Methods of differentiating nerve cells and related compositions and methods of use

Abstract

The present disclosure provides methods of causing pluripotent stem cells, including induced pluripotent stem cells, lineage-specific differentiation into midbrain floor progenitor cells, targeted dopamine (DA) neuronal progenitor cells, and / or DA neurons. Also provided are composition uses thereof, such as composition uses for the treatment of neurodegenerative diseases and conditions, including Parkinson's disease.

Description

[0001] This application is a divisional application of a Chinese invention patent application with an application date of January 13, 2021, application number 202180020647.9, and invention name “Methods for differentiating neural cells and related compositions and methods of use”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 62 / 960,669, filed on January 13, 2020, entitled “METHOD OF DIFFERENTIATING NEURAL CELLS AND RELATED COMPOSITIONS AND METHODS OF USE,” the contents of which are incorporated by reference in their entirety for all purposes. Technical Field

[0004] The present disclosure relates to methods for lineage-specific differentiation of pluripotent stem cells, including induced pluripotent stem cells, into midbrain floor plate progenitor cells, committed dopamine (DA) neuron progenitor cells and / or dopamine (DA) neurons. Compositions of differentiated cells and therapeutic uses thereof are also provided, such as for the treatment of neurodegenerative conditions and diseases, including Parkinson's Disease. Background Art

[0005] It is contemplated that various methods for differentiating pluripotent stem cells into lineage-specific cell populations and the resulting cell compositions can be used for cell replacement therapy in patients with diseases that result in loss of function of directed cell populations. However, in some cases, such methods are limited in their ability to produce cells with consistent physiological characteristics, and cells produced by such methods may be limited in their ability to be transplanted in vivo and to innervate other cells. Improved methods and cell compositions thereof are needed, including providing improved methods for differentiating cells, such as to produce physiologically consistent cells. Summary of the invention

[0006] Provided herein is a method for differentiating neural cells, the method comprising: (a) performing a first incubation, comprising culturing pluripotent stem cells under conditions that produce cell spheres in a non-adherent culture vessel, wherein at the start of the first incubation (day 0), the cells are exposed to (i) a TGF-β / activated Nodal signaling inhibitor; (ii) at least one sonic hedgehog (SHH) signaling activator; (iii) a bone morphogenetic protein (BMP) signaling inhibitor; and (iv) a glycogen synthase kinase 3β (GSK3β) signaling inhibitor; and (b) performing a second incubation, comprising culturing the cells of the sphere under conditions that cause the cells to differentiate neurally in a substrate-coated culture vessel. In some embodiments, the second incubation begins at about day 7.

[0007] In some embodiments, the cells are exposed to the TGF-β / Activin Nodal signaling inhibitor until the day before or on day 7. In some embodiments, the cells are exposed to the TGF-β / Activin Nodal signaling inhibitor until day 6. In some embodiments, the cells are exposed to the TGF-β / Activin Nodal inhibitor starting from day 0 to day 6, including every day.

[0008] In some embodiments, the cells are exposed to the at least one SHH signaling activator until the day on or before day 7. In some embodiments, the cells are exposed to the at least one SHH signaling activator until day 6. In some embodiments, the cells are exposed to the at least one SHH signaling activator starting from day 0 to day 6, including every day.

[0009] In some embodiments, the cells are exposed to the BMP signaling inhibitor until the day on or before day 11. In some embodiments, the cells are exposed to the BMP signaling inhibitor until day 10. In some embodiments, the cells are exposed to the BMP signaling inhibitor starting from day 0 to day 10, including every day.

[0010] In some embodiments, the cells are exposed to the GSK3β signaling inhibitor until the day on or before day 13. In some embodiments, the cells are exposed to the GSK3β signaling inhibitor until day 12. In some embodiments, the cells are exposed to the GSK3β signaling inhibitor starting from day 0 to day 12, including every day.

[0011] In some embodiments, culturing cells under conditions that cause neural differentiation of cells in any of the methods provided includes exposing the cells to (i) brain-derived neurotrophic factor (BDNF); (ii) ascorbic acid; (iii) glial cell-derived neurotrophic factor (GDNF); (iv) dibutyryl cyclic AMP (dbcAMP); (v) transforming growth factor β-3 (TGFβ3) (collectively referred to as "BAGCT"); and (vi) Notch signaling inhibitors. In some embodiments, cells are exposed to BAGCT and Notch signaling inhibitors starting on day 11. In some embodiments, cells are exposed to BAGCT and Notch signaling inhibitors starting on day 11 until neural differentiation cells are harvested. In some embodiments, cells are exposed to BAGCT and Notch signaling inhibitors starting on day 11 until just or about day 18. In some embodiments, cells are exposed to BAGCT and Notch signaling inhibitors starting on day 11 until just or about day 25.

[0012] Provided herein is a method for differentiating neural cells, the method comprising: (a) performing a first incubation comprising culturing pluripotent stem cells in a non-adherent culture vessel under conditions that produce cell spheres, wherein at the beginning of the first incubation day (day 0), the cells are exposed to (i) a TGF-β / activin Nodal signaling inhibitor until day 6, including every day; (ii) at least one sonic hedgehog (SHH) signaling activator until day 6, including every day; (iii) a bone morphogenetic protein (BMP) signaling inhibitor until day 6, including every day; and (iv) a glycogen synthase kinase 3β (GSK3β) signaling inhibitor until day 6, including every day; and (b) starting from day 7, performing a second incubation comprising culturing pluripotent stem cells in a non-adherent culture vessel under conditions that produce cell spheres, wherein at the beginning of the first incubation day (day 0), the cells are exposed to (i) a TGF-β / activin Nodal signaling inhibitor until day 6, including every day; (ii) at least one sonic hedgehog (SHH) signaling activator until day 6, including every day; (iii) a bone morphogenetic protein (BMP) signaling inhibitor until day 6, including every day; and (iv) a glycogen synthase kinase 3β (GSK3β) signaling inhibitor until day 6, including every day; and (b) performing a second incubation comprising culturing pluripotent stem cells in a non-adherent culture vessel under conditions that produce cell spheres, wherein at the beginning of the first incubation day (day 0), the cells are exposed to (i) a TGF-β / activin Nodal signaling inhibitor until day 6, including every day; (ii) at least one sonic hedgehog (SHH) signaling activator until day 6, including every day; The invention relates to a method for incubating the spheroid cells for neural differentiation, comprising culturing the cells in a culture vessel coated with a substrate selected from laminin, collagen, nestin, heparin sulfate proteoglycans, and combinations thereof, wherein starting from day 7, the cells are exposed to (i) a BMP signaling inhibitor and (ii) a GSK3β signaling inhibitor; and starting from day 11, the cells are exposed to (i) brain-derived neurotrophic factor (BDNF); (ii) ascorbic acid; (iii) glial cell line-derived neurotrophic factor (GDNF); (iv) dibutyryl cyclic AMP (dbcAMP); (v) transforming growth factor β-3 (TGFβ3) (collectively referred to as “BAGCT”); and (vi) a Notch signaling inhibitor.

[0013] In some methods of any of the methods provided, the method further comprises harvesting the neural differentiated cells. In some embodiments, the harvesting is performed at or after about day 16. In some embodiments, the harvesting is performed between about day 16 and about day 30. In some embodiments, the harvesting is performed between about day 16 and about day 27. In some embodiments, the harvesting is performed between day 18 and day 25. In some embodiments, the harvesting is performed at or about day 18. In some embodiments, the harvesting is performed at or about day 25.

[0014] In some embodiments, the neural differentiated cells are committed dopaminergic neuron progenitor cells. In some embodiments, the dopaminergic neuron progenitor cells are capable of innervating host tissue after transplantation into a subject.

[0015] In some embodiments, prior to performing the second incubation, the spheres are dissociated to produce a cell suspension, and the cells of the cell suspension are cultured in the substrate-coated culture vessel. In some embodiments, the dissociation is performed when the sphere cells express at least one of PAX6 and OTX2. In some embodiments, the dissociation is performed when the sphere cells express OTX2. In some embodiments, the dissociation is performed when the sphere cells express PAX6. In some embodiments, the dissociation is performed when the sphere cells express PAX6 and OTX2. In some embodiments, the dissociation is performed at about day 7.

[0016] In some embodiments, the culture vessel is selected from the group consisting of: a plate, a culture dish, a flask, and a bioreactor. In some embodiments, the culture vessel is a plate. In some embodiments, the plate is a 6-well plate, a 12-well plate, a 24-well plate, or a 96-well plate. In some embodiments, the plate is a microplate. In some embodiments, the microplate is a 6-well plate. In some embodiments, the microplate is a 24-well plate. In some embodiments, the culture vessel is an Aggrewell plate. TM plate.

[0017] In some embodiments, the culture of pluripotent stem cells on day 0 includes about 0.1×10 6 cells / cm 2 About 2×10 6 cells / cm 2 , about 0.1×10 6 cells / cm 2 About 1×10 6 cells / cm 2 , about 0.1×10 6 cells / cm 2About 0.8×10 6 cells / cm 2 , about 0.1×10 6 cells / cm 2 About 0.6×10 6 cells / cm 2 , about 0.1×10 6 cells / cm 2 About 0.4×10 6 cells / cm 2 , about 0.1×10 6 cells / cm 2 About 0.2×10 6 cells / cm 2 , about 0.2×10 6 cells / cm 2 About 2×10 6 cells / cm 2 , about 0.2×10 6 cells / cm 2 About 1×10 6 cells / cm 2 , about 0.2×10 6 cells / cm 2 About 0.8×10 6 cells / cm 2 , about 0.2×10 6 cells / cm 2 About 0.6×10 6 cells / cm 2 , about 0.2×10 6 cells / cm 2 About 0.4×10 6 cells / cm 2 , about 0.4×10 6 cells / cm 2 About 2×10 6 cells / cm 2 , about 0.4×10 6 cells / cm 2 About 1×10 6 cells / cm 2 , about 0.4×10 6 cells / cm 2 About 0.8×10 6 cells / cm 2 , about 0.4×10 6 cells / cm 2 About 0.6×10 6 cells / cm 2 , about 0.6×106 cells / cm 2 About 2×10 6 cells / cm 2 , about 0.6×10 6 cells / cm 2 About 1×10 6 cells / cm 2 , about 0.6×10 6 cells / cm 2 About 0.8×10 6 cells / cm 2 , about 0.8×10 6 cells / cm 2 About 2×10 6 cells / cm 2 , about 0.8×10 6 cells / cm 2 About 1×10 6 cells / cm 2 or about 1.0×10 6 cells / cm 2 About 2×10 6 cells / cm 2 In some embodiments, the culture of pluripotent stem cells on day 0 includes about 0.1×10 6 cells / cm 2 About 2×10 6 cells / cm 2 In some embodiments, the culture of pluripotent stem cells on day 0 includes about 0.1×10 6 cells / cm 2 About 1×10 6 cells / cm 2 In some embodiments, the culture of pluripotent stem cells on day 0 includes about 0.1×10 6 cells / cm 2 About 0.8×10 6 cells / cm 2 In some embodiments, the culture of pluripotent stem cells on day 0 includes about 0.1×10 6 cells / cm 2 About 0.6×10 6 cells / cm 2 In some embodiments, the culture of pluripotent stem cells on day 0 includes about 0.1×10 6 cells / cm 2 About 0.4×10 6 cells / cm 2 In some embodiments, the culture of pluripotent stem cells on day 0 includes about 0.1×106 cells / cm 2 About 0.2×10 6 cells / cm 2 In some embodiments, the culture of pluripotent stem cells on day 0 includes about 0.2×10 6 cells / cm 2 About 2×10 6 cells / cm 2 In some embodiments, the culture of pluripotent stem cells on day 0 includes about 0.2×10 6 cells / cm 2 About 1×10 6 cells / cm 2 In some embodiments, the culture of pluripotent stem cells on day 0 includes about 0.2×10 6 cells / cm 2 About 0.8×10 6 cells / cm 2 In some embodiments, the culture of pluripotent stem cells on day 0 includes about 0.2×10 6 cells / cm 2 About 0.6×10 6 cells / cm 2 In some embodiments, the culture of pluripotent stem cells on day 0 includes about 0.2×10 6 cells / cm 2 About 0.4×10 6 cells / cm 2 In some embodiments, the culture of pluripotent stem cells on day 0 includes about 0.4×10 6 cells / cm 2 About 2×10 6 cells / cm 2 In some embodiments, the culture of pluripotent stem cells on day 0 includes about 0.4×10 6 cells / cm 2 About 1×10 6 cells / cm 2 In some embodiments, the culture of pluripotent stem cells on day 0 includes about 0.4×10 6 cells / cm 2 About 0.8×10 6 cells / cm 2 In some embodiments, the culture of pluripotent stem cells on day 0 includes about 0.4×10 6 cells / cm 2 About 0.6×10 6 cells / cm 2In some embodiments, the culture of pluripotent stem cells on day 0 includes about 0.6×10 6 cells / cm 2 About 2×10 6 cells / cm 2 In some embodiments, the culture of pluripotent stem cells on day 0 includes about 0.6×10 6 cells / cm 2 About 1×10 6 cells / cm 2 In some embodiments, the culture of pluripotent stem cells on day 0 includes about 0.6×10 6 cells / cm 2 About 0.8×10 6 cells / cm 2 In some embodiments, the culture of pluripotent stem cells on day 0 includes about 0.8×10 6 cells / cm 2 About 2×10 6 cells / cm 2 In some embodiments, the culture of pluripotent stem cells on day 0 includes about 0.8×10 6 cells / cm 2 About 1×10 6 cells / cm 2 In some embodiments, the culture of pluripotent stem cells on day 0 includes about 1.0×10 6 cells / cm 2 About 2×10 6 cells / cm 2 .

[0018] In some embodiments, the culture of the pluripotent stem cells on day 0 is performed in a 6-well plate. In some embodiments, the culture of the pluripotent stem cells on day 0 is performed in a 6-well plate and includes about 1×10 6 pluripotent stem cells / well is about 20×10 6 pluripotent stem cells / well, about 1×10 6 15×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 1×10 6 10×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 1×10 6 5×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 5×10 6 pluripotent stem cells / well is about 20×10 6 pluripotent stem cells / well, about 5×10 6 15×10 pluripotent stem cells / well6 pluripotent stem cells / well, about 5×10 6 10×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 10×10 6 pluripotent stem cells / well is about 20×10 6 pluripotent stem cells / well, about 10×10 6 15×10 pluripotent stem cells / well 6 pluripotent stem cells / well or about 15×10 6 pluripotent stem cells / well is about 20×10 6 In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 6-well plate and includes about 1×10 6 pluripotent stem cells / well is about 20×10 6 In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 6-well plate and includes about 1×10 6 15×10 pluripotent stem cells / well 6 In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 6-well plate and includes about 1×10 6 10×10 pluripotent stem cells / well 6 In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 6-well plate and includes about 1×10 6 5×10 pluripotent stem cells / well 6 In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 6-well plate and includes about 5×10 6 pluripotent stem cells / well is about 20×10 6 In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 6-well plate and includes about 5×10 6 15×10 pluripotent stem cells / well 6 In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 6-well plate and includes about 5×10 6 10×10 pluripotent stem cells / well 6 In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 6-well plate and includes about 10×10 6 pluripotent stem cells / well is about 20×10 6 In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 6-well plate and includes about 10×10 6 15×10 pluripotent stem cells / well6 In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 6-well plate and includes about 15×10 6 pluripotent stem cells / well is about 20×10 6 pluripotent stem cells / well.

[0019] In some embodiments, the culture of the pluripotent stem cells on day 0 is performed in a 24-well plate. In some embodiments, the culture of the pluripotent stem cells on day 0 is performed in a 24-well plate and includes about 1×10 5 5×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 1×10 5 1×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 1×10 5 5×10 pluripotent stem cells / well 5 pluripotent stem cells / well, about 5×10 5 5×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 5×10 5 1×10 pluripotent stem cells / well 6 pluripotent stem cells / well or about 1×10 6 5×10 pluripotent stem cells / well 6 In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 24-well plate and includes about 1×10 5 5×10 pluripotent stem cells / well 6 In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 24-well plate and includes about 1×10 5 1×10 pluripotent stem cells / well 6 In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 24-well plate and includes about 1×10 5 5×10 pluripotent stem cells / well 5 In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 24-well plate and includes about 5×10 5 5×10 pluripotent stem cells / well 6 In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 24-well plate and includes about 5×10 5 1×10 pluripotent stem cells / well 6 In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 24-well plate and includes about 1×10 65×10 pluripotent stem cells / well 6 pluripotent stem cells / well.

[0020] In some embodiments, the culture of the pluripotent stem cells at day 0 includes a number of cells sufficient to produce a sphere having about 1,000 cells to about 5,000 cells, such as 2,000 cells to about 3,000 cells, at about day 7. In some embodiments, the culture of the pluripotent stem cells at day 0 includes a number of cells sufficient to produce a sphere having about 1,000 cells to about 5,000 cells at about day 7. In some embodiments, the culture of the pluripotent stem cells at day 0 includes a number of cells sufficient to produce a sphere having about 2,000 cells to about 3,000 cells at about day 7. In some embodiments, the culture of the pluripotent stem cells at day 0 includes a number of cells sufficient to produce a sphere having about 2,000 cells at about day 7. In some embodiments, the culture of the pluripotent stem cells at day 0 includes a number of cells sufficient to produce a sphere having about 3,000 cells at about day 7.

[0021] In some embodiments, the culture vessel is processed to reduce or eliminate cell adhesion. In some embodiments, the culture vessel is processed to reduce cell adhesion. In some embodiments, the culture vessel is processed to eliminate cell adhesion. In some embodiments, processing the culture vessel includes incubating the culture vessel with pluronic acid. In some embodiments, before the first incubation, the non-adherent culture vessel is exposed to a surfactant. In some embodiments, the surfactant is pluronic acid.

[0022] In some embodiments, the non-adherent culture vessel has a low or ultra-low attachment surface. In some embodiments, the non-adherent culture vessel has a low attachment surface. In some embodiments, the non-adherent culture vessel has an ultra-low attachment surface. In some embodiments, the substrate is a basement membrane protein. In some embodiments, the substrate is selected from one or more of laminin, collagen, entactin, heparin sulfate proteoglycans and combinations thereof. In some embodiments, the substrate is a recombinant protein. In some embodiments, the substrate is a recombinant laminin. In some embodiments, the substrate-coated culture vessel is exposed to poly-L-ornithine before being used to culture the cells.

[0023] In some embodiments, the TGF-β / activin Nodal signaling inhibitor is SB431542. In some embodiments, the cells are exposed to SB431542 at a concentration of about 1 μM to about 20 μM, about 5 μM to about 15 μM, or about 8 μM to about 12 μM. In some embodiments, the cells are exposed to SB431542 at a concentration of about 1 μM to about 20 μM. In some embodiments, the cells are exposed to SB431542 at a concentration of about 5 μM to about 15 μM. In some embodiments, the cells are exposed to SB431542 at a concentration of about 8 μM to about 12 μM. In some embodiments, the cells are exposed to SB431542 at a concentration of about 10 μM.

[0024] In some embodiments, the at least one SHH signaling activator is a SHH protein or purmorphamine. In some embodiments, the at least one SHH signaling activator is a SHH protein. In some embodiments, the at least one SHH signaling activator is purmorphamine. In some embodiments, the at least one SHH signaling activator includes two SHH signaling activators selected from SHH protein and purmorphamine. In some embodiments, the at least one SHH signaling activator is two SHH signaling activators selected from SHH protein and purmorphamine. In some embodiments, the cell is exposed to a concentration of about 10 ng / mL to 500 ng / mL, about 20 ng / mL to about 400 ng / mL, about 50 ng / mL to about 200 ng / mL, or about 75 ng / mL to about 150 ng / mL of SHH. In some embodiments, the cell is exposed to a concentration of about 10 ng / mL to 500 ng / mL of SHH. In some embodiments, the cell is exposed to a concentration of about 20 ng / mL to 400 ng / mL of SHH. In some embodiments, the cells are exposed to SHH at a concentration of about 50 ng / mL to 200 ng / mL. In some embodiments, the cells are exposed to SHH at a concentration of about 75 ng / mL to 150 ng / mL. In some embodiments, the cells are exposed to SHH at a concentration of about 100 ng / mL.

[0025] In some embodiments, the cell is exposed to purmorphamine at a concentration of about 1 μM to about 20 μM, about 5 μM to about 15 μM, or about 8 μM to about 12 μM. In some embodiments, the cell is exposed to purmorphamine at a concentration of about 1 μM to about 20 μM. In some embodiments, the cell is exposed to purmorphamine at a concentration of about 5 μM to about 15 μM. In some embodiments, the cell is exposed to purmorphamine at a concentration of about 8 μM to about 12 μM. In some embodiments, the cell is exposed to purmorphamine at a concentration of about 10 μM.

[0026] In some embodiments, the cells are exposed to SHH at a concentration of about 100 ng / mL and purmorphamine at a concentration of about 10 μM.

[0027] In some embodiments, the BMP signaling inhibitor is LDN193189. In some embodiments, the cells are exposed to LDN193189 at a concentration of about 10nM to 500nM, about 20nM to about 400nM, about 50nM to about 200nM, or about 75nM to about 150nM. In some embodiments, the cells are exposed to LDN193189 at a concentration of about 10nM to 500nM. In some embodiments, the cells are exposed to LDN193189 at a concentration of about 20nM to 400nM. In some embodiments, the cells are exposed to LDN193189 at a concentration of about 50nM to 200nM. In some embodiments, the cells are exposed to LDN193189 at a concentration of about 75nM to 150nM. In some embodiments, the cells are exposed to LDN193189 at a concentration of about 100nM.

[0028] In some embodiments, the GSK3β signaling inhibitor is CHIR99021. In some embodiments, the cell is exposed to CHIR99021 at a concentration of about 0.1 μM to about 5 μM, about 0.5 μM to about 4 μM, or about 1 μM to about 3 μM. In some embodiments, the cell is exposed to CHIR99021 at a concentration of about 0.1 μM to about 5 μM. In some embodiments, the cell is exposed to CHIR99021 at a concentration of about 0.5 μM to about 4 μM. In some embodiments, the cell is exposed to CHIR99021 at a concentration of about 1 μM to about 3 μM. In some embodiments, the cell is exposed to CHIR99021 at a concentration of about 2 μM.

[0029] In some embodiments, the cells are exposed to GDNF at a concentration of about 1 ng / mL to about 100 ng / mL, about 5 ng / mL to about 80 ng / mL, about 10 ng / mL to about 60 ng / mL, or about 15 ng / mL to about 30 ng / mL. In some embodiments, the cells are exposed to GDNF at a concentration of about 1 ng / mL to about 100 ng / mL. In some embodiments, the cells are exposed to GDNF at a concentration of about 5 ng / mL to about 80 ng / mL. In some embodiments, the cells are exposed to GDNF at a concentration of about 10 ng / mL to about 60 ng / mL. In some embodiments, the cells are exposed to GDNF at a concentration of about 15 ng / mL to about 30 ng / mL. In some embodiments, the cells are exposed to GDNF at a concentration of about 20 ng / mL.

[0030] In some embodiments, the cells are exposed to BDNF at a concentration of about 1 ng / mL to about 100 ng / mL, about 5 ng / mL to about 80 ng / mL, about 10 ng / mL to about 60 ng / mL, or about 15 ng / mL to about 30 ng / mL. In some embodiments, the cells are exposed to BDNF at a concentration of about 1 ng / mL to about 100 ng / mL. In some embodiments, the cells are exposed to BDNF at a concentration of about 5 ng / mL to about 80 ng / mL. In some embodiments, the cells are exposed to BDNF at a concentration of about 10 ng / mL to about 60 ng / mL. In some embodiments, the cells are exposed to BDNF at a concentration of about 15 ng / mL to about 30 ng / mL. In some embodiments, the cells are exposed to BDNF at a concentration of about 20 ng / mL.

[0031] In some embodiments, the cell is exposed to dbcAMP at a concentration of about 0.1 mM to 5 mM, about 0.2 mM to about 4 mM, about 0.3 mM to about 3 mM, or about 0.4 mM to about 2 mM. In some embodiments, the cell is exposed to dbcAMP at a concentration of about 0.1 mM to 5 mM. In some embodiments, the cell is exposed to dbcAMP at a concentration of about 0.2 mM to 4 mM. In some embodiments, the cell is exposed to dbcAMP at a concentration of about 0.3 mM to 3 mM. In some embodiments, the cell is exposed to dbcAMP at a concentration of about 0.4 mM to 2 mM. In some embodiments, the cell is exposed to dbcAMP at a concentration of about 0.5 mM.

[0032] In some embodiments, the cells are exposed to ascorbic acid at a concentration of about 0.05mM to about 5mM, about 0.1mM to about 1mM, or about 0.2mM to about 0.5mM. In some embodiments, the cells are exposed to ascorbic acid at a concentration of about 0.05mM to about 5mM. In some embodiments, the cells are exposed to ascorbic acid at a concentration of about 0.1mM to about 2mM. In some embodiments, the cells are exposed to ascorbic acid at a concentration of about 0.2mM to about 0.5mM. In some embodiments, the cells are exposed to ascorbic acid at a concentration of about 0.2mM.

[0033] In some embodiments, the cells are exposed to TGFβ3 at a concentration of about 0.1 ng / mL to about 5 ng / mL, about 0.3 ng / mL to about 3 ng / mL, or about 0.5 ng / mL to about 2 ng / mL. In some embodiments, the cells are exposed to TGFβ3 at a concentration of about 0.1 ng / mL to about 5 ng / mL. In some embodiments, the cells are exposed to TGFβ3 at a concentration of about 0.3 ng / mL to about 3 ng / mL. In some embodiments, the cells are exposed to TGFβ3 at a concentration of about 0.5 ng / mL to about 2 ng / mL. In some embodiments, the cells are exposed to TGFβ3 at a concentration of about 1 ng / mL.

[0034] In some embodiments, the Notch signaling inhibitor is DAPT. In some embodiments, the cell is exposed to a concentration of about 1 μM to about 20 μM, about 5 μM to about 15 μM, or about 8 μM to about 12 μM DAPT. In some embodiments, the cell is exposed to a concentration of about 1 μM to about 20 μM DAPT. In some embodiments, the cell is exposed to a concentration of about 5 μM to about 15 μM DAPT. In some embodiments, the cell is exposed to a concentration of about 8 μM to about 12 μM DAPT. In some embodiments, the cell is exposed to a concentration of about 10 μM DAPT.

[0035] In some embodiments of any of the embodiments provided, the culture in the first incubation and / or the second incubation is carried out in a culture medium with serum or a serum substitute. In some embodiments of any of the embodiments provided, the culture in the first incubation is carried out in a culture medium with serum or a serum substitute. In some embodiments of any of the embodiments provided, the culture in the second incubation is carried out in a culture medium with serum or a serum substitute. In some embodiments, the culture in the first incubation and the second incubation is carried out in a culture medium with serum or a serum substitute. In some embodiments, the cells are cultured in a culture medium with serum or a serum substitute from about day 0 to about day 10. In some embodiments, the serum or serum substitute accounts for about 5% (v / v) of the culture medium. In some embodiments, the serum or serum substitute accounts for about 2% (v / v) of the culture medium. In some embodiments, the culture medium comprises about 5% serum or serum substitute (v / v) from about day 0 to about day 1, and comprises about 2% serum substitute (v / v) from about day 2 to about day 10. In some embodiments, the culture medium contains a serum substitute. In some embodiments, the serum replacement does not contain fetal bovine serum (FBS). In some embodiments, the serum replacement is KnockOut TM Serum replacement.

[0036] In some embodiments, the cells are cultured in the absence of serum for the duration of the culture.

[0037] In some embodiments, the cells are exposed to a Rho-associated protein kinase (ROCK) signaling inhibitor during one or more days of the first incubation and the second incubation. In some embodiments, during the process of the method, the ROCK signaling inhibitor is added at least once a week. In some embodiments, the cells are exposed to a ROCK signaling inhibitor on day 0, day 7, day 16, and / or day 20. In some embodiments, the cells are exposed to a Rho-associated protein kinase (ROCK) signaling inhibitor on day 0. In some embodiments, the cells are exposed to a Rho-associated protein kinase (ROCK) signaling inhibitor on day 7. In some embodiments, the cells are exposed to a Rho-associated protein kinase (ROCK) signaling inhibitor on day 16. In some embodiments, the cells are exposed to a Rho-associated protein kinase (ROCK) signaling inhibitor on day 20. In some embodiments, the cells are exposed to a Rho-associated protein kinase (ROCK) signaling inhibitor on day 0, day 7, day 16, and day 20. In some embodiments, the ROCK inhibitor is Y-27632. In some embodiments, the cells are exposed to a ROCK inhibitor at a concentration of about 1 μM to about 20 μM, about 5 μM to about 15 μM, or about 8 μM to about 12 μM. In some embodiments, the cells are exposed to a ROCK inhibitor at a concentration of about 1 μM to about 20 μM. In some embodiments, the cells are exposed to a ROCK inhibitor at a concentration of about 5 μM to about 15 μM. In some embodiments, the cells are exposed to a ROCK inhibitor at a concentration of about 8 μM to about 12 μM. In some embodiments, the cells are exposed to a ROCK inhibitor at a concentration of about 10 μM.

[0038] In some embodiments, during the cultivation period of the first incubation and / or the second incubation, at least about 50% of the culture medium is replaced every day or every other day or every three days. In some embodiments, at least about 50% of the culture medium is replaced every day. In some embodiments, at least about 50% of the culture medium is replaced every other day. In some embodiments, at least about 50% of the culture medium is replaced every three days. In some embodiments, at least about 50% of the culture medium is replaced every day during the first incubation. In some embodiments, at least about 50% of the culture medium is replaced every other day during the first incubation. In some embodiments, at least about 50% of the culture medium is replaced every three days during the first incubation. In some embodiments, at least about 50% of the culture medium is replaced every day during the second incubation. In some embodiments, at least about 50% of the culture medium is replaced every other day during the second incubation. In some embodiments, at least about 50% of the culture medium is replaced every three days during the second incubation. In some embodiments, at least about 50% of the culture medium is replaced every day during the first incubation and the second incubation. In some embodiments, at least about 50% of the culture medium is replaced every other day during the first incubation and the second incubation. In some embodiments, at least about 50% of the culture medium is replaced every three days during the first incubation and the second incubation.

[0039] In some embodiments, the method comprises dissociating the spheroids prior to the second incubation. In some embodiments, the spheroids are dissociated by enzymatic dissociation. In some embodiments, the method comprises dissociating the spheroids prior to the second incubation and the spheroids are dissociated by enzymatic dissociation. In some embodiments, the spheroids are dissociated by enzymatic dissociation, the enzymatic dissociation comprising using an enzyme selected from the group consisting of: Accutase TM , dispase, collagenase and combinations thereof. In some embodiments, the spheroids are dissociated by enzymatic dissociation, the enzymatic dissociation comprising using Accutase TM In some embodiments, the spheroids are treated with Accutase TM Dissociation.

[0040] In some embodiments, after spheroid dissociation, cells of the dissociated spheroids are cultured on a substrate-coated culture vessel, such as by transferring the cells to a substrate-coated culture vessel. In some embodiments, after spheroid dissociation, cells of the dissociated spheroids are cultured on a substrate-coated culture vessel, such as by transferring the cells to a substrate-coated culture vessel at a concentration of about 0.1×10 6 cells / cm 2 About 2×10 6 cells / cm 2 , about 0.1×10 6 cells / cm 2 About 1×106 cells / cm 2 , about 0.1×10 6 cells / cm 2 About 0.8×10 6 cells / cm 2 , about 0.1×10 6 cells / cm 2 About 0.6×10 6 cells / cm 2 , about 0.1×10 6 cells / cm 2 About 0.4×10 6 cells / cm 2 , about 0.1×10 6 cells / cm 2 About 0.2×10 6 cells / cm 2 , about 0.2×10 6 cells / cm 2 About 2×10 6 cells / cm 2 , about 0.2×10 6 cells / cm 2 About 1×10 6 cells / cm 2 , about 0.2×10 6 cells / cm 2 About 0.8×10 6 cells / cm 2 , about 0.2×10 6 cells / cm 2 About 0.6×10 6 cells / cm 2 , about 0.2×10 6 cells / cm 2 About 0.4×10 6 cells / cm 2 , about 0.4×10 6 cells / cm 2 About 2×10 6 cells / cm 2 , about 0.4×10 6 cells / cm 2 About 1×10 6 cells / cm 2 , about 0.4×10 6 cells / cm 2 About 0.8×10 6 cells / cm 2 , about 0.4×10 6cells / cm 2 About 0.6×10 6 cells / cm 2 , about 0.6×10 6 cells / cm 2 About 2×10 6 cells / cm 2 , about 0.6×10 6 cells / cm 2 About 1×10 6 cells / cm 2 , about 0.6×10 6 cells / cm 2 About 0.8×10 6 cells / cm 2 , about 0.8×10 6 cells / cm 2 About 2×10 6 cells / cm 2 , about 0.8×10 6 cells / cm 2 About 1×10 6 cells / cm 2 or about 1.0×10 6 cells / cm 2 About 2×10 6 cells / cm 2 In some embodiments, after spheroid dissociation, cells of the dissociated spheroids are cultured on a substrate-coated culture vessel. In some embodiments, after spheroid dissociation, cells of the dissociated spheroids are cultured on a substrate-coated culture vessel, such as by transferring the cells to a substrate-coated culture vessel at a concentration of about 0.4×10 6 cells / cm 2 About 0.8×10 6 cells / cm 2 In some embodiments, transferring the dissociated spheroid cells to the substrate-coated vessel occurs on about day 7.

[0041] In some embodiments, the spheroid cells from the first incubation are not dissociated prior to transferring the spheroid cells to the substrate-coated culture vessel. In some embodiments, the second incubation comprises culturing the spheroid cells from the first incubation without first dissociating the cells to form a cell suspension.

[0042] In some embodiments of any of the embodiments provided, the method further comprises preparing the harvested cells with a cryoprotectant. In some embodiments, the cryoprotectant is selected from glycerol, propylene glycol, dimethyl sulfoxide (DMSO), or a combination thereof. In some embodiments, the cryoprotectant comprises DMSO. In some embodiments, the cryoprotectant is DMSO. In some embodiments, the harvested cells are prepared with about 10% DMSO.

[0043] In some embodiments, the pluripotent stem cell is an embryonic stem (ES) cell, an induced pluripotent stem cell (iPSC) or a combination thereof. In some embodiments, the pluripotent stem cell is an embryonic stem (ES) cell. In some embodiments, the pluripotent stem cell is an induced pluripotent stem cell (iPSC). In some embodiments, the pluripotent stem cell is an embryonic stem (ES) cell. In some embodiments, the ES cell is a mouse or human embryonic stem cell. In some embodiments, the ES cell is a mouse embryonic stem cell. In some embodiments, the pluripotent stem cell is a human embryonic stem cell. In some embodiments, the pluripotent stem cell is an induced pluripotent stem cell. In some embodiments, iPSC is a mouse or human induced pluripotent stem cell. In some embodiments, the pluripotent stem cell is a mouse induced pluripotent stem cell. In some embodiments, the pluripotent stem cell is a human induced pluripotent stem cell.

[0044] In some embodiments, the pluripotent stem cells are autologous to the subject to be treated with the neural differentiation cells. In some embodiments, the subject to be treated suffers from Parkinson's disease. In some embodiments, the subject to be treated is suspected of suffering from Parkinson's disease. In some embodiments, the subject to be treated suffers from Parkinson's syndrome (Parkinsonism). In some embodiments, the subject to be treated is suspected of suffering from Parkinson's syndrome.

[0045] In some embodiments, the pluripotent stem cells are allogeneic to the subject to be treated with the neural differentiation cells. In some embodiments, the neural differentiation cells are low immunogenic. In some embodiments, the pluripotent stem cells are engineered to (a) remove genes encoding one or more of polymorphic HLA-A / -B / -C and HLA class II molecules; and (b) provide genes encoding one or more of PD-L1, HLA-G, and CD47. In some embodiments, the pluripotent stem cells are engineered to remove genes encoding polymorphic HLA-A / -B / -C and HLA class II molecules. In some embodiments, the pluripotent stem cells are engineered to provide genes encoding PD-L1, HLA-G, and CD47. In some embodiments, the pluripotent stem cells are engineered to provide genes encoding PD-L1, HLA-G, and CD47 in the AAVS1 safe harbor locus. In some embodiments, the pluripotent stem cells are engineered to remove genes encoding polymorphic HLA-A / -B / -C and HLA class II molecules and provide genes encoding PD-L1, HLA-G, and CD47 into the AAVS1 safe harbor locus.

[0046] Also provided herein are therapeutic compositions produced by any of the methods provided.

[0047] In some embodiments, the cells of the therapeutic composition express EN1 and / or CORIN. In some embodiments, the cells of the therapeutic composition express EN1. In some embodiments, the cells of the therapeutic composition express CORIN. In some embodiments, the cells of the therapeutic composition express EN1 and CORIN. In some embodiments, the cells of the composition express EN1 and CORIN, and less than 10% of the total cells in the composition express TH.

[0048] Also provided herein is a therapeutic composition comprising committed dopamine neuron progenitor cells (DDPCs) derived from pluripotent stem cells, wherein the cells in the composition express EN1 and CORIN, and less than 10% of the total cells in the composition express TH.

[0049] In some embodiments, less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the total cells in the therapeutic composition express TH. In some embodiments, less than 10% of the total cells in the therapeutic composition express TH. In some embodiments, less than 5% of the total cells in the therapeutic composition express TH. In some embodiments, less than 3% of the total cells in the therapeutic composition express TH. In some embodiments, less than 1% of the total cells in the therapeutic composition express TH.

[0050] In some embodiments, greater than 10%, greater than 15%, or greater than 20% of the total cells in the therapeutic composition express TH. In some embodiments, greater than 10% of the total cells in the therapeutic composition express TH. In some embodiments, greater than 15% of the total cells in the therapeutic composition express TH. In some embodiments, greater than 20% of the total cells in the therapeutic composition express TH. In some embodiments, about 10% of the total cells in the therapeutic composition express TH. In some embodiments, about 15% of the total cells in the therapeutic composition express TH. In some embodiments, about 20% of the total cells in the therapeutic composition express TH.

[0051] In some embodiments, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% of the total cells in the composition express EN1. In some embodiments, at least about 15% of the total cells in the composition express EN1. In some embodiments, at least about 20% of the total cells in the composition express EN1. In some embodiments, at least about 25% of the total cells in the composition express EN1. In some embodiments, at least about 30% of the total cells in the composition express EN1. In some embodiments, at least about 35% of the total cells in the composition express EN1. In some embodiments, at least about 40% of the total cells in the composition express EN1. In some embodiments, at least about 45% of the total cells in the composition express EN1. In some embodiments, at least about 50% of the total cells in the composition express EN1. In some embodiments, at least about 55% of the total cells in the composition express EN1. In some embodiments, at least about 60% of the total cells in the composition express EN1. In some embodiments, at least about 65% of the total cells in the composition express EN1. In some embodiments, at least about 70% of the total cells in the composition express EN1. In some embodiments, at least about 75% of the total cells in the composition express EN1. In some embodiments, at least about 80% of the total cells in the composition express EN1.

[0052] In some embodiments, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% of the total cells in the composition express CORIN. In some embodiments, at least about 15% of the total cells in the composition express CORIN. In some embodiments, at least about 20% of the total cells in the composition express CORIN. In some embodiments, at least about 25% of the total cells in the composition express CORIN. In some embodiments, at least about 30% of the total cells in the composition express CORIN. In some embodiments, at least about 35% of the total cells in the composition express CORIN. In some embodiments, at least about 40% of the total cells in the composition express CORIN. In some embodiments, at least about 45% of the total cells in the composition express CORIN. In some embodiments, at least about 50% of the total cells in the composition express CORIN. In some embodiments, at least about 55% of the total cells in the composition express CORIN. In some embodiments, at least about 60% of the total cells in the composition express CORIN. In some embodiments, at least about 65% of the total cells in the composition express CORIN. In some embodiments, at least about 70% of the total cells in the composition express CORIN. In some embodiments, at least about 75% of the total cells in the composition express CORIN. In some embodiments, at least about 80% of the total cells in the composition express CORIN.

[0053] In some embodiments, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% of the total cells in the composition express EN1 and CORIN. In some embodiments, at least about 15% of the total cells in the composition express EN1 and CORIN. In some embodiments, at least about 20% of the total cells in the composition express EN1 and CORIN. In some embodiments, at least about 25% of the total cells in the composition express EN1 and CORIN. In some embodiments, at least about 30% of the total cells in the composition express EN1 and CORIN. In some embodiments, at least about 35% of the total cells in the composition express EN1 and CORIN. In some embodiments, at least about 40% of the total cells in the composition express EN1 and CORIN. In some embodiments, at least about 45% of the total cells in the composition express EN1 and CORIN. In some embodiments, at least about 50% of the total cells in the composition express EN1 and CORIN. In some embodiments, at least about 55% of the total cells in the composition express EN1 and CORIN. In some embodiments, at least about 60% of the total cells in the composition express EN1 and CORIN. In some embodiments, at least about 65% of the total cells in the composition express EN1 and CORIN. In some embodiments, at least about 70% of the total cells in the composition express EN1 and CORIN. In some embodiments, at least about 75% of the total cells in the composition express EN1 and CORIN. In some embodiments, at least about 80% of the total cells in the composition express EN1 and CORIN.

[0054] In some embodiments, the cells of the therapeutic composition express EN1, and less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the total cells in the therapeutic composition express TH. In some embodiments, the cells of the therapeutic composition express EN1, and less than 10% of the total cells in the therapeutic composition express TH. In some embodiments, the cells of the therapeutic composition express EN1, and less than 5% of the total cells in the therapeutic composition express TH. In some embodiments, the cells of the therapeutic composition express EN1, and less than 3% of the total cells in the therapeutic composition express TH. In some embodiments, the cells of the therapeutic composition express EN1, and less than 1% of the total cells in the therapeutic composition express TH.

[0055] In some embodiments, the cells of the therapeutic composition express CORIN, and less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the total cells in the therapeutic composition express TH. In some embodiments, the cells of the therapeutic composition express CORIN, and less than 10% of the total cells in the therapeutic composition express TH. In some embodiments, the cells of the therapeutic composition express CORIN, and less than 5% of the total cells in the therapeutic composition express TH. In some embodiments, the cells of the therapeutic composition express CORIN, and less than 3% of the total cells in the therapeutic composition express TH. In some embodiments, the cells of the therapeutic composition express CORIN, and less than 1% of the total cells in the therapeutic composition express TH.

[0056] In some embodiments, the cells of the therapeutic composition express EN1 and CORIN, and less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the total cells in the therapeutic composition express TH. In some embodiments, the cells of the therapeutic composition express EN1 and CORIN, and less than 10% of the total cells in the therapeutic composition express TH. In some embodiments, the cells of the therapeutic composition express EN1 and CORIN, and less than 5% of the total cells in the therapeutic composition express TH. In some embodiments, the cells of the therapeutic composition express EN1 and CORIN, and less than 3% of the total cells in the therapeutic composition express TH. In some embodiments, the cells of the therapeutic composition express EN1 and CORIN, and less than 1% of the total cells in the therapeutic composition express TH.

[0057] Also provided herein is a therapeutic composition comprising a committed dopamine neuron progenitor cell (DDPC) derived from a pluripotent stem cell, wherein the cells of the composition express EN1 and less than 10% of the total cells in the composition express TH. In some embodiments, less than 5% of the total cells in the therapeutic composition express TH. In some embodiments, less than 3% of the total cells in the therapeutic composition express TH. In some embodiments, less than 1% of the total cells in the therapeutic composition express TH.

[0058] Also provided herein is a therapeutic composition comprising a committed dopamine neuron progenitor cell (DDPC) derived from a pluripotent stem cell, wherein the cells of the composition express CORIN and less than 10% of the total cells in the composition express TH. In some embodiments, less than 5% of the total cells in the therapeutic composition express TH. In some embodiments, less than 3% of the total cells in the therapeutic composition express TH. In some embodiments, less than 1% of the total cells in the therapeutic composition express TH.

[0059] Also provided herein is a therapeutic composition comprising a committed dopamine neuron progenitor cell (DDPC) derived from a pluripotent stem cell, wherein the cells of the composition express EN1 and CORIN and less than 10% of the total cells in the composition express TH. In some embodiments, less than 5% of the total cells in the therapeutic composition express TH. In some embodiments, less than 3% of the total cells in the therapeutic composition express TH. In some embodiments, less than 1% of the total cells in the therapeutic composition express TH.

[0060] Also provided herein is a therapeutic composition comprising committed dopamine neuron progenitor cells (DDPCs) derived from pluripotent stem cells, wherein the therapeutic composition exhibits less than about 2.5×10 -2 The ratio of counts per million (CPM) TH to CPM GAPDH.

[0061] Also provided herein is a therapeutic composition comprising committed dopamine neuron progenitor cells (DDPCs) derived from pluripotent stem cells, wherein the therapeutic composition exhibits one or more of the following: (a) greater than about 2×10 -5 (b) greater than about 5×10 -4 (c) greater than about 4×10 -3 The ratio of CPM HES1 to CPM GAPDH is greater than about 8×10 -3 The ratio of CPM PSRC1 to CPM GAPDH is greater than about 2×10 -3 The ratio of CPM NEK6 to CPM GAPDH is greater than about 1×10 -2 The ratio of CPM IQGAP3 to CPM GAPDH is greater than about 8×10 -4 The ratio of CPM USP44 to CPM GAPDH; (h) is greater than about 3×10 -3 The ratio of CPM CEP55 to CPM GAPDH is greater than about 2×10 -2 The ratio of CPM KIF20A to CPMGAPDH is greater than about 2×10 -2 The ratio of CPM AURKA to CPM GAPDH; (k) is less than about 1×10 -3 The ratio of CPM CALCA to CPM GAPDH; (l) less than about 1×10 -3 The ratio of CPM GLRA2 to CPM GAPDH (m) is less than about 1×10 -1The ratio of CPM MAPT to CPM GAPDH; (n) is less than about 5×10 -2 The ratio of CPM CAMK2B to CPM GAPDH; (o) less than about 1×10 -2 The ratio of CPM SYT13 to CPM GAPDH (p) is less than about 1×10 -3 The ratio of CPM LHFPL4 to CPM GAPDH (q) is less than about 5×10 -3 The ratio of CPM RET to CPM GAPDH; (r) is less than about 1×10 -2 The ratio of CPM KCND3 to CPM GAPDH (s) is less than about 5×10 -2 The ratio of CPM NSG2 to CPM GAPDH is less than about 2×10 -2 The ratio of CPM SNAP25 to CPM GAPDH.

[0062] In some embodiments, the ratio of counts per million (CPM) NEUROG1 to CPM GAPDH is greater than about 2×10 -5 In some embodiments, the ratio of CPM EDN3 to CPM GAPDH is greater than about 5×10 -4 In some embodiments, the ratio of CPM HES1 to CPM GAPDH is greater than about 4×10 -3 In some embodiments, the ratio of CPM PSRC1 to CPM GAPDH is greater than about 8×10 -3 In some embodiments, the ratio of CPM NEK6 to CPM GAPDH is greater than about 2×10 -3 In some embodiments, the ratio of CPM IQGAP3 to CPM GAPDH is greater than about 1×10 -2 In some embodiments, the ratio of CPM USP44 to CPM GAPDH is greater than about 8×10 -4 In some embodiments, the ratio of CPM CEP55 to CPM GAPDH is greater than about 3×10 -3 In some embodiments, the ratio of CPM KIF20A to CPM GAPDH is greater than about 2×10 -2 In some embodiments, the ratio of CPM AURKA to CPM GAPDH is greater than about 2×10 -2 In some embodiments, the ratio of CPM CALCA to CPMGAPDH is greater than about 1×10 -3 In some embodiments, the ratio of CPM GLRA2 to CPM GAPDH is less than about 1×10-3 In some embodiments, the ratio of CPM MAPT to CPM GAPDH is less than about 1×10 -1 In some embodiments, the ratio of CPM CAMK2B to CPM GAPDH is greater than about 5×10 -2 In some embodiments, the ratio of CPM SYT13 to CPM GAPDH is less than about 1×10 -2 In some embodiments, the ratio of CPM LHFPL4 to CPM GAPDH is less than about 1×10 -3 In some embodiments, the ratio of CPM RET to CPM GAPDH is less than about 5×10 -3 In some embodiments, the ratio of CPMKCND3 to CPM GAPDH is less than about 1×10 -2 In some embodiments, the ratio of CPM NSG2 to CPM GAPDH is less than about 5×10 -2 In some embodiments, the ratio of CPM SNAP25 to CPM GAPDH is less than about 2×10 -2 .

[0063] In some embodiments, the therapeutic composition exhibits from about 2 to 20 of (a)-(t). In some embodiments, the therapeutic composition exhibits from about 2 to 15 of (a)-(t). In some embodiments, the therapeutic composition exhibits from about 2 to 10 of (a)-(t). In some embodiments, the therapeutic composition exhibits from about 2 to 5 of (a)-(t). In some embodiments, the therapeutic composition exhibits from about 5 to 20 of (a)-(t). In some embodiments, the therapeutic composition exhibits from about 5 to 10 of (a)-(t). In some embodiments, the therapeutic composition exhibits from about 10 to 20 of (a)-(t). In some embodiments, the therapeutic composition exhibits from about 5 to 15 of (a)-(t). In some embodiments, the therapeutic composition exhibits from about 15 to 20 of (a)-(t).

[0064] In some embodiments, the therapeutic composition exhibits at least 2 of (a)-(t). In some embodiments, the therapeutic composition exhibits at least 3 of (a)-(t). In some embodiments, the therapeutic composition exhibits at least 4 of (a)-(t). In some embodiments, the therapeutic composition exhibits at least 5 of (a)-(t). In some embodiments, the therapeutic composition exhibits at least 6 of (a)-(t). In some embodiments, the therapeutic composition exhibits at least 7 of (a)-(t). In some embodiments, the therapeutic composition exhibits at least 8 of (a)-(t). In some embodiments, the therapeutic composition exhibits at least 9 of (a)-(t). In some embodiments, the therapeutic composition exhibits at least 10 of (a)-(t). In some embodiments, the therapeutic composition exhibits at least 11 of (a)-(t). In some embodiments, the therapeutic composition exhibits at least 12 of (a)-(t). In some embodiments, the therapeutic composition exhibits at least 13 of (a)-(t). In some embodiments, the therapeutic composition exhibits at least 14 of (a)-(t). In some embodiments, the therapeutic composition exhibits at least 15 of (a)-(t). In some embodiments, the therapeutic composition exhibits at least 16 of (a)-(t). In some embodiments, the therapeutic composition exhibits at least 17 of (a)-(t). In some embodiments, the therapeutic composition exhibits at least 18 of (a)-(t). In some embodiments, the therapeutic composition exhibits at least 19 of (a)-(t). In some embodiments, the therapeutic composition exhibits at least 20 of (a)-(t).

[0065] In some embodiments, the therapeutic composition exhibits 2 of (a)-(t). In some embodiments, the therapeutic composition exhibits 3 of (a)-(t). In some embodiments, the therapeutic composition exhibits 4 of (a)-(t). In some embodiments, the therapeutic composition exhibits 5 of (a)-(t). In some embodiments, the therapeutic composition exhibits 6 of (a)-(t). In some embodiments, the therapeutic composition exhibits 7 of (a)-(t). In some embodiments, the therapeutic composition exhibits 8 of (a)-(t). In some embodiments, the therapeutic composition exhibits 9 of (a)-(t). In some embodiments, the therapeutic composition exhibits 10 of (a)-(t). In some embodiments, the therapeutic composition exhibits 11 of (a)-(t). In some embodiments, the therapeutic composition exhibits 12 of (a)-(t). In some embodiments, the therapeutic composition exhibits 13 of (a)-(t). In some embodiments, the therapeutic composition exhibits 14 of (a)-(t). In some embodiments, the therapeutic composition exhibits 15 of (a)-(t). In some embodiments, the therapeutic composition exhibits 16 of (a)-(t). In some embodiments, the therapeutic composition exhibits 17 of (a)-(t). In some embodiments, the therapeutic composition exhibits 18 of (a)-(t). In some embodiments, the therapeutic composition exhibits 19 of (a)-(t). In some embodiments, the therapeutic composition exhibits 20 of (a)-(t).

[0066] In some embodiments, the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, the therapeutic composition exhibits greater than about 1×10 -4 In some embodiments, the therapeutic composition exhibits a ratio of EN1 to CPM GAPDH of greater than about 2×10 -2 In some embodiments, the therapeutic composition exhibits a CORIN to GAPDH ratio of greater than about 1×10 -4 The ratio of CPM EN1 to CPM GAPDH is greater than about 2 × 10 -2 In some embodiments, the ratio of CPM EN1 to CPM GAPDH is between about 1.5×10 -3 Up to 1×10 -2 In some embodiments, the ratio of CPM CORIN to CPM GAPDH is between about 5×10 -2 Up to 5×10 -1between.

[0067] In some embodiments, the cells in the composition express TH. In some embodiments, less than 10% of the total cells in the composition express TH. In some embodiments, the therapeutic composition exhibits less than about 3×10 -2 In some embodiments, the ratio of CPM TH to CPM GAPDH is between about 1×10 -3 Up to 2.5×10 -2 In some embodiments, about 2% to about 10%, about 2% to about 8%, about 2% to about 6%, about 2% to about 4%, about 4% to about 10%, about 4% to about 8%, about 4% to about 6%, about 6% to about 10%, about 6% to about 8%, or about 8% to 10% of the total cells in the composition express TH. In some embodiments, about 2% to about 10% of the total cells in the composition express TH. In some embodiments, about 2% to about 8% of the total cells in the composition express TH. In some embodiments, about 2% to about 6% of the total cells in the composition express TH. In some embodiments, about 2% to about 4% of the total cells in the composition express TH. In some embodiments, about 4% to about 10% of the total cells in the composition express TH. In some embodiments, about 4% to about 8% of the total cells in the composition express TH. In some embodiments, about 4% to about 6% of the total cells in the composition express TH. In some embodiments, about 6% to about 10% of the total cells in the composition express TH. In some embodiments, about 6% to about 8% of the total cells in the composition express TH. In some embodiments, about 8% to about 10% of the total cells in the composition express TH. In some embodiments, the percentage of total cells in the composition that are positive for TH expression is or is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, or any value between any of the foregoing values. In some embodiments, 1% of the total cells in the composition express TH. In some embodiments, 2% of the total cells in the composition express TH. In some embodiments, 3% of the total cells in the composition express TH. In some embodiments, 4% of the total cells in the composition express TH. In some embodiments, 5% of the total cells in the composition express TH. In some embodiments, 6% of the total cells in the composition express TH. In some embodiments, 7% of the total cells in the composition express TH. In some embodiments, 8% of the total cells in the composition express TH. In some embodiments, 9% of the total cells in the composition express TH.

[0068] In some embodiments, at least about 15% of the cells in the therapeutic composition express EN1. In some embodiments, at least about 20% of the cells in the therapeutic composition express EN1. In some embodiments, at least about 25% of the cells in the therapeutic composition express EN1. In some embodiments, at least about 30% of the cells in the therapeutic composition express EN1. In some embodiments, at least about 35% of the cells in the therapeutic composition express EN1. In some embodiments, at least about 40% of the cells in the therapeutic composition express EN1. In some embodiments, at least about 45% of the cells in the therapeutic composition express EN1. In some embodiments, at least about 50% of the cells in the therapeutic composition express EN1. In some embodiments, at least about 55% of the cells in the therapeutic composition express EN1. In some embodiments, at least about 60% of the cells in the therapeutic composition express EN1. In some embodiments, at least about 65% of the cells in the therapeutic composition express EN1. In some embodiments, at least about 70% of the cells in the therapeutic composition express EN1. In some embodiments, at least about 75% of the cells in the therapeutic composition express EN1. In some embodiments, at least about 80% of the cells in the therapeutic composition express EN1.

[0069] In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 15% of the cells in the therapeutic composition express EN1. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 20% of the cells in the therapeutic composition express EN1. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 25% of the cells in the therapeutic composition express EN1. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 30% of the cells in the therapeutic composition express EN1. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 35% of the cells in the therapeutic composition express EN1. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 40% of the cells in the therapeutic composition express EN1. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 45% of the cells in the therapeutic composition express EN1. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 50% of the cells in the therapeutic composition express EN1. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 55% of the cells in the therapeutic composition express EN1. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 60% of the cells in the therapeutic composition express EN1. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 65% of the cells in the therapeutic composition express EN1. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 70% of the cells in the therapeutic composition express EN1. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 75% of the cells in the therapeutic composition express EN1. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 80% of the cells in the therapeutic composition express EN1.

[0070] In some embodiments, at least about 15% of the cells in the therapeutic composition express CORIN. In some embodiments, at least about 20% of the cells in the therapeutic composition express CORIN. In some embodiments, at least about 25% of the cells in the therapeutic composition express CORIN. In some embodiments, at least about 30% of the cells in the therapeutic composition express CORIN. In some embodiments, at least about 35% of the cells in the therapeutic composition express CORIN. In some embodiments, at least about 40% of the cells in the therapeutic composition express CORIN. In some embodiments, at least about 45% of the cells in the therapeutic composition express CORIN. In some embodiments, at least about 50% of the cells in the therapeutic composition express CORIN. In some embodiments, at least about 55% of the cells in the therapeutic composition express CORIN. In some embodiments, at least about 60% of the cells in the therapeutic composition express CORIN. In some embodiments, at least about 65% of the cells in the therapeutic composition express CORIN. In some embodiments, at least about 70% of the cells in the therapeutic composition express CORIN. In some embodiments, at least about 75% of the cells in the therapeutic composition express CORIN. In some embodiments, at least about 80% of the cells in the therapeutic composition express CORIN.

[0071] In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 15% of the cells in the therapeutic composition express CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 20% of the cells in the therapeutic composition express CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 25% of the cells in the therapeutic composition express CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 30% of the cells in the therapeutic composition express CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 35% of the cells in the therapeutic composition express CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 40% of the cells in the therapeutic composition express CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 45% of the cells in the therapeutic composition express CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 50% of the cells in the therapeutic composition express CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 55% of the cells in the therapeutic composition express CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 60% of the cells in the therapeutic composition express CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 65% of the cells in the therapeutic composition express CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 70% of the cells in the therapeutic composition express CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 75% of the cells in the therapeutic composition express CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 80% of the cells in the therapeutic composition express CORIN.

[0072] In some embodiments, at least about 15% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 20% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 25% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 30% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 35% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 40% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 45% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 50% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 55% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 60% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 65% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 70% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 75% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 80% of the cells in the therapeutic composition express EN1 and CORIN.

[0073] In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 15% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 20% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 25% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 30% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 35% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 40% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 45% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 50% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 55% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 60% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 65% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 70% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 75% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 80% of the cells in the therapeutic composition express EN1 and CORIN.

[0074] In some embodiments, less than 3%, 2%, 1%, or 0.5% of the total cells in the therapeutic composition are serotonergic cells. In some embodiments, less than 1% of the total cells in the therapeutic composition are serotonergic cells. In some embodiments, less than 0.5% of the total cells in the therapeutic composition are serotonergic cells.

[0075] In some embodiments, less than 1%, 0.5%, 0.25%, or 0.05% of the total cells in the therapeutic composition are pluripotent stem cells. In some embodiments, less than 0.5% of the total cells in the therapeutic composition are pluripotent stem cells. In some embodiments, less than 0.05% of the total cells in the therapeutic composition are pluripotent stem cells.

[0076] In some embodiments, at least about 70%, 75%, 80%, 85%, 90%, or 95% of the total cells in the therapeutic composition are alive. In some embodiments, at least about 70% of the total cells in the therapeutic composition are alive. In some embodiments, at least about 75% of the total cells in the therapeutic composition are alive. In some embodiments, at least about 80% of the total cells in the therapeutic composition are alive. In some embodiments, at least about 85% of the total cells in the therapeutic composition are alive. In some embodiments, at least about 90% of the total cells in the therapeutic composition are alive. In some embodiments, at least about 95% of the total cells in the therapeutic composition are alive.

[0077] In some embodiments, the directed cells are capable of transplanting and innervating other cells in vivo.

[0078] In some embodiments, the therapeutic composition comprises at least 5 million total cells, at least 10 million total cells, at least 15 million total cells, at least 20 million total cells, at least 30 million total cells, at least 40 million total cells, at least 50 million total cells, at least 100 million total cells, at least 150 million total cells, or at least 200 million total cells. In some embodiments, the therapeutic composition comprises at least 5 million total cells. In some embodiments, the therapeutic composition comprises at least 10 million total cells. In some embodiments, the therapeutic composition comprises at least 15 million total cells. In some embodiments, the therapeutic composition comprises at least 20 million total cells. In some embodiments, the therapeutic composition comprises at least 30 million total cells. In some embodiments, the therapeutic composition comprises at least 40 million total cells.

[0079] In some embodiments, the therapeutic composition comprises about 5 million total cells, about 10 million total cells, about 15 million total cells, about 20 million total cells, about 30 million total cells, about 40 million total cells, about 50 million total cells, about 100 million total cells, about 150 million total cells, or about 200 million total cells. In some embodiments, the therapeutic composition comprises about 5 million total cells. In some embodiments, the therapeutic composition comprises about 10 million total cells. In some embodiments, the therapeutic composition comprises about 15 million total cells. In some embodiments, the therapeutic composition comprises about 20 million total cells. In some embodiments, the therapeutic composition comprises about 30 million total cells. In some embodiments, the therapeutic composition comprises about 40 million total cells.

[0080] In some embodiments, the therapeutic composition is about 0.5mL to 5mL. In some embodiments, the therapeutic composition is about 0.5mL. In some embodiments, the therapeutic composition is about 0.75mL. In some embodiments, the therapeutic composition is about 1.0mL. In some embodiments, the therapeutic composition is about 1.25mL. In some embodiments, the therapeutic composition is about 1.5mL. In some embodiments, the therapeutic composition is about 1.75mL. In some embodiments, the therapeutic composition is about 2.0mL. In some embodiments, the therapeutic composition is about 2.5mL. In some embodiments, the therapeutic composition is about 3.0mL. In some embodiments, the therapeutic composition is about 3.5mL. In some embodiments, the therapeutic composition is about 4.0mL. In some embodiments, the therapeutic composition is about 4.5mL. In some embodiments, the therapeutic composition is about 5.0mL.

[0081] In some embodiments, the therapeutic composition comprises a cryoprotectant. In some embodiments, the cryoprotectant is glycerol, propylene glycol, dimethyl sulfoxide (DMSO), or a combination thereof. In some embodiments, the cryoprotectant is DMSO. In some embodiments, the therapeutic composition comprises about 10% DMSO.

[0082] In some embodiments, the composition is used to treat a neurodegenerative disease or condition in a subject. In some embodiments, the neurodegenerative disease or condition includes the loss of dopaminergic neurons. In some embodiments, the neurodegenerative disease or condition includes the loss of dopaminergic neurons in the substantia nigra (SN). In some embodiments, the neurodegenerative disease or condition includes the loss of dopaminergic neurons in the SN compact (SNc). In some embodiments, the neurodegenerative disease or condition is Parkinson's disease. In some embodiments, the neurodegenerative disease or condition is Parkinson's syndrome.

[0083] Also provided herein is a method of treatment comprising administering to a subject a therapeutically effective amount of any of the therapeutic compositions provided herein.

[0084] In some embodiments, the number of cells administered to a subject is about 0.25×10 6 cells to about 20×10 6 cells, about 0.25×10 6 cells to about 15×10 6 cells, about 0.25×10 6 cells to about 10×10 6 cells, about 0.25×10 6 cells to about 5×10 6 cells, about 0.25×10 6 cells to about 1×10 6 cells, about 0.25×10 6 cells to about 0.75×10 6 cells, about 0.25×10 6 cells to about 0.5×10 6 cells, about 0.5×10 6 cells to about 20×10 6 cells, about 0.5×10 6 cells to about 15×10 6 cells, about 0.5×10 6 cells to about 10×10 6 cells, about 0.5×10 6 cells to about 5×10 6 cells, about 0.5×10 6 cells to about 1×10 6 cells, about 0.5×10 6 cells to about 0.75×10 6 cells, about 0.75×10 6 cells to about 20×10 6 cells, about 0.75×10 6 cells to about 15×10 6 cells, about 0.75×10 6 cells to about 10×10 6 cells, about 0.75×10 6 cells to about 5×10 6 cells, about 0.75×10 6 cells to about 1×10 6 cells, about 1×10 6cells to about 20×10 6 cells, about 1×10 6 cells to about 15×10 6 cells, about 1×10 6 cells to about 10×10 6 cells, about 1×10 6 cells to about 5×10 6 cells, about 5×10 6 cells to about 20×10 6 cells, about 5×10 6 cells to about 15×10 6 cells, about 5×10 6 cells to about 10×10 6 cells, about 10×10 6 cells to about 20×10 6 cells, about 10×10 6 cells to about 15×10 6 cells or about 15×10 6 cells to about 20×10 6 In some embodiments, the number of cells administered to a subject is about 0.25×10 6 cells to about 20×10 6 In some embodiments, the number of cells administered to a subject is about 0.5×10 6 cells to about 15×10 6 In some embodiments, the number of cells administered to a subject is about 1×10 6 cells to about 10×10 6 cells.

[0085] In some embodiments, the number of cells administered to a subject is about 5×10 6 In some embodiments, the number of cells administered to a subject is about 10×10 6 In some embodiments, the number of cells administered to a subject is about 15×10 6 In some embodiments, the number of cells administered to a subject is about 20×10 6 In some embodiments, the number of cells administered to a subject is about 25×10 6 In some embodiments, the number of cells administered to a subject is about 30×10 6 cells.

[0086] In some embodiments, cells are administered to a subject at a concentration of about 50,000 cells / microliter to about 150,000 cells / microliter. In some embodiments, cells are administered to a subject at a concentration of about 50,000 cells / microliter. In some embodiments, cells are administered to a subject at a concentration of about 75,000 cells / microliter. In some embodiments, cells are administered to a subject at a concentration of about 100,000 cells / microliter. In some embodiments, cells are administered to a subject at a concentration of about 125,000 cells / microliter. In some embodiments, cells are administered to a subject at a concentration of about 150,000 cells / microliter.

[0087] In some embodiments, the subject suffers from a neurodegenerative disease or condition. In some embodiments, the neurodegenerative disease or condition includes the loss of dopaminergic neurons. In some embodiments, the subject has lost at least 50% of dopaminergic neurons. In some embodiments, the subject has lost at least 50% of dopaminergic neurons in the substantia nigra (SN). In some embodiments, the subject has lost at least 50% of dopaminergic neurons in the SN compact part (SNc). In some embodiments, the neurodegenerative disease or condition is Parkinson's syndrome. In some embodiments, the neurodegenerative disease or condition is Parkinson's disease. In some embodiments, the neurodegenerative disease or condition is idiopathic Parkinson's disease.

[0088] In some embodiments, the administering comprises delivering the cells of the composition by stereotactic injection. In some embodiments, the administering comprises delivering the cells of the composition by catheter. In some embodiments, the cells are delivered to the striatum of the subject. In some embodiments, the subject is administered about 3×10 6 cells / hemisphere to 15×10 6 cells / hemisphere. In some embodiments, a subject is administered about 3×10 6 cells / hemisphere. In some embodiments, a subject is administered about 4×10 6 cells / hemisphere. In some embodiments, a subject is administered about 5×10 6 cells / hemisphere. In some embodiments, a subject is administered about 6×10 6 cells / hemisphere. In some embodiments, a subject is administered about 7×10 6 cells / hemisphere. In some embodiments, a subject is administered about 8×10 6 cells / hemisphere. In some embodiments, a subject is administered about 9×10 6 In some embodiments, the subject is administered about 10×10 6cells / hemisphere. In some embodiments, the subject is administered about 11×10 6 cells / hemisphere. In some embodiments, a subject is administered about 12×10 6 cells / hemisphere. In some embodiments, a subject is administered about 13×10 6 cells / hemisphere. In some embodiments, the subject is administered about 14×10 6 cells / hemisphere. In some embodiments, the subject is administered about 15×10 6 cells / hemisphere.

[0089] In some embodiments, the cells of the therapeutic composition are autologous to the subject.

[0090] In some embodiments, the cells of the therapeutic composition are allogeneic to the subject. In some embodiments, the cells of the therapeutic composition are low immunogenic. In some embodiments, the cells of the therapeutic composition are engineered to (a) remove genes encoding one or more of polymorphic HLA-A / -B / -C and HLA class II molecules; and (b) provide genes encoding one or more of PD-L1, HLA-G, and CD47. In some embodiments, the cells of the therapeutic composition are engineered to remove genes encoding polymorphic HLA-A / -B / -C and HLA class II molecules. In some embodiments, the cells of the therapeutic composition are engineered to provide genes encoding PD-L1, HLA-G, and CD47. In some embodiments, the cells of the therapeutic composition are engineered to provide genes encoding PD-L1, HLA-G, and CD47 in the AAVS1 safe harbor locus. In some embodiments, the cells of the therapeutic composition are engineered to remove genes encoding polymorphic HLA-A / -B / -C and HLA class II molecules and provide genes encoding PD-L1, HLA-G, and CD47 into the AAVS1 safe harbor locus.

[0091] Also provided herein is the use of any composition provided herein for treating Parkinson's syndrome. Also provided herein is the use of any composition provided herein for treating Parkinson's disease.

[0092] Also provided herein are compositions as described herein for use in formulating a medicament for treating a subject suffering from a neurodegenerative disease or condition.

[0093] Also provided herein is any pharmaceutical composition as described herein for use in treating a subject suffering from a neurodegenerative disease or condition.

[0094] In some embodiments, the neurodegenerative disease or condition comprises the loss of dopaminergic neurons. In some embodiments, the subject has lost at least 50%, at least 60%, at least 70%, or at least 80% of dopaminergic neurons, optionally in the substantia nigra (SN), optionally in the SN pars compacta (SNc). In some embodiments, the neurodegenerative disease or condition is Parkinson's syndrome. In some embodiments, the neurodegenerative disease or condition is Parkinson's disease, optionally idiopathic Parkinson's disease.

[0095] In some embodiments, the cells of the composition are autologous to the subject. In some embodiments, the cells of the composition are allogeneic to the subject. In some embodiments, the cells of the therapeutic composition are low immunogenic. In some embodiments, the cells of the therapeutic composition are engineered to (a) remove genes encoding one or more of the polymorphic HLA-A / -B / -C and HLA class II molecules; and (b) provide genes encoding one or more of PD-L1, HLA-G, and CD47, optionally provided to the AAVS1 safe harbor locus. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Figure 1 . An exemplary non-adherent protocol for differentiating pluripotent stem cells into committed dopamine (DA) neuron progenitor cells or DA neurons is shown.

[0097] Figure 2 . Shown are FOXA2, TH and DAPI expression of differentiated cells derived from spheres with approximately 500, 1,000, 2,000, 3,000, 10,000 or 15,000 cells at day 25.

[0098] Figure 3 . Shown are FOXA2, TH and DAPI expression of differentiated cells grown in the presence or absence of serum at day 10.

[0099] Figure 4 . An exemplary adherence protocol for differentiating pluripotent stem cells into committed dopamine (DA) neuron progenitor cells or DA neurons is shown.

[0100] Figure 5 . Shown are the FOXA2, TH and DAPI expressions of differentiated cells derived from an exemplary adherent method or an exemplary non-adherent method at day 25.

[0101] Figure 6 . Shown is the tyrosine hydroxylase (TH) gene expression at day 18 and day 25 of cells differentiated by an exemplary non-adherent method.

[0102] Figure 7. Shown are dopamine (DA) and norepinephrine (NE) production (ng / million assayed cells) of cells cultured to day 75.

[0103] Fig. 8A . Shown are FOXA2 and TH gene expression in cells differentiated by an exemplary non-adherent method at different differentiation days.

[0104] Figure 8B . Shown are EN1 and CORIN gene expression in cells differentiated by an exemplary non-adherent method at different differentiation days.

[0105] Figure 8C . Shown are PAX6 and SOX2 gene expression in cells differentiated by an exemplary non-adherent method at different differentiation days.

[0106] I. definition

[0107] Unless otherwise defined, all specialized terms, symbols, and other technical and scientific terms or terminology used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ease of reference, and the inclusion of such definitions herein should not be construed as indicating a substantial difference from those generally understood in the art.

[0108] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. For example, "a" or "an" means "at least one" or "one or more." It should be understood that the aspects and variations described herein include "consisting of" and / or "consisting essentially of" the aspects and variations.

[0109] Throughout this disclosure, various aspects of the claimed subject matter are presented in the format of a range. It should be understood that the description of the range format is only for convenience and brevity and should not be interpreted as a rigid limitation on the scope of the claimed subject matter. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, in the case of providing a range of values, it should be understood that each intermediate value between the upper and lower limits of the range and any other specified value or intermediate value within the specified range are included in the claimed subject matter. The upper and lower limits of these smaller ranges can be independently included in the smaller range, and are also included in the claimed subject matter, with any clearly excluded limits in the specified range as a condition. When the specified range includes one or two limits, the scope excluding any one or two of the included limits is also included in the claimed subject matter. Regardless of the width of the range, this applies.

[0110] As used herein, the term "about" refers to the usual error range of each value that is readily known. References herein to an "about" value or parameter include (and describe) embodiments for that value or parameter itself. For example, a description referring to "about X" includes a description of "X".

[0111] As used herein, the statement that cell or cell group is " positive " for specific marker refers to that specific marker (usually surface marker) can be detected on cell or in cell.When mentioning surface marker, the term refers to as by flow cytometry, for example, by using the antibody dyeing with the specific binding of this marker and detecting the antibody detected as there is surface expression, wherein this dyeing can be detected by flow cytometry, its level is substantially higher than the dyeing detected by the control of isotype matching under other same conditions, and / or its level is substantially similar to the level of cells known to be positive for this marker, and / or its level is substantially higher than the level of cells known to be negative for this marker.When mentioning the marker in cell, such as transcription product or translation product, the term refers to for example the presence of detectable transcription product or translation product, wherein the level of product detected is substantially higher than the level detected by the control under other same conditions, and / or its level is substantially similar to the level of cells known to be positive for this marker, and / or its level is substantially higher than the level of cells known to be negative for this marker.

[0112] As used herein, the statement that cell or cell group is " negative " for specific marker refers to the absence of specific marker (usually surface marker) on cell or in cell substantial detectable existence.When mentioning surface marker, the term refers to as by flow cytometry, for example, by using the antibody dyeing with the marker specific binding and detecting the antibody detected as there is no surface expression, wherein not by flow cytometry, with substantially higher than the level detected by the control of isotype matching under other same conditions, and / or with substantially lower than the level of known cells positive for the marker, and / or with substantially similar level compared to the level of known cells negative for the marker, dyeing is detected.When mentioning the marker in cell, such as transcription product or translation product, the term refers to for example the absence of detectable transcription product or translation product, wherein not with substantially higher than the level detected by the control under other same conditions, and / or with substantially lower than the level of known cells positive for the marker, and / or with substantially similar level compared to known cells negative for the marker, product is detected.

[0113] The term "expression" or "expressed" as used herein when referring to a gene refers to the transcription product and / or translation product of the gene. The expression level of a DNA molecule in a cell can be determined based on the amount of the corresponding mRNA present in the cell or the amount of the protein encoded by the DNA produced by the cell (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 18.1-18.88).

[0114] As used herein, the term "stem cell" refers to a cell characterized by the ability to self-renew through cell mitosis and the potential to differentiate into tissues or organs. Among mammalian stem cells, embryonic stem cells and somatic stem cells can be distinguished. Embryonic stem cells are present in blastocysts and produce embryonic tissues, while somatic stem cells are present in adult tissues for the purpose of tissue regeneration and repair.

[0115] As used herein, the term "adult stem cell" refers to undifferentiated cells found in individuals after embryonic development. Adult stem cells multiply by cell division to replenish dead cells and regenerate damaged tissues. Adult stem cells can divide and produce another cell like itself or produce a more differentiated cell. Although adult stem cells are associated with the expression of pluripotency markers such as Rex1, Nanog, Oct4 or Sox2, they do not have the ability of pluripotent stem cells to differentiate into cell types of all three germ layers.

[0116] As used herein, the terms "induced pluripotent stem cells", "iPS" and "iPSC" refer to pluripotent stem cells artificially obtained (e.g., by artificial manipulation) from non-pluripotent cells. "Non-pluripotent cells" can be cells with lower self-renewal and differentiation potentials than pluripotent stem cells. Cells with lower potential can be, but are not limited to, adult stem cells, tissue-specific progenitor cells, primary cells or secondary cells.

[0117] As used herein, the term "pluripotent" or "pluripotency" refers to cells that are capable of producing progeny that can, under appropriate conditions, differentiate into cell types that collectively exhibit characteristics associated with cell lineages from the three germ layers (endoderm, mesoderm, and ectoderm). Pluripotent stem cells can contribute to the organization of prenatal, postnatal, or adult organisms.

[0118] As used herein, the term "pluripotent stem cell characteristics" refers to cell characteristics that distinguish pluripotent stem cells from other cells. The expression or non-expression of certain combinations of molecular markers is an example of pluripotent stem cell characteristics. More specifically, human pluripotent stem cells can express at least some, optionally all, of the markers from the following non-limiting list: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49 / 6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Lin28, Rex1, and Nanog. Cell morphology associated with pluripotent stem cells is also a characteristic of pluripotent stem cells.

[0119] As used herein, the term "reprogramming" refers to a process of dedifferentiating non-pluripotent cells into cells that exhibit characteristics of pluripotent stem cells.

[0120] As used herein, the term "adherent culture vessel" refers to a culture vessel to which cells can attach via extracellular matrix molecules, etc., and from which it is necessary to use an enzyme (e.g., trypsin, dispase, etc.) to separate the cells. "Adherent culture vessel" is very different from such a culture vessel, in which cell attachment is reduced and it is not necessary to use an enzyme to remove the cells from the culture vessel.

[0121] As used herein, the term "non-adherent culture vessel" refers to a culture vessel with reduced or limited cell attachment (such as for a period of time). Non-adherent culture vessels can contain low-attachment or ultra-low-attachment surfaces, such as can be achieved by treating the surface with a substance that prevents cell attachment, such as a hydrogel (e.g., a neutrally charged hydrogel and / or a hydrophilic hydrogel) and / or a surfactant (e.g., pluronic acid). Non-adherent culture vessels can contain circular or concave holes, and / or microwells (e.g., Aggrewells TM In some embodiments, the non-adherent culture vessel is Aggrewell TMFor non-adherent culture vessels, it may not be necessary to use enzymes to remove cells from the culture vessel.

[0122] As used herein, the term "cell culture" may refer to an in vitro cell population that exists outside an organism. Cell cultures may be established from primary cells isolated from a cell bank or an animal, or from secondary cells derived from one of these sources and immortalized to obtain long-term in vitro cultures.

[0123] As used herein, the terms "culture", "culturing", "grow", "growing", "maintain", "maintaining", "expand", "expanding", etc., when referring to the cell culture itself or the culture process, can be used interchangeably, meaning cells maintained in vitro (e.g., ex vivo) under conditions suitable for survival. The cultured cells are allowed to survive, and the culture can cause the cells to grow, differentiate or divide.

[0124] As used herein, a composition refers to any mixture of two or more products, substances or compounds (including cells). It can be a solution, a suspension, a liquid, a powder, a paste, an aqueous solution, a non-aqueous solution or any combination thereof.

[0125] The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use, such as pharmaceutical use in a mammalian subject (e.g., a human). A pharmaceutical composition typically comprises an effective amount of an active agent (e.g., a cell) and a carrier, excipient, or diluent. The carrier, excipient, or diluent is typically a pharmaceutically acceptable carrier, excipient, or diluent, respectively.

[0126] "Pharmaceutically acceptable carrier" refers to ingredients in a pharmaceutical formulation other than the active ingredient that are non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0127] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0128] As used herein, a "subject" is a mammal, such as a human or other animal, and typically is a human. DETAILED DESCRIPTION

[0129] The present disclosure relates to methods for lineage-specific differentiation of pluripotent stem cells (PSCs), such as embryonic stem (ES) cells or induced pluripotent stem cells (iPSCs). Methods for directing lineage-specific differentiation of PSCs or iPSCs into midbrain floor plate progenitor cells, directed dopamine (DA) neuron progenitor cells (DDPCs) and / or dopamine (DA) neurons are specifically described. Differentiated cells prepared using the methods provided herein are further contemplated for various uses, including but not limited to use as therapeutic agents for reversing diseases or injuries in which dopamine neurons are deficient in patients.

[0130] Provided herein is a method for making pluripotent stem cells (PSCs) such as embryonic stem (ES) cells or induced pluripotent stem cells (iPSCs) lineage-specifically differentiate into midbrain floor plate progenitor cells, directed dopamine (DA) neuron progenitor cells and / or dopamine (DA) neurons. In some aspects, PSCs differentiate into midbrain floor plate progenitor cells. In some aspects, such midbrain floor plate progenitor cells further differentiate into directed dopamine (DA) neuron progenitor cells. In some embodiments, directed dopaminergic precursor cells are cells that differentiate into dopamine neurons and cannot differentiate into non-dopamine neuron cells. In some aspects, such directed dopamine (DA) neuron progenitor cells further differentiate into dopamine (DA) neurons. In some aspects, PSCs differentiate into midbrain floor plate progenitor cells, differentiate into directed dopamine (DA) neuron progenitor cells, and finally differentiate into dopamine (DA) neurons.

[0131] The provided embodiments address issues associated with features of Parkinson's disease (PD), including the selective degeneration of midbrain dopamine (mDA) neurons in the patient's brain. Because PD symptoms are primarily due to the selective loss of DA neurons in the ventral substantia nigra of the midbrain, PD is considered amenable to cell replacement therapy strategies.

[0132] The challenge in developing cell-based PD therapies has been to identify appropriate cell sources for neuronal replacement. The search for an appropriate cell source has been decades long, and many potential sources for DA neuron replacement have been proposed. Kriks, Protocols for generating ES cell-derived dopamine neurons in Development and engineering of dopamine neurons (ed. Pasterkamp, ​​RJ, Smidt and Burbach) Landes Biosciences (2008); Fitzpatrick et al., Antioxid. Redox. Signal. (2009) 11: 2189-2208. Several of these sources have progressed to early clinical trials, including catecholaminergic cells from the adrenal medulla, carotid body grafts, or retinal pigment epithelial cells of the capsule. Madrazo et al., N. Engl. J. Med. (1987) 316:831-34; Arjona et al., Neurosurgery (2003) 53:321-28; Spheramine trial Bakay et al., Front Biosci. (2004) 9:592-602. However, those trials largely failed to show clinical efficacy and resulted in poor long-term survival of transplanted cells and low DA release.

[0133] Another approach is to transplant fetal midbrain DA neurons, such as those performed in over 300 patients worldwide. Brundin et al., Prog. Brain Res. (2010) 184:265-94; Lindvall, & Kokaia, J. Clin. Invest (2010) 120:29-40. Therapies using human fetal tissue in these patients have demonstrated evidence of DA neuron survival and in vivo DA release up to 10 or 20 years after transplantation in some patients. However, in many patients, fetal tissue transplants cannot replace the function of dopamine neurons. In addition, fetal tissue transplants are plagued by challenges, including low quantity and quality of donor tissue, ethical and practical issues regarding tissue procurement, and poorly defined heterogeneity of transplanted cells, which are some of the factors that contribute to variable clinical outcomes. Mendez et al. Nature Med. (2008); Kordower et al. N. Engl. J. Med. (1995) 332: 1118-24; and Piccini et al. Nature Neuroscience (1999) 2: 1137-40. Hypotheses for the limited efficacy observed in human fetal transplantation trials include that fetal transplantation may not provide sufficient numbers of cells at the correct stage of development and that fetal tissue is difficult to define by cell type and by variables regarding the stage and quality of each tissue sample. Bjorklund et al. Lancet Neurol. (2003) 2: 437-45. Another contributing factor may be the host's inflammatory response to the transplant. Ibid.

[0134] Cells derived from stem cells, such as pluripotent stem cells (PSC), are considered to be the source of cells used in regenerative medicine. Pluripotent stem cells are capable of self-renewal and produce all cells of body tissues. PSC includes two major categories of cells: embryonic stem (ES) cells and induced pluripotent stem cells (iPSC). ES cells are derived from the inner cell mass of preimplantation embryos and can be maintained indefinitely and expanded in vitro with their pluripotent state. Romito and Cobellis, Stem Cells Int. (2016) 2016: 9451492. iPSC can be obtained by reprogramming adult somatic cells ("dedifferentiation") to become more like ES cells, including the ability to expand and differentiate into all three germ layers indefinitely. Ibid.

[0135] Pluripotent stem cells, such as ES cells, have been tested as a source for generating transplantable cells. Early studies in the 1990s using mouse ES cells demonstrated the feasibility of deriving specific lineages from pluripotent cells (including neurons) in vitro. Okabe et al., Mech. Dev. (1996) 59:89-102; Bain et al., Dev. Biol. (1995) 168 v342-357. Based on developmental insights from early explant studies, directed differentiation strategies were used to generate midbrain DA neurons. Lee et al., Nat. Biotechnol. (2000) 18 v675-679; Ye et al., Cell (1998) 93:755-66. However, these efforts did not produce cell populations containing a high percentage of midbrain DA neurons or cells that were able to restore neuronal function in vivo. In addition, the resulting populations also contained a mixture of cell types other than midbrain DA neurons.

[0136] Existing strategies for using human PSCs (hPSCs) for cell therapy have not been completely satisfactory. DA neurons derived from human PSCs generally exhibit poor in vivo performance and cannot compensate for endogenous losses of neuronal function. Tabar et al. Nature Med. (2008) 14:379-81; Lindvall and Kokaia, J. Clin. Invest (2010) 120:29-40.

[0137] Recently, preclinical studies that first differentiated human ES cells into midbrain basal intermediates and then further differentiated into DA neurons demonstrated in vivo survival and resulted in restoration of motor deficits in animal models. Krik et al., Nature (2011) 480:547–51; Kirkeby et al., Cell Rep. (2012) 1:703-14. Despite these advances, the use of embryonic stem cells remains plagued by ethical issues and the possibility that such cells may form tumors in patients. Finally, in the case of allogeneic stem cell transplantation, ES cell-derived grafts may provoke an immune response in patients.

[0138] Using induced pluripotent stem cells (iPSCs), rather than cells from ES sources, has the advantage of avoiding ethical issues. In addition, obtaining iPSCs from patients to be treated (i.e., patients receiving autologous cell transplantation) avoids the risk of immune rejection inherent in embryonic stem cells. As previous studies have revealed, poor standardization of transplanted cell materials leads to high variability, it is necessary to produce a large number of standardized cells, such as new methods for autologous stem cell transplantation. Lindvall and Kokaia, J. Clin. Invest (2010) 120: 29-40.

[0139] A study is currently underway in which human iPSCs are differentiated into DA neuron precursors and transplanted into the human striatum. However, the ability of these cells to survive, engraft, and innervate other cells in vivo has not been reported. Takahashi, Brain Res. (2017) 230:213–26 (2017); Cyranoski, D., Nature News (2018).

[0140] Therefore, existing strategies have not been shown to successfully produce differentiated cell populations for implantation procedures to restore neuronal function in vivo. Provided herein are methods for differentiating PSCs into directed dopaminergic neuron progenitor cells (DDPCs) and / or DA neuron cells. Specifically, the provided methods are based on the following findings: In the presence of SB, LDN, SHH, PUR, and CHIR, PSC culture as non-adherent cells is initiated to generate spheres, followed by further incubation of the cells of the spheres on substrate-coated plates to produce differentiated cells with superior properties. Other aspects of the provided methods include harvesting cells when the cells become directed dopaminergic (dopamine neuron) precursor cells, which are cells that can differentiate into dopaminergic neurons but cannot differentiate into non-dopaminergic neurons. In some embodiments of the provided methods, such cells are cells that are differentiated according to the provided methods and harvested at or about day 18.

[0141] In some cases, the methods described herein include culturing PSCs into spheres in non-adherent culture (i.e., suspension culture) for about 7 days, then dissociating the spheres and continuing to culture (i.e., as adherent culture) until harvest if the dissociated cells are on a plate, such as a substrate-coated plate. The provided methods of differentiating cells in non-adherent culture have advantages over alternative methods, such as methods in which the entire differentiation process is performed by cell adherent culture.

[0142] In some cases, compared with the differentiation method not involving cell non-adherent culture, the non-adherent culture of the provided method is advantageous, because during the non-adherent culture, the variability of one or more substrates and / or one or more reagents is reduced or eliminated on the influence of cell differentiation. Specifically, adherent culture may involve coating of plate with one or more substrates or reagents, and such coating may be uneven, thereby causing one or more substrates and / or reagents to provide the variability of adherent cells, especially in the method that the whole differentiation process is carried out by cell adherent culture. Substrate and / or reagent provide the variability of the cell produced by the differentiation method that can increase harvest to such variability of adherent cells. In contrast, substrate and / or reagent may be unnecessary in non-adherent culture, or be evenly dispersed in the whole non-adherent culture. In the latter case, the cultured cells (such as spheroids) are surrounded by culture medium on all sides, so that the cells are equally exposed to any substrate and / or reagent in the culture medium. Therefore, in the aspect of the provided embodiment, the cell produced by the differentiation method including non-adherent culture can show reduced variability.

[0143] Compared with the method that does not include non-adherent culture, non-adherent culture method described herein is also advantageous, because non-adherent culture allows the interaction between cells to increase. Specifically, the cells in the culture of adherent (e.g., monolayer) only contact other cells on its side surface. In contrast, the cells in the culture of non-adherent (e.g., suspension) can contact other cells on the entire surface. By more faithfully reproducing the physiological environment, the intercellular contact of such increase in non-adherent culture may be advantageous. For example, the increase of intercellular contact allowed by the non-adherent culture method described herein can raise the intercellular protein network, as observed in vivo.

[0144] In some respects, compared with alternative methods, such as the method that the whole differentiation process is carried out by cell adherence culture, the method related to the non-adherent culture method provided as described herein provides the simplicity and efficiency of cultivating a large number of cells.For example, in adherence (such as monolayer) culture, the number of cells cultivated increases along with the surface area of ​​the culture surface on two-dimensional scale (i.e. the length and width of the culture surface).By contrast, in non-adherence (such as suspension) culture, the number of cells cultivated increases along with the volume of culture vessel or the volume of culture medium contained therein on three-dimensional scale.Therefore, in some cases, non-adherent culture is more economical and effective, because compared with adherence culture, using less resources (such as culture vessel and reagent), more cells can be generated, or both are available.Like this, the non-adherent culture method provided can make scale-up possible in manufacturing and production process.

[0145] In addition, a particular benefit is associated with a cell differentiation method comprising only about 7 days of non-adherent culture. The non-adherent culture of about 7 days described herein produces spheroids. If non-adherent culture is allowed to proceed for more than 7 days, the incremental size of the spheroid may limit mass transfer and generate a gradient of reagents (e.g., morphogens) across the diameter of the spheroid. A large number of reagents (e.g., morphogens) gradients across the diameter of the spheroid are undesirable because different cells of the spheroid will be exposed to different concentrations of reagents (e.g., morphogens), resulting in variable cell responses and differentiation. Therefore, the method described herein comprising only about 7 days or up to 7 days of non-adherent culture is advantageous because the concentration changes of reagents (such as morphogens) to which the spheroid cells are exposed are minimized.

[0146] In some aspects, limiting the non-adherent culture component of the methods described herein to about 7 days also helps ensure consistent differentiation of cultured cells. This is because long-term (e.g., greater than 7 days) differentiation of PSCs in non-adherent (e.g., suspension) culture allows PSCs to establish a microenvironment that allows self-renewal in a pluripotent state. Therefore, the methods described herein ensure consistent and efficient differentiation of cultured cells by reducing or eliminating the chance of PSCs persisting in culture.

[0147] In aspects of the methods provided, cells produced by the methods described herein are advantageous in that they exhibit expression of A9-specific markers, demonstrating their fate as A9 dopamine neurons and suitability for transplantation, engraftment, and innervation of other cells in vivo.

[0148] Specifically, the cells produced by the methods described herein, including cells harvested at the 18th day of differentiation, express EN1 and CORIN, genes expressed by A9 progenitor cells during fetal development. The cells harvested at the 25th day of exemplary differentiation methods described herein, compared with the cells harvested at the 18th day, also show the downward regulation of mitosis-related genes, indicating that the stereotype of specific cell type fate increases with the increase of culture time. In addition, compared with the 18th day, the cells produced by the methods described herein show the expression of neurite growth-related genes up-regulated at the 25th day. Neurite growth-related genes participate in the development of new neuron projection and synapse generation. Therefore, this discovery shows that, with the increase of culture time, the cells differentiated by the exemplary methods described herein become more stereotyped as neuron fate. In a word, these characteristics show that the cells harvested at the 18th day are stereotyped dopaminergic neuron progenitor cells. Thus, cells generated by the methods described herein, including differentiated cells harvested at day 18, may be advantageous compared to cells differentiated by other methods and / or harvested at different days (i.e., later days) of the differentiation protocol because they are committed to a DA neuron fate, but are less differentiated than at later days.

[0149] The findings herein also demonstrate that cells harvested at day 18 appear to express many markers of DA neuron precursors, but their genetic expression suggests that they may be less committed to a DA neuron fate than cells harvested at day 25. In some embodiments, cells harvested at day 18 may therefore represent an ideal time point for harvesting cells for subsequent in vivo transplantation. Specifically, one or more of the ability to engraft, innervate, or improve efficacy may be improved compared to cells differentiated by other methods and / or harvested at a different day (i.e., later day) of the differentiation protocol.

[0150] In some embodiments, cells harvested at or about day 18 of the methods described herein, or therapeutic compositions containing the cells described herein, can exhibit an increased ability to engraft and / or innervate other cells compared to cells harvested at a later time (e.g., day 25) in the differentiation method. In some embodiments, cells harvested at day 18 can also exhibit improved efficacy in vivo due to their differentiated state and neuronal commitment. For example, cells harvested at day 18 can exhibit improved engraftment and / or innervation, improved efficacy, or both compared to cells harvested at day 25.

[0151] In some embodiments, the cells harvested by the provided differentiation method show a therapeutic effect for treating neurodegenerative diseases. In some embodiments, the ability of differentiated cells to treat neurodegenerative diseases can be determined in an animal model of neurodegenerative diseases. In some embodiments, the neurodegenerative disease is Parkinson's disease. In some embodiments, an animal model of Parkinson's disease is used to screen differentiated cells harvested by the provided method. Any known and available animal model of Parkinson's disease can be used for screening. In some embodiments, the animal model is an injury model, in which the animal receives a unilateral stereotaxic injection of 6-hydroxydopamine (6-OHDA) to the substantia nigra. In some embodiments, the animal model is an injury model, in which the animal receives a unilateral stereotaxic injection of 6-OHDA to the medial forebrain bundle. In some embodiments, a therapeutic composition containing differentiated cells (e.g., harvested cells, such as cells on the 18th day) produced by the provided method is implanted in the substantia nigra of an animal model. In some embodiments, behavioral assays are performed to screen the therapeutic effects of implantation on animal models. In some embodiments, behavioral assays include monitoring amphetamine-induced circling behavior. In some embodiments, differentiated cells demonstrate a therapeutic effect for treating a neurodegenerative disease if such cells are determined to reduce, decrease, or reverse brain damage in a Parkinson's disease model in such model.

[0152] In addition, unlike previously reported methods, the differentiated cells produced by the methods described herein exhibit physiological consistency. Importantly, this physiological consistency is maintained in cells differentiated from different subjects. Therefore, this method reduces the variability within and between subjects and allows better predictability of cell behavior in vivo. These benefits are relevant to successful treatment strategies, particularly in the case of autologous stem cell transplantation, where cells are generated separately for each patient. Such reproducibility benefits between different subjects can also make it possible to scale up in manufacturing and production processes.

[0153] All publications, including patent documents, scientific papers, and databases, mentioned in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication was individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in patents, applications, published applications, and other publications incorporated herein by reference, the definition set forth herein takes precedence over the definition incorporated herein by reference.

[0154] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0155] II. Methods for cell differentiation

[0156] Provided herein is a method for differentiating neural cells, the method comprising: (1) performing a first incubation comprising culturing pluripotent stem cells in a non-adherent culture vessel under conditions that produce cell spheres, wherein at the beginning of the first incubation (day 0), the cells are exposed to (i) a TGF-β / activin Nodal signaling inhibitor; (ii) at least one sonic hedgehog (SHH) signaling activator; (iii) a bone morphogenetic protein (BMP) signaling inhibitor; and (iv) a glycogen synthase kinase 3β (GSK3β) signaling inhibitor; and (b) performing a second incubation comprising culturing the cells of the sphere in a substrate-coated culture vessel under conditions that cause the cells to undergo neural differentiation.

[0157] Provided are methods of differentiating neural cells, such as by subjecting iPSCs to a cell culture method that induces their differentiation into midbrain floor plate progenitor cells, committed dopamine (DA) neuron progenitor cells, and / or dopamine (DA) neurons.

[0158] As described herein, iPSCs are generated from fibroblasts of human patients with Parkinson's disease. In the first incubation, iPSCs are then differentiated into midbrain floor plate precursors and grown as spheres in non-adherent culture starting from day 0 by exposure to small molecules, such as LDN, SB, PUR, SHH, CHIR, and combinations thereof. The resulting spheres are then transferred to adherent culture as part of the second incubation, optionally after sphere dissociation, and then exposed to additional small molecules (e.g., LDN, CHIR, BDNF, GDNF, ascorbic acid, dbcAMP, TGFβ3, DAPT, and combinations thereof) to induce further differentiation into implantable committed DA neuron progenitors or DA neurons.

[0159] A. Sample and cell preparation

[0160] In embodiments of the provided methods, pluripotent stem cells are differentiated into midbrain floor plate progenitor cells, committed dopamine (DA) neuron progenitor cells and / or dopamine (DA) neurons. Various sources of pluripotent stem cells can be used in the methods, including embryonic stem (ES) cells and induced pluripotent stem cells (iPSCs).

[0161] In some aspects, pluripotency refers to cells that can produce offspring, which can be differentiated into cell types that jointly show characteristics related to the cell lineage from three germ layers (endoderm, mesoderm and ectoderm) under appropriate conditions. Pluripotent stem cells can contribute to the tissue of prenatal, postnatal or adult organisms. Standard tests accepted in the art, such as the ability to form teratomas in 8-12 week-old SCID mice, can be used to establish the pluripotency of cell colonies. However, the identification of various pluripotent stem cell characteristics can also be used to identify pluripotent cells. In some aspects, pluripotent stem cells can be distinguished from other cells by specific characteristics, including by expressing or not expressing a combination of certain molecular markers. More specifically, human pluripotent stem cells can express at least some of the markers from the following non-limiting list, optionally all: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49 / 6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Lin28, Rex1 and Nanog. In some aspects, the pluripotent stem cell characteristic is a cell morphology associated with pluripotent stem cells.

[0162] In some embodiments, pluripotent stem cells are induced pluripotent stem cells (iPSCs) artificially obtained from non-pluripotent cells. In some aspects, non-pluripotent cells are cells with lower self-renewal and differentiation potential than pluripotent stem cells. iPSCs can be generated by a process called reprogramming, in which non-pluripotent cells are effectively "dedifferentiated" into an embryonic stem cell-like state by engineering them to express genes such as OCT4, SOX2, and KLF4. Takahashi and Yamanaka Cell (2006) 126: 663-76.

[0163] The method of generating iPSC is known. For example, in 2006 (Takahashi and Yamanaka) reported mouse iPSC, and in late 2007 (Takahashi et al. and Yu et al.) reported human iPSC. Mouse iPSC shows the important characteristics of pluripotent stem cells, including the expression of stem cell markers, the formation of tumors containing cells from all three germ layers, and the ability to facilitate many different tissues when injected into mouse embryos at the very early stage of development. Human iPSC also expresses stem cell markers and can produce cell characteristics of all three germ layers.

[0164] In some embodiments, the PSC (e.g., iPSC) is autologous to the subject to be treated, i.e., the PSC is derived from the same subject to which the differentiated cells are administered. In some embodiments, non-pluripotent cells (e.g., fibroblasts) derived from a patient with Parkinson's disease (PD) are reprogrammed into iPSCs prior to differentiation into neural and / or neuronal cells. In some embodiments, fibroblasts can be reprogrammed into iPSCs by transforming fibroblasts with genes (OCT4, SOX2, NANOG, LIN28, and KLF4) cloned into a plasmid (e.g., see, Yu et al., Science DOI: 10.1126 / science.1172482). In some embodiments, non-pluripotent fibroblasts derived from a patient with PD are reprogrammed into iPSCs prior to differentiation into committed DA neuron progenitors and / or DA neurons, such as by reprogramming the cells using a non-integrating Sendai virus (e.g., using CTS TM CytoTune TM-iPS2.1 Sendai Reprogramming Kit). In some embodiments, the resulting differentiated cells are then administered to the patient from whom they were obtained in an autologous stem cell transplant. In some embodiments, the PSCs (e.g., iPSCs) are allogeneic to the subject to be treated, i.e., the PSCs are derived from an individual different from the subject to which the differentiated cells are to be administered. In some embodiments, non-pluripotent cells (e.g., fibroblasts) derived from another individual (e.g., an individual who does not suffer from a neurodegenerative disorder such as Parkinson's disease) are reprogrammed into iPSCs prior to differentiation into committed DA neuron progenitors and / or DA neurons. In some embodiments, the cells are reprogrammed using a non-integrating Sendai virus (e.g., using a CTS TM CytoTune TM -iPS2.1 Sendai Reprogramming Kit), at least partially achieving reprogramming. In some embodiments, the resulting differentiated cells are then administered to an individual who is not the same individual from whom the differentiated cells were obtained (e.g., allogeneic cell therapy or allogeneic cell transplantation).

[0165] In any of the embodiments provided, the PSCs described herein (e.g., allogeneic cells) can be genetically engineered to be low immunogenic. Methods for reducing immunogenicity are known and include eliminating polymorphic HLA-A / -B / -C and HLA class II molecule expression and introducing immunomodulatory factors PD-L1, HLA-G, and CD47 into the AAVS1 safe harbor locus in differentiated cells. Han et al., PNAS (2019) 116(21): 10441-46. Therefore, in some embodiments, the PSCs described herein are engineered to delete highly polymorphic HLA-A / -B / -C genes and introduce immunomodulatory factors, such as PD-L1, HLA-G, and / or CD47 into the AAVS1 safe harbor locus.

[0166] In some embodiments, PSCs (e.g., iPSCs) are cultured in the absence of feeder cells until they reach 80-90% confluence, at which point they are harvested and further cultured for differentiation (day 0). In one aspect of the methods described herein, once the iPSCs reach 80-90% confluence, they are washed in phosphate buffered saline (PBS) and enzymatically dissociated, such as with Accutase TM Enzymatic dissociation is performed until the cells are easily dislodged from the surface of the culture vessel. The dissociated iPSCs are then resuspended in culture medium for downstream differentiation into committed DA neuron progenitors and / or DA neurons.

[0167] In some embodiments, PSCs are resuspended in a basal induction medium. In some embodiments, the basal induction medium is formulated to contain a 1:1 ratio of NeurobasalTM culture medium and DMEM / F12 medium supplemented with N-2 and B27 supplements, non-essential amino acids (NEAA), GlutaMAX TM , L-glutamine, β-mercaptoethanol and insulin. In some embodiments, the basal induction medium is further supplemented with serum replacement, Rho-associated protein kinase (ROCK) inhibitors and various small molecules for differentiation. In some embodiments, PSCs are resuspended in the same medium in which they will be cultured for at least a portion of the first incubation.

[0168] B. Non-adherent culture

[0169] The provided method includes culturing PSCs (e.g., iPSCs) by incubating with certain molecules (e.g., small molecules) to induce their differentiation into midbrain floor plate progenitors, directed dopamine (DA) neuron progenitors and / or dopamine (DA) neurons. Specifically, the provided embodiments include incubating PSCs for the first time under non-adherent conditions in the presence of certain molecules (e.g., small molecules) to produce spheres, which in some aspects can improve the consistency of producing physiologically relevant cells for implantation. In some embodiments, the method includes performing a first incubation, including culturing pluripotent stem cells in a non-adherent culture vessel under conditions that produce cell spheres, wherein at the beginning of the first incubation (day 0), the cells are exposed to (i) a TGF-β / activin Nodal signaling inhibitor; (ii) at least one sonic hedgehog (SHH) signaling activator; (iii) a bone morphogenetic protein (BMP) signaling inhibitor; and (iv) a glycogen synthase kinase 3β (GSK3β) signaling inhibitor;

[0170] In some embodiments, non-adherent culture vessels are culture vessels with low or ultra-low attachment surfaces, such as to inhibit or reduce cell attachment. In some embodiments, culturing cells in non-adherent culture vessels does not prevent all cells in the culture from attaching to the surface of the culture vessel.

[0171] In some embodiments, non-adherent culture vessel is a culture vessel with an ultra-low attachment surface. In some respects, the ultra-low attachment surface can suppress cell attachment within a period of time. In some embodiments, the ultra-low attachment surface can suppress cell attachment within the necessary time period for obtaining the fusion growth of the same cell type on the adherent surface. In some embodiments, the ultra-low attachment surface is coated or processed with a material that prevents cell attachment, such as a hydrogel layer (for example, a neutrally charged hydrogel layer and / or a hydrophilic hydrogel layer). In some embodiments, the non-adherent culture vessel is coated or processed with a surfactant before incubation for the first time. In some embodiments, the surfactant is pluronic acid.

[0172] In some embodiments, the non-adherent culture vessel is a plate, a culture dish, a flask or a bioreactor. In some embodiments, the non-adherent culture vessel is a plate, such as a multi-well plate. In some embodiments, the non-adherent culture vessel is a 6-well plate or a 24-well plate. In some embodiments, the hole of the multi-well plate also includes micropores. In some any embodiments provided, the non-adherent culture vessel, such as a multi-well plate, has a circular or concave hole and / or micropores. In any embodiments provided, the non-adherent culture vessel, such as a multi-well plate, has no corners or seams.

[0173] In some embodiments, non-adherent culture vessels allow three-dimensional formation of cell aggregates. In some embodiments, iPSC is cultured in non-adherent culture vessels, such as multi-well plates, to produce cell aggregates (e.g., spheres). In some embodiments, iPSC is cultured in non-adherent culture vessels, such as multi-well plates, to produce cell aggregates (e.g., spheres) on about the 7th day of the method. In some embodiments, cell aggregates (e.g., spheres) express at least one of PAX6 and OTX2 on or before about the 7th day of the method.

[0174] In some embodiments, the first incubation comprises culturing the pluripotent stem cells in a non-adherent culture vessel under conditions that produce spheroids.

[0175] In some embodiments, the number of PSCs plated on day 0 of the method is about 0.1×10 6 cells / cm 2 About 2×10 6 cells / cm 2 , about 0.1×10 6 cells / cm 2 About 1×10 6 cells / cm 2 , about 0.1×10 6 cells / cm 2 About 0.8×10 6 cells / cm 2 , about 0.1×10 6 cells / cm 2 About 0.6×10 6 cells / cm 2 , about 0.1×10 6 cells / cm 2 About 0.4×10 6 cells / cm 2 , about 0.1×10 6 cells / cm 2 About 0.2×10 6 cells / cm2 , about 0.2×10 6 cells / cm 2 About 2×10 6 cells / cm 2 , about 0.2×10 6 cells / cm 2 About 1×10 6 cells / cm 2 , about 0.2×10 6 cells / cm 2 About 0.8×10 6 cells / cm 2 , about 0.2×10 6 cells / cm 2 About 0.6×10 6 cells / cm 2 , about 0.2×10 6 cells / cm 2 About 0.4×10 6 cells / cm 2 , about 0.4×10 6 cells / cm 2 About 2×10 6 cells / cm 2 , about 0.4×10 6 cells / cm 2 About 1×10 6 cells / cm 2 , about 0.4×10 6 cells / cm 2 About 0.8×10 6 cells / cm 2 , about 0.4×10 6 cells / cm 2 About 0.6×10 6 cells / cm 2 , about 0.6×10 6 cells / cm 2 About 2×10 6 cells / cm 2 , about 0.6×10 6 cells / cm 2 About 1×10 6 cells / cm 2 , about 0.6×10 6 cells / cm 2 About 0.8×10 6 cells / cm 2 , about 0.8×10 6 cells / cm 2About 2×10 6 cells / cm 2 , about 0.8×10 6 cells / cm 2 About 1×10 6 cells / cm 2 or about 1.0×10 6 cells / cm 2 About 2×10 6 cells / cm 2 In some embodiments, the number of cells plated on the substrate-coated culture vessel is about 0.4×10 6 cells / cm 2 About 0.8×10 6 cells / cm 2 .

[0176] In some embodiments, the number of PSCs plated on day 0 of the method is about 1×10 5 pluripotent stem cells / well is about 20×10 6 pluripotent stem cells / well, about 1×10 5 15×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 1×10 5 10×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 1×10 5 5×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 1×10 5 1×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 1×10 5 5×10 pluripotent stem cells / well 5 pluripotent stem cells / well, about 5×10 5 pluripotent stem cells / well is about 20×10 6 pluripotent stem cells / well, about 5×10 5 15×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 5×10 5 10×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 5×10 5 5×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 5×10 5 1×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 1×10 6 pluripotent stem cells / well is about 20×106 pluripotent stem cells / well, about 1×10 6 15×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 1×10 6 10×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 1×10 6 5×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 5×10 6 pluripotent stem cells / well is about 20×10 6 pluripotent stem cells / well, about 5×10 6 15×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 5×10 6 10×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 10×10 6 pluripotent stem cells / well is about 20×10 6 pluripotent stem cells / well, about 10×10 6 15×10 pluripotent stem cells / well 6 pluripotent stem cells / well or about 15×10 6 pluripotent stem cells / well is about 20×10 6 pluripotent stem cells / well.

[0177] In some embodiments, the number of PSCs plated in a 6-well plate on day 0 of the method is about 1×10 6 pluripotent stem cells / well is about 20×10 6 pluripotent stem cells / well, about 1×10 6 15×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 1×10 6 10×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 1×10 6 5×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 5×10 6 pluripotent stem cells / well is about 20×10 6 pluripotent stem cells / well, about 5×10 6 15×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 5×10 6 10×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 10×10 6 pluripotent stem cells / well is about 20×106 pluripotent stem cells / well, about 10×10 6 15×10 pluripotent stem cells / well 6 pluripotent stem cells / well or about 15×10 6 pluripotent stem cells / well is about 20×10 6 pluripotent stem cells / well.

[0178] In some embodiments, the number of PSCs plated in a 24-well plate on day 0 of the method is about 1×10 5 5×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 1×10 5 1×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 1×10 5 5×10 pluripotent stem cells / well 5 pluripotent stem cells / well, about 5×10 5 5×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 5×10 5 1×10 pluripotent stem cells / well 6 pluripotent stem cells / well or about 1×10 6 5×10 pluripotent stem cells / well 6 pluripotent stem cells / well.

[0179] In some days, the number of PSCs plated on day 0 of the method is a number of cells sufficient to produce a cell sphere containing about 1,000 to about 5,000 cells, or about 2,000 to about 3,000 cells. In some days, the number of PSCs plated on day 0 of the method is a number of cells sufficient to produce a cell sphere containing about 1,000 to about 5,000 cells. In some days, the number of PSCs plated on day 0 of the method is a number of cells sufficient to produce a cell sphere containing about 2,000 to about 3,000 cells. In some days, the number of PSCs plated on day 0 of the method is a number of cells sufficient to produce a cell sphere containing about 2,000 cells. In some days, the number of PSCs plated on day 0 of the method is a number of cells sufficient to produce a cell sphere containing about 3,000 cells. In some embodiments, spheres containing the desired number are produced by the method on or before about day 7.

[0180] In some embodiments of the methods provided herein, the first incubation includes culturing pluripotent stem cells in a non-adherent culture vessel under conditions that produce cell spheroids. In some embodiments, the first incubation is from about day 0 to about day 6. In some embodiments, the first incubation includes culturing pluripotent stem cells in a culture medium ("medium"). In some embodiments, the first incubation includes culturing pluripotent stem cells in a culture medium from about day 0 to about day 6. In some embodiments, the first incubation includes culturing pluripotent stem cells in a culture medium to induce PSC differentiation into midbrain floor plate progenitor cells.

[0181] In some embodiments, the culture medium is also supplemented with a serum replacement that contains minimal components of non-human origin (e.g., KnockOut TM Serum replacement). In some embodiments, during at least a portion of the first incubation, a 5% (v / v) serum replacement is provided in the culture medium. In some embodiments, on days 0 and 1, a 5% (v / v) serum replacement is provided in the culture medium. In some embodiments, during at least a portion of the first incubation, a 2% (v / v) serum replacement is provided in the culture medium. In some embodiments, from day 2 to day 6, a 2% (v / v) serum replacement is provided in the culture medium. In some embodiments, on days 0 and 1, a 5% (v / v) serum replacement is provided in the culture medium and from day 2 to day 6, a 2% (v / v) serum replacement is provided.

[0182] In some embodiments, the culture medium is also supplemented with a small molecule, such as any of the small molecules described above. In some embodiments, the small molecule is selected from the group consisting of: a Rho-associated protein kinase (ROCK) inhibitor, a TGF-β / activin Nodal signaling inhibitor, at least one sonic hedgehog (SHH) signaling activator, a bone morphogenetic protein (BMP) signaling inhibitor, a glycogen synthase kinase 3β (GSK3β) signaling inhibitor, and combinations thereof.

[0183] In some embodiments, on one or more days when the cells are passaged, the culture medium is supplemented with a Rho-associated protein kinase (ROCK) inhibitor. In some embodiments, on each day of the cell passage, the culture medium is supplemented with a ROCK inhibitor. In some embodiments, the culture medium is supplemented with a ROCK inhibitor at day 0.

[0184] In some embodiments, the cells are exposed to a ROCK inhibitor at a concentration of about 1 μM to about 20 μM, about 5 μM to about 15 μM, or about 8 μM to about 12 μM. In some embodiments, the cells are exposed to a ROCK inhibitor at a concentration of about 1 μM to about 20 μM. In some embodiments, the cells are exposed to a ROCK inhibitor at a concentration of about 5 μM to about 15 μM. In some embodiments, the cells are exposed to a ROCK inhibitor at a concentration of about 8 μM to about 12 μM. In some embodiments, the cells are exposed to a ROCK inhibitor at a concentration of about 10 μM.

[0185] In some embodiments, the ROCK inhibitor is selected from the group consisting of: Fasudil, Ripasudil, Netarsudil, RKI-1447, Y-27632, GSK429286A, Y-30141, and combinations thereof. In some embodiments, the ROCK inhibitor is a small molecule. In some embodiments, the ROCK inhibitor selectively inhibits p160ROCK. In some embodiments, the ROCK inhibitor is Y-27632, having the formula:

[0186]

[0187] In some embodiments, the cells are exposed to Y-27632 at a concentration of about 10 μM. In some embodiments, the cells are exposed to Y-27632 at a concentration of about 10 μM on day 0.

[0188] In some embodiments, the culture medium is supplemented with a TGF-β / activin Nodal signaling inhibitor. In some embodiments, the culture medium is supplemented with a TGF-β / activin Nodal signaling inhibitor until about day 7 (e.g., day 6 or day 7). In some embodiments, from about day 0 to day 6, including every day, the culture medium is supplemented with a TGF-β / activin Nodal signaling inhibitor.

[0189] In some embodiments, the cells are exposed to a concentration of about 1 μM to about 20 μM, about 5 μM to about 15 μM, or about 8 μM to about 12 μM TGF-β / activin Nodal signaling inhibitor. In some embodiments, the cells are exposed to a concentration of about 1 μM to about 20 μM TGF-β / activin Nodal signaling inhibitor. In some embodiments, the cells are exposed to a concentration of about 5 μM to about 15 μM TGF-β / activin Nodal signaling inhibitor. In some embodiments, the cells are exposed to a concentration of about 8 μM to about 12 μM TGF-β / activin Nodal signaling inhibitor. In some embodiments, the cells are exposed to a concentration of about 10 μM TGF-β / activin Nodal signaling inhibitor.

[0190] In some embodiments, the TGF-β / activin Nodal signaling inhibitor is a small molecule. In some embodiments, the TGF-β / activin Nodal signaling inhibitor is capable of reducing or blocking transforming growth factor beta (TGFβ) / activin Nodal signaling. In some embodiments, the TGF-β / activin Nodal signaling inhibitor inhibits ALK4, ALK5, ALK7, or a combination thereof. In some embodiments, the TGF-β / activin Nodal signaling inhibitor inhibits ALK4, ALK5, and ALK7. In some embodiments, the TGF-β / activin Nodal signaling inhibitor does not inhibit ALK2, ALK3, ALK6, or a combination thereof. In some embodiments, the inhibitor does not inhibit ALK2, ALK3, or ALK6. In some embodiments, the TGF-β / activin Nodal signaling inhibitor is SB431542 (e.g., CAS 301836-41-9, with a molecular formula of C22H18N4O3 and a name of 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide), having the following formula:

[0191]

[0192] In some embodiments, the cells are exposed to SB431542 at a concentration of about 10 μM. In some embodiments, the cells are exposed to SB431542 at a concentration of about 10 μM until about day 7. In some embodiments, the cells are exposed to SB431542 at a concentration of about 10 μM from about day 0 to about day 6, including every day.

[0193] In some embodiments, the culture medium is supplemented with at least one sonic hedgehog (SHH) signaling activator. SHH refers to a protein that is one of at least three proteins in a family of mammalian signaling pathways, called hedgehogs, another being the desert hedgehog (DHH), and the third being the Indian hedgehog (IHH). Shh interacts with at least two transmembrane proteins by interacting with the transmembrane molecules Patched (PTC) and Smoothened (SMO). In some embodiments, the culture medium is supplemented with at least one SHH signaling activator until about day 7 (e.g., day 6 or day 7). In some embodiments, from about day 0 to day 6, including every day, the culture medium is supplemented with at least one SHH signaling activator.

[0194] In some embodiments, the at least one SHH signaling activator is a SHH protein. In some embodiments, the at least one SHH signaling activator is a recombinant SHH protein. In some embodiments, the at least one SHH signaling activator is a recombinant mouse SHH protein. In some embodiments, the at least one SHH signaling activator is a recombinant human SHH protein. In some embodiments, the at least one SHH signaling activator is a recombinant N-terminal fragment of a full-length murine sonic hedgehog protein capable of binding to an SHH receptor to activate SHH. In some embodiments, the at least one SHH signaling activator is a C25II SHH protein.

[0195] In some embodiments, the cell is exposed to a concentration of about 10 ng / mL to about 500 ng / mL, about 20 ng / mL to 400 μg / mL, about 30 ng / mL to about 300 ng / mL, about 40 ng / mL to about 200 ng / mL, or about 50 ng / mL to about 100 ng / mL (including each end value). In some embodiments, the cell is exposed to a concentration of about 50 ng / mL to about 100 ng / mL (including each end value). In some embodiments, the cell is exposed to a concentration of about 100 ng / mL of at least one SHH signaling activator. In some embodiments, the cell is exposed to about 100 ng / mL of SHH protein. In some embodiments, the cell is exposed to about 100 ng / mL of recombinant SHH protein. In some embodiments, the cell is exposed to about 100 ng / mL of recombinant mouse SHH protein. In some embodiments, the cells are exposed to about 100 ng / mL of C25II SHH protein.

[0196] In some embodiments, the cells are exposed to a recombinant SHH protein at a concentration of about 10 ng / mL. In some embodiments, the cells are exposed to a recombinant SHH protein at a concentration of about 10 ng / mL until about day 7 (e.g., day 6 or day 7). In some embodiments, the cells are exposed to a recombinant SHH protein at a concentration of about 10 ng / mL from about day 0 to about day 6, including every day.

[0197] In some embodiments, the cell is exposed to a concentration of about 1 μM to about 20 μM, about 5 μM to about 15 μM, or about 8 μM to about 12 μM of the at least one SHH signaling activator. In some embodiments, the cell is exposed to a concentration of about 1 μM to about 20 μM of the at least one SHH signaling activator. In some embodiments, the cell is exposed to a concentration of about 5 μM to about 15 μM of the at least one SHH signaling activator. In some embodiments, the cell is exposed to a concentration of about 8 μM to about 12 μM of the at least one SHH signaling activator. In some embodiments, the cell is exposed to a concentration of about 10 μM of the at least one SHH signaling activator.

[0198] In some embodiments, the at least one SHH signaling activator is an activator of the hedgehog receptor Smoothened. In some embodiments, the at least one SHH signaling activator is a small molecule. In some embodiments, the at least one SHH signaling activator is purmorphamine (eg, CAS 483367-10-8), having the following formula:

[0199]

[0200] In some embodiments, the cells are exposed to purmorphamine at a concentration of about 10 μM. In some embodiments, the cells are exposed to purmorphamine at a concentration of about 10 μM until day 7 (e.g., day 6 or day 7). In some embodiments, the cells are exposed to purmorphamine at a concentration of about 10 μM from about day 0 to about day 6, including every day.

[0201] In some embodiments, the at least one SHH signaling activator is SHH protein and purmorphamine. In some embodiments, cells are exposed to a certain concentration of SHH protein and purmorphamine until about the 7th day (e.g., the 6th day or the 7th day). In some embodiments, cells are exposed to SHH protein and purmorphamine from about day 0 to about day 6, including every day. In some embodiments, cells are exposed to a certain concentration of 100ng / mL SHH protein and 10μM purmorphamine until about day 7 (e.g., the 6th day or the 7th day). In some embodiments, cells are exposed to 100ng / mL SHH protein and 10μM purmorphamine from about day 0 to about day 6, including every day.

[0202] In some embodiments, the culture medium is supplemented with a BMP signaling inhibitor. In some embodiments, the culture medium is supplemented with a BMP signaling inhibitor until about day 7 (e.g., day 6 or day 7). In some embodiments, from about day 0 to day 6, including every day, the culture medium is supplemented with a BMP signaling inhibitor.

[0203] In some embodiments, cells are exposed to a concentration of about 0.01 μM to about 5 μM, about 0.05 μM to about 1 μM, or about 0.1 μM to about 0.5 μM (including each end value) of a BMP signaling inhibitor. In some embodiments, cells are exposed to a concentration of about 0.01 μM to about 5 μM of a BMP signaling inhibitor. In some embodiments, cells are exposed to a concentration of about 0.05 μM to about 1 μM of a BMP signaling inhibitor. In some embodiments, cells are exposed to a concentration of about 0.1 μM to about 0.5 μM of a BMP signaling inhibitor. In some embodiments, cells are exposed to a concentration of about 0.1 μM of a BMP signaling inhibitor.

[0204] In some embodiments, the BMP signaling inhibitor is a small molecule. In some embodiments, the BMP signaling inhibitor is selected from LDN193189 or K02288. In some embodiments, the BMP signaling inhibitor can inhibit "Small Mothers Against Decapentaplegic" SMAD signaling. In some embodiments, the BMP signaling inhibitor inhibits ALK1, ALK2, ALK3, ALK6 or a combination thereof. In some embodiments, the BMP signaling inhibitor inhibits ALK1, ALK2, ALK3 and ALK6. In some embodiments, the BMP signaling inhibitor inhibits BMP2, BMP4, BMP6, BMP7 and activin cytokine signals, and subsequently inhibits SMAD phosphorylation of Smad1, Smad5 and Smad8. In some embodiments, the BMP signaling inhibitor is LDN193189. In some embodiments, the BMP signaling inhibitor is LDN193189 (e.g., IUPAC name 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline, chemical formula C25H22N6), having the following formula:

[0205]

[0206] In some embodiments, the cells are exposed to LDN193189 at a concentration of about 0.1 μM. In some embodiments, the cells are exposed to LDN193189 at a concentration of about 0.1 μM until about day 7 (e.g., day 6 or day 7). In some embodiments, the cells are exposed to LDN193189 at a concentration of about 0.1 μM from about day 0 to about day 6, including every day.

[0207] In some embodiments, the culture medium is supplemented with a GSK3β signaling inhibitor. In some embodiments, the culture medium is supplemented with a GSK3β signaling inhibitor until about day 7 (e.g., day 6 or day 7). In some embodiments, from about day 0 to day 6, including every day, the culture medium is supplemented with a GSK3β signaling inhibitor.

[0208] In some embodiments, the cell is exposed to a GSK3β signaling inhibitor at a concentration of about 0.1 μM to about 10 μM, about 0.5 μM to about 8 μM, or about 1 μM to about 4 μM, or about 2 μM to about 3 μM (including each end value). In some embodiments, the cell is exposed to a GSK3β signaling inhibitor at a concentration of about 0.1 μM to about 10 μM. In some embodiments, the cell is exposed to a GSK3β signaling inhibitor at a concentration of about 0.5 μM to about 8 μM. In some embodiments, the cell is exposed to a BMP signaling inhibitor at a concentration of about 1 μM to about 4 μM. In some embodiments, the cell is exposed to a BMP signaling inhibitor at a concentration of about 2 μM to about 3 μM. In some embodiments, the cell is exposed to a GSK3β signaling inhibitor at a concentration of about 2 μM.

[0209] In some embodiments, the GSK3β signaling inhibitor is selected from the group consisting of: lithium ion, valproic acid, iodinated tuberculocidin, naproxen, famotidine, curcumin, olanzapine, CHIR99012, and combinations thereof. In some embodiments, the GSK3β signaling inhibitor is a small molecule. In some embodiments, the GSK3β signaling inhibitor inhibits glycogen synthase kinase 3β enzyme. In some embodiments, the GSK3β signaling inhibitor inhibits GSK3α. In some embodiments, the GSK3β signaling inhibitor regulates TGF-β and MAPK signaling. In some embodiments, the GSK3β signaling inhibitor is a wingless / integrated (Wnt) signaling agonist. In some embodiments, the GSK3β signaling inhibitor has an IC50 of 6.7nM for human GSK3β. In some embodiments, the GSK3β signaling inhibitor is CHIR99021 (e.g., "3-[3-(2-carboxyethyl)-4-methylpyrrole-2-methylene]-2-indolone" or the IUPAC name 6-(2-(4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino)ethylamino)nicotinonitrile), having the formula:

[0210]

[0211] In some embodiments, the cells are exposed to CHIR99021 at a concentration of about 2.0 μM. In some embodiments, the cells are exposed to CHIR99021 at a concentration of about 2.0 μM until about day 7 (e.g., day 6 or day 7). In some embodiments, the cells are exposed to CHIR99021 at a concentration of about 2.0 μM from about day 0 to about day 6, including every day.

[0212] In some embodiments, at least about 50% of the culture medium is replaced every day from about day 2 to about day 6. In some embodiments, about 50% of the culture medium is replaced every day, every other day, or every three days from about day 2 to about day 6. In some embodiments, about 50% of the culture medium is replaced every day from about day 2 to about day 6. In some embodiments, at least about 75% of the culture medium is replaced on day 1. In some embodiments, about 100% of the culture medium is replaced on day 1. In some embodiments, the replacement culture medium contains about twice as concentrated small molecule as compared to the concentration of small molecule in the culture medium on day 0.

[0213] In some embodiments, the first incubation comprises culturing pluripotent stem cells in a "basal induction medium". In some embodiments, the first incubation comprises culturing pluripotent stem cells in a basal induction medium from about day 0 to about day 6. In some embodiments, the first incubation comprises culturing pluripotent stem cells in a basal induction medium to induce differentiation of PSCs into midbrain floor plate progenitor cells.

[0214] In some embodiments, the basal induction medium is formulated to contain Neurobasal TM culture medium and DMEM / F12 medium supplemented with N-2 and B27 supplements, non-essential amino acids (NEAA), GlutaMAX TM , L-glutamine, β-mercaptoethanol and insulin. In some embodiments, the basal induction medium is further supplemented with any of the above-mentioned small molecules.

[0215] C. Transfer or dissociation of spheroids

[0216] In some embodiments, cell aggregates (eg, spheroids) generated after a first incubation of culturing pluripotent stem cells in a non-adherent culture vessel are transferred or dissociated prior to a second incubation of cells on a substrate (adherent culture).

[0217] In some embodiments, a first incubation is performed to produce cell aggregates (e.g., spheres) expressing at least one of PAX6 and OTX2. In some embodiments, the first incubation produces cell aggregates (e.g., spheres) expressing PAX6 and OTX2. In some embodiments, the first incubation produces cell aggregates (e.g., spheres) at about day 7 or before of the methods provided herein. In some embodiments, the first incubation produces cell aggregates (e.g., spheres) expressing at least one of PAX6 and OTX2 at about day 7 or before of the methods provided herein. In some embodiments, the first incubation produces cell aggregates (e.g., spheres) expressing at least one of PAX6 and OTX2 at about day 7 or before of the methods provided herein. In some embodiments, the first incubation produces cell aggregates (e.g., spheres) expressing PAX6 and OTX2 at about day 7 or before of the methods provided herein.

[0218] In some embodiments, before the cells are incubated for the second time on the substrate, the cell aggregates (e.g., spheres) produced by the first incubation are dissociated. In some embodiments, the cell aggregates (e.g., spheres) produced by the first incubation are dissociated to produce a cell suspension. In some embodiments, the cell suspension produced by dissociation is a single cell suspension. In some embodiments, dissociation is performed when the spheroid cells express at least one of PAX6 and OTX2. In some embodiments, dissociation is performed when the spheroid cells express PAX6 and OTX2. In some embodiments, dissociation is performed on about the 7th day. In some embodiments, cell aggregates (e.g., spheres) are dissociated by enzymatic dissociation. In some embodiments, the enzyme is selected from the group consisting of: Accutase TM , dispase, collagenase and combinations thereof. In some embodiments, the enzyme comprises Accutase TM In some embodiments, the enzyme is Accutase TM In some embodiments, the enzyme is dispase. In some embodiments, the enzyme is collagenase.

[0219] In some embodiments, the cell aggregate or the cell suspension produced therefrom is transferred to a substrate-coated culture vessel for a second incubation. In some embodiments, the cell aggregate or the cell suspension produced therefrom is transferred to a substrate-coated culture vessel after the cell aggregate (e.g., spheroid) is dissociated. In some embodiments, the transfer is performed immediately after the dissociation. In some embodiments, the transfer is performed on about the 7th day.

[0220] In some embodiments, the cell aggregates (e.g., spheres) are not dissociated before the second incubation. In some embodiments, the cell aggregates (e.g., spheres) are transferred as a whole to a substrate-coated culture vessel for a second incubation. In some embodiments, the transfer is performed when the sphere cells express at least one of PAX6 and OTX2. In some embodiments, the transfer is performed when the sphere cells express PAX6 and OTX2. In some embodiments, the transfer is performed on about the 7th day.

[0221] In some embodiments, transfer to an adherent culture vessel. In some embodiments, the culture vessel is a plate, a culture dish, a flask or a bioreactor. In some embodiments, the culture vessel is coated with a substrate. In some embodiments, the substrate is a basement membrane protein. In some embodiments, the substrate is selected from laminin, collagen, nestin, heparin sulfate proteoglycans and combinations thereof. In some embodiments, the substrate is laminin. In some embodiments, the substrate is recombinant. In some embodiments, the substrate is recombinant laminin. In some embodiments, the substrate-coated culture vessel is exposed to poly-L-ornithine before being used to culture cells. In some embodiments, the substrate-coated culture vessel is a 6-well plate or a 24-well plate. In some embodiments, the substrate-coated culture vessel is a 6-well plate. In some embodiments, the substrate-coated culture vessel is a 24-well plate.

[0222] D. Adherent culture

[0223] In some embodiments, the method comprises a second incubation of the spheroid cells transferred to the substrate-coated culture vessel. In some embodiments, culturing the spheroid cells under adherent conditions in the substrate-coated culture vessel induces them to differentiate into midbrain floor plate progenitor cells, committed dopamine (DA) neuron progenitor cells and / or dopamine (DA) neurons.

[0224] In some embodiments, the second incubation includes culturing cells of the sphere in a culture vessel coated with a substrate comprising laminin, collagen, nestin, heparin sulfate proteoglycans, and combinations thereof, wherein starting from day 7, the cells are exposed to (i) a BMP signaling inhibitor and (ii) a GSK3β signaling inhibitor; and starting from day 11, the cells are exposed to (i) brain-derived neurotrophic factor (BDNF); (ii) ascorbic acid; (iii) glial cell-derived neurotrophic factor (GDNF); (iv) dibutyryl cyclic AMP (dbcAMP); (v) transforming growth factor β-3 (TGFβ3); and (vi) a Notch signaling inhibitor. In some embodiments, the method further includes harvesting the differentiated cells.

[0225] In some embodiments, the substrate-coated culture vessel is a culture vessel with a surface to which cells can attach. In some embodiments, the substrate-coated culture vessel is a culture vessel with a surface to which a large number of cells attach. In some embodiments, the substrate is a basement membrane protein. In some embodiments, the substrate is laminin, collagen, entactin, heparin sulfate proteoglycans, or a combination thereof. In some embodiments, the substrate is laminin. In some embodiments, the substrate is collagen. In some embodiments, the substrate is entactin. In some embodiments, the substrate is heparin sulfate proteoglycans. In some embodiments, the substrate is a recombinant protein. In some embodiments, the substrate is a recombinant laminin. In some embodiments, the substrate-coated culture vessel is exposed to poly-L-ornithine. In some embodiments, the substrate-coated culture vessel is exposed to poly-L-ornithine before being used for cell culture.

[0226] In some embodiments, the substrate-coated culture vessel is a plate, a culture dish, a flask or a bioreactor. In some embodiments, the substrate-coated culture vessel is a plate, such as a multi-well plate. In some embodiments, the substrate-coated culture vessel is a plate. In some embodiments, the substrate-coated culture vessel is a 6-well plate or a 24-well plate. In some embodiments, the substrate-coated culture vessel is a culture dish. In some embodiments, the substrate-coated culture vessel is a flask. In some embodiments, the substrate-coated culture vessel is a bioreactor.

[0227] In some embodiments, the substrate-coated culture vessel allows monolayer cell culture. In some embodiments, the cell of the cell aggregate (such as spheroid) produced by the first incubation is cultivated in the form of monolayer culture on the substrate-coated plate. In some embodiments, the cell of the cell aggregate (such as spheroid) produced by the first incubation is cultivated to produce a positive monolayer cell culture for one or more of LMX1A, FOXA2, EN1, CORIN and their combination. In some embodiments, the cell of the cell aggregate (such as spheroid) produced by the first incubation is cultivated to produce a monolayer cell culture, wherein at least some cells are positive for EN1 and CORIN. In some embodiments, the cell of the cell aggregate (such as spheroid) produced by the first incubation is cultivated to produce a monolayer cell culture, wherein at least some cells are TH+. In some embodiments, at least some cells are TH+ before about the 25th day or at about the 25th day. In some embodiments, cells derived from cell aggregates (e.g., spheres) produced by the first incubation are cultured to produce a monolayer cell culture in which at least some of the cells are TH+FOXA2+. In some embodiments, at least some of the cells are TH+FOXA2+ before or at about day 25.

[0228] In the methods provided herein, the second incubation comprises culturing the cells of the sphere in a substrate-coated culture vessel under conditions that induce neural differentiation of the cells. In some embodiments, the cells of the sphere are plated on the substrate-coated culture vessel at about day 7.

[0229] In some embodiments, the number of cells plated on the substrate-coated culture vessel is about 0.1×10 6 cells / cm 2 About 2×10 6 cells / cm 2 , about 0.1×10 6 cells / cm 2 About 1×10 6 cells / cm 2 , about 0.1×10 6 cells / cm 2 About 0.8×10 6 cells / cm 2 , about 0.1×10 6 cells / cm 2 About 0.6×10 6 cells / cm 2 , about 0.1×10 6 cells / cm 2 About 0.4×10 6 cells / cm2 , about 0.1×10 6 cells / cm 2 About 0.2×10 6 cells / cm 2 , about 0.2×10 6 cells / cm 2 About 2×10 6 cells / cm 2 , about 0.2×10 6 cells / cm 2 About 1×10 6 cells / cm 2 , about 0.2×10 6 cells / cm 2 About 0.8×10 6 cells / cm 2 , about 0.2×10 6 cells / cm 2 About 0.6×10 6 cells / cm 2 , about 0.2×10 6 cells / cm 2 About 0.4×10 6 cells / cm 2 , about 0.4×10 6 cells / cm 2 About 2×10 6 cells / cm 2 , about 0.4×10 6 cells / cm 2 About 1×10 6 cells / cm 2 , about 0.4×10 6 cells / cm 2 About 0.8×10 6 cells / cm 2 , about 0.4×10 6 cells / cm 2 About 0.6×10 6 cells / cm 2 , about 0.6×10 6 cells / cm 2 About 2×10 6 cells / cm 2 , about 0.6×10 6 cells / cm 2 About 1×10 6 cells / cm 2 , about 0.6×10 6 cells / cm 2About 0.8×10 6 cells / cm 2 , about 0.8×10 6 cells / cm 2 About 2×10 6 cells / cm 2 , about 0.8×10 6 cells / cm 2 About 1×10 6 cells / cm 2 or about 1.0×10 6 cells / cm 2 About 2×10 6 cells / cm 2 In some embodiments, the number of cells plated on the substrate-coated culture vessel is about 0.4×10 6 cells / cm 2 About 0.8×10 6 cells / cm 2 .

[0230] In some embodiments, the second incubation starts from about day 7 until the cells are harvested. In some embodiments, the cells are harvested on or after about day 16. In some embodiments, the cells are harvested between about day 16 and about day 30. In some embodiments, the cells are harvested between about day 18 and about day 25. In some embodiments, the cells are harvested on about day 18. In some embodiments, the cells are harvested on about day 25. In some embodiments, the second incubation is from about day 7 until about day 18. In some embodiments, the second incubation is from about day 7 until about day 25.

[0231] In some embodiments, the second incubation comprises culturing cells derived from cell aggregates (eg, spheroids) in culture medium ("medium").

[0232] In some embodiments, the second incubation comprises culturing the cells in the culture medium starting from about day 7 until harvesting or collecting. In some embodiments, the cells are cultured in the culture medium to produce committed dopamine (DA) neuron progenitor cells or dopamine (DA) neurons.

[0233] In some embodiments, the culture medium is also supplemented with a serum replacement that contains minimal components of non-human origin (e.g., KnockOut TMIn some embodiments, from about day 7 to about day 10, the culture medium is supplemented with a serum replacement. In some embodiments, the culture medium is supplemented with about 2% (v / v) serum replacement. In some embodiments, from about day 7 to about day 10, the culture medium is supplemented with about 2% (v / v) serum replacement.

[0234] In some embodiments, the culture medium is further supplemented with a small molecule. In some embodiments, the small molecule is selected from the group consisting of a Rho-associated protein kinase (ROCK) inhibitor, a bone morphogenetic protein (BMP) signaling inhibitor, a glycogen synthase kinase 3β (GSK3β) signaling inhibitor, and combinations thereof.

[0235] In some embodiments, on one or more days when the cells are passaged, the culture medium is supplemented with a Rho-associated protein kinase (ROCK) inhibitor. In some embodiments, on each day of the cell passage, the culture medium is supplemented with a ROCK inhibitor. In some embodiments, the culture medium is supplemented with a ROCK inhibitor on the 7th day, the 16th day, the 20th day, or a combination thereof. In some embodiments, the culture medium is supplemented with a ROCK inhibitor on the 7th day. In some embodiments, the culture medium is supplemented with a ROCK inhibitor on the 16th day. In some embodiments, the culture medium is supplemented with a ROCK inhibitor on the 20th day. In some embodiments, the culture medium is supplemented with a ROCK inhibitor on the 7th and 16th days. In some embodiments, the culture medium is supplemented with a ROCK inhibitor on the 16th and 20th days. In some embodiments, the culture medium is supplemented with a ROCK inhibitor on the 7th, 16th, and 20th days.

[0236] In some embodiments, the cells are exposed to a ROCK inhibitor at a concentration of about 1 μM to about 20 μM, about 5 μM to about 15 μM, or about 8 μM to about 12 μM. In some embodiments, the cells are exposed to a ROCK inhibitor at a concentration of about 1 μM to about 20 μM. In some embodiments, the cells are exposed to a ROCK inhibitor at a concentration of about 5 μM to about 15 μM. In some embodiments, the cells are exposed to a ROCK inhibitor at a concentration of about 8 μM to about 12 μM. In some embodiments, the cells are exposed to a ROCK inhibitor at a concentration of about 10 μM.

[0237] In some embodiments, the ROCK inhibitor is fasudil, ripasudil, naftadil, RKI-1447, Y-27632, GSK429286A, Y-30141, or a combination thereof. In some embodiments, the ROCK inhibitor is a small molecule. In some embodiments, the ROCK inhibitor selectively inhibits p160ROCK. In some embodiments, the ROCK inhibitor is Y-27632, having the following formula:

[0238]

[0239] In some embodiments, the cells are exposed to Y-27632 at a concentration of about 10 μM. In some embodiments, the cells are exposed to Y-27632 at a concentration of about 10 μM on day 7, day 16, day 20, or a combination thereof. In some embodiments, the cells are exposed to Y-27632 at a concentration of about 10 μM on day 7. In some embodiments, the cells are exposed to Y-27632 at a concentration of about 10 μM on day 16. In some embodiments, the cells are exposed to Y-27632 at a concentration of about 10 μM on day 20. In some embodiments, the cells are exposed to Y-27632 at a concentration of about 10 μM on day 7 and day 16. In some embodiments, the cells are exposed to Y-27632 at a concentration of about 10 μM on day 16 and day 20. In some embodiments, the cells are exposed to Y-27632 at a concentration of about 10 μM on day 7, day 16, and day 20.

[0240] In some embodiments, the culture medium is supplemented with a BMP signaling inhibitor. In some embodiments, the culture medium is supplemented with a BMP signaling inhibitor from about day 7 until about day 11 (e.g., day 10 or day 11). In some embodiments, from about day 7 to day 10, including every day, the culture medium is supplemented with a BMP signaling inhibitor.

[0241] In some embodiments, cells are exposed to a concentration of about 0.01 μM to about 5 μM, about 0.05 μM to about 1 μM, or about 0.1 μM to about 0.5 μM (including each end value) of a BMP signaling inhibitor. In some embodiments, cells are exposed to a concentration of about 0.01 μM to about 5 μM of a BMP signaling inhibitor. In some embodiments, cells are exposed to a concentration of about 0.05 μM to about 1 μM of a BMP signaling inhibitor. In some embodiments, cells are exposed to a concentration of about 0.1 μM to about 0.5 μM of a BMP signaling inhibitor. In some embodiments, cells are exposed to a concentration of about 0.1 μM of a BMP signaling inhibitor.

[0242] In some embodiments, the BMP signaling inhibitor is a small molecule. In some embodiments, the BMP signaling inhibitor is LDN193189 or K02288. In some embodiments, the BMP signaling inhibitor is capable of inhibiting "small maternal antagonism to Drosophila skin growth factor" SMAD signaling. In some embodiments, the BMP signaling inhibitor inhibits ALK1, ALK2, ALK3, ALK6, or a combination thereof. In some embodiments, the BMP signaling inhibitor inhibits ALK1, ALK2, ALK3, and ALK6. In some embodiments, the BMP signaling inhibitor inhibits BMP2, BMP4, BMP6, BMP7, and activin cytokine signals, and subsequently inhibits SMAD phosphorylation of Smad1, Smad5, and Smad8. In some embodiments, the BMP signaling inhibitor is LDN193189. In some embodiments, the BMP signaling inhibitor is LDN193189 (e.g., IUPAC name 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline, chemical formula C25H22N6), having the following formula:

[0243]

[0244] In some embodiments, the cells are exposed to LDN193189 at a concentration of about 0.1 μM. In some embodiments, the cells are exposed to LDN193189 at a concentration of about 0.1 μM starting from about day 7 until about day 11 (e.g., day 10 or day 11). In some embodiments, the cells are exposed to LDN193189 at a concentration of about 0.1 μM from about day 7 to about day 10, including every day.

[0245] In some embodiments, the culture medium is supplemented with a GSK3β signaling inhibitor. In some embodiments, the culture medium is supplemented with a GSK3β signaling inhibitor from about day 7 until about day 13 (e.g., day 12 or day 13). In some embodiments, from about day 7 to day 12, including every day, the culture medium is supplemented with a GSK3β signaling inhibitor.

[0246] In some embodiments, the cell is exposed to a GSK3β signaling inhibitor at a concentration of about 0.1 μM to about 10 μM, about 0.5 μM to about 8 μM, or about 1 μM to about 4 μM, or about 2 μM to about 3 μM (including each end value). In some embodiments, the cell is exposed to a GSK3β signaling inhibitor at a concentration of about 0.1 μM to about 10 μM. In some embodiments, the cell is exposed to a GSK3β signaling inhibitor at a concentration of about 0.5 μM to about 8 μM. In some embodiments, the cell is exposed to a GSK3β signaling inhibitor at a concentration of about 1 μM to about 4 μM. In some embodiments, the cell is exposed to a GSK3β signaling inhibitor at a concentration of about 2 μM to about 3 μM. In some embodiments, the cell is exposed to a GSK3β signaling inhibitor at a concentration of about 2 μM.

[0247] In some embodiments, the GSK3β signaling inhibitor is selected from lithium ions, valproic acid, iodinated tuberculocidin, naproxen, famotidine, curcumin, olanzapine, CHIR99012 or a combination thereof. In some embodiments, the GSK3β signaling inhibitor is a small molecule. In some embodiments, the GSK3β signaling inhibitor inhibits glycogen synthase kinase 3β enzyme. In some embodiments, the GSK3β signaling inhibitor inhibits GSK3α. In some embodiments, the GSK3β signaling inhibitor regulates TGF-β and MAPK signaling. In some embodiments, the GSK3β signaling inhibitor is a wingless / integrated (Wnt) signaling agonist. In some embodiments, the GSK3β signaling inhibitor has an IC50 of 6.7nM for human GSK3β. In some embodiments, the GSK3β signaling inhibitor is CHIR99021 (e.g., "3-[3-(2-carboxyethyl)-4-methylpyrrole-2-methylene]-2-indolone" or the IUPAC name 6-(2-(4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino)ethylamino)nicotinonitrile), having the formula:

[0248]

[0249] In some embodiments, the cells are exposed to CHIR99021 at a concentration of about 2.0 μM. In some embodiments, the cells are exposed to CHIR99021 at a concentration of about 2.0 μM starting from about day 7 until about day 13 (e.g., day 12 or day 13). In some embodiments, the cells are exposed to CHIR99021 at a concentration of about 2.0 μM from about day 7 to about day 12, including every day.

[0250] In some embodiments, the culture medium is supplemented with brain-derived neurotrophic factor (BDNF). In some embodiments, the culture medium is supplemented with BDNF starting from about day 11. In some embodiments, the culture medium is supplemented with BDNF starting from about day 11 until harvest or collection. In some embodiments, the culture medium is supplemented with BDNF from about day 11 to day 18. In some embodiments, the culture medium is supplemented with BDNF from about day 11 to day 25.

[0251] In some embodiments, the cells are exposed to BDNF at a concentration of about 1 ng / mL to 100 ng / mL, about 5 ng / mL to about 50 ng / mL, about 10 ng / mL to about 30 ng / mL. In some embodiments, the cells are exposed to BDNF at a concentration of about 10 ng / mL to about 30 ng / mL. In some embodiments, the cells are exposed to BDNF at a concentration of about 20 ng / mL.

[0252] In some embodiments, the culture medium is supplemented with 20 ng / mL BDNF starting at about day 11. In some embodiments, the culture medium is supplemented with 20 ng / mL BDNF starting at about day 11 until harvest or collection. In some embodiments, the culture medium is supplemented with about 20 ng / mL BDNF from about day 11 to day 18. In some embodiments, the culture medium is supplemented with about 20 ng / mL BDNF from about day 11 to day 25.

[0253] In some embodiments, the culture medium is supplemented with glial cell line derived neurotrophic factor (GDNF). In some embodiments, the culture medium is supplemented with GDNF starting from about day 11. In some embodiments, the culture medium is supplemented with GDNF starting from about day 11 until harvest or collection. In some embodiments, the culture medium is supplemented with GDNF from about day 11 to day 18. In some embodiments, the culture medium is supplemented with GDNF from about day 11 to day 25.

[0254] In some embodiments, the cells are exposed to GDNF at a concentration of about 1 ng / mL to about 100 ng / mL, about 5 ng / mL to about 50 ng / mL, about 10 ng / mL to about 30 ng / mL. In some embodiments, the cells are exposed to GDNF at a concentration of about 10 ng / mL to about 30 ng / mL. In some embodiments, the cells are exposed to GDNF at a concentration of about 20 ng / mL.

[0255] In some embodiments, the culture medium is supplemented with 20 ng / mL GDNF starting at about day 11. In some embodiments, the culture medium is supplemented with 20 ng / mL GDNF starting at about day 11 until harvest or collection. In some embodiments, the culture medium is supplemented with about 20 ng / mL GDNF from about day 11 to day 18. In some embodiments, the culture medium is supplemented with about 20 ng / mL GDNF from about day 11 to day 25.

[0256] In some embodiments, the culture medium is supplemented with ascorbic acid. In some embodiments, the culture medium is supplemented with ascorbic acid starting from about day 11. In some embodiments, the culture medium is supplemented with ascorbic acid starting from about day 11 until harvest or collection. In some embodiments, the culture medium is supplemented with ascorbic acid from about day 11 to day 18. In some embodiments, the culture medium is supplemented with ascorbic acid from about day 11 to day 25.

[0257] In some embodiments, the cells are exposed to ascorbic acid at a concentration of about 0.05 mM to about 5 mM, about 0.1 mM to about 1 mM, about 0.2 mM to about 0.5 mM (including each end value). In some embodiments, the cells are exposed to ascorbic acid at a concentration of about 0.05 mM to about 5 mM (including each end value). In some embodiments, the cells are exposed to ascorbic acid at a concentration of about 0.1 mM to about 1 mM (including each end value). In some embodiments, the cells are exposed to ascorbic acid at a concentration of about 0.2 mM.

[0258] In some embodiments, the culture medium is supplemented with about 0.2 mM ascorbic acid starting at about day 11. In some embodiments, the culture medium is supplemented with 0.2 mM ascorbic acid starting at about day 11 until harvest or collection. In some embodiments, the culture medium is supplemented with about 0.2 mM ascorbic acid from about day 11 to day 18. In some embodiments, the culture medium is supplemented with about 0.2 mM ascorbic acid from about day 11 to day 25.

[0259] In some embodiments, the culture medium is supplemented with dibutyryl cyclic AMP (dbcAMP). In some embodiments, the culture medium is supplemented with dbcAMP starting from about day 11. In some embodiments, the culture medium is supplemented with dbcAMP starting from about day 11 until harvest or collection. In some embodiments, the culture medium is supplemented with dbcAMP from about day 11 to day 18. In some embodiments, the culture medium is supplemented with dbcAMP from about day 11 to day 25.

[0260] In some embodiments, the cells are exposed to dbcAMP at a concentration of about 0.05 mM to about 5 mM, about 0.1 mM to about 3 mM, about 0.2 mM to about 1 mM (including each end value). In some embodiments, the cells are exposed to dbcAMP at a concentration of about 0.1 mM to about 3 mM (including each end value). In some embodiments, the cells are exposed to dbcAMP at a concentration of about 0.2 mM to about 1 mM (including each end value). In some embodiments, the cells are exposed to dbcAMP at a concentration of about 0.5 mM.

[0261] In some embodiments, the culture medium is supplemented with about 0.5 mM dbcAMP starting at about day 11. In some embodiments, the culture medium is supplemented with 0.5 mM dbcAMP starting at about day 11 until harvest or collection. In some embodiments, the culture medium is supplemented with about 0.5 mM dbcAMP from about day 11 to day 18. In some embodiments, the culture medium is supplemented with about 0.5 mM dbcAMP from about day 11 to day 25.

[0262] In some embodiments, the culture medium is supplemented with transforming growth factor β3 (TGFβ3). In some embodiments, the culture medium is supplemented with TGFβ3 starting from about day 11. In some embodiments, the culture medium is supplemented with TGFβ3 starting from about day 11 until harvest or collection. In some embodiments, the culture medium is supplemented with TGFβ3 from about day 11 to day 18. In some embodiments, the culture medium is supplemented with TGFβ3 from about day 11 to day 25.

[0263] In some embodiments, the cells are exposed to a concentration of about 0.1 ng / mL to 10 ng / mL, about 0.5 ng / mL to about 5 ng / mL, or about 1.0 ng / mL to about 2.0 ng / mL of TGFβ3. In some embodiments, the cells are exposed to a concentration of about 1.0 ng / mL to about 2.0 ng / mL of TGFβ3 (including each end value). In some embodiments, the cells are exposed to a concentration of about 1 ng / mL of TGFβ3.

[0264] In some embodiments, the culture medium is supplemented with about 1 ng / mL TGFβ3 starting on about day 11. In some embodiments, the culture medium is supplemented with 1 ng / mL TGFβ3 starting on about day 11 until harvest or collection. In some embodiments, the culture medium is supplemented with about 1 ng / mL TGFβ3 from about day 11 to day 18. In some embodiments, the culture medium is supplemented with about 1 ng / mL TGFβ3 from about day 11 to day 25.

[0265] In some embodiments, the culture medium is supplemented with a Notch signaling inhibitor. In some embodiments, the culture medium is supplemented with a Notch signaling inhibitor starting from about day 11. In some embodiments, the culture medium is supplemented with a Notch signaling inhibitor starting from about day 11 until harvest or collection. In some embodiments, the culture medium is supplemented with a Notch signaling inhibitor from about day 11 to day 18. In some embodiments, the culture medium is supplemented with a Notch signaling inhibitor from about day 11 to day 25.

[0266] In some embodiments, the Notch signaling inhibitor is selected from cowanin, PF-03084014, L685458, LY3039478, DAPT, or a combination thereof. In some embodiments, the Notch signaling inhibitor inhibits gamma secretase. In some embodiments, the Notch signaling inhibitor is a small molecule. In some embodiments, the Notch signaling inhibitor is DAPT, having the following formula:

[0267]

[0268] In some embodiments, the cells are exposed to DAPT at a concentration of about 1 μM to about 20 μM, about 5 μM to about 15 μM, or about 8 μM to about 12 μM. In some embodiments, the cells are exposed to DAPT at a concentration of about 1 μM to about 20 μM. In some embodiments, the cells are exposed to DAPT at a concentration of about 5 μM to about 15 μM. In some embodiments, the cells are exposed to DAPT at a concentration of about 8 μM to about 12 μM. In some embodiments, the cells are exposed to DAPT at a concentration of about 10 μM.

[0269] In some embodiments, the culture medium is supplemented with about 10 μM DAPT starting on about day 11. In some embodiments, the culture medium is supplemented with 10 μM DAPT starting on about day 11 until harvest or collection. In some embodiments, the culture medium is supplemented with about 10 μM DAPT from about day 11 to day 18. In some embodiments, the culture medium is supplemented with about 10 μM DAPT from about day 11 to day 25.

[0270] In some embodiments, starting from about day 11, the culture medium is supplemented with about 20 ng / mL BDNF, about 20 ng / mL GDNF, about 0.2 mM ascorbic acid, about 0.5 mM dbcAMP, about 1 ng / mL TGFβ3, and about 10 μM DAPT. In some embodiments, starting from about day 11 until harvest or collection, the culture medium is supplemented with about 20 ng / mL BDNF, about 20 ng / mL GDNF, about 0.2 mM ascorbic acid, about 0.5 mM dbcAMP, about 1 ng / mL TGFβ3, and about 10 μM DAPT. In some embodiments, from about day 11 to day 18, the culture medium is supplemented with about 20 ng / mL BDNF, about 20 ng / mL GDNF, about 0.2 mM ascorbic acid, about 0.5 mM dbcAMP, about 1 ng / mL TGFβ3, and about 10 μM DAPT. In some embodiments, from about day 11 to day 25, the culture medium is supplemented with about 20 ng / mL BDNF, about 20 ng / mL GDNF, about 0.2 mM ascorbic acid, about 0.5 mM dbcAMP, about 1 ng / mL TGFβ3, and about 10 μM DAPT.

[0271] In some embodiments, serum replacement is provided in the culture medium from about day 7 to about day 10. In some embodiments, serum replacement is provided in the culture medium at 2% (v / v) from day 7 to day 10.

[0272] In some embodiments, from about day 7 to about day 16, at least about 50% of the culture medium is replaced every day. In some embodiments, from about day 7 to about day 16, about 50% of the culture medium is replaced every day, every other day, or every three days. In some embodiments, from about day 7 to about day 16, about 50% of the culture medium is replaced every day. In some embodiments, starting from about day 17, at least about 50% of the culture medium is replaced every day, every other day, or every three days. In some embodiments, starting from about day 17, at least about 50% of the culture medium is replaced every other day. In some embodiments, starting from about day 17, about 50% of the culture medium is replaced every other day. In some embodiments, starting from about day 17, about 50% of the culture medium is replaced every other day. In some embodiments, starting from about day 17, about 50% of the culture medium is replaced every other day. In some embodiments, the replacement culture medium contains about twice as concentrated small molecules as compared to the concentration of small molecules in the culture medium at day 0.

[0273] In some embodiments, the second incubation comprises culturing cells derived from cell aggregates (e.g., spheres) in a "basal induction medium." In some embodiments, the second incubation comprises culturing cells derived from cell aggregates (e.g., spheres) in a "maturation medium." In some embodiments, the second incubation comprises culturing cells derived from cell aggregates (e.g., spheres) in a basal induction medium and then in a maturation medium.

[0274] In some embodiments, the second incubation comprises culturing the cells in a basal induction medium from about day 7 to about day 10. In some embodiments, the second incubation comprises culturing the cells in a maturation medium starting from about day 11. In some embodiments, the second incubation comprises culturing the cells in a basal induction medium from about day 7 to about day 10, and then culturing the cells in a maturation medium starting from about day 11. In some embodiments, the cells are cultured in a maturation medium to produce committed dopamine (DA) neuron progenitor cells or dopamine (DA) neurons.

[0275] In some embodiments, the basal induction medium is formulated to contain Neurobasal TM culture medium and DMEM / F12 medium supplemented with N-2 and B27 supplements, non-essential amino acids (NEAA), GlutaMAX TM , L-glutamine, β-mercaptoethanol and insulin. In some embodiments, the basal induction medium is further supplemented with any of the molecules described in Section II.

[0276] In some embodiments, the maturation medium is formulated to contain N-2 and B27 supplements, non-essential amino acids (NEAA), and GlutaMAX TM Neurobasal TM Culture medium. In some embodiments, the maturation medium is further supplemented with any of the molecules described in Section II.

[0277] In some embodiments, cells are cultured in a basal induction medium from about the 7th day to about the 11th day (e.g., the 10th day or the 11th day). In some embodiments, cells are cultured in a basal induction medium from about the 7th day to the 10th day, including every day. In some embodiments, cells are cultured in a maturation medium from about the 11th day. In some embodiments, cells are cultured in a basal induction medium from about the 7th day to about the 10th day, and then cells are cultured in a maturation medium from about the 11th day. In some embodiments, cells are cultured in a maturation medium from about the 11th day until harvest or cells are collected. In some embodiments, cells are harvested between the 16th day and the 27th day. In some embodiments, cells are harvested between the 18th day and the 25th day. In some embodiments, cells are harvested on the 18th day. In some embodiments, cells are harvested on the 25th day.

[0278] E. Harvesting, collecting and preparing differentiated cells

[0279] In embodiments of the provided methods, the neutrally differentiated cells produced by the methods provided herein can be harvested or collected, such as for use in formulating and using the cells. In some embodiments, the provided methods for producing differentiated cells, such as for use as cell therapy in the treatment of neurodegenerative diseases, can include formulating cells, such as formulating differentiated cells produced by the provided methods described herein. In some embodiments, a cell dose comprising differentiated cells (e.g., committed DA neuron progenitor cells or DA neurons) is provided as a composition or formulation, such as a pharmaceutical composition or formulation. Such compositions can be used according to the provided methods, such as for preventing or treating neurodegenerative disorders, including Parkinson's disease.

[0280] In some cases, processing cells in one or more steps to manufacture, generate or produce cell therapy and / or differentiated cells can include cell preparations, such as preparations of differentiated cells produced by the methods. In some cases, cells can be prepared in an amount for dosage administration, such as for single unit dosage administration or multiple dosage administration.

[0281] In certain embodiments, one or more compositions of differentiated cells are formulated. In specific embodiments, after one or more compositions are produced, one or more compositions of differentiated cells are formulated. In some embodiments, the one or more compositions have previously been cryopreserved and stored and thawed prior to administration.

[0282] In certain embodiments, the differentiated cells include committed DA neuron progenitor cells. In some embodiments, the formulated composition of differentiated cells is a composition enriched in committed DA neuron progenitor cells. In certain embodiments, the differentiated cells include DA neurons. In some embodiments, the formulated composition of differentiated cells is a composition enriched in DA neurons.

[0283] In certain embodiments, the cells are cultured for a minimum or maximum duration or amount of time. In certain embodiments, the cells are cultured for a minimum duration or amount of time. In certain embodiments, the cells are cultured for a maximum duration or amount of time. In some embodiments, the cells are differentiated for at least 16 days. In some embodiments, the cells are differentiated for 16 to 30 days. In some embodiments, the cells are differentiated for 16 to 27 days. In some embodiments, the cells are differentiated for 18 to 25 days. In some embodiments, the cells are differentiated for about 18 days. In some embodiments, the cells are differentiated for about 25 days.

[0284] In certain embodiments, the cells are cultured for the shortest or longest duration or amount of time. In certain embodiments, the cells are cultured for the shortest duration or amount of time. In certain embodiments, the cells are cultured for the longest duration or amount of time. In some embodiments, the cells are harvested after at least 16 days in culture. In some embodiments, the cells are harvested between 16 and 30 days in culture. In some embodiments, the cells are harvested between 16 and 27 days in culture. In some embodiments, the cells are harvested between 18 and 25 days in culture. In some embodiments, the cells are harvested after about 18 days in culture. In some embodiments, the cells are harvested after about 25 days in culture.

[0285] In some embodiments, the cells are formulated in a pharmaceutically acceptable buffer, which in some aspects may include a pharmaceutically acceptable carrier or excipient. In some embodiments, the processing includes replacing the medium with a pharmaceutically acceptable medium or a formulation buffer that is desired to be applied to a subject. In some embodiments, the processing step may include washing the differentiated cells to replace the cells in a pharmaceutically acceptable buffer, which may include one or more optional pharmaceutically acceptable carriers or excipients. Examples of such drug forms, including pharmaceutically acceptable carriers or excipients, may be any drug form described below together with acceptable forms for applying cells and compositions to a subject. In some embodiments, the pharmaceutical composition contains an amount effective to treat or prevent neurodegenerative conditions or diseases (e.g., Parkinson's disease), such as a therapeutically effective amount or a preventive effective amount of cells.

[0286] "Pharmaceutically acceptable carrier" refers to ingredients in a pharmaceutical formulation other than the active ingredient that are non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0287] In some aspects, the selection of carriers is determined in part by specific cells and / or the method of administration. Therefore, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain a preservative. Suitable preservatives can include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. Preservatives or mixtures thereof are generally present in an amount of about 0.0001% to about 2% of the total composition weight. Carriers are described, for example, by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinyl pyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG).

[0288] In some aspects, a buffer is included in the composition. Suitable buffers include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffers is used. The buffer or a mixture thereof is typically present in an amount of about 0.001% to about 4% of the total composition weight. Methods for preparing an administrable pharmaceutical composition are known. Exemplary methods are described in more detail, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st edition (May 1, 2005).

[0289] The formulation may include an aqueous solution. The formulation or composition may also contain one or more active ingredients that can be used for a specific indication, disease or condition to be treated with the cells, preferably those active ingredients that have complementary activities to the cells, wherein the respective activities do not adversely affect each other. Such active ingredients are suitably present in combination in an amount effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further comprises other pharmaceutically active agents or drugs, such as carbidopa-levodopa (e.g., levodopa), dopamine agonists (e.g., pramipexole, ropinirole, rotigotine, and apomorphine), MAO B inhibitors (e.g., selegiline, rasagiline, and safinamide), catechol O-methyltransferase (COMT) inhibitors (e.g., entacapone and tolcapone), anticholinergics (e.g., benztropine and trihexylphenidyl), amantadine, and the like.

[0290] In some embodiments, compositions are provided in the form of sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions or viscous compositions, and the liquid preparations can be buffered to selected pH in some respects.Liquid compositions can include carriers, and the carriers can be solvents or dispersion media containing, for example, water, saline, phosphate buffered saline, polyols (for example, glycerol, propylene glycol, liquid polyethylene glycol) and their suitable mixtures. Aseptic injection solutions can be prepared by mixing cells in solvents, such as mixing with suitable carriers, diluents or excipients such as sterile water, saline, glucose, dextrose, etc. According to route of administration and required preparation, compositions can contain auxiliary substances, such as wetting agents, dispersants or emulsifiers (for example methylcellulose), pH buffers, gelling or viscosity enhancing additives, preservatives and / or coloring agents. In some respects, suitable preparations can be prepared with reference to standard texts.

[0291] Various additives that enhance the stability and sterility of the composition may be added, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of microbial action may be ensured by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, and sorbic acid). Prolonged absorption of injectable drug forms may be achieved by using agents that delay absorption, such as aluminum monostearate and gelatin.

[0292] In some embodiments, the preparation buffer contains a cryopreservative. In some embodiments, cells are prepared with a cryopreservation solution containing 1.0% to 30% DMSO solution, such as 5% to 20% DMSO solution or 5% to 10% DMSO solution. In some embodiments, the cryopreservation solution is or contains, for example, PBS containing 20% ​​DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. In some embodiments, the cryopreservation solution is or contains, for example, at least or about 7.5% DMSO. In some embodiments, the processing step may include washing the differentiated cells to replace the cells in the cryopreservation solution. In some embodiments, cells are frozen, e.g., cryopreserved or cryoprotected, in a medium and / or solution with a final concentration of at or about 12.5%, 12.0%, 11.5%, 11.0%, 10.5%, 10.0%, 9.5%, 9.0%, 8.5%, 8.0%, 7.5%, 7.0%, 6.5%, 6.0%, 5.5%, or 5.0% DMSO, or 1% to 15%, 6% to 12%, 5% to 10%, or 6% to 8% DMSO. In certain embodiments, the cells are frozen, e.g., cryopreserved or cryoprotected, in a medium and / or solution with a final concentration of at or about 5.0%, 4.5%, 4.0%, 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.25%, 1.0%, 0.75%, 0.5%, or 0.25% HSA, or 0.1% to -5%, 0.25% to 4%, 0.5% to 2%, or 1% to 2% HSA.

[0293] In a specific embodiment, the composition of differentiated cells is prepared, cryopreserved, and then stored for a period of time. In certain embodiments, the cryopreserved cells prepared are stored until the cells are released for administration. In a specific embodiment, the cryopreserved cells prepared are stored for 1 day to 6 months, 1 month to 3 months, 1 day to 14 days, 1 day to 7 days, 3 days to 6 days, 6 months to 12 months or longer than 12 months. In some embodiments, the cells are cryopreserved and stored for about or less than 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days. In certain embodiments, the cells are thawed and applied to a subject after storage.

[0294] In some embodiments, one or more processing steps are used for preparation, including washing, diluting or concentrating cells. In some embodiments, the processing may include diluting or concentrating cells to a desired concentration or number, such as a composition in a unit dose form of the number of cells administered in a given dose or part thereof. In some embodiments, the processing step may include reducing the volume, thereby increasing the cell concentration as needed. In some embodiments, the processing step may include increasing the volume, thereby reducing the cell concentration as needed. In some embodiments, the processing includes adding a certain volume of formulation buffer to the differentiated cells. In some embodiments, the volume of the formulation buffer is about 1 μL to 5000 μL, such as at least or about at least or about or just 5 μL, 10 μL, 20 μL, 50 μL, 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, 1000 μL, 2000 μL, 3000 μL, 4000 μL or 5000 μL.

[0295] The container can generally contain cells to be administered, such as one or more unit doses thereof. The unit dose can be the amount or number of cells to be administered to a subject, or twice (or more) the number of cells to be administered. It can be the minimum dose or the lowest possible dose of cells to be administered to a subject.

[0296] In some embodiments, such cells produced by the methods, or compositions comprising such cells, are administered to a subject to treat a neurodegenerative disease or condition.

[0297] F. Example Process

[0298] As described above, by the methods provided herein, pluripotent stem cells can be differentiated into lineage-specific cell populations, including committed DA progenitor cells and DA neurons. These cells can then be used for cell replacement therapy. As described above, by the methods herein, in some embodiments, pluripotent stem cells are differentiated into midbrain floor plate progenitor cells, and the resulting sphere cells are further differentiated into committed dopamine (DA) neuron progenitor cells and / or dopamine (DA) neurons. In some embodiments, pluripotent stem cells are differentiated into committed DA neuron progenitor cells. In some embodiments, pluripotent stem cells are differentiated into DA neurons. In some embodiments, pluripotent stem cells are embryonic stem cells. In some embodiments, pluripotent stem cells are induced pluripotent stem cells.

[0299] In some embodiments, embryonic stem cells differentiate into midbrain floor plate progenitor cells and then differentiate into committed dopamine (DA) neuron progenitor cells and / or dopamine (DA) neurons. In some embodiments, embryonic stem cells differentiate into committed DA neuron progenitor cells. In some embodiments, embryonic stem cells differentiate into DA neurons.

[0300] In some embodiments, induced pluripotent stem cells are differentiated into midbrain floor plate progenitor cells, and then differentiated into committed dopamine (DA) neuron progenitor cells and / or dopamine (DA) neurons. In some embodiments, induced pluripotent stem cells are differentiated into committed DA neuron progenitor cells. In some embodiments, induced pluripotent stem cells are differentiated into DA neurons.

[0301] In some embodiments, the method includes (a) performing a first incubation comprising culturing pluripotent stem cells in a non-adherent culture vessel under conditions that produce cell spheroids, wherein at the beginning of the first incubation (day 0), the cells are exposed to (i) a TGF-β / activin Nodal signaling inhibitor; (ii) at least one sonic hedgehog (SHH) signaling activator; (iii) a bone morphogenetic protein (BMP) signaling inhibitor; and (iv) a glycogen synthase kinase 3β (GSK3β) signaling inhibitor; and (b) performing a second incubation comprising culturing the cells of the spheroid in a substrate-coated culture vessel under conditions that induce neural differentiation of the cells.

[0302] In some embodiments, culturing cells under conditions that induce neural differentiation of the cells comprises exposing the cells to (i) brain-derived neurotrophic factor (BDNF); (ii) ascorbic acid; (iii) glial cell-derived neurotrophic factor (GDNF); (iv) dibutyryl cyclic AMP (dbcAMP); (v) transforming growth factor β-3 (TGFβ3); and (vi) a Notch signaling inhibitor.

[0303] In some embodiments, the method includes (a) performing a first incubation, including culturing pluripotent stem cells under conditions that produce cell spheres in a plate having multiple wells, wherein at the beginning of the first incubation (day 0), the cells are exposed to (i) a TGF-β / activin Nodal signaling inhibitor; (ii) at least one sonic hedgehog (SHH) signaling activator; (iii) a bone morphogenetic protein (BMP) signaling inhibitor; (iv) a glycogen synthase kinase 3β (GSK3β) signaling inhibitor; and (v) a serum replacement; (b) dissociating cells of the sphere to produce a cell suspension; (c) transferring cells of the cell suspension to a laminin-coated culture vessel; (d) performing a second incubation, including culturing cells of the sphere in a laminin-coated culture vessel under conditions that induce neural differentiation of the cells; and (e) harvesting neural differentiated cells. In some embodiments, the second incubation includes culturing cells in the presence of a serum replacement. In some embodiments, culturing cells under conditions that induce neural differentiation of the cells comprises exposing the cells to (i) brain-derived neurotrophic factor (BDNF); (ii) ascorbic acid; (iii) glial cell-derived neurotrophic factor (GDNF); (iv) dibutyryl cyclic AMP (dbcAMP); (v) transforming growth factor β-3 (TGFβ3); and (vi) a Notch signaling inhibitor.

[0304] In some embodiments, the cells are exposed to a TGF-β / activin Nodal inhibitor (e.g., SB431542 or "SB") from day 0 to about day 7 (e.g., day 6 or day 7). In some embodiments, the cells are exposed to a TGF-β / activin Nodal inhibitor (e.g., SB431542 or "SB") from day 0 to day 6, including every day. In some embodiments, the cells are exposed to the at least one SHH signaling activator (e.g., SHH protein and purmorphamine, collectively referred to as "SHH / PUR") from day 0 to about day 7 (e.g., day 6 or day 7). In some embodiments, the cells are exposed to the at least one SHH signaling activator (e.g., SHH protein and purmorphamine, collectively referred to as "SHH / PUR") from day 0 to day 6, including every day. In some embodiments, the cells are exposed to a BMP signaling inhibitor (e.g., LDN193189 or "LDN") from day 0 to about day 11 (e.g., day 10 or day 11). In some embodiments, the cells are exposed to a BMP signaling inhibitor (e.g., LDN193189 or "LDN") from day 0 to day 10, including every day. In some embodiments, the cells are exposed to a GSK3β signaling inhibitor (e.g., CHIR99021 or "CHIR") from day 0 to about day 13 (e.g., day 12 or day 13). In some embodiments, the cells are exposed to a GSK3β signaling inhibitor (e.g., CHIR99021 or "CHIR") from day 0 to day 12.

[0305] In some embodiments, the cells are (i) exposed to a TGF-β / activin Nodal signaling inhibitor from day 0 to about day 7 (e.g., day 6 or day 7); (ii) exposed to at least one sonic hedgehog (SHH) signaling activator from day 0 to about day 7 (e.g., day 6 or day 7); (iii) exposed to a bone morphogenetic protein (BMP) signaling inhibitor from day 0 to about day 11 (e.g., day 10 or day 11); and (iv) exposed to a glycogen synthase kinase 3β (GSK3β) signaling inhibitor from day 0 to about day 13 (e.g., day 12 or day 13). In some embodiments, the cells are (i) exposed to SB from day 0 to about day 7 (e.g., day 6 or day 7); (ii) exposed to SHH / PUR from day 0 to about day 7 (e.g., day 6 or day 8); (iii) exposed to LDN from day 0 to about day 11 (e.g., day 10 or day 11); and (iv) exposed to CHIR from day 0 to about day 13 (e.g., day 12 or day 13). In some embodiments, the cells are (i) exposed to a TGF-β / activin Nodal signaling inhibitor from day 0 to day 6, including each day; (ii) exposed to at least one sonic hedgehog (SHH) signaling activator from day 0 to day 6, including each day; (iii) exposed to a bone morphogenetic protein (BMP) signaling inhibitor from day 0 to day 10, including each day; and (iv) exposed to a glycogen synthase kinase 3β (GSK3β) signaling inhibitor from day 0 to day 12, including each day. In some embodiments, the cells are (i) exposed to SB from day 0 to day 6, including each day; (ii) exposed to SHH / PUR from day 0 to day 6, including each day; (iii) exposed to LDN from day 0 to day 10, including each day; and (iv) exposed to CHIR from day 0 to day 12, including each day.

[0306] In some embodiments, cells are exposed to brain-derived neurotrophic factor (BDNF) from day 11. In some embodiments, cells are exposed to ascorbic acid. In some embodiments, cells are exposed to glial cell-derived neurotrophic factor (GDNF) from day 11. In some embodiments, cells are exposed to dibutyryl cyclic AMP (dbcAMP) from day 11. In some embodiments, cells are exposed to transforming growth factor β-3 (TGFβ3) from day 11. In some embodiments, cells are exposed to Notch signaling inhibitors (e.g., DAPT) from day 11. In some embodiments, starting from day 11, cells are exposed to (i) brain-derived neurotrophic factor (BDNF); (ii) ascorbic acid; (iii) glial cell-derived neurotrophic factor (GDNF); (iv) dibutyryl cyclic AMP (dbcAMP); (v) transforming growth factor β-3 (TGFβ3); and (vi) Notch signaling inhibitors (e.g., DAPT) (collectively referred to as "BAGCT / DAPT"). In some embodiments, cells are exposed to BAGCT / DAPT starting on day 11 until harvest or collection. In some embodiments, cells are exposed to BAGCT / DAPT from day 11 to day 18. In some embodiments, cells are exposed to BAGCT / DAPT from day 11 to day 25.

[0307] In some embodiments, the cell is exposed to a Rho-associated protein kinase (ROCK) inhibitor at day 0. In some embodiments, the cell is exposed to a Rho-associated protein kinase (ROCK) inhibitor at day 7. In some embodiments, the cell is exposed to a Rho-associated protein kinase (ROCK) inhibitor at day 16. In some embodiments, the cell is exposed to a Rho-associated protein kinase (ROCK) inhibitor at day 20. In some embodiments, the cell is exposed to a Rho-associated protein kinase (ROCK) inhibitor at day 0, day 7, day 16, and day 20. In some embodiments, on the day of cell passage, the cell is exposed to a ROCK inhibitor. In some embodiments, the cell is passaged at day 0, day 7, day 16, day 20, or a combination thereof. In some embodiments, the cell is passaged at day 0, day 7, day 16, and day 20.

[0308] In some embodiments, cells are cultured in a basal induction medium comprising DMEM / F-12 and Neurobasal medium (e.g., 1:1 ratio), which is supplemented with N2, B27, non-essential amino acids (NEAA), Glutamax, L-glutamine, β-mercaptoethanol, and insulin. In some embodiments, cells are cultured in a basal induction medium from about day 0 to about day 10. In some embodiments, the basal induction medium is used to differentiate pluripotent stem cells into midbrain floor plate progenitor cells.

[0309] In some embodiments, cells are cultured in a maturation medium comprising Neurobasal medium supplemented with N2, B27, non-essential amino acids (NEAA) and Glutamax. In some embodiments, cells are cultured in a basal induction medium from about day 11 until harvest or collection. In some embodiments, cells are cultured in a basal induction medium from about day 11 to day 18. In some embodiments, the maturation medium is used to differentiate midbrain floor plate progenitor cells into directed dopamine (DA) neuron progenitor cells. In some embodiments, cells are cultured in a basal induction medium from about day 11 to day 25. In some embodiments, the maturation medium is used to differentiate midbrain floor plate progenitor cells into dopamine (DA) neurons.

[0310] In some embodiments, culture medium is supplemented with small molecules as described above, including SB, SHH / PUR, LDN, CHIR, BAGCT / DAPT and ROCKi. In some embodiments, culture medium is replaced every day or every other day. In some embodiments, culture medium is replaced every day. In some embodiments, culture medium is replaced every other day. In some embodiments, culture medium is replaced every day from about day 0 to about day 17 (e.g., day 16 or day 18). In some embodiments, culture medium is replaced every other day from about day 18 until harvest or collection. In some embodiments, culture medium is replaced every day from about day 0 to about day 17 (e.g., day 16 or day 18), and then culture medium is replaced every other day from about day 18 until harvest or collection.

[0311] In some embodiments, serum replacement is provided in the culture medium from about day 0 to about day 10 (e.g., day 9 or day 11). In some embodiments, on day 0 and day 1, 5% (v / v) serum replacement is provided in the culture medium. In some embodiments, on day 2 to day 10, 2% (v / v) serum replacement is provided in the culture medium. In some embodiments, on day 0 and day 1, 5% (v / v) serum replacement is provided in the culture medium and from day 2 to day 10, 2% (v / v) serum replacement is provided in the culture medium. In some embodiments, after day 10, no serum replacement is provided in the culture medium.

[0312] In some embodiments, at least about 50% or at least about 75% of the culture medium is replaced. In some embodiments, at least about 50% of the culture medium is replaced. In some embodiments, at least about 75% of the culture medium is replaced. In some embodiments, about 100% of the culture medium is replaced.

[0313] In some embodiments, about 50% or about 75% of the culture medium is replaced. In some embodiments, about 50% of the culture medium is replaced. In some embodiments, about 75% of the culture medium is replaced. In some embodiments, about 100% of the culture medium is replaced.

[0314] In some embodiments, the culture medium is supplemented with a small molecule selected from SB, SHH / PUR, LDN, CHIR, BAGCT / DAPT, ROCKi, or a combination thereof. In some embodiments, when about 50% of the culture medium is replaced, the concentration of each small molecule is doubled compared to its concentration on day 0.

[0315] In some embodiments, cells are harvested between about the 16th day and about the 30th day. In some embodiments, cells are harvested between about the 16th day and about the 27th day. In some embodiments, cells are harvested between about the 18th day and about the 25th day. In some embodiments, cells are harvested at about the 18th day. In some embodiments, cells are harvested at about the 25th day. In some embodiments, the harvested cells are prepared with a cryopreservative, such as DMSO. In some embodiments, the harvested cells produced by the method are cryopreserved before use. In some embodiments, such cryopreserved cells are thawed before use or administration to a subject, such as a human patient suffering from a neurodegenerative disease or condition (such as Parkinson's disease).

[0316] In some embodiments, the composition comprising the cells generated by the methods provided herein is used to treat a neurodegenerative disease or condition, such as Parkinson's disease. In some embodiments, the composition of the cells generated by any method described herein is administered to a subject suffering from Parkinson's disease. In some embodiments, the composition of the cells generated by any method described herein is administered by stereotactic injection, for example, with a catheter. In some embodiments, the composition of the cells generated by any method described herein is administered to the striatum of a Parkinson's disease patient.

[0317] III. Compositions and formulations

[0318] Provided herein are therapeutic compositions containing differentiated cells that are committed dopamine (DA) neuron progenitor cells. Also provided herein are therapeutic compositions containing differentiated cells produced by any of the methods provided, such as any of the methods described in Section II. In some embodiments, the differentiated cells produced by any of the methods described herein are committed dopamine (DA) neuron progenitor cells.

[0319] In some embodiments, the differentiated cells in the provided therapeutic compositions, including cells produced by any of the methods described herein, are capable of producing dopamine (DA). In some embodiments, the differentiated cells in the provided therapeutic compositions, including cells produced by any of the methods described herein, do not produce or substantially do not produce norepinephrine (NE). Thus, in some embodiments, the differentiated cells in the provided therapeutic compositions, including cells produced by any of the methods described herein, are capable of producing DA, but do not produce or substantially do not produce NE.

[0320] In some embodiments, committed dopamine (DA) neuron progenitor cells express EN1. In some embodiments, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% of the total cells in the composition express EN1. In some embodiments, at least about 20% of the cells in the therapeutic composition express EN1. In some embodiments, at least about 25% of the cells in the therapeutic composition express EN1. In some embodiments, at least about 30% of the cells in the therapeutic composition express EN1. In some embodiments, at least about 35% of the cells in the therapeutic composition express EN1. In some embodiments, at least about 40% of the cells in the therapeutic composition express EN1. In some embodiments, at least about 45% of the cells in the therapeutic composition express EN1. In some embodiments, at least about 50% of the cells in the therapeutic composition express EN1. In some embodiments, at least about 55% of the cells in the therapeutic composition express EN1. In some embodiments, at least about 60% of the cells in the therapeutic composition express EN1. In some embodiments, at least about 65% of the cells in the therapeutic composition express EN1. In some embodiments, at least about 70% of the cells in the therapeutic composition express EN1. In some embodiments, at least about 75% of the cells in the therapeutic composition express EN1. In some embodiments, at least about 80% of the cells in the therapeutic composition express EN1.

[0321] In some embodiments, the therapeutic composition exhibits a -4 In some embodiments, the ratio of CPM EN1 to CPM GAPDH is between about 1.5×10 -3 Up to 1×10 -2 between.

[0322] In some embodiments, the committed dopamine (DA) neuron progenitor cells express CORIN. In some embodiments, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% of the total cells in the composition express CORIN. In some embodiments, at least about 20% of the cells in the therapeutic composition express CORIN. In some embodiments, at least about 25% of the cells in the therapeutic composition express CORIN. In some embodiments, at least about 30% of the cells in the therapeutic composition express CORIN. In some embodiments, at least about 35% of the cells in the therapeutic composition express CORIN. In some embodiments, at least about 40% of the cells in the therapeutic composition express CORIN. In some embodiments, at least about 45% of the cells in the therapeutic composition express CORIN. In some embodiments, at least about 50% of the cells in the therapeutic composition express CORIN. In some embodiments, at least about 55% of the cells in the therapeutic composition express CORIN. In some embodiments, at least about 60% of the cells in the therapeutic composition express CORIN. In some embodiments, at least about 65% of the cells in the therapeutic composition express CORIN. In some embodiments, at least about 70% of the cells in the therapeutic composition express CORIN. In some embodiments, at least about 75% of the cells in the therapeutic composition express CORIN. In some embodiments, at least about 80% of the cells in the therapeutic composition express CORIN.

[0323] In some embodiments, the therapeutic composition exhibits a -4 In some embodiments, the ratio of CPM CORIN to CPM GAPDH is between about 5×10 -2 Up to 5×10 -1 between.

[0324] In some embodiments, the committed dopamine (DA) neuron progenitor cells express EN1 and CORIN. In some embodiments, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% of the total cells in the composition express EN1 and CORIN. In some embodiments, at least about 20% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 25% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 30% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 35% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 40% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 45% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 50% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 55% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 60% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 65% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 70% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 75% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 80% of the cells in the therapeutic composition express EN1 and CORIN.

[0325] In some embodiments, the therapeutic composition exhibits (a) greater than about 1×10 -4 10-10 counts per million (CPM) EN1 to CPM GAPDH ratio; and (b) greater than about 2×10 -2 In some embodiments, the ratio of CPM EN1 to CPM GAPDH is between about 1.5×10 -3 Up to 1×10 -2 and the ratio of CPM CORIN to CPMGAPDH is between about 5×10 -2 Up to 5×10 -1 between.

[0326] In some embodiments, less than 10% of committed dopamine (DA) neuron progenitor cells express TH. In some embodiments, committed dopamine (DA) neuron progenitor cells express low levels of TH. In some embodiments, committed dopamine (DA) neuron progenitor cells do not express TH. In some embodiments, committed dopamine (DA) neuron progenitor cells express TH at a lower level than cells harvested or collected on other days. In some embodiments, some committed dopamine (DA) neuron progenitor cells express EN1 and CORIN and less than 10% of the cells express TH.

[0327] In some embodiments, less than 8% of cells express TH. In some embodiments, less than 5% of cells express TH. In some embodiments, about 2% to 10%, about 2% to 8%, about 2% to 6%, about 2% to 4%, about 4% to 10%, about 4% to 8%, about 4% to 6%, about 6% to 10%, about 6% to 8%, or about 8% to 10% of the total cells in the composition express TH.

[0328] In some embodiments, the therapeutic composition exhibits less than about 3×10 -2 In some embodiments, the ratio of CPM TH to CPM GAPDH is between about 1×10 -3 Up to 2.5×10 -2 between.

[0329] In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 20% of the cells in the therapeutic composition express EN1. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 25% of the cells in the therapeutic composition express EN1. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 30% of the cells in the therapeutic composition express EN1. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 35% of the cells in the therapeutic composition express EN1. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 40% of the cells in the therapeutic composition express EN1. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 45% of the cells in the therapeutic composition express EN1. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 50% of the cells in the therapeutic composition express EN1. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 55% of the cells in the therapeutic composition express EN1. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 60% of the cells in the therapeutic composition express EN1. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 65% of the cells in the therapeutic composition express EN1. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 70% of the cells in the therapeutic composition express EN1. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 75% of the cells in the therapeutic composition express EN1. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 80% of the cells in the therapeutic composition express EN1.

[0330] In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 20% of the cells in the therapeutic composition express CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 25% of the cells in the therapeutic composition express CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 30% of the cells in the therapeutic composition express CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 35% of the cells in the therapeutic composition express CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 40% of the cells in the therapeutic composition express CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 45% of the cells in the therapeutic composition express CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 50% of the cells in the therapeutic composition express CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 55% of the cells in the therapeutic composition express CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 60% of the cells in the therapeutic composition express CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 65% of the cells in the therapeutic composition express CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 70% of the cells in the therapeutic composition express CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 75% of the cells in the therapeutic composition express CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 80% of the cells in the therapeutic composition express CORIN.

[0331] In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 20% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 25% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 30% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 35% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 40% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 45% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 50% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 55% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 60% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 65% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 70% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 75% of the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, less than 10% of the total cells in the composition express TH, and at least about 80% of the cells in the therapeutic composition express EN1 and CORIN.

[0332] In some embodiments, the differentiated cells produced by any of the methods described herein are dopamine (DA) neurons (e.g., midbrain-destined DA neurons). In some embodiments, midbrain-destined dopamine (DA) neurons are FOXA2+ / TH+ at harvest. In some embodiments, midbrain-destined dopamine (DA) neurons are FOXA2+ / TH+ before or at about day 18. In some embodiments, midbrain-destined dopamine (DA) neurons are FOXA2+ / TH+ before or at about day 25.

[0333] In some embodiments, the therapeutic compositions described herein comprise committed dopamine progenitor cells (DDPCs) derived from pluripotent stem cells. In some embodiments, the therapeutic compositions produced by any of the methods described herein exhibit one or more of the following: (a) greater than about 2×10 -5 (b) greater than about 5×10 -4 The ratio of EDN3 to CPM GAPDH is greater than about 4×10 -3 The ratio of HES1 to CPM GAPDH is greater than about 8×10 -3 The ratio of PSRC1 to CPM GAPDH is greater than about 2×10 -3 The ratio of NEK6 to CPM GAPDH is greater than about 1×10 -2 The ratio of IQGAP3 to CPM GAPDH is greater than about 8×10 -4 The ratio of USP44 to CPM GAPDH; (h) greater than about 3×10 -3 The ratio of CEP55 to CPM GAPDH is greater than about 2×10 -2 The ratio of KIF20A to CPMGAPDH is greater than about 2×10 -2 The ratio of AURKA to CPM GAPDH; (k) is less than about 1×10 -3 The ratio of CALCA to CPM GAPDH; (l) less than about 1×10 -3 The ratio of GLRA2 to CPM GAPDH (m) is less than about 1×10 -1 The ratio of MAPT to CPM GAPDH; (n) is less than about 5×10 -2 The ratio of CAMK2B to CPM GAPDH; (o) less than about 1×10 -2 The ratio of SYT13 to CPM GAPDH (p) is less than about 1×10 -3 The ratio of LHFPL4 to CPM GAPDH (q) is less than about 5×10 -3 The ratio of RET to CPM GAPDH; (r) is less than about 1×10 -2 The ratio of KCND3 to CPM GAPDH is less than about 5×10 -2 The ratio of NSG2 to CPM GAPDH is less than about 2×10 -2 The ratio of SNAP25 to CPM GAPDH.

[0334] In some embodiments, (a) the ratio of CPM NEUROG1 to CPM GAPDH is between about 5×10 -5 About 5×10 -3 (b) The ratio of CPM EDN3 to CPM GAPDH is between about 1×10 -3 About 5×10 -2 (c) The ratio of CPM HES1 to CPM GAPDH is between about 5×10 -3 About 5×10 -2 (d) The ratio of CPM PSRC1 to CPM GAPDH is between about 1×10 -2 About 5×10 -2 (e) The ratio of CPM NEK6 to CPM GAPDH is between about 2×10 -2 About 5×10 -1 (f) The ratio of IQGAP3 to CPM GAPDH is between about 1.5×10 -2 About 1×10 -1 (g) The ratio of CPM USP44 to CPMGAPDH is between about 5×10 -3 About 5×10 -2 (h) The ratio of CPM CEP55 to CPM GAPDH is between about 4×10 -3 About 5×10 -2 (i) The ratio of CPM KIF20A to CPM GAPDH is between 2.5×10 -2 About 2×10 -1 (j) The ratio of CPM AURKA to CPM GAPDH is between about 2.6×10 -2 About 2×10 -1 (k) The ratio of CPM CALCA to CPM GAPDH is between about 1×10 -5 About 1×10 -3 (l) The ratio of CPM GLRA2 to CPM GAPDH is between about 1×10 -6 About 1×10 -3 The ratio of (m)CPM MAPT to CPM GAPDH is between about 5×10 -3 About 1×10 -1 (n) The ratio of CPM CAMK2B to CPM GAPDH is between about 1×10 -3 About 5×10 -2 (o) The ratio of CPM SYT13 to CPM GAPDH is between about 1×10 -3 About 1×10-2 (p) The ratio of CPM LHFPL4 to CPM GAPDH is between about 1×10 -4 About 1×10 -3 (q) The ratio of CPM RET to CPM GAPDH is between about 1×10 -4 About 4×10 -3 (r) The ratio of CPM KCND3 to CPM GAPDH is between about 1×10 -3 About 1×10 -2 The ratio of (s)CPM NSG2 to CPMGAPDH is between about 1×10 -3 About 2.4×10 -2 and / or (t) the ratio of CPM SNAP25 to CPM GAPDH is between about 1×10 -3 About 1×10 -2 between.

[0335] Methods for measuring or assessing gene expression or gene products (including transcription and / or translation products) include those methods described in Section I. In addition, in some embodiments, measuring or assessing gene expression or gene products is or includes assessing, measuring, determining and / or quantifying the level, amount or concentration of a gene product (transcription and / or translation) in a sample.

[0336] In some embodiments, gene expression is or includes the process of using gene information for the synthesis of gene product.Therefore, in some embodiments, gene product is any biomolecule assembled, generated and / or synthesized with the information of gene encoding, and can include polynucleotides and / or polypeptides.In specific embodiments, assessing, measuring and / or determining gene expression is or includes determining or measuring the level, amount or concentration of gene product.In certain embodiments, the level, amount or concentration of gene product can be converted (for example, standardized) or directly analyzed (for example, original).

[0337] In some embodiments, gene products are or include proteins encoded and / or expressed by genes, i.e., polypeptides. In specific embodiments, gene product encoding is confined and / or exposed to the cell surface protein. In some embodiments, protein is soluble protein. In certain embodiments, protein is secreted by cells. In specific embodiments, gene expression is the amount, level and / or concentration of proteins encoded by genes. In certain embodiments, one or more protein gene products are measured by any suitable method known in the art. Suitable methods for assessing, measuring, determining and / or quantifying the level, amount or concentration of one or more protein gene products include, but are not limited to, immunoassays, nucleic acid-based or protein-based aptamer technology, HPLC (high-precision liquid chromatography), peptide sequencing (such as Edman degradation sequencing or mass spectrometry (e.g., MS / MS), optionally in combination with HPLC), and any of the aforementioned microarray adaptations (including nucleic acid, antibody or protein-protein (i.e., non-antibody) arrays) for detection. In some embodiments, immunoassays are or include methods or assays based on immune responses, such as detecting antibodies or antigen-binding antibody fragments and gene products for detecting proteins in combination. Immunoassays include, but are not limited to, quantitative immunocytochemistry or immunohistochemistry, ELISA (including direct, indirect, sandwich, competitive, multiplex and portable ELISA (see, e.g., U.S. Pat. No. 7,510,687), Western blotting (including one-, two- or higher-dimensional blotting or other chromatographic means, optionally including peptide sequencing), enzyme immunoassay (EIA), RIA (radioimmunoassay) and SPR (surface plasmon resonance).

[0338] In certain embodiments, gene product is a polynucleotide encoded by a gene, such as mRNA or protein. In some embodiments, gene product is a polynucleotide expressed and / or encoded by a gene. In certain embodiments, polynucleotide is RNA. In some embodiments, gene product is messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA, small nuclear RNA, small nucleolar RNA, antisense RNA, long non-coding RNA, microRNA, Piwi interaction RNA, small interfering RNA and / or short hairpin RNA. In specific embodiments, gene product is mRNA.

[0339] In a specific embodiment, the amount or level of the RNA gene product is assessed, measured, determined and / or quantified, including the step of generating, polymerizing and / or deriving cDNA polynucleotides and / or cDNA oligonucleotides from the RNA gene product. In certain embodiments, the RNA gene product is assessed, measured, determined and / or quantified by directly assessing, measuring, determining and / or quantifying cDNA polynucleotides and / or cDNA oligonucleotides derived from the RNA gene product.

[0340] In certain embodiments, the amount or level of a polynucleotide in a sample can be assessed, measured, determined, and / or quantified by any suitable method known in the art. For example, in some embodiments, the amount or level of a polynucleotide gene product can be assessed, measured, determined, and / or quantified by polymerase chain reaction (PCR), including reverse transcriptase (rt) PCR, droplet digital PCR, real-time and quantitative PCR (qPCR) methods (including, for example, Molecular beacons, LIGHTUP TM SCORPION TM , See, e.g., U.S. Pat. Nos. 5,538,848; 5,925,517; 6,174,670; 6,329,144; 6,326,145 and 6,635,427); northern blotting; e.g., Southern blotting of reverse transcription products and derivatives; array-based methods, including blot arrays, microarrays, or in situ synthesis arrays; and sequencing, e.g., sequencing by synthesis, pyrosequencing, dideoxy sequencing, or sequencing by ligation, or any other method known in the art, such as discussed in Shendure et al., Nat. Rev. Genet. 5:335-44 (2004) or Nowrousian, Euk. Cell 9(9):1300-1310 (2010), including such as 454, ABI and In certain embodiments, the level of nucleic acid gene products is measured by quantitative PCR (qPCR) methods, such as qRT-PCR. In some embodiments, qRT-PCR uses three sets of nucleic acids per gene, where the three nucleic acids include a primer pair and a probe that binds between the target nucleic acid regions to which the primers bind - commercially known as Determination method.

[0341] In certain embodiments, the expression of two or more genes is measured or evaluated simultaneously. In certain embodiments, multiplex PCR, such as multiplex rt-PCR evaluation or multiplex quantitative PCR (qPCR), is used to measure, determine and / or quantify the level, amount or concentration of two or more gene products. In some embodiments, a microarray (e.g., and In some embodiments, microarrays are used to evaluate, measure, determine and / or quantify the level, amount or concentration of two or more gene products. In some embodiments, microarrays are used to evaluate, measure, determine and / or quantify the level, amount or concentration of cDNA polynucleotides derived from RNA gene products. In some embodiments, the expression of one or more gene products (e.g., polynucleotide gene products) is determined by sequencing the gene product and / or by sequencing the cDNA polynucleotides derived from the gene product. In some embodiments, sequencing is performed by non-Sanger sequencing methods and / or next generation sequencing (NGS) technology. The example of next generation sequencing technology includes but is not limited to massively parallel signature sequencing (MPSS), Polony sequencing, pyrophosphate sequencing, reversible dye terminator sequencing, SOLiD sequencing, ion semiconductor sequencing, DNA nanoball sequencing, Helioscope single molecule sequencing, single molecule real-time (SMRT) sequencing, single molecule real-time (RNAP) sequencing and nanopore DNA sequencing.

[0342] In some embodiments, NGS technology is RNA sequencing (RNA-Seq). In specific embodiments, the expression of the one or more polynucleotide gene products is measured, determined and / or quantified by RNA-Seq. RNA-Seq, also known as whole transcriptome shotgun sequencing, determines the presence and quantity of RNA in a sample. The RNA sequencing method has been adapted to the most common DNA sequencing platforms [HiSeq system (Illumina), 454 genome sequencer FLX system (Roche), Applied Biosystems SOLiD (Life Technologies), IonTorrent (Life Technologies). These platforms require RNA to be initially reverse transcribed into cDNA. In contrast, the single-molecule sequencer HeliScope (Helicos BioSciences) can use RNA as a template for sequencing. The principle verification of direct RNA sequencing on the PacBio RS platform has also been demonstrated (Pacific Bioscience). In some embodiments, the one or more RNA gene products are evaluated, measured, determined and / or quantified by RNA-seq. In some embodiments, RNA-seq is RNA-seq based on tags. In tag-based methods, each transcript is represented by a unique tag. Initially, the tag-based method was developed as a sequence-based method for measuring transcript abundance and identifying differentially expressed genes, assuming that the number of tags (counts) directly corresponds to the abundance of mRNA molecules. The complexity of obtaining sample reduction by sequencing a limited area is crucial for making the Sanger-based method affordable. When NGS technology becomes available, a large number of reads that can be generated promote the analysis of differential gene expression. In the tag-based method, transcript length deviations such as quantification of gene expression levels observed for shotgun methods will not be encountered. By definition, all tag-based methods are chain-specific. In a specific embodiment, one or more RNA gene products are evaluated, measured, determined and / or quantified by RNA-seq based on tags.

[0343] In some embodiments, RNA-seq is shotgun RNA-seq. Many protocols have been described for shotgun RNA-seq, but they share many common steps: fragmentation (which can occur at the RNA level or the cDNA level), conversion of RNA to cDNA (performed by oligo dT or random primers), second strand synthesis, ligation of adapter sequences at the 3' and 5' ends (at the RNA or DNA level), and final amplification. In some embodiments, RNA-seq can focus only on polyadenylated RNA molecules (mainly mRNA, but also some lncRNA, snoRNA, pseudogenes, and histones) if poly(A)+ RNA is selected before fragmentation, or can also include non-polyadenylated RNA if no selection is made. In the latter case, ribosomal RNA (more than 80% of the total RNA pool) needs to be depleted before fragmentation. Therefore, it is clear that differences in capturing the mRNA portion of the transcriptome result in partial overlap in the types of transcripts detected. Moreover, different protocols can affect the abundance and distribution of sequencing reads. This makes it difficult to compare the results of experiments performed with different library preparation protocols.

[0344] In some embodiments, RNA is obtained from each sample, fragmented and used to generate complementary DNA (cDNA) sample, such as a cDNA library for sequencing. Reading can be processed and compared with the human genome, and the expected mapping number of each gene / isotype is estimated and used to measure the reading count. In some embodiments, the reading count is normalized according to the length of gene / isotype and the number of readings in the library, to obtain, for example, the FPKM normalized according to the length of gene / isotype and the number of readings in the library, to obtain the number of fragments (FPKM) of exons per thousand bases in every million mapping readings according to gene length and total mapping readings. In some respects, normalization between samples is realized by normalization, such as the 75th quantile normalization, wherein each sample is scaled by the median value of the 75th quantile from all samples, for example, to obtain quantile-normalized FPKM (FPKQ) value. FPKQ value can be logarithmic transformation (log2).

[0345] In some embodiments, RNA is obtained from each sample, fragmented and used to generate complementary DNA (cDNA) samples, such as cDNA libraries for sequencing. The reads can be processed and compared to the human genome, and the expected number of mappings for each gene / isoform is estimated and used to determine the read counts. In some embodiments, the read counts are normalized according to the length of the gene / isoform and the number of reads in the library. In some embodiments, the read counts are provided in the form of counts per million (CPM).

[0346] In some embodiments, relative gene expression is measured by comparing the CPM of the target gene with the CPM of a housekeeping gene. In some embodiments, the housekeeping gene is GAPDH. In some embodiments, the relative gene expression of the target gene is determined as the ratio of the CPM of the target gene to the CPM of a housekeeping gene (e.g., GAPDH).

[0347] In some embodiments, among any of the compositions provided are pharmaceutical compositions containing a pharmaceutically acceptable carrier. In some embodiments, a cell dose comprising cells produced by any of the methods disclosed herein is provided in the form of a composition or formulation, such as a pharmaceutical composition or formulation. Such compositions can be used according to the provided methods, articles, and / or provided compositions, such as for preventing or treating diseases, conditions, and disorders, such as neurodegenerative disorders.

[0348] The term "pharmaceutical formulation" refers to a preparation which is in such form that the biological activity of the active ingredient contained therein is effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

[0349] "Pharmaceutically acceptable carrier" refers to ingredients in a pharmaceutical formulation other than the active ingredient that are non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0350] In some aspects, the selection of carriers is determined in part by specific cells or agents and / or methods of administration. Therefore, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain a preservative. Suitable preservatives can include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. Preservatives or mixtures thereof are typically present in an amount of about 0.0001% to about 2% of the total composition weight. Carriers are described, for example, by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinyl pyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG).

[0351] In some aspects, a buffer is included in the composition. Suitable buffers include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffers is used. The buffer or a mixture thereof is typically present in an amount of about 0.001% to about 4% of the total composition weight. Methods for preparing an administrable pharmaceutical composition are known. Exemplary methods are described in more detail, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st edition (May 1, 2005).

[0352] The formulation or composition may also contain one or more active ingredients that can be used for specific indications, diseases or conditions that are being prevented or treated with the cell or medicament, wherein the respective activities do not adversely affect each other. Such active ingredients are appropriately present in combination in an amount effective for the intended purpose. Therefore, in some embodiments, the pharmaceutical composition also includes other pharmaceutically active agents or drugs, such as carbidopa-levodopa (e.g., levodopa), dopamine agonists (e.g., pramipexole, ropinirole, rotigotine and apomorphine), MAO B inhibitors (e.g., selegiline, rasagiline and safinamide), catechol O-methyltransferase (COMT) inhibitors (e.g., entacapone and tolcapone), anticholinergics (e.g., benztropine and trihexyphenidyl), amantadine, etc. In some embodiments, the medicament or cell is administered in the form of a salt, such as a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable acid addition salts include those derived from inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid and sulfuric acid, and organic acids, such as tartaric acid, acetic acid, citric acid, malic acid, lactic acid, fumaric acid, benzoic acid, glycolic acid, gluconic acid, succinic acid and aryl sulfonic acids, e.g. p-toluenesulfonic acid.

[0353] The formulation or composition may also be administered in combination with another therapeutic modality useful for the specific indication, disease or condition being prevented or treated with the cell or agent, wherein the activities of each do not adversely affect each other. Thus, in some embodiments, the pharmaceutical composition is administered in combination with deep brain stimulation (DBS).

[0354] In some embodiments, the pharmaceutical composition contains an amount that effectively treats or prevents a disease or condition, such as a therapeutically effective amount or a preventively effective amount of a medicament or cell. In some embodiments, the therapeutic or preventive efficacy is monitored by regular assessment of the treated subject. For repeated administration over a few days or longer, as appropriate, repeated treatment occurs until the desired inhibition of disease symptoms occurs. However, other dosage regimens may also be useful and can be determined. The desired dose can be delivered by administering the composition by a single bolus, by administering the composition by multiple boluses, or by administering the composition by continuous infusion.

[0355] The agent or cell can be administered by any suitable means, for example, by stereotactic injection (e.g., using a catheter). In some embodiments, a given dose is administered by a single bolus of the cell or agent. In some embodiments, the cell or agent is administered by multiple bolus injections, for example, over a period of several months or years. In some embodiments, the agent or cell can be administered by stereotactic injection into the brain, such as in the striatum.

[0356] For the prevention or treatment of disease, the appropriate dosage may depend on the type of disease to be treated, the type of one or more agents, the type of cell or recombinant receptor, the severity and course of the disease, whether the agent or cell is administered for preventive or therapeutic purposes, previous therapy, the subject's clinical history and response to the agent or cell, and the judgment of the attending physician. In some embodiments, the composition is appropriately administered to the subject at one time or over a series of treatments.

[0357] The cells or agents can be administered using standard administration techniques, formulations and / or devices. Formulations and devices, such as syringes and vials, for storing and administering compositions are provided. With regard to cells, administration can be autologous. For example, non-pluripotent cells (e.g., fibroblasts) can be obtained from a subject and administered to the same subject after reprogramming and differentiation. When administering a therapeutic composition (e.g., a pharmaceutical composition containing a medicament for the treatment or improvement of the symptoms of a disease or disorder (such as a neurodegenerative disorder)), it is usually formulated into a unit dose injection form (solution, suspension, emulsion). Formulations include those for stereotactic administration, such as formulations administered to the brain (e.g., striatum).

[0358] Composition is provided in the form of sterile liquid preparation in some embodiments, for example isotonic aqueous solution, suspension, emulsion, dispersion or viscous composition, and described liquid preparation can be buffered to selected pH in some respects.Liquid preparation is easier to prepare than gel, other viscous composition and solid composition usually.In addition, liquid composition is slightly more convenient to use, especially by injection.On the other hand, viscous composition can be prepared in suitable viscosity range, to provide longer contact period with specific tissue.Liquid or viscous composition can comprise carrier, and described carrier can be the solvent or dispersion medium containing for example water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and their suitable mixture.

[0359] Sterile injection solutions can be prepared by incorporating the agent or cells into a solvent, such as mixing with a suitable carrier, diluent or excipient such as sterile water, physiological saline, glucose, dextrose and the like.

[0360] Formulations for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

[0361] IV. Treatment

[0362] Provided herein are methods for treating a disease or condition of a subject in need thereof using any of the compositions provided. In specific embodiments, the composition is produced by the methods provided herein. Such methods and uses include methods of treatment and uses, for example, including administering therapeutic cells or compositions containing therapeutic cells to a subject suffering from a disease, condition or illness. In some embodiments, the disease or condition is a neurodegenerative disease or condition. In some embodiments, the cell or its pharmaceutical composition is administered in an effective amount to achieve treatment of the disease or condition. Uses include the use of the cell or its pharmaceutical composition in such methods and treatments, and in the preparation of a drug in order to implement such methods of treatment. In some embodiments, the method thus treats a disease or condition or illness of a subject.

[0363] The present disclosure relates to methods for lineage-specific differentiation of pluripotent stem cells (PSCs), including embryonic stem (ES) cells and induced pluripotent stem cells (iPSCs), for use in neurodegenerative diseases. Specifically, the methods, compositions, and uses thereof provided herein contemplate the differentiation of pluripotent stem cells for administration to subjects exhibiting dopamine (DA) neuron loss, including Parkinson's disease.

[0364] Parkinson's disease (PD) is the second most common neurodegenerative disease, affecting an estimated 4-5 million patients worldwide. This number is expected to more than double by 2030. PD is the second most common neurodegenerative condition after Alzheimer's disease, affecting approximately 1 million patients in the United States, with 60,000 new patients diagnosed each year. There is currently no cure for PD, the pathology of which is characterized by the selective loss of midbrain DA neurons in the substantia nigra. The fundamental characteristic of PD is therefore the progressive, severe and irreversible loss of midbrain dopamine (DA) neurons, ultimately leading to motor dysfunction.

[0365] In some embodiments, the subject suffers from a neurodegenerative disease. In some embodiments, the neurodegenerative disease includes the loss of dopamine neurons in the brain. In some embodiments, the subject has lost dopamine neurons in the substantia nigra (SN). In some embodiments, the subject has lost dopamine neurons in the substantia nigra compacta (SNc). In some embodiments, the subject exhibits rigidity, bradykinesia, postural reflex impairment, resting tremor, or a combination thereof. In some embodiments, the subject exhibits an abnormal [18F]-L-DOPA PET scan. In some embodiments, the subject exhibits [18F]-DG-PET evidence of Parkinson's disease-related patterns (PDRP).

[0366] In some embodiments, the neurodegenerative disease is Parkinson's syndrome. In some embodiments, the neurodegenerative disease is Parkinson's disease. In some embodiments, the neurodegenerative disease is idiopathic Parkinson's disease. In some embodiments, the neurodegenerative disease is a familial form of Parkinson's disease. In some embodiments, the subject suffers from mild Parkinson's disease. In some embodiments, the subject has a Movement Disorder Association-Unified Parkinson's Disease Rating Scale (MDS-UPDRS) motor score of less than or equal to 32. In some embodiments, the subject suffers from moderate or late Parkinson's disease. In some embodiments, the subject suffers from mild Parkinson's disease. In some embodiments, the subject has an MDS-UPDRS motor score between 33 and 60.

[0367] In some embodiments, according to the provided methods and / or provided articles or compositions, a dose of cells is administered to a subject. In some embodiments, the size or timing of the dose is determined according to the subject's specific disease or condition. In some cases, the size or timing of the dose for a specific disease can be determined empirically in view of the provided description.

[0368] In some embodiments, the dose of cells is administered to the striatum of the subject. In some embodiments, the dose of cells is administered to one hemisphere of the striatum of the subject. In some embodiments, the dose of cells is administered to both hemispheres of the striatum of the subject.

[0369] In some embodiments, the dose of cells administered to a subject is about 5×10 6 In some embodiments, the dose of cells administered to a subject is about 10×10 6 In some embodiments, the dose of cells administered to a subject is about 15×10 6 In some embodiments, the dose of cells administered to a subject is about 20×10 6 In some embodiments, the dose of cells administered to a subject is about 25×10 6 In some embodiments, the dose of cells administered to a subject is about 30×10 6 cells.

[0370] In some embodiments, the cell dose includes just about 250,000 cells per hemisphere to just about 20 million cells per hemisphere, just about 500,000 cells per hemisphere to just about 20 million cells per hemisphere, just about 1 million cells per hemisphere to just about 20 million cells per hemisphere, just about 5 million cells per hemisphere to just about 20 million cells per hemisphere, just about 10 million cells per hemisphere to just about 20 million cells per hemisphere, cells per hemisphere, from just or about 15 million cells per hemisphere to just or about 20 million cells per hemisphere, from just or about 250,000 cells per hemisphere to just or about 15 million cells per hemisphere, from just or about 500,000 cells per hemisphere to just or about 15 million cells per hemisphere, from just or about 1 million cells per hemisphere to just or about 15 million cells per hemisphere, from just or about 5 million cells per hemisphere to just or about 15 million cells per hemisphere, 0,000 cells per hemisphere to just or about 15 million cells per hemisphere, just or about 250,000 cells per hemisphere to just or about 10 million cells per hemisphere, just or about 500,000 cells per hemisphere to just or about 10 million cells per hemisphere, just or about 1 million cells per hemisphere to just or about 10 million cells per hemisphere, just or about 5 million cells per hemisphere to just or about 10 million cells per hemisphere, just or about 250,000 cells per hemisphere to just or about 10 million cells per hemisphere or about 5 million cells, from just or about 500,000 cells per hemisphere to just or about 5 million cells per hemisphere, from just or about 1 million cells per hemisphere to just or about 5 million cells per hemisphere, from just or about 250,000 cells per hemisphere to just or about 1 million cells per hemisphere, from just or about 500,000 cells per hemisphere to just or about 1 million cells per hemisphere, or from just or about 250,000 cells per hemisphere to just or about 500,000 cells per hemisphere.

[0371] In some embodiments, the cell dose is between just or about 1 million cells per hemisphere and just or about 30 million cells per hemisphere. In some embodiments, the cell dose is between just or about 5 million cells per hemisphere and just or about 20 million cells per hemisphere. In some embodiments, the cell dose is between just or about 10 million cells per hemisphere and just or about 15 million cells per hemisphere.

[0372] In some embodiments, the cell dose is about 3×10 6 cells / hemisphere to 15×10 6 cells / hemisphere. In some embodiments, the cell dose is about 3×10 6cells / hemisphere. In some embodiments, the cell dose is about 4×10 6 cells / hemisphere. In some embodiments, the cell dose is about 5×10 6 cells / hemisphere. In some embodiments, the cell dose is about 6×10 6 cells / hemisphere. In some embodiments, the cell dose is about 7×10 6 cells / hemisphere. In some embodiments, the cell dose is about 8×10 6 cells / hemisphere. In some embodiments, the cell dose is about 9×10 6 cells / hemisphere. In some embodiments, the cell dose is about 10×10 6 cells / hemisphere. In some embodiments, the cell dose is about 11×10 6 cells / hemisphere. In some embodiments, the cell dose is about 12×10 6 cells / hemisphere. In some embodiments, the cell dose is about 13×10 6 cells / hemisphere. In some embodiments, the cell dose is about 14×10 6 cells / hemisphere. In some embodiments, the cell dose is about 15×10 6 cells / hemisphere.

[0373] In some embodiments, the number of cells administered to a subject is about 0.25×10 6 The total number of cells is about 20 × 10 6 Total cells, about 0.25×10 6 The total number of cells is about 15 × 10 6 Total cells, about 0.25×10 6 The total number of cells is about 10 × 10 6 Total cells, about 0.25×10 6 The total number of cells is about 5×10 6 Total cells, about 0.25×10 6 The total number of cells is about 1×10 6 Total cells, about 0.25×10 6 The total number of cells was about 0.75 × 10 6 Total cells, about 0.25×10 6 The total number of cells is about 0.5×10 6 Total cells, about 0.5×10 6 The total number of cells is about 20 × 10 6 Total cells, about 0.5×10 6 The total number of cells is about 15 × 10 6 Total cells, about 0.5×10 6The total number of cells is about 10 × 10 6 Total cells, about 0.5×10 6 The total number of cells is about 5×10 6 Total cells, about 0.5×10 6 The total number of cells is about 1×10 6 Total cells, about 0.5×10 6 The total number of cells was about 0.75 × 10 6 Total cells, about 0.75×10 6 The total number of cells is about 20 × 10 6 Total cells, about 0.75×10 6 The total number of cells is about 15 × 10 6 Total cells, about 0.75×10 6 The total number of cells is about 10 × 10 6 Total cells, about 0.75×10 6 The total number of cells is about 5×10 6 Total cells, about 0.75×10 6 The total number of cells is about 1×10 6 Total cells, about 1×10 6 The total number of cells is about 20 × 10 6 Total cells, about 1×10 6 The total number of cells is about 15 × 10 6 Total cells, about 1×10 6 The total number of cells is about 10 × 10 6 Total cells, about 1×10 6 The total number of cells is about 5×10 6 Total cells, about 5×10 6 The total number of cells is about 20 × 10 6 Total cells, about 5×10 6 The total number of cells is about 15 × 10 6 Total cells, about 5×10 6 The total number of cells is about 10 × 10 6 Total cells, about 10×10 6 The total number of cells is about 20 × 10 6 Total cells, about 10×10 6 The total number of cells is about 15 × 10 6 Total cells or about 15 × 10 6 The total number of cells is about 20 × 10 6 Total cells.

[0374] In certain embodiments, a single population of cells or cell subtypes is administered to a subject in a range of about 5 million cells per hemisphere to about 20 million cells per hemisphere, or any value between these ranges. The dosage may vary depending on the disease or condition and / or patient and / or other treatment-specific attributes.

[0375] In some embodiments, the patient is administered multiple doses, and each dose or the total dose can be within any of the aforementioned values. In some embodiments, the cell dose comprises administering just or about 5 million cells per hemisphere to about 20 million cells per hemisphere, including each end value.

[0376] In some embodiments, a dose of cells (eg, differentiated cells) is administered to a subject as a single dose, or is administered only once over a two-week, one-month, three-month, six-month, one-year, or longer period.

[0377] In the case of stem cell transplantation, the administration of a given "dose" encompasses the administration of a given amount or number of cells in the form of a single composition and / or a single uninterrupted administration, for example in the form of a single injection or continuous infusion, and also encompasses the administration of a given amount or number of cells in divided doses or in the form of multiple compositions, with multiple separate compositions or infusions provided over a specific time period (such as a day). Thus, in some cases, the dose is a single or continuous administration of a specified number of cells given or initiated at a single time point. However, in some cases, the dose is administered by multiple injections or infusions within a single period, such as by multiple infusions within a one-day period.

[0378] Thus, in some aspects, the dose of cells is administered in a single pharmaceutical composition. In some embodiments, the dose of cells is administered in multiple compositions that together contain the dose of cells.

[0379] In some embodiments, the dose of cells can be administered by administering a plurality of compositions or solutions, such as a first and a second, optionally more compositions or solutions, each containing some of the dose of cells. In some aspects, a plurality of compositions each containing a different cell population and / or cell subtype are administered individually or independently, optionally over a period of time.

[0380] In some embodiments, the administration of the composition or dosage, such as the administration of multiple cell compositions, includes separate administration of cell compositions. In some aspects, separate administration is performed simultaneously, or sequentially in any order.

[0381] In some embodiments, the subject receives multiple doses of cells, such as two or more doses or multiple consecutive doses of cells. In some embodiments, two doses are administered to the subject. In some embodiments, multiple consecutive doses are administered after the first dose so that one or more additional doses are administered after the consecutive doses are administered. In some aspects, the number of cells administered to the subject with the additional dose is the same or similar to the first dose and / or consecutive dose. In some embodiments, the one or more additional doses are greater than the previous dose.

[0382] In some aspects, the size of the first and / or subsequent doses is determined based on one or more criteria, such as the subject's response to previous treatments, e.g., disease stage and / or the subject's likelihood or incidence of developing an adverse outcome (e.g., movement disorder).

[0383] In some embodiments, the cell dose is generally large enough to be effective to ameliorate disease symptoms.

[0384] In some embodiments, cells are administered at a desired dose, which in some aspects includes the desired dose or number of cells or cell types and / or the desired ratio of cell types. In some embodiments, the cell dose is based on the total number of cells (or number per kg body weight) required in a single population or a single cell type (e.g., TH+ or TH-). In some embodiments, the dose is based on a combination of such features, such as the total number of cells required, the desired ratio, and the total number of cells required in a single population.

[0385] Thus, in some embodiments, the dose is based on a desired total fixed dose of cells and a desired ratio, and / or based on a desired fixed dose of one or more (eg, each) of the individual subtypes or subpopulations.

[0386] In certain embodiments, the number and / or concentration of cells refers to the number of TH-negative cells. In other embodiments, the number and / or concentration of cells refers to the number or concentration of all cells administered.

[0387] In some embodiments, the cell is administered with a desired dose, and in some aspects, the desired dose includes the desired dose or number of cells or cell types and / or the desired ratio of cell types. Therefore, in some embodiments, the cell dose is based on the total number of cells and the required ratio of a single colony or hypotype. In some embodiments, the cell dose is based on the required total number (or number per kg body weight) of cells in a single colony or a single cell type. In some embodiments, the dosage is based on a combination of such features, such as the total number of cells required, the required ratio, and the total number of cells required in a single colony.

[0388] Thus, in some embodiments, the dose is based on a desired total fixed dose of cells and a desired ratio, and / or based on a desired fixed dose of one or more (eg, each) of the individual subtypes or subpopulations.

[0389] In certain embodiments, the number and / or concentration of cells refers to the number of TH-negative cells. In other embodiments, the number and / or concentration of cells refers to the number or concentration of all cells administered.

[0390] In some aspects, the size of the dose is determined based on one or more criteria, such as the subject's response to previous treatments, e.g., disease type and / or stage and / or the likelihood or incidence of the subject developing toxic consequences (e.g., movement disorders).

[0391] V. Products and kits

[0392] Also provided are articles, systems, devices and kits that can be used to perform the provided methods. Also provided are articles, comprising: (i) one or more reagents for differentiating pluripotent stem cells into midbrain floor plate progenitor cells, committed dopamine (DA) neuron progenitor cells and / or dopamine (DA) neurons; and (ii) instructions for using the one or more reagents to perform any of the methods described herein.

[0393] In some embodiments of any such embodiments, the reagent for differentiation is or includes a small molecule capable of inhibiting TGF-β / activin Nodal signaling. In some embodiments of any such embodiments, the reagent for differentiation is or includes SB431542. In some embodiments of any such embodiments, the reagent for differentiation is or includes a small molecule capable of activating SHH signaling. In some embodiments of any such embodiments, the reagent for activating SHH signaling is or includes SHH. In some embodiments of any such embodiments, the reagent for activating SHH signaling is or includes purmorphamine. In some embodiments of any such embodiments, the reagent for activating SHH signaling is or includes SHH and purmorphamine. In some embodiments of any such embodiments, the reagent for differentiation is or includes a small molecule capable of inhibiting BMP signaling. In some embodiments of any such embodiments, the reagent for inhibiting BMP signaling is LDN193189. In some embodiments of any such embodiments, the reagent for differentiation is or includes a small molecule capable of inhibiting GSK3β signaling. In some embodiments of any such embodiments, the reagent is or includes CHIR99021. In some embodiments of any such embodiments, the reagent for differentiation is or includes one or more of BDNF, GDNF, dbcAMP, ascorbic acid, TGFβ3, and DAPT. In one embodiment, the reagents in the kit can be in the form of a solution, can be frozen, or can be lyophilized.

[0394] Also provided are articles of manufacture comprising (i) any composition described herein; and (ii) instructions for administering the composition to a subject.

[0395] In some embodiments, the article or kit includes one or more containers (usually multiple containers), packaging materials, and a label or package insert on or accompanying the one or more containers and / or packages, typically including instructions for use, such as instructions for reagents for differentiation of pluripotent cells, such as iPSCs into midbrain floor plate progenitor cells, directed dopamine (DA) neuron progenitor cells and / or dopamine (DA) neurons, and instructions for performing any of the methods provided herein. In some aspects, provided articles contain reagents for differentiation and / or maturation of cells, such as any reagents described in any step of Part II and Part III, for example, at one or more steps of a manufacturing process.

[0396] Also provided are articles and kits containing differentiated cells, such as those generated using the methods provided herein, and optionally instructions for use, such as instructions for administration. In some embodiments, the instructions provide guidance or specify methods for assessing whether a subject is likely or suspected to be likely to respond and / or the extent or level of response after administration of differentiated cells expressing a recombinant receptor to treat a disease or condition prior to receiving cell therapy. In some aspects, the article may contain a dose of differentiated cells or a composition of differentiated cells.

[0397] Articles provided herein contain packaging materials. Packaging materials for packaging provided materials are well known to those skilled in the art. See, for example, U.S. Patent Nos. 5,323,907, 5,052,558 and 5,033,252, each of which is incorporated herein in its entirety. Examples of packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, disposable laboratory supplies, such as pipette tips and / or plastic plates, or bottles. Articles or test kits may include a device to facilitate the distribution of materials or to facilitate use in a high throughput or large-scale manner, such as to facilitate use in robotic equipment. Typically, the packaging does not react with the compositions contained therein.

[0398] In some embodiments, reagents and / or cell compositions are packaged separately. In some embodiments, each container can have a single compartment. In some embodiments, other components of an article or test kit are packaged separately, or are packaged together in a single compartment.

[0399] VI. Exemplary embodiments

[0400] Among the embodiments provided are:

[0401] 1. A method for differentiating neural cells, the method comprising:

[0402] (a) performing a first incubation, comprising culturing pluripotent stem cells in a non-adherent culture vessel under conditions that produce cell spheroids, wherein at the beginning of the first incubation (day 0), the cells are exposed to (i) a TGF-β / activin Nodal signaling inhibitor; (ii) at least one sonic hedgehog (SHH) signaling activator; (iii) a bone morphogenetic protein (BMP) signaling inhibitor; and (iv) a glycogen synthase kinase 3β (GSK3β) signaling inhibitor; and

[0403] (b) performing a second incubation comprising culturing the cells of the sphere in a substrate-coated culture vessel under conditions that cause neural differentiation of the cells.

[0404] 2. The method of embodiment 1, wherein the second incubation begins at about day 7.

[0405] 3. The method of embodiment 1 or embodiment 2, wherein the cells are exposed to the TGF-β / Activin Nodal signaling inhibitor until the day before or on day 7.

[0406] 4. A method according to any one of embodiments 1-3, wherein the cells are exposed to the TGF-β / Activin Nodal inhibitor from day 0 to day 6, including every day.

[0407] 5. The method of any one of embodiments 1-4, wherein the cells are exposed to the at least one SHH signaling activator until the day before or on day 7.

[0408] 6. A method according to any one of embodiments 1-5, wherein the cells are exposed to the at least one SHH signaling activator from day 0 to day 6, including every day.

[0409] 7. The method of any one of embodiments 1-6, wherein the cells are exposed to the BMP signaling inhibitor until the day before or on day 11.

[0410] 8. A method according to any one of embodiments 1-7, wherein the cells are exposed to the BMP signaling inhibitor from day 0 to day 10, including every day.

[0411] 9. The method of any one of embodiments 1-8, wherein the cells are exposed to the GSK3β signaling inhibitor until the day before or on day 13.

[0412] 10. The method of any one of embodiments 1-9, wherein the cells are exposed to the GSK3b signaling inhibitor starting from day 0 to day 12, including every day.

[0413] 11. A method according to any one of embodiments 1-10, wherein culturing the cells under conditions that cause neural differentiation of the cells includes exposing the cells to (i) brain-derived neurotrophic factor (BDNF); (ii) ascorbic acid; (iii) glial cell-derived neurotrophic factor (GDNF); (iv) dibutyryl cyclic AMP (dbcAMP); (v) transforming growth factor β-3 (TGFβ3) (collectively referred to as "BAGCT"); and (vi) a Notch signaling inhibitor.

[0414] 12. The method of any one of embodiments 1-11, wherein the cells are exposed to BAGCT and the Notch signaling inhibitor starting from day 11.

[0415] 13. A method according to any one of embodiments 1-12, wherein the cells are exposed to BAGCT and the Notch signaling inhibitor starting from day 11 until the neural differentiated cells are harvested, optionally until day 18, optionally until day 25.

[0416] 14. A method for differentiating neural cells, the method comprising:

[0417] (a) performing a first incubation, comprising culturing pluripotent stem cells in a non-adherent culture vessel under conditions that produce cell spheroids, wherein at the beginning of the first incubation day (day 0), the cells are exposed to (i) a TGF-β / activin Nodal signaling inhibitor until day 6, including every day; (ii) at least one sonic hedgehog (SHH) signaling activator until day 6, including every day; (iii) a bone morphogenetic protein (BMP) signaling inhibitor until day 6, including every day;

[0418] and (iv) glycogen synthase kinase 3β (GSK3β) signaling inhibitor by day 6,

[0419] Includes every day; and

[0420] (b) starting from day 7, performing a second incubation to allow neural differentiation of the cells of the sphere, comprising culturing the cells in a culture vessel coated with a substrate selected from laminin, collagen, entactin, heparin sulfate proteoglycans, and combinations thereof, wherein starting from day 7, the cells are exposed to (i) a BMP signaling inhibitor and (ii) a GSK3β signaling inhibitor; and starting from day 11, the cells are exposed to (i) brain-derived neurotrophic factor (BDNF); (ii) ascorbic acid;

[0421] (iii) glial cell line-derived neurotrophic factor (GDNF); (iv) dibutyryl cyclic AMP

[0422] (dbcAMP); (v) transforming growth factor β-3 (TGFβ3) (collectively referred to as

[0423] "BAGCT"); and (vi) a Notch signaling inhibitor.

[0424] 15. The method according to any one of embodiments 1-14, further comprising harvesting the neural differentiated cells.

[0425] 16. The method of embodiment 15, wherein the harvesting is performed on or after about day 16.

[0426] 17. The method of embodiment 15 or embodiment 16, wherein the harvesting occurs between about day 16 and about day 30.

[0427] 18. The method according to any one of embodiments 15-17, wherein the harvesting is performed between day 18 and day 25.

[0428] 19. The method according to any one of embodiments 15-18, wherein the harvesting is performed on or about day 18.

[0429] 20. The method of any one of embodiments 15-18, wherein the harvesting is performed on or about day 25.

[0430] 21. A method according to any one of embodiments 1-20, wherein the neural differentiated cells are committed dopaminergic neuron progenitor cells.

[0431] 22. A method according to embodiment 21, wherein the dopaminergic neuron progenitor cells are capable of innervating the host tissue after being transplanted into a subject.

[0432] 23. A method according to any one of embodiments 1-22, wherein, prior to the second incubation, the spheres are dissociated to produce a cell suspension, and the cells of the cell suspension are cultured in the substrate-coated culture vessel.

[0433] 24. The method of embodiment 23, wherein the dissociation is performed when the spheroid cells express at least one of PAX6 and OTX2.

[0434] 25. The method of embodiment 23 or embodiment 24, wherein the dissociation is performed on about day 7.

[0435] 26. The method according to any one of embodiments 1-25, wherein the culture vessel is selected from the group consisting of: a plate, a culture dish, a flask, and a bioreactor.

[0436] 27. The method of any one of embodiments 1-26, wherein the culture vessel is a plate.

[0437] 28. A method according to embodiment 27, wherein the plate is a 6-well plate, a 12-well plate, a 24-well plate or a 96-well plate.

[0438] 29. A method according to embodiment 27 or embodiment 28, wherein the plate is a microwell plate.

[0439] 30. A method according to embodiment 29, wherein the microwell plate is a 6-well plate.

[0440] 31. A method according to embodiment 29, wherein the microwell plate is a 24-well plate.

[0441] 32. The method according to any one of embodiments 1-31, wherein the culture vessel is Aggrewell TM plate.

[0442] 33. The method of any one of embodiments 29-32, wherein the culture of pluripotent stem cells at day 0 comprises about 0.1×10 6 cells / cm 2 About 2×10 6 cells / cm 2 , about 0.1×10 6 cells / cm 2 About 1×10 6 cells / cm 2 , about 0.1×10 6 cells / cm 2 About 0.8×10 6 cells / cm 2 , about 0.1×106 cells / cm 2 About 0.6×10 6 cells / cm 2 , about 0.1×10 6 cells / cm 2 About 0.4×10 6 cells / cm 2 , about 0.1×10 6 cells / cm 2 About 0.2×10 6 cells / cm 2 , about 0.2×10 6 cells / cm 2 About 2×10 6 cells / cm 2 , about 0.2×10 6 cells / cm 2 About 1×10 6 cells / cm 2 , about 0.2×10 6 cells / cm 2 About 0.8×10 6 cells / cm 2 , about 0.2×10 6 cells / cm 2 About 0.6×10 6 cells / cm 2 , about 0.2×10 6 cells / cm 2 About 0.4×10 6 cells / cm 2 , about 0.4×10 6 cells / cm 2 About 2×10 6 cells / cm 2 , about 0.4×10 6 cells / cm 2 About 1×10 6 cells / cm 2 , about 0.4×10 6 cells / cm 2 About 0.8×10 6 cells / cm 2 , about 0.4×10 6 cells / cm 2 About 0.6×10 6 cells / cm 2 , about 0.6×10 6 cells / cm 2 About 2×10 6cells / cm 2 , about 0.6×10 6 cells / cm 2 About 1×10 6 cells / cm 2 , about 0.6×10 6 cells / cm 2 About 0.8×10 6 cells / cm 2 , about 0.8×10 6 cells / cm 2 About 2×10 6 cells / cm 2 , about 0.8×10 6 cells / cm 2 About 1×10 6 cells / cm 2 or about 1.0×10 6 cells / cm 2 About 2×10 6 cells / cm 2 .

[0443] 34. The method according to any one of embodiments 29-33, wherein the culture of pluripotent stem cells on day 0 is performed in a 6-well plate and comprises about 1×10 6 pluripotent stem cells / well is about 20×10 6 pluripotent stem cells / well, about 1×10 6 15×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 1×10 6 10×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 1×10 6 5×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 5×10 6 pluripotent stem cells / well is about 20×10 6 pluripotent stem cells / well, about 5×10 6 15×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 5×10 6 10×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 10×10 6 pluripotent stem cells / well is about 20×10 6 pluripotent stem cells / well, about 10×10 6 pluripotent stem cells

[0444] / hole is about 15×10 6pluripotent stem cells / well or about 15×10 6 pluripotent stem cells / well is about 20×10 6 pluripotent stem cells / well.

[0445] 35. The method of any one of embodiments 29-34, wherein the culture of day 0 pluripotent stem cells is performed in a 24-well plate and comprises about 1×10 5 pluripotent stem cells /

[0446] The hole is about 5×10 6 pluripotent stem cells / well, about 1×10 5 1×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 1×10 5 5×10 pluripotent stem cells / well 5 pluripotent stem cells / well, about 5×10 5 5×10 pluripotent stem cells / well 6 pluripotent stem cells / well, about 5×10 5 1×10 pluripotent stem cells / well 6 pluripotent stem cells / well or about 1×10 6 5×10 pluripotent stem cells / well 6 pluripotent stem cells / well.

[0447] 36. The method of any one of embodiments 29-35, wherein the culture of day 0 pluripotent stem cells comprises a sufficient number of cells to produce spheres comprising about 1,000 cells to about 5,000 cells or about 2,000 cells to about 3,000 cells on about day 7.

[0448] 37. The method of any one of embodiments 29-36, wherein the culture of day 0 pluripotent stem cells comprises a sufficient number of cells to produce spheres comprising about 2,000 cells at about day 7.

[0449] 38. The method of any one of embodiments 29-36, wherein the culture of day 0 pluripotent stem cells comprises a sufficient number of cells to produce spheres comprising about 3,000 cells at about day 7.

[0450] 39. The method of any one of embodiments 1-38, wherein the culture vessel is treated to reduce or eliminate cell adhesion.

[0451] 40. The method of claim 39, wherein the treating the culture vessel comprises incubating the culture vessel with pluronic acid.

[0452] 41. The method of any one of embodiments 1-40, wherein the non-adherent culture vessel is exposed to a surfactant prior to the first incubation.

[0453] 42. The method of embodiment 41, wherein the surfactant is pluronic acid.

[0454] 43. A method according to any one of embodiments 1-42, wherein the non-adherent culture vessel comprises a low or ultra-low attachment surface.

[0455] 44. According to the method according to any one of embodiments 1-43, the non-adherent culture vessel comprises an ultra-low attachment surface.

[0456] 45. A method according to any one of embodiments 1-44, wherein the substrate is a basement membrane protein.

[0457] 46. ​​The method of any one of embodiments 1-45, wherein the substrate is selected from one or more of laminin, collagen, entactin, heparin sulfate proteoglycans, and combinations thereof.

[0458] 47. The method of any one of embodiments 1-46, wherein the substrate is a recombinant protein.

[0459] 48. The method of any one of embodiments 1-47, wherein the substrate is recombinant laminin.

[0460] 49. The method of any one of embodiments 1-48, wherein the substrate-coated culture vessel is exposed to poly-L-ornithine prior to being used to culture the cells.

[0461] 50. The method of any one of embodiments 1-49, wherein the TGF-β / Activin Nodal signaling inhibitor is SB431542.

[0462] 51. A method according to embodiment 50, wherein the cells are exposed to SB431542 at a concentration of about 1 μM to about 20 μM, about 5 μM to about 15 μM, or about 8 μM to about 12 μM, optionally about 10 μM.

[0463] 52. The method according to any one of embodiments 1-51, wherein the at least one SHH

[0464] The signal transduction activator was SHH or purmorphamine.

[0465] 53. The method according to any one of embodiments 1-51, wherein the at least one SHH

[0466] The signaling activator comprises two SHH signaling activators selected from SHH protein and purmorphamine.

[0467] 54. A method according to embodiment 52 or embodiment 53, wherein the cells are exposed to SHH at a concentration of about 10 ng / mL to 500 ng / mL, about 20 ng / mL to about 400 ng / mL, about 50 ng / mL to about 200 ng / mL, or about 75 ng / mL to about 150 ng / mL, optionally about 100 ng / mL.

[0468] 55. The method of any one of embodiments 52-54, wherein the cell is exposed to purmorphamine at a concentration of about 1 μM to about 20 μM, about 5 μM to about 15 μM, or about 8 μM to about 12 μM, optionally about 10 μM.

[0469] 56. The method of any one of embodiments 1-55, wherein the BMP signaling inhibitor is LDN193189.

[0470] 57. A method according to embodiment 56, wherein the cells are exposed to LDN193189 at a concentration of about 10 nM to 500 nM, about 20 nM to about 400 nM, about 50 nM to about 200 nM, or about 75 nM to about 150 nM, optionally about 100 nM.

[0471] 58. The method of any one of embodiments 1-57, wherein the GSK3β signaling inhibitor is CHIR99021.

[0472] 59. The method of embodiment 58, wherein the cells are exposed to CHIR99021 at a concentration of about 0.1 μM to about 5 μM, about 0.5 μM to about 4 μM, or about 1 μM to about 3 μM, optionally about 2 μM.

[0473] 60. The method of any one of embodiments 11-59, wherein the cells are exposed to a concentration of about 1 ng / mL to about 100 ng / mL, about 5 ng / mL to about 80 ng / mL, about 10 ng / mL to about 60 ng / mL, or about 15 ng / mL to about 30 ng / mL, optionally about

[0474] 20ng / mL of GDNF.

[0475] 61. The method of any one of embodiments 11-60, wherein the cells are exposed to a concentration of about 1 ng / mL to about 100 ng / mL, about 5 ng / mL to about 80 ng / mL, about 10 ng / mL to about 60 ng / mL, or about 15 ng / mL to about 30 ng / mL, optionally about

[0476] 20ng / mL of BDNF.

[0477] 62. The method of embodiments 11-61, wherein the cells are exposed to a concentration of about 0.1 mM to 5 mM, about 0.2 mM to about 4 mM, about 0.3 mM to about 3 mM, or about

[0478] 0.4 mM to about 2 mM, optionally about 0.5 mM dbcAMP.

[0479] 63. The method of any one of embodiments 11-62, wherein the cells are exposed to ascorbic acid at a concentration of about 0.05 mM to about 5 mM, about 0.1 mM to about 1 mM, or about 0.2 mM to about 0.5 mM, optionally about 0.2 mM.

[0480] 64. A method according to any one of embodiments 11-63, wherein the cells are exposed to TGFβ3 at a concentration of about 0.1 ng / mL to about 5 ng / mL, about 0.3 ng / mL to about 3 ng / mL, or about 0.5 ng / mL to about 2 ng / mL, optionally about 1 ng / mL.

[0481] 65. A method according to any one of embodiments 11-64, wherein the Notch signaling inhibitor is DAPT.

[0482] 66. The method of any one of embodiments 11-65, wherein the cells are exposed to DAPT at a concentration of about 1 μM to about 20 μM, about 5 μM to about 15 μM, or about 8 μM to about 12 μM, optionally about 10 μM.

[0483] 67. The method according to any one of embodiments 1-66, wherein the culturing in the first incubation and / or the second incubation is performed in a culture medium comprising serum or a serum replacement.

[0484] 68. The method of any one of embodiments 1-67, wherein the culturing in the first incubation and the second incubation is performed in a culture medium comprising serum or a serum replacement.

[0485] 69. The method of any one of embodiments 1-68, wherein the cells are cultured in a medium comprising serum or a serum replacement from about day 0 to about day 10.

[0486] 70. The method of any one of embodiments 67-69, wherein the serum or serum replacement comprises about 5% (v / v) of the culture medium.

[0487] 71. The method of any one of embodiments 67-70, wherein the serum or serum replacement comprises about 2% (v / v) of the culture medium.

[0488] 72. The method of any one of embodiments 67-71, wherein the culture medium comprises about 5% serum or serum replacement (v / v) from about day 0 to about day 1, and about 2% serum replacement (v / v) from about day 2 to about day 10.

[0489] 73. The method of any one of embodiments 67-72, wherein the culture medium comprises a serum replacement.

[0490] 74. The method of any one of embodiments 67-73, wherein the serum replacement does not contain fetal bovine serum (FBS).

[0491] 75. The method of any one of embodiments 67-74, wherein the serum replacement is KnockOut TM Serum replacement.

[0492] 76. The method of any one of embodiments 1-66, wherein the cells are cultured in the absence of serum for the duration of the culture.

[0493] 77. A method according to any one of embodiments 1-76, wherein the cells are exposed to a Rho-associated protein kinase (ROCK) signaling inhibitor on day 0, day 7, day 16 and / or day 20.

[0494] 78. A method according to any one of embodiments 1-77, wherein the cells are exposed to a Rho-associated kinase protein (ROCK) signaling inhibitor on day 0, day 7, day 16 and day 20.

[0495] 79. The method of embodiment 77 or embodiment 78, wherein the ROCK inhibitor is Y-27632.

[0496] 80. The method of any one of embodiments 77-79, wherein the cells are exposed to the ROCK inhibitor at a concentration of about 1 μM to about 20 μM, about 5 μM to about 15 μM, or about 8 μM to about 12 μM, optionally about 10 μM.

[0497] 81. The method of any one of embodiments 1-80, wherein at least about 50% of the culture medium is replaced every day, every other day, or every three days.

[0498] 82. The method of any one of embodiments 23-81, wherein the spheroids are dissociated by enzymatic dissociation.

[0499] 83. The method of any one of embodiments 23-82, wherein the spheroids are dissociated by enzymatic dissociation comprising using an enzyme selected from the group consisting of: Accutase TM , dispase, collagenase and combinations thereof.

[0500] 84. The method of any one of embodiments 23-83, wherein the spheroids are dissociated by enzymatic dissociation comprising using Accutase TM .

[0501] 85. The method according to any one of embodiments 1-84, wherein the method comprises transferring about 0.1×10 6 cells / cm 2 About 2×10 6 cells / cm 2 , about 0.1×10 6 cells / cm 2 About 1×10 6 cells / cm 2 , about 0.1×10 6 cells / cm 2 About 0.8×10 6 cells / cm 2 , about 0.1×10 6 cells / cm 2 About 0.6×10 6 cells / cm 2 , about 0.1×10 6 cells / cm 2 About 0.4×10 6 cells / cm 2 , about 0.1×10 6 cells / cm 2 About 0.2×10 6 cells / cm 2 , about 0.2×10 6 cells / cm 2 About 2×10 6 cells / cm 2 , about 0.2×10 6 cells / cm 2 About 1×10 6 cells / cm 2 , about 0.2×10 6 cells / cm 2 About 0.8×10 6 cells / cm2 , about 0.2×10 6 cells / cm 2 About 0.6×10 6 cells / cm 2 , about 0.2×...

Claims

1. A method for differentiating neural cells, the method comprising: (a) performing a first incubation comprising culturing pluripotent stem cells in a non-adherent culture vessel under conditions that produce cell spheroids, wherein starting from day 0, the cells are exposed to (i) a TGF-β / Activin Nodal signaling inhibitor, wherein the TGF-β / Activin Nodal signaling inhibitor is SB431542; (ii) at least one sonic hedgehog (SHH) signaling activator, wherein the at least one SHH signaling activator is SHH or purmorphamine; or wherein the at least one SHH signaling activator comprises two SHH signaling activators selected from SHH and purmorphamine; (iii) a bone morphogenetic protein (BMP) signaling inhibitor, wherein the BMP signaling inhibitor is LDN193189; and (iv) a glycogen synthase kinase 3β (GSK3β) signaling inhibitor, wherein the GSK3β signaling inhibitor is CHIR99021, wherein day 0 corresponds to the initiation of said first incubation, and wherein starting from day 0 to day 6, inclusive, said cells are exposed to said TGF-β / Activin Nodal inhibitor, said at least one SHH signaling activator, said BMP signaling inhibitor, and said GSK3β signaling inhibitor; and (b) performing a second incubation comprising culturing the cells of the sphere in a substrate-coated culture vessel under conditions that cause neural differentiation of the cells.

2. The method of claim 1, wherein the second incubation begins on day 7.

3. The method of claim 1 or claim 2, wherein the cells: (i) exposure to said TGF-β / Activin Nodal signaling inhibitor until the day before or on day 7; (ii) exposure to the at least one SHH signaling activator until the day before or on day 7; (iii) exposure to the BMP signaling inhibitor until the day before or on day 11; (iv) exposing to the BMP signaling inhibitor starting from day 0 to day 10, inclusive; (v) exposure to the GSK3β signaling inhibitor until the day before or on day 13; (vi) exposure to the GSK3β signaling inhibitor starting from day 0 to day 12, inclusive; (vii) exposure to transforming growth factor β-3 (TGFβ3) (collectively referred to as "BAGCT") and a Notch signaling inhibitor starting on day 11; and / or (viii) exposure to BAGCT and the Notch signaling inhibitor starting from day 11, Until harvesting of neural differentiated cells, optionally until day 18, optionally until day 25.

4. The method of any one of claims 1-3, wherein culturing the cells under conditions that cause neural differentiation of the cells comprises exposing the cells to (i) brain-derived neurotrophic factor (BDNF); (ii) ascorbic acid; (iii) glial cell line-derived neurotrophic factor (GDNF); (iv) dibutyryl cyclic AMP (dbcAMP); (v) transforming growth factor β-3 (TGFβ3) (collectively referred to as "BAGCTs"); and (vi) Notch signaling inhibitors.

5. The method according to claim 1, wherein: The second incubation begins on day 7 and includes culturing the cells in a culture vessel coated with a substrate selected from laminin, collagen, entactin, heparin sulfate proteoglycans and combinations thereof, wherein starting from day 7, the cells are exposed to (i) a BMP signaling inhibitor and (ii) a GSK3β signaling inhibitor; and starting from day 11, the cells are exposed to (i) brain-derived neurotrophic factor (BDNF); (ii) ascorbic acid; (iii) glial cell line-derived neurotrophic factor (GDNF); (iv) dibutyryl cyclic AMP (dbcAMP); (v) transforming growth factor β-3 (TGFβ3) (collectively referred to as "BAGCT"); and (vi) a Notch signaling inhibitor.

6. The method of any one of claims 1-5, further comprising harvesting the neural differentiated cells, optionally wherein the harvesting is performed on or after about day 16, wherein the harvesting is performed between about day 16 and about day 30, wherein the harvesting is performed between day 18 and day 25, and / or wherein the harvesting is performed on or about day 18 or about day 25.

7. The method of any one of claims 1-6, wherein the neural differentiated cells are committed dopaminergic neuron progenitor cells, optionally wherein the dopaminergic neuron progenitor cells are capable of innervating host tissue after transplantation into a subject.

8. The method according to any one of claims 1 to 7, wherein: Prior to performing the second incubation, the spheroids are dissociated to produce a cell suspension, and the cells of the cell suspension are cultured in the substrate-coated culture vessel, optionally wherein the dissociation is performed when the spheroid cells express at least one of PAX6 and OTX2, and / or wherein the dissociation is performed at about day 7, and / or wherein the spheroids are dissociated by enzymatic dissociation.

9. The method of any one of claims 2-8, wherein the Notch signaling inhibitor is DAPT.

10. The method according to any one of claims 1 to 9, wherein the culturing in the first incubation and / or the second incubation is performed in a medium comprising serum or a serum replacement, or wherein the cells are cultured in the absence of serum for the duration of the culturing.

11. The method of any one of claims 1-10, wherein the cells are exposed to a Rho-associated protein kinase (ROCK) signaling inhibitor on day 0, day 7, day 16 and / or day 20, optionally wherein the ROCK inhibitor is Y-27632.

12. The method of any one of claims 1-11, wherein at least about 50% of the culture medium is replaced every day, every other day, or every three days.

13. The method of any one of claims 5-12, further comprising formulating the harvested cells with a cryoprotectant, optionally further comprising cryopreserving the harvested cells, and optionally thawing the cells prior to use.

14. The method of any one of claims 1-13, wherein the pluripotent stem cells are embryonic stem (ES) cells, induced pluripotent stem cells (iPSCs), or a combination thereof, and / or wherein the pluripotent stem cells are human induced pluripotent stem cells, and / or wherein the pluripotent stem cells are autologous to a subject to be treated with the neural differentiated cells or are allogeneic to a subject to be treated with the neural differentiated cells, and / or wherein the pluripotent stem cells are low immunogenic, optionally wherein the pluripotent stem cells are engineered to (a) remove genes encoding one or more of polymorphic HLA-A / -B / -C and HLA class II molecules; and (b) provide genes encoding one or more of PD-L1, HLA-G, and CD47, optionally into the AAVS1 safe harbor locus.

15. A method of providing a therapeutic composition, the method comprising: Differentiating neural cells using the method according to any one of claims 1 to 14, and A therapeutic composition comprising differentiated cells is formulated, optionally wherein the cells of the composition express EN1 and CORIN and less than 10% of the total cells in the composition express TH, and / or wherein the composition comprises dopamine neuron progenitor cells (DDPCs) derived from pluripotent stem cells, wherein the therapeutic composition exhibits one or more of the following: (a) greater than about 2×10 -5 The ratio of counts per million (CPM) NEUROG1 to CPM GAPDH; (b) greater than about 5×10 -4 The ratio of CPM EDN3 to CPM GAPDH; (c) greater than about 4×10 -3 The ratio of CPM HES1 to CPM GAPDH; (d) greater than about 8×10 -3 The ratio of CPM PSRC1 to CPM GAPDH; (e) greater than about 2×10 -3 The ratio of CPM NEK6 to CPM GAPDH; (f) greater than about 1×10 -2 The ratio of CPM IQGAP3 to CPM GAPDH; (g) greater than about 8×10 -4 The ratio of CPM USP44 to CPM GAPDH; (h) greater than about 3×10 -3 The ratio of CPM CEP55 to CPM GAPDH; (i) greater than about 2×10 -2 The ratio of CPM KIF20A to CPM GAPDH; (j) greater than about 2×10 -2 The ratio of CPM AURKA to CPM GAPDH; (k) is less than about 1×10 -3 The ratio of CPM CALCA to CPM GAPDH; (l) Less than about 1×10 -3 The ratio of CPM GLRA2 to CPM GAPDH; (m) is less than about 1×10 -1 The ratio of CPM MAPT to CPM GAPDH; (n) is less than about 5×10 -2 The ratio of CPM CAMK2B to CPM GAPDH; (o) Less than about 1×10 -2 The ratio of CPM SYT13 to CPM GAPDH; (p) is less than about 1×10 -3 The ratio of CPM LHFPL4 to CPM GAPDH; (q) is less than about 5×10 -3 The ratio of CPM RET to CPM GAPDH; (r) is less than about 1×10 -2 The ratio of CPM KCND3 to CPM GAPDH; (s) is less than about 5×10 -2 The ratio of CPM NSG2 to CPM GAPDH; and (t) is less than about 2×10 -2 The therapeutic composition comprises a CPM SNAP25 to CPM GAPDH ratio, optionally wherein the therapeutic composition exhibits between 2 and 20 of (a)-(t).

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