Methods for Differentiating Neural Cells and Related Compositions and Methods of Use - Patent application

Abstract

The present disclosure provides methods for lineage-specific differentiation of pluripotent stem cells, including induced pluripotent stem cells, into floor plate midbrain progenitor cells, committed dopamine (DA) neuron progenitor cells, and / or DA neurons, as well as compositions and uses thereof for treating neurodegenerative diseases and conditions, including, for example, Parkinson's disease.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Application No. 62 / 960,669, filed January 13, 2020, entitled “METHOD OF DIFFERENTIATING NEURAL CELLS AND RELATED COMPOSITIONS AND METHODS OF USE,” the entire contents of which are incorporated by reference for all purposes.

[0002] FIELD OF THEINVENTION The present disclosure relates to methods for lineage-specific differentiation of pluripotent stem cells, including induced pluripotent stem cells, into floor plate midbrain progenitor cells, determined dopamine (DA) neuronal progenitor cells, and / or dopamine (DA) neurons. Also provided are compositions of the differentiated cells and therapeutic uses thereof for treating neurodegenerative conditions and diseases, including, for example, Parkinson's disease. [Background technology]

[0003] Various methods for differentiating pluripotent stem cells into lineage-specific cell populations and the resulting cell compositions are intended to be utilized in cell replacement therapy for patients with diseases, resulting in loss of function of defined cell populations.However, in some cases, such methods are limited in their ability to generate cells with consistent physiological characteristics, and the cells obtained by such methods may have limited ability to engraft and innervate other cells in vivo.There is a need for improved methods and cell compositions thereof, including providing improved methods for differentiating cells to generate physiologically consistent cells. Summary of the Invention

[0004] Provided herein are methods for differentiating neural cells, comprising: (a) performing a first incubation comprising culturing pluripotent stem cells in a non-adherent culture vessel under conditions that generate cell spheroids, the first incubation comprising exposing the cells to (i) an inhibitor of TGF-β / Activin-Nodal signaling, (ii) at least one activator of Sonic Hedgehog (SHH) signaling, (iii) an inhibitor of bone morphogenetic protein (BMP) signaling, and (iv) an inhibitor of glycogen synthase kinase 3 beta (GSK3β) signaling beginning at the beginning of the first incubation (day 0); and (b) performing a second incubation comprising culturing the cells of the spheroids in a substrate-coated culture vessel under conditions that cause neural differentiation of the cells. In some embodiments, the second incubation begins at about day 7.

[0005] In some embodiments, the cells are exposed to an inhibitor of TGF-β / activin-nodal signaling until day 7 or the day before day 7. In some embodiments, the cells are exposed to an inhibitor of TGF-β / activin-nodal signaling until day 6. In some embodiments, the cells are exposed to an inhibitor of TGF-β / activin-nodal signaling starting on day 0 and continuing through day 6, inclusive.

[0006] In some embodiments, the cells are exposed to at least one activator of SHH signaling until day 7 or the day before day 7. In some embodiments, the cells are exposed to at least one activator of SHH signaling until day 6. In some embodiments, the cells are exposed to at least one activator of SHH signaling starting on day 0 until day 6, inclusive.

[0007] In some embodiments, the cells are exposed to the inhibitor of BMP signaling until day 11 or the day before day 11. In some embodiments, the cells are exposed to the inhibitor of BMP signaling until day 10. In some embodiments, the cells are exposed to the inhibitor of BMP signaling starting on day 0 until day 10, inclusive.

[0008] In some embodiments, the cells are exposed to an inhibitor of GSK3β signaling until day 13 or the day before day 13. In some embodiments, the cells are exposed to an inhibitor of GSK3β signaling until day 12. In some embodiments, the cells are exposed to an inhibitor of GSK3β signaling starting on day 0 until day 12, inclusive.

[0009] In some embodiments, culturing the cells under conditions to neuronally differentiate the cells in any of the methods provided 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 beta-3 (TGFβ3) (collectively, "BAGCT"), and (vi) an inhibitor of Notch signaling. In some embodiments, the cells are exposed to BAGCT and the inhibitor of Notch signaling beginning on day 11. In some embodiments, the cells are exposed to BAGCT and the inhibitor of Notch signaling beginning on day 11 until harvesting the neuronally differentiated cells. In some embodiments, the cells are exposed to BAGCT and the inhibitor of Notch signaling beginning on day 11 until at or about day 18. In some embodiments, cells are exposed to inhibitors of BAGCT and Notch signaling beginning on day 11 until or about day 25.

[0010] A method for differentiating neural cells, comprising: (a) performing a first incubation comprising culturing pluripotent stem cells in a non-adherent culture vessel under conditions to generate cell spheroids, the first incubation beginning on a start date (day 0) of the first incubation, exposing the cells to (i) an inhibitor of TGF-β / activin-nodal signaling until day 6, inclusive; (ii) at least one activator of Sonic Hedgehog (SHH) signaling until day 6, inclusive; (iii) an inhibitor of bone morphogenetic protein (BMP) signaling until day 6, inclusive; and (iv) an inhibitor of glycogen synthase kinase 3 beta (GSK3β) signaling until day 6, inclusive; and (b) beginning on day 7, exposing the cells to an inhibitor of laminin, collagen, endosomes, or endothelial cells. Provided herein are methods for neurodifferentiating cells of the spheroids, comprising culturing the cells in a culture vessel coated with a substrate selected from among tactin, heparin sulfate proteoglycan, and combinations thereof, and performing a second incubation for neurodifferentiating the cells of the spheroids, wherein starting on day 7, the cells are exposed to (i) an inhibitor of BMP signaling and (ii) an inhibitor of GSK3β signaling, and starting on day 11, the second incubation is performed 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 beta-3 (TGFβ3) (collectively, "BAGCT"), and (vi) an inhibitor of Notch signaling.

[0011] In some of any of the provided methods, the method further comprises harvesting the neurally differentiated cells. In some embodiments, the harvesting occurs on or after about day 16. In some embodiments, the harvesting occurs between about day 16 and about day 30. In some embodiments, the harvesting occurs between about day 16 and about day 27. In some embodiments, the harvesting occurs between day 18 and day 25. In some embodiments, the harvesting occurs at or about day 18. In some embodiments, the harvesting occurs at or about day 25.

[0012] In some embodiments, the neurally differentiated cells are committed dopaminergic neuronal progenitor cells. In some embodiments, the dopaminergic progenitor cells are capable of innervating a host tissue upon transplantation into a subject.

[0013] In some embodiments, before the second incubation, the spheroids are dissociated to generate a cell suspension, and the cells of the cell suspension are cultured in a substrate-coated culture vessel. In some embodiments, dissociation occurs when the spheroid cells express at least one of PAX6 and OTX2. In some embodiments, dissociation occurs when the spheroid cells express OTX2. In some embodiments, dissociation occurs when the spheroid cells express PAX6. In some embodiments, dissociation occurs when the spheroid cells express PAX6 and OTX2. In some embodiments, dissociation occurs at about day 7.

[0014] In some embodiments, the culture vessel is selected from the group consisting of a plate, a 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 microwell plate. In some embodiments, the microwell plate is a 6-well plate. In some embodiments, the microwell plate is a 24-well plate. In some embodiments, the culture vessel is an Aggrewell™ plate.

[0015] In some embodiments, the culture of pluripotent stem cells on 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 ~Approx. 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 ~Approx. 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 ~Approx. 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 ~Approx. 1×10 6 cells / cm 2 , about 0.2×10 6 cells / cm2 ~about 0.8×10 6 cells / cm 2 , about 0.2×10 6 cells / cm 2 ~Approx. 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 ~Approx. 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 ~Approx. 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 ~Approx. 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 ~Approx. 1×10 6 cells / cm 2 , or approximately 1.0 × 10 6 cells / cm 2 ~about 2×10 6 cells / cm 2In some embodiments, the culture of pluripotent stem cells on day 0 comprises about 0.1 x 10 6 cells / cm 2 ~Approx. 2×10 6 cells / cm 2 In some embodiments, the culture of pluripotent stem cells on day 0 comprises about 0.1 x 10 6 cells / cm 2 ~Approx. 1×10 6 cells / cm 2 In some embodiments, the culture of pluripotent stem cells on day 0 comprises about 0.1 x 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 comprises about 0.1 x 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 comprises about 0.1 x 10 6 cells / cm 2 ~Approx. 0.4×10 6 cells / cm 2 In some embodiments, the culture of pluripotent stem cells on day 0 comprises about 0.1 x 10 6 cells / cm 2 ~Approx. 0.2×10 6 cells / cm 2 In some embodiments, the culture of pluripotent stem cells on day 0 comprises about 0.2×10 6 cells / cm 2 ~Approx. 2×10 6 cells / cm 2 In some embodiments, the culture of pluripotent stem cells on day 0 comprises about 0.2×10 6 cells / cm 2 ~Approx. 1×10 6 cells / cm 2 In some embodiments, the culture of pluripotent stem cells on day 0 comprises about 0.2×10 6 cells / cm 2 ~about 0.8×10 6 cells / cm 2In some embodiments, the culture of pluripotent stem cells on day 0 comprises about 0.2×10 6 cells / cm 2 ~Approx. 0.6×10 6 cells / cm 2 In some embodiments, the culture of pluripotent stem cells on day 0 comprises 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 comprises 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 comprises about 0.4×10 6 cells / cm 2 ~Approx. 1×10 6 cells / cm 2 In some embodiments, the culture of pluripotent stem cells on day 0 comprises 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 comprises about 0.4×10 6 cells / cm 2 ~Approx. 0.6×10 6 cells / cm 2 In some embodiments, the culture of pluripotent stem cells on day 0 comprises 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 comprises about 0.6×10 6 cells / cm 2 ~Approx. 1×10 6 cells / cm 2 In some embodiments, the culture of pluripotent stem cells on day 0 comprises about 0.6×10 6 cells / cm 2 ~about 0.8×10 6 cells / cm 2In some embodiments, the culture of pluripotent stem cells on day 0 comprises 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 comprises about 0.8×10 6 cells / cm 2 ~Approx. 1×10 6 cells / cm 2 In some embodiments, the culture of pluripotent stem cells on day 0 comprises about 1.0 x 10 6 cells / cm 2 ~about 2×10 6 cells / cm 2 Includes.

[0016] In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 6-well plate. In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 6-well plate and is approximately 1×10 6 Pluripotent stem cells / well ~ approx. 20 x 10 6 Pluripotent stem cells / well, approximately 1 x 10 6 Pluripotent stem cells / well ~ approx. 15 x 10 6 Pluripotent stem cells / well, approximately 1 x 10 6 Pluripotent stem cells / well ~ approx. 10 x 10 6 Pluripotent stem cells / well, approximately 1 x 10 6 Pluripotent stem cells / well ~ 5 x 10 6 Pluripotent stem cells / well, approximately 5 x 10 6 Pluripotent stem cells / well ~ approx. 20 x 10 6 Pluripotent stem cells / well, approximately 5 x 10 6 Pluripotent stem cells / well ~ approx. 15 x 10 6 Pluripotent stem cells / well, approximately 5 x 10 6 Pluripotent stem cells / well ~ approx. 10 x 10 6 Pluripotent stem cells / well, approximately 10 x 10 6 Pluripotent stem cells / well ~ approx. 20 x 10 6 Pluripotent stem cells / well, approximately 10 x 10 6 Pluripotent stem cells / well ~ approx. 15 x 10 6 Pluripotent stem cells / well, or approximately 15 x 10 6Pluripotent stem cells / well ~ approx. 20 x 10 6 In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 6-well plate and contains approximately 1×10 6 Pluripotent stem cells / well ~ approx. 20 x 10 6 In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 6-well plate and contains approximately 1×10 6 Pluripotent stem cells / well ~ approx. 15 x 10 6 In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 6-well plate and contains approximately 1×10 6 Pluripotent stem cells / well ~ approx. 10 x 10 6 In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 6-well plate and contains approximately 1×10 6 Pluripotent stem cells / well ~ 5 x 10 6 In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 6-well plate and contains approximately 5×10 6 Pluripotent stem cells / well ~ approx. 20 x 10 6 In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 6-well plate and contains approximately 5×10 6 Pluripotent stem cells / well ~ approx. 15 x 10 6 In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 6-well plate and contains approximately 5×10 6 Pluripotent stem cells / well ~ approx. 10 x 10 6 In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 6-well plate and contains approximately 10×10 6 Pluripotent stem cells / well ~ approx. 20 x 10 6 In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 6-well plate and contains approximately 10×10 6 Pluripotent stem cells / well ~ approx. 15 x 10 6In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 6-well plate and contains approximately 15×10 6 Pluripotent stem cells / well ~ approx. 20 x 10 6 Contains pluripotent stem cells / well.

[0017] In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 24-well plate. In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 24-well plate and is about 1×10 5 Pluripotent stem cells / well ~ 5 x 10 6 Pluripotent stem cells / well, approximately 1 x 10 5 Pluripotent stem cells / well ~ approx. 1 x 10 6 Pluripotent stem cells / well, approximately 1 x 10 5 Pluripotent stem cells / well ~ 5 x 10 5 Pluripotent stem cells / well, approximately 5 x 10 5 Pluripotent stem cells / well ~ 5 x 10 6 Pluripotent stem cells / well, approximately 5 x 10 5 Pluripotent stem cells / well ~ approx. 1 x 10 6 Pluripotent stem cells / well, or approximately 1 x 10 6 Pluripotent stem cells / well ~ 5 x 10 6 In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 24-well plate and contains approximately 1×10 5 Pluripotent stem cells / well ~ 5 x 10 6 In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 24-well plate and contains approximately 1×10 5 Pluripotent stem cells / well ~ approx. 1 x 10 6 In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 24-well plate and contains approximately 1×10 5 Pluripotent stem cells / well ~ 5 x 10 5 In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 24-well plate and contains approximately 5×10 5 Pluripotent stem cells / well ~ 5 x 10 6In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 24-well plate and contains approximately 5×10 5 Pluripotent stem cells / well ~ approx. 1 x 10 6 In some embodiments, the culture of pluripotent stem cells on day 0 is performed in a 24-well plate and contains approximately 1×10 6 Pluripotent stem cells / well ~ 5 x 10 6 Contains pluripotent stem cells / well.

[0018] In some embodiments, the culture of pluripotent stem cells on day 0 includes a sufficient number of cells to generate spheroids having about 1,000 cells to about 5,000 cells, for example, about 2,000 cells to about 3,000 cells, on about day 7. In some embodiments, the culture of pluripotent stem cells on day 0 includes a sufficient number of cells to generate spheroids having about 1,000 cells to about 5,000 cells on about day 7. In some embodiments, the culture of pluripotent stem cells on day 0 includes a sufficient number of cells to generate spheroids having about 2,000 cells to about 3,000 cells on about day 7. In some embodiments, the culture of pluripotent stem cells on day 0 includes a sufficient number of cells to generate spheroids having about 2,000 cells on about day 7. In some embodiments, the culture of pluripotent stem cells on day 0 includes a sufficient number of cells to generate spheroids having about 3,000 cells on about day 7.

[0019] In some embodiments, the culture vessel is treated to reduce or eliminate cell adhesion. In some embodiments, the culture vessel is treated to reduce cell adhesion. In some embodiments, the culture vessel is treated to eliminate cell adhesion. In some embodiments, the treatment of the culture vessel comprises incubating the culture vessel with pluronic acid. In some embodiments, prior to the first incubation, the non-adherent culture vessel is exposed to a detergent. In some embodiments, the detergent is pluronic acid.

[0020] 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 proteoglycan, and combinations thereof. In some embodiments, the substrate is a recombinant protein. In some embodiments, the substrate is recombinant laminin. In some embodiments, the substrate-coated culture vessel is exposed to poly-L-ornithine prior to use for culturing cells.

[0021] In some embodiments, the inhibitor of TGF-β / activin-nodal signaling is SB431542. In some embodiments, 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, cells are exposed to SB431542 at a concentration of about 1 μM to about 20 μM. In some embodiments, cells are exposed to SB431542 at a concentration of about 5 μM to about 15 μM. In some embodiments, cells are exposed to SB431542 at a concentration of about 8 μM to about 12 μM. In some embodiments, cells are exposed to SB431542 at a concentration of about 10 μM.

[0022] In some embodiments, the at least one activator of SHH signaling is SHH protein or palmorphamin. In some embodiments, the at least one activator of SHH signaling is SHH protein. In some embodiments, the at least one activator of SHH signaling is palmorphamin. In some embodiments, the at least one activator of SHH signaling comprises two activators of SHH signaling selected from SHH protein and palmorphamin. In some embodiments, the at least one activator of SHH signaling is two activators of SHH signaling selected from SHH protein and palmorphamin. In some embodiments, the cells are exposed to SHH at a concentration of about 10 ng / mL to about 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. In some embodiments, the cells are exposed to SHH at a concentration of about 10 ng / mL to about 500 ng / mL. In some embodiments, the cells are exposed to SHH at a concentration of about 20 ng / mL to 400 ng / mL. 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.

[0023] In some embodiments, the cells are exposed to palmorphamin 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 palmorphamin at a concentration of about 1 μM to about 20 μM. In some embodiments, the cells are exposed to palmorphamin at a concentration of about 5 μM to about 15 μM. In some embodiments, the cells are exposed to palmorphamin at a concentration of about 8 μM to about 12 μM. In some embodiments, the cells are exposed to palmorphamin at a concentration of about 10 μM.

[0024] In some embodiments, the cells are exposed to SHH at a concentration of about 100 ng / mL and palmorfamine at a concentration of about 10 μM.

[0025] In some embodiments, the inhibitor of BMP signaling is LDN193189. In some embodiments, the cells are exposed to LDN193189 at a concentration of about 10 nM to about 500 nM, about 20 nM to about 400 nM, about 50 nM to about 200 nM, or about 75 nM to about 150 nM. In some embodiments, the cells are exposed to LDN193189 at a concentration of about 10 nM to 500 nM. In some embodiments, the cells are exposed to LDN193189 at a concentration of about 20 nM to 400 nM. In some embodiments, the cells are exposed to LDN193189 at a concentration of about 50 nM to 200 nM. In some embodiments, the cells are exposed to LDN193189 at a concentration of about 75 nM to 150 nM. In some embodiments, the cells are exposed to LDN193189 at a concentration of about 100 nM.

[0026] In some embodiments, the inhibitor of GSK3β signaling is CHIR99021. In some embodiments, 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. In some embodiments, the cells are exposed to CHIR99021 at a concentration of about 0.1 μM to about 5 μM. In some embodiments, the cells are exposed to CHIR99021 at a concentration of about 0.5 μM to about 4 μM. In some embodiments, the cells are exposed to CHIR99021 at a concentration of about 1 μM to about 3 μM. In some embodiments, the cells are exposed to CHIR99021 at a concentration of about 2 μM.

[0027] In some embodiments, 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, cells are exposed to GDNF at a concentration of about 1 ng / mL to about 100 ng / mL. In some embodiments, cells are exposed to GDNF at a concentration of about 5 ng / mL to about 80 ng / mL. In some embodiments, cells are exposed to GDNF at a concentration of about 10 ng / mL to about 60 ng / mL. In some embodiments, cells are exposed to GDNF at a concentration of about 15 ng / mL to about 30 ng / mL. In some embodiments, cells are exposed to GDNF at a concentration of about 20 ng / mL.

[0028] In some embodiments, 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, cells are exposed to BDNF at a concentration of about 1 ng / mL to about 100 ng / mL. In some embodiments, cells are exposed to BDNF at a concentration of about 5 ng / mL to about 80 ng / mL. In some embodiments, cells are exposed to BDNF at a concentration of about 10 ng / mL to about 60 ng / mL. In some embodiments, cells are exposed to BDNF at a concentration of about 15 ng / mL to about 30 ng / mL. In some embodiments, cells are exposed to BDNF at a concentration of about 20 ng / mL.

[0029] In some embodiments, the cells are 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 cells are exposed to dbcAMP at a concentration of about 0.1 mM to 5 mM. In some embodiments, the cells are exposed to dbcAMP at a concentration of about 0.2 mM to 4 mM. In some embodiments, the cells are exposed to dbcAMP at a concentration of about 0.3 mM to 3 mM. In some embodiments, the cells are exposed to dbcAMP at a concentration of about 0.4 mM to 2 mM. In some embodiments, the cells are exposed to dbcAMP at a concentration of about 0.5 mM.

[0030] In some embodiments, the cells are exposed to ascorbin 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. In some embodiments, the cells are exposed to ascorbin at a concentration of about 0.05 mM to about 5 mM. In some embodiments, the cells are exposed to ascorbin at a concentration of about 0.1 mM to about 2 mM. In some embodiments, the cells are exposed to ascorbin at a concentration of about 0.2 mM to about 0.5 mM. In some embodiments, the cells are exposed to ascorbin at a concentration of about 0.2 mM.

[0031] 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.

[0032] In some embodiments, the inhibitor of Notch signaling is DAPT. 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.

[0033] In some of any of the embodiments provided, the culture in the first incubation and / or the second incubation is in a medium with serum or serum replacement. In some of any of the embodiments provided, the culture in the first incubation is in a medium with serum or serum replacement. In some of any of the embodiments provided, the culture in the second incubation is in a medium with serum or serum replacement. In some embodiments, the culture in the first incubation and the second incubation is in a medium with serum or serum replacement. In some embodiments, the cells are cultured in a medium with serum or serum replacement from about day 0 to about day 10. In some embodiments, the serum or serum replacement comprises about 5% (v / v) of the medium. In some embodiments, the serum or serum replacement comprises about 2% (v / v) of the medium. In some embodiments, the 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. In some embodiments, the medium contains a serum replacement. In some embodiments, the serum replacement does not contain fetal bovine serum (FBS). In some embodiments, the serum replacement is KnockOut™ serum replacement.

[0034] In some embodiments, the cells are cultured in the absence of serum for the duration of the culture period.

[0035] In some embodiments, the cells are exposed to an inhibitor of Rho-associated protein kinase (ROCK) signaling for one or more days of the first and second incubation. In some embodiments, the inhibitor of ROCK signaling is added at least once a week during the course of the method. In some embodiments, the cells are exposed to an inhibitor of ROCK signaling on day 0, day 7, day 16, and / or day 20. In some embodiments, the cells are exposed to an inhibitor of Rho-associated protein kinase (ROCK) signaling on day 0. In some embodiments, the cells are exposed to an inhibitor of Rho-associated protein kinase (ROCK) signaling on day 7. In some embodiments, the cells are exposed to an inhibitor of Rho-associated protein kinase (ROCK) signaling on day 16. In some embodiments, the cells are exposed to an inhibitor of Rho-associated protein kinase (ROCK) signaling on day 20. In some embodiments, the cells are exposed to an inhibitor of Rho-associated protein kinase (ROCK) signaling on days 0, 7, 16, and 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.

[0036] In some embodiments, during the first and / or second incubation, at least about 50% of the medium is changed daily or every other day or every second day. In some embodiments, at least about 50% of the medium is changed daily. In some embodiments, at least about 50% of the medium is changed every other day. In some embodiments, at least about 50% of the medium is changed every second day. In some embodiments, at least about 50% of the medium is changed every day during the first incubation. In some embodiments, at least about 50% of the medium is changed every other day during the first incubation. In some embodiments, at least about 50% of the medium is changed every second day during the first incubation. In some embodiments, at least about 50% of the medium is changed every day during the second incubation. In some embodiments, at least about 50% of the medium is changed every day during the second incubation. In some embodiments, at least about 50% of the medium is changed every other day during the second incubation. In some embodiments, during the second incubation, at least about 50% of the medium is changed every 2 days. In some embodiments, during the first and second incubation, at least about 50% of the medium is changed every day. In some embodiments, during the first and second incubation, at least about 50% of the medium is changed every other day. In some embodiments, during the first and second incubation, at least about 50% of the medium is changed every 2 days.

[0037] In some embodiments, the method includes dissociating the spheroids before the second incubation. In some embodiments, the spheroids are dissociated by enzymatic dissociation. In some embodiments, the method includes dissociating the spheroids before the second incubation, and the spheroids are dissociated by enzymatic dissociation. In some embodiments, the spheroids are dissociated by enzymatic dissociation including the use of an enzyme selected from actase, dispase, collagenase, or a combination thereof. In some embodiments, the spheroids are dissociated by enzymatic dissociation including the use of actase. In some embodiments, the spheroids are dissociated by actase.

[0038] In some embodiments, after dissociation of the spheroids, the cells of the dissociated spheroids are cultured in a substrate-coated culture vessel, such as by transferring the cells to a substrate-coated culture vessel. In some embodiments, after dissociation of the spheroids, the cells of the dissociated spheroids are cultured in a substrate-coated culture vessel, such as by transferring the cells to a substrate-coated culture vessel. 6 cells / cm 2 ~about 2×10 6 cells / cm 2 , about 0.1×10 6 cells / cm 2 ~Approx. 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 ~Approx. 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 ~Approx. 0.2×10 6 cells / cm 2 , about 0.2×10 6 cells / cm 2 ~about 2×106 cells / cm 2 and about 0.2×10 6 cells / cm 2 ~ about 1×10 6 cells / cm 2 and about 0.2×10 6 cells / cm 2 ~ about 0.8×10 6 cells / cm 2 and about 0.2×10 6 cells / cm 2 ~ about 0.6×10 6 cells / cm 2 and about 0.2×10 6 cells / cm 2 ~ about 0.4×10 6 cells / cm 2 and about 0.4×10 6 cells / cm 2 ~ about 2×10 6 cells / cm 2 and about 0.4×10 6 cells / cm 2 ~ about 1×10 6 cells / cm 2 and about 0.4×10 6 cells / cm 2 ~ about 0.8×10 6 cells / cm 2 and about 0.4×10 6 cells / cm 2 ~ about 0.6×10 6 cells / cm 2 and about 0.6×10 6 cells / cm 2 ~ about 2×10 6 cells / cm 2 and about 0.6×10 6 cells / cm 2 ~ about 1×10 6 cells / cm 2 and about 0.6×10 6 cells / cm 2 ~ about 0.8×10 6 cells / cm 2 and about 0.8×10 6 cells / cm 2 ~ about 2×10 6 cells / cm 2 and about 0.8×10 6 cells / cm 2 ~ about 1×106 cells / cm 2 , or approximately 1.0 × 10 6 cells / cm 2 ~Approx. 2×10 6 cells / cm 2 In some embodiments, after dissociation of the spheroids, the cells of the dissociated spheroids are cultured in a substrate-coated culture vessel, such as by transferring the cells to the 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, the cells of the dissociated spheroids are cultured in a substrate-coated culture vessel, such as by transferring the cells to the substrate-coated culture vessel at a concentration of about 1000 μg / ml. In some embodiments, the transfer of the cells of the dissociated spheroids to the substrate-coated vessel occurs on about day 7.

[0039] In some embodiments, the cells of the spheroids from the first incubation are not dissociated prior to implanting the spheroid cells into the substrate-coated culture vessel. In some embodiments, the second incubation comprises culturing the cells of the spheroids from the first incubation without first dissociating the cells to form a cell suspension.

[0040] In some of any of the provided embodiments, the method further comprises formulating a cryoprotectant to the collected cells. 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 collected cells are formulated with about 10% DMSO.

[0041] In some embodiments, the pluripotent stem cells are embryonic stem (ES) cells, induced pluripotent stem cells (iPSCs), or a combination thereof. In some embodiments, the pluripotent stem cells are embryonic stem (ES) cells. In some embodiments, the pluripotent stem cells are induced pluripotent stem cells (iPSCs). In some embodiments, the pluripotent stem cells are embryonic stem (ES) cells. In some embodiments, the ES cells are mouse or human embryonic stem cells. In some embodiments, the ES cells are mouse embryonic stem cells. In some embodiments, the pluripotent stem cells are human embryonic stem cells. In some embodiments, the pluripotent stem cells are induced pluripotent stem cells. In some embodiments, the iPSCs are mouse or human induced pluripotent stem cells. In some embodiments, the pluripotent stem cells are mouse induced pluripotent stem cells. In some embodiments, the pluripotent stem cells are human induced pluripotent stem cells.

[0042] In some embodiments, the pluripotent stem cells are autologous to the subject being treated with the neurally differentiated cells. In some embodiments, the subject being treated has Parkinson's disease. In some embodiments, the subject being treated is suspected of having Parkinson's disease. In some embodiments, the subject being treated has Parkinsonism. In some embodiments, the subject being treated is suspected of having Parkinsonism.

[0043] In some embodiments, the pluripotent stem cells are allogeneic to the subject to be treated with the neurally differentiated cells. In some embodiments, the neurally differentiated cells are hypoimmunogenic. In some embodiments, the pluripotent stem cells are genetically 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 genetically engineered to remove genes encoding polymorphic HLA-A / -B / -C and HLA class II molecules. In some embodiments, the pluripotent stem cells are genetically engineered to provide genes encoding PD-L1, HLA-G, and CD47. In some embodiments, the pluripotent stem cells are genetically engineered to provide genes encoding PD-L1, HLA-G, and CD47 at the AAVS1 safe harbor locus. In some embodiments, pluripotent stem cells are genetically engineered to remove genes encoding polymorphic HLA-A / -B / -C and HLA class II molecules, and to provide genes encoding PD-L1, HLA-G, and CD47 at the AAVS1 safe harbor locus.

[0044] Also provided herein are therapeutic compositions produced by any of the provided methods.

[0045] 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.

[0046] Also provided herein is a therapeutic composition comprising determined dopamine neuron progenitor cells (DDPCs) derived from pluripotent stem cells, wherein cells in the composition express EN1 and CORIN, and less than 10% of the total cells in the composition express TH.

[0047] 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.

[0048] In some embodiments, more than 10%, more than 15%, or more than 20% of the total cells in the therapeutic composition express TH. In some embodiments, more than 10% of the total cells in the therapeutic composition express TH. In some embodiments, more than 15% of the total cells in the therapeutic composition express TH. In some embodiments, more 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.

[0049] 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 all cells in the composition express EN1. In some embodiments, at least about 55% of all cells in the composition express EN1. In some embodiments, at least about 60% of all cells in the composition express EN1. In some embodiments, at least about 65% of all cells in the composition express EN1. In some embodiments, at least about 70% of all cells in the composition express EN1. In some embodiments, at least about 75% of all cells in the composition express EN1. In some embodiments, at least about 80% of all cells in the composition express EN1.

[0050] 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.

[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 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 all cells in the composition express EN1 and CORIN. In some embodiments, at least about 45% of all cells in the composition express EN1 and CORIN. In some embodiments, at least about 50% of all cells in the composition express EN1 and CORIN. In some embodiments, at least about 55% of all cells in the composition express EN1 and CORIN. In some embodiments, at least about 60% of all cells in the composition express EN1 and CORIN. In some embodiments, at least about 65% of all cells in the composition express EN1 and CORIN. In some embodiments, at least about 70% of all cells in the composition express EN1 and CORIN. In some embodiments, at least about 75% of all cells in the composition express EN1 and CORIN. In some embodiments, at least about 80% of all cells in the composition express EN1 and CORIN.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] Also provided herein is a therapeutic composition comprising committed dopamine neuron progenitor cells (DDPCs) derived from pluripotent stem cells, wherein the cells of the composition express EN1, and wherein 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.

[0056] Also provided herein is a therapeutic composition comprising committed dopamine neuron progenitor cells (DDPCs) derived from pluripotent stem cells, wherein the cells of the composition express CORIN, and wherein 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.

[0057] Also provided herein is a therapeutic composition comprising committed dopamine neuron progenitor cells (DDPCs) derived from pluripotent stem cells, wherein the cells of the composition express EN1 and CORIN, and wherein 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] A therapeutic composition comprising committed dopamine neuronal progenitor cells (DDPCs) derived from pluripotent stem cells, the composition comprising approximately 2.5×10 -2 Also provided herein are compositions that exhibit a counts per million (CPM) / CPM ratio of TH to GAPDH of less than 0.01.

[0059] Also provided herein is a therapeutic composition containing committed dopamine neuronal progenitor cells (DDPCs) derived from pluripotent stem cells, the composition exhibiting one or more of the following: (a) about 2×10 -5 (b) Counts per million (CPM) / CPM ratio of NEUROG1 to GAPDH of approximately 5 × 10 -4 (c) CPM / CPM ratio of EDN3 to GAPDH of approximately 4 × 10 -3 (d) CPM / CPM ratio of HES1 to GAPDH of approximately 8 × 10 -3 (e) CPM / CPM ratio of PSRC1 to GAPDH of approximately 2 × 10 -3 (f) CPM / CPM ratio of NEK6 to GAPDH of approximately 1 × 10 -2 The CPM / CPM ratio of IQGAP3 to GAPDH, (g) was approximately 8 × 10 -4 The CPM / CPM ratio of USP44 to GAPDH (h) was approximately 3 × 10 -3 (i) CPM / CPM ratio of CEP55 to GAPDH of approximately 2 × 10 -2 (j) CPM / CPM ratio of KIF20A to GAPDH of approximately 2 × 10 -2The CPM / CPM ratio of AURKA to GAPDH is approximately 1 × 10 -3 (l) CPM / CPM ratio of CALCA to GAPDH of less than 1 × 10 -3 CPM / CPM ratio of GLRA2 to GAPDH (m) of less than 1 × 10 -1 CPM / CPM ratio of MAPT to GAPDH less than (n) approx. 5 × 10 -2 (o) CPM / CPM ratio of CAMK2B to GAPDH of less than 1 × 10 -2 CPM / CPM ratio of SYT13 to GAPDH less than approx. 1 × 10 -3 CPM / CPM ratio of LHFPL4 to GAPDH (q) of approximately 5 × 10 -3 CPM / CPM ratio of RET to GAPDH (r) of approximately 1 × 10 -2 CPM / CPM ratio of KCND3 to GAPDH less than (s) approx. 5 × 10 -2 CPM / CPM ratio of NSG2 to GAPDH of less than 2 × 10 -2 CPM / CPM ratio of SNAP25 to GAPDH less than 1.

[0060] In some embodiments, the counts per million (CPM) / CPM ratio of NEUROG1 to GAPDH is about 2×10 -5 In some embodiments, the CPM / CPM ratio of EDN3 to GAPDH is about 5×10 -4 In some embodiments, the CPM / CPM ratio of HES1 to GAPDH is about 4×10 -3 In some embodiments, the CPM / CPM ratio of PSRC1 to GAPDH is about 8×10 -3 In some embodiments, the CPM / CPM ratio of NEK6 to GAPDH is about 2×10 -3 In some embodiments, the CPM / CPM ratio of IQGAP3 to GAPDH is about 1 x 10 -2 In some embodiments, the CPM / CPM ratio of USP44 to GAPDH is about 8×10 -4In some embodiments, the CPM / CPM ratio of CEP55 to GAPDH is about 3×10 -3 In some embodiments, the CPM / CPM ratio of KIF20A to GAPDH is about 2×10 -2 In some embodiments, the CPM / CPM ratio of AURKA to GAPDH is about 2×10 -2 In some embodiments, the CPM / CPM ratio of CALCA to GAPDH is about 1×10 -3 In some embodiments, the CPM / CPM ratio of GLRA2 to GAPDH is less than about 1×10 -3 In some embodiments, the CPM / CPM ratio of MAPT to GAPDH is less than about 1×10 -1 In some embodiments, the CPM / CPM ratio of CAMK2B to GAPDH is less than about 5×10 -2 In some embodiments, the CPM / CPM ratio of SYT13 to GAPDH is less than about 1×10 -2 In some embodiments, the CPM / CPM ratio of LHFPL4 to GAPDH is about 1×10 -3 In some embodiments, the CPM / CPM ratio of RET to GAPDH is less than about 5×10 -3 In some embodiments, the CPM / CPM ratio of KCND3 to GAPDH is less than about 1×10 -2 In some embodiments, the CPM / CPM ratio of NSG2 to GAPDH is less than about 5×10 -2 In some embodiments, the CPM / CPM ratio of SNAP25 to GAPDH is less than about 2×10 -2 is less than.

[0061] In some embodiments, the therapeutic composition exhibits about 2-20 of (a)-(t). In some embodiments, the therapeutic composition exhibits about 2-15 of (a)-(t). In some embodiments, the therapeutic composition exhibits about 2-10 of (a)-(t). In some embodiments, the therapeutic composition exhibits about 2-5 of (a)-(t). In some embodiments, the therapeutic composition exhibits about 5-20 of (a)-(t). In some embodiments, the therapeutic composition exhibits about 5-15 of (a)-(t). In some embodiments, the therapeutic composition exhibits about 5-10 of (a)-(t). In some embodiments, the therapeutic composition exhibits about 10-20 of (a)-(t). In some embodiments, the therapeutic composition exhibits about 5-15 of (a)-(t). In some embodiments, the therapeutic composition exhibits about 15-20 of (a)-(t).

[0062] In some embodiments, the therapeutic composition exhibits at least two of (a)-(t). In some embodiments, the therapeutic composition exhibits at least three of (a)-(t). In some embodiments, the therapeutic composition exhibits at least four of (a)-(t). In some embodiments, the therapeutic composition exhibits at least five of (a)-(t). In some embodiments, the therapeutic composition exhibits at least six of (a)-(t). In some embodiments, the therapeutic composition exhibits at least seven of (a)-(t). In some embodiments, the therapeutic composition exhibits at least eight of (a)-(t). In some embodiments, the therapeutic composition exhibits at least nine of (a)-(t). In some embodiments, the therapeutic composition exhibits at least ten of (a)-(t). In some embodiments, the therapeutic composition exhibits at least eleven of (a)-(t). In some embodiments, the therapeutic composition exhibits at least twelve of (a)-(t). In some embodiments, the therapeutic composition exhibits at least thirteen 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).

[0063] In some embodiments, the therapeutic composition exhibits two of (a)-(t). In some embodiments, the therapeutic composition exhibits three of (a)-(t). In some embodiments, the therapeutic composition exhibits four of (a)-(t). In some embodiments, the therapeutic composition exhibits five of (a)-(t). In some embodiments, the therapeutic composition exhibits six of (a)-(t). In some embodiments, the therapeutic composition exhibits seven of (a)-(t). In some embodiments, the therapeutic composition exhibits eight of (a)-(t). In some embodiments, the therapeutic composition exhibits nine of (a)-(t). In some embodiments, the therapeutic composition exhibits ten of (a)-(t). In some embodiments, the therapeutic composition exhibits eleven of (a)-(t). In some embodiments, the therapeutic composition exhibits twelve of (a)-(t). In some embodiments, the therapeutic composition exhibits thirteen 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).

[0064] In some embodiments, the cells in the therapeutic composition express EN1 and CORIN. In some embodiments, the therapeutic composition comprises about 1×10 -4 In some embodiments, the therapeutic composition exhibits a counts per million (CPM) / CPM ratio of EN1 to GAPDH of about 2×10 -2 In some embodiments, the therapeutic composition exhibits a ratio of CORIN to GAPDH of about 1×10 -4The CPM / CPM ratio of EN1 to GAPDH was greater than 2 × 10 -2 In some embodiments, the CPM / CPM ratio of CORIN to GAPDH is greater than about 1.5×10. -3 ~1×10 -2 In some embodiments, the CPM / CPM ratio of CORIN to GAPDH is about 5×10 -2 ~5×10 -1 It is.

[0065] 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 comprises about 3×10 -2 In some embodiments, the CPM / CPM ratio of TH to GAPDH is less than about 1×10 -3 ~2.5×10 -2In 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 expression of TH is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, or any value between any of the foregoing. 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.

[0066] In some embodiments, at least about 15% of the cells of the therapeutic composition express EN1. In some embodiments, at least about 20% of the cells of the therapeutic composition express EN1. In some embodiments, at least about 25% of the cells of the therapeutic composition express EN1. In some embodiments, at least about 30% of the cells of the therapeutic composition express EN1. In some embodiments, at least about 35% of the cells of the therapeutic composition express EN1. In some embodiments, at least about 40% of the cells of the therapeutic composition express EN1. In some embodiments, at least about 45% of the cells of the therapeutic composition express EN1. In some embodiments, at least about 50% of the cells of the therapeutic composition express EN1. In some embodiments, at least about 55% of the cells of the therapeutic composition express EN1. In some embodiments, at least about 60% of the cells of the therapeutic composition express EN1. In some embodiments, at least about 65% of the cells of the therapeutic composition express EN1. In some embodiments, at least about 70% of the cells of the therapeutic composition express EN1. In some embodiments, at least about 75% of the cells of the therapeutic composition express EN1. In some embodiments, at least about 80% of the cells of the therapeutic composition express EN1.

[0067] 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.

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

[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 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.

[0070] In some embodiments, at least about 15% of the cells of the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 20% of the cells of the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 25% of the cells of the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 30% of the cells of the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 35% of the cells of the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 40% of the cells of the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 45% of the cells of the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 50% of the cells of the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 55% of the cells of the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 60% of the cells of the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 65% of the cells of the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 70% of the cells of the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 75% of the cells of the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 80% of the cells of the therapeutic composition express EN1 and 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 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.

[0072] 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.

[0073] 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.

[0074] In some embodiments, at least about 70%, 75%, 80%, 85%, 90%, or 95% of the total cells in the therapeutic composition are viable. In some embodiments, at least about 70% of the total cells in the therapeutic composition are viable. In some embodiments, at least about 75% of the total cells in the therapeutic composition are viable. In some embodiments, at least about 80% of the total cells in the therapeutic composition are viable. In some embodiments, at least about 85% of the total cells in the therapeutic composition are viable. In some embodiments, at least about 90% of the total cells in the therapeutic composition are viable. In some embodiments, at least about 95% of the total cells in the therapeutic composition are viable.

[0075] In some embodiments, committed cells are capable of engrafting and innervating other cells in vivo.

[0076] 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.

[0077] 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.

[0078] In some embodiments, the therapeutic composition is about 0.5 mL to 5 mL. In some embodiments, the therapeutic composition is about 0.5 mL. In some embodiments, the therapeutic composition is about 0.75 mL. In some embodiments, the therapeutic composition is about 1.0 mL. In some embodiments, the therapeutic composition is about 1.25 mL. In some embodiments, the therapeutic composition is about 1.5 mL. In some embodiments, the therapeutic composition is about 1.75 mL. In some embodiments, the therapeutic composition is about 2.0 mL. In some embodiments, the therapeutic composition is about 2.5 mL. In some embodiments, the therapeutic composition is about 3.0 mL. In some embodiments, the therapeutic composition is about 3.5 mL. In some embodiments, the therapeutic composition is about 4.0 mL. In some embodiments, the therapeutic composition is about 4.5 mL. In some embodiments, the therapeutic composition is about 5.0 mL.

[0079] 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.

[0080] In some embodiments, the composition is for use in treating a neurodegenerative disease or condition in a subject. In some embodiments, the neurodegenerative disease or condition comprises loss of dopaminergic neurons. In some embodiments, the neurodegenerative disease or condition comprises loss of dopaminergic neurons in the substantia nigra (SN). In some embodiments, the neurodegenerative disease or condition comprises loss of dopaminergic neurons in the substantia nigra (SN pars compacta (SNc)). In some embodiments, the neurodegenerative disease or condition is Parkinson's disease. In some embodiments, the neurodegenerative disease or condition is parkinsonism.

[0081] Also provided herein are methods of treatment that include administering to a subject a therapeutically effective amount of any of the therapeutic compositions provided herein.

[0082] In some embodiments, the number of cells administered to a subject is about 0.25×10 6 Cells ~ approx. 20×10 6 cells, approximately 0.25 x 10 6 Cells ~ approx. 15 x 10 6 cells, approximately 0.25 x 10 6 Cells ~ approx. 10×10 6 cells, approximately 0.25 x 10 6 Cells ~ approx. 5 x 10 6 cells, approximately 0.25 x 10 6 cells ~ approx. 1 x 10 6 cells, approximately 0.25 x 10 6 Cells ~ approx. 0.75×10 6 cells, approximately 0.25 x 10 6 Cells ~ approx. 0.5×10 6 cells, approximately 0.5 x 10 6 Cells ~ approx. 20×10 6 cells, approximately 0.5 x 10 6 Cells ~ approx. 15 x 10 6 cells, approximately 0.5 x 10 6 Cells ~ approx. 10×10 6 cells, approximately 0.5 x 10 6 Cells ~ approx. 5 x 10 6 cells, approximately 0.5 x 10 6 cells ~ approx. 1 x 10 6 cells, approximately 0.5 x 10 6 Cells ~ approx. 0.75×10 6 cells, approximately 0.75×10 6 Cells ~ approx. 20×10 6 cells, approximately 0.75×10 6 Cells ~ approx. 15 x 10 6 cells, approximately 0.75×10 6 Cells ~ approx. 10×10 6 cells, approximately 0.75×10 6 Cells ~ approx. 5 x 10 6 cells, approximately 0.75×10 6 cells ~ approx. 1 x 10 6 cells, approximately 1 x 10 6 Cells ~ approx. 20×106 cells, approximately 1 x 10 6 Cells ~ approx. 15 x 10 6 cells, approximately 1 x 10 6 Cells ~ approx. 10×10 6 cells, approximately 1 x 10 6 Cells ~ approx. 5 x 10 6 cells, approximately 5 x 10 6 Cells ~ approx. 20×10 6 cells, approximately 5 x 10 6 Cells ~ approx. 15 x 10 6 cells, approximately 5 x 10 6 Cells ~ approx. 10×10 6 cells, approximately 10 x 10 6 Cells ~ approx. 20×10 6 cells, approximately 10 x 10 6 Cells ~ approx. 15 x 10 6 cells, or approximately 15 x 10 6 Cells ~ approx. 20×10 6 In some embodiments, the number of cells administered to a subject is about 0.25×10 -6 Cells ~ approx. 20×10 6 In some embodiments, the number of cells administered to a subject is about 0.5×10 -6 Cells ~ approx. 15 x 10 6 In some embodiments, the number of cells administered to a subject is about 1×10 -6 Cells ~ approx. 10×10 6 It is a cell.

[0083] 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 It is a cell.

[0084] In some embodiments, the cells are administered to the subject at a concentration of about 50,000 cells / microliter to about 150,000 cells / microliter. In some embodiments, the cells are administered to the subject at a concentration of about 50,000 cells / microliter. In some embodiments, the cells are administered to the subject at a concentration of about 75,000 cells / microliter. In some embodiments, the cells are administered to the subject at a concentration of about 100,000 cells / microliter. In some embodiments, the cells are administered to the subject at a concentration of about 125,000 cells / microliter. In some embodiments, the cells are administered to the subject at a concentration of about 150,000 cells / microliter.

[0085] In some embodiments, the subject has a neurodegenerative disease or condition. In some embodiments, the neurodegenerative disease or condition comprises loss of dopaminergic neurons. In some embodiments, the subject has at least 50% loss of dopaminergic neurons. In some embodiments, the subject has at least 50% loss of dopaminergic neurons in the substantia nigra (SN). In some embodiments, the subject has at least 50% loss of dopaminergic neurons in the substantia nigra pars compacta (SNc). In some embodiments, the neurodegenerative disease or condition is Parkinsonism. 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.

[0086] In some embodiments, administering comprises delivering cells of the composition by stereotactic injection. In some embodiments, administering comprises delivering cells of the composition through a catheter. In some embodiments, the cells are delivered to the striatum of the subject. In some embodiments, about 3×10 6 cells / hemisphere~15×10 6 In some embodiments, about 3×10 cells / hemisphere are administered to a subject. 6 In some embodiments, about 4×10 cells / hemisphere are administered to a subject. 6In some embodiments, about 5×10 cells / hemisphere are administered to a subject. 6 In some embodiments, about 6×10 cells / hemisphere are administered to a subject. 6 In some embodiments, about 7×10 cells / hemisphere are administered to a subject. 6 In some embodiments, about 8×10 cells / hemisphere are administered to a subject. 6 In some embodiments, about 9×10 cells / hemisphere are administered to a subject. 6 In some embodiments, about 10×10 cells / hemisphere are administered to a subject. 6 In some embodiments, about 11×10 cells / hemisphere are administered to a subject. 6 In some embodiments, about 12×10 cells / hemisphere are administered to a subject. 6 In some embodiments, about 13×10 cells / hemisphere are administered to a subject. 6 In some embodiments, about 14×10 cells / hemisphere are administered to a subject. 6 In some embodiments, about 15×10 cells / hemisphere are administered to a subject. 6 Cells / hemisphere are administered to the subject.

[0087] In some embodiments, the cells of the therapeutic composition are autologous to the subject.

[0088] In some embodiments, the cells of the therapeutic composition are allogeneic to the subject. In some embodiments, the cells of the therapeutic composition are hypoimmunogenic. In some embodiments, the cells of the therapeutic composition are genetically engineered to (a) remove genes encoding one or more of polymorphisms 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 genetically engineered to remove genes encoding polymorphisms HLA-A / -B / -C and HLA class II molecules. In some embodiments, the cells of the therapeutic composition are genetically engineered to provide genes encoding PD-L1, HLA-G, and CD47. In some embodiments, the cells of the therapeutic composition are genetically engineered to provide genes encoding PD-L1, HLA-G, and CD47 at the AAVS1 safe harbor locus. In some embodiments, the cells of the therapeutic composition are genetically engineered to remove genes encoding polymorphic HLA-A / -B / -C and HLA class II molecules, and to provide genes encoding PD-L1, HLA-G, and CD47 at the AAVS1 safe harbor locus.

[0089] Also provided herein is the use of any of the compositions provided herein for treating Parkinsonism. Also provided herein is the use of any of the compositions provided herein for treating Parkinson's disease.

[0090] Also provided herein are compositions described herein for formulating a medicament for treating a subject having a neurodegenerative disease or condition.

[0091] Also provided herein is any pharmaceutical composition described herein for use in treating a subject having a neurodegenerative disease or condition.

[0092] In some embodiments, the neurodegenerative disease or condition comprises loss of dopaminergic neurons. In some embodiments, the subject has at least 50%, at least 60%, at least 70%, or at least 80% loss of dopaminergic neurons, optionally in the substantia nigra (SN), optionally in the substantia nigra pars compacta (SNc). In some embodiments, the neurodegenerative disease or condition is Parkinsonism. In some embodiments, the neurodegenerative disease or condition is Parkinson's disease, optionally idiopathic Parkinson's disease.

[0093] In some embodiments, the cells of the composition are autologous to the subject. In some embodiments, the cells of the therapeutic composition are allogeneic to the subject. In some embodiments, the cells of the therapeutic composition are hypoimmunogenic. In some embodiments, the cells of the therapeutic composition are genetically 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 at the AAVS1 safe harbor locus. [Brief description of the drawings]

[0094] [Figure 1] An exemplary non-adherent protocol for differentiating pluripotent stem cells into committed dopamine (DA) neuronal progenitor cells or DA neurons is shown.

[0095] [Diagram 2] Shown is the expression of FOXA2, TH, and DAPI by differentiated cells at day 25 derived from spheroids with approximately 500, 1,000, 2,000, 3,000, 10,000, or 15,000 cells.

[0096] [Diagram 3] Expression of FOXA2, TH, and DAPI by differentiated cells at day 10 grown with or without serum replacement is shown.

[0097] [Figure 4] An exemplary adherent protocol for differentiating pluripotent stem cells into committed dopamine (DA) neuronal progenitor cells or DA neurons is shown.

[0098] [Diagram 5] 1 shows expression of FOXA2, TH, and DAPI by differentiated cells at day 25 from an exemplary adherent or an exemplary non-adherent method.

[0099] [Figure 6] 1 shows expression of the tyrosine hydroxylase (TH) gene at days 18 and 25 by cells differentiated by an exemplary non-adherent method.

[0100] [Figure 7] Dopamine (DA) and norepinephrine (NE) production (ng / million cells assayed) by cells cultured up to day 75 is shown.

[0101] [Figure 8A] 1 shows the expression of FOXA2 and TH genes in cells differentiated by an exemplary non-adherent method at various days of differentiation.

[0102] [Figure 8B] 1 shows the expression of EN1 and CORIN genes in cells differentiated by an exemplary non-adherent method at various days of differentiation.

[0103] [Figure 8C] 1 shows the expression of PAX6 and SOX2 genes in cells differentiated by an exemplary non-adherent method at various days of differentiation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0104] I. Definition Unless otherwise defined, all technical terms, notations, and other technical and scientific terms or terminology used herein are intended to have the same meaning as commonly understood by those of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be construed as representing something substantially different to what is generally understood in the art.

[0105] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more." It is understood that the embodiments and variations described herein include "consisting of" and / or "consisting essentially of" embodiments and variations.

[0106] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity, and should not be construed as rigidly limiting the scope of the claimed subject matter. Thus, the description of a range should be construed as specifically disclosing all possible subranges as well as individual numerical values ​​within that range. For example, when a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, and any other specified or intervening value in the specified range, is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, and are also encompassed by the claimed subject matter, subject to any specifically excluded limits within the specified range. When a specified range includes one or both of the limits, ranges excluding either or both of those inclusive limits are also encompassed by the claimed subject matter. This applies regardless of the breadth of the range.

[0107] As used herein, the term "about" refers to a normal error range for each value that is easily recognized. Reference herein to a value or parameter of "about" includes (and describes) an embodiment that is directed to the value or parameter itself. For example, a description that refers to "about X" includes a description of "X."

[0108] As used herein, the statement that a cell or cell population is "positive" for a particular marker refers to the detectable presence of a particular marker, typically a surface marker, on or within the cell.When referring to a surface marker, the term refers to the presence of surface expression detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting the antibody, and the staining is detectable by flow cytometry at a level substantially higher than that detected by performing the same procedure with an isotype-matched control under otherwise identical conditions, and / or at a level substantially similar to that of cells known to be positive for the marker, and / or at a level substantially higher than that of cells known to be negative for the marker.When referring to an intracellular marker, such as a transcription product or translation product, the term refers to the presence of a detectable transcription or translation product, for example, the product is detected at a level substantially higher than that detected by performing the same procedure with a control under otherwise identical conditions, and / or at a level substantially similar to that of cells known to be positive for the marker, and / or at a level substantially higher than that of cells known to be negative for the marker.

[0109] As used herein, the statement that a cell or cell population is "negative" for a particular marker refers to the absence of detectable presence of a particular marker, typically a surface marker, on or within the cell.When referring to a surface marker, the term refers to the absence of surface expression detected by flow cytometry, for example, by staining an antibody that specifically binds to the marker and detecting the antibody, and the staining is not detected by flow cytometry at a level substantially higher than that detected by performing the same procedure with an isotype-matched control under otherwise identical conditions, and / or at a level substantially lower than that of cells known to be positive for the marker, and / or at a level substantially similar to that of cells known to be negative for the marker.When referring to an intracellular marker, such as a transcription product or translation product, the term refers to the absence of detectable transcription or translation product, for example, the product is not detected at a level substantially higher than that detected by performing the same procedure with a control under otherwise identical conditions, and / or at a level substantially lower than that of cells known to be positive for the marker, and / or at a level substantially similar to that of cells known to be negative for the marker.

[0110] The term "expression" or "expressed" as used herein with respect to a gene refers to the transcription and / or translation product of that gene. The expression level of a DNA molecule in a cell can be determined based on the amount of corresponding mRNA present in the cell or the amount of protein encoded by the DNA produced by the cell (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 18.1-18.88).

[0111] As used herein, the term "stem cell" refers to a cell characterized by the ability for self-renewal by cell division and the potential to differentiate into tissues or organs. Among mammalian stem cells, embryonic and somatic stem cells can be distinguished. Embryonic stem cells are present in the blastocyst and give rise to embryonic tissues, whereas somatic stem cells are present in adult tissues for the purpose of tissue regeneration and repair.

[0112] As used herein, the term "adult stem cell" refers to undifferentiated cells found in an individual after embryonic development. Adult stem cells proliferate by cell division to replenish dying cells and regenerate damaged tissues. Adult stem cells have the ability to divide and create other cells similar to themselves or create more differentiated cells. 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 to differentiate into all three germ layers of cell types of pluripotent stem cells.

[0113] As used herein, the terms "induced pluripotent stem cell", "iPS" and "iPSC" refer to pluripotent stem cells artificially obtained (e.g., through artificial manipulation) from non-pluripotent cells. "Non-pluripotent cells" may be cells that have less ability to self-renew and differentiate than pluripotent stem cells. Less capable cells may be, but are not limited to, adult stem cells, tissue-specific progenitor cells, primary or secondary cells.

[0114] As used herein, the term "pluripotent" or "pluripotency" refers to cells that have the capacity, under appropriate conditions, to give rise to progeny that can undergo differentiation 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 tissues of prenatal, postnatal, or adult organisms.

[0115] As used herein, the term "pluripotent stem cell characteristics" refers to cellular characteristics that distinguish pluripotent stem cells from other cells. The expression or non-expression of a particular combination of molecular markers is an example of a pluripotent stem cell characteristic. More specifically, human pluripotent stem cells may express at least some, and 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 pluripotent stem cell characteristic.

[0116] As used herein, the term "reprogramming" refers to the process of dedifferentiating a non-pluripotent cell into a cell that exhibits characteristics of a pluripotent stem cell.

[0117] As used herein, the term "adherent culture vessel" refers to a culture vessel to which cells can attach, such as via extracellular matrix molecules, and which requires the use of enzymes (e.g., trypsin, dispase, etc.) to detach the cells from the culture vessel. An "adherent culture vessel" is in contrast to a culture vessel in which cell attachment is reduced and does not require the use of enzymes to remove the cells from the culture vessel.

[0118] As used herein, the term "non-adherent culture vessel" refers to a culture vessel in which cell attachment is reduced or limited, for example, for a period of time. Non-adherent culture vessels may have low- or ultra-low-attachment surfaces, which may be achieved, for example, by treating the surface with substances such as hydrogels (e.g., neutrally charged and / or hydrophilic hydrogels) and / or surfactants (e.g., pluronic acid) to prevent cell attachment. Non-adherent culture vessels may have rounded or concave wells and / or microwells (e.g., Aggrewell™). In some embodiments, the non-adherent culture vessel is an Aggrewell™ plate. Non-adherent culture vessels may not require the use of enzymes to remove cells from the culture vessel.

[0119] As used herein, the term "cell culture" may refer to an in vitro population of cells present outside of 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 for long-term in vitro culture.

[0120] As used herein, the terms "culture," "cultivate," "grow," "grow," "maintain," "maintaining," "proliferation," "proliferating," and the like, when referring to cell culture itself or the process of culturing, may be used interchangeably to mean that cells are maintained outside the body (e.g., ex vivo) under conditions suitable for survival. Cultured cells may be allowed to survive, and as a result of the culturing, the cells may grow, differentiate, or divide.

[0121] As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells, which may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

[0122] The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use, such as in a mammalian subject (e.g., a human). A pharmaceutical composition typically comprises an effective amount of an active agent (e.g., cells) and a carrier, excipient, or diluent. The carrier, excipient, or diluent is typically a pharma- ceutically acceptable carrier, excipient, or diluent, respectively.

[0123] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0124] The term "package insert" is used to refer to instructions typically included in the commercial packaging of a therapeutic product that contain information regarding the indications, uses, dosage, administration, concomitant therapy, contraindications, and / or warnings regarding the use of such therapeutic product.

[0125] As used herein, a "subject" is a mammal, such as a human or other animal, typically a human.

[0126] Detailed Description The present disclosure relates to a method for lineage-specific differentiation of pluripotent stem cells (PSCs), such as embryonic stem (ES) cells or induced pluripotent stem cells (iPSCs). Specifically, a method is described for inducing lineage-specific differentiation of PSCs or iPSCs into floor plate midbrain progenitor cells, committed dopamine (DA) neuron progenitor cells (DDPCs), and / or dopamine (DA) neurons. The differentiated cells produced using the methods provided herein are further contemplated for various uses, including but not limited to, as therapeutic agents for reversing disease, or damage, or loss of dopamine neurons in patients.

[0127] Provided herein are methods for lineage-specific differentiation of pluripotent stem cells (PSCs), such as embryonic stem (ES) cells or induced pluripotent stem cells (iPSCs), into floor-plate midbrain progenitor cells, committed dopamine (DA) neuron progenitor cells, and / or dopamine (DA) neurons. In some aspects, PSCs are differentiated into floor-plate midbrain progenitor cells. In some aspects, such floor-plate midbrain progenitor cells are further differentiated into committed dopamine (DA) neuron progenitor cells. In some embodiments, committed dopaminergic progenitor cells are cells that differentiate into dopamine neurons and cannot differentiate into non-dopamine neuron cells. In some aspects, such committed dopamine (DA) neuron progenitor cells are further differentiated into dopamine (DA) neurons. In some aspects, PSCs are differentiated into floor-plate midbrain progenitor cells, committed dopamine (DA) neuron progenitor cells, and finally into dopamine (DA) neurons.

[0128] Provided embodiments address problems associated with Parkinson's disease (PD), a hallmark of PD that involves selective degeneration of midbrain dopamine (mDA) neurons in the brains of patients. Because PD symptoms result primarily from selective loss of DA neurons in the substantia nigra of the ventral midbrain, PD is believed to be amenable to cell replacement therapy strategies.

[0129] A challenge in developing cell-based therapies for PD has been the identification of a suitable cell source for use in neuronal replacement. The search for a suitable cell source has been ongoing for several decades, 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 (eds. Pasterkamp, ​​RJ, Smidt, & Burbach) Landes Biosciences (2008); Fitzpatrick, et al., Antioxid. Redox. Signal. (2009) 11: 2189-2208. Some of these sources, including catecholaminergic cells from the adrenal medulla, carotid body grafts, or encapsulated retinal pigment epithelial cells, have progressed to early clinical trials. 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, these trials showed little clinical efficacy, poor long-term survival, and low DA release from the transplanted cells.

[0130] Another approach has been the transplantation of fetal midbrain DA neurons, which has been performed in over 300 patients worldwide, for example. Brundin, et al., Prog. Brain Res. (2010) 184: 265-94; Lindvall, & Kokaia, J. Clin. Invest (2010) 120: 29-40. Therapy using human fetal tissue in these patients has shown 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 transplantation has failed to replace DA neuron function. Furthermore, fetal tissue transplantation suffers from challenges including low quantity and poor quality of donor tissue, ethical and practical issues surrounding tissue acquisition, and the poorly defined and heterogeneous nature of the transplanted cells, which are some of the factors that contribute to the variability of 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 transplants may not provide sufficient numbers of cells at the correct developmental stage, and that fetal tissues are very poorly defined by cell type and variable with respect to stage and quality of each tissue sample. Bjorklund, et al. Lancet Neurol. (2003) 2:437-45. An inflammatory host response to the graft may be an additional contributing factor. Id.

[0131] Stem cell-derived cells, such as pluripotent stem cells (PSCs), are contemplated as a source of cells for applications in regenerative medicine. Pluripotent stem cells have the capacity to undergo self-renewal and give rise to all the cells of the body's tissues. PSCs include two broad categories of cells: embryonic stem (ES) cells and induced pluripotent stem cells (iPSCs). ES cells are derived from the inner cell mass of preimplantation embryos and can be maintained indefinitely and propagated in a pluripotent state in vitro. Romito and Cobellis, Stem Cells Int. (2016) 2016:9451492. iPSCs can be obtained by reprogramming ("dedifferentiating") adult somatic cells to make them more ES cell-like, including having the capacity to proliferate indefinitely and differentiate into all three germ layers. Ibid.

[0132] Pluripotent stem cells, such as ES cells, have been tested as a source for generating cells capable of engraftment. Early studies in the 1990s using mouse ES cells demonstrated the feasibility of obtaining specific lineages, including neurons, from pluripotent cells in vitro. Okabe, et al., Mech. Dev. (1996) 59: 89-102; Bain, et al., Dev. Biol. (1995) 168 v 342-357. Based on developmental knowledge gained from early explant studies, directed differentiation strategies were used to generate midbrain DA neurons. Lee, et al., Nat. Biotechnol. (2000) 18 v 675-679; Ye, et al., Cell (1998) 93: 755-66. However, these attempts did not yield cell populations with a high percentage of midbrain DA neurons or cells that could restore neuronal function in vivo. Furthermore, the resulting population contained a mixture of cell types in addition to midbrain DA neurons.

[0133] Existing strategies for using human PSCs (hPSCs) for cell therapy have not been entirely satisfactory. DA neurons derived from human PSCs have generally shown poor in vivo performance and failed to compensate for the endogenous loss of neuronal function. Tabar, et al. Nature Med. (2008) 14: 379-81;; Lindvall and Kokaia, J. Clin. Invest (2010) 120: 29-40.

[0134] More recently, preclinical studies in which human ES cells were first differentiated into midbrain floor intermediates and then further differentiated into DA neurons showed in vivo survival and led to the recovery 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 is plagued by ethical issues, as well as the possibility that such cells may form tumors in patients. Finally, ES cell-derived grafts may provoke immune responses in patients in the setting of allogeneic stem cell transplantation.

[0135] The use of induced pluripotent stem cells (iPSCs) rather than ES-derived cells has the advantage of avoiding ethical issues. Furthermore, the risk of immune rejection inherent in the use of embryonic stem cells is avoided if the iPSCs are derived from the patient being treated (i.e., the patient undergoes an autologous cell transplant). Previous studies have revealed that poor standardization of the transplanted cell material contributes to high variability, so new methods are needed to generate substantial numbers of standardized cells for autologous stem cell transplants and the like. Lindvall and Kokaia, J. Clin. Invest (2010) 120:29-40.

[0136] Studies are currently underway to differentiate human iPSCs into DA neuron precursors and transplant them into the human striatum. However, the ability of these cells to survive, engraft, and innervate other cells in vivo has not yet been reported. Takahashi, Brain Res. (2017) 230: 213-26 (2017); Cyranoski, D., Nature News (2018).

[0137] Thus, existing strategies have yet to prove successful in generating populations of differentiated cells for use in engraftment procedures to restore neuronal function in vivo. Provided herein are methods for differentiating PSCs into committed dopaminergic neuronal progenitor cells (DDPCs) and / or DA neuronal cells. Specifically, the provided methods are based on the finding that initiating culture of PSCs as non-adherent cells in the presence of SB, LDN, SHH, PUR, and CHIR to generate spheroids, followed by further incubation of the cells of the spheroids on a substrate-coated plate, generates differentiated cells with superior properties. Further aspects of the provided methods include collecting cells at a time when the cells are committed dopaminergic (dopamine neuronal) progenitor 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 differentiated according to the provided methods and collected at or about day 18.

[0138] In some cases, the methods described herein involve culturing PSCs in non-adherent culture (i.e., suspension culture) for about 7 days into spheroids, then dissociating the spheroids and continuing to culture until collection if the dissociated cells are present on a plate, e.g., a substrate-coated plate (i.e., as an adherent culture). The provided methods of differentiating cells in non-adherent culture entail advantages over alternative methods, such as methods in which the entire process of differentiation is performed by adherent culture of cells.

[0139] In some cases, the non-adherent culture of the provided methods is advantageous compared to differentiation methods that do not involve non-adherent culture of cells, since the effect of variability of one or more substrates and / or one or more reagents on cell differentiation during non-adherent culture is reduced or eliminated. Specifically, the adherent culture may involve coating a plate with one or more substrates or reagents, and such coating may be non-uniform, thereby variably providing one or more substrates and / or reagents to the adherent cells, particularly in methods in which the entire differentiation process is performed by adherent culture of cells. Such variably providing substrates and / or reagents to the adherent cells may increase the variability of the collected cells generated by the differentiation method. In contrast, substrates and / or reagents may not be required in non-adherent culture, or may be uniformly distributed throughout the non-adherent culture. In the latter case, the cultured cells (e.g., spheroids) are surrounded by culture medium on all sides so that they are evenly exposed to any substrates and / or reagents in the culture medium. Thus, in aspects of the provided embodiments, cells generated by differentiation methods involving non-adherent culture may exhibit reduced variability.

[0140] In addition, the non-adherent culture method described herein is advantageous compared to methods that do not include non-adherent culture, since non-adherent culture can increase cell-cell interactions. In particular, cells in adherent (e.g., monolayer) cultures only contact other cells on their outer surfaces. In contrast, cells in non-adherent (e.g., suspension) cultures can contact other cells over their entire surface. Such increased cell-cell contact in non-adherent culture can be advantageous by more closely reproducing a physiological environment. For example, the increased cell-cell contact enabled by the non-adherent culture method described herein can upregulate the intercellular protein network as observed in vivo.

[0141] In some embodiments, the methods involving the non-adherent culture methods provided herein provide for easy and efficient culture of a larger number of cells compared to alternative methods, such as those in which the entire differentiation process is carried out by adherent culture of cells. For example, in adherent (e.g., monolayer) culture, the number of cells cultured increases in two-dimensional scale (i.e., the length and width of the culture surface) along with the surface area of ​​the culture surface. In contrast, in non-adherent (e.g., suspension) culture, the number of cells cultured increases in three-dimensional scale along with the volume of the culture vessel, or the volume of culture medium contained therein. Thus, in some cases, non-adherent culture is more economical and efficient in that fewer resources (e.g., culture vessels and reagents) are used, more cells can be generated, or both, compared to adherent culture. In this way, the non-adherent culture methods provided can enable scale-up in manufacturing and production processes.

[0142] Further, a particular advantage relates to the method of differentiating cells, which comprises non-adherent culture for only about 7 days. Non-adherent culture for about 7 days as described herein produces spheroids. If non-adherent culture is allowed to proceed beyond 7 days, the increase in size of the spheroid may limit mass transfer and result in a gradient of reagent (e.g., morphogen) across the diameter of the spheroid. Substantial gradient of reagent (e.g., morphogen) across the diameter of the spheroid is undesirable, since different cells of the spheroid will be exposed to different concentrations of reagent (e.g., morphogen), which will result in variable cell responses and differentiation. Thus, the method described herein, which comprises non-adherent culture for only about 7 days or up to 7 days, is advantageous, since the variation in concentration of reagent, such as morphogen, to which the cells of the spheroid are exposed is minimized.

[0143] Also, in some embodiments, limiting the non-adherent culture component of the methods described herein to about 7 days helps ensure consistent differentiation of cultured cells. This is because prolonged (e.g., more than 7 days) differentiation of PSCs in non-adherent (e.g., suspension) culture can allow PSCs to establish a microenvironment that allows them to self-renew in a pluripotent state. Thus, the methods described herein ensure consistent and effective differentiation of cultured cells by reducing or eliminating the chances that PSCs will persist in culture.

[0144] In embodiments of the methods provided, cells generated by the methods described herein are advantageous because they exhibit expression of A9-specific markers, demonstrate a fate as A9 dopamine neurons, and are suitable for transplantation, engraftment, and innervation of other cells in vivo.

[0145] In particular, cells generated by the methods described herein, including cells collected on day 18 of differentiation, express EN1 and CORIN, genes expressed by A9 progenitor cells during fetal development. Compared to cells collected on day 18 of the exemplary differentiation methods described herein, cells collected on day 25 also show downregulation in mitosis-related genes, indicating increased commitment to specific cell type fates with increasing time in culture. Furthermore, cells generated by the methods described herein show upregulation of expression of neurite outgrowth-related genes on day 25 compared to day 18. Neurite outgrowth-related genes are involved in the development of new neuronal projections and synapse formation. Thus, this finding indicates that cells differentiated by the exemplary methods described herein become more committed to neuronal fates with increasing time in culture. Taken together, these characteristics indicate that cells collected on day 18 are committed to dopaminergic neuronal progenitor cells. Thus, cells generated by the methods described herein, including differentiated cells collected on day 18, may be advantageous compared to cells differentiated by other methods and / or cells collected on different (i.e., later) days of the differentiation protocol, as they are committed to a DA neuron fate but are less differentiated than on subsequent days.

[0146] The findings herein also demonstrate that although cells collected on day 18 appear to express many markers of DA neuron precursors, their genetic expression may be less committed to a DA neuron fate than cells collected on day 25. Thus, in some embodiments, cells collected on day 18 may represent an ideal time point to collect 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 cells collected at different (i.e., later) days of the differentiation protocol.

[0147] In some embodiments, cells collected at or about day 18 of the methods described herein, or therapeutic compositions containing same, may exhibit improved ability to engraft and / or innervate other cells compared to cells collected at a later time point (e.g., day 25) of the differentiation method. In some embodiments, cells collected on day 18 may also exhibit improved efficacy in vivo due to the state of differentiation and neuronal commitment. For example, cells collected on day 18 may exhibit improved engraftment and / or innervation, improved efficacy, or both, compared to cells collected on day 25.

[0148] In some embodiments, the cells collected by the provided differentiation methods exhibit therapeutic efficacy for treating a neurodegenerative disease. In some embodiments, the ability of the differentiated cells to treat a neurodegenerative disease may be determined in an animal model of the neurodegenerative disease. In some embodiments, the neurodegenerative disease is Parkinson's disease. In some embodiments, the differentiated cells collected by the provided methods are screened using an animal model of Parkinson's disease. Any known and available animal model of Parkinson's disease can be used for screening. In some embodiments, the animal model is a lesion model in which 6-hydroxydopamine (6-OHDA) is unilaterally stereotactically injected into the substantia nigra of the animal. In some embodiments, the animal model is a lesion model in which 6-OHDA is unilaterally stereotactically injected into the medial forebrain bundle of the animal. In some embodiments, a therapeutic composition containing differentiated cells generated by the provided methods, such as cells collected on day 18, is transplanted into the substantia nigra of the animal model. In some embodiments, a behavioral assay is performed to screen the therapeutic efficacy of transplantation into the animal model. In some embodiments, the behavioral assay includes monitoring amphetamine-induced rotation behavior. In some embodiments, the differentiated cells demonstrate therapeutic efficacy for treating a neurodegenerative disease if determined to reduce, diminish, or reverse brain pathology in a Parkinson's model in the model.

[0149] Moreover, unlike previously reported methods, the differentiated cells generated by the methods described herein show physiological consistency. Importantly, this physiological consistency is maintained across differentiated cells from different subjects. Thus, this method reduces both intra- and inter-subject variability, allowing better prediction of cell behavior in vivo. These advantages are relevant to the success of therapeutic strategies, especially in the context of autologous stem cell transplantation, where cells are generated separately for each patient. Such reproducibility advantages between different subjects can also allow for scale-up in manufacturing and production processes.

[0150] All publications, including patent documents, scientific literature, and databases, referred to 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. To the extent that a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in a patent, patent application, published application, or other publication incorporated herein by reference, the definition set forth herein shall take precedence over the definition incorporated herein by reference.

[0151] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. II. Methods for Differentiating Cells

[0152] 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 to generate cell spheroids, wherein beginning at the beginning of the first incubation (day 0), the first incubation comprises exposing the cells to (i) an inhibitor of TGF-β / Activin-Nodal signaling, (ii) at least one activator of Sonic Hedgehog (SHH) signaling, (iii) an inhibitor of bone morphogenetic protein (BMP) signaling, and (iv) an inhibitor of Glycogen Synthase Kinase 3β (GSK3β) signaling; and (b) performing a second incubation comprising culturing cells of the spheroids in a substrate-coated culture vessel under conditions to cause neural differentiation of the cells.

[0153] Methods of differentiating neural cells are provided, such as by subjecting iPSCs to cell culture methods that induce differentiation into floor plate midbrain progenitor cells, committed dopamine (DA) neuron progenitor cells, and / or dopamine (DA) neurons.

[0154] As described herein, iPSCs were generated from fibroblasts of human patients with Parkinson's disease. In the first incubation, starting from day 0, the iPSCs were then differentiated into midbrain floor plate precursors by exposure to small molecules such as LDN, SB, PUR, SHH, CHIR, and combinations thereof, and grown as spheroids in non-adherent culture. The resulting spheroids were then transferred to adherent culture as part of the second incubation, optionally after dissociating the spheroids, 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 engraftable committed DA neuron precursors or DA neurons. A. Sample and Cell Preparation

[0155] In embodiments of the provided methods, pluripotent stem cells are differentiated into floor plate midbrain progenitor cells, committed dopamine (DA) neuron progenitor cells, and / or dopamine (DA) neurons. Various sources of pluripotent stem cells, including embryonic stem (ES) cells and induced pluripotent stem cells (iPSCs), can be used in the methods.

[0156] In some embodiments, pluripotency refers to cells that have the ability, under appropriate conditions, to give rise to progeny that can undergo differentiation 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 tissues of prenatal, postnatal, or adult organisms. Standard art-accepted tests, such as the ability to form teratomas in 8-12 week old SCID mice, can be used to establish the pluripotency of a cell population. However, identification of various pluripotent stem cell characteristics can also be used to identify pluripotent cells. In some embodiments, pluripotent stem cells can be distinguished from other cells by specific characteristics, including the expression or non-expression of certain combinations of molecular markers. More specifically, human pluripotent stem cells may express at least some, and 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. In some embodiments, a characteristic of a pluripotent stem cell is a cell morphology associated with a pluripotent stem cell.

[0157] In some embodiments, pluripotent stem cells are artificially obtained from non-pluripotent cells, induced pluripotent stem cells (iPSCs). In some aspects, non-pluripotent cells are cells that have less ability to self-renew and differentiate than pluripotent stem cells. iPSCs can be generated by a process known as reprogramming, which effectively "dedifferentiates" non-pluripotent cells to an embryonic stem cell-like state by genetically engineering them to express genes such as OCT4, SOX2, and KLF4. Takahashi and Yamanaka Cell (2006) 126:663-76.

[0158] Methods for generating iPSCs are known. For example, mouse iPSCs were reported in 2006 (Takahashi and Yamanaka), and human iPSCs were reported in late 2007 (Takahashi et al. and Yu et al.). Mouse iPSCs exhibit key characteristics of pluripotent stem cells, including expression of stem cell markers, formation of tumors containing cells from all three germ layers, and the ability to contribute to many different tissues when injected into mouse embryos at the very early stage of development. Human iPSCs also express stem cell markers and can generate cells characteristic of all three germ layers.

[0159] In some embodiments, the PSCs (e.g., iPSCs) are autologous to the subject being treated, i.e., the PSCs are 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 to become iPSCs prior to differentiation into neural cells and / or neurons. In some embodiments, fibroblasts can be reprogrammed to iPSCs by transforming the fibroblasts with genes (OCT4, SOX2, NANOG, LIN28, and KLF4) cloned into plasmids (see, e.g., Yu, et al., Science DOI:10.1126 / science.1172482). In some embodiments, non-pluripotent fibroblasts derived from a patient with PD are reprogrammed to become iPSCs before differentiating into committed DA neuron progenitor cells and / or DA neurons, such as by using a non-integrating Sendai virus to reprogram the cells (e.g., using the CTS™ CytoTune™-iPS 2.1 Sendai Reprogramming Kit). In some embodiments, the resulting differentiated cells are then administered to the patient from whom they were derived in an autologous stem cell transplant. In some embodiments, the PSCs (e.g., iPSCs) are allogeneic to the subject being treated, i.e., the PSCs are derived from a different individual to whom the differentiated cells are administered. In some embodiments, non-pluripotent cells (e.g., fibroblasts) derived from another individual (e.g., an individual not having a neurodegenerative disorder such as Parkinson's disease) are reprogrammed to become iPSCs before differentiating into committed DA neuron progenitor cells and / or DA neurons. In some embodiments, reprogramming is accomplished, at least in part, by using a non-integrating Sendai virus to reprogram the cells (e.g., using the CTS™ CytoTune™-iPS 2.1 Sendai Reprogramming Kit). In some embodiments, the resulting differentiated cells are then administered to an individual that is not the same individual from which the differentiated cells were derived (e.g., allogeneic cell therapy or allogeneic cell transplantation).

[0160] In any of the embodiments provided, the PSCs (e.g., allogeneic cells) described herein can be genetically engineered to be less immunogenic. Methods for reducing immunogenicity are known and include abolishing expression of polymorphic HLA-A / -B / -C and HLA class II molecules, and introducing immune regulators PD-L1, HLA-G, and CD47 into the AAVS1 safe harbor locus in differentiated cells. Han et al., PNAS (2019) 116 (21): 10441-46. Thus, in some embodiments, the PSCs described herein are genetically engineered to delete the highly polymorphic HLA-A / -B / -C genes and introduce immune regulators such as PD-L1, HLA-G, and / or CD47 into the AAVS1 safe harbor locus.

[0161] 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 iPSCs reach 80-90% confluence, they are washed with phosphate-buffered saline (PBS) and subjected to enzymatic dissociation, such as with Accutase™, until the cells can be easily detached from the surface of the culture vessel. The dissociated iPSCs are then resuspended in culture medium for downstream differentiation into committed DA neuron progenitor cells and / or DA neurons.

[0162] In some embodiments, the PSCs are resuspended in a basal induction medium. In some embodiments, the basal induction medium is formulated to contain Neurobasal™ medium and DMEM / F12 medium in a 1:1 ratio, supplemented with N-2 and B27 supplements, non-essential amino acids (NEAA), GlutaMAX™, 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, the PSCs are resuspended in the same medium in which they are cultured for at least part of the first incubation. B. Non-adherent culture

[0163] The methods provided include culturing PSCs (e.g., iPSCs) by incubation with specific molecules (e.g., small molecules) to induce differentiation into floor plate midbrain progenitor cells, committed dopamine (DA) neuron progenitor cells, and / or dopamine (DA) neurons. Specifically, the embodiments provided include a first incubation of PSCs under non-adherent conditions in the presence of specific molecules (e.g., small molecules) to generate spheroids, which in some aspects can improve the consistency of generation of physiologically relevant cells for transplantation. In some embodiments, the method includes performing a first incubation involving culturing pluripotent stem cells in a non-adherent culture vessel under conditions to generate cell spheroids, wherein beginning at the beginning of the first incubation (day 0), the first incubation exposes the cells to (i) an inhibitor of TGF-β / Activin-Nodal signaling, (ii) at least one activator of Sonic Hedgehog (SHH) signaling, (iii) an inhibitor of bone morphogenetic protein (BMP) signaling, and (iv) an inhibitor of glycogen synthase kinase 3β (GSK3β) signaling.

[0164] In some embodiments, a non-adherent culture vessel is a culture vessel that has a low or ultra-low attachment surface, e.g., to inhibit or reduce cell attachment. In some embodiments, culturing cells in a non-adherent culture vessel does not prevent all cells of the culture from attaching to the surface of the culture vessel.

[0165] In some embodiments, the non-adherent culture vessel is a culture vessel with an ultra-low attachment surface. In some aspects, the ultra-low attachment surface can inhibit cell attachment for a period of time. In some embodiments, the ultra-low attachment surface can inhibit cell attachment for a period of time required to grow the same cell type confluently on the adherent surface. In some embodiments, the ultra-low attachment surface is coated or treated with a substance to prevent cell attachment, such as a hydrogel layer (e.g., a neutrally charged and / or hydrophilic hydrogel layer). In some embodiments, the non-adherent culture vessel is coated or treated with a surfactant prior to the first incubation. In some embodiments, the surfactant is pluronic acid.

[0166] In some embodiments, the non-adherent culture vessel is a plate, dish, flask, or 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 or 24-well plate. In some embodiments, the wells of the multi-well plate further comprise microwells. In some of any of the provided embodiments, the non-adherent culture vessel, such as a multi-well plate, has round or concave wells and / or microwells. In any of the provided embodiments, the non-adherent culture vessel, such as a multi-well plate, has no corners or seams.

[0167] In some embodiments, the non-adherent culture vessel allows three-dimensional formation of cell aggregates. In some embodiments, the iPSCs are cultured in a non-adherent culture vessel, such as a multi-well plate, to generate cell aggregates (e.g., spheroids). In some embodiments, the iPSCs are cultured in a non-adherent culture vessel, such as a multi-well plate, to generate cell aggregates (e.g., spheroids) at about day 7 of the method. In some embodiments, the cell aggregates (e.g., spheroids) express at least one of PAX6 and OTX2 at or by about day 7 of the method.

[0168] In some embodiments, the first incubation comprises culturing the pluripotent stem cells in a non-adherent culture vessel under conditions that produce cell spheroids.

[0169] In some embodiments, the number of PSCs plated on day 0 of the method is about 0.1 x 10 6 cells / cm 2 ~Approx. 2×10 6 cells / cm 2 , about 0.1×10 6 cells / cm 2 ~Approx. 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 ~Approx. 0.4×10 6 cells / cm 2 , about 0.1×10 6 cells / cm 2 ~Approx. 0.2×10 6 cells / cm 2 , about 0.2×10 6 cells / cm 2 ~Approx. 2×10 6 cells / cm 2 , about 0.2×106 cells / cm 2 ~Approx. 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 ~Approx. 0.4×10 6 cells / cm 2 , about 0.4×10 6 cells / cm 2 ~Approx. 2×10 6 cells / cm 2 , about 0.4×10 6 cells / cm 2 ~Approx. 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 ~Approx. 2×10 6 cells / cm 2 , about 0.6×10 6 cells / cm 2 ~Approx. 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 ~Approx. 2×10 6 cells / cm 2 , about 0.8×10 6 cells / cm 2 ~Approx. 1×10 6 cells / cm 2 , or approximately 1.0 × 106 cells / cm 2 ~Approx. 2×10 6 cells / cm 2 In some embodiments, the number of cells plated onto the substrate-coated culture vessel is about 0.4×10 6 cells / cm 2 ~about 0.8×10 6 cells / cm 2 It is.

[0170] In some embodiments, the number of PSCs plated on day 0 of the method is about 1 x 10 5 Pluripotent stem cells / well ~ approx. 20 x 10 6 Pluripotent stem cells / well, approximately 1 x 10 5 Pluripotent stem cells / well ~ approx. 15 x 10 6 Pluripotent stem cells / well, approximately 1 x 10 5 Pluripotent stem cells / well ~ approx. 10 x 10 6 Pluripotent stem cells / well, approximately 1 x 10 5 Pluripotent stem cells / well ~ 5 x 10 6 Pluripotent stem cells / well, approximately 1 x 10 5 Pluripotent stem cells / well ~ approx. 1 x 10 6 Pluripotent stem cells / well, approximately 1 x 10 5 Pluripotent stem cells / well ~ 5 x 10 5 Pluripotent stem cells / well, approximately 5 x 10 5 Pluripotent stem cells / well ~ approx. 20 x 10 6 Pluripotent stem cells / well, approximately 5 x 10 5 Pluripotent stem cells / well ~ approx. 15 x 10 6 Pluripotent stem cells / well, approximately 5 x 10 5 Pluripotent stem cells / well ~ approx. 10 x 10 6 Pluripotent stem cells / well, approximately 5 x 10 5 Pluripotent stem cells / well ~ 5 x 10 6 Pluripotent stem cells / well, approximately 5 x 10 5 Pluripotent stem cells / well ~ approx. 1 x 10 6 Pluripotent stem cells / well, approximately 1 x 10 6 Pluripotent stem cells / well ~ approx. 20 x 10 6 Pluripotent stem cells / well, approximately 1 x 10 6Pluripotent stem cells / well ~ approx. 15 x 10 6 Pluripotent stem cells / well, approximately 1 x 10 6 Pluripotent stem cells / well ~ approx. 10 x 10 6 Pluripotent stem cells / well, approximately 1 x 10 6 Pluripotent stem cells / well ~ 5 x 10 6 Pluripotent stem cells / well, approximately 5 x 10 6 Pluripotent stem cells / well ~ approx. 20 x 10 6 Pluripotent stem cells / well, approximately 5 x 10 6 Pluripotent stem cells / well ~ approx. 15 x 10 6 Pluripotent stem cells / well, approximately 5 x 10 6 Pluripotent stem cells / well ~ approx. 10 x 10 6 Pluripotent stem cells / well, approximately 10 x 10 6 Pluripotent stem cells / well ~ approx. 20 x 10 6 Pluripotent stem cells / well, approximately 10 x 10 6 Pluripotent stem cells / well ~ approx. 15 x 10 6 Pluripotent stem cells / well, or approximately 15 x 10 6 Pluripotent stem cells / well ~ approx. 20 x 10 6 Pluripotent stem cells / well.

[0171] 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 ~ approx. 20 x 10 6 Pluripotent stem cells / well, approximately 1 x 10 6 Pluripotent stem cells / well ~ approx. 15 x 10 6 Pluripotent stem cells / well, approximately 1 x 10 6 Pluripotent stem cells / well ~ approx. 10 x 10 6 Pluripotent stem cells / well, approximately 1 x 10 6 Pluripotent stem cells / well ~ 5 x 10 6 Pluripotent stem cells / well, approximately 5 x 10 6 Pluripotent stem cells / well ~ approx. 20 x 10 6 Pluripotent stem cells / well, approximately 5 x 10 6 Pluripotent stem cells / well ~ approx. 15 x 10 6 Pluripotent stem cells / well, approximately 5 x 10 6 Pluripotent stem cells / well ~ approx. 10 x 106 Pluripotent stem cells / well, approximately 10 x 10 6 Pluripotent stem cells / well ~ approx. 20 x 10 6 Pluripotent stem cells / well, approximately 10 x 10 6 Pluripotent stem cells / well ~ approx. 15 x 10 6 Pluripotent stem cells / well, or approximately 15 x 10 6 Pluripotent stem cells / well ~ approx. 20 x 10 6 Pluripotent stem cells / well.

[0172] In some embodiments, the number of PSCs plated in a 24-well plate on day 0 of the method is about 1×10 5 Pluripotent stem cells / well ~ 5 x 10 6 Pluripotent stem cells / well, approximately 1 x 10 5 Pluripotent stem cells / well ~ approx. 1 x 10 6 Pluripotent stem cells / well, approximately 1 x 10 5 Pluripotent stem cells / well ~ 5 x 10 5 Pluripotent stem cells / well, approximately 5 x 10 5 Pluripotent stem cells / well ~ 5 x 10 6 Pluripotent stem cells / well, approximately 5 x 10 5 Pluripotent stem cells / well ~ approx. 1 x 10 6 Pluripotent stem cells / well, or approximately 1 x 10 6 Pluripotent stem cells / well ~ 5 x 10 6 Pluripotent stem cells / well.

[0173] On some days, the number of PSCs plated on day 0 of the method is a number of cells sufficient to generate cell spheroids containing about 1,000 cells to about 5,000 cells or about 2,000 cells to about 3,000 cells. On some days, the number of PSCs plated on day 0 of the method is a number of cells sufficient to generate cell spheroids containing about 1,000 cells to about 5,000 cells. On some days, the number of PSCs plated on day 0 of the method is a number of cells sufficient to generate cell spheroids containing about 2,000 cells to about 3,000 cells. On some days, the number of PSCs plated on day 0 of the method is a number of cells sufficient to generate cell spheroids containing about 2,000 cells. On some days, the number of PSCs plated on day 0 of the method is a number of cells sufficient to generate cell spheroids containing about 3,000 cells. In some embodiments, spheroids containing the desired number are produced by the method at or by about day 7.

[0174] In some embodiments of the methods provided herein, the first incubation comprises culturing the pluripotent stem cells in a non-adherent culture vessel under conditions to generate cell spheroids. In some embodiments, the first incubation is from about day 0 to about day 6. In some embodiments, the first incubation comprises culturing the pluripotent stem cells in a culture medium ("medium"). In some embodiments, the first incubation comprises culturing the pluripotent stem cells in medium from about day 0 to about day 6. In some embodiments, the first incubation comprises culturing the pluripotent stem cells in medium to induce differentiation of the PSCs into floor plate midbrain progenitor cells.

[0175] In some embodiments, the medium is supplemented with a serum replacement that contains minimal non-human derived components (e.g., KnockOut™ serum replacement). In some embodiments, the serum replacement is provided to the medium at 5% (v / v) for at least a portion of the first incubation. In some embodiments, the serum replacement is provided to the medium at 5% (v / v) on days 0 and 1. In some embodiments, the serum replacement is provided to the medium at 2% (v / v) for at least a portion of the first incubation. In some embodiments, the serum replacement is provided to the medium at 2% (v / v) on days 2-6. In some embodiments, the serum replacement is provided to the medium at 5% (v / v) on days 0 and 1, and at 2% (v / v) on days 2-6.

[0176] In some embodiments, the medium is further supplemented with a small molecule, such as any of those described above. In some embodiments, the small molecule is selected from the group consisting of a Rho-associated protein kinase (ROCK) inhibitor, an inhibitor of TGF-β / activin-nodal signaling, at least one activator of Sonic Hedgehog (SHH) signaling, an inhibitor of bone morphogenetic protein (BMP) signaling, an inhibitor of glycogen synthase kinase 3β (GSK3β), and combinations thereof.

[0177] In some embodiments, the medium is supplemented with a Rho-associated protein kinase (ROCK) inhibitor for one or more days when the cells are passaged. In some embodiments, the medium is supplemented with a ROCK inhibitor every day the cells are passaged. In some embodiments, the medium is supplemented with a ROCK inhibitor on day 0.

[0178] 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.

[0179] 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, which has the following formula: [ka]

[0180] In some embodiments, the cells are exposed to Y-27632 at a concentration of about 10 μM. In some embodiments, on day 0, the cells are exposed to Y-27632 at a concentration of about 10 μM.

[0181] In some embodiments, the medium is supplemented with an inhibitor of TGF-β / activin-nodal signaling. In some embodiments, the medium is supplemented with an inhibitor of TGF-β / activin-nodal signaling until about day 7 (e.g., day 6 or day 7). In some embodiments, the medium is supplemented with an inhibitor of TGF-β / activin-nodal signaling from about day 0 to day 6, inclusive.

[0182] In some embodiments, the cells are exposed to an inhibitor of TGF-β / activin-nodal signaling 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 an inhibitor of TGF-β / activin-nodal signaling at a concentration of about 1 μM to about 20 μM. In some embodiments, the cells are exposed to an inhibitor of TGF-β / activin-nodal signaling at a concentration of about 5 μM to about 15 μM. In some embodiments, the cells are exposed to an inhibitor of TGF-β / activin-nodal signaling at a concentration of about 8 μM to about 12 μM. In some embodiments, the cells are exposed to an inhibitor of TGF-β / activin-nodal signaling at a concentration of about 10 μM.

[0183] In some embodiments, the inhibitor of TGF-β / activin-nodal signaling is a small molecule. In some embodiments, the inhibitor of TGF-β / activin-nodal signaling can reduce or block transforming growth factor beta (TGFβ) / activin-nodal signaling. In some embodiments, the inhibitor of TGF-β / activin-nodal signaling inhibits ALK4, ALK5, ALK7, or a combination thereof. In some embodiments, the inhibitor of TGF-β / activin-nodal signaling inhibits ALK4, ALK5, and ALK7. In some embodiments, the inhibitor of TGF-β / activin-nodal signaling 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 inhibitor of TGF-β / activin-nodal signaling is SB431542 (e.g., CAS 301836-41-9, molecular formula: C22H18N4O3, and name: 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide), which has the following formula: [ka]

[0184] In some embodiments, cells are exposed to SB431542 at a concentration of about 10 μM. In some embodiments, cells are exposed to SB431542 at a concentration of about 10 μM until about day 7. In some embodiments, cells are exposed to SB431542 at a concentration of about 10 μM from about day 0 to about day 6, inclusive.

[0185] In some embodiments, the medium is supplemented with at least one activator of Sonic Hedgehog (SHH) signaling. SHH refers to a protein that is one of at least three proteins in a mammalian signaling pathway family called Hedgehog, another protein is Desert Hedgehog (DHH), while a third protein is 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 medium is supplemented with at least one activator of SHH signaling until about day 7 (e.g., day 6 or day 7). In some embodiments, the medium is supplemented with at least one activator of SHH signaling from about day 0 to day 6, inclusive.

[0186] In some embodiments, the at least one activator of SHH signaling is a SHH protein. In some embodiments, the at least one activator of SHH signaling is a recombinant SHH protein. In some embodiments, the at least one activator of SHH signaling is a recombinant mouse SHH protein. In some embodiments, the at least one activator of SHH signaling is a recombinant human SHH protein. In some embodiments, the at least one activator of SHH signaling is a recombinant N-terminal fragment of a full-length mouse Sonic Hedgehog protein capable of binding to a SHH receptor to activate SHH. In some embodiments, the at least one activator of SHH signaling is a C25II SHH protein.

[0187] In some embodiments, the cells are exposed to at least one activator of SHH signaling at a concentration of about 10 ng / mL to about 500 ng / mL, about 20 ng / mL to about 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 (inclusive). In some embodiments, the cells are exposed to at least one activator of SHH signaling at a concentration of about 50 ng / mL to about 100 ng / mL (inclusive). In some embodiments, the cells are exposed to at least one activator of SHH signaling at a concentration of about 100 ng / mL. In some embodiments, the cells are exposed to about 100 ng / mL of SHH protein. In some embodiments, the cells are exposed to about 100 ng / mL of recombinant SHH protein. In some embodiments, the cells are 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.

[0188] In some embodiments, the cells are exposed to recombinant SHH protein at a concentration of about 10 ng / mL. In some embodiments, the cells are exposed to 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 recombinant SHH protein at a concentration of about 10 ng / mL from about day 0 to about day 6, inclusive.

[0189] In some embodiments, the cells are exposed to at least one activator of SHH signaling 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 at least one activator of SHH signaling at a concentration of about 1 μM to about 20 μM. In some embodiments, the cells are exposed to at least one activator of SHH signaling at a concentration of about 5 μM to about 15 μM. In some embodiments, the cells are exposed to at least one activator of SHH signaling at a concentration of about 8 μM to about 12 μM. In some embodiments, the cells are exposed to at least one activator of SHH signaling at a concentration of about 10 μM.

[0190] In some embodiments, the at least one activator of SHH signaling is an activator of the hedgehog receptor smoothened. In some embodiments, the at least one activator of SHH signaling is a small molecule. In some embodiments, the at least one activator of SHH signaling is palmorfamine (e.g., CAS483367-10-8), which has the following formula: [ka]

[0191] In some embodiments, the cells are exposed to palmorfamine at a concentration of about 10 μM. In some embodiments, the cells are exposed to palmorfamine at a concentration of about 10 μM until about day 7 (e.g., day 6 or day 7). In some embodiments, the cells are exposed to palmorfamine at a concentration of about 10 μM from about day 0 to about day 6, inclusive.

[0192] In some embodiments, at least one activator of SHH signaling is SHH protein and palmorphamin. In some embodiments, the cells are exposed to a concentration of SHH protein and palmorphamin until about day 7 (e.g., day 6 or day 7). In some embodiments, the cells are exposed to SHH protein and palmorphamin from about day 0 to about day 6, inclusive. In some embodiments, the cells are exposed to a concentration of 100 ng / mL SHH protein and 10 μM palmorphamin until about day 7 (e.g., day 6 or day 7). In some embodiments, the cells are exposed to 100 ng / mL SHH protein and 10 μM palmorphamin from about day 0 to about day 6, inclusive.

[0193] In some embodiments, the medium is supplemented with an inhibitor of BMP signaling. In some embodiments, the medium is supplemented with an inhibitor of BMP signaling until about day 7 (e.g., day 6 or day 7). In some embodiments, the medium is supplemented with an inhibitor of BMP signaling from about day 0 to day 6, inclusive.

[0194] In some embodiments, the cells are exposed to a concentration of the inhibitor of BMP signaling 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 inclusive. In some embodiments, the cells are exposed to a concentration of the inhibitor of BMP signaling of about 0.01 μM to about 5 μM. In some embodiments, the cells are exposed to a concentration of the inhibitor of BMP signaling of about 0.05 μM to about 1 μM. In some embodiments, the cells are exposed to a concentration of the inhibitor of BMP signaling of about 0.1 μM to about 0.5 μM. In some embodiments, the cells are exposed to a concentration of the inhibitor of BMP signaling of about 0.1 μM.

[0195] In some embodiments, the inhibitor of BMP signaling is a small molecule. In some embodiments, the inhibitor of BMP signaling is selected from LDN193189 or K02288. In some embodiments, the inhibitor of BMP signaling can inhibit "Small Mothers Against Decapentaplegic" SMAD signaling. In some embodiments, the inhibitor of BMP signaling inhibits ALK1, ALK2, ALK3, ALK6, or a combination thereof. In some embodiments, the inhibitor of BMP signaling inhibits ALK1, ALK2, ALK3, and ALK6. In some embodiments, the inhibitor of BMP signaling inhibits BMP2, BMP4, BMP6, BMP7, and activin cytokine signals, and subsequently inhibits SMAD phosphorylation of Smad1, Smad5, and Smad8. In some embodiments, the inhibitor of BMP signaling is LDN193189. In some embodiments, the inhibitor of BMP signaling is LDN193189, which has the following formula (e.g., IUPAC name 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline, which has the chemical formula C25H22N6): [ka]

[0196] 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, inclusive.

[0197] In some embodiments, the medium is supplemented with an inhibitor of GSK3β signaling. In some embodiments, the medium is supplemented with an inhibitor of GSK3β signaling until about day 7 (e.g., day 6 or day 7). In some embodiments, the medium is supplemented with an inhibitor of GSK3β signaling from about day 0 to day 6, inclusive.

[0198] In some embodiments, the cells are exposed to an inhibitor of GSK3β signaling 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 inclusive. In some embodiments, the cells are exposed to an inhibitor of GSK3β signaling at a concentration of about 0.1 μM to about 10 μM. In some embodiments, the cells are exposed to an inhibitor of GSK3β signaling at a concentration of about 0.5 μM to about 8 μM. In some embodiments, the cells are exposed to an inhibitor of BMP signaling at a concentration of about 1 μM to about 4 μM. In some embodiments, the cells are exposed to an inhibitor of BMP signaling at a concentration of about 2 μM to about 3 μM. In some embodiments, the cells are exposed to an inhibitor of GSK3β signaling at a concentration of about 2 μM.

[0199] In some embodiments, the inhibitor of GSK3β signaling is selected from the group consisting of lithium ion, valproic acid, iodotubercidin, naproxen, famotidine, curcumin, olanzapine, CHIR99012, and combinations thereof. In some embodiments, the inhibitor of GSK3β signaling is a small molecule. In some embodiments, the inhibitor of GSK3β signaling inhibits glycogen synthase kinase 3β enzyme. In some embodiments, the inhibitor of GSK3β signaling inhibits GSK3α. In some embodiments, the inhibitor of GSK3β signaling modulates TGF-β and MAPK signaling. In some embodiments, the inhibitor of GSK3β signaling is an agonist of wingless / integrated (Wnt) signaling. In some embodiments, the inhibitor of GSK3β signaling has an IC50=6.7 nM for human GSK3β. In some embodiments, the inhibitor of GSK3β signaling is CHIR99021 (e.g., “3-[3-(2-carboxyethyl)-4-methylpyrrole-2-methylidenyl]-2-indolinone”, or IUPAC name 6-(2-(4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino)ethylamino)nicotinonitrile), which has the following formula: [ka]

[0200] 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, inclusive.

[0201] In some embodiments, at least about 50% of the medium is replaced daily from about day 2 to about day 6. In some embodiments, about 50% of the medium is replaced daily, every other day, or every third day from about day 2 to about day 6. In some embodiments, about 50% of the medium is replaced daily from about day 2 to about day 6. In some embodiments, at least about 75% of the medium is replaced on day 1. In some embodiments, about 100% of the medium is replaced on day 1. In some embodiments, the replacement medium contains about 2-fold concentrated small molecules compared to the concentration of the small molecules in the medium on day 0.

[0202] In some embodiments, the first incubation comprises culturing the pluripotent stem cells in a "basic induction medium." In some embodiments, the first incubation comprises culturing the pluripotent stem cells in a basal induction medium from about day 0 to about day 6. In some embodiments, the first incubation comprises culturing the pluripotent stem cells in a basal induction medium to induce differentiation of the PSCs into floor plate midbrain progenitor cells.

[0203] In some embodiments, the basal induction medium is formulated to contain Neurobasal™ medium and DMEM / F12 medium in a 1:1 ratio, supplemented with N-2 and B27 supplements, non-essential amino acids (NEAA), GlutaMAX™, L-glutamine, β-mercaptoethanol, and insulin. In some embodiments, the basal induction medium is further supplemented with any of the small molecules as described above. C. Transfer or dissociation of spheroids

[0204] In some embodiments, cell aggregates (e.g., spheroids) generated after a first incubation of pluripotent stem cells in a non-adherent culture vessel are displaced or dissociated prior to a second incubation of the cells on the substrate (adherent culture).

[0205] In some embodiments, the first incubation is performed to generate cell aggregates (e.g., spheroids) expressing at least one of PAX6 and OTX2. In some embodiments, the first incubation generates cell aggregates (e.g., spheroids) expressing PAX6 and OTX2. In some embodiments, the first incubation generates cell aggregates (e.g., spheroids) at or by about day 7 of the methods provided herein. In some embodiments, the first incubation generates cell aggregates (e.g., spheroids) expressing at least one of PAX6 and OTX2 at or by about day 7 of the methods provided herein. In some embodiments, the first incubation generates cell aggregates (e.g., spheroids) expressing PAX6 and OTX2 at or by about day 7 of the methods provided herein.

[0206] In some embodiments, the cell aggregates (e.g., spheroids) generated by the first incubation are dissociated prior to a second incubation of the cells on the substrate. In some embodiments, the cell aggregates (e.g., spheroids) generated by the first incubation are dissociated to generate a cell suspension. In some embodiments, the cell suspension generated by dissociation is a single cell suspension. In some embodiments, the dissociation occurs when the spheroid cells express at least one of PAX6 and OTX2. In some embodiments, the dissociation occurs when the spheroid cells express PAX6 and OTX2. In some embodiments, the dissociation occurs at about day 7. In some embodiments, the cell aggregates (e.g., spheroids) are dissociated by enzymatic dissociation. In some embodiments, the enzyme is selected from the group consisting of actase, dispase, collagenase, and combinations thereof. In some embodiments, the enzyme comprises actase. In some embodiments, the enzyme is actase. In some embodiments, the enzyme is dispase. In some embodiments, the enzyme is collagenase.

[0207] In some embodiments, the cell aggregates or a cell suspension generated therefrom are transferred to a substrate-coated culture vessel for a second incubation. In some embodiments, the cell aggregates (e.g., spheroids) or a cell suspension generated therefrom are transferred to a substrate-coated culture vessel after dissociation of the cell aggregates (e.g., spheroids). In some embodiments, the transfer is performed immediately after dissociation. In some embodiments, the transfer is performed on about day 7.

[0208] In some embodiments, the cell aggregates (e.g., spheroids) are not dissociated before the second incubation. In some embodiments, the entire cell aggregates (e.g., spheroids) are transferred to a substrate-coated culture vessel for the second incubation. In some embodiments, the transfer is performed when the spheroid cells express at least one of PAX6 and OTX2. In some embodiments, the transfer is performed when the spheroid cells express PAX6 and OTX2. In some embodiments, the transfer is performed at about day 7.

[0209] In some embodiments, the cells are transferred to an adherent culture vessel. In some embodiments, the culture vessel is a plate, dish, flask, or bioreactor. In some embodiments, the culture vessel is substrate-coated. In some embodiments, the substrate is a basement membrane protein. In some embodiments, the substrate is selected from laminin, collagen, entactin, heparin sulfate proteoglycan, 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 optionally exposed to poly-L-ornithine before being used to culture the cells. In some embodiments, the substrate-coated culture vessel is a 6-well or 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. D. Adhesive culture

[0210] In some embodiments, the method includes performing a second incubation of the transferred spheroid cells in a substrate-coated culture vessel. In some embodiments, the cells of the spheroid are cultured in the substrate-coated culture vessel under adherent conditions to induce differentiation into floor plate midbrain progenitor cells, committed dopamine (DA) neuron progenitor cells, and / or dopamine (DA) neurons.

[0211] In some embodiments, the second incubation involves culturing the cells of the spheroids in a culture vessel coated with a substrate comprising laminin, collagen, entactin, heparin sulfate proteoglycan, or a combination thereof, and exposing the cells to (i) an inhibitor of BMP signaling and (ii) an inhibitor of GSK3β signaling starting on day 7, and 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 beta-3 (TGFβ3), and (vi) an inhibitor of Notch signaling starting on day 11. In some embodiments, the method further comprises harvesting the differentiated cells.

[0212] In some embodiments, the substrate-coated culture vessel is a culture vessel having a surface to which cells can adhere. In some embodiments, the substrate-coated culture vessel is a culture vessel having a surface to which a substantial number of cells adhere. In some embodiments, the substrate is a basement membrane protein. In some embodiments, the substrate is laminin, collagen, entactin, heparin sulfate proteoglycan, 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 proteoglycan. In some embodiments, the substrate is a recombinant protein. In some embodiments, the substrate is 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 prior to use for cell culture.

[0213] In some embodiments, the substrate-coated culture vessel is a plate, dish, flask, or 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 or 24-well plate. In some embodiments, the substrate-coated culture vessel is a dish. In some embodiments, the substrate-coated culture vessel is a flask. In some embodiments, the substrate-coated culture vessel is a bioreactor.

[0214] In some embodiments, the substrate-coated culture vessel allows monolayer cell culture. In some embodiments, cells derived from the cell aggregates (e.g., spheroids) generated by the first incubation are cultured in monolayer culture on the substrate-coated plate. In some embodiments, cells derived from the cell aggregates (e.g., spheroids) generated by the first incubation are cultured to generate a monolayer culture of cells positive for one or more of LMX1A, FOXA2, EN1, CORIN, and combinations thereof. In some embodiments, cells derived from the cell aggregates (e.g., spheroids) generated by the first incubation are cultured to generate a monolayer culture of cells in which at least some of the cells are positive for EN1 and CORIN. In some embodiments, cells derived from the cell aggregates (e.g., spheroids) generated by the first incubation are cultured to generate a monolayer culture of cells in which at least some of the cells are TH+. In some embodiments, at least some of the cells become TH+ by or at about day 25. In some embodiments, cells derived from the cell aggregates (e.g., spheroids) generated by the first incubation are cultured to generate a monolayer culture of cells in which at least some of the cells are TH+FOXA2+. In some embodiments, at least some of the cells become TH+FOXA2+ by or at about day 25.

[0215] In the methods provided herein, the second incubation involves culturing the cells of the spheroids in a substrate-coated culture vessel under conditions that induce neuronal differentiation of the cells. In some embodiments, the cells of the spheroids are plated on the substrate-coated culture vessel on about day 7.

[0216] In some embodiments, the number of cells plated onto the substrate-coated culture vessel is about 0.1×10 6 cells / cm 2 ~Approx. 2×106 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 / 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 ~Approx. 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 ~Approx. 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 ~Approx. 1×10 6 cells / cm 2 , or approximately 1.0 × 10 6 cells / cm 2 ~about 2×10 6 cells / cm 2 In some embodiments, the number of cells plated onto the substrate-coated culture vessel is about 0.4×10 6 cells / cm 2 ~about 0.8×10 6 cells / cm 2 It is.

[0217] In some embodiments, the second incubation is from about day 7 until the cells are harvested. In some embodiments, the cells are harvested after about day 16. In some embodiments, the cells are harvested from about day 16 to about day 30. In some embodiments, the cells are harvested from about day 18 to 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 to about day 18. In some embodiments, the second incubation is from about day 7 to about day 25.

[0218] In some embodiments, the second incubation involves culturing cells derived from the cell aggregates (eg, spheroids) in culture medium ("medium").

[0219] In some embodiments, the second incubation involves culturing the cells in medium from about day 7 until harvest or recovery. In some embodiments, the cells are cultured in medium to generate committed dopamine (DA) neuronal progenitor cells or dopamine (DA) neurons.

[0220] In some embodiments, the medium is supplemented with a serum replacement that contains minimal non-human derived components (e.g., KnockOut™ serum replacement). In some embodiments, the medium is supplemented with serum replacement from about day 7 to about day 10. In some embodiments, the medium is supplemented with about 2% (v / v) serum replacement. In some embodiments, the medium is supplemented with about 2% (v / v) serum replacement from about day 7 to about day 10.

[0221] In some embodiments, the medium is further supplemented with a small molecule, which in some embodiments is selected from the group consisting of a Rho-associated protein kinase (ROCK) inhibitor, an inhibitor of bone morphogenetic protein (BMP) signaling, an inhibitor of glycogen synthase kinase 3β (GSK3β), and combinations thereof.

[0222] In some embodiments, the medium is supplemented with a Rho-associated protein kinase (ROCK) inhibitor for one or more days when the cells are passaged. In some embodiments, the medium is supplemented with a ROCK inhibitor every day the cells are passaged. In some embodiments, the medium is supplemented with a ROCK inhibitor on day 7, day 16, day 20, or a combination thereof. In some embodiments, the medium is supplemented with a ROCK inhibitor on day 7. In some embodiments, the medium is supplemented with a ROCK inhibitor on day 16. In some embodiments, the medium is supplemented with a ROCK inhibitor on day 20. In some embodiments, the medium is supplemented with a ROCK inhibitor on days 7 and 16. In some embodiments, the medium is supplemented with a ROCK inhibitor on days 16 and 20. In some embodiments, the medium is supplemented with a ROCK inhibitor on days 7, 16, and 20.

[0223] 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.

[0224] In some embodiments, the ROCK inhibitor is fasudil, ripasudil, netarsudil, 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, which has the following formula: [ka]

[0225] 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 days 7 and 16. In some embodiments, the cells are exposed to Y-27632 at a concentration of about 10 μM on days 16 and 20. In some embodiments, the cells are exposed to Y-27632 at a concentration of about 10 μM on days 7, 16, and 20.

[0226] In some embodiments, the medium is supplemented with an inhibitor of BMP signaling. In some embodiments, the medium is supplemented with an inhibitor of BMP signaling from about day 7 to about day 11 (e.g., day 10 or day 11). In some embodiments, the medium is supplemented with an inhibitor of BMP signaling from about day 7 to day 10 (inclusive).

[0227] In some embodiments, the cells are exposed to a concentration of the inhibitor of BMP signaling 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 inclusive. In some embodiments, the cells are exposed to a concentration of the inhibitor of BMP signaling of about 0.01 μM to about 5 μM. In some embodiments, the cells are exposed to a concentration of the inhibitor of BMP signaling of about 0.05 μM to about 1 μM. In some embodiments, the cells are exposed to a concentration of the inhibitor of BMP signaling of about 0.1 μM to about 0.5 μM. In some embodiments, the cells are exposed to a concentration of the inhibitor of BMP signaling of about 0.1 μM.

[0228] In some embodiments, the inhibitor of BMP signaling is a small molecule. In some embodiments, the inhibitor of BMP signaling is LDN193189 or K02288. In some embodiments, the inhibitor of BMP signaling can inhibit "Small Mothers Against Decapentaplegic" SMAD signaling. In some embodiments, the inhibitor of BMP signaling inhibits ALK1, ALK2, ALK3, ALK6, or a combination thereof. In some embodiments, the inhibitor of BMP signaling inhibits ALK1, ALK2, ALK3, and ALK6. In some embodiments, the inhibitor of BMP signaling inhibits BMP2, BMP4, BMP6, BMP7, and activin cytokine signals, and subsequently inhibits SMAD phosphorylation of Smad1, Smad5, and Smad8. In some embodiments, the inhibitor of BMP signaling is LDN193189. In some embodiments, the inhibitor of BMP signaling is LDN193189, which has the following formula (e.g., IUPAC name 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline, which has the chemical formula C25H22N6): [ka]

[0229] 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 from about day 7 to 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, inclusive.

[0230] In some embodiments, the medium is supplemented with an inhibitor of GSK3β signaling. In some embodiments, the medium is supplemented with an inhibitor of GSK3β signaling from about day 7 to about day 13 (e.g., day 12 or day 13). In some embodiments, the medium is supplemented with an inhibitor of GSK3β signaling from about day 7 to day 12 (inclusive).

[0231] In some embodiments, the cells are exposed to an inhibitor of GSK3β signaling 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 inclusive. In some embodiments, the cells are exposed to an inhibitor of GSK3β signaling at a concentration of about 0.1 μM to about 10 μM. In some embodiments, the cells are exposed to an inhibitor of GSK3β signaling at a concentration of about 0.5 μM to about 8 μM. In some embodiments, the cells are exposed to an inhibitor of GSK3β signaling at a concentration of about 1 μM to about 4 μM. In some embodiments, the cells are exposed to an inhibitor of GSK3β signaling at a concentration of about 2 μM to about 3 μM. In some embodiments, the cells are exposed to an inhibitor of GSK3β signaling at a concentration of about 2 μM.

[0232] In some embodiments, the inhibitor of GSK3β signaling is selected from lithium ion, valproic acid, iodotubercidin, naproxen, famotidine, curcumin, olanzapine, CHIR99012, or a combination thereof. In some embodiments, the inhibitor of GSK3β signaling is a small molecule. In some embodiments, the inhibitor of GSK3β signaling inhibits glycogen synthase kinase 3β enzyme. In some embodiments, the inhibitor of GSK3β signaling inhibits GSK3α. In some embodiments, the inhibitor of GSK3β signaling modulates TGF-β and MAPK signaling. In some embodiments, the inhibitor of GSK3β signaling is an agonist of Wingless / Integrated (Wnt) signaling. In some embodiments, the inhibitor of GSK3β signaling has an IC50=6.7 nM for human GSK3β. In some embodiments, the inhibitor of GSK3β signaling is CHIR99021 (e.g., “3-[3-(2-carboxyethyl)-4-methylpyrrole-2-methylidenyl]-2-indolinone”, or IUPAC name 6-(2-(4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino)ethylamino)nicotinonitrile), which has the following formula: [ka]

[0233] 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 from about day 7 to 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, inclusive.

[0234] In some embodiments, the medium is supplemented with Brain-Derived Neurotrophic Factor (BDNF). In some embodiments, the medium is supplemented with BDNF beginning at about day 11. In some embodiments, the medium is supplemented with BDNF from about day 11 until harvesting or recovery. In some embodiments, the medium is supplemented with BDNF from about day 11-18. In some embodiments, the medium is supplemented with BDNF from about day 11-25.

[0235] In some embodiments, cells are exposed to BDNF at a concentration of about 1 ng / mL to about 100 ng / mL, about 5 ng / mL to about 50 ng / mL, or about 10 ng / mL to about 30 ng / mL. In some embodiments, cells are exposed to BDNF at a concentration of about 10 ng / mL to about 30 ng / mL. In some embodiments, cells are exposed to BDNF at a concentration of about 20 ng / mL.

[0236] In some embodiments, the medium is supplemented with about 20 ng / mL of BDNF beginning at about day 11. In some embodiments, the medium is supplemented with 20 ng / mL of BDNF from about day 11 until harvest or recovery. In some embodiments, the medium is supplemented with about 20 ng / mL of BDNF from about day 11-18. In some embodiments, the medium is supplemented with about 20 ng / mL of BDNF from about day 11-25.

[0237] In some embodiments, the medium is supplemented with glial cell line derived neurotrophic factor (GDNF). In some embodiments, the medium is supplemented with GDNF beginning at about day 11. In some embodiments, the medium is supplemented with GDNF from about day 11 until harvest or recovery. In some embodiments, the medium is supplemented with GDNF from about day 11-18. In some embodiments, the medium is supplemented with GDNF from about day 11-25.

[0238] In some embodiments, 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, cells are exposed to GDNF at a concentration of about 10 ng / mL to about 30 ng / mL. In some embodiments, cells are exposed to GDNF at a concentration of about 20 ng / mL.

[0239] In some embodiments, the medium is supplemented with about 20 ng / mL of GDNF beginning at about day 11. In some embodiments, the medium is supplemented with 20 ng / mL of GDNF from about day 11 until harvest or recovery. In some embodiments, the medium is supplemented with about 20 ng / mL of GDNF from about day 11-18. In some embodiments, the medium is supplemented with about 20 ng / mL of GDNF from about day 11-25.

[0240] In some embodiments, the medium is supplemented with ascorbic acid. In some embodiments, the medium is supplemented with ascorbic acid beginning at about day 11. In some embodiments, the medium is supplemented with ascorbic acid from about day 11 until harvest or recovery. In some embodiments, the medium is supplemented with ascorbic acid from about day 11-18. In some embodiments, the medium is supplemented with ascorbic acid from about day 11-25.

[0241] 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 (inclusive). In some embodiments, the cells are exposed to ascorbic acid at a concentration of about 0.05 mM to about 5 mM (inclusive). In some embodiments, the cells are exposed to ascorbic acid at a concentration of about 0.1 mM to about 1 mM (inclusive). In some embodiments, the cells are exposed to ascorbic acid at a concentration of about 0.2 mM.

[0242] In some embodiments, the medium is supplemented with about 0.2 mM ascorbic acid beginning at about day 11. In some embodiments, the medium is supplemented with about 0.2 mM ascorbic acid from about day 11 until harvest or recovery. In some embodiments, the medium is supplemented with about 0.2 mM ascorbic acid from about day 11-18. In some embodiments, the medium is supplemented with about 0.2 mM ascorbic acid from about day 11-25.

[0243] In some embodiments, the medium is supplemented with dibutyryl cyclic AMP (dbcAMP). In some embodiments, the medium is supplemented with dbcAMP beginning at about day 11. In some embodiments, the medium is supplemented with dbcAMP from about day 11 until harvest or recovery. In some embodiments, the medium is supplemented with dbcAMP from about day 11-18. In some embodiments, the medium is supplemented with dbcAMP from about day 11-25.

[0244] In some embodiments, cells are exposed to dbcAMP at a concentration of about 0.05 mM to 5 mM, about 0.1 mM to about 3 mM, about 0.2 mM to about 1 mM (inclusive). In some embodiments, cells are exposed to dbcAMP at a concentration of about 0.1 mM to about 3 mM (inclusive). In some embodiments, cells are exposed to dbcAMP at a concentration of about 0.2 mM to about 1 mM (inclusive). In some embodiments, cells are exposed to dbcAMP at a concentration of about 0.5 mM.

[0245] In some embodiments, the medium is supplemented with about 0.5 mM dbcAMP beginning at about day 11. In some embodiments, the medium is supplemented with about 0.5 mM dbcAMP from about day 11 until harvest or recovery. In some embodiments, the medium is supplemented with about 0.5 mM dbcAMP from about day 11-18. In some embodiments, the medium is supplemented with about 0.5 mM dbcAMP from about day 11-25.

[0246] In some embodiments, the medium is supplemented with transforming growth factor beta 3 (TGFβ3). In some embodiments, the medium is supplemented with TGFβ3 beginning at about day 11. In some embodiments, the medium is supplemented with TGFβ3 from about day 11 until harvest or recovery. In some embodiments, the medium is supplemented with TGFβ3 from about day 11-18. In some embodiments, the medium is supplemented with TGFβ3 from about day 11-25.

[0247] In some embodiments, cells are exposed to TGFβ3 at 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. In some embodiments, cells are exposed to TGFβ3 at a concentration of about 1.0 ng / mL to about 2.0 ng / mL, inclusive. In some embodiments, cells are exposed to TGFβ3 at a concentration of about 1 ng / mL.

[0248] In some embodiments, the medium is supplemented with about 1 ng / mL TGFβ3 beginning at about day 11. In some embodiments, the medium is supplemented with 1 ng / mL TGFβ3 from about day 11 until harvest or recovery. In some embodiments, the medium is supplemented with about 1 ng / mL TGFβ3 from about day 11-18. In some embodiments, the medium is supplemented with about 1 ng / mL TGFβ3 from about day 11-25.

[0249] In some embodiments, the medium is supplemented with an inhibitor of Notch signaling. In some embodiments, the medium is supplemented with an inhibitor of Notch signaling beginning at about day 11. In some embodiments, the medium is supplemented with an inhibitor of Notch signaling from about day 11 until harvest or recovery. In some embodiments, the medium is supplemented with an inhibitor of Notch signaling from about day 11-18. In some embodiments, the medium is supplemented with an inhibitor of Notch signaling from about day 11-25.

[0250] In some embodiments, the inhibitor of Notch signaling is selected from Cowanin, PF-03084014, L685458, LY3039478, DAPT, or a combination thereof. In some embodiments, the inhibitor of Notch signaling inhibits gamma secretase. In some embodiments, the inhibitor of Notch signaling is a small molecule. In some embodiments, the inhibitor of Notch signaling is DAPT, which has the formula: [ka]

[0251] 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.

[0252] In some embodiments, the medium is supplemented with about 10 μM DAPT beginning at about day 11. In some embodiments, the medium is supplemented with 10 μM DAPT from about day 11 until harvest or recovery. In some embodiments, the medium is supplemented with about 10 μM DAPT from about day 11-18. In some embodiments, the medium is supplemented with about 10 μM DAPT from about day 11-25.

[0253] In some embodiments, beginning at about day 11, the 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 until harvest or recovery, the 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 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 about day 25, the 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.

[0254] In some embodiments, serum replacement is provided to the medium from about day 7 to about day 10. In some embodiments, serum replacement is provided to the medium at 2% (v / v) from day 7 to day 10.

[0255] In some embodiments, from about day 7 to about day 16, at least about 50% of the medium is replaced daily. In some embodiments, from about day 7 to about day 16, about 50% of the medium is replaced daily, every other day, or every second day. In some embodiments, from about day 7 to about day 16, about 50% of the medium is replaced daily. In some embodiments, starting at about day 17, at least about 50% of the medium is replaced every other day. In some embodiments, starting at about day 17, at least about 50% of the medium is replaced every other day. In some embodiments, starting at about day 17, about 50% of the medium is replaced every other day. In some embodiments, starting at about day 17, at least about 50% of the medium is replaced every other day. In some embodiments, the replacement medium contains small molecules concentrated about 2-fold compared to the concentration of small molecules in the medium on day 0.

[0256] In some embodiments, the second incubation involves culturing cells derived from the cell aggregates (e.g., spheroids) in a "basic induction medium." In some embodiments, the second incubation involves culturing cells derived from the cell aggregates (e.g., spheroids) in a "maturation medium." In some embodiments, the second incubation involves culturing cells derived from the cell aggregates (e.g., spheroids) in a basal induction medium and then in a maturation medium.

[0257] In some embodiments, the second incubation involves culturing the cells in basal induction medium from about day 7 to about day 10. In some embodiments, the second incubation involves culturing the cells in maturation medium beginning at about day 11. In some embodiments, the second incubation involves culturing the cells in basal induction medium from about day 7 to about day 10, and then in maturation medium beginning at about day 11. In some embodiments, the cells are cultured in maturation medium to generate committed dopamine (DA) neuronal progenitor cells or dopamine (DA) neurons.

[0258] In some embodiments, the basal induction medium is formulated to contain Neurobasal™ medium and DMEM / F12 medium in a 1:1 ratio, supplemented with N-2 and B27 supplements, non-essential amino acids (NEAA), GlutaMAX™, 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.

[0259] In some embodiments, the maturation medium is formulated to contain Neurobasal™ medium supplemented with N-2 and B27 supplements, non-essential amino acids (NEAA), and GlutaMAX™. In some embodiments, the maturation medium is further supplemented with any of the molecules described in Section II.

[0260] In some embodiments, the cells are cultured in basal induction medium from about day 7 to about day 11 (e.g., day 10 or day 11). In some embodiments, the cells are cultured in basal induction medium from about day 7 to about day 10, inclusive. In some embodiments, the cells are cultured in maturation medium beginning on about day 11. In some embodiments, the cells are cultured in basal induction medium from about day 7 to about day 10, and then the cells are cultured in maturation medium beginning on about day 11. In some embodiments, the cells are cultured in maturation medium from about day 11 until harvest or recovery. In some embodiments, the cells are harvested between day 16 and day 27. In some embodiments, the cells are harvested between day 18 and day 25. In some embodiments, the cells are harvested on day 18. In some embodiments, the cells are harvested on day 25. E. Differentiated Cell Collection, Harvesting, and Formulation

[0261] In embodiments of the methods provided, the neutrally differentiated cells generated by the methods provided herein can be collected or harvested, such as for formulation and use of the cells. In some embodiments, the methods provided for generating differentiated cells for use as cell therapy, such as in the treatment of neurodegenerative diseases, can include formulation of the cells, such as formulation of differentiated cells obtained from the methods provided herein. In some embodiments, a dose of cells including 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 methods provided, such as in the prevention or treatment of neurodegenerative disorders, including Parkinson's disease.

[0262] In some cases, the cells are processed in one or more steps to manufacture, produce, or generate a cell therapy, and / or the differentiated cells may include a formulation of the cells, such as a formulation of the differentiated cells obtained from the method. In some cases, the cells can be formulated in an amount for dosage administration, such as a single unit dosage administration or multiple dosage administration.

[0263] In certain embodiments, the one or more compositions of differentiated cells are formulated. In certain embodiments, the one or more compositions of differentiated cells are formulated after the one or more compositions are produced. In some embodiments, the one or more compositions are previously cryopreserved and stored, and are thawed before administration.

[0264] In certain embodiments, the differentiated cells comprise 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 comprise DA neurons. In some embodiments, the formulated composition of differentiated cells is a composition enriched in DA neurons.

[0265] 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 differentiated for at least 16 days. In some embodiments, the cells are differentiated for 16-30 days. In some embodiments, the cells are differentiated for 16-27 days. In some embodiments, the cells are differentiated for 18-25 days. In some embodiments, the cells are differentiated for about 18 days. In some embodiments, the cells are differentiated for about 25 days.

[0266] 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 harvested after at least 16 days of culture. In some embodiments, the cells are harvested after 16-30 days of culture. In some embodiments, the cells are harvested after 16-27 days of culture. In some embodiments, the cells are harvested after 18-25 days of culture. In some embodiments, the cells are harvested after about 18 days of culture. In some embodiments, the cells are harvested after about 25 days of culture.

[0267] In some embodiments, the cells are formulated in a pharma- ceutically acceptable buffer, which in some aspects may include a pharma- ceutically acceptable carrier or excipient. In some embodiments, the processing includes replacing the culture medium with a pharma- ceutically acceptable or desired medium or formulation buffer for administration to a subject. In some embodiments, the processing step may involve washing the differentiated cells and replacing the cells in a pharma- ceutically acceptable buffer, which may include one or more optional pharma- ceutically acceptable carriers or excipients. Examples of such pharmaceutical forms, including pharma- ceutically acceptable carriers or excipients, may be any of those described below in conjunction with forms acceptable for administering the cells and compositions to a subject. The pharmaceutical composition in some embodiments contains an amount of cells effective to treat or prevent a neurodegenerative condition or disease (e.g., Parkinson's disease), such as a therapeutically effective or prophylactically effective amount.

[0268] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0269] In some embodiments, the choice of carrier is determined in part by the specific cells and / or by the method of administration. Thus, there are a variety of suitable formulations. For example, the pharmaceutical composition may contain a preservative. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some embodiments, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. 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, citric acid, 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 polyvinylpyrrolidone; 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 non-ionic surfactants such as polyethylene glycol (PEG).

[0270] In some embodiments, a buffering agent is included in the composition. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some embodiments, a mixture of two or more buffering agents is used. The buffering agent or mixture is typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0271] The formulation may comprise an aqueous solution. The formulation or composition may also contain more than one active ingredient useful for the specific indication, disease, or condition being treated with the cells, preferably those with complementary activities to the cells, where the respective activities do not adversely affect each other. Such active ingredients are suitably present in combination in amounts effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further comprises other pharma- ceutical 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.

[0272] The compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH in some aspects. Liquid compositions may include a carrier, which may be a solvent or dispersion medium, including, for example, water, saline, phosphate buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof. Sterile injectable solutions may be prepared by incorporating the cells in a solvent, such as a mixture with a suitable carrier, diluent, or excipient, such as sterile water, saline, glucose, dextrose, and the like. The compositions may contain auxiliary substances, such as wetting agents, dispersing agents, or emulsifying agents (e.g., methylcellulose), pH buffers, gelling or thickening additives, preservatives, and / or coloring agents, depending on the desired route of administration and preparation. In some aspects, standard texts may be consulted to prepare suitable preparations.

[0273] Various additives can be added to enhance the stability and sterility of the composition, including antibacterial preservatives, antioxidants, chelating agents, and buffers. Prevention of microbial action can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. The use of agents that delay absorption, such as aluminum monostearate and gelatin, can allow the injectable pharmaceutical form to absorb continuously.

[0274] In some embodiments, the formulation buffer contains a cryopreservative. In some embodiments, the cells are formulated with a cryopreservative solution containing 1.0% to 30% DMSO solution, such as a 5% to 20% DMSO solution or a 5% to 10% DMSO solution. In some embodiments, the cryopreservative solution is or contains, for example, PBS containing 20% ​​DMSO and 8% human serum albumin (HSA), or other suitable cell freezing medium. In some embodiments, the cryopreservative solution is or contains, for example, at least or about 7.5% DMSO. In some embodiments, the processing step may involve washing the differentiated cells to replace the cells in the cryopreservative solution. In some embodiments, the cells are frozen, e.g., cryopreserved or cryoprotected, in medium and / or solution with a final concentration of 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 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 between 1% and 15%, 6% and 12%, 5% and 10%, or 6% and 8% DMSO. In certain embodiments, the cells are frozen, e.g., cryopreserved or cryoprotected, in 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%-5%, 0.25%-4%, 0.5%-2%, or 1%-2% HSA.

[0275] In certain embodiments, compositions of differentiated cells are formulated, cryopreserved, and then stored for a period of time. In certain embodiments, the formulated and cryopreserved cells are stored until the cells are released for administration. In certain embodiments, the formulated and cryopreserved cells 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 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, 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 after storage and administered to a subject.

[0276] In some embodiments, the formulation is performed using one or more processing steps, including washing, diluting, or concentrating the cells. In some embodiments, the processing may include diluting or concentrating the cells to a desired concentration or number, such as a unit dosage composition that includes the number of cells for administration of a given dose or a portion thereof. In some embodiments, the processing step may include decreasing the volume, thereby increasing the concentration of the cells, if desired. In some embodiments, the processing step may include increasing the volume, thereby decreasing the concentration of the cells, if desired. In some embodiments, the processing includes adding a volume of formulation buffer to the differentiated cells. In some embodiments, the volume of the formulation buffer is about 1 μL to 5000 μL, for example, at least or about 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.

[0277] The container can generally contain one or more unit doses of cells to be administered, for example. A 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 lowest dose or the lowest possible dose of cells to be administered to a subject.

[0278] In some embodiments, cells produced by such methods, or compositions comprising such cells, are administered to a subject to treat a neurodegenerative disease or condition. F. Exemplary Process

[0279] As described 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 in cell replacement therapy. As described by the methods provided herein, in some embodiments, pluripotent stem cells are differentiated into floor plate midbrain progenitor cells, and the resulting spheroid 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.

[0280] In some embodiments, embryonic stem cells are differentiated into floor plate midbrain progenitor cells, and then differentiated into committed dopamine (DA) neuron progenitor cells and / or dopamine (DA) neurons. In some embodiments, embryonic stem cells are differentiated into committed DA neuron progenitor cells. In some embodiments, embryonic stem cells are differentiated into DA neurons.

[0281] In some embodiments, induced pluripotent stem cells are differentiated into floor plate midbrain 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.

[0282] In some embodiments, methods are provided herein that involve: (a) performing a first incubation comprising culturing pluripotent stem cells in a non-adherent culture vessel under conditions to generate cell spheroids, where the first incubation comprises exposing the cells, beginning at a start of the first incubation (day 0), to (i) an inhibitor of TGF-β / Activin-Nodal signaling, (ii) at least one activator of Sonic Hedgehog (SHH) signaling, (iii) an inhibitor of bone morphogenetic protein (BMP) signaling, and (iv) an inhibitor of Glycogen Synthase Kinase 3β (GSK3β) signaling; and (b) performing a second incubation comprising culturing the cells of the spheroids in a substrate-coated culture vessel under conditions to induce neuronal differentiation of the cells.

[0283] In some embodiments, culturing the cells under conditions that induce neuronal differentiation of the cells involves 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 beta-3 (TGFβ3), and (vi) an inhibitor of Notch signaling.

[0284] In some embodiments, the method includes (a) performing a first incubation comprising culturing pluripotent stem cells in a plate having microwells under conditions to generate cell spheroids, beginning at a start of the first incubation (day 0), treating the cells with (i) an inhibitor of TGF-β / Activin-Nodal signaling, (ii) at least one activator of Sonic Hedgehog (SHH) signaling, (iii) an inhibitor of bone morphogenetic protein (BMP) signaling, (iv) an inhibitor of glycogen synthase. The method includes performing a first incubation, exposing the cells to an inhibitor of GSK3β signaling and (v) a serum replacement, (b) dissociating the cells of the spheroids to generate a cell suspension, (c) transferring the cells of the cell suspension to a laminin-coated culture vessel, (d) performing a second incubation, comprising culturing the cells of the spheroids in the laminin-coated culture vessel under conditions that induce neuronal differentiation of the cells, and (e) collecting the neuronally differentiated cells. In some embodiments, the second incubation involves culturing the cells in the presence of a serum replacement. In some embodiments, culturing the cells under conditions that induce neuronal differentiation of the cells involves 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 beta-3 (TGFβ3), and (vi) an inhibitor of Notch signaling.

[0285] In some embodiments, the cells are exposed to an inhibitor of TGF-β / activin-nodal signaling (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 an inhibitor of TGF-β / activin-nodal signaling (e.g., SB431542 or "SB") from day 0 to about day 6 (inclusive). In some embodiments, the cells are exposed to at least one activator of SHH signaling (e.g., SHH protein and palmorphamin, collectively "SHH / PUR") from day 0 to about day 7 (e.g., day 6 or day 7). In some embodiments, the cells are exposed to at least one activator of SHH signaling (e.g., SHH protein and palmorphamin, collectively "SHH / PUR") from day 0 to about day 6 (inclusive). In some embodiments, the cells are exposed to an inhibitor of BMP signaling (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 an inhibitor of BMP signaling (e.g., LDN193189 or "LDN") from day 0 to day 10 (inclusive). In some embodiments, the cells are exposed to an inhibitor of GSK3β signaling (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 an inhibitor of GSK3β signaling (e.g., CHIR99021 or "CHIR") from day 0 to day 12.

[0286] In some embodiments, the cells are exposed to (i) an inhibitor of TGF-β / activin-nodal signaling from day 0 to about day 7 (e.g., day 6 or day 7), (ii) at least one activator of Sonic Hedgehog (SHH) signaling from day 0 to about day 7 (e.g., day 6 or day 7), (iii) an inhibitor of bone morphogenetic protein (BMP) signaling from day 0 to about day 11 (e.g., day 10 or day 11), and (iv) an inhibitor of glycogen synthase kinase 3β (GSK3β) signaling from day 0 to about day 13 (e.g., day 12 or day 13). In some embodiments, cells are exposed to (i) SB from day 0 to about day 7 (e.g., day 6 or 7), (ii) SHH / PUR from day 0 to about day 7 (e.g., day 6 or 8), (iii) LDN from day 0 to about day 11 (e.g., day 10 or 11), and (iv) CHIR from day 0 to about day 13 (e.g., day 12 or 13). In some embodiments, the cells are exposed to an inhibitor of TGF-β / activin-nodal signaling on days 0-6, inclusive; (ii) at least one activator of Sonic Hedgehog (SHH) signaling on days 0-6, inclusive; (iii) an inhibitor of bone morphogenetic protein (BMP) signaling on days 0-10, inclusive; and (ii) an inhibitor of glycogen synthase kinase 3β (GSK3β) signaling on days 0-12, inclusive. In some embodiments, the cells are exposed to (i) SB on days 0-6, inclusive; (ii) SHH / PUR on days 0-6, inclusive; (iii) LDN on days 0-10, inclusive; and (iv) CHIR on days 0-12, inclusive.

[0287] In some embodiments, beginning on day 11, the cells are exposed to brain-derived neurotrophic factor (BDNF). In some embodiments, the cells are exposed to ascorbic acid. In some embodiments, beginning on day 11, the cells are exposed to glial cell line-derived neurotrophic factor (GDNF). In some embodiments, beginning on day 11, the cells are exposed to dibutyryl cyclic AMP (dbcAMP). In some embodiments, beginning on day 11, the cells are exposed to transforming growth factor beta-3 (TGFβ3). In some embodiments, beginning on day 11, the cells are exposed to an inhibitor of Notch signaling (e.g., DAPT). In some embodiments, beginning on day 11, 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 beta-3 (TGFβ3), and (vi) an inhibitor of Notch signaling (e.g., DAPT) (collectively "BAGCT / DAPT"). In some embodiments, cells are exposed to BAGCT / DAPT beginning on day 11 until harvest or recovery. In some embodiments, cells are exposed to BAGCT / DAPT from day 11-18. In some embodiments, cells are exposed to BAGCT / DAPT from day 11-25.

[0288] In some embodiments, the cells are exposed to a Rho-associated protein kinase (ROCK) inhibitor on day 0. In some embodiments, the cells are exposed to a Rho-associated protein kinase (ROCK) inhibitor on day 7. In some embodiments, the cells are exposed to a Rho-associated protein kinase (ROCK) inhibitor on day 16. In some embodiments, the cells are exposed to a Rho-associated protein kinase (ROCK) inhibitor on day 20. In some embodiments, the cells are exposed to a Rho-associated protein kinase (ROCK) inhibitor on days 0, 7, 16, and 20. In some embodiments, the cells are exposed to a ROCK inhibitor on the day the cells are passaged. In some embodiments, the cells are passaged on days 0, 7, 16, 20, or a combination thereof. In some embodiments, the cells are passaged on days 0, 7, 16, and 20.

[0289] In some embodiments, the cells are cultured in a basal induction medium comprising DMEM / F-12 and Neurobasil medium (e.g., in a 1:1 ratio) supplemented with N2, B27, non-essential amino acids (NEAA), Glutamax, L-glutamine, β-mercaptoethanol, and insulin. In some embodiments, the cells are cultured in the basal induction medium from about day 0 to about day 10. In some embodiments, the basal induction medium is for differentiating pluripotent stem cells into floor plate midbrain progenitor cells.

[0290] In some embodiments, the cells are cultured in maturation medium comprising Neurobasal medium supplemented with N2, B27, non-essential amino acids (NEAA), and Glutamax. In some embodiments, the cells are cultured in basal induction medium from about day 11 until harvest or recovery. In some embodiments, the cells are cultured in basal induction medium from about day 11-18. In some embodiments, the maturation medium is for differentiating floor plate midbrain progenitor cells into committed dopamine (DA) neuron progenitor cells. In some embodiments, the cells are cultured in basal induction medium from about day 11-25. In some embodiments, the maturation medium is for differentiating floor plate midbrain progenitor cells into dopamine (DA) neurons.

[0291] In some embodiments, the medium is supplemented with small molecules as described above, including SB, SHH / PUR, LDN, CHIR, BAGCT / DAPT, and ROCKi. In some embodiments, the medium is changed daily or every other day. In some embodiments, the medium is changed daily. In some embodiments, the medium is changed every other day. In some embodiments, the medium is changed daily from about day 0 to about day 17 (e.g., day 16 or day 18). In some embodiments, the medium is changed every other day from about day 18 until harvest or collection. In some embodiments, the medium is changed daily from about day 0 to about day 17 (e.g., day 16 or day 18), then every other day from about day 18 until harvest or collection.

[0292] In some embodiments, serum replacement is provided to the medium from about day 0 to about day 10 (e.g., day 9 or day 11). In some embodiments, serum replacement is provided to the medium at 5% (v / v) on days 0 and 1. In some embodiments, serum replacement is provided to the medium at 2% (v / v) on days 2-10. In some embodiments, serum replacement is provided to the medium at 5% (v / v) on days 0 and 1, and at 2% (v / v) on days 2-10. In some embodiments, serum replacement is not provided to the medium after day 10.

[0293] In some embodiments, at least about 50% or at least about 75% of the medium is replaced. In some embodiments, at least about 50% of the medium is replaced. In some embodiments, at least about 75% of the medium is replaced. In some embodiments, about 100% of the medium is replaced.

[0294] In some embodiments, about 50% or about 75% of the medium is replaced. In some embodiments, about 50% of the medium is replaced. In some embodiments, about 75% of the medium is replaced. In some embodiments, about 100% of the medium is replaced.

[0295] In some embodiments, the medium is supplemented with small molecules selected from SB, SHH / PUR, LDN, CHIR, BAGCT / DAPT, ROCKi, or combinations thereof. In some embodiments, upon replacement of about 50% of the medium, the concentration of each small molecule is doubled compared to the concentration on day 0.

[0296] In some embodiments, the cells are harvested at about day 16 to about day 30. In some embodiments, the cells are harvested at about day 16 to about day 27. In some embodiments, the cells are harvested at about day 18 to about day 25. In some embodiments, the cells are harvested at about day 18. In some embodiments, the cells are harvested at about day 25. In some embodiments, the harvested cells are formulated with a cryopreservative, e.g., DMSO. In some embodiments, the harvested cells produced by the method are cryopreserved prior to use. In some embodiments, such cryopreserved cells are thawed prior to use or administration to a subject, e.g., a human patient having a neurodegenerative disease or condition, such as Parkinson's disease.

[0297] In some embodiments, compositions comprising cells produced by the methods provided herein are used to treat neurodegenerative diseases or conditions, such as Parkinson's disease. In some embodiments, compositions of cells produced by any of the methods described herein are administered to a subject with Parkinson's disease. In some embodiments, compositions of cells produced by any of the methods described herein are administered by stereotactic injection, such as by catheter. In some embodiments, compositions of cells produced by any of the methods described herein are administered to the striatum of a subject with Parkinson's disease. III. Compositions and Formulations

[0298] Provided herein is a therapeutic composition containing differentiated cells that are committed dopamine (DA) neuron progenitor cells.Also provided herein is a therapeutic composition containing differentiated cells produced by any of the provided methods, 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.

[0299] In some embodiments, the differentiated cells in the therapeutic compositions provided, including those produced by any of the methods described herein, can produce dopamine (DA). In some embodiments, the differentiated cells in the therapeutic compositions provided, including those produced by any of the methods described herein, do not produce, or do not substantially produce, norepinephrine (NE). Thus, in some embodiments, the differentiated cells in the therapeutic compositions provided, including those produced by any of the methods described herein, can produce DA, but do not produce, or do not substantially produce, NE.

[0300] In some embodiments, the committed dopamine (DA) neuronal 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 of the therapeutic composition express EN1. In some embodiments, at least about 25% of the cells of the therapeutic composition express EN1. In some embodiments, at least about 30% of the cells of the therapeutic composition express EN1. In some embodiments, at least about 35% of the cells of the therapeutic composition express EN1. In some embodiments, at least about 40% of the cells of the therapeutic composition express EN1. In some embodiments, at least about 45% of the cells of the therapeutic composition express EN1. In some embodiments, at least about 50% of the cells of the therapeutic composition express EN1. In some embodiments, at least about 55% of the cells of the therapeutic composition express EN1. In some embodiments, at least about 60% of the cells of the therapeutic composition express EN1. In some embodiments, at least about 65% of the cells of the therapeutic composition express EN1. In some embodiments, at least about 70% of the cells of the therapeutic composition express EN1. In some embodiments, at least about 75% of the cells of the therapeutic composition express EN1. In some embodiments, at least about 80% of the cells of the therapeutic composition express EN1.

[0301] In some embodiments, the therapeutic composition comprises about 1×10 -4 In some embodiments, the CPM / CPM ratio of EN1 to GAPDH is greater than about 1.5×10 -3 ~1×10 -2 It is.

[0302] 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 of the therapeutic composition express CORIN. In some embodiments, at least about 25% of the cells of the therapeutic composition express CORIN. In some embodiments, at least about 30% of the cells of the therapeutic composition express CORIN. In some embodiments, at least about 35% of the cells of the therapeutic composition express CORIN. In some embodiments, at least about 40% of the cells of the therapeutic composition express CORIN. In some embodiments, at least about 45% of the cells of the therapeutic composition express CORIN. In some embodiments, at least about 50% of the cells of the therapeutic composition express CORIN. In some embodiments, at least about 55% of the cells of the therapeutic composition express CORIN. In some embodiments, at least about 60% of the cells of the therapeutic composition express CORIN. In some embodiments, at least about 65% of the cells of the therapeutic composition express CORIN. In some embodiments, at least about 70% of the cells of the therapeutic composition express CORIN. In some embodiments, at least about 75% of the cells of the therapeutic composition express CORIN. In some embodiments, at least about 80% of the cells of the therapeutic composition express CORIN.

[0303] In some embodiments, the therapeutic composition comprises about 1×10 -4 In some embodiments, the CPM / CPM ratio of CORIN to GAPDH is greater than about 5×10 -2 ~5×10 -1 It is.

[0304] 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 of the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 25% of the cells of the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 30% of the cells of the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 35% of the cells of the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 40% of the cells of the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 45% of the cells of the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 50% of the cells of the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 55% of the cells of the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 60% of the cells of the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 65% of the cells of the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 70% of the cells of the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 75% of the cells of the therapeutic composition express EN1 and CORIN. In some embodiments, at least about 80% of the cells of the therapeutic composition express EN1 and CORIN.

[0305] In some embodiments, the therapeutic composition comprises: (a) about 1×10 -4(b) a counts per million (CPM) / CPM ratio of EN1 to GAPDH of approximately 2 × 10 -2 In some embodiments, the CPM / CPM ratio of CORIN to GAPDH is greater than about 1.5×10. -3 ~1×10 -2 and the CPM / CPM ratio of CORIN to GAPDH is approximately 5 × 10 -2 ~5×10 -1 It is.

[0306] In some embodiments, less than 10% of the committed dopamine (DA) neuron progenitor cells express TH. In some embodiments, the committed dopamine (DA) neuron progenitor cells express low levels of TH. In some embodiments, the committed dopamine (DA) neuron progenitor cells do not express TH. In some embodiments, the committed dopamine (DA) neuron progenitor cells express TH at a lower level than cells collected or harvested on other days. In some embodiments, some of the committed dopamine (DA) neuron progenitor cells express EN1 and CORIN, and less than 10% of the cells express TH.

[0307] In some embodiments, less than 8% of the cells express TH. In some embodiments, less than 5% of the cells express TH. In some embodiments, about 2%-10%, about 2%-8%, about 2%-6%, about 2%-4%, about 4%-10%, about 4%-8%, about 4%-6%, about 6%-10%, about 6%-8%, or about 8%-10% of the total cells in the composition express TH.

[0308] In some embodiments, the therapeutic composition comprises about 3×10 -2 In some embodiments, the CPM / CPM ratio of TH to GAPDH is less than about 1×10 -3 ~2.5×10 -2 It is.

[0309] 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.

[0310] 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.

[0311] 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.

[0312] In some embodiments, the differentiated cells generated by any of the methods described herein are dopamine (DA) neurons (e.g., midbrain-fate DA neurons). In some embodiments, the midbrain-fate dopamine (DA) neurons are FOXA2+ / TH+ at the time of collection. In some embodiments, the midbrain-fate dopamine (DA) neurons become FOXA2+ / TH+ by or at about day 18. In some embodiments, the midbrain-fate dopamine (DA) neurons become FOXA2+ / TH+ by or at about day 25.

[0313] 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) about 2×10 -5 (b) Counts per million (CPM) / CPM ratio of NEUROG1 to GAPDH of approximately 5 × 10 -4 (c) CPM ratio of EDN3 to GAPDH of approximately 4 × 10 -3 (d) CPM ratio of HES1 to GAPDH of approximately 8 × 10 -3 (e) CPM ratio of PSRC1 to GAPDH of approximately 2 × 10 -3 (f) CPM ratio of NEK6 to GAPDH of approximately 1 × 10 -2 The CPM ratio of IQGAP3 to GAPDH (g) is approximately 8 × 10 -4 The CPM ratio of USP44 to GAPDH (h) is approximately 3 × 10 -3 (i) CPM ratio of CEP55 to GAPDH of approximately 2 × 10 -2 CPM ratio of KIF20A to GAPDH, (j) approximately 2 × 10 -2 The CPM ratio of URKA to AGAPDH (k) is approximately 1 × 10 -3 (l) CPM ratio of CALCA to GAPDH of less than 1 × 10 -3 CPM ratio of GLRA2 to GAPDH (m) of less than 1 × 10 -1CPM ratio of MAPT to GAPDH less than (n) approx. 5 × 10 -2 (o) CPM ratio of CAMK2B to GAPDH of less than about 1 × 10 -2 CPM ratio of SYT13 to GAPDH of less than 1 × 10 -3 CPM ratio of LHFPL4 to GAPDH (q) of approximately 5 × 10 -3 CPM ratio of RET to GAPDH (r) of less than 1 × 10 -2 CPM ratio of KCND3 to GAPDH less than (s) approx. 5 × 10 -2 CPM ratio of NSG2 to GAPDH of less than 2 × 10 -2 CPM ratio of SNAP25 to GAPDH less than 1.

[0314] In some embodiments, (a) the CPM / CPM ratio of NEUROG1 to GAPDH is about 5×10 -5 ~Approx. 5×10 -3 (b) The CPM / CPM ratio of EDN3 to GAPDH is approximately 1 × 10 -3 ~Approx. 5×10 -2 (c) The CPM / CPM ratio of HES1 to GAPDH is approximately 5 × 10 -3 ~Approx. 5×10 -2 (d) The CPM / CPM ratio of PSRC1 to GAPDH is approximately 1 × 10 -2 ~Approx. 5×10 -2 (e) The CPM / CPM ratio of NEK6 to GAPDH is approximately 2 × 10 -2 ~Approx. 5×10 -1 (f) The CPM / CPM ratio of IQGAP3 to GAPDH is approximately 1.5 × 10 -2 ~Approx. 1×10 -1 (g) The CPM / CPM ratio of USP44 to GAPDH is approximately 5 × 10 -3 ~Approx. 5×10 -2 (h) The CPM / CPM ratio of CEP55 to GAPDH is approximately 4 × 10 -3 ~Approx. 5×10 -2 (i) The CPM / CPM ratio of KIF20A to GAPDH is approximately 2.5 × 10 -2 ~about 2×10-1 (j) The CPM / CPM ratio of AURKA to GAPDH is approximately 2.6 × 10 -2 ~Approx. 2×10 -1 The CPM / CPM ratio of (k)CALCA to GAPDH is approximately 1 × 10 -5 ~Approx. 1×10 -3 (l) The CPM / CPM ratio of GLRA2 to GAPDH is approximately 1 × 10 -6 ~Approx. 1×10 -3 The CPM / CPM ratio of (m)MAPT to GAPDH is approximately 5 × 10 -3 ~Approx. 1×10 -1 (n) The CPM / CPM ratio of CAMK2B to GAPDH is approximately 1 × 10 -3 ~Approx. 5×10 -2 (o) The CPM / CPM ratio of SYT13 to GAPDH is approximately 1 × 10 -3 ~Approx. 1×10 -2 The CPM / CPM ratio of (p)LHFPL4 to GAPDH is approximately 1 × 10 -4 ~Approx. 1×10 -3 The CPM / CPM ratio of (q)RET to GAPDH is approximately 1 × 10 -4 ~Approx. 4×10 -3 The CPM / CPM ratio of (r)KCND3 to GAPDH is approximately 1 × 10 -3 ~Approx. 1×10 -2 The CPM / CPM ratio of (s)NSG2 to GAPDH is approximately 1 × 10 -3 ~Approx. 2.4×10 -2 and / or the CPM / CPM ratio of (t)SNAP25 to GAPDH is about 1 × 10 -3 ~Approx. 1×10 -2 It is.

[0315] Methods of measuring or assessing gene expression or gene products (including transcription and / or translation products) include those described in Chapter I. Additionally, 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.

[0316] In some embodiments, gene expression is or includes the process in which the information of a gene is used in the synthesis of a gene product. Thus, in some embodiments, a gene product is any biomolecule that is assembled, produced, and / or synthesized using the information encoded by a gene, and may include polynucleotides and / or polypeptides. In certain embodiments, the evaluation, measurement, and / or determination of gene expression is or includes the determination or measurement of the level, amount, or concentration of a gene product. In certain embodiments, the level, amount, or concentration of a gene product may be transformed (e.g., normalized) or analyzed directly (e.g., raw).

[0317] In some embodiments, the gene product is or includes a protein, i.e., a polypeptide encoded and / or expressed by a gene. In certain embodiments, the gene product encodes a protein that is localized and / or exposed on the surface of a cell. In some embodiments, the protein is a soluble protein. In certain embodiments, the protein is secreted by the cell. In certain embodiments, gene expression is the amount, level, and / or concentration of a protein encoded by a gene. In certain embodiments, one or more protein gene products are measured by any suitable means known in the art. Suitable methods for assessing, measuring, determining, and / or quantifying the level, amount, or concentration, or further protein gene products, include, but are not limited to, immunoassays, nucleic acid-based or protein-based aptamer technology, HPLC (high performance liquid chromatography), peptide sequencing (e.g., Edman degradation sequencing or mass spectrometry (e.g., MS / MS), optionally coupled to HPLC), and detection by microarray adaptations of any of the foregoing, including nucleic acid, antibody, or protein-protein (i.e., non-antibody) arrays. In some embodiments, an immunoassay is or includes a method or assay that detects a protein based on an immunological reaction, for example, by detecting the binding of an antibody or an antigen-binding antibody fragment to a gene product. Immunoassays include, but are not limited to, quantitative immunocytochemistry or immunohistochemistry, ELISA (including direct, indirect, sandwich, competitive, multiple, and portable ELISA (see, for example, U.S. Pat. No. 7,510,687), Western blotting (including one-dimensional, two-dimensional, or higher order blotting or other chromatographic means, optionally including peptide sequencing), enzyme immunoassay (EIA), RIA (radioimmunoassay), and SPR (surface plasmon resonance).

[0318] In certain embodiments, the gene product is a polynucleotide, such as an mRNA or a protein, encoded by a gene. In some embodiments, the gene product is a polynucleotide expressed and / or encoded by a gene. In certain embodiments, the polynucleotide is an RNA. In some embodiments, the gene product is a messenger RNA (mRNA), a transfer RNA (tRNA), a ribosomal RNA, a small nuclear RNA, a small nucleolar RNA, an antisense RNA, a long non-coding RNA, a microRNA, a Piwi-interacting RNA, a small interfering RNA, and / or a short hairpin RNA. In certain embodiments, the gene product is an mRNA.

[0319] In certain embodiments, assessing, measuring, determining, and / or quantifying the amount or level of an RNA gene product comprises generating, polymerizing, and / or obtaining a cDNA polynucleotide and / or a cDNA oligonucleotide 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 the cDNA polynucleotide and / or cDNA oligonucleotide derived from the RNA gene product.

[0320] In certain embodiments, the amount or level of a polynucleotide in a sample may be assessed, measured, determined, and / or quantified by any suitable means known in the art. For example, in some embodiments, the amount or level of a polynucleotide gene product may 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, TAQMAN®, Molecular Beacons, LIGHTUP™, SCORPION™, SIMPLEPROBES®; see, for example, U.S. Patent No. 5,538,848 ... Nos. 5,925,517, 6,174,670, 6,329,144, 6,326,145, and 6,635,427); Northern blotting; Southern blotting of, e.g., reverse transcription products and derivatives; array-based methods, including blot arrays, microarrays, or in situ synthesized arrays; and sequencing, e.g., sequencing by synthesis, pyrosequencing, dideoxysequencing, or ligation, or shendure sequencing. et al., Nat. Rev. Genet. 5:335-44 (2004) or any other method known in the art, such as those discussed in Nowrousian, Euk. Cell 9(9):1300-1310 (2010), including specific platforms such as HELICOS®, ROCHE® 454, ILLUMINA® / SOLEXA®, ABI SOLiD®, and POLONATOR® sequencing. In certain embodiments, the levels of nucleic acid gene products are measured by quantitative PCR (qPCR) methods, such as qRT-PCR. In some embodiments, qRT-PCR uses a set of three nucleic acids for each gene, which contain primer pairs with probes that bind between the regions of the target nucleic acid to which the primers bind, which is commercially known as the TAQMAN® assay.

[0321] In certain embodiments, the expression of two or more of the 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, microarrays (e.g., AFFYMETRIX®, AGILENT®, and ILLUMINA®-style arrays) 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 products and / or by sequencing cDNA polynucleotides derived from the gene products. In some embodiments, the sequencing is performed by non-Sanger sequencing methods and / or next generation sequencing (NGS) technologies. Examples of next generation sequencing technologies include, but are not limited to, Massively Parallel Signature Sequencing (MPSS), polony sequencing, pyrosequencing, reversible dye terminator sequencing, SOLiD sequencing, ion semiconductor sequencing, DNA nanoball sequencing, heliscope single molecule sequencing, single molecule real time (SMRT) sequencing, single molecule real time (RNAP) sequencing, and nanopore DNA sequencing.

[0322] In some embodiments, the NGS technology is RNA sequencing (RNA-Seq). In certain embodiments, the expression of one or more polynucleotide gene products is measured, determined, and / or quantified by RNA-Seq. RNA-Seq, also called whole transcriptome shotgun sequencing, determines the presence and amount of RNA in a sample. RNA sequencing methods are adapted to the most common DNA sequencing platforms [HiSeq systems (Illumina), 454 Genome Sequencer FLX System (Roche), Applied Biosystems SOLiD (Life Technologies), IonTornet (Life Technologies). These platforms require RNA to be reverse transcribed into cDNA first. Conversely, the single molecule sequencer HeliScope (Helicos BioSciences) can use RNA as a template for sequencing. Proof of principle has also been shown for direct RNA sequencing on the PacBio RS platform (PacificBioscience). In some embodiments, one or more RNA gene products are evaluated, measured, determined, and / or quantified by RNA-seq. In some embodiments, the RNA-seq is tag-based RNA-seq. In tag-based methods, each transcript is represented by a unique tag. Initially, tag-based approaches were developed as sequence-based methods to measure transcript abundance and identify differentially expressed genes, assuming that the number of tags (count) corresponds directly to the abundance of mRNA molecules. Reducing the complexity of the samples obtained by sequencing defined regions was essential to make Sanger-based methods affordable. When NGS technology became available, it became possible to generate a large number of reads, which facilitated differential gene expression analysis. Tag-based methods do not encounter transcript length bias in quantification of gene expression levels, as observed for shotgun methods. All tag-based methods are, by definition, strand-specific.In certain embodiments, one or more RNA gene products are assessed, measured, determined, and / or quantified by tag-based RNA-seq.

[0323] In some embodiments, RNA-seq is shotgun RNA-seq. Numerous protocols have been described for shotgun RNA-seq, which have many steps in common: fragmentation (which may be performed at the RNA or 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 is performed by analysing polyadenylated RNA molecules (mainly mRNA, but also some lncRNA, snRNA, etc.) if poly(A)+RNA is selected before fragmentation. Fragmentation may focus only on RNAs (oRNAs, pseudogenes, and even histones) or, if no selection is performed, may also include non-polyadenylated RNAs. In the latter case, ribosomal RNA (>80% of the total RNA pool) needs to be depleted before fragmentation. Thus, differences in the capture of the mRNA part of the transcriptome are evident with partial overlap in the types of transcripts detected. Furthermore, different protocols may affect the abundance and distribution of sequenced reads. This makes it difficult to compare the results of experiments with different library preparation protocols.

[0324] In some embodiments, RNA is obtained from each sample, fragmented, and used to generate a complementary DNA (cDNA) sample, such as a cDNA library for sequencing. The reads may be processed and aligned to the human genome, and used to estimate the expected number of mappings per gene / isoform and determine the read count. In some embodiments, the read count is normalized by the length of the gene / isoform and the number of reads in the library to obtain a normalized FPKM, for example, by the length of the gene / isoform and the number of reads in the library to obtain fragments per kilobase of exon per million mapped reads (FPKM) according to the gene length and the total reads mapped. In some aspects, normalization between samples is achieved by normalization, such as, for example, 75th quantile normalization, where each sample is scaled by the median of the 75th quantile from all samples to obtain a quantile-normalized FPKM (FPKQ) value. The FPKQ value may be log-transformed (log2).

[0325] In some embodiments, RNA from each sample is obtained, fragmented, and used to generate complementary DNA (cDNA) samples, such as cDNA libraries, for sequencing. The reads may be processed and aligned to the human genome, and the expected number of mappings per gene / isoform is estimated and used to determine read counts. In some embodiments, the read counts are normalized by the length of the gene / isoform and the number of reads in the library. In some embodiments, the read counts are provided as counts per million (CPM).

[0326] In some embodiments, the relative gene expression is measured by comparing the CPM of target gene with the CPM of housekeeping gene. In some embodiments, the housekeeping gene is GAPDH. In some embodiments, the relative gene expression of target gene is determined as the CPM / CPM ratio of target gene to housekeeping gene (e.g., GAPDH).

[0327] In some embodiments, any of the compositions provided include pharmaceutical compositions that contain a pharma- ceutically acceptable carrier. In some embodiments, a dose of cells, including cells produced by any of the methods disclosed herein, is provided as a composition or formulation, such as a pharmaceutical composition or formulation. Such compositions can be used according to the methods, products, and / or compositions provided, for example, in the prevention or treatment of diseases, conditions, and disorders, such as neurodegenerative disorders.

[0328] The term "pharmaceutical formulation" refers to a preparation that is in a form that effectively utilizes the biological activity of the active ingredients contained therein and does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0329] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0330] In some embodiments, the choice of carrier is determined in part by the specific cell or agent and / or by the method of administration. Thus, there are a variety of suitable formulations. For example, the pharmaceutical composition may contain a preservative. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some embodiments, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. 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, citric acid, 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 polyvinylpyrrolidone; 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 non-ionic surfactants such as polyethylene glycol (PEG).

[0331] In some embodiments, a buffering agent is included in the composition. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some embodiments, a mixture of two or more buffering agents is used. The buffering agent or mixture is typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0332] The formulation or composition may also contain more than one active ingredient useful for the specific indication, disease, or condition prevented or treated with the cells or agent, provided that the respective activities do not adversely affect each other. Such active ingredients are suitably present in combination in amounts effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further comprises other pharma- ceutical 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. In some embodiments, the agent or cell is administered in the form of a salt, e.g., a pharma- ceutical acceptable salt. Suitable pharma- ceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid, and sulfuric acid, and from 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 arylsulfonic acids, e.g., p-toluenesulfonic acid.

[0333] The formulation or composition may also be administered in combination with another form of therapy useful for the particular indication, disease, or condition being prevented or treated with the cells or agent, provided that the respective activities do not adversely affect each other. Thus, in some embodiments, the pharmaceutical composition is administered in combination with deep brain stimulation (DBS).

[0334] In some embodiments, the pharmaceutical composition contains an amount of the agent or cells effective for treating or preventing a disease or condition, such as a therapeutically or prophylactically effective amount. In some embodiments, the therapeutic or prophylactic effectiveness is monitored by periodically evaluating the treated subject. In the case of repeated administration over several days or longer, depending on the condition, the treatment is repeated until the symptoms of the disease are suppressed as desired. However, other administration regimes may be useful and may be determined. The desired dosage may be delivered by a single bolus administration of the composition, by multiple boluses of the composition, or by continuous infusion administration of the composition.

[0335] The agent or cells 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 cells or agent. In some embodiments, it is administered by multiple boluses of cells or agent, for example, over a period of months or years. In some embodiments, the agent or cells can be administered by stereotactic injection into the brain, such as the striatum.

[0336] When preventing or treating a disease, the appropriate dosage may depend on the type of disease being treated, the type of agent, the type of cells or recombinant receptor, the severity and course of the disease, whether the agent or cells are administered for prophylactic or therapeutic purposes, medical history, the subject's clinical history and response to the agent or cells, and the discretion of the attending physician. The compositions, in some embodiments, are suitably administered to the subject at one time or over a series of treatments.

[0337] The cells or agents may be administered using standard administration techniques, formulations, and / or devices. Formulations and devices such as syringes and vials for storing and administering the compositions are provided. For cells, administration may 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 therapeutic compositions (e.g., pharmaceutical compositions containing genetically reprogrammed and / or differentiated cells, or agents for treating or ameliorating symptoms of diseases or disorders, such as neurodegenerative disorders), they are generally formulated into unit dose injectable forms (solutions, suspensions, emulsions). Formulations include those for stereotactic administration into the brain (e.g., striatum), etc.

[0338] The compositions in some embodiments are provided as sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH in some aspects. Liquid preparations are usually easier to prepare than gels, other viscous compositions, and solid compositions. In addition, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within an appropriate viscosity range to contact with specific tissues for longer periods of time. Liquid or viscous compositions can include a carrier, which can be a solvent or dispersion medium, including, for example, water, saline, phosphate buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

[0339] Sterile injectable solutions can be prepared by incorporating the agent or cells in a solvent, such as a mixture with a suitable carrier, diluent, or excipient, such as sterile water, physiological saline, glucose, dextrose, or the like.

[0340] The formulations to be used for in vivo administration are generally sterile. Sterility can be readily accomplished, for example, by filtration through sterile filtration membranes. IV. Treatment method

[0341] Provided herein is a method of using any of the provided compositions in a subject in need thereof to treat a disease or condition. In certain embodiments, the composition is produced by the methods provided herein. Such methods and uses include, for example, therapeutic methods and uses involving administering therapeutic cells or compositions containing same to a subject having a disease, condition, or disorder. In some embodiments, the disease or condition is a neurodegenerative disease or condition. In some embodiments, the cells or pharmaceutical composition thereof are administered in an amount effective to provide treatment of the disease or disorder. Uses include such methods and treatments, as well as the use of the cells or pharmaceutical composition thereof in the preparation of a medicament for carrying out such therapeutic methods. In some embodiments, the method thereby treats a disease or condition or disorder in a subject.

[0342] 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 provided herein contemplate the differentiation of pluripotent stem cells for administration to subjects exhibiting loss of dopamine (DA) neurons, including Parkinson's disease.

[0343] Parkinson's disease (PD) is the second most common neurodegenerative disorder, estimated to affect 4-5 million patients worldwide. This number is predicted to more than double by 2030. PD is the second most common neurodegenerative disorder after Alzheimer's disease, affecting approximately 1 million patients in the United States, with 60,000 new cases diagnosed each year. Currently, there is no cure for PD, which is pathologically characterized by selective loss of midbrain DA neurons in the substantia nigra. Thus, the cardinal feature of PD is the progressive, severe, and irreversible loss of midbrain dopamine (DA) neurons that ultimately leads to life-disabling motor dysfunction.

[0344] In some embodiments, the subject has a neurodegenerative disease. In some embodiments, the neurodegenerative disease comprises loss of dopamine neurons in the brain. In some embodiments, the subject has loss of dopaminergic neurons in the substantia nigra (SN). In some embodiments, the subject has loss of dopaminergic neurons in the substantia nigra pars compacta (SNc). In some embodiments, the subject exhibits rigidity, bradykinesia, impaired postural reflexes, resting tremor, or a combination thereof. In some embodiments, the subject exhibits abnormal [ 18 [F]-L-DOPA PET scan. In some embodiments, the subject is screened for Parkinson's Disease Related Pattern (PDRP) [ 18 F]-DG-PET evidence is shown.

[0345] In some embodiments, the neurodegenerative disease is Parkinsonism. 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 familial Parkinson's disease. In some embodiments, the subject has mild Parkinson's disease. In some embodiments, the subject has a Movement Disorder Society-Unified Parkinson's Disease Rating Scale (MDS-UPDRS) motor score of 32 or less. In some embodiments, the subject has moderate or advanced Parkinson's disease. In some embodiments, the subject has mild Parkinson's disease. In some embodiments, the subject has an MDS-UPDRS motor score of 33-60.

[0346] In some embodiments, the dose of cells according to the method provided and / or according to the product or composition provided is administered to the subject.In some embodiments, the size or timing of the dose is determined according to the specific disease or condition in the subject.In some cases, the size or timing of the dose for a specific disease can be empirically determined, taking into account the explanation provided.

[0347] 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 subject.

[0348] 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 It is a cell.

[0349] In some embodiments, the dose of cells is from 250,000 or about 250,000 cells / hemisphere to 20 million or about 20 million cells / hemisphere, 500,000 or about 500,000 cells / hemisphere to 20 million or about 20 million cells / hemisphere, 1 million or about 1 million cells / hemisphere to 20 million or about 20 million cells / hemisphere, 5 million or about 5 million cells / hemisphere to 20 million or about 20 million cells / hemisphere, 10 million or about 10 million cells / hemisphere to 20 million or about 20 million cells / hemisphere , 15 million or about 15 million cells / hemisphere to 20 million or about 20 million cells / hemisphere, 250,000 or about 250,000 cells / hemisphere to 15 million or about 15 million cells / hemisphere, 500,000 or about 500,000 cells / hemisphere to 15 million or about 15 million cells / hemisphere, 1 million or about 1 million cells / hemisphere to 15 million or about 15 million cells / hemisphere, 5 million or about 5 million cells / hemisphere to 15 million or about 15 million cells / hemisphere, 10 million or about 10 million cells / hemisphere ~15 million or about 15 million cells / hemisphere, 250,000 or about 250,000 cells / hemisphere ~10 million or about 10 million cells / hemisphere, 500,000 or about 500,000 cells / hemisphere ~10 million or about 10 million cells / hemisphere, 1 million or about 1 million cells / hemisphere ~10 million or about 10 million cells / hemisphere, 5 million or about 5 million cells / hemisphere ~10 million or about 10 million cells / hemisphere, 250,000 or about 250,000 cells / hemisphere ~5 million or about 5 0 million cells / hemisphere, 500,000 or about 500,000 cells / hemisphere to 5 million or about 5 million cells / hemisphere, 1 million or about 1 million cells / hemisphere to 5 million or about 5 million cells / hemisphere, 250,000 or about 250,000 cells / hemisphere to 1 million or about 1 million cells / hemisphere, 500,000 or about 500,000 cells / hemisphere to 1 million or about 1 million cells / hemisphere, or 250,000 or about 250,000 cells / hemisphere to 500,00 or about 500,00 cells / hemisphere.

[0350] In some embodiments, the dose of cells is between 1 million or about 1 million cells / hemisphere and 30 million or about 30 million cells / hemisphere. In some embodiments, the dose of cells is between 5 million or about 5 million cells / hemisphere and 20 million or about 20 million cells / hemisphere. In some embodiments, the dose of cells is between 10 million or about 10 million cells / hemisphere and 15 million or about 15 million cells / hemisphere.

[0351] In some embodiments, the dose of cells is about 3×10 6 cells / hemisphere~15×10 6 In some embodiments, the dose of cells is about 3×10 6 In some embodiments, the dose of cells is about 4×10 6 In some embodiments, the dose of cells is about 5×10 6 In some embodiments, the dose of cells is about 6×10 6 In some embodiments, the dose of cells is about 7×10 6 In some embodiments, the dose of cells is about 8×10 6 In some embodiments, the dose of cells is about 9×10 6 In some embodiments, the dose of cells is about 10×10 6 In some embodiments, the dose of cells is about 11 x 10 6 In some embodiments, the dose of cells is about 12×10 6 In some embodiments, the dose of cells is about 13×10 6 In some embodiments, the dose of cells is about 14×10 6 In some embodiments, the dose of cells is about 15×10 6 cells / hemisphere.

[0352] In some embodiments, the number of cells administered to a subject is about 0.25×10 6 Total cells ~ approx. 20 x 106 Total cells, approximately 0.25 x 10 6 Total cells ~ approx. 15 x 10 6 Total cells, approximately 0.25 x 10 6 Total cells ~ approx. 10 x 10 6 Total cells, approximately 0.25 x 10 6 Total cells ~ approx. 5 x 10 6 Total cells, approximately 0.25 x 10 6 Total cells ~ approx. 1 x 10 6 Total cells, approximately 0.25 x 10 6 Total cells ~ approx. 0.75 x 10 6 Total cells, approximately 0.25 x 10 6 Total cells ~ approx. 0.5 x 10 6 Total cells, approximately 0.5 x 10 6 Total cells ~ approx. 20 x 10 6 Total cells, approximately 0.5 x 10 6 Total cells ~ approx. 15 x 10 6 Total cells, approximately 0.5 x 10 6 Total cells ~ approx. 10 x 10 6 Total cells, approximately 0.5 x 10 6 Total cells ~ approx. 5 x 10 6 Total cells, approximately 0.5 x 10 6 Total cells ~ approx. 1 x 10 6 Total cells, approximately 0.5 x 10 6 Total cells ~ approx. 0.75 x 10 6 Total cells, approximately 0.75 x 10 6 Total cells ~ approx. 20 x 10 6 Total cells, approximately 0.75 x 10 6 Total cells ~ approx. 15 x 10 6 Total cells, approximately 0.75 x 10 6 Total cells ~ approx. 10 x 10 6 Total cells, approximately 0.75 x 10 6 Total cells ~ approx. 5 x 10 6 Total cells, approximately 0.75 x 10 6 Total cells ~ approx. 1 x 10 6 Total cells, approximately 1 x 10 6 Total cells ~ approx. 20 x 10 6 Total cells, approximately 1 x 10 6 Total cells ~ approx. 15 x 10 6 Total cells, approximately 1 x 106 Total cells ~ approx. 10 x 10 6 Total cells, approximately 1 x 10 6 Total cells ~ approx. 5 x 10 6 Total cells, approximately 5 x 10 6 Total cells ~ approx. 20 x 10 6 Total cells, approximately 5 x 10 6 Total cells ~ approx. 15 x 10 6 Total cells, approximately 5 x 10 6 Total cells ~ approx. 10 x 10 6 Total cells, approximately 10 x 10 6 Total cells ~ approx. 20 x 10 6 Total cells, approximately 10 x 10 6 Total cells ~ approx. 15 x 10 6 total cells, or approximately 15 x 10 6 Total cells ~ approx. 20 x 10 6 All cells in the organism.

[0353] In certain embodiments, individual populations of cells, or subtypes of cells, are administered to a subject at a dose ranging from about 5 million cells / hemisphere to about 20 million cells / hemisphere, or any value therebetween. Dosages may vary depending on attributes, particularly the disease or disorder and / or the patient and / or other treatments.

[0354] In some embodiments, the patient is administered multiple doses, and each of the doses or total doses can be within any of the values ​​set forth above. In some embodiments, the dose of cells comprises administration of 5 million or about 5 million cells / hemisphere to about 20 million cells, inclusive.

[0355] In some embodiments, a dose of cells, e.g., differentiated cells, is administered to a subject as a single dose or is administered only once within a period of two weeks, one month, three months, six months, one year, or more.

[0356] In the context of stem cell transplantation, administration of a given "dose" includes administration of a given amount or number of cells as a single composition and / or a single continuous administration, for example, as a single injection or continuous infusion, and also includes administration of a given amount or number of cells as a split dose or as multiple compositions provided in multiple individual compositions or infusions over a specified period of time, such as one day.Thus, in some situations, a dose is a single or continuous administration of a specified number of cells given or initiated at a single time point.However, in some situations, a dose is administered in multiple injections or infusions in a single period of time, such as by multiple infusions over a single day.

[0357] Thus, in some aspects, the dose of cells is administered in a single pharmaceutical composition, hi some embodiments, the dose of cells is administered in multiple compositions that jointly contain the dose of cells.

[0358] In some embodiments, a dose of cells may be administered by administration of multiple compositions or solutions, e.g., a first and a second, optionally more, each containing a portion of the cells of the dose. In some aspects, multiple compositions, each containing different populations and / or subtypes of cells, are administered separately or independently, optionally within a certain period of time.

[0359] In some embodiments, administration of a composition or dose, e.g., administration of multiple cellular compositions, involves separate administration of the cellular compositions, which in some aspects occur simultaneously or sequentially, in any order.

[0360] In some embodiments, the subject is administered multiple doses of cells, for example, two or more doses or multiple consecutive doses. In some embodiments, two doses are administered to the subject. In some embodiments, the first dose is followed by multiple consecutive doses, such that an additional dose is administered after the administration of the consecutive dose. In some aspects, the number of cells administered to the subject in the additional dose is the same or similar to the first dose and / or the consecutive dose. In some embodiments, the additional dose is greater than the previous dose.

[0361] In some embodiments, 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 treatment, e.g., the stage of the disease and / or the likelihood or incidence of the subject developing an adverse event, e.g., dyskinesia.

[0362] In some embodiments, the dose of cells is generally large enough to be effective in ameliorating a symptom of the disease.

[0363] In some embodiments, the cells are administered at a desired dosage, which in some aspects includes a desired dose or number of cells or cell types and / or a desired ratio of cell types. In some embodiments, the dosage of cells is based on the desired total number (or number per kg of body weight) of cells in each population or of each cell type (e.g., TH+ or TH-). In some embodiments, the dosage is based on a combination of such features, such as the desired number of total cells, the desired ratio, and the desired total number of cells in each population.

[0364] Thus, in some embodiments, dosage is based on a desired fixed dose and desired ratios of total cells and / or based on a desired fixed dose of one or more of the individual subtypes or subpopulations, e.g., each.

[0365] In certain embodiments, the number and / or concentration of cells refers to the number of TH-negative cells, while in other embodiments, the number and / or concentration of cells refers to the number or concentration of all cells administered.

[0366] In some embodiments, the cells are administered at a desired dosage, which in some aspects includes a desired dose or number of cells or cell types and / or a desired ratio of cell types. Thus, the dosage of cells is in some embodiments based on the total number of cells and the desired ratio of individual populations or subtypes. In some embodiments, the dosage of cells is based on the desired total number (or number per kg body weight) of cells in individual populations or individual cell types. In some embodiments, the dosage is based on a combination of such features, such as the desired number of total cells, the desired ratio, and the desired total number of cells in individual populations.

[0367] Thus, in some embodiments, dosage is based on a desired fixed dose and desired ratios of total cells and / or based on a desired fixed dose of one or more of the individual subtypes or subpopulations, e.g., each.

[0368] In certain embodiments, the number and / or concentration of cells refers to the number of TH-negative cells, while in other embodiments, the number and / or concentration of cells refers to the number or concentration of all cells administered.

[0369] In some embodiments, the size of the dose is determined based on one or more criteria, such as the subject's response to previous treatments, e.g., the type and / or stage of the disease, and / or the likelihood or incidence of the subject developing a toxic event, e.g., dyskinesia. V. Products and Kits

[0370] Also provided are articles of manufacture, systems, devices, and kits useful for practicing the provided methods, including (i) one or more reagents for differentiating pluripotent stem cells into floor plate midbrain progenitor cells, committed dopamine (DA) neuron progenitor cells, and / or dopamine (DA) neurons, and (ii) instructions for use of the one or more reagents for practicing any of the methods described herein.

[0371] In some of any such embodiments, the reagent for differentiation is or includes a small molecule capable of inhibiting TGF-β / activin-nodal signaling. In some of any such embodiments, the reagent for differentiation is or includes SB431542. In some of any such embodiments, the reagent for differentiation is or includes a small molecule capable of activating SHH signaling. In some of any such embodiments, the reagent for activating SHH signaling is or includes SHH. In some of any such embodiments, the reagent for activating SHH signaling is or includes palmorphamin. In some of any such embodiments, the reagent for activating SHH signaling is or includes SHH and palmorphamin. In some of any such embodiments, the reagent for differentiation is or includes a small molecule capable of inhibiting BMP signaling. In some of any such embodiments, the reagent for inhibiting BMP signaling is LDN193189. In some of any such embodiments, the reagent for differentiation is or includes a small molecule capable of inhibiting GSK3β signaling. In some of any such embodiments, the reagent is or includes CHIR99021. In some of any such embodiments, the reagent for differentiation is or includes one or more of BDNF, GDNF, dbcAMP, ascorbic acid, TGFβ3, and DAPT. The reagents in the kit, in one embodiment, may be in solution, frozen, or lyophilized.

[0372] Also provided is an article of manufacture that includes (i) any of the compositions described herein and (ii) instructions for administering the composition to a subject.

[0373] In some embodiments, the article or kit comprises one or more containers, typically multiple containers, packaging material, and a label or insert on or attached to the container and / or packaging that generally includes instructions for use, e.g., instructions for reagents for differentiating pluripotent cells, e.g., differentiating iPSCs into floor plate midbrain progenitor cells, committed dopamine (DA) neuron progenitor cells, and / or dopamine (DA) neurons, and instructions for performing any of the methods provided herein. In some aspects, the article of manufacture provided includes reagents for differentiating and / or maturing cells at one or more steps in the manufacturing process, e.g., any of the reagents described in any of the steps in Sections II and III.

[0374] Also provided are articles of manufacture and kits that include differentiated cells, such as those produced using the methods provided herein, and, optionally, instructions for use, e.g., administration instructions. In some embodiments, the instructions provide instructions or specify methods for assessing whether a subject is likely or likely to respond after administration of differentiated cells expressing a recombinant receptor for treating a disease or disorder, and / or the extent or level of response, prior to undergoing cell therapy. In some aspects, the articles of manufacture may include a dose or composition of differentiated cells.

[0375] The products provided herein include packaging materials. Packaging materials for use in packaging the materials provided are well known to those skilled in the art. For example, see 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 consumables, such as pipette tips and / or plastic plates, or bottles. The products or kits may include devices for facilitating the distribution of materials or for facilitating the use in high throughput or large scale, for example, for facilitating the use in robotic equipment. Typically, packaging does not react with the composition contained therein.

[0376] In some embodiments, the reagents and / or cell compositions are packaged separately. In some embodiments, each container may have a single compartment. In some embodiments, other components of the product or kit are packaged separately or together in a single compartment. VI. ILLUSTRATIVE EMBODIMENTS

[0377] Among the embodiments provided are the following: 1. A method for differentiating neural cells, comprising: (a) performing a first incubation comprising culturing pluripotent stem cells in a non-adherent culture vessel under conditions to generate cell spheroids, wherein beginning at the beginning of the first incubation (day 0), the cells are exposed to (i) an inhibitor of TGF-β / Activin-Nodal signaling, (ii) at least one activator of Sonic Hedgehog (SHH) signaling, (iii) an inhibitor of bone morphogenetic protein (BMP) signaling, and (iv) an inhibitor of glycogen synthase kinase 3β (GSK3β) signaling; (b) performing a second incubation, comprising culturing the cells of the spheroids in a substrate-coated culture vessel under conditions that induce neuronal differentiation of the cells; A method comprising: 2. The method of embodiment 1, wherein said second incubation begins at about day 7. 3. The method of embodiment 1 or embodiment 2, wherein the cells are exposed to the inhibitor of TGF-β / activin-nodal signaling until day 7 or before day 7. The method of any one of embodiments 1-3, wherein the cells are exposed to an inhibitor of TGF-β / activin-nodal starting on day 4.0 through day 6, inclusive. 5. The method according to any one of the preceding embodiments, wherein the cells are exposed to at least one activator of SHH signaling until the 7th day or the day before the 7th day. The method of any one of embodiments 1 to 5, wherein the cells are exposed to at least one activator of the SHH signaling starting on day 6.0 through day 6, inclusive. 7. The method of any one of embodiments 1-6, wherein the cells are exposed to the inhibitor of BMP signaling until the 11th day or the day before the 11th day. The method of any one of embodiments 1-7, wherein the cells are exposed to the inhibitor of BMP signaling starting on day 8.0 through day 10, inclusive. 9. The method according to any one of embodiments 1 to 8, wherein the cells are exposed to the inhibitor of GSK3β signaling until the 13th day or the day before the 13th day. The method of any one of embodiments 1-9, wherein the cells are exposed to the inhibitor of GSK3b signaling starting on day 10.0 through day 12, inclusive. 11. The method of any one of embodiments 1-10, wherein culturing the cell under conditions that cause neural differentiation of the cell comprises exposing the cell 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 beta-3 (TGFβ3) (collectively, "BAGCT"), and (vi) an inhibitor of Notch signaling. 12. The method of any one of embodiments 1-11, wherein the cells are exposed to BAGCT and the inhibitor of Notch signaling starting on day 11. 13. The method of any one of embodiments 1-12, wherein the cells are exposed to BAGCT and the inhibitor of Notch signaling starting on day 11 until harvesting of the neuronally differentiated cells, optionally until day 18, and optionally until day 25. 14. A method for differentiating neural cells, comprising: (a) performing a first incubation comprising culturing pluripotent stem cells in a non-adherent culture vessel under conditions to generate cell spheroids, the first incubation beginning on a start date (day 0) of the first incubation, exposing the cells to (i) an inhibitor of TGF-β / Activin-Nodal signaling until day 6, inclusive; (ii) at least one activator of Sonic Hedgehog (SHH) signaling until day 6, inclusive; (iii) an inhibitor of bone morphogenetic protein (BMP) signaling until day 6, inclusive; and (iv) an inhibitor of glycogen synthase kinase 3β (GSK3β) signaling until day 6, inclusive; (b) performing a second incubation for neuronal differentiation of the cells of the spheroids beginning on day 7, comprising culturing the cells in a culture vessel coated with a substrate selected from laminin, collagen, entactin, heparin sulfate proteoglycan, and combinations thereof, wherein beginning on day 7, the cells are exposed to (i) an inhibitor of BMP signaling and (ii) an inhibitor of GSK3β signaling, and beginning on 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 beta-3 (TGFβ3) (collectively, "BAGCT"), and (vi) an inhibitor of Notch signaling; A method comprising: 15. The method of any one of embodiments 1 to 14, further comprising collecting the neuronally differentiated cells. 16. The method of embodiment 15, wherein said harvesting occurs on or after about day 16. 17. The method of embodiment 15 or embodiment 16, wherein said harvesting occurs from about day 16 to about day 30. 18. The method of any one of embodiments 15-17, wherein said harvesting is performed on days 18 to 25. 19. The method of any one of embodiments 15-18, wherein said harvesting is performed on or about day 18. 20. The method of any one of embodiments 15-18, wherein said harvesting is performed on or about the 25th day. 21. The method according to any one of embodiments 1 to 20, wherein said neuronally differentiated cells are committed dopaminergic neuronal progenitor cells. 22. The method of embodiment 21, wherein the dopaminergic neuronal progenitor cells are capable of innervating a host tissue upon transplantation into a subject. 23. The method according to any one of the preceding embodiments, wherein prior to carrying out the second incubation, the spheroids are dissociated to produce a cell suspension, and cells of the cell suspension are cultured in a substrate-coated culture vessel. 24. The method of embodiment 23, wherein the dissociation is performed when the spheroid cells express at least one of PAX6 and OTX2. 25. The method of embodiment 23 or embodiment 24, wherein the dissociation is performed on about day 7. 26. The method according to any one of the preceding embodiments, wherein the culture vessel is selected from the group consisting of a plate, a dish, a flask, and a bioreactor. 27. The method according to any one of embodiments 1 to 26, wherein the culture vessel is a plate. 28. The method of embodiment 27, wherein the plate is a 6-well plate, a 12-well plate, a 24-well plate, or a 96-well plate. 29. The method of embodiment 27 or embodiment 28, wherein the plate is a microwell plate. 30. The method of embodiment 29, wherein the microwell plate is a 6-well plate. 31. The method of embodiment 29, wherein the microwell plate is a 24-well plate. 32. The method of any one of embodiments 1-31, wherein the culture vessel is an Aggrewell™ plate. On day 33, the pluripotent stem cell culture yielded approximately 0.1 × 10 6 cells / cm 2 ~about 2×10 6 cells / cm 2 , about 0.1×10 6 cells / cm 2 ~Approx. 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 / 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 / cm2 ~Approx. 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 ~Approx. 2×10 6 cells / cm 2 , about 0.8×10 6 cells / cm 2 ~Approx. 1×10 6 cells / cm 2 , or approximately 1.0 × 10 6 cells / cm 2 ~Approx. 2×10 6 cells / cm 2 The method according to any one of embodiments 29 to 32, comprising: 34. The pluripotent stem cells on day 0 were cultured in a 6-well plate, with approximately 1 x 10 6 Pluripotent stem cells / well ~ approx. 20 x 10 6 Pluripotent stem cells / well, approximately 1 x 10 6 Pluripotent stem cells / well ~ approx. 15 x 10 6 Pluripotent stem cells / well, approximately 1 x 10 6 Pluripotent stem cells / well ~ approx. 10 x 10 6 Pluripotent stem cells / well, approximately 1 x 10 6 Pluripotent stem cells / well ~ 5 x 10 6 Pluripotent stem cells / well, approximately 5 x 10 6 Pluripotent stem cells / well ~ approx. 20 x 10 6 Pluripotent stem cells / well, approximately 5 x 10 6 Pluripotent stem cells / well ~ approx. 15 x 10 6 Pluripotent stem cells / well, approximately 5 x 10 6 Pluripotent stem cells / well ~ approx. 10 x 10 6 Pluripotent stem cells / well, approximately 10 x 10 6 Pluripotent stem cells / well ~ approx. 20 x 10 6 Pluripotent stem cells / well, approximately 10 x 10 6 Pluripotent stem cells / well ~ approx. 15 x 10 6 Pluripotent stem cells / well, or approximately 15 x 10 6 Pluripotent stem cells / well ~ approx. 20 x 106 The method of any one of embodiments 29-33, comprising pluripotent stem cells / well. 35. The day 0 pluripotent stem cells were cultured in a 24-well plate, with approximately 1 x 10 5 Pluripotent stem cells / well ~ 5 x 10 6 Pluripotent stem cells / well, approximately 1 x 10 5 Pluripotent stem cells / well ~ approx. 1 x 10 6 Pluripotent stem cells / well, approximately 1 x 10 5 Pluripotent stem cells / well ~ 5 x 10 5 Pluripotent stem cells / well, approximately 5 x 10 5 Pluripotent stem cells / well ~ 5 x 10 6 Pluripotent stem cells / well, approximately 5 x 10 5 Pluripotent stem cells / well ~ approx. 1 x 10 6 Pluripotent stem cells / well, or approximately 1 x 10 6 Pluripotent stem cells / well ~ 5 x 10 6 The method of any one of embodiments 29-34, comprising pluripotent stem cells / well. 36. The method of any one of embodiments 29 to 35, wherein the culture of pluripotent stem cells on day 0 comprises a sufficient number of cells to generate spheroids on about day 7 comprising about 1,000 cells to about 5,000 cells or about 2,000 cells to about 3,000 cells. 37. The method of any one of embodiments 29-36, wherein the day 0 pluripotent stem cell culture comprises a sufficient number of cells to generate spheroids comprising about 2,000 cells on about day 7. 38. The method of any one of embodiments 29-36, wherein the day 0 pluripotent stem cell culture comprises a sufficient number of cells to generate spheroids comprising about 3,000 cells on about day 7. 39. The method according to any one of embodiments 1 to 38, wherein the culture vessel is treated to reduce or eliminate cell adhesion. 40. The method of claim 39, wherein treating the culture vessel comprises incubating the culture vessel with pluronic acid. 41. The method according to any one of embodiments 1 to 40, wherein prior to said first incubation, said non-adherent culture vessel is exposed to a surfactant. 42. The method of embodiment 41, wherein the surfactant is pluronic acid. 43. The method of any one of embodiments 1 to 42, wherein the non-adherent culture vessel comprises a low or ultra-low attachment surface. 44. The method of any one of embodiments 1-43, wherein the non-adherent culture vessel comprises an ultra-low attachment surface. 45. The method of any one of the preceding embodiments, wherein the substrate is a basement membrane protein. 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 proteoglycan, and combinations thereof. 47. The method of any one of embodiments 1 to 46, wherein the substrate is a recombinant protein. 48. The method of any one of embodiments 1 to 47, wherein the substrate is recombinant laminin. 49. The method according to any one of the preceding embodiments, wherein the substrate-coated culture vessel is exposed to poly-L-ornithine before being used to culture the cells. 50. The method of any one of embodiments 1 to 49, wherein the inhibitor of TGF-β / activin-nodal signaling is SB431542. 51. The method of 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. 52. The method according to any one of embodiments 1 to 51, wherein the at least one activator of SHH signaling is SHH or palmorfamine. 53. The method according to any one of the preceding embodiments, wherein the at least one activator of SHH signaling comprises two activators of SHH signaling selected from the SHH protein and palmorphamin. 54. The method of embodiment 52 or embodiment 53, wherein the cells are exposed to SHH at a concentration of about 10 ng / mL to about 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. 55. The method of any one of embodiments 52 to 54, wherein the cells are exposed to palmorfamine 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. 56. The method of any one of embodiments 1 to 55, wherein the inhibitor of BMP signaling is LDN193189. 57. The method of embodiment 56, wherein the cells are exposed to LDN193189 at a concentration of about 10 nM to about 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. 58. The method of any one of embodiments 1 to 57, wherein the inhibitor of GSK3β signaling is CHIR99021. 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. 60. The method of any one of embodiments 11 to 59, wherein 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, optionally about 20 ng / mL. 61. The method of any one of embodiments 11 to 60, wherein 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, optionally about 20 ng / mL. 62. The method of any one of embodiments 11 to 61, wherein the cells are 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, optionally about 0.5 mM. 63. The method of any one of embodiments 11 to 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. 64. The method of any one of embodiments 11 to 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. 65. The method of any one of embodiments 11-64, wherein the inhibitor of Notch signaling is DAPT. 66. The method of any one of embodiments 11 to 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. 67. The method according to any one of the preceding embodiments, wherein the culturing in the first incubation and / or the second incubation is carried out in a medium containing serum or a serum substitute. 68. The method according to any one of the preceding embodiments, wherein the culturing in the first incubation and in the second incubation is carried out in a medium containing serum or a serum substitute. 69. The method of any one of embodiments 1 to 68, wherein the cells are cultured in a medium containing serum or a serum replacement from about day 0 to about day 10. 70. The method of any one of embodiments 67 to 69, wherein the serum or serum replacement constitutes about 5% (v / v) of the medium. 71. The method according to any one of embodiments 67 to 70, wherein the serum or serum replacement constitutes about 2% (v / v) of the medium. 72. The method according to any one of embodiments 67 to 71, wherein the 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. 73. The method according to any one of embodiments 67 to 72, wherein the culture medium contains a serum replacement. 74. The method of any one of embodiments 67 to 73, wherein the serum replacement does not contain fetal bovine serum (FBS). 75. The method of any one of embodiments 67-74, wherein the serum replacement is KnockOut™ serum replacement. 76. The method of any one of embodiments 1-66, wherein the cells are cultured in the absence of serum during the culture period. 77. The method of any one of embodiments 1-76, wherein the cells are exposed to an inhibitor of Rho-associated protein kinase (ROCK) signaling on days 0, 7, 16, and / or 20. 78. The method of any one of embodiments 1 to 77, wherein the cells are exposed to an inhibitor of Rho-associated kinase protein (ROCK) signaling on days 0, 7, 16, and 20. 79. The method of embodiment 77 or embodiment 78, wherein the ROCK inhibitor is Y-27632. 80. The method of any one of embodiments 77-79, wherein 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, optionally about 10 μM. 81. The method of any one of embodiments 1-80, wherein at least about 50% of the medium is replaced every day, or every other day, or every third day. 82. The method of any one of embodiments 23 to 81, wherein the spheroids are dissociated by enzymatic dissociation. 83. The method according to any one of embodiments 23 to 82, wherein the spheroids are dissociated by enzymatic dissociation comprising the use of an enzyme selected from the group consisting of actase, dispase, collagenase, and combinations thereof. 84. The method of any one of embodiments 23 to 83, wherein the spheroids are dissociated by enzymatic dissociation comprising the use of actase. 85. Approximately 0.1×10 6 cells / cm 2 ~about 2×10 6 cells / cm 2 , about 0.1×10 6 cells / cm 2 ~Approx. 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 ~Approx. 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 ~Approx. 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 ~Approx. 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 ~Approx. 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 ~Approx. 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 ~Approx. 0.6×10 6 cells / cm 2 , about 0.6×10 6 cells / cm 2 ~Approx. 2×10 6 cells / cm 2 , about 0.6×10 6 cells / cm 2 ~Approx. 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 ~Approx. 2×10 6 cells / cm 2 , about 0.8×10 6 cells / cm 2 ~Approx. 1×10 6 cells / cm 2 , or approximately 1.0 × 10 6 cells / cm 2 ~Approx. 2×10 6 cells / cm 2 85. The method of any one of embodiments 1-84, comprising transferring the cells to the substrate-coated culture vessel. 86. Approximately 0.4×10 6 cells / cm 2 ~about 0.8×10 6 cells / cm 2 86. The method of any one of embodiments 1-85, comprising transferring the cells to the substrate-coated culture vessel. 87. The method of embodiment 85 or embodiment 86, wherein the transfer occurs on about day 7. 88. The method of any one of embodiments 15 to 8...

Claims

1. 1. A method for differentiating neural cells, comprising: (a) performing a first incubation comprising culturing pluripotent stem cells in a non-adherent culture vessel under conditions to generate cell spheroids, wherein starting on day 0, the cells are: (i) an inhibitor of TGF-β / activin-nodal signaling, wherein the inhibitor of TGF-β / activin-nodal signaling is SB431542; (ii) at least one activator of Sonic Hedgehog (SHH) signaling, wherein the at least one activator of SHH signaling is SHH or palmorphamin; or the at least one activator of SHH signaling comprises two activators of SHH signaling selected from SHH and palmorphamin; (iii) an inhibitor of bone morphogenetic protein (BMP) signaling, wherein the inhibitor of BMP signaling is LDN193189; and (iv) an inhibitor of glycogen synthase kinase 3β (GSK3β) signaling, wherein the inhibitor of GSK3β signaling is CHIR99021. performing a first incubation in which the cells are exposed to an inhibitor of TGF-β / activin-nodal, at least one activator of SHH signaling, an inhibitor of BMP signaling, and an inhibitor of GSK3β signaling, wherein day 0 corresponds to the start of the first incubation, and wherein starting on day 0 and up to day 6 (inclusive), the cells are exposed to (b) performing a second incubation, comprising culturing the cells of the spheroids in a substrate-coated culture vessel under conditions that induce neuronal differentiation of the cells; A method comprising:

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

3. (i) exposing the cells to the inhibitor of TGF-β / activin-nodal signaling on or before the 7th day; (ii) exposing said cells to at least one activator of SHH signalling until day 7 or until day 7; (iii) exposing the cells to the inhibitor of BMP signaling until day 11 or before day 11; (iv) exposing the cells to the inhibitor of BMP signaling starting on day 0 through day 10, inclusive; (v) exposing the cells to the inhibitor of GSK3β signaling on or before day 13; (vi) exposing the cells to the inhibitor of GSK3β signaling starting on day 0 through day 12, inclusive; (vii) exposing the cells to an inhibitor of BAGCT and Notch signaling beginning on day 11; and / or (viii) exposing the cells to an inhibitor of BAGCT and Notch signaling until harvesting the neuronally differentiated cells beginning on day 11, and optionally until day 18, and optionally until day 25; The method according to claim 1 or claim 2.

4. 4. The method of any one of claims 1-3, wherein culturing the cells under conditions that induce neuronal 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 beta-3 (TGFβ3) (collectively, "BAGCT"), and (vi) an inhibitor of Notch signaling.

5. 2. The method of claim 1, comprising: beginning on day 7, performing a second incubation for neural differentiation of the cells of the spheroids, the second incubation comprising culturing the cells in a culture vessel coated with a substrate selected from among laminin, collagen, entactin, heparin sulfate proteoglycan, and combinations thereof; beginning on day 7, exposing the cells to (i) an inhibitor of BMP signaling and (ii) an inhibitor of GSK3β signaling; and beginning on day 11, performing a second incubation 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 beta-3 (TGFβ3) (collectively, "BAGCT"), and (vi) an inhibitor of Notch signaling.

6. 6. The method of any one of claims 1 to 5, further comprising harvesting the neuronally differentiated cells, optionally wherein said harvesting occurs on day 16 or later, said harvesting occurs between day 16 and day 30, said harvesting occurs between day 18 and day 25, and / or said harvesting occurs on or at day 18 or day 25.

7. 7. The method of any one of claims 1 to 6, wherein the neuronally differentiated cells are committed dopaminergic neuronal progenitor cells, and optionally the dopaminergic neuronal progenitor cells are capable of innervating a host tissue upon transplantation into a subject.

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

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

10. 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 containing serum or a serum replacement, or the cells are cultured in the absence of serum during the culture period.

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

12. A method according to any one of claims 1 to 11, wherein the first incubation is carried out in a culture medium containing an inhibitor of TGF-β / activin-nodal, at least one activator of SHH signalling, an inhibitor of BMP signalling and an inhibitor of GSK3β signalling, and at least 50% of the culture medium is replaced every day, or every other day, or every third day.

13. 7. The method of claim 6, further comprising formulating a cryoprotectant to the collected cells, optionally cryopreserving the collected cells, and optionally thawing the cells prior to use.

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