CN116635047A

CN116635047A - Method for deriving dopaminergic neurons from pluripotent stem cells

Info

Publication number: CN116635047A
Application number: CN202180053247.8A
Authority: CN
Inventors: 辛西娅·巴姆达德; 凯文·R·伊; 斯科特·T·莫伊; 托马斯·全
Original assignee: Minerva Biotechnologies Corp
Current assignee: Minerva Biotechnologies Corp
Priority date: 2020-06-26
Filing date: 2021-06-28
Publication date: 2023-08-22

Abstract

The application discloses a method for producing dopaminergic neurons from human stem cells, which is carried out by the following steps: vitamins were added to or added to the neural basal medium at about day 20+/-3 of the protocol for differentiation of pluripotent stem cells into dopaminergic neurons.

Description

Method for deriving dopaminergic neurons from pluripotent stem cells

Background

1. Field of the application

The present application relates to a method of differentiating pluripotent stem cells into dopaminergic neurons. The application also relates to the treatment or prevention of diseases associated with the transplantation of the dopaminergic neuron cells thus obtained into a patient.

2. General background and state of the art

The development of stem cell derived dopaminergic neurons for the treatment of parkinson's disease has long been a major area of concern in regenerative medicine. Despite a clinical trial (Jun Takahashi, japan), there are serious technical challenges that have so far prevented FDA approval for human trials in the united states. These technical challenges may make it impractical to treat parkinson's disease using stem cell derived dopaminergic neurons.

One problem 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 terminally differentiated cells that are more functional than cells derived from ipscs. However, regulatory approval requires the generation of a large number of identical ESCs, i.e., master cell banks, for the generation of all investigational new drugs that achieve experimental data, and then for the treatment of patients. Past efforts have shown that ESCs acquire karyotype abnormalities and become unstable after generation of a source cell bank, all required experiments are performed and then used for many passages required for clinical testing in humans. In addition, many countries have now banned the use of embryonic stem cells for research or treatment.

Ipscs are more practical from a regulatory approval perspective, as each treatment is patient-specific and is typically generated by his or her own cells. Thus, there is no master cell bank. However, to date, ipscs have not differentiated into functional cells as well as embryonic stem cells. Cloning limitations are a major problem in differentiating ipscs into desired cell types. That is, one iPSC clone may be able to form neurons, while another clone is unable. More subtly, one clone may form very good neurons or hepatocytes, while other clones may express characteristic molecular markers, but function less than other clones or naturally occurring cells. Many clones often must be tested to determine which clone is capable of differentiating into a particular cell type. There is important scientific evidence supporting the notion that cloning limitations are due to cell fate decisions that stem cells in the original state (primed state stem cell) have made. Cells that have been induced to become pluripotent still retain some molecular markers, such as methylation or acetylation, that limit why cell clones can mature.

These fundamental challenges of developing stem cell derived therapeutics become more problematic in developing dopaminergic neurons for the treatment of parkinson's disease.

The average age of parkinson's disease patients in the first need of treatment was 65 years. If the dopaminergic neurons are derived from donor embryonic stem cells, the patient is required to take the immunosuppressant for a period of time to prevent rejection of the donor cells. For patients, this is not a good age for receiving immunosuppressants.

In addition, current methods for generating dopaminergic neurons from stem cells produce neurons with very low implantation rates. For parkinson's disease, it is thought that 100,000 cells need to be implanted to obtain therapeutic benefit. Due to the lower implantation rate, more cellular neurons than 10X-100X need to be transplanted to achieve therapeutic benefit. This means that 1,000,000-10,000,000 cells need to be transplanted, which is a technical challenge for differentiating stem cells into dopaminergic neurons using existing methods. It is reported that when dopaminergic neurons are generated from human iPS cells, only about 3% of the yield is true dopaminergic neurons. To obtain pure populations of dopaminergic neurons from ipscs, researchers need to sort cells for specific molecular markers (such as Corin and LRTM 1) early in the differentiation process. These researchers showed that dopaminergic neurons or precursors thereof sorted for corin+ and lrtm1+ had a higher percentage of TH positive cells and also showed about 10-fold greater engraftment potential (purer population) than unsorted cells (samta and Takahashi 2016, doi:10.1038/ncomms 13097). Nevertheless, dopaminergic neurons or precursors thereof must be transplanted at an early time point, day 28, and even then only about 10% of the transplanted cells are present 3 months after the transplantation.

To overcome the problem of low implantation rate of stem cell derived dopaminergic neurons, dopaminergic neurons were transplanted early between day 15 and day 32 of differentiation, when they were in the precursor stage. Experiments have shown that implantation of immature neurons greatly increases implantation rates, probably because the host brain provides the unknown factors required for effective implantation. However, transplantation of early progenitor cells has attracted attention from regulatory authorities such as the USFDA.

Regulatory authorities in the USFDA and other countries require characterization of cells for implantation. For example, characterization of dopaminergic neurons intended for use in the treatment of parkinson's disease will include demonstration of dopamine production by the cells. However, early cells implanted (about day 15-20) to ensure adequate implantation and expansion have not secreted dopamine, or even the final molecular markers identifying them as dopaminergic neurons. In addition, the early cell population may contain pluripotent stem cells that may generate teratomas in the brain of the recipient.

If the USFDA applies the same acceptance criteria to cells treating parkinson's disease as they apply to other cell therapies, it is difficult to see how early implantation of dopaminergic neurons or precursors thereof is acceptable. The FDA may be expected to require that cells for therapeutic use meet certain regulatory standards. That is, the cells produced need to reproducibly express specific molecular markers and demonstrate efficacy, e.g., secretion of a specific range of dopamine. Current methods for generating stem cell derived dopaminergic neurons plus early engraftment make it impossible to fully characterize the cell products and show efficacy prior to engraftment.

Thus, development methods (including formulations) that efficiently and reproducibly induce stem cell differentiation into dopaminergic neurons or precursors thereof that increase survival, increase implantation potential, increase yield, and also secrete increased amounts of dopamine would be an improvement over the prior art. It would be a significant improvement over the prior art if methods were developed to increase the efficiency, purity, yield, and/or dopamine secretion from human iPS cells. Dopaminergic neurons derived from ipscs would eliminate the need to treat patients with immunosuppressants and would eliminate the need for a master cell bank of embryo donor cells.

Current strategies for parkinson's disease cell therapy are differentiation of stem cells into precursors of dopaminergic neurons and transplantation into appropriate areas of the brain prior to final maturation into dopamine-producing neurons. The reason for early transplantation of dopaminergic neurons or precursors thereof is that the local environment within the brain provides an unknown factor that is required for the precursors to mature into the final maturation step of functional dopaminergic neurons with nerve transport capacity, implantation and production and secretion of dopamine.

Currently, stem cell-derived dopaminergic neurons, or more specifically their precursors, need to be implanted into the brain prior to full development. Experiments indicate that early transplantation yields higher implantation rates and more benefits, possibly due to increased dopamine production. Dopaminergic neurons or precursors are transplanted at a pre-dopamine production stage, such that unknown factors in the local environment of the brain induce proper maturation to the dopamine production stage.

A disadvantage of the early implantation of dopaminergic neurons or progenitors is that the cells cannot be fully characterized. For treating humans with cell therapies, the US FDA requires that cells be characterized and "released" for administration to humans only if certain predetermined criteria are met. Standards such as the presence of a defined percentage of cells expressing certain molecular markers and the production of specific amounts of dopamine from 1M cells are expected based on FDA regulatory requirements for other cell therapies.

It would therefore be a significant improvement over the prior art if stem cell derived dopaminergic neurons could be reliably and reproducibly cultured in vitro to a stage where a high percentage of transplanted cells express definitive molecular markers and produce substantial amounts of dopamine. Finally, these cells should exhibit the ability to implant into the brain in vitro.

Thus, identifying the factors provided by the brain that induce the dopaminergic neuron maturation step, and the time frame in which the dopaminergic neuron precursor should be contacted with those factors would be a significant improvement over the prior art.

Disclosure of Invention

The present invention relates to a method for producing dopaminergic neurons from human stem cells, comprising the step of adding vitamins to or increasing the concentration of vitamins for a neural basal medium on about day 20+/-3 of a regimen for differentiating pluripotent stem cells into dopaminergic neurons. The scheme may be scheme a. The vitamin may be vitamin a such as retinol, retinol acetate, 9-cis retinoic acid, 13-cis retinoic acid, or all-trans retinoic acid. Vitamin a may be dissolved in lipid-rich formulations such as human serum albumin, albumax, non-human serum albumin. In one embodiment, the final concentration of vitamin a may be 1uM to 3uM.

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

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 200nM to 110uM.

In any of the above methods, the pluripotent stem cells to be differentiated may have been cultured in NME7-AB or WNT 3A. In another aspect, the pluripotent stem cells to be differentiated may be in an initial state.

According to the above, the produced dopaminergic neurons may be characterized by greater than 30%, 100%, 500% or 1000% more dopamine than the dopaminergic neurons produced by a differentiation regimen without the addition or augmentation of vitamins.

According to the above, the produced dopaminergic neurons are characterized by greater than 30%, 100%, 500% or 1000% more neurites than the dopaminergic neurons produced by a differentiation regimen without the addition or augmentation of vitamins.

The present invention also relates to a method of increasing the likelihood of successful implantation of dopaminergic neurons into a subject in need thereof, comprising administering dopaminergic neurons obtained in the above-described method to said subject.

The present invention also relates to a method of treating a central nervous system disorder in a patient in need of implantation of dopamine-producing nerve cells comprising implanting into a human in need thereof the dopaminergic neurons obtained in the method described above. The central nervous system disorder is parkinson's disease, huntington's disease, multiple sclerosis or alzheimer's disease. Damage to the central and peripheral nervous systems can also be treated by implanting neurons, dopaminergic neurons for central nervous system conditions, and other types of neurons that treat peripheral nerve damage to the site of the damage.

Drawings

The patent or application contains at least one drawing which is drawn in color. Copies of this patent or patent application publication with color drawings will be provided by the office upon request and payment of the necessary fee.

The present invention will become more fully understood from the detailed description given herein below and the accompanying drawings, which are given by way of illustration only and thus are not limitative of the present invention, and wherein;

figures 1A-1E show schematic diagrams of four different protocols for differentiating pluripotent stem cells into dopaminergic neurons. Fig. 1A is a schematic illustration of a protocol disclosed in patent application US2018/0094242A1 (the disclosure of a medium for differentiating pluripotent stem cells into dopaminergic neurons, the contents of which are incorporated herein by reference), and is referred to herein as protocol a. FIG. 1B is a schematic representation of a new development by the inventors, herein referred to as scheme B, wherein pyridoxal is added at a final concentration of 11uM from day 21 and beyond, such that the total concentration in the medium reaches about 21uM pyridoxal. FIG. 1C is a schematic representation of a new development by the inventors, herein referred to as regimen C, wherein various forms of vitamin A, vitamin B, and in some cases vitamin C, are added to the basal nerve medium from day 21 and beyond; FIG. 1D shows a schematic of protocol C.2, wherein from day 21 and onwards, basal neural medium is exchanged for basal neural medium that does not contain pyridoxal but contains pyridoxine at a final concentration of 16uM plus retinol at a final concentration of 1.2uM and retinol acetate at a final concentration of 0.17 uM. FIG. 1E is a schematic representation of an optimization protocol, protocol D, wherein a basal medium containing about 10uM pyridoxal is supplemented with additional 11uM pyridoxal, 1.2uM retinol, 0.17uM retinol acetate, 61uM 2-phosphate-ascorbic acid, and 11uM L-ascorbic acid on and after day 21.

FIGS. 2A-2L show fluorescence photographs of three different types of pluripotent stem cells, cultured in different media, and then differentiated to attempt to generate dopaminergic neurons, taken on day 24 of protocol A. FIGS. 2A-2D show human embryonic stem cells, HES3 commercially available cells (referred to herein as hES _E8-HES3 ) The cells have been cultured in E8 medium prior to differentiation. Figures 2E-2H show that human induced pluripotent stem cells (referred to herein as iPS _E8-A6 ) The cells have been cultured in E8 medium prior to differentiation. Figures 2I-2L show that human induced pluripotent stem cells (referred to herein as iPS _NME7-6E ) Is a fluorescent photograph of the cells before differentiation, already at NME7 _AB Culturing in an initial culture medium. FIGS. 2A, 2E and 2I show cells stained for the presence of GIRK2 (G protein regulated inward rectifying potassium channel 2), GIRK2 expressed in dopaminergic neurons. FIGS. 2B, 2F and 2J showCells stained for the presence of TH (tyrosine hydroxylase), which is considered the gold standard for the identification of dopaminergic neurons, are shown. FIGS. 2C, 2G and 2K show cells stained for the presence of Tuj1 (neuron-specific class Ill B-tubulin), tuj1 being a pan-nerve marker. Fig. 2D, 2H and 2L show the superposition of all three markers.

Figures 3A-3L show fluorescence photographs of three different types of pluripotent stem cells, cultured in different media, and then differentiated to attempt to generate dopaminergic neurons, taken on day 24 of protocol c.2. FIGS. 3A-3D show human embryonic stem cells, HES3 commercially available cells (referred to herein as hES _E8-HES3 ) The cells have been cultured in E8 medium prior to differentiation. Figures 3E-3H show that human induced pluripotent stem cells (referred to herein as iPS _E8-A6 ) The cells have been cultured in E8 medium prior to differentiation. Figures 3I-3L show that human induced pluripotent stem cells (referred to herein as iPS _NME7-6E ) Is a fluorescent photograph of the cells before differentiation, already at NME7 _AB Culturing in an initial culture medium. FIGS. 3A, 3E and 3I show cells stained for the presence of GIRK2 (G protein regulated inward rectifying potassium channel 2) expressed in dopaminergic neurons. Figures 3B, 3F and 3J show cells stained for the presence of TH (tyrosine hydroxylase), which is considered the gold standard for identifying dopaminergic neurons. FIGS. 3C, 3G and 3K show cells stained for the presence of Tuj1 (neuron-specific class Ill B-tubulin), tuj1 being a pan-nerve marker. Figures 3D, 3H and 3L show the superposition of all three markers.

FIGS. 4A-4H show fluorescent photographs of pluripotent stem cells taken on day 60, differentiated according to either protocol A or protocol C.2. Some photographs show cells differentiated according to scheme c.2, but WNT3A was added to pluripotent stem cell medium at 100ng/mL for 48 hours before differentiation began. Figures 4A, 4B, 4E, 4F, 4G and 4H show iPS reprogrammed by using the epi method _NME7-N7B Image of cells differentiated from primary stem cells. Fig. 4C and 4D show the use of iPS _E8-A6 Stem cell differentiationIs a cell image of a cell. FIGS. 4A-4B differentiate according to scheme A. FIGS. 4E-4F differentiate according to scheme C.2. FIGS. 4C-4D and 4G-4H differentiated according to protocol C.2 except that WNT3A was added to the corresponding pluripotent medium at 100ng/mL for 48 hours before the differentiation protocol was initiated. Figures 4A, 4E, 4C and 4G show cells stained for the presence of DAT (dopamine active transporter) and Tuj 1. FIGS. 4B, 4F, 4D and 4H show cells stained for the presence of GIRK2, TH and Tuj 1.

Fig. 5A-5F show fluoroscopic photographs of a scratch assay, also known as a scar or wound healing assay, for assessing the implantable ability of neurons. The starting stem cells are in NME7 _AB Initial Stem cell "iPS" cultured in Medium _NME7-6E ", or is an originating stem cell" iPS "cultured in E8 medium _E8-A6 ". The cells shown differentiated into dopaminergic neurons and grew to confluence according to protocol c.2, either on day 13 or day 15. Mechanical scratches were made on the cell area to form gaps. The rate at which neurite outgrowth closes this gap is monitored and correlated with the implant potential. Green fluorescence is a measure of dopamine uptake, from labeled dopamine. Figures 5A-5C show iPS _NME7-6E Photographs of the derivative neurons. Figures 5D-5F show iPS _E8-A6 Photographs of the derivative neurons.

FIGS. 6A-6D show 800,000 cells/cm plated from day 11 of the protocol over a period of 30 th to 60 th days after initiation of differentiation ² Graph of secreted dopamine and its metabolites. Fig. 6A-6B show dopamine secreted from cells differentiated into dopaminergic neurons according to scheme a. Figures 6C-6D show dopamine secreted from cells differentiated into dopaminergic neurons according to scheme c.2. Fig. 6A, 6C show a slave iPS _E8-A6 Dopaminergic neurons derived from stem cells in an originating state. Fig. 6B, 6D show a slave iPS _NME7-6E Dopaminergic neurons derived from stem cells in an initial state.

FIG. 7 shows a graph of the amounts of dopamine and its metabolites secreted by a variable number of cells and measured on day 60 or day 40, in the case indicated, where horizontal striped bars indicate according to the formulaCase a differentiated stem cells in the original state, the cross-hatched bars indicate stem cells in the original state differentiated according to scheme c.2, the vertical bars indicate stem cells in the original state differentiated according to scheme a, and the solid black bars indicate stem cells in the original state differentiated according to scheme c.2. It should be noted that cell number refers to the number of cells per cm at day 11 of the protocol ² Cell number plated.

Fig. 8 is a table showing the amounts of dopamine and its metabolites secreted by dopaminergic neurons derived from different numbers of human stem cells. The table is organized according to the starting stem cell type. The stem cells are originating embryo HES3 cells and originating induced pluripotent stem cell iPS _E8-A6 Initial induced pluripotent stem cell (iPS) generated by using Sendai virus _NME7-6E "or initial induced pluripotent stem cell" iPS generated by using epiomal technique _NME7-N7B ". In some cases, as indicated in the table, stem cells were cultured in their respective media to which WNT3A was added at 100ng/mL for 48 hours before differentiation began.

Fig. 9 is a diagram showing the cloning of iPS from the episomal initial clone _NME7-N7B Table of the amount of secreted dopamine and its metabolites. The number of cells plated on day 11 and the number of days for measuring dopamine secretion varied. In addition, in some cases, WNT3A was added to the medium at 100ng/mL for 48 hours before differentiation began, as indicated.

Fig. 10 is a diagram showing the cloning of iPS from the episomal initial clone _NME7-6E Table of the amount of secreted dopamine and its metabolites. The number of cells plated on day 11 and the number of days for measuring dopamine secretion varied. In addition, in some cases, WNT3A was added to the medium at 100ng/mL for 48 hours before differentiation began, as indicated.

Fig. 11A-11K show fluorescence photographs taken at 20X magnification on confocal microscopy of human iPS cells differentiated into dopaminergic neurons according to the protocol described herein as protocol a, although modifications were made to introduce vitamin a in the form of retinol and retinol acetate into the medium on day 24, i.e., around day 20 of the protocol, until cell harvest. These cells served as controls for studying the effect of adding various forms of vitamin B6 around day 20 of the protocol. FIG. 11A shows a fluorescence photograph of cells stained for GIRK 2. Fig. 11B shows a fluorescence photograph of cells stained for TH (tyrosine hydroxylase). Fig. 11C shows a fluorescence photograph of cells stained for Tuj 1. FIG. 11D shows a fluorescence photograph of cells stained with Hoechst dye to reveal nuclei. Fig. 11E shows a bright field image. FIG. 11F shows a fluorescence photograph of a superposition of GIRK2, TH, tuj1 and Hoechst. Fig. 11G shows a fluorescence photograph of cells stained for DAT (dopamine transporter). Fig. 11H shows a fluorescence photograph of cells stained for Tuj 1. FIG. 11I shows a fluorescence photograph of cells stained with Hoechst dye to reveal nuclei. Fig. 11J shows a bright field image. FIG. 11K shows a fluorescence photograph of a DAT, tuj1 and Hoechst overlay.

Figures 12A-12K show fluorescence photographs taken at 20X magnification on confocal microscopy of human iPS cells differentiated into dopaminergic neurons according to the protocol described herein as protocol a, except that vitamin a in the form of retinol and retinol acetate was introduced into the medium on day 20 or so until cell harvest. In this experiment vitamin B6 in the form of pyridoxine was added to a final concentration of 16 uM. FIG. 12A shows a fluorescence photograph of cells stained for GIRK 2. Fig. 12B shows a fluorescence photograph of cells stained for TH (tyrosine hydroxylase). Fig. 12C shows a fluorescence photograph of cells stained for Tuj 1. FIG. 12D shows a fluorescence photograph of cells stained with Hoechst dye to reveal nuclei. Fig. 12E shows a bright field image. FIG. 12F shows a fluorescence photograph of a superposition of GIRK2, TH, tuj1 and Hoechst. Fig. 12G shows a fluorescence photograph of cells stained for DAT (dopamine transporter). Fig. 12H shows a fluorescence photograph of cells stained for Tuj 1. FIG. 12I shows a fluorescence photograph of cells stained with Hoechst dye to reveal nuclei. Fig. 12J shows a bright field image. FIG. 12K shows a fluorescence photograph of a DAT, tuj1 and Hoechst overlay.

Figures 13A-13K show fluorescence photographs taken at 20X magnification on confocal microscopy of human iPS cells differentiated into dopaminergic neurons according to the protocol described herein as protocol a, except that vitamin a in the form of retinol and retinol acetate was introduced into the medium on day 20 or so until cell harvest. In this experiment vitamin B6 in the form of pyridoxal was added to a final concentration of 11 uM. FIG. 13A shows a fluorescence photograph of cells stained for GIRK 2. Fig. 13B shows a fluorescence photograph of cells stained for TH (tyrosine hydroxylase). Fig. 13C shows a fluorescence photograph of cells stained for Tuj 1. FIG. 13D shows a fluorescence photograph of cells stained with Hoechst dye to reveal nuclei. Fig. 13E shows a bright field image. FIG. 13F shows a fluorescence photograph of a superposition of GIRK2, TH, tuj1 and Hoechst. Fig. 13G shows a fluorescence photograph of cells stained for DAT (dopamine transporter). Fig. 13H shows a fluorescence photograph of cells stained for Tuj 1. FIG. 13I shows a fluorescence photograph of cells stained with Hoechst dye to reveal nuclei. Fig. 13J shows a bright field image. FIG. 13K shows a fluorescence photograph of a DAT, tuj1 and Hoechst overlay.

Figures 14A-14K show fluorescence photographs taken at 20X magnification on confocal microscopy of human iPS cells differentiated into dopaminergic neurons according to the protocol described herein as protocol a, except that vitamin a in the form of retinol and retinol acetate was introduced into the medium on day 20 or so until cell harvest. In this experiment, vitamin B6 in the form of pyridoxal-5' -phosphate (also known as PLP) was added to a final concentration of 20 uM. FIG. 14A shows a fluorescence photograph of cells stained for GIRK 2. Fig. 14B shows a fluorescence photograph of cells stained for TH (tyrosine hydroxylase). Fig. 14C shows a fluorescence photograph of cells stained for Tuj 1. FIG. 14D shows a fluorescence photograph of cells stained with Hoechst dye to reveal nuclei. Fig. 14E shows a bright field image. FIG. 14F shows a fluorescence photograph of a superposition of GIRK2, TH, tuj1 and Hoechst. Fig. 14G shows a fluorescence photograph of cells stained for DAT (dopamine transporter). Fig. 14H shows a fluorescence photograph of cells stained for Tuj 1. FIG. 14I shows a fluorescence photograph of cells stained with Hoechst dye to reveal nuclei. Fig. 14J shows a bright field image. FIG. 14K shows a fluorescence photograph of a DAT, tuj1 and Hoechst overlay.

Figures 15A-15K show fluorescence photographs taken at 20X magnification on confocal microscopy of human iPS cells differentiated into dopaminergic neurons according to the protocol described herein as protocol a, except that vitamin a in the form of retinol and retinol acetate was introduced into the medium on day 20 or so until cell harvest. In this experiment, vitamin B6 was added as all three forms of pyridoxine-HCL, pyridoxal and pyridoxal-5' -phosphate (also known as PLP). FIG. 15A shows a fluorescence photograph of cells stained for GIRK 2. Fig. 15B shows a fluorescence photograph of cells stained for TH (tyrosine hydroxylase). Fig. 15C shows a fluorescence photograph of cells stained for Tuj 1. FIG. 15D shows a fluorescence photograph of cells stained with Hoechst dye to reveal nuclei. Fig. 15E shows a bright field image. FIG. 15F shows a fluorescence photograph of a superposition of GIRK2, TH, tuj1 and Hoechst. Fig. 15G shows a fluorescence photograph of cells stained for DAT (dopamine transporter). Fig. 15H shows a fluorescence photograph of cells stained for Tuj 1. FIG. 15I shows a fluorescence photograph of cells stained with Hoechst dye to reveal nuclei. Fig. 15J shows a bright field image. FIG. 15K shows a fluorescence photograph of a DAT, tuj1 and Hoechst overlay.

Fig. 16A-16E show fluorescence photographs taken at 20X magnification on confocal microscopy of human iPS cells differentiated into dopaminergic neurons according to the protocol described herein as protocol a, except that retinol and retinol acetate were added to the differentiation medium at around day 20, such that the effect of the addition of different forms of vitamin B6 could be observed. Fig. 16A shows a fluorescence photograph of cells at a final concentration of pyridoxine-HCL to 16uM added from day 20. Fig. 16B shows a fluorescence photograph of cells at the final concentration of pyridoxal to 11uM added from day 20. Fig. 16C shows a fluorescence photograph of cells at a final concentration of pyridoxal-5' -phosphate added to 20uM from day 20. Fig. 16D shows a fluorescence photograph of cells when all three B6 vitamins (including pyridoxine, pyridoxal, and pyridoxal-5' -phosphate) were added together. Fig. 16E shows a control experiment according to scheme a, except that vitamin a in the form of retinol and retinol acetate was added on day 20.

Fig. 17 is a graph of the amount of dopamine and its metabolites (measured by HPLC) from 200,000 cells present in conditioned medium, which was collected on day 30, day 40, day 50 or day 60. The medium is not withdrawn from a single cell source. Instead, a separate experiment was allowed to proceed until the day the medium was withdrawn for analysis. This experiment used protocol C, where retinol and retinol acetate were added to each condition on and after day 20. The form of vitamin B added to the basal nerve medium varies. In this experiment, the basal medium contained about 10uM pyridoxal. In the case of pyridoxine addition, pyridoxal is omitted from the basal medium.

Fig. 18A-18I show fluorescence photographs taken at 20X magnification on confocal microscopy of human iPS cells differentiated into dopaminergic neurons on day 24. These photographs are photographs of control experiments in which cells differentiated according to scheme a. FIG. 18A shows a fluorescence photograph of cells stained for GIRK 2. Fig. 18B shows a fluorescence photograph of cells stained for TH (tyrosine hydroxylase). Fig. 18C shows a fluorescence photograph of cells stained for Tuj 1. FIG. 18D shows a fluorescence photograph of cells stained with Hoechst dye to reveal nuclei. FIG. 18E shows a fluorescence photograph of a superposition of GIRK2, TH, tuj1 and Hoechst. Fig. 18F shows a fluorescence photograph of cells stained for DAT (dopamine transporter). Fig. 18G shows a fluorescence photograph of cells stained for Tuj 1. FIG. 18H shows a fluorescence photograph of cells stained with Hoechst dye to reveal nuclei. FIG. 18I shows a fluorescence photograph of a DAT, tuj1 and Hoechst overlay.

Figures 19A-19I show fluorescence photographs taken at 20X magnification on confocal microscopy of human iPS cells differentiated into dopaminergic neurons on day 24. These photographs are of control experiments in which cells differentiated according to scheme a except that pyridoxal was added to the medium at a final concentration of 11uM on day 20; this modified scheme is referred to herein as scheme B. In this way one can see the additional effect of adding various forms of vitamin a. FIG. 19A shows a fluorescence photograph of cells stained for GIRK 2. Fig. 19B shows a fluorescence photograph of cells stained for TH (tyrosine hydroxylase). Fig. 19C shows a fluorescence photograph of cells stained for Tuj 1. FIG. 19D shows a fluorescence photograph of cells stained with Hoechst dye to reveal nuclei. FIG. 19E shows a fluorescence photograph of a superposition of GIRK2, TH, tuj1 and Hoechst. Fig. 19F shows a fluorescence photograph of cells stained for DAT (dopamine transporter). Fig. 19G shows a fluorescence photograph of cells stained for Tuj 1. FIG. 19H shows a fluorescence photograph of cells stained with Hoechst dye to reveal nuclei. FIG. 19I shows a fluorescence photograph of a DAT, tuj1 and Hoechst overlay.

Fig. 20A-20I show fluorescence photographs taken at 20X magnification on confocal microscopy of human iPS cells differentiated into dopaminergic neurons according to protocol B on day 24. In this experiment, in addition to pyridoxal being added to the medium from day 20, two forms of vitamin a were added. Retinol was added to a final concentration of 0.7uM and retinol acetate was added to a final concentration of 0.6 uM. FIG. 20A shows a fluorescence photograph of cells stained for GIRK 2. Fig. 20B shows a fluorescence photograph of cells stained for TH (tyrosine hydroxylase). Fig. 20C shows a fluorescence photograph of cells stained for Tuj 1. FIG. 20D shows a fluorescence photograph of cells stained with Hoechst dye to reveal nuclei. FIG. 20E shows a fluorescence photograph of a superposition of GIRK2, TH, tuj1 and Hoechst. Fig. 20F shows a fluorescence photograph of cells stained for DAT (dopamine transporter). Fig. 20G shows a fluorescence photograph of cells stained for Tuj 1. FIG. 20H shows a fluorescence photograph of cells stained with Hoechst dye to reveal nuclei. FIG. 20I shows a fluorescence photograph of a DAT, tuj1 and Hoechst overlay.

Fig. 21A-21I show fluorescence photographs taken at 20X magnification on confocal microscopy of human iPS cells differentiated into dopaminergic neurons according to protocol B on day 24. In this experiment, in addition to pyridoxal being added to the medium from day 20, vitamin a was also added in the following form: 9-cis retinoic acid was added to a final concentration of 0.446 uM; 13-cis retinoic acid was added to a final concentration of 0.446 uM; and all-trans retinoic acid was added to a final concentration of 0.446 uM. FIG. 21A shows a fluorescence photograph of cells stained for GIRK 2. Fig. 21B shows a fluorescence photograph of cells stained for TH (tyrosine hydroxylase). Fig. 21C shows a fluorescence photograph of cells stained for Tuj 1. FIG. 21D shows a fluorescence photograph of cells stained with Hoechst dye to reveal nuclei. FIG. 21E shows a fluorescence photograph of a superposition of GIRK2, TH, tuj1 and Hoechst. Fig. 21F shows a fluorescence photograph of cells stained for DAT (dopamine transporter). Fig. 21G shows a fluorescence photograph of cells stained for Tuj 1. FIG. 21H shows a fluorescence photograph of cells stained with Hoechst dye to reveal nuclei. FIG. 21I shows a fluorescence photograph of a DAT, tuj1 and Hoechst overlay.

Fig. 22A-22I show fluorescence photographs taken at 20X magnification on confocal microscopy of human iPS cells differentiated into dopaminergic neurons according to protocol B on day 24. In this experiment, in addition to pyridoxal being added to the medium from day 20, all-trans retinoic acid was also added to a final concentration of 1.33 uM. FIG. 22A shows a fluorescence photograph of cells stained for GIRK 2. Fig. 22B shows a fluorescence photograph of cells stained for TH (tyrosine hydroxylase). Fig. 22C shows a fluorescence photograph of cells stained for Tuj 1. FIG. 22D shows a fluorescence photograph of cells stained with Hoechst dye to reveal nuclei. FIG. 22E shows a fluorescence photograph of a superposition of GIRK2, TH, tuj1 and Hoechst. Fig. 22F shows a fluorescence photograph of cells stained for DAT (dopamine transporter). Fig. 22G shows a fluorescence photograph of cells stained for Tuj 1. FIG. 22H shows a fluorescence photograph of cells stained with Hoechst dye to reveal nuclei. FIG. 22I shows a fluorescence photograph of a DAT, tuj1 and Hoechst overlay.

Fig. 23A-23E show fluorescence photographs taken at 20X magnification on confocal microscopy of human iPS cells differentiated into dopaminergic neurons on day 24. FIG. 23A shows a fluorescence photograph of cells differentiated according to protocol A. Fig. 23B shows a fluorescence photograph of cells differentiated according to protocol B, but on day 20, vitamin a in the form of retinol (0.7 uM) and retinol acetate (0.6 uM) was added in addition to pyridoxal. Fig. 23C shows a fluorescence photograph of cells differentiated according to protocol B, but on day 20, vitamin a in the form of 9-cis retinoic acid, 13-cis retinoic acid and all-trans retinoic acid was added in addition to pyridoxal, at a final concentration of 0.446uM each. Fig. 23D shows a fluorescence photograph of cells differentiated according to protocol B, but at day 20, vitamin a in the form of all-trans retinoic acid was added to a final concentration of 1.33uM in addition to pyridoxal. Fig. 23E shows a control experiment in which cells differentiated according to scheme B, which differs from scheme a in that pyridoxal was added to a final concentration of 11uM on and after day 20.

Fig. 24A-24I show fluorescence photographs taken at 20X magnification on confocal microscopy of human iPS cells differentiated into dopaminergic neurons according to protocol B on day 24. In this experiment, in addition to pyridoxal being added to the medium from day 20, two forms of vitamin a were added, wherein vitamin a was dissolved in 2mg/mL Albumax. Retinol was added to a final concentration of 1.2uM and retinol acetate was added to a final concentration of 0.17 uM. FIG. 24A shows a fluorescence photograph of cells stained for GIRK 2. Fig. 24B shows a fluorescence photograph of cells stained for TH (tyrosine hydroxylase). Fig. 24C shows a fluorescence photograph of cells stained for Tuj 1. FIG. 24D shows a fluorescence photograph of cells stained with Hoechst dye to reveal nuclei. FIG. 24E shows a fluorescence photograph of a superposition of GIRK2, TH, tuj1 and Hoechst. Fig. 24F shows a fluorescence photograph of cells stained for DAT (dopamine transporter). Fig. 24G shows a fluorescence photograph of cells stained for Tuj 1. FIG. 24H shows a fluorescence photograph of cells stained with Hoechst dye to reveal nuclei. FIG. 24I shows a fluorescence photograph of a DAT, tuj1 and Hoechst overlay.

Figures 25A-25I show fluorescence photographs taken at 20X magnification on confocal microscopy of human iPS cells differentiated into dopaminergic neurons according to protocol B on day 24. In this experiment, in addition to pyridoxal being added to the medium from day 20, two forms of vitamin a plus two forms of vitamin C were added. Vitamin A was dissolved in 2mg/mL Albumax. Retinol was added to a final concentration of 1.2uM and retinol acetate was added to a final concentration of 0.17 uM. Vitamin C was added as 61uM 2-phospho-ascorbic acid and 110uM L-ascorbic acid. FIG. 25A shows a fluorescence photograph of cells stained for GIRK 2. Fig. 25B shows a fluorescence photograph of cells stained for TH (tyrosine hydroxylase). Fig. 25C shows a fluorescence photograph of cells stained for Tuj 1. FIG. 25D shows a fluorescence photograph of cells stained with Hoechst dye to reveal nuclei. FIG. 25E shows a fluorescence photograph of a superposition of GIRK2, TH, tuj1 and Hoechst. Fig. 25F shows a fluorescence photograph of cells stained for DAT (dopamine transporter). Fig. 25G shows a fluorescence photograph of cells stained for Tuj 1. FIG. 25H shows a fluorescence photograph of cells stained with Hoechst dye to reveal nuclei. FIG. 25I shows a fluorescence photograph of a DAT, tuj1 and Hoechst overlay.

Fig. 26A-26I show fluorescence photographs taken at 20X magnification on confocal microscopy of human iPS cells differentiated into dopaminergic neurons according to protocol B on day 24. In this experiment, in addition to pyridoxal being added to the medium from day 20, vitamin A was added as all-trans retinoic acid to a final concentration of 1.33uM, wherein the vitamin was dissolved in 2mg/mL Albumax. FIG. 26A shows a fluorescence photograph of cells stained for GIRK 2. Fig. 26B shows a fluorescence photograph of cells stained for TH (tyrosine hydroxylase). Fig. 26C shows a fluorescence photograph of cells stained for Tuj 1. FIG. 26D shows a fluorescence photograph of cells stained with Hoechst dye to reveal nuclei. FIG. 26E shows a fluorescence photograph of a superposition of GIRK2, TH, tuj1 and Hoechst. Fig. 26F shows a fluorescence photograph of cells stained for DAT (dopamine transporter). Fig. 26G shows a fluorescence photograph of cells stained for Tuj 1. FIG. 26H shows a fluorescence photograph of cells stained with Hoechst dye to reveal nuclei. FIG. 26I shows a fluorescence photograph of a DAT, tuj1 and Hoechst overlay.

Fig. 27A-27I show fluorescence photographs taken at 20X magnification on confocal microscopy of human iPS cells differentiated into dopaminergic neurons according to protocol B on day 24. In this experiment, in addition to pyridoxal being added to the medium from day 20, vitamin A was added as all-trans retinoic acid to a final concentration of 1.33uM, wherein the vitamin was dissolved in 2mg/mL Albumax. Vitamin C was added as 61uM 2-phospho-ascorbic acid and 110uM L-ascorbic acid. FIG. 27A shows a fluorescence photograph of cells stained for GIRK 2. Fig. 27B shows a fluorescence photograph of cells stained for TH (tyrosine hydroxylase). Fig. 27C shows a fluorescence photograph of cells stained for Tuj 1. FIG. 27D shows a fluorescence photograph of cells stained with Hoechst dye to reveal nuclei. FIG. 27E shows a fluorescence photograph of a superposition of GIRK2, TH, tuj1 and Hoechst. Fig. 27F shows a fluorescence photograph of cells stained for DAT (dopamine transporter). Fig. 27G shows a fluorescence photograph of cells stained for Tuj 1. FIG. 27H shows a fluorescence photograph of cells stained with Hoechst dye to reveal nuclei. FIG. 27I shows a fluorescence photograph of a DAT, tuj1 and Hoechst overlay.

Detailed Description

Definition of the definition

The terms used in the present specification generally have their ordinary meanings in the art in the context of the present application and in the specific context of the use of each term. Certain terms are discussed below or elsewhere in the specification to provide additional guidance to the practitioner in describing the compositions and methods of the application and how to make and use them.

In the present application, "a" and "an" are used to refer to both single and multiple objects.

As used herein, "about" or "substantially" generally provides room to avoid 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 is not limited to the recited number of amino acids. It may involve the addition or subtraction of a small amount of amino acids from the N-terminus or the C-terminus, provided that there is functional activity (such as binding activity thereof). The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, depending in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" may mean within 3 or more than 3 standard deviations. Alternatively, "about" may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude of a value, e.g., within 5 times a value, or within 2 times a value.

As used herein, the term "population of cells" or "population of cells" refers to a group of at least two cells. In non-limiting examples, the population of cells can include at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000 cells. The population may be a pure population comprising one cell type, such as a population of dopaminergic neurons, or a population of undifferentiated stem cells. Alternatively, the cell population may comprise more than one cell type, e.g. a mixed cell population.

As used herein, "amino acid" refers to all naturally occurring L-a-amino acids. This definition is intended to include norleucine, ornithine and homocysteine.

As used herein, a "carrier" includes a pharmaceutically acceptable carrier, excipient, or stabilizer that is non-toxic to the cells or mammals to which it is exposed at the dosages and concentrations employed. The pharmaceutically acceptable carrier is often an aqueous pH buffered solution. Examples of pharmaceutically acceptable carriers include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; a low molecular weight (less than about 10 residues) polypeptide; proteins such as 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 dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or nonionic surfactants, such as Polyethylene glycol (PEG) and->

As used herein, the term "contacting" one or more cells with a compound (e.g., one or more inhibitors, activators, and/or inducers) refers to providing the compound in a location that allows one or more cells to enter the compound. The contacting may be accomplished using any suitable method. For example, contacting may be achieved by adding the compound in concentrated form to the cells or cell population (e.g., in the context of cell culture) to achieve the desired concentration. Contacting may also be accomplished by including the compound as a component of the formulation medium.

As used herein, the term "culture medium" refers to a liquid that covers cells in a culture vessel (such as a petri dish, a multi-well plate, etc.) and contains nutrients that nourish and support the cells. The medium may also contain growth factors added to produce the desired change in the cells.

As used herein, an "effective amount of an agent that inhibits an NME family protein" refers to an amount of an agent that is effective in blocking the activation interaction between an NME family protein and its cognate receptor, such as.

As used herein, "in combination" administration with one or more other therapeutic agents includes simultaneous (concurrent) and sequential administration in any order.

As used herein, the term "induced pluripotent stem cells" or "ipscs" refers to a type of pluripotent stem cells formed by introducing certain embryonic genes, such as but not limited to OCT4, SOX2, and KLF4 transgenes (see, e.g., takahashi and Yamanaka Cell 126,663-676 (2006), incorporated herein by reference), into somatic cells.

As used herein, "pluripotent" stem cells refer to stem cells that can differentiate into other cell types, wherein the number of different cell types is limited.

As used herein, "Primary Stem cells [ ]stem cells) "are those stem cells that are similar to the cells of the cell mass within the blastula and share quantifiable characteristics. The primary stem cells have quantifiable differences in the expression of certain genes as compared to the originating stem cells, which are similar to cells from the blastula ectodermal portion and share traits and characteristics. Notably, the initial stem cells of female origin have two active X chromosomes, known as XaXa, while the later-originating stem cells of female origin have one X chromosome inactivated.

As used herein, "neural basal medium" refers to a medium that allows for long-term maintenance of the normal phenotype and growth of neuronal cells, and maintains a pure population of neuronal cells without the need for an astrocyte feeder layer.

As used herein, the "NME family protein" or "NME family member protein" numbered 1-10 are proteins that are combined together because they all have at least one NDPK (nucleotide biphosphate kinase) domain. In some cases, the NDPK domain does not play a role in being able to catalyze the conversion of ATP to ADP. NME proteins have previously been called NM23 proteins, numbered H1 and H2. Recently, up to ten (10) NME family members have been identified. Herein, the terms NM23 and NME are interchangeable. Herein, the terms NME1, NME2, NME5, NME6, NME7, NME8 and NME9 are used to refer to native proteins as well as NME variants. In some cases, these variants are more soluble, expressed better in E.coli, or more soluble than the native sequence protein. For example, NME7 as used in the specification may mean a native protein or variant, such as NME7AB with excellent commercial applicability, as the variation allows for high yield expression of soluble properly folded proteins in e. NME7AB consists mainly of NME7A and B domains, but lacks a majority of the DM10 domain, which DM10 domain is located at the N-terminus of the native protein. "NME1" as referred to herein is interchangeable with "NM 23-H1". It is also intended that the invention is not limited by the exact sequence of the NME protein. NME7 as referred to herein is intended to mean a molecular weight of about 42kDa native NME7. NME7 as referenced herein _AB By native or recombinant NME7 lacking the DM-10 domain is meant having a molecular weight of about 33kDa, or an alternative native variant NME7-X1 likewise lacking the DM-10 domain, having a molecular weight of about 31kDa.

As used herein, the term "NME7 _AB "," NME7AB "and" NME-AB "are used interchangeably.

As used herein, "pharmaceutically acceptable carrier and/or diluent" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional medium or agent is incompatible with the active ingredient, its use in therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the compositions.

It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the mammalian subject to be treated; each unit contains a predetermined amount of active material calculated to produce the desired therapeutic effect in combination with the desired pharmaceutical carrier. The specification of the dosage unit form of the invention is determined by and directly depends on the following factors: (a) The unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) limitations inherent in the art of compounding such active materials for the treatment of diseases in living subjects suffering from disease conditions and impaired physical health.

The primary active ingredient is compounded in dosage unit form with an effective amount of a suitable pharmaceutically acceptable carrier for convenient and effective administration. For example, unit dosage forms may contain the primary active compound in an amount ranging from 0.5 μg to about 2000 mg. The active compound is typically present in the carrier at about 0.5 μg/ml, expressed as a ratio. In the case of compositions containing supplementary active ingredients, the dosage is determined by reference to the usual dosage and mode of administration of the ingredients.

As used herein, a "pluripotency marker" is one that increases in expression when a cell returns to a state that is less mature than the starting cell. Pluripotency markers include OCT4, SOX2, NANOG, KLF4, KLF2, tra 1-60, tra 1-81, SSEA4 and REX-1 as well as other markers previously described and those currently being discovered. For example, fibroblasts express undetectable or low levels of these multipotent markers, but express a fibroblast differentiation marker called CD 13. To determine whether a cell becomes less mature than the starting cell, the difference in expression levels of the pluripotency marker between the starting cell and the resulting cell can be measured.

As used herein, "pluripotent" stem cells refer to stem cells that can differentiate into all three lineages, endodermal, ectodermal and mesodermal, to differentiate into any cell type in vivo, but which are incapable of producing a whole organism. Totipotent stem cells are stem cells that can differentiate or mature into a whole organism (such as a human). With respect to embryonic pluripotent stem cells, they are cells derived from the inner cell mass of blastula. Typical markers of pluripotency are OCT4, KLF4, NANOG, tra 1-60, tra 1-81 and SSEA4.

As used herein, an "originating stem cell" is a cell that is similar to and shares traits and characteristics with cells from the blastula ectodermal portion.

As used herein, a cell population of "semi-dopaminergic neuronal state" or "pre-dopaminergic neuronal state" or "dopaminergic neuronal precursor" refers to a cell population in which some or all cells have the morphological characteristics and dopamine expression levels of dopaminergic neurons, however the cell population contains at least some cells that are not fully mature dopaminergic neurons.

As used herein, the term "stem cell" refers to a cell that has the ability to divide indefinitely in culture and produce specialized cells.

As used herein, "treatment" is a method for achieving a beneficial or desired clinical result. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "treatment" may also mean an extension of survival compared to the expected survival in the absence of treatment. "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder and those of the disorder to be prevented. By "moderating" a disease is meant that the extent of the disease state and/or the time course of the adverse clinical manifestations is reduced and/or progressed is slowed or prolonged compared to the case where no treatment is being performed.

Differentiation of pluripotent stem cells into dopaminergic neurons

In this work, the inventors explored two main directions of investigation: 1) Comparing the potential of the primary stem cells to differentiate into functional dopaminergic neurons with the stem cells in their original state; and 2) the effect of time-dependent addition of various factors, including vitamins produced in the brain of the developing embryo, on functional dopaminergic neuron differentiation.

We first used a protocol derived from US2018/0094242A1 (referred to herein as protocol a) to direct the differentiation of human iPS cells into dopaminergic neurons (fig. 1A and example 1). In these experiments, the starting iPSC was either in the earliest initial state, grown in NME7-AB initial medium (Carter et al 2016), or in a later initial state, grown in E8 medium containing FGF 2. Using protocol A and starting from hESC or hiPSC in the original state, cultured in FGF 2-containing E8 medium, the resulting dopaminergic neurons secrete only about 2-3ng/mL of dopamine or its metabolites per 800,000 cells/cm on day 60 of protocol ² . In contrast, using scheme a but starting from the initial hiPSC, on day 60, in one case they secreted 8.45ng/mL of dopamine and metabolite per cm2 from 400,000 cells, and in another case they secreted 5.85ng/mL of dopamine and metabolite per 800,000 cells/cm ² . These results are consistent with the following: in the naive state, NME 7-AB-grown cells differentiated better into dopaminergic neurons than the naive stem cells.

Previous studies have shown that dopaminergic neuron precursors implanted in the brain at about day 16 to day 28 after initiation of differentiation are better implanted than implanted cells after complete maturation to about days 40-60 and have more therapeutic benefit. These results indicate that in the brain environment, progenitor cells are able to mature into functional dopaminergic neurons. These results are also consistent with the following: no maturation factors provided by the brain are required at the early stages of differentiation into neurons or dopaminergic neuron precursors. We therefore strive to determine which factors produced in the brain may be important for the development of dopaminergic neurons, and also to determine the time frame for contacting dopaminergic neuron precursors with these factors. We found empirically that dopaminergic neuron progenitor cells must be implanted into the brain at about the same time (this is between day 16 and day 28 of the differentiation regimen) by the addition of candidate factors (samta and Takahashi 2016, doi: 10.1038/ncoms 13097). The effect of candidate factors added alone or in combination on molecular marker expression, implantation and dopamine secretion was evaluated.

Several factors produced in the brain are thought to be important for neural differentiation. Some candidate factors that may induce maturation into dopaminergic neurons that are provided by the brain in the final step are vitamin A [ Qingmu et al 2018, DOI:10.1080/21691401.2018.1436552; engberg et al, stem Cells 2010;28:1498-1509; JD Brenner, 2007, doi:10.1016/j.pnp bp.2007.07.001], vitamin B [ Carlos Alberto Calder Mn-Ospina, mauricio Orlando Nava-Mesa, doi:10.1111/cns.13207, guilarte,2006Journal of Neurochemistry,DOI:10.1111/j.1471-4159.1987.tb04111.X, peraza et al, 2018,BMC Neuroscience ], vitamin C [ 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 are in various forms and several vitamins are reported to increase in brain levels during neurodevelopmental processes. However, the results of the study are often conflicting. Several scientific studies have concluded that retinoic acid, either vitamin a or its derivatives, blocks neural differentiation, while other studies suggest that they may be required for neural differentiation. Similarly, various types of vitamin B are reported to be beneficial for neural differentiation, while others report that they inhibit neural differentiation.

We assessed the effects of various forms of B vitamins (especially the neurotropic tissue B vitamins B1, B6 and B12), various forms of vitamin A and vitamin C on the development of dopaminergic neurons derived from hiPS cells. In addition to the rather insoluble vitamin a, we also evaluated the effect of various high lipid density additives on dissolved vitamin a, such as serum albumin and serum albumin substitutes, including commercially available Albumax.

We have performed a series of experiments comparing the prior art with the methods and compositions of the present invention. In one aspect, we compared standard naive stem cells to primary stem cells of NME7-AB growth differentiated into dopaminergic neurons. On the other hand, we compared protocol a with our modifications, protocols B, C, c.2 and D (fig. 1A-1E), where the addition of various vitamins and other components began around day 20+/-3 of the protocol. The resulting cells were analyzed at various time points for the presence of appropriate molecular markers, characteristic morphology, length of neurite outgrowth as an indicator of implantation potential, and most importantly, the amount of dopamine produced and secreted.

In scheme c.2, on day or so, the neural basal medium was exchanged for a medium that did not contain pyridoxal but contained pyridoxine and two forms of vitamin a. The following experiments compare stem cells differentiated into dopaminergic neurons according to either scheme a or scheme c.2. On day 24, immunofluorescent staining was performed to detect the presence of molecular markers of dopaminergic neuron progenitor cells generated using either protocol A (FIG. 2A-FIG. 2L) or protocol C.2 (FIG. 3A-FIG. 3L). GIRK2 (G protein regulated inward rectifying potassium channel 2) is expressed in dopaminergic neurons, while Tuj1 (neuron specific class Ill B-tubulin) is a pan-neural marker. TH (tyrosine hydroxylase) is considered the gold standard for identifying dopaminergic neurons because it is an enzyme that catalyzes the conversion of L-tyrosine to L-3, 4-dihydroxyphenylalanine, which is the rate-limiting step in dopamine synthesis. DAT (dopamine active transporter) is equally important because it is a transmembrane protein that pumps dopamine from the synapse back into the cytosol. Both differentiation protocols produced cells positive for all appropriate molecular markers. However, cells generated using scheme c.2 formed an interconnected neuronal network with longer projections and more connectivity than cells generated by scheme a.

The morphological differences were also evident in immunofluorescence studies performed on day 60. Previously at NME7 _AB While ipscs grown in and then differentiated according to scheme a lack the interconnected network characteristics of axonal projections and functional neurons, the same cells differentiated according to scheme c.2 had the desired morphology (fig. 4A-4B and fig. 4E-4F). It should be noted that in this experiment, the second initial clone iPSC was used _NME7-N7B . This second initial clone was generated by epiomal reprogramming in NME7-AB medium using the core pluripotent factor OCT4/SOX2/KLF 4/c-Myc. Another clone used in these experiments, iPSC _NME7-6E Sendai virus generation (Carter et al 2016) was used and has previously been demonstrated to be able to differentiate into functional cardiomyocytes and hepatocytes. Differentiation of stem cells in an originating state, such as iPS, using protocol A _E8-A6 Cells failed repeatedly before reaching day 60. However, we found that if ipscs grown in E8 were cultured in E8 plus β -catenin, an agonist (such as WNT 3A) for 48 hours before beginning differentiation, they exhibited increased survival on day 60 and possibly improved morphology. However, the morphology and expression pattern of DAT, TH and GIRK2 are still not as good as in NME7 _AB Ipscs grown in WNT3A for 48 hours and then differentiated according to scheme c.2 (figures 4C-4D and 4G-4H) were added. The primary stem cells differentiated according to scheme c.2 form a network with the desired morphology. The complexity of the morphology of neurons, such as branch density and grouping pattern, is highly correlated with the function of neurons.

One impediment to the therapeutic use of stem cell-derived dopaminergic neurons is the current low implantation rate. It is estimated that for therapeutic benefit, at least 100,000 functional dopaminergic neurons need to be implanted into the brain of a patient. The first order in vitro method of assessing implantation may be wound healingThe assay, also known as scratch test. Previously at NME7 _AB Or ipscs grown in E8 were differentiated using protocol c.2 until the cells were confluent, and then scored or scarred. Derived from the previous NME7 _AB Dopaminergic neurons of ipscs grown in medium generated neurite projections that closed the gap within 6 days, with fewer and shorter projections for cells grown in E8 medium (fig. 5A-5F). Green fluorescence is a measure of dopamine uptake from labeled dopamine mimetics.

One of the most important measures of dopaminergic neuronal function is their ability to make and secrete dopamine. HPLC analysis was performed at a range of cell densities and at different stages of the differentiation protocol to quantify secreted dopamine and its metabolites. The originating human iPS cells differentiated into dopaminergic neurons according to protocol a secreted up to 10ng/mL of dopamine and its metabolites at day 60 (fig. 6A), whereas the naive stem cells differentiated using protocol a secreted about 7ng/mL at this time point (fig. 6B). In sharp contrast, stem cells in the original state differentiated using protocol C.2 secreted nearly 40ng/mL on day 60 (FIG. 6C), whereas stem cells in the original state secreted nearly 60ng/mL of dopamine and its metabolites (FIG. 6D). In the experiment shown in FIG. 6, the measured amount of dopamine and its metabolites was measured from 400,000 cells/cm ² Secreted and measured on day 60 after initiation of differentiation. Fig. 7 shows a graph of the amounts of dopamine and its metabolites secreted by a variable number of cells and measured on day 60 or day 40, in the case indicated, with horizontal and vertical striped bars indicating cells differentiated using protocol a, and cross-hatched and filled bars indicating cells differentiated using protocol c.2. Horizontal striped bars and cross-hatched bars indicate the use of naive stem cells, and vertical striped bars and solid bars indicate the use of naive stem cells. In comparison to protocol c.2, more than 40 experiments (fig. 8, 9 and 10) performed using protocol a showed that cells differentiated into dopaminergic neurons using protocol c.2 produce cells that secrete 10-fold more dopamine than those produced using protocol a on average. Furthermore, by NME7 _AB The cells differentiated from the primary cells reproducibly produced the most dopamine.

Next, we have attempted to further investigate the effect of various forms of vitamin B6 on stem cell differentiation into dopaminergic neurons. Recall that in the previous set of experiments, either the naive or the naive stem cells differentiated into dopaminergic neurons according to protocol a or protocol c.2, wherein on day or so, the neural basal medium was exchanged such that it no longer contained pyridoxal, but pyridoxine plus retinol and retinol acetate.

In this next set of experiments we followed protocol a except that on day 20 or so, the neural basal medium was exchanged for medium containing approximately 1.2uM retinol and 0.17uM retinol acetate, and then various other forms of vitamin B6 were added (fig. 1, example 2). The resulting cells were analyzed for cell morphology, yield of TH and DAT positive cells relative to Tuj1 positivity, and number and length of neural projections as an indicator of implantation potential.

The B1 (thiamine), B6 (pyridoxine) and B12 (cobalamin) sub-sets of vitamins are referred to as neurotropic B vitamins. The level of B6 increases during gestational brain development. Pyrrolol is a dietary form of B6. The PLP form of vitamin B6 (pyridoxal phosphate) is a biologically active form of vitamin B6, which is necessary for synthesis of neurotransmitters, such as for the synthesis of dopamine from levodopa. In the medium, pyridoxine may be metabolized to form pyridoxal-5' -phosphate. B12 is reported to play a role in the synthesis of myelin.

We found that in vitro wound healing experiments, the addition of certain forms of vitamin B6 to basal neural differentiation media greatly improved the quality of dopaminergic neurons derived from stem cells, greatly increased the amount of dopamine they secreted, and significantly increased implantation. The time, concentration and various forms of vitamin B are key factors in the in vitro differentiation and maturation of dopaminergic neurons from human stem cells.

We assessed the effect of various forms of vitamin B6 on differentiation of dopaminergic neurons from human iPS cells. Since initial experiments showed that initial NME7-AB human iPS cells differentiated better to dopaminergic neurons than hipscs grown in FGF 2-containing E8 medium, these experiments were performed on only naive stem cells and then repeated using naive stem cells. A differentiation protocol called protocol a (fig. 1) was followed until day 20. From day 21, 1.2uM of vitamin A in the form of retinol and 0.17uM of vitamin A in the form of retinol acetate were added to the basal medium, as well as vitamin C as ascorbic acid-2-phosphate at a final concentration of about 200 nM. Negative controls without additional vitamin B added are shown in fig. 11A-11K. It is important to note that the neural basal medium used under all conditions contains 10uM pyridoxal, so this low concentration of pyridoxal is present from the beginning of differentiation.

In addition to vitamins a and C, which are constant under all conditions, various forms of vitamin B6 were added: 16uM pyridoxine (FIGS. 12A-12K), or 11uM pyridoxal (FIGS. 13A-13K), or 20uM pyridoxal-5 '-phosphate (also known as PLP (FIGS. 14A-14K)), or all of these vitamin B's. All additional B vitamins added to the control medium are shown in fig. 15A-15K. Comparison of control medium with addition of various vitamin B6 forms is shown in fig. 16A-16E. To assess the quality of the resulting dopaminergic neurons, we examined: a) Tyrosine hydroxylase, an enzyme that mediates the conversion of L-tyrosine to L-3, 4-dihydroxyphenylalanine, is the rate limiting step in dopamine synthesis, for the percentage of GIRK2 positive cells that are also positive for TH (tyrosine hydroxylase); this percentage should be high as an indicator of the yield of the desired cell type (dopaminergic neurons); b) Percentage of TUJ positive neurons that were also TH positive; TUJ is a pleiotropic marker for neurons; only those neurons that are TH positive are true dopaminergic neurons; c) Percentage of TUJ positive cells that were also positive for DAT (dopamine transporter); d) The shape of the cell body should have an elongated triangle characteristic of neurons; and e) the length and number of nerve projections associated with TH and DAT positives. For implantation, the length and number of nerve projections are considered to be the most important factors. Examination of the photographs of figures 12-16 shows that increasing the amount of various forms of vitamin B around day 21 of the differentiation regimen greatly enhances differentiation of dopaminergic neurons based on morphology and percent yield. Recall that in our pair In the group, as shown in fig. 16E, 2 forms of vitamins a and C were added. Referring to fig. 16A-16E, the percentage of cells that were positive for TH and DAT visualized by Hoechst dye and had many interconnected and long nerve projections showed that the addition of pyridoxal-5-phosphate, a biologically active form of vitamin B6, or pyridoxal (a direct precursor to the biologically active form) or a combination of all vitamin B6 forms, starting from about day 21 of the differentiation regimen greatly increased dopamine production and implantation potential by generating many long and interconnected nerve projections. Fig. 17 is a graph of the amount of dopamine and its metabolites (measured by HPLC) from only 200,000 cells present in conditioned medium, which was collected on day 30, day 40, day 50 or day 60. The medium is not withdrawn from a single cell source. Instead, a separate experiment was allowed to proceed until the day the medium was withdrawn for analysis. This experiment used protocol C, where retinol and retinol acetate were added to each condition on and after day 20. The form of vitamin B added to the basal nerve medium varies. In this experiment, the basal medium contained about 10uM pyridoxal. In the case of pyridoxine addition (scheme C.2), pyridoxal was omitted from the basal medium. It is important to note that in fig. 17, "NBM" refers to the nerve basal medium, but vitamin a in the form of retinol and retinol acetate is also added, so the effect of adding more vitamin B in various forms from about day 20 alone can be compared. 200,000 cells/cm measured by HPLC on days 30, 40, 50 and 60 ² The amount of dopamine and its metabolites secreted into the medium (fig. 17). The figure shows that the peak of dopamine secretion is around day 50 of the differentiation regimen. As can be seen in the figure, the highest amounts of dopamine were from retinol, retinol acetate and vitamin C added to the nerve base medium on day 20, alone or with 11uM pyridoxal, or with all three B6 forms, with 11uM pyridoxal, 20uM pyridoxal-5' -phosphate and 16uM pyridoxine added. We note that the amount of dopamine produced and the ability to implant into a living brain region may be two different amounts of dopaminergic neurons that are good for implantationDegree. Although controls (nerve basal medium plus retinol and retinol acetate) produced large amounts of dopamine, they did not produce dopaminergic neurons with many long interconnected projections critical for implantation.

In one aspect of the invention, the addition of pyridoxine or pyridoxine-HCL to the differentiation medium is started on days 16-30 or so and continued until implantation or final testing, which may be on days 40-60. In one aspect of the invention, the addition of pyridoxine or pyridoxine-HCl to the differentiation medium is started on day 20+/-3 or so and continued until implantation or final testing, which may be on day 40-day 60. In one aspect, pyridoxine is added to the differentiation medium at day 20+/-3 to a concentration of 5.0uM to 25.0 uM. In another aspect, it is added to a final concentration of 10.0uM to 30.0 uM. In another aspect, it is added to a final concentration of 10.0uM to 20.0 uM. In yet another aspect, it is added to a final concentration of 15.0 uM. In another aspect of the invention, pyridoxine is present at a concentration of 5.0uM to 15.0uM from the beginning of differentiation. On the other hand, pyridoxine was increased to a final concentration of 10uM-30uM on day 16-30 or so and continued until cell harvest. In another aspect of the invention, pyridoxal is added to the differentiation medium on days 16-30 or so and continued until implantation or final testing, which may be on days 40-60. In another aspect of the invention, pyridoxal is added to the differentiation medium beginning at around day 20+/-3 and continuing until implantation or final testing, which may be at day 40-day 60. In one aspect, pyridoxal is added such that the final concentration in the differentiation medium is 10uM-40uM. In another aspect, it is added to a final concentration of 10uM-30.0 uM. In another aspect, it is added to a final concentration of 15uM-30 uM. In yet another aspect, it is added to a final concentration of 21 uM. In another aspect of the invention pyridoxal is present at a concentration of 5.0uM-15.0uM from the start of differentiation. On the other hand, pyridoxal was increased to a final concentration of 10uM-30uM on day 16-day 30 or so, and continued until cell harvest. In another aspect of the invention, pyridoxal-5' -phosphate, a biologically active form of vitamin B6, is added to the differentiation medium on days 16-30 or so and continued until implantation or final testing, which may be on days 25-60. In another aspect of the invention, pyridoxal-5' -phosphate is added to the differentiation medium beginning at around day 20+/-3 and continuing until implantation or final testing, which may be at day 30-day 60. In one aspect, pyridoxal-5' -phosphate is added to a final concentration of 5.0uM to 50.0 uM. In another aspect, it is added to a final concentration of 10.0uM to 30.0 uM. In yet another aspect, it is added to a final concentration of 20.0 uM. In another aspect of the invention pyridoxal-5' -phosphate is present at a concentration of 5.0 to 15.0uM from the start of differentiation. In another aspect, pyridoxal-5' -phosphate is present at a concentration of 5.0uM to 25.0uM from the start of differentiation. On the other hand, pyridoxal-5' -phosphate was increased to a final concentration of 10uM-30uM on day 16-day 30 or so and continued until cell harvest. On the other hand, pyridoxal-5' -phosphate was increased to a final concentration of 10uM-40uM on day 16-day 30 or so and continued until cell harvest.

In yet another aspect of the invention, these B vitamins are added together to the differentiation medium on days 16-30, more preferably on days 20+/-3 or so, and continued until implantation or final testing, which may be days 40-60, where the total final concentration of B vitamins is 5uM-140uM. In another aspect of the invention, the total final concentration of B vitamins is 15uM-100uM. In another aspect of the invention, the total final concentration of B vitamins is 40uM-70uM. In another aspect of the invention, the total final concentration of B vitamins is 50uM-55uM. In another aspect of the invention, the total final concentration of B vitamins is 10uM to 30uM. In one aspect, pyridoxal is present in the dopaminergic neuron differentiation medium from the beginning at about 10uM, and increases to a final total concentration of 20uM on day 20+/-3, pyridoxine is added together to a final concentration of 15uM on about day 20+/-3, and pyridoxal-5' -phosphate is added to a final concentration of 20uM on about day 20+/-3.

Vitamin A

The number of publications reporting that vitamin a inhibits and vitamin a promotes neural differentiation is substantially equal [2011Gudas and Wagner J Cell Physiol, 2011, month 2; 226:322-330; khillan et al Nutrients 2014doi:10.3390/nu6031209; ole Isacson Molecular and Cellular Neuroscience, volume 45, phase 3, month 11 2010; 258-266]. Retinoic acid binds to specific Retinoic Acid Receptors (RARs) in the nucleus and induces the expression of genes involved in stem cell differentiation and more particularly neural differentiation. Rarα is a retinoic acid receptor that drives the development of dopaminergic neurons. Thus, agonists of rarα (such as BMS 753) may be added to the post-culture medium to replace or supplement various forms of vitamin a.

We found that the form of vitamin a is beneficial for the maturation of dopaminergic neurons, however, the time, concentration and type of vitamin a added to the basal medium are important factors. We modified protocol a, starting with the addition of various forms of vitamin a to basal neural differentiation medium at around day 20+/-3 and continuing until cells were harvested at day 30, 40, 50 or 60. Vitamin a, retinol, retinoic Acid (RA), its active metabolite, 9 cis-RA, all-trans RA (atRA), 13 cis-RA and/or retinol acetate are added to the basal neural differentiation medium. We have found that the addition of vitamin A and/or its active metabolites greatly improves the production of dopaminergic neurons from stem cells in terms of phenotype, expression of appropriate molecular markers, implantation, and dopamine production.

In this set of experiments, naive human iPS cells were used. These pluripotent stem cells were cultured in minimal medium with NME7-AB as the sole added growth factor (Carter et al 2016). The control was protocol A (FIGS. 18A-18I), and to see only the effect of the added vitamin A form, we used a modified protocol A, called protocol B, in which about 10uM pyridoxal was present in the basal medium from the beginning of differentiation, but an additional 10-11uM of pyridoxal or pyridoxal-HCL was added starting on day 2+/-3 until cell harvest (FIGS. 19A-19I). Vitamin a in the form of retinol at a final concentration of 0.7uM and vitamin a in the form of retinol acetate at a final concentration of 0.6uM were added to the differentiated stem cells of protocol B on day 20+/-3 or so (fig. 20A-20I). In another set of experiments, the added vitamin A forms on day 20 were 9-cis, 13-cis and all-trans retinoic acid, with final concentrations of 0.446uM each (FIGS. 21A-21I). In another set of experiments, only all-trans retinoic acid was added to a final concentration of 1.33uM (fig. 22A-22I). Fig. 23A-23E show a comparison between two controls, regimen a without addition of vitamin B6 or vitamin a around day 20 (fig. 23A), regimen B with addition of another 11uM to increase pyridoxal form of vitamin B6 around day 20 (fig. 23E), and with addition of vitamin a in the form of retinol and retinol acetate (fig. 23B), or with addition of vitamin a in the form of 9-cis, 13-cis and all-trans retinoic acid (fig. 23C), or with addition of vitamin a in the form of all-trans retinoic acid (fig. 23D). As can be seen in the figures, the addition of retinol and retinol acetate (fig. 23B) or 9-cis, 13-cis and all-trans retinoic acid (fig. 23C) both increased the number, length and interconnectivity of nerve projections compared to the control (fig. 23A and 23E). However, the morphology of the nerve cell bodies stained with GIRK2, a higher percentage of TUJ positive cells that bound also to TH positive and DAT positive, indicates that the addition of retinol and retinol acetate would result in higher engraftment rates.

In one aspect of the invention, retinol acetate and/or retinoic acid is added to the differentiation medium beginning at about day 16-day 30 and continuing until implantation or final testing, which may be at day 40-day 60. In another aspect of the invention, they are added to the differentiation medium beginning at around day 20+/-3 and continuing until implantation or final testing, which may be at day 40-day 60. In one aspect, vitamin A and/or derivatives thereof are added to the basal medium to a final combined concentration of 0.5uM to 5.0 uM. In another aspect, vitamin A and/or derivatives thereof are added to the basal medium to a final combined concentration of 1.0uM to 3.0 uM. In another aspect, retinol is added to the basal medium at a final concentration of 0.5uM to 5.0 uM. In yet another aspect, retinol is added to the basal medium at a final concentration of 1.0uM to 2.0uM, and retinol acetate is also added at a final concentration of 0.1uM to 1.0 uM. In yet another aspect, retinol is added to the basal medium at a final concentration of 1.0uM to 3.0uM, and retinol acetate is also added at a final concentration of 0.1uM to 1.2 uM. The basal medium to which vitamin a and/or its derivatives are added may be a neural differentiation basal medium, including but not limited to neural basal medium (thermo fisher) and NeuroCult (StemCell Technologies).

Because vitamin a is fat-soluble, lipids or albumin may optionally be added to the basal medium upon addition of vitamin a or its derivatives. The basal neural medium we used contained some BSA, however for human use we sought a non-bovine alternative to BSA. In addition, it is expected that additional vitamin a requires additional lipids to aid in dissolution. In this set of experiments, vitamin a was first dissolved in Albumax and then added to the differentiation medium as described in protocol B, starting around day 20 +/-3. Recall that regimen B included the addition of additional 11uM pyridoxal beginning at around day 20 +/-3. FIGS. 24A-24I show confocal microscopy images of cells obtained on day 24, where vitamin A in the form of retinol (1.2 uM) and retinol acetate (0.17 uM) dissolved in 2mg/mL Albumax was added to the medium beginning around day 20. Figures 25A-25I show confocal microscopy images of the resulting cells, wherein vitamin C in the form of 2-phospho-ascorbic acid at a final concentration of 61uM and in the form of L-ascorbic acid at a final concentration of 110uM was added to the differentiation medium around day 20 in addition to the retinol and retinol acetate mentioned above. In another set of experiments, vitamin a in the form of all-trans retinoic acid was added to the medium of protocol B around day 20 to a final concentration of 1.33uM and dissolved in Albumax (fig. 26A-26I). FIGS. 27A-27I show confocal microscopy images of the resulting cells, in which vitamin C in the form of 2-phospho-ascorbic acid at a final concentration of 61uM and in the form of L-ascorbic acid at a final concentration of 110uM was added to the differentiation medium on day 20 or so in addition to the all-trans retinoic acid mentioned above.

In one aspect of the invention, vitamin C is added to the differentiation medium on day 16-day 30 or so of differentiation. On the other hand, vitamin C was added to the differentiation medium around day 20. In one aspect, vitamin C is added to a final concentration of 200nM-110 uM. In another aspect, vitamin C is added to a final concentration of 1uM-100 uM. In yet another aspect, vitamin C is added to a final concentration of 50uM-75 uM. In one aspect, the vitamin C is in the form of 2-phospho-ascorbic acid. In another aspect, the vitamin C is in the form of L-ascorbic acid. In yet another aspect, two forms of vitamin C are added. In another aspect of the invention, vitamin C is present in the differentiation medium at a final concentration of 100nM to 500nM from the beginning of differentiation. In another aspect of the invention, vitamin C is present at a concentration of 100nM to 500nM from the beginning of differentiation and increases to 50uM to 70uM on day 16 to about day 30 or from about day 20.

Fig. 5 shows a photograph of a wound healing assay (also known as a scratch test), which is considered an in vitro replacement for in vivo implantation. Stem cells differentiated into dopaminergic neurons according to scheme c.2. In one case, the starting stem cells are in an initial state and are grown in NME7-AB initial medium without any FGF2 or other growth factors (FIGS. 5A-5C). In another case, the starting stem cells are in an original state and are grown in E8 medium containing FGF2 (FIGS. 5D-5F). Six (6) days after scratch was made, the number of nerve projections and the length of the nerve projections of the resulting cells were analyzed on day 21. On day 21, the projections of neuronal production derived from naive stem cells were 10-12 times that of naive stem cells, which was a 1000% to 1200% increase. Those projections are 5-7 times longer than those generated in stem cells in the original state, which is a 500% to 700% increase. Thus, the simulated implantation increased from 500% to 1200% due to the change to naive stem cells alone.

Improvements over the prior art are also measured in terms of yield and purity of the resulting population. Recall that the conventionally known methods for obtaining stem cell derived dopaminergic neurons for the treatment of parkinson's disease require sorting of the cells on day 14 to obtain a semi-pure population. Such conventional methods indicate that the purified population implanted into the rat brain is 10 times better than the impure population. Fig. 4 compares the yield and purity of primary stem cells differentiated into dopaminergic neurons using protocol a relative to protocol c.2. The percentage of cells in the population that were positive for the four (4) key markers GIRK2, TH, DAT and Tuj1 and displayed neurological morphology determined the percentage of purity of the population. Hoechst dye stains the nuclei of all cells and Tuj1 is a general stain for many types of neurons, but only those neurons positive for Tuj1, GIRK2 (A9 neuron's marker), TH (catalytic reaction to produce dopamine) and DAT (dopamine transporter) are actually dopaminergic neurons. Of the cells differentiated according to protocol a, only about 5% were positive for both GIRK2 and TH, as the red plus green overlay was yellow (fig. 4B). In contrast, cells differentiated according to protocol c.2 had neurological morphology, and 80% -90% of Tuj1 positive cells were DAT positive as well (fig. 4E), and about 70% were both GIRK2 and TH positive (fig. 4F). Scheme c.2 induced a more than 10-fold (1000%) increase in the yield and purity of dopaminergic neurons.

In another experiment, the primary stem cells were differentiated according to protocol a (fig. 18A-18I). Here, the percentage of cells positive for GIRK2, TH, DAT and Tuj1 and having neuromorphic morphology was less than 35%. FIGS. 24A-24I show the same starting cells differentiated according to protocol C, with additional pyridoxal plus retinol and retinol acetate added on day 21. The percentage of cells positive for GIRK2, TH, DAT and Tuj1 was 90% -100%. Fig. 25 shows a photograph of the same starting cells differentiated according to scheme D, which differs from the scheme shown in fig. 24 in that vitamin C in the form of 2-phosphoascorbic acid and L-ascorbic acid is also added from day 21. As can be seen in fig. 25A-25I, nearly 100% of the cells had neuromorphic and were positive for GIRK2, TH, DAT and Tuj 1. Thus, the percentage improvement between scheme a and the scheme described in the description of fig. 24 is 250%. The percentage improvement between scheme a and scheme D (fig. 25) was 290%.

Another feature of dopaminergic neurons that is critical to their use as therapeutic agents for the treatment of parkinson's disease is their ability to secrete dopamine. A direct comparison of the amounts of dopamine and its metabolites produced was quantified between: 1) Primary stem cells versus stem cells in an originating state; and 2) scheme A (Prior Art) and scheme of the invention (scheme C.2) wherein at about day 24 and after scheme A, pyridoxal in the basal medium is exchanged for pyridoxine and vitamin A in the form of retinol and retinol acetate is added. Figures 6 and 7 show graphs of the amounts of dopamine and its metabolites secreted into the conditioned medium at a specific day after the initiation of differentiation as measured by HPLC (Vanderbilt University). Protocol A was used first and starting from stem cells in the original state at 400,000 cells/cm ² 1.34ng/mL of dopamine and its metabolites were measured on day 40 and 13.4ng/mL on day 60 (FIG. 6A). Using protocol A with naive stem cells, 1.3ng/mL was measured on day 40 and 5.85ng/mL was measured on day 60 (FIG. 6B). In contrast, using protocol C.2 with stem cells in the original state, 33.4ng/mL of dopamine and its metabolites were measured on day 40, and 15.6ng/mL was measured on day 60 (FIG. 6C). Using protocol C.2 with naive stem cells, 43.0ng/mL of dopamine and its metabolites were measured on day 40 and 54.1ng/mL on day 60 (FIG. 6D). The secretion of dopamine and its metabolites was 25-fold or 2500% increased over protocol a on day 40 using protocol c.2, when using the naive stem cells, and 33-fold or 3300% when using the naive stem cells. In fig. 7, the amount of dopamine secreted by a variable number of cells on day 60 or day 40 (where indicated) is plotted. Using naive stem cells, at 800,000 cells/cm ² On day 60, cells differentiated according to scheme c.2 produced about 10 times more dopamine (54 ng/mL versus 5.8 ng/mL) than the same cells differentiated according to scheme a. When only half the number of cells were present at 400,000 cells/cm ² When plated at a density of 2.0-2.6 times greater than the dopamine produced by the same cells differentiated using scheme c.2. Comparing the amount of dopamine produced by the naive stem cells according to scheme c.2 (54 ng/mL from 800K cells on day 60) relative to the compositions and methods of the present invention for the naive cells according to the current prior art and scheme a (3 ng/mL from 800K cells on day 60), the naive stem cells and scheme c.2 produced 18 times (1800%) the prior art.

In one aspect of the invention, variant B is modified such that, starting at around day 20+/-3 of the differentiation protocol, the differentiation medium is supplemented with retinol (which is added to a final concentration of 0.5uM to 2.0 uM) and retinol acetate (which is added to a final concentration of 0.1 to 1.5 uM). In another aspect of the invention, retinol is added to a final concentration of 0.7-1.2uM, and retinol acetate is added to a final concentration of 0.17-0.6 uM. In yet another aspect of the invention, a combination of retinol and retinol acetate is added such that the final concentration of the combination is 1.0uM to 2.5uM. In yet another aspect of the invention, the combined final concentration is 1.33uM.

In one aspect, bovine serum albumin is added. In another aspect, human serum albumin is added. In yet another aspect, lipid-rich human serum albumin is added. In yet another aspect, albumax (a lipid-rich bovine albumin) or similar lipid-rich human albumin is added. The lipid-rich albumin may be added to a final molar concentration of 10.0uM-40.0 uM. The final molar concentration may be 10.0uM to 15.0uM. In one aspect, vitamin a and/or its derivatives are dissolved in an alcohol/water mixture and evaporated under vacuum to form a film. The films were then mixed with BSA or HSA solutions at 37 ℃ for 30 minutes to dissolve the lipids.

Vitamin C is expressed at high levels in the fetal brain at the later stages of neural development. Vitamin C is reported to be involved in upregulation of Nurr1, nurr1 being critical for midbrain neural differentiation and possibly a key factor in dopaminergic neuron maturation. In one aspect of the invention, the tri-sodium salt of vitamin C2-phosphate-L-ascorbate is added to the differentiation medium on days 16-30 or so and is continued until implantation or final testing, which may be on days 40-60. In another aspect of the invention, the trisodium salt of vitamin C2-phosphate-L-ascorbate is added to the differentiation medium at day 20+/-3 and persists until implantation or final testing, which may be at day 40-day 60. In one aspect, vitamin C2-phosphate-L-ascorbate trisodium salt is added to the differentiation medium to a final concentration of 40.0uM to 100.0 uM. In another aspect, it is added to a final concentration of 50.0uM-70.0 uM. In yet another aspect, it is added to a final concentration of 60.0uM-65.0 uM. In another aspect of the invention, vitamin C ascorbic acid is added to the differentiation medium on days 16-21 or so and continued until implantation or final testing, which may be on days 40-60. In yet another aspect of the invention, vitamin C ascorbic acid is added to the differentiation medium at day 20+/-3 and persisted until implantation or final testing, which may be at day 40-day 60. In one aspect, ascorbic acid is added to the differentiation medium to a concentration of 5.0uM to 20.0 uM. In another aspect, it is added to a final concentration of 10.0uM-15.0uM. In yet another aspect, it is added to a final concentration of 12.0uM-14.0 uM.

In one aspect of the invention, vitamin C in the form of 2-phospho-ascorbic acid is added to the differentiation medium around day 20 to a final concentration of 25uM-100 uM. In another aspect of the invention, it is added to a final concentration of 40-75 uM. In a preferred embodiment, it is added to a final concentration of 61 uM. In one aspect of the invention, vitamin C in the form of L-ascorbic acid is added to the differentiation medium around day 20 to a final concentration of 1uM-120 uM. In another aspect of the invention, it is added to a final concentration of 5-100 uM. In a preferred embodiment, it is added to a final concentration of 11 uM. In a preferred embodiment, one or more forms of vitamin C are added to the medium of protocol B at about day 20+/-3 to a final concentration of 50-75 uM. In a more preferred embodiment, the two forms of vitamin C are ascorbic acid 2-phosphate and L-ascorbic acid.

In one aspect of the invention, the above mentioned vitamins are added to the basal neural medium at a given concentration together with the lipid-rich albumin and the stem cells differentiate into dopaminergic neurons and are cultured in this medium on days 16-30 or so, in particular on day 20+/-3, until terminal differentiation or implantation, which may be between day 30 and day 60 after the start of differentiation.

In addition to we found key vitamins, their metabolites and lipid-rich albumin (which when added to differentiated stem cells increases and enhances differentiation to dopaminergic neurons), we found that use of naive stem cells further increased and enhanced differentiation to dopaminergic neurons.

In a preferred embodiment, the stem cells differentiate into dopaminergic neurons according to scheme C.

In a more preferred embodiment, the stem cells differentiate into dopaminergic neurons according to scheme D, wherein the stem cells (preferably NME 7-AB-grown naive stem cells) are located in a neural basal medium, which is supplemented by the addition of 11uM pyridoxal or 20uM pyridoxal-5' -phosphate, 1.2uM retinol and 0.17uM retinol acetate (dissolved in a lipid rich formulation) and 61uM 2-phosphoascorbic acid and 11uM L-ascorbic acid on day 20+/-3 or so.

In yet another aspect of the invention, the protocol of the 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 comprising NME 7-AB.

In yet another aspect of the present invention, the 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 culture medium containing WNT 3A.

The improvement over the prior art described herein is the addition of specific vitamins and other factors in various forms at specific concentrations to the basal neural differentiation medium. In one aspect of the invention, the addition or increase in concentration of vitamin a, vitamin B and/or vitamin C begins at the beginning 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 continued until cell harvest. In yet another aspect of the invention, they are added about 18-23 days after initiation of differentiation and continued until cell harvest. In one effective embodiment, the candidate factor is added on day 20 or 21. We have found that these vitamins A, B and C are dopaminergic maturation factors and can be added to several different basal neural differentiation media including, but not limited to, neural basal media (ThermoFisher), neuroCult (StemCell Technologies) or other neural differentiation basal media.

In some examples shown herein, the basis of protocol A (FIG. 1, example 1) was employed until around day 20+/-3, at which time specific vitamins were added to the basal neural differentiation medium. The addition of these factors on day 20+/-3 or so greatly increases the yield and function of stem cell-derived dopaminergic neurons, including increased implantation and dopamine secretion, while allowing fully functional dopaminergic neurons to mature in vitro.

Methods of treating neurodegenerative diseases, conditions or injuries

Dopaminergic neurons differentiated in vitro may be used to treat neurodegenerative diseases. The compositions and methods described herein are applicable to the generation of other types of neurons from stem cells. The differentiated dopaminergic neurons can be used to treat any condition that benefits from successful implantation of dopaminergic neurons in the central nervous system, such as neurodegenerative diseases. Other types of neurons may be produced from stem cells using the methods of the invention for treating other conditions, such as those caused by injury to the spinal cord, for example. Neurons, such as sensory neurons, motor neurons, or interneurons, may be generated from stem cells according to the methods of the invention. These neurons may also be used to treat peripheral nerve injuries, which may include complete or partial transection of a nerve by stretching, cutting (tearing), compression, shearing, or squeezing. The presently disclosed subject matter provides a method of treating a neurodegenerative disease comprising administering an effective amount of the presently disclosed differentiated dopaminergic neurons to a subject suffering from a neurodegenerative disease.

Non-limiting examples of neurodegenerative diseases include parkinson's disease, huntington's disease, alzheimer's disease, and multiple sclerosis. Other neurotrophic B vitamins can be added to the regimens described herein. For example, when neurons are generated for the treatment of multiple sclerosis, vitamin B12 that contributes to myelin production may be added to the differentiation medium.

In particular, the neurodegenerative disease is parkinson's disease. Major motor signs of parkinson's disease include, but are not limited to, hand, arm, leg, mandibular and facial tremor, bradykinesia or slow motion, stiffness or rigidity of the extremities and torso, and unstable posture or impaired balance and coordination.

In certain embodiments, the neurodegenerative disease is parkinson's disease, which refers to a disease associated with insufficient dopamine in the basal ganglia, which is part of the brain that controls movement. Symptoms include tremor, bradykinesia (extremely slow movement), flexed posture, unstable posture and stiffness. Non-limiting examples of parkinson's disease include corticobasal degeneration, dementia with lewy bodies, multiple system atrophy and progressive supranuclear palsy.

The differentiated dopaminergic neurons of the present disclosure may be administered or provided to a subject either systemically or directly for use in the treatment or prevention of neurodegenerative diseases. In certain embodiments, the differentiated dopaminergic neurons of the present disclosure are injected directly into an organ of interest, e.g., the Central Nervous System (CNS) or the Peripheral Nervous System (PNS). In certain embodiments, the differentiated dopaminergic neurons of the present disclosure are injected directly into the striatum.

The differentiated dopaminergic neurons of the present disclosure may be administered in any physiologically acceptable vehicle. Also provided are pharmaceutical compositions comprising the differentiated dopaminergic neurons of the present disclosure and a pharmaceutically acceptable vehicle. The differentiated dopaminergic neurons and the pharmaceutical compositions comprising said cells disclosed herein may be administered by local injection, in situ (OT) injection, systemic injection, intravenous injection or parenteral administration. In certain embodiments, the differentiated dopaminergic neurons of the present disclosure are administered to a subject suffering from a neurodegenerative disease by in situ (OT) injection.

The differentiated dopaminergic neurons and pharmaceutical compositions comprising said cells of the present disclosure may conveniently be provided as a sterile liquid formulation, e.g. an isotonic aqueous solution, suspension, emulsion, dispersion or viscous composition, which may be buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. In addition, the liquid composition is somewhat more convenient to administer, particularly by injection. In another aspect, the adhesive composition may be formulated within an appropriate viscosity range to provide longer contact times with specific tissues. The liquid or viscous composition may comprise 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.), and suitable mixtures thereof. Sterile injectable solutions can be prepared by incorporating the compositions of the presently disclosed subject matter (e.g., compositions comprising the presently disclosed differentiated dopaminergic neurons) in the required amounts of the other ingredients, as appropriate. Such compositions may be admixed with suitable carriers, diluents or excipients such as sterile water, physiological saline, dextrose and the like. The composition may also be lyophilized. The compositions may contain auxiliary substances such as wetting, dispersing or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity-enhancing additives, preservatives, flavoring agents, coloring agents and the like, depending on the route of administration and the desired formulation. Reference may be made to standard text, such as "REMINGTON' SPHARMACEUTICAL SCIENCE", 17 th edition, 1985, incorporated herein by reference, for the preparation of suitable formulations without undue experimentation.

Various additives may be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffering agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, and the like). Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents which delay absorption (e.g., aluminum monostearate and gelatin). However, any vehicle, diluent or additive used in accordance with the presently disclosed subject matter must be compatible with the presently disclosed differentiated dopaminergic neurons.

If desired, the viscosity of the composition may be maintained at a selected level using a pharmaceutically acceptable thickener. Methylcellulose may be used because it is readily and economically available and easy to use. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomers, and the like. The concentration of the thickener depends on the agent selected. It is important to use an amount that will achieve the selected viscosity. The choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., 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).

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 efficacy of the differentiated dopaminergic neurons of the present disclosure. This does not present any problem to the skilled person of chemical and pharmaceutical principles or can be easily avoided by reference to standard texts or by simple experiments (without undue experimentation) in light of the present disclosure and the literature cited herein.

In certain non-limiting embodiments, the cells and precursors described herein are included in a composition that further includes a biocompatible scaffold or matrix, e.g., a biocompatible three-dimensional scaffold that promotes tissue regeneration when the cells are implanted or transplanted into a subject. In certain non-limiting embodiments, the biocompatible scaffold comprises an extracellular matrix material, a synthetic polymer, a cytokine, collagen, a polypeptide or protein, a polysaccharide, including fibronectin, laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparin sulfate, chondroitin sulfate, agarose or gelatin, and/or a hydrogel. (see, e.g., U.S. publication nos. 2015/0159135, 2011/0296542, 2009/01234633, and 2008/0268019, each of which is incorporated by reference in its entirety). In certain embodiments, the composition further comprises a growth factor for promoting maturation of the implanted/transplanted cells into midbrain DA cells.

One consideration with respect to the therapeutic use of the differentiated dopaminergic neurons of the present disclosure is the number of cells required to achieve optimal effect. Optimal effects include, but are not limited to, refilling of CNS and/or PNS regions of a subject suffering from a neurodegenerative disease, and/or improvement of CNS and/or PNS function in a subject.

In certain embodiments, an effective amount of the presently disclosed differentiated dopaminergic neurons is an amount sufficient to repopulate CNS and/or PNS regions of a subject suffering from a neurodegenerative disease. In certain embodiments, an effective amount of the differentiated dopaminergic neurons of the present disclosure is an amount sufficient to improve the function of the CNS and/or PNS of a subject suffering from a neurodegenerative disease, e.g., 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 of a normal human.

The number of cells to be administered will vary depending on the subject being treated. In certain embodiments, about 1x10 ⁴ Up to about 1x10 ¹⁰ About 1x10 ⁴ Up to about 1x10 ⁵ About 1x10 ⁵ Up to about 1x10 ⁹ About 1x10 ⁵ Up to about 1x10 ⁶ About 1x10 ⁵ Up to about 1x10 ⁷ About 1x 106 to about 1x10 ⁷ About 1x10 ⁶ Up to about 1x10 ⁸ About 1x10 ⁷ Up to about 1x10 ⁸ About 1x10 ⁸ Up to about 1x10 ⁹ About 1x10 ⁸ Up to about 1x10 ¹⁰ Or about 1x10 ⁹ Up to about 1x10 ¹⁰ The differentiated dopaminergic neurons of the present disclosure are administered to a subject. In certain embodiments, about 1x10 ⁵ Up to about 1x10 ⁷ The differentiated dopaminergic neurons disclosed herein are administered to a subject suffering from a neurodegenerative disease. In certain embodiments, about 1x10 ⁶ Up to about 1x10 ⁷ The differentiated dopaminergic neurons disclosed herein are administered to a subject suffering from a neurodegenerative disease. The manner in which the precise determination of how much is considered to be an effective dose may be based on individual factors of each subject, including their body shape, age, sex, weight and condition of the particular subject. Dosages can be readily determined by one of ordinary skill in the art in light of the present disclosure and knowledge in the art.

Examples

Example 1-scheme A

In protocol A, cells were plated onto Geltrex coated plates in nerve basal medium (Thermo Fisher # 21103049), B-27 without vitamin A (Thermo Fisher # 12587010), N2 supplement (Stem Cell Technologies # 07156), 2mM Glutamax (Thermo Fisher # 35050061), 250nM LDN193189 (Selleck Chemicals # S7507), 10.8. Mu.M SB431542 (Selleck Chemicals # S1067), 500ng/ml SHH (R & D Sytsems # 464-SH-200), 0.7. Mu.M CHIR99021 (R & D Systems # 4423), 10. Mu. M Y27632 (Selleck Chemicals #S1049). On days 1 and 3, the medium was replaced with fresh neural basal medium containing B-27 without vitamin A, N2 supplement, 2mM Glutamax, 250nM LDN193189, 10.8. Mu.M SB431542, 500ng/ml SHH, 0.7. Mu.M CHIR 99021. On days 4 and 6, the medium was replaced with fresh neural basal medium containing B-27 without vitamin A, N2 supplement, 2mM Glutamax, 250nM LDN193189, 10.8. Mu.M SB431542, 500ng/ml SHH, 7.5. Mu.M CHIR 99021. On days 7 and 9, the medium was replaced with fresh neural basal medium containing B-27 without vitamin A, N2 supplement, 2mM Glutamax, 7.5. Mu.M CHIR 99021. On day 10, the medium was replaced with fresh nerve basal medium containing B-27 without vitamin A, 2mM Glutamax, 3. Mu.M CHIR99021, 20ng/mL BDNF (Peprotech#450-02), 200nM ascorbic acid (Sigma Aldrich#A 4403), 20ng/mL GDNF (Peprotech#450-10), 1ng/mL TGF beta 3 (Peprotech#100-36E), 500nM cAMP (Peprotech# 1698950). On day 11, cells were re-plated into day 10 medium with 10 μ M 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). On days 12 to 60, the medium was changed daily to a neural basal medium containing B-27 without vitamin A, 2mM Glutamax, 20ng/mL BDNF, 200nM ascorbic acid, 20ng/mL GDNF, 1ng/mL TGFβ3, 500nM cAMP, 10. Mu.M DAPT (Selleck Chem #S2215).

The nerve basal medium (Thermo Fisher # 21103049) is indicated to contain: amino acids of 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; 0.028571420mM choline chloride, 0.008385744mM calcium D-pantothenate, 0.009070295mM folic acid, 0.032786883mM nicotinamide, 0.019607844mM pyridoxal hydrochloride, 0.0010638298mM riboflavin, 0.011869436mM thiamine hydrochloride, 5.0184503E-6mM vitamin B12, 0.04mM i-inositol; inorganic salts of calcium chloride (CaCl 2) (anhydrous), ferric nitrate (Fe (NO 3) 3' 9H 2O), magnesium chloride (anhydrous), potassium chloride (KCl), sodium bicarbonate (NaHCO 3), sodium chloride (NaCl), sodium dihydrogen phosphate (NaH 2PO 4-H2O), zinc sulfate (ZnSO 4-7H 2O); d-glucose (dextrose), HEPES, phenol red, other components of sodium pyruvate.

EXAMPLE 2 investigation of the Effect of adding vitamin B6 form to regimen A

In this set of experiments we used basal neural medium, which contained 10uM pyridoxal plus 1.2uM retinol and 0.17uM retinol acetate, beginning at about day 20 and after. Increased levels of various B vitamins were added near the time (i.e., around day 20) at which the investigator found that implantation into the host brain increased implantation rates. On day 20+/-3, we add: pyridoxine to a final concentration of between 5 and 25 uM; pyridoxal to a final concentration of between 5 and 20 uM; pyridoxal-5' -phosphate (in biologically active form) to a final concentration of between 10 and 40; or all three B vitamins in combination. It was empirically determined that the optimal concentration of pyridoxine was about 10-20uM. Figures 12A-12K show the effect of pyridoxine added to a final concentration of 16 uM. The optimal concentration of pyridoxal is empirically determined to be about 5-20uM. Figures 13A-13K show the effect of pyridoxal added to a final concentration of 11 uM. The optimal concentration of pyridoxal-5' -phosphate was empirically determined to be about 10-40uM. FIGS. 14A-14K show the effect of pyridoxal-5' -phosphate added to a final concentration of 20uM. Figures 15A-15K show the effect of all three vitamins B added together.

EXAMPLE 3 scheme B

Based on the results of example 2, we limited the type and concentration of vitamin B6 added around day 20 so that we could next study the type and concentration of vitamin a, which may or may not improve the purity/yield, implantation or amount of dopamine secreted by the dopaminergic neurons derived from the stem cells.

In protocol B, cells were plated into neural basal medium, B-27 without vitamin A, N2 supplement, 2mM Glutamax, 250nM LDN193189, 10.8. Mu.M SB431542, 500ng/ml SHH, 0.7. Mu.M CHIR99021, 10. Mu. M Y27632 on Geltrex-coated plates. On days 1 and 3, the medium was replaced with fresh neural basal medium containing B-27 without vitamin A, N2 supplement, 2mM Glutamax, 250nM LDN193189, 10.8. Mu.M SB431542, 500ng/ml SHH, 0.7. Mu.M CHIR 99021. On days 4 and 6, the medium was replaced with fresh neural basal medium containing B-27 without vitamin A, N2 supplement, 2mM Glutamax, 250nM LDN193189, 10.8. Mu.M SB431542, 500ng/ml SHH, 7.5. Mu.M CHIR 99021. On days 7 and 9, the medium was replaced with fresh neural basal medium containing B-27 without vitamin A, N2 supplement, 2mM Glutamax, 7.5. Mu.M CHIR 99021. On day 10, the medium was replaced with fresh nerve basal medium containing B-27 without vitamin A, 2mM Glutamax, 3. Mu.M CHIR99021, 20ng/mL BDNF (Peprotech#450-02), 200nM ascorbic acid (Sigma Aldrich#A 4403), 20ng/mL GDNF (Peprotech#450-10), 1ng/mL TGF beta 3 (Peprotech#100-36E), 500nM cAMP (Peprotech# 1698950). On day 11, cells were re-plated into day 10 medium with 10 μ M Y27632 on plates coated with 15 μg poly-L-ornithine (Sigma Aldrich#P4957)/10 μg laminin (Sigma Aldrich#L2020)/1 μg fibronectin (Thermo Fisher#33016-015). On days 12 to 20, the medium was changed daily to a neural basal medium containing B-27 without vitamin A, 2mM Glutamax, 20ng/mL BDNF, 200nM ascorbic acid, 20ng/mL GDNF, 1ng/mL TGFβ3, 500nM cAMP, 10. Mu.M DAPT (Selleck Chem #S2215). On days 21 to 60 or until cell harvest, the medium was changed daily to a neural basal medium containing B-27 without vitamin A, 2mM Glutamax, 20ng/mL BDNF, 200nM ascorbic acid, 20ng/mL GDNF, 1ng/mL TGFβ3, 500nM cAMP, 10. Mu.M DAPT plus 11. Mu.M pyridoxal (Sigma Aldrich P1930).

EXAMPLE 4 investigation of the Effect of adding various forms of vitamin A to regimen B

On day 20 or thereabout of differentiation according to protocol B, various forms of vitamin a were added over a range of concentrations. Retinol was added from day 20 at a concentration ranging from 0.1 to 1.5uM. Retinol acetate was added at a concentration ranging from 0.1 to 1.5uM from day 20. Retinoic acid in the 9-cis, 13-cis and/or all-trans forms is added such that the final concentration (whether added separately or together) is about 1.5-2.0uM. The results are shown in fig. 18-23. Empirically determined, the optimal condition for dopaminergic neuron differentiation is the addition of both retinol and retinol acetate to a final combined concentration of about 2uM on and after day 20.

EXAMPLE 5 investigation of the Effect of adding various forms of vitamin A dissolved in a lipid-rich formulation to regimen B with or without vitamin C

Vitamin a is known to be quite insoluble. Thus, we tested the addition of various forms of vitamin a after dissolution in lipid-rich formulations. We tested the dissolution of vitamin A in 2mg/mL BSA or Albumax (which may be substituted with human serum albumin). In addition to the addition of various forms of dissolved vitamin a, we tested even more addition of vitamin C in the form of 2-phospho-ascorbic acid or L-ascorbic acid. The results of these studies are shown in fig. 24-27.

EXAMPLE 6 scheme C

In protocol C, protocol A was followed up to day 20+/-3 except that on day 11, the surface to which differentiated cells were re-plated contained 10ug/mL instead of 1ug/mL laminin. According to scheme C, on day 20 or so, medium was supplemented: vitamin B6 in which 16uM is in the form of pyridoxine, 11uM is in the form of pyridoxal, 20uM is in the form of pyridoxal-5' -phosphate, or all together; and 0.7 to 1.2uM in the form of retinol and 0.17 to 0.6uM in the form of retinol acetate, or 0.446uM each in the form of 9-cis retinoic acid, 13-cis retinoic acid and all-trans retinoic acid or 1.33uM in the form of all-trans retinoic acid; and 61uM in the form of 2-phospho-ascorbic acid and 110uM in the form of L-ascorbic acid.

EXAMPLE 7 scheme C.2

In protocol C.2, protocol A was followed up to day 20+/-3 except that on day 11, the surface to which differentiated cells were re-plated contained 10ug/mL instead of 1ug/mL laminin. According to scheme c.2, on day or so, the neural basal medium was exchanged for a neural basal medium that did not contain pyridoxal but contained 16uM vitamin B6 in the form of pyridoxine and 1.2uM vitamin a in the form of retinol and 0.17uM vitamin a in the form of retinol acetate.

Example 8-scheme D

In protocol D, protocol A was followed up to day 20+/-3 except that on day 11, the surface to which differentiated cells were re-plated contained 10ug/mL instead of 1ug/mL laminin. According to protocol D, medium was supplemented around and after day 20: 11uM pyridoxal, 1.2uM retinol and 0.17uM retinol acetate, and 61uM vitamin C in the form of 2-phospho-ascorbic acid and 11uM vitamin C in the form of L-ascorbic acid. See fig. 3-10 for quantification of stem cells differentiated into dopaminergic neurons according to protocol c.2.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described specifically herein. Such equivalents are intended to be encompassed by the scope of the claims.

Claims

1. A method of producing dopaminergic neurons from human stem cells, comprising the step of adding vitamins to or increasing the concentration of vitamins for a neural basal medium on about day 20+/-3 of a regimen for differentiating pluripotent stem cells into dopaminergic neurons.

2. The method of claim 1, wherein the scheme is scheme a.

3. The method of claim 1, wherein the vitamin is vitamin a.

4. The method of claim 3, wherein the vitamin a is in the form of retinol.

5. The method of claim 3, wherein the vitamin a is in the form of retinol acetate.

6. The method of claim 3, wherein the vitamin a is in the form of 9-cis retinoic acid, 13-cis retinoic acid, or all-trans retinoic acid.

7. The method of claim 3, wherein the vitamin a is dissolved in a lipid-rich formulation.

8. The method of claim 7, wherein the lipid-enriched formulation is human serum albumin.

9. The method of claim 7, wherein the lipid-enriched formulation is Albumax.

10. The method of claim 7, wherein the lipid-enriched formulation is non-human serum albumin.

11. The method of claim 3, wherein the final concentration of vitamin a is 1uM to 3uM.

12. The method of claim 1, wherein the vitamin is vitamin B6.

13. The method of claim 12, wherein the vitamin B6 is in the form of pyridoxine.

14. The method of claim 12, wherein the vitamin B6 is in the form of pyridoxal.

15. The method of claim 12, wherein the vitamin B6 is in the form of pyridoxal-5' -phosphate, also known as PLP.

16. The method of claim 12, wherein the final concentration of vitamin B6 is 10uM to 30uM.

17. The method of claim 1, wherein the vitamin is vitamin C.

18. The method of claim 17, wherein the vitamin C is in the form of 2-phosphoric acid-ascorbic acid.

19. The method of claim 17, wherein the vitamin C is in the form of L-ascorbic acid.

20. The method of claim 17, wherein the final concentration of vitamin C is 200nM to 110uM.

21. The method according to claim 1, wherein the pluripotent stem cells to be differentiated have been cultured in NME 7-AB.

22. The method of claim 1, wherein the pluripotent stem cells to be differentiated have been cultured in WNT 3A.

23. The method of claim 1, wherein the pluripotent stem cells to be differentiated are in an initial state.

24. The method of claim 1, wherein the dopaminergic neurons produced are characterized by greater than 30% more dopamine than the dopaminergic neurons produced by a differentiation regimen without the addition or augmentation of vitamins.

25. The method of claim 24, wherein the dopaminergic neurons produced are characterized by greater than 100% more dopamine than dopaminergic neurons produced by a differentiation regimen without the addition or augmentation of vitamins.

26. The method of claim 25, wherein the dopaminergic neurons produced are characterized by greater than 500% more dopamine than dopaminergic neurons produced by a differentiation regimen without the addition or augmentation of vitamins.

27. The method of claim 26, wherein the dopaminergic neurons produced are characterized by greater than 1000% more dopamine than the dopaminergic neurons produced by a differentiation regimen without the addition or augmentation of vitamins.

28. The method of claim 1, wherein the dopaminergic neurons produced are characterized by greater than 30% more neurite formation than dopaminergic neurons produced by a differentiation regimen without the addition or augmentation of vitamins.

29. The method of claim 28, wherein the dopaminergic neurons produced are characterized by greater than 100% more neurite formation than dopaminergic neurons produced by a differentiation regimen without the addition or augmentation of vitamins.

30. The method of claim 29, wherein the dopaminergic neurons produced are characterized by greater than 500% more neurite formation than dopaminergic neurons produced by a differentiation regimen without the addition or augmentation of vitamins.

31. The method of claim 30, wherein the dopaminergic neurons produced are characterized by greater than 1000% more neurite formation than dopaminergic neurons produced by a differentiation regimen without the addition or augmentation of vitamins.

32. A method of increasing the likelihood of successful implantation of a dopaminergic neuron in a subject in need thereof, comprising administering to the subject the dopaminergic neuron obtained in the method of claim 1.

33. A method of treating a central nervous system disorder in a patient in need of implantation of dopamine-producing nerve cells comprising implanting dopaminergic neurons obtained in the method of claim 1 into a human in need thereof.

34. The method of claim 33, wherein the central nervous system disorder is parkinson's disease, huntington's disease, multiple sclerosis, or alzheimer's disease.