A method for deriving dopaminergic neurons from pluripotent stem cells.

Abstract

The present application discloses a method for producing dopaminergic neurons from human stem cells by adding or increasing the vitamin concentration in neurobasal medium around day 20±3 of the protocol for differentiating pluripotent stem cells into dopaminergic neurons.

Description

[Technical Field]

[0001] This application relates to a method for differentiating pluripotent stem cells into dopaminergic neurons. Furthermore, this application relates to the treatment or prevention of diseases associated with transplanting the resulting dopaminergic neurons into patients. [Background technology]

[0002] The development of stem cell-derived dopaminergic neurons for treating Parkinson's disease is attracting attention as a major area of ​​regenerative medicine. While one clinical trial has been conducted to date (Jun Takahashi, Japan), significant technical challenges remain, and FDA approval for human trials has not yet been obtained in the United States. These technical challenges may make the treatment of Parkinson's disease with stem cell-derived dopaminergic neurons impractical.

[0003] One issue is that embryonic stem cells (ESCs) differentiate more readily into the desired cell type than induced pluripotent stem cells (iPSCs). Embryonic stem cells often produce more functionally advanced cells than iPSC-derived cells. However, regulatory approval requires creating a large stock of the same ESCs, which will be used to generate all the investigational drugs that enable experimental data and subsequently to treat patients, forming a master cell bank. Efforts to date have revealed that after generating a source cell bank, performing the necessary experiments, and undergoing the many passages required for use in human clinical trials, ESCs acquire karyotype abnormalities and become unstable. In addition, many countries currently prohibit the use of embryonic stem cells for research or treatment.

[0004] iPSCs are more practical from a regulatory approval standpoint because each procedure is patient-specific and typically generated from the patient's own cells. Therefore, there is no master cell bank. However, iPSCs to date have not differentiated into functional cells, similar to embryonic stem cells. Clonal limitations are a major problem in differentiating iPSCs into desired cell types. In other words, one iPSC clone may be able to form nerve cells, while another cannot. Even more precisely, one clone may form excellent nerve or hepatocyte cells, while another, even if expressing characteristic molecular markers, may not function the same way as other clones or naturally occurring cells. Often, many clones need to be tested to determine which clones can differentiate into a particular cell type. There is significant scientific evidence supporting the idea that clonal limitations stem from cell fate determination already made by the priming stem cells. Cells induced to become pluripotent still retain molecular markers, such as methylation or acetylation, that limit what their cell clones can mature into.

[0005] These fundamental challenges in developing stem cell-derived therapies pose further problems in the development of dopaminergic neurons for the treatment of Parkinson's disease.

[0006] On average, Parkinson's disease patients require treatment for the first time at age 65. If the dopaminergic neurons originate from donor embryonic stem cells, the patient needs to take immunosuppressants for a period to prevent donor cell rejection. This age is not suitable for administering immunosuppressants.

[0007] Furthermore, current methods for generating dopaminergic neurons from stem cells produce neurons with very low engraftment rates. In the case of Parkinson's disease, it is thought that 100,000 cells need to engraft to achieve a therapeutic effect. Due to the low engraftment rate, it is necessary to transplant 10 to 100 times the number of cells to achieve a therapeutic effect. In other words, 1,000,000 to 10,000,000 cells need to be transplanted, which is a technically difficult challenge with existing methods for differentiating stem cells into dopaminergic neurons. It has been reported that when dopaminergic neurons are generated from human iPS cells, only about 3% of the yield are true dopaminergic neurons. To obtain a pure population of dopaminergic neurons from iPSCs, researchers have had to select cells using specific molecular markers such as Corin and LRTM1 in the early stages of differentiation. These researchers found that dopaminergic neurons or their precursors, selected for Corin+ and LRTM1+, had a higher proportion of TH-positive cells and exhibited approximately 10 times greater engraftment ability compared to the pure population of unselected cells (Samata and Takahashi 2016, DOI:10.1038 / ncomms13097). Nevertheless, these dopaminergic neurons or their precursors needed to be transplanted as early as day 28, and even then, only about 10% were present at 3 months post-transplant.

[0008] To overcome the problem of low engraftment rates of stem cell-derived dopaminergic neurons, dopaminergic neurons are transplanted at an early stage, between day 15 and day 32 of differentiation, when they are in the progenitor phase. Experiments have shown that engrafting immature neurons significantly improves the engraftment rate, which is thought to be due to the provision of unknown factors necessary for efficient engraftment from the host brain. However, the transplantation of early progenitor cells raises concerns among regulatory authorities such as the USFDA.

[0009] Like regulatory authorities in other countries, the USFDA requires the characterization of cells for transplantation. For example, characterizing dopaminergic neurons for the treatment of Parkinson's disease would require demonstrating that the cells produce dopamine. However, early cells (around 15-20 days old) transplanted to ensure proper engraftment and growth have not yet secreted dopamine, or even the final molecular markers that identify them as dopaminergic neurons. Furthermore, the early cell population may contain pluripotent stem cells, which could potentially cause teratomatic tumors in the recipient's brain.

[0010] If the USFDA applies the same acceptance criteria to cells for treating Parkinson's disease as it does to other cell therapies, it seems highly unlikely that early transplantation of dopaminergic neurons or their precursors would be accepted. The FDA is likely to require therapeutic cells to meet certain release criteria. In other words, the manufactured cells would need to exhibit efficacy, such as reproducibly expressing specific molecular markers and secreting a specific range of dopamine. Current methods for generating stem cell-derived dopaminergic neurons, and early transplantation, make it impossible to fully understand the characteristics and titer of the cell products before transplantation.

[0011] Therefore, developing a method, including a formulation, that efficiently and reproducibly induces stem cells and differentiates them into dopaminergic neurons or their precursors, resulting in improved viability, engraftment ability, yield, and increased dopamine secretion would be an improvement in this field. A method that improves any of the efficiency, purity, yield, and / or dopamine secreted from human iPS cells would represent a significant improvement over the current situation. iPSC-derived dopaminergic neurons would eliminate the need to treat patients with immunosuppressants and the need for master cell banks of embryonic donor cells.

[0012] Currently, in the cell therapy for Parkinson's disease, a strategy is taken to differentiate stem cells into precursors of dopaminergic neurons and finally transplant them into the appropriate site of the brain before they mature into dopamine-producing neurons. The reason for transplanting dopaminergic neurons or their precursors early is that the local environment in the brain provides unknown factors necessary for the precursors to have the ability of nerve transport, engraft, and finally mature into functional dopaminergic neurons that produce and secrete dopamine.

[0013] Currently, stem cell-derived dopaminergic neurons, more specifically their precursors, need to be engrafted into the brain before they are fully developed. According to experiments, early transplantation increases the engraftment rate and more benefits can be obtained, which is probably due to the increased production of dopamine. Dopaminergic neurons or precursors are transplanted at a stage before dopamine production so that unknown factors in the local environment of the brain can induce appropriate maturation to the dopamine production stage.

[0014] The drawback of the method for early engraftment of dopaminergic neurons or precursor cells is that the characteristics of the cells cannot be fully understood. When treating humans with cell therapy, the US FDA requires evaluating the characteristics of the cells and "releasing" them for administration to humans only when they meet certain predefined criteria. Based on the FDA's release criteria requirements for other cell therapies, criteria such as the defined proportion of cells expressing specific molecular markers and the defined dopamine production amount by 1M cells are expected.

[0015] Therefore, if stem cell-derived dopaminergic neurons can be cultured in vitro with high reliability and reproducibility until a stage where a high proportion of the transplanted cells express definitive molecular markers and a considerable amount of dopamine is produced, it will be a significant advancement in the art. Finally, these cells should demonstrate their ability to engraft into the brain in vitro.

[0016] Therefore, identifying the brain-provided factors that induce the maturation steps of dopaminergic neurons, as well as the timeframe in which dopaminergic neuron precursors should come into contact with these factors, would represent a significant improvement over the current state of this field. [Overview of the Initiative]

[0017] The present invention relates to a method for producing dopaminergic neurons from human stem cells, comprising the step of adding a vitamin to a neuronal basal medium or increasing the vitamin concentration around day 20±3 of a protocol for differentiating pluripotent stem cells into dopaminergic neurons. The protocol may be Protocol A. The vitamin may be vitamin A, such as retinol, retinol acetate, 9-cisretinoic acid, 13-cisretinoic acid, or all-trans retinoic acid. Vitamin A may be solubilized in a lipid-rich formulation such as human serum albumin, Albumax, or non-human serum albumin. In one embodiment, the final concentration of vitamin A may be 1 μM to 3 μM.

[0018] Alternatively, according to the above, the vitamin may be vitamin B6. Vitamin B6 may be in the form of pyridoxal-5'-phosphate, also known as pyridoxine, pyridoxal, or PLP. In one embodiment, the final concentration of vitamin B6 may be 10 μM to 30 μM.

[0019] Alternatively, according to the above, the vitamin may be vitamin C. Vitamin C may be in the form of 2-phospho-ascorbic acid or L-ascorbic acid. In one embodiment, the final concentration of vitamin C may be 200 nM to 110 uM.

[0020] In any of the above methods, the differentiated pluripotent stem cells may be cultured in NME7-AB or WNT3A. In other embodiments, the differentiated pluripotent stem cells may be untreated.

[0021] As a result, the produced dopaminergic neurons may be characterized by expressing 30% or more, 100% or more, 500% or more, or 1000% or more of dopamine compared to dopaminergic neurons produced by differentiation protocols that do not add or increase vitamins.

[0022] Based on the above, the produced dopaminergic neurons are characterized by forming 30%, 100%, 500%, or 1000% more neurites compared to dopaminergic neurons produced using differentiation protocols that do not involve the addition or increase of vitamins.

[0023] The present invention also covers methods to improve the likelihood of successful engraftment of dopaminergic neurons to a target, which include administering the dopaminergic neurons obtained by the above method to the target.

[0024] The present invention also covers a method for treating central nervous system disorders in patients for whom dopamine-producing neuronal engraftment is desired, comprising engrafting dopaminergic neurons obtained by the above method into the individual in need. Central nervous system disorders include Parkinson's disease, Huntington's disease, multiple sclerosis, or Alzheimer's disease. Damage to the central and peripheral nervous systems may further be treated by engrafting neurons at the site of injury, dopaminergic neurons for central nervous system disorders, and other types of neurons for peripheral nerve damage. [Brief explanation of the drawing]

[0025] The patent or application file must include at least one drawing created in color. A copy of this patent or patent application publication, including the color drawing, will be provided by the relevant office after the request and payment of the required fees.

[0026] The present invention will be better understood from the given detailed description herein and the accompanying drawings, which are given only as examples, and will not be limited thereto. [Figure 1A] Figure 1A is a schematic diagram of four different protocols used to differentiate pluripotent stem cells into dopaminergic neurons. Figure 1A is a schematic diagram of the protocol published in patent application US2018 / 0094242A1 (the disclosure of a culture medium for differentiating pluripotent stem cells into dopaminergic neurons is incorporated herein by reference), which is referred to herein as Protocol A. [Figure 1B] This is a schematic diagram of four different protocols used to differentiate pluripotent stem cells into dopaminergic neurons. Figure 1B is a schematic diagram of a novel and improved protocol developed by the inventors, which is referred to here as Protocol B. In this protocol, pyridoxal is added at a final concentration of 11 μM from day 21 onward, bringing the total pyridoxal concentration in the culture medium to approximately 21 μM. [Figure 1C] This is a schematic diagram of four different protocols used to differentiate pluripotent stem cells into dopaminergic neurons. Figure 1C is a schematic diagram of a novel and improved protocol developed by the inventors, which is referred to here as Protocol C, in which various forms of vitamin A, vitamin B, and possibly vitamin C are added to the basal neuronal culture medium from day 21 onward. [Figure 1D] This is a schematic diagram of four different protocols used to differentiate pluripotent stem cells into dopaminergic neurons. Figure 1D shows a schematic diagram of protocol C.2, in which, from day 21 onward, the underlying neuronal medium is replaced with one containing pyridoxine at a final concentration of 16 μM, retinol at a final concentration of 1.2 μM, and retinyl acetate at a final concentration of 0.17 μM, without pyridoxal. [Figure 1E]This is a schematic diagram of four different protocols used to differentiate pluripotent stem cells into dopaminergic neurons. Figure 1E is a schematic diagram of Protocol D, the optimized protocol, in which, from day 21 onward, the basal medium containing approximately 10 μM pyridoxal is further supplemented with 11 μM pyridoxal, 1.2 μM retinol, 0.17 μM retinyl acetate, 61 μM ascorbic acid 2-phosphate, and 11 μM L-ascorbic acid. [Figure 2] Images A-L show fluorescence images taken on day 24 of Protocol A, in which three different types of pluripotent stem cells cultured in different media were differentiated to attempt to generate dopaminergic neurons. Images A-D are fluorescence images of commercially available HES3 cells, here referred to as hESE8-HES3, which are human embryonic stem cells cultured in E8 medium before differentiation. Images E-H are fluorescence images of human induced pluripotent stem cells, here referred to as iPSE8-A6, which were cultured in E8 medium before differentiation. Images I-L show fluorescence images of human induced pluripotent stem cells, here referred to as iPSNME7-6E, which were cultured in NME7AB untreated medium before differentiation. Images A, E, and I show cells stained for the presence of GIRK2 (G protein-regulated inward-rectifier potassium channel 2), which is expressed in dopaminergic neurons. B, F, and J show cells stained for the presence of TH (tyrosine hydroxylase), which is considered the gold standard in identifying dopaminergic neurons. C, G, and K show cells stained for the presence of Tuj1 (neuron-specific class II B-tubulin), a panneuronal marker. D, H, and L show superimposed images of all three markers. [Figure 3]Images A-L show fluorescence images taken on day 24 of protocol C.2, in which three different types of pluripotent stem cells cultured in different media were differentiated to attempt to generate dopaminergic neurons. Images A-D show fluorescence images of commercially available HES3 cells, here referred to as hESE8-HES3, human embryonic stem cells cultured in E8 medium before differentiation. Images E-H show fluorescence images of human induced pluripotent stem cells, here referred to as iPSE8-A6, cultured in E8 medium before differentiation. Images I-L show fluorescence images of human induced pluripotent stem cells, here referred to as iPSNME7-6E, cultured in NME7AB untreated medium before differentiation. Images A, E, and I show cells stained for the presence of GIRK2 (G-protein-regulated inward-rectifier potassium channel 2), which is expressed in dopaminergic neurons. Images B, F, and J show cells stained for the presence of TH (tyrosine hydroxylase), which is considered the gold standard in the identification of dopaminergic neurons. C, G, and K show cells stained for the presence of Tuj1 (neuron-specific class II B-tubulin), a panneuronal marker. D, H, and L show the three markers superimposed. [Figure 4]Images A–H show fluorescence images taken at day 60 of pluripotent stem cells differentiated according to either protocol A or protocol C.2. Some images show cells differentiated according to protocol C.2, but with 100 ng / mL of WNT3A added to the pluripotent stem cell medium for 48 hours prior to differentiation initiation. Images A, B, E, F, G, and H show cells differentiated from iPSNME7-N7B naive stem cells reprogrammed by episome synthesis. Images C and D show cells differentiated from iPSE8-A6 stem cells. Images A–B were differentiated according to protocol A. Images E–F were differentiated according to protocol C.2. Images C–D and G–H were differentiated according to protocol C.2, except that 100 ng / mL of WNT3A was added to their respective pluripotent medium for 48 hours prior to initiating the differentiation protocol. Images A, E, C, and G show cells stained for the presence of DAT (dopamine-active transporter) and Tuj1. B, F, D, and H show cells stained for the presence of GIRK2, TH, and Tuj1. [Figure 5] Images A–F show fluorescence images of a scratch assay, also known as a scar or wound healing assay, which assesses the engraftment capacity of neurons. The starting stem cells are naive stem cells "iPSNME7-6E" cultured in NME7AB medium, or priming-state stem cells "iPSE8-A6" cultured in E8 medium. The cells shown were differentiated into dopaminergic neurons according to protocol C.2 and proliferated to confluence on day 13 or 15. Mechanical scratches were applied across the cell field to create gaps. The rate at which neurite growth bridged these gaps was monitored and correlated with engraftment capacity. Green fluorescence is an indicator of dopamine uptake from labeled dopamine. Images A–C show images of neurons derived from iPSNME7-6E. Images D–F show images of neurons derived from iPSE8-A6. [Figure 6]Graphs A-D show the secretion levels of dopamine and its metabolites from 800,000 cells / cm2 sown on day 11 of the protocol, from day 30 to day 60 after differentiation initiation. Graphs A-B show dopamine secreted from cells differentiated into dopaminergic neurons according to protocol A. Graphs C-D show dopamine secreted from cells differentiated into dopaminergic neurons according to protocol C.2. Graphs A and C show dopaminergic neurons derived from stem cells in the iPSE8-A6 primed state. Graphs B and D show dopaminergic neurons derived from stem cells in the iPSNME7-6E untreated state. [Figure 7] This graph shows the amount of dopamine and its metabolites secreted by a variable number of cells, measured on day 60 or day 40, where indicated. Horizontal bars represent priming stem cells differentiated according to Protocol A, crosshatched bars represent priming stem cells differentiated according to Protocol C.2, vertical bars represent naive stem cells differentiated according to Protocol A, and solid black bars represent naive stem cells differentiated according to Protocol C.2. Note that the cell count is the number of cells sown on day 11 of the protocol, expressed per 1 cm². [Figure 8] This table shows the amounts of dopamine and its metabolites secreted by various numbers of dopaminergic neurons derived from human stem cells. The table is organized according to the starting stem cell type. The stem cells were either primed embryonic "HES3" cells, primed induced pluripotent stem cells iPSE8-A6 cells, untreated induced pluripotent stem cells "iPSNME7-6E" generated with Sendai virus, or untreated induced pluripotent stem cells "iPSNME7-N7B" generated using episome technology. In some cases, as shown in the table, the stem cells were cultured for 48 hours in their respective media supplemented with 100 ng / mL of WNT3A before differentiation began. [Figure 9]This table shows the amounts of dopamine and its metabolites secreted by the episomal naive clone iPSNME7-N7B. The number of cells seeded on day 11 and the date on which dopamine secretion is measured are varied. Additionally, where indicated, WNT3A was added to the culture medium at 100 ng / mL for 48 hours prior to the start of differentiation. [Figure 10] This table shows the amounts of dopamine and its metabolites secreted by the episomal naive clone iPSNME7-6E. The number of cells seeded on day 11 and the date on which dopamine secretion is measured are varied. Additionally, where indicated, WNT3A was added to the culture medium at 100 ng / mL for 48 hours prior to the start of differentiation. [Figure 11-1] Images A-K show fluorescence images of human iPS cells, differentiated into dopaminergic neurons on day 24 according to the protocol described herein as Protocol A, taken at 20x magnification using a confocal microscope, with the addition of vitamin A in the form of retinol and retinyl acetate, introduced into the culture medium around day 20 of the protocol until cell harvesting. These cells served as controls to investigate the effects of adding various forms of vitamin B6 around day 20 of the protocol. A shows a fluorescence image of cells stained for GIRK2. B shows a fluorescence image of cells stained for TH, tyrosine hydroxylase. C shows a fluorescence image of cells stained for Tuj1. D shows a fluorescence image of cells stained with Hoechst staining so that the nucleus is visible. E shows a bright-field image. F shows a superimposed fluorescence image of GIRK2, TH, Tuj1, and Hoechst. [Figure 11-2]Images A-K show fluorescence images of human iPS cells, differentiated into dopaminergic neurons on day 24 according to the protocol described herein as Protocol A, taken at 20x magnification using a confocal microscope, with the modification that vitamin A in the form of retinol and retinyl acetate was introduced into the culture medium around day 20 of the protocol until cell harvesting. These cells served as controls to investigate the effects of adding various forms of vitamin B6 around day 20 of the protocol. G shows fluorescence images of cells stained for DAT, a dopamine transporter protein. H shows fluorescence images of cells stained for Tuj1. I shows fluorescence images of cells stained with Hoechst stain so that the nucleus is visible. J shows a bright-field image. K shows a superimposed fluorescence image of DAT, Tuj1, and Hoechst. [Figure 12-1] A–K show fluorescence images taken at 20x magnification on a confocal microscope of human iPS cells at day 24 of differentiation into dopaminergic neurons, according to the protocol described herein as Protocol A, except that vitamin A in the form of retinol and retinyl acetate was introduced into the culture medium around day 20 until cell harvesting. In this experiment, vitamin B6 in the form of pyridoxine was added until the final concentration was 16 μM. A shows a fluorescence image of cells stained for GIRK2. B shows a fluorescence image of cells stained for TH, tyrosine hydroxylase. C shows a fluorescence image of cells stained for Tuj1. D shows a fluorescence image of cells stained with Hoechst stain so that the nucleus is visible. E in Figure 12-1 shows a bright-field image. F shows a superimposed fluorescence image of GIRK2, TH, Tuj1, and Hoechst. [Figure 12-2]A–K show fluorescence images taken at 20x magnification on a confocal microscope of human iPS cells at day 24 of differentiation into dopaminergic neurons, according to the protocol described herein as Protocol A, except that vitamin A in the form of retinol and retinyl acetate was introduced into the culture medium around day 20 until cell harvesting. In this experiment, vitamin B6 in the form of pyridoxine was added until the final concentration was 16 μM. G shows a fluorescence image of cells stained for DAT, a dopamine transporter protein. H shows a fluorescence image of cells stained for Tuj1. I shows a fluorescence image of cells stained with Hoechst stain so that the nucleus is visible. J shows a bright-field image. K shows a superimposed fluorescence image of DAT, Tuj1, and Hoechst. [Figure 13-1] A–K show fluorescence images taken at 20x magnification on a confocal microscope of human iPS cells at day 24 of differentiation into dopaminergic neurons, according to the protocol described herein as Protocol A, except that vitamin A in the form of retinol and retinyl acetate was introduced into the culture medium around day 20 until cell harvesting. In this experiment, vitamin B6 in the form of pyridoxal was added until the final concentration was 11 μM. A shows a fluorescence image of cells stained for GIRK2. B shows a fluorescence image of cells stained for TH, tyrosine hydroxylase. C shows a fluorescence image of cells stained for Tuj1. D shows a fluorescence image of cells stained with Hoechst stain so that the nucleus is visible. E shows a bright-field image. F shows a superimposed fluorescence image of GIRK2, TH, Tuj1, and Hoechst. [Figure 13-2]A–K show fluorescence images taken at 20x magnification on a confocal microscope of human iPS cells at day 24 of differentiation into dopaminergic neurons, according to the protocol described herein as Protocol A, except that vitamin A in the form of retinol and retinyl acetate was introduced into the culture medium around day 20 until cell harvesting. In this experiment, vitamin B6 in the form of pyridoxal was added until the final concentration was 11 μM. G shows a fluorescence image of cells stained for DAT, a dopamine transporter protein. H shows a fluorescence image of cells stained for Tuj1. I shows a fluorescence image of cells stained with Hoechst stain so that the nucleus is visible. J shows a bright-field image. K shows a superimposed fluorescence image of DAT, Tuj1, and Hoechst. [Figure 14-1] A–K show fluorescence images taken at 20x magnification on a confocal microscope of human iPS cells at day 24 of differentiation into dopaminergic neurons, according to the protocol described herein as Protocol A, except that vitamin A in the form of retinol and retinyl acetate was introduced into the culture medium around day 20 until cell harvesting. In this experiment, vitamin B6 in the form of pyridoxal-5'-phosphate, also known as PLP, was added until the final concentration was 20 μM. A shows a fluorescence image of cells stained for GIRK2. B shows a fluorescence image of cells stained for TH, tyrosine hydroxylase. C shows a fluorescence image of cells stained for Tuj1. D shows a fluorescence image of cells stained with Hoechst stain so that the nucleus is visible. E shows a bright-field image. F shows a superimposed fluorescence image of GIRK2, TH, Tuj1, and Hoechst. [Figure 14-2]A–K show fluorescence images taken at 20x magnification on a confocal microscope of human iPS cells at day 24 of differentiation into dopaminergic neurons, according to the protocol described herein as Protocol A, except that vitamin A in the form of retinol and retinyl acetate was introduced into the culture medium around day 20 until cell harvesting. In this experiment, vitamin B6 in the form of pyridoxal-5'-phosphate, also known as PLP, was added until the final concentration was 20 μM. G shows a fluorescence image of cells stained for DAT, a dopamine transporter protein. H shows a fluorescence image of cells stained for Tuj1. I shows a fluorescence image of cells stained with Hoechst stain so that the nucleus is visible. J shows a bright-field image. K shows a superimposed fluorescence image of DAT, Tuj1, and Hoechst. [Figure 15-1] A–K show fluorescence images taken at 20x magnification on a confocal microscope of human iPS cells at day 24 of differentiation into dopaminergic neurons, according to the protocol described herein as Protocol A, except that vitamin A in the forms of retinol and retinyl acetate was introduced into the culture medium around day 20 until cell harvesting. In this experiment, all three forms of vitamin B6 were added as pyridoxine-HCl, pyridoxal, and pyridoxal-5'-phosphate, also known as PLP. A shows a fluorescence image of cells stained for GIRK2. B shows a fluorescence image of cells stained for TH, tyrosine hydroxylase. C shows a fluorescence image of cells stained for Tuj1. D shows a fluorescence image of cells stained with Hoechst stain so that the nucleus is visible. E shows a bright-field image. shows a superimposed fluorescence image of GIRK2, TH, Tuj1, and Hoechst. [Figure 15-2]A–K show fluorescence images taken at 20x magnification on a confocal microscope of human iPS cells at day 24 of differentiation into dopaminergic neurons, according to the protocol described herein as Protocol A, except that vitamin A in the forms of retinol and retinyl acetate was introduced into the culture medium around day 20 until cell harvesting. In this experiment, all three forms of vitamin B6 were added as pyridoxine-HCl, pyridoxal, and pyridoxal-5'-phosphate, also known as PLP. G shows fluorescence images of cells stained for DAT, a dopamine transporter protein. H shows fluorescence images of cells stained for Tuj1. I shows fluorescence images of cells stained with Hoechst stain so that the nucleus is visible. J shows a bright-field image. K shows a superimposed fluorescence image of DAT, Tuj1, and Hoechst. [Figure 16] Figures A-E show fluorescence images of human iPS cells at day 24 of differentiation into dopaminergic neurons, taken at 20x magnification using a confocal microscope, according to the protocol described herein as Protocol A, except that retinol and retinyl acetate were introduced into the differentiation medium around day 20 to observe the effects of various forms of vitamin B6. Figure 16A shows fluorescence images of cells added from day 20 until a final concentration of 16 μM of pyridoxine-HCl was reached. Figure 16B shows fluorescence images of cells added from day 20 until a final concentration of 11 μM of pyridoxal was reached. Figure 16C shows fluorescence images of cells added from day 20 until a final concentration of 20 μM of pyridoxal-5'-phosphate was reached. Figure 16D shows fluorescence images of cells added when all three forms of vitamin B6, including pyridoxine, pyridoxal, and pyridoxal-5'-phosphate, were added. Figure 16E shows a control experiment following Protocol A, except that vitamin A in the forms of retinol and retinyl acetate was added on day 20. [Figure 17]This graph shows the amounts of dopamine and its metabolites, measured by HPLC, present in prepared media from 200,000 cells collected on days 30, 40, 50, or 60. The media were not taken from a single cell source. The media were used in separate experiments until the day the media was collected for analysis. This experiment used Protocol C, in which retinol and retinyl acetate were added under each condition from around day 20 onwards. The form of vitamin B added to the basal neuronal medium was altered. In this experiment, the basal medium contained approximately 10 μM pyridoxal. Under the pyridoxine-added conditions, pyridoxal was omitted from the basal medium. [Figure 18-1] Images A-I show fluorescence images of human iPS cells at day 24 of differentiation into dopaminergic neurons, taken at 20x magnification using a confocal microscope. These images are from a control experiment where cells were differentiated according to protocol A. A shows a fluorescence image of cells stained for GIRK2. B shows a fluorescence image of cells stained for TH and tyrosine hydroxylase. C shows a fluorescence image of cells stained for Tuj1. D shows a fluorescence image of cells stained with Hoechst stain so that the nucleus is visible. E shows a fluorescence image with GIRK2, TH, Tuj1, and Hoechst stained superimposed. [Figure 18-2] Images A-I show fluorescence images of human iPS cells at 24 days after differentiation into dopaminergic neurons, taken at 20x magnification using a confocal microscope. These images are from a control experiment where cells were differentiated according to protocol A. Image F shows fluorescence images of cells stained for DAT, a dopamine transporter protein. Image G shows fluorescence images of cells stained for Tuj1. Image H shows fluorescence images of cells stained with Hoechst stain so that the nucleus is visible. Image I shows fluorescence images with DAT, Tuj1, and Hoechst superimposed. [Figure 19-1]Images A-I show fluorescence images of human iPS cells at day 24 of differentiation into dopaminergic neurons, taken at 20x magnification using a confocal microscope. These images are from another control experiment in which cells were differentiated according to Protocol A, except that pyridoxal was added to the culture medium at a final concentration of 11 μM on day 20. This modified protocol is referred to here as Protocol B. Thus, additional effects can be observed by adding various forms of vitamin A. A shows a fluorescence image of cells stained for GIRK2. B shows a fluorescence image of cells stained for TH, tyrosine hydroxylase. C shows a fluorescence image of cells stained for Tuj1. D shows a fluorescence image of cells stained with Hoechst stain so that the nucleus is visible. E shows a fluorescence image with GIRK2, TH, Tuj1, and Hoechst overlaid. [Figure 19-2] Images A-I show fluorescence images of human iPS cells at day 24 of differentiation into dopaminergic neurons, taken at 20x magnification using a confocal microscope. These images are from another control experiment in which cells were differentiated according to Protocol A, except that pyridoxal was added to the culture medium at a final concentration of 11 μM on day 20. This modified protocol is referred to here as Protocol B. Thus, additional effects can be observed by adding various forms of vitamin A. F shows fluorescence images of cells stained for DAT, a dopamine transporter protein. G shows fluorescence images of cells stained for Tuj1. H shows fluorescence images of cells stained with Hoechst stain so that the nucleus is visible. I shows fluorescence images with DAT, Tuj1, and Hoechst overlaid. [Figure 20-1]Images A-I show fluorescence images of human iPS cells at day 24 of differentiation into dopaminergic neurons, taken at 20x magnification using a confocal microscope, according to protocol B. In this experiment, pyridoxal was added to the culture medium from day 20 onward, in addition to two forms of vitamin A. Retinol was added until the final concentration was 0.7 μM, and retinyl acetate was added until the final concentration was 0.6 μM. A shows a fluorescence image of cells stained for GIRK2. B shows a fluorescence image of cells stained for TH, tyrosine hydroxylase. C shows a fluorescence image of cells stained for Tuj1. D shows a fluorescence image of cells stained with Hoechst stain so that the nucleus is visible. E shows a superimposed fluorescence image of GIRK2, TH, Tuj1, and Hoechst. [Figure 20-2] Images A-I show fluorescence images of human iPS cells at day 24 of differentiation into dopaminergic neurons, taken at 20x magnification using a confocal microscope, according to protocol B. In this experiment, pyridoxal was added to the culture medium from day 20 onward, in addition to two forms of vitamin A. Retinol was added until the final concentration was 0.7 μM, and retinyl acetate was added until the final concentration was 0.6 μM. Image F shows fluorescence images of cells stained for DAT, a dopamine transporter protein. Image G shows fluorescence images of cells stained for Tuj1. Image H shows fluorescence images of cells stained with Hoechst stain so that the nucleus is visible. Image I shows fluorescence images with DAT, Tuj1, and Hoechst stained superimposed. [Figure 21-1]Images A-I show fluorescence images of human iPS cells at day 24 of differentiation into dopaminergic neurons, taken at 20x magnification using a confocal microscope, according to protocol B. In this experiment, in addition to the addition of pyridoxal to the culture medium from day 20 onward, the following forms of vitamin A were added: 9-cisretinoic acid until the final concentration reached 0.446 μM, 13-cisretinoic acid until the final concentration reached 0.446 μM, and all-trans retinoic acid until the final concentration reached 0.446 μM. A shows a fluorescence image of cells stained for GIRK2. B shows a fluorescence image of cells stained for TH and tyrosine hydroxylase. C shows a fluorescence image of cells stained for Tuj1. D shows a fluorescence image of cells stained with Hoechst stain so that the nucleus is visible. E shows a superimposed fluorescence image of GIRK2, TH, Tuj1, and Hoechst. [Figure 21-2] Images A-I show fluorescence images of human iPS cells at day 24 of differentiation into dopaminergic neurons, taken at 20x magnification using a confocal microscope, according to protocol B. In this experiment, pyridoxal was added to the culture medium from day 20 onward, and the following forms of vitamin A were added: 9-cisretinoic acid until the final concentration reached 0.446 μM, 13-cisretinoic acid until the final concentration reached 0.446 μM, and all-trans retinoic acid until the final concentration reached 0.446 μM. F shows fluorescence images of cells stained for DAT, a dopamine transporter protein. G shows fluorescence images of cells stained for Tuj1. H shows fluorescence images of cells stained with Hoechst stain so that the nucleus is visible. I shows fluorescence images of cells stained with DAT, Tuj1, and Hoechst overlaid. [Figure 22-1]Images A-I show fluorescence images of human iPS cells at day 24 of differentiation into dopaminergic neurons, taken at 20x magnification using a confocal microscope, according to protocol B. In this experiment, pyridoxal was added to the culture medium from day 20 onward, and all-trans retinoic acid was added until the final concentration reached 1.33 μM. A shows a fluorescence image of cells stained for GIRK2. B shows a fluorescence image of cells stained for TH and tyrosine hydroxylase. C shows a fluorescence image of cells stained for Tuj1. D shows a fluorescence image of cells stained with Hoechst stain so that the nucleus is visible. E shows a fluorescence image with GIRK2, TH, Tuj1, and Hoechst stained superimposed. [Figure 22-2] Images A-I show fluorescence images of human iPS cells at day 24 of differentiation into dopaminergic neurons, taken at 20x magnification using a confocal microscope, according to protocol B. In this experiment, pyridoxal was added to the culture medium from day 20 onward, and all-trans retinoic acid was added until the final concentration reached 1.33 μM. Image F shows fluorescence images of cells stained for DAT, a dopamine transporter protein. Image G shows fluorescence images of cells stained for Tuj1. Image H shows fluorescence images of cells stained with Hoechst stain so that the nucleus is visible. Image I shows fluorescence images with DAT, Tuj1, and Hoechst stained superimposed. [Figure 23]Images A-E show fluorescence images of human iPS cells at 24 days after differentiation into dopaminergic neurons, taken under a confocal microscope at 20x magnification. A shows a fluorescence image of cells differentiated according to protocol A. B shows a fluorescence image of cells differentiated according to protocol B, but on day 20, in addition to pyridoxal, vitamin A in the form of retinol (0.7 μM) and retinyl acetate (0.6 μM) was added. C shows a fluorescence image of cells differentiated according to protocol B, but on day 20, in addition to pyridoxal, vitamin A in the form of 9-cisretinoic acid, 13-cisretinoic acid, and all-trans retinoic acid was added, each at a final concentration of 0.446 μM. D shows a fluorescence image of cells differentiated according to protocol B, but on day 20, in addition to pyridoxal, all-trans form of vitamin A was added at a final concentration of 1.33 μM. Protocol E is a control experiment in which cells were differentiated according to Protocol B, and differs from Protocol A in that pyridoxal was added to a final concentration of 11 μM from day 20 onwards. [Figure 24-1] Images A-I show fluorescence images of human iPS cells at day 24 of differentiation into dopaminergic neurons, taken at 20x magnification using a confocal microscope, according to protocol B. In this experiment, pyridoxal was added to the culture medium from day 20 onward, as well as two types of vitamin A solubilized in albumax at 2 mg / mL. Retinol was added until the final concentration was 1.2 μM, and retinyl acetate was added until the final concentration was 0.17 μM. A shows a fluorescence image of cells stained for GIRK2. B shows a fluorescence image of cells stained for TH and tyrosine hydroxylase. C shows a fluorescence image of cells stained for Tuj1. D shows a fluorescence image of cells stained with Hoechst stain so that the nucleus is visible. E shows a superimposed fluorescence image of GIRK2, TH, Tuj1, and Hoechst. [Figure 24-2]Images A-I show fluorescence images of human iPS cells at day 24 of differentiation into dopaminergic neurons, taken at 20x magnification using a confocal microscope, according to protocol B. In this experiment, pyridoxal was added to the culture medium from day 20 onward, and two types of vitamin A solubilized in albumax at 2 mg / mL were added. Retinol was added until the final concentration was 1.2 μM, and retinyl acetate was added until the final concentration was 0.17 μM. Image F shows fluorescence images of cells stained for DAT, a dopamine transporter protein. Image G shows fluorescence images of cells stained for Tuj1. Image H shows fluorescence images of cells stained with Hoechst stain so that the nucleus is visible. Image I shows fluorescence images of cells stained with DAT, Tuj1, and Hoechst overlaid. [Figure 25-1] Images A-I show fluorescence images of human iPS cells at day 24 of differentiation into dopaminergic neurons, taken at 20x magnification using a confocal microscope, according to protocol B. In this experiment, in addition to the addition of pyridoxal to the culture medium from day 20 onward, two forms of vitamin A and two forms of vitamin C were added. Vitamin A was solubilized in Albumax at 2 mg / mL. Retinol was added to a final concentration of 1.2 μM, and retinyl acetate to a final concentration of 0.17 μM. Vitamin C was added as 61 μM of 2-phospho-ascorbic acid and 110 μM of L-ascorbic acid. A shows a fluorescence image of cells stained for GIRK2. B shows a fluorescence image of cells stained for TH, tyrosine hydroxylase. C shows a fluorescence image of cells stained for Tuj1. D shows a fluorescence image of cells stained with Hoechst stain so that the nucleus is visible. E shows a superimposed fluorescence image of GIRK2, TH, Tuj1, and Hoechst. [Figure 25-2]Images A-I show fluorescence images of human iPS cells at day 24 of differentiation into dopaminergic neurons, taken at 20x magnification using a confocal microscope, according to protocol B. In this experiment, in addition to the addition of pyridoxal to the culture medium from day 20 onward, two forms of vitamin A and two forms of vitamin C were added. Vitamin A was solubilized in Albumax at 2 mg / mL. Retinol was added until the final concentration was 1.2 μM, and retinyl acetate until the final concentration was 0.17 μM. Vitamin C was added as 61 μM of 2-phospho-ascorbic acid and 110 μM of L-ascorbic acid. Image F shows fluorescence images of cells stained for DAT, a dopamine transporter protein. Image G shows fluorescence images of cells stained for Tuj1. Image H shows fluorescence images of cells stained with Hoechst stain so that the nucleus is visible. Image I shows fluorescence images with DAT, Tuj1, and Hoechst overlaid. [Figure 26-1] Images A-I show fluorescence images of human iPS cells at day 24 of differentiation into dopaminergic neurons, taken at 20x magnification using a confocal microscope, according to protocol B. In this experiment, pyridoxal was added to the culture medium from day 20 onward, and vitamin A, which was solubilized in Albumax at 2 mg / mL, was added as all-trans retinoic acid until the final concentration reached 1.33 μM. A shows a fluorescence image of cells stained for GIRK2. B shows a fluorescence image of cells stained for TH and tyrosine hydroxylase. C shows a fluorescence image of cells stained for Tuj1. D shows a fluorescence image of cells stained with Hoechst stain so that the nucleus is visible. E shows a fluorescence image with GIRK2, TH, Tuj1, and Hoechst stained superimposed. [Figure 26-2]Images A-I show fluorescence images of human iPS cells at day 24 of differentiation into dopaminergic neurons, taken at 20x magnification using a confocal microscope, according to protocol B. In this experiment, pyridoxal was added to the culture medium from day 20 onward, and vitamin A, which was solubilized in Albumax at 2 mg / mL, was added as all-trans retinoic acid until the final concentration reached 1.33 μM. Image F shows fluorescence images of cells stained for DAT and dopamine transporter protein. Image G shows fluorescence images of cells stained for Tuj1. Image H shows fluorescence images of cells stained with Hoechst stain so that the nucleus is visible. Image I shows fluorescence images with DAT, Tuj1, and Hoechst stained superimposed. [Figure 27-1] Images A-I show fluorescence images of human iPS cells at day 24 of differentiation into dopaminergic neurons, taken at 20x magnification using a confocal microscope, according to protocol B. In this experiment, pyridoxal was added to the culture medium from day 20 onward, and vitamin A, which had been solubilized in Albumax at 2 mg / mL, was added as all-trans retinoic acid until the final concentration reached 1.33 μM. Vitamin C was added until 61 μM of 2-phospho-ascorbic acid and 110 μM of L-ascorbic acid were present. A shows a fluorescence image of cells stained for GIRK2. B shows a fluorescence image of cells stained for TH and tyrosine hydroxylase. C shows a fluorescence image of cells stained for Tuj1. D shows a fluorescence image of cells stained with Hoechst stain so that the nucleus is visible. E shows a fluorescence image with GIRK2, TH, Tuj1, and Hoechst stained superimposed. [Figure 27-2]Figures A-I show fluorescence images of human iPS cells at day 24 of differentiation into dopaminergic neurons, taken at 20x magnification using a confocal microscope, according to protocol B. In this experiment, pyridoxal was added to the culture medium from day 20 onward, and vitamin A, which was solubilized in Albumax at 2 mg / mL, was added as all-trans retinoic acid until the final concentration reached 1.33 μM. Vitamin C was added until the concentrations reached 61 μM of 2-phospho-ascorbic acid and 110 μM of L-ascorbic acid. Figure 27-2F shows fluorescence images of cells stained for DAT and dopamine transporter proteins. Figure 27-2G shows fluorescence images of cells stained for Tuj1. Figure 27-2H shows fluorescence images of cells stained with Hoechst stain so that the nuclei are visible. Figure 27-2I shows fluorescence images of cells stained with DAT, Tuj1, and Hoechst overlaid. [Modes for carrying out the invention]

[0027] definition

[0028] Terms used herein generally have their usual meanings in the art, within the context of the invention, and in the specific context in which each term is used. Specific terms are described below or elsewhere in the specification to provide additional guidance to those skilled in the art when describing the compositions and methods of the invention, as well as their manufacturing methods and uses.

[0029] In this application, "a" and "an" are used to refer to both a single object and multiple objects.

[0030] As used herein, “about” or “substantially” generally gives room for not being limited to an exact number. For example, as used in the context of the length of a polypeptide sequence, “about” or “substantially” indicates that the polypeptide should not be limited to the number of amino acids mentioned. It may include several amino acids added to or removed from the N-terminus or C-terminus, as long as functional activity, such as binding activity, exists. The terms “about” or “approximately” mean within an acceptable margin of error of a particular value as determined by those skilled in the art, which depends in part on how that value is measured or determined, for example, on the limitations of the measurement system. For example, “about” may mean within three or more standard deviations for practices in the art. Alternatively, “about” may mean a range of up to 20% of a given value, e.g., up to 10%, up to 5%, or up to 1%. Alternatively, particularly with respect to biological systems or biological processes, the term may mean within one order of magnitude of a given value, e.g., within five times, or within two times.

[0031] As used herein, the term “population of cells” or “cell population” refers to a group of at least two cells. In non-limiting examples, a cell population may contain 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, or at least about 1000 cells. A population may be a pure population containing one cell type, such as a population of dopaminergic neurons, or it may be a population of undifferentiated stem cells. Alternatively, a population may contain more than one cell type, such as a mixed cell population.

[0032] As used herein, “amino acid” and “amino acids” refer to all naturally occurring L-α-amino acids. This definition includes norleucine, ornithine, and homocysteine.

[0033] As used herein, “carrier” includes pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to cells or mammals to which they are exposed at the doses and concentrations employed. Often, pharmaceutically acceptable carriers are pH-buffered aqueous solutions. Examples of pharmaceutically acceptable carriers include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid; proteins such as low molecular weight (less than about 10 residues) polypeptides, serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.

[0034] As used herein, the term “contact” a cell(s) with a compound (e.g., one or more inhibitors, activators, and / or inducers) means providing the compound to a position accessible to the cell(s). Contact may be carried out using any suitable method. For example, to achieve a desired concentration, contact may be carried out by adding a concentrated form of the compound to cells or a population of cells, for example, in the context of cell culture. Contact may further be carried out by including the compound as a component of a formulated culture medium.

[0035] As used herein, the term “culture medium” refers to the liquid that surrounds cells in a culture vessel, such as a petri dish or multiwell plate, and which contains nutrients to nourish and support the cells. The culture medium may further contain growth factors that are added to bring about desired changes in the cells.

[0036] As used herein, “an effective amount of drug that inhibits an NME family member protein” refers to an effective amount of drug that inhibits the activating interaction between an NME family member protein and its corresponding receptor.

[0037] As used herein, “combined” administration with one or more further therapeutic agents includes concurrent and con successive administration in any order.

[0038] As used herein, the term “induced pluripotent stem cell” or “iPSC” refers to a type of pluripotent stem cell formed by introducing specific embryonic genes (such as, but not limited to, OCT4, SOX2, and KLF4 transgenes) into somatic cells (see, for example, Takahashi and Yamanaka Cell 126, 663-676 (2006), incorporated herein by reference).

[0039] As used herein, “pluripotent” stem cells mean stem cells that can differentiate into other cell types, with a limited number of different cell types.

[0040] As used herein, “naive stem cells” are those that resemble and share quantitative characteristics with cells in the inner mass of a blastocyst. Compared to primed stem cells, naive stem cells exhibit quantitative differences in the expression of certain genes and share characteristics and properties similar to cells in the epiblast region of a blastocyst. Note that female-derived naive stem cells have two active X chromosomes, known as XaXa, whereas female-derived late-primed stem cells have one X chromosome inactivated.

[0041] As used herein, “neuronal basal medium” means a medium that can maintain the normal phenotype and growth of nerve cells over a long period of time and can maintain a pure population of nerve cells without requiring an astrocyte feeder layer.

[0042] As used herein, “NME family proteins” or “NME family member proteins” are proteins numbered 1 through 10, grouped together by having at least one NDPK (nucleotide diphosphate kinase) domain. In some cases, the NDPK domain is non-functional in that it can catalyze the conversion of ATP to ADP. NME proteins were formerly known as NM23 proteins and were numbered H1 and H2. In recent years, as many as 10 NME family members have been identified. In this specification, the terms NM23 and NME are interchangeable. In this specification, the terms NME1, NME2, NME5, NME6, NME7, NME8, and NME9 are used to refer to NME variants as well as native proteins. In some cases, these variants are highly soluble, express well in E. coli, or are more soluble than the native sequence protein. For example, NME7 as used herein may mean a native protein or a variant such as NME7AB, which has excellent commercial applicability because it enables high-yield expression of a soluble and properly folded protein in E. coli. AB It is mainly composed of the NME7 A and B domains, but most of the DM10 domain at the N-terminus of the native protein is missing. "NME1" as referred to herein is interchangeable with "NM23-H1". The present invention is further intended not to be limited by the exact sequence of the NME protein. NME7 as referred to herein is intended to mean native NME7 having a molecular weight of about 42 kDa. AB This is intended to mean either a natural or recombinant NME7 lacking the DM-10 domain with a molecular weight of approximately 33 kDa, or an alternative natural mutant NME7-X1 also lacking the DM-10 domain with a molecular weight of approximately 31 kDa.

[0043] As used herein, the term "NME7 AB"NME7AB" and "NME-AB" are interchangeable.

[0044] As used herein, “pharmaceutically acceptable carriers and / or diluents” include all solvates, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders, etc. The use of such media and agents for active pharmaceutical ingredients is known in the art. Unless any conventional media or agent is compatible with the active ingredient, their use in the therapeutic composition is intended. Supplementary active ingredients may also be incorporated into the composition.

[0045] Formulating parenteral compositions in unit dosage forms offers particular advantages for facilitating administration and ensuring dose uniformity. Unit dosage forms as used herein refer to physically distinct units suitable as unitary dosages for the mammalian subject being treated, each unit containing a predetermined amount of active material calculated to produce a desired therapeutic effect associated with the required pharmaceutical carrier. The specifications relating to the unit dosage forms of the present invention are determined and directly depend on (a) the inherent properties of the active material and the specific therapeutic effects to be achieved, and (b) the inherent limitations in the technique of formulating such active materials for the treatment of diseases in living subjects having impaired physical health.

[0046] The main active ingredient is formulated in the form of a dosage unit, along with an appropriate pharmaceutically acceptable carrier, for convenient and effective administration. A unit dosage form may contain, for example, an amount of the main active compound ranging from 0.5 μg to approximately 2000 mg. Expressed as a percentage, the active compound is generally present in the carrier at approximately 0.5 μg / ml. For compositions containing auxiliary active ingredients, the dosage is determined by referring to the usual dose and method of administration of those ingredients.

[0047] As used herein, “pluripotency markers” are genes and proteins whose expression increases when cells revert to a less mature state than the initial cells. Pluripotency markers include OCT4, SOX2, NANOG, KLF4, KLF2, Tra 1-60, Tra 1-81, SSEA4, and REX-1, as well as those previously described and currently discovered. For example, fibroblasts may not be detectable or express these pluripotency markers at low levels, but they express a fibroblast differentiation marker called CD13. To determine whether cells are less mature than the initial cells, the difference in pluripotency marker expression levels between the initial cells and the resulting cells can be measured.

[0048] As used herein, “pluripotent” stem cells refer to stem cells that can differentiate into all three germ cells—endoderm, ectoderm, and mesoderm—and into any cell type in the body, but cannot produce a complete organism. Totipotent stem cells are cells that can differentiate into or mature into a complete organism, such as a human. Embryonic pluripotent stem cells are cells derived from the inner cell mass of a blastocyst. Typical pluripotency markers are OCT4, KLF4, NANOG, Tra 1-60, Tra 1-81, and SSEA4.

[0049] As used herein, “stimulated stem cells” are cells that resemble, and share with the phenotypes and characteristics of, the cells of the epidermal portion of a blastocyst.

[0050] As used herein, a “semi-dopaminergic neuronal state,” “pre-dopaminergic neuronal state,” or “dopaminergic neuron precursor” of a cell population refers to a cell population in which some or all of the cells have the morphological features and dopamine expression levels of dopaminergic neurons, but the cell population contains at least some cells that are not fully mature dopaminergic neurons.

[0051] As used herein, the term “stem cell” refers to a cell that, under culture conditions, divides irregularly and has the ability to produce specialized cells.

[0052] As used herein, “treatment” is an approach to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, symptom relief, reduction of disease extent, a stable (e.g., non-exacerbating) state of disease, delay or slowing of disease progression, improvement or mitigation of disease status, and remission (either partial or total), whether detectable or undetectable. “Treatment” may further mean extending survival time compared to the expected survival time without treatment. “Treatment” refers to both therapeutic treatments and preventive or relapse-preventing measures. Those requiring treatment include those who already have a disability as well as those for whom disability will be prevented. To “mitigate” a disease means that the degree of disease status and / or undesirable clinical symptoms are reduced and / or the time course of progression is slowed or prolonged compared to without treatment.

[0053] Differentiation of pluripotent stem cells into dopaminergic neurons

[0054] In this part of the study, the inventors explored two main research themes: 1) a comparison of the differentiation potential of naive stem cells and priming-state stem cells into dopaminergic neurons, and 2) the effects of time-dependent addition of various factors, including vitamins, produced in the brain of developing embryos, on the differentiation of functional dopaminergic neurons.

[0055] The inventors first differentiated human iPS cells into dopaminergic neurons using a protocol derived from US2018 / 0094242A1, which is referred to herein as Protocol A (Figure 1A and Example 1). In these experiments, the starting iPSCs were either in the earliest untreated state cultured in NME7-AB untreated medium (Carter et al 2016) or in the later priming state cultured in FGF2-containing E8 medium. When starting with either priming hESCs or hiPSCs cultured in FGF2-containing E8 medium using Protocol A, the resulting dopaminergic neurons reached 800,000 cells / cm³ at day 60 of the protocol. 2 Only about 2-3 ng / mL of dopamine or its metabolites were secreted per unit area. In contrast, when using Protocol A, starting from untreated hiPSCs, the cell count reached 400,000 cells / cm³ on day 60 in some cases. 2 8.45 ng / mL of dopamine and metabolites are secreted per unit area, and in another case, 800,000 cells / cm³. 2 5.85 ng / mL of dopamine and metabolites were secreted per cell. These results are consistent with the idea that cells cultured in NME7-AB in an untreated state differentiate better into dopaminergic neurons than stem cells in a primed state.

[0056] Previous studies have shown that transplanting dopaminergic neuron precursors into the brain approximately 16–28 days after differentiation initiation resulted in better engraftment and higher therapeutic efficacy than transplantation 40–60 days after full maturity. These results suggest that the progenitor cells were able to mature into functional dopaminergic neurons within the brain environment. These results are further consistent with the idea that maturation factors supplied by the brain are not required in the early stages of differentiation into neurons or dopaminergic neuron precursors. Therefore, we aimed to determine which factors produced in the brain may be important for the development of dopaminergic neurons and to determine the timeframe for exposing dopaminergic neuron precursors to these factors. Candidate factors were added between days 16 and 28 of the differentiation protocol at approximately the same point in time when researchers empirically determined that dopaminergic neuron precursor cells needed to be transplanted into the brain (Samata and Takahashi 2016, DOI:10.1038 / ncomms13097). The effects of individually or in combination of candidate molecular markers on expression, engraftment, and dopamine secretion were evaluated.

[0057] Several factors produced within the brain are suggested to be important for neural differentiation. Some of the candidate brain-supplied factors that can induce the final stage of maturation into dopaminergic neurons include vitamin A [Qing mu et al 2018, DOI:10.1080 / 21691401.2018.1436552; Engberg et al, Stem Cells 2010;28:1498-1509; JD Bremner, 007, doi:10.1016 / j.pnpbp.2007.07.001] and vitamin B [Carlos Alberto Calderon-Ospina, Mauricio Orlando Nava-Mesa, doi:10.1111 / cns.13207, Guilarte, 2006 Journal of Neurochemistry, DOI:10.1111 / j.1471-4159.1987.tb04111.x, Peraza et al]. Examples include vitamin A [V.Bagga et al 2008, Cell Transplantation; Xi-Biao He et al 2015, Stem Cells, doi:10.1002 / stem.1932], and vitamin D [Luan et al 2018, Mol Neurobiol, doi:10.1007 / s12035-017-0497-3]. These vitamins exist in various forms, and some have been reported to increase in brain levels during neuronal development. However, the results of studies are often contradictory. Some academic studies have concluded that vitamin A, or its derivative retinoic acid, blocks neuronal differentiation, while others suggest that it may be necessary for neuronal differentiation. Similarly, various vitamin Bs have been reported to be effective in neuronal differentiation, while others have been reported to inhibit it.

[0058] The inventors evaluated the effects of various forms of B vitamins, particularly the neurostimulant vitamins B1, B6, B12, and vitamin A, as well as vitamin C, on the development of dopaminergic neurons derived from hiPS cells. In addition to the relatively poorly soluble vitamin A, the effects of various high-lipid-density additives for solubilizing vitamin A, such as serum albumin containing commercially available Albumax and serum albumin substitutes, were evaluated.

[0059] The inventors conducted a series of experiments comparing the state of the art with the methods and compositions of the present invention. They compared standard priming stem cells with naive stem cells cultured with NME7-AB that differentiated into dopaminergic neurons. They also compared Protocol A with improved protocols B, C, C.2, and D (Figures 1A to 1E), which involved the addition of various vitamins and other components starting around 20±3 days into the protocol. The resulting cells were analyzed at various time points for the presence of appropriate molecular markers, characteristic morphology, neurite outgrowth (an indicator of engraftment potential), and, most importantly, dopamine production and secretion.

[0060] In Protocol C.2, around the 20th day, the neural basal medium was replaced with one containing pyridoxine and two forms of vitamin A instead of pyridoxal. In the following experiments, stem cells differentiated to become dopaminergic neurons according to Protocol A or Protocol C.2 were compared. On the 24th day, immunofluorescence staining was performed to detect the presence of molecular markers of dopaminergic neuron progenitor cells generated using Protocol A (A - L in Figure 2) or Protocol C.2 (A - L in Figure 3). GIRK2 (G - protein - coupled inwardly - rectifying potassium channel 2) is expressed in dopaminergic neurons, and Tuj1 (neuron - specific class Ill B - tubulin) is a pan - neural marker. TH (tyrosine hydroxylase) is an enzyme that converts L - tyrosine to L - 3,4 - dihydroxyphenylalanine, which is the rate - limiting step in dopamine synthesis, and is thus regarded as the gold standard in the identification of dopaminergic neurons. DAT (dopamine active transporter) is also important and is a transmembrane protein that transports dopamine back from the synapse to the cytoplasm. In both differentiation protocols, cells with all appropriate molecular markers positive were produced. However, the cells produced using Protocol C.2 formed a network of interconnected neurons with longer processes and better connectivity than the cells produced from Protocol A.

[0061] This morphological difference is further revealed in the immunofluorescence test performed on the 60th day. iPSCs AB cultured beforehand with NME7 S and differentiated according to Protocol A lack the characteristics of axonal projections and interconnected networks of functional neurons, while the same cells differentiated according to Protocol C.2 have a desirable morphology (A - B and E - F in Figure 4). In this experiment, the second naive clone, iPSCs NME7-N7BNote that the following was used. This second untreated clone was generated by episomal reprogramming using the core pluripotency factor OCT4 / SOX2 / KLF4 / c-Myc in NME7-AB medium. Another clone used in these experiments is iPSC. NME7-6E iPS cells were previously shown to be generated using Sendai virus (Carter et al 2016) and capable of differentiating into functional cardiomyocytes and hepatocytes. E8-A6 When protocol A was used to differentiate priming stem cells like those in NME7, cells repeatedly failed before reaching day 60. However, culturing iPSCs grown in E8 with agonists such as E8+β-catenin and WNT3A for 48 hours before initiating differentiation showed increased day 60 survival and potentially improved morphology. However, the morphology and expression patterns of DAT, TH, and GIRK2 were different from those of NME7. AB These naive stem cells were cultured in WNT3A for 48 hours and were inferior to iPSCs differentiated according to protocol C.2 (Figures 4C-D and 4G-H). Naive stem cells differentiated according to protocol C.2 formed networks with the desired morphology. Morphological complexity, such as neuronal branching density and grouping patterns, correlates highly with neuronal function.

[0062] One factor hindering the therapeutic use of stem cell-derived dopaminergic neurons is the current low engraftment rate. It is estimated that at least 100,000 functional dopaminergic neurons need to engraft in the patient's brain to achieve therapeutic effect. A primary in vitro method for assessing engraftment rate would be a wound healing assay, also known as the scratch test. (NME7) AB iPSCs cultured in either E8 were differentiated using protocol C.2 until the cells became confluent, after which scratching or wounding was performed. ABiPSC-derived dopaminergic neurons pre-cultured in culture medium developed nerves and axons within 6 days, filling the gaps, while cells cultured in E8 medium had fewer and shorter processes (Figure 5A-F). Green fluorescence indicates dopamine uptake from labeled dopamine mimetic.

[0063] One of the most important indicators of dopaminergic neuron function is the ability to produce and secrete dopamine. HPLC analysis to quantify secreted dopamine and its metabolites was performed at various cell densities and different stages of differentiation protocols. Priming human iPSC cells differentiated into dopaminergic neurons according to Protocol A secreted as much as 10 ng / mL of dopamine and its metabolites by day 60 (Figure 6A), while untreated stem cells differentiated using Protocol A secreted only about 7 ng / mL at that point (Figure 6B). In contrast, priming stem cells differentiated using Protocol C2 secreted nearly 40 ng / mL of dopamine and its metabolites at day 60 (Figure 6C), and untreated stem cells secreted nearly 60 ng / mL of dopamine and its metabolites (Figure 6D). In the experiment shown in Figure 6, the measured amounts of dopamine and its metabolites were measured per 1 cm³. 2Dopamine was secreted from approximately 400,000 cells per cell and measured at 60 days after the start of differentiation. Figure 7 shows a graph of the amounts of dopamine and its metabolites secreted by a variable number of cells, measured at 60 or 40 days. Where shown, horizontal and vertical bars represent cells differentiated using Protocol A, and crosshatched and filled bars represent cells differentiated using Protocol C.2. Horizontal and crosshatched bars indicate the use of primed stem cells, while vertical and filled bars indicate the use of untreated stem cells. In more than 40 experiments conducted using Protocol A compared to Protocol C.2 (Figures 8, 9, and 10), cells differentiated into dopaminergic neurons using Protocol C.2 were shown to produce cells that secreted more than 10 times the amount of dopamine on average compared to cells produced using Protocol A. Furthermore, NME7 AB Cells differentiated from naive cells consistently produced the highest amount of dopamine.

[0064] Next, we attempted to further investigate the effects of various forms of vitamin B6 on stem cell differentiation into dopaminergic neurons. It should be recalled that in the previous series of experiments, either primed or untreated stem cells were differentiated into dopaminergic neurons according to either Protocol A or Protocol C.2, in which the underlying culture medium was replaced around day 20 with a medium containing pyridoxine, retinol, and retinyl acetate, instead of pyridoxal.

[0065] In this subsequent series of experiments, Protocol A was followed, except that around day 20, the underlying neurogenesis medium was replaced with a medium containing approximately 1.2 μM retinol and 0.17 μM retinyl acetate, followed by the addition of various other forms of vitamin B6 (Figure 1, Example 2). The resulting cells were analyzed for cell morphology, the yield of TH and DAT-positive cells relative to Tuj1-positive cells, and the number and length of neurites as indicators of engraftment potential.

[0066] The B vitamin subgroups, B1 (thiamine), B6 ​​(pyridoxine), and B12 (cobalamin), are collectively known as the neurotropic vitamin B group. B6 levels increase during brain development in pregnancy. Pyrodoxine is a type of food-derived form of B6. The PLP form of vitamin B6 (pyridoxal phosphate) is a biologically active form of vitamin B6 required for the synthesis of neurotransmitters, such as dopamine from L-DOPA. In culture media, pyridoxine can be metabolized to form pyridoxal-5'-phosphate. B12 has been reported to be involved in myelin synthesis.

[0067] The inventors discovered that adding a certain form of vitamin B6 to a basic neuronal differentiation medium significantly improved the quality of stem cell-derived dopaminergic neurons, greatly increased the amount of dopamine secreted, and greatly improved the engraftment rate in in vitro wound healing experiments. The timing, concentration, and various forms of vitamin B intake are important factors in the differentiation and maturation of dopaminergic neurons from human stem cells in vitro.

[0068] The inventors evaluated the effects of various forms of vitamin B6 on the differentiation of human iPS cells into dopaminergic neurons. Initial experiments showed that naive NME7-AB human iPS cells differentiated better into dopaminergic neurons than hiPSCs cultured in E8 medium containing FGF2; therefore, these experiments were performed only with untreated stem cells and later repeated using primed stem cells. A differentiation protocol called Protocol A (Figure 1) was followed until day 20. From day 21 onward, vitamin A was added to the basal medium at 1.2 μM in the form of retinol, 0.17 μM in the form of retinyl acetate, and vitamin C was added to the basal medium at a final concentration of approximately 200 nM in the form of ascorbic acid-2-phosphate. Negative controls without additional vitamin B are shown in Figures 11A-K. It is important to note that the neuronal basal medium used under all conditions contained 10 μM pyridoxal, and this low concentration of pyridoxal was present from the start of differentiation.

[0069] In addition to vitamins A and C, which are constant under all conditions, 16 μM pyridoxine (Figure 12 A-K), 11 μM pyridoxal (Figure 13 A-K), or 20 μM pyridoxal-5'-phosphate known as PLP (Figure 14 A-K), or all B vitamins were added. All of the additional B vitamins added to the control medium are shown in Figure 15 A-K. A comparison of the control medium with the addition of various forms of vitamin B6 is shown in Figure 16 A-E. To evaluate the quality of the resulting dopaminergic neurons, we examined the following: a) the percentage of GIRK2-positive cells that were also positive for TH, tyrosine hydroxylase, the enzyme that mediates the conversion of L-tyrosine to L-3,4-dihydroxyphenylalanine, a rate-limiting step in dopamine synthesis. This percentage should be high as an indicator of the yield of dopaminergic neurons, which are the desired cell type. b) The percentage of TUJ-positive neurons that were also TH-positive. TUJ is a pleiotropic expression marker of neurons, and only TH-positive ones are true dopaminergic neurons. c) The percentage of TUJ-positive cells that were also positive for DAT, a dopamine transporter protein. d) The cell body shape should be the elongated triangular shape characteristic of neurons. e) The length and number of neurites associated with TH positivity and DAT positivity. The length and number of neurites are considered the most important factors for engraftment. Examining the photographs in Figures 12-16 shows that increasing the amount of various forms of vitamin B around day 21 of the differentiation protocol greatly promotes differentiation into dopaminergic neurons based on morphology and yield. Note that in the control group shown in Figure 16E, two forms of vitamin A and C were added.Referring to Figures 16A-E, the percentage of TH-positive and DAT-positive cells with numerous interconnected long neurites, visualized by Hoechst dye, indicates that adding bioactive forms of vitamin B6, pyrodoxal-5-phosphate, or the bioactive immediate precursor pyridoxal, or a combination of all forms of vitamin B6, from around day 21 of the differentiation protocol generates many long interconnected neurites, significantly increasing dopamine yield and engraftment ability. Figure 17 is a graph showing the amounts of dopamine and its metabolites, measured by HPLC, present in prepared media from only 200,000 cells collected on days 30, 40, 50, or 60. The media were not taken from a single cell source. More precisely, it was possible to continue separate experiments until the day the media was taken for analysis. In this experiment, Protocol C was used, in which retinol and retinyl acetate were added to all conditions from around day 20 onwards. The vitamin B added to the basal neuronal culture medium varied in form. In this experiment, the basal medium contained approximately 10 μM of pyridoxal. Under the condition where pyridoxine was added, protocol C.2, pyridoxal was omitted from the basal medium. In Figure 17, "NBM" refers to the basal neuronal culture medium, but it is important to note that since vitamin A was also added in the form of retinol and retinyl acetate, only the effects of adding vitamin B in various forms after day 20 can be compared. 200,000 cells / cm². 2The amount of dopamine and its metabolites secreted into the culture medium by the cells on days 30, 40, 50, and 60 was measured by HPLC (Figure 17). This graph shows that there is a peak in dopamine secretion around day 50 of the differentiation protocol. As can be seen from the figure, it is clear that the highest amount of dopamine is obtained when the neuronal basal medium is supplemented with retinol, retinyl acetate, and vitamin C around day 20, or with 11 μM pyridoxal, or when all three B6 forms are added together: 11 μM pyridoxal, 20 μM pyridoxal-5'-phosphate, and 16 μM pyridoxine. It should be noted that the amount of dopamine produced and the ability to engraft in a region of the living brain appear to be two different measures of dopaminergic neurons suitable for transplantation. While controls, basal neural media, and retinol and retinyl acetate produce large amounts of dopamine, they do not generate dopaminergic neurons with the many long, interconnected processes essential for engraftment.

[0070] In one aspect of the present invention, pyridoxine or pyridoxine-HCl is added to the differentiation medium starting around day 16 and continuing until day 30, which may be the day of transplantation or final examination, which may be day 40 to 60. In another aspect of the present invention, pyridoxine or pyridoxine-HCl is added to the differentiation medium starting around day 20 ± 3, which may be the day of transplantation or final examination, which may be day 40 to 60. In one aspect, pyridoxine is added to the differentiation medium until the final concentration reaches 5.0 μM to 25.0 μM by day 20 + / - 3. In another aspect, pyridoxine is added until the final concentration reaches 10.0 μM to 30.0 μM. In yet another aspect, pyridoxine is added until the final concentration reaches 10.0 μM to 20.0 μM. In yet another aspect, pyridoxine is added until the final concentration reaches 15.0 μM. In yet another aspect of the present invention, pyridoxine is present at a concentration of 5.0 μM to 15.0 μM from the start of differentiation. In another embodiment, pyridoxine is increased to a final concentration of 10 μM to 30 μM around day 16 to 30 and continued until cell harvesting. In yet another embodiment of the present invention, pyridoxal is added to the differentiation medium around day 16 to 30 and continued until the day of transplantation or final examination, which may be day 40 to 60. In yet another embodiment of the present invention, pyridoxal is added to the differentiation medium around day 20 ± 3 and continued until the day of transplantation or final examination, which may be day 40 to 60. In one embodiment, pyridoxal is added to the differentiation medium to a final concentration of 10 μM to 40 μM. In another embodiment, pyridoxal is added until the final concentration is 10 μM to 30 μM. In yet another embodiment, pyridoxal is added until the final concentration is 15 μM to 30 μM. In yet another embodiment, pyridoxal is added until the final concentration is 21 μM. In another aspect of the present invention, pyridoxal is present at a concentration of 5.0 μM to 15.0 μM from the start of differentiation. In another aspect, pyridoxal is increased to a final concentration of 10 μM to 30 μM around day 16 to 30 and continued until cell harvesting. In another aspect of the present invention, pyridoxal-5'-phosphate, the bioactive form of vitamin B6, is added to the differentiation medium around day 16 to 30 and continued until the day of transplantation or final examination, which may be day 25 to 60.In another aspect of the present invention, pyridoxal-5'-phosphate is added to the differentiation medium around day 20±3 and continues until the day of transplantation or final examination, which may be day 30 to 60. In one aspect, pyridoxal-5'-phosphate is added until the final concentration reaches 5.0 μM to 50.0 μM. In another aspect, pyridoxal-5'-phosphate is added until the final concentration reaches 10.0 μM to 30.0 μM. In yet another aspect, pyridoxal-5'-phosphate is added until the final concentration reaches 20.0 μM. In yet another aspect of the present invention, pyridoxal-5'-phosphate is present at a concentration of 5.0 μM to 15.0 μM from the start of differentiation. In yet another aspect, pyridoxal-5'-phosphate is present at a concentration of 5.0 μM to 25.0 μM from the start of differentiation. In other embodiments, pyridoxal-5'-phosphate is increased to a final concentration of 10 μM to 30 μM around day 16 to 30 and continued until cell collection. In other embodiments, pyridoxal-5'-phosphate is increased to a final concentration of 10 μM to 40 μM around day 16 to 30 and continued until cell collection.

[0071] In another aspect of the present invention, these B vitamins are added collectively to the differentiation medium around day 16 to 30, more preferably around day 20±3, and this continues until the day of transplantation or final test, which may be day 40 to 60, with a total final concentration of B vitamins of 5 μM to 140 μM. In another aspect of the present invention, the total final concentration of B vitamins is 15 μM to 100 μM. In another aspect of the present invention, the total final concentration of B vitamins is 40 μM to 70 μM. In another aspect of the present invention, the total final concentration of B vitamins is 50 μM to 55 μM. In another aspect of the present invention, the total final concentration of B vitamins is 10 μM to 30 μM. On the one hand, pyridoxal is present in the dopaminergic neuron differentiation medium at approximately 10 μM from the start, increasing to a final total concentration of 20 μM on day 20 ± 3. Along with this, pyridoxine is added until its final concentration reaches 15 μM around day 20 ± 3, and pyridoxal-5'-phosphate is added until its final concentration reaches 20 μM around day 20 ± 3.

[0072] Vitamin A

[0073] The fact that vitamin A inhibits and promotes neurogenic differentiation has been reported in virtually equal numbers of publications [2011 Gudas and Wagner J Cell Physiol 2011 Feb;226:322-330; Khillan et al Nutrients 2014 doi:10.3390 / nu6031209; Ole Isacson Molecular and Cellular Neuroscience Vol 45, Issue 3, November 2010;258-266]. Retinoic acid binds to specific retinoic acid receptors (RARs) in the nucleus, inducing the expression of genes involved in stem cell differentiation, more specifically, neurogenic differentiation. RARα is a retinoic acid receptor that promotes the development of dopaminergic neurons. Therefore, RARα agonists such as BMS753 may be added to late culture media in place of or in addition to various vitamin As.

[0074] The inventors discovered that while the form of vitamin A is beneficial for the maturation of dopaminergic neurons, the timing, concentration, and type of vitamin A added to the basal culture medium are significant factors. The inventors modified Protocol A by adding various forms of vitamin A to the basal neuronal differentiation medium around day 20 ± 3, continuing until cell harvesting occurred on either day 30, 40, 50, or 60. Vitamin A, retinol, and its active metabolites—retinoic acid (RA), 9-cis-RA, all-trans-RA (atRA), 13-cis-RA, and / or retinyl acetate—were added to the basal neuronal differentiation medium. The inventors found that the addition of vitamin A and / or its active metabolites significantly improved the generation of dopaminergic neurons from stem cells in terms of phenotype, expression of appropriate molecular markers, engraftment, and the amount of dopamine produced.

[0075] In this series of experiments, untreated human iPS cells were used. These pluripotent stem cells were cultured in minimal medium supplemented with NME7-AB as the sole growth factor (Carter et al 2016). Protocol A (Figure 18 A-I) was used as the control. To confirm only the effect of added vitamin A, the inventors adopted a modified version of Protocol A, which they named Protocol B. In this protocol, approximately 10 μM pyridoxal was present in the basal medium from the start of differentiation, and an additional 10-11 μM pyridoxal or pyridoxal HCl was added from day 2+ / -3 until cell harvesting (Figure 19 A-I). Vitamin A was added to differentiated stem cells of Protocol B around day 20 ± 3 in the form of retinol at a final concentration of 0.7 μM and retinyl acetate at a final concentration of 0.6 μM (Figure 20 A-I). In another experimental group, the forms of vitamin A added around day 20 were 9-cis, 13-cis, and all-trans retinoic acid, each with a final concentration of 0.446 μM (Figure 21 A-I). In yet another experimental group, only all-trans retinoic acid was added until the final concentration reached 1.33 μM (Figure 22 A-I). Figures 23 A-E show a comparison between two controls: Protocol A (Figure 23 A), in which neither vitamin B6 nor vitamin A was added around day 20, and Protocol B (Figure 23 E), in which the pyridoxal form of vitamin B6 was further increased by the addition of 11 μM around day 20, and protocols in which vitamin A was added in the form of retinol and retinyl acetate (Figure 23 B), or in the form of 9-cis, 13-cis, and all-trans retinoic acid (Figure 23 C), or in the form of all-trans retinoic acid (Figure 23 D). As shown in the figures, the addition of retinol and retinyl acetate (Figure 23B), or the addition of 9-cis, 13-cis, and all-trans retinoic acid (Figure 23C), increased the number, length, and interconnectivity of neurites compared to the control (Figure 23A, Figure 23E), respectively. However, when the morphology of nerve cell bodies stained with GIRK2 was combined with a high proportion of TUJ-positive cells that were both TH-positive and DAT-positive, it was observed that the engraftment rate increased with the addition of retinol and retinyl acetate.

[0076] In one embodiment of the present invention, retinol, retinyl acetate, and / or retinoic acid are added to the differentiation medium from around day 16 to around day 30 and continued until the day of transplantation or final examination, which may be day 40 to 60. In another embodiment of the present invention, these are added to the differentiation medium from around day 20 ± 3 and continued until the day of transplantation or final examination, which may be day 40 to 60. In one embodiment, vitamin A and / or its derivatives are added to the basal medium until the final combined concentration is 0.5 μM to 5.0 μM. In another embodiment, vitamin A and / or its derivatives are added to the basal medium until the final combined concentration is 1.0 μM to 3.0 μM. In yet another embodiment, retinol is added to the basal medium at a final concentration of 0.5 μM to 5.0 μM. In yet another embodiment, retinol is added to the basal medium at a final concentration of 1.0 μM to 2.0 μM, and retinyl acetate is added at a final concentration of 0.1 μM to 1.0 μM. In yet another embodiment, retinol is added to the basal medium at a final concentration of 1.0 μM to 3.0 μM, and retinyl acetate is added at a final concentration of 0.1 μM to 1.2 μM. The basal medium to which vitamin A and / or its derivatives are added can be a basal medium for neural differentiation, including but not limited to Neural Basal Media (ThermoFisher) or NeuroCult (StemCell Technologies).

[0077] Since vitamin A is fat-soluble, optionally, when vitamin A or its derivatives are added, lipids or albumin may be added to the underlying culture medium. The underlying neuronal medium used by the inventors contained some BSA, but for human use, a non-bovine alternative to BSA was sought. Additional vitamin A is expected to require additional lipids to aid solubility. In this series of experiments, vitamin A was first solubilized with Albumax and then added to the differentiation medium around day 20±3, as described in Protocol B. Note that Protocol B includes the addition of 11 μM pyridoxal around day 20+ / -3. Figures 24A-I show confocal microscopy images of cells obtained on day 24, where vitamin A, in the form of retinol (1.2 μM) and retinyl acetate (0.17 μM), solubilized in 2 mg / mL Albumax, was added to the medium around day 20. Figures 25A-I show confocal microscope images of cells obtained when, in addition to the aforementioned retinol and retinyl acetate, vitamin C was added to the differentiation medium around day 20 in the form of 2-phospho-ascorbic acid until the final concentration reached 61 μM, and then in the form of L-ascorbic acid until the final concentration reached 110 μM. In another experimental group, vitamin A in the form of all-trans retinoic acid solubilized in Albumax was added to the medium of Protocol B around day 20 until the final concentration reached 1.33 μM (Figures 26A-I). Figures 27A-I show confocal microscope images of cells obtained when, in addition to the aforementioned all-trans retinoic acid and retinyl acetate, vitamin C was added to the differentiation medium around day 20 in the form of 2-phospho-ascorbic acid until the final concentration reached 61 μM, and then in the form of L-ascorbic acid until the final concentration reached 110 μM.

[0078] In one aspect of the present invention, vitamin C is added to the differentiation medium around day 16 to day 30 of differentiation. In another aspect, vitamin C is added to the differentiation medium around day 20. In one aspect, vitamin C is added until the final concentration reaches 200 nM to 110 μM. In another aspect, vitamin C is added until the final concentration reaches 1 μM to 100 μM. In yet another aspect, vitamin C is added until the final concentration reaches 50 μM to 75 μM. In one aspect, vitamin C is in the form of 2-phospho-ascorbic acid. In another aspect, vitamin C is in the form of L-ascorbic acid. In yet another aspect, both forms of vitamin C are added. In yet another aspect of the present invention, vitamin C is present in the differentiation medium from the start of differentiation at a final concentration of 100 nM to 500 nM. In another aspect of the present invention, vitamin C is present at a concentration of 100 nM to 500 nM from the start of differentiation and increases to 50 uM to 70 uM around day 16 to 30, or around day 20.

[0079] Figure 5 shows a photograph of a wound healing assay, also known as the scratch test, which is considered an in vitro surrogate for in vivo engraftment. Stem cells were differentiated to become dopaminergic neurons according to protocol C.2. In some cases, the initiating stem cells were untreated stem cells grown in NME7-AB untreated medium without FGF2 or other growth factors (Figure 5A-C). In other cases, the initiating stem cells were primed stem cells grown in E8 medium containing FGF2 (Figure 5D-F). On day 21, six days after wounding, the number and length of neurites in the obtained cells were analyzed. On day 21, neurons derived from untreated stem cells produced 10-12 times, or 1000-1200%, more neurites than those from primed stem cells. The length of these neurites was 5-7 times, or 500-700%, greater than that of neurites produced by primed stem cells. Therefore, the simulated engraftment rate improves by 500% to 1200% simply by changing to untreated stem cells.

[0080] Furthermore, improvements in state-of-the-art technology are measured in terms of the yield and purity of the resulting population. It should be recalled that conventional methods for obtaining stem cell-derived dopaminergic neurons for the treatment of Parkinson's disease require cell sorting on day 14 to obtain a semi-pure population. Such conventional methods have shown that purified populations engraft in rat brains 10 times better than impure populations. Figure 4 compares the yield and purity of naive stem cells differentiated into dopaminergic neurons using Protocol A with those using Protocol C.2. The purity of the population is determined by the percentage of cells exhibiting neuronal morphology and being positive for four key markers: GIRK2, TH, DAT, and Tuj1. While Hoechst dye stains the nucleus of all cells, and Tuj1 is a common stain for many types of neurons, only those positive for Tuj1, GIRK2 (a marker for A9 neurons), TH (catalyzes the reaction that produces dopamine), and DAT (dopamine transporter protein) are actually dopaminergic neurons. Of the cells differentiated according to Protocol A, only about 5% were positive for both GIRK2 and TH (Figure 4B, where the areas overlaid with green on red are yellow). In contrast, cells differentiated according to Protocol C.2 exhibited neuronal morphology, with 80% to 90% of Tuj1-positive cells being DAT-positive (Figure 4E), and approximately 70% being positive for both GIRK2 and TH (Figure 4F). Protocol C.2 increased the yield and purity of dopaminergic neurons by more than 10 times (1000%).

[0081] In another experiment, naive stem cells were differentiated according to protocol A (Figures 18A-I). Here, the percentage of cells positive for GIRK2, TH, DAT, and Tuj1 and exhibiting neuronal morphology was less than 35%. Figures 24A-24I show the same starting cells differentiated according to protocol C, with pyridoxal added on day 21, and further retinol and retinyl acetate added. The percentage of cells positive for GIRK2, TH, DAT, and Tuj1 was 90%-100%. Figure 25 shows photographs of the same starting cells differentiated according to protocol D, which differs from the protocol shown in Figure 24 in that vitamin C is further added in the form of 2-phospho-ascorbic acid and L-ascorbic acid after day 21. As seen in Figures 25A-25I, virtually 100% of the cells exhibit neuronal morphology and are positive for GIRK2, TH, DAT, and Tuj1. Therefore, the improvement rate between Protocol A and the protocol described in Figure 24 is 250%. The improvement rate between Protocol A and Protocol D (Figure 25) is 290%.

[0082] Another important characteristic of dopaminergic neurons for their usefulness as therapeutic agents in the treatment of Parkinson's disease is their ability to secrete dopamine. Direct comparisons of dopamine and its metabolite production were quantified between 1) naive stem cells versus primed stem cells, and 2) Protocol A of the most advanced technology, and Protocol C.2 of the present invention, in which pyridoxal in the basal medium is replaced with pyridoxine and vitamin A is added in the form of retinol and retinyl acetate from approximately 24 days after the start of differentiation. Graphs of the amounts of dopamine and its metabolites secreted into the conditioned medium at specific days after the start of differentiation, as measured by HPLC (Vanderbilt University), are shown in Figures 6 and 7. First, using Protocol A, starting with primed stem cells, 1 cm 2When plated at a cell density of 400,000 cells per cell, dopamine and its metabolites were measured at 1.34 ng / mL on day 40 and 13.4 ng / mL on day 60 (Figure 6A). When protocol A was used on untreated stem cells, 1.3 ng / mL was measured on day 40 and 5.85 ng / mL on day 60 (Figure 6B). In contrast, when protocol C.2 was used on primed stem cells, dopamine and its metabolites were measured at 33.4 ng / mL on day 40 and 15.6 ng / mL on day 60 (Figure 6C). When protocol C.2 was used on untreated stem cells, dopamine and its metabolites were measured at 43.0 ng / mL on day 40 and 54.1 ng / mL on day 60 (Figure 6D). When using Protocol C.2, the secretion of dopamine and its metabolites on day 40 increased 25-fold (2500%) when using primed stem cells and 33-fold (3300%) when using untreated stem cells, compared to when using Protocol A. Figure 7 graphs the amount of dopamine secreted from various numbers of cells on days 60 and 40. 2 Using untreated stem cells plated at a density of 800,000 cells per centimeter, at day 60, cells differentiated according to Protocol C.2 produced approximately 10 times more dopamine (54 ng / mL vs. 5.8 ng / mL) than the same cells differentiated according to Protocol A. Half of these cells produced 400,000 cells / cm³. 2 When plated, cells differentiated by Protocol C.2 produced approximately 2.0 to 2.6 times more dopamine than the same cells differentiated by Protocol A. Comparing the amount of dopamine produced according to current state-of-the-art technology, primed cells, and Protocol A (3 ng / mL from 800K cells at day 60) with the amount of dopamine produced by untreated stem cells according to the composition and method of the present invention and Protocol C.2 (54 ng / mL from 800K cells at day 60), naive cells and Protocol C.2 produced 18 times, or 1800%, more dopamine than state-of-the-art technology.

[0083] In one aspect of the present invention, Protocol B is modified so that from around day 20±3 of the differentiation protocol, retinol is added to the differentiation medium until the final concentration is 0.5 μM to 2.0 μM, and retinyl acetate is added until the final concentration is 0.1 to 1.5 μM. In another aspect of the present invention, retinol is added until the final concentration is 0.7 to 1.2 μM, and retinyl acetate is added until the final concentration is 0.17 to 0.6 μM. In yet another aspect of the present invention, the combination of retinol and retinyl acetate is added so that the final concentration of the combination is 1.0 μM to 2.5 μM. In yet another aspect of the present invention, the final concentration of the combination is 1.33 μM.

[0084] In one embodiment, bovine serum albumin is added. In another embodiment, human serum albumin is added. In yet another embodiment, lipid-rich human serum albumin is added. In yet another embodiment, Albumax, i.e., lipid-rich bovine albumin or similar lipid-rich human albumin, is added. Lipid-rich albumin may be added until the final molar concentration is 10.0 μM to 40.0 μM. The final molar concentration may be 10.0 μM to 15.0 μM. In one embodiment, vitamin A and / or its derivatives are dissolved in an alcohol / water mixture and evaporated under vacuum to form a thin film. This thin film is then mixed with a solution of BSA or HSA at 37°C for 30 minutes to dissolve the lipids.

[0085] Vitamin C is expressed at high levels in the fetal brain during the later stages of neurodevelopment. Vitamin C has been reported to be involved in the increased expression of Nurr1, which is important for the differentiation of midbrain neurons and may be an important factor in the maturation of dopaminergic neurons. In one aspect of the present invention, vitamin C 2-phospho-L-ascorbate trisodium salt is added to the differentiation medium around day 16 to 30 and continued until the day of transplantation or final examination, which may be day 40 to 60. In another aspect of the present invention, vitamin C 2-phospho-L-ascorbate trisodium salt is added to the differentiation medium around day 20+ / -3 and continued until the day of transplantation or final examination, which may be day 40 to 60. In one aspect, vitamin C 2-phospho-L-ascorbate trisodium salt is added to the differentiation medium until the final concentration reaches 40.0 μM to 100.0 μM. In another embodiment, vitamin C 2-phospho-L-ascorbic acid trisodium salt is added until the final concentration reaches 50.0 μM to 70.0 μM. In yet another embodiment, vitamin C 2-phospho-L-ascorbic acid trisodium salt is added until the final concentration reaches 60.0 μM to 65.0 μM. In yet another embodiment of the present invention, vitamin C ascorbic acid is added to the differentiation medium around day 16 to 21 and continues until the day of transplantation or final examination, which may be day 40 to 60. In yet another embodiment of the present invention, vitamin C ascorbic acid is added to the differentiation medium around day 20+ / -3 and continues until the day of transplantation or final examination, which may be day 40 to 60. In one embodiment, ascorbic acid is added to the differentiation medium until the final concentration reaches 5.0 μM to 20.0 μM. In another embodiment, ascorbic acid is added until the final concentration reaches 10.0 μM to 15.0 μM. In another embodiment, ascorbic acid is added until the final concentration reaches 12.0 μM to 14.0 μM.

[0086] In one embodiment of the present invention, vitamin C in the form of 2-phospho-ascorbic acid is added to the differentiation medium around day 20 until the final concentration reaches 25 μM to 100 μM. In another embodiment of the present invention, vitamin C is added until the final concentration reaches 40 to 75 μM. In a preferred embodiment, vitamin C is added until the final concentration reaches 61 μM. In one embodiment of the present invention, vitamin C in the form of L-ascorbic acid is added to the differentiation medium around day 20 until the final concentration reaches 1 μM to 120 μM. In another embodiment of the present invention, vitamin C is added until the final concentration reaches 5 to 100 μM. In a preferred embodiment, vitamin C is added until the final concentration reaches 11 μM. In a preferred embodiment, one or more forms of vitamin C are added to the medium of Protocol B around day 20 + / - 3 until the final concentration reaches 50 to 75 μM. In a more preferred embodiment, the two forms of vitamin C are 2-phospho-ascorbic acid and L-ascorbic acid.

[0087] In one aspect of the present invention, the aforementioned vitamin is added to a basal neuronal culture medium at a given concentration along with lipid-rich albumin, and the stem cells are differentiated into dopaminergic neurons. These cells are then cultured in this medium for approximately 16 to 30 days, specifically from 20±3 days until the day of final differentiation or transplantation, which can occur between 30 and 60 days after the start of differentiation.

[0088] In addition to discovering key vitamins, their metabolites, and lipid-rich albumin that increase and promote differentiation into dopaminergic neurons when added to differentiated stem cells, it was found that using untreated stem cells further increases and promotes differentiation into dopaminergic neurons.

[0089] In a preferred embodiment, stem cells are differentiated into dopaminergic neurons according to protocol C.

[0090] In a more preferred embodiment, the stem cells are differentiated into dopaminergic neurons according to protocol D, where the stem cells, preferably naive stem cells cultured in NME7-AB, are in a neuronal base medium, which is supplemented around day 20 ± 3 by adding 11 μM pyridoxal or 20 μM pyridoxal-5'-phosphate, 1.2 μM retinol, and 0.17 μM retinyl acetate solubilized in a lipid-rich formulation, and by adding 61 μM 2-phospho-ascorbic acid and 11 μM L-ascorbic acid.

[0091] In yet another aspect of the present invention, the protocols of the present invention, including Protocol B, Protocol C, Protocol C.2, or Protocol D, are applied to pluripotent stem cells cultured in a pluripotent stem cell medium containing NME7-AB.

[0092] In yet another aspect of the present invention, a protocol of the present invention, including protocol B, protocol C, protocol C.2, or protocol D, is applied to pluripotent stem cells cultured in a pluripotent stem cell medium containing WNT3A.

[0093] The improvements in the cutting-edge technologies described herein involve adding specific vitamins and other factors in various forms and concentrations to the underlying neuronal differentiation medium. In one aspect of the invention, the addition or increase in concentration of vitamins A, B, and / or C begins with the initiation of differentiation and continues throughout the differentiation process. In another aspect of the invention, they are added 16–30 days after the initiation of differentiation and continue until cell harvesting. In yet another aspect of the invention, they are added around 18–23 days after the initiation of differentiation and continue until cell harvesting. In one effective embodiment, candidate factors were added on day 20 or 21. These vitamins A, B6, and C, which have been identified as dopaminergic maturation factors, can be added to several different underlying neuronal differentiation media, including but not limited to Neural Basal Media (ThermoFisher) and NeuroCult (StemCell Technologies).

[0094] In some of the examples presented herein, the basis of Protocol A (Figure 1, Example 1) was utilized until around day 20±3, when specific vitamins were added to the underlying neuronal differentiation medium. The addition of these factors around day 20±3 significantly improved the yield and functionality of stem cell-derived dopaminergic neurons, including increased engraftment rates and dopamine secretion, and simultaneously enabled the maturation of fully functional dopaminergic neurons in vitro.

[0095] Methods for treating neurodegenerative disorders, diseases, or injuries.

[0096] In vitro differentiated dopaminergic neurons may be used to treat neurodegenerative disorders. The compositions and methods expressed herein are applicable to generating other types of neurons from stem cells. Differentiated dopaminergic neurons may be used to treat diseases such as neurodegenerative disease conditions that benefit from the successful engraftment of dopaminergic neurons in the central nervous system. Other types of neurons can be generated from stem cells using the methods of the present invention for the treatment of other diseases, such as those caused by damage to the spinal cord, etc. Neurons such as sensory neurons, motor neurons, or interneurons may be generated from stem cells according to the methods of the present invention. These neurons may also be used to treat peripheral nerve injuries, which may include transection of all or part of a nerve due to stretching, transection (laceration), compression, shearing, or collapse. The inventive features of the present disclosure provide a method for treating a neurodegenerative disorder, comprising the step of administering an effective amount of the differentiated dopaminergic neurons of the present disclosure to a subject suffering from a neurodegenerative disorder.

[0097] Non-limiting examples of neurodegenerative disorders include Parkinson's disease, Huntington's disease, Alzheimer's disease, and multiple sclerosis. Other neuronutrient B vitamins may be added to the protocols described herein. For example, vitamin B12, which assists myelin production, may be added to the differentiation medium when treatment neurons for multiple sclerosis are being generated.

[0098] Specifically, the neurodegenerative disease is Parkinson's disease. The main motor symptoms of Parkinson's disease include, but are not limited to, tremors of the hands, arms, legs, jaw, and face; slowing or dulling of movement; rigidity or stiffness of the limbs and trunk; postural instability; or loss of balance and coordination.

[0099] In some embodiments, the neurodegenerative disease is a parkinsonism disease, which refers to a disorder associated with a deficiency of dopamine in the basal ganglia, the part of the brain that controls movement. In some embodiments, symptoms include tremor, bradykinesia (extreme slowness of movement), flexion, postural instability, and tonicity. Non-exclusive examples of parkinsonism diseases include corticobasal degeneration, Lewy body dementia, multiple system atrophy, and progressive supranuclear palsy.

[0100] The differentiated dopaminergic neurons of this disclosure may be administered or provided systemically or directly to a subject to treat or prevent neurodegenerative disorders. In some embodiments, the differentiated dopaminergic neurons of this disclosure are injected directly into an organ of the subject (e.g., the central nervous system (CNS) or the peripheral nervous system (PNS). In some embodiments, the differentiated dopaminergic neurons of this disclosure are injected directly into the striatum.

[0101] The differentiated dopaminergic neurons of this disclosure can be administered in any physiologically acceptable vehicle. Pharmaceutical compositions comprising the differentiated dopaminergic neurons of this disclosure and a pharmaceutically acceptable vehicle are also provided. The differentiated dopaminergic neurons of this disclosure, and pharmaceutical compositions comprising such cells, can be administered by local injection, orthotopic (OT) injection, systemic injection, intravenous injection, or parenteral administration. In one embodiment, the differentiated dopaminergic neurons of this disclosure are administered via orthotopic (OT) injection to a subject suffering from a neurodegenerative disorder.

[0102] The differentiated dopaminergic neurons and pharmaceutical compositions comprising such cells of the present disclosure may be conveniently provided as sterile liquid formulations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH. Liquid preparations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Furthermore, liquid compositions are somewhat more convenient to administer, particularly by injection. Viscous compositions, on the other hand, can be formulated within a suitable viscosity range to further extend the contact period with specific tissues. Liquid or viscous compositions may contain a carrier, which may be a solvent or dispersion medium containing, for example, water, saline, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), or suitable mixtures thereof. Sterile injectable solutions can be prepared by incorporating the compositions of the invention-specific features of the present disclosure, such as the compositions comprising the differentiated dopaminergic neurons of the present disclosure, into the required amount of a suitable solvent containing, optionally, various amounts of other components. Such compositions may be miscible with suitable carriers, diluents, or excipients such as sterile water, physiological saline, glucose, or dextrose. The compositions may also be lyophilized. Depending on the administration route and the desired preparation, the compositions may contain auxiliary substances such as wetting agents, dispersants, emulsifiers (e.g., methylcellulose), pH buffers, gelling or viscosity-enhancing additives, preservatives, fragrances, and colorants. Standard texts incorporated herein by reference, such as the 17th edition of "REMINGTON'S PHARMACEUTICAL SCIENCE" from 1985, may be consulted to prepare suitable preparations without requiring excessive experimentation.

[0103] Various additives can be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of microbial activity can be ensured by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. Long-term absorption of injectable pharmaceutical forms can be achieved by using absorption-delaying agents, such as aluminum monostearate or gelatin. However, according to the inventive features of this disclosure, any vehicle, diluent, or additive used must be compatible with the differentiated dopaminergic neurons of this disclosure.

[0104] The viscosity of the composition can be maintained at a selected level, if desired, using a pharmaceutically acceptable thickener. Methylcellulose can be used because it is readily and economically available and readily acts in conjunction with other thickeners. Other suitable thickeners include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, and carbomer. The concentration of the thickener may depend on the selected agent. The important point is to use an amount that achieves the desired viscosity. The selection of a suitable carrier and other additives will depend on the precise route of administration and the specific dosage form, e.g., the properties of the liquid dosage form (e.g., whether the composition is formulated as a solution, suspension, gel, or another liquid form such as a time-release form or a liquid-filled form).

[0105] Those skilled in the art will recognize that the components of the composition should be selected to be chemically inert and not affect the viability or potency of the differentiated dopaminergic neurons of this disclosure. This problem can be easily avoided by those skilled in the chemical and pharmaceutical fields by referring to standard texts from this disclosure and the documents cited herein, or by simple experiments (without excessive experimentation).

[0106] In some non-limiting embodiments, the cells and precursors described herein are comprised in a composition further comprising a biocompatible scaffold or matrix, for example, a biocompatible three-dimensional scaffold that promotes tissue regeneration when cells are transplanted or engrafted in a subject. In some non-limiting embodiments, the biocompatible scaffold comprises extracellular matrix material, synthetic polymers, cytokines, collagen, polypeptides or proteins, polysaccharides including fibronectin, laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparin sulfate, chondroitin sulfate, agarose or gelatin, and / or hydrogels. (See, for example, U.S. Public Appeals No. 2015 / 0159135, No. 2011 / 0296542, No. 2009 / 0123433, No. 2008 / 0268019, the contents of each of these are incorporated in whole by reference.) In some embodiments, the composition further comprises growth factors for promoting the maturation of transplanted / engrafted cells into midbrain DA cells.

[0107] One consideration regarding the therapeutic use of differentiated dopaminergic neurons in this disclosure is the quantity of cells required to achieve the optimal effect. The optimal effect includes, but is not limited to, regrowth of CNS and / or PNS regions in subjects suffering from neurodegenerative disorders, and / or improvement of the function of the CNS and / or PNS in the subject.

[0108] In some embodiments, an effective amount of the differentiated dopaminergic neurons of the Disclosure is sufficient to regrow CNS and / or PNS regions in a subject suffering from a neurodegenerative disorder. In some embodiments, an effective amount of the differentiated dopaminergic neurons of the Disclosure is sufficient to improve the function of the CNS and / or PNS in a subject suffering from a neurodegenerative disorder, for example, the improved function may be about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, or about 100% of the function of the CNS and / or PNS in a normal person.

[0109] The amount of cells administered varies depending on the target being treated. In one embodiment, approximately 1 × 10⁻⁶ 4 ~Approx. 1×10 10 , about 1×10 4 ~Approx. 1×10 5 , about 1×10 5 ~Approx. 1×10 9 , about 1×10 5 ~Approx. 1×10 6 , about 1×10 5 ~Approx. 1×10 7 , about 1×10 6 ~Approx. 1×10 7 , about 1×10 6 ~Approx. 1×10 8 , about 1×10 7 ~Approx. 1×10 8 , about 1×10 8 ~Approx. 1×10 9 , about 1×10 8 ~Approx. 1×10 10 , or approximately 1 x 10 9 ~Approx. 1×10 10 The differentiated dopaminergic neurons disclosed herein are administered to the subject. In one embodiment, approximately 1 × 10⁻⁶ 5 ~Approx. 1×10 7 The differentiated dopaminergic neurons of this disclosure are administered to subjects suffering from neurodegenerative disorders. In one embodiment, approximately 1 × 10⁻⁶ 6 ~Approx. 1×10 7 The differentiated dopaminergic neurons of this disclosure are administered to subjects suffering from neurodegenerative disorders. The precise determination of what is considered an effective dose may depend on individual factors for each subject, such as size, age, sex, weight, and condition. The dosage can be readily determined by a person skilled in the art from this disclosure and knowledge of the art. [Examples]

[0110] Example 1 - Protocol A

[0111] In Protocol A, cells were seeded on Geltrex-coated plates in NeuroBasal medium (Thermo Fisher #21103049), B-27 without vitamin A (Thermo Fisher #12587010), N2 supplement (Stem Cell Technologies #07156), 2 mM Glutamax (Thermo Fisher #35050061), 250 nM LDN193189 (Selleck Chemicals #S7507), 10.8 μM SB431542 (Selleck Chemicals #S1067), 500 ng / ml SHH (R&D Systems #464-SH-200), 0.7 μM CHIR99021 (R&D Systems #4423), and 10 μM Y27632 (Selleck Chemicals #S1049). On days 1 and 3, the medium was replaced with fresh NeuroBasal medium containing vitamin A-free B-27, N2 supplement, 2 mM Glutamax, 250 nM LDN193189, 10.8 μM SB431542, 500 ng / ml SHH, and 0.7 μM CHIR99021. On days 4 and 6, the medium was replaced with fresh NeuroBasal medium containing vitamin A-free B-27, N2 supplement, 2 mM Glutamax, 250 nM LDN193189, 10.8 μM SB431542, 500 ng / ml SHH, and 7.5 μM CHIR99021. On days 7 and 9, the culture medium was replaced with fresh NeuroBasal medium containing vitamin A-free B-27, N2 supplement, 2 mM Glutamax, and 7.5 μM CHIR99021. On day 10, the culture medium was replaced with fresh NeuroBasal medium containing vitamin A-free B-27, 2 mM Glutamax, 3 μM CHIR99021, 20 ng / mL BDNF (Peprotech #450-02), 200 nM ascorbic acid (Sigma Aldrich #A4403), 20 ng / mL GDNF (Peprotech #450-10), 1 ng / mL TGFβ3 (Peprotech #100-36E), and 500 nM cAMP (Peprotech #1698950).On day 11, cells were re-seed in 10-day medium containing 10 μM Y27632 on plates coated with 15 μg poly-L-ornithine (Sigma Aldrich #P4957) / 1 μg laminin (Sigma Aldrich #L2020) / 1 μg fibronectin (Thermo Fisher #33016-015). From days 12 to 60, the medium was replaced daily with NeuroBasal medium containing vitamin A-free B-27, 2 mM Glutamax, 20 ng / mL BDNF, 200 nM ascorbic acid, 20 ng / mL GDNF, 1 ng / mL TGFβ3, 500 nM cAMP, and 10 μM DAPT (Selleck Chem #S2215).

[0112] NeuroBasal medium (Thermo Fisher #21103049) contains the following amino acids: glycine, L-alanine, L-arginine hydrochloride, L-asparagine-H2O, 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, L-valine, 0.028571420 mM; choline chloride, 0.008385744 mM; calcium D-pantothenate, 0.009070295 mM; folic acid, 0.032786883 mM; niacinamide, 0.019607844 mM; and pyridoxal hydrochloride, 0.0010638298 mM. The product is indicated to contain riboflavin, 0.011869436 mM; thiamine hydrochloride, 5.0184503E-6 mM; vitamin B12, 0.04 mM; i-inositol (a group of vitamins); calcium chloride (CaCl2) (anhydrous); iron(III) nitrate (Fe(NO3)3”9H2O); magnesium chloride (anhydrous); potassium chloride (KCl); sodium bicarbonate (NaHCO3); sodium chloride (NaCl); monosodium phosphate (NaH2PO4-H2O); zinc sulfate (ZnSO4-7H2O); and other components such as D-glucose (dextrose), HEPES, phenol red, and sodium pyruvate.

[0113] Example 2 - Investigation of the effect of adding a form of vitamin B6 to Protocol A

[0114] In this series of experiments, a baseline neuronal medium containing 10 μM pyridoxal, 1.2 μM retinol, and 0.17 μM retinyl acetate was used, starting around day 20. Increased levels of various vitamin Bs were added around day 20, when researchers discovered that transplantation into the host brain increased engraftment. At day 20 + / - 3, pyridoxine was added to a final concentration between 5 and 25 μM, pyridoxal to a final concentration between 5 and 20 μM, the bioactive form of pyridoxal-5'-phosphate, or a combination of all three vitamin Bs to a final concentration between 10 and 40 μM. The optimal concentration of pyridoxine was empirically determined to be approximately 10–20 μM. Figures 12A–K show the effect of pyridoxine added to a final concentration of 16 μM. The optimal concentration of pyridoxal was empirically determined to be approximately 5–20 μM. Figures 13A-K show the effect of pyridoxal added until a final concentration of 11 μM is reached. It was empirically determined that the optimal concentration of pyridoxal-5'-phosphate is approximately 10-40 μM. Figures 14A-K show the effect of pyridoxal-5'-phosphate added until a final concentration of 20 μM is reached. Figures 15A-K show the effect of all three added vitamin B compounds.

[0115] Example 3 - Protocol B

[0116] Based on the results of Example 2, the type and concentration of vitamin B6 added around day 20 were restricted, and then the type and concentration of vitamin A that could potentially improve purity / yield, engraftment rate, or the amount of dopamine secreted by stem cell-derived dopaminergic neurons could be investigated.

[0117] In Protocol B, cells were seeded on Geltrex-coated plates in NeuroBasal medium containing B-27 without vitamin A, N2 supplement, 2 mM Glutamax, 250 nM LDN193189, 10.8 μM SB431542, 500 ng / ml SHH, 0.7 μM CHIR99021, and 10 μM Y27632. On days 1 and 3, the medium was replaced with fresh NeuroBasal medium containing B-27 without vitamin A, N2 supplement, 2 mM Glutamax, 250 nM LDN193189, 10.8 μM SB431542, 500 ng / ml SHH, and 0.7 μM CHIR99021. On days 4 and 6, the culture medium was replaced with fresh NeuroBasal medium containing vitamin A-free B-27, N2 supplement, 2 mM Glutamax, 250 nM LDN193189, 10.8 μM SB431542, 500 ng / ml SHH, and 7.5 μM CHIR99021. On days 7 and 9, the culture medium was replaced with fresh NeuroBasal medium containing vitamin A-free B-27, N2 supplement, 2 mM Glutamax, and 7.5 μM CHIR99021. On day 10, the culture medium was replaced with fresh NeuroBasal medium containing vitamin A-free B-27, 2 mM Glutamax, 3 μM CHIR99021, 20 ng / mL BDNF (Peprotech #450-02), 200 nM ascorbic acid (Sigma Aldrich #A4403), 20 ng / mL GDNF (Peprotech #450-10), 1 ng / mL TGFβ3 (Peprotech #100-36E), and 500 nM cAMP (Peprotech #1698950). On day 11, the cells were re-seed in 10-day medium containing 10 μM Y27632 on plates coated with 15 μg of poly-L-ornithine (Sigma Aldrich #P4957) / 10 μg of laminin (Sigma Aldrich #L2020) / 1 μg of fibronectin (Thermo Fisher #33016-015).From days 12 to 20, the culture medium was replaced daily with NeuroBasal medium containing vitamin A-free B-27, 2 mM Glutamax, 20 ng / mL BDNF, 200 nM ascorbic acid, 20 ng / mL GDNF, 1 ng / mL TGFβ3, 500 nM cAMP, and 10 μM DAPT (Selleck Chem #S2215). From days 21 to 60, or until cell harvesting, the culture medium was replaced daily with NeuroBasal medium containing vitamin A-free B-27, 2 mM Glutamax, 20 ng / mL BDNF, 200 nM ascorbic acid, 20 ng / mL GDNF, 1 ng / mL TGFβ3, 500 nM cAMP, 10 μM DAPT, plus 11 μM pyridoxal (Sigma Aldrich P1930).

[0118] Example 4 - Investigating the effects of adding various forms of vitamin A to Protocol B.

[0119] According to Protocol B, various forms of vitamin A were added at or around day 20 of differentiation, across a range of concentrations. Retinol was added from around day 20 in a concentration range of 0.1–1.5 μM. Retinyl acetate was added from around day 20 in a concentration range of 0.1–1.5 μM. 9-cis, 13-cis, and / or all-trans retinoic acids were added individually or together until the final concentration reached approximately 1.5–2.0 μM. The results are shown in Figures 18–23. Empirically, it was determined that the optimal conditions for dopaminergic neuron differentiation were the addition of retinol and retinyl acetate together from day 20 onward, up to a final combined concentration of approximately 2 μM.

[0120] Example 5 - The effect of adding various forms of solubilized vitamin A to a lipid-rich formulation, with or without adding vitamin C, to Protocol B.

[0121] Vitamin A is known to be extremely insoluble. Therefore, we tested the addition of various forms of vitamin A after solubilization in lipid-rich formulations. We tested the solubilization of vitamin A in Albumax, which can be substituted with 2 mg / mL BSA or human serum albumin. In addition to adding various forms of solubilized vitamin A, we also tested the further addition of vitamin C in the form of 2-phospho-ascorbic acid or L-ascorbic acid. The results of these tests are shown in Figures 24 to 27.

[0122] Example 6 - Protocol C

[0123] In Protocol C, the culture medium followed Protocol A until day 20 + / - 3 days later, with the exception that on day 11, the surface on which differentiating cells were reseeded contained 10 ug / mL of laminin instead of 1 ug / mL. In accordance with Protocol C, around day 20, the medium was supplemented with vitamin B6 in the form of pyridoxine at 16 uM, pyridoxal at 11 uM, pyridoxal-5'-phosphate at 20 uM, or all of them combined; vitamin A in the form of retinol at 0.7–1.2 uM and retinyl acetate at 0.17–0.6 uM, or 9-cis-retinoic acid, 13-cis-retinoic acid, and all-trans retinoic acid at 0.446 uM each, or all-trans retinoic acid at 1.33 uM; and vitamin C in the form of 2-phospho-ascorbic acid at 61 uM and L-ascorbic acid at 110 uM.

[0124] Example 7 - Protocol C.2

[0125] Protocol C.2 followed Protocol A up to day 20 + / - 3 days, with the exception that on day 11, the surface on which differentiating cells are reseeded contains 10 ug / mL of laminin instead of 1 ug / mL. According to Protocol C.2, around day 20, the neuronal base medium was replaced with one containing 16 uM of vitamin B6 in the form of pyridoxine, 1.2 uM of retinol, and 0.17 uM of retinyl acetate, without containing pyridoxal.

[0126] Example 8 - Protocol D

[0127] Protocol D followed Protocol A until day 20 + / - 3 days, with the exception that on day 11, the surface on which differentiating cells were reseeded contained 10 ug / mL of laminin instead of 1 ug / mL. In accordance with Protocol D, starting around day 20, the medium was supplemented with pyridoxal at 11 uM, retinol at 1.2 uM, and retinyl acetate at 0.17 uM, and vitamin C in the form of 2-phospho-ascorbic acid at 61 uM and L-ascorbic acid at 11 uM. For quantification of stem cells differentiated into dopaminergic neurons according to Protocol C.2, see Figures 3–10.

[0128] Those skilled in the art will recognize many equivalents to the specific embodiments of the invention described herein, or will be able to identify such equivalents without resorting to routine experimentation. Such equivalents are intended to be included within the scope of the claims.

Claims

1. A method for producing dopaminergic neurons from human stem cells, the method comprising the step of adding vitamin A to a neuronal basal medium or increasing the concentration of vitamin A around 20 ± 3 days of a protocol for differentiating pluripotent stem cells into dopaminergic neurons, wherein the produced dopaminergic neurons secrete more than 30% more dopamine and its metabolites than dopaminergic neurons produced by the same differentiation protocol without the addition or increase of vitamin A.

2. The method according to claim 1, wherein the vitamin A is in the form of retinol and / or retinyl acetate.

3. The method according to claim 2, wherein the total concentration of retinol and / or retinyl acetate is 1.0 μM to 3.0 μM.

4. The method according to claim 2, wherein the total concentration of the combination of retinol and / or retinyl acetate is 1.33 μM.

5. The method according to claim 2, wherein the total concentration of the combination of retinol and / or retinyl acetate is 2 μM.

6. The method according to any one of claims 1 to 5, further comprising the step of adding CHIR99021 or increasing the concentration of CHIR99021 on the fourth day.

7. The method according to any one of claims 1 to 5, further comprising the step of adding vitamin B6 or increasing the vitamin B6 concentration around 20 ± 3 days of the differentiation protocol.

8. The method according to claim 7, wherein the vitamin B6 is in the form of pyridoxine.

9. The method according to claim 7, wherein the vitamin B6 is in the form of pyridoxal.

10. The method according to claim 7, wherein the vitamin B6 is in the form of pyridoxal-5'-phosphate, also known as PLP.

11. The method according to claim 7, wherein the total concentration of the vitamin B6 is 10 μM to 30 μM.

12. The method according to any one of claims 1 to 5, further comprising the step of adding vitamin C or increasing the vitamin C concentration around 20 ± 3 days of the differentiation protocol.

13. The method according to claim 12, wherein the vitamin C is in the form of 2-phosphoascorbic acid.

14. The method according to claim 12, wherein the vitamin C is in the form of L-ascorbic acid.

15. The method according to claim 12, wherein the total concentration of vitamin C is 200 nM to 110 uM.

16. The method according to any one of claims 1 to 5, wherein the pluripotent stem cells to be differentiated are cultured in a medium containing NM23 protein.

17. The method according to any one of claims 1 to 5, wherein the pluripotent stem cells to be differentiated are cultured in a medium containing NME7-AB.

18. The method according to any one of claims 1 to 5, wherein the pluripotent stem cells to be differentiated are cultured in a medium containing WNT3A.

19. The method according to any one of claims 1 to 5, characterized in that the dopaminergic neurons produced form more than 30% more neurites than dopaminergic neurons produced by a differentiation protocol that does not add or increase vitamins.

20. The method according to any one of claims 1 to 5, characterized in that the dopaminergic neurons produced form more than 100% more neurites than dopaminergic neurons produced by a differentiation protocol that does not add or increase vitamins.

21. A method for producing dopaminergic neurons from human stem cells, the method comprising the step of adding vitamin A to a neuronal basal medium or increasing the concentration of vitamin A around 20 ± 3 days of a protocol for differentiating pluripotent stem cells into dopaminergic neurons, characterized in that the produced dopaminergic neurons form more than 30% more neurites than dopaminergic neurons produced by a differentiation protocol in which vitamin A is not added or increased.

22. The method according to claim 21, wherein the vitamin A is in the form of retinol and / or retinyl acetate, and the total concentration of the vitamin A is 1.0 μM to 2.5 μM.

23. The aforementioned differentiation protocol is On day 0, the process involves plating human stem cells in a culture medium containing LDN193189, SB431542, Sonic Hedgehog, and 0.7 μM CHIR99021. On the fourth day, the process involves increasing the concentration of CHIR99021 to 7.5 μM. The method according to claim 21 or 22, including the method according to claim 21 or 22.

24. The method according to claim 21 or 22, further comprising the step of adding vitamin B6 to the neuronal basal medium or increasing the vitamin B6 concentration around 20 ± 3 days of a protocol for differentiating pluripotent stem cells into dopaminergic neurons.

25. The method according to claim 24, wherein the vitamin B6 has a final concentration of 10 μM to 30 μM.

26. The method according to claim 24, further comprising the step of adding vitamin C to the neuronal basal medium or increasing the vitamin C concentration around 20 ± 3 days of the protocol for differentiating pluripotent stem cells into dopaminergic neurons.

27. The method according to claim 24, wherein the pluripotent stem cells to be differentiated are cultured in a medium containing NM23 protein.

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