Methods and compositions of dopaminergic cells for treating parkinson's disease

Abstract

This disclosure relates to methods and compositions for the treatment of Parkinson's disease, a neurodegenerative disorder characterized by a loss of dopaminergic neurons. In particular, this disclosure provides formulations of dopaminergic cells demonstrated to possess a therapeutic impact on both motor and non-motor symptoms of the disease.

Description

METHODS AND COMPOSITIONS OF DOPAMINERGIC CELLS FOR TREATING PARKINSON'S DISEASECROSS-REFERENCE

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 578,734, filed August 25, 2023, and U.S. Provisional Application No. 63 / 561,232, filed March 4, 2024, the entire contents of each of which are incorporated herein by reference.FIELD

[0002] This disclosure relates generally to methods and compositions for treating Parkinson’s disease with dopaminergic cells.BACKGROUND

[0003] Parkinson’s disease is a chronic and progressive movement disorder characterized by both motor and non-motor symptoms. Motor symptoms include tremors, stiffness, and bradykinesia, while non-motor symptoms can involve cognitive decline, mood disorders, and autonomic dysfunction.

[0004] The etiology of Parkinson’s disease is not fully understood, but it is known to involve the degeneration of dopaminergic neurons, leading to a reduction in dopamine levels. Traditional treatments for Parkinson's disease have largely focused on managing symptoms with pharmacological agents such as Levodopa, which aim to increase dopamine levels or mimic its effects. These treatments, while beneficial, often have limited efficacy, significant side effects, and do not halt disease progression.

[0005] Cell therapy approaches have recently emerged as a promising avenue for treatment of Parkinson’s, aiming to replace or regenerate lost dopaminergic neurons. However, previous attempts with these approaches have encountered challenges, such as, inefficient cell integration, limited duration of effects, and unpredictability in patient outcomes. Furthermore, it remains a challenge to not only halt the progression of Parkinson’s disease but also to achieve actual improvement in motor and non-motor functions. As such, there remains a significant unmet need for therapeutic strategies that not only manage symptoms but reverse the progression of Parkinson's disease provide sustained, long-term benefits.SUMMARY

[0006] This disclosure relates to methods and compositions for treating Parkinson’s disease. More particularly, this disclosure relates to methods and compositions of dopaminergic cells that are demonstrated with Phase 1 clinical data to have a sustained, therapeutic effect on subjects with Parkinson’s disease. Unlike prior approaches, the methods and compositions described herein not only provide temporary relief of symptoms of Parkinson’s disease but also lead to a marked and sustained improvement in both motor and non-motor functions, with clinical data demonstrating enduring effects lasting at least 52 weeks without diminution.

[0007] In one aspect, provided herein is a method for treating Parkinson’s disease in a subject comprising: administering an effective quantity of a population of dopaminergic cells to the subject. In some embodiments, the administering results in an improvement in a motor function or a non-motor function, or a combination thereof, of the subject as compared to a control or a baseline of the subject prior to the administering.

[0008] In some embodiments, the effective quantity of the population of dopaminergic cells comprises between about 1.0xl0A6 to about 1.2xlOA7 dopaminergic cells. In some embodiments, the effective quantity of the population of dopaminergic cells comprises about 1.8xlOA6 dopaminergic cells. In some embodiments, the effective quantity of the population of dopaminergic cells comprises about 5.4xlOA6 dopaminergic cells.

[0009] In some embodiments, greater than about 90% of the dopaminergic cells in the population of dopaminergic cells administered to the subject are positive for FOXA2. In some embodiments, fewer than about 2% of the dopaminergic cells in the population of dopaminergic cells administered to the subject are positive for PAX6. In some embodiments, fewer than about 2% of the dopaminergic cells in the population of dopaminergic cells administered to the subject are positive for CRABP1. In some embodiments, fewer than about 12% of the dopaminergic cells in the population of dopaminergic cells administered to the subject are positive for Ki67.

[0010] In some embodiments, between about 70% to about 80% of the dopaminergic cells in the population of dopaminergic cells administered to the subject are viable.

[0011] In some embodiments, the population of dopaminergic cells comprises a capacity for producing dopamine that, when evaluated in vitro using liquid chromatography -tandem mass spectrometry (LC-MS / MS), provides an area under the concentration-time curve (AUC) that is equal to or greater than 11.2 ng x day / mL.

[0012] In some embodiments, the administering comprises delivering the population of the dopaminergic neurons to the posterior putamen of the subject. In some embodiments, the administering comprises delivering a first portion of the population of dopaminergic cells to the left hemisphere of subject’s posterior putamen and a second portion of the population of dopaminergic cells to the right hemisphere of the subject’s posterior putamen. In some embodiments, the first portion is about half of the effective quantity of the population of dopaminergic cells and the second portion is about half of the effective quantity of the population of dopaminergic cells.

[0013] In some embodiments, the dopaminergic cells are delivered to the subject in a therapeutic composition, and wherein the population of dopaminergic cells in the therapeutic composition is at a concentration of about 71,000 cells / pL to about 123,000 cells / pL.

[0014] In some embodiments, the improvement is in at least one motor function and at least one non-motor function of the subject. In some embodiments, the improvement in motor function is determined based, at least in part, on a change in the subject’s Movement Disorder Society-Sponsored Revision of the Unified Parkinson's Disease Rating Scale (MDS UPDRS) Part II Score as compared to the control or the baseline of the subject prior to the administering. In some embodiments, the improvement in motor function is determined based, at least in part, on a change in the subject’s Movement Disorder Society-Sponsored Revision of the Unified Parkinson's Disease Rating Scale (MDS UPDRS) Part III Score as compared to the control or the baseline of the subject prior to the administering. In some embodiments, the improvement in motor function is determined based, at least in part, on a change in the subject’s Unified Dyskinesia Rating Scale (UDysRS) Objective Subscore as compared to the control or the baseline of the subject prior to the administering. In some embodiments, the improvement in motor function is determined based, at least in part, on a change in an ON score and / or OFF score of the subject as compared to the control or the baseline of the subject prior to the administering. In some embodiments, the improvement in non-motor function is measured by one or more assays selected from the group consisting of a sleep-quality assessment, a cognitive assessment, a neuropsychological test, a mood evaluation, an autonomic function test, an imaging test, and other standardized non-motor function evaluation. In some embodiments, the baseline is a measurement of the subject’s motor function and non-motor function before the administering. In some embodiments, the improvement is indicative of a reversal in progression of Parkinson’s disease. In some embodiments, the improvement in non-motor function is determined, at least in part, on a change in the subject’s Parkinson’s Disease Non-Motor Symptom Scale (PD NMSS) score ascompared to the control or the baseline of the subject prior to the administering. In some embodiments, the improvement in non-motor function is determined, at least in part, on a change in the subject’s 39-item Parkinson’s Disease Questionnaire (PDQ-39) score as compared to the control or the baseline of the subject prior to the administering. In some embodiments, the improvement in non-motor function is determined, at least in part, on a change in the subject’s Neuropsychiatric Inventory Questionnaire (NPI-Q) score as compared to the control or the baseline of the subject prior to the administering. In some embodiments, the improvement in non-motor function is determined, at least in part, on a change in the subject’s Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) score as compared to the control or the baseline of the subject prior to the administering. In some embodiments, the improvement in non-motor function is determined, at least in part, on a change in the subject’s Frontal Systems Behavior Scale (FrSBe) score as compared to the control or the baseline of the subject prior to the administering.

[0015] In some embodiments, the administering comprises delivering the population of dopaminergic cells to the subject with a stereotactic-guided delivery system.

[0016] In some embodiments, the method further comprises administering an immunosuppressive regimen to the subject, wherein the immunosuppressive regimen comprises basiliximab, methylprednisolone, and tacrolimus. In some embodiments, the basiliximab is administered at about 20 mg intravenously intraoperatively and post-operative at about 4 days after the administering of the population of dopaminergic cells; the methylprednisolone is administered at about 500 mg intravenously prior to administering the population of dopaminergic cells; and the tacrolimus is administered at about one day after the administering of the population of dopaminergic cells. In some embodiments, the methylprednisolone is further administered at about 5 mg daily following the administering of the population of dopaminergic cells.

[0017] In some embodiments, the improvement is detectable at about 12 weeks following the administering of the population of dopaminergic cells. In some embodiments, the improvement persists for a duration of at least one year. In some embodiments, the improvement persists for a duration of at least 1.5 years or at least 2 years. In some embodiments, the improvement persists after removal of the immunosuppressive regimen.

[0018] In some embodiments, the population of dopaminergic cells are derived from pluripotent stem cells that were differentiated into dopaminergic cells in vitro. In some embodiments, the population of dopaminergic cells comprise midbrain dopaminergic neurons or precursors thereof. In some embodiments, the midbrain dopaminergic neurons orprecursors thereof are derived from floor plate progenitor cells. In some embodiments, the floor plate progenitor cells are derived from pluripotent stem cells.

[0019] In some embodiments, the Parkinson’s disease is advanced Parkinson’s disease.

[0020] In another aspect, provided herein is a therapeutic composition comprising: an effective quantity of a population of dopaminergic cells for treating Parkinson’s disease; and a cell delivery solution.

[0021] In some embodiments, at least 90% of the dopaminergic cells in the population of dopaminergic cells are positive for FOXA2. In some embodiments, fewer than about 2% of the dopaminergic cells in the population of dopaminergic cells are positive for PAX6. In some embodiments, fewer than about 2% of the dopaminergic cells in the population of dopaminergic cells are positive for CRABP1. In some embodiments, fewer than about 12% of the dopaminergic cells in the population of dopaminergic cells are positive for Ki67.

[0022] In some embodiments, between about 70% to about 80% of the dopaminergic cells in the population of dopaminergic cells are viable.

[0023] In some embodiments, the population of dopaminergic cells comprise a capacity for producing dopamine that, when evaluated in vitro using liquid chromatography -tandem mass spectrometry (LC-MS / MS), provides an area under the concentration-time curve (AUC) that is equal to or greater than 11.2 ng x day / mL.

[0024] In some embodiments, the population of dopaminergic cells in the delivery solution is at a concentration of about 71,000 cells / pL to about 123,000 cells / pL.

[0025] In some embodiments, the cell delivery solution comprises: (a) one or more energy source components; (b) one or more pH buffers; (c) one or more salts; and (d) one or more stabilizing agents. In some embodiments, the one or more stabilizing agents are selected from the group consisting of recombinant albumin (rHSA), Dextran, and Poloxamer. In some embodiments, the one or more energy source components comprise a sugar. In some embodiments, the sugar is dextrose.

[0026] In some embodiments, the population of dopaminergic cells comprises at least 0.9 million dopaminergic cells. In some embodiments, the population of dopaminergic cells comprises at least 2.7 million dopaminergic cells. In some embodiments, the population of dopaminergic cells comprises at least 5.4xlOA6 dopaminergic cells.

[0027] In some embodiments, the Parkinson’s disease is advance Parkinson’s disease. In some embodiments, the therapeutic composition is used in the treatment of Parkinson’s disease.

[0028] In yet another aspect, provided herein is a vessel comprising any of the therapeutic compositions described herein. In some embodiments, the vessel comprises a cryovial. In some embodiments, the vessel comprises an aseptic technology (AT) vial.BRIEF DESCRIPTION OF DRAWINGS

[0029] FIG. 1 is a schematic of a Phase 1 clinical trial study design for evaluating the safety, tolerability, and efficacy of the dopaminergic neurons in the treatment of Parkinson’s disease.

[0030] FIG. 2 shows an exemplary surgical approach for administering effective quantities of dopaminergic neurons to subjects in need thereof.

[0031] FIGS. 3A-3B show exemplary data of a comparative analysis of 18F-DOPAPET Data in subjects treated with dopaminergic neurons. FIG. 3A shows a voxel-based analysis of the 18F-DOPAPET data that was conducted. The image displays voxel clusters with significant (P < 0.05) group level changes between baseline and 1 year (positive, blue and negative, orange). FIG. 3B is a box plot showing change in striatal-to-occipital ratio (SOR) from baseline limited to significant voxel clusters. Changes within the significant voxel clusters for the caudate and putamen are combined for each subject using volume weighted means. Lines represent the median change for the cohort, circles represent the mean, the box represents the lower and upper quartiles and the whiskers represent the extremes.

[0032] FIGS. 4A-4D show exemplary clinical data demonstrating therapeutic efficacy of specific doses of dopaminergic neurons for the treatment of Parkinson’s disease. FIG. 4A shows boxplots of changes from baseline in Cohort A (left panel) and Cohort B (right panel) in Movement Disorder Society-Sponsored Revision of the Unified Parkinson’s Disease Rating Scale (MDS-UPDRS) Part III score (OFF) after administering dopaminergic neurons. The y-axis represents the change in OFF time, while the x-axis denotes time in weeks. Symbols including lines, circles, boxes, and whiskers denote the median, mean, quartiles, and extremes, respectively. FIG. 4B shows boxplots of changes from baseline in Cohort A (left panel) and Cohort B (right panel) in patient reported ON time without troublesome dyskinesia. The change in ON time from baseline is plotted on the y-axis, with time in weeks on the x-axis. Symbols including lines, circles, boxes, and whiskers denote the median, mean, quartiles, and extremes, respectively. FIG. 4C shows boxplots of changes from baseline in Cohort A (left panel) and Cohort B (right panel) in patient-reported OFF time. The y-axis reflects the change in OFF time, and the x-axis marks time in weeks. Symbols including lines, circles, boxes, and whiskers denote the median, mean, quartiles, and extremes,respectively. FIG. 4D shows boxplots of changes from baseline in Cohort A (left panel) and Cohort B (right panel) in patient reported ON time with troublesome dyskinesia. The y-axis charts the change in ON time, and the x-axis represents time in weeks. Symbols including lines, circles, boxes, and whiskers denote the median, mean, quartiles, and extremes, respectively.

[0033] FIG. 5 shows exemplary clinical data demonstrating therapeutic efficacy of specific doses of dopaminergic neurons for the treatment of Parkinson’s disease as measured by a neuropsychological evaluation using the Neuropsychiatric Inventory Questionnaire (NPI-Q). The figure shows boxplots of changes from baseline in Cohort A (low dose, left panel) and Cohort B (high dose, right panel) in NPI-Q after administering dopaminergic neurons. The y-axis represents the NPI-Q score, while the x-axis denotes time in months post-transplantation of the dopaminergic neurons. Symbols including lines, diamonds, boxes, and whiskers denote the median, mean, quartiles, and extremes, respectively.

[0034] FIGS. 6A-6B show exemplary clinical data demonstrating therapeutic efficacy of specific doses of dopaminergic neurons for the treatment of Parkinson’s disease as measured by a neuropsychological evaluation using the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS). FIG. 6A shows bar graphs of changes from baseline in RBANS categories for Cohort A (low dose) at baseline and at twelve months after administering dopaminergic neurons. FIG. 6B shows bar graphs of changes from baseline in RBANS categories for Cohort A (high dose) at baseline and at twelve months after administering dopaminergic neurons. The y-axis represents the RBANS score, while the x- axis denotes RBANS categories corresponding to immediate memory, visuospatial / constructional, language, attention, and delayed memory, and the total score. The mean (median) is denoted atop each bar. Symbols including lines and whiskers denote the median and extremes, respectively.

[0035] FIGS. 7A-7B show exemplary clinical data demonstrating therapeutic efficacy of specific doses of dopaminergic neurons for the treatment of Parkinson’s disease as measured by a neuropsychological evaluation using the Frontal Systems Behavior Scale (FrSBe). FIG. 7A shows bar graphs of changes from baseline in FrSBe categories for Cohort A (low dose) at baseline and at twelve months after administering dopaminergic neurons. FIG. 7B shows bar graphs of changes from baseline in FrSBe categories for Cohort A (high dose) at baseline and at twelve months after administering dopaminergic neurons. The y-axis represents the RBANS score, while the x-axis denotes FrSBe categories corresponding to apathy, dishinibition, and executive, and the total score. Raw scores were converted to T-scores(mean 50, SD 10), and T-scores >65 were considered clinically significant). The mean (median) is denoted atop each bar. Symbols including lines and whiskers denote the median and extremes, respectively.

[0036] FIG. 8 shows exemplary clinical data demonstrating therapeutic efficacy of specific doses of dopaminergic neurons for the treatment of PD motor symptoms as measured by Movement Disorders Society-Unified Parkinson’s Disease Rating Scale (MDS-UPDRS) Part II scores. Baseline MDS-UPDRS Part II scores were (mean [median]) 10.8 (10.0) in the low-dose cohort and 12.7 (11.0) in the high-dose cohort. MDS-UPDRS Part II contains 18 items with a subscore range of 0 to 52 points; higher scores indicate more severe motor symptoms. Horizontal lines represent median; diamonds represent mean; boxes represent Quartile 1 (QI) and Quartile 3 (Q3); whiskers represent extreme values. CfB means change from baseline. IS means immunosuppression.

[0037] FIG. 9 shows exemplary clinical data demonstrating therapeutic efficacy of specific doses of dopaminergic neurons for the treatment of PD motor symptoms as measured by Movement Disorders Society-Unified Parkinson’s Disease Rating Scale (MDS-UPDRS) Part III OFF scores. Baseline MDS-UPDRS Part III OFF scores were (mean [median]) 43.2 (44.0) in the low-dose cohort and 49.0 (51.0) in the high-dose cohort. MDS-UPDRS Part III contains 18 items with a subscore range of 0 to 132 points; higher scores indicate more severe motor symptoms; higher scores indicate more severe motor symptoms. Horizontal lines represent median; diamonds represent mean; boxes represent Quartile 1 (QI) and Quartile 3 (Q3); whiskers represent extreme values. CfB means change from baseline. IS means immunosuppression.

[0038] FIGS. 10A-10B shows exemplary clinical data demonstrating therapeutic efficacy of specific doses of dopaminergic neurons for the treatment of PD motor symptoms as measured by Hauser / PD Diary ON and OFF times. FIG. 10A shows exemplary data of patient-reported Hauser Diary Good ON Times. Baseline adjusted Hauser diary Good ON times were (mean [median]) 12.0 hours (13.2) in the low-dose cohort and 10.9 hours (10.4) in the high-dose cohort. Good ON state is the sum of ON state without dyskinesia and ON state with non-troublesome dyskinesia. Horizontal lines represent median; diamonds represent mean; boxes represent QI and Q3; whiskers represent extreme values. CfB, change from baseline; IS, immunosuppression; QI, quartile 1; Q3, quartile 3. FIG. 10B shows exemplary data of patient-reported Hauser Diary Good ON Times. Baseline adjusted Hauser diary OFF times were (mean [median]) 3.2 hours (2.6) in the low-dose cohort and 5.0 hours (5.6) in thehigh-dose cohort. Horizontal lines represent median; diamonds represent mean; boxes represent QI and Q3; whiskers represent extreme values. CfB, change from baseline; IS, immunosuppression; QI, quartile 1; Q3, quartile 3.

[0039] FIG. 11 shows exemplary clinical data demonstrating therapeutic efficacy of specific doses of dopaminergic neurons for the treatment of PD motor symptoms as measured by the Unified Dyskinesia Rating Scale (UDysRS). Baseline UDysRS objective subscores were (mean [median]) 10.6 (14.0) in the low-dose cohort and 2.3 (0.0) in the high-dose cohort. The UDysRS Objective Subscore is made up of Part 3 (Impairment) and Part 4 (Disability). The objective subscale consists of 11 items on a scale of 0 (normal) to 4 (severe), with a total possible score of 44; higher scores represent more severe symptoms. Horizontal lines represent median; diamonds represent mean; boxes represent QI and Q3; whiskers represent extreme values. CfB, change from baseline; IS, immunosuppression; QI, quartile 1; Q3, quartile 3.

[0040] FIG. 12 is a schematic of an exemplary dual-imaging strategy for assessing engraftment, survival, and function of transplanted dopaminergic neurons. In particular, FIG. 12 outlines the process and objectives of using Positron Emission Tomography (PET) and Magnetic Resonance Imaging (MRI) to assess the engraftment of dopaminergic neurons in the brain.

[0041] FIG. 13 illustrates an exemplary voxel-based group analysis method for evaluating engraftment, survival, and function of transplanted dopaminergic neurons.

[0042] FIG. 14 shows exemplary clinical image data of mean 18F-DOPA uptake as measured by positron emission tomography (PET). The mean image shows average 18F- DOPA uptake at baseline (prior to treatment), 12 months, and 18 months post administration. Mean image = Sum (all SOR-1 images at each visit) / sample size.

[0043] FIG. 15 shows exemplary image data of 18F-DOPA uptake at 12 and 18 months as measured by positron emission tomography (PET). Voxels with an increase in 18F-DOPA uptake are depicted in orange; voxels with a decrease 18F-DOPA uptake are depicted in blue.

[0044] FIGS. 16A-16B shows exemplary data show of 18F-DOPA uptake in the putamen and caudate, respectively. In particular, FIG. 16A shows a bar graph of changes from baseline in 18F-DOPA uptake in the putamen at 18 months post administration. FIG. 16B shows a bar graph of changes from baseline in 18F-DOPA uptake in the caudate at 18 months post administration.

[0045] FIG 17 shows exemplary Magnetic Resonance Imaging (MRI) image data taken from a subject administered dopaminergic neurons at baseline, 6 months, 12 months, and 18 months post administration.

[0046] FIGS. 18A-18B show exemplary clinical data demonstrating the therapeutic efficacy of specific doses of dopaminergic neurons for the treatment of PD motor symptoms as measured by Movement Disorders Society-Unified Parkinson’s Disease Rating Scale (MDS-UPDRS) Part III ON scores. FIG. 18A shows boxplots of MDS-UPDRS Part III ON scores measured from 0 (baseline, z.e., before transplantation) to 24 months posttransplantation. FIG. 18B shows boxplots of MDS-UPDRS Part III OFF scores measured from 0 (baseline, z.e., before transplantation) to 24 months post-transplantation. The y-axis indicates the MDS-UPDRS Part III score, while the x-axis indicates time in months. Mean (SD) scores are shown above each boxplot. Symbols including lines, diamonds, boxes, and whiskers indicate the median, mean, quartiles, and extremes, respectively.

[0047] FIG. 19 shows exemplary clinical data demonstrating the therapeutic efficacy of specific doses of dopaminergic neurons for the treatment of PD symptoms as measured by Movement Disorders Society-Unified Parkinson’s Disease Rating Scale (MDS-UPDRS) Part II scores. The figure shows boxplots of MDS-UPDRS Part II scores before (baseline, z.e., before transplantation) and after administering dopaminergic neurons up to 24 months. The y- axis indicates MDS-UPDRS Part II score, while the x-axis indicates time in months. Mean (SD) scores are shown above each boxplot. Symbols including lines, diamonds, boxes, and whiskers indicate the median, mean, quartiles, and extremes, respectively.

[0048] FIGS. 20A-20B show exemplary clinical data demonstrating the therapeutic efficacy of specific doses of dopaminergic neurons for the treatment of PD motor symptoms as measured by the Unified Dyskinesia Rating Scale (UDysRS) scores. FIG. 20A shows boxplots of UDysRS Objective Subscores before and after administering dopaminergic neurons. FIG. 20B shows boxplots of UDysRS Historical Subscores before and after administering dopaminergic neurons. The y-axis represents the respective UDysRS subscore, while the x-axis denotes time in months post-transplantation. Baseline scores are indicated at 0 months (pre-transplantation). Mean (SD) scores are shown above each boxplot. Symbols including lines, diamonds, boxes, and whiskers denote the median, mean, quartiles, and extremes, respectively.

[0049] FIGS. 21A-21B show exemplary clinical data demonstrating the therapeutic efficacy of specific doses of dopaminergic neurons for the treatment of PD symptoms as measured by patient-reported Parkinson’s Disease (PD) Diary scores. FIG. 21A showsboxplots of adjusted PD Diary Good ON Time before (0 months) and after administering dopaminergic neurons up to 24 months. FIG. 21B shows boxplots of adjusted PD Diary OFF Time before (0 months) and after administering dopaminergic neurons up to 24 months. The y-axis indicates time in hours and the x-axis indicates months. Baseline scores are indicated at 0 months. Mean (SD) scores are shown above or below each boxplot. Symbols including lines, diamonds, boxes, and whiskers denote the median, mean, quartiles, and extremes, respectively.

[0050] FIG. 22 shows exemplary clinical data demonstrating the therapeutic efficacy of specific doses of dopaminergic neurons for the treatment of PD non-motor symptoms as measured by the Neuropsychiatric Inventory Questionnaire (NPI-Q) Total Score. The figure shows boxplots of NPI-Q Total Scores at baseline (0 months), 12 months, and 24 months after administering dopaminergic neurons. The y-axis represents the NPI-Q Total Score, while the x-axis denotes time in months post-transplantation. Mean (SD) scores are shown above each boxplot. Symbols including lines, diamonds, boxes, and whiskers denote the median, mean, quartiles, and extremes, respectively.DETAILED DESCRIPTION

[0051] Aspects of the present disclosure relate to methods, populations of dopaminergic cells, compositions, and devices for treating Parkinson’s disease in a subject in a subject. In particular, the methods, methods, compositions, and devices for treating Parkinson’s disease in a subject include a population of dopaminergic cells. In some embodiments, administration of the population of dopaminergic cells to a subject that has Parkinson’s disease results in an improvement in motor function and / or non-motor function of the subject as compared to a control or baseline.

[0052] Parkinson’s disease is a neurodegenerative disorder leading to motor and non- motor manifestations and characterized by extensive degeneration of dopaminergic neurons in the nigrostriatal system. The motor manifestations of Parkinson’s disease are attributable to the degeneration of dopaminergic neurons within the substantia nigra, which in turn causes a dopamine deficiency in the striatum. Motor symptoms of Parkinson’s disease include tremor, hypokinesia (e.g., bradykinesia, akinesia, rigidity), postural instability, abnormal gait and swallowing disturbances. Non-motor symptoms include autonomic and neuropsychiatric disturbances such as anosmia, or sleep abnormalities. While effective management of a patient with Parkinson’s disease is possible during the initial stages of disease, progression ofParkinson’s disease into advanced stages over time can lead to severely debilitating complications.

[0053] In the embodiments described herein, the methods described herein include administration of populations of dopaminergic cells for the treatment of Parkinson’s disease in a subject. The population of dopaminergic cells for use with the methods described is capable of producing the neurotransmitter dopamine. Increased availability of dopamine in subjects having Parkinson’s disease can improve motor and non-motor function of the subject as compared to subjects that have not been administered the population of dopaminergic cells, or to a baseline of disease progression.

[0054] The embodiments described herein use novel populations of dopaminergic cells, which are broadly applicable to treat Parkinson’s disease in a subject. Such novel populations and methods of use are based, at least in part, on the surprising and unexpected observation from clinical data that administration of certain quantities of dopaminergic cells to a subject having Parkinson’s disease results in not only improvements of motor and nonmotor function, but in a reversal of disease progression. Remarkably, this unexpected outcome persists in subjects for at least 52 weeks without diminishment, which is in contrast to the typical decline in both motor and non-motor function seen in Parkinson’s disease. Thus, the methods described herein present an improvement in the treatment of Parkinson’s disease.

[0055] Furthermore, the clinical trial data reported herein demonstrates that populations of dopaminergic cells, derived in vitro as described herein, effectively engraft, survive, and function as dopamine producers in the brain of a subject to alleviate symptoms of Parkinson’s disease. Previous efforts to produce dopaminergic cells have exhibited inconsistencies in cell characteristics, functionality, and maturity, leading to unpredictable transplantation outcomes. In the absence of clinical data or thorough in vivo experimentation, there’s inherent uncertainty regarding the ability of in vitro produced dopaminergic cells to successfully integrate, survive, and restore function in Parkinson’s disease-affected brains of subjects, making the determination of a therapeutically effective cell dose exceedingly difficult. However, this disclosure fills that knowledge gap. The remarkable performance of the disclosed quantities of a population of dopaminergic cells described herein, evidenced by a significant improvement of both motor and non-motor Parkinson’s disease symptoms for at least 52 weeks post-transplantation, underscores the innovative and unique attributes of the cell doses and methods described herein.

[0056] Utilizing embodiments of this disclosure can help ensure efficacy in treating subjects with Parkinson’s disease. Administering too few dopaminergic cells would fail to correct the dopamine deficit in Parkinson’s disease, while an excessive number of dopaminergic cells risks neurotransmitter imbalance, leading to complications like dyskinesias, mood disturbances, or other neurological effects. Overloading with dopaminergic cells can also compromise the viability and function of both existing and newly introduced cells. Advantageously, provided herein are quantities of dopaminergic cells that strike a balance between desirable therapeutic outcomes and minimized risk.

[0057] Although the disclosure describes various exemplary alternatives and implementations as provided herein, it should be understood that the various features, aspects, and functionality described in one or more of the individual alternatives are not limited in their applicability to the particular alternative with which they are described. Instead, they can be applied alone or in various combinations to one or more of the other alternatives of the disclosure, whether the alternatives are described or whether the features are presented as a part of the described alternative. The breadth and scope of the present disclosure should not be limited by any exemplary alternatives described or shown herein.I. Definitions

[0058] The following definitions supplement those in the art and are directed to the present disclosure only. The following definitions are not to be imputed to any related or unrelated case, e.g., to any commonly owned patent or patent application. Although some methods and materials similar or equivalent to those described herein can be used to practice features of the disclosure, some preferred materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0059] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0060] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0061] As used herein, the articles “a,” “an,” and “the” are used herein to refer to one or to more than one (z.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0062] The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives.

[0063] As used herein, the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In some instances, the term “about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1% of a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0064] As used herein, the terms “administration,” “administering” and variants thereof refer to the introduction of a composition or therapeutic agent (e.g., a population of dopaminergic cells) into a subject. Administration includes concurrent and sequential introduction of the composition or therapeutic agent. Administration of the composition or therapeutic agent (e.g., a population of dopaminergic cells) into a subject is by any suitable route, including local injection or surgical implantation. A suitable route of administration allows the composition or the agent to perform its intended function. Administration also includes the administration by another. The administration can also be performed locally. For instance, a composition or therapeutic agent (e.g., a population of dopaminergic cells) can be administered by surgical implantation into a tissue, including the use of a device or vessel.

[0065] As used herein, the term “and / or” should be understood to mean either one, or both of, or any combination of the alternatives.

[0066] As used herein, the term “basiliximab” refers to an immunosuppressive monoclonal antibody that binds an IL-2 receptor identified under CAS Registry No. 179045- 86-4. Basiliximab is also known as Simulect. Basiliximab is a chimeric mouse-human antibody that blocks IL-2 binding to the IL-2 receptor in immune cells.

[0067] As used herein, the term “delivery solution” or “cell delivery solution” is any solution that is added to a container containing cells so that the cells can be administered to a subject. The cell delivery solution can contain no, minimal or trace amounts ofcryoprotectant and / or cell wash solution, or other components that are not desired for administration. The delivery solution can be used to reconstitute a population of cells (e.g., a population of dopaminergic cells) for administration following thawing of cells prior to administration or clinical use.

[0068] As used herein, the term “differentiation,” and any grammatical variants thereof, refers to a process whereby an unspecialized cell (e.g., an induced pluripotent stem cell (iPSC) or a pluripotent stem cell) acquires the features of a specialized cell such as a neuron. Differentiation can be controlled by the interaction of a cell’s genes with the physical and chemical conditions outside the cell, usually through signaling pathways involving proteins embedded in the cell surface. The term “directed differentiation” refers to a manipulation of cell culture conditions to induce an unspecialized cell (e.g., an induced pluripotent stem cell (iPSC) or a pluripotent stem cell) differentiation into a particular (for example, desired) cell type, such as neural, neural crest, cranial placode, and non-neural ectoderm precursors.

[0069] As used herein, the term “dopaminergic cell” refers to a cell capable of producing the neurotransmitter dopamine. The dopaminergic cell can be a dopaminergic neuron, or a dopaminergic progenitor cell. Exemplary dopaminergic cells include, but are not limited to, engraftable midbrain dopaminergic neurons, midbrain dopaminergic neurons, authentic midbrain dopamine neurons, midbrain dopaminergic neuron progenitor cells, floor-plate derived dopaminergic neurons, authentic midbrain dopaminergic neurons, dopaminergic neuron progenitor cells, and dopaminergic neuron precursor cells.

[0070] As used herein, an “effective amount,” “therapeutically effective amount,” or “effective quantity” refers to an amount sufficient to affect a beneficial or desired clinical result upon treatment. An effective amount can be administered to a subject in at least one dose. In terms of treatment, an effective amount includes an amount that is sufficient to palliate, ameliorate, stabilize, reverse or slow the progression of a disease (e.g., Parkinson’s disease), or otherwise reduce the pathological consequences of the disease. The effective amount can vary on a case-by-case basis. Several factors are typically taken into account when determining an appropriate dosage to achieve an effective amount. These factors include age, sex and weight of the subject, the condition being treated, the severity of the condition and the form and effective concentration of the population of dopaminergic cells administered. Exemplary effective quantities of the population of dopaminergic cells are described herein.

[0071] As used herein, an “immunosuppressive regimen” refers to a treatment regimen that inhibits or prevents an immune response to a foreign material in a subject. Theimmunosuppressive regimen can include immunosuppressive agents that inhibit immune cell activation, disrupt proliferation, or suppress inflammation.

[0072] As used herein, an “improvement” in response to administration of a treatment refers to the betterment of at least one parameter of disease progression. Improvement of disease progression can be the maintenance of a subject’s disease state when the type of disease (e.g., Parkinson’s disease) is a disease that gets progressively worse as a subject ages. Improvement of disease progression can be a reduction in disease progression, or it can include a reversal of symptoms associated with disease progression. In other words, improvement includes a reduction in the symptoms associated with the disease.Improvements of disease progression be determined based on methods known in the art. A person skilled in the art can determine the proper methods based on the type of diseases (e.g., Parkinson’s disease) being evaluated.

[0073] As used herein, the term “ / / / vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments exemplified, but are not limited to, test tubes and cell cultures.

[0074] As used herein, the term “ / / / vivo” refers to the natural environment (e.g., an animal or a cell) and to processes or reactions that occur within a natural environment, such as embryonic development, cell differentiation, neural tube formation, etc.

[0075] As used herein, the term “methylprednisolone” refers to a synthetic immunosuppressive steroid identified under CAS Registry No. 83-43-2, as well as under the PubChem Compound Identifier No. 6741. Methylprednisolone is also known as Depo- Medrol, Medrol, and Solu-Medrol.

[0076] As used herein, the term “Movement Disorder Society Unified Parkinson’s Disease Rating Scale” or “MDS-UPDRS” refers to a standardized scale used to evaluate various aspects of Parkinson’s disease. The MDS-UPDRS score can include evaluation of including non-motor and / or motor experiences of daily living, as well as a motor evaluation and characterization of the extent and burden of Parkinson’s disease across various populations. The MDS-UPDRS can be used in a clinical setting as well as in research. The MDS-UPDRS includes different parts, each one evaluating different aspects of Parkinson’s disease.

[0077] As used herein, the “MDS-UPDRS ON score” or the “ON score” refers to the MDS-UPDRS score of a patient during a period when a therapy (e.g., a population of dopaminergic cells) is effective, and symptoms of a disease (e.g., Parkinson’s disease) are under control. As used herein, the “MDS-UPDRS OFF score” or the “OFF score” refers tothe MDS-UPDRS score of a patient during a period when the effects a therapy (e.g., a population of dopaminergic cells) have worn off, and at least one symptom of a disease e.g., Parkinson’s disease) has returned.

[0078] As used herein, the term “neuron” refers to a nerve cell, the principal functional units of the nervous system of a subject. A neuron typically includes a cell body and its processes - an axon and at least one dendrite. Neurons transmit information to other neurons or cells by releasing neurotransmitters at synapses. In the context of the cells that are used in the methods and compositions described herein, a neuron includes cells that express one or more marker that is indicative of a neuron. Such markers include, but are not limited to, engrailed- 1 (EN1), orthodenticle homeobox 2 (OTX2), tyrosine hydroxylase (TH), nuclear receptor related- 1 protein (NURR1), forkhead box protein A2 (FOXA2), and LIM homeobox transcription factor 1 alpha (LMX1 A), PITX3, LM03, SNCA, ADCAP1, CHRNA4, and GIRK2.

[0079] As used herein, the term “Parkinson’s disease” refers to a neurodegenerative disorder characterized by extensive degeneration of dopaminergic neurons in the substantia nigra region in the brain. Parkinson’s disease manifest in changes of both motor and nonmotor functions. Exemplary changes in motor functions that are symptomatic of Parkinson’s disease include, but are not limited to, tremor, hypokinesia, postural instability, abnormal gait and swallowing disturbances. Exemplary changes in non-motor functions that are symptomatic of Parkinson’s disease include, but are not limited to, autonomic and neuropsychiatric disturbances such as anosmia or sleep abnormalities. Parkinson’s disease can be characterized as early Parkinson’s disease, or it can be characterized as advanced Parkinson’s disease, with more severe symptoms in the more advanced stages of the disease.

[0080] As used herein, the terms “patient,” “subject,” “individual,” and the like are used interchangeably and refer to any animal, or cells thereof, whether in vitro or in situ, amenable to the compositions and methods described herein. In some instances, the patient, subject or individual is a human.

[0081] As used herein, a “pluripotent stem cell” is a cell that has the ability to differentiate into cells from any of the three germ layers of an organism (endoderm, mesoderm or ectoderm), but not into extra-embryonic tissues like the placenta. The pluripotency may be incomplete or partial, in that the pluripotent cell may form cells of all three germ layers but may not exhibit all the characteristics of completely pluripotent cells. An “induced pluripotent stem cell” or “iPSC” refers to any pluripotent stem cell artificiallyderived from a non-pluripotent cell, typically an adult somatic cell, by inducing a “forced” expression of specific genes.

[0082] As used herein, the term “population” of cells or “cell population” refers to a group of at least two cells. In non-limiting examples, a cell population can include at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, or more cells. The population may be a pure population comprising one cell type. Alternatively, the population may include more than one cell type, for example a mixed cell population.

[0083] As used herein, a statement that a cell or population of cells is “positive” for a particular marker, or “expresses” a particular marker, refers to the detectable presence on or in the cell of a particular marker, for example, a surface marker or an intracellular marker, such as transcription factors. When referring to a surface marker, the term refers to the presence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is detectable by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control or fluorescence minus one (FMO) gating control under otherwise identical conditions and / or at a level substantially similar to that for cell known to be positive for the marker, and / or at a level substantially higher than that for a cell known to be negative for the marker.

[0084] As used herein, a statement that a cell or population of cells is “negative” for a particular marker, or fails to express a particular marker or gene, refers to the absence of substantial detectable presence on or in the cell of a particular marker, such as a surface marker or an intracellular marker, such as transcription factors. When referring to a surface marker, the term refers to the absence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is not detected by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control or fluorescence minus one (FMO) gating control under otherwise identical conditions, and / or at a level substantially lower than that for cell known to be positive for the marker, or at a level substantially similar as compared to that for a cell known to be negative for the marker.

[0085] As used herein, the term “progenitor cell,” and its grammatical equivalents, refers to a descendant of a stem cell that can further differentiate into specialized cell types within a particular cell lineage.

[0086] As used herein, the term “stem cell” refers to a cell with the ability to divide for indefinite periods in culture and to give rise to specialized cells.

[0087] As used herein, the term “substantially” or “essentially” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In some instances, the terms “essentially the same” or “substantially the same” refer a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that is about the same as a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0088] As used herein, the term “tacrolimus” refers to an immunosuppressive drug identified under CAS Registry No. 104987-11-3, as well as under the PubChem Compound Identifier No. 445643. Tacrolimus is also known as FK506 and Prograf. Tacrolimus can suppress T-cell activation primarily by binding to an FK506 binding protein (FKBP) immunophilin receptor, FKBP 12, that in turn inhibits calcineurin phosphatase activity and reduces production of IL-2 and activation of immune cells. Tacrolimus can also modulate the immune response in the nervous system.

[0089] As used herein, the term “treating” or “treatment” refers to clinical intervention in an attempt to alter the disease course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Therapeutic effects of treatment include, without limitation, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, reversal of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. By preventing or reversing progression of a disease or disorder, a treatment can prevent deterioration due to a disorder in an affected or diagnosed subject or a subject suspected of having the disorder, but also a treatment may prevent the onset of the disorder or a symptom of the disorder in a subject at risk for the disorder or suspected of having the disorder.

[0090] As used herein, the term “vessel” refers to any suitable container that can contain a therapeutic agent (e.g., a population of dopaminergic cells). In some cases, the vessel can be just used for storage of the therapeutic agent, or it can be used during administration of the therapeutic agent to a subject.

[0091] Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example,I, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.II. Population of Dopaminergic Cells

[0092] Certain aspects of the disclosure provide populations of dopaminergic cells. In some embodiments, the population of dopaminergic cells is suitable for use in the treatment of Parkinson’s disease.

[0093] In some embodiments, the population of dopaminergic cells includes dopaminergic neurons. In some embodiments, the dopaminergic neurons include one or more of a midbrain dopaminergic neuron, a floor-plate-derived dopaminergic neuron, and an authentic midbrain dopaminergic neuron. In some embodiments, the population of dopaminergic cells includes a dopaminergic progenitor cell or a dopaminergic precursor cell.

[0094] In some embodiments, the population of dopaminergic cells is positive for FOXA2. In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of the population of dopaminergic cells is positive for FOXA2. In some embodiments, at least 80% of the population of dopaminergic cells is positive for FOXA2. In some embodiments, at least 90% of the population of dopaminergic cells is positive for FOXA2. In some embodiments, at least 95% of the population of dopaminergic cells is positive for FOXA2. In some embodiments, at least 98% of the population of dopaminergic cells is positive for FOXA2. In some embodiments, at least 99% of the population of dopaminergic cells is positive for FOXA2.

[0095] In some embodiments, only a percentage of the population of dopaminergic cells is positive for PAX2. In some embodiments, less than about 20%, less than about 15%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the population of dopaminergic cells is positive for PAX2. In someembodiments, less than about 10% of the population of dopaminergic cells is positive for PAX2. In some embodiments, less than about 5% of the population of dopaminergic cells is positive for PAX2. In some embodiments, less than about 4% of the population of dopaminergic cells is positive for PAX2. In some embodiments, less than about 3% of the population of dopaminergic cells is positive for PAX2. In some embodiments, less than about 2% of the population of dopaminergic cells is positive for PAX2. In some embodiments, less than about 1% of the population of dopaminergic cells is positive for PAX2. In some embodiments, the population of dopaminergic cells is negative for PAX2.

[0096] In some embodiments, only a percentage of the population of dopaminergic cells is positive for CRABP1. In some embodiments, less than about 20%, less than about 15%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the population of dopaminergic cells is positive for CRABP1. In some embodiments, less than about 10% of the population of dopaminergic cells is positive for CRABP1. In some embodiments, less than about 5% of the population of dopaminergic cells is positive for CRABP1. In some embodiments, less than about 4% of the population of dopaminergic cells is positive for CRABP1. In some embodiments, less than about 3% of the population of dopaminergic cells is positive for CRABP1. In some embodiments, less than about 2% of the population of dopaminergic cells is positive for CRABP1. In some embodiments, less than about 1% of the population of dopaminergic cells is positive for CRABP1. In some embodiments, the population of dopaminergic cells is negative for CRABP1.

[0097] In some embodiments, only a percentage of the population of dopaminergic cells is positive for Ki67. In some embodiments, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 18%, less than about 15%, less than about 12%, less than about 10%, less than about 8%, or less than about 5% of the population of dopaminergic cells is positive for Ki67. In some embodiments, less than about 30% of the population of dopaminergic cells is positive for Ki67. In some embodiments, less than about 20% of the population of dopaminergic cells is positive for Ki67. In some embodiments, less than about 15% of the population of dopaminergic cells is positive for Ki67. In some embodiments, less than about 12% of the population of dopaminergic cells is positive for Ki67. In some embodiments, less than about 10% of the population of dopaminergic cells is positive for Ki67. In some embodiments, less than about 5% of the population of dopaminergic cells ispositive for Ki67. In some embodiments, the population of dopaminergic cells is negative for Ki67.

[0098] In some embodiments, a certain amount of the dopaminergic cells in the population of dopaminergic cells are viable. In some embodiments, about 10% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 50%, or about 50% to about 100% of the dopaminergic cells in the population of dopaminergic cells are viable. In some embodiments, about 50% to about 100% of the dopaminergic cells in the population of dopaminergic cells are viable. In some embodiments, about 60% to about 90% of the dopaminergic cells in the population of dopaminergic cells are viable. In some embodiments, about 70% to about 80% of the dopaminergic cells in the population of dopaminergic cells are viable. In some embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or more of the dopaminergic cells in the population of dopaminergic cells are viable. In some embodiments, at least about 90% of the dopaminergic cells in the population of dopaminergic cells are viable. In some embodiments, at least about 80% of the dopaminergic cells in the population of dopaminergic cells are viable. In some embodiments, at least about 70% of the dopaminergic cells in the population of dopaminergic cells are viable. In some embodiments, at least about 50% of the dopaminergic cells in the population of dopaminergic cells are viable.

[0099] In some embodiments, the population of dopaminergic cells have a capacity for producing dopamine. In some embodiments, the population of dopaminergic cells have the capacity for producing dopamine at a rate of at least about 25 ng / day / mL, about 20 ng / day / mL, about 15 ng / day / mL, about 12 ng / day / mL, about 10 ng / day / mL, about 8 ng / day / mL, about 6 ng / day / mL, about 4 ng / day / mL, or about 2 ng / day / mL. In some embodiments, the population of dopaminergic cells have the capacity for producing dopamine at a rate of at least about 20 ng / day / mL. In some embodiments, the population of dopaminergic cells have the capacity for producing dopamine at a rate of at least about 15 ng / day / mL. In some embodiments, the population of dopaminergic cells have the capacity for producing dopamine at a rate of at least about 11.2 ng / day / mL. In some embodiments, the population of dopaminergic cells have the capacity for producing dopamine at a rate of at least about 8 ng / day / mL.

[0100] The capacity of the population of dopaminergic cells for producing dopamine may be measured by any suitable means known in the art. In some embodiments, the populationof dopaminergic cells have a capacity for producing dopamine that, when evaluated in vitro using liquid chromatography-tandem mass spectrometry (LC-MS / MS), provides an area under the concentration-time curve (AUC) that is at least about 25 ng / day / mL, about 20 ng / day / mL, about 15 ng / day / mL, about 12 ng / day / mL, about 10 ng / day / mL, about 8 ng / day / mL, about 6 ng / day / mL, about 4 ng / day / mL, or about 2 ng / day / mL. In some embodiments, the population of dopaminergic cells have a capacity for producing dopamine that, when evaluated in vitro using liquid chromatography-tandem mass spectrometry (LC- MS / MS), provides an area under the concentration-time curve (AUC) that is at least about 20 ng / day / mL. In some embodiments, the population of dopaminergic cells have a capacity for producing dopamine that, when evaluated in vitro using liquid chromatography -tandem mass spectrometry (LC-MS / MS), provides an area under the concentration-time curve (AUC) that is at least about 15 ng / day / mL. In some embodiments, the population of dopaminergic cells have a capacity for producing dopamine that, when evaluated in vitro using liquid chromatography -tandem mass spectrometry (LC-MS / MS), provides an area under the concentration-time curve (AUC) that is at least about 11.2 ng / day / mL. In some embodiments, the population of dopaminergic cells have a capacity for producing dopamine that, when evaluated in vitro using liquid chromatography-tandem mass spectrometry (LC- MS / MS), provides an area under the concentration-time curve (AUC) that is at least about 8 ng / day / mL.

[0101] In some embodiments, the population of dopaminergic cells includes about 1 x 10A4 to about 1 x 10Al 0 dopaminergic cells, about 1 x 10A4 to about 1 x 10A5 dopaminergic cells, about 1 x 10A5 to about 1 x 10A9 dopaminergic cells, about 1 x 10A5 to about 1 x 10A6 dopaminergic cells, about 1 x 10A5 to about 1 x 10A7 dopaminergic cells, about 1 x 10A6 to about 1 x 10A7 dopaminergic cells, about 1 x 10A6 to about 1 x 10A8 dopaminergic cells, about 1 x 10A7 to about 1 x 10A8, from about 1 x 10A8 to about 1 x 10A9 dopaminergic cells, about 1 x 10A8 to about 1 x 10Al 0 dopaminergic cells, or about 1 x 10A9 to about 1 x 10Al 0 dopaminergic cells. In some embodiments, the population of cells includes at least 0.25 million dopaminergic cells, at least 0.5 million dopaminergic cells, at least 0.75 million dopaminergic cells, at least 1 million dopaminergic cells, at least 1.25 million dopaminergic cells, at least 1.5 million dopaminergic cells, at least 1.75 million dopaminergic cells, at least 2 million dopaminergic cells, at least 2.25 million dopaminergic cells, at least 2.5 million dopaminergic cells, at least 2.75 million dopaminergic cells, at least 3 million dopaminergic, at least 3.25 million dopaminergic cells, at least 3.5 million dopaminergic cells, at least 3.75 million dopaminergic cells, at least 4 million dopaminergic cells, at least 4.5 milliondopaminergic cells, at least 5 million dopaminergic cells, at least 5.5 million dopaminergic cells, at least 6 million dopaminergic cells, at least 7 million dopaminergic, at least 8 million dopaminergic cells, at least 9 million dopaminergic cells, or at least 10 million or more dopaminergic cells.

[0102] In some embodiments, the population of dopaminergic cells includes at least about 1.0 xlOA6 to about 1.2 xlOA7 dopaminergic cells. In some embodiments, the population of dopaminergic cells includes at least about 0.9 million dopaminergic cells. In some embodiments, the population of dopaminergic cells includes at least about 1.8 million dopaminergic cells. In some embodiments, the population of dopaminergic cells includes at least about 2.7 million dopaminergic cells. In some embodiments, the population of dopaminergic cells includes at least about 5.4 million dopaminergic cells. In some embodiments, the population of dopaminergic cells includes more than 5.4 million dopaminergic cells.

[0103] In some embodiments, the population of therapeutic cells is suitable for treating Parkinson’s disease in a subject. In some embodiments, the Parkinson’s disease is early Parkinson’s disease. In some embodiments, the Parkinson’s disease is advanced Parkinson’s disease. In some embodiments, the subject is a human.Production of Dopaminergic Cells

[0104] In some embodiments, the population of dopaminergic cells (e.g., a population of dopaminergic cells described herein) is prepared by differentiating stem cells into dopaminergic cells in vitro. In some embodiments, the population of dopaminergic cells comprises midbrain dopaminergic neurons or progenitors thereof. Accordingly, in some embodiments, the population of dopaminergic cells is prepared by differentiating stem cells into midbrain dopaminergic neurons or progenitors thereof in vitro. In some embodiments, the preparation of the midbrain dopaminergic neurons or progenitors thereof involves differentiating stem cells into neural precursor cells in vitro. In some embodiments, the preparation of the midbrain dopaminergic neurons or progenitors thereof further comprises differentiating the neural precursor cells into floor plate progenitor cells in vitro. In some embodiments, the preparation of the midbrain dopaminergic neurons or progenitors thereof further comprises differentiating the floor plate progenitor cells into the midbrain dopaminergic neurons or progenitors thereof in vitro.

[0105] In some embodiments, differentiating stem cells into dopaminergic cells includes contacting the stem cells with at least one inhibitor of Small Mothers AgainstDecapentaplegic (SMAD) signaling (referred to as “SMAD inhibitor”), at least one activator of Sonic hedgehog (SHH) signaling (referred to as “SHH activator”), and at least one activator of wingless (Wnt) signaling (referred to as “Wnt activator”) to obtain a population of cells that expresses at least one marker indicating a dopaminergic cell.

[0106] In some embodiments, the at least one marker indicating a dopaminergic cell is selected from EN1, FOX1A, LMX1A, OTX2, NURR1, TH, PITX3, LM03, SNCA, ADCAP1, CHRNA4, GIRK2, and FOXA2. In some embodiments, the at least one marker indicating a dopaminergic cell is FOXA2. In some embodiments, the at least one marker indicating a dopaminergic cell comprises TH.

[0107] In some embodiments, the population of dopaminergic cells is prepared by differentiating stem cells into midbrain dopaminergic neurons or precursors thereof in vitro. In some embodiments, differentiating the stem cells into midbrain dopaminergic neurons or precursors thereof involves a neural induction process, whereby the stem cells are differentiated into neural progenitor cells. In some embodiments, the neural induction process involves contacting the stem cells with Dual SMAD inhibitors (e.g., Noggin and SB431542), for example, as described in Chambers, 2009, Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling, Nat Biotechnol; 27(3): 275-280; and also in W02010096496A2, each of which are incorporated by reference.

[0108] In some embodiments, the preparation of midbrain dopaminergic neurons or precursors thereof further includes contacting stem cells or neural progenitor cells with an activator of SHH signaling. In some embodiments, the differentiation of the stem cells or the neural progenitor cells into midbrain dopaminergic neurons or precursors thereof involves the differentiation of the stem cells or the neural progenitor cells into midbrain floor plate progenitor cells. In some embodiments, the differentiation of the stem cells or the neural progenitor cells into midbrain floor plate progenitor cells involves contacting the stem cells or the neural progenitor cells with an activator of SHH signaling in combination with an activator of Wnt signaling, for example, as described in WO2013067362A1, which is incorporated by reference. Accordingly, in some embodiments, this disclosure provides dopaminergic cells (e.g., midbrain dopaminergic neurons or precursors thereof) that were generated in vitro using a floor-plate base differentiation strategy, for example, as described in Kriks, 2011, Floor plate-derived dopamine neurons from hESCs efficiently engraft in animal models of Parkinson’s disease, Nature; 480(7378): 547-551, which is incorporated by reference.

[0109] In some embodiments, the population of dopaminergic cells is prepared by differentiating stem cells into midbrain dopaminergic neurons or progenitors thereof by a two-step Wnt activation pathway, for example, as described in Kim et. al., 2021, Biphasic activation of Wnt signaling facilitates the derivation of midbrain dopamine neurons from hESCs for translational use, Cell Stem Cell, 28(2): 343-355; and in WO2016196661A1, each of which are incorporated by reference. In some embodiments, the Wnt signaling pathway is activated by contacting the cells with an activator of Wnt. In some embodiments, the activator of Wnt comprises a GSK3 inhibitor. In some embodiments, the Wnt activator comprises CHIR99021, referred to as Chir. In some embodiments, the population of dopaminergic cells is prepared by differentiating stem cells into midbrain dopaminergic neurons or progenitors thereof by contacting the stem cells with a Wnt activator at an initial concentration of about 0.7 pM of Chir and then increasing the concentration to about 7.0 pM of Chir at about day 3 of differentiation. In some embodiments, the differentiation is performed with a fully defined base media. In some embodiments, the base media comprises E8 medium.

[0110] In some embodiments, the concentration of the at least one Wnt activator is increased during its exposure to the cells. In some embodiments, said increase of the concentration of the at least one Wnt activator is initiated about 4 days from initial exposure of the stem cells to the at least one SMAD inhibitor. In some embodiments, the concentration of the at least one Wnt activator is increased by about 300% to about 1000%. In some embodiments, the cells are exposed to the at least one Wnt activator with the increased concentration for at least about 7 days. In some embodiments, at least one additional Wnt activator is added to increase the overall concentration of the Wnt activator.

[0111] In some embodiments, differentiating stem cells into dopaminergic cells further includes contacting the cells with at least one activator of fibroblast growth factor (FGF) signaling, also known as an FGF activator.

[0112] In some embodiments, the initial contact of the cells with the at least one activator of FGF signaling is at least about 5 days from the initial contact of the cells with the at least one inhibitor of SMAD signaling. In some embodiments, the initial exposure of the cells to the at least one FGF activator is at least about 10 days from initial exposure of the stem cells to the at least one SMAD inhibitor. In some embodiments, the exposure of the cells to the at least one FGF activator prolongs the expression of EN1 by the dopaminergic cells.

[0113] In some embodiments, differentiating stem cells into dopaminergic cells further includes contacting the cells with dopaminergic cell lineage-specific activator or inhibitor, such as, BDNF, GDNF, cAMP, TGFP, ascorbic acid (AA), and / or DAPT.(i) Stem cells

[0114] In some embodiments, the stem cells are pluripotent stem cells. In some embodiments, the pluripotent stem cells are embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), or a combination thereof. In some embodiments, the stem cells are multipotent stem cells. Non-limiting examples of stem cells that can be used to produce a population of dopaminergic cells include, but are not limited to, human, nonhuman primate or rodent nonembryonic stem cells, embryonic stem cells, induced nonembryonic pluripotent cells and engineered pluripotent cells. In some embodiments, the stem cells are human stem cells. Non-limiting examples of human stem cells include human embryonic stem cells (hESC), human pluripotent stem cell (hPSC), human induced pluripotent stem cells (hiPSC), human parthenogenetic stem cells, primordial germ cell-like pluripotent stem cells, epiblast stem cells, F-class pluripotent stem cells, somatic stem cells, cancer stem cells, or any other cell capable of lineage specific differentiation. In certain embodiments, the stem cells are human embryonic stem cells (hESCs). In some embodiments, the stem cells are human induced pluripotent stem cells (hiPSCs).

[0115] In some embodiments, the stem cells or a progeny cell thereof contains an introduced heterologous nucleic acid, where said nucleic acid may encode a desired nucleic acid or protein product or have informational value (see, for example, U.S. Patent No. 6,312,911, which is incorporated by reference in its entirety). Non-limiting examples of protein products include markers detectable via in vivo imaging studies, for example receptors or other cell membrane proteins. Non-limiting examples of markers include fluorescent proteins (such as green fluorescent protein (GFP), blue fluorescent protein (EBFP, EBFP2, Azurite, mKalamal), cyan fluorescent protein (ECFP, Cerulean, CyPet, mTurquoise2), and yellow fluorescent protein derivatives (YFP, Citrine, Venus, YPet, EYFP)), b-galactosidase (LacZ), chloramphenicol acetyltransferase (cat), neomycin phosphotransferase (neo), enzymes (such as oxidases and peroxidases), and antigenic molecules. In some embodiments, the reporter can be driven by a recombinant promoter of a dopaminergic cell marker gene, for example, NURR1. fii) SMAD Inhibitors

[0116] In some embodiments, the at least one SMAD inhibitor includes at least one TGFp / Activin-Nodal inhibitor. In some embodiments, the at least one TGFp / Activin-Nodalinhibitor is selected from inhibitors of ALK5, inhibitors of ALK4, inhibitors of ALK7, and combinations thereof). In some embodiments, the TGFp / Activin-Nodal inhibitor includes an inhibitor of ALK5. In some embodiments, the TGFp / Activin-Nodal inhibitor is a small molecule selected from SB431542, derivatives thereof, and mixtures thereof. In some embodiments, the TGFp / Activin-Nodal inhibitor includes SB431542. In some embodiments, the TGFp / Activin-Nodal inhibitor includes a derivative of SB431542. In some embodiments, the derivative of SB431542 is A83-01.

[0117] In some embodiments, the at least one SMAD inhibitor includes at least one BMP inhibitor. In some embodiments, the at least one BMP inhibitor includes a small molecule selected from LDN193189, Noggin, dorsomorphin, derivatives thereof, and mixtures thereof. In some embodiments, the at least one BMP inhibitor includes LDN193189. In some embodiments, the at least one BMP inhibitor includes Noggin.

[0118] In some embodiments, the stem cells are exposed to one SMAD inhibitor, e.g., one TGFp / Activin-Nodal inhibitor. In some embodiments, the one TGFp / Activin-Nodal inhibitor is SB431542 or A83-01. In some embodiments, the stem cells are exposed to two SMAD inhibitors. In some embodiments, the two SMAD inhibitors are a TGFp / Activin- Nodal inhibitor and a BMP inhibitor. In some embodiments, the stem cells are exposed to SB431542 or A83-01, and LDN193189 or Noggin. In some embodiments, the stem cells are exposed to SB431542 and Noggin.

[0119] In some embodiments, the stem cells are exposed to or contacted with at least one SMAD inhibitor for at least about 5 days, or at least about 10 days. In some embodiments, the stem cells are contacted with or exposed to the at least one SMAD inhibitor for up to about 5 days, or up to about 10 days. In some embodiments, the stem cells are contacted with or exposed to the at least one SMAD inhibitor for between about 5 days and about 10 days. In some embodiments, the stem cells are contacted with or exposed to the at least one SMAD inhibitor for about 5 days. In some embodiments, the stem cells are contacted with or exposed to the at least one SMAD inhibitor for 7 days. In some embodiments, the cells are contacted with or exposed to the at least one SMAD inhibitor from day 0 through day 6. In some embodiments, the at least one SMAD inhibitor is added every day or every other day to a cell culture medium comprising the stem cells from day 0 through day 6. In some embodiments, the at least one SMAD inhibitor is added every day (daily) to a cell culture medium comprising the stem cells from day 0 to day 6.

[0120] In some embodiments, the cells are contacted with or exposed to a TGFp / Activin- Nodal inhibitor. In some embodiments, the concentration of the TGFp / Activin-Nodalinhibitor contacted with or exposed to the cells is between about 1 pM and about 20 pM, between about 1 pM and about 10 pM, between about 1 pM and about 15 pM, between about 10 pM and about 15 pM, between about 5 pM and about 10 pM, between about 5 pM and about 15 pM, between about 5 pM and about 20 pM, or between about 15 pM and about 20 pM. In some embodiments, the concentration of the TGFp / Activin-Nodal inhibitor contacted with or exposed to the cells is between about 1 pM and about 10 pM. In some embodiments, the concentration of the TGFp / Activin-Nodal inhibitor contacted with or exposed to the cells is about 5 pM to about 10 pM. In some embodiments, the concentration of the TGFp / Activin- Nodal inhibitor contacted with or exposed to the cells is about 10 pM. In some embodiments, the TGFp / Activin-Nodal inhibitor includes SB431542 or a derivative thereof (e.g., A83-01). In some embodiments, the TGFp / Activin-Nodal inhibitor includes SB431542.

[0121] In some embodiments, the cells are contacted with or exposed to a BMP inhibitor. In some embodiments, the concentration of the BMP inhibitor contacted with or exposed to the cells is between about 50 nM and about 500 nM, or between about 100 nM and about 500 nM, or between about 200 nM and about 500 nM, or between about 200 and about 300 nM, or between about 200 nM and about 400 nM, or between about 100 nM and about 250 nM, or between about 100 nM and about 250 nM, or between about 200 nM and about 250 nM, or between about 250 nM and about 300 nM. In some embodiments, the concentration of the BMP inhibitor contacted with or exposed to the cells is between about 200 nM and about 300 mM. In some embodiments, the concentration of the BMP inhibitor contacted with or exposed to the cells is about 150 nM, about 200 nM, about 250 nM, about 300 nM, or about 350 nM. In some embodiments, the concentration of the BMP inhibitor contacted with or exposed to the cells is about 250 nM. In some embodiments, the BMP inhibitor includes LDN193189 or a derivative thereof. In some embodiments, the BMP inhibitor includes LDN193189. In some embodiments, the BMP inhibitor includes Noggin. In some embodiments, the cells are contacted with or exposed to Noggin. In some embodiments, the concentration of Noggin contacted with or exposed to the cells is between about 200 ng / mL to about 800 ng / mL, or between 300 ng / mL to about 700 ng / mL, or between 400 ng / mL to about 600 ng / mL. In some embodiments, the concentration of Noggin contacted with or exposed to the cells is about 300 ng / mL, about 400 ng / mL, about 500 ng / mL, about 600 ng / mL, or about 600 ng / mL.

[0122] In some embodiments, the cells are contacted with or exposed to the TGFp / Activin-Nodal inhibitor and the BMP inhibitor simultaneously. In some embodiments, the stem cells are contacted with or exposed to the TGFp / Activin-Nodal inhibitor and theBMP inhibitor for 7 days. In some embodiments, the cells are contacted with or exposed to the TGFp / Activin-Nodal inhibitor and the BMP inhibitor from day 0 through day 6. In some embodiments, the TGFp / Activin-Nodal inhibitor and the BMP inhibitor are added every day or every other day to a cell culture medium comprising the stem cells from day 0 through day 6. In some embodiments, the TGFp / Activin-Nodal inhibitor and the BMP inhibitor are added every day (daily) to a cell culture medium comprising the stem cells from day 0 to day 6.(Hi) Wnt Activators

[0123] In some embodiments, the at least one Wnt activator lowers GSK3P for activation of Wnt signaling. Thus, in some embodiments, the Wnt activator is a GSK3P inhibitor. In some embodiments, the at least one Wnt activator is a small molecule selected from CHIR99021, Wnt3A, Wntl, Wnt5a, BIO, CHIR98014, Lithium, 3F8, derivatives thereof, and mixtures thereof. In some embodiments, the at least one Wnt activator includes CHIR99021 or a derivative thereof. In some embodiments, the at least one Wnt activator includes CHIR99021.

[0124] In some embodiments, the cells are contacted with or exposed to the at least one Wnt activator for at least about 5 days, at least about 10 days, at least about 15 days, or at least about 20 days. In some embodiments, the cells are contacted with or exposed to the at least one Wnt activator for up to about 5 days, up to about 10 days, up to about 15 days, or up to about 20 days. In some embodiments, the cells are contacted with or exposed to the at least one Wnt activator for between about 5 days and about 20 days, between about 5 days and about 15 days, between about 10 days and about 20 days, between about 5 days and about 15 days, or between about 10 days and about 15 days.

[0125] In some embodiments, the cells are contacted with the at least one Wnt activator for between about 10 days and about 15 days. In some embodiments, the cells are contacted with the at least one Wnt activator for about 10 days. In some embodiments, the stem cells are contacted with the at least one activator of Wnt signaling for 12 days.

[0126] In some embodiments, the cells are contacted with the at least one Wnt activator from day 0 through day 11. In some embodiments, the at least one Wnt activator is added every day or every other day to a cell culture medium comprising the cells from day 0 through day 11. In some embodiments, the at least one Wnt activator is added every day (daily) to a cell culture medium comprising the cells from day 0 through day 11.

[0127] In some embodiments, the concentration of the at least Wnt activator is increased during its exposure to the cells (also referred to as “Wnt Boost”). In some embodiments, the increase or Wnt Boost is initiated at least about 2 days, at least about 4 days, or at least about5 days from the initial exposure of the cells to the at least one Wnt activator. In some embodiments, the increase or Wnt Boost is initiated about 4 days from the initial exposure of the cells to the at least one Wnt activator.

[0128] In some embodiments, the cells are contacted with or exposed to the increased concentration of the at least one Wnt activator for at least about 5 days, or at least about 10 days. In some embodiments, the cells are contacted with or exposed to the increased concentration of the at least one Wnt activator for at least about 5 days. In some embodiments, the cells are contacted with the increased concentration of the at least one Wnt activator for up to about 5 days, up to about 10 days, or up to about 15 days. In some embodiments, the cells are contacted with the increased concentration of the at least one Wnt activator for up to about 10 days.

[0129] In some embodiments, the cells are contacted with or exposed to the increased concentration of the at least one Wnt activator for between about 5 days and about 15 days, or between about 5 days and about 10 days, or between about 10 days and about 15 days. In some embodiments, the cells are contacted with or exposed to the increased concentration of the at least one Wnt activator for between about 5 days and about 10 days. In some embodiments, the cells are contacted with or exposed to the increased concentration of the at least one Wnt activator for about 5 days, about 10 days, or about 15 days. In some embodiments, the cells are contacted with or exposed to the increased concentration of the at least one Wnt activator for about 5 days. In some embodiments, the cells are contacted with or exposed to the increased concentration of the at least one Wnt activator for 6 days. In some embodiments, the cells are contacted with or exposed to the increased concentration of the at least one Wnt activator from day 4 through day 9. In some embodiments, the cells are contacted with or exposed to the increased concentration of the at least one Wnt activator for about 10 days. In some embodiments, the cells are contacted with or exposed to the increased concentration of the at least one Wnt activator for 8 days. In some embodiments, the cells are contacted with or exposed to the increased concentration of the at least one Wnt activator from day 4 through day 11.

[0130] In some embodiments, the initial concentration of the at least one Wnt activator contacted with or exposed to the cells prior to the Wnt Boost is less than about 5 pM, less than about 3 pM, or less than about 1 pM, including, but not limited to, between about 0.01 pM and about 5 pM, between about 0.01 pM and about 3 pM, between about 0.05 pM and about 3 pM, between about 0.1 pM and about 3 pM, between about 0.5 pM and about 3 pM, between about 0.5 pM and about 2 pM, or between about 0.5 pM and about 1 pM. In someembodiments, the initial concentration of the at least one Wnt activator contacted with or exposed to the cells prior to the Wnt Boost is less than about 1 pM, e.g., about 0.1 pM, about 0.2 pM, about 0.3 pM, about 0.4 pM, about 0.5 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, about 0.9 pM, or about 1 pM. In some embodiments, the initial concentration of the at least one Wnt activator contacted with or exposed to the cells prior to the Wnt boost is about 0.5 pM. In some embodiments, the initial concentration of the at least one Wnt activator contacted with or exposed to the cells prior to the Wnt boost is about 0.7 pM.

[0131] In some embodiments, the increased concentration of the at least one Wnt activator post the Wnt Boost is about 3 pM or greater, about 5 pM or greater, about 10 pM or greater, about 15 pM or greater, or about 20 pM or greater. In some embodiments, the increased concentration of the at least one Wnt activator post the Wnt Boost is between about 3 pM and about 15 pM, between about 3 pM and about 10 pM, or between about 5 pM and about 10 pM. In some embodiments, the increased concentration of the at least one Wnt activator post the Wnt Boost is about 3 pM, about 3.5 pM, about 4 pM, about 4.5 pM, about 5 pM, about 5.5 pM, about 6 pM, about 6.5 pM, about 7 pM, about 7.5 pM, about 8 pM, about 8.5 pM, about 9 pM, about 9.5 pM, or about 10 pM. In some embodiments, the increased concentration of the at least one Wnt activator post the Wnt Boost is about 3 pM. In some embodiments, the increased concentration of the at least one Wnt activator post the Wnt boost is about 7 pM. In some embodiments, the increased concentration of the at least one Wnt activator post the Wnt Boost is about 7.5 pM.

[0132] In some embodiments, the concentration of the at least one Wnt activator is increased from the initial concentration contacted with or exposed to the cells by between about 50% and about 2000%, or between about 100% and about 1500%, or between about 150% and about 1500%, or between about 200% and about 1500%, or between about 250% and about 1500%, or between about 300% and about 1500%, or between about 300% and about 1000%, or between about 300% and about 400%, or between about 500% and about 1000%, or between about 800% and about 1000%, or between about 900% and about 1000%, or between about 950% and about 1000. In some embodiments, the concentration of the at least one Wnt activator is increased from the initial concentration contacted with or exposed to the cells by between about 300% and about 1000%. In some embodiments, the concentration of the at least one Wnt activator is increased from the initial concentration contacted with or exposed to the cells by between about 300% and about 400%. In some embodiments, the concentration of the at least one Wnt activator is increased from the initial concentration contacted with or exposed to the cells by between about 900% and about1000%. In some embodiments, the concentration of the at least one Wnt activator is increased from the initial concentration contacted with or exposed to the cells by about 300%, about 350%, about 400%, about 450%, about 500%, about 550%, about 600%. 650%, about 700%, about 750%, about 800%, about 850%, about 900%, about 950%, about 1000%, about 1050%, or about 1100%. In some embodiments, the concentration of the at least one Wnt activator is increased from the initial concentration contacted with or exposed to the cells by about 300%. In some embodiments, the concentration of the at least one Wnt activator is increased from the initial concentration contacted with or exposed to the cells by about 350%. In some embodiments, the concentration of the at least one Wnt activator is increased from the initial concentration contacted with or exposed to the cells by about 950%. In some embodiments, the concentration of the at least one Wnt activator is increased from the initial concentration contacted with or exposed to the cells by about 1000%.( iv) SHH Activators

[0133] In some embodiments, the SHH activator includes a SHH protein, a PTC antagonist, a Smoothened (SMO) agonist, or a combination thereof. In some embodiments, the SHH protein includes a recombinant SHH, a purified SHH, or a combination thereof. In some embodiments, the recombinant SHH isan SHH N-terminal fragment. In some embodiments, the recombinant SHH is SHH C25II. In some embodiments, the SMO agonist includes purmorphamine.

[0134] In some embodiments, the cells are contacted with or exposed to the at least one SHH activator for at least about 5 days, or at least about 10 days. In some embodiments, the cells are contacted with or exposed to the at least one SHH activator for up to about 5 days, or up to about 10 days. In some embodiments, the cells are contacted with or exposed to the at least one SHH activator for between about 5 days and about 10 days. In some embodiments, the cells are contacted with or exposed to the at least one SHH activator for about 5 days. In some embodiments, the cells are contacted with or exposed to the at least one SHH activator for 7 days. In some embodiments, the cells are contacted with or exposed to the at least one SHH activator from day 0 through day 6. In some embodiments, the at least one SHH activator is added every day or every other day to a cell culture medium comprising the cells from day 0 through day 6. In some embodiments, the at least one SHH activator is added every day (daily) to a cell culture medium comprising the cells from day 0 through day 6.

[0135] In some embodiments, the concentration of the at least one SHH activator contacted with or exposed to the cells is between about 50 ng / mL and about 1000 ng / mL, between about 100 ng / mL and about 1000 ng / mL, between about 20 ng / mL and about 1000ng / mL, between about 300 ng / mL and about 1000 ng / mL, between about 400 ng / mL and about 1000 ng / mL, between about 500 ng / mL and about 1000 ng / mL, between about 400 ng / mL and about 800 ng / mL, between about 400 ng / mL and about 700 ng / mL, between about 400 ng / mL and about 600 ng / mL, or between about 500 ng / mL and about 600 ng / mL. In some embodiments, the concentration of the at least one SHH activator contacted with or exposed to the cells is between about 400 ng / mL and about 600 ng / mL. In some embodiments, the concentration of the at least one SHH activator contacted with or exposed to the cells is about 400 ng / mL, about 450 ng / mL, about 500 ng / mL, about 550 ng / mL, or about 600 ng / mL. In some embodiments, the concentration of the at least one SHH activator contacted with or exposed to the cells is about 500 ng / mL. fvi F GF Activators

[0136] In some embodiments, the FGF activator is capable of causing expansion of the midbrain and upregulating midbrain gene expression. In some embodiments, the FGF activator is selected from FGF8a, FGF17, FGF18, FGF2, FGF4, derivatives thereof, and combinations thereof. In some embodiments, the FGF activator includes or is FGF 18.

[0137] In some embodiments, the cells are contacted with or exposed to the at least one FGF activator for at least about 1 day, at least about 3 days, at least about 5 days, at least about 8 days, or at least about 10 days. In some embodiments, the cells are contacted with or exposed to the at least one FGF activator for up to about 5 days, or up to about 10 days, or up to about 15 days, or up to about 20 days. In some embodiments, the cells are contacted with or exposed to the at least one FGF activator for between about 1 days and about 20 days, between about 1 day and about 15 days, or between about 5 days and about 20 days, or between about 5 days and about 15 days, or between about 5 days and about 10 days, or between about 10 days and about 20 days. In some embodiments, the cells are contacted with or exposed to the at least one FGF activator for between about 5 days and about 10 days. In some embodiments, the cells are contacted with or exposed to the at least one FGF activator for about 3 days, about 5 days, or about 8 days. In some embodiments, the cells are contacted with or exposed to the at least one FGF activator for about 5 days.

[0138] In some embodiments, the initial contact of the cells with or the initial exposure of the cells to the at least one FGF activator is at least about 5 days, or at least about 10 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In some embodiments, the initial contact of the cells with or the initial exposure of the cells to the at least one FGF activator is no later than about 5 days, no later than about 10 days, or no later than about 15 days from the initial contact of the cells with orthe initial exposure of the cells to the at least one SMAD inhibitor. In some embodiments, the initial contact of the cells with or the initial exposure of the cells to the at least one FGF activator is between about 5 days and about 15 days, between about 5 days and about 10 days, or between about 10 days and about 15 days, from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In some embodiments, the initial contact of the cells with or the initial exposure of the cells to the at least one FGF activator is between about 5 days and about 10 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In some embodiments, the initial contact of the cells with or the initial exposure of the cells to the at least one FGF activator is about 10 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In some embodiments, the initial contact of the cells with or the initial exposure of the cells to the at least one FGF activator is 9 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In some embodiments, the initial contact of the cells with or the initial exposure of the cells to the at least one FGF activator is 10 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In some embodiments, the initial contact of the cells with or the initial exposure of the cells to the at least one FGF activator is 12 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor.

[0139] In some embodiments, the initial contact of the cells with or the initial exposure of the cells to the at least one FGF activator is about 5 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor, and the cells are contacted with the at least FGF activator for about 3 days. In some embodiments, the initial contact of the cells with or the initial exposure of the cells to the at least one FGF activator is about 5 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor, and the cells are contacted with the at least FGF activator for about 5 days. In some embodiments, the initial contact of the cells with or the initial exposure of the cells to the at least one FGF activator is about 10 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor, and the cells are contacted with the at least FGF activator for about 3 days. In some embodiments, the initial contact of the cells with or the initial exposure of the cells to the at least one FGF activator is about 10 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor, and the cells are contacted with the at least FGF activator for about 5 days. In some embodiments, the initial contact of the cells with or theinitial exposure of the cells to the at least one FGF activator is 12 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor, and the cells are contacted with the at least FGF activator for 5 days.

[0140] In some embodiments, the concentration of the at least one FGF activator contacted with or exposed to the cells is between about 10 ng / mL and about 500 ng / mL, between about 50 ng / mL and about 500 ng / mL, between about 100 ng / mL and about 500 ng / mL, between about 100 ng / mL and about 400 ng / mL, between about 100 ng / mL and about 300 ng / mL, between about 100 ng / mL and about 200 ng / mL, between about 100 ng / mL and about 250 ng / mL. In some embodiments, the concentration of the at least one FGF activator contacted with or exposed to the cells is between about 100 ng / mL and about 200 ng / mL. In some embodiments, the concentration of the at least one FGF activator contacted with or exposed to the cells is about 100 ng / mL. In some embodiments, concentration of the at least one FGF activator contacted with or exposed to the cells is about 200 ng / mL.

[0141] In some non-limiting embodiments, the stem cells are contacted with or exposed to at least one TGFp / Activin-Nodal inhibitor (e.g., SB431542, e.g., at a concentration of about 10 mM), at least one BMP inhibitor (e.g., Noggin, e.g., at a concentration of about 500 ng / mL), and at least one SHH activator (e.g., SHH C25II, e.g., a concentration of about 500 ng / mL) for about 5 days (e.g., 7 days, e.g., from day 0 to day 6), and the cells are contacted with the at least one Wnt activator (e.g., CHIR99021, e.g., at a concentration of about 0.7 pM for 5 days (e.g., 4 days ,e.g., from day 0 to day 3), and at a concentration of about 7.5 pM for about 5 days (e.g., 6 days, e.g., from day 4 to day 9), and at a concentration of about 3 pM for about 2 days (e.g., from day 10 to day 11). The cells are optionally contacted with or exposed to the at least one FGF activator (e.g., FGF 18, e.g., at a concentration of about 100 ng / ml), wherein the initial contact of the cells with the at least one FGF activator is about 10 days (e.g., 10 days or 12 days) from the initial contact of the cells with the at least one SMAD inhibitor, and the cells are contacted with the at least one FGF activator for about 5 days (e.g., 5 days (from day 12 to day 16) or 7 days (e.g., from day 10 to day 16).( vi) Dopaminergic Neuron Lineage -Specific Activators and Inhibitors

[0142] In some embodiments, the cells are further contacted with at least one dopaminergic neuron lineage specific activator or inhibitor. In some embodiments, the at least one dopaminergic neuron lineage-specific activator or inhibitor includes L-glutamine, brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), Cyclic adenosine monophosphate (cAMP), Transforming growth factor beta (TGFB, for example, TGFB3), ascorbic acid (AA), and DAPT (also known as, N-[(3,5-Difluorophenyl)acetyl]- L-alanyl-2-phenyl]glycine-l,l-dimethylethyl ester; LY-374973, N-[N- (3,5- Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester; or N-[N- (3,5- difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester).

[0143] In some embodiments, the cells are contacted with the at least one dopaminergic neuron lineage specific activator or inhibitor for at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 10 or more days. In some embodiments, the cells are contacted with the at least one dopaminergic neuron lineage specific activator or inhibitor for between about 2 days and about 20 days, between about 3 days and about 19 days, between about 4 days and about 18 days, between about 5 days and about 17 days, between about 6 days and about 16 days, between about 7 days and about 15 days, between about 8 days and about 15 days, between about 9 days and about 14 days, or between about 10 days and about 13 days. In In some embodiments, the cells are contacted with the at least one dopaminergic neuron lineage activator or inhibitor for up to about 2, up to about 3, up to about 4, up to about 5, up to about 6, up to about 7, up to about 8, up to about 9, or up to about 10 days or more days. In some embodiments, the cells are contacted with the at least one dopaminergic neuron lineage specific activator or inhibitor for about 4 days, about 5 days, about 6 days, about 7 days, or about 8 days.

[0144] In some embodiments, the cells are contacted with L-glutamine at a concentration of between about 0.5 mM and about 5 mM, or between about 1 mM and about 5 mM, or between about 1.5 mM and about 2.5 mM, between about 1 mM and about 2 mM, about 0.5 mM and about 500 mM, or between about 1 mM and about 300 mM, or between about 1 mM and about 250 mM, or between about 2 mM and about 200 mM. In some embodiments, the cells are contacted with L-glutamine at a concentration of about 2 mM. In some embodiments, the cells are contacted with L-glutamine at a concentration of about 20 mM. In some embodiments, the cells are contacted with L-glutamine at a concentration of about 100 mM. In some embodiments, the cells are contacted with L-glutamine at a concentration of about 200 mM. In some embodiments, the cells are contacted with L-glutamine at a concentration of about 300 mM.

[0145] In some embodiments, the cells are contacted with BDNF at a concentration of between about 5 ng / ml and about 50 ng / mL, or between about 10 ng / ml and about 50 ng / mL, or between about 10 ng / ml and about 40 ng / mL, or between about 20 ng / ml and about 50 ng / mL, or between about 20 ng / ml and about 40 ng / mL, or between about 10 ng / ml and about 30 ng / mL, or between about 10 ng / ml and about 20 ng / mL, or between about 20 ng / ml andabout 30 ng / mL. In some embodiments, the cells are contacted with BDNF at a concentration of about 20 ng / mL.

[0146] In some embodiments, the cells are contacted with ascorbic acid (AA) at a concentration of between about 50 nM and about 500 nM, or between about 100 nM and about 500 nM, or between about 100 nM and about 400 nM, or between about 200 nM and about 400 nM, or between about 200 nM and about 300 nM, or between about 100 nM and about 300 nM. In some embodiments, the cells are contacted with AA at a concentration of about 200 nM.

[0147] In some embodiments, the cells are contacted with GDNF at a concentration of between about 5 ng / ml and about 50 ng / mL, or between about 10 ng / ml and about 50 ng / mL, or between about 10 ng / ml and about 40 ng / mL, or between about 20 ng / ml and about 50 ng / mL, or between about 20 ng / ml and about 40 ng / mL, or between about 10 ng / ml and about 30 ng / mL, or between about 10 ng / ml and about 20 ng / mL, or between about 20 ng / ml and about 30 ng / mL. In some embodiments, the cells are contacted with GDNF at a concentration of about 20 ng / mL.

[0148] In some embodiments, the cells are contacted with cAMP at a concentration of between about 200 pM and about 800 pM, or between about 200 pM and about 700 pM, or between about 300 pM and about 700 pM, or between about 300 pM and about 600 pM, or between about 400 pM and about 600 pM, or between about 450 pM and about 550 pM. In some embodiments, the cells are contacted with cAMP at a concentration of about 500 pM.

[0149] In some embodiments, the cells are contacted with TGFB3 at a concentration of between about 0.01 ng / ml and about 5 ng / mL, or between about 0.1 ng / ml and about 4 ng / mL, or between about 0.5 ng / ml and about 5 ng / mL, or between about 1 ng / ml and about 3 ng / mL, or between about 1 ng / ml and about 2 ng / mL. In some embodiments, the cells are contacted with TGFP3 at a concentration of about 1 ng / mL.(vii) Dopaminergic Cell Markers

[0150] In some embodiments, the method includes obtaining a cell population of the differentiated cells, wherein at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the differentiated cells express at least one marker indicating a dopaminergic cell. Non-limiting examples of markers indicating a dopaminergic cell include engrailed- 1 (EN1), orthodenticle homeobox 2 (OTX2), tyrosine hydroxylase (TH), nuclear receptor related- 1 protein (NURR1), forkhead box protein A2 (FOXA2), and LIM homeobox transcription factor 1 alpha (LMX1 A), PITX3, LM03, SNCA, ADCAP1, CHRNA4, andGIRK2. In some embodiments, the at least one marker indicating a dopaminergic cell is FOXA2.

[0151] In some embodiments, the differentiated cells express the at least one marker indicating a dopaminergic cell at least about 10 days (e.g., about 15 days, about 20 days, about 30 days, about 40 days, or about 50 days) from the initial contact of the cells with the at least one SMAD inhibitor.

[0152] In some embodiments, a population of dopaminergic cells are prepared by differentiating stem cells in vitro. In some embodiments, the population of dopaminergic cells comprise midbrain dopaminergic neurons or precursors thereof. In some embodiments, the midbrain dopaminergic neurons or precursors thereof express one or more markers indicating the midbrain dopaminergic neuron or precursor thereof. Non-limiting examples of markers indicating a midbrain dopaminergic neuron or precursor thereof include engrailed- 1 (EN1), orthodenticle homeobox 2 (OTX2), tyrosine hydroxylase (TH), nuclear receptor related- 1 protein (NURR1), forkhead box protein A2 (FOXA2), and LIM homeobox transcription factor 1 alpha (LMX1 A), PITX3, LM03, SNCA, ADCAP1, CHRNA4, and GIRK2. In some embodiments, the midbrain dopaminergic neurons or precursors thereof express a combination of markers indicating the midbrain dopaminergic neuron or precursor thereof. In some embodiments, the combination of markers include TH and FOXA2.

[0153] The treatment of the cells with at least FGF activator can lead to sustained expression of EN1. EN1 is a survival factor for midbrain dopaminergic neurons during development, and continues to exert neuroprotective and physiological function in adult midbrain dopaminergic neurons. As such, cells with sustained expression of EN1 can develop into functionally dopaminergic cells upon further development and maturation. In some embodiments, the differentiated cells have a detectable level of expression of EN1 at least about 10 days, at least about 15 days, at least about 16 days, at least about 20 days, at least about 25 days, at least about 27 days, at least about 30 days, at least about 35 days, at least about 40 days, at least about 45 days, at least about 50 days, at least about 60 days, at least about 70 days, at least about 80 days, or at least about 90 days from the initial contact of the stem cells to the at least one SMAD inhibitor. In some embodiments, the differentiated cells have a detectable level of expression of EN1 about 30 days from the initial contact of the stem cells to the at least one SMAD inhibitor. In some embodiments, the differentiated cells have a detectable level of expression of EN1 about 40 days from the initial contact of the stem cells to the at least one SMAD inhibitor.

[0154] In some embodiments, the dopaminergic cells derived from the differentiation of stem cells do not express or have a low expression of at least one marker selected from PAX6, EMX2, LHX2, SMA, CRABP1, Ki67 SIX1, PITX2, SIM1, POU4F1, PHOX2A, BARHL1, BARHL2, GBX2, HOXA2, HOXB2, POU5F1, NANOG, and combinations thereof. In some embodiments, the dopaminergic cells derived from the differentiation of stem cells do not express or have a low expression of PAX6, CRABP1, and / or Ki67.(viii) Cell Culture Medium

[0155] In some embodiments, the above-described inhibitors and activators are added to a cell culture medium comprising the cells. Suitable cell culture media include, but are not limited to, Knockout® Serum Replacement (KSR) medium, Neurobasal® medium (NB), N2 medium, B-27 medium, and Essential 8® / Essential 6® (E8ZE6) medium, and combinations thereof. KSR medium, NB medium, N2 medium, B-27 medium, and E8ZE6 medium are commercially available. KSR medium is a defined, serum-free formulation optimized to grow and maintain undifferentiated hESCs in culture.

[0156] In some embodiments, the cell culture medium is a KSR medium. In some embodiments, a KSR medium includes Knockout DMEM, Knockout Serum Replacement, L- Glutamine, Pen / Strep, MEM, and 13 -mercaptoethanol. In some embodiments, 1 liter of KSR medium includes 820 mL of Knockout DMEM, 150 mL of Knockout Serum Replacement, 10 mL of 200 mM L-Glutamine, 10 mL of Pen / Strep, 10 mL of 10 mM MEM, and 55 mM of 13- mercaptoethanol.

[0157] In some embodiments, the cell culture medium is an E8ZE6 medium. In some embodiments, an E8ZE6 cell culture medium includes DMEM / F12, ascorbic acid, selenium, insulin, NaHCOs, transferrin, FGF2 and TGFp. The E8ZE6 medium differs from a KSR medium in that E8ZE6 medium does not include an active BMP or Wnt ingredient. Thus, in some embodiments, when an E8ZE6 medium is used to culture the presently disclosed population of stem cells to differentiate into a population of dopaminergic cells, at least one inhibitor of SMAD signaling (e.g., those inhibiting BMP) is not required to be added to the E8ZE6 medium.Selection and Characterization of Dopaminergic Cells

[0158] In some embodiments, the population of dopaminergic cells (e.g., a population of dopaminergic cells described herein) is selected for and characterized after differentiation from stem cells.

[0159] In some embodiments, selecting for the population of dopaminergic cells includes isolating and propagating cells that express at least one dopaminergic cell marker gene. In some embodiments, selecting the population of dopaminergic cells for includes, at least in part, selecting dopaminergic cells which express FOXA2. Other dopaminergic cell marker genes that may be use for detection include, but are not limited to, EN1, OTX2, TH, NURR1, LMX1A, PITX3, LM03, SNCA, ADCAP1, CHRNA4, and GIRK2. Expression of the at least one dopaminergic cell marker gene can be detected by any suitable method known in the art such as, for example, flow cytometry, fluorescence activated cell sorting (FACS) or magnetic bead-based sorting.

[0160] In some embodiments, selecting for the population of dopaminergic cells further includes isolating and propagating cells that do not express a detectable level of at least one marker gene selected from PAX6, EMX2, LHX2, SMA, SIX1, CRABP1, Ki67, PITX2, SIM1, POU4F1, PHOX2A, BARHL1, BARHL2, GBX2, H0XA2, HOXB2, POU5F1, NANOG, or any combinations thereof.

[0161] In some embodiments, selecting for the population of dopaminergic cells includes isolating and propagating cells expressing at least one selectable marker gene. Expression of the selectable marker gene can be detected by any suitable method known in the art, such as for example, FACS or culturing in a selection media. Non-limiting examples of selectable marker genes include, but are not limited to, fluorescent proteins (such as green fluorescent protein (GFP), blue fluorescent protein (EBFP, EBFP2, Azurite, mKalamal), cyan fluorescent protein (ECFP, Cerulean, CyPet, mTurquoise2), and yellow fluorescent protein derivatives (YFP, Citrine, Venus, YPet, EYFP)), b-galactosidase (LacZ), chloramphenicol acetyltransferase (cat), neomycin phosphotransferase (neo), enzymes (such as oxidases and peroxidases), and antigenic molecules. In some embodiments, the selectable marker gene can be driven by a promoter of a dopaminergic cell marker gene.

[0162] In some embodiments, characterizing the population of dopaminergic cells includes assessing the viability of the population of dopaminergic cells. The viability of the cells can be assessed by any suitable method known in the art. For instance, the viability of the cells can be assessed by morphological analysis, or by staining the cells with a solution to identify dead cells, such as a staining solution containing Acridine Orange and Propidium Iodide (AO / PI).

[0163] In some embodiments, characterizing the population of dopaminergic cells includes determining a capacity for production of dopamine of the dopaminergic cells. Methods for detecting proteins production (e.g., dopamine production) by a cell are wellknown in the art. Non-limiting examples include, but are not limited to, immunoassays, enzyme immunoassays (EIA), radioimmunoassays (RIA), antigen capture assays, two- antibody sandwich assays, Western blot analysis, enzyme linked immunosorbent assays (ELISA), colorimetric assays, chemiluminescent assays, fluorescence assays, immunohistochemistry assays, chromatography, liquid chromatography, size exclusion chromatography, high performance liquid chromatography (HPLC), gas chromatography, mass spectrometry, tandem mass spectrometry, microscopy, microfluidic chip-based assays, and surface plasmon resonance. In some embodiments, the capacity for production of dopamine of the dopaminergic cells is determined in vitro using liquid chromatographytandem mass spectrometry (LC-MS / MS). In some embodiments, determining the capacity for production of dopamine of the dopaminergic cells includes determining an area under the concentration-time curve (AUC) produced by in vitro LC-MS / MS.III. Compositions

[0164] Certain aspects of the disclosure provide therapeutic compositions that include a population of dopaminergic cells. In some embodiments, the therapeutic composition includes an effective quantity of a population of dopaminergic cells for treating Parkinson’s disease, and a delivery solution.

[0165] In some embodiments, the therapeutic composition includes an effective quantity of a population of dopaminergic cells (e.g., a population of dopaminergic cells described herein). In some embodiments, the population of dopaminergic cells includes dopaminergic neurons. In some embodiments, the dopaminergic neurons include one or more of a midbrain dopaminergic neuron, a floor-plate-derived dopaminergic neuron, and an authentic midbrain dopaminergic neuron. In some embodiments, the population of dopaminergic cells includes a dopaminergic progenitor cell or a dopaminergic precursor cell. In some embodiments, the dopaminergic cells comprise midbrain dopaminergic neurons or progenitors thereof. In some embodiments, the midbrain dopaminergic neurons, or progenitors thereof, are derived from midbrain floor plate progenitor cells, whereby the midbrain floor plate progenitor cells are derived from stem cells in vitro.

[0166] In some embodiments, the therapeutic composition includes a population of dopaminergic cells that are positive for FOXA2. In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of the population of dopaminergic cells is positive for FOXA2. In some embodiments, at least 80% of thepopulation of dopaminergic cells is positive for F0XA2. In some embodiments, at least 90% of the population of dopaminergic cells is positive for FOXA2. In some embodiments, at least 95% of the population of dopaminergic cells is positive for FOXA2. In some embodiments, at least 98% of the population of dopaminergic cells is positive for FOXA2. In some embodiments, at least 99% of the population of dopaminergic cells is positive for FOXA2.

[0167] In some embodiments, the therapeutic composition includes a population of dopaminergic cells that are positive for TH. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the population of dopaminergic cells is positive for TH. In some embodiments, about 10% of the population of dopaminergic cells is positive for TH. In some embodiments, about 15% of the population of dopaminergic cells is positive for TH. In some embodiments, about 17% of the population of dopaminergic cells is positive for TH.

[0168] In some embodiments, the therapeutic composition includes a population of dopaminergic cells where only a percentage of the population of dopaminergic cells are positive for PAX2. In some embodiments, less than about 20%, less than about 15%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the population of dopaminergic cells is positive for PAX2. In some embodiments, less than about 10% of the population of dopaminergic cells is positive for PAX2. In some embodiments, less than about 5% of the population of dopaminergic cells is positive for PAX2. In some embodiments, less than about 4% of the population of dopaminergic cells is positive for PAX2. In some embodiments, less than about 3% of the population of dopaminergic cells is positive for PAX2. In some embodiments, less than about 2% of the population of dopaminergic cells is positive for PAX2. In some embodiments, less than about 1% of the population of dopaminergic cells is positive for PAX2. In some embodiments, the population of dopaminergic cells is negative for PAX2.

[0169] In some embodiments, the therapeutic composition includes a population of dopaminergic cells where only a percentage of the population of dopaminergic cells are positive for CRABP1. In some embodiments, less than about 20%, less than about 15%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the population of dopaminergic cells is positive for CRABP1. In someembodiments, less than about 10% of the population of dopaminergic cells is positive for CRABP1. In some embodiments, less than about 5% of the population of dopaminergic cells is positive for CRABP1. In some embodiments, less than about 4% of the population of dopaminergic cells is positive for CRABP1. In some embodiments, less than about 3% of the population of dopaminergic cells is positive for CRABP1. In some embodiments, less than about 2% of the population of dopaminergic cells is positive for CRABP1. In some embodiments, less than about 1% of the population of dopaminergic cells is positive for CRABP1. In some embodiments, the population of dopaminergic cells is negative for CRABP1.

[0170] In some embodiments, the therapeutic composition includes a population of dopaminergic cells where only a percentage of the population of dopaminergic cells are positive for Ki67. In some embodiments, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 18%, less than about 15%, less than about 12%, less than about 10%, less than about 8%, or less than about 5% of the population of dopaminergic cells is positive for Ki67. In some embodiments, less than about 30% of the population of dopaminergic cells is positive for Ki67. In some embodiments, less than about 20% of the population of dopaminergic cells is positive for Ki67. In some embodiments, less than about 15% of the population of dopaminergic cells is positive for Ki67. In some embodiments, less than about 12% of the population of dopaminergic cells is positive for Ki67. In some embodiments, less than about 10% of the population of dopaminergic cells is positive for Ki67. In some embodiments, less than about 5% of the population of dopaminergic cells is positive for Ki67. In some embodiments, the population of dopaminergic cells is negative for Ki67.

[0171] In some embodiments, the therapeutic composition includes a population of dopaminergic cells that includes dopaminergic cells that are viable. In some embodiments, about 10% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 50%, or about 50% to about 100% of the dopaminergic cells in the population of dopaminergic cells are viable. In some embodiments, about 50% to about 100% of the dopaminergic cells in the population of dopaminergic cells are viable. In some embodiments, about 60% to about 90% of the dopaminergic cells in the population of dopaminergic cells are viable. In some embodiments, about 70% to about 80% of the dopaminergic cells in the population of dopaminergic cells are viable. In some embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at leastabout 98%, or at least about 99% or more of the dopaminergic cells in the population of dopaminergic cells are viable. In some embodiments, at least about 90% of the dopaminergic cells in the population of dopaminergic cells are viable. In some embodiments, at least about 80% of the dopaminergic cells in the population of dopaminergic cells are viable. In some embodiments, at least about 70% of the dopaminergic cells in the population of dopaminergic cells are viable. In some embodiments, at least about 50% of the dopaminergic cells in the population of dopaminergic cells are viable.

[0172] In some embodiments, the therapeutic composition includes a population of dopaminergic cells have a capacity for producing dopamine. In some embodiments, the population of dopaminergic cells have the capacity for producing dopamine at a rate of at least about 25 ng / day / mL, about 20 ng / day / mL, about 15 ng / day / mL, about 12 ng / day / mL, about 10 ng / day / mL, about 8 ng / day / mL, about 6 ng / day / mL, about 4 ng / day / mL, or about 2 ng / day / mL. In some embodiments, the population of dopaminergic cells have the capacity for producing dopamine at a rate of at least about 20 ng / day / mL. In some embodiments, the population of dopaminergic cells have the capacity for producing dopamine at a rate of at least about 15 ng / day / mL. In some embodiments, the population of dopaminergic cells have the capacity for producing dopamine at a rate of at least about 11.2 ng / day / mL. In some embodiments, the population of dopaminergic cells have the capacity for producing dopamine at a rate of at least about 8 ng / day / mL.

[0173] In some embodiments, the population of dopaminergic cells have a capacity for producing dopamine that, when evaluated in vitro using liquid chromatography -tandem mass spectrometry (LC-MS / MS), provides an area under the concentration-time curve (AUC) that is at least about 25 ng / day / mL, about 20 ng / day / mL, about 15 ng / day / mL, about 12 ng / day / mL, about 10 ng / day / mL, about 8 ng / day / mL, about 6 ng / day / mL, about 4 ng / day / mL, or about 2 ng / day / mL. In some embodiments, the population of dopaminergic cells have a capacity for producing dopamine that, when evaluated in vitro using liquid chromatographytandem mass spectrometry (LC-MS / MS), provides an area under the concentration-time curve (AUC) that is at least about 20 ng / day / mL. In some embodiments, the population of dopaminergic cells have a capacity for producing dopamine that, when evaluated in vitro using liquid chromatography-tandem mass spectrometry (LC-MS / MS), provides an area under the concentration-time curve (AUC) that is at least about 15 ng / day / mL. In some embodiments, the population of dopaminergic cells have a capacity for producing dopamine that, when evaluated in vitro using liquid chromatography-tandem mass spectrometry (LC- MS / MS), provides an area under the concentration-time curve (AUC) that is at least about11.2 ng / day / mL. In some embodiments, the population of dopaminergic cells have a capacity for producing dopamine that, when evaluated in vitro using liquid chromatography -tandem mass spectrometry (LC-MS / MS), provides an area under the concentration-time curve (AUC) that is at least about 8 ng / day / mL.

[0174] The number of dopaminergic cells in the therapeutic composition can be any suitable and efficacious number. In some embodiments, the therapeutic composition includes a population of dopaminergic cells that includes about 1 x 10A4 to about 1 x10Al 0 dopaminergic cells, about 1 x 10A4 to about 1 x 10A5 dopaminergic cells, about 1 x 10A5 to about 1 x 10A9 dopaminergic cells, about 1 x 10A5 to about 1 x 10A6 dopaminergic cells, about 1 x 10A5 to about 1 x 10A7 dopaminergic cells, about 1 x 10A6 to about 1 x 10A7 dopaminergic cells, about 1 x 10A6 to about 1 x 10A8 dopaminergic cells, about 1 x 10A7 to about 1 x 10A8, from about 1 x 10A8 to about 1 x 10A9 dopaminergic cells, about 1 x 10A8 to about 1 x 10Al 0 dopaminergic cells, or about 1 x 10A9 to about 1 x 10Al 0 dopaminergic cells. In some embodiments, the population of cells includes at least 0.25 million dopaminergic cells, at least 0.5 million dopaminergic cells, at least 0.75 million dopaminergic cells, at least 1 million dopaminergic cells, at least 1.25 million dopaminergic cells, at least 1.5 million dopaminergic cells, at least 1.75 million dopaminergic cells, at least 2 million dopaminergic cells, at least 2.25 million dopaminergic cells, at least 2.5 million dopaminergic cells, at least 2.75 million dopaminergic cells, at least 3 million dopaminergic, at least 3.25 million dopaminergic cells, at least 3.5 million dopaminergic cells, at least 3.75 million dopaminergic cells, at least 4 million dopaminergic cells, at least 4.5 million dopaminergic cells, at least 5 million dopaminergic cells, at least 5.5 million dopaminergic cells, at least 6 million dopaminergic cells, at least 7 million dopaminergic, at least 8 million dopaminergic cells, at least 9 million dopaminergic cells, or at least 10 million or more dopaminergic cells.

[0175] In some embodiments, the therapeutic composition includes a population of dopaminergic cells that includes at least about 1.0 xlOA6 to about 1.2 xlOA7 dopaminergic cells. In some embodiments, the therapeutic composition includes a population of dopaminergic cells that includes at least about 0.9 million dopaminergic cells. In some embodiments, the therapeutic composition includes a population of dopaminergic cells that includes at least about 1.8 million dopaminergic cells. In some embodiments, the therapeutic composition includes a population of dopaminergic cells that includes at least about 2.7 million dopaminergic cells. In some embodiments, the therapeutic composition includes a population of dopaminergic cells that includes at least about 5.4 million dopaminergic cells.In some embodiments, the therapeutic composition includes a population of dopaminergic cells that includes more than 5.4 million dopaminergic cells.

[0176] In some embodiments, the therapeutic composition includes a population of dopaminergic cells at a concentration of about 71,000 cells / pL to about 123000 cells / pL, about 75,000 cells / pL to about 115,000 cells / pL, about 80,000 cells / pL to about 110,000 cells / pL, about 85,000 cells / pL to about 105,000 cells / pL, or about 90,000 cells / pL to about 100,000 cells / pL. In some embodiments, the population of dopaminergic cells are at a concentration of about 71,000 cells / pL to about 123000 cells / pL. In some embodiments, the population of dopaminergic cells are at a concentration of about 75,000 cells / pL, about 80,000 cells / pL, about 85,000 cells / pL, about 90,000 cells / pL, about 95,000 cells / pL, about 100,000 cells / pL, about 105,000 cells / pL, about 110,000 cells / pL, about 115,000 cells / pL, or about 120,000 cells / pL. In some embodiments, the population of dopaminergic cells are at a concentration of about 75,000 cells / pL, about 80,000 cells / pL, about 85,000 cells / pL, about 90,000 cells / pL, about 95,000 cells / pL, about 100,000 cells / pL, about 105,000 cells / pL, about 110,000 cells / pL, about 115,000 cells / pL, or about 120,000 cells / pL. In some embodiments, the population of dopaminergic cells are at a concentration of about 90,000 cells / pL. In some embodiments, the population of dopaminergic cells are at a concentration of about 100,000 cells / pL. In some embodiments, the population of dopaminergic cells are at a concentration of about 110,000 cells / pL.

[0177] In some embodiments, the therapeutic composition includes an effective quantity of a population of therapeutic cells for treating Parkinson’s disease in a subject. In some embodiments, the Parkinson’s disease is early Parkinson’s disease. In some embodiments, the Parkinson’s disease is advanced Parkinson’s disease. In some embodiments, the subject is a human.

[0178] In some embodiments, the therapeutic composition includes a population of dopaminergic cells that is allogenic to a subject that is administered the therapeutic composition. In some embodiments, the therapeutic composition includes a population of dopaminergic cells that is autologous to a subject that is administered the therapeutic composition.Delivery Solutions

[0179] In some embodiments, the therapeutic composition includes a population of dopaminergic cells (e.g., a population of dopaminergic cells described herein), and a delivery solution that includes one or more energy components, one or more pH buffers, one or moresalts, one or more stabilizing agents, or any combination thereof, for example, a delivery solution as described in International Application No PCT / US23 / 21961, which is incorporated by reference.

[0180] In some embodiments, the therapeutic composition includes a population of dopaminergic cells (e.g., a population of dopaminergic cells described herein), and a delivery solution that includes one or more energy components. An energy component is any component that can provide chemical energy to one or more cells. In some embodiments, the one or more energy components include a sugar. Exemplary sugars that can be included in the delivery solution include, but are not limited to, dextrose, fructose, galactose, glucose, lactose, maltose, and sucrose. In some embodiments, the sugar is dextrose. The delivery solution can include any amount of the one or more energy sources that achieve a desired effect (e.g., treatment of Parkinson’s disease). In some embodiments, the delivery solution includes one or more energy sources at a concentration of about 24.5 mM to about 24.8 mM, about 24.4 mM to about 24.9 mM, about 24.3 mM to about 25.0 mM, about 24.1 mM to about 25.2 mM, about 23.9 mM to about 25.4 mM, about 23.7 mM to about 25.6 mM. In some embodiments, the delivery solution includes one or more energy sources at a concentration of about 23.5 mM, about 23 mM, about 22 mM, about 21 mM, about 20 mM or less. In some embodiments, the delivery solution includes one or more energy sources at a concentration of about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM or more.

[0181] In some embodiments, the therapeutic composition includes a population of dopaminergic cells (e.g., a population of dopaminergic cells described herein), and a delivery solution that includes one or more pH buffers. The one or more pH buffers can include any suitable buffering agent, such as a zwitterionic organic chemical buffering agent, examples of which include, but are not limited to, 4-(2-hy droxy ethyl)- 1 -piperazineethanesulfonic acid (HEPES), sodium bicarbonate, 4-Morpholinepropanesulfonic acid, 3 -propanesulfonic acid (MOPS), and 2-(N-morpholino)ethanesulfonic acid (MES). The delivery solution can include any amount of the one or more pH buffers that achieves a desired effect (e.g., treatment of Parkinson’s disease in a subject). In some embodiments, the delivery solution includes one or more pH buffers at a concentration of about 10.6 mM to about 10.9 mM, about 10.5 mM to about 11.0 mM, about 10.4 mM to about 11.1 mM, about 10.2 mM to about 11.3 mM, about 10.0 mM to about 11.5 mM, or about 9.8 mM to about 11.7 mM. In some embodiments, the delivery solution includes one or more pH buffers at a concentration of about 9.5 mM, about 9 mM, about 8 mM, about 7 mM, about 6 mM or less, about 12 mM,about 13 mM, about 14 mM, about 15 mM, or about 16 mM. In some embodiments, the one or more pH buffers include one or more of 4-(2-hy droxy ethyl)- 1 -piperazineethanesulfonic acid (HEPES), sodium bicarbonate, 4-Morpholinepropanesulfonic acid, 3 -propanesulfonic acid (MOPS), and 2-(N-morpholino)ethanesulfonic acid (MES).

[0182] In some embodiments, the therapeutic composition includes a population of dopaminergic cells (e.g., a population of dopaminergic cells described herein), and a delivery solution that includes one or more salts. In some embodiments, the one or more salts include one or more of calcium chloride, magnesium chloride, potassium chloride, sodium phosphate monobasic, or sodium chloride.

[0183] In some embodiments, the one or more salts include calcium chloride. The delivery solution can include calcium chloride in any amount suitable to achieve the desired effect (e.g., treatment of Parkinson’s disease). In some embodiments, the delivery solution includes calcium chloride at a concentration of about 1.6 mM to about 1.9 mM, about 1.5 mM to about 2.0 mM, about 1.4 mM to about 2.1 mM, about 1.3 mM to about 2.2 mM, about 1.1 mM to about 2.4 mM, about 0.9 mM to about 2.6 mM, or about 0.7 mM to about 2.8 mM. In some embodiments, the delivery solution includes calcium chloride at a concentration of about 0.5 mM, about 0.3 mM, or about 0.1 mM or less. In some embodiments, the delivery solution includes calcium chloride at a concentration of about 3 mM, about 3.5 mM, about 4 mM, or about 5 mM or more.

[0184] In some embodiments, the one or more salts include magnesium chloride, delivery solution can include magnesium chloride in any amount suitable to achieve the desired effect (e.g., treatment of Parkinson’s disease). In some embodiments, the delivery solution includes magnesium chloride at a concentration of about 0.7 mM to about 1.0 mM, about 0.6 mM to about 1.1 mM, about 0.5 mM to about 1.2 mM, about 0.4 mM to about 1.4 mM, about 0.3 mM to about 1.6 mM, about 0.2 mM to about 1.9 mM, or about 0.1 mM to about 2.2 mM. In some embodiments, the delivery solution includes magnesium chloride at a concentration of about 0.05 mM or less, about 2.5 mM, or about 3.0 mM or more.

[0185] In some embodiments, the one or more salts include potassium chloride. The delivery solution can include potassium chloride in any amount suitable to achieve the desired effect (e.g., treatment of Parkinson’s disease). In some embodiments, the delivery solution includes potassium chloride at a concentration of about 5.1 mM to about 5.4 mM, about 5.0 mM to about 5.5 mM, about 4.9 mM to about 5.7 mM, about 4.8 mM to about 5.8 mM, about 4.6 mM to about 6.0 mM, about 4.4. mM to about 6.2 mM, or about 4.2 mM to about 6.4 mM. In some embodiments, the delivery solution includes calcium chloride at aconcentration of about 4.0 mM, about 3.5 mM, or about 3.0 mM or less. In some embodiments, the delivery solution includes calcium chloride at a concentration of about 6.5 mM, about 7.0 mM, about 7.5 mM, or about 8.0 mM or more.

[0186] In some embodiments, the one or more salts include sodium phosphate monobasic. The delivery solution can include sodium phosphate monobasic in any amount suitable to achieve the desired effect (e.g., treatment of Parkinson’s disease). In some embodiments, the delivery solution includes sodium phosphate monobasic at a concentration of about 0.88 mM to about 0.91 mM, about 0.87 mM to about 0.92 mM, about 0.86 mM to about 0.93 mM, about 0.85 mM to about 0.94 mM, about 0.83 mM to about 0.96 mM, or about 0.81 mM to about 0.98 mM. In some embodiments, the delivery solution includes sodium phosphate monobasic at a concentration of about 0.8 mM, about 0.75 mM, or about 0.7 mM or less. In some embodiments, the delivery solution includes sodium phosphate monobasic at a concentration of about 1.0 mM, about 1.05 mM, or about 1.1 mM or more.

[0187] In some embodiments, the one or more salts include sodium chloride. The delivery solution can include sodium chloride in any amount suitable to achieve the desired effect (e.g., treatment of Parkinson’s disease). In some embodiments, the delivery solution includes sodium chloride at a concentration of about 119 mM to about 122 mM, about 118 mM to about 123 mM, about 117 mM to about 124 mM, about 115 mM to about 126 mM, or about 113 mM to about 128 mM. In some embodiments, the delivery solution includes sodium chloride at a concentration of about 110 mM, about 105 mM, or about 100 mM or less. In some embodiments, the delivery solution includes sodium chloride at a concentration of about 130 mM, about 135 mM, or about 140 mM or more. In some embodiments, the delivery solution includes sodium chloride at a concentration of about 74 mM to about 77 mM, about 73 mM to about 78 mM, about 72 mM to about 79 mM, about 70 mM to about 81 mM, about 68 mM to about 83 mM, or about 66 mM to about 85 mM. In some embodiments, the delivery solution includes sodium chloride at a concentration of about 65 mM, about 60 mM, or about 55 mM or less. In some embodiments, the delivery solution includes sodium chloride at a concentration of about 90 mM, about 95 mM, or about 100 mM or more. In some embodiments, the delivery solution includes sodium chloride at a concentration of about 93 mM to about 96 mM, about 92 mM to about 97 mM, about 91 mM to about 98 mM, about 90 mM to about 99 mM, about 88 mM to about 101 mM, about 85 mM to about 103 mM, or about 83 mM to about 105 mM. In some embodiments, the delivery solution includes sodium chloride at a concentration of about 80 mM, about 75 mM,or about 70 mM or less. In some embodiments, the delivery solution includes sodium chloride at a concentration of about 105 mM, about 110 mM or about 115 mM or more.

[0188] In some embodiments, the therapeutic composition includes a population of dopaminergic cells (e.g., a population of dopaminergic cells described herein), and a delivery solution that includes one or more stabilizing agents. A stabilizing agent is to any component that can act to reduce or prevent degradation of other solution components. The one or more stabilizing agents can include any suitable stabilizing agent, such, but not limited to one or more of a protein, such as one or more albumins, such as recombinant albumin (rHSA), Dextran (including Dextran 40, as one example), Pol oxamer (including Pol oxamer 188 as one example). Additionally, the one or more stabilizing agents can include one or more of the following, which also may be an excipient of the delivery solution: polyethylene glycol, carboxymethyl cellulose, hyaluronic acid, starches, acrylates, methacrylates, polyvinyl alcohols, polyethylene oxides, polypropylene oxides, polyacrylates, polyvinylpyrrolidone, polymethacrylate, poly lactic-co-glycolic acids, polyacrylamides, polylactides, chitosans, gums, guar gums, xantham gums, carrageenans, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, cyclodextrin derivatives, beta-cyclodextrin derivatives, alginates, calcium alginates, and stearates. In some embodiments, the one or more stabilizing agents include one or more of recombinant albumin (rHSA), Dextran, and Poloxamer.

[0189] In some embodiments, the one or more stabilizing agents include recombinant albumin (rHSA). The delivery solution can include rHSA in any amount suitable to achieve the desired effect (e.g., treatment of Parkinson’s disease). In some embodiments, the delivery solution includes rHSA at a concentration of about 0.07 w / w% to about 0.09 w / w%, about 0.06 w / w% to about 0.1 w / w%, about 0.05 w / w% to about 0.11 w / w%, about 0.04 w / w% to about 0.12 w / w%, about 0.03 w / w% to about 0.13 w / w%, about 0.02 w / w% to about 0.15 w / w%, or about 0.01 w / w% to about 0.17 w / w%. In some embodiments, the delivery solution includes rHSA at a concentration of about 0.005 w / w% or less, about 0.2 w / w%, or about 0.25 w / w% or more. In some embodiments, the delivery solution includes rHSA at a concentration of about 0.08 w / w% to about 0.11 w / w%, about 0.07 w / w% to about 0.12 w / w%, about 0.06 w / w% to about 0.13 w / w%, about 0.05 w / w% to about 0.14 w / w%, about 0.04 w / w% to about 0.16 w / w%, about 0.03 w / w% to about 0.18 w / w%, about 0.02 w / w% to about 0.2 w / w%, about 0.01 w / w% 10 about 0.22 w / w%, or about 0.05 w / w% or less. In some embodiments, the delivery solution includes rHSA at a concentration of about 0.25 w / w%, or about 0.3 w / w%, or about 0.35 w / w% or more. In some embodiments, the deliverysolution includes rHSA at a concentration of about 6.50 w / w% to about 6.8 w / w%, about 6.4 w / w% to about 6.9 w / w%, about 6.3 w / w% to about 7.0 w / w%, about 6.1 w / w% to about 7.2 w / w%, about 5.9 w / w% to about 7.4 w / w%, or about 5.7 w / w% to about 7.6 w / w%. In some embodiments, the delivery solution includes rHSA at a concentration of about 5.5 w / w%, about 5.0 w / w%, or about 4.5 w / w% or less. In some embodiments, the delivery solution includes rHSA at a concentration of about 8.0 w / w%, or about 8.5 w / w%, or about 9.0 w / w% or more.

[0190] In some embodiments, the one or more stabilizing agents include Dextran. The delivery solution can include Dextran in any amount suitable to achieve the desired effect (e.g., treatment of Parkinson’s disease). In some embodiments, the delivery solution includes Dextran at a concentration of about 17.27 w / w% to about 17.30 w / w%, about 17.26 w / w% to about 17.31 w / w%, about 17.24 w / w% to about 17.33 w / w%, about 17.2 w / w% to about 17.35 w / w%, about 17.1 w / w% to about 17.4 w / w%, or about 17.0 w / w% to about 17.5 w / w%. In some embodiments, the delivery solution includes Dextran at a concentration of about 16.5 w / w%, about 16.0 w / w%, or about 15.0 w / w% or less. In some embodiments, the delivery solution includes Dextran at a concentration of about 18.0 w / w%, about 18.5 w / w%, or about 19.0 w / w% or more. In some embodiments, the delivery solution includes Dextran at a concentration of about 13.02 w / w% to about 13.05 w / w%, about 13.0 w / w% to about 13.1 w / w%, about 12.9 w / w% to about 13.2 w / w%, about 12.7 w / w% to about 13.4 w / w%, about 12.5 w / w% to about 13.5 w / w%, or about 12.2 w / w% to about 13.8 w / w%. In some embodiments, the delivery solution includes Dextran at a concentration of about 12.0 w / w%, about 11.5 w / w%, or about 11.0 w / w% or less. In some embodiments, the delivery solution includes Dextran at a concentration of about 14.0 w / w%, about 14.5 w / w%, or about 15.0 w / w% or more.

[0191] In some embodiments, the one or more stabilizing agents include Poloxamer. The delivery solution can include Poloxamer in any amount suitable to achieve the desired effect (e.g., treatment of Parkinson’s disease). In some embodiments, the delivery solution includes Poloxamer at a concentration of about 0.07 w / w% to about 0.09 w / w%, about 0.06 w / w% to about 0.1 w / w%, about 0.05 w / w% to about 0.11 w / w%, about 0.04 w / w% to about 0.12 w / w%, about 0.03 w / w% to about 0.13 w / w%, about 0.02 w / w% to about 0.15 w / w%, or about 0.01 w / w% to about 0.17 w / w%. In some embodiments, the delivery solution includes Poloxamer at a concentration of about 0.005 w / w% or less, about 0.2 w / w%, or about 0.25 w / w% or more.

[0192] In certain embodiments, the therapeutic composition includes a population of dopaminergic cells (e.g., a population of dopaminergic cells described herein), a delivery solution, and a cryoprotectant. Exemplary cryoprotectants include, but are not limited to, dimethylsulfoxide (DMSO), glycerol, polyethylene glycol, sucrose, trehalose, dextrose, or combinations thereof.

[0193] In certain embodiments, the therapeutic composition includes a population of dopaminergic cells (e.g., a population of dopaminergic cells described herein), a delivery solution, and a biocompatible scaffold or matrix. In some embodiments, the biocompatible scaffold or matrix includes one or more of extracellular matrix material, synthetic polymers, cytokines, collagen, polypeptides or proteins, polysaccharides including fibronectin, laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparin sulfate, chondroitin sulfate, agarose or gelatin, and hydrogel.

[0194] In some embodiments, there are several components that are not included in the delivery solution and can be excluded wholly or below detectable limits. Some examples of components that can be excluded are: certain components of animal origin; certain stabilizing agents such as human serum albumin (HSA); certain salts such as zinc sulfate, sodium bicarbonate, and ferric nitrate; certain pH indicators such as Phenol Red; certain sources of energy such as sodium pyruvate; certain amino acids such as Glycine, L-Alanine, L- Arginine hydrochloride, L-Asparagine-H2O, L-Glutamine, L-Cysteine, L-Histidine hydrochloride- H2O, L-Isoleucine, L-Leucine, L-Lysine hydrochloride, L-Methionine, L-Phenylalanine, L- Proline, L- Serine, L-Threonine, L-Tryptophan, L-Tyrosine, and L- Valine; and certain vitamins such as Ascorbic Acid, Choline Chloride, D-Calcium pantothenate, Folic Acid, Niacinamide, Pyridoxal hydrochloride, Riboflavin, Thiamine hydrochloride, Vitamin B 12 and i-Inositol.

[0195] In some embodiments, the cell delivery solution includes an energy source component, such as, D-Glucose (Dextrose), one or more pH buffers, such as, HEPES, a pol oxamer, such as, Pol oxamer 188, a dextran, such as, dextran 40, and recombinant albumin, in combination with one or more salts. In some embodiments, the one or more salts are selected from calcium chloride, magnesium chloride, potassium chloride, sodium chloride, and sodium phosphate monobasic. In some embodiments, the one or more salts include calcium chloride, magnesium chloride, potassium chloride, sodium chloride, and sodium phosphate monobasic. For example, in one embodiment, the cell delivery solution comprises D-Glucose (Dextrose) at a concentration of about 24 mM, Poloxamer 188 at a concentration of about 0.08% w / w, dextran 40 at a concentration of about 17% w / w, HEPES at aconcentration of about 10 mM, recombinant human serum albumin at a concentration of about 0.08% w / w, calcium chloride at a concentration of about 2 mM, magnesium chloride at a concentration of about 0.8 mM, potassium chloride at a concentration of about 5 mM, sodium chloride at a concentration of about 83 mM, and sodium phosphate monobasic at a concentration of about 0.89 mM.

[0196] In some embodiments, the therapeutic composition includes a population of dopaminergic cells (e.g., a population of dopaminergic cells described herein), and a delivery solution. In some embodiments, the delivery solution comprises a transplantation media. Transplantation media refers to a chemically defined, research-grade mixture of Neurobasal Medium, L-Glutamine, human serum albumin, and L- Ascorbic Acid. In some embodiments, the L-Glutamine is at a concentration of about 2 mM. In some embodiments, the L- Glutamine is at a concentration of about 200 mM. In some embodiments, the human serum albumin at a concentration of about 0.1% w / w. In some embodiments, the L- Ascorbic Acid at a concentration of about 200 pM. In another embodiment, the cell delivery solution comprises Neurobasal Medium, L-Glutamine at a concentration of about 2 mM, human serum albumin at a concentration of about 0.1% w / w, and L- Ascorbic Acid at a concentration of about 200 pM. In yet another embodiment, the cell delivery solution comprises Neurobasal Medium, L-Glutamine at a concentration of about 200 mM, human serum albumin at a concentration of about 0.1% w / w, and L-Ascorbic Acid at a concentration of about 200 pM.Preparation for Administration

[0197] In some embodiments, the therapeutic composition (e.g., a therapeutic composition described herein) is prepared for administration to a subject having Parkinson’s disease.

[0198] In some embodiments, preparation of the therapeutic composition (e.g., a therapeutic composition described herein) for administration includes preparing a cell suspension. In some embodiments, the cell suspension includes an effective quantity of a population of dopaminergic cells (e.g., a population of dopaminergic cells described herein) in suspension in a delivery solution. In addition to the population of dopaminergic cells and the delivery solution, the cell suspension can also include additional components such as, for example, a cell wash buffer. Exemplary cell wash buffers include, but are not limited to, phosphate buffer saline (PBS), compositions comprising 4-(2 -hydroxy ethyl)- 1- piperazineethanesulfonic acid (HEPES), Bio-Plex Pro™ Cell Signaling Wash Buffer, andCultrex™ 3-D Cell Wash Buffer. The cell suspension can also contain trace amounts of cryoprotectant and / or cell wash solution that were not fully removed after a suspension step.

[0199] In some embodiments, preparing the cell suspension includes washing the cells before resuspension of the cells in the delivery solution. Washing the cells can remove unwanted components from the suspension, such as components used for culturing or cryopreserving the population of cells that are not suitable for administration. The cells can be washed with the delivery solution or with a wash buffer.

[0200] The delivery solution and / or the wash buffer can contain no, minimal or trace amounts of cryoprotectant and / or cell wash solution, which the cells were stored and / or washed in prior to contact with the delivery solution during resuspension, that were not fully removed from a vessel containing the cell after a supernatant discard process, which can occur after an optional centrifuge step of the container, which can optionally form a cell pellet or concentrated cell solution. This supernatant discard process reduces the concentration and / or removes components of a stored sample that are not desired for injection, such as the cryoprotectant. The cell delivery solution can be used to reconstitute the cell solution following thawing of cells prior to administration for clinical use.

[0201] During preparation, the therapeutic composition can be formulated to constitute various qualities, such as a particular pH, osmolarity or density. In some embodiments, the therapeutic composition is formulated to have a pH level of about 5.5 to about 9.0, or a pH level of about 6.0 to about 8.0, or a pH level of about 6.4 to about 7.8, or a pH level of about 6.8 to about 7.6, or a pH level of about 7.0 to about 7.5, or a pH level of about 7.2 to about 7.4. In some embodiments, the therapeutic composition is formulated to have an osmolarity of about 100 to about 700 mOsm / L, an osmolarity of about 150 to about 500 mOsm / L, an osmolarity of about 200 to about 500 mOsm / L, an osmolarity of about 225 to about 400 mOsm / L, an osmolarity of about 250 to about 350 mOsm / L, an osmolarity of about 270 to about 325 mOsm / L, or an osmolarity of about 280 to about 300 mOsm / L. In some embodiments, the therapeutic composition is formulated to have a density of about 1.00 to about 1.30 g / mL, a density of about 1.02 to about 1.20 g / mL, a density of about 1.04 to about 1.15, a density of about 1.05 to about 1.11 g / mL, g / mL, a density of about 1.07 to about 1.09 g / mL, or a density of about 1.08 g / mL. In some embodiments, the therapeutic composition is formulated to have a relatively low viscosity. In some embodiments, the therapeutic composition is formulated to have a good cell compatibility such that the delivery solution are substantially not cytotoxic. The therapeutic composition can also be configured to have asufficient shelf life at typical or standard storage conditions, making it ready to use for clinical applications.

[0202] These qualities can influence the ability of the delivery solution to maintain a population of dopaminergic cells (e.g., a population of dopaminergic cells) in suspension in the therapeutic composition. The population of dopaminergic cells dispersed within a liquid can remain dispersed for 0 to 104 hours or more. The shelf-life of the population of dopaminergic cells dispersed or suspended within a liquid in accordance with the present disclosure is up to about 104 hours. More specifically, in this disclosure the liquid can be the delivery solution that can maintain the population of dopaminergic cells dispersed within it either without agitation (mixing) and / or homogenization or after agitation (mixing) and / or homogenization, for up to about 15 minutes, up to about 30 minutes, up to about 45 minutes, up to about 1 hour, up to about 90 minutes, up to about 2 hours, up to about 4 hours, up to about 6 hours, up to about 8 hours, up to about 12 hours, up to about 16 hours, up to about 20 hours, up to about 24 hours, up to about 30 hours, up to about 36 hours, up to about 42 hours, up to about 48 hours, up to about 56 hours, up to about 64 hours, up to about 72 hours, up to about 80 hours, up to about 88 hours, up to about 96 hours, up to about 104 hours, or more.

[0203] The delivery solution can also be warmed or cooled to any appropriate temperature (e.g., about room temperature, or about 37°C, or about 4°C, or about 0°C, or about 2°C to about 8°C, or about 1°C to about 10°C, or about 0°C to about 12°C) before contact with any cells while preparing the therapeutic composition.

[0204] The therapeutic composition can be prepared for any suitable mode of administration. For instance, the therapeutic composition can be prepared for surgical implantation or injection, e.g., local injection into the brain.

[0205] In some embodiments, the therapeutic composition comprises an effective quantity of a population of dopaminergic cells for treating Parkinson’s disease and a cell delivery solution. In some embodiments, the population of dopaminergic cells are included in the delivery solution is at a concentration of about 71,000 cells / pL to about 123000 cells / pL, which is, advantageously, optimized to reduce variability in the quantity of dopaminergic cells administered to a subject when the quantity of the population of dopaminergic cells is administered with a cell delivery device as described, for example, in WO2021 / 211518, which is incorporated by reference.

[0206] In some embodiments, preparing the therapeutic composition for administration further includes loading the therapeutic composition into a dose delivery device, such as a syringe or any other device able to deliver a solution to a subject. After loading thetherapeutic composition into the delivery device, the therapeutic composition can then be administered (e.g., injected) into a subject.IV. Method of Treatment

[0207] Certain aspects of the disclosure provide methods of treating Parkinson’s disease in a subject. In some embodiments, the method includes administering an effective quantity of a population of dopaminergic cells e.g., a population of dopaminergic cells described herein), or a therapeutic composition that includes an effective quantity of the population of dopaminergic cells.

[0208] In some embodiments, the population of dopaminergic cells includes dopaminergic neurons or a dopaminergic progenitor cell. In some embodiments, the dopaminergic neurons include one or more of a midbrain dopaminergic neuron, a floor-plate- derived dopaminergic neuron, and an authentic midbrain dopaminergic neuron. In some embodiments, the dopaminergic cells comprise midbrain dopaminergic neurons or progenitors thereof. In some embodiments, the midbrain dopaminergic neurons, or progenitors thereof, are derived from midbrain floor plate progenitor cells, whereby the midbrain floor plate progenitor cells are derived from stem cells in vitro.

[0209] In some embodiments, the method includes administering an effective quantity of a population of dopaminergic cells that are positive for FOXA2. In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of the population of dopaminergic cells is positive for FOXA2. In some embodiments, at least 80% of the population of dopaminergic cells is positive for FOXA2. In some embodiments, at least 90% of the population of dopaminergic cells is positive for FOXA2. In some embodiments, at least 95% of the population of dopaminergic cells is positive for FOXA2. In some embodiments, at least 98% of the population of dopaminergic cells is positive for FOXA2. In some embodiments, at least 99% of the population of dopaminergic cells is positive for FOXA2.

[0210] In some embodiments, the method includes administering an effective quantity of a population of dopaminergic cells that are positive for TH. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the population of dopaminergic cells is positive for TH. In some embodiments, about 10% of the population of dopaminergic cells is positive for TH. In some embodiments, about 15% of the populationof dopaminergic cells is positive for TH. In some embodiments, about 17% of the population of dopaminergic cells is positive for TH.

[0211] In some embodiments, the method includes administering an effective quantity of a population of dopaminergic cells where only a percentage of the population of dopaminergic cells are positive for PAX2. In some embodiments, less than about 20%, less than about 15%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the population of dopaminergic cells is positive for PAX2. In some embodiments, less than about 10% of the population of dopaminergic cells is positive for PAX2. In some embodiments, less than about 5% of the population of dopaminergic cells is positive for PAX2. In some embodiments, less than about 4% of the population of dopaminergic cells is positive for PAX2. In some embodiments, less than about 3% of the population of dopaminergic cells is positive for PAX2. In some embodiments, less than about 2% of the population of dopaminergic cells is positive for PAX2. In some embodiments, less than about 1% of the population of dopaminergic cells is positive for PAX2. In some embodiments, the population of dopaminergic cells is negative for PAX2.

[0212] In some embodiments, the method includes administering an effective quantity of a population of dopaminergic cells where only a percentage of the population of dopaminergic cells are positive for CRABP1. In some embodiments, less than about 20%, less than about 15%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the population of dopaminergic cells is positive for CRABP1. In some embodiments, less than about 10% of the population of dopaminergic cells is positive for CRABP1. In some embodiments, less than about 5% of the population of dopaminergic cells is positive for CRABP1. In some embodiments, less than about 4% of the population of dopaminergic cells is positive for CRABP1. In some embodiments, less than about 3% of the population of dopaminergic cells is positive for CRABP1. In some embodiments, less than about 2% of the population of dopaminergic cells is positive for CRABP1. In some embodiments, less than about 1% of the population of dopaminergic cells is positive for CRABP1. In some embodiments, the population of dopaminergic cells is negative for CRABP1.

[0213] In some embodiments, the method includes administering an effective quantity of a population of dopaminergic cells where only a percentage of the population of dopaminergic cells are positive for Ki67. In some embodiments, less than about 50%, lessthan about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 18%, less than about 15%, less than about 12%, less than about 10%, less than about 8%, or less than about 5% of the population of dopaminergic cells is positive for Ki67. In some embodiments, less than about 30% of the population of dopaminergic cells is positive for Ki67. In some embodiments, less than about 20% of the population of dopaminergic cells is positive for Ki67. In some embodiments, less than about 15% of the population of dopaminergic cells is positive for Ki67. In some embodiments, less than about 12% of the population of dopaminergic cells is positive for Ki67. In some embodiments, less than about 10% of the population of dopaminergic cells is positive for Ki67. In some embodiments, less than about 5% of the population of dopaminergic cells is positive for Ki67. In some embodiments, the population of dopaminergic cells is negative for Ki67.

[0214] In some embodiments, the method includes administering an effective quantity of a population of dopaminergic cells that includes viable dopaminergic cells. In some embodiments, about 10% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 50%, or about 50% to about 100% of the dopaminergic cells in the population of dopaminergic cells are viable. In some embodiments, about 50% to about 100% of the dopaminergic cells in the population of dopaminergic cells are viable. In some embodiments, about 60% to about 90% of the dopaminergic cells in the population of dopaminergic cells are viable. In some embodiments, about 70% to about 80% of the dopaminergic cells in the population of dopaminergic cells are viable. In some embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or more of the dopaminergic cells in the population of dopaminergic cells are viable. In some embodiments, at least about 90% of the dopaminergic cells in the population of dopaminergic cells are viable. In some embodiments, at least about 80% of the dopaminergic cells in the population of dopaminergic cells are viable. In some embodiments, at least about 70% of the dopaminergic cells in the population of dopaminergic cells are viable. In some embodiments, at least about 50% of the dopaminergic cells in the population of dopaminergic cells are viable.

[0215] In some embodiments, the method includes administering an effective quantity of a population of dopaminergic cells that have a capacity for producing dopamine. In some embodiments, the population of dopaminergic cells have the capacity for producing dopamine at a rate of at least about 25 ng / day / mL, about 20 ng / day / mL, about 15 ng / day / mL,about 12 ng / day / mL, about 10 ng / day / mL, about 8 ng / day / mL, about 6 ng / day / mL, about 4 ng / day / mL, or about 2 ng / day / mL. In some embodiments, the population of dopaminergic cells have the capacity for producing dopamine at a rate of at least about 20 ng / day / mL. In some embodiments, the population of dopaminergic cells have the capacity for producing dopamine at a rate of at least about 15 ng / day / mL. In some embodiments, the population of dopaminergic cells have the capacity for producing dopamine at a rate of at least about 11.2 ng / day / mL. In some embodiments, the population of dopaminergic cells have the capacity for producing dopamine at a rate of at least about 8 ng / day / mL.

[0216] In some embodiments, the population of dopaminergic cells have a capacity for producing dopamine that, when evaluated in vitro using liquid chromatography -tandem mass spectrometry (LC-MS / MS), provides an area under the concentration-time curve (AUC) that is at least about 25 ng / day / mL, about 20 ng / day / mL, about 15 ng / day / mL, about 12 ng / day / mL, about 10 ng / day / mL, about 8 ng / day / mL, about 6 ng / day / mL, about 4 ng / day / mL, or about 2 ng / day / mL. In some embodiments, the population of dopaminergic cells have a capacity for producing dopamine that, when evaluated in vitro using liquid chromatographytandem mass spectrometry (LC-MS / MS), provides an area under the concentration-time curve (AUC) that is at least about 20 ng / day / mL. In some embodiments, the population of dopaminergic cells have a capacity for producing dopamine that, when evaluated in vitro using liquid chromatography-tandem mass spectrometry (LC-MS / MS), provides an area under the concentration-time curve (AUC) that is at least about 15 ng / day / mL. In some embodiments, the population of dopaminergic cells have a capacity for producing dopamine that, when evaluated in vitro using liquid chromatography-tandem mass spectrometry (LC- MS / MS), provides an area under the concentration-time curve (AUC) that is at least about 11.2 ng / day / mL. In some embodiments, the population of dopaminergic cells have a capacity for producing dopamine that, when evaluated in vitro using liquid chromatography -tandem mass spectrometry (LC-MS / MS), provides an area under the concentration-time curve (AUC) that is at least about 8 ng / day / mL.

[0217] The number of dopaminergic cells in the effective quantity of the population of dopaminergic cells administered in the methods described herein can be any suitable and efficacious number. In some embodiments, the effective quantity of the population of dopaminergic cells includes about 1 x 10A4 to about 1 x 10A10 dopaminergic cells, about 1 x 10A4 to about 1 x 10A5 dopaminergic cells, about 1 x 10A5 to about 1 x 10A9 dopaminergic cells, about 1 x 10A5 to about 1 x 10A6 dopaminergic cells, about 1 x 10A5 to about 1 x 10A7 dopaminergic cells, about 1 x 10A6 to about 1 x 10A7 dopaminergic cells, about 1 x 10A6 toabout 1 x 10A8 dopaminergic cells, about 1 x 10A7 to about 1 x 10A8, from about 1 x 10A8 to about 1 x 10A9 dopaminergic cells, about 1 x 10A8 to about 1 x 10Al 0 dopaminergic cells, or about 1 x 10A9 to about 1 x 10Al 0 dopaminergic cells. In some embodiments, the effective quantity of the population of dopaminergic cells includes at least 0.25 million dopaminergic cells, at least 0.5 million dopaminergic cells, at least 0.75 million dopaminergic cells, at least 1 million dopaminergic cells, at least 1.25 million dopaminergic cells, at least 1.5 million dopaminergic cells, at least 1.75 million dopaminergic cells, at least 2 million dopaminergic cells, at least 2.25 million dopaminergic cells, at least 2.5 million dopaminergic cells, at least 2.75 million dopaminergic cells, at least 3 million dopaminergic, at least 3.25 million dopaminergic cells, at least 3.5 million dopaminergic cells, at least 3.75 million dopaminergic cells, at least 4 million dopaminergic cells, at least 4.5 million dopaminergic cells, at least 5 million dopaminergic cells, at least 5.5 million dopaminergic cells, at least 6 million dopaminergic cells, at least 7 million dopaminergic, at least 8 million dopaminergic cells, at least 9 million dopaminergic cells, or at least 10 million or more dopaminergic cells.

[0218] In some embodiments, the effective quantity of the population of dopaminergic cells includes at least about 1.0 xlOA6 to about 1.2 xlOA7 dopaminergic cells. In some embodiments, the effective quantity of the population of dopaminergic cells includes at least about 0.9 million dopaminergic cells. In some embodiments, the therapeutic composition includes a population of dopaminergic cells that includes at least about 1.8 million dopaminergic cells. In some embodiments, the effective quantity of the population of dopaminergic cells includes at least about 2.7 million dopaminergic cells. In some embodiments, the effective quantity of the population of dopaminergic cells includes at least about 5.4 million dopaminergic cells.

[0219] In some embodiments, administering the effective quantity of the population of dopaminergic cells includes delivering the population of dopaminergic cells to the posterior putamen of the subject. In some embodiments, administering the effective quantity of the population of dopaminergic cells includes delivering the population of dopaminergic cells to the post commissural putamen of the subject. The putamen is a round structure located at the based on the forebrain, and together with the caudate nucleus, it forms the dorsal striatum. The putamen primarily regulates movement at various stages. The putamen can also be subdivided into the left putamen and the right putamen, based on the location of the putamen in the left and right hemispheres of the brain. In some embodiments, a first portion of the effective quantity of the population of dopaminergic cells is delivered to the left hemisphere of the subject’s posterior putamen and a second portion of the effective quantity of thepopulation of dopaminergic cells is delivered to the right hemisphere of the subject’s posterior putamen. In some embodiments, the first portion is about half of the effective quantity of the population of dopaminergic cells and the second portion is about half of the effective quantity of the population of dopaminergic cells.

[0220] In some embodiments, the effective quantity of the population of dopaminergic cells is delivered to the subject in a therapeutic composition (e.g., a therapeutic composition described herein). In some embodiments, the therapeutic composition includes a population of dopaminergic cells at a concentration of about 71,000 cells / pL to about 123000 cells / pL, about 75,000 cells / pL to about 115,000 cells / pL, about 80,000 cells / pL to about 110,000 cells / pL, about 85,000 cells / pL to about 105,000 cells / pL, or about 90,000 cells / pL to about 100,000 cells / pL. In some embodiments, the population of dopaminergic cells are at a concentration of about 71,000 cells / pL to about 123000 cells / pL. In some embodiments, the population of dopaminergic cells are at a concentration of about 75,000 cells / pL, about 80,000 cells / pL, about 85,000 cells / pL, about 90,000 cells / pL, about 95,000 cells / pL, about 100,000 cells / pL, about 105,000 cells / pL, about 110,000 cells / pL, about 115,000 cells / pL, or about 120,000 cells / pL. In some embodiments, the population of dopaminergic cells are at a concentration of about 75,000 cells / pL, about 80,000 cells / pL, about 85,000 cells / pL, about 90,000 cells / pL, about 95,000 cells / pL, about 100,000 cells / pL, about 105,000 cells / pL, about 110,000 cells / pL, about 115,000 cells / pL, or about 120,000 cells / pL. In some embodiments, the population of dopaminergic cells are at a concentration of about 90,000 cells / pL. In some embodiments, the population of dopaminergic cells are at a concentration of about 100,000 cells / pL. In some embodiments, the population of dopaminergic cells are at a concentration of about 110,000 cells / pL. In some embodiments, the population of dopaminergic cells is allogenic to the subject. In some embodiments, the population of dopaminergic cells is autologous to a subject.

[0221] In some embodiments, the population of dopaminergic cells are derived from stem cells that were differentiated into dopaminergic cells in vitro (e.g., by a method described herein).

[0222] In some embodiments, the Parkinson’s disease is early Parkinson’s disease. In some embodiments, the Parkinson’s disease is advanced Parkinson’s disease. In some embodiments, the subject is a human.Immunosuppressive Regimen

[0223] In some embodiments, the method for treating a disease, such as Parkinson’s disease, in a subject using a cell therapy, such as those described herein, further includes administering an immunosuppressive regimen to the subject. In some embodiments, the immunosuppressive regimen is administered prior to, during, after administration of the effective quantity of the population of therapeutic cells (e.g., dopaminergic cells), or a combination thereof. In some embodiments, the immunosuppressive regime includes basiliximab, methylprednisolone, and tacrolimus.

[0224] In some embodiments, the basiliximab is administered at about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, or about 50 mg. In some embodiments, the basiliximab is administered at about 10 mg. In some embodiments, the basiliximab is administered at about 20 mg. In some embodiments, the basiliximab is administered at about 30 mg. In some embodiments, the basiliximab is administered intravenously intraoperatively and post-operative. In some embodiments, the basiliximab is administered at about 1 day, at about 2 days, at about 3 days, at about 4 days, at about 5 days, at about 6 days, at about 7 days, at about 8 days, at about 9 days, at about 10 days, at about 11 days, at about 12 days, at about 13 days, or at about 14 days after the administering of the population of therapeutic cells (e.g., dopaminergic cells). In some embodiments, the basiliximab is administered at about 2 days after the administering of the population of therapeutic cells (e.g., dopaminergic cells). In some embodiments, the basiliximab is administered at about 4 days after the administering of the population of therapeutic cells (e.g., dopaminergic cells). In some embodiments, the basiliximab is administered at about 6 days after the administering of the population of therapeutic cells (e.g., dopaminergic cells).

[0225] In some embodiments, the methylprednisolone is administered at about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 50 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 900 mg, or about 1000 mg. In some embodiments, the methylprednisolone is administered at about 300 mg. In some embodiments, the methylprednisolone is administered at about 500 mg. In some embodiments, the methylprednisolone is administered at about 700 mg. In some embodiments, the methylprednisolone is administered prior to administering the populationof therapeutic cells (e.g., dopaminergic cells). In some embodiments, the methylprednisolone is further administered following the administering of the population of therapeutic cells (e.g., dopaminergic cells). In some embodiments, the methylprednisolone is further administered weekly following the administering of the population of therapeutic cells (e.g., dopaminergic cells). In some embodiments, the methylprednisolone is further administered daily following the administering of the population of therapeutic cells (e.g., dopaminergic cells). In some embodiments, the methylprednisolone is further administered at about 5 mg daily following the administering of the population of therapeutic cells (e.g., dopaminergic cells).

[0226] In some embodiments, the tacrolimus is administered after the administering of the population of therapeutic cells (e.g., dopaminergic cells). In some embodiments, the tacrolimus is administered at about 1 day, at about 2 days, at about 3 days, at about 4 days, at about 5 days, at about 6 days, at about 7 days, at about 8 days, at about 9 days, at about 10 days, at about 11 days, at about 12 days, at about 13 days, or at about 14 days after the administering of the population of therapeutic cells (e.g., dopaminergic cells). In some embodiments, the tacrolimus is administered at about 1 day after the administering of the population of therapeutic cells (e.g., dopaminergic cells). In some embodiments, the tacrolimus is administered at about 2 days after the administering of the population of therapeutic cells (e.g., dopaminergic cells). In some embodiments, the tacrolimus is administered at about 3 days after the administering of the population of therapeutic cells (e.g., dopaminergic cells).

[0227] In a particular embodiment, the immunosuppressive regimen includes administering the basiliximab at about 20 mg intravenously intraoperatively and postoperative at about 4 days after the administering of the population of therapeutic cells (e.g., dopaminergic cells), administering the methylprednisolone at about 500 mg intravenously prior to administering the population of therapeutic cells (e.g., dopaminergic cells), and administering the tacrolimus at about one day after the administering of the population of therapeutic cells (e.g., dopaminergic cells). In some embodiments, the methylprednisolone is further administered at about 5 mg daily following the administering of the population of therapeutic cells (e.g., dopaminergic cells)Methods of Administration

[0228] Any suitable method of administration can be used to administer the population of dopaminergic cells and / or the immunosuppressive regimen in accordance with the methodsdescribed herein. For instance, the population of dopaminergic cells and / or the immunosuppressive regimen can be administered by injection (e.g., intravenous injection) or surgical implantation. In some embodiments, the population of dopaminergic cells and / or the immunosuppressive regimen is administered systemically. In some embodiments, the population of dopaminergic cells and / or the immunosuppressive regimen is administered locally. In some embodiments, administering the population of dopaminergic cells and / or the immunosuppressive regimen is performed by delivering the population of dopaminergic cells and / or the immunosuppressive regimen to the posterior putamen of the subject.

[0229] In some embodiments, administering includes delivering the population of dopaminergic cells to the subject with a stereotactic-guided delivery system. An exemplary stereotactic-guided delivery system is described in, for example, the published PCT application, WO 2021 / 211518 Al, which is incorporated herein by reference.

[0230] In some embodiments, the stereotactic-guided delivery system is configured to reduce backflow of a therapeutic composition that includes the population of dopaminergic cells (e.g., a therapeutic composition described herein) out of a target site (e.g., a putamen) during injection of the therapeutic composition into the target site. In some embodiments, the stereotactic-guided delivery system includes a needed, a plunger and a stereotactic frame. In some embodiments, the needle may be arranged to retract while the plunger advances. The needle may create a cavity in the tissue for the therapeutic substance. As the therapeutic composition is ejected from the needle, the needle can be retracted, thereby providing a volume of space in the created cavity for the therapeutic substance to inhabit.

[0231] In some embodiments, the stereotactic-guided delivery system is configured to help reduce cell settling or particle settling within the needle lumen. In some embodiments, the diameter of the needle lumen is less than 1 mm. In some embodiments, the ratio of the needle lumen diameter to the cell or particle diameter is less than 100: 1.

[0232] In some embodiments, the stereotactic-guided delivery system is configured to help a user to control the ejection rate of the therapeutic substance. With some therapeutic substances, such as certain types of cells (e.g., dopaminergic cells), a slower ejection rate may help to reduce shear or other harmful effects on the cells, which may result in a higher viability of dopaminergic cells delivered. A slower ejection rate may also reduce the risk of brain tissue trauma. In some embodiments, the device actuator is a rotary actuator. In some embodiments, multiple complete turns of the rotary actuator are needed to deliver a total target volume.

[0233] In some embodiments, the stereotactic-guided delivery system is configured to improve dose assurance. In some embodiments, the therapeutic composition is contained in a needle lumen that is relatively small and has a constant diameter. In the case of a population of dopaminergic cells, such an arrangement may help the cells to move in tandem with its fluid solution, which may help to ensure delivery of a larger portion of the cells. In some embodiments, such an arrangement may help to reduce cell settling.

[0234] In some embodiments, the stereotactic-guided delivery system is configured to help reduce waste of the therapeutic substance that may arise during loading of the substance into the delivery device. In some embodiments, the delivery device is configured to be front- loaded with the therapeutic substance. In some embodiments, the stereotactic-guided delivery system may include an air vent arrangement to permit front-loading. In some embodiments, the stereotactic-guided delivery system may include an arrangement for priming the system after the therapeutic substance has been loaded to remove air from the stereotactic-guided delivery system prior to use, to prevent injection of air into the target site.

[0235] In some embodiments, the stereotactic-guided delivery system includes an indicator comprising only mechanical components. Such an arrangement may permit the stereotactic-guided delivery system to be more portable and easier to sterilize due to a lack of electrical components.Assessment of Treatment and Disease Progression

[0236] In some embodiments, administering the effective quantity of the population of cells results in an improvement in a motor function or a non-motor function, or a combination thereof, of the subject. In some embodiments, administering the effective quantity of the population of cells results in an improvement in a motor function or a non-motor function, or a combination thereof, of the subject as compared to a control or a baseline of the subject prior to the administering. Exemplary detects in motor function that can be improved by administration of the effective quantity of the population of dopaminergic cells include, but are not limited to, tremor, bradykinesia or slowness of movement, rigidity or stiffness of the limbs, trunk and postural instability, and impaired balance and coordination. Exemplary detects in non-motor function that can be improved by administration of the effective quantity of the population of dopaminergic cells include, but are not limited to, autonomic dysfunction, neuropsychiatric problems, and sensory and sleep difficulties. In some embodiments, the improvement is in at least one motor function and at least one non-motor function of the subject.

[0237] Any suitable method for assessing motor and non-motor function in subjects having Parkinson’s disease can be used to assess improvement in response to administration of the effective quantity of the population of dopaminergic cells. Exemplary methods of assessing motor or non-motor function of subjects having Parkinson’s disease include, without limitation, single-photon emission computerized tomography (SPECT), magnetic resonance imaging (MRI), positron emission tomography (PET), computerized tomography (CT), dopamine transporter (DAT) scan, ultrasound, biomarker testing, the Movement Disorder Society-Sponsored Revision of the Unified Parkinson’s Disease Rating Scale (MDS UPDRS), the Total Unified Parkinson’s Disease Rating Scale (Total UPDRS), the Anticipatory Care Plan, the Clinical algorithm: Assessment of mild cognitive impairment in Parkinson’s (PD-MCI), the Epworth Sleepiness Scale (ESS), the Hospital Anxiety and Depression Scale (HADS), the Lindop Parkinson’s Physiotherapy Assessment Scale, the Modified Bradykinesia Rating Scale (MBRS), the Montreal Cognitive Assessment (MOCA), the Non motor symptoms questionnaire, the “Nil by Mouth” medication dose calculator and guideline, the Parkinson’s Disease Driving Questionnaire, the Parkinson’s Disease Fatigue Scale Parkinson’s Disease Questionnaire (PDQ-39), the Parkinson’s Disease Sleep Scale (PDSS), the Unified Dyskinesia Rating Scale (UDysRS), the Unified Dystonia Rating Scale (UDRS), the Unified Parkinson's Disease Rating Scale (UPDRS), the Parkinson’s Disease Non-Motor Symptom Scale (PD NMSS), the Neuropsychiatric Inventory Questionnaire (NPI-Q), the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS), and the Frontal Systems Behavior Scale (FrSBe).

[0238] In some embodiments, the improvement in motor function is determined based, at least in part, on a change in the subject’s Movement Disorder Society-Sponsored Revision of the Unified Parkinson's Disease Rating Scale (MDS UPDRS) Part II Score as compared to the control or the baseline of the subject prior to the administering. In some embodiments, the improvement in motor function is determined based, at least in part, on a change in the subject’s Movement Disorder Society-Sponsored Revision of the Unified Parkinson's Disease Rating Scale (MDS UPDRS) Part III Score as compared to the control or the baseline of the subject prior to the administering, for example, as described in Sanchez-Ferro, 2018, Minimal Clinically Important Difference for UPDRS-III in Daily Practice, Mov Disord Clin Pract; Jul- Aug; 5(4): 448-450, which is incorporated by reference. In some embodiments, the improvement in motor function is determined based, at least in part, on a change in an ON score and / or OFF score of the subject as compared to the control or the baseline of the subject prior to the administering, for example, as described in Hauser, 2014, Minimal clinicallyimportant difference in Parkinson’s disease as assessed in pivotal trials of pramipexole extended release, Parkinsons Dis; 2014:467131, which is incorporated by reference. In some embodiments, the improvement in motor function is determined based, at least in part, on a change in an ON score of the subject as compared to the control or the baseline of the subject prior to the administering. In some embodiments, the improvement in motor function is determined based, at least in part, on a change in an OFF score of the subject as compared to the control or the baseline of the subject prior to the administering.

[0239] In some embodiments, the improvement in motor function is determined based, at least in part, on a change in the subject’s Unified Dyskinesia Rating Scale (UDysRS) Objective Subscores as compared to the control or the baseline of the subject prior to the administering. This method provides an objective measure of dyskinesia. This method further provides a quantifiable measure of the motor complications associated with the disease and its treatments, which can be used to track the progression of the disease, the effects of medication, or the impact of therapeutic interventions.

[0240] In some embodiments, the improvement in non-motor function is measured by one or more assays selected from the group consisting of a sleep-quality assessment, a cognitive assessment, a neuropsychological test, a mood evaluation, an autonomic function test, an imaging test, and other standardized non-motor function evaluation.

[0241] In some embodiments, the improvement in non-motor function is determined, at least in part, on a change in the subject’s Parkinson’s Disease Non-Motor Symptom Scale (PD NMSS) score as compared to the control or the baseline of the subject prior to the administering. In some embodiments, the improvement in non-motor function is determined, at least in part, on a change in the subject’s PD NMSS score as described in, for example, Ray Chaudhuri K, Rojo JM, Schapira AHV, et al. PLoS One. 2013;8(2):e57221, which is incorporated by reference. The PD NMSS is scored across various domains on a range of 0 to 360, with lower scores indicating improvement. In some embodiments, the change in the subject’s PD NMSS is a change in the score of one or more PD NMSS domains. In some embodiments, the change in the subject’s PD NMSS is a change in the total PD NMSS score.

[0242] In some embodiments, the improvement in non-motor function is determined, at least in part, on a change in the subject’s 39-item Parkinson’s Disease Questionnaire (PDQ- 39) score as compared to the control or the baseline of the subject prior to the administering. In some embodiments, the improvement in non-motor function is determined, at least in part, on a change in the subject’s PDQ-39 score as described in, for example, Balestrino R, Hurtado-Gonzalez CA, Stocchi F, et al. NPJ Parkinsons Dis. 2019;5:26, which isincorporated by reference. The PDQ-39 is scored across various domains on a range of 0 to 100, with lower scores indicating improvement. In some embodiments, the change in the subject’s PDQ-39 is a change in the score of one or more PDQ-39 domains. In some embodiments, the change in the subject’s PDQ-39 is a change in the total PDQ-39 score.

[0243] In some embodiments, the improvement in non-motor function is determined, at least in part, on a change in the subject’s Neuropsychiatric Inventory Questionnaire (NPI-Q) score as compared to the control or the baseline of the subject prior to the administering. In some embodiments, the improvement in non-motor function is determined, at least in part, on a change in the subject’s NPI-Q score as described in, for example, Cummings JL. The Neuropsychiatric Inventory Questionnaire: Background and Administration. 1994. (Accessible at alz.org / media / documents / npiq-questionnaire.pdf). The NPI-Q is scored on a range of 0 to 96, with lower scores indicating improvement.

[0244] In some embodiments, the improvement in non-motor function is determined, at least in part, on a change in the subject’s Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) score as compared to the control or the baseline of the subject prior to the administering. In some embodiments, the improvement in non-motor function is determined, at least in part, on a change in the subject’s RBANS score as described in, for example, Yang C, Garrett-Mayer E, Schneider JS, Gollomp SM, Tilley BC. Mov Disord. 2009;24(10): 1453-60, which is incorporated by reference. The RBANS is scored across various domains on a range of 40 to 60, with higher scores indicating improvement. In some embodiments, the change in the subject’s RBANS is a change in the score of one or more RBANS domains. In some embodiments, the change in the subject’s RBANS is a change in the total RBANS score.

[0245] In some embodiments, the improvement in non-motor function is determined, at least in part, on a change in the subject’s Frontal Systems Behavior Scale (FrSBe) score as compared to the control or the baseline of the subject prior to the administering. In some embodiments, the improvement in non-motor function is determined, at least in part, on a change in the subject’s FrSBe score as described in, for example, Cabrera S, Edelstein K, Mason WP, Tartaglia MC. Neurooncol Pract. 2016;3(2): 113-9, which is incorporated by reference. The RBANS is scored across various domains, with lower scores indicating improvement. In some embodiments, the change in the subject’s FrSBe is a change in the score of one or more FrSBe domains. In some embodiments, the change in the subject’s FrSBe is a change in the total FrSBe score.

[0246] In some embodiments, the baseline is a measurement of the subject’s motor function and non-motor function before administering of the effective quantity of the population of dopaminergic cells.

[0247] In some embodiments, the improvement is indicative of remission of progression of Parkinson’s disease in the subject. In some embodiments, the improvement is indicative of a reversal in progression of Parkinson’s disease in the subject.

[0248] In some embodiments, the improvement is detectable after a certain period following administration of the population of dopaminergic cells. In some embodiments, the improvement is detectable at about 1 weeks, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 15 weeks, about 18 weeks, about 21 weeks, about 24 weeks, about 27 weeks, about 30 weeks, about 33 weeks, about 36 weeks, about 39 weeks, about 42 weeks, about 45 weeks, about 48 weeks, or about 52 weeks or more following the administering of the population of dopaminergic cells. In some embodiments, the improvement is detectable at about 4 weeks following the administering of the population of dopaminergic cells. In some embodiments, the improvement is detectable at about 12 weeks following the administering of the population of dopaminergic cells. In some embodiments, the improvement is detectable at about 24 weeks following the administering of the population of dopaminergic cells.

[0249] In some embodiments, the improvement persists for a duration of at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 1 year, at least about 1.5 years (18 months), at least about 2 years (24 months), at least about 2.5 years (30 months), or at least about 3 years (36 months) or more. In some embodiments, the improvement persists for a duration of at least about 6 months. In some embodiments, the improvement persists for a duration of at least about 1 year. In some embodiments, the improvement persists for a duration of at least about 1.5 years. In some embodiments, the improvement persists for a duration of at least about 2 years. In some embodiments, the improvement persists after removal of the immunosuppressive regimen. In some embodiments, the improvement persists for at least 6 months year after removal of the immunosuppressive regimen. In some embodiments, the improvement persists for at least 1 year after removal of the immunosuppressive regimen.V. Vessels

[0250] Certain aspects of the disclosure provide for vessels comprising a population of dopaminergic cells, or a composition comprising a population of dopaminergic cells described herein. The vessel can be a storage vessel or vessel used during administration of the population of dopaminergic cells or the composition. In some embodiments, the vessel includes a cryovial. In some embodiments, the vessel includes an aseptic technology (AT) vial. Other non-limiting examples of vessels which may be used include syringes, fine glass tubes, stereotactic needles and cannulas.VI. Kits

[0251] Certain aspects of the disclosure provide for kits for treating Parkinson’s disease comprising a population of dopaminergic cells, or a composition comprising a population of dopaminergic cells suitable for administration to a subject, including any of the populations of dopaminergic cells and compositions described herein.

[0252] In some embodiments, the kit includes instructional material for the use of said composition. In some embodiments, the instructional material includes instructions of preparing the composition or the population of dopaminergic cells for administration into a subject. Such instructions can include, but are not limited to, instructions for preparing or storing the population of dopaminergic cells or composition, diluting the population of dopaminergic cells, adding additional additives to the treatment, or combining the composition with an additional therapeutic agent. In some embodiments, the instructional material includes instructions for administering the population of dopaminergic cells or the composition to a subject.

[0253] In some embodiments, the kit further includes an applicator for administering a population of dopaminergic cells or a composition comprising a population of dopaminergic cells described herein. The applicator can be any device suitable for administration of the population of dopaminergic cells or composition described herein to a subject, including, but not limited to, a hypodermic syringe, a needle, a balloon-dilating catheter, a pipette, and the like. In some embodiments, the applicator is a stereotactic-guided delivery system. The applicator can also be a single-use or multiple-use administration device, and can be included in the kit as a pre-filled delivery system with, e.g., a therapeutic composition comprising the population of dopaminergic cells.EXAMPLES

[0254] The Example below provides clinical data supporting an effective cell therapy approach for treating Parkinson’s disease (PD). Specifically, this Example provides comprehensive findings from an extensive Phase 1 study conducted across various centers and sites to treat PD by administering specific doses of dopaminergic neurons. This study was aimed at evaluating the safety, tolerability, and efficacy of the dopaminergic neurons in PD treatment.Example 1: Phase 1 Clinical Trial Study Design and Results Demonstrating the Safety, Tolerability, and Efficacy of Administering Dopaminergic Neurons in Human Subjects with Parkinson's DiseasePhase 1 clinical trial study design:

[0255] Participants diagnosed with PD were sequentially recruited into a Phase 1 study (NCT04802733). The open-label, non-randomized study was conducted to assess safety, tolerability, and initial efficacy of specific doses of dopaminergic neurons for PD treatment.

[0256] FIG. 1 is a schematic of the Phase 1 clinical trial study design. The study was conducted on 12 participants with PD. The first five participants were enrolled into a low- dose group, receiving a treatment comprising ~0.9 million dopaminergic neurons administered to the posterior putamen of each hemisphere (-1.8 million dopaminergic neurons in total, per participant) referred to herein as “Cohort A”. Subsequent participants were enrolled into a high-dose group, receiving a treatment comprising -2.7 million dopaminergic neurons administered to the posterior putamen of each hemisphere (-5.4 million dopaminergic in total, per participant) referred to herein as “Cohort B”. The enrolled participants were between the ages of 50 and 78 years old (Canada) and 60 to 78 years old (US) with PD-related motor symptoms inadequately relieved by standard treatment. Baseline evaluations were conducted days before the dopaminergic neurons were administered.Dopaminergic neurons prepared for administration:

[0257] The participants were administered cell solutions containing dopaminergic cells (e.g., midbrain dopaminergic neuronal precursor cells), which were generated, in vitro, from human pluripotent stem cells using a floor plate-based differentiation strategy as described herein. The dopaminergic neurons were generated and frozen in cryovials for storage and transportation to clinical facilities.

[0258] Frozen cryovials of dopaminergic neurons were prepared for delivery by thawing the frozen vials of dopaminergic neurons and combining the dopaminergic neurons with a cell delivery solution. The dopaminergic neurons were combined with the cell delivery solution such that the final concentration of dopaminergic neurons in the cell delivery solution was 1 x 10A8 ± 10% (cells / mL). After preparing the cell delivery solution, cell identity / purity markers from the dopaminergic neurons were assessed by flow cytometry. The flow cytometry data (not show) demonstrated that approximately 91.1% (2.9% standard deviation, “SD”) of the dopaminergic neurons were FOXA2 positive; approximately 1.8% (1.3% SD) of the dopaminergic neurons were PAX6 positive; approximately 2.2% (1.8% SD) of the dopaminergic neurons were CRABP1 positive; approximately 11.8% (2.7% SD) of the dopaminergic neurons were Ki67 positive. The expression of TH was evaluated separately, i.e., from thawed cell cultures of the dopaminergic neurons. Flow cytometry data showed that approximately 17.3% (2.6% SD) of the dopaminergic neurons were TH positive.

[0259] In addition, cell viability and cell concentration were assessed with an automated cell counter using a staining solution containing Acridine Orange and Propidium Iodide (AO / PI) to identify dead cells. Following preparation of cells for delivery, approximately 77.2% (6.5% SD) of the dopaminergic neurons were viable.

[0260] The release of dopamine from stem-cell derived dopaminergic neurons was previously evaluated on cell culture supernatants using liquid chromatography -tandem mass spectrometry (LC-MS / MS). This analysis revealed an area under the curve (AUC) that is greater than or equal to 11.2 ng x day / mL for the release of dopamine.

[0261] The dopaminergic neurons were administered surgically, and the participants were put on an immunosuppressive regimen as described below. Participants were evaluated periodically. At twelve months post-administration, safety, tolerability, and initial efficacy of the dopaminergic neurons was evaluated. Additional evaluations were planned for 15, 18, 21 and 24 months post-administration.

[0262] The immunosuppressive regimen was initiated intraoperatively and continued post-operatively for 1 year after administration of the dopaminergic neurons. In particular, participants were initiated on an immunosuppressive regimen of basiliximab 20 mg intravenously intraoperatively and post-operative on Day 4; methylprednisolone 500 mg intravenously prior to surgery, methylprednisolone 250 mg the day after surgery, then tapered to oral prednisone and continued at 5 mg daily for 1 year; and tacrolimus taken orally beginning on the day after surgery (Day 1) and then adjusted to a target trough blood level of 4-7 ng / mL for a period of 1 year.Surgical approach

[0263] FIG. 2 shows an exemplary surgical approach for administering effective quantities of dopaminergic neurons to subjects in need thereof. Prepared solutions of dopaminergic neurons (at a concentration of 1 x 10A8 ± 10% cells / mL) were administered in a single session of stereotactically guided surgical injection into the posterior putamen 203 of both the left and right hemispheres through a separate burr hole 205 for each side. The solutions of dopaminergic neurons were administered through each burr hole 205 along three tracks 207, or passes, using a cannula. Three deposits of the cell delivery solution were deposited per track 207, for a total of nine cell deposits in each putamen 203. For Cohort A, each deposit comprised about 1-1.5 pL of cell solution for a total of about 3.5 pL per track in order to provide about 0.9 million dopaminergic neurons to the posterior putamen 203 of each hemisphere. For Cohort B, each deposit comprised about 3 4 pL of cell delivery solution for a total of about 11 pL at provide about 2.7 million dopaminergic neurons to the posterior putamen 203 of each hemisphere.Safety and tolerability analysis

[0264] One objective of the clinical study was to assess safety and tolerability of administering dopaminergic neurons. The assessment included measuring the incidence of serious adverse events (SAEs) at 1-year after administering doses of dopaminergic neurons.

[0265] Safety of dopaminergic neurons was defined based on the following criteria for Cohort A and Cohort B: Two or fewer participants in each cohort developing two or more serious adverse events (SAEs) related to surgery, the presence of transplanted cells, or immunosuppression; two or fewer participants in each cohort developing a tumor or abnormal tissue overgrowth attributed to the presence of the transplanted cells; two or fewer participants in each cohort developing an intracerebral hemorrhage considered to be lifethreatening; and one or zero deaths occurring within the participants of either Cohort A or Cohort B.

[0266] In addition, the following secondary endpoints were assessed at Year 1 : changes from baseline in striatal fluorodopa (18F-DOPA) uptake using positron emission tomography (PET); changes from baseline in the International Parkinson and Movement Disorder Society (MDS)-Unified Parkinson’s Disease Rating Scale (UPDRS) motor sub-score in the OFF medication state; changes from baseline in the number of hours in the ON state withouttroublesome dyskinesia, OFF state and ON state with troublesome dyskinesia. And, feasibility, as defined by intra-operative delivery of at least 50% of the intended number of deposits per brain in >50% of subjects.

[0267] The study planned to assess changes from baseline at two years post-transplant in the following areas: the International Parkinson and Movement Disorder Society (MDS)- Unified Parkinson’s Disease Rating Scale (UPDRS) motor sub-score in the OFF medication state; the number of hours in the ON state without troublesome dyskinesia; the incidence of Serious Adverse Events (SAEs) at two years post-transplant, and the incidence and type of Adverse Events (AEs) at one and two years post-transplant. Feasibility within the study was defined by the successful intraoperative delivery of at least 50% of the intended total number of deposits per brain in more than 50% of the participants.Exploratory non-motor outcomes

[0268] Another objective of the clinical study was to assess the impact of administering dopaminergic neurons up to 18 months post-transplantation (6 months post discontinuation of the immunosuppressive regimen).

[0269] Exploratory non-motor outcomes are reported as mean scores, which were measured at baseline and 18 months post-transplantation on the Parkinson’s Disease NonMotor Symptom Scale (PD NMSS), and 39-item Parkinson’s Disease Questionnaire (PDQ- 39).

[0270] A formal neuropsychological evaluation, which included the Neuropsychiatric Inventory Questionnaire (NPI-Q), Repeatable Battery for the Assessment of Neuropsychological Status (RBANS), and Frontal System Behavior Scale (FrSBe), was performed at baseline and at 12 months post-transplantation.Results of safety and tolerability analysis

[0271] Patient disposition: Patient baseline characteristics were comparable across both cohorts (see, Table 1) All participants received their planned dosage. All participants (N=12) were included in the safety and evaluable populations.Table 1. Baseline characteristics.Safety

[0272] The study met its safety objective. There was no reports of an AE or SAE related to the dopaminergic neurons (see Table 2). There was 1 SAE of transient seizure attributed to the surgical procedure reported in Cohort B.Table 2. Summary of treatment-emergent serious adverse events (TESAEs) at Year 1.

[0273] Of the twelve subjects, eleven reported TEAEs (66 events in total; Table 3). MostTEAEs were mild to moderate in severity. Only one severe TEAE was reported. There were no discontinuations or deaths in the study. There were no graft-induced dyskinesias seen in either cohort. A summary of TEAEs is provided in Table 3.Table 3. Summary of treatment-emergent adverse events (TEAEs).Engraftment survival, and function of administered dopaminergic neurons

[0274] Engraftment, survival, and function of transplanted dopaminergic neurons was assessed by 18F-DOPA uptake. In the majority of subjects, there was an observed increase in 18F-DOPA uptake in the posterior putamen (transplant site) (FIGS. 3A and 3B). Furthermore, a decrease in 18F-DOPA was observed in the caudate nucleus (distant from the site of transplantation), which can be attributed to the progression of PD. Taken together, these data provide evidence of engraftment, survival, and cell function of dopaminergic neurons administered for the treatment of PD.

[0275] FIGS. 3A and 3B show exemplary data of a comparative analysis of 18F-DOPA PET Data in subjects treated with dopaminergic neurons. The comparative analysis is an analysis of 18F-DOPAPET Data in subjects as compared to their corresponding baselines. FIG. 3A shows a voxel-based analysis of the 18F-DOPAPET data that was conducted. The image displays voxel clusters with low p-value (P < 0.05) group level changes between baseline and 1 year (voxels with increase in orange, voxels with decrease in blue). FIG. 3B is a box plot showing change in striatal-to-occipital ratio (SOR) from baseline limited to low p- value voxel clusters. Changes within the significant voxel clusters for the caudate and putamen are combined for each subject using volume weighted means. Lines represent the median change for the cohort, circles represent the mean, the box represents the lower and upper quartiles and the whiskers represent the extremes. Taken together, these data demonstrate that dopaminergic neurons, administered to subjects for the treatment of PD, successfully engraft and survive 52 weeks post transplantation.Administered doses of dopaminergic neurons improved motor and non-motor function in subjects with PD

[0276] The following section provides clinical data showing the efficacy of high and low doses of dopaminergic neurons for improving motor and non-motor function in subjects with PD.

[0277] Movement Disorder Society Unified Parkinson’s Disease Rating Scale (MDS- UPDRS) Part III OFF were assessed to evaluate changes in subject motor function. The “OFF” state refers to the time when effects of medication have worn off, and symptoms return, thus offering an evaluation of the patient’s motor symptoms in the absence of medication. Accordingly, a change in the MDS-UPDRS Part III score (OFF medication) reflects a change in motor function in a subject with Parkinson's Disease.

[0278] Additionally, patient-reported via Hauser / PD Diary ON and OFF times were evaluated to assess the effectiveness of the cell therapy for improving motor and non-motor function. The ON time refers to the periods when the subject’s symptoms were well- controlled, and the medication was effective. During these times, motor symptoms such as tremors, stiffness, or bradykinesia were reduced. The OFF time refers to the periods when the medication was not working effectively, and the symptoms return. OFF times can be characterized by the re-emergence of motor symptoms. By reporting patient-reported ON and OFF times, this study provided insight into how treatment with high or low doses of dopaminergic neurons controlled symptoms of PD. Accordingly, the assessment of ON and OFF times provided an assessment on severity and types of motor symptoms as well as fluctuations throughout the day.

[0279] MDS-UPDRS Part III OFF, along with patient-reported ON and OFF times, were recorded for subjects treated in Cohort A and Cohort B and compared against baseline measurements, z.e., measurements obtained from the corresponding subjects prior to treatment with dopaminergic neurons (see FIGS. 4A-4D and Table 4). A reduction in MDS- UPDRS Part III score (OFF) was observed in PD patients following treatment with dopaminergic neurons (FIG. 4A), indicating an improvement in motor function which was more pronounced in the high dose group, and suggesting a reversal of PD-related effects on motor function. Additionally, an increase in patient-reported ON time following treatment was also observed (FIG. 4B), indicating an ability of dopaminergic neurons to reverse unwanted PD symptoms and sustain this effect for at least 52 weeks post-treatment. Furthermore, patient-reported OFF time was diminished in comparison to corresponding baseline scores after the therapy (FIG. 4C), indicating the potential of dopaminergic neurons to reverse symptoms of PD for a duration of at least 52 weeks post-treatment. Patient- reported ON time with troublesome dyskinesia also remained low to negligible (FIG. 4D), further affirming the safety and tolerability of dopaminergic neurons in PD treatment.

[0280] Collectively, these data demonstrate the remarkable capacity of effective quantities of dopaminergic neurons may decelerate the advancement of PD symptoms, andmay also reverse some of its manifestations on motor and non-motor function. These data further demonstrate the remarkable effects were maintained with sustained efficacy for a period extending up to 52 weeks, emphasizing the treatment’s potential for both symptom reversal and long-term efficacy. Furthermore, a comparative analysis between the two cohorts underlined that the enhanced outcomes in clinical assessments were more pronounced in Cohort B (high dose) in contrast to Cohort A (low dose), accentuating the potential dosedependent efficiency of the therapeutic strategy detailed herein.Table 4. Change in clinical outcomes at Year 1 from baseline.Administered doses of dopaminergic neurons also improved in exploratory non-motor function outcomes in subjects with PD

[0281] Mean total PD NMSS scores were found to increase in Cohort A (low dose) from baseline at 18 months post-transplantation of the dopaminergic cells (Table 5), indicating worsening, while the PD NMSS scores remained stable for Cohort B (high dose) (Table 5). On the PDQ-39, summary index scores were stable in the Cohort A (low dose) and decreased in the Cohort B (high dose) (Table 6), indicating improvement in the latter group.Table 5. Change in Parkinson’s Disease Non-Motor Symptom Scale (or PD NMSS) score at 18 months from baseline.Table 6. Change in 39-item Parkinson’s Disease Questionnaire (or PDQ-39), score at 18 months from baseline.

[0282] In the formal neuropsychological evaluation, the mean NPI-Q scores were stable between baseline and 12 months in the Cohort A (low dose) and decreased over the same period in the Cohort B (high dose) (FIG. 5), indicating improvement. Mean scores on most RBANS domains were stable at 12 months for Cohort A (low dose) (FIG. 6A). However, Cohort B (high dose) showed increases on the immediate memory and delayed memory RBANS domains, indicating improvement in these domains, while also showing a moderate decrease on the attention RBANS domain, indicating worsening in this domain (FIG. 6B). Moreover, increases in the apathy and executive domains of the FrSBe were observed posttransplantation in Cohort A (low dose), indicating worsening in these domains (FIG. 7A). In Cohort B (high dose), lower scores, indicating improvement, were observed across all FrSBe domains (FIG. 7B).

[0283] Taken together, this Example provides Phase 1 clinical trial data demonstrating the safety and tolerability of certain doses of dopaminergic neurons at 1-year post-transplantation for PD treatment. The feasibility of stereotactic surgical delivery and a therapeutic regimen was further demonstrated, with all participants receiving the planned dose of neurons andenduring the 1-year immunosuppression regimen without significant issues. Additionally, 18F-DOPAPET imaging provided evidence of survival, and engraftment, of the transplanted neurons in the posterior putamen. Clinical analysis revealed tangible improvements, notably a reduction in the MDS-UPDRS Part III (OFF) score, and a decrease in OFF time and an increase in ON time without troublesome dyskinesia, with no occurrence of graft-induced dyskinesias through the first year. While efficacy trends were discernible across both cohorts, Cohort B (high dose) exhibited a greater magnitude of changes, reinforcing the potential of this innovative therapeutic approach in offering a dose-dependent, treatment for PD.

[0284] These results also demonstrate that administration of certain doses of dopaminergic cells led to stability or improvement in exploratory non-motor, quality of life, and psychiatric outcomes. While improvements in neuropsychological measures were observed for both cohorts, Cohort B (high dose) showed greater trends towards improvement on the NPI-Q, RBANS, and FrSBe at 12 months, suggesting positive outcomes in neuropsychiatric symptoms, cognitive functioning, and frontal lobe-related behaviors. Cohort B also demonstrated stability of PD NMSS and PDQ-39 scores at 18 months (6 months post discontinuation of immunosuppression), suggesting controlled non-motor symptom severity and health-related quality of life. The results from the exploratory non-motor outcomes further support the potential of this dopaminergic cell transplantation therapeutic approach in offering a dose-dependent, treatment for PD.Administered doses of dopaminergic neurons led to observed, sustained improvements in exploratory motor function outcomes in subjects with PD

[0285] To evaluate improvement in motor function, MDS-UPDRS Part II scores were evaluated. In particular, MDS-UPDRS Part II scores were evaluated for subjects treated with dopaminergic neurons and compared against measurements obtained from the corresponding subjects prior to treatment with low (Cohort A) and high (Cohort B) doses of dopaminergic neurons. A reduction in mean total MDS-UPDRS Part II score was observed in PD patients from Cohort B following treatment with dopaminergic neurons (FIG. 8). This reduction was observed at 18 months post treatment, demonstrating a sustained improvement and reversal of disease progression.

[0286] MDS-UPDRS Part III OFF scores were also evaluated up to 18 months post treatment. The MDS-UPDRS Part III OFF score were recorded for subjects treated in Cohort A and Cohort B and compared against baseline measurements, i.e., measurements obtainedfrom the corresponding subjects prior to treatment with dopaminergic neurons (FIG. 9). A reduction in MDS-UPDRS Part III score (OFF) was observed in PD patients following treatment with dopaminergic neurons, indicating an improvement in motor function which was more pronounced in the high dose group (Cohort B), suggesting a reversal of PD-related effects on motor function. The reduction in MDS-UPDRS Part III score (OFF) was observed at 18 months post treatment, and 6 months after the removal of immunosuppression.

[0287] Additionally, Hauser / PD Diary ON and OFF times were recorded for the subjects treated in Cohort A and Cohort B. Subjects demonstrated a marked increase in Good ON Times when compared to their respective baseline scores (FIG. 10A), which were recorded immediately prior to the initiation of the cell therapy treatment. These improvements were sustained over an extended period, with the therapeutic effects evident up to 18 months posttreatment. Concurrently, there was a notable reduction in the Hauser Diary Off Times (FIG. 10B), indicating a decrease in periods of diminished motor function commonly associated with PD. These results underscore the long-term efficacy and potential of certain doses of dopaminergic neurons as a treatment for PD.

[0288] In addition, the clinical efficacy of low and high (Cohort A and Cohort B, respectively) doses of dopaminergic neurons were evaluated using the UDysRS Objective Subscores. These scores were reported and compared with baseline scores established prior to treatment. For subjects that received a low or high dose of dopaminergic neurons a reduction in their UDysRS Objective Subscores was observed (FIG. 11). This reduction indicates an improvement in dyskinesia symptoms, demonstrating the potential of dopaminergic neuron transplantation for treating motor symptoms in PD patients.

[0289] These results demonstrate that administration of certain doses of dopaminergic cells led to stability or improvement in exploratory motor function outcomes up to 18 months post administration.Evidence of sustained survival and function of engrafted dopaminergic neurons 6 months after cessation of immunosuppression and 18 months post transplantation

[0290] To assess engraftment, survival, and function of transplanted dopaminergic neurons a combination of Positron Emission Tomography (PET) and Magnetic Resonance Imaging (MRI) imaging techniques were employed.

[0291] The survival of transplanted dopaminergic neurons was assessed at 12 and 18 months following transplantation. FIG. 12 and FIG. 13 outline an exemplary strategy for evaluating the engraftment, survival, and function of transplanted neurons.

[0292] MRI was used to determine the anatomical location and integrity of transplanted neurons. Concurrently, PET imaging, specifically through the measurement of 18F- fluorodopa (18F-DOPA) uptake, was used to provide a functional assessment of the dopaminergic activity and viability of the transplanted neurons. By employing voxel-based brain mapping analysis, this provides a precise quantification of 18F-DOPA uptake changes over time and enabled the identification of specific regions of interest where neuronal engraftment and survival were observed. This dual-imaging approach, leveraged strengths of MRI and PET to provide a robust and detailed assessment of the long-term survival and functional integration of the transplanted dopaminergic neurons. This approach is particularly useful for monitoring disease progression and therapeutic outcomes of a neurodegenerative disease, such as PD.

[0293] FIG. 14 shows exemplary imaging data that demonstrate engraftment, survival and function of administered dopaminergic neurons. The image represents a mean image of a plurality of images taken at 12 and 18 months post transplantation as compared to a baseline.

[0294] FIG. 15 shows exemplary imaging data of 18F-DOPA uptake at 12 and 18 months post transplantation. The data show that compared with baseline, group-level analysis (N=12) of 18F-DOPA uptake at 12 and 18 months post transplantation revealed clusters of increased 18F-DOPAPET signal within the striatal hypothesis testing space. Furthermore, the data show that 18F-DOPA uptake was stable or increased in the putamen at 18 months post transplantation (6 months post discontinuation of immunosuppression)

[0295] FIGS. 16A and 16B show exemplary clinical data of 18F-DOPA uptake in the putamen and caudate, respectively. In the putamen, increases in mean SOR-1 were observed in the largest clusters identified within the left and right putamen at 18 months post transplantation (n=ll) (FIG. 16A). These clusters are interpreted to be associated with administered dopaminergic neurons. At 18 months post transplantation, participants were off immunosuppression for 6 months. Decreases in mean SOR-1 were observed in the largest clusters identified within the left and right caudate at 18 months post transplantation (n=l 1) (FIG. 16B). Decreased 18F-DOPA uptake in the caudate is interpreted to indicate continued neurodegeneration associated with Parkinson’s disease pathology.

[0296] No evidence of intracerebral hemorrhage, mass, lesion, and / or cellular overgrowth Transplanted cells indiscernible on MRI Expected finding of bilateral tracks accompanied by surrounding gliosis (FIG. 17).

[0297] Taken together, these clinical results demonstrate successful neuron engraftment, sustained survival, and functionality up to six months following the cessation of immunosuppression therapy. Furthermore, these findings demonstrate the potential of a dualimaging approach for monitoring therapeutic outcomes in neurodegenerative diseases.Evidence of sustained survival and function of engrafted dopaminergic neurons 12 months after cessation of immunosuppression and 24 months post transplantation

[0298] To further assess the long-term safety, tolerability, and efficacy of dopaminergic neuron transplantation in subjects with Parkinson's disease, participants from both Cohort A (low dose, n=5) and Cohort B (high dose, n=7) were evaluated at 24 months posttransplantation. The results from this follow-up assessment provide clinical evidence of successful neuron engraftment, sustained survival, and functionality 12 months following the cessation of immunosuppression therapy. The following exemplary results provide a comprehensive analysis of motor function, dyskinesias, daily symptom fluctuations, and nonmotor symptoms at the 24-month timepoint. Specifically, changes in Movement Disorder Society-Sponsored Revision of the Unified Parkinson’s Disease Rating Scale (MDS-UPDRS) Part III and Part II scores were measured to evaluate motor function, Unified Dyskinesia Rating Scale (UDysRS) scores were measured to assess dyskinesias, PD diary scores were measured to capture daily symptom fluctuations, and Neuropsychiatric Inventory Questionnaire (NPI-Q) scores were measured to evaluate non-motor symptoms. In addition, treatment emergent adverse events were continuously reported and monitored.

[0299] FIGS. 18A and 18B show the progression of MDS-UPDRS Part III ON scores over 24 months for both low dose (n=5) and high dose (n=7) cohorts. The high dose cohort demonstrated a sustained reduction in scores as measured before transplantation (24.3) to 24 months (20.7), indicating a persistent improvement in motor function. The low dose cohort showed a more modest improvement.

[0300] FIG. 19 shows the changes in MDS-UPDRS Part II scores, which reflected improved motor experiences of daily living. Both cohorts showed improvements, with the high dose group demonstrating a more pronounced and sustained effect.

[0301] The impact on dyskinesias, a common side effect of long-term levodopa treatment, was assessed using the UDysRS Objective Subscores. FIGS. 20A-20B shows exemplary experimental results of UDysRS Subscores from 0 (before transplantation) to 24 months post-transplantation. Overall, UDysRS scores did not substantially worsen for either low dose or the high dose cohort.

[0302] FIGS. 21A and 21B show exemplary patient-reported outcomes via PD Diary scores. FIG. 21A shows an increase in Good ON Time for both cohorts, with the high dose group showing a more pronounced improvement. FIG. 21B demonstrates a reduction in OFF Time, particularly in the high dose group, decreasing from 5.0 hours before transplantation to 3.1 hours at 24 months post transplantation.

[0303] Non-motor symptoms were evaluated using the NPLQ Total Score, as shown in FIG. 22. Both cohorts showed improvements in neuropsychiatric symptoms, with the high dose group demonstrating a more substantial improvement, decreasing from 6.4 pretransplantation to 4.0 at 24 months.

[0304] Taken together, these results demonstrate that the therapeutic effects of transplanted dopaminergic neurons persist for at least 24 months post-transplantation, with the high dose cohort generally showing more pronounced and sustained improvements across both motor and non-motor symptoms. These findings suggest successful engraftment, survival, and continued functionality of the transplanted neurons, even 12 months after the cessation of immunosuppression therapy.EQUIVALENTS AND SCOPE, INCORPORATION BY REFERENCE

[0305] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. It is understood that modifications which do not substantially affect the activity of the various embodiments of this disclosure are also provided within the description of the disclosure provided herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims.

[0306] In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one,more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure also includes embodiments in which more than one, or all of the group members, are present in, employed in, or otherwise relevant to a given product or process.

[0307] Furthermore, it is to be understood that the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the claims or from relevant portions of the description is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

[0308] Where elements are presented as lists, e.g., in Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the disclosure, or aspects of the embodiments, is / are referred to as comprising particular elements, features, steps, etc., certain embodiments of the disclosure or aspects of the embodiments consist, or consist essentially of, such elements, features, steps, etc. Thus, for each embodiment of the disclosure that comprises one or more elements, features, steps, etc., the disclosure also provides embodiments that consist or consist essentially of those elements, features, steps, etc.

[0309] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and / or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and / or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of thesubrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

[0310] In addition, it is to be understood that any particular embodiment of the present disclosure may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and / or methods of the disclosure, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.

[0311] Throughout this disclosure various publications, patents, and sequence database entries are mentioned. The disclosures of these publications, patents, and sequence database entries, including those items listed above, are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0312] Although the disclosure has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the scope of the disclosure. Accordingly, the above examples are intended to illustrate but not limit the present disclosure.

Claims

CLAIMS1. A method for treating Parkinson’s disease in a subject comprising: administering an effective quantity of a population of dopaminergic cells to the subject, wherein the administering results in an improvement in a motor function or a nonmotor function, or a combination thereof, of the subject as compared to a control or a baseline of the subject prior to the administering.

2. The method of claim 1, wherein the effective quantity of the population of dopaminergic cells comprises between about 1.0xl0A6 to about 1.2xlOA7 dopaminergic cells.

3. The method of claim 1 or 2, wherein the effective quantity of the population of dopaminergic cells comprises about 1.8xlOA6 dopaminergic cells.

4. The method of any one of claims 1 to 3, wherein the effective quantity of the population of dopaminergic cells comprises about 5.4xlOA6 dopaminergic cells.

5. The method of any one of claims 1 to 4, wherein greater than about 90% of the dopaminergic cells in the population of dopaminergic cells administered to the subject are positive for FOXA2.

6. The method of any one of claims 1 to 5, wherein fewer than about 2% of the dopaminergic cells in the population of dopaminergic cells administered to the subject are positive for PAX6.

7. The method of any one of claims 1 to 6, wherein fewer than about 2% of the dopaminergic cells in the population of dopaminergic cells administered to the subject are positive for CRABP1.

8. The method of any one of claims 1 to 7, wherein fewer than about 12% of the dopaminergic cells in the population of dopaminergic cells administered to the subject are positive for Ki67.

9. The method of any one of claims 1 to 8, wherein between about 70% to about 80% of the dopaminergic cells in the population of dopaminergic cells administered to the subject are viable.

10. The method of any one of claims 1 to 9, wherein the population of dopaminergic cells comprises a capacity for producing dopamine that, when evaluated in vitro using liquid chromatography -tandem mass spectrometry (LC-MS / MS), provides an area under a concentration-time curve (AUC) that is equal to or greater than 11.2 ng x day / mL.

11. The method of any one of claims 1 to 10, wherein the administering comprises delivering the population of dopaminergic cells to the posterior putamen of the subject.

12. The method of any of claims 11, wherein the administering comprises delivering a first portion of the population of dopaminergic cells to the left hemisphere of subject’s posterior putamen and a second portion of the population of dopaminergic cells to the right hemisphere of the subject’s posterior putamen.

13. The method of claim 12, wherein the first portion is about half of the effective quantity of the population of dopaminergic cells and the second portion is about half of the effective quantity of the population of dopaminergic cells.

14. The method of any one of claims 1 to 13, wherein the dopaminergic cells are delivered to the subject in a therapeutic composition, and wherein the population of dopaminergic cells in the therapeutic composition is at a concentration of about 71,000 cells / pL to about 123,000 cells / pL.

15. The method of any one of claims 1 to 14, wherein the improvement is in at least one motor function and at least one non-motor function of the subject.

16. The method of any one of claims 1 to 15, wherein the improvement in motor function is determined based, at least in part, on a change in one or more of:the subject’s Movement Disorder Society-Sponsored Revision of the Unified Parkinson's Disease Rating Scale (MDS UPDRS) Part II or Part III Score as compared to the control or the baseline of the subject prior to the administering; and the subject’s Unified Dyskinesia Rating Scale (UDysRS) Objective Subscore as compared to the control or the baseline of the subject prior to the administering.

17. The method of any one of claims 1 to 16, wherein the improvement in motor function is determined based, at least in part, on a change in an ON score and / or OFF score of the subject as compared to the control or the baseline of the subject prior to the administering.

18. The method of any one of claims 1 to 17, wherein the improvement in non-motor function is measured by one or more assays selected from the group consisting of a sleepquality assessment, a cognitive assessment, a neuropsychological test, a mood evaluation, an autonomic function test, an imaging test, and other standardized non-motor function evaluation.

19. The method of any one of claims 1 to 18, wherein the improvement in non-motor function is determined, at least in part, on a change in the subject’s Parkinson’s Disease Non-Motor Symptom Scale (PD NMSS) score as compared to the control or the baseline of the subject prior to the administering.

20. The method of any one of claims 1 to 19, wherein the improvement in non-motor function is determined, at least in part, on a change in the subject’s 39-item Parkinson’s Disease Questionnaire (PDQ-39) score as compared to the control or the baseline of the subject prior to the administering.

21. The method of any one of claims 1 to 20, wherein the improvement in non-motor function is determined, at least in part, on a change in the subject’s Neuropsychiatric Inventory Questionnaire (NPI-Q) score as compared to the control or the baseline of the subject prior to the administering.

22. The method of any one of claims 1 to 21, wherein the improvement in non-motor function is determined, at least in part, on a change in the subject’s Repeatable Battery for theAssessment of Neuropsychological Status (RBANS) score as compared to the control or the baseline of the subject prior to the administering.

23. The method of any one of claims 1 to 22, wherein the improvement in non-motor function is determined, at least in part, on a change in the subject’s Frontal Systems Behavior Scale (FrSBe) score as compared to the control or the baseline of the subject prior to the administering.

24. The method of any one of claims 1 to 23, wherein the baseline is a measurement of the subject’s motor function and non-motor function before the administering.

25. The method of any one of claims 1 to 24, wherein the improvement is indicative of a reversal in progression of Parkinson’s disease.

24. The method of any one of claims 1 to 25, wherein the administering comprises delivering the population of dopaminergic cells to the subject with a stereotactic-guided delivery system.

27. The method of any one of claims 1 to 26, wherein the method further comprises administering an immunosuppressive regimen to the subject, wherein the immunosuppressive regimen comprises basiliximab, methylprednisolone, and tacrolimus.

28. The method of claim 27, wherein: the basiliximab is administered at about 20 mg intravenously intraoperatively and post-operative at about 4 days after the administering of the population of dopaminergic cells; the methylprednisolone is administered at about 500 mg intravenously prior to administering the population of dopaminergic cells; and the tacrolimus is administered at about one day after the administering of the population of dopaminergic cells.

29. The method of claim 28, wherein: the methylprednisolone is further administered at about 5 mg daily following the administering of the population of dopaminergic cells.

30. The method of any one of claims 1 to 29, wherein the improvement is detectable at about 12 weeks following the administering of the population of dopaminergic cells.

31. The method of any one of claims 1 to 30, wherein the improvement persists for a duration of at least one year.

32. The method of any one of claims 1 to 31, wherein the improvement persists for a duration of at least 1.5 years or at least 2 years.

33. The method of any one of claims 27 to 32, wherein the improvement persists after removal of the immunosuppressive regimen.

34. The method of any one of claims 1 to 33, wherein the population of dopaminergic cells are derived from pluripotent stem cells that were differentiated into dopaminergic cells in vitro.

35. The method of any one of claims 1 to 34, wherein the population of dopaminergic cells comprise midbrain dopaminergic neurons, or precursors thereof.

36. The method of claim 35, wherein the midbrain dopaminergic neurons, or precursors thereof, are derived from floor plate progenitor cells.

37. The method of any one of claims 1 to 36, wherein the Parkinson’s disease is advanced Parkinson’s disease.

38. A therapeutic composition comprising: an effective quantity of a population of dopaminergic cells for treating Parkinson’s disease; and a cell delivery solution.

39. The therapeutic composition of claim 38, wherein at least 90% of the dopaminergic cells in the population of dopaminergic cells are positive for FOXA2.

40. The therapeutic composition of claim 38 or 39, wherein fewer than about 2% of the dopaminergic cells in the population of dopaminergic cells are positive for PAX6.

41. The therapeutic composition of any one of claims 38 to 40, wherein fewer than about 2% of the dopaminergic cells in the population of dopaminergic cells are positive for CRABP1.

42. The therapeutic composition of any one of claims 38 to 41, wherein fewer than about 12% of the dopaminergic cells in the population of dopaminergic cells are positive for Ki67.

43. The therapeutic composition of any one of claims 38 to 42, wherein between about 70% to about 80% of the dopaminergic cells in the population of dopaminergic cells are viable.

44. The therapeutic composition of any one of claims 38 to 43, wherein the population of dopaminergic cells comprise a capacity for producing dopamine that, when evaluated in vitro using liquid chromatography-tandem mass spectrometry (LC-MS / MS), provides an area under a concentration-time curve (AUC) that is equal to or greater than 11.2 ng x day / mL.

45. The therapeutic composition of any one of claims 38 to 44, wherein the population of dopaminergic cells in the delivery solution is at a concentration of about 71,000 cells / pL to about 123000 cells / pL.

46. The therapeutic composition of any of claims 38 to 45, wherein the population of dopaminergic cells comprise midbrain dopaminergic neurons, or precursors thereof.

47. The therapeutic composition of claim 46, wherein the midbrain dopaminergic neurons, or precursors thereof, are derived from floor plate progenitor cells in vitro.

48. The therapeutic composition of claim 47, wherein the floor plate progenitor cells are derived from pluripotent stem cells in vitro.

49. The therapeutic composition of any one of claims 38 to 48, wherein the cell delivery solution comprises:(a) one or more energy source components;(b) one or more pH buffers;(c) one or more salts; and(d) one or more stabilizing agents, wherein the one or more stabilizing agents are selected from the group consisting of recombinant albumin (rHSA), Dextran, and Poloxamer.

50. The therapeutic composition of claim 49, wherein the one or more energy source components comprise a sugar.

51. The therapeutic composition of claim 50, wherein the sugar is dextrose.

52. The therapeutic composition of any one of claims 38 to 51, wherein the population of dopaminergic cells comprises at least 0.9 million dopaminergic cells.

53. The therapeutic composition of any one of claims 38 to 52, wherein the population of dopaminergic cells comprises at least 2.7 million dopaminergic cells.

54. The therapeutic composition of any one of claims 38 to 53, therein the population of dopaminergic cells comprises at least 5.4xlOA6 dopaminergic cells.

55. The therapeutic composition of any one of claims 38 to 54, wherein the Parkinson’s disease is advance Parkinson’s disease.

56. A vessel comprising the therapeutic composition of any one of claims 38 to 55.

57. The vessel of claim 56, wherein the vessel comprises a cryovial.

58. The vessel of claim 56 or 57, wherein the vessel comprises an aseptic technology (AT) vial.

59. Use of the therapeutic composition of any one of claims 38 to 55 in the treatment ofParkinson’s disease.

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