Dopaminergic Precursor Cells and Methods of Use

Abstract

Provided herein are midbrain dopaminergic neuron progenitor cells that can be used to treat brain disorders. Improved mono-SMAD methods are provided that can be used to differentiate pluripotent cells into midbrain dopaminergic (DA) neurons or midbrain neuron precursors. In some aspects, methods are provided for mono-SMAD culture protocols and culture periods that can be used to generate dopaminergic neuron progenitor cells with significantly improved properties for treating brain disorders, such as Parkinson's disease. Methods of treating Parkinson's disease and other brain disorders with midbrain dopaminergic neuron progenitor cells are also provided.

Description

[Technical field]

[0001] Claiming priority This application claims the benefit of priority to U.S. Provisional Application No. 63 / 171,837, filed April 7, 2021, and U.S. Provisional Application No. 63 / 275,691, filed November 4, 2021, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] 1. Field of the invention The present invention relates generally to the fields of molecular biology and medicine, and more specifically to methods for producing neuronal progenitor cells from pluripotent stem cells and related treatment methods.

[0003] 2. Description of Related Technology Parkinson's disease (PD) is a debilitating neurodegenerative disorder that manifests as movement disorders due to a loss of A9 midbrain dopaminergic (mDA) neurons and subsequent reduction in dopamine neuron signaling. Current PD treatments, including dopaminergic drug therapy and deep brain stimulation (DBS), address the improvement of motor symptoms.

[0004] The clinical and societal costs of PD are predicted to rise, and current treatment options show significant limitations. As the population continues to age, PD is predicted to increase dramatically, with a conservative estimate of over 14 million patients worldwide by 2040 (Dorsey & Bloem, 2018). Although PD patients may present with a wide range of non-motor characteristics, the defining symptom is progressive movement disorders due to dopaminergic dysfunction in the striatum secondary to a loss of dopaminergic substantia nigra neurons. Current treatments are symptomatic and primarily focused on reversing movement disorders.

[0005] Dopamine agonist delivery typically provides only mild to moderate relief. Treatment with L-dopa requires careful dosing, is usually only effective for 4-6 years, and often results in dyskinesias. For example, oral L-DOPA or dopaminergic agonists may provide initial relief of motor symptoms, but after 5-10 years most patients experience debilitating motor fluctuations and dyskinesias (Ahlskog & Muenter, 2001). DBS requires the use of invasive implants, has well-known neuropsychiatric side effects, and is typically effective for less than 10 years. DBS stimulation of the subthalamic nucleus (STN) or internal segment of the globus pallidus can compensate for DA loss in some patients, but this approach is primarily indicated for younger patients who do not exhibit cognitive decline and requires regular battery replacement. None of these treatments address the underlying disease pathology, the progressive loss of mDA neurons.

[0006] Cell transplantation therapy has been tested, but these efforts have been accompanied by significant limitations. Cell-based therapy to replace lost cells and provide relief of PD motor symptoms for 10–15 years is a major goal for the treatment of PD. Studies using fetal tissue have been conducted (Brundin et al., 1986; Doucet et al., 1989; Freeman, Sanberg, et al., 1995). Studies using fetal ventral midbrain (fVM) cells as a source of midbrain neural progenitor cells have been conducted (Barker et al., 2013), but the effective results among several different studies involving cell transplantation have been mixed or inconsistent (e.g., Barbuti et al., 2021; Barker, Drouin-Ouellet, & Parmar, 2015).

[0007] Replacement therapy involving administration of fVM cells presents both ethnic and technical limitations. Dopamine neurons from rodent and human fetal ventral midbrain (hfVM) donor tissue can be therapeutically beneficial when grafted into the dopamine-depleted striatum of experimental animals (Steinbeck & Studer, 2015; Wianny & Vezoli, 2017). Some patients in open-label hfVM trials (Freeman, Olanow, et al., 1995; Lindvall et al., 1990) have shown clinical improvement. However, although predefined secondary endpoints (Unified Parkinson's Disease Rating Scale, UPDRS) showed statistically significant benefits in younger (age < 60 years, (Freed et al., 2001)) or less impaired (UPDRS < 49 on OFF, (Olanow et al., 2003)) subjects, randomized double-blind placebo-controlled clinical trials showed that these benefits were too variable to meet the primary endpoint of the study. In addition, some patients developed graft-induced dyskinesias (GIDs) (Freed et al., 2001; Hagell et al., 2002; Ma et al., 2002), which may be related to pre-existing L-DOPA-induced dyskinesias and the grafts containing serotonergic cells along with the desired dopaminergic neurons (Hagell & Cenci, 2005; Lane & Smith, 2010). These findings prompted a reevaluation of the approach. More recently, the European collaborative consortium TRANSEURO revisited fetal transfer in an open-label study (NCT01898390) in 11 patients at a relatively early disease stage who had not developed L-DOPA-induced dyskinesia prior to grafting ( Barker & consortium, 2019 ).

[0008] There is currently no treatment that can stop or reverse the disease process, so there is a large unmet need for new effective PD treatments. Due to limited donor tissue availability and the ethical issues of using fetal tissue, fVM treatment is not considered useful for widespread clinical use, and therefore other cell sources such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are being investigated. There is clearly a need for new and improved methods of generating dopaminergic neurons from pluripotent cells that can be used, for example, to treat PD. Summary of the Invention [Problem to be solved by the invention]

[0009] In some aspects, the present disclosure overcomes limitations of the prior art by providing cultures of dopaminergic (DA) progenitor cells, preferably progenitor midbrain dopaminergic (mDA) cell cultures, with improved properties for treating disease or engraftment in mammalian subjects. Methods for generating such DA progenitor cell cultures from pluripotent stem cells, e.g., induced pluripotent stem (iPS) cells, are provided. In contrast to previous experiments using the mono-SMAD method described in WO2018 / 035214, where comparable patterning of DA progenitor cells was observed at several time points toward day 37 of differentiation culture, the present disclosure is based in part on the discovery that DA progenitor cells utilized after about 360-456 hours, more preferably about 384-432 hours, of differentiation culture using the mono-SMAD method can surprisingly exhibit superior properties for therapeutic applications, such as treating Parkinson's disease (PD). As shown in the examples below, these cells differentiated under these conditions for these periods were observed to show dramatic improvements in engraftment and innervation in vivo using the 6-OHDA athymic nude rat model of PD. These periods in the mono-SMAD differentiation method provided herein are therefore believed to be critical for generating cultures of cells with dramatically improved therapeutic potential. Behavioral experiments in rats receiving cells administered to the striatum resulted in improved treatment and in vivo recovery of PD symptoms. Cell maturity for survival and efficacy of transplanted mDA progenitor cells, immature neurons, and postmitotic neurons was examined in hemiparkinsonian rats. Midbrain DA progenitor cells were significantly superior to immature or mature neurons in terms of survival, innervation, and efficacy. Homotopic (intrasubstantia nigra) engraftment demonstrated that mDA progenitor cells had a greater capacity than immature neurons to innervate forebrain structures over long distances. Clear dose responses in structure and in function were observed when progenitor cells were evaluated over a wide dose range. Although the grafts were iPSC-derived, no teratomas or significant cell proliferation were observed. These data support the use of human iPSC-derived mDA progenitor cells for transplantation to treat PD.Also provided are methods of treating brain disorders, such as PD, using the cell cultures or DA progenitor cells provided herein. [Means for solving the problem]

[0010] In some aspects, cryopreserved single cell suspensions containing iPSC-derived midbrain DA neuron progenitor cells (e.g., "FCDI DAPC-1" or D17 cells) are provided. DA progenitor cells can be generated using about 360-456 hours, more preferably about 384-432 hours, of differentiation in culture using the mono-SMAD method provided herein. These cells can be derived from allogeneic human iPS cells or iPS cell lines via directed differentiation to obtain a population of DA neuron progenitor cells. As shown in the Examples below, such DA neuron progenitor cells were observed to have phenotypic markers (e.g., Figures 1 and 2) and developmental potential similar to dopamine neuron precursors found in the substantia nigra region of the developing midbrain (e.g., Figures 3, 13, and 14). FCDI DAPC-1 was observed to lack forebrain neurons and a notable number of residual iPSCs that may be detrimental for therapeutic use (e.g., Figures 5, 6, and Table 3). Unlike other DA cells considered for therapeutic use, FCDI DAPC-1 are a proliferative neural progenitor population as demonstrated by EdU incorporation (Figure 7). FCDI DAPC-1 showed superior engraftment and innervation, features associated with improved recovery in the 6-OHDA athymic rat model of PD (Figures 9, 10, and 14).

[0011] In some embodiments, dopaminergic neuronal precursor cells (e.g., FCDI DAPC-1) can be produced by culturing pluripotent stem cells, e.g., iPS cells, using a mono-SMAD method in which the cells are cultured under differentiation conditions for about 360-456 hours, more preferably about 384-432 hours. Mono-SMAD differentiation methods (also referred to as "mono-SMAD inhibition" or "mono-SMADi" methods) are described, for example, in WO2018 / 035214, which is incorporated herein by reference in its entirety. Mono-SMADDi methods can provide advantages over methods that require inhibition of SMAD signaling using two or more SMAD inhibitors. Generally, mono-SMAD methods involve the use of only one SMAD inhibitor (i.e., a single SMAD inhibitor, not a second SMAD inhibitor). Mono-SMAD methods may include: (i) adjusting the addition of Wnt agonist (e.g., CHIR99021) on days 2 or 3, (ii) reoptimizing the CHIR concentration (e.g., about 0.5-3.0 μM, 0.7-3 μM, 1-2.5 μM, 1.25-2.25 μM, about 1.25 μM to greater than about 2 μM, or about 1.55, 1.65, 1.75 μM, or using any range derivable therebetween), and / or (iii) including a MEK inhibitor (e.g., PD0325901) in the differentiation medium on days 3 to 5. Methods may include, for example, aspects (i and ii), (ii and iii), (i and iii), or (i, ii and iii) above. In some embodiments, the cells are exposed to a BMP inhibitor (e.g., dorsomorphin or LDN-193189), but the cells are not exposed to a TGF-β inhibitor, such as SB431542. The cells are exposed to midbrain DA neurons or FOXA2 + / LMX1A + The cells may be differentiated into cells. These methods may be used for the generation of mDA progenitor cells from iPS cell lines using medium containing only a single SMAD inhibitor (e.g., only dorsomorphin, or only LDN-193189).

[0012] One aspect of the disclosure is a method for the treatment of cancer, comprising administering to the patient a signal transduction modulator comprising: (a) Small Mothers Against Decapentaplegic (Small Mothers Against Decapentaplegic); 1. A culture comprising midbrain dopaminergic (mDA) neuron progenitor cells generated by culturing human pluripotent cells in the presence of (a) a first inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling, (b) at least one activator of Sonic Hedgehog (SHH) signaling, and (c) at least one activator of Wingless (Wnt) signaling, wherein the method does not include culturing the human pluripotent cells in the presence of a second inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling, the human pluripotent cells are cultured under differentiation-inducing conditions for about 360 to about 456 hours, and the cells are then refrigerated or cryopreserved, and the midbrain dopaminergic progenitor cells express both Forkhead box protein A2 (FOXA2) and LIM homeobox transcription factor 1 (LMX1) (FOXA2). + / LMX1 +In some embodiments, the human pluripotent cells are cultured for about 384 to about 432 hours under conditions inducing differentiation. In some embodiments, the mDA neuron progenitor cells do not express NURR1. The mDA neuron progenitor cells may express Forkhead box protein A2 (FOXA2), LIM homeobox transcription factor 1 (LMX1) and EN1. The mDA neuron progenitor cells may further express OTX2. In some embodiments, Forkhead box protein A2 (FOXA2) and LIM homeobox transcription factor 1 (LMX1) are co-expressed by about 60% to about 100% or about 85% to about 95% or more of the mDA neuron progenitor cells. In some embodiments, about 65-75% of the mDA neuron precursors co-express both FOXA2 and LMX1. In some embodiments, the midbrain dopaminergic progenitor cells express (FOXA2, LMX1A, ETV5, and EN1) and the midbrain dopaminergic progenitor cells do not express (NURR1, TH, CALB1, BARHL1, and GRIK2). In some embodiments, the mDA neuron progenitor cells include proliferating or dividing cells. In some embodiments, at least about 40% or more or about 50-75% of the mDA neuron progenitor cells are proliferating or dividing. The culture may further include about 5% or less serotonergic neuron progenitor cells. The serotonergic neuron progenitor cells may express BARLH1. The culture may further include glial progenitor cells. The glial progenitor cells may express GLAST, SLC13A, CD44, and / or hGFAP. The inhibitor of SMAD signaling may be a BMP inhibitor, such as, for example, LDN-193189, dorsomorphin, DMH-1, or noggin. In some embodiments, the BMP inhibitor is LDN-193189.LDN-193189 may be present at a concentration of about 0.2 μM to about 4 μM, more preferably about 1 μM to about 4 μM, about 1 μM to about 3 μM, about 0.5 μM to about 4 μM, about 0.5 μM to about 2 μM, about 0.2 μM to about 4 μM, about 0.2 μM to about 2 μM, or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 μM, or any range derivable therebetween. In some embodiments, the SMAD signaling inhibitor is a TGFβ inhibitor. The TGFβ inhibitor may be SB431542. In some embodiments, SB431542 is present at a concentration of about 1-20 μM, about 5-15 μM, about 9-11 μM, or about 10 μM. The pluripotent cells may be cultured with the SMAD inhibitor on days 1-15, 1-16, or 1-17 of culture. The pluripotent cells may be cultured with the SMAD inhibitor on days 1-17 of culture. The pluripotent cells may be cultured with the SMAD inhibitor substantially continuously or daily for 15, 16, or 17 days. The pluripotent cells may be cultured with the SMAD inhibitor substantially continuously or daily for 17 days. The SMAD inhibitor may be present at a concentration of about 50-2000 or 50-500 nM. The SMAD inhibitor may be present at a concentration of about 180-240 nM. The method may further comprise contacting the pluripotent cells with a MEK inhibitor. In some embodiments, the MEK inhibitor is PD0325901. PD0325901 may be present at a concentration of about 0.25-2.5 μM. The MEK inhibitor may be contacted with the pluripotent cells for about 1-3 days, or on days 1-3, 2-4, 3-5, or on days 1, 2, 3, 4, or 5 after initiation of contact with the inhibitor of SMAD signaling. In some embodiments, the MEK inhibitor is contacted with the pluripotent cells for about 24 to about 48 hours after initiation of contact with the inhibitor of SMAD signaling. In some embodiments, the MEK inhibitor is contacted with the pluripotent cells daily or substantially continuously for about 3-4 days beginning about 1-2 days after initiation of contact with the inhibitor of SMAD signaling. In some embodiments, the MEK inhibitor is contacted with the pluripotent cells on days 2-5 or 3-6 after initiation of contact with the inhibitor of SMAD signaling on day 1.The activator of Wnt signaling may be a GSK3 inhibitor. In some embodiments, the GSK3 inhibitor is CHIR99021. CHIR99021 may be present at a concentration of about 1.5-2 μM, about 1.5-1.7 μM, about 1.6-1.7 μM, or about 1.65 μM. In some embodiments, CHIR99021 is present at a concentration of about 4-7 μM 9-17 days after initiation of contact with the inhibitor of SMAD signaling. The activator of Wnt signaling may be contacted with the pluripotent cells for 1-3 days after initiation of contact with the inhibitor of SMAD signaling. The activator of Wnt signaling may be contacted with the pluripotent cells within 24-48 hours after initiation of contact with the inhibitor of SMAD signaling. In some embodiments, the pluripotent cells are cultured with the activator of Wnt signaling substantially continuously or daily for 14, 15, or about 16 days. In some embodiments, the activator of Wnt signaling is contacted with the pluripotent cells 2-17 days after initiation of contact with the inhibitor of SMAD signaling. In some embodiments, the activator of SHH signaling is purmorphamine or C25II Shh. The method may further comprise contacting the pluripotent cells with two activators of SHH signaling. The two activators of SHH signaling may be purmorphamine and C25II Shh. In some embodiments, the at least one activator of SHH signaling is contacted with the pluripotent cells on the same day as initiation of contact with the inhibitor of SMAD signaling or within 24-48 hours after initiation of contact with the inhibitor of SMAD signaling. In some embodiments, the at least one activator of SHH signaling is contacted with the pluripotent cells 1-7 days with or after initiation of contact with the inhibitor of SMAD signaling. The method may further comprise contacting the pluripotent cells with FGF-8. In some embodiments, FGF-8 is not contacted with the pluripotent cells on the same day as initiation of contact with the inhibitor of SMAD signaling. In some embodiments, FGF-8 is contacted with the pluripotent cells 9-17 days or 11-17 days after initiation of contact with the inhibitor of SMAD signaling. FGF-8 may be present at a concentration of about 50-200 ng / mL.The pluripotent cells may comprise an antibiotic resistance transgene under the control of a neuronal promoter. The method may further comprise selecting for neural cells, midbrain DA neurons or mDA neuron progenitors derived from the pluripotent cells by contacting the cells with an antibiotic, a chemotherapeutic agent, a DNA crosslinking agent, a DNA synthesis inhibitor or a mitosis inhibitor. The method may further comprise contacting the pluripotent cells with an antibiotic or a chemotherapeutic agent (e.g., mitomycin C). In some embodiments, mitomycin C is contacted with the pluripotent cells on days 27, 28, 29 and / or 30 after initiation of contact with the inhibitor of SMAD signaling. In some embodiments, the antibiotic is G418 (geneticin). The method may further comprise culturing or incubating the pluripotent cells in medium containing a ROCK inhibitor prior to initiation of contact with the inhibitor of SMAD signaling. The method may further comprise contacting the pluripotent cells with blebbistatin. Blebbistatin may be contacted with the cells on days 5 and 17 of differentiation. In some embodiments, mDA dopaminergic progenitor cells do not express NURR1, MAP2, and TH. Nevertheless, mDA dopaminergic progenitor cells may retain the potential to express NURR1, MAP2, and / or TH, for example, after further growth or differentiation in the future. In some embodiments, mDA dopaminergic progenitor cells express EN1. Although both PITX2 and PITX3 have been observed in mature dopaminergic neurons, mDA dopaminergic progenitor cells may express low levels or substantially no expression of these markers. In some embodiments, mDA dopaminergic progenitor cells express GBX2, OTX1, OTX2, ETV5, CORIN, and / or DCX. In some embodiments, the pluripotent cells are human induced pluripotent stem (iPS) cells. LMX1 can be LIM homeobox transcription factor 1 alpha (LMX1A). In some embodiments, about 5% or less (eg, less than about 1% or less than 0.5%) of the cells in the cell composition are serotonergic cells or serotonergic precursor cells.The method may further comprise incubating the human pluripotent cells in the presence of a DNase or endonuclease (e.g., DNase I or Benzonase®). DNase I or Benzonase® may be present at a concentration of about 10-20 U / mL or about 10-15 U / mL. For example, DNase I Benzonase® may be added on day 17 of culture, e.g., to reduce cell clumping in a cell preparation, e.g., a single cell preparation. In some embodiments, the human pluripotent cells are cultured in the presence of an endonuclease on at least one of days 4-6 after initiation of contact with an inhibitor of SMAD signaling. The human pluripotent cells may be cultured in the presence of an endonuclease on day 5 after initiation of contact with an inhibitor of SMAD signaling. The culture may be contained in a container means. In some embodiments, the midbrain dopaminergic neurons or midbrain dopaminergic neuron progenitor cells are comprised in a pharmaceutical preparation. The pharmaceutical preparation is formulated for injection. In some embodiments, the pharmaceutical preparation comprises a hyaluronic acid matrix. The culture may contain about 2,500 cells / μL to about 150,000 cells / μL, about 2,500 cells / μL to about 100,000 cells / μL, about 10,000 cells / μL to about 150,000 cells / μL, about 40,000 cells / μL to about 100,000 cells / μL, or about 15,000-45,000 cells / μL. The cells may be midbrain dopaminergic neuronal progenitor cells or DAPC-1 cells. The culture may contain about 1×10 cells. 6 From about 25 x 10 6 , more preferably about 3×10 6 Pieces to about 9 x 10 6In some embodiments, about 10% or less of the cells in the culture are serotonergic progenitor cells, more preferably about 7% or less. In some embodiments, about 5% or less of the cells in the culture are serotonergic progenitor cells. In some embodiments, about 5% or less of the cells in the culture express SERT and TPH2. As shown in the Examples below, the cultures contain approximately 5% serotonergic cells (serotonergic progenitor cells) based on the expression of SERT and TPH2 at day 14, and it was observed that no serotonergic neurons remained after engraftment. In some preferred embodiments, the total number of serotonergic cells is about 5% or less, while in some embodiments, the cultures may contain about 6%, 7%, 8% or more serotonergic cells. In some embodiments, about 0.1-5% or less of the cells in the culture express FOXG1, and / or wherein about 0.1-5% or less of the cells in the culture express PAX6. In some embodiments, less than about 1% of the cells in the culture express FOXG1, and / or wherein less than about 1% of the cells in the culture express PAX6.

[0013] Another aspect of the disclosure relates to a method of treating a disease in a mammalian subject comprising administering to the subject a therapeutically effective amount of the culture described above or herein, for example, preferably wherein the culture is administered to the brain of the subject. The mammalian subject may be a human. The disease may be a disease of the central nervous system (CNS). In some embodiments, the disease is Parkinson's disease (PD) or Parkinson plus syndrome (PPS). In some embodiments, the culture comprises mDA progenitor cells that express Engrailed 1 (EN1) but do not express NURR1. In some embodiments, the culture comprises dopaminergic neurons that are not fully differentiated. The culture is administered to the striatum, e.g., putamen or substantia nigra, of the subject. In some embodiments, the culture is administered to more than one location in the striatum or putamen of the subject. The culture is administered to multiple sites and / or with multiple needles in the striatum or putamen of the subject. The culture may be included in a pharmaceutical composition. The pharmaceutical composition may comprise a hyaluronic acid matrix. In some embodiments, the culture comprises about 15,000-45,000 cells / μL or about 2×10 5 , 2.5×10 5 , 3×10 5 , 4×10 5 , 4.5×10 5 The cultures contain approximately 1×10 cells or any range derivable therebetween. 6 From about 25 × 10 6 , more preferably about 3×10 6 From about 9 × 10 6The culture may contain about 2,500 cells / μL to about 150,000 cells / μL, about 10,000 cells / μL to about 150,000 cells / μL, about 40,000 cells / μL to about 100,000 cells / μL, or about 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000 cells / μL, or any range derivable therebetween. In some embodiments, the subject has Parkinson's disease, and the subject shows improvement in at least one motor symptom after administration of the culture. In some embodiments, the subject shows a reduction in one or more of tremor, muscle rigidity, bradykinesia, falls, dizziness, movement freezing, muscle spasms, or dystonia. The midbrain dopaminergic progenitor cells may at least partially reinnervate the striatum or putamen of the subject. In some embodiments, the midbrain dopaminergic progenitor cells show limited, little or no proliferation after administration. Although the midbrain dopaminergic progenitor cells may include at least some cells that are still dividing or proliferating, the midbrain dopaminergic progenitor cells may continue to differentiate after administration. In some embodiments, less than about 1%, preferably less than 0.5%, of the cells in the cell culture are serotonergic cells. In some embodiments, at least 80% of the administered cells differentiate into differentiated cells that express both FOXA2 and LMX1 after administration to the subject. In some embodiments, at least 85% of the differentiated cells express both FOXA2 and LMX1. In some embodiments, at least about 60% of the administered cells express both FOXA2 and LMX1. The culture may be cryogenically frozen (e.g., cryogenically frozen in liquid nitrogen) prior to administration. For example, the cells may be cryogenically frozen for storage and then brought to room temperature where the cells are administered to a subject.Differentiated cells expressing FOXA2 and LMX1 may further express at least one marker selected from the group consisting of Engrailed (EN1), tyrosine kinase (TH), orthodenticle homeobox 2 (OTX2), nuclear receptor-associated 1 protein (NURR1), neuron-specific class III beta tubulin (Tuj1), TTF3, paired-like homeodomain 3 (PITX3), achaete-scute complex (ASCL), early B-cell factor 1 (EBF-1), early B-cell factor 3 (EBF-3), transthyretin (TTR), synapsin, dopamine transporter (DAT) and G protein-coupled inwardly rectifying potassium channel (Kir3.2 / GIRK2), CD142, DCSM1, CD63 and CD99. In some embodiments, the differentiated cells expressing FOXA2 and LMX1 or FOXA2 and TH further express Engrailed, PITX3 and NURR1. In some embodiments, about 10-25% of the cells in the cell culture co-express FOXA2 and tyrosine hydroxylase (TH). The pluripotent cells can be human induced pluripotent stem (iPS) cells. In some embodiments, LMX1 is LIM homeobox transcription factor 1 alpha (LMX1A). In some embodiments, less than about 5%, less than about 1%, or less than 0.5% of the cells in the cell composition are serotonergic cells. In some embodiments, the administration does not result in gliosis in the host. The administration does not result in or essentially no growth or proliferation of non-neuronal cells in the subject's brain. The administration can result in engraftment of mDA progenitor cells in the subject's brain and / or innervation of at least a portion of the subject's brain by mDA progenitor cells. The administration can be via injection (e.g., stereotaxic injection).

[0014] One aspect of the present disclosure is a method for culturing human cells expressing both Forkhead box protein A2 (FOXA2) and LIM homeobox transcription factor 1 (LMX1) comprising culturing human pluripotent cells in the presence of the following signal transduction modulators: (a) a first inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling, (b) at least one activator of Sonic Hedgehog (SHH) signaling, and (c) at least one activator of Wingless (Wnt) signaling. + / LMX1 +The present invention relates to an in vitro method of preparing a cell composition comprising a human pluripotent cell (a human pluripotent cell), the method not including culturing the human pluripotent cell in the presence of a second inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling, the human pluripotent cell being cultured under differentiation-inducing conditions for about 360 to about 456 hours, or any range derivable therein, and the cells then being refrigerated or cryopreserved. In some embodiments, the human pluripotent cell is cultured under differentiation-inducing conditions for about 384 to about 432 hours. In some embodiments, the human cell does not express NURR1. The human cell may express Forkhead box protein A2 (FOXA2), LIM homeobox transcription factor 1 (LMX1), and Engrailed homeobox 1 (EN1). The human cell may further express OTX2. In some embodiments, Forkhead box protein A2 (FOXA2) is expressed by about 85-95% of the cells. In some embodiments, FOXA2 and LIM homeobox transcription factor 1 (LMX1) are co-expressed by about 65% to about 85% or more, or about 65% to about 75% of human cells. The inhibitor of SMAD signaling can be a BMP inhibitor (e.g., LDN-193189, dorsomorphin, DMH-1, or noggin). In some embodiments, the BMP inhibitor is LDN-193189. LDN-193189 may be present, for example, at a concentration of about 0.2 μM to about 4 μM, or at a concentration of about 1 μM to about 3 μM, about 0.5 μM to about 4 μM, about 0.5 μM to about 2 μM, about 0.2 μM to about 4 μM, about 0.2 μM to about 2 μM, or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 μM, or any range derivable therebetween. In some embodiments, the SMAD signaling inhibitor is a TGFβ inhibitor (e.g., SB431542). SB431542 may be present at a concentration of about 1-20 μM, 5-15 μM, 9-11 μM, or about 10 μM. The pluripotent cells may be cultured with an inhibitor of a SMAD on days 1-15, 1-16, or 1-17 of culture. In some embodiments, the pluripotent cells are cultured with an inhibitor of a SMAD on days 1-17 of culture.The pluripotent cells may be cultured with the inhibitor of SMADs substantially continuously or daily for 15, 16 or 17 days. In some embodiments, the pluripotent cells are cultured with the inhibitor of SMADs substantially continuously or daily for 17 days. The inhibitor of SMADs may be present at a concentration of about 50-2000 or about 50-500 nM. In some embodiments, the inhibitor of SMADs is present at a concentration of about 180-240 nM. The method may further include contacting the pluripotent cells with a MEK inhibitor (e.g., PD0325901). PD0325901 may be present at a concentration of about 0.25-2.5 μM. In some embodiments, the MEK inhibitor is contacted with the pluripotent cells for about 1-3 days, or on days 1-3, 2-4, 3-5, or on days 1, 2, 3, 4 or 5 after initiation of contact with the inhibitor of SMAD signaling. In some embodiments, the MEK inhibitor is contacted with the pluripotent cells for about 24 to about 48 hours after initiation of contact with the inhibitor of SMAD signaling. In some embodiments, the MEK inhibitor is contacted with the pluripotent cells daily or substantially continuously for about 3-4 days beginning about 1-2 days after initiation of contact with the inhibitor of SMAD signaling. In some embodiments, the MEK inhibitor is contacted with the pluripotent cells on days 2-5 or 3-6 after initiation of contact with the inhibitor of SMAD signaling on day 1. The activator of Wnt signaling can be a GSK3 inhibitor (e.g., CHIR99021). In some embodiments, CHIR99021 is present at a concentration of about 1.5-1.7 μM, about 1.6-1.7 μM, about 1.65 μM, or any range derivable therebetween. In some embodiments, CHIR99021 is present at a concentration of about 4-7 μM on days 9-17 after initiation of contact with the inhibitor of SMAD signaling. In some embodiments, the activator of Wnt signaling is contacted with the pluripotent cells for 1-3 days after initiation of contact with the inhibitor of SMAD signaling. The activator of Wnt signaling may be contacted with the pluripotent cells within 24-48 hours after initiation of contact with the inhibitor of SMAD signaling. In some embodiments, the pluripotent cells are cultured with the activator of Wnt signaling substantially continuously or daily for 14, 15, or about 16 days.In some embodiments, the activator of Wnt signaling is contacted with the pluripotent cells 2-17 days after initiation of contact with the inhibitor of SMAD signaling. The activator of SHH signaling can be palmorphamin or C25II Shh. The method can further include contacting the pluripotent cells with two activators of SHH signaling (e.g., palmorphamin and C25II Shh). In some embodiments, the at least one activator of SHH signaling is contacted with the pluripotent cells on the same day as initiation of contact with the inhibitor of SMAD signaling or within 24-48 hours after initiation of contact with the inhibitor of SMAD signaling. The at least one activator of SHH signaling can be contacted with the pluripotent cells 1-7 days with or after initiation of contact with the inhibitor of SMAD signaling. The method can further include contacting the pluripotent cells with FGF-8. In some embodiments, FGF-8 is not contacted with the pluripotent cells on the same day as initiation of contact with the inhibitor of SMAD signaling. In some embodiments, FGF-8 is contacted with the pluripotent cells 9-17 days or 11-17 days after initiation of contact with the inhibitor of SMAD signaling. FGF-8 may be present at a concentration of about 50-200 ng / mL. The pluripotent cells may comprise an antibiotic resistance transgene under the control of a neuronal promoter. The method may further comprise selecting for neural cells, midbrain DA neurons or mDA progenitor cells derived from the pluripotent cells by contacting the cells with an antibiotic, a chemotherapeutic agent, a DNA crosslinking agent, a DNA synthesis inhibitor or a mitosis inhibitor. The method may further comprise contacting the pluripotent cells with an antibiotic or a chemotherapeutic agent. The chemotherapeutic agent may be mitomycin C. In some embodiments, mitomycin C is contacted with the pluripotent cells 27, 28 and / or 29 days after initiation of contact with the inhibitor of SMAD signaling. In some embodiments, the antibiotic is G418 (geneticin). The method may further comprise culturing or incubating the pluripotent cells in medium comprising a ROCK inhibitor prior to initiating contact with the inhibitor of SMAD signaling.The method may further comprise contacting the pluripotent cells with blebbistatin.In some embodiments, blebbistatin is contacted with the cells on days 5 and 17 of differentiation. In some embodiments, at least 40%, at least 60%, at least 80%, or at least 85% of the human pluripotent cells are differentiated and express both FOXA2 and LMX1. In some embodiments, about 10-25% of the human pluripotent cells are differentiated and express both FOXA2 and tyrosine hydroxylase (TH). The pluripotent cells can be human induced pluripotent stem (iPS) cells. In some embodiments, the LMX1 is LIM homeobox transcription factor 1 alpha (LMX1A). In some embodiments, the differentiated cells expressing FOXA2 and LMX1 or FOXA2 and TH further express at least one marker selected from the group consisting of orthodenticle homeobox 2 (OTX2), nuclear receptor-associated 1 protein (NURR1), neuron-specific class III beta tubulin (Tuj1), TTF3, paired-like homeodomain 3 (PITX3), achaete-scute complex (ASCL), early B-cell factor 1 (EBF-1), early B-cell factor 3 (EBF-3), transthyretin (TTR), synapsin, dopamine transporter (DAT) and G protein-coupled inwardly rectifying potassium channel (Kir3.2 / GIRK2), CD142, DCSM1, CD63, and CD99. FOXA2. + / LMX1 + The cells may further express Engrailed (EN1). FOXA2 + / LMX1 + The cells may further express EN1, Pax8 and ETV5. In some embodiments, FOXA2 + / LMX1 + The cells do not express NURR1. FOXA2 + / LMX1 +The cells may express GBX2, OTX1, OTX2, ETV5, CORIN and / or DCX. In some embodiments, less than about 1%, preferably 5% or less of the cells in the cell composition are serotonergic cells. The method may further comprise incubating the human pluripotent cells in the presence of a DNase or endonuclease. The endonuclease may be DNase I or Benzonase®. The DNase I or Benzonase® may be present at a concentration of about 10-20 U / mL, or at a concentration of about 10-15 U / mL, or any range derivable therebetween. In some embodiments, the human pluripotent cells are cultured in the presence of an endonuclease for at least one of the four to six days after initiation of contact with the inhibitor of SMAD signaling. In some embodiments, the human pluripotent cells are cultured in the presence of an endonuclease for the fifth day after initiation of contact with the inhibitor of SMAD signaling.

[0015] In some embodiments, cells differentiated using the mono-SMAD method provided herein for longer or shorter periods than the ranges mentioned above are provided. For example, in addition to D17 cells, cells at later stages of differentiation, such as D24 cells and / or D37 cells, are provided herein and can be administered to a subject to treat neurological or brain diseases. In some embodiments, cultures containing cells differentiated using the mono-SMAD method provided herein for at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37 days or any range derivable therein are provided and can be, for example, included in pharmaceutical compositions or used for in vitro testing (e.g., toxicology testing, drug screening, electrophysiological testing, etc.) or used to treat neurological diseases in vivo. In some embodiments, provided herein are cells differentiated using the mono-SMAD methods provided herein for about 12, 13, 14, 15, 16 days, or any range of time derivable therein, and it is expected that such cells can be administered to a mammalian subject to treat a neurological disease described herein, such as PD. As shown in the Examples below, D17, D24 and D37 cells can express the following cell markers, as follows: [Table 1]

[0016] Another aspect of the present disclosure relates to a culture comprising midbrain dopaminergic neurons or midbrain dopaminergic neuron progenitor cells produced by the methods described above or herein. The culture may be contained in a container means. In some embodiments, the midbrain dopaminergic neurons or midbrain dopaminergic neuron progenitor cells are contained in a pharmaceutical preparation. The pharmaceutical preparation may be formulated for injection.

[0017] Another aspect of the present disclosure is: (a) a FOXA2 + / LMX1A+ The present invention relates to a method of screening a test compound, comprising contacting a cell or mDA precursor cell (e.g., D17 cell) described above or herein with a test compound, and (b) measuring the function, physiology, or viability of the cell. Measuring can include testing for a toxicological response or for a change in an electrophysiological response of the cell. In some embodiments, the cell is a midbrain dopaminergic neuron or a midbrain dopaminergic neuron precursor cell.

[0018] Additional conditions and methods that may be used in combination with the present invention may be found, for example, in US 2015 / 0265652, US 2015 / 0010514, and WO2013 / 067362, which are incorporated by reference in their entireties. Additional methods of purifying pluripotent cells or promoting their differentiation into neurons or midbrain DA neurons that may be used in combination with the present invention include, for example, Kirkeby et al. (2012), Kriks, et al. (2011), Chung, et al. (2011), Xi et al. (2012), Young et al. (2014), Jaeger et al. (2011), Jiang et al. (2012), and US2016 / 0177260.

[0019] As used herein, "days of differentiation" refers to the number of days of incubation of cells in the medium, with the initiation of exposure of pluripotent cells to differentiation medium being day 1. In some preferred embodiments, the differentiation medium on day 1 contains a single SMAD inhibitor. Prior to incubation or culture in differentiation medium, the cells may be incubated, for example, in medium comprising or consisting of Essential 8™ Basal Medium and Essential 8™ Supplement (Thermo Fisher Scientific, Waltham, Mass.) for 1, 2 or 3 days prior to incubation in differentiation medium (i.e., day 0, day -1 and / or day -2), optionally with the addition of a ROCK inhibitor (e.g., H1152 at about 0.25-5 μM, 0.5, 0.75, 1, 1.25, 1.5, 2, 3, 4 or any range derivable therebetween, e.g., on day -2), and / or blebbestatin (e.g., at a concentration of about 0.1-20 μM, more preferably about 1.25-5 μM or about 2.5 μM).

[0020] As used herein, "essentially free" with respect to a specified component is used herein to mean that any specified component is not intentionally formulated into the composition and / or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintentional contaminant of the composition is preferably less than 0.01%. Most preferred are compositions in which the amount of the specified component cannot be detected using standard analytical methods.

[0021] As used herein, the designation "a" or "an" may mean one or more. When used in the claim(s), when used in conjunction with the word "comprising," the word "a" or "an" may mean one or more than one.

[0022] Although the use of "or" in the claims is used to mean "and / or" unless expressly indicated to refer to only alternatives or the options are mutually exclusive, the present disclosure supports a definition that refers to only one alternative and "and / or." As used herein, "another" may mean at least a second or more.

[0023] The term "about" is used throughout this application to indicate that a value includes error due to the inherent variation that exists for the device, the method used to determine the value, or the variation that exists in the study subject.

[0024] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0025] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0026] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief description of the drawings]

[0027] [Figure 1] Flow cytometry of the final product, FOXA2.

[0028] [Diagram 2] Purity of DA precursors (FOXA2+ / LMX1+).

[0029] [Diagram 3] The differentiation potential of DA neurons (NURR1+ / S100β-).

[0030] [Figure 4] Neuronal differentiation potential (MAP2+ / nestin-).

[0031] [Figure 5A-B] Forebrain neurons. FCDI DAPC-1 cells were stained with (Figure 5A) anti-PAX6 (Biolegend #901301) or (Figure 5B) anti-FOXG1. iCell GABA Neuron (FCDI) are shown as positive controls; these are cells patterned with a forebrain phenotype, are primarily GABAergic, and contain a subpopulation of PAX6+ neurons, as well as a subpopulation of FOXG1+ neurons.

[0032] [Figure 6] RT-QPCR of remaining iPSCs.

[0033] [Figure 7A-C] FCDI DAPC-1 consist of proliferating cells. (Figure 7A) Time course of EdU incorporation throughout FCDI DAPC-1 production. Approximately half of the FCDI DAPC-1 cells are proliferating. (Figure 7B) Time course of EdU incorporation into the FOXA2+ population of FCDI DAPC-1 in post-thaw cultures. Proliferation decreases as cells differentiate from the mDA precursor stage into mature DA neurons. (Figure 7C) EdU incorporated into mDA precursor cells over 24 hours on day 17 is retained in Nurr1+ cells for 12 days after precursor maturation.

[0034] [Figure 8] Amphetamine rotation in nude rats. Rotations shown are the average of all recordings taken. Error bars represent the standard error of the mean, n = 10-12 animals per group.

[0035] [Figure 9] Striatal reinnervation 6 months after transplantation. Reinnervation is shown using staining for TH positive cells. Although there was no statistical difference in the number of TH+ cells in the grafts at D17 and 24D, it was observed that striatal innervation by D17 cells was better than that by D24 cells.

[0036] [Figure 10] Intra-nigral grafts innervate the striatum. When cells were injected directly into the subtantia nigra, D17 grafts showed better innervation to the medial forebrain bundle and striatum compared to D24 cells.

[0037] [Figure 11] Time course of qPCR progenitor markers. Progenitor markers differ slightly between D17 and D24 cells. Lmx1, Pitx2, Nurr1 and Pitx3 are highly expressed in D24 cells, whereas En-1, Pax8, ETV5 and Glast are highly expressed in D17 cells.

[0038] [Figure 12] Time course of qPCR markers. Maturation markers are also differentially expressed, with AQP4 and tyrosine hydroxylase (TH) being expressed at higher levels in D24 cells compared to D17 cells.

[0039] [Figure 13] Comparison of immunocytochemistry (ICC) between D17 and D24 cultures.

[0040] [Figure 14] Violin plot of gene expression.

[0041] [Figure 15] Amphetamine rotation using alternative cell types. Rotations shown are the average of all recordings taken. Error bars represent the standard error of the mean, n = 4-10 animals.

[0042] [Figure 16A-C] TH and Ki67 are percentages of the cell population. Percentage of hNuc was calculated by dividing the number of hNuc+ cells by 450,000 injected cells, and TH and Ki67 are percentages of engrafted hNuc in the same graft. Results are shown for hNuc (Figure 16A), TH (Figure 16B) and Ki67 (Figure 16C). Data from rat tissue slices are shown. The percentages of each population are listed in the title of each graph (hNuc in total input, TH in total hNuc counted and Ki67 in total hNuc counted).

[0043] [Figure 17A-C] Stereological analysis of hNuc, TH and Ki67. Every 12th section (1 / 2 of the series) was stained with hNucli, TH or hKi67 and quantified by unbiased stereology. The graft area was outlined and counted in each animal. Each graph has its own Y-axis. Figure 17A shows the number of hNuc positive cells for each animal in each test group, the mean and standard error of the mean (SEM). Figure 17B shows the number of TH positive cells for each animal in each test group, including the mean and SEM. Figure 17C shows the number of Ki67 positive cells for each animal in each test group, including the mean and SEM.

[0044] [Figure 18A-C] Panel 1 (top) shows FoxA2 expression by flow cytometry in cells generated with 1.50 uM CHIR (Figure 18A), 1.75 uM CHIR (Figure 18B) and 2.00 uM CHIR (Figure 18C). Panel 2 (bottom) shows FoxA2 (y-axis) / Lmx (x-axis) expression by flow cytometry.

[0045] [Figure 19] Expression of genes in cells generated after various days of differentiation as measured using qPCR.

[0046] [Figure 20A-J]Characterization of D17 progenitors in vivo and analysis of function, survival and innervation. (Fig. 20A) Time-based analysis of d-amphetamine-induced rotation measured before surgery and 2, 4 and 6 months after engraftment. (Fig. 20B) Stereological estimates of hNuclei-ir cells contained in grafts with low, medium, high or maximum possible dose. Stereological estimates of TH-ir cells (Fig. 20C) and quantification of stereological estimates in each group (Fig. 20D). (E) Representative images of graft sections stained with hGFAP (scale bar 200*m) and (F) 5-HT (scale bar 1 mm (inset 25*m)). Alternative images containing low, medium, high and maximum possible dose grafts of DAB-treated (Fig. 20G) hNuclei and (Fig. 20H) TH, or immunofluorescent triple-labeled (Fig. 20I) hNuclei / TH / FoxA2 (green / red / blue) and (Fig. 20J) TH / Girk2 / calbindin (green / red / blue). Scale bar = 500*m.

[0047] [Figure 21A-B] In vitro differentiation and gene expression. (FIG. 21A) Schematic of differentiation and transplantation. MMC=mitomycin c. (FIG. 21B) qPCR comparing mRNA expression of target and off-target regions, cell type and neuronal maturation markers at days 17, 24 and 37 of iPSC-mDA differentiation. Three biological replicates were analyzed in technical triplicate for each treatment time point. Mean Ct values ​​are presented for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ΔCt). Error bars are SEM. Significance is shown in Table 7.

[0048] [Figure 22A-B]In vitro protein expression. (Fig. 22A) Flow cytometry comparing immunoreactive populations of mDA target markers at 17, 24 and 37 days of iPSC-mDA differentiation. Quantification of positive cell populations of hepatocytes, indicated as FOXA2+, FOXA2+ / LMX1+, NURR1+, MAP2+ and FOXA2+ / TH+. Three biological replicates were analyzed at each time point (mean ± SEM). (Fig. 22B) Immunocytochemistry comparing immunoreactive populations of target and off-target markers at 17, 24 and 37 days of iPSC-mDA differentiation. Images are representative of three biological replicates analyzed at each time point.

[0049] [Fig. 23A-E] Graft survival and function. (FIG. 23A) Time-based analysis of d-amphetamine-induced turning measured before surgery and 2, 4, and 6 months after engraftment. At 4 months after engraftment, P<0.0005 for D17 and P<0.005 for G418, and at 6 months after engraftment, P<0.0005 for D17 and D24 and P<0.05 for G418. Data were analyzed by mixed ANOVA with Tukey adjustment, error bars are SEM. Comparisons were made with vehicle groups. (FIG. 23B) Representative graft sections stained with hNuclei and (FIG. 23C) hKi-67, with the borders of the grafts outlined in black. Unbiased stereological quantification of (Fig. 23D) hNuclei-ir (P<0.001 for D17 vs. D37 / G418, P<0.0005 and P<0.005 for D24 vs. D37 / G418, respectively) and (Fig. 23E) hKi-67-ir cells (P<0.05 for D17 vs. D37, P<0.01 for D17 vs. G418, P<0.05 for D24 vs. D37). Scale bars = 500 μM in (Fig. 23B) and 50 μM in (Fig. 23C, inset). hNuclei estimates were analyzed by one-way ANOVA with Tukey adjustment and error bars represent SD. hKi-67 estimates were analyzed by Kruskal-Wallis test and Dwass-Steele-Critchlow-Fligner post hoc.

[0050] [Fig. 24A-D] Visualization of dopaminergic phenotype in vivo. (FIG. 24A) Representative graft-containing sections stained with DAB-treated TH. Quantification of TH-ir cells (FIG. 24B) contained within the grafts after 6 months in vivo (P<0.001 and P<0.005 for D17 vs. D37 / G418, P<0.0005 and P<0.01 for D24 vs. D37 / G418, respectively). (FIG. 24C) Optical density of graft-derived TH-ir fibers. Significant P values ​​were calculated for D17 vs. D24, D37, and G418 (P<0.0005, P<0.001, P<05), D24 vs. D37 (P<01), and G418 vs. D37 (P<0.0005). (FIG. 24D) Immunofluorescence triple labeling of TH / FOXA2 / hNuclei (green / red / blue). Scale bar (A)=500 μM, (D)=20 μM.

[0051] [Diagram 25] Long-range innervation of grafted cells transplanted into the substantia nigra. Representative computerized inverted micrographs of hNCAM immunoreactivity in coronal sections extending from the forebrain to the graft site within the substantia nigra. DAB-processed images were inverted and adjusted to show the extent of innervation, and all enhancements were applied in the same manner to each sample. AC=anterior commissure, AON=anterior olfactory nucleus, cc=corpus callosum, CPu=caudate-putamen, Fr=frontal cortex, NAc=nucleus accumbens, PrL=prelimbic area, Sept=septum, T=graft, Tu=olfactory tubercle.

[0052] [Fig. 26A-F]Quantitative analysis of D17 progenitor function, survival and innervation in vivo. (FIG. 26A) Time-based analysis of d-amphetamine-induced rotation measured before surgery and 2, 4 and 6 months after engraftment. At 4 months after engraftment, MDF was P<0.0001 and high dose was P<0.0005, and at 6 months after engraftment, MDF and high dose were P<0.0001 and medium dose was P<0.005, as analyzed by mixed ANOVA with Tukey adjustment. Comparisons were made with the vehicle group. (FIG. 26B) Stereological estimates of hNuclei-ir cells (visualized in FIG. 26E) contained in grafts with low, medium, high or "maximum possible" doses. P<0.0001 for all comparisons by one-way ANOVA with Tukey adjustment. (FIG. 26C) Stereological estimates of TH-ir cells (visualized in FIG. 26F) contained in grafts of low, medium, high or "maximum possible" doses. Analyzed by one-way ANOVA with Tukey adjustment, MFD was P<0.0001 for all groups, and P<0.005 and P<0.05 for the high dose group versus the medium and low dose groups, respectively. (FIG. 26D) Quantification of optical density of graft-induced TH. One-way ANOVA with Tukey adjustment showed P<0.0001 for MFD versus the medium and low doses, and P<0.05 for MFD versus the high dose versus the medium and low doses. One-way or mixed-effect ANOVA with Tukey adjustment for histological and behavioral data, respectively, error bars represent SD or SEM for histological and behavioral data, respectively. Images of the low dose group are from rats with grafts that substantially survived. Scale bar=500 μM.

[0053] [Figure 27A-C]Correlation of dopaminergic phenotype with behavioral recovery and visualization of mDA subtypes. (Fig. 27A) Estimated number of TH-ir cells and TH optical density measurements plotted against absolute value of magnitude of net change in d-amphetamine-induced rotation and fitted to a logarithmic regression curve. Linear regression of low / medium or high / "maximum possible" dose and behavioral recovery. Alternative images containing immunofluorescent triple-labeled (Fig. 27B) hNuclei / TH / FOXA2 (blue / green / red) and (Fig. 27C) TH / GIRK2 / calbindin (green / red / blue) grafts of low, medium, high and maximum possible doses.

[0054] [Fig. 28A-F] Non-dopaminergic cell types observed in the grafts. Representative photomicrographs (Fig. 28A) and stereological estimates (Fig. 28B) of grafts stained with hKi-67 in each group. Representative images of grafts stained with (Fig. 28C) hGFAP (glia), (Fig. 28D), Iba1 (microglia), and (Fig. 28E) 5-HT (serotonergic neurons). Scale bars: (Fig. 28A) = 100 μM, (Fig. 28C) = 200 μM, (Fig. 28D) = 500 μM, (Fig. 28E) = 1 mm (E, inset) = 25 μM. P<0.05 for medium dose vs low dose, all others P<0.005 by Kruskal-Wallis test with Dwass-Steele-Critchlow-Fligner method.

[0055] [Figure 29] Visualization of protein expression in vitro. Immunocytochemistry comparing immunoreactive populations of mDA target and off-target markers at days 17, 24 and 37 of iPSC-mDA differentiation. Images are representative of three biological replicates analyzed at each time point.

[0056] [Fig. 30A-B]Short-term engraftment. Coronal sections containing bilateral G418, D37, D24 or D17 striatal grafts in intact rats 3 months after injection stained with (FIG. 30A) hNCAM or (FIG. 30B) TH.

[0057] [Fig. 31A-I] Single cell gene expression in vitro. Single cell qPCR (Fluidigm) comparing mRNA expression at days 17, 24 and 36 of iPSC-mDA differentiation of targeted markers: A) FoxA2, ​​B) LMX1A, C) NURR1, D) TH, E) CALB1, F) ETV5, G) EN1, H) BARHL1 and I) GIRK2. 96 individual cells were assessed for each treatment time point. Log2 expression values ​​of each cell are represented as single markers on the graph. Error bars are SEM.

[0058] [Diagram 32] FCDI DAPC-1 flow cytometry assays of potentially dangerous non-target cell markers FOXG1+ and PAX6+ cells demonstrate a very low percentage of forebrain neuronal precursors.

[0059] [Diagram 33] FCDI DAPC-1 qPCR assay of serotonergic cell populations from 0-19 DPT.

[0060] [Diagram 34] FCDI DAPC-1 qPCR assay of SERT at 14 DPT shows consistently low expression in batches.

[0061] [Diagram 35] FCDI DAPC-1 ICC assay of the serotonergic marker 5-HT supports the qPCR results of SERT and TPH2. Representative images show ICC staining for 5-HT (red) at 1, 8, 15 and 20-DPT time points. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0062] Description of exemplary embodiments In some aspects, the present invention overcomes limitations in the prior art by providing compositions and methods for differentiating pluripotent cells, such as induced pluripotent stem cells, into dopaminergic (DA) neuronal progenitor cells that may exhibit significantly improved properties for the treatment of brain disorders in vivo. The methods include differentiating pluripotent cells under mono-SMAD conditions in the presence of a single SMAD inhibitor (a "mono-SMAD inhibitor") for a specified period of time, such as about 360-456 hours or more preferably about 384-432 hours. In general, and in contrast to previous dual-SMAD methods, the mono-SMAD method involves the use of only one SMAD inhibitor as opposed to dual-SMAD methods that utilize two SMAD inhibitors. In contrast to previous studies (Ganat et al., 2012, Qiu et al., 2017) that concluded that immature neurons expressing NURR1 were more effective than less mature progenitor cells that did not express NURR1, midbrain dopaminergic (mDA) progenitor cells are provided herein that do not express NURR1 and exhibit superior efficacy in vivo (e.g., for the treatment of PD) compared to mDA progenitor cells expressing NURR1 (e.g., D17 cells). As shown in the Examples below, cell cultures comprising midbrain DA neuron progenitor cells differentiated for these specified time periods were surprisingly observed to exhibit superior properties in vivo compared to cell cultures differentiated for other time periods using these mono-SMAD methods, and significant improvements in engraftment and innervation were observed using these cells for the treatment of a rat model of PD, resulting in increased functional recovery. Related cell cultures and methods for treating brain diseases (e.g., PD) are also provided.

[0063] In some aspects, PD is treated in a subject by administering cell replacement therapy of mDA cells differentiated from induced pluripotent stem cells (iPSCs). As shown in the examples below, it has been observed that mDA progenitor cells, in contrast to iPSC-derived postmitotic mDA neurons, produce superior results for treatments, including cell transplantation therapy, of brain diseases such as PD. The effect of cell maturation on graft survival and efficacy was examined by engrafting mDA progenitor cells (cryopreserved on day 17 of differentiation, D17), immature neurons (D24) and postmitotic neurons (D37) into immunocompromised hemiparkinsonian rats. It was observed that D17 progenitor cells were significantly superior to immature D24 or mature D37 neurons in terms of cell survival, fiber elongation and beneficial effects on motor impairment in vivo. The observed engraftment within the substantia nigra to the ventral midbrain demonstrated that D17 cells had a greater ability to innervate over longer distances to forebrain structures, including the striatum, than D24 cells. When D17 cells were examined over a wide dose range (7,500-450,000 cells injected per striatum), a clear dose response was observed in terms of the number of surviving neurons, innervation, and functional recovery. Importantly, although these grafts were derived from iPSCs, no teratoma formation or significant outgrowth of other cells was observed in any of the animals. These data support the use of these iPSC-derived D17 mDA progenitor cells for clinical therapeutic treatment of PD.

[0064] I. Definition "Pluripotency" or "pluripotent" refers to a stem cell or undifferentiated cell that has the potential to differentiate into all of the cells that make up one or more tissues or organs, for example, into any of the three germ layers: endoderm (e.g., the lining of the stomach, gastrointestinal tract, lungs), mesoderm (e.g., muscle, bone, blood, urogenital tract), or ectoderm (e.g., epithelial tissue, nervous system).

[0065] "Induced pluripotent stem cells", commonly abbreviated as iPS cells or iPSCs, refer to a type of pluripotent stem cell that is artificially prepared from a non-pluripotent cell, typically an adult somatic cell or a fully differentiated cell, such as a fibroblast, hematopoietic cell, muscle cell, neuron, epithelial cell, etc., by introducing or contacting the non-pluripotent cell with a reprogramming factor.

[0066] "Embryonic stem (ES) cells" are pluripotent stem cells derived from the early embryo.

[0067] An "adherent culture" is a culture in which cells or aggregates of cells adhere to a surface.

[0068] "Suspension culture" refers to a culture in which cells or aggregates of cells replicate while suspended in a liquid medium.

[0069] "Essentially free" of exogenously added components refers to a medium that does not contain or is essentially free of a particular component from a source other than cells in the medium. "Essentially free" of exogenously added growth factors or signaling inhibitors, such as TGFβ, bFGF, TGFβ superfamily signaling inhibitors, can mean minimal or undetectable amounts of the exogenously added components. For example, a medium or environment that is essentially free of TGFβ or bFGF can contain less than 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, 0.001 ng / mL or any range derivable therebetween. For example, a medium or environment that is essentially free of signal transduction inhibitors may contain 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, 0.005, 0.001 μM or any range derivable therebetween.

[0070] "Differentiation" is the process by which a less specialized cell forms at least one new, more specialized cell type progeny, e.g., a stem cell can differentiate into a neuronal progenitor cell, which can differentiate into a DA neuron.

[0071] The term "aggregation-promoting medium" means any medium that enhances cell aggregate formation, without any limitation on the mode of action.

[0072] The term "aggregates", or embryoid bodies, refers to homogeneous or heterogeneous clusters of cells, including differentiated cells, partially differentiated cells and / or pluripotent stem cells, cultured in suspension.

[0073] "Neurons" or "neuronal cells" or "neuronal cell types" or "neuronal lineages" may include any neuronal lineage cells and may be understood to refer to cells at any stage of neuronal ontogeny without any limitations, unless otherwise specified. For example, neurons may include both neuronal progenitor cells and / or mature neurons. "Neuronal cells" or "neuronal cell types" and "neuronal lineages" may include any neuronal lineages and / or any stage of neuronal ontogeny without any limitations, unless otherwise specified. For example, neuronal cells may include neuronal progenitor cells, glial progenitor cells, mature neurons and / or glia.

[0074] A "gene," "polynucleotide," "coding region," "sequence," "segment," or "fragment" that "encodes" a particular protein is a nucleic acid molecule that, when placed under the control of appropriate control sequences, is transcribed in vitro or in vivo, and optionally also translated into a gene product, e.g., a polypeptide. The coding region may be present in either cDNA, genomic DNA, or RNA form. If in DNA form, the nucleic acid molecule may be single-stranded (i.e., the sense strand) or double-stranded.

[0075] The term "transgene" refers to a gene, nucleic acid, or polynucleotide that has been introduced into a cell or organism, either artificially or by natural means, such as an exogenous nucleic acid. An exogenous nucleic acid may be from a different organism or cell, or may be one or more additional copies of a nucleic acid that is naturally present in the organism or cell.

[0076] The term "promoter" is used herein in its ordinary sense to refer to a nucleotide region containing DNA regulatory sequences, where the regulatory sequences are derived from a gene capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding sequence.

[0077] As used herein, "mesencephalic DA neuron progenitor cells", "mDA neuron progenitor cells", "mDA neuron progenitor cells" and "mDA progenitor cells" are used interchangeably and refer to neuronal progenitor cells that express FoxA2, ​​Lmx1 and EN1 (midbrain specific markers), but the cells do not express Nurr1. Mesencephalic DA neuron progenitor cells can express one or more of: GBX2, OTX2, ETV5, DBX1TPH2, TH, BARHL1, SLC6A4, GATA2, NR4A2, GAD1, DCX, NXK6-1, RBFOX3, KCNJ6, CORIN, CD44, SPRY1, FABP7, SLC17A7, OTX1 and / or FGFR3. In some embodiments, mDA progenitor cells express TH, e.g., mDA progenitor cells cannot express TH, but may retain the ability to express TH after further differentiation. mDA progenitor cells can express select genes at different stages of differentiation.

[0078] "Neural stem cells (NSCs)" are pluripotent cells capable of self-renewal, potentially unlimited proliferation, and can generate progeny cells that can terminally differentiate into neurons, astrocytes, and / or oligodendrocytes. Non-stem cell progeny of NSCs are called neural progenitor cells. "Neural progenitor cells" are precursor cells that have the ability to proliferate and differentiate into more than one cell type. Neural progenitor cells can be unipotent, bipotent, or multipotent. The defining characteristic of neural progenitor cells is that, unlike stem cells, they have limited proliferation capacity and do not exhibit self-renewal. "Neural progenitor cells" (NPCs) refer to a mixed population of cells consisting of all undifferentiated progeny of neural stem cells, including both neural progenitor cells and neural stem cells. The term neural progenitor cells can be used to describe a mixed population of NSCs and neural progenitor cells derived from embryonic stem cells or induced pluripotent stem cells.

[0079] II. SMAD Inhibitors for Mono-SMAD Inhibition In some aspects, the pluripotent cells are differentiated for a period of about 360-456 hours, more preferably about 384-432 hours, using the mono-SMAD method to produce a culture of neural cells. In the mono-SMAD method, a single SMAD inhibitor, such as a single BMP signaling inhibitor or a single TGF-β signaling inhibitor, is used to inhibit SMAD signaling in a method of converting pluripotent cells (e.g., iPS cells, ES cells) into neuronal cells, such as midbrain dopaminergic cells. In general, and in contrast to other dual-SMAD methods of differentiation, the mono-SMAD differentiation method utilizes only a single SMAD inhibitor, and no second SMAD inhibitor is included in the differentiation medium. For example, in some aspects, the pluripotent cells are converted into a population of neuronal progenitor cells, including midbrain DA neuron progenitor cells, where differentiation occurs in a medium containing a single BMP signaling inhibitor. In some embodiments, the BMP inhibitor is LDN-193189, dorsomorphin, or DMH-1. Non-limiting examples of inhibitors of BMP signaling include Dorsomorphin, dominant negative BMP, truncated BMP receptor, soluble BMP receptor, BMP receptor-Fc chimera, Noggin, LDN-193189, Follistatin, Chordin, Gremlin, Cerberus / DAN family proteins, Ventropin, High-dose Activin and Amnionless. In some embodiments, nucleic acid, antisense, RNAi, siRNA or other genetic methods can be used to inhibit BMP signaling. As used herein, BMP signaling inhibitors may simply be referred to as "BMP inhibitors". The BMP inhibitor may be included in the differentiation medium on days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and / or 17 of differentiation, or any range derivable therein (e.g., days 1-17, 1-16, 1-15, 2-15, etc.). In some embodiments, the BMP inhibitor is included in the differentiation medium on all days 1-17 of differentiation. Nonetheless, it is anticipated that the BMP inhibitor may be removed from the differentiation medium at specific times, such as days 1, 2, or 3 as described above.In some embodiments, a BMP inhibitor is optionally not included in the differentiation medium on days 11-17, and in some preferred embodiments a BMP inhibitor is included in the differentiation medium on days 1-10. The mono-SMAD method is further discussed in WO2018 / 035214.

[0080] In some embodiments, the BMP inhibitor is LDN-193189, dorsomorphin, DMH-1, or noggin. For example, cells can be cultured in medium containing about 1-2500, 1-2000, or 1-1,000 nM LDN-193189 (e.g., about 10-500, 50-500, 50-300, 50, 100, 150, 200, 250, 300, 500, 750, 1000, 1250, 1500, 1750, 2000, 2250, or about 2500 nM LDN-193189, or any range derivable therebetween). In some embodiments, cells may be cultured in medium containing about 0.1 to 10 μM dorsomorphin (e.g., about 0.1 to 10, 0.5 to 7.5, 0.75 to 5, 0.5 to 3, 1 to 3, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.55, 1.6, 1.65, 1.7, 1.75, 2, 2.25, 2.5, 2.75, 3, or about 2 μM dorsomorphin, or any range derivable therebetween), in some embodiments, cells may be cultured in medium containing about 1 μM DMH-1 (e.g., about 0.2 to 8, 0.5 to 2, or about 1 μM DMH-1, or any range derivable therebetween). LDN-193189, Dorsomorphin and DMH-1 can be successfully used in mono-SMAD inhibition method to produce midbrain dopaminergic neurons or mDA progenitor cells from iPS cells. In some embodiments, the BMP inhibitor is K02288 or DMH2.

[0081] In some aspects, TGFβ inhibitors can be used to inhibit SMADs in a mono-SMAD method to generate midbrain dopaminergic neurons or mDA progenitor cells from pluripotent cells, such as iPS cells. For example, in some embodiments, the differentiation medium comprises a TGFβ signaling inhibitor. Non-limiting examples of TGFβ signaling include LDN-193189, SB-525334, GW788388, A-83-01, GW6604, IN-1130, K i26894, LY2157299, LY364947 (HTS-466284), A-83-01, LY550410, LY573636, LY580276, NPC-30345, SB-431542, SB-505124, SD-093, Sml6, SM305, SX-007, Antp-Sm2A, and LY2109761. For example, the TGFβ inhibitor in the differentiation medium can be SB431542. In some aspects, cells are cultured in medium containing about 0.1 to 100 μM SB431542 (e.g., between about 1 to 100, 10 to 80, 15 to 60, 20 to 50, or about 40 μM SB431542). As used herein, TGFβ signaling inhibitors, including TGFβ receptor inhibitors, may be simply referred to as "TGFβ inhibitors." In some embodiments, TGFβ inhibitors are not included in the differentiation medium. In some embodiments, TGFβ inhibitors (e.g., SB431542) are included in the differentiation medium on days 1 to 3 or days 1, 2, 3, and / or 4 as mono-SMAD inhibitors. As shown in the Examples below, in some embodiments, BMP inhibitors are used as mono-SMAD inhibitors since it has been observed that these compounds result in superior differentiation of pluripotent cells into midbrain DA neurons or mDA progenitor cells compared to the use of TGFβ inhibitors.

[0082] III. Contains MEK inhibitor In some aspects, a MEK inhibitor is included in the differentiation medium, for example in combination with a BMP inhibitor or a mono-SMAD inhibitor, to produce midbrain dopaminergic neurons or mDA progenitor cells from pluripotent cells, such as iPS cells. In some embodiments, the MEK inhibitor is PD0325901. Non-limiting examples of MEK inhibitors that can be used include PD0325901, trametinib (GSK1120212), selumetinib (AZD6244), pimasertib (AS-703026), MEK162, cobimetinib, PD184352, PD173074, BIX 02189, AZD8330, and PD98059. For example, in some embodiments, the methods include culturing the cells in the presence of between about 0.1 and 10 μM (e.g., between about 0.1 and 5, 0.5 and 3, or 0.5 and 1.5 μM) of a MEK inhibitor, such as PD0325901. In some embodiments, the cells are contacted with the MEK inhibitor (e.g., PD0325901) on days 3, 4, 5, or days 3-5 of differentiation.

[0083] Thus, in certain aspects, differentiating the cells comprises culturing a population of pluripotent cells in a medium comprising a BMP inhibitor, an activator of Sonic Hedgehog (SHH) signaling, an activator of Wnt signaling, a MEK inhibitor, or a combination of the foregoing, wherein the medium does not contain exogenously added FGF8b. In some cases, a TGFβ inhibitor can be used instead of a BMP inhibitor. In some embodiments, the method does not include purifying cells using a DA-specific marker. In some aspects, the pluripotent cells comprise a resistance gene under the control of a neuronal promoter that can be used for purifying neuronal cells (e.g., neuronal cells expressing an antibiotic resistance gene survive exposure to antibiotics, while non-neuronal cells die).

[0084] In some embodiments, mesencephalic DA neuron progenitor cells may be produced by a method comprising: obtaining a population of pluripotent cells, differentiating the cells into a neural lineage cell population in a medium containing a MEK inhibitor (e.g., PD0325901), where the medium does not contain exogenously added FGF8b on day 1 of differentiation, and further differentiating the cells of the neural lineage cell population to provide an enriched population of mesencephalic DA neurons or mDA progenitor cells. In some embodiments, it has been observed that inclusion of FGF8 (e.g., FGF8b) in the differentiation medium on day 1 may in some cases interfere with or prevent differentiation of cells into mesencephalic DA neuron progenitor cells. In some embodiments, FGF8 may be included in the differentiation medium, optionally on a later day of differentiation, such as on day 9, 10, 11, 12, 13, 14, 15, 16, 17, or any range derivable therebetween, for example, preferably where contacting of the pluripotent cells is initiated on day 1 with a single SMAD inhibitor in the differentiation medium.

[0085] IV. Contains Wnt activators or GSK inhibitors In some aspects, a Wnt activator (e.g., a GSK3 inhibitor) is included in the differentiation medium, e.g., in combination with a BMP inhibitor or a mono-SMAD inhibitor, to generate midbrain dopaminergic neuronal progenitor cells from pluripotent cells, e.g., iPS cells. In some embodiments, the pluripotent cells are differentiated into a population of neuronal cells that include midbrain DA neurons or mDA progenitor cells, where the differentiation is in a medium that includes at least a first activator of Wnt signaling.

[0086] Various Wnt activators or GSK3 inhibitors can be used in various aspects of the present disclosure. For example, the activator of WNT signaling can be a glycogen synthase kinase 3 (GSK3) inhibitor. Non-limiting examples of GSK3 inhibitors include NP031112, TWS119, SB216763, CHIR-98014, AZD2858, AZD1080, SB415286, LY2090314 and CHIR99021. In some embodiments, the pluripotent cells are contacted with a single SMAD inhibitor that is not SB415286. In some embodiments, the activator of Wnt signaling is CHIR99021. Thus, in some aspects, culture media for use according to embodiments comprises about 0.1 to about 10 μM CHIR99021 (e.g., between about 0.1 to 5, 0.5 to 5, 0.5 to 3, between about 1.25 to greater than 2.25, about 1.25, 1.5, 1.55, 1.65, 1.7, 1.75, 1.8, 1.9, 2.0 or about 1.75 μM CHIR99021, or any range derivable therebetween). In some preferred embodiments, about 1.6-1.7 μM or about 1.65 μM CHIR99021 is used.

[0087] In some preferred embodiments, optionally a Wnt activator (e.g., a GSK3 inhibitor) is not included in the differentiation medium on day 1 of differentiation. In some embodiments, a Wnt activator or a GSK inhibitor (e.g., CHR99021) is included in the differentiation medium on days 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and / or 17, or any combination or all of these days. For example, in some embodiments, a Wnt activator or a GSK inhibitor is included in the differentiation medium on days 2-17 or days 3-17.

[0088] V. Sonic Hedgehog Activator In some aspects, an activator of Sonic Hedgehog (SHH) signaling is included in the differentiation medium, e.g., in combination with a BMP inhibitor or a mono-SMAD inhibitor, to generate midbrain dopaminergic neurons or mDA progenitor cells from pluripotent cells, e.g., iPS cells. In some embodiments, the Sonic Hedgehog activator is Sonic Hedgehog (Shh) or a mutant Shh. Shh can be, e.g., a human or mouse protein or can be derived from human or mouse Shh. For example, in some embodiments, Shh is a mutant mouse Shh protein, e.g., mouse C25II Shh or human C24II Shh. In some embodiments, the differentiation medium includes both Shh (e.g., C25II Shh) and a small molecule activator of SHH, such as palmorfamine. Without being bound by any theory, activators of Shh and / or Sonic Hedgehog can promote neural floor plate differentiation.

[0089] In some embodiments, mDA progenitor cells are generated from pluripotent cells by a method comprising culturing the pluripotent cells in a medium comprising at least a first activator of SHH signaling. For example, the activator of SHH signaling can be a recombinant SHH polypeptide (or a portion thereof) or a small molecule activator. In certain aspects, the activator of SHH can be Shh C25II, palmorfamine or a palmorfamine analog (e.g., a smoothened agonist, e.g., SAG-1 or 3-chloro-N-[(1r,4r)-4-(methylamino)cyclohexyl]-N-[3-(pyridin-4-yl)benzyl]benzo[b]thiophene-2-carboxamide). Thus, in certain aspects, the culture medium for use according to the embodiments comprises about 0.1 to 10 μM palmorfamine (e.g., between about 0.1 to 20, 0.5 to 10, 0.5 to 5, or about 2 μM palmorfamine). In further aspects, the culture medium comprises about 1 to 1,000 ng / ml Shh C25II (e.g., about 10 to 1,000, 10 to 500, 50 to 500, or about 100 ng / ml Shh C25II). In some embodiments, the activator of SHH signaling comprises both Shh C25II and palmorphamin. For example, cells may be cultured in medium comprising about 0.1 to 10 μM palmorphamin and about 1 to 1,000 ng / ml Shh C25II. The SHH activator(s) (e.g., Shh C25II and palmorphamin) may be included in the differentiation medium on days 1, 2, 3, 4, 5, 6, and / or 7. In some embodiments, the SHH activator is omitted from the differentiation medium on day 1. For example, in various embodiments, the SHH activator(s) is included in the differentiation medium on days 1-6 or 2-7.

[0090] Thus, in certain embodiments, pluripotent cells may be differentiated and cultured for 1 to 6 days in an adherent culture system using DMEM / F12 medium containing B27 supplement, 1 to 3000 or 1 to 1000 nM LDN-193189 (or 0.1 to 100 μM SB431542), 0.1 to 50 μM palmorfamine, 1 to 1,000 ng / ml Shh C25II, and 0.1 to 10 μM CHIR99021. In one embodiment, the medium may contain B27 supplement, 200 nM LDN-193189 (or 10 μM SB431542), 2 μM palmorfamine, 100 ng / ml Shh C25II, and 1.25 μM CHIR99021. In some embodiments, a MEK inhibitor is included in the media after 1-2 days (eg, the MEK inhibitor is included on days 2-4 or days 2, 3 and / or 4 of differentiation).

[0091] VI. Sources of Pluripotent Stem Cells Pluripotent stem cells can be used in the methods disclosed herein for neural induction. The methods and compositions disclosed herein can be used, for example, to generate midbrain DA neuron progenitor cells with improved therapeutic properties (e.g., for the treatment of neurodegenerative diseases, such as PD).

[0092] The term "pluripotent stem cells" or "pluripotent cells" refers to cells that can give rise to cells of all three germ layers, i.e., endoderm, mesoderm, and ectoderm. Although theoretically pluripotent stem cells can differentiate into any cell of the body, experimental determination of pluripotency is typically based on differentiation of pluripotent cells into several cell types of each germ layer. In some embodiments, the pluripotent stem cells are embryonic stem (ES) cells derived from the inner cell mass of a blastocyst. In other embodiments, the pluripotent stem cells are induced pluripotent stem cells derived from reprogramming somatic cells. In some embodiments, the pluripotent stem cells are embryonic stem cells derived from somatic cell nuclear transfer. Pluripotent stem cells can be obtained or derived from healthy subjects (e.g., healthy humans) or subjects with disease (e.g., neurodegenerative disease, Parkinson's disease, etc.).

[0093] A. Embryonic stem cells Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of a blastocyst. ES cells can be isolated by removing the outer trophectoderm layer of a developing embryo and then culturing the inner cell mass on a feeder layer of non-proliferating cells. Under appropriate conditions, colonies of proliferating undifferentiated ES cells are produced. The colonies can be transferred, dissociated into individual cells, and then replated on a fresh feeder layer. The replated cells continue to proliferate, producing new colonies of undifferentiated ES cells. The new colonies are then transferred, dissociated, replated, and allowed to proliferate. This process of "subculturing" or "passaging" undifferentiated ES cells can be repeated to produce cell lines containing undifferentiated ES cells (e.g., as described in U.S. Pat. Nos. 5,843,780, 6,200,806, and 7,029,913). A "primary cell culture" is a culture of cells obtained directly from a tissue, such as the inner cell mass of a blastocyst. A "subculture" is any culture derived from a primary cell culture.

[0094] Methods for obtaining mouse ES cells are well known. In one method, preimplantation blastocysts from mouse strain 129 are treated with mouse antisera to remove the trophectoderm, and the inner cell mass is cultured in medium containing fetal bovine serum on a feeder layer of chemically inactivated mouse embryonic fibroblast cells. Colonies of undifferentiated ES cells that develop are passaged in the presence of fetal bovine serum on a mouse embryonic fibroblast feeder layer to produce a population of ES cells. In some methods, mouse ES cells can be grown in the absence of a feeder layer by adding the cytokine leukemia inhibitory factor (LIF) to serum-containing culture medium (Smith, 2000). In another method, mouse ES cells can be grown in serum-free medium in the presence of bone morphogenetic protein and LIF (Ying et al., 2003).

[0095] Human ES cells can be obtained from blastocysts using previously described methods (Thomson et al., 1995; Thomson et al., 1998; Thomson and Marshall, 1998; Reubinoff et al, 2000.). In one method, day 5 human blastocysts are exposed to rabbit anti-human spleen cell antiserum and then to a 1:5 dilution of guinea pig complement to lyse the trophectoderm cells. After removing the lysed trophectoderm cells from the intact inner cell mass, the inner cell mass is cultured on a feeder layer of gamma-inactivated mouse embryonic fibroblasts in the presence of fetal bovine serum. After 9 to 15 days, the clumps of cells derived from the inner cell mass are chemically (e.g., exposure to trypsin) or mechanically dissociated and replated on a feeder layer of mouse embryonic fibroblasts in fresh medium containing fetal bovine serum. Upon further expansion, colonies with undifferentiated morphology are selected by micropipette, mechanically dissociated into clumps, and replated (see U.S. Patent No. 6,833,269). ES-like morphology is characterized as small colonies with apparently high nuclear to cytoplasmic ratios and prominent nucleoli. The resulting ES cells can be routinely passaged by short trypsinization or by selection of individual colonies with a micropipette. In some methods, human ES cells can be grown without serum by culturing the ES cells on a feeder layer of fibroblasts in the presence of basic fibroblast growth factor (Amit et al., 2000). In other examples, human ES cells can be grown without a feeder cell layer by culturing the cells on a protein matrix, such as Matrigel™ or laminin, in the presence of a “conditioned” medium containing basic fibroblast growth factor (Xu et al., 2001). The medium may be preconditioned by co-culturing fibroblasts.

[0096] Methods for isolating rhesus monkey and common marmoset ES cells are also well known (Thomson and Marshall, 1998; Thomson et al., 1995; Thomson and Odorico, 2000).

[0097] Another source of ES cells are established ES cell lines. Various mouse and human ES cell lines are well known, and conditions for their growth and proliferation have been defined. For example, the mouse CGR8 cell line was established from the inner cell mass of mouse line 129 embryos, and cultures of CGR8 cells can grow in the presence of LIF without a feeder layer. As a further example, human ES cell lines H1, H7, H9, H13 and H14 were established by Thomson et al. (2000). In addition, subclones H9.1 and H9.2 of the H9 line were developed. It is expected that virtually any ES or stem cell line known in the art can be used with the present disclosure, such as those described in Yu and Thomson, 2008, which is incorporated herein by reference.

[0098] Sources of ES cells can include blastocysts, cells derived from culturing the inner cell mass of blastocysts, and cells obtained from cultures of established cell lines. Thus, as used herein, the term "ES cells" can refer to the inner cell mass of blastocysts, ES cells obtained from cultures of inner cell mass, and ES cells obtained from cultures of ES cell lines.

[0099] B. Induced pluripotent stem cells Induced pluripotent stem (iPS) cells have the characteristics of ES cells, but are obtained by reprogramming differentiated somatic cells. Induced pluripotent stem cells have been obtained by various methods. In one method, adult human dermal fibroblasts are transfected with the transcription factors Oct4, Sox2, c-Myc, and Klf4 using retroviral transduction (Takahashi et al., 2006, 2007). The transfected cells are plated on SNL feeder cells (a mouse cell fibroblast cell line that produces LIF) in medium supplemented with basic fibroblast growth factor (bFGF). After approximately 25 days, colonies resembling human ES cell colonies appear in the culture. The ES cell-like colonies are picked and expanded on feeder cells in the presence of bFGF. In some preferred embodiments, the iPS cells are human iPS cells.

[0100] Induced pluripotent stem cells are morphologically similar to human ES cells and express various human ES cell markers. When grown under conditions known to result in the differentiation of human ES cells, the induced pluripotent stem cells differentiate accordingly. For example, induced pluripotent stem cells can differentiate into cells with neuronal structures and neuronal markers. It is expected that virtually any iPS cell or cell line can be used with the present disclosure, including, for example, those described in Yu and Thomson, 2008. As will be appreciated by those skilled in the art, various iPS cell lines have been generated and iPS cells derived from these established cell lines can be used in various embodiments of the present disclosure.

[0101] In another method, human fetal or neonatal fibroblasts are transfected with four genes Oct4, Sox2, Nanog and Lin28 using lentiviral transduction (Yu et al., 2007). 12-20 days after infection, colonies with human ES cell morphology become visible. The colonies are picked and expanded. The induced pluripotent stem cells forming the colonies are morphologically similar to human ES cells, express various human ES cell markers, and form teratomas with neural tissue, cartilage and intestinal epithelium after injection into mice.

[0102] Methods for preparing induced pluripotent stem cells from mouse cells are also well known (Takahashi and Yamanaka, 2006). Induction of iPS cells typically requires expression of or exposure to at least one member from the Sox family and at least one member from the Oct family. Sox and Oct are believed to be central to the transcriptional regulatory hierarchy that specifies ES cell identity. For example, Sox can be Sox-1, Sox-2, Sox-3, Sox-15 or Sox-18, and Oct can be Oct-4. Additional factors such as Nanog, Lin28, Klf4 or c-Myc can increase reprogramming efficiency, and a specific set of reprogramming factors can be a set that includes Sox-2, Oct-4, Nanog and optionally Lin-28, or Sox-2, Oct4, Klf and optionally c-Myc.

[0103] iPS cells, such as ES cells, have characteristic antigens that can be identified or confirmed by immunohistochemistry or flow cytometry using antibodies against SSEA-1, SSEA-3 and SSEA-4 (Developmental Studies Hybridoma Bank, National Institute of Child Health and Human Development, Bethesda Md.) and TRA-1-60 and TRA-1-81 (Andrews et al., 1987). Pluripotency of embryonic stem cells can be determined, for example, at approximately 0.5-10×10 cells. 6 By injecting 10 μg / ml into the hind leg muscle of 8-12 week old male SCID mice, teratoma development can be confirmed, demonstrating at least one cell type of each of the three germ layers.

[0104] iPS cells can be generated using somatic cells modified to express reprogramming factors including Oct family members and Sox family members, e.g., Oct4 and Sox2 in combination with Klf or Nanog, as described above. Somatic cells can be any somatic cells that can be induced to pluripotency, such as, for example, fibroblasts, keratinocytes, hematopoietic cells, mesenchymal cells, liver cells, gastric cells, or beta cells. In some embodiments, T cells can also be used as a source of somatic cells for reprogramming (see, e.g., WO 2010 / 141801, incorporated herein by reference).

[0105] Reprogramming factors can be expressed from expression cassettes contained in one or more vectors, such as integrating vectors, RNA viral vectors that do not integrate into chromosomes (see U.S. Patent Application No. 13 / 054,022, incorporated herein by reference), or episomal vectors, such as EBV element-based systems (see, e.g., WO 2009 / 149233, Yu et al., 2009, incorporated herein by reference). In further aspects, reprogramming proteins or RNAs (e.g., mRNA or miRNA) can be directly introduced into somatic cells by protein or RNA transfection (Yakubov et al., 2010).

[0106] C. Embryonic stem cells derived from somatic cell nuclear transfer Pluripotent stem cells can be prepared by means of somatic cell nuclear transfer, in which a donor nucleus is transferred into a spindle-less oocyte. Stem cells produced by nuclear transfer are genetically identical to the donor nucleus. Methods for generating embryonic stem cells derived from somatic cell nuclear transfer are provided in Tachibana et al., 2013. As used herein, the term "ES cells" refers to embryonic stem cells derived from an embryo containing a fertilized nucleus, and embryonic stem cells produced by nuclear transfer are referred to as "NT-ESCs."

[0107] VII. Differentiation medium A differentiation medium according to certain aspects of the present disclosure may be prepared using a medium used to culture animal cells as its basal medium. In some embodiments, the differentiation medium is used to differentiate pluripotent cells into midbrain dopaminergic neuron progenitor cells (e.g., D17 cells) using only a single BMP inhibitor or a single TGF-beta inhibitor. For example, a differentiation medium used to promote differentiation of pluripotent cells (e.g., into midbrain dopaminergic progenitor cells) may contain a single BMP inhibitor (e.g., LDN-193189 or dorsomorphin, e.g., on days 1-17 of differentiation, an activator of sonic hedgehog (SHH) signaling (e.g., palmorphamin, human C25II SHH or mouse C24II SHH). In some embodiments, a single TGFβ inhibitor (e.g., SB-431542, for example, days 1-4) may be used in place of a single BMP inhibitor, although in some embodiments, a single BMP inhibitor may have reduced inhibition of FOXA2 compared to the use of a single TGF-β inhibitor. + / LMX1A + In some embodiments, FGF-8 (e.g., FGF-8b) is not included in the differentiation medium on the first day or on days 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any combination thereof (e.g., days 1-8), for example, in some embodiments, FGF-8 is included in the differentiation medium on days 9, 10, 11, 12, 13, 14, 15, 16, and 17, or any combination thereof. In various embodiments, the differentiation culture may contain TGFβ and bFGF, or either one, the differentiation medium is essentially free of TGFβ and bFGF.

[0108] In certain aspects, methods of differentiation according to embodiments include passaging of cells through a range of media conditions, e.g., cells are cultured in: - in adherent culture in a medium containing a single BMP inhibitor (or a TGFβ inhibitor), an activator of Sonic Hedgehog (SHH) signaling and an activator of Wnt signaling, - in suspension in a medium containing a single BMP inhibitor (or TGFβ inhibitor), an activator of SHH signaling and an activator of Wnt signaling, wherein the cell aggregates are formed; - in adherent culture in Neurobasal medium containing B27 supplement, L-glutamine, BDNF, GDNF, TGFβ, ascorbic acid, dibutyryl cAMP and DAPT (optionally without exogenously added retinol or retinoic acid) for maturation It is cultivated in.

[0109] As the basal medium, any chemically defined medium may be used, such as Basal Medium Eagle (BME), BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, Iscove's Modified Dulbecco's Medium (IMDM), Medium 199, Eagle's MEM, αMEM, DMEM, Ham, RPMI 1640 and Fisher's Medium, modifications or combinations thereof, in which TGFβ and bFGF may or may not be present.

[0110] In further embodiments, the cell differentiation environment may also contain supplements such as B-27 supplement, insulin, transferrin and selenium (ITS) supplement, L-glutamine, NEAA (non-essential amino acids), P / S (penicillin / streptomycin), N2 supplement (5 μg / mL insulin, 100 μg / mL transferrin, 20 nM progesterone, 30 nM selenium, 100 μM putrescine (Bottenstein, and Sato, 1979 PNAS USA 76, 514-517), and / or β-mercaptoethanol (β-ME). It is contemplated that additional factors may or may not be added, including, but not limited to, fibronectin, laminin, heparin, heparin sulfate, retinoic acid.

[0111] Growth factors may or may not be added to the differentiation medium. In addition to or instead of the factors outlined above, growth factors such as members of the epidermal growth factor family (EGF), members of the fibroblast growth factor family (FGF) including FGF2 and / or FGF8, members of the platelet-derived growth factor family (PDGF), transforming growth factor (TGF) / bone morphogenetic protein (BMP) / growth and differentiation factor (GDF) family antagonists, etc. may be used at various steps of the process. In some embodiments, FGF-8 is included in the differentiation medium as described herein. Other factors that may or may not be added to the differentiation medium include molecules that can activate or inactivate signaling through the Notch receptor family, including but not limited to proteins of the delta-like and Jagged families and other inhibitors of Notch processing or cleavage, such as gamma secretase inhibitors and DAPT. Other growth factors include members of the insulin-like growth factor family (IGF), the wingless-related (WNT) factor family, and the hedgehog factor family.

[0112] Additional factors may be added to the aggregate formation and / or differentiation medium to promote proliferation and survival of neural stem / progenitor cells, as well as neuronal survival and differentiation. These neurotrophic factors include, but are not limited to, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 / 5 (NT-4 / 5), interleukin 6 (IL-6), ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), cardiotrophins, members of the transforming growth factor (TGF) / bone morphogenetic protein (BMP) / growth and differentiation factor (GDF) family, the glial-derived neurotrophic factor (GDNF) family, including, but not limited to, neurturin, neublastin / artemin, and persephin, and factors related to and including hepatocyte growth factor. Neural cultures terminally differentiated to form postmitotic neurons may also contain a mitotic inhibitor or a mixture of mitotic inhibitors, including but not limited to 5-fluoro 2'-deoxyuridine, mitomycin C, and / or cytosine β-D-arabinofuranoside (Ara-C).

[0113] The medium may be a serum-containing medium or a serum-free medium. Serum-free medium refers to a medium that does not contain raw or unpurified serum, and may therefore include medium that contains purified blood-derived components or animal tissue-derived components (e.g., growth factors). In terms of preventing contamination with heterologous animal-derived components, the serum may be from the same animal as the stem cell(s). In some embodiments, the medium is a defined medium, which does not contain serum or other animal tissue-derived components (e.g., medium conditioned with irradiated mouse fibroblasts or irradiated fibroblast feeder cells).

[0114] The medium may or may not contain a serum replacement. Serum replacements include materials that suitably contain albumin (e.g., lipid-rich albumin, albumin substitutes such as recombinant albumin, vegetable starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, or equivalents thereof. For example, serum replacements can be prepared by the methods disclosed in WO 98 / 30679. Alternatively, commercially available materials can be used for added convenience. Commercially available materials include Knockout Serum Replacement (KSR) and Chemically-defined Lipid concentrate (Gibco).

[0115] The medium may also contain fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamin(s), growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, and inorganic salts. The concentration of 2-mercaptoethanol may be, for example, about 0.05 to 1.0 mM, particularly about 0.1 to 0.5 or 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.5, 0.8, 1, 1.5, 2, 2.5, 5, 7.5, 10 mM, or any intermediate value, but the concentration is not specifically limited thereto, as long as it is suitable for culturing stem cell(s).

[0116] In some embodiments, pluripotent stem cells are cultured in medium prior to aggregate formation (e.g., prior to being dissociated into single cells or small aggregates to induce aggregate formation) to improve neural induction and floor plate patterning. In certain embodiments of the invention, stem cells may be cultured in the absence of feeder cells, feeder cell extracts and / or serum.

[0117] B.Culture conditions Culture vessels used to culture the cell(s) include, but are not limited to: flasks, tissue culture flasks, spinner flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, microslides, chamber slides, tubes, trays, CellSTACK® Chambers, culture bags, and roller bottles, so long as the cells can be cultured therein. Cells can be cultured in volumes of about 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 800, 1000, 1500 mL or more, or any range derivable therebetween, depending on the needs of the culture. In certain embodiments, the culture vessel can be a bioreactor, which can refer to any device or system that supports a biologically active environment. The bioreactor may have a volume of about 2, 4, 5, 6, 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 500 liters, 1, 2, 4, 6, 8, 10, 15 cubic meters or more, or any range derivable therebetween.

[0118] The culture vessel surface may be prepared with or without cell attachment. The cell-attached culture vessel may be coated with any material for cell attachment, such as an extracellular matrix (ECM), to improve the adhesion of the vessel surface to the cells. The substrate used for cell attachment may be any material intended to attach stem cells or feeder cells (if used). Non-limiting substrates for cell attachment include collagen, gelatin, poly-L-lysine, poly-D-lysine, poly-L-ornithine, laminin, vitronectin and fibronectin, as well as mixtures thereof, such as Engelbreth-Holm-Swarm mouse sarcoma cell-derived protein mixtures (e.g., Matrigel™ or Geltrex) and dissolved cell membrane preparations (Klimanskaya et al., 2005). In some embodiments, the cell-attached culture vessel is coated with a cadherin protein, such as epithelial cadherin (E-cadherin).

[0119] Other culture conditions may be appropriately determined. For example, the culture temperature may be about 30 to 40° C., for example, about 31, 32, 33, 34, 35, 36, 37, 38, 39° C. or higher, but is not limited thereto. 2 The concentration may be about 1 to 10%, such as about 2 to 7%, or any range derivable therebetween. The oxygen partial pressure may be about 1, 5, 8, 10, 20% or more, or any range derivable therebetween.

[0120] Adherent culture may be used in certain aspects. Optionally, cells may be cultured in the presence of feeder cells. When feeder cells are used, stromal cells, such as fetal fibroblasts, may be used as feeder cells (see, for example, Manipulating the Mouse Embryo A Laboratory Manual (1994), Gene Targeting, A Practical Approach (1993), Martin (1981); Evans et al. (1981), Jainchill et al., (1969), Nakano et al., (1996), Kodama et al. (1982), and WO 01 / 088100 and WO 2005 / 080554). In some embodiments, feeder cells are not included in the cell culture medium, and cells may be cultured using defined conditions.

[0121] In other embodiments, suspension cultures may be used. Suspension cultures that may be used include suspension cultures on carriers (Fernandes et al., 2007) or gel / biopolymer encapsulation (US Patent Publication No. 2007 / 0116680). Suspension cultures of stem cells generally include cultures of cells (e.g., stem cells) in non-adherent conditions with respect to feeder cells (if used) in culture vessels or medium. Suspension cultures of stem cells generally include dissociated cultures of stem cells and aggregate suspension cultures of stem cells. Dissociated cultures of stem cells include cultures of suspended stem cells, e.g., small cell aggregates composed of a single stem cell or multiple stem cells (e.g., about 2 to 400 cells). If dissociated culture is continued, the cultured dissociated cells will usually form larger aggregates of stem cells, whereby aggregate suspension cultures can be produced or utilized. Aggregation suspension culture methods include the embryoid body culture method (see Keller et al., 1995) and the SFEB (serum-free embryoid body) method (Watanabe et al., 2005; WO 2005 / 123902).

[0122] C. Pluripotent Stem Cell Culture Methods for preparing and culturing pluripotent stem cells, e.g., ES cells, can be found in standard textbooks and reviews of cell biology, tissue culture, and development, including teratocarcinoma and embryonic stem cells: Guide to Techniques in Mouse Development (1993), Embryonic Stem Cell Differentiation in vitro (1993), Properties and uses of Embryonic Stem Cells: Prospects for Application to Human Biology and Gene Therapy (1998), all of which are incorporated herein by reference. Standard methods commonly used in tissue culture are described in Animal Cell Culture (1987), Gene Transfer Vectors for Mammalian Cells (1987), and Current Protocols in Molecular Biology and Short Protocols in Molecular Biology (1987 & 1995).

[0123] After the somatic cells have been introduced or contacted with the reprogramming factors, the cells can be cultured in a medium sufficient to maintain their pluripotency and undifferentiated state. The culture of induced pluripotent stem (iPS) cells can use a variety of media and techniques developed for culturing primate pluripotent stem cells, embryonic stem cells, or iPS cells, as described, for example, in U.S. Patent Publication Nos. 2007 / 0238170, 2003 / 0211603, and 2008 / 0171385, which are hereby incorporated by reference. It is understood that additional methods for culturing and maintaining pluripotent stem cells can be used, as known to those skilled in the art.

[0124] In certain embodiments, undefined conditions may be used, for example, pluripotent cells may be cultured on fibroblast feeder cells or in media exposed to fibroblast feeder cells to maintain the stem cells in an undifferentiated state. Alternatively, pluripotent cells may be cultured and maintained in an essentially undifferentiated state using defined feeder-free culture systems, such as TeSR medium (Ludwig et al., 2006a, Ludwig et al., 2006b) or E8 medium (Chen et al., 2011, PCT / US2011 / 046796). Feeder-free culture systems and media may be used to culture and maintain pluripotent cells. These approaches allow human pluripotent stem cells to maintain an essentially undifferentiated state without the need for a mouse fibroblast "feeder layer."

[0125] A variety of matrix components can be used to culture, maintain or differentiate human pluripotent stem cells. For example, as described by Ludwig et al. (2006a, 2006b), which are incorporated by reference in their entirety, collagen IV, fibronectin, laminin and vitronectin in combination can be used to coat culture surfaces as a means of providing a solid support for pluripotent cell growth.

[0126] Matrigel™ can also be used to provide a substrate for cell culture and maintenance of human pluripotent stem cells. Matrigel™ is a gelatinous protein mixture secreted by mouse tumor cells and is commercially available from BD Biosciences (New Jersey, USA). This mixture resembles the complex extracellular environment found in many tissues and is used by cell biologists as a substrate for cell culture. In some embodiments, E-cadherin (e.g., recombinant E-cadherin substratum) is provided as a substrate for the culture and maintenance of pluripotent cells, e.g., human pluripotent cells or human iPS cells. Related methods are shown, for example, in Nagaoka et al. (2010).

[0127] D. Single Cell Passage In some embodiments of pluripotent stem cell culture, when the culture vessel is full, the colonies are split into aggregated cells or even into single cells by a method suitable for dissociation, and the cells are then placed into a new culture vessel for passaging. Passaging or splitting of cells is a technique that allows cells to survive and grow under culture conditions for an extended period of time. Cells are typically passaged when they are about 70%-100% confluent.

[0128] Single cell dissociation of pluripotent stem cells followed by single cell passaging can be used in the present method with several advantages, such as promoting cell proliferation, allowing automation of cell sorting, defined seeding for differentiation and culture procedures and clonal expansion. For example, progeny cells clonally derived from single cells can be homogenous in genetic makeup and / or cell cycle synchronized, allowing increased targeted differentiation. Exemplary methods of single cell passaging are described in US 2008 / 0171385, which is incorporated herein by reference.

[0129] In certain embodiments, pluripotent stem cells can be dissociated into single individual cells, or combinations of single individual cells and into small cell clusters containing 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cells. Dissociation can be achieved by mechanical force or by cell dissociation agents, such as chelating agents, sodium citrate (Na Citrate), or enzymes, such as trypsin, trypsin EDTA, Accutase, TrypLE Select, etc. Cell dissociation can be achieved using chemical separation (e.g., using chelators or enzymes) and / or mechanical agitation to dissociate the cells.

[0130] Based on the source of pluripotent stem cells and the need for expansion, dissociated cells may be transferred individually or in small clusters to new culture vessels at split ratios such as about 1:2, 1:4, 1:5, 1:6, 1:8, 1:10, 1:20, 1:40, 1:50, 1:100, 1:150, 1:200 or less or any range derivable therebetween. Split ratios for suspension cell lines may be performed in the volume of cultured cell suspension. Passage intervals may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 days or less or any range derivable therebetween. For example, achievable split ratios with various enzymatic passaging protocols can be 1:2 every 3-7 days, 1:3 every 4-7 days, and 1:5 to 1:10 approximately every 7 days, 1:50 to 1:100 every 7 days. If higher split ratios are used, the passaging interval can be extended to at least 12-14 days or any period without excessive cell loss due to spontaneous differentiation or cell death.

[0131] In certain aspects, single cell passaging may be in the presence of a small molecule effective to increase cloning efficiency and cell survival time, such as a ROCK inhibitor or a myosin II inhibitor. ROCK inhibitors or myosin II inhibitors, such as Y-27632, HA-1077, H-1152, or blebbistatin, may be used at effective concentrations, such as about 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 to about 100 μM or more, or any range derivable therebetween.

[0132] E. Stem Cell Differentiation Methods are provided herein for generating mDA progenitor cells with improved therapeutic properties (e.g., for treating Parkinson's disease). Differentiation of pluripotent stem cells can be induced in various ways, e.g., in adherent colonies or by forming cell aggregates, e.g., in low-adhesion environments, where the aggregates are called embryoid bodies (EBs). Molecular and cellular morphological signals and events within EBs mimic many aspects of the natural ontogeny process of such cells in developing embryos. Methods for directing cells to neuronal differentiation are provided, for example, in US Publication No. 2012 / 0276063, which is incorporated herein by reference. Further details and specific protocols for DA neuronal differentiation are provided in PCT Publication No. WO2013 / 067362, which is incorporated herein by reference.

[0133] Embryoid bodies (EBs) are aggregates of cells that may be derived from pluripotent stem cells, e.g. ES cells or iPS cells, and have been studied with mouse embryonic stem cells. To recapitulate some cues inherent to in vivo differentiation, three-dimensional aggregates (i.e., embryoid bodies) can be generated as an intermediate step. At the beginning of cell aggregation, differentiation can be initiated and cells begin to recapitulate embryonic development to a limited extent. They cannot form trophectoderm tissues (including the placenta), but can generate virtually all other types of cells present in the organism. Neural differentiation can be promoted following aggregate formation.

[0134] Cell aggregation can be forced by hanging drop, plating on non-tissue culture treated plates or spinner flasks, both methods that prevent cells from attaching to the surface to form typical colony growth. ROCK inhibitors or myosin II inhibitors can be used before, during or after aggregate formation to culture pluripotent stem cells.

[0135] Pluripotent stem cells can be seeded into aggregation-promoting medium using any method known in the art of cell culture. For example, pluripotent stem cells can be seeded into aggregation-promoting medium as single colonies or as groups of clones, and pluripotent stem cells can also be seeded as essentially individual cells. In some embodiments, pluripotent stem cells are dissociated into essentially individual cells using mechanical or enzymatic methods known in the art. By way of non-limiting example methods, pluripotent stem cells can be exposed to proteolytic enzymes that destroy connections between the cells and the culture surface and between the cells themselves. Enzymes that can be used to individualize pluripotent stem cells for aggregate formation and differentiation include, but are not limited to, trypsin, such as TrypLE, in its various commercially available formulations, or a mixture of enzymes, such as Accutase®. In certain embodiments, pluripotent cells can be added or seeded into culture medium for culture formation on a culture surface as essentially individual (or dispersed) cells.

[0136] For example, dispersed pluripotent cells can be seeded into culture medium. In these embodiments, the culture surface can be essentially composed of any material that is compatible with standard sterile cell culture methods in the art, e.g., a non-adherent surface. The culture surface can additionally include a matrix component as described herein. In some embodiments, the matrix component can be applied to the culture surface prior to contacting the surface with the cells and medium.

[0137] Substrates such as collagen, fibronectin, vitronectin, laminin, matrigel, etc. can be used to induce differentiation. Differentiation can also be induced without resuming proliferation (i.e., without dissociating the neurospheres) by placing the cells in suspension in the presence of proliferation-inducing growth factors.

[0138] In some embodiments, cells are cultured on a fixed substrate in a culture medium. A proliferation-inducing growth factor can then be administered to the cells. The proliferation-inducing growth factor can cause the cells to attach to the substrate (e.g., polyornithine-treated plastic or glass), plate, and begin to differentiate into various cell types.

[0139] V. Non-static culture In certain aspects, non-static cultures can be used for the culture and differentiation of pluripotent stem cells. Non-static cultures can be any culture in which cells are kept at a controlled movement rate, for example, using shaking, rotating or agitating platforms or culture vessels, particularly large volume rotating bioreactors. In some embodiments, rocker tables can be used. Agitation can improve the circulation of nutrients and cellular waste products, and can also control cell aggregation by providing a more uniform environment. For example, the rotation speed can be set to at least or at most about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100 rpm, or any range derivable therebetween. The incubation period in non-static culture for pluripotent stem cells, cell aggregates, differentiated stem cells or progeny cells derived therefrom may be about 4 hours, 8 hours, 16 hours, or 1, 2, 3, 4, 5, 6 days, or 1, 2, 3, 4, 5, 6, 7 weeks or more, or any range derivable therein.

[0140] VI. Genetic modification and purification of cells In some embodiments, the cells provided herein, such as mDA progenitor cells, may be genetically modified. A cell is said to be "genetically modified" or "transgenic" if a polynucleotide has been transferred to the cell by any suitable means of artificial manipulation, or if the cell is a descendant of an original cell in which the polynucleotide has been inherited and modified. In some embodiments, the cell may contain an antibiotic resistance gene, such as under the control of a neuronal promoter, such as the MAP2 promoter. For example, in some embodiments, the marker gene is an antibiotic resistance gene, and neuronal cells may be purified by exposing the cell culture to an antibiotic, thereby killing cells that have not differentiated into neuronal cells. For example, cells expressing the neomycin gene under the control of the MAP2 promoter may be exposed to G418 to kill non-neuronal cells. Additional methods that may be used with the present invention are described in U.S. Patent Application No. 14 / 664,245, which is incorporated by reference herein in its entirety and without any disclaimer.

[0141] In some embodiments, a population of cells containing dopaminergic neurons can be purified by exposing the cells to a mitotic inhibitor or chemotherapeutic agent that kills dividing cells. For example, in some embodiments, a population of cells containing immature midbrain DA neurons (e.g., D27-D31 cells) produced by the methods of the invention can be purified, for example, by contacting the cells with mitomycin C to kill dividing cells.

[0142] VII. Uses of Dopaminergic Neurons and Dopaminergic Neuronal Precursors The mDA precursor cells (e.g., D17 cells) provided herein can be used in a variety of applications, including, but not limited to, in vivo cell transplantation or implantation; in vitro screening of cytotoxic compounds, carcinogens, mutagens growth / regulatory factors, pharmaceutical compounds, etc.; elucidation of mechanisms of neurodegeneration; studying which drugs and / or growth factors activate the mechanisms; gene therapy; and production of biologically active products.

[0143] A. Test Compound Screening The midbrain DA precursors (e.g., D17 cells) provided herein can be used to screen factors (e.g., solvents, small molecule drugs, peptides and polynucleotides) or environmental conditions (e.g., culture conditions or manipulations) that affect the characteristics of the DA neurons or mDA precursor cells provided herein.

[0144] In some applications, stem cells (differentiated or undifferentiated) may be used to screen for factors that promote maturation of cells along a neural lineage or promote the growth and maintenance of such cells in long-term culture. For example, candidate neural maturation or growth factors may be tested by adding them to stem cells in different wells and then determining any phenotypic changes that occur with the desired criteria for further culture and use of the cells.

[0145] Screening applications of the present disclosure include testing of pharmaceutical compounds in drug research. Standard methods of testing are provided, for example, in In Vitro Methods in Pharmaceutical Research, Academic Press, 1997). In certain aspects of the embodiment, cells produced by the methods detailed herein can be used as test cells for standard drug screening and toxicity assays (e.g., testing to identify, validate, and characterize function or test delivery of therapeutic molecules for treating cell lineage-specific diseases), as previously performed on primary neurons in short-term culture. Evaluating the activity of a candidate pharmaceutical compound generally involves combining neurons provided in certain aspects of the invention with the candidate compound, determining any changes in the electrophysiology, morphology, marker phenotype, or metabolic activity of the cells resulting from the compound (compared to untreated cells or to cells treated with an inactive compound), and then comparing the correlation between the effect of the compound and the observed changes. Screening may be performed either because the compound is designed to have a pharmacological effect on neuronal cells, or because a compound designed to have an effect elsewhere may have unintended neurological side effects. Two or more drugs can be tested in combination (by combining with cells simultaneously or sequentially) to detect possible drug-drug interactions.

[0146] In some applications, compounds may be screened or tested for potential neurotoxicity. Cytotoxicity may be initially determined by effects on cell viability, survival time, morphology, or leakage of enzymes into the culture medium. In some embodiments, testing is performed to determine whether a compound(s) affects cell function (e.g., neurotransmission or electrophysiology) without causing toxicity.

[0147] B. Treatment of Disorders of the Central Nervous System 1. Diseases of the central nervous system The dopaminergic neurons and mDA precursor cells (e.g., D17 cells) provided herein can be transplanted to regenerate neural cells in individuals with central nervous system (CNS) diseases. In some embodiments, the mDA precursor cells produced by the methods of the present invention can be administered to a subject to treat CNS diseases (e.g., administered to the brain or midbrain, e.g., caudate nucleus, putamen, or substantia nigra to treat Parkinson's disease). Such diseases include, but are not limited to, neurodegenerative diseases, e.g., Parkinsonism.

[0148] As used herein, the term "parkinsonism" refers to a group of diseases that are all related to a lack of dopamine in the basal ganglia, the part of the brain that regulates movement. Symptoms include tremor, bradykinesia (extremely slow movement), flexed posture, postural instability and rigidity. A diagnosis of parkinsonism requires the presence of at least two of these symptoms, one of which must be tremor or bradykinesia. The most common form of parkinsonism is idiopathic or classic Parkinson's disease (PD), but a significant minority of diagnoses, about 15 percent of the total, may have one of the Parkinson Plus Syndromes (PPS). These syndromes, also known as atypical parkinsonism, include corticobasal degeneration, Lewy body disease, multiple systematrophy and progressive supranuclear palsy. Parkinson's disease generally involves the dysfunction and death of important nerve cells in the brain, primarily in an area of ​​the brain called the substantia nigra. Many of these important nerve cells produce dopamine. When these neurons die, the amount of dopamine decreases and prevents a person from regulating movement normally. The intestinal tract also has dopamine cells that degenerate in Parkinson's patients, which may be an important causative factor in the gastrointestinal symptoms that are part of the disease. The exact symptoms experienced by an individual may vary from person to person. The main motor signs of Parkinson's disease include: tremors of the hands, arms, legs, jaw and face; bradykinesia or slow movement; stiffness and rigidity of the limbs and trunk; and postural instability or loss of balance and coordination.

[0149] In some embodiments, iPSC-derived mDA progenitor cells (e.g., D17 cells) may show improved properties for clinical treatment of PD compared to other iPSC-derived mature mDA neurons. iPSC-derived mDA neurons differentiated through floor plate intermediates can engraft into athymic rats with unilateral 6-hydroxydopamine (6-OHDA) lesions, survive for long periods, and reduce or reverse drug-induced locomotor asymmetry (Hiller et al., 2020; Wakeman et al., 2017). Cells at various stages of development have been transplanted previously (Bye, Thompson, & Parish, 2012; Kirkeby et al., 2012; Kriks et al., 2011; Niclis et al., 2017).

[0150] In some embodiments, the mDA progenitor cells provided herein may exhibit superior properties for clinical treatment of diseases, such as PD. As shown in the examples below, 1) an iPSC system and differentiation process was developed that resulted in the generation of clinically usable mDA progenitor cells; 2) intrastriatal grafts of iPSC-derived mDA progenitor cells (cryopreserved in vitro at day 17) in immunocompromised rats completely reversed 6-OHDA-induced locomotor asymmetry, survived in large numbers, and densely reinnervated the host striatum, outperforming grafts of cells cryopreserved at days 24 and 37; 3) D17 progenitor cells were cryopreserved in vitro at day 17, and 4) D17 progenitor cells were cryopreserved in vitro at day 17, and 5) D17 progenitor cells were cryopreserved in vivo at day 17, and 6) D17 progenitor cells were cryopreserved in vivo at day 17, and 7) D17 progenitor cells were cryopreserved in vivo at day 17, and 8) D17 progenitor cells were cryopreserved in vivo at day 17, and 9) D17 progenitor cells were cryopreserved in vivo at day 17, and 10) D17 progenitor cells were cryopreserved in vivo at day 17, and 11) D17 progenitor cells were cryopreserved in vivo at day 17, and 12) D17 progenitor cells were cryopreserved in vivo at day 17, and 13) D17 progenitor cells were cryopreserved in vivo at day 17, and 14) D17 progenitor cells were cryopreserved in vivo at day 17, and 15) D17 progenitor cells were cryopreserved in They were observed to mature in vivo and maintain the appropriate mDA lineage, 4) D17 and D24 grafts placed in the substantia nigra showed long-distance axonal growth to multiple host targets normally innervated by the telencephalic central dopamine system, 5) high doses of D17 progenitor cells resulted in earlier and more complete functional recovery than low doses with corresponding increases in cell survival and graft-derived TH innervation, and 6) neither teratomas nor excessive proliferation of cells were observed when iPSCs subjected to our differentiation protocol were transplanted. The mDA progenitor cells provided herein may exhibit one or more or all of the above advantages listed above when used clinically.

[0151] In some embodiments, mDA progenitor cells (e.g., D17 cells) are administered to patients to treat brain diseases or brain injuries involving the death of dopaminergic neurons, such as Parkinson's disease (PD). As shown in the examples below, mDA progenitor cells have been observed to engraft, innervate, and functionally effective in vivo using an animal model of PD (i.e., hemiparkinsonian rats). mDA progenitor or precursor cells (cryopreserved at day 17, "D17"), immature mDA neurons ("D24"), and purified mDA neurons ("D37") have been tested and compared to commercially available R&D grade purified mDA neurons (D38, "G418") (Hiller et al., 2020; Wakeman et al., 2017). D17 or D24 cells were observed to provide long-distance innervation when grafted into the substantia nigra (SN). D17 mDA progenitor cells were observed to have the most robust survival and fiber outgrowth, and dose-ranging experiments were used to determine the lowest dose that resulted in early onset of functional recovery in hemiparkinsonian rats. These results demonstrated that the mDA progenitor cells provided herein can be used to treat PD in mammalian subjects, e.g., humans.

[0152] It is expected that various dosages of the mDA precursor cells (e.g., D17 cells) disclosed herein can be administered therapeutically to a mammalian subject, e.g., a human, for example, from about 2,500 cells / μL to about 150,000 cells / μL, from about 10,000 cells / μL to about 150,000 cells / μL, from about 40,000 cells / μL to about 100,000 cells / μL, from about 15,000 cells / μL to about 45,000 cells / μL, about 3×10 cells / μL, or from about 2×10 cells / μL to about 3×10 cells / μL. 6 ~9×10 6 pieces, cells 2500, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 1×10 4 , 2×10 4 , 3×10 4 , 4×10 4 , 5×10 4 , 6×104 , 7×10 4 , 8×10 4 , 9×10 4 , 1×10 5 , 1.1×10 5 , 1.2×10 5 , 1.3×10 5 , 1.4×10 5 Or 1.5 x 10 5 10 cells / μL of midbrain dopaminergic neuron progenitor cells, or any range derivable therebetween, can be administered to a mammalian subject, e.g., a human. The total number of cells administered to a mammalian subject, e.g., a human patient, is about 1×10 5 From about 100×10 6 It is expected that the total number of cells can range from 10 to 100, and can be selected by a clinician based on the symptoms and other characteristics of the subject. Preferably, the cells are administered to the subject's brain. For example, the mDA precursor cells can be administered to the subject's striatum, such as the putamen or substantia nigra. In some cases, it may be sufficient to administer the mDA precursor cells to one location in the subject's brain. In other embodiments, the mDA precursor cells are administered to multiple sites in the subject's brain (e.g., the striatum or putamen) and / or with multiple needles. In human subjects, it is expected that administration of mDA cells at multiple sites in the striatum can promote more extensive innervation by mDA precursor cells in some cases.

[0153] 2. Method of administering cells The cells provided herein can be administered to a subject either locally or systemically. In some preferred embodiments, mDA progenitor cells (e.g., D17 cells) are administered to the subject's brain. Methods for administering DA neurons to a subject, such as stereotactic administration to the brain, are well known in the art and can be applied to the cells and cell cultures provided herein. When a patient receives cells derived from his or her own cells, this is called autologous transplantation, and such transplants have a low probability of rejection.

[0154] An exemplary method of administering stem cells or differentiated neuronal cells to a subject, particularly a human subject, includes injecting or transplanting cells into a target site (e.g., the striatum and / or substantia nigra) of the subject. The mDA progenitor cells can be inserted into a delivery device that facilitates induction by injection or transplantation of cells into a subject. Such a delivery device can include a tube, e.g., a catheter, for injecting cells and liquids into the body of a recipient subject. In a preferred embodiment, the tube additionally has a needle, e.g., a syringe, through which the cells of the present invention can be introduced into a subject at a desired location. The stem cells can be inserted into such a delivery device, e.g., a syringe, in various forms. For example, the cells can be suspended in a solution, can be cell aggregates, or alternatively can be embedded in a support matrix when contained in such a delivery device.

[0155] The support matrix into which the stem cells, neurons or neuronal progenitor cells can be incorporated or embedded can include a matrix that is recipient-compatible and degrades into products that are not harmful to the recipient. The support matrix can be a natural (e.g., hyaluronic acid, collagen, etc.) and / or synthetic biodegradable matrix. Synthetic biodegradable matrices that can be used include synthetic polymers such as polyanhydrides, polyorthoesters, and polylactic acids. In some embodiments, dopaminergic neurons (e.g., dopaminergic neurons that are not fully differentiated) are embedded in a hyaluronic acid matrix and administered to a subject to treat a neurodegenerative disease (e.g., Parkinson's disease).

[0156] As used herein, the term "solution" includes pharma- ceutically acceptable carriers or diluents in which the cells of the present invention remain viable. Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer, solvents and / or dispersion media. The use of such carriers and diluents is well known in the art. The solution is preferably sterile and fluid to the extent that easy syringability exists.

[0157] Preferably, the solution is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms, such as bacteria and fungi. In some embodiments, the solution containing mDA progenitor cells (e.g., D17 cells) is administered to a patient in a sterile solution of BSS PLUS (Alcon, Fort Worth, TX). If desired, a preservative or antibiotic may be included in the pharmaceutical composition for administration. The solution of the present invention can be prepared by incorporating the mDA neuron progenitor cells described herein into a pharma- ceutically acceptable carrier or diluent, and optionally other ingredients.

[0158] 3. Dosage and Administration In one aspect, the methods described herein provide methods for enhancing engraftment of neuronal progenitor cells (e.g., D17 cells) or DA neurons in a subject. In some embodiments, the subject is a mammal, e.g., a human.

[0159] The composition is administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The amount and timing of administration will depend on the subject being treated, the capacity of the subject's system to utilize the active ingredient, and the degree of therapeutic effect desired. The exact amount of each active ingredient required to be administered will depend on the judgment of the practitioner and may be unique to each patient or subject. The suitable dosage range may depend on the route of administration, and various administration methods may be used.

[0160] Various dosages of the mDA precursor cells (e.g., D17 cells) disclosed herein can be administered to a mammalian subject for treatment, for example, from about 2,500 cells / μL to about 150,000 cells / μL, from about 10,000 cells / μL to about 150,000 cells / μL, from about 40,000 cells / μL to about 100,000 cells / μL, from about 15,000-45,000 cells / μL, or about 1×10 cells / μL. 6 ~9×10 6 pcs / μL, approx. 2500, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 1×10 4 , 2×10 4 , 3×10 4 , 4×10 4 , 5×104 , 6×10 4 , 7×10 4 , 8×10 4 , 9×10 4 , 1×10 5 , 1.1×10 5 , 1.2×10 5 , 1.3×10 5 , 1.4×10 5 Or 1.5 x 10 5 The total number of cells administered to a mammalian subject, e.g., a human patient, may be about 1×10 5 From about 100×10 6 It is expected that the total number of cells may range from 10 to 100, and may be selected by a clinician based on the subject's symptoms and other characteristics. In some embodiments, the mDA progenitor cells are administered via injection into the brain or central nervous system of a mammalian subject, preferably a human patient (e.g., into a single site or multiple sites in the brain, such as the striatum or putamen).

[0161] 4. Effectiveness The efficacy of a given treatment for enhancing DA neuron engraftment can be determined by one of skill in the art. However, as the term is used herein, a treatment is considered to be "effective treatment" if, for example, any one or all of the signs or symptoms of insufficient DA neuron engraftment are beneficially altered, other clinically observed symptoms are improved, or even, for example, at least 10% reversed, following treatment with a cell population as described herein. Efficacy can also be measured by the absence of individual deterioration as assessed by hospitalization, need for medical intervention (i.e., disease progression is halted), or frequency of engraftment failure. Methods for measuring these indicators are well known to those of skill in the art and / or described herein. Treatment includes any treatment of a disease in an individual or animal (some non-limiting examples of which include humans or mammals), including: (1) inhibiting the disease, e.g., preventing engraftment failure, or (2) alleviating the disease, e.g., causing a reduction in one or more symptoms. An effective amount for the treatment of a disease means an amount that is sufficient to cause a treatment or therapeutic benefit for the disease when administered to a mammal in need thereof. The efficacy of the agent can be determined by assessing physical indicators, such as DA neuron engraftment, e.g., tremor, bradykinesia, flexed posture, balance and coordination. In some embodiments, engraftment or neurofunction can be measured in vivo (e.g., in humans) using PET scans to detect metabolism, activity, dopaminergic neurotransmission (e.g., using PET tracers to image the dopaminergic system). Efficacy can be assessed in animal models of Parkinson's disease, for example, by performing behavioral tests, such as the step test or cylinder test.

[0162] C. Commercial, Therapeutic and Research Distribution For purposes of manufacture, distribution and use, neural cells, such as the midbrain DA neuron progenitor cells described herein, may be supplied in the form of a cell culture or suspension, in an isotonic vehicle or culture medium, optionally frozen for ease of transport or storage.

[0163] The mDA progenitor cells described herein can be provided using various reagent systems, including, for example, sets or combinations of cells present at any time during manufacture, distribution or use. The cell set can include any combination of two or more cell populations described in this disclosure, including but not limited to combinations with programming-derived cells (neuronal lineage cells, their precursors and subtypes), undifferentiated stem cells or other differentiated cell types. The cell populations of the set can share the same genome or its genetically modified form. Each cell type of the set can be packaged together or in separate containers, in the same facility or at different locations, at the same time or at different times, under the control of the same entity or different entities that share a business relationship. EXAMPLES

[0164] IV. [Example] The following examples are included to demonstrate preferred embodiments of the invention. It should be understood by those of skill in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to work well in the practice of the invention, and thus can be considered to constitute preferred modes of practicing the invention. However, those of skill in the art should understand, in light of this disclosure, that many changes can be made to the specific embodiments that are disclosed and still obtain the same or similar results without departing from the spirit and scope of the invention.

[0165] [Example 1] Materials and methods for producing cell cultures Midbrain neuron differentiation of VTN-TN expanded human induced pluripotent stem (iPS) cell lines in Essential 8 medium was performed with small molecule and growth factor induction using various differentiation medium compositions and schedules detailed in Table 1. In general, iPS cells were cultured in D1 DA neuron induction medium on day 1, D2 neuron induction medium on day 2, and D3-D4 DA induction medium on days 3 and 4. On day 5, cells were dissociated with TrypLE for 15 min and collected in DA quench medium before transferring cells into spinner flask suspension culture to form aggregates in D5 DA neuron aggregate formation medium.

[0166] On day 6, the aggregates were allowed to settle, approximately 66% of the medium was removed, and the aggregates were fed in DA neuron induction medium. On days 7–16, the aggregates were fed daily in DA neuron aggregate maintenance medium, and medium was changed from day 11 to day 16. On day 17, the aggregates were dissociated into a single cell suspension by TrypLE and plated on Matrigel in D17 DA neuron aggregate plating medium. On days 18, 20, and 22, medium was changed to Dopa neuron maturation medium. On day 24, cells were dissociated using Accutase and plated in DA neuron maturation plating medium. The following day, medium was changed to Dopa neuron maturation medium.

[0167] On days 27 and 29, the medium was changed to DA neuron maturation medium plus mitomycin C. On day 31, cells were dissociated with Accutase and re-plated in DA neuron maturation plating medium on poly-L-ornithine (PLO) / laminin-coated flasks. Cells were then fed with Dopa neuron maturation medium on days 32, 34, and 36. On days 37 or 38, cells were dissociated again with Accutase and subjected to analysis or cryopreserved for later use. [Table 2] JPEG2024513912000003.jpg244170JPEG2024513912000004.jpg245170JPEG2024513912000005.jpg206170

[0168] Midbrain neuron differentiation of VTN-TN expanded human induced pluripotent stem (iPS) cell lines in Essential 8 medium was performed with small molecule and growth factor induction using various differentiation medium compositions and schedules detailed in Table 2. In general, iPS cells were cultured in D1 DA neuron induction medium on day 1, D2 neuron induction medium on day 2, and D3-D4 DA induction medium on days 3 and 4. On day 5, cells were dissociated with TrypLE for 15 min and collected in DA quench medium before transferring cells into spinner flask suspension culture to form aggregates in D5 DA neuron aggregate formation medium.

[0169] On days 6 and 7, aggregates were allowed to settle, approximately 66% of the medium was removed, and aggregates were fed on D6-7 DA induction medium (days 6 and 7). On days 8-10, aggregates were fed daily on D8-10 DA aggregate maintenance medium. On days 11-16, aggregates were fed daily on D11-16 DA aggregate maintenance medium. On day 17, aggregates were dissociated into a single cell suspension by TrypLE and placed in quench for 15 min (DA quench medium 1), then washed and cryopreserved in cryopreservation medium. Cryopreserved cells were stored in the vapor phase of liquid nitrogen.

[0170] [Table 3] JPEG2024513912000007.jpg240170JPEG2024513912000008.jpg228170

[0171] [Example 2] mDA precursor patterning Efficient patterning of mDA precursors generally requires obtaining a highly enriched population of mDA neurons at the end of the manufacturing process, as measured by the percentage of cells co-expressing FoxA2 and Lmx1 on method day 17. If the majority of cells on day 17 are not mDA precursors, the resulting neurons will either have a large population of non-mesencephalic phenotype neurons or have an outgrowth of proliferative cells that typically results in neuronal detachment or difficulty or inability to purify post-mitotic neurons.

[0172] These mono-SMAD experiments were repeated with the modification that Benzonase® (endonuclease, EMD Millipore) was included in the incubation at a concentration of 10 U / mL on day 5. Inclusion of Benzonase® in the incubation on day 5 was observed to reduce or prevent excessive clumping in aggregate formation.

[0173] [Example 3] Flow cytometry assay of FoxA2 / Lmx1 co-expression FoxA2 / Lmx1 co-expression is a key readout for successful dopamine neuron precursor patterning, therefore an intracellular flow cytometry assay was developed that is less subjective and variable than results derived using cell counting software run on immunocytochemistry images. This assay can accurately quantify the percentage of cells co-expressing FoxA2 and Lmx1 from days 17 to 24 of treatment, with results correlating to counts from the analyzed ICC images. Precursor patterning is considered successful if cells are >65% FoxA2+ / Lmx1+ at day 17.

[0174] [Example 4] Dopamine release from iPSC-mDA neurons iPSC line "K" (21534.101) was differentiated to completion (day 37) and cryopreserved. Cells were thawed and cultured at high density (8.8 × 10 5 / cm 2). Cells were fed with maturation medium without DAPT every 3rd day for a total of 14 days. On the day of the assay, cells were washed and incubated with HBSS (with or without 56 mM KCl) for 30 min. Dopamine concentration in the release solution was determined using a competitive dopamine ELISA kit (Eagle Biosciences). No dopamine release was detected from iPSC-derived forebrain neurons (iCell Neurons). Conversely, iPSC-mDA cells induced using the optimized mono-SMADi method (DA Therapy Neurons) secreted at least as much dopamine as cells induced using the optimized dual-SMAD method (iCell DopaNeurons). Thus, the cells are capable of performing the main functional attributes of mature dopamine neurons.

[0175] [Example 5] Electrical activity of iPSC-mDA neurons Cryopreserved iPSC-mDA neurons were thawed and plated on PEI-coated 48-well multielectrode array (MEA) plates. Cells were cultured according to the protocol applied by FUJIFILM Cellular Dynamics, Inc., "Measuring synchronous neuronal activity on the Maestro multielectrode array" in U.S. Patent Application No. 14 / 830,162. Neurons generated by the optimized mono-SMADi protocol (DA Therapy) demonstrated similar electrical activity, including mean firing rate (mFR), bursts (macro-BPMS) and connectivity, compared to cells generated by the optimized dual-SMADi protocol (iCell Dopa G100). Mean firing rate (mFR), frequency, and connectivity, as well as burst strength, increased over time and plateaued approximately 16 days after thawing. Time raster plots showed clear interspike intervals, high burst strength and bursting at all electrodes in the well, demonstrating high electrical activity.

[0176] [Example 6] Quantitative gene expression profile of FCDI DAPC-1 neurons On day 37 of the method, RNA was extracted from four batches of iPSC-mDA cells induced using the optimized mono-SMADi method (batches 1-4) and one batch of iPSC-mDA cells induced using the optimized dual-SMADi protocol (iCell DopaNeurons). After RNA isolation, real-time quantitative polymerase chain reaction (PCR) was performed using TaqMan Gene Expression Assays (Applied Biosystems) and results were expressed as relative expression to the GAPDH control. Values ​​<10 -4 are considered background (shaded box). Expression of midbrain and mDA neuron markers was similar between batches and between cells generated using different protocols. Markers of non-midbrain regions or non-mDA cell types were low and similar between mono-SMADi and dual-SMADi derived cells. Results are shown in Figures 12 and 13.

[0177] [Example 7] Engraftment of iPSC-DA precursors and neurons in a rat model of Parkinson's disease iPSC line "K" was differentiated using the optimized mono-SMADi protocol and cryopreserved at different stages of the differentiation process (days 17, 24 and 37). In addition, iPSC-mDA cells derived using the optimized dual-SMADi protocol (iCell Dopa) were cryopreserved on day 37 of the method. Cells were thawed and transplanted bilaterally into the striatum of 6-OHDA-treated but asymptomatic nude (RNU) rats (n=3 per group) (4.5×10 per injection). 5Cells). After 3 months, cell engraftment and innervation were assessed by histology of coronal sections. Although neuronal engraftment and innervation were observed in all four groups (Human NCAM staining), iPSC-DA progenitor cells (day 17) and immature mDA neurons (day 24) had much larger grafts and significant innervation compared to the more mature mono-SMADi and dual-SMADi derived mDA neurons (day 37 and day 37, Cell Dopa, respectively). In addition, numerous DA neurons (TH+) were observed in the precursor and immature DA neuron grafts. Ki67 staining revealed few proliferating cells in the grafts in the day 37 cells, and few Ki67+ cells in the grafts in the day 17 and day 24 cells. No tumors, nerve growth, or other adverse effects were observed in any of these animals. These results suggest that cells removed early (days 17-24) in the optimized mono-SMADi differentiation method can engraft and innervate better than more mature cells. The results are shown in Figure 9.

[0178] [Example 8] The above experiment was repeated using various CHIR concentrations, and the results are shown in Figure 18. As shown in the results, significant improvements were observed when using CHIR99021 concentrations of about 1.5 to about 1.75 μM.

[0179] [Example 9] Characterization of mDA progenitor cells A cryopreserved single cell suspension containing iPSC-derived midbrain dopamine neuronal progenitor cells ("FCDI DAPC-1") was generated via the methods described in the Examples above. Cells were derived from an allogeneic human iPSC line (FCDI designation 21534.101) via directed differentiation to obtain a population of dopaminergic neuronal progenitor cells.

[0180] FOXA2 flow cytometry assay was performed on mDA progenitor cells generated as described in the above examples. FOXA2 flow cytometry assay indicated that mDA progenitor cells showed correct floor plate patterning of FCDI DAPC-1. The results are shown in Figure 1.

[0181] FOXA2 / LMX flow cytometry assays revealed co-expression of FOXA2 and LMX in FCDI DAPC-1 mDA progenitor cells. Parallel ICC staining was performed for comparison, and co-expressing cells were observed to appear yellow. The results are shown in Figure 2.

[0182] After 12 days of post-thaw culture, FCDI DAPC-1 mDA progenitor cells have the potential to differentiate into immature DA neurons, as demonstrated by NURR1 expression. Parallel ICC staining was also performed. The results are shown in Figure 3.

[0183] A MAP2 / Nestin flow cytometry assay was used to confirm the percentage of cells with the potential to become mature (post-mitotic) neurons 14 days after thawing. Results from a representative batch are shown in Figure 4. Mutually exclusive nestin co-staining was included for better separation and gating of the MAP+ population. [Table 4] JPEG2024513912000010.jpg100170 [Table 5] JPEG2024513912000012.jpg47170

[0184] Dopamine secretion by cells similar to FCDI DAPC-1 ("PD Therapy Cells", a variant of an earlier method) was measured after 5 weeks of culture in maturation medium. The concentration of dopamine released into HBSS upon 30 minutes of incubation was measured. High values ​​were obtained after cell depolarization (HBSS + 56 mM KCl).

[0185] FCDI DAPC-1 cells were stained with anti-PAX6 (Biolegend #901301) (Figure 5A) or anti-FOXG1 (Figure 5B). iCell GABA Neuron (FCDI) are shown as a positive control; these are cells patterned with a forebrain phenotype, are primarily GABAergic, and contain a subpopulation of PAX6+ neurons, as well as a subpopulation of FOXG1+ neurons. Results are shown in Figure 5A-B.

[0186] RT-QPCR assays of REX1, TDGF1 and NODAL can detect spiking inhibitory postsynaptic currents (iPSCs) in DA precursor cells (FCDI DAPC-1 method). The REX1 assay is the most sensitive, reproducibly detecting one iPSC per 100,000 cells of the FCDI DAPC-1 method. The results are shown in Figure 6. [Table 6]

[0187] As shown above, dopaminergic neuronal progenitor cells displayed phenotypic markers (Figures 1 and 2) and developmental potential similar to dopamine neuronal precursors found in the substantia nigra region of the developing midbrain (Figures 3, 4). FCDI DAPC-1 lack significant forebrain neurons and residual iPSCs that may be detrimental to therapeutic applications (Figures 5A-B, 6, and Table 3). Importantly, unlike other DA cell therapeutic products, FCDI DAPC-1 were observed to be proliferative progenitor cells, as demonstrated by EdU incorporation (Figure 7).

[0188] [Example 10] Reduction of motor impairment in vivo in PD animal models 6-OHDA-lesioned athymic nude rats (RNUCrl: NIH-Foxn1), an animal model of Parkinson's disease (PD), rnu) were used for further studies. These animals displayed significant motor impairments, which can be observed using the amphetamine-induced rotation test (Blesa et al., 2014; Campos et al., 2013; Deumens et al., 202; Vermilyea, et al., 2018). Dopaminergic progenitor neurons (D19) produced as described in the above examples were administered to the substantia nigra of mice to determine whether this reduced the animals' motor impairments using the amphetamine-induced rotation test. As discussed below, mature D37 neurons did not improve the animals' motor impairments, but administration of D17 and D19 dopaminergic progenitor neurons to mice was able to completely reverse motor impairments by 6 months in vivo.

[0189] Rats with unilateral lesions in the nigrostriatal dopamine system (e.g., induced by injection of neurotoxins, e.g., 6-hydroxydopamine, overexpression of α-synuclein or toxic synuclein protofibrils) were used as an experimental model to mimic the loss of dopamine neurons seen in Parkinson's disease. The amphetamine rotation test is commonly used to monitor the degree of motor dysfunction induced by the lesion, and this test has also become a standard tool to demonstrate transplant-induced functional recovery or to demonstrate the efficacy of neuroprotective interventions aimed at preserving or restoring DA neuron function. This test is described, for example, in Wakeman et al., 2017.

[0190] Amphetamine rotation was tested in the rat PD model described above. As shown in Figure 8, administration of day 17 (D17) dopaminergic neuronal progenitor cells resulted in the alleviation of motor symptoms in rats by 6 months, as observed in the amphetamine rotation test. D24 immature neurons improved motor performance, but the effect of D24 neurons appeared to be less than that of D17 neurons, which was particularly noted at 4 and 6 months.

[0191] Immunohistochemical staining of brain slices was performed on brain slices 6 months after administration of neurons into the striatum of rats. Increased NCAM expression was observed after administration of D17 or D24 neurons compared to mature D37 or iCell® Dopa neurons. These results indicate that precursor (D17) and immature (D24) mDA neurons are more capable of engraftment than mature (D37) mDA neurons.

[0192] Striatal reinnervation was observed 6 months after transplantation. Striatal innervation of D17 cells appeared to be the best compared to other neurons tested. D17 and D24 cells showed a marked improvement in innervation compared to D37 or iCell® Dopa neurons. Results are shown in FIG. 9, and an example of intranigral innervation of grafts to the striatum is shown in FIG. 10.

[0193] Progenitor markers were measured in D17, D24 and D37 cells using qPCR. Comparing D17 and D24 cells, Lmx1, Nurr1 and Pitx3 are expressed at higher levels in D24 cells, while En-1, Pax8, ETV5 and Glast are expressed at higher levels in D17 cells (Figure 11). Maturation markers were also measured in cells, with AQP4 and tyrosine hydroxylase (TH) being expressed at higher levels in D24 cells compared to D17 cells (Figure 12). Additional data on normalized expression of different genes in different cell types generated after various differentiation periods (D17, D24 and D37 time points) are shown in Figure 19. These results are consistent with increased differentiation of D24 cells into mature dopaminergic neurons compared to D17 dopaminergic neuronal progenitor cells. Immunocytochemistry was also performed on D24 and D27 cells, and the results are shown in Figure 13. The results of immunocytochemistry (ICC) experiments were consistent with the qPCR findings.

[0194] Functional testing of alternative cell types showed that administration of D19 "intermediate" dopaminergic cells was able to completely reverse motor deficits at 6 months. These cells were plated on LN521 in D17 plating medium, then fed neuronal maturation medium minus DAPT D16-18, and frozen at D19, but with modifications in which benzonase was added to D5 and D17 quench medium, according to the methods described in Table 2, until D15, when the CHIR concentration was changed from 1.75 to 1.65 μM. D19 animals began to show functional improvement at 4 months, and this group showed rapid improvement compared to reaggregates (D17 cells that were dissociated, reaggregated to a smaller size overnight, and frozen at D18) or their control cells (D17 cells from which reaggregates were created). Reaggregates and their control cells maintained motor deficits at 4 months, before improvement was realized. The total number of cells injected into each animal averaged 290k per animal for D19, 333k per animal for reaggregates, and 369k per animal for reaggregate controls. Multiple animals (N=3) were tested in each of the reaggregate and reaggregate control groups. The D19 animal group had N=10 animals. The results are shown in FIG.

[0195] [Example 11] Expression of D17 dopaminergic precursor neurons Brain slices were stained for the presence of human nuclei (hNuc), tyrosine hydroxylase (TH) and Ki67 6 months after engraftment. TH is involved in the production of dopamine by dopaminergic neurons and dopaminergic neuronal precursor neurons. Ki67 is a gene involved in cell proliferation. h-Nuc is a genetic marker expressed by neuronal precursor cells and was measured to assess whether further cell expansion occurs after engraftment. The results are shown in Figure 16. A full series of 40 μm coronal sections stained for HuNuclei using the DAB method were counted at 60x magnification using a Stereo Investigator optical fractionator (Microbrightfield Bioscience, version 10.40). The stereological parameters for TH (every 12th consecutive section) and HuNuclei (every 12th consecutive section) were frame size (75 μm×75 μm) and grid size (250 μm×250 μm) to count 9% of the total graft area with an average CE=0.13 for HuNuclei (Gundersen m=1), and frame size (80 μm×80 μm) and grid size (225 μm×225 μm) to count 12.6% of the total graft area with an average CE=0.17 for TH (Gundersen m=1). Percentages were calculated based on the calculated number of hNuc, TH, and Ki67 positive cells in each graft. The calculated total number of cells divided by the total number of cells input yields the percent positive.

[0196] As shown in Figure 16A-C, each group average showed over 100% positivity of hNuc, indicating cell expansion after engraftment. At 6 months of sacrifice, the Ki67 positive population averaged less than 1% of the hNuc population, excluding D18 and reaggregates. This low percentage of Ki67 supports the notion that cells no longer proliferate 6 months after engraftment, but does not reflect the proliferative potential of engrafted cells early after the engraftment date. Having an average hNuc positivity of over 100% in all groups suggests that the proliferative cell types early after engraftment have changed to final cell types that no longer proliferate but retain markers of human origin. The percentage of TH positive cells is much lower than previously seen in this animal study. The group average is around 10-15%, whereas previously, the inventors have experienced average TH+ percentages in the range of 20-30%.

[0197] Stereological analysis of hNuc, TH and Ki67 was performed. Every 12th section (1 / 2 of the series) was stained with hNuclie, TH or hKi67 and quantified by unbiased stereology. In each animal, the grafted areas were outlined and counted. Each graph has its own Y-axis. The results are shown in Figures 17A-C.

[0198] The number of hNuc positive cells for each animal in each test group, including the mean and standard error of the mean (SEM), is shown in Figure 17A. The use of this marker demonstrates that the cells that are hNuc-ir are of human origin (injected test material). The D17 T75 fresh group shows the greatest extent of engrafted hNuc+ cells compared to all other groups. All other groups appear to have consistent cell engraftment in all animals within that group. The averages for each group vary by sample. Analysis using a one-way ANOVA test indicates that there is statistical significance in the mean number of hNuc+ cells for the D17 T75 fresh and D19 groups (p=0.0385).

[0199] The number of TH positive cells for each animal in each test group, including the mean and SEM, is shown in Figure 17B. TH-ir positive cells represent a cell type capable of producing dopamine, and the cells are from the test material due to the ablation performed prior to transplantation. With the exception of the D17 T75 6 hr group (only one animal had staining when quantified), all groups showed similar numbers of engrafted TH+ cells, averaging approximately 60,000 cells. One-way ANOVA test indicates that there is no statistical difference between these treatment groups for TH engraftment.

[0200] Figure 17C shows the number, mean and SEM of Ki67 positive cells for each animal in each group. Ki67-ir cells represent a cell type capable of division / spreading. The specific antibody used in this assay is human specific and binds only to cells of human origin. These results show that the administered cells display a very low rate of cell proliferation.

[0201] In vivo behavioral improvements were observed in 6-OHDA-lesioned animals administered D17 cells. In vivo characterization of D17 precursors and analysis of function, survival and innervation are shown in Figures 20A-J. Time-based analysis of d-amphetamine-induced rotation measured before surgery and 2, 4 and 6 months after engraftment (Figure 20A). Stereological estimates of hNuclei-ir cells contained in grafts with low, medium, high or maximum possible dose (Figure 20B). Quantification of stereological estimates of TH-ir cells (Figure 20C) and in each group (Figure 20D) was performed. Graft sections showed positive staining for hGFAP (Figure 20E), 5-HT (Figure 20F). Low, medium, high and maximum possible doses of D17 cells were imaged for hNuclei (Figure 20G), TH (Figure 20H), immunofluorescent triple-labeled hNuclei / TH / FoxA2 (Figure 20I) and TH / Girk2 / calbindin (Figure 20J). These results demonstrate that D17 cells can be administered to restore behavioral capabilities in vivo, as observed using an animal model of Parkinson's disease.

[0202] [Example 12] Materials and Methods The following methods were used in the experiments described in Examples 10-12.

[0203] Lesion and engraftment: Female nude rats underwent 6-OHDA lesions at 8-9 weeks of age. Neurotoxin was administered directly into the medial forebrain bundle while rats were anesthetized in a stereotaxic apparatus. Rats were tested every 3 weeks after lesion with amphetamine and scored for rotational speed measured using a Rotometer. Animals showing successful lesion (>5 rotations / min in 30 min) were randomly distributed into experimental treatment groups based on amphetamine rotational data to receive cells or vehicle control. Freshly prepared cells were injected directly into the striatum of rats in a volume of 3 μL (40,000 cells per animal) at a concentration of 150,000 cells / μL.

[0204] Rotation measurements: After lesion, animals showed rotational behavior (circling) towards the lesioned side, indicating a successful lesion. This behavior was induced using amphetamine, which increases the amount of dopamine in the brain. After allowing rats to acclimate to the chamber for 5 min, rotations were tracked for 90 min, binned every 5 min, and the mean net number of rotations per minute was calculated. Amphetamine rotations were measured every 2 months after engraftment (Figure 1). Apomorphine injections were used to track rotations in the opposite direction of the lesioned hemisphere. Apomorphine-induced rotations were tracked for 60 min and measured every 3 months after engraftment (Figure 2).

[0205] Postmortem analysis: Rats (6 months) were anesthetized and perfused transcardially with ice-cold 0.9% saline followed by 4% paraformaldehyde. Brains were removed and postfixed 18-24 hours later in 4% paraformaldehyde before being mounted and submerged in a sucrose gradient (10%, 20%, 30%). All brains were sectioned in 40 μm coronal sections in a freezing sledge microtome and processed for immunohistochemistry using 3,3'-diaminobenzidine (DAB) with nickel enhancement where applicable or fluorescent immunocytochemistry. Stereological parameters for TH and HuNuclei (1 / 2 of a series) were frame size (80 μm × 80 μm) and grid size (350 μm × 350 μm) to count 9% of the total area with an average CE = 0.14 for TH (Gundersen m = 1) counted at 60x magnification. Sections containing the grafts (every 12th section) were stained with Ki-67. The total area of ​​the grafts (100%) was counted using an Olympus BX61. The number of Ki67+ cells was calculated as 12× the sum of the number of Ki67+ cells counted in the five sections.

[0206] [Example 13] Characterization of mDA progenitor cells in vitro Previous transplantation studies utilized research grade iPSC-derived mDA neurons, and cells generated using a variation of the same differentiation protocol (Hiller et al., 2020; Wakeman et al., 2017). The next phase in the development of cell therapy took steps to transition to a method suitable for cGMP manufacturing and clinical use. A clinical grade human iPSC line was used. An iPSC master cell bank and a working cell bank were manufactured under cGMP conditions. The early stages of iPSC-mDA differentiation were adapted by altering the timing and concentration of small molecule inhibitors. To address safety and regulatory concerns, raw materials used for the differentiation method were of clinical grade when possible. iPSC-mDA neurons differentiated to the most advanced maturation stage (D37) were enriched by a previously described differentiation method (Hiller et al., 2020) that uses a low concentration of mitomycin C to remove proliferating cells (Figure 21A). This approach circumvented the need for a drug selection cassette used for R&D grade G418 cells. mDA precursor (D17) and immature (D24) mDA neurons cannot be enriched with mitomycin C because they remain proliferative, so the primary goal of this experiment was to determine whether an adapted differentiation method (without an enrichment step) would be suitable to prevent unwanted cell proliferation in grafted D17 and D24 cells.

[0207] Previous studies have provided evidence that human iPS-mDA neurons can express high levels of regional midbrain markers, as well as low levels of forebrain and hindbrain markers (Hiller et al., 2020; Wakeman et al., 2017). A similar gene expression panel was used to characterize cells generated using the differentiation method adapted for translational use (Figure 21B, Table 5). All stages of differentiation (days 17, 24, and 37) expressed high levels of regional midbrain markers OTX2, FOXA2, and LMX1A. EN1 was most highly expressed at D17, decreased by D24, and maintained this expression level until D37. More mature mDA markers (NURR1, TH, DAP, GIRK, CALB) were expressed at very low levels or not at all at D17 and showed a gradual increase from D24 to D37. PITX3 expression was highest at D24. ETV5 and SPRY1, markers reported to predict good engraftment (Kirkeby et al., 2017), were expressed at all stages, whereas CNPY1 had low expression at D17 and D24 and was nearly undetectable at D37. Expression levels of markers of non-mDA cell types, e.g., motor (PHOX2A, HB9), cholinergic (CHAT), glutamatergic (VGLUT1), GABAergic (GAD1) ​​and serotonergic (SERT) neurons, were low / not expressed at all stages of differentiation. The most highly expressed off-target marker was GLAST, indicating that some astrocyte precursors were present in the cultures. Consistent with the presence of STN neurons expressing some of the same molecular markers as mDA neurons (Kee et al., 2017; Nouri & Awatramani, 2017), expression of DBX1, PITX2 and BARHL1 was observed at all stages of differentiation. The hindbrain marker HOXA2 was not expressed, and low levels of forebrain markers were detected across D17 to 37. Flow cytometry demonstrated that <1% of D17 cells expressed FOXG1 or PAX6, indicating a lack of forebrain neuronal precursors.BRN3A, which is expressed in the red nucleus of the midbrain (Agarwala, Sanders, & Ragsdale, 201; Wallen et al., 1999), was also detected. At all time points tested (D17, D24, and D37), the neural stem cell marker SOX1 was not expressed, indicating that the cultured cells had passed the stem cell differentiation stage. At each of the three time points, the neuronal precursor marker DCX was expressed, while the expression of the more mature neural marker NEUN increased from D17 to D37. [Table 7] JPEG2024513912000015.jpg39170

[0208] We then used flow cytometry to examine the mDA population at the protein level (Figure 22A) and single-cell PCR to examine the mDA population at the RNA level (Figure 31A-I). The percentage of FOXA2 immunoreactive (ir) cells remained high (>80%) from D17 to D37, while co-expression of FOXA2 and LMX1 was approximately 70% at D17 and increased to over 90% by D24. This population of FOXA2 / LMX1-ir cells remained high (about 85%) in D37 cultures. As expected, and consistent with the qPCR results, more mature markers such as NURR1, MAP2, and TH were not detected in D17 samples. The total population percentages for each of these three markers increased over time, with approximately 20% immunoreactive in D24 samples and 50% (NURR1, FOXA2 / TH) or 90% (MAP2) immunoreactive in D37 samples, respectively. Immunocytochemistry was used to visually confirm these cell populations (Figure 22B, Figure 29). Consistent with the flow cytometry results, LMX1A and FOXA2 were co-expressed in a high percentage of cells at each developmental time point. Also consistent with flow cytometry, NURR1- and TH-ir cells were absent at D17, while faint amounts were seen by D24, with numerous cells and distinct, well-defined cells also observed at D37. MAP2 was not detectable in D17 samples, but expression increased over time with robust MAP2-ir at D37. Conversely, nestin-ir cells were abundant at both D17 and D24, but nearly undetectable at D37. STN markers BARHL1 and PITX2 were detected at all time points, with only a few immunoreactive cells at D17 and an increasing number of cells detected over time. A small percentage of D37 cells expressed BARHL1, suggesting that STN neurons are a minor subset of NURR1-ir cells and that immature mDA neurons are significantly more numerous.

[0209] Taken together, these data indicate that the differentiation protocol results in the production of cultures with a predominantly midbrain phenotype, including cells from regions close to the SN, including the STN and red nucleus cells. Furthermore, minimal contamination of forebrain or hindbrain cells was observed. D17 cells were observed to be at a progenitor stage and did not express NURR1 or other markers characteristic of mature mDA neurons, other than EN1.

[0210] [Example 14] Effect of cell maturity on graft survival and function To assess the effect of cell maturity on graft survival, grafts of D17, D24, D37, or G418 cells were injected bilaterally into the striatum of intact athymic rats. Three months after transplantation (Figure 30A-B), coronal sections stained with human-specific neural cell adhesion molecule (hNCAM) revealed relatively small G418 and D37 grafts, with only a few hNCAM-ir fibers innervating the host striatum. In contrast, large hNCAM-ir grafts and their processes were visualized in animals engrafted with either D17 or D24 cells. Although all grafts contained TH-ir neurons, only D17 grafts were cytoarchitecturally arranged in a manner similar to that characteristically seen in fVM transplanted material, with dopaminergic cell bodies localized to the periphery of the graft (L. Thompson, Barraud, Andersson, Kirik, & Bjorklund, 2005).

[0211] After observing cells produced by the modified differentiation protocol surviving in the brains of immunocompromised intact rats, we performed a long-term functional study. Rats with unilateral 6-OHDA-induced medial forebrain bundle (MFB) lesions confirmed by repeated d-amphetamine-induced rotations were implanted with vehicle control or D17, D24, D37 or G418 cells (150,000 cells / μL, 3 μL, n=9-11 rats / group) and sacrificed 6 months after injection. A summary table (Table 4) describes the histological and behavioral findings for each cell type and dosing group. [Table 8]

[0212] To demonstrate the functional capacity of each cell type, d-amphetamine-induced rotation tests were performed at baseline (10-11 weeks after 6-OHDA lesion), 2, 4, and 6 months after transplantation (Figure 24A). Hemiparkinsonian rats receiving vehicle or D37 grafts failed to demonstrate functional recovery. Mixed-effects ANOVA with Tukey's post-hoc test revealed that rats receiving D17, D24, or G418 cells exhibited significant (P<0.005, P<0.005, P<0.05) recovery of motor asymmetry by 6 months after injection. In addition, animals receiving D17 grafts displayed complete (P<0.0005) normalization of rotation by 4 months after injection. These surprising results demonstrate the superiority of D17 cells in promoting functional recovery in vivo, as observed using an animal model of PD.

[0213] To quantify graft survival of each cell type, human specific nuclei (hNuclei) were counted in graft sections using unbiased stereological analysis (Figure 23B, Figure 23D). Means (±SD) of 304,303±140,487 hNuclei-ir cells in the D17 group, 266,956±95,419 in the D24 group, 52,623±22,955 in the D37 group, and 108,093±188,944 in the G418 group were estimated based on these experiments, representing 67.6%, 59.3%, 11.7%, and 24.0% of the grafted cells, respectively. One-way ANOVA with Tukey post hoc adjustment demonstrated better engraftment and survival of grafts composed of D17 (P<0.005, P<0.01) and D24 (P<0.005, P<0.05) cells compared with D37 and D418, respectively.

[0214] Excessive levels of proliferation preclude any cell type from clinical use due to increased risk of development of intracranial simple teratomas or overgrowth of lineage-restricted cells (e.g., neuronal precursors). Stereological estimates of human-specific Ki-67 (hKi-67) revealed median (±IQR) values ​​of 3,412±1,391 hKi-67-ir cells in D17 grafts, 1,858±2,275 in D24 grafts, 0±180 in D37 grafts, and 352±697 in G418 grafts, representing only 1.2%, 0.6%, 0.0%, and 0.6% of hNuclei-ir cells, respectively (Figure 23C, Figure 23E). Using Kruskal-Wallis rank sum tests with Dwass-Steele-Critchlow-Fligner post hoc tests, we detected significant differences in the percentage number of hNuclei-ir cells between D17 and G418 (P<0.01) or D37 (P<0.05), as well as in the total number of hKi-67-ir cells between D24 and D37 (P<0.05) and between D17 and D37 (P<0.005). These findings demonstrate that cell grafts transplanted early in differentiation contained more post-transplantation proliferating cells. D17 and D24 grafts were quantitatively similar in size at 3 and 6 months, suggesting any volumetric expansion associated with proliferation subsided soon after transplantation. Critically, there was no evidence of teratomas or growths compressing adjacent brain regions.

[0215] Stereological analysis was used to estimate the number of TH-ir cells in each graft, and a mean (±SD) of 79,061 ± 44,167 TH-ir cells were observed in D17 grafts, 67,830 ± 25,944 in D24 grafts, 9,318 ± 5,523 in D37 grafts, and 20,355 ± 23,452 in G418 grafts, representing 24.0%, 25.5%, 6.1%, and 23.5% of the estimated hNuclei-ir cells, respectively (Figure 24A-B). The TH-ir population was significantly greater in D17 (P<0.0001, P<0.005), and D24 (P<0.0005 and P<0.01) grafts compared to D37 and G418 grafts, respectively, by one-way ANOVA with Tukey's post-hoc test. There was also a significant difference in the TH-ir cells generated between D17 and D37 (P<0.05).

[0216] To assess the ability of each cell type to reinnervate the host striatum with TH-ir axons, we measured TH optical density in the striatum, excluding the grafts. Using the TH-denervated striatum of vehicle-treated animals and the contralateral intact striatum as reference points, data were scaled from 0 to 1 and converted to optical density units (ODU) based on the minimum and maximum values ​​obtained, respectively (Figure 24C). We calculated a mean (±SD) of 0.46±0.14 ODU in D17-treated animals, 0.29±0.03 ODU in D24-treated rats, 0.13±0.09 ODU in D37-treated rats, and 0.33±0.03 ODU in G418-treated rats. D17 grafts had significantly more TH-ir treatment than any other cell type (P<0.0005, P<0.0001, P<0.05 compared to D24, D37, and G418, respectively), whereas both D24 (P<0.001) and G418 (P<0.0005) cells had significantly more TH-ir treatment than D37 grafts, as shown using one-way ANOVA with Tukey post-hoc adjustment. Collectively, these data demonstrate that cells grafted early in development (i.e., D17) contain enriched TH populations and neurite outgrowth.

[0217] FOXA2 plays an important role in the induction and maintenance of authentic mDA neurons (Domanskyi, Alter, Vogt, Gass, & Vinnikov, 2014; Kittappa, Chang, Awatramani, & McKay, 2007). Using immunofluorescence co-labeling, we determined FOXA2 expression in hNuclei / TH-ir neurons (Figure 24D), showing that the majority of transplanted cells expressed FOXA2. A substantial subset of hNuclei / FOXA2-ir cells also expressed TH, confirming the authentic mDA phenotype.

[0218] [Example 15] Long-range, site-specific innervation by mDA precursor cells The ability to innervate over long distances is extremely useful for promoting therapeutic responses from administration of stem cell grafts to treat PD in the human brain. To evaluate these capabilities of cells, we grafted D17 or D24 cells into the SN of rats and examined whether they formed long-distance projections to natural targets in the forebrain. Six months after grafting, hNCAM immunoreactivity was assessed in coronal sections to identify fibers emanating from the grafts and their targets (Figure 25). Projections from D24 grafts innervated A10 structures in the prelimbic cortex, olfactory tubercle, anterior olfactory nucleus, septum, and nucleus accumbens, with fibers scattered in the striatum, an A9 target. We observed a remarkably dense innervation by D17 grafts to these same A9 and A10 targets, in addition to the forebrain cortex (A10). In both D17 and D24 grafted animals, we observed hNCAM-ir fibers in most rostral brain regions examined (approximately 7-8 mm from the rostral aspect of most of the grafts in the SN) and demonstrated the ability to project fibers over long distances. These results demonstrated the superiority of D17 cells in innervating natural targets over long distances.

[0219] [Example 16] Dose response with mDA progenitors D17 grafts demonstrated the most robust efficacy, viability, and dopaminergic phenotypic expression without problematic proliferation and were selected by the inventors for further study. To determine the optimal dosing strategy, the concentration of D17 cells was titrated from the amount used in the initial testing. Unilaterally parkinsonian athymic rats received a maximum feasible dose (MFD) of 150,000 cells / μL, high dose (40,000s cells / μL), medium dose (10,000 cells / μL), low dose (25,000 cells / μL) or vehicle control of 3 μL of striatal grafts (n=8-11 / group). Motor asymmetries were assessed by d-amphetamine-induced rotations every 2 months after transplantation for 6 months, at which point the rats were sacrificed and the brains evaluated histologically.

[0220] We observed a clear dose response in all behavioral and histological analyses. Rats receiving vehicle or low doses of transplanted cells failed to demonstrate functional recovery in the d-amphetamine-induced rotation test. Mixed-effects ANOVA with Tukey post-hoc test revealed that rats receiving medium (P=0.002), high (P<0.0001) or "maximum possible" (P<0.0001) doses displayed complete normalization of motor asymmetry by 6 months after transplantation (Figure 26A). Of note, high (P=0.0002) or maximum possible (P<0.0001) doses were useful in normalizing rotation as early as 4 months after injection. Furthermore, extensive innervation in rats of the two highest dose groups resulted in overcompensation of d-amphetamine-induced rotation, resulting in circling in the opposite direction to that seen before grafting (Figure 26A).

[0221] Quantification of hNuclei staining of grafts (FIG. 26B,E) showed that the number of viable cells correlated directly with dose, with estimated means (±SD) of 611,588±53,377 viable cells in MFD-treated animals, 214,898±91,906 in high-dose animals, 36,848±18,816 in mid-dose animals, and 4,604±5,904 in low-dose animals. Significant differences were calculated by one-way ANOVA with Tukey post-hoc test for MFD compared to low, mid, and high doses, and high dose compared to mid and low doses (P<0.0001 for all comparisons).

[0222] We also quantified the number of TH-ir cells within each graft (Figure 26C,F) using unbiased stereological analysis. As expected, the number of TH-ir cells correlated directly with dose, with estimated means (±SD) of 59,929±18,927k TH-ir cells in MFD grafts, 19,973±5,759 in high-dose grafts, 6,400±4,709 in medium-dose grafts, and 1,087±1,471 TH-ir cells in low-dose grafts, representing 10.2%, 10.0%, 15.0%, and 7.5% of estimated hNucleiir cells, respectively. Significant differences were calculated using one-way ANOVA with Tukey post-hoc test for MFD compared to low, medium and high doses (P<0.0001), and for high dose compared to medium (P=0.03) and low dose (P=0.02).

[0223] To evaluate the ability of each cell type to replenish host tissues with TH-ir treatment, we measured and processed TH optical density in the striatum as described above. The density of processes reinnervating the striatum correlated directly with dose, with mean (±SD) calculated as 0.51±0.04 ODU, 0.36±0.16 ODU, 0.13±0.06 ODU, and 0.09±0.12 ODU in the MFD, high, medium, and low dose groups, respectively (Figure 26D). Significant differences were found by one-way ANOVA with Tukey post-hoc test when comparing MFD with low (P<0.0001), medium (P<0.0001), and high (p<0.05) doses, and for high dose compared with medium (p<0.0001) and low (P<0.0001).

[0224] In initial evaluation, the low-dose group displayed no behavioral modification despite containing 4,604 ± 5,904 hNuclei-ir cells and 1,087 ± 1,471 TH-ir cells. Further examination revealed five rats with few surviving grafts that did not recover motor asymmetry. In contrast, rats with substantial amounts of surviving grafts (containing 1,827, 2,068, and 4,100 TH-ir cells) recovered to various degrees (18%, 49%, and 85% reduced rotations, respectively) by 6 months after transplantation. To further probe the behavioral effects of different doses of D17 mDA precursor, behavioral recovery was plotted against the number of TH-ir cells and TH optical density (Figure 27A). In view of the nonlinear quality of the data, logarithmic regression was used to evaluate these correlations. The r of TH optical density was 2 = 0.3625 (P < 0.0005) and r 2 A result of r = 0.4887 (P < 0.00001) was observed, indicating a moderate correlation with functional recovery. When we divided the data into low / medium and high / MFD groups, the TH optical density (r 2 = 0.6340, P < 0.0005) and TH-ir cells (r 2= 0.3618, P < 0.05), a linear relationship was observed in the low / intermediate groups. These analyses indicated that although there was a clear ceiling effect for both measures of dopaminergic phenotype, graft-derived innervation was a more robust indicator of overall graft function at the low dose.

[0225] [Example 17] Characterization of the mDA phenotype in vivo To confirm the mDA phenotype, immunofluorescence triple labeling of the grafts was performed 6 months after injection study (Figure 27B). The majority of grafted cells expressed TH / FOXA2, and most TH co-expressing cells were localized at the border of the graft. In addition, many hNuclei-ir cells expressing TH / GIRK2 (62.6±2.9%) were observed, with a smaller percentage of TH / calbindin-ir (31.8±1.7%) cells (Figure 27C), demonstrating that the dopaminergic subtypes of both A9 and A10 were consistent with the long-distance innervation pattern by D17 cells grafted to the SN. Some GIRK2-ir cells (3.3±1.2%) that did not express TH were observed, which may be of paracerebellar peduncular or paranigra origin. These results support the observation that D17 cells produce superior innervation of long-distance targets compared to other cells.

[0226] [Example 18] Tests for proliferation, gliosis or serotonergic contamination Conclusively, as determined by unbiased stereological analysis performed on hKi-67 stained sections (Figure 28A,B), consistent with our previous studies, low levels of continued proliferation were seen in the grafts after 6 months. We estimated 1,006±18,927k TH-ir cells in MFD grafts, 1,038±741 in high-dose grafts, 532±745 in mid-dose grafts, and 0±5 hKi-67-ir cells in low-dose grafts, representing 0.4%, 0.4%, 1.2%, and 0.0% of the estimated hNuclei-ir cells, respectively. We calculated significant differences using Kruskal-Wallis and Dwass-Steele-Critchlow-Fligner methods for MFD compared with high (P=0.003), medium (P=0.004) and low (P=0.003) doses, and low dose compared with high (P=0.003) and medium (P=0.04) dose groups for total number of hKi-67-ir cells, and for percentage of low dose compared with high and "maximum possible dose" (P<0.05). Again, we reported no evidence of teratoma formation.

[0227] To assess the degree of astrocytosis within the grafts, sections were stained for human-specific GFAP (Figure 28C). We observed an immunoreactive pattern roughly resembling long fibers running through the graft body with some astrocytic bodies, consistent with GLAST expression detected by qPCR and similar to murine fVM grafts (LH Thompson, Kirik, & Bjorklund, 2008). We assessed Iba1-ir to determine if there was an elevated microglial response to the xenografts. In general, Iba1-ir was not prominent, except near the injection site in the cortex adjacent to the craniotomy, at the dural puncture site, and near the periphery of the graft, where animals showed slightly increased immunoreactivity and / or elevated microglia. Iba1-ir microglia with reactive morphology were observed near or within the graft (one animal in the medium dose group had more intense Ibal-ir in the graft), and some animals had a population of microglia with hypertrophic processes and more intense staining near the dorsal aspect of the graft adjacent to the craniotomy and at the dural puncture site (Figure 28D). We found that D17 grafts contained very few serotonergic (5-HT) cells (Figure 28E), and MFD grafts had an estimated 277 ± 194 5-HT-ir cells (0.04% of estimated hNuclei-ir cells). Collectively, these data indicate a general lack of development of off-target cell types or host gliosis.

[0228] [Example 19] iPSC-derived mDA progenitor cells for the treatment of PD As shown in the above experiments, grafts of mature (D37 / G418) neurons were clearly different from grafts of immature neurons (D24) and precursors (D17) in terms of behavioral effects and histological characteristics. Differences in graft size were evident as early as 3 months after injection based on hNCAM- and TH-immunostaining, with mature (D37 / G418) neurons forming thin pencil-shaped grafts and younger (D17 / D24) cells forming relatively large grafts. Six months after grafting, we observed a stronger dopaminergic phenotype in D17 and D24 grafts compared to D37 or G418, which also reflected a complete reversal of d-amphetamine-induced locomotor asymmetry in D17- and D24-grafted rats. For all cell types and doses, grafted cells expressed TH / FOXA2, confirming continued maturation 6 months after grafting in vivo and giving rise to mature mDA neurons derived from transplanted precursors and immature neurons. When we grafted D17 and D24 cells into the substantia nigra, we observed preferential innervation of both A9 and A10 targets over long distances. These findings are consistent with earlier observations in fVM and ESC-VM tissues (Cardoso et al., 2018; Grealish et al., 2014) and are supported by the above experiments showing both TH / GIRK2-ir and TH / calbindin-ir cells in the grafts. GIRK2 and CALBINDIN are routinely used to differentiate A9 and A10 mDA, and these populations can also be further defined using sequencing and / or high multiplexing. The ability of grafted iPSC mDA cells to project over long distances in the rat brain indicates that these approaches can be applied to the human putamen.

[0229] A striking difference in the outgrowth of graft-derived TH-immunoreactive fibers into the host striatum was observed depending on the differentiation protocol used. Rats grafted with D17, D24, and G418 cells exhibited TH-ir fibers that encompassed the entire striatum, whereas rats grafted with D37 cells did not exhibit graft-induced behavioral recovery and showed few graft-derived TH-ir axons innervating the host. In fact, high-magnification images revealed that although these grafts contained TH-ir cells and fibers, these axons abruptly terminated upon reaching the outermost edge of the D37 graft. The comparable numbers of TH-ir cells in D17 and D24 grafts, and the comparable numbers of TH-ir cells in D37 and G418 grafts, suggest that the propensity of D17 and G418 cells to innervate the host likely underlies these functions. Indeed, it was observed that the behavioral outcomes of animals with large (67,800 TH-ir cells) D24 grafts were similar to the small (6,400 TH-ir cells) D17 grafts, and without wishing to be bound by any theory, this finding is expected to be due to a similar reinnervation of the host striatum. Regression analysis showed a ceiling effect for the number of D17 TH-ir cells and these treatments, but even at lower doses, innervation correlated higher than the number of TH-ir cells with behavioral recovery. These results observed in rats are particularly important for obtaining improved therapeutic responses in humans, as they are important for the development of a more robust and more efficient treatment of the putamen (3.96 cm in PD patients). 3(Yin et al., 2009)) is considerably larger than the rat striatum. Many more TH-ir cells and their processing are needed to produce clinical benefit in humans, and alternatively or in combination, cells can be deposited at multiple sites along multiple tracks in an array that aids in global reinnervation of the putamen, possibly without the reduced return associated with large grafts seen in rats. The indication here that the 17 mDA precursor is effective at a wide range of doses indicates that clinicians may have some latitude in utilizing various surgical techniques to administer the cells. Additional studies can be performed to further optimize dosing regimens in humans, and it is expected that similar therapeutic results will be observed.

[0230] Proliferating cells were only occasionally observed in grafts of mature cells (G481, D37), consistent with previous observations (Hiller et al., 2020; Wakeman et al., 2017). D17 and D24 grafts contained more hKi-67 cells than grafts of G418 / D37 cells, but the number of proliferating cells as a percentage of viable graft cells was low (<1,000 per 100,000 hNuclei-ir in D17 / D24 grafts), demonstrating that no purification step is necessary to prevent unwanted cell proliferation. Furthermore, hKi-67-ir cells were not present in clusters indicative of active cell division in any of the grafted rats. Another safety concern is the occurrence of GID, which has been reported in a subset of patients transplanted with fVM (Freed et al., 201; Hagell & Cenci, 2005; Olanow et al., 2009), and it has been suggested that aberrant grafting of serotonergic neurons contributes to the development of GID. As further evidence for the safety of iPSC-derived mDA progenitor grafts, we did not observe numbers of serotonergic neurons approaching those postulated to induce GID (Carlsson et al., 2009).

[0231] The transplantation studies described above using stem cell-derived mDA cells utilize, in some embodiments, the developmental stages of precursors and immature neurons. Without wishing to be bound by any theory, it is expected that the mDA progenitor cells provided herein may exhibit many of the beneficial effects of fetal tissues that have been successfully used in clinical trials. Although it is difficult to directly compare developmental stages of cells used in different studies due to different protocols, many of the cells focused efforts to adapt for translational use incorporate exposure to neuromaturation factors, such as BDNF, GDNF, TGF-β3 and / or DAPT (Doi et al., 2020; Kim et al., 2021; Kirkeby et al., 2017; Song et al., 2020). In studies directly comparing different developmental stages, the conclusion has been that NURR1+ immature neurons are more effective than less mature precursors (Ganat et al., 2012; Qiu et al., 2017). In contrast, the above studies demonstrate that D17 cells exposed to mDA patterning factors (SMAD inhibition, SHH, WNT, FGF8) and cryopreserved prior to NURR1 expression produce grafts that outperform the same cells cultured for an additional week with maturation factors (D24, NURR1+ / -). Cells at both maturation stages engraft in similar numbers and mature into mDA neurons, suggesting that the difference in performance is not simply a difference in proliferation potential, and without wishing to be bound by any theory, it may result from differences in innervation, A9 patterning, and / or other early mDA maturation signals received in vivo. Single cell sequencing of grafted cells can be used to further analyze other non-dopaminergic cells contained in the grafts. Importantly, it was observed that D17 cells were appropriately patterned and did not require exposure to maturation factors prior to transplantation to "lock in" to the mDA patterning or to prevent proliferation of undesirable (e.g., serotonergic) cell types.

[0232] Without wishing to be bound by any theory, the data presented above support the notion that if mDA neurons or precursor cells are too mature at the time of grafting into the striatum, they typically survive less well and have less pronounced behavioral effects. The above studies also demonstrate that relatively small grafts of D17 precursors can produce sufficient dopaminergic innervation to induce behavioral recovery in hemiparkinsonian rats. These data support the notion that a relatively small total number of cells can be injected into a small number of locations in the striatum of each patient, which may result in the treatment of PD, and also show that favorable clinical safety can be observed.

[0233] Some other mDA progenitor cells tested in clinical trials were derived from ESCs (NCT04802733) (Piao et al., 2021) or iPSCs (JMA-IIA00384, UMIN000033564) (Doi et al., 2020). The above data indicate that the mDA progenitor cells provided herein (e.g., D17 cells) can be administered to patients to treat PD. If desired, the mDA progenitor cells may be administered in combination with an immunosuppressant drug or regimen, and / or dopamine replacement therapy, if desired. In some embodiments, dopamine replacement therapy is not administered to the patient after administration of the mDA progenitor cells. It is expected that the mDA progenitor cells provided herein (e.g., D17 cells) when administered to select PD patients can achieve significant clinical benefit using dopamine cell replacement therapy in a carefully selected group.

[0234] [Example 20] Levels of off-target cell types Incorrect patterning during midbrain DA precursor differentiation may result in dangerous off-target cell types, such as neuronal precursors with a forebrain (rostral) phenotype and serotonergic cells. Forebrain-type cells are of particular concern because previous DA neuron differentiation protocols often result in neural outgrowth that includes neuronal precursors with rostral (FOXG1+) and / or lateral (PAX6+) cell types that can form rosette structures in vivo and have been observed to persist for several months after engraftment. Thus, cultures were tested for off-target or non-dopaminergic cell types.

[0235] FCDI DAPC-1 (day 17 DA precursor) cells were differentiated and cryopreserved as described in Example 1 (Table 2). Cells were thawed and washed with DPBS prior to flow cytometry or qPCR analysis of day 17 precursor cells (day 0 post-thaw, 0 DPT). Alternatively, cells were thawed and cultured in DA maturation medium for analysis of cells at later time points (7-20 days post-thaw, 7-20 DPT) to assess expression of markers expressed in more mature cells.

[0236] Flow cytometry assays were used to monitor FOXG1 and PAX6 expression upon thawing. Flow cytometry of FOXG1 and PAX6 was performed on six representative run batches, each thawed at one or two separate time points (n=9 total). On average, FCDI DAPC-1 was 0.1% FOXG1+ with a standard deviation (SD) of 0.1% and 0.4% PAX6+ with a SD of 0.7% upon thawing, confirming that FCDI DAPC-1 lacks markers of these off-target cell types (Figure 32). Based on flow cytometry assays of off-target cell markers FOXG1+ and PAX6+ expressing potentially dangerous cells, the cell cultures contain a very low percentage of such forebrain neuronal precursors. The results are shown in Table 5 below. [Table 9]

[0237] In significant quantities, the inclusion of serotonergic cells in the graft may be potentially dangerous and contribute to graft-induced dyskinesias (Carlsson et al., 2009). Definitive markers of serotonergic cells include serotonin (5-HT), tryptophan hydroxylase-2 (TPH), the rate-limiting enzyme for 5-HT synthesis, and the 5-HT transporter (SERT). These markers are only expressed in mature cells, so assays were not performed on FCDI DAPC-1 immediately after thawing (0 DPT). There are no known definitive markers for serotonergic cell precursors. To determine the earliest time point at which serotonergic cells can be detected, FCDI DAPC-1 were evaluated at 0 DPT (0 days after thawing), 7 DPT, 14 DPT, and 19-20 DPT using qPCR and immunohistochemistry (ICC). As a positive control for serotonergic marker expression by qPCR, total RNA samples from human pons, a brain region that harbors serotonergic cells, were included.

[0238] Expression levels of SERT and TPH2 were observed to be lower in FCDI DAPC-1 both at 0 DPT and throughout maturation in culture compared to the pons (Figure 33). Results are shown in Table 6 below. SERT expression increased significantly between 0 DPT and 7 DPT, then decreased significantly after 14 DPT. TPH2 showed a gradual increase in expression from 0 DPT to 19 DPT. For both markers, peak expression was seen at either 7 DPT or 14 DPT, and expression at 14 DPT was consistent across different DAPC-1 batches (Figure 34). Results are shown in Table 7 below. [Table 10] [Table 11]

[0239] Log less than 0.01 as a marker 2Normalized expression values ​​are considered to be very low expression, with values ​​below 0.001 considered to be below detection. These results show that cells in FCDI DAPC-1 cultures express very low levels of the serotonergic markers SERT and TPH2, as shown by the use of qPCR.

[0240] To determine the percentage of serotonergic cells, we performed ICC staining for 5-HT in cells along the time course of post-thaw culture. The results are shown in Table 8 below. Essentially no 5-HT+ cells were observed at 1 day post-thaw (1 DPT). Significant populations of 5-HT+ cells were observed at 8 DPT, 15 DPT, and 20 DPT. Quantification using high content imaging software (Molecular Devices ImageXpress) showed the percentage of serotonergic neurons to be approximately 0.2% (0 DPT), 3.4% (8 DPT), 2.1% (15 DPT), and 5.6% (20 DPT). This data demonstrates that DAPC-1 contains approximately 5% serotonergic neuronal precursors that can be visualized as mature (post-mitotic) serotonergic neurons by 8 DPT. This percentage does not increase over time. [Table 12]

[0241] [Example 21] Materials and Methods The following materials and methods were used in Examples 13-20.

[0242] Statistical analysis: Statistical analysis was performed with SAS (Stereologic and Behavioral Results in Dosing Studies) or Prism (version 9.1.2, GraphPad). Graphs were generated in Prism. Immunohistochemical data were analyzed using one-way ANOVA with Tukey post-hoc test, except for hKi-67, which was analyzed by Kruskal-Wallis test with Dwass-Steele-Critchlow-Fligner post-hoc method. Behavioral data were analyzed by mixed-effects ANOVA with Tukey post-hoc test. Histological data are expressed as mean ± sD, except for hKi-67 (median ± IQR). Median percentages were reported for TH- or hKi-678-ir cells as a percentage of hNuclei-ir cells in each animal. Rotation was reported as mean ± SEM.

[0243] Cell Differentiation: Research use G418 neurons (iCell DopaNeurons, Fujifilm Cellular Dynamics, Inc.) were derived as previously described (Hiller et al., 2020; Wakeman et al., 2017) utilizing engineered iPSC lines to allow G418 drug selection of neurons during differentiation and to allow cryopreservation of neurons at day 38 of the method. For clinical development, non-engineered iPSC lines reprogrammed using procedures and reagents appropriate for cell therapy development were selected and expanded into master cell banks (MCBs) and working cell banks (WCBs) in a cGMP manufacturing facility (Waisman Biomanufacturing, Madison, WI). The iPSC-mDA differentiation protocol was adjusted for this iPSC line, including streamlining the SMAD signaling inhibition (LDN-193189, Reprocell) and shifting the GSK-3 inhibition (CHIR99021, Reprocell) one day later to method day 2 and adjusting to a higher concentration at this timing. The raw material was improved to be suitable for clinical development, including the use of GMP grade Shh C25II, BDNF, GDNF and TGFβ3 (Bio-techne). D37 neurons were purified during the process using mitomycin C (Tocriis, 150 ng / mL on method days 27 and 29) as previously described (Hiller et al., 2020) and cryopreserved in CryoStor (Biolife Solutions) on method day 37. D17 progenitors were cryopreserved at day 17 of the method, without exposure to maturation medium (Kriks et al., 2011) or mitomycin C treatment, by dissociating precursor aggregates with CTS TrypLE Select Enzyme (Thermo). D24 immature neurons were cryopreserved later in the method (day 24 of the method) after plating in maturation medium for 1 week, but without mitomycin C treatment. Cells used to compare iPSC DA maturation stages were produced in the laboratory using a manufacturing process adapted for clinical translation.The D17 cells used in the dose-ranging study were generated in a controlled, non-graded, clean laboratory using the same methods.

[0244] qPCR: Cells were thawed and lysed in Buffer RLT Plus (Qiagen) containing 1:100 beta-mercaptoethanol. Total RNA was extracted using the RNeasy Plus kit (Qiagen). cDNA was generated using the High Capacity RNA-to-cDNA Kit (ThermoFisher) with an RNA input of 500 ng. Quantitative polymerase chain reaction (qPR) was performed on a LightCycler480 (Roche) using TaqMan Gene Expression Master Mix (ThermoFisher), TaqMan assays (see Table 5 for a list of assays) and a cDNA input of 2.5 ng. Values ​​are expressed relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Three biological replicates were analyzed in technical triplicate for each treatment time point.

[0245] Flow cytometry: Cells were thawed as previously described (Wakeman et al., 2017). Cells were centrifuged, stained with GhostDye510 (Tonbo Biosciences), fixed with 4% paraformaldehyde, and washed with buffer (2% FBS in DPBS). Cells were stained with primary antibodies in 1x BD Perm / Wash (BD Biosciences) + 0.2%m Triton X-100 (except for Map2 staining, which did not contain Triton X-100) at 4°C (see Table 6 for a list of antibodies and diluents) and labeled with secondary antibodies (where appropriate) at room temperature. Flow cytometry was performed on a MACSQuant® Analyzer 10 flow cytometer (Miltenyi Biotec). Three biological replicates were analyzed at each maturation time point.

[0246] Immunocytochemistry: Cells were thawed and seeded at 170,000 cells / well in 96-well plates, cultured overnight, and fixed with 4% paraformaldehyde. Cells were stained with primary antibodies in staining buffer (2% FBS, 2% donkey serum, 0.2% Triton X-100 in DPBS) at 4°C (see Table 6 for list of antibodies and diluents) and labeled with secondary antibodies (where appropriate) and Hoechst (ThermoFisher) at room temperature. Cells were analyzed with an ImageXpress High Content Imager (Molecular Devices) at 10x magnification. Three biological replicates were analyzed for each time point.

[0247] Animal Procedures: All animal procedures were performed in accordance with the Institutional Animal Care and Use Committee at Rush University Medical Center.

[0248] Lesion induction and transplantation: Female athymic nude (rnu) rats were acclimated for 1 week after receipt. Rats aged 9-10 weeks (170-200 g) received a unilateral injection of 6-OHDA (15 mg in 3 μL of 0.5% ascorbic acid) into the right MFB (anterior / posterior [AP]: -4.0 mm, medial / lateral [ML]: -1.3 mm from bregma, dorsal / ventral [DV]: -7.0 mm from dura). Animals with confirmed lesions by 10 weeks post-lesion received striatal (AP: +0.5 mm, ML: ±3.0 mm from bregma, DV: -5.3 mm from dura) injections of iPSC-mDA cells (n=8-11 / group) and were sacrificed 3 or 6 months after transplantation. Cryopreserved cells were thawed and cells were counted by trypan blue exclusion. Cells were centrifuged and resuspended at the appropriate density for injection. Intranigral grafts were placed at AP: +0.5 mm, ML: -3.0 mm from bregma, DV: -5.0 mm from dura. Injection volume was 3 μL in all experiments. A concentration of 150,000 cells / μL was used for cell maturity comparison and intranigral experiments, and 2,500, 10,000, 30,000 or 150,000 cells / μL were used for dose ranging experiments.

[0249] d-Amphetamine-induced rotations: Animals received an intraperitoneal injection of d-amphetamine (2.5 mg / kg, Sigma) and were placed in a harness in a translucent chamber and connected to a Rotometer system (San Diego Instruments). Net ipsilateral (clockwise) rotations were reported over a 10-40 min period following d-amphetamine administration.

[0250] Tissue processing: Tissues were processed and immunohistological and stereological analyses were performed as previously described (Hiller et al., 2020). Briefly, rats were anesthetized with a ketamine / xalazine mixture and perfused with saline followed by 4% paraformaldehyde. Brains were removed, mounted in a sucrose gradient, and sectioned at 40 μM on a sliding microtome. Free-floating sections were stained using antibody concentrations for immunofluorescence triple labeling or DAB treatment listed in Table 6. Sections were mounted on glass gelatin-coated slides, cover slipped, and imaged.

[0251] Stereoanalysis: Coverslip-covered slides were analyzed by unbiased stereoanalysis (StereoInvestigator v10.40, MBF biosciences). In cell maturation comparison experiments, 5.22% of the total graft area was explored in half of the series (1 / 12 of serial sections) of tissue stained with TH, hNuclei or hKi-67. In dose-ranging experiments, 5.22% of TH-ir and hNuclei-ir grafts, 28.4% of hKi-67-ir grafts or 20.3% of 5-HT-ir grafts were explored in half of the series (1 / 12 of serial sections) of stained tissue. In animals in the mid or low dose groups with a Gundersen m=1 coefficient of error ≥ 0.45 or with no cells counted in either the hNuclei- or TH-stained sections, an additional half of the series (1 / 12 of serial sections) was stained and re-explored using the same parameters. Estimates were then calculated for the entire series (1 / 6 of the consecutive sections) and the results averaged.

[0252] Optical density: Grayscale images of seven (±3 of the center of the graft) coronal sections stained with TH were analyzed for each animal. In each section, a contour was drawn around the striatum excluding the graft body and the mean pixel density of the area was recorded using ImageJ. Values ​​were averaged for each animal and data were rescaled considering the minimum point in the denervated striatum as 0 and the maximum point in the intact striatum as 1. Data sets for cell maturity comparison and dose ranging experiments were rescaled separately.

[0253] Quantification of mDA subtypes: Graft sections from four mFD animals were stained with TH / GIRK2 / calbindin and imaged with a Nikon Eclipse Ti2 confocal microscope equipped with a Nikon A1RHD camera using NIS Elements AR software (version 5.10.01) and saved as tiff files. Markers in 53–80 cells in each graft were quantified by z-stack using ImageJ (version 1.53a).

[0254] qPCR assays of serotonergic cell populations 0-19 DPT: RT-PCR assays for SERT and TPH2 in six FCDI DAPC-1 batches at thawing and in culture for 7-, 14- or 19-DPT. Shading represents different batches at the corresponding time points. The pons is the positive control brain region. The mean ΔCq (Cq ASSAY -Cq GAPDH ) and standard deviations are shown in the table.

[0255] All methods disclosed and claimed herein can be made and practiced without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described with respect to preferred embodiments, it will be understood by those skilled in the art that modifications can be made to the methods and steps or sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be understood that certain agents that are both chemically and physiologically related can be substituted for the agents described herein while still achieving the same or similar results. 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Claims

1. 1. A culture comprising midbrain dopaminergic (mDA) neuronal progenitor cells for use in treating Parkinsonism, Parkinson's Disease (PD) or Parkinson Plus Syndrome (PPS) in a mammalian subject, said treating comprising administering a therapeutically effective amount of the culture to the subject; mDA neuron progenitor cells express the following signaling modulators: (a) The first inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling; (b) at least one activator of Sonic Hedgehog (SHH) signaling; and (c) at least one activator of wingless (Wnt) signaling and wherein the human pluripotent cells are cultured in the presence of The method does not include culturing the human pluripotent cells in the presence of a second inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling; The human pluripotent cells are cultured with a SMAD inhibitor substantially continuously or daily for 17 days, and then the cells are refrigerated or cryopreserved; Midbrain dopaminergic progenitor cells express both forkhead box protein A2 (FOXA2) and LIM homeobox transcription factor 1 (LMX1) (FOXA2 + / LMX1 + cell), Culture.

2. The culture of claim 1, wherein the culture comprises between 1e6 and 25e6 cells.

3. The culture of claim 1, wherein the culture comprises between 3e6 and 9e6 cells.

4. mDA neuronal progenitor cells do not express NURR1, and / or the mDA neuron progenitor cells express forkhead box protein A2 (FOXA2), LIM homeobox transcription factor 1 (LMX1) and EN1, where optionally the mDA neuron progenitor cells further express OTX2; and / or Forkhead box protein A2 (FOXA2) and LIM homeobox transcription factor 1 (LMX1) are co-expressed by 60% to 100% or 85% to 95% or more of the mDA neuron precursor cells, optionally wherein 65-75% of the mDA neuron precursors co-express both FOXA2 and LMX1; and / or mesencephalic dopaminergic progenitor cells express FOXA2, LMX1A, ETV5 and EN1, and mesencephalic dopaminergic progenitor cells do not express NURR1, TH, CALB1, BARHL1 and GRIK2; The culture of claim 1.

5. the mDA neuron progenitor cells comprise proliferating or dividing cells, optionally wherein at least about 40% or more of the mDA neuron progenitor cells are proliferating or dividing, optionally wherein 50-75% of the mDA neuron progenitor cells are proliferating or dividing, and / or the culture further comprises about 5% or less serotonergic neuronal progenitor cells, optionally wherein the serotonergic neuronal progenitor cells express BARLH1; and / or the culture further comprises glial progenitor cells, optionally wherein the glial progenitor cells express GLAST, SLC13A, CD44 and / or hGFAP; The culture of claim 1.

6. the inhibitor of SMAD signaling is a BMP inhibitor, optionally wherein the BMP inhibitor is LDN-193189, dorsomorphin, DMH-1 or noggin; wherein optionally the BMP inhibitor is LDN-193189, optionally wherein LDN-193189 is present at a concentration of 0.2 μM to 4 μM, or 1 μM to 4 μM, optionally wherein LDN-193189 is present at a concentration of 1 μM to 3 μM, optionally wherein LDN-193189 is present at a concentration of 0.5 μM to 4 μM, optionally wherein LDN-193189 is present at a concentration of 0.5 μM to 2 μM, or wherein LDN-193189 is present at a concentration of 0.2 μM to 4 μM, optionally wherein LDN-193189 is present at a concentration of 0.2 μM to 2 μM; A culture according to any one of claims 1 to 5.

7. and / or the SMAD signaling inhibitor is a TGFβ inhibitor, optionally wherein the TGFβ inhibitor is SB431542, optionally wherein SB431542 is present at a concentration of 1-20 μM or 5-15 μM or 10 μM; and / or the inhibitor of SMAD is present at a concentration of 50-2000 or 50-500 nM, optionally wherein the inhibitor of SMAD is present at a concentration of 180-240 nM; A culture according to any one of claims 1 to 5.

8. the method further comprises contacting the pluripotent cell with a MEK inhibitor, optionally wherein the MEK inhibitor is PD0325901, optionally wherein PD0325901 is present at a concentration of 0.25-2.5 μM; and / or The MEK inhibitor is contacted with the pluripotent cells about 1-3 days, or 1-3 days, 2-4 days, 3-5 days, or 1, 2, 3, 4 or 5 days after initiation of contact with the inhibitor of SMAD signaling, optionally wherein the MEK inhibitor is contacted with the pluripotent cells about 24 to about 48 hours after initiation of contact with the inhibitor of SMAD signaling; The MEK inhibitor is contacted with the pluripotent cells daily or substantially continuously for 3-4 days beginning 1-2 days after initiation of contact with the inhibitor of SMAD signaling, optionally wherein the MEK inhibitor is contacted with the pluripotent cells on days 2-5 or 3-6 after initiation of contact with the inhibitor of SMAD signaling on day 1; A culture according to any one of claims 1 to 5.

9. the activator of Wnt signalling is a GSK3 inhibitor, optionally wherein the GSK3 inhibitor is CHIR99021, optionally wherein CHIR99021 is present at a concentration of 1.5-2 μM, 1.5-1.7 μM, 1.6-1.7 μM or 1.65 μM; or CHIR99021 is present at a concentration of 4-7 μM from 9 to 17 days after the initiation of contact with the inhibitor of SMAD signaling; and / or The activator of Wnt signaling is contacted with the pluripotent cells for 1 to 3 days after initiation of contact with the inhibitor of SMAD signaling, where optionally the activator of Wnt signaling is contacted with the pluripotent cells within 24 to 48 hours after initiation of contact with the inhibitor of SMAD signaling, and / or the pluripotent cells are cultured with an activator of Wnt signalling substantially continuously or daily for 14, 15 or about 16 days; and / or The activator of Wnt signaling is contacted with the pluripotent cells 2-17 days after initiation of contact with the inhibitor of SMAD signaling. A culture according to any one of claims 1 to 5.

10. the activator of SHH signaling is palmorphamin or C25II Shh, optionally the method further comprises contacting the pluripotent cell with two activators of SHH signaling, optionally the two activators of SHH signaling are palmorphamin and C25II Shh, and / or at least one activator of SHH signaling is contacted with the pluripotent cells on the same day as the initiation of contact with the inhibitor of SMAD signaling or within 24 to 48 hours after the initiation of contact with the inhibitor of SMAD signaling, optionally wherein at least one activator of SHH signaling is contacted with the pluripotent cells on days 1 to 7 with or after the initiation of contact with the inhibitor of SMAD signaling, and / or The method further comprises contacting the pluripotent cells with FGF-8, optionally wherein FGF-8 is not contacted with the pluripotent cells on the same day as initiation of contact with the inhibitor of SMAD signaling, or wherein FGF-8 is contacted with the pluripotent cells 9-17 days or 11-17 days after initiation of contact with the inhibitor of SMAD signaling, optionally wherein FGF-8 is present at a concentration of 50-200 ng / mL. A culture according to any one of claims 1 to 5.

11. The pluripotent cells contain an antibiotic resistance transgene under the control of a neuronal promoter, and / or the method further comprises selecting for neural cells or midbrain DA neurons derived from the pluripotent cells by contacting the cells with an antibiotic, a chemotherapeutic agent, a DNA crosslinking agent, a DNA synthesis inhibitor, or a mitosis inhibitor; or the method further comprises contacting the pluripotent cells with an antibiotic or chemotherapeutic agent, optionally wherein the chemotherapeutic agent is mitomycin C, optionally wherein the mitomycin C is contacted with the pluripotent cells 27, 28, 29 and / or 30 days after initiation of contact with the inhibitor of SMAD signaling, or the antibiotic is G418 (geneticin), and / or and / or the method further comprises culturing or incubating the pluripotent cells in medium comprising a ROCK inhibitor prior to initiation of contact with the inhibitor of SMAD signaling. the method further comprises contacting the pluripotent cells with blebbistatin, optionally wherein blebbistatin is contacted with the cells on days 5 and 17 of differentiation. A culture according to any one of claims 1 to 5.

12. mDA dopaminergic progenitor cells do not express NURR1, MAP2 and TH, and / or mDA dopaminergic progenitor cells express EN1, or mDA dopaminergic progenitor cells express GBX2, OTX1, OTX2, ETV5, CORIN and / or DCX; and / or the pluripotent cells are human induced pluripotent stem (iPS) cells, and / or LMX1 is LIM homeobox transcription factor 1 alpha (LMX1A), and / or the method further comprises incubating the human pluripotent cells in the presence of a DNase or endonuclease, optionally wherein the endonuclease is DNase I or Benzonase®, optionally wherein the DNase I or Benzonase® is present at a concentration of 10-20 U / mL or 10-15 U / mL; or The human pluripotent cells are cultured in the presence of an endonuclease for at least one of the days 4 to 6 after initiation of contact with an inhibitor of SMAD signaling; or The human pluripotent cells are cultured in the presence of an endonuclease 5 days after initiation of contact with an inhibitor of SMAD signaling. A culture according to any one of claims 1 to 5.

13. the culture comprises midbrain dopaminergic neuron progenitor cells at 2,500 cells / μL to 150,000 cells / μL, 2,500 cells / μL to 100,000 cells / μL, 10,000 cells / μL to 150,000 cells / μL, 40,000 cells / μL to 100,000 cells / μL, or 15,000-45,000 cells / μL; and / or about 10% or less, about 7% or less, or about 5% or less of the cells in the culture are serotonergic progenitor cells, optionally wherein about 5% or less of the cells in the culture express SERT and TPH2. A culture according to any one of claims 1 to 5.

14. the mammalian subject is a human, and / or the culture comprises mDA progenitor cells that express Engrailed but do not express NURR1; and / or The culture is administered to the striatum, e.g., putamen or substantia nigra, of a subject; or The culture is administered to more than one location in the striatum or putamen of the subject; or The culture is administered to multiple sites and / or via multiple needles in the striatum or putamen of the subject; and / or the culture is comprised in a pharmaceutical composition, optionally the pharmaceutical composition comprises a hyaluronic acid matrix; and / or the culture comprises between about 2,500 cells / μL and about 150,000 cells / μL; and / or Administration is via injection, where optionally The injection is a stereotactic injection, A culture according to any one of claims 1 to 5.

15. 6. An in vitro method for preparing a culture comprising midbrain dopaminergic (mDA) neuron progenitor cells according to any one of claims 1 to 5.

16. (a) contacting midbrain dopaminergic (mDA) neuron progenitor cells differentiated by the method of claim 15 with a test compound; and (b) Measuring the function, physiology, or viability of cells A method for screening a test compound comprising:

17. 17. The method of claim 16, wherein said measuring comprises testing for a toxicological response of a cell or for a change in an electrophysiological response.

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