Method for differentiating dopaminergic neurons from stem cells

Abstract

The present disclosure provides methods of differentiating pluripotent stem cells, including inducible pluripotent stem cells, into lineage-specific midbrain floor progenitor cells, decisive dopaminergic neuron progenitor cells, definitive dopaminergic neuron progenitor cells, and / or dopaminergic neuron cells. Also provided are compositions use thereof, such as for the treatment of neurodegenerative diseases and conditions, including Parkinson's disease, as well as articles of manufacture and kits for use thereof.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 472,789, filed June 13, 2023, entitled “METHODS FOR DIFFERENTIATING DOPAMINEERGIC NEURONS FROM STEM CELLS”, the contents of which are incorporated herein by reference in their entirety for all purposes. Technical Field

[0002] This disclosure relates to methods for differentiating pluripotent stem cells, including induced pluripotent stem cells, into lineage-specific midbrain plate progenitor cells, definitive dopaminergic neuronal progenitor cells, definitive dopaminergic neuronal progenitor cells, and / or dopaminergic neurons. Compositions for differentiating cells and their therapeutic uses, such as for treating neurodegenerative conditions and diseases including Parkinson's disease, are also provided, as well as articles thereof and kits. Background Technology

[0003] Parkinson's disease (PD) causes frailty-motor complications, and there is currently no available restorative treatment. PD is the second most common neurodegenerative disease after Alzheimer's, affecting approximately 0.3% of the general population and 1-2% of those aged 65 and older. With the aging populations in developed countries, the prevalence of PD is projected to double or triple. (Cha et al., 2023) J. Mov. Disord 16: 22-41; Rong et al. (2021) Neurology 97: e1986–e1993; Dorsey and Bloem (2018) JAMA Neurol .75:9-10; de Lau and Breteler (2006) Lancet Neurol .5: 525-535).

[0004] Dopamine deficiency, resulting from the progressive loss of dopaminergic neurons in the substantia nigra, is a common feature of Parkinson's disease (PD). By the time of diagnosis, patients have already experienced significant nigrostriatal degeneration. Currently available treatments, such as dopamine replacement therapy (e.g., using levodopa or dopamine agonists), are beneficial to some patients, but have a limited therapeutic window due to side effects and reduced efficacy. Cha et al., Weiss et al. (1971) Lancet 1:1016-1017; Kang and Fahn (1988) Ration.Drug Ther 22: 1-7.

[0005] Cellular replacement therapies aimed at restoring lost dopamine-producing neurons have been developed for many years. A challenge in developing cell-based therapies for Parkinson's disease (PD) has been identifying suitable cell sources for neuronal replacement. One approach is transplantation of fetal midbrain dopamine neurons, such as those performed in over 300 patients worldwide. Brundin et al., Prog.Brain Res . (2010) 184:265-94; Lindvall and Kokaia, J. Clin.Invest (2010) 120:29-40. Therapies using human fetal tissue in these patients have demonstrated evidence of DA neuron survival and in vivo DA release up to 10 or 20 years after transplantation in some patients. However, in many patients, fetal tissue transplantation cannot replace DA neuron function. Furthermore, Parkinson's disease patients treated with fetal cell transplantation sometimes experience transplant-induced motor dysfunction upon discontinuation of treatment. Evidence suggests that this serious side effect is caused by serotonin (5-HT) produced by the transplanted fetal cells. Politis et al., Mov.Disord (2011) 26: 1997-2003. Furthermore, fetal tissue transplantation is plagued by challenges, including low quantity and quality of donor tissue, ethical and practical issues regarding tissue sourcing, and an unclear definition of transplanted cell heterogeneity, all of which contribute to variable clinical outcomes. Mendez et al., Nature Med (2008); Kordower et al., N. Engl. J. Med (1995) 332:1118-24; and Piccini et al., Nature Neuroscience (1999) 2:1137-40. Hypotheses regarding the limited efficacy observed in human fetal transplantation trials include: fetal transplantation may not provide a sufficient number of cells at the correct developmental stage, and fetal tissue is difficult to define by cell type and variables regarding the stage and quality of each tissue sample. Bjorklund et al., Lancet Neurol (2003) 2:437-45. Another contributing factor may be the host's inflammatory response to the graft. Ibid.

[0006] Another approach is to use stem cell-derived cells, such as pluripotent stem cells (PSCs), as the cell source for applications in regenerative medicine. Pluripotent stem cells are capable of self-renewal and generating all the cells of body tissues. PSCs comprise two main categories of cells: embryonic stem (ES) cells and induced pluripotent stem cells (iPSCs). ES cells are derived from the inner cell mass of the pre-implantation embryo and can be maintained indefinitely and expanded in vitro in their pluripotent state. Romito and Cobellis, Stem Cells Int(2016) 2016:9451492. Recently, preliminary results from a Phase I clinical trial involving the implantation of dopaminergic neurons derived from ES cell differentiation into the brains of Parkinson's disease patients (2023 International Conference on Parkinson's Disease and Movement Disorders, Copenhagen, Denmark, August 27-31). Results showed that the strategy was well-tolerated with no serious treatment-related adverse effects. Preliminary efficacy data indicated improvements in motor function. Despite these advances, the use of embryonic stem cells remains hampered by ethical concerns and the potential for such cells to form tumors in patients. Finally, in the case of allogeneic stem cell transplantation, ES cell-derived grafts may induce immune responses in patients.

[0007] Using induced pluripotent stem cells (iPSCs) instead of ES-derived cells offers the advantage of avoiding ethical issues. Furthermore, deriving iPSCs from the patient to be treated (i.e., the patient receiving an autologous cell transplant) avoids the immune rejection risks inherent in the use of embryonic stem cells. iPSCs can be obtained by reprogramming (“dedifferentiating”) adult somatic cells into cells that are more like ES cells, including those with the ability to expand indefinitely and differentiate into all three germ layers. Ibid. Such reprogramming is typically accomplished using “Yamano factors” (Oct 3 / 4, Sox2, Klf4, and Myc family members). See, for example, U.S. Patent 8,530,238.

[0008] Various methods for differentiating pluripotent stem cells into lineage-specific cell populations and the resulting cell compositions are considered for use as cell replacement therapy in patients with diseases leading to the loss of function of the targeted cell populations. However, in some cases, such methods are limited in their ability to produce cells with consistent physiological characteristics, and the cells produced by such methods may be limited in their ability to be transplanted in vivo and to neurally innervate other cells. For example, neural cells obtained by differentiation from pluripotent stem cells may be more readily transplanted into the brain of a treated subject when nerve cells are in an intermediate stage between an early stage (e.g., the progenitor or precursor cell stage) and a late stage (e.g., the differentiated cell stage). Moreover, there is a need to improve the manufacturability of lineage-specific cell populations derived from pluripotent stem cells, for example, for therapeutic purposes, by reducing the time and / or resources required for such manufacturing, including costs. Therefore, improved methods and cell compositions thereof are needed. The present invention addresses these and other needs. Summary of the Invention

[0009] In some embodiments, the present invention provides a method for differentiating pluripotent stem cells into dopaminergic neuronal progenitor cells. In some embodiments, these methods include: (a) performing a first incubation comprising non-adherent culturing of pluripotent stem cells in a first culture dish under conditions that generate cell spheroids, wherein the first incubation comprises: (i) exposing the pluripotent stem cells to at least one TGF-β / activin-Nodal signaling inhibitor and at least one bone morphogenetic protein (BMP) signaling inhibitor for at least one day (day 0) in the absence of: x) a sound hedgehog factor (SHH) signaling activator and y) a glycogen synthase kinase 3β (GSK3β) signaling inhibitor; and (ii) starting from the second day of the first incubation (day 1), exposing the pluripotent stem cells to at least one sound hedgehog factor (SHH) signaling activator and at least one glycogen synthase kinase 3β (GSK3β) signaling inhibitor; and (b) performing a second incubation comprising adherent culturing of spheroids of cells in a second culture dish under conditions that allow the cells to further differentiate into dopaminergic neuronal progenitor cells.

[0010] In some embodiments, the dopaminergic neuron progenitor cells are definitive dopaminergic neuron progenitor cells. In some embodiments, the pluripotent stem cells are induced pluripotent stem cells. In some embodiments, the pluripotent stem cells are autologous to the subject to be treated with dopaminergic neuron progenitor cells.

[0011] In some embodiments, the first incubation also includes exposing the pluripotent stem cells to a ROCK inhibitor (ROCKi) starting from day 0. In some embodiments, during the first incubation, the pluripotent stem cells are not exposed to ROCKi prior to exposure to TGF-β / activin-Nodal signaling inhibitors and bone morphogenetic protein (BMP) signaling inhibitors.

[0012] In some embodiments, the method includes exposing pluripotent stem cells to: (a) a TGF-β / activin-Nodal signaling inhibitor from day 0 until day 4; (b) a BMP signaling inhibitor from day 0 until day 10; (c) a sound hedgehog signaling activator from day 1 until day 6; and (d) a glycogen synthase kinase 3β (GSK3β) signaling inhibitor from day 1 until day 12.

[0013] In some embodiments, the BMP signaling inhibitor is LDN193189. In some embodiments, cells are exposed to LDN193189 at concentrations between about 10 nM and 500 nM, between about 20 nM and about 400 nM, between about 50 nM and about 200 nM, or between about 75 nM and about 150 nM, optionally about 100 nM.

[0014] In some embodiments, the TGF-β / activin-Nodal signaling inhibitor is SB431542. In some embodiments, cells are exposed to SB431542 at concentrations between about 1 µM and about 20 µM, between about 5 µM and about 15 µM, or between about 8 µM and about 12 µM, optionally about 10 µM.

[0015] In some embodiments, the SHH signaling activator is SHH protein or purmorphamine. In some embodiments, the cells are exposed to SHH at concentrations of about 10 ng / mL to 500 ng / mL, about 20 ng / mL to about 400 ng / mL, about 50 ng / mL to about 200 ng / mL, or about 75 ng / mL to about 150 ng / mL, optionally about 100 ng / mL. In some embodiments, the cells are exposed to purmorphamine at concentrations between about 0.1 µM and about 20 µM, between about 0.5 µM and about 10 µM, between about 1 µM and about 5 µM, between about 1 µM and about 3 µM, or between about 1.5 µM and about 2.5 µM, optionally about 2 µM.

[0016] In some embodiments, the GSK3β signaling inhibitor is CHIR99021. In some embodiments, cells are exposed to CHIR99021 at concentrations between about 0.1 µM and about 5 µM, between about 0.5 µM and about 4 µM, between about 0.5 µM and about 2 µM, optionally about 1 µM; and on each day from day 2 to day 12, cells are exposed to CHIR99021 at concentrations between about 0.1 µM and about 5 µM, between about 0.5 µM and about 4 µM, or between about 1 µM and about 3 µM, optionally about 2 µM.

[0017] In some embodiments, the first incubation includes changing the culture medium on one or more days from day 1 to day 6. In some embodiments, the first incubation includes changing the culture medium every day from day 1 to day 6. In some embodiments, the culture medium change includes replacing 50% or about 50% of the culture medium. In some embodiments, the culture medium change includes replacing all or almost all of the culture medium.

[0018] In some embodiments, the second incubation begins on day 7 or approximately day 7. In some embodiments, the cells of the spheroids are dissociated to form a cell suspension prior to the second incubation. In some embodiments, dissociation is performed when the spheroid cells express at least one of PAX6 and OTX2. In some embodiments, dissociation is performed approximately day 7. In some embodiments, the second incubation comprises adhering to and culturing the cell suspension in a second cell culture dish.

[0019] In some embodiments, the second incubation includes exposing the cells of the sphere to a bone morphogenetic protein (BMP) signaling inhibitor and a GSK3β signaling inhibitor. In some embodiments, the second incubation also includes exposing the cells to: (i) brain-derived neurotrophic factor (BDNF); (ii) ascorbic acid; (iii) glial cell-derived neurotrophic factor (GDNF); (iv) dibutyryl cyclic AMP (dbcAMP); (v) transforming growth factor β-3 (TGFβ3) (collectively, “BAGCT”); and (vi) a Notch signaling inhibitor. In some embodiments, the cells are exposed to BAGCT and the Notch signaling inhibitor starting on day 11. In some embodiments, the cells are exposed to BAGCT and the Notch signaling inhibitor starting on day 11 until harvesting dopaminergic neuronal progenitor cells, optionally until day 14, day 15, day 16, or day 17.

[0020] In some embodiments, the second incubation includes changing the culture medium for one or more days from day 7 until harvest or collection. In some embodiments, the second incubation includes changing the culture medium every day from day 7 until harvest or collection. In some embodiments, changing the culture medium for one or more days from day 7 until harvest includes replacing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the culture medium. In some embodiments, about or at least about 50% of the culture medium is replaced on days 1, 2, 3, 4, 5, and 6. In some embodiments, about 50% of the culture medium is replaced on days 1, 2, 3, 4, 5, and 6. In some embodiments, all or almost all of the culture medium is replaced during the culture medium change.

[0021] In some embodiments, the method further includes harvesting dopaminergic neuronal progenitor cells. In some embodiments, the dopaminergic neuronal progenitor cells are harvested on day 14 or later. In some embodiments, the dopaminergic neuronal progenitor cells are harvested between day 14 and day 17.

[0022] In some embodiments, the method further includes formulating the harvested dopaminergic neuronal progenitor cells with a cryoprotectant. In some embodiments, the cryoprotectant is selected from the group consisting of glycerol, propylene glycol, and dimethyl sulfoxide (DMSO). In some embodiments, the method further includes cryopreserving the formulated harvested dopaminergic neuronal progenitor cells and optionally thawing the cells prior to use. In some embodiments, cryopreservation includes controlled-rate freezing.

[0023] In some embodiments, this document provides a therapeutic composition containing dopaminergic neuronal progenitor cells generated using a method comprising: (a) performing a first incubation comprising non-adherent culturing of pluripotent stem cells in a first culture dish under conditions that generate cell spheroids, wherein the first incubation comprises: (i) exposing the pluripotent stem cells to at least one TGF-β / activin-Nodal signaling inhibitor and at least one bone morphogenetic protein (BMP) signaling inhibitor for at least one day (day 0) in the absence of: x) a sound hedgehog factor (SHH) signaling activator and y) a glycogen synthase kinase 3β (GSK3β) signaling inhibitor; and (ii) starting from the second day of the first incubation (day 1), exposing the pluripotent stem cells to at least one sound hedgehog factor (SHH) signaling activator and a glycogen synthase kinase 3β (GSK3β) signaling inhibitor; and (b) performing a second incubation comprising adherent culturing of the cells in a second culture dish under conditions that allow the cells to further differentiate into dopaminergic neuronal progenitor cells.

[0024] In some embodiments, the therapeutic compositions provided herein comprise dopaminergic neuronal progenitor cells that, compared to neurons generated using adherent culture differentiation methods, exhibit one or more characteristics selected from the group consisting of: (a) higher levels of FOXA2 expression; (b) lower levels of PAX6 expression; (c) higher predicted graft size after implantation; (d) higher predicted dopamine production levels after implantation; (e) lower serotonin production; (f) a higher percentage of viable cells; (g) higher levels of CORIN expression; (h) lower levels of PITX2 expression; and (i) lower levels of NKX2.1 expression. In some embodiments, the cells of the therapeutic composition exhibit two or more of these characteristics. In some embodiments, the cells exhibit three, four, five, six, seven, or eight or more of these characteristics. In some embodiments, the cells exhibit all nine listed characteristics.

[0025] In some embodiments, the therapeutic compositions provided herein comprise dopaminergic neuronal progenitor cells exhibiting one or more properties selected from the group consisting of: (a) producing serotonin at levels below 2 nM and increasing less than twice the level of an unstimulated baseline upon stimulation with KCl; (b) more than 90% of the dopaminergic neuronal progenitor cells in the composition being viable; (c) expressing FOXA2 at greater than 100 TPM in a bulk RNA-seq analysis; (d) expressing CORIN at greater than 300 TPM in a bulk RNA-seq analysis; (e) expressing PITX2 at less than 50 TPM in a bulk RNA-seq analysis; (f) expressing NKX2.1 at less than 10 TPM in a bulk RNA-seq analysis; (g) greater than 90% FOXA2-positive cells; and (h) having a GraftTest value of at least 1500. ™ Scoring. In some embodiments, the cells of the therapeutic composition exhibit two or more of the properties listed in this paragraph. In some embodiments, the cells exhibit three, four, five, six, or seven or more of these properties. In some embodiments, the cells exhibit all eight listed properties.

[0026] In some embodiments, the dopaminergic neuron progenitor is a definitive dopaminergic neuron progenitor. In some embodiments, the dopaminergic neuron progenitor is a definitive dopaminergic neuron progenitor. In some embodiments, the dopaminergic neuron progenitor, after transplantation into a subject, enables the host tissue to be neurally innervated.

[0027] In some embodiments, the culture is carried out in microporous containers. In these embodiments, the number of PSCs introduced into the microporous container on day 0 of the method is between about 100 pluripotent stem cells / well and about 5,000 pluripotent stem cells / well. In some embodiments, the number of pluripotent stem cells per well is between about 250 and 3,000, and in some embodiments, the number of PSCs introduced into the microporous container is between about 500 and 1,000 cells per microwell.

[0028] In some embodiments, the day-0 pluripotent stem cell culture contains a number of cells sufficient to produce spheroids containing approximately 1,000 to approximately 9,000 cells or approximately 2,000 to approximately 5,000 cells by approximately day 7. In some embodiments, the day-0 pluripotent stem cell culture contains a number of cells sufficient to produce spheroids containing approximately 2,000 cells by approximately day 7. In some embodiments, the day-0 pluripotent stem cell culture contains a number of cells sufficient to produce spheroids containing approximately 3,000 cells by approximately day 7.

[0029] In some embodiments, cells are exposed to a Rho-associated protein kinase (ROCK) signaling inhibitor on day 0 and / or day 7. In some embodiments, the ROCK inhibitor is Y-27632. In some embodiments, cells are exposed to a ROCK inhibitor at concentrations between about 1 µM and about 20 µM, between about 5 µM and about 15 µM, or between about 8 µM and about 12 µM, optionally about 10 µM. In some embodiments, during the first incubation, pluripotent stem cells are not exposed to ROCKi prior to exposure to the TGF-β / activin-Nodal signaling inhibitor and the bone morphogenetic protein (BMP) signaling inhibitor.

[0030] In some embodiments, the pluripotent stem cells are embryonic stem (ES) cells, induced pluripotent stem cells (iPSCs), or combinations thereof. In some embodiments, the pluripotent stem cells are embryonic stem (ES) cells, optionally mouse or human 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, optionally mouse or human induced pluripotent stem cells. In some embodiments, the pluripotent stem cells are human induced pluripotent stem cells.

[0031] In some implementations, the pluripotent stem cells are autologous to the subject awaiting treatment with neurally differentiated cells. In some implementations, the pluripotent stem cells are allogeneic to the subject awaiting treatment with dopaminergic neuronal progenitor cells.

[0032] In some embodiments, the pluripotent stem cells are derived from healthy human subjects. In some embodiments, the pluripotent stem cells are derived from human subjects suffering from a neurodegenerative disease or condition. In some embodiments, the neurodegenerative disease or condition includes the loss of dopaminergic neurons. In some embodiments, the neurodegenerative disease or condition is Parkinson's syndrome. In some embodiments, the neurodegenerative disease or condition is Parkinson's disease.

[0033] This document also provides therapeutic compositions comprising dopaminergic neuronal progenitor cells generated by any of the methods disclosed herein. In some embodiments, the therapeutic composition comprises dopaminergic neuronal progenitor cells generated by a method comprising: (a) performing a first incubation comprising non-adherent culturing of pluripotent stem cells in a first culture dish under conditions that generate cell spheroids, wherein the first incubation comprises: (i) exposing the pluripotent stem cells, starting from the first day of the first incubation (day 0), to a TGF-β / activin-Nodal signaling inhibitor and a bone morphogenetic protein (BMP) signaling inhibitor in the absence of: x) a sound hedgehog factor (SHH) signaling activator and y) a glycogen synthase kinase 3β (GSK3β) signaling inhibitor; and (ii) exposing the pluripotent stem cells, starting from the second day of the first incubation (day 1), to at least one sound hedgehog factor (SHH) signaling activator and a glycogen synthase kinase 3β (GSK3β) signaling inhibitor; and (b) performing a second incubation comprising adherent culturing of the cells in a second culture dish under conditions that allow the cells to further differentiate into dopaminergic neuronal progenitor cells.

[0034] In some embodiments, the therapeutic composition comprises dopaminergic neuronal progenitor cells that, compared to neurons generated using an adherent culture differentiation method, exhibit one or more characteristics selected from the group consisting of: (a) higher levels of FOXA2 expression; (b) lower levels of PAX6 expression; (c) higher predicted graft size after implantation; (d) higher predicted dopamine production levels after implantation; (e) lower serotonin production; (f) a higher percentage of viable cells; (g) higher levels of CORIN expression; (h) lower levels of PITX2 expression; and (i) lower levels of NKX2.1 expression.

[0035] In some implementations, dopaminergic neuronal progenitor cells can be transplanted in vivo and innervate other cells.

[0036] In some embodiments, dopaminergic neuronal progenitor cells are able to produce dopamine after administration to a subject and subsequent transplantation, optionally wherein the directed cells do not produce or substantially do not produce norepinephrine. In some embodiments, the cells do not produce or substantially do not produce serotonin.

[0037] In some embodiments, at least about 70%, 75%, 80%, 85%, 90%, or 95% of the total cells in the therapeutic composition are live.

[0038] In some embodiments, the therapeutic composition comprises a cryoprotectant. In some embodiments, the cryoprotectant is selected from the group consisting of glycerol, propylene glycol, and dimethyl sulfoxide (DMSO).

[0039] This article also provides populations of differentiated neural cells generated by any of the methods disclosed herein.

[0040] This article also provides therapeutic compositions comprising any differentiated neural cell populations disclosed herein.

[0041] This article also provides a treatment method comprising administering a therapeutically effective amount of any of the therapeutic compositions provided herein to a subject in need.

[0042] This document also provides for the use of any of the compositions disclosed herein in the manufacture of a medicament for treating a subject suffering from a neurodegenerative disease or condition.

[0043] This document also provides any compositions disclosed herein for the treatment of subjects suffering from neurodegenerative diseases or conditions.

[0044] In some embodiments, the neurodegenerative disease or condition includes the loss of dopaminergic neurons. In some embodiments, the subject has lost at least 50%, at least 60%, at least 70%, or at least 80% of dopaminergic neurons, optionally in the substantia nigra (SN) and optionally in the SN compacta (SNc). In some embodiments, the neurodegenerative disease or condition is Parkinson's syndrome. In some embodiments, the neurodegenerative disease or condition is Parkinson's disease, optionally idiopathic Parkinson's disease. In some embodiments, the cells of the composition are autologous to the subject. In some embodiments, the cells of the composition are allogeneic to the subject. Attached Figure Description

[0045] Figure 1 An exemplary non-adherent scheme for differentiating pluripotent stem cells into dopaminergic neuronal progenitors, definitive dopaminergic neuronal progenitors, definitive dopaminergic neuronal progenitors, or dopaminergic neurons is shown.

[0046] Figure 2 The expression of EN1, GBX2, and NKX2-1 markers (per million counts) in cells cultured on day 7 using each of the following methods: 2D adherent culture, 3D condition 1, 3D condition 2, 3D condition 3, and 3D condition 4 is shown.

[0047] Figure 3AThe expression of LMX1A and DAPI on cells cultured using each of the following methods is shown: 2D adherent culture, 3D condition 1, 3D condition 2, 3D condition 3, and 3D condition 4. Figure 3B The graph shows the percentage of cells expressing LMX1A from three cell lines cultured using each of the following methods: 2D adherent culture, 3D condition 1, 3D condition 2, 3D condition 3, and 3D condition 4.

[0048] Figure 4 The relative transcriptome maturity is shown using principal component analysis (PCA) on a training set based on a 2D adherent culture method and a test set as described in Example 1, using a non-adherent 3D culture method. PC1 (x-axis) is representative of transcriptome maturity during differentiation, with lower values ​​corresponding to earlier differentiation time points and higher values ​​corresponding to later differentiation time points. The results span seven training set time points. n =12 samples are shown in the bottom seven rows, while the 3D differentiation test set is shown in the top three rows. The test set data are sorted by their mean PC1 value. The shaded box shows the mean ±1.96×SD of the PC1 score of the 2D differentiation samples on day 20.

[0049] Figure 5 Flow cytometry measurements of key lineage markers in dopaminergic progenitor cells (n=4) obtained using adherent and suspension culture methods are shown. Adherent cells were harvested on day 20, while suspension cells were harvested on day 16. EphB2, a pan-neuronal marker, showed no difference between cells obtained using adherent and suspension culture methods. Figure 5 A). FOXA2 expression is characteristic of the floor plate lineage in brain development, and flow cytometry determined that suspension culture increased FOXA2 expression compared to adherent culture, pointing to a more accurate cell fate of ventral midbrain dopaminergic neurons. () Figure 5 B). PAX6 expression is characteristic of the forebrain lineage in brain development, and flow cytometry showed that PAX6 levels in suspension cultured cells were approximately 10-fold lower than in adherent cultured cells. () Figure 6 C). The error bars are shown as the standard error (SEM) of the mean.

[0050] Figure 6 A shows the predicted graft size of neurons derived from adherent and suspension cultures after implantation into the brains of subjects. Graft size predictions were estimated in a donor background (n=4). Among all donors, the predicted graft size was larger under suspension culture conditions. When grouped by culture conditions, suspension culture was associated with significantly larger predicted graft sizes. The error bars are shown as the standard error (SEM) of the mean. Figure 6 B shows the estimated dopamine release from neurons derived from neuronal progenitor cells obtained using adherent and suspension culture protocols after implantation into the subject's brain. When grouped by culture conditions, there was no significant difference in predicted dopamine release between differentiation conditions (p>0.05). Error bars are shown as the standard error (SEM) of the mean.

[0051] Figure 7 Figure A shows a graph comparing the dopamine concentration (in nM) in the supernatant at baseline and 30 min after KCl treatment between the adherent and suspension culture protocols without normalization. There was no significant difference in initial dopamine release between the adherent and suspension culture protocols (ns p > 0.05). Figure 7 B shows the fold change in dopamine release in the supernatant after stimulation compared to baseline. There was no significant difference between the adherent and suspension culture protocols (ns, p > 0.05). Error bars are shown as the standard error of the mean (SEM). For all plots, n = 4.

[0052] Figure 8 A shows the serotonin concentrations (nM) in the supernatant at baseline and 30 min KCl treatment for both adherent and suspension culture protocols (n=4) without normalization. There was no significant difference in raw serotonin release between the adherent and suspension culture protocols (ns p>0.05). Figure 8 B shows the fold change in serotonin release after stimulation compared to baseline, demonstrating a significant decrease in serotonin production in dopaminergic neuronal progenitors generated using suspension culture conditions compared to cells generated using adherent culture conditions (n=4). Therefore, cells produced using suspension culture generate similar amounts of dopamine while producing less serotonin than cells from adherent conditions. Error bars are shown as the standard error of the mean (SEM). For all plots, n = 4.5. HT = 5-hydroxytryptamine (serotonin).

[0053] Figure 9 The cell viability (n=4) of dopaminergic neuronal progenitors generated using suspension culture is shown compared to that generated using adherent culture, as determined using the "Cell Counting and Viability" protocol on a Nucleocounter NC-200. Cells generated using suspension culture conditions showed a significant increase in the percentage of viable cells. ).

[0054] Figure 10 Two "target" markers, FOXA2, are shown. Figure 10 A) and CORIN ( Figure 10 The expression of B) indicates the presence of dopaminergic neuronal progenitors and shows two “off-target” markers, PITX2 ( Figure 10 C) and NKX2.1 Figure 10 (D) Their increased expression is not characteristic of dopaminergic neuronal progenitors. Therefore, the data in this figure demonstrate that cells produced using the day 1 suspension culture differentiation protocol disclosed herein are more characteristic of dopaminergic neuronal progenitors than cells produced using a differentiation protocol in which both the first and second incubations are performed in adherent culture. Detailed Implementation

[0055] This disclosure relates to lineage-specific differentiation of pluripotent stem cells (PSCs), such as embryonic stem (ES) cells or induced pluripotent stem cells (iPSCs). Specifically, methods are provided for directing the lineage-specific differentiation of PSCs or iPSCs into midbrain basal plate progenitor cells, dopaminergic neuron progenitor cells, decisive dopaminergic neuron progenitor cells (DDPCs), definitive dopaminergic neuron progenitor cells, and / or dopaminergic neurons. Differentiated cells prepared using the methods provided herein are further considered for various uses, including but not limited to, as a therapeutic agent for reversing diseases or damage in patients with a lack of dopamine-producing neurons.

[0056] This article provides methods for specifically differentiating pluripotent stem cells (PSCs), such as embryonic stem (ES) cells or induced pluripotent stem cell (iPSC) lineages, into midbrain basal plate progenitor cells, dopaminergic neuronal progenitor cells (including definitive dopaminergic neuronal progenitor cells), and / or dopaminergic neurons. In some aspects, PSCs differentiate into midbrain basal plate progenitor cells. In some aspects, such midbrain basal plate progenitor cells further differentiate into dopaminergic neuronal progenitor cells, including definitive dopaminergic neuronal progenitor cells. In some embodiments, definitive dopaminergic progenitor cells are cells that differentiate into dopaminergic neurons but cannot differentiate into non-dopaminergic neurons. In some aspects, such definitive dopaminergic neuronal progenitor cells further differentiate into definitive dopaminergic neuronal progenitor cells. In some aspects, PSCs differentiate into midbrain basal plate progenitor cells, differentiate into dopaminergic neurons, definitive dopaminergic neuronal progenitor cells, definitive dopaminergic neuronal progenitor cells, and finally differentiate into dopaminergic neurons.

[0057] The proposed implementation addresses issues related to the manufacture of cells that can be used to treat Parkinson's disease (PD), characterized by the selective degeneration of midbrain dopamine (mDA) neurons in the brain. Because PD symptoms are primarily due to the selective loss of DA neurons in the ventral substantia nigra of the midbrain, cell replacement therapy strategies are considered suitable for PD.

[0058] In some embodiments, the provided method for differentiating PSCs into dopaminergic neuronal progenitors and related cell types is based on the finding that: culture of PSCs as non-adherent cells is initiated on the day of inoculation (Day 0) in the presence of SB and LDN, followed by further incubation with SHH, PUR, and CHIR to generate spheroids on the second day after the first incubation (Day 1), followed by further incubation of the spheroidized cells on a substrate-coated plate, resulting in differentiated cells with superior properties and a faster timeline than other methods. Specifically, it has been found that culturing PSCs according to the method described herein can accelerate the differentiation of PSCs into dopaminergic neuronal progenitors several days faster than other methods. This is beneficial in several ways, including reducing the amount of time and resources (including cost) required to generate iPSC-derived dopaminergic neuronal progenitors with characteristics suitable for therapeutic purposes. Other aspects of the provided method include harvesting cells when they become definitive or definitive dopaminergic neuronal progenitors, said progenitors being cells capable of differentiating into dopaminergic neurons but not into non-dopaminergic neurons. In some embodiments of the provided method, such cells are differentiated according to the provided method and harvested between approximately day 14 and approximately day 17. This is earlier than other methods that harvest definitive dopaminergic neuronal precursor cells or definitive dopaminergic progenitor cells on day 20 or later (including day 25).

[0059] In some cases, the methods described herein involve culturing PSCs into spheroids for approximately 7 days in a non-adherent culture (i.e., suspension culture), then dissociating the spheroids and, if the dissociated cells are on a plate, such as a substrate-coated plate, continuing culture (i.e., as an adherent culture) until harvest. The provided method for cell differentiation using the non-adherent culture method described herein offers advantages over alternative methods, including those described above that involve superior manufacturability, such as methods where the entire differentiation process is carried out via adherent cell culture.

[0060] In some cases, the non-adherent culture of the provided method is advantageous compared to differentiation methods that do not involve non-adherent cell culture because the effect of the variability of one or more substrates and / or reagents on cell differentiation is reduced or eliminated during non-adherent culture. Specifically, adherent culture may involve coating a plate with one or more substrates or reagents, and such coating may be non-uniform, resulting in one or more substrates and / or reagents contributing to the variability of adherent cells, especially in methods where the entire differentiation process is carried out through adherent cell culture. This contribution of substrates and / or reagents to the variability of adherent cells can increase the variability of the harvested cells produced by this differentiation method. In contrast, substrates and / or reagents may be unnecessary in non-adherent culture, or may be uniformly dispersed throughout the non-adherent culture. In the latter case, the cultured cells (e.g., spheroids) are surrounded by culture medium on all sides, such that the cells are equally exposed to any substrates and / or reagents in the culture medium. Therefore, in aspects of the provided embodiments, cells produced by differentiation methods that include non-adherent culture may exhibit reduced variability.

[0061] The non-adherent suspension culture method described herein is also advantageous compared to methods that do not include non-adherent culture because it allows for increased cell-cell interactions. Specifically, in adherent (e.g., monolayer) culture, cells only contact other cells on their lateral surfaces. In contrast, cells in non-adherent (e.g., suspension) culture are able to contact other cells across the entire surface. This increased cell-cell contact in non-adherent culture can be advantageous by more faithfully reproducing the physiological environment. For example, the increased cell-cell contact allowed by the non-adherent culture method described herein can upregulate intercellular protein networks, as observed in vivo.

[0062] In some respects, the non-adherent culture method described herein offers the simplicity and efficiency of culturing large numbers of cells compared to alternative methods, such as those that involve the entire differentiation process through adherent cell culture. For example, in adherent (e.g., monolayer) culture, the number of cells cultured increases on a two-dimensional scale (i.e., the length and width of the culture surface) with the surface area of ​​the culture surface. In contrast, in non-adherent (e.g., suspension) culture, the number of cells cultured increases on a three-dimensional scale with the volume of the culture vessel or the volume of the culture medium contained therein. Therefore, in some cases, non-adherent culture is more economical and efficient because more cells, or both, can be generated using fewer resources (e.g., culture vessels and reagents) compared to adherent culture. Thus, the non-adherent culture method provided enables scalability in manufacturing and production processes.

[0063] In some respects, cells produced using the non-adherent culture methods described herein offer advantages over those produced using adherent and other non-adherent methods. For example, cells produced by exposing pluripotent stem cells to a TGF-β / activin-Nodal signaling inhibitor (e.g., SB431542) and a bone morphogenetic protein (BMP) signaling inhibitor (e.g., LDN193189) for one day (day 0) from the time of cell seeding, and then waiting until the next day (day 1) to expose the cells to at least one sound hedgehog factor (SHH) signaling activator (e.g., SHH protein and / or purinermamine) and a glycogen synthase kinase 3β (GSK3β) signaling inhibitor (e.g., CHIR99021), can have… Advantageously, it exhibited one or more of the following: (i) increased expression of the dopaminergic spectrum marker EN1 on day 7 or about day 7 compared to each of these reagents if cells were exposed to them starting on day 0; (ii) decreased expression of the off-target non-dopaminergic spectrum marker GBX2 on day 7 or about day 7 compared to each of these reagents if cells were exposed to them starting on day 0; and (iii) increased expression of the dopaminergic spectrum marker LMX1A on day 10 or about day 10 compared to each of these reagents if cells were exposed to them starting on day 0. Therefore, in some cases, exposing cells to a TGF-β / activin-Nodal signaling inhibitor (e.g., SB431542) and a BMP signaling inhibitor (e.g., LDN193189) at day 0 seeding, and then waiting until day 1 to expose cells to at least one SHH signaling activator (e.g., SHH protein and / or pomegranate) and a GSK3β signaling inhibitor (e.g., CHIR99021), advantageously results in an increased specificity for the dopaminergic spectrum compared to exposing cells to each of these agents at day 0.

[0064] In some respects, the non-adherent culture method described herein offers advantages in manufacturability over adherent and other non-adherent culture methods, including reduced costs and time involved by accelerating the differentiation timeline compared to adherent culture methods. For example, differentiated dopaminergic neurons derived from PSCs cultured according to the non-adherent culture method described herein can exhibit gene expression profiles, for example, on day 16, comparable to those of differentiated dopaminergic neurons derived from PSCs cultured using adherent culture methods, for example, on day 20 or 21. This can advantageously reduce the amount of resources required, including culture medium, inhibitors, and other supplements, and can also advantageously reduce the amount of time required to produce differentiated dopaminergic neurons derived from PSCs, for example, by allowing harvesting or collection between approximately day 14 and 17, for example, on days 14, 15, 16, or 17, or approximately day 14, 15, 16, or 17.

[0065] Furthermore, specific benefits relate to cell differentiation methods that include only about 7 days of non-adherent culture. The approximately 7-day non-adherent culture described herein produces spheroids. If non-adherent culture is allowed to exceed 7 days, the increasing size of the spheroids may limit mass transfer and create reagent (e.g., morphogenetic) gradients across the spheroid diameter. Large reagent (e.g., morphogenetic) gradients across the spheroid diameter are undesirable because different cells within the spheroid will be exposed to different concentrations of reagents (e.g., morphogenetics), resulting in variable cell responses and differentiation. Therefore, the method described herein, which includes only about 7 days or at most 7 days of non-adherent culture, is advantageous because the concentration variations of reagents (such as morphogenetics) exposed to the spheroid cells are minimized.

[0066] In some respects, limiting the non-adherent culture component of the methods described herein to approximately 7 days also helps ensure uniform differentiation of cultured cells. This is because prolonged differentiation of PSCs in non-adherent (e.g., suspension) culture (e.g., greater than 7 days) allows PSCs to establish a microenvironment that enables self-renewal in a pluripotent state. Therefore, the methods described herein ensure uniform and efficient differentiation of cultured cells by reducing or eliminating the opportunity for PSCs to persist in the culture.

[0067] In some embodiments, cells produced using the methods described herein, or therapeutic compositions containing such cells, may exhibit an enhanced ability to implant and / or innervate other cells compared to cells harvested at a later time in the differentiation process (e.g., day 25). In some embodiments, cells harvested between days 14 and 17 may also exhibit improved efficacy in vivo due to their differentiation state and neuronal settling. For example, cells harvested on day 16 may exhibit improved implantation and / or innervation, improved efficacy, or both, compared to cells harvested on day 25. Therefore, cells differentiated using the methods described herein may exhibit improved implantation and / or innervation, improved efficacy, or both, in addition to improving manufacturability by reducing the time and resources (including costs) involved.

[0068] In some embodiments, cells harvested using the provided differentiation method exhibit therapeutic efficacy for neurodegenerative diseases. In some embodiments, the ability of differentiated cells to treat neurodegenerative diseases can be determined in animal models of neurodegenerative diseases. In some embodiments, the neurodegenerative disease is Parkinson's disease. In some embodiments, an animal model of Parkinson's disease is used to screen differentiated cells harvested using the provided method. Any known and available animal model of Parkinson's disease can be used for screening. In some embodiments, the animal model is an injury model in which the animal receives a unilateral stereotactic injection of 6-hydroxydopamine (6-OHDA) into the substantia nigra. In some embodiments, the animal model is an injury model in which the animal receives a unilateral stereotactic injection of 6-OHDA into the medial forebrain tract. In some embodiments, a therapeutic composition containing differentiated cells produced by the provided method (e.g., harvested cells, such as cells from day 14 to day 17, e.g., day 16 cells) is implanted into the substantia nigra of the animal model. In some embodiments, behavioral assays are performed to screen the therapeutic efficacy of the implantation in the animal model. In some embodiments, the behavioral assays include monitoring amphetamine-induced circling behavior. In some implementations, if differentiated cells are found to reduce, decrease, or reverse brain damage in a Parkinson's syndrome model, such cells exhibit therapeutic effects for neurodegenerative diseases.

[0069] Furthermore, unlike some previously reported methods, the differentiated cells generated by the method described herein exhibit physiological consistency. Importantly, this physiological consistency is maintained in cells differentiated from different subjects. Therefore, this method reduces intra- and inter-subject variability and allows for better predictability of cellular behavior in vivo. These benefits are associated with successful therapeutic strategies, particularly in the case of autologous stem cell transplantation, where cells are generated individually for each patient. Such reproducibility benefits across different subjects also enable scalability in manufacturing and production processes.

[0070] All publications mentioned in this application, including patent documents, scientific papers, and databases, are incorporated herein by reference in their entirety for all purposes, as if each individual publication were incorporated individually by reference. If any definition presented herein contradicts or otherwise is inconsistent with a definition presented in a patent, application, published application, or other publication incorporated herein by reference, the definition presented herein shall prevail over the definition incorporated herein by reference.

[0071] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the topics described.

[0072] A. Definition Unless otherwise defined, all specialized terms, symbols, and other technical and scientific terms or terminology used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some instances, for clarity and / or convenience of reference, terms with commonly understood meanings are defined herein, and the inclusion of these definitions herein should not be construed as indicating a material difference from those commonly understood in the art.

[0073] As used herein, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include plural indicators. For example, “a” or “an” means “at least one” or “one or more.” It should be understood that aspects and variations described herein include “consisting of aspects and variations” and / or “substantially consisting of aspects and variations.”

[0074] Throughout this disclosure, all aspects of the claimed subject matter are presented in a scope format. It should be understood that this scope format is for convenience and brevity only and should not be construed as a rigid limitation on the scope of the claimed subject matter. Therefore, the scope description should be considered as having specifically disclosed all possible sub-scopes and individual values ​​within that scope. For example, in the case of providing a range of values, it should be understood that every intermediate value between the upper and lower limits of the range, as well as any other specified value or intermediate value within that specified range, is covered within the claimed subject matter. The upper and lower limits of these smaller scopes may be independently included within the smaller scopes and are also covered within the claimed subject matter, conditional on any explicitly excluded limit value within the specified scope. When a specified scope includes one or two limits, the scope excluding any one or both of those included limits is also included in the claimed subject matter. This applies regardless of the width of the scope.

[0075] As used herein, the term "about" refers to the typical range of error for individual values ​​that are readily known. References to "about" values ​​or parameters include (and describe) implementations for that value or parameter itself. For example, a description of "about X" includes a description of "X".

[0076] As used herein, the statement that a cell or cell population is “positive” for a particular marker means that the particular marker (typically a surface marker) is present on or detectable within the cell. When referring to a surface marker, the term means the presence of surface expression as detected by flow cytometry, for example by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is detectable by flow cytometry and is substantially higher than that detected under otherwise identical conditions with a type-matched control using the same procedure, and / or its level is substantially similar to that of cells known to be positive for the marker, and / or its level is substantially higher than that of cells known to be negative for the marker. When referring to a marker within the cell, such as a transcription product or translation product, the term means, for example, the presence of a detectable transcription product or translation product, wherein the level of the detected product is substantially higher than that detected under otherwise identical conditions with a control using the same procedure, and / or its level is substantially similar to that of cells known to be positive for the marker, and / or its level is substantially higher than that of cells known to be negative for the marker.

[0077] As used herein, the statement that a cell or cell population is “negative” for a particular marker means that the particular marker (typically a surface marker) is not present on the cell or substantially detectable within the cell. When referring to a surface marker, the term means the absence of surface expression as detected by flow cytometry, for example by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is not detected by flow cytometry at a level substantially higher than that detected by the same procedure with a type-matched control under otherwise identical conditions, and / or at a level substantially lower than that detected by cells known to be positive for the marker, and / or at a level substantially similar to that detected by cells known to be negative for the marker. When referring to markers within the cell, such as transcripts or translation products, the term means, for example, the absence of detectable transcripts or translation products, wherein the product is not detected at a level substantially higher than that detected by the same procedure with a control under otherwise identical conditions, and / or at a level substantially lower than that detected by cells known to be positive for the marker, and / or at a level substantially similar to that detected by cells known to be negative for the marker.

[0078] As used in this article when referring to genes, the terms "expressed" or "expressed" refer to the transcriptional and / or translational products 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 that DNA produced by the cell. RNA sequencing (RNA-seq) is commonly used to determine gene expression levels. See, for example, Conesa et al. (2016). Genome Biology A review of RNAseq methods in 17:13 (https: / / doi.org / 10.1186 / s13059-016-0881-8).

[0079] As used herein, the term "stem cell" refers to a cell characterized by its ability to self-renew through mitosis and its potential to differentiate into tissues or organs. In mammalian stem cells, embryonic stem cells and somatic stem cells can be distinguished. Embryonic stem cells reside in the blastocyst and produce embryonic tissues, while somatic stem cells reside in adult tissues and are used for tissue regeneration and repair.

[0080] As used herein, the term "adult stem cell" refers to undifferentiated cells found in an individual after embryonic development. Adult stem cells multiply through cell division to replenish dying cells and regenerate damaged tissue. Adult stem cells are capable of dividing and producing another cell identical to themselves or producing 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 possess the ability of pluripotent stem cells to differentiate into cell types from all three germ layers.

[0081] As used herein, the terms “induced pluripotent stem cells,” “iPS,” and “iPSC” refer to pluripotent stem cells artificially obtained (e.g., through artificial manipulation) from non-pluripotent cells. “Non-pluripotent cells” can be cells with a lower self-renewal and differentiation potential than pluripotent stem cells. Cells with lower potential can be, but are not limited to, adult stem cells, tissue-specific progenitor cells, primary cells, or secondary cells.

[0082] As used herein, the term "pluripotency" or "pluripotency" refers to cells capable of producing progeny that, under appropriate conditions, differentiate into cell types that collectively exhibit characteristics associated with cell lineages from the three germ layers (endoderm, mesoderm, and ectoderm). Pluripotent stem cells can contribute to the tissue organization of prenatal, postnatal, or adult organisms.

[0083] As used herein, the term "pluripotent stem cell characteristic" refers to the cellular characteristics that distinguish pluripotent stem cells from other cells. The expression or non-expression of certain combinations of molecular markers are examples of pluripotent stem cell characteristics. More specifically, human pluripotent stem cells may express at least some, optionally all, of the following non-limiting list of markers: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49 / 6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Lin28, Rex1, and Nanog. Cell morphology associated with pluripotent stem cells is also a characteristic of pluripotent stem cells.

[0084] As used in this article, the term "reprogramming" refers to the process of dedifferentiating non-pluripotent cells into cells that exhibit characteristics of pluripotent stem cells.

[0085] As used in this article, the terms “differentiated” or “classified” refer to one or more cells that have acquired cell type-specific functions.

[0086] "Neuron precursor cells" are cells that have the tendency to differentiate into neurons or glial cells but do not possess the pluripotent potential of stem cells. Neuron precursors are cells that are characterized as neurons or glial lineages, characterized by expressing one or more marker genes specific to the neuron or glial lineage. The terms "neural" and "neuronal" are used according to their common meaning in the art and are used interchangeably throughout this document.

[0087] As used herein, "dopaminergic cells" or "differentiated dopaminergic cells" refer to cells capable of synthesizing the neurotransmitter dopamine. In some embodiments, dopaminergic cells are A9 dopaminergic cells. The term "A9 dopaminergic cells" refers to the most densely packed group of dopaminergic cells in the human brain, located in the substantia nigra pars compacta of the midbrain of healthy adults.

[0088] As used herein, the terms "dopaminergic neuronal progenitor" and "deterministic dopaminergic progenitor" refer to cells that will differentiate into dopaminergic neurons but cannot differentiate into non-dopaminergic cells. A "deterministic dopaminergic progenitor" is a cell capable of differentiating into dopaminergic neurons independently of its environment. Deterministic dopaminergic progenitors may express Foxa2 or Nurl1. In some embodiments, decisive dopaminergic progenitors do not express significant levels of serotonin.

[0089] As used in this article, “defined dopaminergic progenitor cells” are dopaminergic neuron progenitor cells in a differentiated state after the differentiation stage of decisive dopaminergic neuron progenitor cells.

[0090] As used herein, the term "adherent culture dish" refers to a culture dish in which cells can attach to the dish via extracellular matrix molecules, and where separating the cells from the dish requires the use of enzymes (e.g., trypsin, dispersant, etc.). "Adherent culture dishes" are quite different from such dishes; cell attachment is reduced, and removing the cells from the dish does not require the use of enzymes.

[0091] As used herein, the term "non-adherent culture vessel" refers to a culture vessel in which cell adhesion is reduced or restricted (e.g., for a period of time). Non-adherent culture vessels may contain low- or ultra-low-adherence surfaces, such as those achieved by treating the surfaces with a substance that prevents cell adhesion, such as a hydrogel (e.g., a neutrally charged hydrogel and / or a hydrophilic hydrogel) and / or a surfactant (e.g., prawnic acid). Non-adherent culture vessels may contain round or concave pores, and / or micropores (e.g., Aggrewells). ™ In some implementations, non-adherent culture dishes are Aggrewell. ™ For non-adherent culture dishes, it may not be necessary to use enzymes to remove cells from the culture dish.

[0092] As used herein, the term "cell culture" can refer to an in vitro population of cells existing outside an organism. Cell cultures can be established from primary cells isolated from a cell bank or animal, or from secondary cells derived from one of these sources and immortalized to obtain long-term in vitro cultures.

[0093] As used herein, the terms “culture,” “culturing,” “grow,” “growing,” “maintain,” “maintaining,” “expand,” and “expanding” are used interchangeably when referring to cell cultures themselves or the culturing process, meaning cells maintained in vitro (e.g., in vitro) under conditions suitable for survival. This means that the cultured cells are allowed to survive, and that the culture can induce cell growth, differentiation, or division.

[0094] As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It can be a solution, suspension, liquid, powder, paste, aqueous solution, non-aqueous solution, or any combination thereof.

[0095] The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use, such as in mammalian subjects (e.g., humans). Pharmaceutical compositions typically contain an effective amount of an active agent (e.g., cells) and a carrier, excipient, or diluent. This carrier, excipient, or diluent is typically pharmaceutically acceptable, respectively.

[0096] "Pharmaceutically acceptable carriers" refer to components in a drug formulation that are non-toxic to the subjects, excluding the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0097] The term "instructions for use" is used to refer to instructions that are typically included in the commercial packaging of a therapeutic product, which contain information about the indications, usage, dosage, administration, combination therapy, contraindications, and / or warnings for the use of such therapeutic products.

[0098] As used in this article, “subject” is a mammal, such as a human or other animal, and is usually a human.

[0099] As used in this article, "Day 0" refers to a 24-hour period in which plate seeding / cell inoculation begins at the start of the 24-hour period of Day 0, and "Day 1" refers to the day following Day 0 (also a 24-hour period) starting 24 hours after Day 0. Each subsequent day also refers to a consecutive 24-hour period.

[0100] B. Methods to induce cell differentiation This article provides methods for differentiating stem cells, including induced pluripotent stem cells (iPSCs) and embryonic stem cells, into neural cells. In some embodiments, these methods include performing a first incubation, which involves culturing pluripotent stem cells in a non-adherent culture dish under conditions that produce cell spheroids. The pluripotent stem cells are exposed to at least one TGF-β / activin-Nodal signaling inhibitor and at least one bone morphogenetic protein (BMP) signaling inhibitor for at least one day (day 0) in the absence of: x) a sound hedgehog factor (SHH) signaling activator and y) a glycogen synthase kinase 3β (GSK3β) signaling inhibitor. Starting from the second day after the first incubation (day 1), the pluripotent stem cells are exposed to at least one sound hedgehog factor (SHH) signaling activator and at least one glycogen synthase kinase 3β (GSK3β) signaling inhibitor. The spheroid cell solution is then subjected to a second incubation, which involves culturing the spheroid cells in a substrate-coated culture dish under conditions that induce neural differentiation. In some embodiments, the first incubation includes, for example, non-adherent culture conditions as described in section B.1, and the second incubation includes, for example, adherent culture conditions as described in section B.4. Figure 1 The diagram shows some implementation schemes of the provided method.

[0101] The provided protocols include subjecting iPSCs to cell culture methods that induce their differentiation into midbrain basal plate progenitor cells, dopaminergic neuron progenitor cells, including definitive dopaminergic neuron progenitor cells, definitive dopaminergic neuron progenitor cells, and / or dopaminergic neurons. The provided suspension culture method for neural cell differentiation, referring to the first exposure of cells to the SHH activator and glycogen synthase kinase 3β (GSK3β) signaling inhibitor on day 1 (day 2) of the protocol, is sometimes referred to herein as the "day 1" suspension culture differentiation protocol.

[0102] In some implementations, iPSCs are generated from fibroblasts from human patients with Parkinson's disease. During the first incubation, the iPSCs then differentiate into midbrain lamina precursors and grow as spheroids in non-adherent culture, starting from day 0 (SB, LDN) or day 1 (SHH / PUR, CHIR), by exposure to small molecules such as LDN, SB, PUR, SHH, CHIR, and combinations thereof. The resulting spheroids are then transferred to adherent cultures as part of a second incubation, optionally after spheroid dissociation, and then exposed to additional small molecules (e.g., LDN, CHIR, BDNF, GDNF, ascorbic acid, dbcAMP, TGFβ3, DAPT, and combinations thereof) to induce further differentiation into implantable definitive dopaminergic neuronal progenitors, definitive dopaminergic neuronal progenitors, or dopaminergic neurons.

[0103] 1. Sample and cell preparation In embodiments of the provided method, pluripotent stem cells differentiate into midbrain basal plate progenitor cells, definitive dopaminergic neuron progenitor cells, definitive dopaminergic neuron progenitor cells, and / or dopaminergic neurons. Various sources of pluripotent stem cells, including embryonic stem (ES) cells and induced pluripotent stem cells (iPSCs), can be used in this method.

[0104] In some respects, pluripotency refers to the ability of cells to produce progeny that, under appropriate conditions, differentiate into cell types that collectively exhibit characteristics associated with cell lineages from the three germ layers (endoderm, mesoderm, and ectoderm). Pluripotent stem cells can contribute to the tissue organization 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, the identification of various pluripotent stem cell characteristics can also be used to identify pluripotent cells. In some respects, pluripotent stem cells can be distinguished from other cells by specific characteristics, including by the expression or non-expression of a combination of certain molecular markers. More specifically, human pluripotent stem cells may express at least some, optionally all, of the following non-limiting list of markers: 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 respects, pluripotent stem cell characteristics are associated with cell morphology.

[0105] In some implementations, pluripotent stem cells are induced pluripotent stem cells (iPSCs) artificially obtained from non-pluripotent cells. In some respects, non-pluripotent cells are cells with a lower self-renewal and differentiation potential than pluripotent stem cells. iPSCs can be generated through a process called reprogramming, in which non-pluripotent cells are effectively “dedifferentiated” into an embryonic stem cell-like state by engineering them to express genes such as OCT4, SOX2, and KLF4. (Takahashi and Yamanaka (2006)) Cell 126: 663-76.

[0106] 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. (2007)). Cell 131: 861-872 and Yu et al. (2007) Science318: 1917-1920). Mouse iPSCs exhibit important characteristics of pluripotent stem cells, including expression of stem cell markers, tumor formation containing cells from all three germ layers, and the ability to promote many different tissues when injected into mouse embryos at a very early stage of development. Human iPSCs also express stem cell markers and are able to produce cellular characteristics from all three germ layers.

[0107] In some embodiments, the PSCs (e.g., iPSCs) are autologous to the subject to be treated, meaning the PSCs originate from the same subject to whom the differentiated cells were administered. In some embodiments, non-pluripotent cells (e.g., fibroblasts) derived from patients with Parkinson's disease (PD) are reprogrammed into iPSCs before differentiating into nerve cells and / or neurons. In some embodiments, fibroblasts can be reprogrammed into iPSCs by converting them into fibroblasts with genes cloned into plasmids (OCT4, SOX2, NANOG, LIN28, and KLF4) (e.g., see, Yu et al., Science DOI: 10.1126 / science.1172482). In some embodiments, non-pluripotent fibroblasts derived from patients with PD are reprogrammed into iPSCs before differentiating into definitive dopaminergic neuronal progenitors, definitive dopaminergic neuronal progenitors, and / or dopaminergic neurons, such as by reprogramming cells using non-integrating Sendai virus (e.g., using 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 obtained in an autologous stem cell transplantation. In some embodiments, the PSCs (e.g., iPSCs) are allogeneic to the subject to be treated, i.e., the PSCs originate from an individual different from the subject from whom the differentiated cells will be administered. In some embodiments, non-pluripotent cells (e.g., fibroblasts) derived from another individual (e.g., an individual without a neurodegenerative disease such as Parkinson's disease) are reprogrammed into iPSCs before differentiating into definitive dopaminergic neuronal progenitors, definitive dopaminergic neuronal progenitors, and / or dopaminergic neurons. In some embodiments, cells are reprogrammed using a non-integrating Sendai virus (e.g., using CTS). ™ CytoTune ™ The iPS 2.1 Sendai Reprogramming Kit at least partially enables reprogramming. In some embodiments, the resulting differentiated cells are then administered to an individual from whom the differentiated cells were not derived (e.g., allogeneic cell therapy or allogeneic cell transplantation).

[0108] In any of the embodiments provided, the PSCs described herein (e.g., allogeneic cells) can be genetically engineered to be low in immunogenicity. Methods for reducing immunogenicity are known and include eliminating the expression of polymorphic HLA-A / -B / -C and HLA class II molecules and introducing immunomodulatory factors such as PD-L1, HLA-G, and CD47 into the AAVS1 safe harbor locus in differentiated cells. Han et al., PNAS (2019) 116(21):10441-46. Therefore, in some embodiments, the PSCs described herein are engineered to have a highly polymorphic HLA-A / -B / -C gene deleted and immunomodulatory factors such as PD-L1, HLA-G, and / or CD47 introduced into the AAVS1 safe harbor locus.

[0109] In some implementations, PSCs (e.g., iPSCs) are cultured in the absence of feeder cells until they reach 75%–90% confluence, at which point they are harvested and cultured further for differentiation (day 0). In one aspect of the methods described herein, once the iPSCs reach 75%–90% confluence, they are washed in phosphate-buffered saline (PBS) and enzymatically dissociated, such as with Accutase. ™ Enzymatic dissociation was performed until cells could be easily removed from the surface of the culture dish. The dissociated iPSCs were then resuspended in culture medium for downstream differentiation into definitive dopaminergic neuronal progenitor cells, type-specific dopaminergic neuronal progenitor cells, and / or dopaminergic neurons.

[0110] In some embodiments, PSCs are resuspended in basal induction medium. In some embodiments, the basal induction medium is formulated to contain Neurobasal... ™ The culture medium and DMEM / F12 medium were supplemented with N-2 and B27, non-essential amino acids (NEAA), and GlutaMAX. ™ L-glutamine, β-mercaptoethanol, and insulin. In some embodiments, at inoculation (day 0), the basal induction medium is further supplemented for differentiation with serum substitutes, Rho-associated protein kinase (ROCK) inhibitors, and certain small molecules (e.g., TGF-β / activin-Nodal signaling inhibitors and BMP signaling inhibitors). In some embodiments, PSCs are resuspended in the same medium in which they will be cultured for at least a portion of the initial incubation.

[0111] 2. Non-adherent culture The provided methods include culturing PSCs (e.g., iPSCs) by co-incubating with certain molecules (e.g., small molecules) to induce their differentiation into midbrain basal plate progenitor cells, dopaminergic neuronal progenitor cells, including deterministic dopaminergic neuronal progenitor cells, morphological dopaminergic neuronal progenitor cells, and / or dopaminergic neurons. Specifically, the provided embodiments include performing a first incubation of PSCs under non-adherent conditions at a certain time in the presence of certain molecules (e.g., small molecules) to generate spheroids. This can improve the consistency of the generation of physiologically relevant cells for implantation in some respects and also improves manufacturing by accelerating the timeline for iPSC differentiation into, for example, deterministic dopaminergic neuronal progenitor cells, morphological dopaminergic neuronal progenitor cells, and / or dopaminergic neurons, thereby advantageously reducing the time and amount of resources required for the differentiation process. In some embodiments, these methods include performing a first incubation, which comprises culturing pluripotent stem cells in a non-adherent culture dish under conditions that generate cell spheroids. The first incubation includes (i) exposing pluripotent stem cells to at least one TGF-β / activin-Nodal signaling inhibitor and at least one bone morphogenetic protein (BMP) signaling inhibitor for at least one day (day 0); and (ii) starting from the second day of the first incubation (day 1), exposing pluripotent stem cells to at least one sound hedgehog factor (SHH) signaling activator and at least one glycogen synthase kinase 3β (GSK3β) signaling inhibitor. In some embodiments, day 0 incubation is performed in the absence of: x) a sound hedgehog factor (SHH) signaling activator and y) a glycogen synthase kinase 3β (GSK3β) signaling inhibitor.

[0112] In some embodiments, non-adherent culture dishes are culture dishes with low or ultra-low adhesion surfaces, such as those designed to inhibit or reduce cell adhesion. In some embodiments, culturing cells in non-adherent culture dishes does not prevent all cells in the culture from adhering to the surface of the culture dish.

[0113] In some embodiments, the non-adherent culture vessel is a culture vessel with an ultra-low adhesion surface. In some aspects, the ultra-low adhesion surface can inhibit cell adhesion for a period of time. In some embodiments, the ultra-low adhesion surface can inhibit cell adhesion for the period of time necessary to achieve fusion growth of the same cell type as on an adherent surface. In some embodiments, the ultra-low adhesion surface is coated or treated with a substance that prevents cell adhesion, such as a hydrogel layer (e.g., a neutrally charged hydrogel layer and / or a 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 pranonic acid.

[0114] In some embodiments, non-adherent culture dishes allow for the three-dimensional formation of cell aggregates. In some embodiments, iPSCs are cultured in non-adherent culture dishes, such as multi-well plates, to generate cell aggregates (e.g., spheroids). In some embodiments, iPSCs are cultured in non-adherent culture dishes, such as multi-well plates, at approximately day 7 of the method to generate cell aggregates (e.g., spheroids). In some embodiments, the cell aggregates (e.g., spheroids) express at least one of PAX6 and OTX2 at or before approximately day 7 of the method.

[0115] In some implementations, the first incubation includes culturing pluripotent stem cells in a non-adherent culture dish under conditions that produce cell spheroids.

[0116] In some embodiments, the non-adherent culture vessel is a plate, petri 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 plate or a 24-well plate. In some embodiments, the pores of the multi-well plate also include micropores. In some of the provided embodiments, the non-adherent culture vessel, such as a multi-well plate, has circular or concave holes and / or micropores. In some of the provided embodiments, the non-adherent culture vessel, such as a multi-well plate, has no corners or seams.

[0117] In some implementations, the non-adherent culture vessel is a multi-well plate, where each well contains microwells, and the number of PSCs seeded on day 0 of this method is approximately 3,000 cells per microwell. For example, in an AggreWell 400 24-well plate, each well contains approximately 1,200 microwells, each microwell having a diameter of 400 μM, and approximately 3.6 × 10⁶ cells are seeded per well. 6 The AggreWell 400 6-well plate contains approximately 5900 microwells per well, with each well having a diameter of 400 μM. Therefore, in some embodiments, when using such a 6-well plate, approximately 17.7 × 10⁶ cells are seeded per well. 6 Each micropore contains approximately 3,000 cells, resulting in an average of about 3,000 cells per micropore.

[0118] In some implementations, the number of PSCs inoculated on day 0 of this method is approximately 0.1 × 10⁻⁶. 6 cells / cm 2 Approximately 2×10 6 cells / cm 2 Approximately 0.1 × 10 6 cells / cm 2 To approximately 1×10 6 cells / cm 2 Approximately 0.1 × 10 6cells / cm 2 To approximately 0.8 × 10 6 cells / cm 2 Approximately 0.1 × 10 6 cells / cm 2 To approximately 0.6 × 10 6 cells / cm 2 Approximately 0.1 × 10 6 cells / cm 2 To approximately 0.4 × 10 6 cells / cm 2 Approximately 0.1 × 10 6 cells / cm 2 To approximately 0.2 × 10 6 cells / cm 2 Approximately 0.2 × 10 6 cells / cm 2 Approximately 2×10 6 cells / cm 2 Approximately 0.2 × 10 6 cells / cm 2 To approximately 1×10 6 cells / cm 2 Approximately 0.2 × 10 6 cells / cm 2 To approximately 0.8 × 10 6 cells / cm 2 Approximately 0.2 × 10 6 cells / cm 2 To approximately 0.6 × 10 6 cells / cm 2 Approximately 0.2 × 10 6 cells / cm 2 To approximately 0.4 × 10 6 cells / cm 2 Approximately 0.4 × 10 6 cells / cm 2 Approximately 2×10 6 cells / cm 2 Approximately 0.4 × 10 6 cells / cm 2 To approximately 1×10 6 cells / cm 2 Approximately 0.4 × 10 6 cells / cm 2 To approximately 0.8 × 10 6 cells / cm 2 Approximately 0.4 × 10 6 cells / cm 2 To approximately 0.6 × 10 6 cells / cm2 Approximately 0.6 × 10 6 cells / cm 2 Approximately 2×10 6 cells / cm 2 Approximately 0.6 × 10 6 cells / cm 2 To approximately 1×10 6 cells / cm 2 Approximately 0.6 × 10 6 cells / cm 2 To approximately 0.8 × 10 6 cells / cm 2 Approximately 0.8 × 10 6 cells / cm 2 Approximately 2×10 6 cells / cm 2 Approximately 0.8 × 10 6 cells / cm 2 To approximately 1×10 6 cells / cm 2 or approximately 1.0 × 10 6 cells / cm 2 Approximately 2×10 6 cells / cm 2 In some implementations, the number of cells seeded on substrate-coated culture plates is approximately 0.4 × 10⁻⁶. 6 cells / cm 2 To approximately 0.8 × 10 6 cells / cm 2 .

[0119] In some implementations, the number of PSCs inoculated on platelets on day 0 of this method is approximately 1 × 10⁻⁶. 5 10 pluripotent stem cells / well to approximately 20 × 10⁻⁶ 6 1 multipotent stem cell / well, approximately 1×10 5 15 × 10⁶ pluripotent stem cells / well to approximately 15 × 10⁶ 6 1 multipotent stem cell / well, approximately 1×10 5 10 × 10⁶ pluripotent stem cells / well to approximately 10 × 10⁶ 6 1 multipotent stem cell / well, approximately 1×10 5 pluripotent stem cells / well approximately 5 × 10⁶ 6 1 multipotent stem cell / well, approximately 1×10 5 1 × 10⁶ pluripotent stem cells / well to approximately 1 × 10⁶ 6 1 multipotent stem cell / well, approximately 1×10 5 pluripotent stem cells / well approximately 5 × 10⁶ 5 1 multipotent stem cell / well, approximately 5 × 10 510 pluripotent stem cells / well to approximately 20 × 10⁻⁶ 6 1 multipotent stem cell / well, approximately 5 × 10 5 15 × 10⁶ pluripotent stem cells / well to approximately 15 × 10⁶ 6 1 multipotent stem cell / well, approximately 5 × 10 5 10 × 10⁶ pluripotent stem cells / well to approximately 10 × 10⁶ 6 1 multipotent stem cell / well, approximately 5 × 10 5 pluripotent stem cells / well approximately 5 × 10⁶ 6 1 multipotent stem cell / well, approximately 5 × 10 5 1 × 10⁶ pluripotent stem cells / well to approximately 1 × 10⁶ 6 1 multipotent stem cell / well, approximately 1×10 6 10 pluripotent stem cells / well to approximately 20 × 10⁻⁶ 6 1 multipotent stem cell / well, approximately 1×10 6 15 × 10⁶ pluripotent stem cells / well to approximately 15 × 10⁶ 6 1 multipotent stem cell / well, approximately 1×10 6 10 × 10⁶ pluripotent stem cells / well to approximately 10 × 10⁶ 6 1 multipotent stem cell / well, approximately 1×10 6 pluripotent stem cells / well approximately 5 × 10⁶ 6 1 multipotent stem cell / well, approximately 5 × 10 6 10 pluripotent stem cells / well to approximately 20 × 10⁻⁶ 6 1 multipotent stem cell / well, approximately 5 × 10 6 15 × 10⁶ pluripotent stem cells / well to approximately 15 × 10⁶ 6 1 multipotent stem cell / well, approximately 5 × 10 6 10 × 10⁶ pluripotent stem cells / well to approximately 10 × 10⁶ 6 10 multipotent stem cells / well, approximately 10 × 10 6 10 pluripotent stem cells / well to approximately 20 × 10⁻⁶ 6 10 multipotent stem cells / well, approximately 10 × 10 6 15 × 10⁶ pluripotent stem cells / well to approximately 15 × 10⁶ 6 One pluripotent stem cell / well or approximately 15 × 10⁶ 6 10 pluripotent stem cells / well to approximately 20 × 10⁻⁶ 6 One pluripotent stem cell / well.

[0120] In some implementations, the number of PSCs inoculated on day 0 of this method is approximately 1 × 10⁻⁶. 6 10 × 10⁶ pluripotent stem cells / well to approximately 10 × 10⁶ 6 1 multipotent stem cell / well, approximately 2 × 10 6 pluripotent stem cells / well approximately 8 × 10⁸ 6 1 multipotent stem cell / well, approximately 2.5 × 10⁶ 6pluripotent stem cells / well approximately 5 × 10⁶ 6 One pluripotent stem cell / well or approximately 3 × 10⁶ 6 pluripotent stem cells / well approximately 4 × 10⁻⁶ 6 100 pluripotent stem cells / well. In some embodiments, the number of PSCs seeded on day 0 of this method is or approximately 3.6 × 10⁻⁶. 6 One pluripotent stem cell / well.

[0121] On some days, the number of PSCs seeded on the plate on day 0 of this method is sufficient to produce cell spheroids containing approximately 1,000 to approximately 5,000 cells, or approximately 2,000 to approximately 3,000 cells. On some days, the number of PSCs seeded on the plate on day 0 of this method is sufficient to produce cell spheroids containing approximately 1,000 to approximately 5,000 cells. On some days, the number of PSCs seeded on the plate on day 0 of this method is sufficient to produce cell spheroids containing approximately 2,000 to approximately 3,000 cells. On some days, the number of PSCs seeded on the plate on day 0 of this method is sufficient to produce cell spheroids containing approximately 2,000 cells. On some days, the number of PSCs seeded on the plate on day 0 of this method is sufficient to produce cell spheroids containing approximately 3,000 cells. In some embodiments, the desired number of spheroids is produced by this method on or before day 7.

[0122] In some embodiments of the methods provided herein, the first incubation includes culturing pluripotent stem cells in a non-adherent culture dish under conditions that generate cell spheroids. In some embodiments, the first incubation is from approximately day 0 to approximately day 6. In some embodiments, the first incubation includes culturing pluripotent stem cells in a culture medium (“medium”). In some embodiments, the culture medium for the first incubation is a basal induction medium for inducing PSC differentiation into midbrain basal plate progenitor cells. In some embodiments, the first incubation includes culturing pluripotent stem cells in a culture medium from approximately day 0 to approximately day 6. In some embodiments, the first incubation includes culturing pluripotent stem cells in a culture medium to induce PSC differentiation into midbrain basal plate progenitor cells.

[0123] In some implementations, components containing minimal non-human origin (e.g., KnockOut) are also used. ™The medium is supplemented with a serum substitute. In some embodiments, 5% (v / v) serum substitute is provided in the medium during at least a portion of the first incubation. In some embodiments, 5% (v / v) serum substitute is provided in the medium on days 0 and 1. In some embodiments, 2% (v / v) serum substitute is provided in the medium during at least a portion of the first incubation. In some embodiments, 2% (v / v) serum substitute is provided in the medium from day 2 through day 6. In some embodiments, 5% (v / v) serum substitute is provided in the medium on days 0 and 1, and 2% (v / v) serum substitute is provided from day 2 through day 6.

[0124] In some embodiments, the culture medium is further supplemented with small molecules such as any of the small molecules described above. In some embodiments, the small molecule is selected from the group consisting of: Rho-associated protein kinase (ROCK) inhibitors, TGF-β / activin-Nodal signaling inhibitors, at least one sound hedgehog factor (SHH) signaling activator, bone morphogenetic protein (BMP) signaling inhibitors, glycogen synthase kinase 3β (GSK3β) signaling inhibitors, and combinations thereof.

[0125] In some embodiments, the culture medium is supplemented with a Rho-associated protein kinase (ROCK) inhibitor on one or more days of cell passage. In some embodiments, the culture medium is supplemented with a ROCK inhibitor every day of cell passage. In some embodiments, the culture medium is supplemented with a ROCK inhibitor on day 0. In some embodiments, during the first incubation, the pluripotent stem cells are not exposed to ROCKi before being exposed to TGF-β / activin-Nodal signaling inhibitors and bone morphogenetic protein (BMP) signaling inhibitors.

[0126] In some embodiments, cells are exposed to a ROCK inhibitor at concentrations between about 1 µM and about 20 µM, between about 5 µM and about 15 µM, or between about 8 µM and about 12 µM. In some embodiments, cells are exposed to a ROCK inhibitor at concentrations between about 1 µM and about 20 µM. In some embodiments, cells are exposed to a ROCK inhibitor at concentrations between about 5 µM and about 15 µM. In some embodiments, cells are exposed to a ROCK inhibitor at concentrations between about 8 µM and about 12 µM. In some embodiments, cells are exposed to a ROCK inhibitor at a concentration of 10 µM or about 10 µM.

[0127] In some embodiments, the ROCK inhibitor is selected from the group consisting of: fasudil, ribasudil, netarsudil, RKI-1447, Y-27632, GSK429286A, Y-30141, and combinations thereof. In some embodiments, the ROCK inhibitor is a small molecule. In some embodiments, the ROCK inhibitor selectively inhibits p160ROCK. In some embodiments, the ROCK inhibitor is Y-27632, having the following formula: In some embodiments, cells are exposed to a concentration of Y-27632 of 10 µM or approximately. In some embodiments, cells are exposed to a concentration of Y-27632 of 10 µM or approximately on day 0.

[0128] In some embodiments, the culture medium is supplemented with a TGF-β / activin-Nodal signaling inhibitor. In some embodiments, the culture medium is supplemented with a TGF-β / activin-Nodal signaling inhibitor up to about day 5 (e.g., day 4 or day 5). In some embodiments, the culture medium is supplemented with a TGF-β / activin-Nodal signaling inhibitor from about day 0 until day 4 (inclusive).

[0129] In some embodiments, cells are exposed to a TGF-β / activin-Nodal signaling inhibitor at concentrations between about 1 µM and about 20 µM, between about 5 µM and about 15 µM, or between about 8 µM and about 12 µM. In some embodiments, cells are exposed to a TGF-β / activin-Nodal signaling inhibitor at concentrations between about 1 µM and about 20 µM. In some embodiments, cells are exposed to a TGF-β / activin-Nodal signaling inhibitor at concentrations between about 5 µM and about 15 µM. In some embodiments, cells are exposed to a TGF-β / activin-Nodal signaling inhibitor at concentrations between about 8 µM and about 12 µM. In some embodiments, cells are exposed to a TGF-β / activin-Nodal signaling inhibitor at a concentration of 10 µM or about 10 µM.

[0130] In some embodiments, the culture medium is partially or completely replaced on one or more days of the first incubation, such as each day from day 1 to day 6. In some embodiments, the culture medium replacement involves replacing 50% or about 50% of the culture medium with fresh culture medium, which may be the same as or different from the previous culture medium. In some embodiments, the culture medium replacement involves replacing about 25% to about 75% of the culture medium. In some embodiments, the culture medium for one or more days of the first incubation, such as each day from day 1 to day 6, is added by a 50% culture medium replacement. For example, the culture medium for one or more days from day 1 to day 6 is added by removing about 50% of the previous culture medium and replacing it with an equal volume of new culture medium prepared for that day. Thus, the concentration of certain small molecules contained in the culture medium to be added by the 50% culture medium replacement is contained in the new culture medium at twice (2x) the concentration at which the cells are intended to be exposed on that day. In some implementations, the concentration of TGF-β / activin-Nodal signaling inhibitor contained in the culture medium added on each of the four days from day 1 is twice the concentration of TGF-β / activin-Nodal signaling inhibitor contained in the culture medium on day 0.

[0131] In some embodiments, the culture medium is replaced every day from day 1 to day 4, for example, with a 50% replacement, and the added culture medium contains a TGF-β / activin-Nodal signaling inhibitor at a concentration of about 2 µM to about 40 µM, about 10 µM to about 30 µM, or about 16 µM to about 24 µM. In some embodiments, the culture medium is replaced every day from day 1 to day 4, for example, with a 50% replacement, and the added culture medium contains a TGF-β / activin-Nodal signaling inhibitor at a concentration of about 2 µM to about 40 µM. In some embodiments, the culture medium is replaced every day from day 1 to day 4, for example, with a 50% replacement, and the added culture medium contains a TGF-β / activin-Nodal signaling inhibitor at a concentration of about 10 µM to about 30 µM. In some embodiments, the culture medium is replaced every day from day 1 to day 4, for example, with a 50% replacement, and the added culture medium contains a TGF-β / activin-Nodal signaling inhibitor at a concentration of about 16 µM to about 24 µM. In some implementations, the culture medium is replaced every day from day 1 to day 4, for example, by replacing the medium with 50% of the medium, and the added medium contains a TGF-β / activin-Nodal signaling inhibitor at a concentration of or about 20 µM.

[0132] In some embodiments, the TGF-β / activin-Nodal signaling inhibitor is a small molecule. In some embodiments, the TGF-β / activin-Nodal signaling inhibitor can reduce or block transforming growth factor β (TGFβ) / activin-Nodal signaling. In some embodiments, the TGF-β / activin-Nodal signaling inhibitor inhibits ALK4, ALK5, ALK7, or combinations thereof. In some embodiments, the TGF-β / activin-Nodal signaling inhibitor inhibits ALK4, ALK5, and ALK7. In some embodiments, the TGF-β / activin-Nodal signaling inhibitor does not inhibit ALK2, ALK3, ALK6, or combinations thereof. In some embodiments, the inhibitor does not inhibit ALK2, ALK3, or ALK6. In some embodiments, the TGF-β / activin-Nodal signaling inhibitor is SB431542 (e.g., CAS 301836-41-9, molecular formula C22H18N4O3 and given name 4-[4-(1,3-benzodioxane-5-yl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide), having the following formula: 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 5. In some embodiments, cells are exposed to SB431542 at a concentration between about 8 µM and about 15 µM from about day 0 to about day 4 (inclusive). In some embodiments, cells are exposed to SB431542 at a concentration of 10 µM or about 10 µM from about day 0 to about day 4 (inclusive).

[0133] In some embodiments, the culture medium is supplemented with a BMP signaling inhibitor. In some embodiments, the culture medium is supplemented with a BMP signaling inhibitor up to about day 7 (e.g., day 6 or day 7). In some embodiments, the culture medium is supplemented with a BMP signaling inhibitor from about day 0 until day 6 (inclusive).

[0134] In some embodiments, cells are exposed to a BMP signaling inhibitor at concentrations between about 0.01 µM and about 5 µM, between about 0.05 µM and about 1 µM, between about 0.05 µM and about 0.2 µM, or between about 0.1 µM and about 0.5 µM (inclusive of each endpoint). In some embodiments, cells are exposed to a BMP signaling inhibitor at concentrations between about 0.01 µM and about 5 µM. In some embodiments, cells are exposed to a BMP signaling inhibitor at concentrations between about 0.05 µM and about 1 µM. In some embodiments, cells are exposed to a BMP signaling inhibitor at concentrations between about 0.1 µM and about 0.5 µM. In some embodiments, cells are exposed to a BMP signaling inhibitor at concentrations between about 0.05 µM and about 0.2 µM. In some embodiments, cells are exposed to a BMP signaling inhibitor at concentrations between about 0.08 µM and about 0.15 µM. In some implementations, cells are exposed to a BMP signaling inhibitor at a concentration of about 0.1 µM.

[0135] In some embodiments, the culture medium is at least partially replaced with fresh culture medium, and the concentration of the BMP signaling inhibitor included in the culture medium added each day from day 1 to day 6 during the culture medium replacement is twice the concentration of the BMP signaling inhibitor included in the culture medium on day 0, for example, due to the addition of culture medium via a 50% culture medium replacement. In some embodiments, the culture medium added each day from day 1 to day 6 via a culture medium replacement, such as a 50% culture medium replacement, contains a concentration of BMP signaling inhibitor between about 0.02 µM and about 10 µM, between about 0.1 µM and about 2 µM, between about 0.1 µM and about 0.4 µM, or between about 0.2 µM and about 1 µM (inclusive of each extreme value). In some embodiments, the culture medium added each day from day 1 to day 6 via a culture medium replacement, such as a 50% culture medium replacement, contains a concentration of BMP signaling inhibitor between 0.02 µM and about 10 µM. In some embodiments, the culture medium added during each of the six days from day 1 to day 6 by replacing the medium with, for example, 50% replacement, contains a BMP signaling inhibitor at a concentration between about 0.1 µM and about 2 µM. In some embodiments, the culture medium added during each of the six days from day 1 to day 6 by replacing the medium with, for example, 50% replacement, contains a BMP signaling inhibitor at a concentration between about 0.2 µM and about 1 µM. In some embodiments, the culture medium added during each of the six days from day 1 to day 6 by replacing the medium with, for example, 50% replacement, contains a BMP signaling inhibitor at a concentration between about 0.1 µM and about 0.4 µM. In some embodiments, the culture medium added during each of the six days from day 1 to day 6 by replacing the medium with, for example, 50% replacement, contains a BMP signaling inhibitor at a concentration of about 0.2 µM.

[0136] In some embodiments, the BMP signaling inhibitor is a small molecule. In some embodiments, the BMP signaling inhibitor is selected from LDN193189 or K02288. In some embodiments, the BMP signaling inhibitor is capable of inhibiting SMAD signaling of "small mother antagonistic Drosophila skin growth factor". In some embodiments, the BMP signaling inhibitor inhibits ALK1, ALK2, ALK3, ALK6, or combinations thereof. In some embodiments, the BMP signaling inhibitor inhibits ALK1, ALK2, ALK3, and ALK6. In some embodiments, the BMP signaling inhibitor inhibits BMP2, BMP4, BMP6, BMP7, and activin cytokine signaling, and subsequently inhibits SMAD phosphorylation of Smad1, Smad5, and Smad8. In some embodiments, the BMP signaling inhibitor is LDN193189. In some embodiments, the BMP signaling inhibitor is LDN193189 (e.g., IUPAC name 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline, chemical formula C25H22N6), having the following formula: In some embodiments, cells are exposed to LDN193189 at a concentration of about 0.1 µM. In some embodiments, cells are exposed to LDN193189 at a concentration of about 0.1 µM for up to about day 7 (e.g., day 6 or day 7). In some embodiments, cells are exposed to LDN193189 at a concentration of about 0.1 µM on day 0, and to LDN193189 at a concentration of about 0.08 µM to about 0.15 µM from about day 0 to about day 6 (inclusive). In some embodiments, cells are exposed to LDN193189 at a concentration of about 0.1 µM from about day 0 to about day 6 (inclusive).

[0137] In some embodiments, the culture medium is supplemented with a GSK3β signaling inhibitor. In some embodiments, the culture medium is supplemented with a GSK3β signaling inhibitor from day 1 and up to about day 7 (e.g., day 6 or day 7). In some embodiments, the culture medium is supplemented with a GSK3β signaling inhibitor from about day 1 until day 6 (inclusive). In some embodiments, the cells are not exposed to the GSK3β signaling inhibitor prior to day 1 of the incubation process.

[0138] In some embodiments, cells are exposed to a GSK3β signaling inhibitor at concentrations between about 0.1 µM and about 10 µM, between about 0.5 µM and about 8 µM, between about 0.5 µM and about 2 µM, between about 1 µM and about 4 µM, or between about 2 µM and about 3 µM (inclusive of each end value). In some embodiments, cells are exposed to a GSK3β signaling inhibitor at concentrations between about 0.1 µM and about 10 µM. In some embodiments, cells are exposed to a GSK3β signaling inhibitor at concentrations between about 0.5 µM and about 8 µM. In some embodiments, cells are exposed to a GSK3β signaling inhibitor at concentrations between about 0.5 µM and about 2 µM. In some embodiments, cells are exposed to a GSK3β signaling inhibitor at concentrations between about 1 µM and about 4 µM. In some embodiments, cells are exposed to a GSK3β signaling inhibitor at concentrations between about 2 µM and about 3 µM. In some embodiments, cells are exposed to a GSK3β signaling inhibitor at a concentration of about 1 µM on day 1 and at a concentration of about 2 µM on each of days 2 through 6. In some embodiments, cells are exposed to a GSK3β signaling inhibitor on day 1 at a concentration that is 50% of the concentration of the GSK3β signaling inhibitor exposed to cells on each of days 2 through 6.

[0139] In some embodiments, the culture medium is at least partially replaced with fresh culture medium, and the culture medium added each day from day 1 to day 6 contains a concentration of GSK3β signaling inhibitor that is intended to contact the cells, for example, due to the addition of culture medium via a 50% culture medium replacement. In some embodiments, the concentration of GSK3β signaling inhibitor contained in the culture medium added on day 1 via a 50% culture medium replacement is 50% of the concentration of GSK3β signaling inhibitor in the culture medium added each day from day 2 to day 6 via a 50% culture medium replacement.

[0140] In some embodiments, the culture medium added on each day from day 1 to day 6 by a culture medium replacement, such as a 50% culture medium replacement, independently contains a GSK3β signaling inhibitor at a concentration between about 0.2 µM and about 20 µM, between about 1 µM and about 16 µM, between about 2 µM and about 8 µM, or between about 4 µM and about 6 µM (inclusive of each extreme value). In some embodiments, the culture medium added on day 1 by a culture medium replacement, such as a 50% culture medium replacement, contains a GSK3β signaling inhibitor at a concentration of about 0.1 µM to about 10 µM. In some embodiments, the culture medium added on day 1 by a culture medium replacement, such as a 50% culture medium replacement, contains a GSK3β signaling inhibitor at a concentration of about 0.5 µM to about 8 µM. In some embodiments, the culture medium added on day 1 by a culture medium replacement, such as a 50% culture medium replacement, contains a GSK3β signaling inhibitor at a concentration of about 1 µM to about 4 µM. In some embodiments, on day 1, the added medium contains a GSK3β signaling inhibitor at a concentration of about 1 µM to about 3 µM, for example, a 50% replacement. In some embodiments, on day 1, the added medium contains a GSK3β signaling inhibitor at a concentration of about 1.5 µM to about 2.5 µM, for example, a 50% replacement. In some embodiments, on day 1, the added medium contains a GSK3β signaling inhibitor at a concentration of about 2 µM, for example, a 50% replacement.

[0141] In some embodiments, the culture medium is replaced every day from day 2 to day 6, for example, a 50% replacement, with the added medium containing a GSK3β signaling inhibitor at a concentration of about 0.2 µM to about 20 µM. In some embodiments, the culture medium is replaced every day from day 2 to day 6, for example, a 50% replacement, with the added medium containing a GSK3β signaling inhibitor at a concentration of about 1 µM to about 16 µM. In some embodiments, the culture medium is replaced every day from day 2 to day 6, for example, a 50% replacement, with the added medium containing a GSK3β signaling inhibitor at a concentration of about 2 µM to about 8 µM. In some embodiments, the culture medium is replaced every day from day 2 to day 6, for example, a 50% replacement, with the added medium containing a GSK3β signaling inhibitor at a concentration of about 4 µM to about 6 µM. In some embodiments, the culture medium is replaced every day from day 2 to day 6, for example, a 50% replacement, with the added medium containing a GSK3β signaling inhibitor at a concentration of about 3 µM to about 5 µM. In some implementations, the culture medium is replaced every day from day 2 to day 6, for example, with a 50% replacement of the culture medium, and the added culture medium contains a GSK3β signaling inhibitor at a concentration of about 4 µM.

[0142] In some embodiments, the GSK3β signaling inhibitor is selected from the group consisting of lithium ions, valproic acid, iodocarboxin, naproxen, famotidine, curcumin, olanzapine, CHIR99012, and combinations thereof. In some embodiments, the GSK3β signaling inhibitor is a small molecule. In some embodiments, the GSK3β signaling inhibitor inhibits glycogen synthase kinase 3β. In some embodiments, the GSK3β signaling inhibitor inhibits GSK3α. In some embodiments, the GSK3β signaling inhibitor regulates TGF-β and MAPK signaling. In some embodiments, the GSK3β signaling inhibitor is a wingless / integral (Wnt) signaling agonist. In some embodiments, the GSK3β signaling inhibitor has an IC50 of 6.7 nM against human GSK3β. In some embodiments, the GSK3β signaling inhibitor is CHIR99021 (e.g., "3-[3-(2-carboxyethyl)-4-methylpyrrole-2-methylene]-2-indolone" or the IUPAC name 6-(2-(4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino)ethylamino)nicotinamide), having the following formula: In some embodiments, the culture medium is supplemented with at least one sound hedgehog factor (SHH) signaling activator. SHH refers to a protein that is one of at least three proteins in the mammalian signaling pathway family, called hedgehog factor, another being desert hedgehog factor (DHH), and the third being Indian hedgehog factor (IHH). Shh interacts with at least two transmembrane proteins by interacting with the transmembrane molecules Patched (PTC) and Smoothened (SMO). In some embodiments, the culture medium is supplemented with at least one SHH signaling activator from day 1 and up to about day 7 (e.g., day 6 or day 7). In some embodiments, the culture medium is supplemented with at least one SHH signaling activator from about day 1 until day 6 (inclusive). In some embodiments, cells are not exposed to sound hedgehog factor (SHH) signaling activators prior to day 1 of the differentiation protocol.

[0143] In some embodiments, at least one SHH signaling activator is an SHH protein. In some embodiments, at least one SHH signaling activator is a recombinant SHH protein. In some embodiments, at least one SHH signaling activator is a recombinant mouse SHH protein. In some embodiments, at least one SHH signaling activator is a recombinant human SHH protein. In some embodiments, at least one SHH signaling activator is a recombinant N-terminal fragment of a full-length murine sound hedgehog factor protein capable of binding to the SHH receptor to activate SHH. In some embodiments, at least one SHH signaling activator is a C25II SHH protein.

[0144] In some embodiments, cells are exposed to at least one SHH signaling activator at concentrations between about 10 ng / mL and about 500 ng / mL, between about 20 ng / mL and 400 ng / mL, between about 30 ng / mL and about 300 ng / mL, between about 40 ng / mL and about 200 ng / mL, between about 50 ng / mL and 150 ng / mL, between about 50 ng / mL and about 100 ng / mL, or between about 75 ng / mL and 125 ng / mL (inclusive of each endpoint). In some embodiments, cells are exposed to at least one SHH signaling activator at concentrations between about 75 ng / mL and about 125 ng / mL (inclusive of each endpoint). In some embodiments, cells are exposed to at least one SHH signaling activator at concentrations between about 50 ng / mL and about 100 ng / mL (inclusive of each endpoint). In some embodiments, cells are exposed to at least one SHH signaling activator at a concentration of 100 ng / mL or more. In some embodiments, cells are exposed to SHH protein at a concentration of 100 ng / mL or more. In some embodiments, cells are exposed to recombinant SHH protein at a concentration of 100 ng / mL or more. In some embodiments, cells are exposed to recombinant mouse SHH protein at a concentration of 100 ng / mL or more. In some embodiments, cells are exposed to C25II SHH protein at a concentration of 100 ng / mL or more.

[0145] In some embodiments, cells are exposed to recombinant SHH protein at a concentration of 100 ng / mL or approximately. In some embodiments, cells are exposed to recombinant SHH protein at a concentration of 100 ng / mL or approximately from day 1 to at most day 7 (e.g., day 6 or day 7). In some embodiments, cells are exposed to recombinant SHH protein at a concentration of approximately 100 ng / mL from approximately day 1 to approximately day 6 (inclusive).

[0146] In some embodiments, cells are exposed to at least one SHH signaling activator at a concentration between about 1 µM and about 20 µM, between about 5 µM and about 15 µM, or between about 8 µM and about 12 µM. In some embodiments, cells are exposed to at least one SHH signaling activator at a concentration between about 1 µM and about 20 µM. In some embodiments, cells are exposed to at least one SHH signaling activator at a concentration between about 5 µM and about 15 µM. In some embodiments, cells are exposed to at least one SHH signaling activator at a concentration between about 8 µM and about 12 µM. In some embodiments, cells are exposed to at least one SHH signaling activator at a concentration of about 10 µM.

[0147] In some embodiments, the culture medium is at least partially exchanged, and the culture medium added by replacing the medium each day from day 1 to day 6 contains at least one SHH signaling activator at a concentration twice that of at least one SHH signaling activator intended to contact the cells, for example, this is due to replacing the added medium by 50% of the culture medium.

[0148] In some embodiments, the culture medium is replaced daily from day 1 to day 6, for example, with a 50% replacement. The added culture medium contains at least one SHH signaling activator, such as SHH protein, at a concentration of about 20 ng / mL to about 1 µg / mL, about 40 ng / mL to 800 ng / mL, about 60 ng / mL to about 600 ng / mL, about 80 ng / mL to about 400 ng / mL, about 100 ng / mL to 300 ng / mL, about 100 ng / mL to about 200 ng / mL, or about 150 ng / mL to 250 ng / mL, including each end value. In some embodiments, cells are exposed to at least one SHH signaling activator, such as SHH protein, at a concentration between about 75 ng / mL and about 125 ng / mL (including each end value). In some embodiments, the culture medium is replaced daily from day 1 to day 6, for example, with a 50% replacement. The added culture medium contains at least one SHH signaling activator, such as SHH protein, at a concentration of about 150 ng / mL to about 250 ng / mL (inclusive of each end value). In some embodiments, the culture medium is replaced daily from day 1 to day 6, for example, with a 50% replacement. The added culture medium contains at least one SHH signaling activator, such as SHH protein, at a concentration of about 175 ng / mL to about 225 ng / mL (inclusive of each end value). In some embodiments, the culture medium is replaced daily from day 1 to day 6, for example, with a 50% replacement. The added culture medium contains at least one SHH signaling activator, such as SHH protein, at a concentration of 200 ng / mL or more.

[0149] In some embodiments, at least one SHH signaling activator is an activator of the hedgehog factor receptor Smoothened. In some embodiments, at least one SHH signaling activator is a small molecule. In some embodiments, at least one SHH signaling activator is purinemorphine (e.g., CAS 483367-10-8), having the following formula: In some embodiments, cells are exposed to a concentration of about 2 µM of puromorphamine. In some embodiments, cells are exposed to a concentration of about 2 µM of puromorphamine starting from day 1 and up to day 7 (e.g., day 6 or day 7). In some embodiments, cells are exposed to a concentration of about 2 µM of puromorphamine from about day 1 to about day 6 (inclusive).

[0150] In some embodiments, cells are exposed to puromorphine at concentrations between about 0.1 µM and about 20 µM, between about 0.5 µM and about 10 µM, between about 1 µM and about 5 µM, between about 1 µM and about 3 µM, or between about 1.5 µM and about 2.5 µM. In some embodiments, cells are exposed to puromorphine at a concentration of about 2 µM.

[0151] In some implementations, the culture medium is at least partially exchanged, and the concentration of purine morphine contained in the supplemented culture medium is twice the concentration of purine morphine intended to contact the cells, for example, this is due to the supplemented culture medium being replaced by 50% of the culture medium.

[0152] In some embodiments, the culture medium is replaced every day from day 1 to day 6, for example, with a 50% replacement. The added culture medium contains puremorphine at concentrations of about 0.2 µM to about 40 µM, about 1 µM to about 20 µM, about 2 µM to about 10 µM, about 2 µM to about 6 µM, or about 3 µM to about 5 µM. In some embodiments, the culture medium is replaced every day from day 1 to day 6, for example, with a 50% replacement. The added culture medium contains puremorphine at concentrations of about 3 µM to about 5 µM. In some embodiments, the added culture medium contains puremorphine at concentrations of about 4 µM, with a 50% replacement every day from day 1 to day 6.

[0153] In some embodiments, the at least one SHH signaling activator is SHH protein and puromorphamine. In some embodiments, cells are exposed to SHH protein and puromorphamine from day 1 to at most about day 7 (e.g., day 6 or day 7). In some embodiments, cells are exposed to SHH protein and puromorphamine from about day 1 to about day 6 (inclusive). In some embodiments, cells are exposed to about 100 ng / mL SHH protein and about 2 µM puromorphamine from day 1 to at most about day 7 (e.g., day 6 or day 7). In some embodiments, cells are exposed to about 100 ng / mL SHH protein and about 2 µM puromorphamine from about day 1 to about day 6 (inclusive).

[0154] In some embodiments, the culture medium is replaced every day from day 1 to day 6. In some embodiments, the culture medium replacement includes replacing 50% or about 50% of the culture medium. In some embodiments, the culture medium replacement includes replacing about 25% to about 75% of the culture medium. In some embodiments, about 25% to about 75% of the culture medium is replaced daily from about day 1 to about day 6. In some embodiments, at least about 50% of the culture medium is replaced daily from about day 1 to about day 6. In some embodiments, about 50% of the culture medium is replaced daily, every other day, or every three days from about day 1 to about day 6. In some embodiments, about 50% of the culture medium is replaced daily from about day 1 to about day 6. In some embodiments, about 50% of the culture medium is replaced each day on days 1, 2, 3, 4, 5, and 6. Replacing about 50% of the culture medium is also referred to herein as a "50% culture medium replacement". In some embodiments, the replacement culture medium for each day from day 1 to day 6 contains about twice the concentration of the same small molecules as the culture medium on day 0.

[0155] In some embodiments, the first incubation includes culturing pluripotent stem cells in a basal induction medium from approximately day 0 to approximately day 6. In some embodiments, the first incubation includes culturing pluripotent stem cells in a basal induction medium to induce PSC differentiation into midbrain basal plate progenitor cells.

[0156] In some implementations, the basal induction medium is formulated to contain Neurobasal at a 1:1 ratio. ™ The culture medium and DMEM / F12 medium were supplemented with N-2 and B27, non-essential amino acids (NEAA), and GlutaMAX. ™ L-glutamine, β-mercaptoethanol, and insulin. In some embodiments, the basal induction medium is further supplemented with any of the above small molecules.

[0157] 3. Transfer and / or dissociation of the sphere In some implementations, cell aggregates (e.g., spheroids) generated after the first incubation of pluripotent stem cells cultured in non-adherent culture dishes are transferred or dissociated before the second incubation of cells in adherent culture.

[0158] In some embodiments, a first incubation is performed to produce cell aggregates (e.g., spheroids) expressing at least one of PAX6 and OTX2. In some embodiments, the first incubation produces cell aggregates (e.g., spheroids) expressing both PAX6 and OTX2. In some embodiments, the first incubation produces cell aggregates (e.g., spheroids) on or before approximately day 7 of the method provided herein. In some embodiments, the first incubation produces cell aggregates (e.g., spheroids) expressing at least one of PAX6 and OTX2 on or before approximately day 7 of the method provided herein. In some embodiments, the first incubation produces cell aggregates (e.g., spheroids) expressing both PAX6 and OTX2 on or before approximately day 7 of the method provided herein.

[0159] In some embodiments, cell aggregates (e.g., spheroids) generated during the first incubation are dissociated before a second incubation under adherent conditions. In some embodiments, cell aggregates (e.g., spheroids) generated during the first incubation are dissociated to produce a cell suspension. In some embodiments, the cell suspension produced by dissociation is a single-cell suspension. In some embodiments, dissociation is performed when the spheroid cells express at least one of PAX6 and OTX2. In some embodiments, dissociation is performed when the spheroid cells express both PAX6 and OTX2. In some embodiments, dissociation is performed on approximately day 7. In some embodiments, cell aggregates (e.g., spheroids) are dissociated by enzymatic dissociation. In some embodiments, the enzyme is selected from the group consisting of Accutase. ™ Dispersants, collagenases, and combinations thereof. In some embodiments, the enzyme includes an accutase. In some embodiments, the enzyme is an accutase. In some embodiments, the enzyme is a dispersant. In some embodiments, the enzyme is a collagenase. In some embodiments, the enzyme is a dispersant and a collagenase.

[0160] In some embodiments, the cell aggregates or cell suspensions derived therefrom are transferred to a second culture dish for a second incubation under adherent conditions. In some embodiments, the cell aggregates (e.g., spheroids) or cell suspensions derived therefrom are transferred to a substrate-coated culture dish 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 approximately day 7.

[0161] In some embodiments, the cell aggregates (e.g., spheroids) do not dissociate prior to the second incubation. In some embodiments, the cell aggregates (e.g., spheroids) are transferred integrally to a second culture dish for a second incubation, wherein the cells attach to the second culture dish. 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 both PAX6 and OTX2. In some embodiments, the transfer is performed on approximately day 7.

[0162] 4. Adherent culture In some implementations, the cell aggregates or cell suspensions are subjected to a second incubation, which is carried out in adherent culture, such as... Figure 1 As shown. Cell aggregates or cell suspensions are transferred to a second culture dish, which is treated to allow cell attachment. In some embodiments, the culture dish is a plate, culture dish, flask, or bioreactor. In some embodiments, the culture dish is coated with a substrate. In some embodiments, the substrate is a basement membrane protein. In some embodiments, the substrate is selected from laminin or fragments thereof, collagen, nestin, heparan 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 or fragments thereof. In some embodiments, the substrate is xenogeneic-free. In some embodiments, the substrate is xenogeneic-free laminin or fragments thereof.

[0163] In some embodiments, laminin or fragments thereof comprises an α chain, a β chain, and a γ chain. In some embodiments, the α chain is LAMA1, LAMA2, LAMA3, LAMA4, LAMA5, or a combination thereof. In some embodiments, the β chain is LAMB1, LAMB2, LAMB3, LAMB4, or a combination thereof. In some embodiments, the γ chain is LAMC1, LAMC2, LAMC3, or a combination thereof. In some embodiments, laminin or fragments thereof comprises, as described in Aumailley (2013). Cell Adh Migra Any α-chain, β-chain, and / or γ-chain described in 7(1):48-55.

[0164] In some embodiments, the laminin or a fragment thereof is selected from the group consisting of: laminin 111, laminin 121, laminin 211, laminin 213, laminin 221, laminin 3A32, laminin 3B32, laminin 3A11, laminin 3A21, laminin 411, laminin 421, laminin 423, laminin 511, laminin 521, laminin 522, laminin 523, or a fragment thereof. In some embodiments, the laminin is selected from laminin 521, laminin 111, laminin 511, and laminin 511-E8. In some embodiments, the laminin or a fragment thereof comprises the E8 fragment of LAMA5, the E8 fragment of LAMB1, and the E8 fragment of LAMC1. In some embodiments, the laminin or a fragment thereof is the laminin 511-E8 fragment. See Miyazaki et al. (2012). Nat Commun 3:1236. In some embodiments, the substrate-coated culture dish is exposed to poly-L-ornithine before being used to culture cells.

[0165] In some embodiments, the substrate-coated culture vessel is a plate, petri dish, flask, or bioreactor. In some embodiments, the substrate-coated culture vessel is a 6-well plate, a 12-well plate, or a 24-well plate. In some embodiments, the substrate-coated culture vessel is a 6-well plate. In some embodiments, the substrate-coated culture vessel is a 12-well plate. In some embodiments, the substrate-coated culture vessel is a 24-well plate.

[0166] In some embodiments, these methods include a second incubation of spherical cells transferred to a second culture dish and cultured under adherent conditions. In some embodiments, the cells cultured under adherent conditions are induced to differentiate into midbrain basal plate progenitor cells, dopaminergic neuron progenitor cells, including deterministic dopaminergic neuron progenitor cells, morphological dopaminergic neuron progenitor cells, and / or dopaminergic neurons.

[0167] In some embodiments, the second incubation involves culturing the spheroidal cells in a second culture dish coated with a substrate that promotes cell attachment. Typically, starting on day 7 after the cells are placed in the second culture dish, the cells are exposed to (i) BMP signaling inhibitors and (ii) GSK3β signaling inhibitors; and starting on day 11, the cells are exposed to (i) brain-derived neurotrophic factor (BDNF); (ii) ascorbic acid; (iii) glial cell-derived neurotrophic factor (GDNF); (iv) dibutyryl cyclic AMP (dbcAMP); (v) transforming growth factor β-3 (TGFβ3); and (vi) Notch signaling inhibitors.

[0168] In some embodiments, the second culture dish allows for monolayer cell culture. In some embodiments, cells derived from cell aggregates (e.g., spheroids) produced by the first incubation are cultured in a monolayer on the second culture dish. In some embodiments, cells derived from cell aggregates (e.g., spheroids) produced by the first incubation are cultured to produce a monolayer cell culture that is positive for one or more of LMX1A, FOXA2, EN1, CORIN, and combinations thereof. In some embodiments, cells derived from cell aggregates (e.g., spheroids) produced by the first incubation are cultured to produce a monolayer cell culture in which at least some cells are positive for EN1 and CORIN. In some embodiments, cells derived from cell aggregates (e.g., spheroids) produced by the first incubation are cultured to produce a monolayer cell culture in which at least some cells are TH+. In some embodiments, at least some cells are TH+ before or at approximately day 16. In some embodiments, cells derived from cell aggregates (e.g., spheroids) produced during the first incubation are cultured to produce a monolayer cell culture in which at least some cells are TH+FOXA2+. In some embodiments, at least some cells are TH+FOXA2+ before or at approximately day 16.

[0169] In the method provided herein, the second incubation involves culturing the cells of the spheroids in a second culture dish under conditions that induce neural differentiation. In some embodiments, the cell plates of the spheroids are seeded onto the second culture dish at approximately day 7.

[0170] In some implementations, the number of cells seeded on the second culture dish is approximately 0.1 × 10⁻⁶. 6 cells / cm 2 Approximately 2×106 cells / cm 2 Approximately 0.1 × 10 6 cells / cm 2 To approximately 1.5 × 10 6 cells / cm 2 Approximately 0.1 × 10 6 cells / cm 2 To approximately 1×10 6 cells / cm 2 Approximately 0.1 × 10 6 cells / cm 2 To approximately 0.8 × 10 6 cells / cm 2 Approximately 0.2 × 10 6 cells / cm 2 Approximately 2×10 6 cells / cm 2 Approximately 0.2 × 10 6 cells / cm 2 To approximately 1.5 × 10 6 cells / cm 2 Approximately 0.2 × 10 6 cells / cm 2 To approximately 1×10 6 cells / cm 2 Approximately 0.2 × 10 6 cells / cm 2 To approximately 0.8 × 10 6 cells / cm 2 Approximately 0.4 × 10 6 cells / cm 2 Approximately 2×10 6 cells / cm 2 Approximately 0.4 × 10 6 cells / cm 2 To approximately 1.5 × 10 6 cells / cm 2 Approximately 0.4 × 10 6 cells / cm 2 To approximately 1×10 6 cells / cm 2 Approximately 0.4 × 10 6 cells / cm 2 To approximately 0.8 × 10 6 cells / cm 2 Approximately 0.6 × 10 6 cells / cm 2 Approximately 2×10 6 cells / cm 2 Approximately 0.6 × 10 6cells / cm 2 To approximately 1.5 × 10 6 cells / cm 2 Approximately 0.6 × 10 6 cells / cm 2 To approximately 1×10 6 cells / cm 2 Approximately 0.6 × 10 6 cells / cm 2 To approximately 0.8 × 10 6 cells / cm 2 Approximately 0.8 × 10 6 cells / cm 2 Approximately 2×10 6 cells / cm 2 or approximately 0.8 × 10 6 cells / cm 2 To approximately 1×10 6 cells / cm 2 In some implementations, the number of cells seeded on the second culture dish is approximately 0.6 × 10⁶. 6 cells / cm 2 From approximately 1.0 × 10 6 cells / cm 2 In some embodiments, the number of cells seeded on the second culture dish is 0.8 × 10⁶ or approximately 0.8 × 10⁶. 6 cells / cm 2 .

[0171] In some implementations, the second incubation includes culturing cells derived from cell aggregates (e.g., spheres) in a culture medium (“medium”).

[0172] In some embodiments, the second incubation involves culturing cells derived from cell aggregates in a culture medium from approximately day 7 until harvest or collection. In some embodiments, the culture medium is at least partially exchanged daily from day 8 until harvest or collection. In the culture medium replacement, new culture medium replaces the medium added the previous day; for example, there is a complete or near-complete replacement or exchange of the culture medium each day during these days. In some embodiments, cells are cultured in a culture medium to generate definitive dopaminergic neuronal progenitor cells, stereotyped dopaminergic neuronal progenitor cells, or dopaminergic neurons.

[0173] In some implementations, components containing minimal non-human origin (e.g., KnockOut) are also used. ™The culture medium is supplemented with a serum substitute (serum substitute). In some embodiments, the culture medium is supplemented with a serum substitute from about day 7 to about day 10. In some embodiments, the culture medium is supplemented with about 2% (v / v) of serum substitute. In some embodiments, the culture medium is supplemented with about 2% (v / v) of serum substitute from about day 7 to about day 10.

[0174] In some embodiments, the culture medium is further supplemented with small molecules. In some embodiments, the small molecules are selected from the group consisting of: Rho-associated protein kinase (ROCK) inhibitors, bone morphogenetic protein (BMP) signaling inhibitors, glycogen synthase kinase 3β (GSK3β) signaling inhibitors, and combinations thereof.

[0175] In some embodiments, the culture medium is supplemented with a Rho-associated protein kinase (ROCK) inhibitor one or more days during cell passage. In some embodiments, cells are passaged on day 7 or approximately day 7. In some embodiments, the culture medium is supplemented with a ROCK inhibitor on each day of cell passage, for example, day 7. In some embodiments, the culture medium is supplemented with a ROCK inhibitor on day 7.

[0176] In some embodiments, cells are exposed to a ROCK inhibitor at concentrations between about 1 µM and about 20 µM, between about 5 µM and about 15 µM, or between about 8 µM and about 12 µM. In some embodiments, cells are exposed to a ROCK inhibitor at concentrations between about 1 µM and about 20 µM. In some embodiments, cells are exposed to a ROCK inhibitor at concentrations between about 5 µM and about 15 µM. In some embodiments, cells are exposed to a ROCK inhibitor at concentrations between about 8 µM and about 12 µM. In some embodiments, cells are exposed to a ROCK inhibitor at a concentration of 10 µM or about 10 µM. In some embodiments, cells are exposed to Y-27632 at a concentration of about 10 µM on day 7.

[0177] In some embodiments, the culture medium is supplemented with a BMP signaling inhibitor. In some embodiments, the culture medium is supplemented with a BMP signaling inhibitor from about day 7 to about day 11 (e.g., up to day 10 or day 11). In some embodiments, the culture medium is supplemented with a BMP signaling inhibitor from about day 7 until day 10 (inclusive).

[0178] In some embodiments, cells are exposed to a BMP signaling inhibitor at concentrations between about 0.01 µM and about 5 µM, between about 0.05 µM and about 1 µM, between about 0.05 µM and about 0.2 µM, or between about 0.1 µM and about 0.5 µM (inclusive of each endpoint). In some embodiments, cells are exposed to a BMP signaling inhibitor at concentrations between about 0.01 µM and about 5 µM. In some embodiments, cells are exposed to a BMP signaling inhibitor at concentrations between about 0.05 µM and about 1 µM. In some embodiments, cells are exposed to a BMP signaling inhibitor at concentrations between about 0.05 µM and about 0.2 µM. In some embodiments, cells are exposed to a BMP signaling inhibitor at a concentration of about 0.1 µM. In some embodiments, cells are exposed to LDN193189 at a concentration of about 0.1 µM. In some embodiments, 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, cells are exposed to LDN193189 at a concentration of about 0.1 µM from about day 7 to about day 10 (inclusive).

[0179] In some embodiments, the culture medium is supplemented with a GSK3β signaling inhibitor. In some embodiments, the culture medium is supplemented with a GSK3β signaling inhibitor from about day 7 to about day 13 (e.g., day 12 or day 13). In some embodiments, the culture medium is supplemented with a GSK3β signaling inhibitor from about day 7 until day 12 (inclusive).

[0180] In some embodiments, cells are exposed to a GSK3β signaling inhibitor at concentrations between about 0.1 µM and about 10 µM, between about 0.5 µM and about 8 µM, between about 1 µM and about 4 µM, between about 1.5 µM and about 3 µM, or between about 1.5 µM and about 2.5 µM (inclusive of each end value). In some embodiments, cells are exposed to a GSK3β signaling inhibitor at concentrations between about 0.1 µM and about 10 µM. In some embodiments, cells are exposed to a GSK3β signaling inhibitor at concentrations between about 0.5 µM and about 8 µM. In some embodiments, cells are exposed to a GSK3β signaling inhibitor at concentrations between about 1 µM and about 4 µM. In some embodiments, cells are exposed to a GSK3β signaling inhibitor at concentrations between about 1.5 µM and about 3 µM. In some embodiments, cells are exposed to a GSK3β signaling inhibitor at a concentration of about 2 µM. In some embodiments, cells are exposed to CHIR99021 at a concentration of about 2.0 µM. In some embodiments, cells are exposed to CHIR99021 at a concentration of about 2.0 µM from about day 7 to about day 13 (e.g., up to day 12 or 13). In some embodiments, cells are exposed to CHIR99021 at a concentration of about 2.0 µM from about day 7 to about day 12 (inclusive).

[0181] In some embodiments, the culture medium is supplemented with brain-derived neurotrophic factor (BDNF). In some embodiments, BDNF supplementation begins from approximately day 11. In some embodiments, BDNF supplementation continues from approximately day 11 until harvest or collection. In some embodiments, BDNF supplementation continues from approximately day 11 to approximately day 14, 15, 16, or 17. In some embodiments, BDNF supplementation continues from approximately day 11 until day 14. In some embodiments, BDNF supplementation continues from approximately day 11 until day 15. In some embodiments, BDNF supplementation continues from approximately day 11 until day 16. In some embodiments, BDNF supplementation continues from approximately day 11 until day 17.

[0182] In some embodiments, cells are exposed to BDNF at concentrations between about 1 ng / mL and about 100 ng / mL, between about 5 ng / mL and about 50 ng / mL, and between about 10 ng / mL and about 30 ng / mL. In some embodiments, cells are exposed to BDNF at concentrations between about 10 ng / mL and about 30 ng / mL. In some embodiments, cells are exposed to BDNF at a concentration of about 20 ng / mL.

[0183] In some embodiments, the culture medium is supplemented with 20 ng / mL BDNF starting from approximately day 11. In some embodiments, the culture medium is supplemented with 20 ng / mL BDNF starting from approximately day 11 until harvest or collection. In some embodiments, the culture medium is supplemented with approximately 20 ng / mL BDNF from approximately day 11 to approximately day 14, 15, 16, or 17. In some embodiments, the culture medium is supplemented with approximately 20 ng / mL BDNF from approximately day 11 until day 14. In some embodiments, the culture medium is supplemented with approximately 20 ng / mL BDNF from approximately day 11 until day 15. In some embodiments, the culture medium is supplemented with approximately 20 ng / mL BDNF from approximately day 11 until day 16. In some embodiments, the culture medium is supplemented with approximately 20 ng / mL BDNF from approximately day 11 until day 17.

[0184] In some embodiments, the culture medium is supplemented with glial cell-derived neurotrophic factor (GDNF). In some embodiments, GDNF supplementation begins from approximately day 11. In some embodiments, GDNF supplementation continues from approximately day 11 until harvest or collection. In some embodiments, GDNF supplementation continues from approximately day 11 to approximately day 14, 15, 16, or 17. In some embodiments, GDNF supplementation continues from approximately day 11 until day 14. In some embodiments, GDNF supplementation continues from approximately day 11 until day 15. In some embodiments, GDNF supplementation continues from approximately day 11 until day 16. In some embodiments, GDNF supplementation continues from approximately day 11 until day 17.

[0185] In some embodiments, cells are exposed to GDNF at concentrations between about 1 ng / mL and about 100 ng / mL, between about 5 ng / mL and about 50 ng / mL, and between about 10 ng / mL and about 30 ng / mL. In some embodiments, cells are exposed to GDNF at concentrations between about 10 ng / mL and about 30 ng / mL. In some embodiments, cells are exposed to GDNF at a concentration of about 20 ng / mL.

[0186] In some embodiments, the culture medium is supplemented with 20 ng / mL GDNF starting from approximately day 11. In some embodiments, the culture medium is supplemented with 20 ng / mL GDNF starting from approximately day 11 until harvest or collection. In some embodiments, the culture medium is supplemented with approximately 20 ng / mL GDNF from approximately day 11 to approximately day 14, 15, 16, or 17. In some embodiments, the culture medium is supplemented with approximately 20 ng / mL GDNF from approximately day 11 until day 14. In some embodiments, the culture medium is supplemented with approximately 20 ng / mL GDNF from approximately day 11 until day 15. In some embodiments, the culture medium is supplemented with approximately 20 ng / mL GDNF from approximately day 11 until day 16. In some embodiments, the culture medium is supplemented with approximately 20 ng / mL GDNF from approximately day 11 until day 17.

[0187] In some embodiments, the culture medium is supplemented with ascorbic acid. In some embodiments, ascorbic acid supplementation begins from approximately day 11. In some embodiments, ascorbic acid supplementation continues from approximately day 11 until harvest or collection. In some embodiments, ascorbic acid supplementation continues from approximately day 11 to approximately day 14, 15, 16, or 17. In some embodiments, ascorbic acid supplementation continues from approximately day 11 until day 14. In some embodiments, ascorbic acid supplementation continues from approximately day 11 until day 15. In some embodiments, ascorbic acid supplementation continues from approximately day 11 until day 16. In some embodiments, ascorbic acid supplementation continues from approximately day 11 until day 17.

[0188] In some embodiments, cells are exposed to ascorbic acid at concentrations between about 0.05 mM and about 5 mM, between about 0.1 mM and about 1 mM, and between about 0.2 mM and about 0.5 mM (inclusive of each extreme value). In some embodiments, cells are exposed to ascorbic acid at concentrations between about 0.05 mM and about 5 mM (inclusive of each extreme value). In some embodiments, cells are exposed to ascorbic acid at concentrations between about 0.1 mM and about 1 mM (inclusive of each extreme value). In some embodiments, cells are exposed to ascorbic acid at a concentration of about 0.2 mM.

[0189] In some embodiments, the culture medium is supplemented with about 0.2 mM ascorbic acid starting from about day 11. In some embodiments, the culture medium is supplemented with about 0.2 mM ascorbic acid from about day 11 until harvest or collection. In some embodiments, the culture medium is supplemented with about 0.2 mM ascorbic acid from about day 11 to about day 14, 15, 16, or 17. In some embodiments, the culture medium is supplemented with about 0.2 mM ascorbic acid from about day 11 until day 14. In some embodiments, the culture medium is supplemented with about 0.2 mM ascorbic acid from about day 11 until day 15. In some embodiments, the culture medium is supplemented with about 0.2 mM ascorbic acid from about day 11 until day 16. In some embodiments, the culture medium is supplemented with about 0.2 mM ascorbic acid from about day 11 until day 17.

[0190] In some embodiments, the culture medium is supplemented with dibutyryl cyclic AMP (dbcAMP). In some embodiments, dbcAMP supplementation begins from approximately day 11. In some embodiments, dbcAMP supplementation continues from approximately day 11 until harvest or collection. In some embodiments, dbcAMP supplementation continues from approximately day 11 to approximately day 14, 15, 16, or 17. In some embodiments, dbcAMP supplementation continues from approximately day 11 until day 14. In some embodiments, dbcAMP supplementation continues from approximately day 11 until day 15. In some embodiments, dbcAMP supplementation continues from approximately day 11 until day 16. In some embodiments, dbcAMP supplementation continues from approximately day 11 until day 17.

[0191] In some embodiments, cells are exposed to dbcAMP at concentrations between about 0.05 mM and about 5 mM, between about 0.1 mM and about 3 mM, and between about 0.2 mM and about 1 mM (inclusive of each extreme value). In some embodiments, cells are exposed to dbcAMP at concentrations between about 0.1 mM and about 3 mM (inclusive of each extreme value). In some embodiments, cells are exposed to dbcAMP at concentrations between about 0.2 mM and about 1 mM (inclusive of each extreme value). In some embodiments, cells are exposed to dbcAMP at a concentration of about 0.5 mM.

[0192] In some embodiments, the culture medium is supplemented with about 0.5 mM dbcAMP starting from about day 11. In some embodiments, the culture medium is supplemented with 0.5 mM dbcAMP from about day 11 until harvest or collection. In some embodiments, the culture medium is supplemented with about 0.5 mM dbcAMP from about day 11 to about day 14, 15, 16, or 17. In some embodiments, the culture medium is supplemented with about 0.5 mM dbcAMP from about day 11 until day 14. In some embodiments, the culture medium is supplemented with about 0.5 mM dbcAMP from about day 11 until day 15. In some embodiments, the culture medium is supplemented with about 0.5 mM dbcAMP from about day 11 until day 16. In some embodiments, the culture medium is supplemented with about 0.5 mM dbcAMP from about day 11 until day 17.

[0193] In some embodiments, the culture medium is supplemented with transforming growth factor β3 (TGFβ3). In some embodiments, TGFβ3 supplementation begins from approximately day 11. In some embodiments, TGFβ3 supplementation continues from approximately day 11 until harvest or collection. In some embodiments, TGFβ3 supplementation continues from approximately day 11 to approximately day 14, 15, 16, or 17. In some embodiments, TGFβ3 supplementation continues from approximately day 11 until day 14. In some embodiments, TGFβ3 supplementation continues from approximately day 11 until day 15. In some embodiments, TGFβ3 supplementation continues from approximately day 11 until day 16. In some embodiments, TGFβ3 supplementation continues from approximately day 11 until day 17.

[0194] In some embodiments, cells are exposed to TGFβ3 at concentrations between about 0.1 ng / mL and 10 ng / mL, between about 0.5 ng / mL and about 5 ng / mL, or between about 1.0 ng / mL and about 2.0 ng / mL. In some embodiments, cells are exposed to TGFβ3 at concentrations between about 1.0 ng / mL and about 2.0 ng / mL (inclusive). In some embodiments, cells are exposed to TGFβ3 at a concentration of about 1 ng / mL.

[0195] In some embodiments, the culture medium is supplemented with approximately 1 ng / mL TGFβ3 starting from approximately day 11. In some embodiments, the culture medium is supplemented with approximately 1 ng / mL TGFβ3 from approximately day 11 until harvest or collection. In some embodiments, the culture medium is supplemented with approximately 1 ng / mL TGFβ3 from approximately day 11 to approximately day 14, 15, 16, or 17. In some embodiments, the culture medium is supplemented with approximately 1 ng / mL TGFβ3 from approximately day 11 until day 14. In some embodiments, the culture medium is supplemented with approximately 1 ng / mL TGFβ3 from approximately day 11 until day 15. In some embodiments, the culture medium is supplemented with approximately 1 ng / mL TGFβ3 from approximately day 11 until day 16. In some embodiments, the culture medium is supplemented with approximately 1 ng / mL TGFβ3 from approximately day 11 until day 17.

[0196] In some embodiments, the culture medium is supplemented with a Notch signaling inhibitor. In some embodiments, the culture medium is supplemented with a Notch signaling inhibitor starting from approximately day 11. In some embodiments, the culture medium is supplemented with a Notch signaling inhibitor from approximately day 11 until harvest or collection. In some embodiments, the culture medium is supplemented with a Notch signaling inhibitor from approximately day 11 to approximately day 14, 15, 16, or 17. In some embodiments, the culture medium is supplemented with a Notch signaling inhibitor from approximately day 11 until day 14. In some embodiments, the culture medium is supplemented with a Notch signaling inhibitor from approximately day 11 until day 15. In some embodiments, the culture medium is supplemented with a Notch signaling inhibitor from approximately day 11 until day 16. In some embodiments, the culture medium is supplemented with a Notch signaling inhibitor from approximately day 11 until day 17.

[0197] In some embodiments, the Notch signaling inhibitor is selected from cowanin, PF-03084014, L685458, LY3039478, DAPT, or combinations thereof. In some embodiments, the Notch signaling inhibitor inhibits γ-secretase. In some embodiments, the Notch signaling inhibitor is a small molecule. In some embodiments, the Notch signaling inhibitor is DAPT, having the following formula: In some embodiments, cells are exposed to DAPT at concentrations between about 1 µM and about 20 µM, between about 5 µM and about 15 µM, or between about 8 µM and about 12 µM. In some embodiments, cells are exposed to DAPT at concentrations between about 1 µM and about 20 µM. In some embodiments, cells are exposed to DAPT at concentrations between about 5 µM and about 15 µM. In some embodiments, cells are exposed to DAPT at concentrations between about 8 µM and about 12 µM. In some embodiments, cells are exposed to DAPT at a concentration of about 10 µM.

[0198] In some embodiments, the culture medium is supplemented with about 10 µM DAPT starting from about day 11. In some embodiments, the culture medium is supplemented with 10 µM DAPT from about day 11 until harvest or collection. In some embodiments, the culture medium is supplemented with about 10 µM DAPT from about day 11 to about day 14, 15, 16, or 17. In some embodiments, the culture medium is supplemented with about 10 µM DAPT from about day 11 until day 14. In some embodiments, the culture medium is supplemented with about 10 µM DAPT from about day 11 until day 15. In some embodiments, the culture medium is supplemented with about 10 µM DAPT from about day 11 until day 16. In some embodiments, the culture medium is supplemented with about 10 µM DAPT from about day 11 until day 17.

[0199] In some embodiments, starting from approximately day 11, the culture medium is supplemented with approximately 20 ng / mL BDNF, approximately 20 ng / mL GDNF, approximately 0.2 mM ascorbic acid, approximately 0.5 mM dbcAMP, approximately 1 ng / mL TGFβ3, and approximately 10 µM DAPT. In some embodiments, from approximately day 11 until harvest or collection, the culture medium is supplemented with approximately 20 ng / mL BDNF, approximately 20 ng / mL GDNF, approximately 0.2 mM ascorbic acid, approximately 0.5 mM dbcAMP, approximately 1 ng / mL TGFβ3, and approximately 10 µM DAPT. In some embodiments, from approximately day 11 until approximately day 14, 15, 16, or 17, the culture medium is supplemented with approximately 20 ng / mL BDNF, approximately 20 ng / mL GDNF, approximately 0.2 mM ascorbic acid, approximately 0.5 mM dbcAMP, approximately 1 ng / mL TGFβ3, and approximately 10 µM DAPT. In some embodiments, the culture medium is supplemented with approximately 20 ng / mL BDNF, approximately 20 ng / mL GDNF, approximately 0.2 mM ascorbic acid, approximately 0.5 mM dbcAMP, approximately 1 ng / mL TGFβ3, and approximately 10 µM DAPT from approximately day 11 to day 14. In some embodiments, the culture medium is supplemented with approximately 20 ng / mL BDNF, approximately 20 ng / mL GDNF, approximately 0.2 mM ascorbic acid, approximately 0.5 mM dbcAMP, approximately 1 ng / mL TGFβ3, and approximately 10 µM DAPT from approximately day 11 to day 15. In some embodiments, the culture medium is supplemented with approximately 20 ng / mL BDNF, approximately 20 ng / mL GDNF, approximately 0.2 mM ascorbic acid, approximately 0.5 mM dbcAMP, approximately 1 ng / mL TGFβ3, and approximately 10 µM DAPT from approximately day 11 to day 16. In some implementations, from approximately day 11 to day 17, the culture medium is supplemented with approximately 20 ng / mL BDNF, approximately 20 ng / mL GDNF, approximately 0.2 mM ascorbic acid, approximately 0.5 mM dbcAMP, approximately 1 ng / mL TGFβ3 and approximately 10 µM DAPT.

[0200] In some embodiments, a serum substitute is provided in the culture medium from approximately day 7 to approximately day 10. In some embodiments, a 2% (v / v) serum substitute is provided in the culture medium from day 7 to day 10.

[0201] In some embodiments, the culture medium is replaced daily from approximately day 7 until harvest or collection, such as approximately day 14, 15, 16, or 17. In some embodiments, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the culture medium is replaced daily from approximately day 7 until harvest or collection, such as approximately day 14, 15, 16, or 17. In some embodiments, at least 95%, 96%, 97%, 98%, or 99% of the culture medium is replaced daily from approximately day 7 until harvest or collection, such as approximately day 14, 15, 16, or 17. In some embodiments, at least 98% or 99% of the culture medium is replaced daily from approximately day 7 until harvest or collection, such as approximately day 14, 15, 16, or 17.

[0202] In some embodiments, the second incubation includes culturing cells derived from cell aggregates (e.g., spheroids) in a basal induction medium. In some embodiments, the second incubation includes culturing cells derived from cell aggregates (e.g., spheroids) in a maturation medium. In some embodiments, the second incubation includes culturing cells derived from cell aggregates (e.g., spheroids) in a basal induction medium and then in a maturation medium.

[0203] In some embodiments, the second incubation includes culturing cells in basal induction medium from approximately day 7 to approximately day 10. In some embodiments, the second incubation includes culturing cells in maturation medium starting from approximately day 11. In some embodiments, the second incubation includes culturing cells in basal induction medium from approximately day 7 to approximately day 10, and then culturing cells in maturation medium starting from approximately day 11 until collection or harvest. In some embodiments, cells are cultured in maturation medium to generate deterministic dopaminergic neuronal progenitor cells, definitive dopaminergic neuronal progenitor cells, and / or dopaminergic neurons.

[0204] In some implementations, the basal induction medium is formulated to contain Neurobasal at a 1:1 ratio. ™ The culture medium and DMEM / F12 medium were supplemented with N-2 and B27, non-essential amino acids (NEAA), and GlutaMAX. ™ L-glutamine, β-mercaptoethanol, and insulin. In some embodiments, the basal induction medium is further supplemented with any of the molecules described in Part B.

[0205] In some implementations, the maturation medium is formulated to contain supplements of N-2 and B27, non-essential amino acids (NEAA), and GlutaMAX. ™ Neurobasal ™ Culture medium. In some embodiments, the maturation medium is further supplemented with any of the molecules described in Part B.

[0206] In some embodiments, 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, cells are cultured in basal induction medium from about day 7 until day 10 (inclusive). In some embodiments, cells are cultured in maturation medium starting from about day 11. In some embodiments, cells are cultured in basal induction medium from about day 7 to about day 10, and then cultured in maturation medium starting from about day 11. In some embodiments, cells are cultured in maturation medium starting from about day 11 until harvest or collection of cells.

[0207] In some embodiments, the second incubation begins around day 7 and continues until cell harvesting. In some embodiments, the method further includes harvesting differentiated cells. In some embodiments, differentiated cells are harvested between around day 14 and around day 17. In some embodiments, differentiated cells are harvested on day 14, day 15, day 16, or day 17, or approximately day 14, approximately day 15, approximately day 16, or approximately day 17. In some embodiments, differentiated cells are harvested on day 14 or around day 14. In some embodiments, differentiated cells are harvested on day 15 or around day 15. In some embodiments, differentiated cells are harvested on day 16 or around day 16. In some embodiments, differentiated cells are harvested on day 17 or around day 17. In some embodiments, the second incubation is from around day 7 until around day 14. In some embodiments, the second incubation is from around day 7 until around day 15. In some embodiments, the second incubation is from around day 7 until around day 16. In some implementations, the second incubation period is from about day 7 to about day 17.

[0208] 5. Harvesting, collecting, and preparing differentiated cells In embodiments of the provided methods, neutral differentiated cells generated by the methods provided herein may be harvested or collected, such as for the formulation and use of cells. In some embodiments, the provided methods for generating differentiated cells, such as for use as cell therapy in the treatment of neurodegenerative diseases, may include the formulation of cells, such as the formulation of differentiated cells generated by the methods provided herein. In some embodiments, a cell dose containing differentiated cells (e.g., directed DA neuronal progenitor cells, typed dopaminergic neuronal progenitor cells, or dopaminergic neurons) is provided as a composition or formulation, such as a pharmaceutical composition or formulation. Such compositions may be used according to the provided methods, such as for the prevention or treatment of neurodegenerative diseases, including Parkinson's disease.

[0209] In some cases, processing cells in one or more steps to manufacture, generate, or produce cell therapy and / or differentiated cells may include cell formulations, such as formulations of differentiated cells produced by the methods. In some cases, cells may be formulated in amounts intended for dosage administration, such as for single unit dose administration or multiple dose administration.

[0210] In some embodiments, one or more compositions are formulated to differentiate cells. In particular embodiments, one or more compositions are formulated to differentiate cells after the production of one or more compositions. In some embodiments, the one or more compositions have been previously cryopreserved and stored, and thawed prior to application.

[0211] In some embodiments, the differentiating cells include dopaminergic neuronal progenitor cells, including stereotyped dopaminergic neuronal progenitor cells. In some embodiments, the formulation composition of the differentiating cells is a composition rich in stereotyped dopaminergic neuronal progenitor cells. In some embodiments, the differentiating cells include dopaminergic neurons. In some embodiments, the formulation composition of the differentiating cells is a composition rich in dopaminergic neuronal cells.

[0212] 6. Exemplary Process As previously described, using the methods provided herein, pluripotent stem cells can differentiate into lineage-specific cell populations, including directed dopaminergic progenitor cells and dopaminergic neurons. These cells can then be used for cell replacement therapy. In some embodiments, using the methods described herein, pluripotent stem cells differentiate into midbrain basal plate progenitor cells, and the resulting spherical cells further differentiate into definitive dopaminergic neuronal progenitor cells, type-specific dopaminergic neuronal progenitor cells, and / or dopaminergic neurons. In some embodiments, pluripotent stem cells differentiate into directed dopaminergic neuronal progenitor cells. In some embodiments, pluripotent stem cells differentiate into dopaminergic neurons. In some embodiments, pluripotent stem cells are embryonic stem cells, such as isolated embryonic stem cells. In some embodiments, pluripotent stem cells are induced pluripotent stem cells.

[0213] In some embodiments, embryonic stem cells (e.g., isolated embryonic stem cells) differentiate into midbrain basal plate progenitor cells, and then differentiate into definitive dopaminergic neuron progenitor cells, type-specific dopaminergic neuron progenitor cells, and / or dopaminergic neurons. In some embodiments, embryonic stem cells (e.g., isolated embryonic stem cells) differentiate into definitive dopaminergic neuron progenitor cells. In some embodiments, embryonic stem cells (e.g., isolated embryonic stem cells) differentiate into type-specific dopaminergic neuron progenitor cells. In some embodiments, embryonic stem cells (e.g., isolated embryonic stem cells) differentiate into dopaminergic neurons.

[0214] In some embodiments, induced pluripotent stem cells (iPSCs) differentiate into midbrain basal plate progenitor cells, and then differentiate into definitive dopaminergic neuronal progenitor cells, type-specific dopaminergic neuronal progenitor cells, and / or dopaminergic neurons. In some embodiments, iPSCs differentiate into definitive dopaminergic neuronal progenitor cells. In some embodiments, iPSCs differentiate into type-specific dopaminergic neuronal progenitor cells. In some embodiments, iPSCs differentiate into dopaminergic neurons.

[0215] In some embodiments, the method includes (a) performing a first incubation, which comprises culturing pluripotent stem cells in a non-adherent culture dish under conditions that generate cell spheroids, wherein (i) starting from day 0 of the first incubation, the cells are exposed to a TGF-β / activin-Nodal signaling inhibitor and a bone morphogenetic protein (BMP) signaling inhibitor; and (ii) starting from day 1 of the first incubation, the cells are exposed to at least one sound hedgehog factor (SHH) signaling activator and a glycogen synthase kinase 3β (GSK3β) signaling inhibitor; and (b) performing a second incubation, which comprises culturing the spheroidized cells in a substrate-coated culture dish under conditions that induce cell neural differentiation. In some embodiments, the day 0 incubation is performed in the absence of: (x) a sound hedgehog factor (SHH) signaling activator and (y) a glycogen synthase kinase 3β signaling inhibitor. Because the cells are first exposed to SHH activators and GSK3β signaling inhibitors on day 1 (day 2) of the first incubation, these methods are sometimes referred to in this paper as the “day 1” suspension culture differentiation protocol.

[0216] In some implementations, culturing cells under conditions that induce neural differentiation includes exposing cells to (i) brain-derived neurotrophic factor (BDNF); (ii) ascorbic acid; (iii) glial cell-derived neurotrophic factor (GDNF); (iv) dibutyryl cyclic AMP (dbcAMP); (v) transforming growth factor β-3 (TGFβ3); and (vi) Notch signaling inhibitors.

[0217] In some embodiments, the method includes (a) performing a first incubation comprising culturing pluripotent stem cells in a microporous plate under conditions that generate cell spheroids, wherein (i) starting from day 0 of the first incubation, the cells are exposed to a TGF-β / activin-Nodal signaling inhibitor, a bone morphogenetic protein (BMP) signaling inhibitor, and a serum substitute; and (ii) starting from day 1 of the first incubation, the cells are exposed to at least one sound hedgehog factor (SHH) signaling activator, a glycogen synthase kinase 3β (GSK3β) signaling inhibitor, and a serum substitute; (b) dissociating the cells from the spheroids to generate a cell suspension; (c) transferring the cells from the cell suspension to a laminin-coated culture dish; (d) performing a second incubation comprising culturing the cells from the spheroids in a laminin-coated culture dish under conditions that induce neural differentiation; and (e) harvesting the neurally differentiated cells. In some embodiments, day 0 incubation is performed in the absence of: (x) a sound hedgehog factor (SHH) signaling activator and (y) a glycogen synthase kinase 3β signaling inhibitor. In some embodiments, a second incubation includes culturing cells in the presence of a serum substitute. In some embodiments, culturing cells under conditions that induce neural differentiation includes exposing cells to (i) brain-derived neurotrophic factor (BDNF); (ii) ascorbic acid; (iii) glial cell-derived neurotrophic factor (GDNF); (iv) dibutyryl cyclic AMP (dbcAMP); (v) transforming growth factor β-3 (TGFβ3); and (vi) a Notch signaling inhibitor.

[0218] In some embodiments, cells are exposed to a TGF-β / activin-Nodal inhibitor (e.g., SB431542 or “SB”) from day 0 to approximately day 5 (e.g., day 4 or day 5). In some embodiments, cells are exposed to a TGF-β / activin-Nodal inhibitor (e.g., SB431542 or “SB”) from day 0 until day 4 (inclusive). In some embodiments, cells are exposed to at least one SHH signaling activator (e.g., SHH protein and puromorphine, collectively “SHH / PUR”) from day 1 to approximately day 7 (e.g., day 6 or day 7). In some embodiments, cells are exposed to at least one SHH signaling activator (e.g., SHH protein and puromorphine, collectively “SHH / PUR”) from day 1 until day 6 (inclusive). In some embodiments, cells are exposed to a BMP signaling inhibitor (e.g., LDN193189 or “LDN”) from day 0 to approximately day 11 (e.g., day 10 or day 11). In some embodiments, cells are exposed to a BMP signaling inhibitor (e.g., LDN193189 or “LDN”) from day 0 until day 10 (inclusive). In some embodiments, cells are exposed to a GSK3β signaling inhibitor (e.g., CHIR99021 or “CHIR”) from day 1 to approximately day 13 (e.g., day 12 or day 13). In some embodiments, cells are exposed to a GSK3β signaling inhibitor (e.g., CHIR99021 or “CHIR”) from day 1 until day 12.

[0219] In some embodiments, cells are exposed to (i) a TGF-β / activin-Nodal signaling inhibitor from day 0 to about day 5 (e.g., day 4 or 5); (ii) at least one sound hedgehog factor (SHH) signaling activator from day 1 to about day 7 (e.g., day 6 or 7); (iii) a bone morphogenetic protein (BMP) signaling inhibitor from day 0 to about day 11 (e.g., day 10 or 11); and (iv) a glycogen synthase kinase 3β (GSK3β) signaling inhibitor from day 0 to about day 13 (e.g., day 12 or 13). In some implementations, cells are exposed to SB from day 0 to about day 5 (e.g., day 4 or day 5); (ii) to SHH / PUR from day 1 to about day 7 (e.g., day 6 or day 8); (iii) to LDN from day 0 to about day 11 (e.g., day 10 or day 11); and (iv) to CHIR from day 1 to about day 13 (e.g., day 12 or day 13). In some embodiments, cells are exposed to (i) a TGF-β / activin-Nodal signaling inhibitor from day 0 to day 5 (inclusive); (ii) at least one sound hedgehog factor (SHH) signaling activator from day 1 to day 6 (inclusive); (iii) a bone morphogenetic protein (BMP) signaling inhibitor from day 0 to day 10 (inclusive); and (iv) a glycogen synthase kinase 3β (GSK3β) signaling inhibitor from day 1 to day 12 (inclusive). In some embodiments, cells are exposed to SB from day 0 to day 5 (inclusive); (ii) SHH / PUR from day 1 to day 6 (inclusive); (iii) LDN from day 0 to day 10 (inclusive); and (iv) CHIR from day 1 to day 12 (inclusive).

[0220] In some embodiments, cells are exposed to brain-derived neurotrophic factor (BDNF) starting on day 11. In some embodiments, cells are exposed to ascorbic acid. In some embodiments, cells are exposed to glial cell-derived neurotrophic factor (GDNF) starting on day 11. In some embodiments, cells are exposed to dibutyryl cyclic AMP (dbcAMP) starting on day 11. In some embodiments, cells are exposed to transforming growth factor β-3 (TGFβ3) starting on day 11. In some embodiments, cells are exposed to a Notch signaling inhibitor (e.g., DAPT) starting on day 11. In some embodiments, cells are exposed to (i) brain-derived neurotrophic factor (BDNF); (ii) ascorbic acid; (iii) glial cell-derived neurotrophic factor (GDNF); (iv) dibutyryl cyclic AMP (dbcAMP); (v) transforming growth factor β-3 (TGFβ3); and (vi) a Notch signaling inhibitor (e.g., DAPT) (collectively, “BAGCT / DAPT”) starting on day 11. In some embodiments, cells are exposed to BAGCT / DAPT starting from day 11 until harvest or collection. In some embodiments, cells are exposed to BAGCT / DAPT from day 11 until approximately day 14, 15, 16, or 17. In some embodiments, cells are exposed to BAGCT / DAPT from day 11 until day 14. In some embodiments, cells are exposed to BAGCT / DAPT from day 11 until day 15. In some embodiments, cells are exposed to BAGCT / DAPT from day 11 until day 16. In some embodiments, cells are exposed to BAGCT / DAPT from day 11 until day 17.

[0221] In some embodiments, cells are exposed to a Rho-associated protein kinase (ROCK) inhibitor on day 0. In some embodiments, cells are exposed to a Rho-associated protein kinase (ROCK) inhibitor on day 7. In some embodiments, cells are exposed to a ROCK inhibitor on the day of cell passage. In some embodiments, pluripotent stem cells are not exposed to ROCK inhibitors during the first incubation prior to exposure to TGF-β / activin-Nodal signaling inhibitors and bone morphogenetic protein (BMP) signaling inhibitors. In some embodiments, cells are passaged on days 0 and 7.

[0222] In some embodiments, cells are cultured in a basal induction medium comprising DMEM / F-12 and Neurobasal medium (e.g., a 1:1 ratio), supplemented with N2, B27, non-essential amino acids (NEAA), Glutamax, L-glutamine, β-mercaptoethanol, and insulin. In some embodiments, cells are cultured in the basal induction medium from approximately day 0 to approximately day 10. In some embodiments, the basal induction medium is used to differentiate pluripotent stem cells into midbrain basal plate progenitor cells.

[0223] In some embodiments, cells are cultured in a maturation medium comprising Neurobasal medium supplemented with N2, B27, non-essential amino acids (NEAA), and Glutamax. In some embodiments, cells are cultured in basal induction medium from approximately day 11 until harvest or collection. In some embodiments, cells are cultured in basal induction medium from approximately day 11 to approximately day 14, 15, 16, or 17. In some embodiments, the maturation medium is used to differentiate midbrain basal plate progenitor cells into definitive dopaminergic neuronal progenitor cells, definitive dopaminergic neuronal progenitor cells, and / or dopaminergic neurons. In some embodiments, cells are cultured in basal induction medium from approximately day 11 until day 14. In some embodiments, cells are cultured in basal induction medium from approximately day 11 until day 15. In some embodiments, cells are cultured in basal induction medium from approximately day 11 until day 16. In some embodiments, cells are cultured in basal induction medium from approximately day 11 until day 17. In some implementations, the maturation culture medium is used to differentiate midbrain basal plate progenitor cells into dopaminergic neurons.

[0224] In some embodiments, the culture medium is supplemented with small molecules as described above (including SB, SHH / PUR, LDN, CHIR, BAGCT / DAPT, and ROCKi). In some embodiments, the culture medium is changed daily or every other day. In some embodiments, the culture medium is changed daily. In some embodiments, the culture medium is changed every other day. In some embodiments, the culture medium is changed daily from about day 1 to about day 14, 15, 16, or 17. In some embodiments, the culture medium is changed every other day from about day 1 until harvest or collection, for example, on day 14, 15, 16, or 17. In some embodiments, the culture medium is changed daily from about day 1 until day 6 by a 50% replacement, then daily by a complete or near-complete replacement (e.g., replacing at least 95%, 96%, 97%, 98%, or 99% of the culture medium) until harvest or collection, for example, on day 14, 15, 16, or 17.

[0225] In some embodiments, a serum substitute is provided in the culture medium from approximately day 0 to approximately day 10 (e.g., day 9 or day 11). In some embodiments, 5% (v / v) of a serum substitute is provided in the culture medium on days 0 and 1. In some embodiments, 2% (v / v) of a serum substitute is provided in the culture medium from day 2 through day 10. In some embodiments, 5% (v / v) of a serum substitute is provided in the culture medium on days 0 and 1, and 2% (v / v) of a serum substitute is provided in the culture medium from day 2 through day 10. In some embodiments, no serum substitute is provided in the culture medium after day 10.

[0226] In some embodiments, at least about 50% or at least about 75% of the culture medium is replaced on one or more days during these days. In some embodiments, at least about 50% of the culture medium is replaced on one or more days during these days, for example, from day 1 to day 6. In some embodiments, at least about 75% of the culture medium is replaced on one or more days during these days, for example, from day 7 until harvest or collection, for example, day 14, 15, 16, or 17. In some embodiments, at least 95% of the culture medium is replaced on one or more days during these days, for example, from day 7 until harvest or collection, for example, day 14, 15, 16, or 17. In some embodiments, about 100% of the culture medium is replaced on one or more days during these days, for example, from day 7 until harvest or collection, for example, day 14, 15, 16, or 17.

[0227] In some embodiments, approximately 50% or approximately 75% of the culture medium is replaced. In some embodiments, approximately 50% of the culture medium is replaced. In some embodiments, approximately 75% of the culture medium is replaced. In some embodiments, approximately 100% of the culture medium is replaced.

[0228] In some embodiments, the culture medium is supplemented with small molecules selected from SB, SHH / PUR, LDN, CHIR, BAGCT / DAPT, ROCKi, or combinations thereof. In some embodiments, when approximately 50% of the culture medium is replaced, the concentration of each small molecule is doubled compared to its expected concentration in the combined culture medium in contact with the cells.

[0229] In some embodiments, cells are harvested between approximately day 14 and approximately day 17. In some embodiments, cells are harvested between approximately day 14 and approximately day 16. In some embodiments, cells are harvested between approximately day 15 and approximately day 17. In some embodiments, cells are harvested around day 14. In some embodiments, cells are harvested around day 15. In some embodiments, cells are harvested around day 16. In some embodiments, cells are harvested around day 17. In some embodiments, the harvested cells are formulated with a cryopreservation agent, such as DMSO. In some embodiments, the harvested cells produced by this method are cryopreserved before use. In some embodiments, such cryopreserved cells are thawed before use or administration to a subject, such as a human patient with a neurodegenerative disease or condition (such as Parkinson's disease).

[0230] In some embodiments, compositions comprising cells generated by the methods provided herein are used to treat neurodegenerative diseases or conditions, such as Parkinson's disease. In some embodiments, compositions comprising cells generated by any of the methods described herein are administered to a subject suffering from Parkinson's disease. In some embodiments, compositions comprising cells generated by any of the methods described herein are administered by stereotactic injection, e.g., via a catheter. In some embodiments, compositions comprising cells generated by any of the methods described herein are administered to the striatum of a patient with Parkinson's disease.

[0231] C. Compositions and formulations This document provides therapeutic compositions containing differentiated cells, including dopaminergic neuronal progenitor cells, such as definitive dopaminergic neuronal progenitor cells. This document also provides therapeutic compositions containing differentiated cells generated by any of the methods provided. In some embodiments, the differentiated cells generated by any of the methods described herein are definitive dopaminergic neuronal progenitor cells, definitive dopaminergic neuronal progenitor cells, or dopaminergic neurons. In some embodiments, the therapeutic composition comprises dopaminergic neuronal progenitor cells generated by a method comprising: (a) performing a first incubation comprising non-adherent culturing of pluripotent stem cells in a first culture dish under conditions that generate cell spheroids, wherein the first incubation comprises: (i) exposing the pluripotent stem cells, starting from the first day of the first incubation (day 0), to a TGF-β / activin-Nodal signaling inhibitor and a bone morphogenetic protein (BMP) signaling inhibitor in the absence of: x) a sound hedgehog factor (SHH) signaling activator and y) a glycogen synthase kinase 3β (GSK3β) signaling inhibitor; and (ii) exposing the pluripotent stem cells, starting from the second day of the first incubation (day 1), to at least one sound hedgehog factor (SHH) signaling activator and a glycogen synthase kinase 3β (GSK3β) signaling inhibitor; and (b) performing a second incubation comprising adherent culturing of the cells in a second culture dish under conditions that allow the cells to further differentiate into dopaminergic neuronal progenitor cells.

[0232] In some embodiments, the differentiated cells in the provided therapeutic composition, including cells produced by any of the methods described herein, are capable of producing dopamine (DA). In some embodiments, the differentiated cells in the provided therapeutic composition, including cells produced by any of the methods described herein, do not produce or substantially do not produce norepinephrine (NE). Therefore, in some embodiments, the differentiated cells in the provided therapeutic composition, including cells produced by any of the methods described herein, are capable of producing DA but do not produce or substantially do not produce NE. In some embodiments, the differentiated cells in the provided therapeutic composition, including cells produced by any of the methods described herein, do not produce or substantially do not produce serotonin. Therefore, in some embodiments, the differentiated cells in the provided therapeutic composition, including cells produced by any of the methods described herein, are capable of producing DA but do not produce or substantially do not produce serotonin.

[0233] In some embodiments, the therapeutic composition comprises dopaminergic neuronal progenitor cells that possess one or more improved properties compared to neurons produced using other differentiation methods, such as adherent culture differentiation methods, in their use as a treatment for neurodegenerative diseases such as Parkinson's disease. These improved properties may include, for example, one or more of the following: (a) higher expression of FOXA2; (b) lower expression of PAX6; (c) higher predicted graft size after implantation; (d) production of less serotonin; (e) comprising a higher percentage of viable cells; (f) higher expression of CORIN; (g) lower expression of PITX2; and (h) lower expression of NKX2.1. In some embodiments, the dopaminergic neuronal progenitor cells in the therapeutic composition possess two or more of these properties. In some embodiments, the cells possess three or more of these properties, and in some embodiments, the cells possess four, five, six, or seven or more of these properties. In some embodiments, the cells possess all eight listed properties.

[0234] In some embodiments, the therapeutic compositions provided herein comprise dopaminergic neuronal progenitor cells that, compared to neurons generated using adherent culture differentiation methods, exhibit one or more characteristics selected from the group consisting of: (a) higher levels of FOXA2 expression; (b) lower levels of PAX6 expression; (c) higher predicted graft size after implantation; (d) higher predicted dopamine production levels after implantation; (e) lower serotonin production; (f) a higher percentage of viable cells; (g) higher levels of CORIN expression; (h) lower levels of PITX2 expression; and (i) lower levels of NKX2.1 expression. In some embodiments, the cells of the therapeutic composition exhibit two or more of these characteristics. In some embodiments, the cells exhibit three, four, five, six, seven, or eight or more of these characteristics. In some embodiments, the cells exhibit all nine listed characteristics.

[0235] In some embodiments, the therapeutic composition comprises dopaminergic neuronal progenitor cells having a Graft Test score of at least 1500, calculated as described in U.S. Provisional Application No. 63 / 598,533, filed November 13, 2023, entitled “METHODS OF PREDICTING CHARACTERISTICS OF DIFFERENTIATED NEURONAL CELLS”. In some embodiments, when stimulated with KCl, the dopaminergic neuronal progenitor cells in the therapeutic composition produce serotonin at a level less than two-fold higher than an unstimulated baseline. In some embodiments, more than 99% of the dopaminergic neuronal progenitor cells in the therapeutic composition are viable. In some embodiments, the therapeutic composition comprises dopaminergic neuronal progenitor cells expressing FOXA2 at greater than 100 TPM based on bulk RNAseq analysis. In some embodiments, the dopaminergic neuronal progenitor cells in the therapeutic composition express CORIN at greater than 300 TPM. In some embodiments, the dopaminergic neuronal progenitors in the therapeutic composition express PITX2 at less than 50 TPM. In some embodiments, the dopaminergic neuronal progenitors in the therapeutic composition express NKX2.1 at less than 10 TPM. In some embodiments, the dopaminergic neuronal progenitors in the therapeutic composition possess two or more of these characteristics. In some embodiments, the cells possess three or more of these characteristics, and in some embodiments, the cells possess four, five, six, or more of these characteristics. In some embodiments, the cells possess all seven characteristics listed in this paragraph.

[0236] In some embodiments, the differentiated cells produced by any of the methods described herein are dopaminergic neuronal progenitors, including fate-determining or type-defining dopaminergic neuronal progenitors (e.g., midbrain fate-determining DA neurons). In some embodiments, the midbrain fate-determining or type-defining dopaminergic neuronal progenitors are FOXA2+ / TH+ at harvest. In some embodiments, the midbrain fate-determining or type-defining dopaminergic neuronal progenitors are FOXA2+ / TH+ before or at approximately day 14. In some embodiments, the midbrain fate-determining or type-defining dopaminergic neuronal progenitors are FOXA2+ / TH+ before or at approximately day 15. In some embodiments, the midbrain fate-determining or type-defining dopaminergic neuronal progenitors are FOXA2+ / TH+ before or at approximately day 16. In some implementations, the midbrain fate-determining dopaminergic neuron progenitors or type-specific dopaminergic neuron progenitors are FOXA2+ / TH+ before or around day 17.

[0237] Methods for measuring or evaluating gene expression or gene products (including transcription and / or translation products) include those methods described in Part A. Furthermore, in some embodiments, measuring or evaluating gene expression or gene products is or includes assessing, measuring, determining, and / or quantifying the level, amount, or concentration of gene products (transcription and / or translation) in a sample.

[0238] In some embodiments, gene expression is or includes the process of using genetic information for the synthesis of a gene product. Therefore, in some embodiments, the gene product is any biomolecule assembled, generated, and / or synthesized using information encoded by a gene, and may include polynucleotides and / or polypeptides. In specific embodiments, assessing, measuring, and / or determining gene expression is or includes determining or measuring the level, amount, or concentration of the gene product. In some embodiments, the level, amount, or concentration of the gene product may be transformed (e.g., normalized) or directly analyzed (e.g., raw).

[0239] 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 confined and / or exposed on the cell surface. In some embodiments, the protein is a soluble protein. In some embodiments, the protein is secreted by the cell. In certain embodiments, gene expression is the amount, level, and / or concentration of the protein encoded by the gene. In some embodiments, one or more protein gene products are measured by any suitable method known in the art. Suitable methods for assessing, measuring, determining, and / or quantifying the level, amount, or concentration of one or more protein gene products include, but are not limited to, detection using immunoassays, nucleic acid-based or protein-based aptamer techniques, HPLC (high-precision liquid chromatography), peptide sequencing (such as Edman degradation sequencing or mass spectrometry (e.g., MS / MS, optionally coupled with HPLC), and any of the aforementioned microarray adaptations (including nucleic acid, antibody, or protein-protein (i.e., non-antibody) arrays). In some embodiments, immunoassays are or include methods or assays based on immune reactions, such as detecting proteins by detecting the binding of antibody or antigen-binding antibody fragments to the gene product. Immunoassays include, but are not limited to, quantitative immunocytochemistry or immunohistochemistry, ELISA (including direct, indirect, sandwich, competitive, multiplex, and portable ELISA (see, for example, U.S. Patent 7,510,687), Western blotting (including one-dimensional, two-dimensional, or high-dimensional blotting or other chromatographic methods, optionally including peptide sequencing), enzyme immunoassay (EIA), RIA (radioimmunoassay), and SPR (surface plasmon resonance).

[0240] In some embodiments, the gene product is a polynucleotide encoded by a gene, such as mRNA or a protein. In some embodiments, the gene product is a polynucleotide expressed and / or encoded by a gene. In some embodiments, the polynucleotide is RNA. In some embodiments, the gene product is messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA, small nuclear RNA, small nucleolar RNA, antisense RNA, long noncoding RNA, microRNA, Piwi-interacting RNA, small interfering RNA, and / or short hairpin RNA. In a particular embodiment, the gene product is mRNA.

[0241] In some implementations, RNA gene products are assessed, measured, identified, and / or quantified by directly evaluating, measuring, determining, and / or quantifying cDNA polynucleotides and / or cDNA oligonucleotides derived from RNA gene products.

[0242] In certain embodiments, the amount or level of polynucleotides in a sample can be assessed, measured, determined, and / or quantified by any suitable method known in the art. For example, in some embodiments, the amount or level of polynucleotide gene products can be assessed, measured, determined, and / or quantified by polymerase chain reaction (PCR), including reverse transcriptase (rt) PCR, droplet digital PCR, real-time and quantitative PCR (qPCR) methods (including, for example, TAQMAN). ® Molecular beacons, Lightup ™ SCORPION ™ SIMPLEPROBES ® See, for example, U.S. Patents 5,538,848; 5,925,517; 6,174,670; 6,329,144; 6,326,145 and 6,635,427); northern blotting; for example, Southern blotting of reverse transcripts and their derivatives; array-based methods, including blotting arrays, microarrays or in situ synthetic arrays; and sequencing, such as sequencing-by-synthesis, pyrosequencing, dideoxy sequencing or ligation-by-sequencing, or any other method known in the art, such as those discussed in Shendure et al., Nat. Rev. Genet. 5:335-44 (2004) or Nowrousian, Euk. Cell 9(9): 1300-1310 (2010), including those such as HELICOS ® ROCHE ® 454, ILLUMINA ® / SOLEXA ® ABI SOLiD ® and POLONATOR ® Sequencing and other specific platforms. In certain implementations, the level of nucleic acid gene products is measured using quantitative PCR (qPCR) methods, such as qRT-PCR. In some implementations, qRT-PCR uses a set of three nucleic acids per gene, wherein the three nucleic acids include a primer pair and a probe that binds between the target nucleic acid regions bound by the primers—commercially known as TAQMAN. ® Determination method.

[0243] In certain embodiments, the expression of two or more genes is measured or evaluated simultaneously. In some 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) are used. ® AGILENT ®and ILLUMINA ® Microarrays are used to assess, measure, determine, and / or quantify the level, amount, or concentration of two or more gene products. In some embodiments, microarrays are used to assess, measure, determine, and / or quantify the level, amount, or concentration of cDNA polynucleotides derived from RNA gene products. In some embodiments, the expression of one or more gene products (e.g., polynucleotide gene products) is determined by sequencing the gene product and / or by sequencing the cDNA polynucleotides derived from the gene product. In some embodiments, sequencing is performed using 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 nanosphere sequencing, Helioscope single-molecule sequencing, single-molecule real-time (SMRT) sequencing, single-molecule real-time (RNAP) sequencing, and nanopore DNA sequencing.

[0244] In some implementations, NGS technology is RNA sequencing (RNA-Seq). In specific implementations, the expression of one or more polynucleotide gene products is measured, determined, and / or quantified by RNA-Seq. RNA-Seq, also known as whole transcriptome shotgun sequencing, determines the presence and quantity of RNA in a sample. RNA sequencing methods are compatible with the most common DNA sequencing platforms [HiSeq system (Illumina), FLX 454 genome sequencer system (Roche), Applied Biosystems SOLiD (Life Technologies), IonTorrent (Life Technologies). These platforms require RNA to be initially reverse transcribed into cDNA. Conversely, the single-molecule sequencer HeliScope (Helicos BioSciences) can use RNA as a sequencing template. Validation of the principle for direct RNA sequencing on the PacBio RS platform has also been demonstrated (Pacific Bioscience). In some implementations, one or more RNA gene products are evaluated, measured, determined, and / or quantified by RNA-seq. In some implementations, RNA-seq is tag-based RNA-seq. In tag-based methods, each transcript is represented by a unique tag. Initially, tag-based methods were developed as sequence-based approaches to measure transcript abundance and identify differentially expressed genes, assuming that the number (count) of tags directly corresponds to the abundance of mRNA molecules. The reduced complexity of samples obtained by sequencing defined regions is crucial for making Sanger-based methods affordable. When NGS technology became available, the large number of reads that could be generated facilitated the analysis of differentially expressed genes. Tagged-based methods do not encounter transcript length biases, such as those observed in shotgun sequencing for quantifying gene expression levels. By definition, all tag-based methods are strand-specific. In specific implementations, tag-based RNA-seq is used to assess, measure, identify, and / or quantify one or more RNA gene products.

[0245] In some implementations, RNA-seq is shotgun RNA-seq. Many protocols have been described for shotgun RNA-seq, but they share several common steps: fragmentation (which can occur at the RNA or cDNA level), RNA to cDNA conversion (via oligodT or random primers), second-strand synthesis, ligation of adaptor sequences at the 3' and 5' ends (at the RNA or DNA level), and final amplification. In some implementations, if poly(A)+ RNA is selected prior to fragmentation, RNA-seq can focus solely on polyadenylated RNA molecules (primarily mRNA, but also some lncRNA, snoRNA, pseudogenes, and histones), or, if no selection is made, it can include non-polyadenylated RNA. In the latter case, ribosomal RNA (over 80% of the total RNA library) needs to be depleted before fragmentation. Therefore, it is evident that differences in capturing transcriptome mRNA portions lead to partial overlap in the types of transcripts detected. Moreover, different protocols can affect the abundance and distribution of sequencing reads. This makes it difficult to compare experimental results performed with different library preparation protocols.

[0246] In some implementations, RNA is obtained from each sample, fragmented, and used to generate complementary DNA (cDNA) samples, such as cDNA libraries for sequencing. Reads can be processed and aligned with the human genome, and the expected number of mappings for each gene / isotype can be estimated and used to determine read counts. In some implementations, read counts are normalized according to gene / isotype length and the number of reads in the library to obtain, for example, FPKM normalized according to gene / isotype length and the number of reads in the library, to obtain the number of fragments per thousand bases of exons per million mapped reads (FPKM) based on gene length and total mapped reads. In some aspects, inter-sample normalization is achieved through normalization, such as 75th quantile normalization, where each sample is scaled by the median of the 75th quantile from all samples, for example, to obtain a quantile-normalized FPKM (FPKQ) value. The FPKQ value can be logarithmically transformed (log2).

[0247] In some embodiments, RNA is obtained from each sample, fragmented, and used to generate complementary DNA (cDNA) samples, such as cDNA libraries for sequencing. Reads can be processed and aligned with the human genome, and the expected number of mappings for each gene / isotype can be estimated and used to determine read counts. In some embodiments, read counts are normalized according to gene / isotype length and the number of reads in the library. In some embodiments, read counts are provided in the form of counts per million (CPM).

[0248] In some embodiments, relative gene expression is measured by comparing the CPM of the target gene with the CPM of the housekeeping gene. In some embodiments, the housekeeping gene is GAPDH. In some embodiments, the relative gene expression of the target gene is determined as the ratio of the CPM of the target gene to the CPM of the housekeeping gene (e.g., GAPDH).

[0249] In some embodiments, any of the provided compositions contains a pharmaceutically acceptable carrier. In some embodiments, a cell dose comprising cells produced by any of the methods disclosed herein is provided in the form of a composition or formulation, such as a pharmaceutical composition or formulation. Such compositions may be used, for example for the prevention or treatment of diseases, symptoms, and conditions, such as neurodegenerative diseases, depending on the provided methods, articles, and / or compositions provided.

[0250] The term "pharmaceutical formulation" refers to a preparation in a form in which the bioactivity of the active ingredient contained therein is effective and which does not contain any additional components that would have unacceptable toxicity to the subject to whom the formulation will be administered.

[0251] "Pharmaceutically acceptable carriers" refer to components in a drug formulation that are non-toxic to the subjects, excluding the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0252] In some respects, the choice of carrier is partly determined by the specific cells or drug and / or method of administration. Therefore, a variety of suitable formulations exist. For example, a pharmaceutical composition may contain a preservative. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some respects, mixtures of two or more preservatives are used. The preservative or mixture thereof is typically present in an amount from about 0.0001% to about 2% by weight of the total composition. Carriers are described, for example, in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the doses and concentrations used, and include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl diammonium chloride; benzalkonium chloride; benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methyl or propyl p-hydroxybenzoate; 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 dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming ions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG).

[0253] In some aspects, the composition includes a buffer. Suitable buffers include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffers is used. The buffer or mixture thereof is typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administerable 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 edition (May 1, 2005).

[0254] The formulation or composition may also contain one or more active ingredients that can be used to prevent or treat a specific indication, disease, or condition with which the cells or pharmaceutical agent are being used, wherein the individual activities do not adversely affect each other. Such active ingredients are suitably present in combination in amounts effective for the intended purpose. Therefore, in some embodiments, the pharmaceutical composition also contains other pharmaceutically active agents or drugs, such as carbidopa-levodopa (e.g., levodopa), dopamine agonists (e.g., pramipexole, ropinirole, rotigotine, and apomorphine), MAO B inhibitors (e.g., selegiline, rasagiline, and safenamide), catechol O-methyltransferase (COMT) inhibitors (e.g., entacapone and tocapone), anticholinergic drugs (e.g., bentropine and trihexyphenidyl), amantadine, etc. In some embodiments, the pharmaceutical agent or cells are administered in the form of a salt, such as a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable acid addition salts include those derived from inorganic and organic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid, and sulfuric acid, and organic acids such as tartaric acid, acetic acid, citric acid, malic acid, lactic acid, fumaric acid, benzoic acid, glycolic acid, gluconic acid, succinic acid, and arylsulfonic acids, such as p-toluenesulfonic acid.

[0255] The formulation or composition may also be administered in combination with another form of treatment for a specific indication, disease, or symptom that is being prevented or treated with the cells or agent, wherein the respective activities do not adversely affect each other. Therefore, in some embodiments, the pharmaceutical composition is administered in combination with deep brain stimulation (DBS).

[0256] In some embodiments, the pharmaceutical composition contains an amount effective in treating or preventing the disease or symptom, such as a therapeutically effective amount or a preventatively effective amount of agent or cells. In some embodiments, therapeutic or preventative efficacy is monitored through periodic assessment of the treated subjects. For repeated administration over several days or longer, treatment is repeated as appropriate until the desired suppression of disease symptoms is achieved. However, other dosing regimens may also be useful and can be determined. The required dose can be delivered by a single bolus injection, by multiple bolus injections, or by continuous infusion.

[0257] The drug or cells can be administered by any suitable method, such as stereotactic injection (e.g., using a catheter). In some embodiments, a given dose is administered by a single bolus injection of the cells or drug. In some embodiments, the cells or drug are administered, for example, over several months or years, by multiple bolus injections. In some embodiments, the drug or cells can be administered by stereotactic injection into the brain, such as the striatum.

[0258] For the prevention or treatment of a disease, the appropriate dosage may depend on the type of disease to be treated, the type of one or more agents, the type of cell or recombinant receptor, the severity and course of the disease, whether the agent or cell is administered for preventive or therapeutic purposes, prior therapy, the subject's clinical history and response to the agent or cell, and the judgment of the attending physician. In some embodiments, the composition is appropriately administered to the subject in a single dose or as part of a series of treatments.

[0259] The cells or pharmaceutical agents can be administered using standard application techniques, formulations, and / or devices. Formulations and devices, such as syringes and vials, are provided for storing and administering the compositions. Regarding the cells, administration may be autologous. For example, non-pluripotent cells (e.g., fibroblasts) may be obtained from the 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 improving symptoms of a disease or condition such as neurodegenerative diseases), they are typically formulated as unit-dose injectable forms (solutions, suspensions, emulsions). Formulations include those intended for stereotactic administration, such as administration to the brain (e.g., the striatum).

[0260] In some embodiments, the composition is provided as a sterile liquid formulation, such as an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which in some aspects may be buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are slightly easier to administer, especially by injection. On the other hand, viscous compositions can be formulated within a suitable viscosity range to provide a longer contact period with a particular tissue. Liquid or viscous compositions may contain a carrier, which may be a solvent or dispersion medium containing, for example, water, saline, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

[0261] Sterile injection solutions can be prepared by incorporating drugs or cells into a solvent, such as by mixing with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, or dextrose.

[0262] Formulations intended for internal administration are typically sterile. Sterility can be readily achieved, for example, through filtration using a sterile membrane filter.

[0263] Various additives can be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of microbial action can be ensured by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, and sorbic acid). Extended absorption of injectable drug forms can be achieved by using delayed-absorption agents such as aluminum monostearate and gelatin.

[0264] In some embodiments, the preparation buffer contains a cryopreservation agent. In some embodiments, cells are prepared with a cryopreservation solution containing 1.0% to 30% DMSO, such as 5% to 20% DMSO or 5% to 10% DMSO. In some embodiments, the cryopreservation solution is or contains, for example, PBS containing 20% ​​DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. In some embodiments, the cryopreservation solution is or contains, for example, at least or about 7.5% DMSO. In some embodiments, the processing step may include washing differentiated cells to replace the cells in the cryopreservation solution. In some embodiments, the cells are frozen, for example, cryopreserved or cryoprotected, in a medium and / or solution of DMSO at a final concentration of 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%, or 1% to 15%, 6% to 12%, 5% to 10% or 6% to 8% of DMSO. In a particular embodiment, the cells are frozen, for example, cryopreserved or cryoprotected, in a medium and / or solution with a final concentration of or about 5.0%, 4.5%, 4.0%, 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.25%, 1.0%, 0.75%, 0.5% or 0.25% HSA or 0.1% to -5%, 0.25% to 4%, 0.5% to 2% or 1% to 2% HSA.

[0265] In certain embodiments, the composition of differentiated cells is formulated, cryopreserved, and then stored for a period of time. In some embodiments, the formulated cryopreserved cells are stored in release cells for administration. In certain embodiments, the formulated 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 about or less than 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days. In some embodiments, the cells are thawed after storage and administered to a subject.

[0266] In some embodiments, formulation is performed using one or more processing steps, including washing, diluting, or concentrating cells. In some embodiments, the processing may include diluting or concentrating cells to a desired concentration or number, such as a composition comprising a unit dose of the number of cells to be administered in a given dose or a portion thereof. In some embodiments, the processing steps may include reducing the volume to increase the cell concentration as needed. In some embodiments, the processing steps may include increasing the volume to decrease the cell concentration as needed. 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 from about 1 µL to 5000 µL, such as 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.

[0267] The container typically holds cells to be administered, such as one or more unit doses. This unit dose can be the amount or number of cells to be administered to the subject, or twice (or more) the number of cells to be administered. It can be the minimum or lowest possible dose of cells to be administered to the subject.

[0268] In some embodiments, such cells produced by the method or compositions containing such cells are administered to a subject to treat neurodegenerative diseases or conditions.

[0269] D. Treatment methods This document provides methods for treating a disease or condition in a subject with a need for treatment using any of the provided compositions. In certain embodiments, the composition is produced by the methods provided herein. Such methods and uses include treatment methods and uses, for example, including administering therapeutic cells or compositions containing therapeutic cells to a subject suffering from a disease, condition, or ailment. In some embodiments, the disease or condition is a neurodegenerative disease or ailment. In some embodiments, the cells or a pharmaceutical composition thereof are administered in an effective amount to achieve treatment of the disease or condition. Uses include the use of the cells or a pharmaceutical composition thereof in such methods and treatments, and in the preparation or manufacture of a medicament for performing such treatment methods. In some embodiments, the method thereby treats the disease or condition or ailment of a subject.

[0270] This disclosure relates to methods for the lineage-specific differentiation of pluripotent stem cells (PSCs), including embryonic stem (ES) cells and induced pluripotent stem cells (iPSCs), for use in neurodegenerative diseases. Specifically, the methods, compositions, and uses thereof provided herein contemplate the differentiation of pluripotent stem cells for administration to subjects exhibiting dopaminergic neuronal cell loss, including Parkinson's disease.

[0271] Parkinson's disease (PD) is the second most common neurodegenerative disease, affecting an estimated 4-5 million people worldwide. This number is projected to more than double by 2030. PD is the second most common neurodegenerative disease after Alzheimer's, affecting approximately 1 million people in the United States, with 60,000 new cases diagnosed annually. Currently, there is no cure for PD. The pathological feature of PD is the selective loss of dopaminergic neurons (DA neurons) in the substantia nigra of the midbrain. Therefore, the fundamental characteristic of PD is the progressive, severe, and irreversible loss of midbrain dopaminergic neurons, ultimately leading to motor dysfunction and disability.

[0272] In some implementations, the subject has a neurodegenerative disease. In some implementations, the neurodegenerative disease includes the loss of dopamine neurons in the brain. In some implementations, the subject has lost dopamine neurons in the substantia nigra (SN). In some implementations, the subject has lost dopamine neurons in the pars compacta (SNc). In some implementations, the subject exhibits rigidity, bradykinesia, impaired postural reflexes, resting tremor, or a combination thereof. In some implementations, the subject exhibits abnormal [18F]-L-DOPA PET scans. In some implementations, the subject exhibits [18F]-DG-PET evidence of Parkinson's disease-associated patterns (PDRP).

[0273] In some embodiments, the neurodegenerative disease is Parkinson's syndrome. In some embodiments, the neurodegenerative disease is Parkinson's disease. In some embodiments, the neurodegenerative disease is idiopathic Parkinson's disease. In some embodiments, the neurodegenerative disease is a familial form of Parkinson's disease. In some embodiments, the subject has mild Parkinson's disease. In some embodiments, the subject has a Movement Disorder Association-Unified Parkinson's Disease Rating Scale (MDS-UPDRS) motor score of 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 between 33 and 60.

[0274] In some embodiments, a dose of cells is administered to a subject according to the provided method and / or the provided article or composition. In some embodiments, the dose or timing is determined based on the subject's specific disease or condition. In some cases, given the provided description, the dose or timing for a specific disease can be determined empirically.

[0275] 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.

[0276] In some implementations, the cell dose administered to the subject is approximately 5 × 10⁻⁶. 6 10 × 10⁶ cells. In some implementations, the cell dose administered to the subject is approximately 10 × 10⁶ cells. 6 15 × 10⁶ cells. In some implementations, the cell dose administered to the subject is approximately 15 × 10⁶ cells. 6 10 cells. In some implementations, the cell dose administered to the subject is approximately 20 × 10⁶ cells. 6 10 cells. In some implementations, the cell dose administered to the subject is approximately 25 × 10⁻⁶ cells. 6 100 cells. In some implementations, the cell dose administered to the subject is approximately 30 × 10⁶ cells. 6 Each cell.

[0277] In some embodiments, the cell dose includes exactly or about 250,000 cells per hemisphere to exactly or about 20 million cells per hemisphere, exactly or about 500,000 cells per hemisphere to exactly or about 20 million cells per hemisphere, exactly or about 1 million cells per hemisphere to exactly or about 20 million cells per hemisphere, exactly or about 5 million cells per hemisphere to exactly or about 20 million cells per hemisphere, and exactly or about 10 million cells per hemisphere to exactly or about 20 million cells per hemisphere. Cells, exactly or about 15 million cells per hemisphere to exactly or about 20 million cells per hemisphere, exactly or about 250,000 cells per hemisphere to exactly or about 15 million cells per hemisphere, exactly or about 500,000 cells per hemisphere to exactly or about 15 million cells per hemisphere, exactly or about 1 million cells per hemisphere to exactly or about 15 million cells per hemisphere, exactly or about 5 million cells per hemisphere to exactly or about 15 million cells per hemisphere, exactly or about 100 million cells per hemisphere 0 million cells to exactly or about 15 million cells per hemisphere, exactly or about 250,000 cells per hemisphere to exactly or about 10 million cells per hemisphere, exactly or about 500,000 cells per hemisphere to exactly or about 10 million cells per hemisphere, exactly or about 1 million cells per hemisphere to exactly or about 10 million cells per hemisphere, exactly or about 5 million cells per hemisphere to exactly or about 10 million cells per hemisphere, exactly or about 250,000 cells per hemisphere to exactly... Or approximately 5 million cells, exactly or approximately 500,000 cells per hemisphere to exactly or approximately 5 million cells per hemisphere, exactly or approximately 1 million cells per hemisphere to exactly or approximately 5 million cells per hemisphere, exactly or approximately 250,000 cells per hemisphere to exactly or approximately 1 million cells per hemisphere, exactly or approximately 500,000 cells per hemisphere to exactly or approximately 1 million cells per hemisphere, or exactly or approximately 250,000 cells per hemisphere to exactly or approximately 5 million cells per hemisphere.

[0278] In some embodiments, the cell dose is from exactly or about 1 million cells per hemisphere to exactly or about 30 million cells per hemisphere. In some embodiments, the cell dose is from exactly or about 5 million cells per hemisphere to exactly or about 20 million cells per hemisphere. In some embodiments, the cell dose is from exactly or about 10 million cells per hemisphere to exactly or about 15 million cells per hemisphere.

[0279] In some implementations, the cell dose is approximately 3 × 10⁻⁶. 6 Cells / hemispheric up to 15 × 10 6 One cell per hemisphere. In some embodiments, the cell dose is approximately 3 × 10⁻⁶. 6One cell per hemisphere. In some embodiments, the cell dose is approximately 4 × 10⁻⁶. 6 One cell per hemisphere. In some embodiments, the cell dose is approximately 5 × 10⁻⁶. 6 One cell per hemisphere. In some embodiments, the cell dose is approximately 6 × 10⁻⁶. 6 One cell per hemisphere. In some embodiments, the cell dose is approximately 7 × 10⁻⁶. 6 One cell per hemisphere. In some embodiments, the cell dose is approximately 8 × 10⁻⁶. 6 One cell per hemisphere. In some embodiments, the cell dose is approximately 9 × 10⁻⁶. 6 One cell per hemisphere. In some embodiments, the cell dose is approximately 10 × 10⁻⁶. 6 One cell per hemisphere. In some embodiments, the cell dose is approximately 11 × 10⁻⁶. 6 One cell per hemisphere. In some embodiments, the cell dose is approximately 12 × 10⁻⁶. 6 One cell per hemisphere. In some embodiments, the cell dose is approximately 13 × 10⁻⁶ cells / hemispheric. 6 One cell per hemisphere. In some embodiments, the cell dose is approximately 14 × 10⁻⁶. 6 Cells / hemispheric. In some embodiments, the cell dose is approximately 15 × 10⁻⁶. 6 Cell / hemisphere

[0280] In some implementations, the number of cells administered to the subject is approximately 0.25 × 10⁻⁶. 6 Total cells approximately 20 × 10 6 Total cells, approximately 0.25 × 10⁻⁶ 6 Total cells approximately 15 × 10⁻⁶ 6 Total cells, approximately 0.25 × 10⁻⁶ 6 Total cells approximately 10 × 10 6 Total cells, approximately 0.25 × 10⁻⁶ 6 Total cells approximately 5 × 10 6 Total cells, approximately 0.25 × 10⁻⁶ 6 Total cells approximately 1×10 6 Total cells, approximately 0.25 × 10⁻⁶ 6 Total cells approximately 0.75 × 10⁻⁶ 6 Total cells, approximately 0.25 × 10⁻⁶ 6 Total cells approximately 0.5 × 10⁻⁶ 6 Total cells, approximately 0.5 × 10⁻⁶ 6 Total cells approximately 20 × 10 6 Total cells, approximately 0.5 × 10⁻⁶ 6 Total cells approximately 15 × 10⁻⁶ 6 Total cells, approximately 0.5 × 10⁻⁶ 6Total cells approximately 10 × 10 6 Total cells, approximately 0.5 × 10⁻⁶ 6 Total cells approximately 5 × 10 6 Total cells, approximately 0.5 × 10⁻⁶ 6 Total cells approximately 1×10 6 Total cells, approximately 0.5 × 10⁻⁶ 6 Total cells approximately 0.75 × 10⁻⁶ 6 Total cells, approximately 0.75 × 10⁻⁶ 6 Total cells approximately 20 × 10 6 Total cells, approximately 0.75 × 10⁻⁶ 6 Total cells approximately 15 × 10⁻⁶ 6 Total cells, approximately 0.75 × 10⁻⁶ 6 Total cells approximately 10 × 10 6 Total cells, approximately 0.75 × 10⁻⁶ 6 Total cells approximately 5 × 10 6 Total cells, approximately 0.75 × 10⁻⁶ 6 Total cells approximately 1×10 6 Total cells, approximately 1×10 6 Total cells approximately 20 × 10 6 Total cells, approximately 1×10 6 Total cells approximately 15 × 10⁻⁶ 6 Total cells, approximately 1×10 6 Total cells approximately 10 × 10 6 Total cells, approximately 1×10 6 Total cells approximately 5 × 10 6 Total cells, approximately 5 × 10 6 Total cells approximately 20 × 10 6 Total cells, approximately 5 × 10 6 Total cells approximately 15 × 10⁻⁶ 6 Total cells, approximately 5 × 10 6 Total cells approximately 10 × 10 6 Total cells, approximately 10 × 10 6 Total cells approximately 20 × 10 6 Total cells, approximately 10 × 10 6 Total cells approximately 15 × 10⁻⁶ 6 Total cells or approximately 15 × 10 6 Total cells approximately 20 × 10 6 Total cells.

[0281] In some implementations, a single population of cells or cell subtypes is administered to the subject at a range of approximately 5 million to approximately 20 million cells per hemisphere, or any value between these ranges. The dosage may vary depending on the disease or condition and / or the patient and / or other treatment-specific properties.

[0282] In some implementations, the patient is given multiple doses, and each dose or the total dose may be within any of the aforementioned values. In some implementations, the cell dose includes administration of exactly or about 5 million cells per hemisphere to about 20 million cells per hemisphere, including each end value.

[0283] In some implementations, the cellular dose (e.g., deterministic dopaminergic neuron progenitor cells or type dopaminergic neuron progenitor cells) is administered to the subject as a single dose, or administered only once over a period of two weeks, one month, three months, six months, one year, or longer.

[0284] In the case of stem cell transplantation, the administration of a given “dose” encompasses the administration of a given amount or number of cells in the form of a single composition and / or a single, uninterrupted administration, such as by a single injection or continuous infusion, and also encompasses the administration of a given amount or number of cells in fractional doses or in multiple compositions, provided as multiple separate compositions or infusions over a specific time period (such as a day). Thus, in some cases, the dose is a single or continuous administration of a specified number of cells given or initiated at a single point in time. However, in other cases, the dose is administered by multiple injections or infusions over a single period, such as by multiple infusions over a period of one day.

[0285] Therefore, in some aspects, this dose of cells is administered as a single pharmaceutical composition. In some embodiments, this dose of cells is administered as a combination of multiple compositions that collectively contain this dose of cells.

[0286] In some embodiments, the dose of cells can be administered by applying multiple compositions or solutions, such as the first and second, and optionally more, each containing a certain dose of cells. In some aspects, multiple compositions, each containing different cell populations and / or cell subtypes, are administered alone or independently, optionally over a period of time.

[0287] In some embodiments, the application of the composition or dosage, such as the application of multiple cell compositions, includes the application of a single cell composition. In some aspects, the single applications are performed simultaneously, or sequentially in any order.

[0288] In some embodiments, the subject receives multiple doses of cells, such as two or more doses or multiple consecutive doses of cells. In some embodiments, two doses are administered to the subject. In some embodiments, multiple consecutive doses are administered after the first dose so that one or more additional doses are administered after the consecutive doses. In some aspects, the number of cells administered to the subject with the additional doses is the same as or similar to the first dose and / or consecutive doses. In some embodiments, the one or more additional doses are greater than the previous doses.

[0289] In some respects, 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, for example, disease stage and / or the likelihood or incidence of adverse outcomes (e.g., movement disorders) in the subject.

[0290] In some implementations, the cell dose is typically large enough to effectively improve disease symptoms.

[0291] In some embodiments, cells are administered at a desired dose, 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 cell dose is based on the total number of cells (or the number per kilogram of body weight) required in a single population or a single cell type (e.g., TH+ or TH-). In some embodiments, the dose is based on a combination of such characteristics, such as the desired total number of cells, the desired ratio, and the desired total number of cells in a single population.

[0292] Therefore, in some embodiments, the dose is based on the required total cell fixation dose and the required ratio, and / or based on the required fixation dose of one or more (e.g., each) subtypes or subgroups of the single subtype or subgroup.

[0293] In a particular embodiment, the number and / or concentration of cells refers to the number of TH-negative cells. In other embodiments, the number and / or concentration of cells refers to the total number or concentration of all cells administered.

[0294] In some embodiments, cells are administered at a desired dose, which in some aspects includes a desired dose or number of cells or cell types and / or a desired ratio of cell types. Thus, in some embodiments, the cell dose is based on the total number of cells and the desired ratio of an individual population or subtype. In some embodiments, the cell dose is based on the desired total number of cells (or the number per kilogram of body weight) in a single population or single cell type. In some embodiments, the dose is based on a combination of such characteristics, such as the desired total number of cells, the desired ratio, and the desired total number of cells in a single population.

[0295] Therefore, in some embodiments, the dose is based on the required total cell fixation dose and the required ratio, and / or based on the required fixation dose of one or more (e.g., each) subtypes or subgroups of the single subtype or subgroup.

[0296] In a particular embodiment, the number and / or concentration of cells refers to the number of TH-negative cells. In other embodiments, the number and / or concentration of cells refers to the total number or concentration of all cells administered.

[0297] In some respects, the dosage is determined based on one or more criteria, such as the subject’s response to previous treatment, for example, the type and / or stage of the disease and / or the likelihood or incidence of the subject developing toxic outcomes (e.g., movement disorders).

[0298] E. Products and reagent kits Articles, systems, apparatus, and kits for performing the methods provided are also provided. Articles are also provided comprising: (i) one or more reagents for differentiating pluripotent stem cells into midbrain basal plate progenitor cells, dopaminergic neuron progenitor cells, deterministic dopaminergic neuron progenitor cells, morphological dopaminergic neuron progenitor cells, and / or dopaminergic neurons; and (ii) instructions for using one or more reagents to perform any of the methods described herein.

[0299] In some embodiments of any such implementation, the reagent for differentiation is or includes a small molecule capable of inhibiting TGF-β / activin-Nodal signaling. In some embodiments of any such implementation, the reagent for differentiation is or includes SB431542. In some embodiments of any such implementation, the reagent for differentiation is or includes a small molecule capable of activating SHH signaling. In some embodiments of any such implementation, the reagent for activating SHH signaling is or includes SHH. In some embodiments of any such implementation, the reagent for activating SHH signaling is or includes purinemorphine. In some embodiments of any such implementation, the reagent for activating SHH signaling is or includes SHH and purinemorphine. In some embodiments of any such implementation, the reagent for differentiation is or includes a small molecule capable of inhibiting BMP signaling. In some embodiments of any such implementation, the reagent for inhibiting BMP signaling is LDN193189. In some embodiments of any such implementation, the reagent for differentiation is or includes a small molecule capable of inhibiting GSK3β signaling. In some embodiments, the reagent is or includes CHIR99021. In some embodiments, the reagents used for differentiation are or include one or more of BDNF, GDNF, dbcAMP, ascorbic acid, TGFβ3, and DAPT. In one embodiment, the reagents in the kit may be in solution form, may be frozen, or may be lyophilized.

[0300] Articles of manufacture are also provided, comprising (i) any of the compositions described herein; and (ii) instructions for administering the composition to a subject.

[0301] In some embodiments, the article of manufacture or kit includes one or more containers (typically multiple containers), packaging materials, and labels or packaging inserts on or accompanying the one or more containers and / or packaging. These typically include instructions for use, such as instructions for reagents for pluripotent cell differentiation, such as iPSCs differentiating into midbrain basal plate progenitor cells, definitive dopaminergic neuronal progenitor cells, definitive dopaminergic neuronal progenitor cells, and / or dopaminergic neurons, as well as instructions for performing any of the methods provided herein. In some aspects, the provided article of manufacture contains, for example, reagents used in one or more steps of the manufacturing process for cell differentiation and / or maturation, such as any reagents described in any step of Parts B and C.

[0302] Articles and kits containing differentiated cells, such as those generated using the methods provided herein, are also provided, along with optional instructions for use, such as administration instructions. In some embodiments, the instructions provide guidance or specify methods for assessing, prior to receiving cell therapy, whether a subject is likely or suspected of being able to produce a response and / or the extent or level of such response following administration of differentiated cells expressing recombinant receptors to treat a disease or condition. In some aspects, the articles may contain a dose of differentiated cells or a composition of differentiated cells.

[0303] The articles of manufacture provided herein contain packaging materials. Packaging materials used to package the provided materials are well known to those skilled in the art. See, for example, U.S. Patents 5,323,907, 5,052,558, and 5,033,252, each of which is incorporated herein in its entirety. Examples of packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, disposable laboratory supplies such as pipette tips and / or plastic sheets, or bottles. Articles of manufacture or kits may include a device to facilitate the dispensing of materials or to facilitate use in a high-throughput or large-scale manner, such as for use in robotic equipment. Typically, the packaging does not react with the composition contained therein.

[0304] In some embodiments, the reagents and / or cell compositions are packaged individually. In some embodiments, each container may have a single compartment. In some embodiments, other components of the product or kit are packaged individually or together in a single compartment.

[0305] Example The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0306] A. Example 1: Neuronal Differentiation of iPSCs Induced pluripotent stem cells (iPSCs) are generated from fibroblasts obtained from human donors with Parkinson's disease and undergo a dopaminergic neuron differentiation protocol.

[0307] iPSCs from human donors were maintained by plate seeding in laminin-511 E8-coated 6-well plates. In the absence of feeder cells, iPSCs were developed using mTeSR-based methods. ™ Cells were cultured in a medium containing 1% until they reached approximately 75%–90% confluence. The iPSCs were then washed with sterile PBS and passed through a medium containing Accutase. ™ The iPSCs were separated from the 6-well plates by enzymatic dissociation. The collected iPSCs were then used in subsequent differentiation protocols.

[0308] The collected iPSCs were resuspended in basal induction medium (see below) and used in 24-well AggreWell media. ™ The plates were inoculated under non-adherent conditions. On day 0, the inoculum was 3.6 × 10⁻⁶. 6 iPSCs were seeded at approximately 3,000 cells / well in the supplemental medium described below for each day listed for the differentiation method, e.g., day 0 through day 16. Cells were cultured under non-adherent conditions for 7 days, with the medium replaced as detailed below to form spheroids. On day 7, cells were seeded with Accutase... ™ Enzymatic dissociation was performed to separate the resulting spheres into single cells, and the cells were arranged in a monolayer at a density of 800,000 cells / cm². 2 The concentration of the plate was inoculated onto a plate coated with the laminin 511 E8 fragment for the remaining culture, and further supplemented with nutrients and small molecules as described below.

[0309] Figure 1 Table E1 illustrates an exemplary non-adherent differentiation protocol, depicting the addition of small molecule compounds at different days during the differentiation process. From day 0 to day 10, cells were cultured in a basal induction medium formulated to contain Neurobasal at a 1:1 ratio. ™ Culture medium and DMEM / F12 medium (with N-2 and B27 supplements, non-essential amino acids (NEAA), GlutaMAX) ™ L-glutamine, β-mercaptoethanol, and insulin were added, along with appropriate small molecule compounds. From day 11 to harvest, for example, day 16, the cells were placed in "maturation medium" (containing N-2 and B27 supplements, non-essential amino acids (NEAA), and GlutaMAX). ™ Neurobasal ™ They are cultured in a medium supplemented with appropriate small molecule compounds. The basal induction medium also includes serum substitutes.

[0310] S: Serum substitute; LDN: LDN193189; SB: SB431542; SHH: Recombinant mouse sound hedgehog factor (rmSHH); PUR: Purometamorphine; CHIR: CHIR99021; ROCKi: Y-27632; BDNF: Recombinant human brain-derived neurotrophic factor (rhBDNF); GDNF: Recombinant human glial cell-derived neurotrophic factor (rhGDNF); TGFβ3: Recombinant human transforming growth factor β3 (rhTGFβ3); dbcAMP: Butyryl cyclic AMP; Ascorbic acid: Ascorbic acid; The indicator medium was supplemented with a ROCK inhibitor (Y-27632). On day 0, the basal induction medium was prepared containing 5% serum substitute, 0.1 µM LDN (1×), 10 µM MSB (1×), and 10 µM ROCK inhibitor Y-27632. On day 1, the basal induction medium was prepared containing 5% serum substitute, 0.2 µM LDN (2×), 20 µM SB (2×), 0.2 µg / mL SHH (2×), 4 µM PUR (2×), and 2 µM GSK3β inhibitor CHIR99021, and was added via 50% medium replacement. Since the basal induction medium added from day 1 to day 6 was added via daily 50% medium replacement, the concentrations of small molecule LDN, SHH, and PUR in the basal induction medium were doubled (2×) from day 1 to day 6 compared to a complete medium replacement, and the concentration of small molecule SB was doubled (2×) from day 1 to day 4 compared to a complete medium replacement. Similarly, the basal induction medium for days 2 to 6 was prepared to contain 2% serum substitute and 4 µM CHIR99021. Therefore, the basal induction medium for days 2 to 4 contained 2% serum substitute, 0.2 µM LDN, 20 µM SB, 0.2 µg / mL SHH, 4 µM PUR, and 4 µM CHIR99021, and the basal induction medium for days 5 and 6 contained 2% serum substitute, 0.2 µM LDN, 0.2 µg / mL SHH, 4 µM PUR, and 4 µM CHIR99021.

[0311] As described above, on day 7, cells were transferred to substrate-coated plates using basal induction medium prepared with 2% serum substitute, 0.1 µM LDN, 2 µM CHIR99021, and 10 µM Y-27632. From day 8 to day 10, the medium was completely replaced daily with basal induction medium prepared with 2% serum substitute, 0.1 µM LDN, and 2 µM CHIR99021.

[0312] Starting on day 11, the culture medium was replaced with mature medium, which was prepared containing: 20 ng / mL BDNF, 0.2 mM ascorbic acid, 20 ng / mL GDNF, 0.5 mM dbcAMP, and 1 ng / mL TGFβ3 (collectively referred to as "BAGCT"), 10 µM DAPT, and 2 µM CHIR99021. On day 12, the medium was completely replaced with the same medium preparation containing the same concentrations of small molecule compounds as on day 11. From day 13 until harvest, the medium was replaced daily with mature medium, which was prepared containing the same concentrations of BAGCT and DAPT as on days 11 and 12 (collectively referred to as "BAGCT / DAPT").

[0313] Cells were harvested on day 16.

[0314] B. Example 2: Comparative Analysis of Neuron Differentiation Methods The neuron differentiation method described in Example 1 was compared with the efflorescent neuron differentiation method and three alternative non-efflorescent neuron differentiation methods.

[0315] iPSCs were generated from fibroblasts obtained from human donors with Parkinson's disease and, for comparative purposes, underwent each of the five neuronal differentiation methods until at least day 10.

[0316] The adherent neuron differentiation method (also referred to in this paper as "2D adherent culture") involves culturing iPSCs in mTeSR1-based medium until they reach approximately 75%–90% confluence. The iPSCs are then washed with sterile PBS and differentiated using Accutase. ™ Cells were separated from 6-well plates by enzymatic dissociation. On day -1, cells were inoculated at approximately 300,000 cells / cm² with 10 µM of the ROCK inhibitor Y-27632. 2 Inoculated overnight, each well of a 6-well plate contained 2.9 mg / L of mTeSR1-based medium. 6 Cells were then cultured from day 0 in basal induction medium containing the components shown in Table E2 below, continuing each day listed until day 10. Each day from day 0 to day 10, the medium was completely replaced with the medium shown in Table E2. When indicated, the basal induction medium contained: LDN at a concentration of 0.1 µM LDN; SB at a concentration of 10 µM SB; SHH at a concentration of 0.1 µg / mL; and PUR at a concentration of 2 µM.

[0317] S: Serum substitute; LDN: LDN193189; SB: SB431542; SHH: Recombinant mouse sound hedgehog factor (rmSHH); PUR: Purometamorphine; CHIR: CHIR99021; ROCKi: Y-27632 In addition to three alternative non-adherent methods (referred to as "3D Condition 1", "3D Condition 3", and "3D Condition 4"), a non-adherent neuronal differentiation method as described in Example 1 (also referred to as "3D Condition 2") was performed. Except for the differences indicated in Table E3 below, each of the three alternative non-adherent methods used the same method as 3D Condition 2. Specifically, each of 3D Conditions 1, 2, 3, and 4 was identical except for the small molecules contained in the basal induction medium on day 0 and day 1. For each of 3D Conditions 1, 2, 3, and 4, the basal induction medium on day 2 and thereafter was identical, resulting in the use of the same method and medium except for day 0 and day 1, as shown in Table E3. Specifically, the basal induction medium for 3D Condition 1 prepared for day 1 did not contain CHIR until day 2, while the basal induction media for 3D Conditions 2 and 3 prepared for day 1 each contained different concentrations of CHIR (2 μM for 3D Condition 2 and 4 μM for 3D Condition 3). The basal induction medium for 3D Condition 4 prepared for Day 0 also contained indicated concentrations of SHH, PUR, and CHIR. 3D Condition 4 is a non-adherent neuron differentiation method as described in Example 1 of WO2021 / 146349.

[0318] Cells were differentiated using each of the five differentiation methods, and the expression of various biomarkers was analyzed on days including day 7 and day 10.

[0319] EN1 is a marker of the midbrain dopaminergic spectrum. On day 7, the expression levels of the EN1 dopaminergic spectrum marker were compared between cells derived from each of the five differentiation methods. Figure 2 As shown, cells cultured using the 2D adherent culture method exhibited very low EN1 expression on day 7, the lowest among all five methods tested, while cells cultured using 3D condition 2 exhibited the highest EN1 expression on day 7 among all five differentiation methods, and on average almost three times that of cells cultured using 3D condition 4.

[0320] GBX2 and NKX2-1 are off-target non-dopaminergic biomarkers. For example... Figure 2As shown, on day 7, GBX2 marker expression was relatively low for all five differentiation methods, averaging less than 30 per million for each method, and NKX2-1 marker expression was also very low (less than 30 per million) for all four non-adherent methods (3D conditions 1, 2, 3, and 4). Surprisingly, the non-adherent methods (3D conditions 1, 2, 3, and 4) showed significantly less NKX2-1 marker expression on day 7 than the 2D adherent culture methods (averaging approximately 1 / 10), demonstrating the superior ability of non-adherent methods (including 3D condition 2) to preferentially promote dopaminergic lineage fate.

[0321] LMX1A is a midbrain ventral biomarker that indicates dopaminergic spectrum fate. For example... Figure 3A and Figure 3B As shown, on day 10, LMX1A expression in cells cultured in 3D conditions 1 and 2 was similar to that in cells cultured in 2D adherent culture, and both were higher than that in cells cultured in 3D conditions 3 and 4.

[0322] In summary, the data demonstrate that 3D condition 2 is superior in promoting dopaminergic lineage fate in the early stages of differentiation compared to the 2D adherent culture method and three alternative non-adherent methods (3D conditions 1, 3, and 4). The three alternative non-adherent methods include 3D condition 4, in which cells are exposed to LDN, SB, SHH, PUR, and CHIR starting on day 0.

[0323] Figure 5 The comparison of neuronal marker expression using adherent cell culture and the day 1 suspension culture protocol described herein is shown. Day 1 suspension culture cells were harvested on day 16, while adherent culture cells were harvested on day 20. Figure 5 A shows the expression of the pan-neuronal marker EPHB2, which typically indicates neuronal cell lineage. Neurons obtained using adherent and suspension cultures expressed similar levels of EPHB2, indicating that both differentiation protocols produced neuronal progenitors. Figure 5 B illustrates FOXA2 expression, which is characteristic of the lamina lineage in brain development. The desired dopaminergic neuronal progenitors are of the lamina lineage. Neurons obtained using a cell suspension culture method exhibited higher FOXA2 expression than neurons produced using adherent culture, demonstrating that the suspension culture differentiation protocol more accurately guides cells to the desired midbrain ventral dopaminergic neuronal fate. Figure 5C shows a comparison of PAX6 expression in cells produced using adherent culture versus cells produced using a suspension culture protocol. PAX6 expression in the forebrain lineage during brain development. Cells differentiated using the suspension culture method showed approximately 1 / 10 less PAX6 expression compared to cells produced using adherent culture, further confirming that the suspension culture differentiation method more accurately guides differentiation to the desired midbrain ventral dopaminergic neuron cell fate.

[0324] Cells obtained using the "Day 1" suspension culture differentiation method described herein were then compared with cells produced using an adherent culture differentiation protocol to compare predicted graft size and dopamine production after implantation into the subject's brain. Day 1 suspension culture cells were harvested on day 16, while adherent culture cells were harvested on day 20. Predicted graft size was obtained from GraftTest. ™ The model, obtained using batch RNAseq data, is a computer prediction tool that estimates graft size (the number of human cell nuclei in a rodent's brain hemisphere). It is described in U.S. Provisional Application No. 63 / 598,533, filed November 13, 2023, entitled "METHODS OF PREDICTING CHARACTERISTICS OF DIFFERENTIATED NEURONAL CELLS". The model is trained based on graft characteristics (human cell nuclei), measured 21 days after precursor cells are implanted in the rodent's brain and mature. Importantly, the predictions are underestimations of the total graft size because the model is trained based on cell counts found in one-sixth of the total graft volume in each hemisphere. Each estimate represents the average of several hemispheres. Training data typically includes estimates based on four to five rodents and two hemispheres per rodent. Figure 6 A shows that dopaminergic neuronal progenitors generated using suspension culture differentiation are expected to produce significantly larger grafts after implantation compared to neuronal progenitors generated using adherent culture differentiation.

[0325] Compared to cells generated using the adherent culture differentiation protocol, cells generated using the day-1 suspension culture differentiation method showed predicted dopamine release levels after implantation. Figure 6 In section B, suspension culture cells were harvested on day 16, and adherent culture cells were harvested on day 20. Dopamine release levels were predicted using DopaTest. ™DopaTest is a computer-based predictive tool that uses batch RNA-seq data to estimate the amount of dopamine released by cells after extended culture. It is described in U.S. Provisional Application 63 / 598,533. In the training dataset, dopamine is quantified using liquid chromatography-mass spectrometry (LCMS), and the model is trained using transcriptomic features from precursor-focus time points. Therefore, the predicted dopamine release represents a measure of expected cell performance after long-term culture. The dopamine release estimate is based on the predicted dopamine production (nM) of 1e5 mature dopaminergic neurons. Figure 6 As shown in B, there was no significant difference in predicted dopamine release levels between cells produced using day 1 suspension culture and cells produced using adherent culture.

[0326] Dopamine and serotonin release from cells differentiated using day 1 suspension culture was compared with that from cells produced using adherent culture. Day 1 suspension culture cells were harvested on day 16, while adherent culture cells were harvested on day 20. Cells were then cultured for 60 days in defined neuronal maturation medium. On the day of collection, cells were washed once with Winhan's Balanced Salt Solution (HBSS) and then incubated at 37°C in HBSS for 15 minutes. Triple samples were collected after 15 minutes to determine baseline neurotransmitter release. Cells were then incubated at 37°C in 56 mM potassium chloride (KCl) solution in HBSS to induce neurotransmitter release. Triple samples were collected at 30°C. Figure 7 A shows the dopamine concentrations (nM) in the supernatant after baseline and 30 min KCl treatment for both adherent and suspension culture protocols (n=4) without normalization. There was no significant difference in the initial dopamine release between the differentiation conditions (ns p>0.05). Figure 7 B shows the fold change in dopamine release after stimulation relative to baseline. There was no significant difference in initial dopamine release between the adherent and suspension culture protocols (ns, p > 0.05). Error bars are shown as the standard error of the mean (SEM). For all plots, n = 4.

[0327] Figure 8 A shows the serotonin concentrations (nM) in the supernatant after baseline and 30 min KCl treatment for both adherent and suspension culture protocols (n=4) without normalization. There was no significant difference in raw serotonin release between the adherent and suspension culture protocols (ns p>0.05). Figure 8B shows the normalized serotonin release after KCl stimulation compared to baseline. Data showed that cells differentiated using the day-1 suspension culture method had significantly lower serotonin (5HT) production (n=4) compared to cells differentiated using the adherent culture method. Error bars are shown as the standard error of the mean (SEM). These results demonstrate that the day 1 suspension culture differentiation method yields cells with enhanced safety compared to cells produced using adherent cell culture.

[0328] We then compared the viability of cells differentiated using the day 1 suspension culture method with that of cells differentiated using the adherent cell culture method. Day 1 suspension culture cells were harvested on day 16, while adherent culture cells were harvested on day 20. Cell viability was tested by running the "Cell Counting and Viability" protocol on a Nucleocounter NC-200. Cells were generated using either adherent or suspension culture conditions (n=4). Cells generated using the day 1 suspension culture conditions showed a significant increase in the percentage of viable cells, such as... Figure 9 As shown.

[0329] Cells differentiated using the day-1 suspension culture method were compared with those differentiated using the adherent cell culture method in terms of the expression of two "mid-target" markers and two "off-target" markers. Day-1 suspension culture cells were harvested on day 16, while adherent culture cells were harvested on day 20. RNA-seq analysis showed that cells differentiated using the day-1 suspension culture method exhibited higher levels of the "mid-target" FOXA2 than cells differentiated using the adherent method. Figure 10 A) and CORIN expression ( Figure 10 B), thus indicating that cells produced by day 1 suspension culture were more characteristic of the desired dopaminergic neuronal progenitors than cells produced using adherent culture. Cells produced using the day 1 suspension culture method also expressed significantly less of the "off-target" marker PITX2 (B). Figure 9 C) and NKX2.1 Figure 9 (D) provides further evidence that the day 1 suspension culture differentiation method produces cells that are more accurately guided to the desired dopaminergic neuronal progenitor cell type.

[0330] C. Example 3: Analysis of differentiation time using adherent and non-adherent differentiation methods The results obtained in Example 2 demonstrate that differentiating iPSCs using non-adherent conditions (as described in Example 1) advantageously allows for an accelerated timeline of iPSC differentiation into definitive dopaminergic progenitor cells and / or stereotyped dopaminergic progenitor cells. To confirm this, a study was conducted in which iPSCs were differentiated using the non-adherent method as described in Example 1 (also referred to in this example as “3D differentiated samples”) and harvested on day 16, and gene expression data from those cells were compared with gene expression data obtained from cells differentiated using the 2D adherent culture method of Example 2 and harvested on days 17, 18, 19, 20, 21, 22, and 25 (also referred to in this example as “2D differentiated” samples). The 2D adherent culture method involved culturing cells as described in Example 2 until day 10, and from day 11 until harvest, using the maturation medium as described in Example 1. Cells cultured using the 2D adherent culture method were passaged on day 16 and, if applicable, day 20 in maturation medium also containing a ROCK inhibitor.

[0331] The relative transcriptomic maturity of the cell formulation was assessed by supervised principal component analysis (PCA). Using the aforementioned 2D adherent culture method, the gene set used in the differentiation timeline assay of 2D differentiated dopamine neuron precursor cells (DANPC) was identified. Briefly, batch RNAseq was performed at each of the seven time points corresponding to days 17, 18, 19, 20, 21, 22, and 25 of differentiation. n =12 independent DANPCs (training set). Implemented in R (R Core Team, R: A language and environment for statistical computing, 2021, R Foundation for Statistical Computing, Vienna, Austria). edgeR The library (Robinson et al., Bioinformatics, 2009, 26(1): 139-140) identified genes whose expression was statistically correlated with monotonic changes over time. The statistical significance was then assessed. p The top 50 downregulated genes by score were used as the gene set for supervisory PCA. The first principal component (PC1) of the training set explained approximately 85.8% of the differences in gene expression in the maturity gene set and was subsequently used as a representative of relative transcriptome maturity. Cell formulations with higher PC1 scores were associated with more mature transcriptomes, and vice versa.

[0332] The test set consists of n=Composed of 15 3D differentiated samples, which were differentiated using a non-adherent method as described in Example 1 and analyzed by batch RNAseq on day 16. The transcriptome of the test set was sorted using trained supervised PCA (above), and the predicted PC1 values ​​were compared with those of the training set (see Figure 4 ).

[0333] like Figure 4 As shown, all 16 3D differentiation samples using the non-adherent method as described in Example 1 contained transcriptomes, and the average maturity of these transcriptomes was higher than the average maturity of the 2D differentiation training set using the 2D adherent culture method described above on day 20. Fifteen out of sixteen (93.8%) of the 3D differentiation samples were within the upper limit of the standard deviation of 1.96 × the mean of the 2D differentiated cells on day 20. One 3D differentiation sample was outside this range and was more consistent with the samples from days 21–22. Most of the day 16 3D differentiation samples overlapped with the PC1 distribution of the day 21 2D differentiation training set.

[0334] These data demonstrate that, compared to other methods such as 2D adherent culture, the non-adherent "day 1" suspension culture method of Example 1 accelerates the differentiation timeline of iPSC-derived decisive dopaminergic neuronal progenitors and / or morphologic dopaminergic neuronal progenitors. This is advantageous in several respects, particularly regarding manufacturing, as it reduces the time and amount of resources required to produce iPSC-derived decisive dopaminergic neuronal progenitors and / or morphologic dopaminergic neuronal progenitors suitable for therapeutic use.

[0335] The scope of this invention is not intended to be limited to the specific embodiments disclosed, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods will become apparent from the description and teachings herein. Such changes may be made without departing from the true scope and spirit of this disclosure and are intended to fall within its scope.

Claims

1. A method for differentiating pluripotent stem cells into dopaminergic neuronal progenitor cells, the method comprising: a) Performing a first incubation, said first incubation comprising non-adherent culture of pluripotent stem cells in a first culture dish under conditions that generate cell spheroids, wherein said first incubation comprises: i) Expose the pluripotent stem cells to at least one TGF-β / activin-Nodal signaling inhibitor and at least one bone morphogenetic protein (BMP) signaling inhibitor for at least one day (day 0) in the absence of: x) a sound hedgehog factor (SHH) signaling activator, and y) a glycogen synthase kinase 3β (GSK3β) signaling inhibitor; and ii) Starting from the second day after the first incubation (Day 1), the pluripotent stem cells are exposed to at least one sound hedgehog factor (SHH) signaling activator and at least one glycogen synthase kinase 3β (GSK3β) signaling inhibitor; and b) Perform a second incubation, which includes adhering the cells of the sphere to a second culture dish under conditions that allow the cells to further differentiate into dopaminergic neuronal progenitor cells.

2. The method according to claim 1, wherein the dopaminergic neuron progenitor is a definitive dopaminergic neuron progenitor.

3. The method according to claim 1, wherein the pluripotent stem cell is an induced pluripotent stem cell.

4. The method of claim 1, wherein the pluripotent stem cells are autologous to the subject to be treated with the dopaminergic neuronal progenitor cells.

5. The method of claim 1, wherein the first incubation further comprises exposing the pluripotent stem cells to a ROCK inhibitor (ROCKi) starting from day 0.

6. The method of claim 5, wherein during the first incubation, the pluripotent stem cells are not exposed to ROCKi prior to exposure to the TGF-β / activin-Nodal signaling inhibitor and the bone morphogenetic protein (BMP) signaling inhibitor.

7. The method of claim 1, wherein the method comprises: […] the pluripotent stem cells; a) Exposure to the TGF-β / activin-Nodal signaling inhibitor from day 0 until day 4; b) Exposure to the BMP signaling inhibitor from day 0 until day 10; c) Exposure to the Hedgehog Factor Signaling Activator from day 1 until day 6; and d) Exposure to the glycogen synthase kinase 3β (GSK3β) signaling inhibitor from day 1 until day 12.

8. The method according to claim 1, wherein the BMP signaling inhibitor is LDN193189.

9. The method of claim 8, wherein the cells are exposed to LDN193189 at a concentration between about 10 nM and 500 nM, between about 20 nM and about 400 nM, between about 50 nM and about 200 nM, or between about 75 nM and about 150 nM, optionally about 100 nM.

10. The method of claim 1, wherein the TGF-β / activin-Nodal signaling inhibitor is SB431542.

11. The method of claim 10, wherein the cells are exposed to SB431542 at a concentration between about 1 µM and about 20 µM, between about 5 µM and about 15 µM, or between about 8 µM and about 12 µM, optionally about 10 µM.

12. The method according to claim 1, wherein the SHH signal transduction activator is SHH or purinemorphine.

13. The method of claim 12, wherein the cells are exposed to SHH at a concentration between about 10 ng / mL and 500 ng / mL, between about 20 ng / mL and about 400 ng / mL, between about 50 ng / mL and about 200 ng / mL, or between about 75 ng / mL and about 150 ng / mL, optionally about 100 ng / mL.

14. The method of claim 12, wherein the cells are exposed to purinemorphine at a concentration between about 0.1 µM and about 20 µM, between about 0.5 µM and about 10 µM, between about 1 µM and about 5 µM, between about 1 µM and about 3 µM, or between about 1.5 µM and about 2.5 µM, optionally about 2 µM.

15. The method according to claim 1, wherein the GSK3β signaling inhibitor is CHIR99021.

16. The method of claim 15, wherein the cells are exposed to CHIR99021 at a concentration between about 0.1 µM and about 5 µM, between about 0.5 µM and about 4 µM, between about 0.5 µM and about 2 µM, optionally about 1 µM; and on each day from day 2 to day 12, the cells are exposed to CHIR99021 at a concentration between about 0.1 µM and about 5 µM, between about 0.5 µM and about 4 µM, or between about 1 µM and about 3 µM, optionally about 2 µM.

17. The method of claim 1, wherein the first incubation comprises changing the culture medium on one or more days from day 1 to day 6.

18. The method of claim 17, wherein the first incubation comprises changing the culture medium on each day from day 1 to day 6.

19. The method of claim 1, wherein the second incubation begins on day 7 or about day 7.

20. The method of claim 1, wherein the cells of the sphere are dissociated to produce a cell suspension prior to the second incubation, and the cells of the cell suspension are cultured in an adherent culture dish.

21. The method of claim 1, wherein the second incubation comprises exposing the cells of the sphere to a bone morphogenetic protein (BMP) signaling inhibitor and a GSK3β signaling inhibitor.

22. The method of claim 21, wherein the second incubation further comprises exposing the cells to: (i) brain-derived neurotrophic factor (BDNF); (ii) ascorbic acid; (iii) glial cell-derived neurotrophic factor (GDNF); (iv) dibutyryl cyclic AMP (dbcAMP); (v) transforming growth factor β-3 (TGFβ3) (collectively, "BAGCT"); and (vi) Notch signaling inhibitors.

23. The method of claim 1, wherein the method further comprises harvesting the dopaminergic neuron progenitor cells.

24. The method of claim 23, wherein the dopaminergic neuronal progenitor cells are harvested on day 14 or later.

25. The method of claim 23, wherein the method further comprises preparing the harvested dopaminergic neuronal progenitor cells together with a cryoprotectant.

26. The method of claim 25, wherein the method further comprises cryopreserving the prepared harvested dopaminergic neuronal progenitor cells.

27. A therapeutic composition comprising dopaminergic neuronal progenitor cells generated using a method comprising the following process: a) Performing a first incubation, said first incubation comprising non-adherent culture of pluripotent stem cells in a first culture dish under conditions that generate cell spheroids, wherein said first incubation comprises: i) Expose the pluripotent stem cells to at least one TGF-β / activin-Nodal signaling inhibitor and at least one bone morphogenetic protein (BMP) signaling inhibitor for at least one day (day 0) in the absence of: x) a sound hedgehog factor (SHH) signaling activator, and y) a glycogen synthase kinase 3β (GSK3β) signaling inhibitor; and ii) Starting from the second day after the first incubation (Day 1), the pluripotent stem cells are exposed to at least one sound hedgehog factor (SHH) signaling activator and a glycogen synthase kinase 3β (GSK3β) signaling inhibitor; and b) Perform a second incubation, which includes adhering the cells of the sphere to a second culture dish under conditions that allow the cells to further differentiate into dopaminergic neuronal progenitor cells.

28. The therapeutic composition of claim 27, wherein the therapeutic composition comprises dopaminergic neuronal progenitor cells that, compared with neurons generated using an adherent culture differentiation method, exhibit one or more characteristics selected from the group consisting of: a) Expresses high levels of FOXA2; b) Expressing lower levels of PAX6; c) High predicted graft size after implantation; d) It has a high predictive level of dopamine production after implantation; e) Produces less serotonin; f) Contains a high percentage of live cells; g) Expressing high levels of CORIN; h) expressing low levels of PITX2; and i) Expressing lower levels of NKX2.

1.

29. The therapeutic composition of claim 28, wherein the therapeutic composition comprises dopaminergic neuronal progenitor cells that exhibit three or more characteristics selected from the group thereof, compared to neurons generated using an adherent culture differentiation method.

30. The therapeutic composition of claim 27, wherein the therapeutic composition comprises dopaminergic neuronal progenitor cells exhibiting one or more properties selected from the group consisting of: a) Produces serotonin levels below 2 nM and increases less than twice the baseline level when stimulated with KCl; b) More than 90% of the dopaminergic neuronal progenitor cells in the composition are viable; c) Express FOXA2 at greater than 100 TPM in batch RNAseq analysis; d) Express CORIN at greater than 300 TPM in batch RNAseq analysis; e) Express PITX2 at less than 50 TPM in batch RNAseq analysis; f) NKX2.1 was expressed at less than 10 TPM in batch RNAseq analysis; g) More than 90% of FOXA2-positive cells; and h) GraftTest with at least 1500 ™ score.

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