. Neuronal cells of the present disclosure may exclude 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, Syt16, NeuroD, choline acetyltransferase (ChAT), vesicular ChAT, neurofilament, neuromelanin, Tuj1, Thy1, Chat, GluR (Kainite 1), and Neurod 1.

[0093] The presence of neural cells may be functionally assessed. For example, cells may be assessed according to electrophysiological properties. These assessments may be performed using patch clamp recordings. Other functional characteristics include the ability to fire action potentials, the ability to generate outward currents in response to glycine, GABA, or kainite, and the ability to generate inward currents in response to glutamate.

[0094] Neuronal cells may be assessed and thus identified by the presence of one or more, including two, three, four, five or more, of any of the aforementioned characteristics and / or markers.

[0095] Neuronal cells or cell populations may be assessed for expression of markers characteristic of non-neuronal starting cell populations. In some cases, reprogramming may be assessed by increased expression of neural markers and decreased expression of markers of non-neuronal starting cells.

[0096] The nerve cell may be further defined as a dopaminergic (DA) neuron. The DA neuron may be identified by evaluating the expression variation of proteins and genes (herein, the proteins and genes may be referred to as dopaminergic neuron markers) that are specifically expressed by dopaminergic neurons. The expression variation of the dopaminergic neuron cell marker may be evaluated, for example, by an antigen-antibody reaction to evaluate protein expression, or by quantitative RT-PCR to evaluate gene expression. Examples of the dopaminergic neuron cell marker present in the midbrain include tyrosine hydroxylase (TH), OTX2, FOXA2, LMX1A, LMX1B, PITX3, EN1 and NURR1 genes / proteins.

[0097] Furthermore, 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 confirmed by evaluating dopamine release and responsiveness to oxidative stress and drug stimulation.

[0098] The cells obtained during the process of the production method of the present invention and the dopaminergic neurons of the present invention can be frozen and thawed. The method of freezing and thawing the cells is known in the art and is not particularly limited as long as it does not affect the differentiation ability, 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, detaching them from the culture dish using a cell dispersion liquid (e.g., Accutase (registered trademark) Innovative Cell Technologies), removing the cell dispersion liquid, and suspending the cells in a cryopreservation liquid (e.g., Cell Banker 2 (LSI Medience Corporation)). Examples of the thawing method include a method including a step of thawing in a 37°C incubator, a step of washing the cryopreservation liquid by centrifugation, and a step of suspending in a medium for use. When the cells obtained during the process of the production method of the present invention are frozen and thawed, Nurr1-positive dopaminergic neurons can also be induced from the cells after thawing.

[0099] VII. METHODS OF USE OF NEURON CELLS The method of the present disclosure relates to the production of neural precursor cells that can be used for the treatment of subjects.For example, the cells produced by the method of the present disclosure can be used to treat neurodegenerative diseases.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; Niemann-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 muscular dystrophies, including amyotrophic lateral sclerosis (ALS), multiple sclerosis, Duchenne muscular dystrophy, Becker muscular dystrophy, facioscapulohumeral (Landaugy-Dejerine) muscular dystrophy and limb-girdle muscular dystrophy (LGMD). Also included are neurodegenerative diseases due to 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 of preventing a disease or health-related condition in a subject.

[0100] The present disclosure provides a medicine containing a neuron produced by the method of the present disclosure. As used herein, the term "neuron" is not particularly limited as long as it is a cell obtained by the above-mentioned production method of the present disclosure.

[0101] In the present medicament, neurons may be used as they are, or cell aggregates obtained by concentration through a filter, such as pellets, may be used. Furthermore, the present medicament may be cryopreserved by adding a protective agent such as DMSO (dimethyl sulfoxide). For safer use of the medicament, the medicament may be subjected to treatment under conditions that maintain the function of neurons and denature pathogenic proteins, such as heat treatment and radiation treatment. Furthermore, in order to suppress the proliferation of neurons in an amount greater than necessary, the medicament may be subjected to treatment by a method including, in combination with the above treatment, suppression of proliferation by mitomycin C pretreatment, etc., and a step of introducing a gene for a metabolic enzyme that does not naturally occur in mammals into neurons, and a step of administering the drug in an inactivated form as necessary so that the drug is converted into a toxic substance only in neurons into which the gene for the metabolic enzyme that does not naturally occur in mammals has been introduced, thus eradicating the cells (suicide gene therapy).

[0102] The pharmaceutical agents of the present disclosure are safe and have low toxicity, and therefore can be administered to mammals (e.g., humans, mice, rats, guinea pigs, pigs, and monkeys).

[0103] In the pharmaceutical of the present disclosure, nerve cells may be prepared using the patient's own cells, or cells from a donor with an acceptable tissue compatibility type may be used. If sufficient cells cannot be obtained due to age, constitution, etc., cells embedded in polyethylene glycol or silicone capsules, porous containers, etc. may be transplanted to avoid rejection. The dosage (amount to be transplanted) and administration frequency (number of times to be transplanted) of the pharmaceutical of the present disclosure can be appropriately determined according to the age, weight, symptoms, etc. of the patient to be administered.

[0104] The neuron-containing medicine of the present disclosure can be efficiently engrafted in the patient's body by administration (transplantation) and can efficiently produce (release) dopamine in the patient's body. Therefore, the medicine of the present disclosure is useful for treating diseases caused by a decrease in dopamine production (release), such as neurodegenerative diseases such as Parkinson's disease, Huntington's chorea, Alzheimer's disease, epilepsy, and schizophrenia.

[0105] The neuronal cells of the present disclosure may be used in a method for screening drug compounds, such as compounds for treating neurodegenerative diseases. For example, a test compound can be contacted with the neuron of the present disclosure alone or in combination with other drugs, and the morphological or functional changes of the neuron can be measured to evaluate whether the test compound is useful as a drug. An example of a method for measuring functional changes includes measuring the amount of dopamine produced or released from the neuron. The dopaminergic neuron may be a cell that exhibits the same phenotype as the disease to be treated, and dopaminergic neurons produced by inducing differentiation of stem cells produced from somatic cells derived from the disease are particularly preferred.

[0106] 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, the test compounds may form salts. As salts, salts with physiologically acceptable acids (e.g., inorganic acids, organic acids), bases (e.g., alkali metal salts, alkaline earth metal salts, aluminum salts), etc. are used, and examples of such salts include salts with inorganic acids (e.g., hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), salts with 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, which can be used.

[0107] The pharmaceutical preparations obtained using the above screening methods can be formulated using physiologically acceptable additives according to known methods.

[0108] VIII. Additional Medications It is contemplated that the methods of the present disclosure include administration of an additional agent. The additional agents may be one or more BMP inhibitors such as Noggin, Chordin, Dorsomorphin, LDN-193189 (4-(6-(4-(piperazin-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), and the like; 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-4-quinolin-4-ylpyrazole), and the like; CHIR99021 GSK3β inhibitors such as (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 Purmorphamine (N-(4-morpholinophenyl)-2-(1-naphthyloxy)-9-cyclohexyl-9H-purin-6-amine) and SAG (N-methyl-N'-(3-pyridinylbenzyl)-N'-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane); and specific factors such as Sonic Hedgehog (SHH) and Fibroblast Growth Factor-8 (FGF8). The method may exclude contacting the cells with one or more of the additional agents described herein.The method includes administering to a subject one or more BMP inhibitors, such as chordin, dorsomorphin, noggin, LDN-193189 (4-(6-(4-(piperazin-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), and the like; 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 (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); smoothon agonists such as purmorphamine (N-(4-morpholinophenyl)-2-(1-naphthyloxy)-9-cyclohexyl-9H-purin-6-amine) and SAG (N-methyl-N'-(3-pyridinylbenzyl)-N'-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane); and contact of cells with growth factors such as sonic hedgehog (SHH) and fibroblast growth factor-8 (FGF8) may be removed.

[0109] 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 exclude one or more of an activator of the phosphatidylinositol 3-kinase signaling pathway and an activator of the MAPK signaling pathway. Additional agents can include one or more of: midkine, pleiotrophin, insulin-like growth factor-1, inhibitors of the TGF-β superfamily signaling pathway, A83-01, SB431542, dorsomorphin, inhibitors of the Wnt signaling pathway, activators of the Notch signaling pathway such as PNU-74654, Dickkopf, Delta-1, Delta-2, Delta-3, Delta-4, Jagged-1, Jagged-2, activators of the protein kinase signaling pathway such as forskolin or dibutyryl cAMP, activators of the tyrosine kinase anaplastic lymphoma kinase (ALK), and activators of the insulin-like growth factor (IGF) receptor, as well as 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 remove the contact of the cell with one or more of the activators of the phosphatidylinositol 3-kinase signaling pathway and the activators of the MAPK signaling pathway described herein. The additional agent may remove one or more of the activators of the phosphatidylinositol 3-kinase signaling pathway and the activators of the MAPK signaling pathway.The additional agents can include one or more of: inhibitors of midkine, pleiotrophin, insulin-like growth factor-1, TGF-β superfamily signaling pathways, SB431542, inhibitors of the Wnt signaling pathway, activators of the Notch signaling pathway such as PNU-74654, Dickkopf, Delta-1, Delta-2, Delta-3, Delta-4, Jagged-1, Jagged-2, activators of the protein kinase signaling pathway such as forskolin or dibutyryl cAMP, activators of the tyrosine kinase anaplastic lymphoma kinase (ALK), and activators of the insulin-like growth factor (IGF) receptor, as well as inhibitors of SMAD2, SMAD3, SMAD4, SMAD1, SMAD5, SMAD8, inhibitors of Wnt or LRP binding to Frizzled, or inhibitors of β-catenin stabilization. EXAMPLES

[0110] IX. Working Example The following examples are included to demonstrate preferred aspects of the invention. Those skilled in the art will recognize that the techniques disclosed in the following examples are techniques discovered by the inventors to function well in the practice of the invention, and therefore may be considered to constitute preferred modes for its practice. However, those skilled in the art should recognize in light of this disclosure that many changes can be made in the specific embodiments disclosed without departing from the spirit and scope of the invention and still obtain the same or similar results.

[0111] Example 1: Three-dimensional cell culture for in vitro differentiation of stem or progenitor cells into neural cells A. Materials and Methods 1. Chemicals, proteins and antibodies LDN193189 was provided by Axonmedchem (reference no. 1509) and stored at -20°C in 5 concentrations of 5 mM in DMSO. SB431542 was provided by Abcam (reference no. ab120163) and stored at -20°C in 50 mM in DMSO. FGF-8 (fibroblast growth factor 8, reference no. GFH176-5) was provided by Peprotech and stored at -20°C in 100 μg / mL in pure water. SHH (sonic hedgehog protein, reference no. GFH168-5) was provided by Cell Guidance and stored at -20°C in 100 μg / mL in pure water. Purmorphamine (reference no. SML0868) was provided by Calbiochem and stored at -20°C in 10 mM in pure DMSO. CHIR99021 (reference number ct99021) was provided by Axon Medchem and stored at -20°C at a concentration of 7.5 mM in DMSO. cAMP (cyclic adenosine monophosphate, reference number D0627) was provided by Sigma and stored at -20°C at a concentration of 0.5 M in DMSO. BDNF (brain-derived neurotrophic factor, reference number GFH1-2) was provided by Cell Guidance and stored at -20°C at a concentration of 100 μg / mL in pure water. GDNF (glial cell line-derived neurotrophic factor, reference number GFH2-2) was provided by Cell Guidance and stored at -20°C at a concentration of 100 μg / mL in pure water. TGFB3 (transforming growth factor beta 3, reference number GFH109-2) was provided by Cell Guidance and stored at -20°C at a concentration of 100 μg / mL in pure water. FGF20 (fibroblast growth factor 20, reference number 100-41) was provided by Peprotech and stored at -20°C at a concentration of 100 μg / mL in pure water. Compound E, a γ-secretase inhibitor (reference number CAS 209986-17-4), was provided by Calbiochem and stored at -20°C at a concentration of 5 mM in pure DMSO.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), and goat anti-rabbit IgG-Alexa 488 (Life technologies).

[0112] 2. Embryonic stem cell culture and differentiation Human embryonic stem (hESC) cell line HS420 (kind 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. At 70% confluence, HS420 cells were passaged into 3D cell culture plates in X-VIVO medium (LONZA, ref. BE04-380Q) supplemented with 1% penicillin / streptomycin, 10 μM ROCK inhibitor (Y27632, abcam), 0,5 μM LDN193189 and 10 μM SB431542.

[0113] Dopaminergic neurospheres were produced by using two different 3D cell culture techniques. The first technique is the standard method, "3D-immersion", where spheroids are first forced to aggregate in plastic microwells, then removed and transferred into standard culture plates maintained under agitation (Figure 1A). In the second technique used, called the "3D-AirLiwell" method, spheroids are forced to aggregate in non-adhesive microwells molded with medium-permeable agarose. In contrast to 3D-immersion, spheroids are maintained individually in the microwells without movement, immersion and agitation. A semi-permeable membrane at the bottom of the molded microwells allows their long-term stability and also establishes air / liquid interface conditions favoring gas exchange with air (Figure 1B).

[0114] a. Fabrication of dopaminergic neurospheres by forced aggregation followed by immersion (3Di) - HS420 cells were deposited in supplemented X-VIVO medium in Aggrewell-800™ (Stemcell technologies, 6-well plate, 2 ml per well) at a ratio of 2'200 cells per microwell (the microwell plate used here contains 1'800 microwells per well). To ensure proper distribution of cells in each microwell, the plate was gently moved and placed on a stable support. After 15 min, the plate was incubated at 37°C for 24 h to allow spheroids to form. 24 hours after spheroid formation, spheres were harvested and then transferred to standard 6-well plates in a combination of half X-VIVO medium without ROCK inhibitor and half Neurobasal medium supplemented with 1% penicillin / streptomycin, 1% non-essential amino acids, 1% B-27 supplement (Thermofisher), L-glutamine, 0.5 μM SB431542, 10 μM LDN193189 (dual-SMAD inhibitor cocktail), 100 ng / ml SHH, 2 μM purmorphamine and 100 ng / ml FGF-8. Spheroids were then cultured in 3 ml of medium per well under constant agitation at 37 °C and 5% CO2 under constant agitation (60 rpm, orbital shaker). On day 3, 3 μM CHIR99021 was added. Neurobasal medium was replaced progressively with 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. Remove CHIR99021 on day 13. NOTE: For each medium change, refresh half of the medium using 1,5-2 ml per well.

[0115] b. Fabrication of dopaminergic neurospheres by using 3D-AirLiwell technology (3D-ALi) For neural differentiation, 2200 human pluripotent stem cells per microwell in supplemented X-VIVO medium were deposited at the air-liquid interface onto the substrate material in the molded microwells on the insert (720 microwells per well). To ensure proper distribution of the cells in each microwell, the substrate material was gently moved and placed on a stable support for 15 min. Supplemented X-VIVO medium was added underneath the insert. The medium of the cell suspension added to the microwells was then removed by aspiration with a 0.5 x 16 mm needle, leaving the spheroids only covered with a thin film of residual medium (10), after which the plate was incubated at 37 °C for 24 h.

[0116] 24 h after spheroid formation, the medium under the inserts is replaced with fresh medium made by combining half X-VIVO medium without ROCK inhibitor and half Neurobasal medium supplemented with 1% penicillin / streptomycin, 1% non-essential amino acids, 1% B-27 supplement (Thermofisher), L-glutamine, 0.5 μM SB431542, 10 μM LDN193189 (dual-SMAD inhibitor cocktail), 100 ng / ml SHH, 2 μM purmorphamine and 100 ng / ml FGF-8. Spheroids are then cultured in individualized microwells in a regular incubator at 37 °C and 5% CO2. On day 3, 3 μM CHIR99021 was added. Neurobasal medium was progressively replaced with 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 FGF20, 5 ng / mL TGF-B3 and 1 µM compound E. Remove CHIR99021 on day 13. NOTE: For each medium change, refresh half of the medium using 0,5 ml per well.

[0117] 3. Quantitative RT PCR and RNA Sequencing For qRT-PCR, RNA was extracted from submerged (3D-i) and air-liquid interface (3D-ALi) cultured dopaminergic neurospheres derived from three different cell batches of HS420 cells. The time points for RNA extraction were days 3, 8, 15, 22, 29, 43, and 57. Total RNA isolation was performed using the Qiagen RNeasy kit according to the manufacturer's instructions. RNA concentration was determined by spectrophotometer (Thermo Scientific™ NanoDrop 2000) and RNA quality was verified by 2100 Bioanalyzer (Agilent). Quantitative PCR was performed on a Tecan Freedom Evo by using SYBR Green fluorescence. cDNA was produced from RNA by using the PrimeScript RT Reagent Kit (Takara). All primers were diluted to a concentration of 0.86 μM and used a master mix (Power SYBR Green Master Mix, Thermo Fisher Scientific). To perform RNA sequencing, gene expression was analyzed at 6 weeks of differentiation (=day 43) using the same RNA used for qPCR. TIFF2025512359000002.tif76166

[0118] 4. Dopamine content by HPLC Dopamine production was analyzed by high pressure liquid chromatography (HPLC) after extraction from dopaminergic neurospheres cultured submerged (3D-i) or at the air-liquid interface (3D-ALi) in 0.1 N perchloric acid (HClO4). Cells were lysed in a small volume (250 μL) for 15 min at 4 °C with vigorous vortexing every 5 min. Supernatants were collected after centrifugation and either used immediately for dopamine determination or stored at -20 °C. Catecholamines were measured by HPLC with electrochemical detection in the electrometric mode. Separation of the analytes was performed using a reversed-phase column Symmetry C-18, 5 mm (4.6·150 mm 2) (Waters Corporation) at a flow rate of 1 mL / min in isotonic mode. The electrometric detector parameters, Coulochem III (Thermo scientific), were +200 mV potential for the conditioning cell. For the analytical cell, electrode 1 with +100 mV potential and electrode 2 with -100 mV potential were used.

[0119] 5. Western Blot Total protein was extracted from dopaminergic neurospheres at 1 month of differentiation by homogenization of cells in RIPA buffer (1x) and incubation on ice for 1 hour. Lysates were centrifuged at 13 rpm for 10 minutes and the supernatant was used for Western blot analysis. For each condition, 10, 20, 30 and 40 ug of protein were analyzed by Western blot to assess protein tyrosine hydroxylase expression.

[0120] 6. Electrophysiology with MEA arrays Using a dissection microscope, dopaminergic neurospheres were transferred to the center of the porous MEA device. Eight to four recording electrodes in triplicates were selected for each given condition. An amplifier and data acquisition system for electrophysiological recordings were then used. The signal-to-noise ratio (SNR) was measured as the standard deviation of the voltage during a 30-min recording, and the signal was taken as the average value of the peak-to-peak voltage of spikes recorded during the same 30-min period.

[0121] 7. Plating of neurospheres on extracellular matrix, immunocytochemistry and electron microscopy On day 29, neurospheres were plated on polyornithine / laminin-coated tissue culture plates in mature 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. After 1 week of culture on coverslips, cells on glass coverslips were fixed with 1 mL of 4% paraformaldehyde in PBS for 30 min 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 overnight with primary antibodies in PBS containing 5% bovine serum albumin (Sigma).

[0122] 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 h. Cells were washed three times with PBS and then exposed to DAPI 300 nM in PBS (Sigma) for 15 min at room temperature. After three washes in PBS, cells were rinsed with water and mounted on glass slides using Fluorsave reagent (Merck Millipore). Dopaminergic neurospheres plated on glass coverslips were further dehydrated and then covered with a gold nanolayer (20 nm) and visualized by electron microscopy.

[0123] B. Results First, the homogeneity of dopaminergic neurospheres was examined over a period of 60 days by using the previously described dopaminergic specification protocol. Dopaminergic neurospheres from 3D conventional culture "3D-immersion" were compared with 3D-AirLiwell neurospheres. As a practical note, in the standard method "3D-immersion", spheroids are first forced to aggregate in plastic microwells, then removed and transferred into standard culture plates maintained under agitation. In contrast, in the "3D-AirLiwell" method, spheroids are forced to aggregate in non-adhesive microwells molded with medium-permeable agarose. In contrast to 3D-immersion, spheroids are maintained individually in the microwells without movement, immersion and agitation. A semi-permeable membrane at the bottom of the molded microwells allows their long-term stability and also establishes air / liquid interface conditions favoring gas exchange with air. To more precisely compare and characterize the morphology of dopaminergic spheroids in 3D-AirLiwell versus 3D-immersion, a 2-month dopaminergic differentiation was performed and the changes in their size and shape were followed by light microscopy. As shown in Figure 2, at week 0, there was no significant difference between the two methods in either round shape or size. However, after 2 weeks of culture, dopaminergic neurospheres grown in 3D-AirLiwell were more homogeneous than those grown in 3D-immersion. Software-based quantification of the distribution range of area, circularity, perimeter, circularity and diameter (Figure 2) showed that there was clearly less variability in 3D-AirLiwell. Furthermore, spheroids that were initially round in shape maintained their circular morphology in 3D-AirLiwell but changed to a more heterogeneous and irregular shape in 3D-immersion.

[0124] Next, we monitored dopaminergic specification more quantitatively at different time points by using Q-RT-PCR. In addition to dopaminergic specification, the expression of several markers involved in early or late stages of neural induction was also analyzed, as was cell proliferation at different time points during the two months of differentiation (Figure 3). First of all, no difference was observed in the expression of Ki67, a cell proliferation marker, between the two 3D cell culture methods. Nestin, also an early NPC marker, and βIII-tubulin, a neuronal marker, also showed no significant regulation. These proteins are generally not upregulated in cells during the neural induction process, but are reorganized more in the cytosol to form denser fibers of the cytoskeleton.

[0125] In contrast, and not unexpectedly, the transcription factor Pax-6, which is upregulated during early neural induction, was increased in the "3D-AirLiwell" spheroids from day 15 to the end of differentiation. Together, these findings enhance the ability of 3D-AirLiwell to favor the switch between hESCs and NPCs. With regard to specification markers, Lmx1a (which is an early midbrain marker upregulated in dopaminergic precursors) was also upregulated in 3D-AirLiwell spheroids from week 2 to the end of differentiation. Finally, tyrosine hydroxylase, a marker of mature dopaminergic neurons, showed upregulation in 3D-immersed spheroids. Thus, these Q-RT-PCR results suggest that the 3D-AirLiwell method allows better midbrain region formation of NPCs, although the lack of maturation is clearly confirmed.

[0126] To get a complete picture regarding gene expression, we performed RNA-seq analysis, comparing spheroids grown by both methods at week 6 (=day 43) for three different cell batches of the HS420 cell line. The graph in Figure 4 shows the fold change between 3D-AirLiwell and 3D-immersion, with positive bars meaning upregulation in 3D-AirLiwell and conversely negative bars meaning upregulation in 3D-immersion. The combination of Lmx1a, Otx-2 and FoxA2 is the best association to estimate the initial regional state of NPCs, since dopaminergic NPCs originate from ventral midbrain cells, i.e. triple positive cells. In the graph on the left (Figure 4), these three markers are upregulated in spheroids grown in "3D-AirLiwell". Moreover, other transcription factors involved in dopaminergic neuronal development (e.g., Nurr-1, MSX1, FoxA2) were also upregulated in 3D-ALi, suggesting better dopaminergic regional formation and specification of NPCs. Contrary to that, late dopaminergic markers such as TH, EN-1 (engrailed-1), DRD2 (dopamine receptor), and VMAT2 (dopamine transporter) are upregulated in spheroids grown in standard 3D-immersion method (Figure 4). Furthermore, the expression of other specific markers of maturation was also analyzed. In spheroids cultured in 3D-AirLiwell method, a lack of maturation was confirmed, as shown by the upregulation of all maturation markers (NeuN, neurofilament heavy chain..., Figure 4).

[0127] Furthermore, the expression of cell proliferation markers was also analyzed. As shown in Figure 5, cell proliferation markers were all downregulated in neurospheres cultured by the 3D-ALi method, suggesting that cells cultured by this technique may have a great advantage in terms of a lower tumor risk.

[0128] However, the lack of maturation of dopaminergic neurospheres cultured by "3D-AirLiwell" was also confirmed at the protein level. Dopaminergic differentiation was performed by both 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 low amounts of tyrosine hydroxylase protein in samples of spheroids cultured in "3D-AirLiwell", suggesting a lack of maturation with this novel method.

[0129] Furthermore, dopamine production was analyzed by high pressure liquid chromatography (HPLC) after extraction from dopaminergic neurospheres cultured submerged (3D-i) or at the air-liquid interface (3D-Ali). As expected, the amount of dopamine produced by 3D-ALi dopaminergic neurospheres was less than that produced by 3D-i neurospheres (Figure 7).

[0130] Regarding the electrophysiological aspects of dopaminergic neurospheres, we measured the spontaneous electrical activity generated by neurons differentiated by the two techniques. Both neurospheres cultured in 3D-immersion and 3D-AirLiwell were alive and electrophysiologically active. As shown in the left side of Figure 8, the waveforms of the signals of 3D-AirLiwell neurospheres are more homogeneous and grouped into two similar shapes, whereas those in 3D-immersion are more heterogeneous and grouped into more than four different shapes. Dopaminergic neurospheres cultured in 3D-immersion showed significantly higher spike and burst frequency, amplitude, and higher spike and burst numbers, suggesting more active neuronal networks. However, neurospheres grown in 3D-AirLiwell showed mild and stable activity over time, whereas neurospheres grown in 3D-immersion had higher epileptiform activity (Figure 8).

[0131] Neuronal maturation of dopaminergic neurospheres was induced by plating on extracellular matrix-coated (ECM) plates at the fourth week of differentiation. One week after maturation, immunofluorescence staining was performed with the neuronal marker β-III tubulin and tyrosine hydroxylase (TH), a specific marker for dopaminergic neurons (Figure 9). Both methods yielded abundant neurons around the plated spheres, confirming that the 3D-AirLiwell spheroids retained neuronal maturation properties. Mature neurons on ECM-coated plates were also observed by electron microscopy (Figure 10). This allowed the neuronal network and cell shape to be analyzed with better resolution. In the immersion method, the presence (by DAPI staining and electron microscopy) of invasive large flattened cells (2), suspected to be proliferative NPCs (as they were shown to be nestin+, Pax-6+; data not shown), was observed beneath the neuronal network (1). In contrast and clearly, this cell population was not induced in 3D-AirLiwell, indicating a high level of homogeneity of the cell preparation.

[0132] To further characterize the neurospheres produced by the 3D-Ali and 3D-i methods, single-cell RNA sequencing was performed to determine the phenotype of the various cells in the neurospheres (Figure 13A). The majority of cells in the 3D-Ali neurospheres were in neuronal clusters (86.5%), whereas only half of the cells in the 3D-i neurospheres were in neuronal cell type clusters. The 3D-I neurospheres contained a higher proportion of bone marrow 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) (Figure 13B-C). Thus, 3D dopaminergic differentiation under 3D-Ali conditions yields a more homogenous cell population than under 3D-I conditions. We also found that tyrosine hydroxylase (TH) mRNA was increased under 3D-Ali conditions (Figure 14), but no difference was detected in dopamine content (Figure 15). In the 3D-Ali condition, electrical signals further indicative of stable neurons were also obtained (Figure 16).

[0133] To improve the maturation stage, 2.5% or 10% laminin 511 (Takkara) or Geltrex® (growth factor reduced extracellular matrix, ThermoFisher) are added to the cell culture medium to promote neuronal maturation of spheroids from differentiation day 1 to the end and from day 13 to the end. RGD peptides can also be tested to enhance maturation by adding them to the medium similar to laminin or Geltrex.

[0134] The second strategy consists in 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 in the medium is also tested. These combinations are applied from day 1 to the end of differentiation and from day 13 to the end.

[0135] We tested whether the addition of extracellular matrix and / or hypoxia could improve the development of mature neurons. We tested 2.5% and 10% laminin with normoxia (20% oxygen) or hypoxia (3% oxygen) (Figure 17). As shown in Figure 18, hypoxia appeared to increase and promote dopaminergic neuron maturation at day 29, while laminin had no obvious effect in either normoxia or hypoxia. Furthermore, it was hypothesized that the effect shown by hypoxia could be reproduced by the addition of HIF-1α stabilizer to the cell culture medium. Furthermore, it was hypothesized that other modifications to the cell culture could result in improved neuronal maturation, such as changing the volume of cell culture medium. We found that the ratio of the number of neurospheres to the volume of cell culture medium could result in improved cell maturation. In AirLiwell (6-well plates), seeding 1.5 million cells per well (6-well AirLiwell plate) yields 2350 neurospheres. Under these conditions, 750 μl of cell culture medium is a more effective way to differentiate neurons (Figure 19).

[0136] We also tested the effect of various antioxidants on three-dimensional dopaminergic neuronal differentiation. Figure 20 shows the protocol design with the addition of antioxidants. As shown in Figures 21A-B, antioxidants did not affect overall neuronal differentiation. Equivalent gene expression of neuronal markers b3-tubulin (TUBB3) and MAP2 was observed, as well as equivalent downregulation of proliferation marker ki67. Most of the antioxidants tested reduced TH expression during dopaminergic neuronal differentiation. However, ascorbic acid (vitamin C) was found to result in a significant enhancement of DA neuronal differentiation (Figure 21B).

[0137] It is also contemplated that overexpression of factors that promote dopaminergic neuron maturation can be stably transduced in hESCs (HS420 cell line) to improve dopaminergic maturation. The selected factors 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. Reinforcing this selection, these two transcription factors were weakly expressed in spheroids. Also, many studies have highlighted that Nurr1 and Pitx3 are important for dopaminergic neuron maturation. Of note, a neuron-specific promoter is used to overexpress these two transcription factors. As an example, the Tα1α-tubulin promoter, validated in a previous study, 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. 2006 Mar;24(3):615-23. doi: 10.1634 / stemcells.2005-0226. Epub 2005 Nov 17. PMID: 16293575].

[0138] Example 2 Neuronal differentiation in 3D cell culture To explore the effects of other ALK inhibitors (ALKi) already mentioned, and to explore the possibility of replacing dual-SMAD inhibition in the protocol using 3D-AirLiwell technology, human embryonic stem cell line HS420 was differentiated into dopaminergic neurospheres as already described in Example 1, and the effects of DMH1, DMH2, K02288 and A8301 alone or in combination on hESC neural induction were compared with the combination of LDN193189 and SB431542 compounds. The differentiation results were analyzed by q-PCR after 2 and 4 weeks of dopaminergic differentiation (Figure 23A-D). Cells were exposed to compounds on day 0 of week 1 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 used for the first week of induction as described above, such as purmorphamine, SHH and FGF-8, are added to the medium (Figure 22). DMH1, DMH2, A-8301 and K02288 were found to have comparable activity to LDN / SB (dual Smad inhibition) with respect to general neuronal differentiation. In contrast, the different ALK inhibitors have differential effects on dopaminergic differentiation: DMH1 and DMH2 have approximately equal potency compared to LDN / SB, while A-8301 and K02288 have no significant effect on dopaminergic differentiation. The combination of DMH2 and A-8301 produced a synergistic effect in 2D cell cultures.

[0139] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that modifications can be made to the methods described herein and to the steps or sequence of steps of the methods described herein without departing from the concept, spirit and scope of the present invention. More specifically, it will be apparent that certain agents that are chemically and physiologically related can be substituted for the agents described herein while still achieving the same or similar results. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the present invention as defined by the appended claims.

Claims

1. A method for differentiating stem cells or progenitor cells into nerve cells, comprising the step of culturing the stem cells or progenitor cells in a differentiation composition in microwells to form neurospheres.

2. The method according to claim 1, wherein neurospheres are cultured at the gas-liquid interface in a microwell.

3. The method according to claim 1, wherein the neurospheres are not cultured by 3D immersion culture.

4. The method according to claim 1, wherein neurospheres are individualized within individual microwells.

5. The method according to claim 4, wherein the microwells are contained within a larger culture well.

6. The method according to claim 1, wherein the neurospheres are individualized within individual microwells contained in a larger well, and the neurospheres are cultured at the gas-liquid interface within the microwells.

7. The method according to claim 1, wherein the differentiation composition comprises one or more of DMH1, DMH2, K02288, and A83-01.

8. The method according to claim 1, wherein the differentiation composition does not contain LDN193189 and / or SB431542.

9. The method according to claim 1, wherein the stem cells or progenitor cells include induced pluripotent stem cells (iPSCs) or embryonic stem (ES) cells.

10. The method according to claim 1, wherein stem cells or progenitor cells are cultured in a differentiation composition for about 1 to 7 days.

11. The method according to claim 1, wherein the differentiation composition comprises a Rho kinase (ROCK) inhibitor.

12. The method according to claim 1, wherein the nerve cells are further defined as dopaminergic neurons, glutamatergic, serotonergic, cholinergic, GABAergic, motor neurons, astrocytes, or oligodendrocytes.

13. The method according to claim 12, wherein the nerve cell is a dopaminergic neuron.

14. The method according to claim 1, further comprising exposing cells to a GSK3 (glycogen synthase kinase 3) inhibitor for a certain period of time.

15. The method according to claim 14, wherein the GSK3 inhibitor comprises CHIR99021.

16. The method according to claim 1, further comprising the step of contacting cells with one or more of FHF8, SHH, and purmorphamine for a certain period of time.

17. The method according to claim 1, further comprising the step of contacting cells with one or more of cAMP, GDNF, BDNF, TGFB3, FGF20, HIF-1α stabilizer, ascorbic acid, and a γ-secretase inhibitor for a certain period of time.

18. The method according to claim 1, wherein the differentiation composition comprises an extracellular matrix, or the method further comprises the step of bringing cells, spheroids, or neurospheres into contact with an extracellular matrix for a certain period of time, and the extracellular matrix comprises laminin and / or Geltrex.

19. The method according to claim 1, wherein the differentiation composition comprises an RGD peptide, or the method further comprises the step of bringing cells, spheroids, or neurospheres into contact with an RGD peptide for a certain period of time.

20. The method according to claim 1, wherein cells, spheroids, and / or neurospheres are cultured under hypoxic conditions for a certain period of time, and the hypoxic conditions are 3% oxygen.

21. The method according to claim 1, wherein the ratio of the number of neurospheres to the amount of cell culture medium is approximately 1000 neurospheres per 200 to 500 μl of cell culture medium.

22. The step of culturing cells in microwells includes culturing cells on a substrate material comprising a hydrophilic porous material layer having a plurality of persistent well-shaped depressions on its outer surface, supported by a semipermeable membrane on its inner surface, wherein the well-shaped depressions are 100 pm 2 From approximately 1 mm 2 The opening and approximately 100 pm 2 From approximately 1 mm 2 The method according to claim 1, having a bottom surface.

23. The method according to claim 2, wherein the neurospheres are further defined as dopaminergic progenitor cells expressing Neun, synaptophysin, TH, DRD2, Girk2 and / or VMAT2 at lower levels than neurospheres cultured in 3D immersion culture.

24. The method according to claim 2, wherein the neurospheres are further defined as dopaminergic progenitor cells expressing Neun, synaptophysin, TH, DRD2, Girk2, and VMAT2 at lower levels than neurospheres cultured in 3D immersion culture.

25. The method according to claim 2, wherein the neurospheres are further defined as dopaminergic progenitor cells expressing LMX1A, FOXA2, OTX2 and / or MSX1 at higher levels than those in neurospheres cultured in 3D-immersion culture.

26. The method according to claim 2, wherein the neurospheres are further defined as dopaminergic progenitor cells expressing LMX1A, FOXA2, OTX2, and MSX1 at higher levels than those in neurospheres cultured in 3D immersion culture.

27. The method according to claim 7, wherein the compound is DMH1.

28. The method according to claim 7, wherein the compound is DMH2.

29. The method according to claim 7, wherein the compound is K02288.

30. The method according to claim 7, wherein the compound comprises DMHl and DMH2.

31. The method according to claim 7, wherein the compound comprises K02288 and DMH2.

32. The method according to claim 7, wherein the compound comprises DMH1 and K02288.

33. The method according to claim 1, wherein the compound comprises DMH1, DMH2, and K02288.

34. The method according to claim 1, wherein the compound comprises DMH1, DMH2, and A83-01.

35. Spheroids or neurospheres produced by the method described in claim 1.

36. A population of cells, spheroids, or neurospheres produced by the method described in claim 1.

37. A method for treating a disease in a mammalian subject, comprising the step of administering a therapeutically effective dose to a population of nerve cells, spheroids, or neurospheres as described in claim 36.

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