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

Abstract

The present disclosure relates to methods and compositions for treating Parkinson's disease, a neurodegenerative disorder characterized by loss of dopaminergic neurons. In particular, the present disclosure provides preparations of dopaminergic cells that have been demonstrated to have therapeutic effects on both motor and non-motor symptoms of the disease.

Description

Cross-referencing

[0001] This application claims the 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. Technical Field

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

[0003] Parkinson's disease is a chronic and progressive movement disorder characterized by both motor and nonmotor symptoms. Motor symptoms include tremor, rigidity, and bradykinesia, while nonmotor symptoms can involve cognitive decline, mood disorders, and autonomic dysfunction.

[0004] The causes of Parkinson's disease are not fully understood, but it is known to involve the degeneration of dopaminergic neurons, leading to decreased dopamine levels. Traditional treatments for Parkinson's disease primarily focus on controlling symptoms with pharmacological agents such as levodopa, aiming to increase dopamine levels or mimic its effects. While these treatments are beneficial, they often have limited efficacy, significant side effects, and do not halt disease progression.

[0005] Cellular therapy has recently emerged as a promising approach to treating Parkinson's disease, aiming to replace or regenerate lost dopaminergic neurons. However, previous attempts at these methods have encountered challenges, such as low efficiency of cellular integration, limited duration of action, and unpredictable patient outcomes. Furthermore, achieving not only halting the progression of Parkinson's disease but also realizing substantial improvements in motor and non-motor function remains a challenge. Therefore, a significant unmet need remains for therapeutic strategies that not only control symptoms but also reverse the progression of Parkinson's disease and provide sustained long-term benefits. Summary of the Invention

[0006] This disclosure relates to methods and compositions for treating Parkinson's disease. More specifically, this disclosure relates to methods and compositions using dopaminergic cells, demonstrating, using Phase 1 clinical data, that these methods and compositions using dopaminergic cells have a sustained therapeutic effect on subjects with Parkinson's disease. Unlike existing methods, the methods and compositions described herein not only provide temporary relief of Parkinson's disease symptoms but also induce significant and sustained improvements in motor and non-motor function, with clinical data demonstrating a durable effect lasting at least 52 weeks without diminishing.

[0007] In one aspect, this article provides a method for treating a subject with Parkinson's disease, the method comprising: administering to the subject an effective amount of dopaminergic cell populations. In some embodiments, the administration induces an improvement in the subject's motor function or non-motor function, or a combination thereof, compared to a control or the subject's baseline prior to administration.

[0008] In some embodiments, the effective amount of dopaminergic cell population comprises about 1.0 × 10^6 to about 1.2 × 10^7 dopaminergic cells. In some embodiments, the effective amount of dopaminergic cell population comprises about 1.8 × 10^6 dopaminergic cells. In some embodiments, the effective amount of dopaminergic cell population comprises about 5.4 × 10^6 dopaminergic cells.

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

[0010] In some implementations, approximately 70% to 80% of the dopaminergic cells in the dopaminergic cell population administered to the subject are viable.

[0011] In some implementations, the dopaminergic cell population has the ability to produce dopamine, and when evaluated in vitro using liquid chromatography-tandem mass spectrometry (LC-MS / MS), the ability to produce dopamine provides an area under the concentration-time curve (AUC) equal to or greater than 11.2 ng × day / mL.

[0012] In some embodiments, administration includes delivering a population of dopaminergic neurons to the posterior putamen of the subject. In some embodiments, administration includes delivering a first portion of a population of dopaminergic cells to the left hemisphere of the posterior putamen of the subject and a second portion of the population of dopaminergic cells to the right hemisphere of the posterior putamen of the subject. In some embodiments, the first portion is approximately half the effective amount of the population of dopaminergic cells, and the second portion is approximately half the effective amount of the population of dopaminergic cells.

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

[0014] In some implementations, improvements are reflected in at least one motor function and at least one non-motor function of the subject. In some implementations, improvements in motor function are determined at least in part based on changes in the subject's Part II score of the Unified Motor Disorders Association-sponsored Revised Unified Parkinson's Disease Rating Scale (MDS UPDRS) compared to the baseline of the control or subject before administration. In some implementations, improvements in motor function are determined at least in part based on changes in the subject's Part III score of the Unified Motor Disorders Association-sponsored Revised Unified Parkinson's Disease Rating Scale (MDS UPDRS) compared to the baseline of the control or subject before administration. In some implementations, improvements in motor function are determined at least in part based on changes in the subject's objective section scores of the Unified Motor Disorders Rating Scale (UDysRS) compared to the baseline of the control or subject before administration. In some implementations, improvements in motor function are determined at least in part based on changes in the subject's ON and / or OFF scores compared to the baseline of the control or subject before administration. In some implementations, improvements in nonmotor function are measured by one or more assays selected from a group consisting of: sleep quality assessments, cognitive assessments, neuropsychological tests, mood assessments, autonomic function tests, imaging tests, and other standardized nonmotor function assessments. In some implementations, the baseline is the subject's measurements of motor and nonmotor function prior to administration. In some implementations, the improvement indicates a reversal of Parkinson's disease progression. In some implementations, improvements in nonmotor function are determined at least in part based on changes in the subject's Parkinson's Disease Nonmotor Symptom Scale (PD NMSS) score compared to the baseline of the control or subject prior to administration. In some implementations, improvements in nonmotor function are determined at least in part based on changes in the subject's 39-item Parkinson's Disease Questionnaire (PDQ-39) score compared to the baseline of the control or subject prior to administration. In some implementations, improvements in nonmotor function are determined at least in part based on changes in the subject's Neuropsychiatric Survey Questionnaire (NPI-Q) score compared to the baseline of the control or subject prior to administration. In some implementations, improvements in nonmotor function are determined at least in part based on changes in the subject's Repeatable Bundle of Neuropsychological Status Assessment (RBANS) score compared to a control or subject's baseline before administration. In some implementations, improvements in nonmotor function are determined at least in part based on changes in the subject's Frontal System Behavior Scale (FrSBe) score compared to a control or subject's baseline before administration.

[0015] In some implementations, administration includes delivering a population of dopaminergic cells to the subject using a stereotactic-guided delivery system.

[0016] In some embodiments, the method further includes administering an immunosuppressive regimen to the subject, wherein the immunosuppressive regimen comprises baliximab, methylprednisolone, and tacrolimus. In some embodiments, approximately 4 days after administration of the dopaminergic cell population, baliximab is administered intravenously at approximately 20 mg during and after the procedure; methylprednisolone is administered intravenously at approximately 500 mg prior to administration of the dopaminergic cell population; and tacrolimus is administered approximately one day after administration of the dopaminergic cell population. In some embodiments, methylprednisolone is further administered daily at approximately 5 mg following administration of the dopaminergic cell population.

[0017] In some embodiments, improvement can be detected approximately 12 weeks after administration of the dopaminergic cell population. In some embodiments, the improvement lasts for at least one year. In some embodiments, the improvement lasts for at least 1.5 years or at least 2 years. In some embodiments, the improvement persists after the immunosuppressive regimen is removed.

[0018] In some embodiments, the dopaminergic cell population is derived from pluripotent stem cells that differentiate into dopaminergic cells in vitro. In some embodiments, the dopaminergic cell population comprises midbrain dopaminergic neurons or their precursors. In some embodiments, the midbrain dopaminergic neurons or their precursors are derived from basal plate progenitor cells. In some embodiments, the basal plate progenitor cells are derived from pluripotent stem cells.

[0019] In some implementations, Parkinson's disease is late-stage Parkinson's disease.

[0020] On the other hand, this article provides a therapeutic composition comprising: an effective amount of dopaminergic cell population for treating Parkinson's disease; and a cell delivery solution.

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

[0022] In some implementations, approximately 70% to 80% of the dopaminergic cells in the dopaminergic cell population are viable.

[0023] In some implementations, the dopaminergic cell population has the ability to produce dopamine, and when evaluated in vitro using liquid chromatography-tandem mass spectrometry (LC-MS / MS), the ability to produce dopamine provides an area under the concentration-time curve (AUC) equal to or greater than 11.2 ng × day / mL.

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

[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 stabilizers. In some embodiments, the one or more stabilizers are selected from the group consisting of recombinant albumin (rHSA), dextran, and poloxamer. In some embodiments, the one or more energy source components comprise sugars. In some embodiments, the sugar is dextrose.

[0026] In some embodiments, the dopaminergic cell population contains at least 900,000 dopaminergic cells. In some embodiments, the dopaminergic cell population contains at least 2.7 million dopaminergic cells. In some embodiments, the dopaminergic cell population contains at least 5.4 × 10^6 dopaminergic cells.

[0027] In some embodiments, Parkinson's disease is advanced Parkinson's disease. In some embodiments, the therapeutic composition is used to treat Parkinson's disease.

[0028] In another aspect, this document provides containers comprising any of the therapeutic compositions described herein. In some embodiments, the containers comprise cryogenic vials. In some embodiments, the containers comprise aseptic technique (AT) vials. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of a Phase 1 clinical trial design used to evaluate the safety, tolerability, and efficacy of dopaminergic neurons in the treatment of Parkinson's disease.

[0030] Figure 2 An exemplary surgical method for administering an effective amount of dopaminergic neurons to a subject in need is shown.

[0031] Figures 3A to 3B Exemplary data from a comparative analysis of 18F-DOPA PET data in subjects treated with dopaminergic neurons are shown. Figure 3A Voxel-based analysis of 18F-DOPA PET data is shown. The images show voxel clusters with significant (P<0.05) group-level changes between baseline and 1 year (positive in blue and negative in orange). Figure 3BThis is a box plot showing the variation of striatum-occipital ratio (SOR) relative to baseline, limited to significant voxel clusters. Variations within significant voxel clusters combining the caudate and putamen were calculated using volume-weighted means for each subject. Lines represent the median variation in the cohort, circles represent the mean, boxes represent the lower and upper quartiles, and whisker lines represent extreme values.

[0032] Figures 4A to 4D Exemplary clinical data demonstrate the therapeutic efficacy of specific doses of dopaminergic neurons in treating Parkinson's disease. Figure 4A Box plots show the changes in Part III (OFF) scores of the Modified Unified Parkinson's Disease Rating Scale (MDS-UPDRS) sponsored by the Movement Disorders Association relative to baseline after administration of dopaminergic neurons in cohort A (left) and cohort B (right). The y-axis represents the change in OFF time, while the x-axis represents time in weeks. Symbols including lines, circles, boxes, and whiskers represent the median, mean, quartiles, and extreme values, respectively. Figure 4B Box plots show the variation of ON time (onset time) without disturbing motor dysfunction reported by patients in cohort A (left) and cohort B (right) relative to baseline. ON time variation relative to baseline is plotted on the y-axis, and time in weeks is plotted on the x-axis. Symbols including lines, circles, boxes, and whiskers represent the median, mean, quartiles, and extreme values, respectively. Figure 4C Box plots showing the variation of OFF time reported by patients relative to baseline in cohort A (left) and cohort B (right) are presented. The y-axis reflects the variation of OFF time, and the x-axis marks time in weeks. Symbols including lines, circles, boxes, and whiskers refer to the median, mean, quartiles, and extreme values, respectively. Figure 4D Box plots show the variation of ON time relative to baseline for patients with distressing motor disorders reported by cohort A (left) and cohort B (right). The y-axis plots the variation of ON time, and the x-axis represents time in weeks. Symbols including lines, circles, boxes, and whiskers refer to the median, mean, quartiles, and extreme values, respectively.

[0033] Figure 5 Exemplary clinical data demonstrating the therapeutic efficacy of specific doses of dopaminergic neurons for Parkinson's disease, as measured by neuropsychological evaluation using the Neuropsychiatric Questionnaire (NPI-Q), are presented. The figure shows a box plot of NPI-Q changes relative to baseline after dopaminergic neuron administration in cohort A (low dose, left) and cohort B (high dose, right). The y-axis represents the NPI-Q score, while the x-axis represents time in months following dopaminergic neuron transplantation. Symbols including lines, diamonds, boxes, and whiskers represent the median, mean, quartiles, and extreme values, respectively.

[0034] Figures 6A to 6B Exemplary clinical data are presented demonstrating the therapeutic efficacy of specific doses of dopaminergic neurons for the treatment of Parkinson's disease, as measured by neuropsychological evaluation using the Repeatable Assembly of Neuropsychological States (RBANS). Figure 6A Bar graphs show the changes in RBANS class relative to baseline in cohort A (low dose) at baseline and 12 months after administration of dopaminergic neurons. Figure 6B Bar graphs show the changes in RBANS categories relative to baseline in cohort A (high-dose) at baseline and twelve months after dopaminergic neuron administration. The y-axis represents RBANS scores, while the x-axis refers to RBANS categories corresponding to immediate memory, visuospatial / constructivist, linguistic, attentional, and delayed memory, as well as the total score. The top of each bar represents the mean (median). Symbols including the line and whisker represent the median and extreme values, respectively.

[0035] Figures 7A to 7B Exemplary clinical data are presented demonstrating the therapeutic efficacy of specific doses of dopaminergic neurons in treating Parkinson's disease, as measured by neuropsychological evaluation using the Frontal System Behavior Scale (FrSBe). Figure 7A Bar graphs showing the change in FrSBe class relative to baseline in cohort A (low dose) at baseline and 12 months after administration of dopaminergic neurons are presented. Figure 7B Bar graphs show the changes in FrSBe categories relative to baseline in cohort A (high dose) at baseline and 12 months after dopaminergic neuron administration. The y-axis represents the RBANS score, while the x-axis refers to the FrSBe categories corresponding to emotional blunting, disinhibition, and executive function, as well as the total score. Raw scores were converted to T-scores (mean 50, SD 10), and a T-score ≥65 was considered clinically significant. The top of each bar represents the mean (median). Symbols including the line and whisker represent the median and extreme values, respectively.

[0036] Figure 8Exemplary clinical data are presented demonstrating the therapeutic efficacy of specific doses of dopaminergic neurons for treating motor symptoms of Parkinson's disease (PD), as measured by the Movement Disorders Association Unified Parkinson's Disease Rating Scale (MDS-UPDRS) Part II score. Baseline MDS-UPDRS Part II scores (mean [median]) were 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 sub-scores ranging from 0 to 52; higher scores indicate more severe motor symptoms. Horizontal lines represent the median; diamonds represent the mean; boxes represent the first quartile (Q1) and third quartile (Q3); whiskers represent extreme values. CfB indicates change relative to baseline. IS indicates immunosuppression.

[0037] Figure 9 Exemplary clinical data are presented demonstrating the therapeutic efficacy of specific doses of dopaminergic neurons for treating motor symptoms of Parkinson's disease (PD), as measured by the Movement Disorders Association Unified Parkinson's Disease Rating Scale (MDS-UPDRS) Part III OFF score. The baseline MDS-UPDRS Part III OFF score (mean [median]) was 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 sub-scores ranging from 0 to 132; higher scores indicate more severe motor symptoms. Horizontal lines represent the median; diamonds represent the mean; boxes represent the first quartile (Q1) and third quartile (Q3); whiskers represent extreme values. CfB indicates change relative to baseline. IS indicates immunosuppression.

[0038] Figures 10A to 10B Exemplary clinical data are presented demonstrating the therapeutic efficacy of specific doses of dopaminergic neurons for treating motor symptoms of Parkinson's disease, as measured by the ON and OFF times of the Hauser / PD diary. Figure 10A Exemplary data on patient-reported good ON time in the Hauser diary are shown. The baseline-adjusted good ON time (mean [median]) in the Hauser diary was 12.0 hours (13.2) in the low-dose cohort and 10.9 hours (10.4) in the high-dose cohort. Good ON status is the sum of ON status without motor dysfunction and ON status with non-disturbing motor dysfunction. Horizontal lines represent medians; diamonds represent means; boxes represent Q1 and Q3; whiskers represent extreme values. CfB, change relative to baseline; IS, immunosuppression; Q1, first quartile; Q3, third quartile. Figure 10BExemplary data on patient-reported Hauser diary good ON time are shown. Baseline-adjusted Hauser diary OFF time (mean [median]) was 3.2 hours (2.6) in the low-dose cohort and 5.0 hours (5.6) in the high-dose cohort. Horizontal lines represent the median; diamonds represent the mean; boxes represent Q1 and Q3; whiskers represent extreme values. CfB, change relative to baseline; IS, immunosuppression; Q1, first quartile; Q3, third quartile.

[0039] Figure 11 Exemplary clinical data are presented demonstrating the therapeutic efficacy of specific doses of dopaminergic neurons for motor symptoms of Parkinson's disease (PD), as measured by the Unified Motor Disorder Rating Scale (UDysRS). Baseline UDysRS objective subscale scores (mean [median]) were 10.6 (14.0) in the low-dose cohort and 2.3 (0.0) in the high-dose cohort. The UDysRS objective subscale scores consist of Part 3 (Impairment) and Part 4 (Disability). The objective subscales consist of 11 items on a scale from 0 (normal) to 4 (severity), with a possible total score of 44; higher scores indicate more severe symptoms. Horizontal lines represent medians; diamonds represent means; boxes represent Q1 and Q3; whiskers represent extreme values. CfB, change relative to baseline; IS, immunosuppression; Q1, first quartile; Q3, third quartile.

[0040] Figure 12 This is a schematic diagram of an exemplary dual imaging strategy for assessing the implantation, survival, and function of transplanted dopaminergic neurons. Specifically, Figure 12 This paper outlines the procedures and objectives for evaluating the implantation of dopaminergic neurons in the brain using positron emission tomography (PET) and magnetic resonance imaging (MRI).

[0041] Figure 13 This paper describes an exemplary voxel-based population analysis method for evaluating the implantation, survival, and function of transplanted dopaminergic neurons.

[0042] Figure 14 Exemplary clinical image data of mean 18F-DOPA uptake, as measured by positron emission tomography (PET), are shown. Mean images show the mean 18F-DOPA uptake at baseline (before treatment), 12 months post-treatment, and 18 months post-treatment. Mean images = sum (all SOR-1 images at each visit) / sample size.

[0043] Figure 15Exemplary image data of 18F-DOPA uptake at 12 and 18 months, as measured by positron emission tomography (PET), are shown. Voxels showing increased 18F-DOPA uptake are depicted in orange; voxels showing decreased 18F-DOPA uptake are depicted in blue.

[0044] Figures 16A to 16B Exemplary data on 18F-DOPA uptake in the putamen and caudate nucleus are shown, respectively. Specifically, Figure 16A A bar graph showing the change in 18F-DOPA uptake in the shell core relative to baseline at 18 months post-application is shown. Figure 16B A bar graph showing the change in 18F-DOPA uptake in the caudate nucleus relative to baseline at 18 months post-administration is presented.

[0045] Figure 17 Exemplary magnetic resonance imaging (MRI) image data obtained from subjects who received dopaminergic neurons at baseline, 6 months, 12 months, and 18 months after administration are shown.

[0046] Figures 18A-18B Exemplary clinical data are presented demonstrating the therapeutic efficacy of specific doses of dopaminergic neurons for treating motor symptoms of PD, as measured by the ON score of Part III of the Movement Disorders Association Unified Parkinson's Disease Rating Scale (MDS-UPDRS). Figure 18A Box plots of MDS-UPDRS Part III ON scores measured from 0 (baseline, i.e., pre-transplant) to 24 months post-transplant are shown. Figure 18B Box plots are shown of the MDS-UPDRS Part III OFF scores measured from 0 (baseline, i.e., pre-transplant) to 24 months post-transplant. The y-axis indicates the MDS-UPDRS Part III score, while the x-axis indicates time in months. The mean (SD) score is shown above each box plot. Symbols including lines, diamonds, boxes, and whiskers indicate the median, mean, quartiles, and extreme values, respectively.

[0047] Figure 19Exemplary clinical data demonstrate the therapeutic efficacy of specific doses of dopaminergic neurons in treating PD symptoms, as measured by the Movement Disorders Association Unified Parkinson's Disease Rating Scale (MDS-UPDRS) Part II score. The figure shows box plots of the MDS-UPDRS Part II score before dopaminergic neuron administration (baseline, i.e., pre-transplant) and up to 24 months after administration. The y-axis indicates the MDS-UPDRS Part II score, while the x-axis indicates time in months. The mean (SD) score is shown above each box plot. Symbols including lines, diamonds, squares, and whiskers indicate the median, mean, quartiles, and extreme values, respectively.

[0048] Figures 20A to 20B Exemplary clinical data are presented demonstrating the therapeutic efficacy of specific doses of dopaminergic neurons for treating motor symptoms of PD, as measured by the Unified Movement Disorder Rating Scale (UDysRS) score. Figure 20A Box plots of UDysRS objective sub-item scores before and after administration of dopaminergic neurons are shown. Figure 20B Box plots show the historical UDysRS component scores before and after dopaminergic neuron administration. The y-axis represents the corresponding UDysRS component score, while the x-axis represents time in months post-transplantation. Baseline scores at 0 months (pre-transplantation) are indicated. The mean (SD) score is shown above each box plot. Symbols including lines, diamonds, boxes, and whiskers represent the median, mean, quartiles, and extreme values, respectively.

[0049] Figures 21A to 21B Exemplary clinical data are presented demonstrating the therapeutic efficacy of specific doses of dopaminergic neurons for treating PD symptoms, as measured by patient-reported Parkinson's Disease (PD) diary scores. Figure 21A Box plots show the adjusted good ON time of the PD diary before (0 months) and after 24 months of dopaminergic neuron administration. Figure 21B Box plots showing adjusted PD diary OFF times before dopaminergic neuron administration (0 months) and after 24 months of dopaminergic neuron administration are presented. The y-axis indicates time in hours, and the x-axis indicates time in months. Baseline scores at 0 months are indicated. The mean (SD) score is shown above or below each box plot. Symbols including lines, diamonds, boxes, and whiskers refer to the median, mean, quartiles, and extreme values, respectively.

[0050] Figure 22Exemplary clinical data demonstrate the therapeutic efficacy of specific doses of dopaminergic neurons for treating nonmotor symptoms of Parkinson's disease (PD), as measured by the total score of the Neuropsychiatric Questionnaire (NPI-Q). The figure shows box plots of the total NPI-Q score at baseline (0 months), 12 months after dopaminergic neuron administration, and 24 months after dopaminergic neuron administration. The y-axis represents the total NPI-Q score, while the x-axis represents time in months post-transplantation. The mean (SD) score is shown above each box plot. Symbols including lines, diamonds, squares, and whiskers represent the median, mean, quartiles, and extreme values, respectively. Detailed Implementation

[0051] This disclosure relates to methods, dopaminergic cell populations, compositions, and devices for treating Parkinson's disease in subjects. Specifically, the methods, compositions, and devices for treating Parkinson's disease in subjects include dopaminergic cell populations. In some embodiments, administration of dopaminergic cell populations to a subject with Parkinson's disease, compared to a control or baseline, results in an improvement in the subject's motor and / or non-motor function.

[0052] Parkinson's disease is a neurodegenerative disorder that causes both motor and nonmotor symptoms and is characterized by widespread degeneration of dopaminergic neurons in the substantia nigra-striatal system. The motor symptoms of Parkinson's disease are attributed to the degeneration of dopaminergic neurons in the substantia nigra, which in turn leads to a lack of dopamine in the striatum. Motor symptoms of Parkinson's disease include tremor, decreased motor function (e.g., bradykinesia, apnea, rigidity), postural instability, gait abnormalities, and swallowing disorders. Nonmotor symptoms include autonomic and neuropsychiatric disturbances, such as anosmia or sleep disturbances. While effective management of patients with Parkinson's disease is possible in the early stages, the disease can progress to advanced stages and lead to severe debilitating complications.

[0053] In the embodiments described herein, the method comprises administering a population of dopaminergic cells to treat a subject with Parkinson's disease. The dopaminergic cell population used in conjunction with the described method is capable of producing the neurotransmitter dopamine. Increased dopamine availability in subjects with Parkinson's disease compared to subjects who have not received a population of dopaminergic cells, or compared to a baseline of disease progression, can improve the subject's motor and non-motor functions.

[0054] The implementation method described herein utilizes a novel population of dopaminergic cells, which is broadly applicable for treating Parkinson's disease in subjects. This novel population and method of application are based, at least in part, on surprising and unexpected observations from clinical data, that administration of a specific amount of dopaminergic cells to subjects with Parkinson's disease not only resulted in improvements in motor and non-motor function but also reversed disease progression. Notably, this unexpected outcome persisted in subjects for at least 52 weeks without diminishing, contrary to the typical decline in motor and non-motor function seen in Parkinson's disease. Therefore, the method described herein demonstrates improvement in the treatment of Parkinson's disease.

[0055] Furthermore, the clinical trial data reported herein demonstrate that the in vitro-derived dopaminergic cell populations, as described herein, effectively implant, survive, and act as dopamine producers in the brains of subjects to alleviate Parkinson's disease symptoms. Previous efforts to generate dopaminergic cells have shown inconsistencies in cell characteristics, functionality, and maturity, leading to unpredictable transplant outcomes. In the absence of clinical data or comprehensive in vivo experiments, the inherent uncertainty regarding the ability of in vitro-derived dopaminergic cells to successfully integrate, survive, and restore function in the Parkinson's disease-affected brains of subjects makes determining the therapeutically effective cell dosage extremely difficult. However, this disclosure fills a knowledge gap. The significant performance of the disclosed amount of dopaminergic cell populations, demonstrated by significant improvement in motor and non-motor Parkinson's disease symptoms at least 52 weeks post-transplantation, underscores the innovative and unique nature of the cell dosage and method described herein.

[0056] The embodiments disclosed herein can help ensure the efficacy of treatment for subjects with Parkinson's disease. Administering too few dopaminergic cells will not correct the dopamine deficiency in Parkinson's disease, while an excessive number of dopaminergic cells carries the risk of neurotransmitter imbalance, leading to complications such as motor disorders, mood disturbances, or other neurological effects. Dopaminergic cell overload can also impair the viability and function of existing and newly introduced cells. Advantageously, this document provides the amount of dopaminergic cells that strikes a balance between desired therapeutic outcomes and minimized risks.

[0057] While this disclosure describes various exemplary alternatives and implementations as provided herein, it should be understood that the various features, aspects, and functions described in one or more individual alternatives are not limited in their applicability to the particular alternatives in which they are described. Rather, they may be applied individually or in various combinations to one or more other alternatives of this disclosure, whether the alternatives are described or features are presented as part of the described alternatives. The breadth and scope of this disclosure should not be limited by any exemplary alternatives described or illustrated herein.

[0058] I. Definition

[0059] The following definitions supplement those in the art and are specific to this disclosure. These definitions should not be attributed to any related or unrelated circumstances, such as any jointly owned patents or patent applications. While some methods and materials similar to or equivalent to those described herein may be used to practice the features of this disclosure, preferred materials and methods are described herein. Therefore, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive.

[0060] Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms shall include plural forms and plural terms shall include singular forms.

[0061] It should be understood that the present invention is not limited to the specific methods, schemes, and reagents described herein, and therefore is subject to variation. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined only by the claims.

[0062] As used herein, the articles “a,” “a,” and “the” are used to refer to one or more (i.e., at least one) grammatical objects of that article. For example, “an element” means one element or more elements.

[0063] The use of alternatives (e.g., "or") should be understood to mean any one, both, or any combination of the alternatives.

[0064] As used herein, the term “about” or “approximately” means a quantity, level, value, number, frequency, percentage, size, 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, size, quantity, weight, or length. In some cases, the term “about” or “approximately” means a series of quantities, levels, values, numbers, frequencies, percentages, sizes, quantities, weight, or lengths that are ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%.

[0065] As used herein, the term "application" and its variations refer to the introduction of a composition or therapeutic agent (e.g., a population of dopaminergic cells) into a subject. Application includes the simultaneous and sequential introduction of a composition or therapeutic agent. A composition or therapeutic agent (e.g., a population of dopaminergic cells) is administered to a subject via any suitable route, including local injection or surgical implantation. A suitable route of application allows the composition or agent to perform its intended function. Application also includes administration via another route. Application can also be performed locally. For example, a composition or therapeutic agent (e.g., a population of dopaminergic cells) can be administered by surgical implantation into tissue, including the use of a device or instrument.

[0066] As used herein, the term “and / or” should be understood to mean one or both or any combination of alternatives.

[0067] As used herein, the term "baliximab" refers to an immunosuppressive monoclonal antibody that binds to the IL-2 receptor identified under CAS Registry No. 179045-86-4. Baliximab is also known as Simulect. Baliximab is a chimeric mouse-human antibody that blocks the binding of IL-2 to immune cells.

[0068] As used herein, the term "delivery solution" or "cell delivery solution" is any solution added to a container containing cells so that the cells can be administered to a subject. Cell delivery solutions may be free of, contain minimal or trace amounts of cryoprotectants and / or cell washing solutions, or other components not desired for administration. Delivery solutions may be used to reconstitute cell populations (e.g., dopaminergic cell populations) prior to administration or clinical use after thawing cells.

[0069] As used herein, the term “differentiation” and any grammatical variations thereof refer to the process by which non-specialized cells (e.g., induced pluripotent stem cells (iPSCs) or pluripotent stem cells) acquire characteristics of specialized cells such as neurons. Differentiation can be controlled by the interaction of cellular genes with extracellular physical and chemical conditions, typically through signaling pathways involving proteins embedded on the cell surface. The term “directed differentiation” refers to manipulating cell culture conditions to induce non-specialized cells (e.g., induced pluripotent stem cells (iPSCs) or pluripotent stem cells) to differentiate into specific (e.g., desired) cell types, such as neurons, neural crests, cranial basal plates, and non-neuroectodermal precursors.

[0070] As used herein, the term "dopaminergic cell" refers to a cell capable of producing the neurotransmitter dopamine. A dopaminergic cell can be a dopaminergic neuron or a dopaminergic progenitor cell. Exemplary dopaminergic cells include, but are not limited to, implantable midbrain dopaminergic neurons, midbrain dopaminergic neurons, true midbrain dopaminergic neurons, midbrain dopaminergic neuron progenitor cells, floorplate-derived dopaminergic neurons, true midbrain dopaminergic neurons, dopaminergic neuron progenitor cells, and dopaminergic neuron precursor cells.

[0071] As used herein, “effective quantity,” “therapeutic effective quantity,” or “effective amount” refers to a quantity sufficient to affect a beneficial or desired clinical outcome during treatment. An effective quantity may be administered to a subject at least at one dose. In therapeutic terms, an effective quantity includes a quantity sufficient to alleviate, improve, stabilize, reverse, or slow the progression of a disease (e.g., Parkinson’s disease) or otherwise reduce the pathological consequences of the disease. Effective quantities can vary depending on the circumstances. Several factors are typically considered when determining the appropriate dose to achieve an effective quantity. These factors include the subject’s age, sex, and weight, the condition being treated, the severity of the condition, and the form and effective concentration of the dopaminergic cell population administered. An exemplary effective quantity of dopaminergic cell population is described herein.

[0072] As used in this article, an "immunosuppressive regimen" refers to a treatment regimen that suppresses or prevents a subject's immune response to a foreign substance. Immunosuppressive regimens may include immunosuppressants that inhibit immune cell activation, destroy proliferation, or suppress inflammation.

[0073] As used herein, “improvement” in response to treatment administration refers to an improvement in at least one parameter of disease progression. When the disease type (e.g., Parkinson's disease) is one that progressively worsens with age, improvement in disease progression can be maintaining the subject's disease state. Improvement in disease progression can be a reduction in disease progression or can include a reversal of symptoms associated with disease progression. In other words, improvement includes a reduction in disease-related symptoms. Improvement in disease progression is determined based on methods known in the art. Those skilled in the art can determine appropriate methods based on the type of disease being evaluated (e.g., Parkinson's disease).

[0074] As used herein, the term "in vitro" refers to an artificial environment and the processes or reactions that occur within that environment. Examples of in vitro environments include, but are not limited to, test tubes and cell cultures.

[0075] As used in this article, the term "in vivo" refers to the natural environment (e.g., an animal or a cell) and the processes or reactions that occur in the natural environment, such as embryonic development, cell differentiation, neural tube formation, etc.

[0076] As used herein, the term "methylprednisolone" refers to the synthetic immunosuppressive steroid identified under CAS Registry No. 83-43-2 and PubChem Compound Identification No. 6741. Methylprednisolone is also known as Depo-Medrol, Medrol, and Solu-Medrol.

[0077] As used herein, the term "Movement Disorders Association Unified Parkinson's Disease Rating Scale" or "MDS-UPDRS" refers to a standardized scale used to assess various aspects of Parkinson's disease. MDS-UPDRS scores can include assessments of non-motor and / or motor experiences in daily life, as well as motor assessments and characterizations of the severity and burden of Parkinson's disease across different populations. MDS-UPDRS can be used in clinical settings as well as in research. MDS-UPDRS comprises different sections, each assessing a different aspect of Parkinson's disease.

[0078] As used herein, “MDS-UPDRS ON score” or “ON score” refers to a patient’s MDS-UPDRS score during a period when treatment (e.g., dopaminergic cell population) is effective and the symptoms of the disease (e.g., Parkinson’s disease) are under control. As used herein, “MDS-UPDRS OFF score” or “OFF score” refers to a patient’s MDS-UPDRS score during a period when treatment (e.g., dopaminergic cell population) disappears and at least one symptom of the disease (e.g., Parkinson’s disease) recurs.

[0079] As used herein, the term "neuron" refers to a nerve cell, the primary functional unit of a subject's nervous system. A neuron typically comprises 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 used in the methods and compositions described herein, neurons include cells expressing one or more markers that indicate neurons. Such markers include, but are not limited to, engrailed-1 (EN1), orthodenticle homeobox protein 2 (OTX2), tyrosine hydroxylase (TH), nuclear receptor-associated protein 1 (NURR1), forkhead box protein A2 (FOXA2), and LIM homeobox transcription factor 1α (LMX1A), PITX3, LMO3, SNCA, ADCAP1, CHRNA4, and GIRK2.

[0080] As used herein, the term "Parkinson's disease" refers to a neurodegenerative disorder characterized by widespread degeneration of dopaminergic neurons in the substantia nigra region of the brain. Parkinson's disease manifests as changes in both motor and nonmotor functions. Exemplary changes in motor function as a symptom of Parkinson's disease include, but are not limited to, tremor, hypokinesis, postural instability, gait abnormalities, and swallowing disorders. Exemplary changes in nonmotor function as a symptom of Parkinson's disease include, but are not limited to, autonomic and neuropsychiatric disturbances, such as loss of smell or sleep disturbances. Parkinson's disease can present as early-stage Parkinson's disease or late-stage Parkinson's disease, with symptoms becoming more severe in later stages of the disease.

[0081] As used herein, the terms “patient,” “subject,” “individual,” etc., are used interchangeably and refer to any animal or its cells, whether in vitro or in situ, and apply to the compositions and methods described herein. In some cases, the patient, subject, or individual is a human being.

[0082] As used herein, a "pluripotent stem cell" is a cell that has the ability to differentiate into any of the three germ layers (endoderm, mesoderm, or ectoderm) from an organism, but not into extraembryonic tissues such as the placenta. Pluripotency can be incomplete or partial, as pluripotent cells can form cells from all three germ layers but may not exhibit all the characteristics of a fully pluripotent cell. "Induced pluripotent stem cells" or "iPSCs" refer to any pluripotent stem cell artificially obtained from non-pluripotent cells (usually adult somatic cells) by inducing the "forced" expression of a specific gene.

[0083] As used herein, the term "cell population" or "cell group" refers to a group of at least two cells. In non-limiting examples, a cell population may 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. A population may be a pure population containing only one cell type. Alternatively, a population may include more than one cell type, such as a mixed cell population.

[0084] As used herein, the statement that a cell or cell population is “positive” or “expresses” a particular marker means that the presence of the particular marker (e.g., a surface marker or an intracellular marker, such as a transcription factor) can be detected on or within the cell. When referring to a surface marker, the term means the presence of surface expression as detected by flow cytometry, for example by staining with an antibody that specifically binds to the marker and detecting the antibody, wherein the staining is detectable by flow cytometry and the level is substantially higher than that detected by the same procedure under otherwise identical conditions with an isotype-matched control or a fluorescence-subtracted-one (FMO) gated control, and / or the level is substantially similar to that of cells known to be positive for the marker, and / or the level is substantially higher than that of cells known to be negative for the marker.

[0085] As used herein, the statement that a cell or cell population is “negative” for a particular marker or does not express a particular marker or gene means that there is no substantial detectable presence of the particular marker (such as a surface marker or an intracellular marker, such as a transcription factor) on or within the cell. When referring to a surface marker, the term means that surface expression is not detected as by flow cytometry, for example by staining with an antibody that specifically binds to the marker and detecting the antibody, wherein the level of non-detectable staining by flow cytometry is substantially higher than that detected by the same procedure under otherwise identical conditions with a type-matched control or a fluorescence-subtracted-one (FMO) gated control, and / or its level is substantially lower than that of cells known to be positive for the marker, and / or its level is substantially similar to that of cells known to be negative for the marker.

[0086] As used in this article, the term "progenitor cell" and its grammatical equivalents refer to the offspring of stem cells, which can further differentiate into specialized cell types within a specific cell lineage.

[0087] As used in this article, the term "stem cell" refers to a cell that has the ability to divide indefinitely in culture and produce specialized cells.

[0088] As used herein, the terms “substantially” or “largely” mean approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher of a reference quantity, level, value, number, frequency, percentage, size, weight, or length. In some cases, the terms “substantially the same” or “substantially the same” mean a range of quantities, levels, values, numbers, frequencies, percentages, sizes, weights, or lengths that are approximately the same as a reference quantity, level, value, number, frequency, percentage, size, weight, or length.

[0089] As used herein, the term "tacrolimus" refers to the immunosuppressant identified under CAS Registry No. 104987-11-3 and PubChem Compound Identifier No. 445643. Tacrolimus is also known as FK506 and Prograf. Tacrolimus primarily inhibits T cell activation by binding to the FK506-binding protein (FKBP) immunophile receptor FKBP12, which in turn inhibits calcineurin activity and reduces IL-2 production and immune cell activation. Tacrolimus can also modulate the immune response in the nervous system.

[0090] As used herein, the term "treatment" refers to a clinical intervention that attempts to alter the disease process of the treated individual or cells, and may be performed for prevention or in the course of clinicopathology. The therapeutic effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, slowing the rate of disease progression, reversing disease progression, improving or mitigating the disease state, and alleviating or improving prognosis. By preventing or reversing the progression of the disease or condition, treatment can prevent exacerbations caused by the condition in affected or diagnosed or suspected subjects, and treatment can prevent the onset of the condition or its symptoms in subjects at risk of or suspected of having the condition.

[0091] As used herein, the term "vessel" refers to any suitable container that can hold a therapeutic agent (e.g., a population of dopaminergic cells). In some cases, a vessel may be used solely for storing the therapeutic agent, or it may be used during administration of the therapeutic agent to a subject.

[0092] Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the range format is for convenience and brevity only and should not be construed as a rigid limitation on the scope of the invention. Therefore, the range description should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within those ranges. For example, a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numbers within those ranges, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0093] II. Dopaminergic cell population

[0094] Certain aspects of this disclosure provide dopaminergic cell populations. In some embodiments, dopaminergic cell populations are suitable for treating Parkinson's disease.

[0095] In some embodiments, the dopaminergic cell population includes dopaminergic neurons. In some embodiments, the dopaminergic neurons include one or more of midbrain dopaminergic neurons, lamina-derived dopaminergic neurons, and true midbrain dopaminergic neurons. In some embodiments, the dopaminergic cell population includes dopaminergic progenitor cells or dopaminergic precursor cells.

[0096] In some embodiments, the dopaminergic cell population is FOXA2 positive. 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 dopaminergic cell population is FOXA2 positive. In some embodiments, at least 80% of the dopaminergic cell population is FOXA2 positive. In some embodiments, at least 90% of the dopaminergic cell population is FOXA2 positive. In some embodiments, at least 95% of the dopaminergic cell population is FOXA2 positive. In some embodiments, at least 98% of the dopaminergic cell population is FOXA2 positive. In some embodiments, at least 99% of the dopaminergic cell population is FOXA2 positive.

[0097] In some embodiments, only a certain percentage of the dopaminergic cell population 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 dopaminergic cell population is PAX2 positive. In some embodiments, less than about 10% of the dopaminergic cell population is PAX2 positive. In some embodiments, less than about 5% of the dopaminergic cell population is PAX2 positive. In some embodiments, less than about 4% of the dopaminergic cell population is PAX2 positive. In some embodiments, less than about 3% of the dopaminergic cell population is PAX2 positive. In some embodiments, less than about 2% of the dopaminergic cell population is PAX2 positive. In some embodiments, less than about 1% of the dopaminergic cell population is PAX2 negative.

[0098] In some embodiments, only a certain percentage of the dopaminergic cell population is CRABP1 positive. 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 dopaminergic cell population is CRABP1 positive. In some embodiments, less than about 10% of the dopaminergic cell population is CRABP1 positive. In some embodiments, less than about 5% of the dopaminergic cell population is CRABP1 positive. In some embodiments, less than about 4% of the dopaminergic cell population is CRABP1 positive. In some embodiments, less than about 3% of the dopaminergic cell population is CRABP1 positive. In some embodiments, less than about 2% of the dopaminergic cell population is CRABP1 positive. In some embodiments, less than about 1% of the dopaminergic cell population is CRABP1 negative.

[0099] In some embodiments, only a certain percentage of the dopaminergic cell population is Ki67 positive. 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 dopaminergic cell population is Ki67 positive. In some embodiments, less than about 30% of the dopaminergic cell population is Ki67 positive. In some embodiments, less than about 20% of the dopaminergic cell population is Ki67 positive. In some embodiments, less than about 15% of the dopaminergic cell population is Ki67 positive. In some embodiments, less than about 12% of the dopaminergic cell population is Ki67 positive. In some embodiments, less than about 10% of the dopaminergic cell population is Ki67 positive. In some embodiments, less than about 5% of the dopaminergic cell population is Ki67 positive. In some implementations, the dopaminergic cell population is Ki67 negative.

[0100] In some embodiments, a certain number of dopaminergic cells in the dopaminergic cell population are viable. In some embodiments, approximately 10% to approximately 90%, approximately 20% to approximately 80%, approximately 30% to approximately 80%, approximately 40% to approximately 50%, or approximately 50% to approximately 100% of the dopaminergic cells in the dopaminergic cell population are viable. In some embodiments, approximately 50% to approximately 100% of the dopaminergic cells in the dopaminergic cell population are viable. In some embodiments, approximately 60% to approximately 90% of the dopaminergic cells in the dopaminergic cell population are viable. In some embodiments, approximately 70% to approximately 80% of the dopaminergic cells in the dopaminergic cell population 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 dopaminergic cell population are viable. In some embodiments, at least about 90% of the dopaminergic cells in the dopaminergic cell population are viable. In some embodiments, at least about 80% of the dopaminergic cells in the dopaminergic cell population are viable. In some embodiments, at least about 70% of the dopaminergic cells in the dopaminergic cell population are viable. In some embodiments, at least about 50% of the dopaminergic cells in the dopaminergic cell population are viable.

[0101] In some embodiments, the dopaminergic cell population has the ability to produce dopamine. In some embodiments, the dopaminergic cell population has the ability to produce 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 dopaminergic cell population has the ability to produce dopamine at a rate of at least about 20 ng / day / mL. In some embodiments, the dopaminergic cell population has the ability to produce dopamine at a rate of at least about 15 ng / day / mL. In some embodiments, the dopaminergic cell population has the ability to produce dopamine at a rate of at least about 11.2 ng / day / mL. In some embodiments, the dopaminergic cell population has the ability to produce dopamine at a rate of at least about 8 ng / day / mL.

[0102] The ability of a dopaminergic cell population to produce dopamine can be measured by any suitable method known in the art. In some embodiments, the dopaminergic cell population has the ability to produce dopamine, and when evaluated in vitro using liquid chromatography-tandem mass spectrometry (LC-MS / MS), the ability to produce dopamine provides a concentration-time area (AUC) 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 dopaminergic cell population has the ability to produce dopamine, and when evaluated in vitro using liquid chromatography-tandem mass spectrometry (LC-MS / MS), the ability to produce dopamine provides a concentration-time area (AUC) of at least about 20 ng / day / mL. In some embodiments, the dopaminergic cell population has the ability to produce dopamine, and when evaluated in vitro using liquid chromatography-tandem mass spectrometry (LC-MS / MS), the ability to produce dopamine provides a concentration-time area (AUC) of at least about 15 ng / day / mL. In some embodiments, the dopaminergic cell population has the ability to produce dopamine, and when evaluated in vitro using liquid chromatography-tandem mass spectrometry (LC-MS / MS), the ability to produce dopamine provides a concentration-time area (AUC) of at least about 11.2 ng / day / mL. In some embodiments, the dopaminergic cell population has the ability to produce dopamine, and when evaluated in vitro using liquid chromatography-tandem mass spectrometry (LC-MS / MS), the ability to produce dopamine provides a concentration-time area (AUC) of at least about 8 ng / day / mL.

[0103] In some embodiments, the dopaminergic cell population includes approximately 1×10^4 to approximately 1×10^10 dopaminergic cells, approximately 1×10^4 to approximately 1×10^5 dopaminergic cells, approximately 1×10^5 to approximately 1×10^9 dopaminergic cells, approximately 1×10^5 to approximately 1×10^6 dopaminergic cells, approximately 1×10^5 to approximately 1×10^7 dopaminergic cells, etc. Approximately 1×10^6 to approximately 1×10^7 dopaminergic cells, approximately 1×10^6 to approximately 1×10^8 dopaminergic cells, approximately 1×10^7 to approximately 1×10^8, approximately 1×10^8 to approximately 1×10^9 dopaminergic cells, approximately 1×10^8 to approximately 1×10^10 dopaminergic cells, or approximately 1×10^9 to approximately 1×10^10 dopaminergic cells. In some implementations, the cell population includes at least 250,000 dopaminergic cells, at least 500,000 dopaminergic cells, at least 750,000 dopaminergic cells, at least 1,000,000 dopaminergic cells, at least 1,250,000 dopaminergic cells, at least 1,500,000 dopaminergic cells, at least 1,750,000 dopaminergic cells, at least 2,000,000 dopaminergic cells, at least 2,250,000 dopaminergic cells, at least 2,500,000 dopaminergic cells, at least 2,750,000 dopaminergic cells, and at least 3,000,000 dopaminergic cells. Cells, 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 cells, at least 8 million dopaminergic cells, at least 9 million dopaminergic cells, or at least 10 million or more dopaminergic cells.

[0104] In some embodiments, the dopaminergic cell population comprises at least about 1.0 × 10^6 to about 1.2 × 10^7 dopaminergic cells. In some embodiments, the dopaminergic cell population comprises at least about 900,000 dopaminergic cells. In some embodiments, the dopaminergic cell population comprises at least about 1.8 million dopaminergic cells. In some embodiments, the dopaminergic cell population comprises at least about 2.7 million dopaminergic cells. In some embodiments, the dopaminergic cell population comprises at least about 5.4 million dopaminergic cells. In some embodiments, the dopaminergic cell population comprises more than 5.4 million dopaminergic cells.

[0105] In some embodiments, the therapeutic cell population is adapted to treat the subject's Parkinson's disease. In some embodiments, the Parkinson's disease is early-stage Parkinson's disease. In some embodiments, the Parkinson's disease is late-stage Parkinson's disease. In some embodiments, the subject is a human being.

[0106] Production of dopaminergic cells

[0107] In some embodiments, a population of dopaminergic cells (e.g., the dopaminergic cell population described herein) is prepared by differentiating stem cells into dopaminergic cells in vitro. In some embodiments, the dopaminergic cell population comprises midbrain dopaminergic neurons or their progenitor cells. Therefore, in some embodiments, a population of dopaminergic cells is prepared by differentiating stem cells into midbrain dopaminergic neurons or their progenitor cells in vitro. In some embodiments, the preparation of midbrain dopaminergic neurons or their progenitor cells involves differentiating stem cells into neural progenitor cells in vitro. In some embodiments, the preparation of midbrain dopaminergic neurons or their progenitor cells further includes differentiating neural progenitor cells into basal plate progenitor cells in vitro. In some embodiments, the preparation of midbrain dopaminergic neurons or their progenitor cells further includes differentiating basal plate progenitor cells into midbrain dopaminergic neurons or their progenitor cells in vitro.

[0108] In some implementations, differentiating stem cells into dopaminergic cells involves contacting stem cells with at least one Small Mothers Against Decapentaplegic (SMAD) signaling inhibitor (referred to as "SMAD inhibitor"), at least one sound hedgehog factor (SHH) signaling activator (referred to as "SHH activator"), and at least one wingless (Wnt) signaling activator (referred to as "Wnt activator") to obtain a cell population expressing at least one marker indicating dopaminergic cells.

[0109] In some embodiments, the at least one marker indicating dopaminergic cells is selected from EN1, FOX1A, LMX1A, OTX2, NURR1, TH, PITX3, LMO3, SNCA, ADCAP1, CHRNA4, GIRK2, and FOXA2. In some embodiments, the at least one marker indicating dopaminergic cells is FOXA2. In some embodiments, the at least one marker indicating dopaminergic cells includes TH.

[0110] In some embodiments, a population of dopaminergic cells is prepared by differentiating stem cells in vitro into midbrain dopaminergic neurons or their precursors. In some embodiments, differentiating stem cells into midbrain dopaminergic neurons or their precursors involves a neural induction process, thereby differentiating the stem cells into neural progenitor cells. In some embodiments, the neural induction process involves contacting stem cells with dual SMAD inhibitors (e.g., Noggin and SB431542), as described, for example, 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 WO2010096496A2, each reference incorporated herein by reference.

[0111] In some embodiments, the preparation of midbrain dopaminergic neurons or precursors thereof further includes contacting stem cells or neural progenitor cells with an SHH signaling activator. In some embodiments, differentiating stem cells or neural progenitor cells into midbrain dopaminergic neurons or precursors involves differentiating stem cells or neural progenitor cells into midbrain floorplate progenitor cells. In some embodiments, differentiating stem cells or neural progenitor cells into midbrain floorplate progenitor cells involves contacting stem cells or neural progenitor cells with a combination of an SHH signaling activator and a Wnt signaling activator, for example, as described in WO2013067362A1, which is incorporated herein by reference. Therefore, in some embodiments, this disclosure provides dopaminergic cells (e.g., midbrain dopaminergic neurons or precursors thereof) generated in vitro using a floorplate-based differentiation strategy, for example, as described in Kriks, 2011, Floorplate-derived dopamine neurons from hESCs efficiently engraft in animal models of Parkinson's disease, Nature; 480(7378): 547–551, which is incorporated herein by reference.

[0112] In some embodiments, dopaminergic cell populations are prepared by differentiating stem cells into midbrain dopaminergic neurons or their progenitors via 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 WO2016196661A1, each reference incorporated herein by reference. In some embodiments, the Wnt signaling pathway is activated by contacting cells with a Wnt activator. In some embodiments, the Wnt activator comprises a GSK3 inhibitor. In some embodiments, the Wnt activator comprises CHIR99021, referred to as Chir. In some embodiments, a dopaminergic cell population is prepared by contacting stem cells with a Wnt activator at an initial concentration of about 0.7 μM Chir, followed by increasing the concentration to about 7.0 μM Chir on about day 3 of differentiation, thereby differentiating the stem cells into midbrain dopaminergic neurons or their progenitors. In some embodiments, differentiation is performed using a fully defined basal medium. In some embodiments, the basal medium comprises E8 medium.

[0113] In some embodiments, the concentration of the at least one Wnt activator is increased during its exposure to cells. In some embodiments, the increase in the concentration of the at least one Wnt activator begins approximately 4 days after the initial exposure of stem cells to the at least one SMAD inhibitor. In some embodiments, the concentration of the at least one Wnt activator is increased by approximately 300% to approximately 1000%. In some embodiments, cells are exposed to the at least one Wnt activator at an increased concentration for at least approximately 7 days. In some embodiments, at least one additional Wnt activator is added to increase the total concentration of Wnt activator.

[0114] In some implementations, differentiating stem cells into dopaminergic cells also includes contacting the cells with at least one fibroblast growth factor (FGF) signaling activator (also known as an FGF activator).

[0115] In some embodiments, initial contact of cells with the at least one FGF signaling activator occurs at least 5 days after initial contact with the at least one SMAD signaling inhibitor. In some embodiments, initial cell exposure to the at least one FGF activator occurs at least 10 days after initial exposure of stem cells to the at least one SMAD inhibitor. In some embodiments, cell exposure to the at least one FGF activator prolongs EN1 expression in dopaminergic cells.

[0116] In some implementations, differentiating stem cells into dopaminergic cells also includes contacting the cells with dopaminergic cell lineage-specific activators or inhibitors, such as BDNF, GDNF, cAMP, TGFP, ascorbic acid (AA), and / or DAPT.

[0117] (i) stem cells

[0118] In some embodiments, the stem cell is a pluripotent stem cell. In some embodiments, the pluripotent stem cell is an embryonic stem cell (ESC), an induced pluripotent stem cell (iPSC), or a combination thereof. In some embodiments, the stem cell is a pluripotent stem cell. Non-limiting examples of stem cells that can be used to generate dopaminergic cell populations include, but are not limited to, non-embryonic stem cells, embryonic stem cells, induced non-embryonic pluripotent cells, and engineered pluripotent cells from humans, non-human primates, or rodents. In some embodiments, the stem cell is a human stem cell. Non-limiting examples of human stem cells include human embryonic stem cells (hESC), human pluripotent stem cells (hPSC), human induced pluripotent stem cells (hiPSC), human parthenogenetic stem cells, primordial germ cell-like pluripotent stem cells, ectodermal stem cells, class F pluripotent stem cells, adult stem cells, cancer stem cells, or any other cells capable of lineage-specific differentiation. In some embodiments, the stem cell is a human embryonic stem cell (hESC). In some embodiments, the stem cell is a human induced pluripotent stem cell (hiPSC).

[0119] In some embodiments, stem cells or their progeny cells contain introduced heterologous nucleic acids, wherein said nucleic acids may encode a desired nucleic acid or protein product or have informational value (see, for example, U.S. Patent 6,312,911, which is incorporated herein by reference in its entirety). Non-limiting examples of protein products include biomarkers detectable via in vivo imaging studies, such as receptors or other cell membrane proteins. Non-limiting examples of biomarkers include fluorescent proteins (such as green fluorescent protein (GFP), blue fluorescent proteins (EBFP, EBFP2, azurite, mKalamal), cyan fluorescent proteins (ECFP, Cerulean, CyPet, mTurquoise2), and yellow fluorescent protein derivatives (YFP, Citrine, Venus, YPet, EYFP)), β-galactosidase (LacZ), chloramphenicol acetyltransferase (cat), neomycin phosphotransferase (neo), enzymes (such as oxidases and peroxidases), and antigen molecules. In some embodiments, the reporter factor may be driven by a recombinant promoter of a dopaminergic cell biomarker gene (e.g., NURR1).

[0120] (ii) SMAD inhibitors

[0121] In some embodiments, the at least one SMAD inhibitor comprises at least one TGFp / activin-Nodal inhibitor. In some embodiments, the at least one TGFp / activin-Nodal inhibitor is selected from ALK5 inhibitors, ALK4 inhibitors, ALK7 inhibitors, and combinations thereof. In some embodiments, the TGFp / activin-Nodal inhibitor comprises an ALK5 inhibitor. In some embodiments, the TGFp / activin-Nodal inhibitor is a small molecule selected from SB431542, its derivatives, and mixtures thereof. In some embodiments, the TGFp / activin-Nodal inhibitor comprises SB431542. In some embodiments, the TGFp / activin-Nodal inhibitor comprises a derivative of SB431542. In some embodiments, the derivative of SB431542 is A83-01.

[0122] In some embodiments, at least one SMAD inhibitor comprises at least one BMP inhibitor. In some embodiments, the at least one BMP inhibitor comprises a small molecule selected from LDN193189, Noggin, dorsomorphin, derivatives thereof, and mixtures thereof. In some embodiments, the at least one BMP inhibitor comprises LDN193189. In some embodiments, the at least one BMP inhibitor comprises Noggin.

[0123] In some embodiments, stem cells are exposed to a SMAD inhibitor, such as a TGFp / activin-Nodal inhibitor. In some embodiments, the TGFp / activin-Nodal inhibitor is SB431542 or A83-01. In some embodiments, 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, stem cells are exposed to SB431542 or A83-01, and LDN193189 or Noggin. In some embodiments, stem cells are exposed to SB431542 and Noggin.

[0124] In some embodiments, 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, stem cells are exposed to or contacted with the at least one SMAD inhibitor for up to about 5 days, or up to about 10 days. In some embodiments, stem cells are exposed to or contacted with the at least one SMAD inhibitor for about 5 days to about 10 days. In some embodiments, stem cells are exposed to or contacted with the at least one SMAD inhibitor for about 5 days. In some embodiments, stem cells are exposed to or contacted with the at least one SMAD inhibitor for 7 days. In some embodiments, cells are exposed to or contacted with the at least one SMAD inhibitor from day 0 to day 6. In some embodiments, the at least one SMAD inhibitor is added to the cell culture medium containing stem cells daily or every other day from day 0 to day 6.

[0125] In some embodiments, cells are contacted or exposed to a TGFp / activin-Nodal inhibitor. In some embodiments, the concentration of the TGFp / activin-Nodal inhibitor in contact with or exposure to cells is about 1 μM to about 20 μM, about 1 μM to about 10 μM, about 1 μM to about 15 μM, about 10 μM to about 15 μM, about 5 μM to about 10 μM, about 5 μM to about 15 μM, about 5 μM to about 20 μM, or about 15 μM to about 20 μM. In some embodiments, the concentration of the TGFp / activin-Nodal inhibitor in contact with or exposure to cells is about 1 μM to about 10 μM. In some embodiments, the concentration of the TGFp / activin-Nodal inhibitor in contact with or exposure to cells is about 5 μM to about 10 μM. In some embodiments, the concentration of the TGFp / activin-Nodal inhibitor in contact with or exposure to cells is about 10 μM. 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.

[0126] In some embodiments, cells are contacted or exposed to a BMP inhibitor. In some embodiments, the concentration of the BMP inhibitor in which cells are contacted or exposed is about 50 nM to about 500 nM, or about 100 nM to about 500 nM, or about 200 nM to about 500 nM, or about 200 nM to about 300 nM, or about 200 nM to about 400 nM, or about 100 nM to about 250 nM, or about 100 nM to about 250 nM, or about 200 nM to about 250 nM, or about 250 nM to about 300 nM. In some embodiments, the concentration of the BMP inhibitor in which cells are contacted or exposed is about 200 nM to about 300 mM. In some embodiments, the concentration of the BMP inhibitor in which cells are contacted or exposed 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 exposed to or contacted with 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 head protein. In some embodiments, cells are exposed to or contacted with Noggin. In some embodiments, the concentration of Noggin exposed to or contacted with cells is about 200 ng / mL to about 800 ng / mL, or 300 ng / mL to about 700 ng / mL, or 400 ng / mL to about 600 ng / mL. In some embodiments, the concentration of Noggin exposed to or contacted with cells is about 300 ng / mL, about 400 ng / mL, about 500 ng / mL, about 600 ng / mL, or about 600 ng / mL.

[0127] In some embodiments, cells are simultaneously exposed to or induced to both a TGFp / activator-Nodal inhibitor and a BMP inhibitor. In some embodiments, stem cells are exposed to or induced to both a TGFp / activator-Nodal inhibitor and a BMP inhibitor for 7 days. In some embodiments, cells are exposed to or induced to both a TGFp / activator-Nodal inhibitor and a BMP inhibitor from day 0 to day 6. In some embodiments, the TGFp / activator-Nodal inhibitor and a BMP inhibitor are added to the cell culture medium containing stem cells daily or every other day from day 0 to day 6. In some embodiments, the TGFp / activator-Nodal inhibitor and a BMP inhibitor are added to the cell culture medium containing stem cells daily from day 0 to day 6.

[0128] (iii) Wnt activator

[0129] In some embodiments, the at least one Wnt activator reduces GSK3P to activate Wnt signaling. Therefore, 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, Wnt1, Wnt5a, BIO, CHIR98014, lithium, 3F8, their derivatives, 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.

[0130] In some embodiments, cells are exposed to or contacted with 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, cells are exposed to or contacted with the at least one Wnt activator for at most about 5 days, at most about 10 days, at most about 15 days, or at most about 20 days. In some embodiments, cells are exposed to or contacted with the at least one Wnt activator for about 5 days to about 20 days, about 5 days to about 15 days, about 10 days to about 20 days, about 5 days to about 15 days, or about 10 days to about 15 days.

[0131] In some embodiments, cells are exposed to at least one Wnt activator for about 10 to about 15 days. In some embodiments, cells are exposed to at least one Wnt activator for about 10 days. In some embodiments, stem cells are exposed to at least one Wnt signaling activator for 12 days.

[0132] In some embodiments, the cells are contacted with the at least one Wnt activator from day 0 to day 11. In some embodiments, the at least one Wnt activator is added to the cell culture medium containing the cells daily or every other day from day 0 to day 11. In some embodiments, the at least one Wnt activator is added to the cell culture medium containing the cells daily from day 0 to day 11.

[0133] In some embodiments, the concentration of the at least one Wnt activator is increased during cell exposure (also referred to as "Wnt enhancement"). In some embodiments, the increase or Wnt enhancement begins at least about 2 days, at least about 4 days, or at least about 5 days after initial cell exposure to the at least one Wnt activator. In some embodiments, the increase or Wnt enhancement begins about 4 days after initial cell exposure to the at least one Wnt activator.

[0134] In some embodiments, cells are exposed to or contacted with an increased concentration of the at least one Wnt activator for at least about 5 days or at least about 10 days. In some embodiments, cells are exposed to or contacted with an increased concentration of the at least one Wnt activator for at least about 5 days. In some embodiments, cells are exposed to an 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, cells are exposed to an increased concentration of the at least one Wnt activator for up to about 10 days.

[0135] In some embodiments, cells are exposed to or in increased concentrations of the at least one Wnt activator for about 5 to about 15 days, or about 5 to about 10 days, or about 10 to about 15 days. In some embodiments, cells are exposed to or in increased concentrations of the at least one Wnt activator for about 5 to about 10 days. In some embodiments, cells are exposed to or in increased concentrations of the at least one Wnt activator for about 5 days, about 10 days, or about 15 days. In some embodiments, cells are exposed to or in increased concentrations of the at least one Wnt activator for about 5 days. In some embodiments, cells are exposed to or in increased concentrations of the at least one Wnt activator for 6 days. In some embodiments, cells are exposed to or in increased concentrations of the at least one Wnt activator from day 4 to day 9. In some embodiments, cells are exposed to or in increased concentrations of the at least one Wnt activator for about 10 days. In some embodiments, cells are exposed to or in increased concentrations of the at least one Wnt activator for 8 days. In some implementations, from day 4 to day 11, cells are exposed to or in contact with an increased concentration of the at least one Wnt activator.

[0136] In some embodiments, the initial concentration of the at least one Wnt activator that contacts or exposes the cells prior to Wnt enhancement is less than about 5 μM, less than about 3 μM, or less than about 1 μM, including but not limited to about 0.01 μM to about 5 μM, about 0.01 μM to about 3 μM, about 0.05 μM to about 3 μM, about 0.1 μM to about 3 μM, about 0.5 μM to about 3 μM, about 0.5 μM to about 2 μM, or about 0.5 μM to about 1 μM. In some embodiments, the initial concentration of the at least one Wnt activator that contacts or exposes the cells prior to Wnt enhancement is less than about 1 μM, for example, about 0.1 μM, about 0.2 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, or about 1 μM. In some embodiments, the initial concentration of the at least one Wnt activator exposed to the cells prior to Wnt enhancement is about 0.5 μM. In some embodiments, the initial concentration of the at least one Wnt activator exposed to the cells prior to Wnt enhancement is about 0.7 μM.

[0137] In some embodiments, the increased concentration of the at least one Wnt activator after Wnt enhancement is about 3 μM or higher, about 5 μM or higher, about 10 μM or higher, about 15 μM or higher, or about 20 μM or higher. In some embodiments, the increased concentration of the at least one Wnt activator after Wnt enhancement is about 3 μM to about 15 μM, about 3 μM to about 10 μM, or about 5 μM to about 10 μM. In some embodiments, the increased concentration of the at least one Wnt activator after Wnt enhancement is about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM, about 5 μM, about 5.5 μM, about 6 μM, about 6.5 μM, about 7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM, or about 10 μM. In some embodiments, the increased concentration of the at least one Wnt activator after Wnt enhancement is about 3 μM. In some embodiments, the concentration of the at least one Wnt activator increases by about 7 μM after Wnt enhancement. In some embodiments, the concentration of the at least one Wnt activator increases by about 7.5 μM after Wnt enhancement.

[0138] In some embodiments, the concentration of the at least one Wnt activator is increased from the initial concentration upon contact or exposure to cells by about 50% to about 2000%, or about 100% to about 1500%, or about 150% to about 1500%, or about 200% to about 1500%, or about 250% to about 1500%, or about 300% to about 1500%, or about 300% to about 1000%, or about 300% to about 400%, or about 500% to about 1000%, or about 800% to about 1000%, or about 900% to about 1000%, or about 950% to about 1000%. In some embodiments, the concentration of the at least one Wnt activator is increased from the initial concentration upon contact or exposure to cells by about 300% to about 1000%. In some embodiments, the concentration of the at least one Wnt activator is increased from the initial concentration upon contact or exposure to cells by about 300% to about 400%. In some embodiments, the concentration of the at least one Wnt activator is increased from an initial concentration upon contact or exposure to cells by about 900% to about 1000%. In some embodiments, the concentration of the at least one Wnt activator is increased from an initial concentration upon contact or exposure to 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 an initial concentration upon contact or exposure to cells by about 300%. In some embodiments, the concentration of the at least one Wnt activator is increased from an initial concentration upon contact or exposure to cells by about 350%. In some embodiments, the concentration of the at least one Wnt activator is increased from an initial concentration upon contact or exposure to cells by about 950%. In some embodiments, the concentration of the at least one Wnt activator is increased by about 1000% from the initial concentration at which the cell is exposed or contacted.

[0139] (iv) SHH activator

[0140] In some embodiments, the SHH activator includes the SHH protein, a PTC antagonist, a smoothing agent (SMO) agonist, or a combination thereof. In some embodiments, the SHH protein includes recombinant SHH, purified SHH, or a combination thereof. In some embodiments, the recombinant SHH is the N-terminal fragment of SHH. In some embodiments, the recombinant SHH is SHH C25II. In some embodiments, the SMO agonist includes purmorphamine.

[0141] In some embodiments, cells are exposed to or contacted with the at least one SHH activator for at least about 5 days or at least about 10 days. In some embodiments, cells are exposed to or contacted with the at least one SHH activator for up to about 5 days or up to about 10 days. In some embodiments, cells are exposed to or contacted with the at least one SHH activator for about 5 days to about 10 days. In some embodiments, cells are exposed to or contacted with the at least one SHH activator for about 5 days. In some embodiments, cells are exposed to or contacted with the at least one SHH activator for 7 days. In some embodiments, cells are exposed to or contacted with the at least one SHH activator from day 0 to day 6. In some embodiments, the at least one SHH activator is added to the cell culture medium containing cells daily from day 0 to day 6.

[0142] In some embodiments, the concentration of the at least one SHH activator exposed to or contacting the cells is about 50 ng / mL to about 1000 ng / mL, about 100 ng / mL to about 1000 ng / mL, about 20 ng / mL to about 1000 ng / mL, about 300 ng / mL to about 1000 ng / mL, about 400 ng / mL to about 1000 ng / mL, about 500 ng / mL to about 1000 ng / mL, about 400 ng / mL to about 800 ng / mL, about 400 ng / mL to about 700 ng / mL, about 400 ng / mL to about 600 ng / mL, or about 500 ng / mL to about 600 ng / mL. In some embodiments, the concentration of the at least one SHH activator exposed to or contacting the cells is about 400 ng / mL to about 600 ng / mL. In some embodiments, the concentration of the at least one SHH activator exposed to or contacting 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 exposed to or contacting the cells is about 500 ng / mL.

[0143] (v)FGF activator

[0144] In some embodiments, the FGF activator can induce midbrain amplification and upregulate midbrain gene expression. In some embodiments, the FGF activator is selected from FGF8a, FGF17, FGF18, FGF2, FGF4, their derivatives, and combinations thereof. In some embodiments, the FGF activator includes or includes FGF18.

[0145] In some embodiments, cells are exposed to or contacted with 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, cells are exposed to or contacted with the at least one FGF activator for at most about 5 days, or at most about 10 days, or at most about 15 days, or at most about 20 days. In some embodiments, cells are exposed to or contacted with the at least one FGF activator for about 1 day to about 20 days, about 1 day to about 15 days, or about 5 days to about 20 days, or about 5 days to about 15 days, or about 5 days to about 10 days, or about 10 days to about 20 days. In some embodiments, cells are exposed to or contacted with the at least one FGF activator for about 5 days to about 10 days. In some embodiments, cells are exposed to or contacted with the at least one FGF activator for about 3 days, about 5 days, or about 8 days. In some embodiments, cells are exposed to or contacted with the at least one FGF activator for about 5 days.

[0146] In some embodiments, the initial contact or initial exposure of cells with the at least one FGF activator is at least about 5 days or at least about 10 days from the initial contact or initial exposure of cells with the at least one SMAD inhibitor. In some embodiments, the initial contact or initial exposure of cells with 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 or initial exposure of cells with the at least one SMAD inhibitor. In some embodiments, the initial contact or initial exposure of cells with 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 or initial exposure of cells with the at least one SMAD inhibitor. In some embodiments, the initial contact or initial exposure of cells with the at least one FGF activator is between approximately 5 and 10 days from the initial contact or initial exposure of cells with the at least one SMAD inhibitor. In some embodiments, the initial contact or initial exposure of cells with the at least one FGF activator is approximately 10 days from the initial contact or initial exposure of cells with the at least one SMAD inhibitor. In some embodiments, the initial contact or initial exposure of cells with the at least one FGF activator is 9 days from the initial contact or initial exposure of cells with the at least one SMAD inhibitor. In some embodiments, the initial contact or initial exposure of cells with the at least one FGF activator is 10 days from the initial contact or initial exposure of cells with the at least one SMAD inhibitor. In some embodiments, the initial contact or initial exposure of cells with the at least one FGF activator is 12 days from the initial contact or initial exposure of cells with the at least one SMAD inhibitor.

[0147] In some embodiments, the initial contact or initial exposure of cells with the at least one FGF activator begins approximately 5 days after the initial contact or initial exposure of cells with the at least one SMAD inhibitor, and the cells are then exposed to the at least one FGF activator for approximately 3 days. In some embodiments, the initial contact or initial exposure of cells with the at least one FGF activator begins approximately 5 days after the initial contact or initial exposure of cells with the at least one SMAD inhibitor, and the cells are then exposed to the at least one FGF activator for approximately 5 days. In some embodiments, the initial contact or initial exposure of cells with the at least one FGF activator begins approximately 10 days after the initial contact or initial exposure of cells with the at least one SMAD inhibitor, and the cells are then exposed to the at least one FGF activator for approximately 3 days. In some embodiments, the initial contact or initial exposure of cells with the at least one FGF activator begins approximately 10 days after the initial contact or initial exposure of cells with the at least one SMAD inhibitor, and the cells are then exposed to the at least one FGF activator for approximately 5 days. In some embodiments, the initial contact or initial exposure of cells with the at least one FGF activator begins 12 days after the initial contact or initial exposure of cells with the at least one SMAD inhibitor, and the cells are then exposed to the at least one FGF activator for 5 days.

[0148] In some embodiments, the concentration of the at least one FGF activator exposed to or contacting the cells is about 10 ng / mL to about 500 ng / mL, about 50 ng / mL to about 500 ng / mL, about 100 ng / mL to about 500 ng / mL, about 100 ng / mL to about 400 ng / mL, about 100 ng / mL to about 300 ng / mL, about 100 ng / mL to about 200 ng / mL, or about 100 ng / mL to about 250 ng / mL. In some embodiments, the concentration of the at least one FGF activator exposed to or contacting the cells is about 100 ng / mL to about 200 ng / mL. In some embodiments, the concentration of the at least one FGF activator exposed to or contacting the cells is about 100 ng / mL. In some embodiments, the concentration of the at least one FGF activator exposed to or contacting the cells is about 200 ng / mL.

[0149] In some non-limiting embodiments, stem cells are 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., at a concentration of about 500 ng / mL) for about 5 days (e.g., 7 days, e.g., from day 0 to day 6), and are exposed to at least one Wnt activator (e.g., CHIR99021, e.g., at a concentration of about 0.7 μM for 5 days (e.g., 4 days, e.g., from day 0 to day 3), at a concentration of about 7.5 μM for about 5 days (e.g., 6 days, e.g., from day 4 to day 9), and at a concentration of about 3 μM for about 2 days (e.g., from day 10 to day 11)). Optionally, cells are contacted or exposed to at least one FGF activator (e.g., FGF18, for example, at a concentration of about 100 ng / ml), wherein the initial contact of cells with the at least one FGF activator is about 10 days (e.g., 10 or 12 days) from the initial contact of cells with the at least one SMAD inhibitor, and cells are contacted with 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)).

[0150] (vi) Dopaminergic neuron lineage-specific activators and inhibitors

[0151] 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 β (TGFB, e.g., TGFB3), ascorbic acid (AA), and DAPT (also known as N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethyl ethyl ester; LY-374973, N-[N-(3,5-difluorophenylacetyl)-L-alanyl]-S-phenylglycine tert-butyl ester; or N-[N-(3,5-difluorophenylacetyl)-L-alanyl]-S-phenylglycine tert-butyl ester).

[0152] In some embodiments, cells are exposed to at least one dopaminergic neuron lineage-specific activator or inhibitor for at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, or at least about 10 days or more. In some embodiments, cells are exposed to at least one dopaminergic neuron lineage-specific activator or inhibitor for about 2 days to about 20 days, about 3 days to about 19 days, about 4 days to about 18 days, about 5 days to about 17 days, about 6 days to about 16 days, about 7 days to about 15 days, about 8 days to about 15 days, about 9 days to about 14 days, or about 10 days to about 13 days. In some embodiments, cells are exposed to at least one dopaminergic neuron lineage-specific activator or inhibitor for at most about 2 days, at most about 3 days, at most about 4 days, at most about 5 days, at most about 6 days, at most about 7 days, at most about 8 days, at most about 9 days, or at most about 10 days or more. In some implementations, the cells are exposed to at least one dopaminergic neuron lineage-specific activator or inhibitor for about 4, 5, 6, 7, or 8 days.

[0153] In some embodiments, cells are contacted with L-glutamine at concentrations of: about 0.5 mM to about 5 mM, or about 1 mM to about 5 mM, or about 1.5 mM to about 2.5 mM, about 1 mM to about 2 mM, about 0.5 mM to about 500 mM, or about 1 mM to about 300 mM, or about 1 mM to about 250 mM, or about 2 mM to about 200 mM. In some embodiments, cells are contacted with L-glutamine at a concentration of about 2 mM. In some embodiments, cells are contacted with L-glutamine at a concentration of about 20 mM. In some embodiments, cells are contacted with L-glutamine at a concentration of about 100 mM. In some embodiments, cells are contacted with L-glutamine at a concentration of about 200 mM. In some embodiments, cells are contacted with L-glutamine at a concentration of about 300 mM.

[0154] In some embodiments, cells are contacted with BDNF at concentrations of approximately 5 ng / mL to approximately 50 ng / mL, or approximately 10 ng / mL to approximately 50 ng / mL, or approximately 10 ng / mL to approximately 40 ng / mL, or approximately 20 ng / mL to approximately 50 ng / mL, or approximately 20 ng / mL to approximately 40 ng / mL, or approximately 10 ng / mL to approximately 30 ng / mL, or approximately 10 ng / mL to approximately 20 ng / mL, or approximately 20 ng / mL to approximately 30 ng / mL. In some embodiments, cells are contacted with BDNF at a concentration of approximately 20 ng / mL.

[0155] In some embodiments, cells are contacted with ascorbic acid (AA) at concentrations of about 50 nM to about 500 nM, or about 100 nM to about 500 nM, or about 100 nM to about 400 nM, or about 200 nM to about 400 nM, or about 200 nM to about 300 nM, or about 100 nM to about 300 nM. In some embodiments, cells are contacted with AA at a concentration of about 200 nM.

[0156] In some embodiments, cells are contacted with GDNF at concentrations of approximately 5 ng / mL to approximately 50 ng / mL, or approximately 10 ng / mL to approximately 50 ng / mL, or approximately 10 ng / mL to approximately 40 ng / mL, or approximately 20 ng / mL to approximately 50 ng / mL, or approximately 20 ng / mL to approximately 40 ng / mL, or approximately 10 ng / mL to approximately 30 ng / mL, or approximately 10 ng / mL to approximately 20 ng / mL, or approximately 20 ng / mL to approximately 30 ng / mL. In some embodiments, cells are contacted with GDNF at a concentration of approximately 20 ng / mL.

[0157] In some embodiments, cells are contacted with cAMP concentrations of approximately 200 μM to approximately 800 μM, or approximately 200 μM to approximately 700 μM, or approximately 300 μM to approximately 700 μM, or approximately 300 μM to approximately 600 μM, or approximately 400 μM to approximately 600 μM, or approximately 450 μM to approximately 550 μM. In some embodiments, cells are contacted with cAMP at a concentration of approximately 500 μM.

[0158] In some embodiments, cells are contacted with concentrations of TGFB3 of about 0.01 ng / mL to about 5 ng / mL, or about 0.1 ng / mL to about 4 ng / mL, or about 0.5 ng / mL to about 5 ng / mL, or about 1 ng / mL to about 3 ng / mL, or about 1 ng / mL to about 2 ng / mL. In some embodiments, cells are contacted with TGFP3 at a concentration of about 1 ng / mL.

[0159] (vii) Dopaminergic cell markers

[0160] In some embodiments, the method includes obtaining a population of 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 dopaminergic cells. Non-limiting examples of markers indicating dopaminergic cells include engrailed-1 (EN1), orthodenticle homeobox protein 2 (OTX2), tyrosine hydroxylase (TH), nuclear receptor-associated protein 1 (NURR1), forkhead box protein A2 (FOXA2), and LIM homeobox transcription factor 1α (LMX1A), PITX3, LMO3, SNCA, ADCAP1, CHRNA4, and GIRK2. In some embodiments, the at least one marker indicating dopaminergic cells is FOXA2.

[0161] In some embodiments, differentiated cells express at least one marker indicating dopaminergic cells for 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 time the cells are initially exposed to the at least one SMAD inhibitor.

[0162] In some embodiments, a population of dopaminergic cells is prepared by in vitro differentiation of stem cells. In some embodiments, the population of dopaminergic cells comprises midbrain dopaminergic neurons or precursors thereof. In some embodiments, the midbrain dopaminergic neurons or precursors thereof express one or more markers indicating midbrain dopaminergic neurons or precursors thereof. Non-limiting examples of markers indicating midbrain dopaminergic neurons or precursors thereof include engrailed-1 (EN1), orthodenticle homeobox protein 2 (OTX2), tyrosine hydroxylase (TH), nuclear receptor-associated 1 protein (NURR1), forkhead box protein A2 (FOXA2), and LIM homeobox transcription factor 1α (LMX1A), PITX3, LMO3, SNCA, ADCAP1, CHRNA4, and GIRK2. In some embodiments, the midbrain dopaminergic neurons or precursors thereof express a combination of markers indicating midbrain dopaminergic neurons or precursors thereof. In some embodiments, the combination of markers includes TH and FOXA2.

[0163] Treatment of cells with at least one FGF activator induces sustained expression of EN1. EN1 is a survival factor for midbrain dopaminergic neurons during development and continues to play a neuroprotective and physiological role in adult midbrain dopaminergic neurons. Therefore, cells with sustained EN1 expression can develop into functional dopaminergic cells upon further development and maturation. In some embodiments, differentiated cells exhibit detectable levels of EN1 expression for at least approximately 10 days, at least approximately 15 days, at least approximately 16 days, at least approximately 20 days, at least approximately 25 days, at least approximately 27 days, at least approximately 30 days, at least approximately 35 days, at least approximately 40 days, at least approximately 45 days, at least approximately 50 days, at least approximately 60 days, at least approximately 70 days, at least approximately 80 days, or at least approximately 90 days from the initial exposure of stem cells to the at least one SMAD inhibitor. In some embodiments, differentiated cells exhibit detectable levels of EN1 expression for approximately 30 days from the initial exposure of stem cells to the at least one SMAD inhibitor. In some implementations, approximately 40 days after initial contact of stem cells with the at least one SMAD inhibitor, the differentiated cells exhibit detectable levels of EN1 expression.

[0164] In some embodiments, dopaminergic cells derived from stem cells do not express or express at least one biomarker selected from the following: PAX6, EMX2, LHX2, SMA, CRABP1, Ki67, SIX1, PITX2, SIM1, POU4F1, PHOX2A, BARHL1, BARHL2, GBX2, HOXA2, HOXB2, POU5F1, NANOG, and combinations thereof. In some embodiments, dopaminergic cells derived from stem cells do not express or express at least PAX6, CRABP1, and / or Ki67.

[0165] (viii) Cell culture medium

[0166] In some embodiments, the aforementioned inhibitors and activators are added to the cell culture medium containing the cells. Suitable cell culture media include, but are not limited to, Knockout. ® Serum replacement (KSR) medium, Neurobasal ® Culture media (NB), N2 medium, B-27 medium, and Essential 8 ® / Essential 6 ® (E8 / E6) media and combinations thereof. KSR medium, NB medium, N2 medium, B-27 medium and E8 / E6 medium are commercially available. KSR medium is a defined serum-free preparation optimized to allow the growth and maintenance of undifferentiated hESCs in the culture.

[0167] In some embodiments, the cell culture medium is KSR medium. In some embodiments, KSR medium contains knockout DMEM, knockout serum substitute, L-glutamine, Pen / Strep, MEM, and 13-mercaptoethanol. In some embodiments, 1 liter of KSR medium contains 820 mL of knockout DMEM, 150 mL of knockout serum substitute, 10 mL of 200 mM L-glutamine, 10 mL of Pen / Strep, 10 mL of 10 mM MEM, and 55 mM 13-mercaptoethanol.

[0168] In some embodiments, the cell culture medium is E8 / E6 medium. In some embodiments, the E8 / E6 cell culture medium contains DMEM / F12, ascorbic acid, selenium, insulin, NaHCO3, transferrin, FGF2, and TGFp. The E8 / E6 medium differs from KSR medium in that it does not contain active BMP or Wnt components. Therefore, in some embodiments, when using E8 / E6 medium to culture the stem cell populations disclosed in this invention to differentiate into dopaminergic cell populations, it is not necessary to add at least one SMAD signaling inhibitor (e.g., those that inhibit BMP) to the E8 / E6 medium.

[0169] Selection and characterization of dopaminergic cells

[0170] In some implementations, dopaminergic cell populations (e.g., the dopaminergic cell populations described herein) are selected and characterized after differentiation from stem cells.

[0171] In some embodiments, selecting a dopaminergic cell population includes isolating and propagating cells expressing at least one dopaminergic cell marker gene. In some embodiments, selecting a dopaminergic cell population includes at least partially selecting dopaminergic cells expressing FOXA2. Other dopaminergic cell marker genes that can be detected include, but are not limited to, EN1, OTX2, TH, NURR1, LMX1A, PITX3, LMO3, SNCA, ADCAP1, CHRNA4, and GIRK2. The 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.

[0172] In some embodiments, selecting a dopaminergic cell population also includes isolating and propagating cells that do not express detectable levels of at least one marker gene selected from the following: PAX6, EMX2, LHX2, SMA, SIX1, CRABP1, Ki67, PITX2, SIM1, POU4F1, PHOX2A, BARHL1, BARHL2, GBX2, HOXA2, HOXB2, POU5F1, NANOG, or any combination thereof.

[0173] In some embodiments, selecting a dopaminergic cell population includes isolating and propagating cells expressing at least one selective biomarker gene. The expression of the selective biomarker gene can be detected by any suitable method known in the art, such as FACS or by culturing in a selective medium. Non-limiting examples of selective biomarker genes include, but are not limited to, fluorescent proteins (such as green fluorescent protein (GFP), blue fluorescent proteins (EBFP, EBFP2, azurite, mKalamal), cyan fluorescent proteins (ECFP, Cerulean, CyPet, mTurquoise2), and yellow fluorescent protein derivatives (YFP, Citrine, Venus, YPet, EYFP)), β-galactosidase (LacZ), chloramphenicol acetyltransferase (cat), neomycin phosphotransferase (neo), enzymes (such as oxidases and peroxidases), and antigen molecules. In some embodiments, the selective biomarker gene may be driven by a promoter of a dopaminergic cell biomarker gene.

[0174] In some embodiments, characterizing a dopaminergic cell population includes assessing the viability of the dopaminergic cell population. Cell viability can be assessed by any suitable method known in the art. For example, cell viability can be assessed by morphological analysis or by staining the cells with a solution such as a staining solution containing acridine orange and propidium iodide (AO / PI) to identify dead cells.

[0175] In some embodiments, characterizing dopaminergic cell populations includes determining the ability of dopaminergic cells to produce dopamine. Methods for detecting protein production (e.g., dopamine production) in cells are well known in the art. Non-limiting examples include, but are not limited to, immunoassays, enzyme immunoassays (EIA), radioimmunoassays (RIA), antigen capture assays, double-antibody sandwich assays, Western blot analysis, enzyme-linked immunosorbent assays (ELISA), colorimetric assays, chemiluminescence assays, fluorescence assays, immunohistochemistry, 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 ability of dopaminergic cells to produce dopamine is determined in vitro using liquid chromatography-tandem mass spectrometry (LC-MS / MS). In some embodiments, determining the ability of dopaminergic cells to produce dopamine includes determining the area under the concentration-time curve (AUC) produced by in vitro LC-MS / MS.

[0176] III. Composition

[0177] Certain aspects of this disclosure provide therapeutic compositions comprising dopaminergic cell populations. In some embodiments, the therapeutic composition comprises an effective amount of dopaminergic cell populations for treating Parkinson's disease and a delivery solution.

[0178] In some embodiments, the therapeutic composition comprises an effective amount of a population of dopaminergic cells (e.g., the dopaminergic cell population described herein). In some embodiments, the population of dopaminergic cells comprises dopaminergic neurons. In some embodiments, the dopaminergic neurons comprise one or more of midbrain dopaminergic neurons, lamina-derived dopaminergic neurons, and true midbrain dopaminergic neurons. In some embodiments, the population of dopaminergic cells comprises dopaminergic progenitor cells or dopaminergic precursor cells. In some embodiments, the dopaminergic cells comprise midbrain dopaminergic neurons or their progenitor cells. In some embodiments, the midbrain dopaminergic neurons or their progenitor cells are derived from midbrain lamina progenitor cells, whereby the midbrain lamina progenitor cells are derived from in vitro stem cells.

[0179] In some embodiments, the therapeutic composition comprises a population of FOXA2-positive dopaminergic cells. 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 dopaminergic cell population is FOXA2-positive. In some embodiments, at least 80% of the dopaminergic cell population is FOXA2-positive. In some embodiments, at least 90% of the dopaminergic cell population is FOXA2-positive. In some embodiments, at least 95% of the dopaminergic cell population is FOXA2-positive. In some embodiments, at least 98% of the dopaminergic cell population is FOXA2-positive. In some embodiments, at least 99% of the dopaminergic cell population is FOXA2-positive.

[0180] In some embodiments, the therapeutic composition comprises a TH-positive dopaminergic cell population. 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 dopaminergic cell population are TH-positive. In some embodiments, about 10% of the dopaminergic cell population is TH-positive. In some embodiments, about 15% of the dopaminergic cell population is TH-positive. In some embodiments, about 17% of the dopaminergic cell population is TH-positive.

[0181] In some embodiments, the therapeutic composition comprises a dopaminergic cell population, wherein only a certain percentage of the dopaminergic cell population is PAX2 positive. 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 dopaminergic cell population is PAX2 positive. In some embodiments, less than about 10% of the dopaminergic cell population is PAX2 positive. In some embodiments, less than about 5% of the dopaminergic cell population is PAX2 positive. In some embodiments, less than about 4% of the dopaminergic cell population is PAX2 positive. In some embodiments, less than about 3% of the dopaminergic cell population is PAX2 positive. In some embodiments, less than about 2% of the dopaminergic cell population is PAX2 positive. In some embodiments, less than about 1% of the dopaminergic cell population is PAX2 negative.

[0182] In some embodiments, the therapeutic composition comprises a dopaminergic cell population, wherein only a certain percentage of the dopaminergic cell population is CRABP1 positive. 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 dopaminergic cell population is CRABP1 positive. In some embodiments, less than about 10% of the dopaminergic cell population is CRABP1 positive. In some embodiments, less than about 5% of the dopaminergic cell population is CRABP1 positive. In some embodiments, less than about 4% of the dopaminergic cell population is CRABP1 positive. In some embodiments, less than about 3% of the dopaminergic cell population is CRABP1 positive. In some embodiments, less than about 2% of the dopaminergic cell population is CRABP1 positive. In some embodiments, less than about 1% of the dopaminergic cell population is CRABP1 positive. In some implementations, the dopaminergic cell population is CRABP1 negative.

[0183] In some embodiments, the therapeutic composition comprises a population of dopaminergic cells, wherein only a certain percentage of the dopaminergic cell population is Ki67 positive. 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 dopaminergic cell population is Ki67 positive. In some embodiments, less than about 30% of the dopaminergic cell population is Ki67 positive. In some embodiments, less than about 20% of the dopaminergic cell population is Ki67 positive. In some embodiments, less than about 15% of the dopaminergic cell population is Ki67 positive. In some embodiments, less than about 12% of the dopaminergic cell population is Ki67 positive. In some embodiments, less than about 10% of the dopaminergic cell population is Ki67 positive. In some implementations, less than about 5% of the dopaminergic cell population is Ki67 positive. In some implementations, the dopaminergic cell population is Ki67 negative.

[0184] In some embodiments, the therapeutic composition comprises a population of dopaminergic cells, which 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 are viable. In some embodiments, about 50% to about 100% of the dopaminergic cells in the population are viable. In some embodiments, about 60% to about 90% of the dopaminergic cells in the population are viable. In some embodiments, about 70% to about 80% of the dopaminergic cells in the population 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 dopaminergic cell population are viable. In some embodiments, at least about 90% of the dopaminergic cells in the dopaminergic cell population are viable. In some embodiments, at least about 80% of the dopaminergic cells in the dopaminergic cell population are viable. In some embodiments, at least about 70% of the dopaminergic cells in the dopaminergic cell population are viable. In some embodiments, at least about 50% of the dopaminergic cells in the dopaminergic cell population are viable.

[0185] In some embodiments, the therapeutic composition comprises a population of dopaminergic cells capable of producing dopamine. In some embodiments, the population of dopaminergic cells is capable of 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 is capable of producing dopamine at a rate of at least about 20 ng / day / mL. In some embodiments, the population of dopaminergic cells is capable of producing dopamine at a rate of at least about 15 ng / day / mL. In some embodiments, the population of dopaminergic cells is capable of producing dopamine at a rate of at least about 11.2 ng / day / mL. In some embodiments, the population of dopaminergic cells is capable of producing dopamine at a rate of at least about 8 ng / day / mL.

[0186] In some embodiments, the dopaminergic cell population has the ability to produce dopamine, and when evaluated in vitro using liquid chromatography-tandem mass spectrometry (LC-MS / MS), the ability to produce dopamine provides a concentration-time area (AUC) 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 dopaminergic cell population has the ability to produce dopamine, and when evaluated in vitro using liquid chromatography-tandem mass spectrometry (LC-MS / MS), the ability to produce dopamine provides a concentration-time area (AUC) of at least about 20 ng / day / mL. In some embodiments, the dopaminergic cell population has the ability to produce dopamine, and when evaluated in vitro using liquid chromatography-tandem mass spectrometry (LC-MS / MS), the ability to produce dopamine provides a concentration-time area (AUC) of at least about 15 ng / day / mL. In some embodiments, the dopaminergic cell population has the ability to produce dopamine, and when evaluated in vitro using liquid chromatography-tandem mass spectrometry (LC-MS / MS), the ability to produce dopamine provides a concentration-time area (AUC) of at least about 11.2 ng / day / mL. In some embodiments, the dopaminergic cell population has the ability to produce dopamine, and when evaluated in vitro using liquid chromatography-tandem mass spectrometry (LC-MS / MS), the ability to produce dopamine provides a concentration-time area (AUC) of at least about 8 ng / day / mL.

[0187] The number of dopaminergic cells in the therapeutic composition can be any suitable and effective number. In some embodiments, the therapeutic composition comprises a population of dopaminergic cells including about 1×10^4 to about 1×10^10 dopaminergic cells, about 1×10^4 to about 1×10^5 dopaminergic cells, about 1×10^5 to about 1×10^9 dopaminergic cells, about 1×10^5 to about 1×10^6 dopaminergic cells, and about 1×10^5 to about 1×10^7 dopaminergic cells. 10^6 to about 10^7 dopaminergic cells, about 10^6 to about 10^8 dopaminergic cells, about 10^7 to about 10^8 dopaminergic cells, about 10^8 to about 10^9 dopaminergic cells, about 10^8 to about 10^10 dopaminergic cells, or about 10^9 to about 10^10 dopaminergic cells. In some implementations, the cell population includes at least 250,000 dopaminergic cells, at least 500,000 dopaminergic cells, at least 750,000 dopaminergic cells, at least 1,000,000 dopaminergic cells, at least 1,250,000 dopaminergic cells, at least 1,500,000 dopaminergic cells, at least 1,750,000 dopaminergic cells, at least 2,000,000 dopaminergic cells, at least 2,250,000 dopaminergic cells, at least 2,500,000 dopaminergic cells, at least 2,750,000 dopaminergic cells, and at least 3,000,000 dopaminergic cells. Cells, 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 cells, at least 8 million dopaminergic cells, at least 9 million dopaminergic cells, or at least 10 million or more dopaminergic cells.

[0188] In some embodiments, the therapeutic composition comprises a population of dopaminergic cells comprising at least about 1.0 × 10^6 to about 1.2 × 10^7 dopaminergic cells. In some embodiments, the therapeutic composition comprises a population of dopaminergic cells comprising at least about 900,000 dopaminergic cells. In some embodiments, the therapeutic composition comprises a population of dopaminergic cells comprising at least about 1.8 million dopaminergic cells. In some embodiments, the therapeutic composition comprises a population of dopaminergic cells comprising at least about 2.7 million dopaminergic cells. In some embodiments, the therapeutic composition comprises a population of dopaminergic cells comprising at least about 5.4 million dopaminergic cells. In some embodiments, the therapeutic composition comprises a population of dopaminergic cells comprising more than 5.4 million dopaminergic cells.

[0189] In some embodiments, the therapeutic composition comprises a population of dopaminergic cells at concentrations of about 71,000 cells / µL to about 123,000 cells / µL, about 75,000 cells / µL to about 115,000 cells / µL, about 80,000 cells / µL to about 110,000 cells / µL, about 85,000 cells / µL to about 105,000 cells / µL, or about 90,000 cells / µL to about 100,000 cells / µL. In some embodiments, the concentration of the dopaminergic cell population is about 71,000 cells / µL to about 123,000 cells / µL. In some implementations, the concentration of dopaminergic cell population is approximately 75,000 cells / µL, approximately 80,000 cells / µL, approximately 85,000 cells / µL, approximately 90,000 cells / µL, approximately 95,000 cells / µL, approximately 100,000 cells / µL, approximately 105,000 cells / µL, approximately 110,000 cells / µL, approximately 115,000 cells / µL, or approximately 120,000 cells / µL. In some embodiments, the concentration of the dopaminergic cell population is approximately 75,000 cells / µL, approximately 80,000 cells / µL, approximately 85,000 cells / µL, approximately 90,000 cells / µL, approximately 95,000 cells / µL, approximately 100,000 cells / µL, approximately 105,000 cells / µL, approximately 110,000 cells / µL, approximately 115,000 cells / µL, or approximately 120,000 cells / µL. In some embodiments, the concentration of the dopaminergic cell population is approximately 90,000 cells / µL. In some embodiments, the concentration of the dopaminergic cell population is approximately 100,000 cells / µL. In some embodiments, the concentration of the dopaminergic cell population is approximately 110,000 cells / µL.

[0190] In some embodiments, the therapeutic composition comprises an effective amount of therapeutic cell populations for treating a subject with Parkinson's disease. In some embodiments, the Parkinson's disease is early-stage Parkinson's disease. In some embodiments, the Parkinson's disease is late-stage Parkinson's disease. In some embodiments, the subject is a human being.

[0191] In some embodiments, the therapeutic composition comprises a population of dopaminergic cells allogeneic to the subject to which the therapeutic composition is administered. In some embodiments, the therapeutic composition comprises a population of dopaminergic cells autologous to the subject to which the therapeutic composition is administered.

[0192] Delivery solution

[0193] In some embodiments, the therapeutic composition comprises a population of dopaminergic cells (e.g., the dopaminergic cell population described herein) and a delivery solution comprising one or more energy components, one or more pH buffers, one or more salts, one or more stabilizers, or any combination thereof, such as the delivery solution described in International Application PCT / US23 / 21961, which is incorporated herein by reference.

[0194] In some embodiments, the therapeutic composition comprises a population of dopaminergic cells (e.g., the dopaminergic cell population described herein) and a delivery solution comprising one or more energy components. The energy component is any component capable of providing chemical energy to one or more cells. In some embodiments, the one or more energy components include sugars. Exemplary sugars that may 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 may contain any amount of one or more energy sources to achieve the desired effect (e.g., treatment of Parkinson's disease). In some embodiments, the delivery solution comprises one or more energy sources at concentrations 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, and about 23.7 mM to about 25.6 mM. In some embodiments, the delivery solution contains one or more energy sources with concentrations 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 contains one or more energy sources with concentrations of about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM, or higher.

[0195] In some embodiments, the therapeutic composition comprises a population of dopaminergic cells (e.g., the dopaminergic cell population described herein) and a delivery solution comprising one or more pH buffers. The one or more pH buffers may comprise any suitable buffer, such as a zwitterionic organic chemical buffer, examples of which include, but are not limited to, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), sodium bicarbonate, 4-morpholinopropanesulfonic acid, 3-propanesulfonic acid (MOPS), and 2-(N-morpholino)ethanesulfonic acid (MES). The delivery solution may comprise any amount of one or more pH buffers to achieve the desired effect (e.g., treatment of a subject with Parkinson's disease). In some embodiments, the delivery solution comprises one or more pH buffers at concentrations 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 comprises one or more pH buffers at concentrations 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 comprise one or more of 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), sodium bicarbonate, 4-morpholinopropanesulfonic acid, 3-propanesulfonic acid (MOPS), and 2-(N-morpholino)ethanesulfonic acid (MES).

[0196] In some embodiments, the therapeutic composition comprises a population of dopaminergic cells (e.g., the dopaminergic cell population described herein) and a delivery solution comprising one or more salts. In some embodiments, the one or more salts comprise one or more of calcium chloride, magnesium chloride, potassium chloride, sodium dihydrogen phosphate, or sodium chloride.

[0197] In some embodiments, the one or more salts include calcium chloride. The delivery solution may contain any amount of calcium chloride suitable for achieving the desired effect (e.g., treating Parkinson's disease). In some embodiments, the delivery solution contains calcium chloride at concentrations 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 contains calcium chloride at concentrations of about 0.5 mM, about 0.3 mM, or about 0.1 mM or less. In some embodiments, the delivery solution contains calcium chloride at concentrations of about 3 mM, about 3.5 mM, about 4 mM, or about 5 mM or higher.

[0198] In some embodiments, the one or more salts include magnesium chloride. The delivery solution may contain any amount of magnesium chloride suitable for achieving the desired effect (e.g., treating Parkinson's disease). In some embodiments, the delivery solution contains magnesium chloride at concentrations 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 contains magnesium chloride at concentrations of about 0.05 mM or less, about 2.5 mM, or about 3.0 mM or higher.

[0199] In some embodiments, one or more salts include potassium chloride. The delivery solution may contain any amount of potassium chloride suitable for achieving the desired effect (e.g., treating Parkinson's disease). In some embodiments, the delivery solution contains potassium chloride at concentrations 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 contains calcium chloride at concentrations of about 4.0 mM, about 3.5 mM, or about 3.0 mM or less. In some embodiments, the delivery solution contains calcium chloride at concentrations of about 6.5 mM, about 7.0 mM, about 7.5 mM, or about 8.0 mM or higher.

[0200] In some embodiments, the one or more salts comprise sodium dihydrogen phosphate. The delivery solution may contain any amount of sodium dihydrogen phosphate suitable for achieving the desired effect (e.g., treating Parkinson's disease). In some embodiments, the delivery solution contains sodium dihydrogen phosphate at concentrations 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 contains sodium dihydrogen phosphate at concentrations of about 0.8 mM, about 0.75 mM, or about 0.7 mM or less. In some embodiments, the delivery solution contains sodium dihydrogen phosphate at concentrations of about 1.0 mM, about 1.05 mM, or about 1.1 mM or higher.

[0201] In some embodiments, the one or more salts include sodium chloride. The delivery solution may contain any amount of sodium chloride suitable for achieving the desired effect (e.g., treating Parkinson's disease). In some embodiments, the delivery solution contains sodium chloride at concentrations 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 contains sodium chloride at concentrations of about 110 mM, about 105 mM, or about 100 mM or less. In some embodiments, the delivery solution contains sodium chloride at concentrations of about 130 mM, about 135 mM, or about 140 mM or higher. In some embodiments, the delivery solution contains sodium chloride at concentrations 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 contains sodium chloride at concentrations of about 65 mM, about 60 mM, or about 55 mM or less. In some embodiments, the delivery solution contains chloride at concentrations of about 90 mM, about 95 mM, or about 100 mM or higher. In some embodiments, the delivery solution contains sodium chloride at concentrations 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 contains sodium chloride at a concentration of about 80 mM, about 75 mM, or about 70 mM or less. In some embodiments, the delivery solution contains sodium chloride at a concentration of about 105 mM, about 110 mM, or about 115 mM or higher.

[0202] In some embodiments, the therapeutic composition comprises a population of dopaminergic cells (e.g., the dopaminergic cell population described herein) and a delivery solution containing one or more stabilizers. A stabilizer is any component that can be used to reduce or prevent degradation of other solution components. The one or more stabilizers may include any suitable stabilizers, such as, but not limited to, one or more proteins, such as one or more albumins, such as recombinant albumin (rHSA), dextran (as an example, including dextran 40), and poloxamer (as an example, including poloxamer 188). Additionally, the one or more stabilizers may include one or more of the following substances, which can also be used as excipients in the delivery solution: polyethylene glycol, carboxymethyl cellulose, hyaluronic acid, starch, acrylates, methacrylates, polyvinyl alcohol, polyethylene oxide, polypropylene oxide, polyacrylates, polyvinylpyrrolidone, polymethacrylates, polylactic acid-co-glycolic acid, polyacrylamide, polylactide, chitosan, gums, guar gum, xanthan gum, carrageenan, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, cyclodextrin derivatives, β-cyclodextrin derivatives, alginate, calcium alginate, and stearate. In some embodiments, the one or more stabilizers include one or more of recombinant albumin (rHSA), dextran, and poloxamer.

[0203] In some embodiments, the one or more stabilizers include recombinant albumin (rHSA). The delivery solution may contain any amount of rHSA suitable for achieving the desired effect (e.g., treatment of Parkinson's disease). In some embodiments, the delivery solution contains rHSA at concentrations 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 contains rHSA at concentrations of about 0.005 w / w% or less, about 0.2 w / w%, or about 0.25 w / w% or higher. In some embodiments, the delivery solution contains rHSA at concentrations 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% to about 0.22 w / w%, or about 0.05 w / w% or lower. In some embodiments, the delivery solution contains rHSA at concentrations of about 0.25 w / w%, or about 0.3 w / w%, or about 0.35 w / w% or higher. In some embodiments, the delivery solution contains rHSA at concentrations 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 contains rHSA at concentrations of about 5.5 w / w%, about 5.0 w / w%, or about 4.5 w / w%, or lower. In some embodiments, the delivery solution contains rHSA at concentrations of about 8.0 w / w%, or about 8.5 w / w%, or about 9.0 w / w%, or higher.

[0204] In some embodiments, the one or more stabilizers include dextran. The delivery solution may contain any amount of dextran suitable for achieving the desired effect (e.g., treatment of Parkinson's disease). In some embodiments, the delivery solution contains dextran at concentrations 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 contains dextran at concentrations of about 16.5 w / w%, about 16.0 w / w%, or about 15.0 w / w%, or lower. In some embodiments, the delivery solution contains dextran at a concentration of about 18.0 w / w%, about 18.5 w / w%, or about 19.0 w / w% or higher. In some embodiments, the delivery solution contains 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 contains dextran at a concentration of about 12.0 w / w%, about 11.5 w / w%, or about 11.0 w / w% or lower. In some embodiments, the delivery solution contains dextran at a concentration of about 14.0 w / w%, about 14.5 w / w%, or about 15.0 w / w% or higher.

[0205] In some embodiments, the one or more stabilizers include poloxamer. The delivery solution may contain any amount of poloxamer sugar suitable for achieving the desired effect (e.g., treatment of Parkinson's disease). In some embodiments, the delivery solution contains poloxamer at concentrations 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 contains poloxamer at concentrations of about 0.005 w / w% or less, about 0.2 w / w%, or about 0.25 w / w% or higher.

[0206] In some embodiments, the therapeutic composition comprises a population of dopaminergic cells (e.g., the dopaminergic cell population described herein), a delivery solution, and a cryoprotectant. Exemplary cryoprotectants include, but are not limited to, dimethyl sulfoxide (DMSO), glycerol, polyethylene glycol, sucrose, trehalose, dextrose, or combinations thereof.

[0207] In some embodiments, the therapeutic composition comprises a population of dopaminergic cells (e.g., the dopaminergic cell population described herein), a delivery solution, and a biocompatible scaffold or matrix. In some embodiments, the biocompatible scaffold or matrix comprises one or more of extracellular matrix materials, synthetic polymers, cytokines, collagen, peptides or proteins, polysaccharides (including fibronectin, laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparin sulfate, chondroitin sulfate, agarose, or gelatin), and hydrogels.

[0208] In some implementations, there are several components that are not included in the delivery solution and can be completely excluded or below the detectable limit. Some examples of components that can be excluded are: certain components of animal origin; certain stabilizers, 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 energy sources, 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, nicotinamide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, and i-inositol.

[0209] In some embodiments, the cell delivery solution comprises an energy source component, such as D-glucose (dextrose), one or more pH buffers (such as HEPES), poloxamer (such as poloxamer 188), dextran (such as dextran 40), and a combination of recombinant albumin and one or more salts. In some embodiments, one or more salts are selected from calcium chloride, magnesium chloride, potassium chloride, sodium chloride, and sodium dihydrogen phosphate. In some embodiments, one or more salts include calcium chloride, magnesium chloride, potassium chloride, sodium chloride, and sodium dihydrogen phosphate. 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 a concentration 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 dihydrogen phosphate at a concentration of about 0.89 mM.

[0210] In some embodiments, the therapeutic composition comprises a population of dopaminergic cells (e.g., the dopaminergic cell population described herein) and a delivery solution. In some embodiments, the delivery solution comprises a transplantation medium. The transplantation medium is a chemically defined, research-grade mixture of a neural basal medium, L-glutamine, human serum albumin, and L-ascorbic acid. In some embodiments, the concentration of L-glutamine is about 2 mM. In some embodiments, the concentration of L-glutamine is about 200 mM. In some embodiments, the concentration of human serum albumin is about 0.1% w / w. In some embodiments, the concentration of L-ascorbic acid is about 200 µM. In another embodiment, the delivery solution comprises a neural basal 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 µM. In yet another embodiment, the delivery solution comprises a neural basal culture 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 µM.

[0211] Formulations for administration

[0212] In some embodiments, a therapeutic composition (e.g., the therapeutic composition described herein) is prepared for administration to a subject suffering from Parkinson's disease.

[0213] In some embodiments, the preparation of the therapeutic composition for administration (e.g., the therapeutic composition described herein) includes the preparation of a cell suspension. In some embodiments, the cell suspension contains an effective amount of dopaminergic cell population (e.g., the dopaminergic cell population described herein) suspended in a delivery solution. In addition to the dopaminergic cell population and the delivery solution, the cell suspension may also include additional components, such as, for example, a cell washing buffer. Exemplary cell washing buffers include, but are not limited to, phosphate-buffered saline (PBS), compositions containing 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), and Bio-Plex Pro. ™ Cell signaling washing buffer and Cultrex ™ 3-D cell washing buffer. The cell suspension may also contain trace amounts of cryoprotectant and / or cell washing solution that were not completely removed after the suspension step.

[0214] In some embodiments, preparing the cell suspension includes washing the cells before resuspending them in a delivery solution. Washing the cells can remove unwanted components from the suspension, such as components used for culturing or cryopreserving cell populations unsuitable for application. The cells can be washed with the delivery solution or with a washing buffer.

[0215] The delivery solution and / or wash buffer may be free of, contain minimal or trace amounts of cryoprotectant and / or cell wash solution, and the cells are stored and / or washed with cryoprotectant and / or cell wash solution before contact with the delivery solution during resuspension. The cryoprotectant and / or cell wash solution may not be completely removed from the vessel containing the cells after a supernatant discarding process that may occur after an optional centrifugation step in the container, optionally forming a cell pellet or concentrated cell solution. This supernatant discarding process reduces the concentration of the stored sample and / or removes components from the stored sample that are not intended for injection, such as cryoprotectant. The cell delivery solution can be used to reconstitute the cell solution after cell thawing and before administration for clinical use.

[0216] During preparation, the therapeutic composition can be formulated to possess various properties, such as a specific pH, molar osmotic pressure concentration, 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 a molar osmotic concentration of about 100 mOsm / L to about 700 mOsm / L, about 150 mOsm / L to about 500 mOsm / L, about 200 mOsm / L to about 500 mOsm / L, about 225 mOsm / L to about 400 mOsm / L, about 250 mOsm / L to about 350 mOsm / L, about 270 mOsm / L to about 325 mOsm / L, or about 280 mOsm / L to about 300 mOsm / L. In some embodiments, the therapeutic composition is formulated to have a density of about 1.00 g / mL to about 1.30 g / mL, about 1.02 g / mL to about 1.20 g / mL, about 1.04 g / mL to about 1.15 g / mL, about 1.05 g / mL to about 1.11 g / mL, about 1.07 g / mL to about 1.09 g / mL, or 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 good cell compatibility, such that the delivery solution is substantially non-cytotoxic. The therapeutic composition may also be formulated to have a sufficient shelf life under typical or standard storage conditions to make it ready for clinical use.

[0217] These properties can affect the ability of the delivery solution to maintain dopaminergic cell populations (e.g., dopaminergic cell populations) suspended in the therapeutic composition. Dopaminergic cell populations dispersed in a liquid can remain dispersed for 0 to 104 hours or longer. Dopaminergic cell populations dispersed or suspended in a liquid according to this disclosure have a shelf life of up to about 104 hours. More specifically, in this disclosure, the liquid may be a delivery solution that can maintain a population of dopaminergic cells dispersed therein 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 longer without stirring (mixing) and / or homogenization or after stirring (mixing) and / or homogenization.

[0218] While preparing the therapeutic composition, the delivery solution may be warmed or cooled to any suitable 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 contacting any cells.

[0219] Therapeutic compositions can be prepared for any suitable mode of administration. For example, therapeutic compositions can be prepared for surgical implantation or injection, such as local injection into the brain.

[0220] In some embodiments, the therapeutic composition comprises an effective amount of dopaminergic cell populations for treating Parkinson's disease and a cell delivery solution. In some embodiments, the concentration of the dopaminergic cell populations contained in the delivery solution is from about 71,000 cells / µL to about 123,000 cells / µL. When, for example, an amount of dopaminergic cell populations is administered using a cell delivery device as described by reference in WO2021 / 211518, the concentration is advantageously optimized to reduce variability in the amount of dopaminergic cells administered to the subject.

[0221] In some embodiments, preparing a therapeutic composition for administration further includes loading the therapeutic composition into a dose delivery device, such as a syringe or any other device capable of delivering a solution to a subject. After loading the therapeutic composition into the delivery device, the therapeutic composition can then be administered (e.g., by injection) to the subject.

[0222] IV. Treatment Methods

[0223] Certain aspects of this disclosure provide methods for treating a subject with Parkinson's disease. In some embodiments, the method includes administering an effective amount of a population of dopaminergic cells (e.g., the dopaminergic cell population described herein) or a therapeutic composition comprising an effective amount of a population of dopaminergic cells.

[0224] In some embodiments, the dopaminergic cell population includes dopaminergic neurons or dopaminergic progenitor cells. In some embodiments, the dopaminergic neurons include one or more of midbrain dopaminergic neurons, lamina-derived dopaminergic neurons, and true midbrain dopaminergic neurons. In some embodiments, the dopaminergic cells comprise midbrain dopaminergic neurons or their progenitor cells. In some embodiments, the midbrain dopaminergic neurons or their progenitor cells are derived from midbrain lamina progenitor cells, whereby the midbrain lamina progenitor cells are derived from in vitro stem cells.

[0225] In some embodiments, the method includes administering an effective amount of a FOXA2-positive dopaminergic cell population. 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 dopaminergic cell population is FOXA2-positive. In some embodiments, at least 80% of the dopaminergic cell population is FOXA2-positive. In some embodiments, at least 90% of the dopaminergic cell population is FOXA2-positive. In some embodiments, at least 95% of the dopaminergic cell population is FOXA2-positive. In some embodiments, at least 98% of the dopaminergic cell population is FOXA2-positive. In some embodiments, at least 99% of the dopaminergic cell population is FOXA2-positive.

[0226] In some embodiments, the method includes administering an effective amount of TH-positive dopaminergic cell population. 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 dopaminergic cell population are TH-positive. In some embodiments, approximately 10% of the dopaminergic cell population is TH-positive. In some embodiments, approximately 15% of the dopaminergic cell population is TH-positive. In some embodiments, approximately 17% of the dopaminergic cell population is TH-positive.

[0227] In some embodiments, the method includes administering an effective amount of dopaminergic cell population, wherein only a certain percentage of the dopaminergic cell population is PAX2 positive. 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 dopaminergic cell population is PAX2 positive. In some embodiments, less than about 10% of the dopaminergic cell population is PAX2 positive. In some embodiments, less than about 5% of the dopaminergic cell population is PAX2 positive. In some embodiments, less than about 4% of the dopaminergic cell population is PAX2 positive. In some embodiments, less than about 3% of the dopaminergic cell population is PAX2 positive. In some embodiments, less than about 2% of the dopaminergic cell population is PAX2 positive. In some embodiments, less than about 1% of the dopaminergic cell population is PAX2 negative.

[0228] In some embodiments, the method includes administering an effective amount of dopaminergic cell population, wherein only a certain percentage of the dopaminergic cell population is CRABP1 positive. 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 dopaminergic cell population is CRABP1 positive. In some embodiments, less than about 10% of the dopaminergic cell population is CRABP1 positive. In some embodiments, less than about 5% of the dopaminergic cell population is CRABP1 positive. In some embodiments, less than about 4% of the dopaminergic cell population is CRABP1 positive. In some embodiments, less than about 3% of the dopaminergic cell population is CRABP1 positive. In some embodiments, less than about 2% of the dopaminergic cell population is CRABP1 positive. In some embodiments, less than about 1% of the dopaminergic cell population is CRABP1 positive. In some implementations, the dopaminergic cell population is CRABP1 negative.

[0229] In some embodiments, the method includes administering an effective amount of dopaminergic cell population, wherein only a certain percentage of the dopaminergic cell population is Ki67 positive. 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 dopaminergic cell population is Ki67 positive. In some embodiments, less than about 30% of the dopaminergic cell population is Ki67 positive. In some embodiments, less than about 20% of the dopaminergic cell population is Ki67 positive. In some embodiments, less than about 15% of the dopaminergic cell population is Ki67 positive. In some embodiments, less than about 12% of the dopaminergic cell population is Ki67 positive. In some embodiments, less than about 10% of the dopaminergic cell population is Ki67 positive. In some implementations, less than about 5% of the dopaminergic cell population is Ki67 positive. In some implementations, the dopaminergic cell population is Ki67 negative.

[0230] In some embodiments, the method includes administering an effective amount of a dopaminergic cell population comprising viable dopaminergic cells. In some embodiments, approximately 10% to approximately 90%, approximately 20% to approximately 80%, approximately 30% to approximately 80%, approximately 40% to approximately 50%, or approximately 50% to approximately 100% of the dopaminergic cell population are viable. In some embodiments, approximately 50% to approximately 100% of the dopaminergic cell population are viable. In some embodiments, approximately 60% to approximately 90% of the dopaminergic cell population are viable. In some embodiments, approximately 70% to approximately 80% of the dopaminergic cell population 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 dopaminergic cell population are viable. In some embodiments, at least about 90% of the dopaminergic cells in the dopaminergic cell population are viable. In some embodiments, at least about 80% of the dopaminergic cells in the dopaminergic cell population are viable. In some embodiments, at least about 70% of the dopaminergic cells in the dopaminergic cell population are viable. In some embodiments, at least about 50% of the dopaminergic cells in the dopaminergic cell population are viable.

[0231] In some embodiments, the method includes administering an effective amount of a population of dopaminergic cells capable of producing dopamine. In some embodiments, the population of dopaminergic cells is capable of 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 is capable of producing dopamine at a rate of at least about 20 ng / day / mL. In some embodiments, the population of dopaminergic cells is capable of producing dopamine at a rate of at least about 15 ng / day / mL. In some embodiments, the population of dopaminergic cells is capable of producing dopamine at a rate of at least about 11.2 ng / day / mL. In some embodiments, the population of dopaminergic cells is capable of producing dopamine at a rate of at least about 8 ng / day / mL.

[0232] In some embodiments, the dopaminergic cell population has the ability to produce dopamine, and when evaluated in vitro using liquid chromatography-tandem mass spectrometry (LC-MS / MS), the ability to produce dopamine provides a concentration-time area (AUC) 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 dopaminergic cell population has the ability to produce dopamine, and when evaluated in vitro using liquid chromatography-tandem mass spectrometry (LC-MS / MS), the ability to produce dopamine provides a concentration-time area (AUC) of at least about 20 ng / day / mL. In some embodiments, the dopaminergic cell population has the ability to produce dopamine, and when evaluated in vitro using liquid chromatography-tandem mass spectrometry (LC-MS / MS), the ability to produce dopamine provides a concentration-time area (AUC) of at least about 15 ng / day / mL. In some embodiments, the dopaminergic cell population has the ability to produce dopamine, and when evaluated in vitro using liquid chromatography-tandem mass spectrometry (LC-MS / MS), the ability to produce dopamine provides a concentration-time area (AUC) of at least about 11.2 ng / day / mL. In some embodiments, the dopaminergic cell population has the ability to produce dopamine, and when evaluated in vitro using liquid chromatography-tandem mass spectrometry (LC-MS / MS), the ability to produce dopamine provides a concentration-time area (AUC) of at least about 8 ng / day / mL.

[0233] The number of dopaminergic cells in the effective amount of dopaminergic cell population applied in the methods described herein can be any suitable and effective number. In some embodiments, the effective amount of dopaminergic cell population includes about 1×10^4 to about 1×10^10 dopaminergic cells, about 1×10^4 to about 1×10^5 dopaminergic cells, about 1×10^5 to about 1×10^9 dopaminergic cells, about 1×10^5 to about 1×10^6 dopaminergic cells, and about 1×10^5 to about 1×10^7 dopaminergic cells. Cells, approximately 1×10^6 to approximately 1×10^7 dopaminergic cells, approximately 1×10^6 to approximately 1×10^8 dopaminergic cells, approximately 1×10^7 to approximately 1×10^8, approximately 1×10^8 to approximately 1×10^9 dopaminergic cells, approximately 1×10^8 to approximately 1×10^10 dopaminergic cells, or approximately 1×10^9 to approximately 1×10^10 dopaminergic cells. In some implementations, the effective quantity of dopaminergic cell population includes at least 250,000 dopaminergic cells, at least 500,000 dopaminergic cells, at least 750,000 dopaminergic cells, at least 1,000,000 dopaminergic cells, at least 1,250,000 dopaminergic cells, at least 1,500,000 dopaminergic cells, at least 1,750,000 dopaminergic cells, at least 2,000,000 dopaminergic cells, at least 2,250,000 dopaminergic cells, at least 2,500,000 dopaminergic cells, at least 2,750,000 dopaminergic cells, and at least 3,000,000 dopaminergic cells. Dopaminergic cells, 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 cells, at least 8 million dopaminergic cells, at least 9 million dopaminergic cells, or at least 10 million or more dopaminergic cells.

[0234] In some embodiments, the effective amount of dopaminergic cell population comprises at least about 1.0 × 10^6 to about 1.2 × 10^7 dopaminergic cells. In some embodiments, the effective amount of dopaminergic cell population comprises at least about 900,000 dopaminergic cells. In some embodiments, the therapeutic composition comprises a dopaminergic cell population comprising at least about 1.8 million dopaminergic cells. In some embodiments, the effective amount of dopaminergic cell population comprises at least about 2.7 million dopaminergic cells. In some embodiments, the effective amount of dopaminergic cell population comprises at least about 5.4 million dopaminergic cells.

[0235] In some embodiments, administering an effective amount of dopaminergic cell population includes delivering the dopaminergic cell population to the subject's posterior putamen. In some embodiments, administering an effective amount of dopaminergic cell population includes delivering the dopaminergic cell population to the subject's posterior commissural putamen. The putamen is a round structure located at the base of the forebrain and, together with the caudate nucleus, forms the dorsal striatum. The putamen primarily regulates movement at different stages. Based on its location in the left and right hemispheres of the brain, the putamen can also be subdivided into a left putamen and a right putamen. In some embodiments, a first portion of the effective amount of dopaminergic cell population is delivered to the left hemisphere of the subject's posterior putamen, and a second portion of the effective amount of dopaminergic cell population is delivered to the right hemisphere of the subject's posterior putamen. In some embodiments, the first portion is approximately half the effective amount of dopaminergic cell population, and the second portion is approximately half the effective amount of dopaminergic cell population.

[0236] In some embodiments, an effective amount of dopaminergic cell population is delivered to the subject in the form of a therapeutic composition (e.g., the therapeutic composition described herein). In some embodiments, the therapeutic composition comprises a dopaminergic cell population at a concentration of about 71,000 cells / µL to about 123,000 cells / µL, about 75,000 cells / µL to about 115,000 cells / µL, about 80,000 cells / µL to about 110,000 cells / µL, about 85,000 cells / µL to about 105,000 cells / µL, or about 90,000 cells / µL to about 100,000 cells / µL. In some embodiments, the concentration of the dopaminergic cell population is about 71,000 cells / µL to about 123,000 cells / µL. In some implementations, the concentration of dopaminergic cell population is approximately 75,000 cells / µL, approximately 80,000 cells / µL, approximately 85,000 cells / µL, approximately 90,000 cells / µL, approximately 95,000 cells / µL, approximately 100,000 cells / µL, approximately 105,000 cells / µL, approximately 110,000 cells / µL, approximately 115,000 cells / µL, or approximately 120,000 cells / µL. In some embodiments, the concentration of the dopaminergic cell population is approximately 75,000 cells / µL, approximately 80,000 cells / µL, approximately 85,000 cells / µL, approximately 90,000 cells / µL, approximately 95,000 cells / µL, approximately 100,000 cells / µL, approximately 105,000 cells / µL, approximately 110,000 cells / µL, approximately 115,000 cells / µL, or approximately 120,000 cells / µL. In some embodiments, the concentration of the dopaminergic cell population is approximately 90,000 cells / µL. In some embodiments, the concentration of the dopaminergic cell population is approximately 100,000 cells / µL. In some embodiments, the concentration of the dopaminergic cell population is approximately 110,000 cells / µL. In some embodiments, the dopaminergic cell population is allogeneic to the subject. In some implementations, the dopaminergic cell population is derived from the subject's own body.

[0237] In some implementations, the dopaminergic cell population is derived from stem cells that differentiate into dopaminergic cells in vitro (e.g., through the methods described herein).

[0238] In some implementations, Parkinson's disease is early-stage Parkinson's disease. In some implementations, Parkinson's disease is late-stage Parkinson's disease. In some implementations, the subject is a human being.

[0239] Immunosuppressive regimen

[0240] In some embodiments, the method for treating a subject's disease (such as Parkinson's disease) with cell therapy (such as those described herein) further includes administering an immunosuppressive regimen to the subject. In some embodiments, the immunosuppressive regimen is administered before, during, after, or in combination with an effective amount of therapeutic cell populations (e.g., dopaminergic cell populations). In some embodiments, the immunosuppressive regimen includes bailiximab, methylprednisolone, and tacrolimus.

[0241] In some embodiments, bailiximab is administered at approximately 5 mg, approximately 10 mg, approximately 15 mg, approximately 20 mg, approximately 25 mg, approximately 30 mg, approximately 35 mg, approximately 40 mg, approximately 45 mg, or approximately 50 mg. In some embodiments, bailiximab is administered at approximately 10 mg. In some embodiments, bailiximab is administered at approximately 20 mg. In some embodiments, bailiximab is administered at approximately 30 mg. In some embodiments, bailiximab is administered intravenously during and after surgery. In some embodiments, bailiximab is administered approximately 1 day, approximately 2 days, approximately 3 days, approximately 4 days, approximately 5 days, approximately 6 days, approximately 7 days, approximately 8 days, approximately 9 days, approximately 10 days, approximately 11 days, approximately 12 days, approximately 13 days, or approximately 14 days after administration of the therapeutic cell population (e.g., dopaminergic cell population). In some embodiments, bailiximab is administered approximately 2 days after administration of the therapeutic cell population (e.g., dopaminergic cell population). In some embodiments, baliximab is administered approximately 4 days after administration of the therapeutic cell population (e.g., dopaminergic cell population). In some embodiments, baliximab is administered approximately 6 days after administration of the therapeutic cell population (e.g., dopaminergic cell population).

[0242] In some embodiments, methylprednisolone is administered at doses of 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, methylprednisolone is administered at about 300 mg. In some embodiments, methylprednisolone is administered at about 500 mg. In some embodiments, methylprednisolone is administered at about 700 mg. In some embodiments, methylprednisolone is administered prior to the administration of a therapeutic cell population (e.g., a dopaminergic cell population). In some embodiments, methylprednisolone is further administered after the administration of a therapeutic cell population (e.g., a dopaminergic cell population). In some embodiments, methylprednisolone is further administered weekly after the administration of a therapeutic cell population (e.g., a dopaminergic cell population). In some embodiments, methylprednisolone is administered daily following the administration of the therapeutic cell population (e.g., dopaminergic cell population). In some embodiments, methylprednisolone is administered daily at approximately 5 mg following the administration of the therapeutic cell population (e.g., dopaminergic cell population).

[0243] In some embodiments, tacrolimus is administered after the application of a therapeutic cell population (e.g., a dopaminergic cell population). In some embodiments, tacrolimus is administered approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days after the application of the therapeutic cell population (e.g., a dopaminergic cell population). In some embodiments, tacrolimus is administered approximately 1 day after the application of the therapeutic cell population (e.g., a dopaminergic cell population). In some embodiments, tacrolimus is administered approximately 2 days after the application of the therapeutic cell population (e.g., a dopaminergic cell population). In some embodiments, tacrolimus is administered approximately 3 days after the application of the therapeutic cell population (e.g., a dopaminergic cell population).

[0244] In one particular embodiment, the immunosuppressive regimen includes administering baliximab intravenously at approximately 20 mg approximately 4 days after administration of the therapeutic cell population (e.g., dopaminergic cell population), intravenously administering methylprednisolone at approximately 500 mg prior to administration of the therapeutic cell population (e.g., dopaminergic cell population), and administering tacrolimus approximately 1 day after administration of the therapeutic cell population (e.g., dopaminergic cell population). In some embodiments, methylprednisolone is further administered daily at approximately 5 mg following administration of the therapeutic cell population (e.g., dopaminergic cell population).

[0245] Application method

[0246] Any suitable administration method may be used to administer dopaminergic cell populations and / or immunosuppressive regimens according to the methods described herein. For example, dopaminergic cell populations and / or immunosuppressive regimens may be administered by injection (e.g., intravenous injection) or surgical implantation. In some embodiments, dopaminergic cell populations and / or immunosuppressive regimens are administered systemically. In some embodiments, dopaminergic cell populations and / or immunosuppressive regimens are administered locally. In some embodiments, administration of dopaminergic cell populations and / or immunosuppressive regimens is performed by delivering dopaminergic cell populations and / or immunosuppressive regimens to the posterior putamen of the subject.

[0247] In some implementations, administration includes delivering a population of dopaminergic cells to a subject using a stereotactic guided delivery system. Exemplary stereotactic guided delivery systems are described, for example, in published PCT application WO 2021 / 211518A1, which is incorporated herein by reference.

[0248] In some embodiments, the stereotactic guided delivery system is configured to reduce backflow of the therapeutic composition from the target site during injection of a therapeutic composition containing a population of dopaminergic cells (e.g., the therapeutic composition described herein) into a target site (e.g., the putamen). In some embodiments, the stereotactic guided delivery system includes a needle, a plunger, and a stereotactic frame. In some embodiments, the needle may be arranged to retract as 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 may retract, thereby providing a volume of space within the created cavity for the therapeutic substance to reside.

[0249] In some embodiments, the stereotactic guided delivery system is configured to help reduce cell or particle sedimentation 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.

[0250] In some embodiments, the stereotactic guided delivery system is configured to help the user control the ejection rate of the therapeutic substance. For some therapeutic substances, such as certain types of cells (e.g., dopaminergic cells), a slower ejection rate can help reduce shearing or other harmful effects on the cells, thereby resulting in higher viability of the delivered dopaminergic cells. A slower ejection rate may also reduce the risk of brain tissue injury. In some embodiments, the device actuator is a rotary actuator. In some embodiments, multiple complete rotations of the rotary actuator are required to deliver the total target volume.

[0251] In some embodiments, the stereotactic guided delivery system is configured to improve dose assurance. In some embodiments, the therapeutic composition is contained within a relatively small needle lumen with a constant diameter. In the case of dopaminergic cell populations, such an arrangement facilitates the forward and backward movement of cells with their fluid solution, which helps ensure the delivery of a larger fraction of the cells. In some embodiments, such an arrangement may help reduce cell sedimentation.

[0252] In some embodiments, the stereotactic guided delivery system is configured to help reduce material waste that may occur during the loading of therapeutic material into the delivery device. In some embodiments, the delivery device is configured to load therapeutic material forward. In some embodiments, the stereotactic guided delivery system may include a vent arrangement to allow forward loading. In some embodiments, the stereotactic guided delivery system may include an arrangement for activating the system after the therapeutic material has been loaded to remove air from the stereotactic guided delivery system before use to prevent air injection into the target site.

[0253] In some implementations, the stereotactic guided delivery system includes an indicator that comprises only mechanical components. Because there are no electrical components, this arrangement allows the stereotactic guided delivery system to be more portable and easier to sterilize.

[0254] Assessment of treatment and disease progression

[0255] In some embodiments, administration of an effective amount of the cell population causes improvement in a subject's motor function or non-motor function, or a combination thereof. In some embodiments, administration of an effective amount of the cell population causes improvement in a subject's motor function or non-motor function, or a combination thereof, compared to a control or the subject's baseline before administration. Exemplary tests of motor function that can be improved by administration of an effective amount of dopaminergic cell population include, but are not limited to, tremor, bradykinesia or slow movement, limb stiffness or rigidity, trunk and postural instability, and impaired balance and coordination. Exemplary tests of non-motor function that can be improved by administration of an effective amount of dopaminergic cell population include, but are not limited to, autonomic dysfunction, neuropsychiatric problems, and sensory and sleep difficulties. In some embodiments, the improvement is manifested in at least one motor function and at least one non-motor function of the subject.

[0256] Any appropriate method for assessing motor and nonmotor function in subjects with Parkinson's disease can be used to assess improvement in response to administration of an effective dose of dopaminergic cell population. Exemplary methods for assessing motor or nonmotor function in subjects with Parkinson's disease include, but are not limited to, single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), positron emission tomography (PET), computed tomography (CT), dopamine transporter (DAT) scans, ultrasound, biomarker testing, the Movement Disorders Association-sponsored revised Unified Parkinson's Disease Rating Scale (MDSUPDRS), the Total Unified Parkinson's Disease Rating Scale (UPDRS), pre-planned care plans, Clinical Algorithm: Assessment of Mild Cognitive Impairment in Parkinson's Disease (PD-MCI), the Epworth Somnolence Scale (ESS), the Hospital Anxiety and Depression Scale (HADS), and the Lindop Parkinson's Disease Rating Scale. The Parkinson's Disease Physical Therapy Assessment Scale, Modified Bradykinesia Rating Scale (MBRS), Montreal Cognitive Assessment (MOCA), Nonmotor Symptom Questionnaire, "Do Not Swallow" Medication Dosage Calculator and Guidelines, Parkinson's Disease Driving Questionnaire, Parkinson's Disease Fatigue Scale Parkinson's Disease Questionnaire (PDQ-39), Parkinson's Disease Sleep Scale (PDSS), Unified Motor Disorders Rating Scale (UDysRS), Unified Dystonia Rating Scale (UDRS), Unified Parkinson's Disease Rating Scale (UPDRS), Parkinson's Disease Nonmotor Symptoms Scale (PDNMSS), Neuropsychiatric Questionnaire (NPI-Q), Repeatable Complete Neuropsychological Status Assessment (RBANS), and Frontal System Behavior Scale (FrSBe).

[0257] In some implementations, improvements in motor function are determined at least in part based on changes in the subject's Part II score of the Movement Disorders Association-sponsored Revised Unified Parkinson's Disease Rating Scale (MDS UPDRS) compared to the baseline of the control or subject prior to administration. In some implementations, improvements in motor function are determined at least in part based on changes in the subject's Part III score of the Movement Disorders Association-sponsored Revised Unified Parkinson's Disease Rating Scale (MDS UPDRS) compared to the baseline of the control or subject prior to administration, for example as described in Sánchez-Ferro, 2018, Minimal Clinically Important Difference for UPDRS-III in Daily Practice, MovDisord Clin Pract; July-August; 5(4): 448–450, which is incorporated herein by reference. In some implementations, improvements in motor function are determined at least in part based on changes in the subject's ON and / or OFF scores compared to a control or subject's baseline prior to administration, as described, for example, in Hauser, 2014, Minimal clinically important difference in Parkinson's disease as assessed in pivotal trials of pramipexole extended release, Parkinsons Dis; 2014:467131, which is incorporated herein by reference. In some implementations, improvements in motor function are determined at least in part based on changes in the subject's ON score compared to a control or subject's baseline prior to administration. In some implementations, improvements in motor function are determined at least in part based on changes in the subject's OFF score compared to a control or subject's baseline prior to administration.

[0258] In some implementations, improvements in motor function are determined, at least in part, based on changes in the subject's objective sub-item scores on the Unified Movement Disorder Rating Scale (UDysRS) compared to a control or baseline prior to administration. This method provides an objective measurement of movement disorder. It also provides quantifiable measurements of motor complications associated with the disease and its treatment, which can be used to track disease progression, the effectiveness of pharmacological treatment, or the impact of therapeutic interventions.

[0259] In some implementations, improvements in nonmotor function are measured by one or more assays selected from a group consisting of: sleep quality assessment, cognitive assessment, neuropsychological testing, mood assessment, autonomic nervous system function testing, imaging testing, and other standardized nonmotor function assessments.

[0260] In some implementations, improvement in nonmotor function is determined at least in part based on changes in the subject's Parkinson's Disease Nonmotor Symptom Scale (PD NMSS) score compared to a control or subject's baseline prior to administration. In some implementations, improvement in nonmotor function is determined at least in part based on changes in the subject's PD NMSS score, as described, for example, in Ray Chaudhuri K, Rojo JM, Schapira AHV et al., PLoS One. 2013;8(2):e57221, which is incorporated by reference. PD NMSS scores are taken across different categories on a range of 0 to 360, with lower scores indicating improvement. In some implementations, changes in the subject's PD NMSS are changes in scores across one or more PD NMSS categories. In some implementations, changes in the subject's PD NMSS are changes in the total PD NMSS score.

[0261] In some implementations, improvements in nonmotor function are determined at least in part based on changes in the subject's 39-item Parkinson's Disease Questionnaire (PDQ-39) score compared to a control or the subject's baseline prior to administration. In some implementations, improvements in nonmotor function are determined at least in part based on changes in the subject's PDQ-39 score, as described, for example, in Balestrino R, Hurtado-Gonzalez CA, Stocchi F et al., NPJ Parkinsons Dis. 2019;5:26, which is incorporated by reference. PDQ-39 scores are taken across different categories on a range of 0 to 100, with lower scores indicating improvement. In some implementations, changes in the subject's PDQ-39 are changes in scores across one or more PDQ-39 categories. In some implementations, changes in the subject's PDQ-39 are changes in the total PDQ-39 score.

[0262] In some implementations, improvements in nonmotor function are determined at least in part based on changes in the subject's Neuropsychiatric Inventory Questionnaire (NPI-Q) score compared to a control or baseline prior to administration. As described, for example, in Cummings JL. The Neuropsychiatric Inventory Questionnaire: Background and Administration. 1994. (available at alz.org / media / documents / npiq-questionnaire.pdf). NPI-Q scores are given on a range of 0 to 96, with lower scores indicating improvement.

[0263] In some implementations, improvements in nonmotor function are determined at least in part based on changes in the subject's Repeatable Suite of Neuropsychological Status Assessments (RBANS) score compared to a control or baseline prior to administration. In some implementations, improvements in nonmotor function are determined at least in part based on changes in the subject's RBANS score, as described, for example, in Yang C, Garrett-Mayer E, Schneider JS, Gollomp SM, Tilley BC. MovDisord. 2009;24(10):1453-60, which is incorporated herein by reference. RBANS scores are taken across different categories on a range of 40 to 60, with higher scores indicating improvement. In some implementations, changes in the subject's RBANS are changes in scores across one or more RBANS categories. In some implementations, changes in the subject's RBANS are changes in the total RBANS score.

[0264] In some implementations, improvements in nonmotor function are determined at least in part based on changes in the subject's Frontal Systemic Behavior Scale (FrSBe) score compared to a control or subject's baseline prior to administration. In some implementations, improvements in nonmotor function are determined at least in part based on changes in the subject's FrSBe score, as described, for example, in Cabrera S, Edelstein K, Mason WP, Tartaglia MC. Neurooncol Pract. 2016;3(2):113-9, which is incorporated herein by reference. RBANS scores are performed across different categories, with lower scores indicating improvement. In some implementations, changes in the subject's FrSBe are changes in scores across one or more FrSBe categories. In some implementations, changes in the subject's FrSBe are changes in the total FrSBe score.

[0265] In some implementations, the baseline is a measurement of the subject's motor and non-motor functions prior to the administration of an effective amount of dopaminergic cell population.

[0266] In some implementations, the improvement in remission of Parkinson's disease progression in indicative subjects is enhanced. In some implementations, the improvement in reversal of Parkinson's disease progression in indicative subjects is enhanced.

[0267] In some embodiments, improvement can be detected some time after administration of dopaminergic cell populations. In some embodiments, improvement can be detected approximately 1 week, approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 5 weeks, approximately 6 weeks, approximately 7 weeks, approximately 7 weeks, approximately 8 weeks, approximately 9 weeks, approximately 10 weeks, approximately 11 weeks, approximately 12 weeks, approximately 15 weeks, approximately 18 weeks, approximately 21 weeks, approximately 24 weeks, approximately 27 weeks, approximately 30 weeks, approximately 33 weeks, approximately 36 weeks, approximately 39 weeks, approximately 42 weeks, approximately 45 weeks, approximately 48 weeks, or approximately 52 weeks or longer after administration of dopaminergic cell populations. In some embodiments, improvement can be detected approximately 4 weeks after administration of dopaminergic cell populations. In some embodiments, improvement can be detected approximately 12 weeks after administration of dopaminergic cell populations. In some embodiments, improvement can be detected approximately 24 weeks after administration of dopaminergic cell populations.

[0268] In some embodiments, the improvement lasts 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 longer. In some embodiments, the improvement lasts for a duration of at least about 6 months. In some embodiments, the improvement lasts for a duration of at least about 1 year. In some embodiments, the improvement lasts for a duration of at least about 1.5 years. In some embodiments, the improvement lasts for a duration of at least about 2 years. In some embodiments, the improvement persists after the removal of the immunosuppressive regimen. In some embodiments, the improvement persists for at least 6 months after the removal of the immunosuppressive regimen. In some embodiments, the improvement persists for at least 1 year after the removal of the immunosuppressive regimen.

[0269] V. Vessels

[0270] Certain aspects of this disclosure provide containers containing the dopaminergic cell populations described herein or compositions containing the dopaminergic cell populations described herein. These containers may be storage containers or containers used during administration of the dopaminergic cell populations or compositions. In some embodiments, the containers include cryogenic vials. In some embodiments, the containers include aseptic technique (AT) vials. Other non-limiting examples of usable containers include syringes, thin glass tubes, stereotactic needles, and cannulas.

[0271] VI. Reagent Kit

[0272] Certain aspects of this disclosure provide a kit for treating Parkinson's disease comprising a population of dopaminergic cells suitable for administration to a subject or a composition comprising a population of dopaminergic cells, including any of the dopaminergic cell populations and compositions described herein.

[0273] In some embodiments, the kit includes instructional materials for using the composition. In some embodiments, the instructional materials include instructions for preparing the composition or dopaminergic cell population for administration to a subject. Such instructions may include, but are not limited to, instructions for preparing or storing dopaminergic cell populations or compositions, diluting dopaminergic cell populations, adding additional additives to treatment, or combining the composition with additional therapeutic agents. In some embodiments, the instructional materials include instructions for administering dopaminergic cell populations or compositions to a subject.

[0274] In some embodiments, the kit further includes an applicator for administering the dopaminergic cell populations or compositions comprising dopaminergic cell populations described herein. The applicator can be any device suitable for administering the dopaminergic cell populations or compositions described herein to a subject, including but not limited to hypodermic syringes, needles, balloon dilation catheters, pipettes, etc. In some embodiments, the applicator is a stereotactic guided delivery system. The applicator can also be a single-use or reusable application device and can be included in the kit as a pre-filled delivery system having, for example, a therapeutic composition comprising dopaminergic cell populations.

[0275] Example

[0276] The following examples provide clinical data supporting an effective cell therapy for treating Parkinson's disease (PD). Specifically, this example provides comprehensive findings from extensive Phase 1 studies conducted at various centers and sites to treat PD by administering specific doses of dopaminergic neurons. These studies aimed to evaluate the safety, tolerability, and efficacy of dopaminergic neurons in the treatment of PD.

[0277] Example 1: Demonstrating the safety and tolerability of dopaminergic neurons in human subjects with Parkinson's disease. Design and results of Phase 1 clinical trial on acceptability and efficacy

[0278] Phase 1 clinical trial study design :

[0279] Participants diagnosed with Parkinson's disease (PD) were sequentially recruited into a phase 1 study (NCT04802733). An open-label, non-randomized study was conducted to evaluate the safety, tolerability, and initial efficacy of a specific dose of dopaminergic neurons for the treatment of PD.

[0280] Figure 1 This is a schematic diagram of the Phase 1 clinical trial study design. Twelve participants with Parkinson's disease (PD) were enrolled. The first five participants were enrolled in a low-dose group receiving treatment consisting of approximately 900,000 dopaminergic neurons delivered to the posterior putamen of each hemisphere (approximately 1.8 million dopaminergic neurons per participant in total), referred to in this paper as "Cohort A". Subsequent participants were enrolled in a high-dose group receiving treatment consisting of approximately 2.7 million dopaminergic neurons delivered to the posterior putamen of each hemisphere (approximately 5.4 million dopaminergic neurons per participant in total), referred to in this paper as "Cohort B". Enrolled participants were aged between 50 and 78 years (Canada) and between 60 and 78 years (USA), with PD-related motor symptoms not adequately relieved by standard treatment. Baseline evaluation was performed several days prior to the delivery of dopaminergic neurons.

[0281] Preparation of dopaminergic neurons for administration :

[0282] Participants were administered a cell solution containing dopaminergic cells (e.g., midbrain dopaminergic neuronal precursor cells) derived in vitro from human pluripotent stem cells using a substrate-based differentiation strategy as described herein. Dopaminergic neurons were generated and cryopreserved in vials for storage and transport to clinical facilities.

[0283] Frozen vials of dopaminergic neurons were prepared for delivery by thawing frozen vials and combining the neurons with a cell delivery solution. The combination of dopaminergic neurons with the cell delivery solution resulted in a final concentration of 1 × 10^8 ± 10% (cells / mL) in the cell delivery solution. After preparing the cell delivery solution, cell characteristics / purity markers from the dopaminergic neurons were assessed by flow cytometry. Flow cytometry data (not shown) demonstrated that approximately 91.1% (2.9% standard deviation, “SD”) of the dopaminergic neurons were FOXA2 positive; approximately 1.8% (1.3% SD) were PAX6 positive; approximately 2.2% (1.8% SD) were CRABP1 positive; and approximately 11.8% (2.7% SD) were Ki67 positive. TH expression 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 dopaminergic neurons were TH-positive.

[0284] In addition, dead cells were identified using a staining solution containing acridine orange and propidium iodide (AO / PI), and cell viability and concentration were assessed using an automated cell counter. Approximately 77.2% (6.5% SD) of the dopaminergic neurons were viable after preparation of cells for delivery.

[0285] Previously, liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to evaluate the release of dopamine from stem cell-derived dopaminergic neurons in cell culture supernatants. This analysis revealed that the area under the curve (AUC) of dopamine release was greater than or equal to 11.2 ng × day / mL.

[0286] Dopaminergic neurons were surgically administered, and participants received the immunosuppressive regimen described below. Participants were evaluated periodically. The safety, tolerability, and initial efficacy of the dopaminergic neurons were evaluated at 12 months post-administration. Further evaluations are planned at 15, 18, 21, and 24 months post-administration.

[0287] The immunosuppressive regimen was initiated intraoperatively and continued for one year postoperatively following administration of dopaminergic neurons. Specifically, participants began the following immunosuppressive regimen: intravenous administration of baliximab 20 mg intraoperatively and on postoperative day 4; intravenous administration of methylprednisolone 500 mg preoperatively, followed by methylprednisolone 250 mg on postoperative day 2, then gradually reduced to oral prednisone at 5 mg daily for one year; and oral tacrolimus initiated on postoperative day 1 (day 2), then adjusted to a target trough blood level of 4 ng / mL to 7 ng / mL for one year.

[0288] Surgical methods

[0289] Figure 2 An exemplary surgical method for administering an effective amount of dopaminergic neurons to a subject in need is illustrated. In a single-stage, stereotactic-guided surgical injection, a prepared dopaminergic neuron solution (at a concentration of 1 × 10^8 ± 10% cells / mL) is administered into the posterior putamen 203 of the left and right hemispheres through individual boreholes 205 on each side. Using a cannula, the dopaminergic neuron solution is administered along three tracks 207 or channels through each borehole 205. Three portions of the cell delivery solution are deposited at each track 207, for a total of nine portions of cell deposits in each putamen 203. For cohort A, each portion of the deposit contains approximately 1 μL–1.5 μL of cell solution, for a total of approximately 3.5 μL per track, to deliver approximately 900,000 dopaminergic neurons to the posterior putamen 203 of each hemisphere. For cohort B, each deposit contained approximately 34 μL of cell delivery solution, totaling approximately 11 μL, to deliver approximately 2.7 million dopaminergic neurons to the posterior shell nucleus 203 of each hemisphere.

[0290] Safety and tolerability analysis

[0291] One objective of the clinical study was to evaluate the safety and tolerability of dopaminergic neurons. The evaluation included measuring the incidence of serious adverse events (SAEs) one year after administration of multiple doses of dopaminergic neurons.

[0292] For cohort A and cohort B, the safety of dopaminergic neurons was defined based on the following criteria: two or fewer participants in each cohort developed two or more serious adverse events (SAEs) related to surgery, the presence of transplanted cells, or immunosuppression; two or fewer participants in each cohort developed tumors or abnormal tissue overgrowth attributable to the presence of transplanted cells; two or fewer participants in each cohort developed what was considered life-threatening intracerebral hemorrhage; and one or zero deaths occurred among participants in cohort A or cohort B.

[0293] In addition, the following secondary endpoints were assessed in year 1: changes in striatal fluorodopa (18F-DOPA) uptake relative to baseline using positron emission tomography (PET); changes in the motor subscale score of the International Parkinson's Disease and Movement Disorders Association (MDS)-Unified Parkinson's Disease Rating Scale (UPDRS) relative to baseline while on OFF medication; and changes in the number of hours spent in ON, OFF, and ON states with troublesome movement disorders relative to baseline. Feasibility was also assessed, as defined by delivering at least 50% of the expected number of deposits per brain surgery in >50% of subjects.

[0294] This study plans to assess changes relative to baseline in the following areas two years post-transplantation: the International Parkinson's Disease and Movement Disorders Association (MDS)-Unified Parkinson's Disease Rating Scale (UPDRS) motor subscale score while on OFF medication; the number of hours in ON state without troubling movement disorders; the incidence of serious adverse events (SAEs) two years post-transplantation; and the incidence and type of adverse events (AEs) one and two years post-transplantation. Feasibility in the study is defined as successful intraoperative delivery of at least 50% of the expected total deposits per brain in more than 50% of participants.

[0295] Exploratory non-motor outcomes

[0296] Another objective of the clinical study was to evaluate the effects of administering dopaminergic neurons up to 18 months post-transplantation (6 months after discontinuation of the immunosuppressive regimen).

[0297] The exploratory nonmotor outcome was reported as a mean score, which was measured at baseline and 18 months post-transplantation using the Parkinson's Disease Nonmotor Symptom Scale (PD NMSS) and the 39-item Parkinson's Disease Questionnaire-39 (PDQ-39).

[0298] Formal neuropsychological evaluations were performed at baseline and 12 months post-transplantation, including the Neuropsychiatric Questionnaire (NPI-Q), the Reproducible Complete Neuropsychological Status Assessment (RBANS), and the Frontal System Behavior Scale (FrSBe).

[0299] Results of safety and tolerability analysis

[0300] Patient enrollment: Patients in both cohorts had comparable baseline characteristics (see Table 1). All participants received their planned doses. All participants (N=12) were included in the safety and evaluable population.

[0301] Table 1. Baseline Characteristics .

[0302]

[0303] Security

[0304] This study met its safety objectives. No adverse events (AEs) or subarachnoid injuries (SAEs) related to dopaminergic neurons were reported (see Table 2). One case of SAE attributed to a transient episode following surgery was reported in cohort B.

[0305] Table 2. Summary of serious adverse events (TESAEs) occurring during the first year of treatment. .

[0306]

[0307] Of the 12 participants, 11 reported TEAEs (66 events in total; Table 3). Most TEAEs were mild to moderate in severity. Only one severe TEAE was reported. There were no discontinuations or deaths during the study. No graft-induced motor dysfunctions were observed in either cohort. A summary of TEAEs is provided in Table 3.

[0308] Table 3. Summary of Treatment-Acting Events (TEAEs) .

[0309]

[0310] Implantation, survival, and function of applied dopaminergic neurons

[0311] Implantation, survival, and function of transplanted dopaminergic neurons were assessed using 18F-DOPA uptake. Increased 18F-DOPA uptake was observed in most subjects in the posterior putamen (transplantation site). Figure 3A and Figure 3B Furthermore, a reduction in 18F-DOPA was observed in the caudate nucleus (far from the transplantation site), which could be attributed to the progression of PD. Overall, these data provide evidence for the implantation, survival, and cellular function of dopaminergic neurons administered for the treatment of PD.

[0312] Figure 3A and Figure 3B Exemplary data from a comparative analysis of 18F-DOPA PET data in subjects treated with dopaminergic neurons are shown. The comparative analysis compares the 18F-DOPA PET data in a subject to their corresponding baseline. Figure 3A Voxel-based analysis of 18F-DOPA PET data is shown. The image shows voxel clusters with low p-value (P<0.05) group-level changes between baseline and 1 year (increased voxels are orange, decreased voxels are blue). Figure 3BThis is a box plot showing the variation of striatum-occipital ratio (SOR) relative to baseline for low p-value voxel clusters. Volume-weighted means were used to represent the variation within significant voxel clusters combining the caudate and putamen for each subject. Lines represent the median variation in the cohort, circles represent the mean, boxes represent the lower and upper quartiles, and whisker lines represent extreme values. Overall, these data demonstrate that the dopaminergic neurons administered to subjects to treat PD were successfully engrafted and survived for 52 weeks post-transplantation.

[0313] The administered dose of dopaminergic neurons improved motor and non-motor function in subjects with PD.

[0314] The following sections provide clinical data demonstrating the efficacy of high- and low-dose dopaminergic neurons in improving motor and non-motor function in subjects with PD.

[0315] The Movement Disorders Association Unified Parkinson's Disease Rating Scale (MDS-UPDRS) Part III OFF was assessed to evaluate changes in motor function in subjects. The "OFF" state refers to the time between the loss of efficacy of medication and symptom recurrence, thus providing an assessment of a patient's motor symptoms in the absence of medication. Therefore, changes in the MDS-UPDRS Part III score (OFF with medication) reflect changes in motor function in subjects with Parkinson's disease.

[0316] In addition, the study evaluated patients' reported ON and OFF times via the Hauser / PD diary to assess the effectiveness of cell therapy in improving motor and non-motor function. ON time refers to the period during which a subject's symptoms are well controlled and medication is effective. During these periods, motor symptoms such as tremor, rigidity, or bradykinesia are reduced. OFF time refers to the period during which medication is ineffective and symptoms recur. OFF time can be characterized by the recurrence of motor symptoms. By reporting patient-reported ON and OFF times, this study provides insight into how treatment with high or low doses of dopaminergic neurons controls PD symptoms. Therefore, the assessment of ON and OFF times provides an assessment of the severity and type of motor symptoms and their circadian fluctuations.

[0317] Record the MDS-UPDRS Part III OFF and patient-reported ON and OFF times for treated subjects in cohorts A and B, and compare them with baseline measurements (i.e., measurements obtained from the respective subjects prior to treatment with dopaminergic neurons) (see [reference]). Figures 4A to 4D (and Table 4). A decrease in MDS-UPDRS Part III score (OFF) was observed in PD patients after treatment with dopaminergic neurons. Figure 4AThis indicates improved motor function, which was more pronounced in the high-dose group, and suggests a reversal of the PD-related effects on motor function. Additionally, an increase in patient-reported ON time was observed after treatment. Figure 4B This indicates that dopaminergic neurons can reverse detrimental PD symptoms and maintain this effect for at least 52 weeks after treatment. Furthermore, patients reported a reduction in OFF time compared to the corresponding baseline score after this therapy. Figure 4C This indicates the potential of dopaminergic neurons to reverse PD symptoms for at least 52 weeks after treatment. Patient-reported ON time with troubling motor dysfunction was also kept low to negligible. Figure 4D This further confirms the safety and tolerability of dopaminergic neurons in PD treatment.

[0318] In summary, these data demonstrate the significant ability of effective doses of dopaminergic neurons to slow the progression of PD symptoms and reverse some of their effects on motor and nonmotor function. These data further demonstrate that the significant effects are sustained over a period of up to 52 weeks, highlighting the treatment's potential for symptom reversal and long-term efficacy. Furthermore, the comparative analysis between the two cohorts emphasizes that, unlike cohort A (low dose), the enhanced outcomes in clinical assessment were more pronounced in cohort B (high dose), thus highlighting the potential dose-dependent efficiency of the treatment strategy detailed in this paper.

[0319] Table 4. Changes in clinical outcomes relative to baseline in year 1 .

[0320]

[0321] The administered dose of dopaminergic neurons also improved exploratory nonmotor function outcomes in subjects with PD.

[0322] At 18 months post-dopaminergic cell transplantation, the mean total PD NMSS score increased relative to baseline in cohort A (low dose) (Table 5), indicating worsening, while the PD NMSS score remained stable in cohort B (high dose) (Table 5). Regarding PDQ-39, the total index score remained stable in cohort A (low dose) but decreased in cohort B (high dose) (Table 6), indicating improvement in the latter group.

[0323] Table 5. Changes in Parkinson's Disease Nonmotor Symptom Scale (or PD NMSS) scores relative to baseline at 18 months. .

[0324]

[0325] Table 6. Changes in Parkinson's Disease Questionnaire-39 (PDQ-39) scores relative to baseline at 18 months. .

[0326]

[0327] In formal neuropsychological assessments, the mean NPI-Q score was stable between baseline and 12 months in cohort A (low dose) and decreased within the same period in cohort B (high dose). Figure 5 ), indicating improvement. For cohort A (low dose), the mean score for most RBANS categories remained stable at 12 months. Figure 6A However, cohort B (high dose) showed an increase in the RBANS categories of immediate and delayed memory, indicating improvement in these categories, while also showing a modest decrease in the RBANS category of attention, indicating deterioration in that category. Figure 6B Furthermore, in cohort A (low dose), an increase in emotional apathy and executive function categories was observed in FrSBe post-transplantation, indicating a deterioration in these categories. Figure 7A In cohort B (high dose), lower scores were observed in all FrSBe categories, indicating improvement ( Figure 7B ).

[0328] Overall, this embodiment provides Phase 1 clinical trial data demonstrating the safety and tolerability of certain doses of dopaminergic neurons for PD treatment one year post-transplantation. It further demonstrates the feasibility of the stereotactic surgical delivery and treatment protocol, with all participants receiving the planned dose of neurons and completing a one-year immunosuppressive regimen without significant problems. Additionally, 18F-DOPA PET imaging provided evidence of transplanted neuronal survival and implantation in the posterior putamen. Clinical analysis revealed tangible improvements, particularly a reduction in MDS-UPDRS Part III (OFF) scores, a decrease in OFF time, and an increase in ON time without troubling motor dyskinesia, with no graft-induced motor dyskinesia occurring in the first year. While efficacy trends were discernible between the two cohorts, cohort B (high dose) showed a greater range of variation, reinforcing the potential of this innovative treatment approach in providing dose-dependent therapy for PD.

[0329] These results also demonstrate that administration of certain doses of dopaminergic cells induces stability or improvement in exploratory nonmotor, quality of life, and psychiatric outcomes. While improvements in neuropsychological measures were observed in both cohorts, cohort B (high dose) showed a greater trend toward improvement in NPI-Q, RBANS, and FrSBe at 12 months, indicating positive outcomes in neuropsychiatric symptoms, cognitive function, and frontal lobe-related behaviors. Cohort B also demonstrated stability of PDNMSS and PDQ-39 scores at 18 months (6 months after immunosuppression cessation), indicating controlled nonmotor symptom severity and health-related quality of life. Results from exploratory nonmotor outcomes further support the potential of this dopaminergic cell transplantation therapy in providing dose-dependent treatment for PD.

[0330] The administered dose of dopaminergic neurons induced observed, sustained exploratory movement in subjects with PD. Improvement of motor function outcomes

[0331] To evaluate improvements in motor function, the MDS-UPDRS Part II score was assessed. Specifically, the MDS-UPDRS Part II score was evaluated in subjects treated with dopaminergic neurons and compared with measurements obtained from the respective subjects prior to treatment with low-dose (cohort A) and high-dose (cohort B) dopaminergic neurons. Following treatment with dopaminergic neurons, a decrease in the mean total MDS-UPDRS Part II score was observed in PD patients from cohort B. Figure 8 This reduction was observed 18 months after treatment, demonstrating a sustained improvement and reversal in disease progression.

[0332] The MDS-UPDRS Part III OFF score was also evaluated up to 18 months post-treatment. The MDS-UPDRS Part III OFF scores of treated subjects in cohorts A and B were recorded and compared to baseline measurements (i.e., measurements obtained from the respective subjects prior to treatment with dopaminergic neurons). Figure 9 In patients with Parkinson's disease (PD), a decrease in the MDS-UPDRS Part III (OFF) score, indicating improved motor function, was observed after treatment with dopaminergic neurons. This was more pronounced in the high-dose group (cohort B), suggesting a reversal of PD-related effects on motor function. The decrease in the MDS-UPDRS Part III (OFF) score was observed at 18 months post-treatment and 6 months post-immunosuppression.

[0333] Additionally, the Hauser / PD diary ON and OFF times of treated subjects in cohorts A and B were recorded. When compared to their corresponding baseline scores, subjects showed a significant increase in good ON time (…). Figure 10A These baseline scores were recorded immediately before the start of cell therapy. These improvements persisted over a long period, with treatment effects remaining significant up to 18 months post-treatment. Simultaneously, Hauser diary off-time was significantly reduced. Figure 10B The results indicate a reduction in the period of motor function decline typically associated with PD. These results highlight the long-term efficacy and potential of certain doses of dopaminergic neurons as a treatment for PD.

[0334] In addition, the clinical efficacy of low-dose and high-dose (cohort A and cohort B, respectively) dopaminergic neurons was evaluated using the UDysRS objective subscale score. These scores were reported and compared with baseline scores established before treatment. A decrease in UDysRS objective subscale scores was observed in subjects receiving either low-dose or high-dose dopaminergic neurons. Figure 11This improvement in reducing symptoms of motor dysfunction demonstrates the potential of dopaminergic neuron transplantation for treating motor symptoms in patients with Parkinson's disease (PD).

[0335] These results demonstrate that administration of certain doses of dopaminergic cells leads to stability or improvement in exploratory motor function outcomes up to 18 months after administration.

[0336] The sustained survival of implanted dopaminergic neurons at 6 months after immunosuppression cessation and 18 months after transplantation and Evidence of function

[0337] To assess the implantation, survival, and function of transplanted dopaminergic neurons, a combination of positron emission tomography (PET) and magnetic resonance imaging (MRI) techniques was used.

[0338] Assess the survival of transplanted dopaminergic neurons at 12 and 18 months post-transplantation. Figure 12 and Figure 13 Exemplary strategies for evaluating the implantation, survival, and function of transplanted neurons are outlined.

[0339] MRI was used to determine the anatomical location and integrity of the transplanted neurons. Simultaneously, PET imaging was used, specifically by measuring 18F-fluoridepa (18F-DOPA) uptake, to provide a functional assessment of the dopaminergic activity and viability of the transplanted neurons. This provided precise quantification of 18F-DOPA uptake over time through voxel-based brain atlas analysis and enabled the identification of specific regions of interest where neuronal implantation and survival were observed. This dual imaging approach leverages the strengths of MRI and PET to provide a robust and detailed assessment of the long-term survival and functional integration of transplanted dopaminergic neurons. This method is particularly useful for monitoring disease progression and treatment outcomes in neurodegenerative diseases such as Parkinson's disease (PD).

[0340] Figure 14 Exemplary imaging data demonstrating the implantation, survival, and function of the applied dopaminergic neurons are shown. The image represents the average of multiple images taken at 12 and 18 months post-transplantation compared to baseline.

[0341] Figure 15 Exemplary imaging data of 18F-DOPA uptake at 12 and 18 months post-transplantation are presented. Cluster-level analysis (N=12) of 18F-DOPA uptake at 12 and 18 months post-transplantation, compared to baseline, revealed clusters of increased 18F-DOPA PET signal within the striatal hypothesis testing space. Furthermore, the data showed that 18F-DOPA uptake in the putamen remained stable or increased at 18 months post-transplantation (6 months after immunosuppression cessation).

[0342] Figure 16A and Figure 16B Exemplary clinical data on 18F-DOPA uptake in the putamen and caudate nucleus are presented separately. In the putamen, an increase in mean SOR-1 was observed in the largest clusters identified in the left and right putamen at 18 months post-transplantation (n=11). Figure 16A These clusters were interpreted as being associated with applied dopaminergic neurons. Participants discontinued immunosuppression for 6 months at 18 months post-transplantation. A mean reduction in SOR-1 was observed in the largest clusters identified in the left and right caudate nuclei at 18 months post-transplantation (n=11). Figure 16B The reduced uptake of 18F-DOPA in the caudate nucleus is interpreted as an indication of persistent neurodegeneration associated with Parkinson's disease pathology.

[0343] There was no evidence of intracranial hemorrhage, mass, lesion, and / or cellular overgrowth. The transplanted cells were unidentifiable on MRI. The expected finding was bilateral orbital gliosis with peripheral gliosis. Figure 17 ).

[0344] Overall, these clinical results demonstrate successful neuronal implantation, sustained survival, and functionality up to six months after discontinuation of immunosuppressive therapy. Furthermore, these findings demonstrate the potential of dual-imaging methods for monitoring treatment outcomes in neurodegenerative diseases.

[0345] The sustained survival of implanted dopaminergic neurons at 12 months after immunosuppression cessation and 24 months after transplantation and Evidence of function

[0346] To further evaluate the long-term safety, tolerability, and efficacy of dopaminergic neuron transplantation in subjects with Parkinson's disease, participants from cohort A (low dose, n=5) and cohort B (high dose, n=7) were evaluated at 24 months post-transplantation. Results from this follow-up assessment provided clinical evidence of successful neuronal engraftment, continued survival, and function 12 months after cessation of immunosuppressive therapy. The following exemplary results provide a comprehensive analysis of motor function, movement disorders, daily symptom fluctuations, and non-motor symptoms at the 24-month time point. Specifically, changes in Part III and Part II scores of the Modified Unified Parkinson's Disease Rating Scale (MDS-UPDRS), sponsored by the Movement Disorders Association, were measured to assess motor function; the Unified Movement Disorder Rating Scale (UDysRS) score was measured to assess movement disorders; the PD diary score was measured to capture daily symptom fluctuations; and the Neuropsychiatric Questionnaire (NPI-Q) score was measured to assess non-motor symptoms. Furthermore, adverse events occurring during the treatment period were continuously reported and monitored.

[0347] Figure 18A and Figure 18BThe progression of the MDS-UPDRS Part III ON score over 24 months is shown in the low-dose cohort (n=5) and the high-dose cohort (n=7). The high-dose cohort showed a sustained decrease in the score as measured from pre-transplant (24.3) to 24 months (20.7), indicating a sustained improvement in motor function. The low-dose cohort showed a more moderate improvement.

[0348] Figure 19 The changes in MDS-UPDRS Part II scores, reflecting improvements in daily living motor skills, are shown. Both cohorts showed improvement, with the high-dose group exhibiting a more significant and sustained effect.

[0349] The impact on movement disorders (a common side effect of long-term levodopa treatment) was assessed using the UDysRS objective subscale score. Figures 20A to 20B Exemplary experimental results for UDysRS sub-scores from 0 months (pre-transplant) to 24 months post-transplant are shown. Overall, there was no significant deterioration in UDysRS scores in either the low-dose or high-dose cohorts.

[0350] Figure 21A and Figure 21B An example patient's outcome is shown via PD diary score report. Figure 21A The study showed an increase in good ON time for both cohorts, with the high-dose group exhibiting a more significant improvement. Figure 21B It showed a reduction in OFF time, especially in the high-dose group, from 5.0 hours before transplantation to 3.1 hours 24 months after transplantation.

[0351] Non-motor symptoms were assessed using the NPI-Q total score, such as Figure 22 As shown in the figure. Both cohorts showed improvement in neuropsychiatric symptoms, with the high-dose group demonstrating a more substantial improvement, decreasing from 6.4 pre-transplant to 4.0 at 24 months.

[0352] Overall, these results demonstrate that the therapeutic effect of transplanted dopaminergic neurons persists for at least 24 months post-transplantation, with the high-dose cohort typically showing more significant and sustained improvements in both motor and non-motor symptoms. These findings indicate successful implantation, survival, and continued functionality of the transplanted neurons, even 12 months after cessation of immunosuppressive therapy.

[0353] By referencing the incorporated equivalent schemes and scope

[0354] Those skilled in the art will recognize that many equivalents of the specific embodiments described herein can be determined using only conventional experiments. It should be understood that modifications are also provided within the specification provided herein that do not materially affect the operation of various embodiments of this disclosure. The scope of this disclosure is not intended to be limited to the foregoing description, but rather as set forth in the appended claims.

[0355] In the claims, unless indicated otherwise or otherwise apparent from the context, articles such as “a,” “an,” and “the” may mean one or more. Unless indicated otherwise or otherwise apparent from the context, a claim or description including “or” among one or more members of that group is considered satisfied if one, more, or all of the group members are present in a given product or method, for use in or otherwise related to the given product or method. This disclosure includes embodiments in which exactly one member of the group is present in a given product or method, for use in or otherwise related to the given product or method. This disclosure also includes embodiments in which more than one or all of the group members are present in a given product or method, for use in or otherwise related to the given product or method.

[0356] Furthermore, it should be understood that this disclosure covers all variations, combinations, and arrangements in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more claims or relevant portions of the specification are introduced into another claim. For example, any claim dependent on another claim may be modified to include one or more limitations found in any other claim dependent on the same basic claim. Furthermore, where the claims describe a composition, it should be understood that, unless otherwise instructed or unless a contradiction or inconsistency would be obvious to a person of ordinary skill in the art, this includes methods of using the composition for any purpose disclosed herein, and methods of preparing the composition according to any preparation method disclosed herein or other methods known in the art.

[0357] When elements are presented as a list, such as in a Markush group format, it should be understood that each subgroup of these elements is also disclosed, and any one or more elements may be removed from that group. It should be understood that, generally, when an aspect of this disclosure or an embodiment is referred to as including a particular element, feature, step, etc., certain embodiments or aspects of embodiments of this disclosure consist of or are substantially composed of such elements, features, steps, etc. Therefore, for each embodiment of this disclosure that includes one or more elements, features, steps, etc., this disclosure also provides an embodiment composed of or substantially composed of those elements, features, steps, etc.

[0358] Where a range is given, endpoints are included. Furthermore, it should be understood that unless otherwise indicated or otherwise apparent from the context and / or understanding of a person skilled in the art, values ​​expressed as ranges may be assumed to be any specific value within the range specified in different embodiments of this disclosure up to one-tenth of the unit of the lower limit of that range. It should also be understood that unless otherwise indicated or otherwise apparent from the context and / or understanding of a person skilled in the art, values ​​expressed as ranges may be assumed to be any subrange within a given range, wherein the endpoints of the subranges are expressed with the same precision as one-tenth of the unit of the lower limit of that range.

[0359] Furthermore, it should be understood that any particular embodiment of this disclosure may be expressly excluded from any one or more claims. Given a scope, any value within that scope may be expressly excluded from any one or more claims. Any embodiment, element, feature, application, or aspect of the composition and / or method of this disclosure may be excluded from any one or more claims. For the sake of brevity, not all embodiments in which one or more elements, features, objects, or aspects are expressly excluded herein.

[0360] Throughout this disclosure, references are made to various publications, patents, and sequence database entries. The disclosures of these publications, patents, and sequence database entries (including those listed above) are hereby incorporated herein by reference in their entirety, as if each individual publication or patent were expressly and individually incorporated by reference. In the event of any conflict, any definition herein, including that herein, shall prevail.

[0361] Although this disclosure has been described with reference to the examples provided above, it should be understood that various modifications may be made without departing from the scope of this disclosure. Therefore, the examples above are intended to illustrate and not limit this disclosure.

Claims

1. A method for treating Parkinson's disease in a subject, the method comprising: An effective amount of dopaminergic cell population was administered to the subject. The administration resulted in an improvement in motor function or non-motor function, or a combination thereof, in the subject compared to a control or the subject's baseline prior to the administration.

2. The method according to claim 1, wherein the effective amount of dopaminergic cell population comprises about 1.0 × 10^6 to about 1.2 × 10^7 dopaminergic cells.

3. The method according to claim 1 or 2, wherein the effective amount of dopaminergic cell population comprises about 1.8 × 10^6 dopaminergic cells.

4. The method according to any one of claims 1 to 3, wherein the effective amount of dopaminergic cell population comprises about 5.4 × 10^6 dopaminergic cells.

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

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

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

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

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

10. The method according to any one of claims 1 to 9, wherein the dopaminergic cell population has the ability to produce dopamine, and when evaluated in vitro using liquid chromatography-tandem mass spectrometry (LC-MS / MS), the ability to produce dopamine provides an area under the concentration-time curve (AUC) equal to or greater than 11.2 ng × day / mL.

11. The method according to any one of claims 1 to 10, wherein the administration comprises delivering the dopaminergic cell population to the posterior shell nucleus of the subject.

12. The method of claim 11, wherein the administration comprises delivering a first portion of the dopaminergic cell population to the left hemisphere of the subject's posterior shell nucleus and delivering a second portion of the dopaminergic cell population to the right hemisphere of the subject's posterior shell nucleus.

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

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

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

16. The method according to any one of claims 1 to 15, wherein the improvement in motor function is determined at least in part based on changes in one or more of the following: Compared to the baseline of the control group or the subject prior to the administration, the subject's Part II or Part III score on the Modified Unified Parkinson's Disease Rating Scale (MDS UPDRS), sponsored by the Movement Disorders Association; and The subjects' objective sub-item scores on the Unified Movement Disorder Rating Scale (UDysRS) were compared with the baseline of the control or the subjects prior to the administration.

17. The method according to any one of claims 1 to 16, wherein the improvement in motor function is determined at least in part based on changes in the subject's ON and / or OFF scores compared to the baseline of the control or the subject prior to the administration.

18. The method according to any one of claims 1 to 17, wherein the improvement in said nonmotor function is measured by one or more assays selected from the group consisting of: sleep quality assessment, cognitive assessment, neuropsychological testing, emotion assessment, autonomic nervous system function testing, imaging testing, and other standardized nonmotor function assessments.

19. The method according to any one of claims 1 to 18, wherein the improvement in nonmotor function is determined at least in part based on the change in the subject's Parkinson's Disease Nonmotor Symptom Scale (PD NMSS) score compared with the baseline of the control or the subject prior to the administration.

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

21. The method according to any one of claims 1 to 20, wherein the improvement in nonmotor function is determined at least in part based on the change in the subject's Neuropsychiatric Questionnaire (NPI-Q) score compared with the baseline of the control or the subject prior to the administration.

22. The method according to any one of claims 1 to 21, wherein the improvement in nonmotor function is determined at least in part based on the change in the subject's Repeatable Suite of Neuropsychological Status Assessment (RBANS) score compared with the baseline of the control or the subject prior to the administration.

23. The method according to any one of claims 1 to 22, wherein the improvement in non-motor function is determined at least in part based on the change in the subject's Frontal System Behavior Scale (FrSBe) score compared with the baseline of the control or the subject prior to the administration.

24. The method according to any one of claims 1 to 23, wherein the baseline is a measurement of the subject's motor and non-motor functions prior to the administration.

25. The method according to any one of claims 1 to 24, wherein the improvement indicates a reversal of the progression of Parkinson's disease.

26. The method of any one of claims 1 to 25, wherein the administration comprises delivering the dopaminergic cell population to the subject using 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 baliximab, methylprednisolone, and tacrolimus.

28. The method of claim 27, wherein: Approximately 4 days after the administration of the dopaminergic cell population, the baliximab was administered intravenously at approximately 20 mg during and after the operation. Prior to the administration of the aforementioned dopaminergic cell population, approximately 500 mg of methylprednisolone was administered intravenously; and Tacrolimus was administered approximately one day after the administration of the dopaminergic cell population.

29. The method according to claim 28, wherein: Following the administration of the dopaminergic cell population, methylprednisolone is further administered daily at approximately 5 mg.

30. The method according to any one of claims 1 to 29, wherein the improvement can be detected approximately 12 weeks after the administration of the dopaminergic cell population.

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

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

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

34. The method according to any one of claims 1 to 33, wherein the dopaminergic cell population is derived from pluripotent stem cells that differentiate into dopaminergic cells in vitro.

35. The method according to any one of claims 1 to 34, wherein the dopaminergic cell population comprises midbrain dopaminergic neurons or their precursors.

36. The method of claim 35, wherein the midbrain dopaminergic neuron or its precursor is derived from basal progenitor cells.

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

38. A therapeutic composition comprising: Effective amounts of dopaminergic cell populations for the treatment of Parkinson's disease; and Cell delivery solution.

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

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

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

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

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

44. The therapeutic composition according to any one of claims 38 to 43, wherein the dopaminergic cell population has the ability to produce dopamine, and the ability to produce dopamine provides an area under the concentration-time curve (AUC) equal to or greater than 11.2 ng × day / mL when evaluated in vitro using liquid chromatography-tandem mass spectrometry (LC-MS / MS).

45. The therapeutic composition according to any one of claims 38 to 44, wherein the concentration of the dopaminergic cell population in the delivery solution is from about 71,000 cells / µL to about 123,000 cells / µL.

46. ​​The therapeutic composition according to any one of claims 38 to 45, wherein the dopaminergic cell population comprises midbrain dopaminergic neurons or precursors thereof.

47. The therapeutic composition of claim 46, wherein the midbrain dopaminergic neuron or its precursor is derived from in vitro floorplate progenitor cells.

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

49. The therapeutic composition according to 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 buffer solutions; (c) one or more salts; and (d) One or more stabilizers, The one or more stabilizers mentioned therein are selected from the group consisting of recombinant albumin (rHSA), dextran, and poloxamer.

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

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

52. The therapeutic composition according to any one of claims 38 to 51, wherein the dopaminergic cell population comprises at least 900,000 dopaminergic cells.

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

54. The therapeutic composition according to any one of claims 38 to 53, wherein the dopaminergic cell population comprises at least 5.4 × 10^6 dopaminergic cells.

55. The therapeutic composition according to any one of claims 38 to 54, wherein the Parkinson's disease is advanced Parkinson's disease.

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

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

58. The vessel according to claim 56 or 57, wherein the vessel comprises an aseptic technique (AT) vial.

59. Use of the therapeutic composition according to any one of claims 38 to 55 in the treatment of Parkinson's disease.

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