Enhancement of neuronal differentiation of ventral midbrain nervous system progenitor cells

Abstract

The present invention relates to a method for inducing the differentiation of ventral midbrain NSCs into neurons, which comprises contacting a cell population containing ventral midbrain NSCs with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, wherein the ventral midbrain NSCs co-express the markers FOXA2, LMX1A, EN1, OTX2, and SOX2.

Description

Technical Field

[0001] The present invention generally relates to the field of stem cells such as human embryonic stem cells. A method for obtaining stem cell-derived nervous system cells is provided. Specifically, a method for obtaining stem cell-derived ventral midbrain nervous system cells for the treatment of Parkinson's disease is provided.

Background Art

[0002] The prospect of using human pluripotent stem cells (hPSCs) for the treatment of various pathologies is considered very promising. Treatments include cell replacement therapies for neurological pathologies such as Parkinson's disease and stroke. However, for such treatments to be feasible, the development of in vitro methods for artificially producing stem cell-derived products for delivering them to the central nervous system (CNS) is necessary. The differentiation of hPSCs into defined cell types is a difficult process to control, and often the progeny generated from in vitro protocols are heterogeneous. Typically, when differentiating into nervous system cells, the mixture of cell types produced includes neurons (and various neuron subtypes such as glutamatergic and dopaminergic neurons among them), glia, neural stem cells (NSCs), as well as other non-neural cells (e.g., meningeal stromal cells). Such heterogeneous cultures are suboptimal for disease modeling studies or many cell replacement therapies.

[0003] Cell replacement therapy for Parkinson's disease is a major example. In Parkinson's disease, A9 ventral midbrain dopaminergic neurons (vmDA) are lost, and in order to restore the lost function, this is almost certainly the only cell type that must be transplanted. However, currently, all academic scientific publications and all ongoing human clinical trials are transplanting, in addition to vmDA, a population of multipotent precursor cells that results in a mixed cell population in vivo. This mixed population includes cell types such as proliferative NSCs, vascular leptomeningeal cells (VLMCs), non-vmDA neurons, and astrocytes. Non-dopaminergic neurons do not restore function in Parkinson's disease, and some (i.e., serotonergic neurons), as seen in clinical trials transplanting human fetal cells, produce negative gain-of-function behaviors in patients, and more generally, these non-vmDA carry unknown safety and efficacy risks. As a result, there is a need to provide a way to ensure that patients are treated with ventral midbrain neurons or their precursors.

[0004] Accordingly, an object of the present invention is to overcome the foregoing problems, and in particular, to provide a method capable of directing the differentiation of cells into ventral midbrain neurons. SUMMARY OF THE INVENTION

[0005] The object outlined above is achieved by aspects of the present invention. In addition, the present invention may also solve further problems that become apparent from the disclosure of the exemplary embodiments.

[0006] In a first aspect, the present invention provides a method comprising contacting a cell population comprising ventral midbrain NSCs with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. Specifically, this method is for directing the differentiation of ventral midbrain NSCs into neurons, which means that the developmental fate of ventral midbrain nervous system cells is affected towards a particular outcome, namely neurons. However, the differentiation into neurons is not necessarily complete, and the cells do not necessarily have to develop to their final fate within the methods disclosed herein. Preferably, cells at the precursor stage are directed in vitro towards differentiation into neurons, which is suitable for administration to a patient and further develops in vivo from the precursor stage to reach a neuronal fate. The inventors have found that the differentiation and production of ventral midbrain neurons from cell populations such as human PSCs can be improved by the combined inhibition of the MEK signaling pathway and the NOTCH signaling pathway. In particular, the inventors have surprisingly found that the effect of MEK pathway inhibition can be enhanced by the combined addition of a NOTCH pathway antagonist / inhibitor. Specifically, this combined inhibition reduces the proportion of late proliferating cells (representing off-target lineages), reduces the proportion of non-neuronal cells such as stromal cells, e.g., VLMCs, and increases the proportion of neurons. Importantly, the inventors have found that the timing of inhibiting MEK and NOTCH signaling with respect to the developmental stage of the cells has an impact on the outcome. In a preferred embodiment, the nervous system cells at the time of inhibiting MEK and NOTCH signaling are at a stage where the nervous system cells comprise a mixture of neural stem cells, neuroblast intermediate progenitor cells, and a small number of neurons. In a preferred embodiment, the ventral midbrain NSCs prior to inhibition of MEK signaling are neural induced, ventralized, and caudalized. In a preferred embodiment, at least 5% of the cell population comprising ventral midbrain NSCs co-express the markers FOXA2, LMX1A, EN1, OTX2, and SOX2. A cell population comprising such a mixture of ventral midbrain nervous system cells can arise from any method. However, in one embodiment, the cell population is neural induced, caudalized, and ventralized prior to inhibiting MEK signaling and NOTCH signaling.The cell population may be derived from pluripotent cells such as PSCs. Thus, in a preferred embodiment, the cell population of PSCs is exposed to an inhibitor of SMAD protein signaling, an activator of SHH signaling, and an inhibitor of Wnt signaling, and the cell population is further contacted with an activator of FGF signaling, etc., and is neurally induced, ventralized, and caudalized according to well-known methods. A cell population of neural-induced cells such as ventral midbrain NSCs may be obtained, for example, according to a differentiation method that lasts for 16 days from the start of neural induction, and the resulting cell population mainly contains neural system stem cells. When the cell population is further cultured (for example, up to 8 days at most), more neural system stem cells are further developed into neuroblast intermediate progenitor cells, but by contacting the cells with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, the chance of directing further differentiation of ventral midbrain neural system cells into neurons is still maintained. In the aforementioned method of neural induction of PSCs, it has been found that cells over-cultured for too long, such as more than 28 days, may be difficult to harvest, especially due to the formation of a network where neurites cannot be separated.

[0007] One of ordinary skill in the art will recognize that the optimal timing for contacting the cell population with the inhibitors may vary depending on the specific protocol used to differentiate PSCs into ventral midbrain NSCs. However, the inventors have identified the expression profiles of cell populations containing ventral midbrain NSCs that result in improved neurons. Thus, in a preferred embodiment, when the cell population contacts an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, 40-60% of the cell population containing ventral midbrain NSCs expresses ASCL1, 45-65% of the cell population containing ventral midbrain NSCs expresses KI67, less than 10-15% of the cell population containing ventral midbrain NSCs expresses INA, 2-5% of the cell population containing ventral midbrain NSCs is INA+ / SOX2-, and 80-95% of the cell population containing ventral midbrain NSCs expresses SOX2. Thus, in embodiments where the cell population differentiates into a cell population containing ventral midbrain NSCs, the cell population is differentiated until the cell population acquires the aforementioned expression profile when the cell population contacts an inhibitor of MEK signaling and an inhibitor of NOTCH signaling.

[0008] The in vitro cell population obtained according to this method may be used for the treatment of Parkinson's disease, and the higher purity cell product may provide a reduction in surgical procedure time and cranial injections. Furthermore, the improved purity of the product and the reduction of impurities are expected to provide an enhanced safety and potential efficacy profile.

[0009] Another aspect of the present invention relates to an inhibitor of MEK signaling for use in the treatment of Parkinson's disease in a subject administered a therapeutically effective amount of ventral midbrain NSCs. Specifically, ventral midbrain NSCs co-express the markers FOXA2, LMX1A, EN1, OTX2, and SOX2. The inventors contemplate that the effects of MEK and NOTCH inhibition demonstrated in vitro may be directly linked to the effect of directing the differentiation of administered ventral midbrain NSCs into neurons in vivo. Accordingly, the inventors believe that after administering a cell population containing ventral midbrain NSCs to a subject, e.g., by surgery, the administered cells can be further directed in their differentiation into neurons, thereby reducing the proportion of late proliferating cells and non-neuronal cells (such as VLMCs) and increasing the proportion of neurons. In one embodiment, the inhibitor of MEK signaling is midostaurin, a clinically tested small molecule that acts as a MEK inhibitor and crosses the blood-brain barrier. In one embodiment, the ventral midbrain NSCs are co-administered with the inhibitor of MEK signaling or are treated with the inhibitor prior to transplantation.

[0010] Similar aspects of the invention relate to an inhibitor of MEK signaling, an inhibitor of NOTCH signaling, and a cell population comprising ventral midbrain NSCs for use in combination in the treatment of Parkinson's disease, and a composition comprising a cell population comprising ventral midbrain NSCs, an inhibitor of MEK signaling, and an inhibitor of NOTCH signaling for the treatment of Parkinson's disease. Herein, the inventors contemplate that ventral midbrain NSCs will be co-administered with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. The ventral midbrain NSCs may or may not have been treated in vitro with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling according to the methods disclosed herein prior to co-administration. Even short exposures to inhibitors of MEK and NOTCH signaling direct a cell population comprising ventral midbrain NSCs towards a neuronal fate, and even a single co-administration of the cells and inhibitors is likely to be sufficient to direct further cell maturation in vivo, thus demonstrating a reduction in the required dosage of administration to a patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0011]

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Mode for Carrying Out the Invention

[0012] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The practice of the present invention employs, unless otherwise suggested, conventional methods of chemistry, biochemistry, biophysics, molecular biology, cell biology, genetics, immunology, and pharmacology known to those of ordinary skill in the art.

[0013] It should be noted that all headings and subheadings are used herein for convenience only and should in no way be construed as limiting the invention.

[0014] The use of any and all examples or exemplary language (e.g., "such as") provided in this specification is merely intended to better clarify the present invention and, unless otherwise claimed, does not limit the scope of the present invention.

[0015] Definitions General Definitions As used herein, "a," "an," or "the" can mean one or more than one. Unless otherwise indicated herein, terms presented in the singular include the plural in multiple situations.

[0016] Also, as used herein, "and / or" refers to any and all possible combinations of one or more of the associated listed items, as well as the absence of combinations when interpreted in the alternative ("or"), and includes them. Further, the present invention also contemplates that, in some embodiments of the present invention, any feature or combination of features described herein can be excluded or omitted.

[0017] Stem Cells "Stem cells" should be understood as undifferentiated cells that have the ability to differentiate and proliferate (especially the ability of self-renewal), while maintaining the ability to differentiate. Stem cells include classifications such as pluripotent stem cells, multipotent stem cells, unipotent stem cells, and the like according to their differentiation ability.

[0018] As used herein, the term "pluripotent stem cell" (PSC) refers to a stem cell that can be cultured in vitro and has the ability to differentiate into any cell lineage belonging to the three germ layers (ectoderm, mesoderm, endoderm), as well as into lineage-restricted undifferentiated stem cells that have typically lost the ability to form several cell types, lineages, or developmental regions (plural) via gene editing.

[0019] Pluripotent stem cells can be induced or isolated from fertilized eggs, somatic cell nuclear transfer embryos, germline stem cells, stem cells in tissues, somatic cells, and the like. Examples of pluripotent stem cells (PSCs) include embryonic stem cells (ESCs), embryonic germ cells (EG cells), induced pluripotent stem cells (iPSCs), and the like.

[0020] As used herein, the term "induced pluripotent stem cell" (also known as iPS cell or iPSC) means a type of pluripotent stem cell that can be generated directly from a cell that is not a PSC and has a nucleus. By introducing the products of a specific set of pluripotency-related genes, non-pluripotent cells can be converted into pluripotent stem cells.

[0021] As used herein, the term "embryonic stem cell" means a pluripotent stem cell derived from the inner cell mass of a blastocyst. Pluripotent embryonic stem cells may also be derived from, for example, parthenogenetic organisms as described in WO 2003 / 046141. In addition, embryonic stem cells can be produced from a single blastomere or by culturing the inner cell mass obtained without destroying the embryo. Embryonic stem cells are available from a given tissue and are also commercially available. Preferably, the methods and products of the present invention are based on human PSCs, i.e., human induced pluripotent stem cells or stem cells derived from human embryonic stem cells including parthenogenetic organisms.

[0022] As used herein, the term "multipotent stem cell" means a stem cell that has the potency to differentiate into multiple types of tissues or cells, but not all types, and is typically limited to one germ layer. Neural stem cells are an example of a multipotent stem cell limited to the nervous system.

[0023] As used herein, the term "unipotent stem cell" means a stem cell that has the potency to differentiate into only one specific cell type.

[0024] As used herein, the term "in vitro" means that cells are provided and maintained outside the body of a human or animal, such as in a flask, multi-well, or Petri dish. Thus, the cells will be cultured in a cell culture medium.

[0025] As used herein, the term "non-natural" means that cells that are derived from pluripotent stem cells and may be of human origin are artificial constructs that do not exist in nature. Generally, in the field of stem cell therapy, the goal is to provide cells that closely resemble the cells of the human body. However, it may not always be possible to mimic the development that pluripotent stem cells undergo during the embryonic and fetal stages to such an extent that the mature cells are indistinguishable from the natural cells of the human body. In essence, in one embodiment of the present invention, the cells are artificial.

[0026] As used herein, the term "artificial" with respect to cells may include materials that are naturally occurring in nature but have been modified into constructs that are not naturally occurring. This includes human stem cells that differentiate into non-naturally occurring cells that mimic the cells of the human body.

[0027] Protocol Throughout this application, the terms "method" and "protocol" may be used interchangeably when referring to a process for culturing or differentiating cells.

[0028] As used herein, the terms "day" with respect to a protocol and similarly the number of days in vitro (DIV) refer to the specific time for performing a particular step during a differentiation procedure.

[0029] Generally, and unless otherwise stated, "Day 0" refers to the start of the protocol, which may include, for example, but is not limited to, plating the stem cells, transferring the stem cells to an incubator, or contacting the stem cells in the current cell culture medium with a compound prior to transplantation of the stem cells. Typically, the start of the protocol involves, for example, by plating or incubating, and / or first contacting the undifferentiated stem cells with a compound (s) that affects the undifferentiated stem cells in such a way that the differentiation process is initiated, such as by transferring the undifferentiated stem cells to another cell culture medium and / or container, but is not limited thereto.

[0030] When referring to "cells" in a method, it means all cells of a cell population, regardless of cell type.

[0031] When referring to "Day X", such as Day 1, Day 2, etc., this is relative to the start of the protocol on Day 0. Those skilled in the art will recognize that, unless otherwise specified, the exact time for performing the steps may vary. Thus, "Day X" is meant to encompass a time span such as ±10 hours, ±8 hours, ±6 hours, ±4 hours, ±2 hours, or ±1 hour.

[0032] Culturing of Stem Cells As used herein, the term "culturing" refers to a continuous procedure that is used throughout the method to maintain the viability of cells at various stages. After the cells of interest are isolated, for example, but not limited to, from a biological tissue or embryo, they are subsequently maintained under carefully controlled conditions. These conditions vary depending on each cell type, but generally consist of a suitable container equipped with a substrate and / or medium that supplies essential nutrients (amino acids, carbohydrates, vitamins, minerals), growth factors, hormones, and gases (CO2, O2), and regulates the physicochemical environment (pH buffer, osmotic pressure, temperature).

[0033] As used herein, the term "cell culture medium" refers to a liquid or gel designed to support the growth of cells. Cell culture media generally contain an appropriate energy source and compounds that regulate the cell cycle.

[0034] As used herein, the term "incubator" refers to any suitable incubator that can support cell culture. Non-limiting examples include culture dishes, Petri dishes, and plates (such as microtiter plates, microplates, deep well plates, etc. of 6-well, 24-well, 48-well, 96-well, 384-well, 9600-well and the like), flasks, chamber slides, tubes, cell factories, roller bottles, spinner flasks, hollow fibers, microcarriers, or beads.

[0035] As used herein, the term "providing stem cells" when referred to in a protocol means transferring to a different environment, such as obtaining a batch of cells by the methods as described above and optionally seeding the cells onto a new substrate. Those skilled in the art will readily recognize that stem cells are vulnerable to such transfers, that the procedures require care, and that maintaining the stem cells in the original cell culture medium may facilitate more sustainable transfer of the cells before replacing the cell culture medium with another cell culture medium more suitable for further differentiation processes.

[0036] Differentiation of Stem Cells As used herein, the term "expresses" in relation to a gene or protein refers to the presence of an RNA molecule that can be detected using assays such as reverse transcription quantitative polymerase chain reaction (RT-qPCR), RNA sequencing, and the like, and / or a protein that can be detected using antibody-based assays such as flow cytometry, immunocytochemistry / immunofluorescence, and the like. Depending on the sensitivity and specificity of the assay, a gene or protein may be considered expressed when at least one molecule is detected, for example, in RNA sequencing, or a detection limit above background / noise level may be defined in relation to a control sample such as in flow cytometry. One of ordinary skill in the art will readily understand that when referring to the "expression" of a cell population, such as "a cell population expressing X% of marker Y", it means that X% of the cells in the cell population express marker Y.

[0037] As used herein, the term "co-expresses" means that individual cells express multiple markers.

[0038] As used herein, the term "marker" refers to a naturally occurring identifiable expression produced by a cell, which can be correlated with a particular property of the cell. In a preferred embodiment, the marker is gene expression or proteomic expression that can be detected and correlated with the identity of the cell. A marker may be referred to as being due to a gene, which can be readily translated into the expression of the corresponding mRNA and protein.

[0039] As used herein, when used with respect to any marker such as a surface protein or transcription factor disclosed herein, the terms "negative" or "-" refer to a marker that is not expressed in a cell or cell population, while the terms "weak" or "low" refer to a marker that is expressed at a reduced level in a cell as compared to the average expression of the marker in a cell population or as compared to a reference sample.

[0040] As used herein, the terms "positive" or "+" when used with respect to any marker such as a surface protein or transcription factor disclosed herein, refer to a marker that is expressed in a cell or cell population, while the terms "high" or "strong" refer to a marker that is expressed at an increased level in a cell as compared to the average expression of the marker in a cell population or as compared to a reference sample.

[0041] As used herein, the term "differentiation" broadly refers to the process by which a cell progresses from an immature state to a less immature state, or from an immature state to a mature state, or from an undifferentiated state or a state different from the intended differentiation state to a specific differentiation state, which may occur continuously during the implementation of the method. The term "differentiation" with respect to pluripotent stem cells refers to the process by which a cell progresses from an undifferentiated state to a specific differentiation state, i.e., from an immature state to a less immature state or a final state. Changes in cell interactions and maturation occur when a cell loses markers of undifferentiated cells or acquires markers of differentiated cells. Loss or acquisition of a single marker can indicate that a cell has "fully differentiated" or "terminally differentiated". A "terminally differentiated" cell is at the final stage of a developmental lineage and cannot differentiate further.

[0042] As used herein, the term "contact" with respect to culturing or differentiating cells means exposing the cells to a particular compound, for example, by placing the particular compound in a location where it can touch the cells, to produce "contacted" cells. Contact may be achieved using any suitable means. Non-limiting examples of contact include adding the compound to the cell culture medium of the cells. Cell contact is assumed to occur as long as the cells and the particular compound are in proximity, for example, as long as the compound is present in a suitable concentration in the cell culture medium.

[0043] As used herein, the term "inhibitor" refers to a compound that reduces, suppresses, or downregulates a process such as a signaling pathway that can promote cell differentiation.

[0044] As used herein, the term "activator" refers to a compound that induces, stimulates, or upregulates a process such as a signaling pathway that can promote cell differentiation.

[0045] Stem cell-derived products As used herein, the term "differentiated cell" refers to a cell such as a pluripotent stem cell that has progressed from an undifferentiated state to a less immature state. Differentiated cells may be, for example, less immature specialized cells such as progenitor cells, or may be fully matured into a specialized / terminal cell type.

[0046] As used herein, the term "cell population" refers to a plurality of cells in the same culture. The cell population may be, for example, a mixture of cells at various developmental stages, such as different types of cells, or cells at various maturation stages towards the same or similar specialized characteristics, or may be a more homogeneous composition of cells having a common marker.

[0047] As used herein, the term "nervous system cell population" refers to a cell population that includes nervous system cells.

[0048] As used herein, the terms "genetically modified" and "genetically engineered" with respect to a cell may be used interchangeably and refer to a cell that has been subjected to an artificial manipulation, modification, or recombination of DNA or other nucleic acid molecules in order to change the characteristics (phenotype) of the cell. Such cells are no longer considered to be naturally occurring cells. In the case of genetically modified stem cells, the traits resulting from gene editing persist even when the stem cells further differentiate into specialized cells, and thus the specialized cells are genetically modified and in an artificial, i.e., non-naturally occurring, state. An example of a genetically modified stem cell is an HLA-deficient stem cell, which is also called a universal donor cell and is intended to overcome the problem of graft rejection. A method for obtaining HLA-deficient stem cells is disclosed in International Publication No. WO 2020 / 260563.

[0049] neuroectodermal cell As used herein, the term "neural" refers to the nervous system.

[0050] As used herein, the term "neural cell" (unless otherwise specified) refers to a cell whose natural counterpart forms part of the natural ectodermal germ layer, more specifically the neuroectoderm, and encompasses cells at any stage of development within this germ layer, from neural stem cells to neurons and other terminally differentiated cell types (e.g., glial cells), i.e., cell stages such as the neural stem cell stage and the neuroblast stage. Thus, neurons and their precursors are considered a specific type of neural cell.

[0051] As used herein, the terms "neuron" and "nerve cell" may be used interchangeably to refer to post-mitotic nervous system cells that have ultimately differentiated into specialized cells. Neurons are characterized by the expression of marker INA, or other equivalent markers such as ELAVL3, ELAVL4 (typically detected with antibody HuC / D), RBFOX3 (typically detected with antibody NeuN), STMN2, NCMA1, or other such broad neuron markers.

[0052] As used herein, the terms "neural stem cell" or "NSC" and "neural progenitor cell" or "NPC" are terms used interchangeably to refer to self-renewing multipotent cells of the nervous system that can give rise to numerous more specialized cells of the CNS and PNS. NSCs and NPCs typically express transcription factors such as SOX2, NES, PAX6, SOX1, OTX2, OTX1, NKX6.1, OLIG2, NKX2.2, FOXG1, FOXA2, or LMX1A.

[0053] As used herein, the term "neuroblast" refers to intermediate progenitor cells that are typically multipotent or simply unipotent and can self-renew only to a limited extent. Neuroblasts ultimately give rise to terminally differentiated cell types such as neurons. The terms "neuroblast" and "intermediate precursor cell" and "intermediate progenitor cell" and "radial glial cell" may be used interchangeably.

[0054] As used herein, the term "neuroblast" or "intermediate progenitor cell" means a cell that expresses a gene associated with this stage, such as any of the ASCL1, SOX4, MASH1, EOMES, NHLH1, NEUROD or NEUROG gene families, or other such genes. These cells are cells that are destined to become neurons.

[0055] As used herein, the terms "neuron progenitor," "precursor of a neuron," and "neuron precursor" may be used interchangeably and refer to nervous system cells that have the potential or tendency to further specialize into neurons. The terms "neuron progenitor" and "non-natural neuron progenitor" may be used interchangeably.

[0056] The nervous system cells according to the present invention may have a specific local identity, such as cells specific to the midbrain.

[0057] As used herein, the term "forebrain" refers to the rostral region of the neural tube and the CNS that gives rise to structures including the cerebral cortex and the striatum.

[0058] As used herein, the term "midbrain" refers to the neural tube (on the rostral-caudal axis) and the medial region of the CNS that gives rise to structures including the substantia nigra.

[0059] As used herein, the term "ventral midbrain" with respect to a cell means a nervous system cell having certain characteristics of nervous system cells that occur naturally in the ventral midbrain. Typically, ventral midbrain nervous system cells are characterized by the expression of certain markers such as FOXA2 and LMX1A. Specifically, as used herein, the term "ventral midbrain nervous system stem cell" refers to a nervous system stem cell having the characteristics of nervous system stem cells that occur naturally in the ventral midbrain. Ventral midbrain NSCs or NPCs may be characterized by the co-expression of the markers FOXA2, LMX1A, EN1, OTX2, and SOX2.

[0060] As used herein, the terms "hindbrain" and "spinal cord" refer to the caudal region of the neural tube that is caudal to the isthmic organizer.

[0061] As used herein, the terms "dopaminergic (DA) cells" or "dopaminergic neurons" or "dopamine neurons" refer to cells capable of synthesizing the neurotransmitter dopamine.

[0062] As used herein, the term "stromal cells" refers to cells having the ability to become connective tissue cells or fibroblast-identical cells.

[0063] As used herein, the term "VLMC" means vascular leptomeningeal cells and is considered a type of stromal cell present in the CNS.

[0064] As used herein, the term "glial cells" refers to cells that perform support and protection for neurons, which are non-neuronal cells in the central nervous system (brain and spinal cord) and the peripheral nervous system that do not produce electrical impulses. Examples include astrocytes and oligodendrocytes and their precursors, glial progenitor cells, or glioblasts.

[0065] Directing differentiation towards ventral midbrain neurons In a general aspect of the invention, there is provided a method comprising contacting a cell population comprising ventral midbrain NSCs with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. Specifically, in one embodiment, the method is for directing the differentiation of ventral midbrain NSCs into neurons. In a further embodiment, the method is for differentiating ventral midbrain NSCs into ventral midbrain neurons. In a preferred embodiment, the ventral midbrain NSCs are neuroinduced, ventralized, and caudalized. In one embodiment, the cell population is neuroinduced, ventralized, and caudalized to obtain ventral midbrain NSCs. In one embodiment, the method is in vitro.

[0066] The method is intended to increase the number of cells in a cell population having a neuronal fate. As used herein, the term "neuronal fate" with respect to a cell means that the developmental fate of that cell becomes restricted to develop into a neuron.

[0067] As used herein, the term "direct differentiation" means to affect the developmental fate of a cell towards a particular outcome. Directing the differentiation of a cell is not necessarily a continuous process, and the cell does not necessarily have to develop to its final fate during the methods disclosed herein. In a preferred embodiment, the differentiation of the cell is directed in vitro such that the cell may later develop in vivo towards a particular fate, particularly a neuronal fate.

[0068] Thus, in a general embodiment, the method is for increasing the proportion of nervous system cells that differentiate into neurons. In one embodiment, the method is for decreasing the proportion of nervous system cells that differentiate into stromal cells such as vascular leptomeningeal cells (VLMCs), non-vmDA neurons, and / or astrocytes.

[0069] As used herein, the term "MEK" refers to MAPK / ERK kinase, and the term "MEK signaling" refers to the activation of the MAPK / ERK pathway (also known as the Ras-Raf-MEK-ERK pathway). As used herein, the term "inhibitor of MEK signaling" (MEKi) refers to any compound that inactivates the MAPK / ERK pathway. In one embodiment, the inhibitor of MEK signaling inhibits MKK1 (MEK1) and MKK2 (MEK2). In one embodiment, the inhibitor of MEK signaling inhibits the activation and downstream signaling of MEK. In one embodiment, the inhibitor of MEK signaling is a potent inhibitor that suppresses the phosphorylation of ERK. This applies to all aspects of the present invention.

[0070] As used herein, the term "NOTCH" refers to the signaling pathway of the Notch receptor. As used herein, the term "inhibitor of NOTCH signaling" (NOTCHi) refers to any compound that inactivates the NOTCH pathway. In one embodiment, the inhibitor of NOTCH signaling acts in this manner by targeting gamma-secretase or the gamma-secretase complex. This applies to all aspects of the present invention. The inventors have further found that contacting a cell population with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling results in a synergistic effect for directing the cell population towards a ventral midbrain neuron fate.

[0071] In one embodiment, at least 5%, such as at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of a cell population comprising ventral midbrain NSCs co-express the markers FOXA2, LMX1A, EN1, OTX2, and SOX2. The inventors have found that the effect of increased neuronal differentiation by inhibition of MEK signaling is particularly prominent in NSCs that are neuroinduced, ventralized, and caudalized to the extent of co-expressing the markers FOXA2, LMX1A, EN1, OTX2, and SOX2.

[0072] The inventors have identified the expression profile of a cell population containing ventral midbrain NSCs that provides improved neurons. In one embodiment, 0.5 - 65%, 5 - 65%, 10 - 65%, 15 - 65%, 20 - 65%, 25 - 65%, 30 - 65%, 35 - 65%, or 40 - 65%, preferably 30 - 65%, more preferably 40 - 65% of the cell population containing ventral midbrain NSCs expresses ASCL1 when the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. In one embodiment, 25 - 65%, 30 - 65%, 35 - 65%, 40 - 65%, or 45 - 65%, preferably 45 - 65% of the cell population containing ventral midbrain NSCs expresses KI67 when the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. In one embodiment, less than 20%, preferably less than 15% of the cell population containing ventral midbrain NSCs expresses INA when the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. In one embodiment, 0 - 20%, 0.1 - 20%, 0 - 15%, 0.1 - 15%, 5 - 20%, 10 - 20%, 5 - 15%, or 10 - 15%, preferably 10 - 15% of the cell population containing ventral midbrain NSCs expresses INA when the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. In one embodiment, less than 10%, preferably less than 5% of the cell population containing ventral midbrain NSCs is INA+ / SOX2- when the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. In one embodiment, 0 - 10%, 0 - 5%, 1 - 5%, or 2 - 5%, preferably 2 - 5% of the cell population containing ventral midbrain NSCs is INA+ / SOX2- when the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. In one embodiment, 75 - 95%, preferably 80 - 95% of the cell population containing ventral midbrain NSCs expresses SOX2 when the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling.In a preferred embodiment, all of the above are applied, i.e., when the cell population contacts an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, 40-60% of the cell population containing ventral midbrain NSCs expresses ASCL1, 45-65% of the cell population containing ventral midbrain NSCs expresses KI67, 10-15% of the cell population containing ventral midbrain NSCs expresses INA, 2-5% of the cell population containing ventral midbrain NSCs is INA+ / SOX2-, and 80-95% of the cell population containing ventral midbrain NSCs expresses SOX2. In a preferred embodiment, the cell population does not contact an inhibitor of NOTCH signaling, such as DAPT, before having the aforementioned preferred expression profile.

[0073] In one embodiment, the cell population containing ventral midbrain NSCs is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling before more than 70% or more than 60% of the cell population further develops into intermediate nervous system precursors. In another embodiment, the cell population containing ventral midbrain NSCs is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling before more than 20%, 15%, 10%, or 5% of the cell population further develops into intermediate nervous system precursors or neurons. Specifically, in another embodiment, the cell population containing ventral midbrain NSCs is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling before more than 20%, 15%, 10%, or 5% of the cell population expresses one of the markers ASCL1 and INA.

[0074] As used herein, the term "intermediate nervous system precursor" refers to a cell in a developmental stage between a nervous system stem cell and a neuron. Ventral midbrain nervous system cells at this stage are characterized by the expression of the marker ASCL1. The inventors have found that the chance to efficiently direct the development of the cell population into neurons is before the cells mature to the stage where the cells become neurons or other terminal nervous system cells (such as astrocytes, stromal cells such as VLMCs).

[0075] In one embodiment, the inhibitor of MEK signaling is selected from PD0325901, trametinib (GSK1120212), selumetinib (AZD6244), pimasertib (AS703026), MEK162, cobimetinib, PD184352, PD173074, BIX02189, AZD8330, PD318088, refametinib, and PD98059. As used herein, PD0325901 refers to a small molecule with the chemical name N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide (CAS number 391210-10-9), which is a potent inhibitor of MKK1 (MEK1) and MKK2 (MEK2).

[0076] In one embodiment, the concentration of the inhibitor of MEK signaling is at least 5 μM, preferably at least 10 μM. In one embodiment, the concentration of the inhibitor of MEK signaling is 5 μM to 100 μM, 5 μM to 50 μM, 5 μM to 40 μM, or 5 μM to 30 μM.

[0077] In one embodiment, the inhibitor of NOTCH signaling is selected from DAPT, avagacestat, PF-03084014, and LY450139. As used herein, the term "DAPT" refers to a small molecule with the chemical name (2S)-N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine 1,1-dimethylethyl ester (CAS number 208255-80-5). As used herein, the term "avagacestat" refers to a compound having CAS number 1146699-66-2. As used herein, the term "PF-03084014" refers to a compound having CAS number 1290543-63-3. As used herein, the terms "LY450139" and "semagacestat" may be used interchangeably and refer to a compound having CAS number 425386-60-3.

[0078] In one embodiment, the concentration of the inhibitor of NOTCH signaling is at least 1 μM, preferably at least 10 μM. In a further embodiment, the concentration of the inhibitor of NOTCH signaling is from 1 μM to 100 μM, from 1 μM to 50 μM, from 1 μM to 40 μM, or from 1 μM to 30 μM.

[0079] In one embodiment, the cell population is contacted with an inhibitor of MEK signaling for at least 1 / 2 hour, 1 hour, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours, or 18 hours, or 24 hours, or at least 2 days, 3 days, 4 days, 5 days, or 6 days. In one embodiment, the cell population is contacted with an inhibitor of MEK signaling for from 1 / 2 hour to 15 days, from 1 / 2 hour to 10 days, from 1 / 2 hour to 5 days, from 1 / 2 hour to 2 days, from 1 hour to 2 days, from 3 hours to 2 days, from 6 hours to 2 days, from 12 hours to 2 days, from 18 hours to 2 days, or from 1 to 2 days, or from about 2 to about 15 days. In one embodiment, the cell population is contacted with an inhibitor of NOTCH signaling for at least 1 / 2 hour, 1 hour, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours, or 18 hours, or 24 hours, or at least 2 days, 3 days, 4 days, 5 days, or 6 days. In one embodiment, the cell population is contacted with an inhibitor of NOTCH signaling for from 1 / 2 hour to 15 days, from 1 / 2 hour to 10 days, from 1 / 2 hour to 5 days, from 1 / 2 hour to 2 days, from 1 hour to 2 days, from 3 hours to 2 days, from 6 hours to 2 days, from 12 hours to 2 days, from 18 hours to 2 days, or from 1 to 2 days, or from about 2 to about 15 days.

[0080] In one embodiment, the inhibitor of MEK signaling and the inhibitor of NOTCH signaling are administered to the cells at least every 48 hours, every 36 hours, preferably at least every 30 hours, more preferably at least every 24 hours. In one embodiment, the inhibitor of MEK signaling and the inhibitor of NOTCH signaling are replaced at least every 12 - 36 hours, preferably at least every 18 - 30 hours, more preferably at least every 24 hours. In a preferred embodiment, the cell population is ensured to be sufficiently exposed to the inhibitor during the time of contact with the inhibitor. In one embodiment, the cell population is continuously exposed, for example, by sufficient medium replacement every 24 hours. Alternatively, the cell population may be sufficiently exposed to the inhibitor via a biomaterial or substance that ensures continuous release of the inhibitor.

[0081] In one embodiment, the cell population is brought into contact with the inhibitor of MEK signaling and the inhibitor of NOTCH signaling at least partially simultaneously. In one embodiment, the cell population is brought into contact with the inhibitor of MEK signaling and the inhibitor of NOTCH signaling simultaneously.

[0082] According to the present invention, in order for late administration to a patient to be suitable and for further development in vivo to result in a neuronal fate, the cells are directed in vitro towards differentiation into neurons. Administration to a patient is likely not to occur immediately after the method of directing differentiation towards neuronal development. Thus, in one embodiment, the cell population is cryopreserved after inhibition of MEK and NOTCH signaling. In one embodiment, the cell population is cryopreserved within 4 days, such as within 3 days, within 2 days, within 1 day, within 12 hours, within 6 hours, or within 3 hours after the end of inhibition of MEK and NOTCH signaling. In one embodiment, the cell population is cryopreserved immediately after inhibition of MEK and NOTCH signaling. In one embodiment, the cell population is cryopreserved in DMSO or using a cryoprotectant that does not contain DMSO. Those skilled in the art will understand that such cryoprotectants are commercially available and that techniques for cryopreserving cell populations, such as those containing nervous system cells, are well known.

[0083] According to the present invention, contacting a cell population comprising ventral midbrain NSCs with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling directs the cell population towards a ventral midbrain neuronal fate. Thus, the cell population is contacted with one or both of the inhibitors when at least a portion of the cells have differentiated into ventral midbrain NSCs. Nevertheless, prior to this stage, the cells may be contacted with one or both of the inhibitors to achieve a similar or other effect. However, in a preferred embodiment, the cell population does not contact an inhibitor of MEK signaling or an inhibitor of NOTCH signaling before the cell population is neuroinduced, ventralized, and caudalized. In a preferred embodiment, the cell population does not contact an inhibitor of NOTCH signaling, such as DAPT, before contacting the cell population with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling according to the method.

[0084] The inventors have identified the expression profile of a cell population containing NSCs that are particularly suitable for directing differentiation into neurons. Accordingly, aspects of the invention also include an in vitro method for directing the differentiation of ventral midbrain NSCs into neurons, which comprises contacting a cell population containing ventral midbrain NSCs with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, wherein 0.5 to 66%, preferably 40 to 60%, of the cell population containing ventral midbrain NSCs expresses ASCL1, 25 to 65%, preferably 45 to 65%, of the cell population containing ventral midbrain NSCs expresses KI67, 0 to 20%, preferably 10 to 15%, of the cell population containing ventral midbrain NSCs expresses INA, 0 to 10%, preferably 2 to 5%, of the cell population containing ventral midbrain NSCs is INA+ / SOX2−, and 75 to 95%, preferably 80 to 95%, of the cell population containing ventral midbrain NSCs expresses SOX2. Those skilled in the art will understand that the embodiments mentioned above apply equally to this particular aspect.

[0085] Differentiation of PSCs into ventral midbrain NSCs General aspects regarding methods for directing the differentiation of ventral midbrain NSCs into neurons require a cell population containing ventral midbrain NSCs as a starting material. The cell population containing ventral midbrain NSCs may be obtained by any suitable method. Thus, in one embodiment, the method includes an initial step of differentiating the cell population into ventral midbrain NSCs. In one embodiment, the cell population is derived from PSCs. In such embodiments, the cell population is neuroinduced, ventralized, and caudalized to obtain ventral midbrain NSCs. Thus, in one embodiment, the cell population is cultured to induce differentiation into ventral midbrain NSCs before contacting the cell population with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling.

[0086] In one embodiment, before contacting the cell population with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, the cell population is contacted with an inhibitor of Small Mothers Against Decapentaplegic (SMAD) protein signaling, an activator of sonic hedgehog (SHH) signaling, an activator of wingless (Wnt) signaling, and / or an activator of fibroblast growth factor (FGF) signaling, and optionally ascorbic acid and / or optionally brain-derived neurotrophic factor (BDNF).

[0087] In one embodiment for differentiating PSCs into ventral midbrain NSCs, the cell population is differentiated into ventral midbrain nervous system cells by neuroinducing the cell population by contacting the cell population with an inhibitor of SMAD protein signaling, such as at least two SMAD protein signaling inhibitors. As used herein, the term "SMAD protein signaling" refers to the Small Mothers Against Decapentaplegic (SMAD) protein signaling pathway. One of ordinary skill in the art will recognize that contacting PSCs with one or more inhibitors of the SMAD signaling pathway is a robust technique for differentiating cells into the nervous system. In one embodiment, the inhibitor of the SMAD signaling pathway is selected from noggin, LY364947, SB431542, RepSox, and LDN-193189, or similar compounds.

[0088] In one embodiment for differentiating PSCs into ventral midbrain NSCs, the cell population is differentiated into ventral midbrain neural stem cells by inducing ventralization of the cell population. As used herein, the terms "ventralization" and "ventral patterning" may be used interchangeably and also refer to the process by which pluripotent cells assume a ventral gene expression identity equivalent to that of cells of the embryo or embryonic structure, i.e., the neural tube. In one embodiment, the PSCs are ventralized by contacting the cells with an activator of the SHH signaling pathway. As used herein, the term "activator of Sonic Hedgehog signaling" refers to any molecule or compound having the ability to activate the SHH signaling pathway. Activation of the SHH pathway is well known to be involved in the induction and maintenance of ventral neural tube structures. In one embodiment, the activator of SHH signaling is selected from SHH, purmorphamine, and SAG, or similar compounds.

[0089] In one embodiment for differentiating PSCs into ventral midbrain NSCs, the cell population is differentiated into ventral midbrain neural cells by inducing caudalization of the cell population. As used herein, the term "caudalization" refers to the process by which pluripotent cells assume a caudal gene expression identity equivalent to that of cells of the embryo or embryonic structure, i.e., the neural tube. In one embodiment, the PSCs are caudalized by contacting the cells with an activator of the Wnt signaling pathway. As used herein, the term "activator of Wnt signaling" refers to any molecule or compound capable of activating the Wnt signaling pathway. Inhibitors of Wnt signaling are well known to be involved in the caudalization of neural cells. In one embodiment, the activator of Wnt signaling reduces GSK3-beta for activation of Wnt signaling. Thus, in certain embodiments, the Wnt activator is an inhibitor of GSK3-beta. In one embodiment, the activator of Wnt signaling is selected from CHIR99021 and recombinant Wnt protein.

[0090] In one embodiment for differentiating PSCs into ventral midbrain NSCs, the cell population is differentiated into ventral midbrain nervous system cells by further inducing caudalization of the cell population. Thus, in one embodiment, the cell population is contacted with an activator of fibroblast growth factor (FGF). In one embodiment, the activator of FGF signaling is FGF8b.

[0091] In certain embodiments, the concentration of the inhibitor(s) of SMAD protein signaling is 1 μM to 50 μM, the activator of SHH signaling is 200 ng / ml to 800 ng / ml, the inhibitor of Wnt signaling is 0.1 μM to 1 μM, the activator of FGF signaling is 10 ng / ml to 200 ng / ml, ascorbic acid is 50 μM to 500 μM, and / or BDNF is 1 ng / ml to 50 ng / ml.

[0092] In a preferred embodiment, the PSCs are human embryonic stem cells or human induced pluripotent stem cells.

[0093] A further aspect of the invention is an in vitro method for directing the differentiation of a cell population of PSCs into ventral midbrain neurons, the method comprising culturing a cell population of PSCs and contacting the cell population of PSCs with an inhibitor of SMAD protein signaling, an inhibitor of Wnt signaling, an activator of SHH signaling, an activator of FGF signaling, optionally ascorbic acid, and optionally BDNF to obtain a cell population comprising ventral midbrain NSCs, and further contacting the cell population comprising ventral midbrain NSCs with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling to direct differentiation into ventral midbrain neurons.

[0094] In one embodiment, the cell population of pluripotent stem cells is contacted with an inhibitor of SMAD protein signaling for 5 to 9 days. In certain embodiments, the cell population is contacted with an inhibitor of SMAD protein signaling from day 0 for 5 to 9 days.

[0095] In one embodiment, a cell population of PSCs is contacted with an activator of SHH signaling for 5 to 9 days, for example, 7 to 9 days, preferably 9 days. In a preferred embodiment, the cell population is contacted with an inhibitor of Wnt signaling from day 0 for 5 to 9 days. In one embodiment, the concentration of the activator of SHH signaling is 200 ng / ml to 800 ng / ml.

[0096] In one embodiment, a cell population of pluripotent stem cells is contacted with an inhibitor of Wnt signaling for 5 to 9 days, for example, 7 to 9 days, preferably 9 days. In a preferred embodiment, the cell population is contacted with an inhibitor of Wnt signaling from day 0 for 5 to 9 days. In one embodiment, the concentration of the activator of Wnt signaling is 0.1 μM to 1 μM.

[0097] In one embodiment, after terminating the contact of the cell population of PSCs with an inhibitor of SMAD protein signaling, an inhibitor of Wnt signaling, and / or an activator of SHH signaling, the cell population is contacted with an activator of FGF signaling for 7 to 12 days. In another embodiment, the cell population is contacted with an activator of FGF signaling from day 5 to 9 for 7 to 12 days, or at the end of the contact with an inhibitor of SMAD protein signaling, an inhibitor of Wnt signaling, and / or an activator of SHH signaling. In a particular embodiment, the cell population is contacted with an inhibitor of SMAD protein signaling, an activator of SHH signaling, and an inhibitor of Wnt signaling from day 0 to 9, and then the cell population is contacted with an activator of FGF signaling from day 9 to 16. In one embodiment, the activator of FGF signaling is FGF8b. In one embodiment, the concentration of the activator of FGF signaling is 10 ng / ml to 200 ng / ml.

[0098] In one embodiment, the cell population is contacted with ascorbic acid for 5 to 7 days from day 10 or 11. In one embodiment, the concentration of ascorbic acid is 10 μM to 400 μM, preferably 100 μM to 300 μM, preferably 150 μM to 250 μM, more preferably about 200 μM.

[0099] In one embodiment, the cell population is contacted with BDNF for 5 to 7 days starting from the 10th or 11th day. In one embodiment, the concentration of BDNF is 1 ng / ml to 40 ng / ml, preferably 10 ng / ml to 40 ng / ml, preferably 15 ng / ml to 30 ng / ml, more preferably about 20 ng / ml.

[0100] In one embodiment, before contacting the cell population with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, the cell population is differentiated into ventral midbrain NSCs for 14 to 24 days, such as 15 to 20 days. In one embodiment, the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling for 1 / 2 hour to 15 days, 1 / 2 hour to 10 days, 1 / 2 hour to 5 days, 1 / 2 hour to 2 days, 1 hour to 2 days, 3 hours to 2 days, 6 hours to 2 days, 12 hours to 2 days, 18 hours to 2 days, or 1 to 2 days, or about 2 to about 10 days. In one embodiment, the cell population is contacted with inhibitors of MEK and NOTCH 0 to 10 days after the cell population has ceased to be in contact with an activator of FGF signaling.

[0101] In a preferred embodiment, while differentiating the cell population into ventral midbrain NSCs, the cell population is not contacted with an inhibitor of NOTCH signaling such as DAPT.

[0102] In one embodiment, before contacting the cell population with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, the cell population containing ventral midbrain NSCs is cultured in a suitable culture medium for 6 to 8 days starting from the 16th day without contacting the cell population with an inhibitor of SMAD protein signaling, an inhibitor of Wnt signaling, an activator of SHH signaling, an activator of FGF signaling, or an inhibitor of NOTCH signaling such as DAPT. Those skilled in the art will recognize that the cells will continue to differentiate during further culturing of the cell population without using such components.

[0103] In an alternative embodiment, instead of contacting the cell population with an activator of FGF, the cell population is contacted with an inhibitor of Wnt signaling for at least 14 days. Then, in such embodiments, 0 to 10 days after the cell population no longer contacts the inhibitor of Wnt signaling, the cells are contacted with inhibitors of MEK and NOTCH.

[0104] In one embodiment, the cell population is harvested 28 days prior to the initiation of differentiation of the cell population into ventral midbrain NSCs (27 days prior, 26 days prior, or 25 days prior, preferably 26 days prior, etc.).

[0105] In one embodiment, the cell population is harvested on the 28th, 27th, 26th, or 25th day, preferably the 25th day, from the initiation of differentiation of the cell population into ventral midbrain NSCs.

[0106] As used herein, the term "harvested" means that the cells are collected and transferred to a new environment. This may be that the cells are re - seeded into a new in vitro culture system. This may also, in one embodiment, be that the cell population is cryopreserved at the time of harvest so that the cells do not further develop into neurons. Thus, in one embodiment, the cell population is cryopreserved after inhibition of MEK signaling and inhibition of NOTCH signaling. In one embodiment, the cell population is cryopreserved within 4 days, such as within 3 days, 2 days, 1 day, 12 hours, 6 hours, 3 hours, or 1 hour after the end of inhibition of MEK signaling and inhibition of NOTCH signaling. In one embodiment, the cell population is cryopreserved within 4 days, such as within 3 days, 2 days, 1 day, 12 hours, 6 hours, or 3 hours after harvest.

[0107] In one embodiment, the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling at least partially simultaneously. In certain embodiments, the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling simultaneously.

[0108] In one embodiment, the cell population is cultured in two-dimensional culture. In a further embodiment, the cell population is initially plated on a substrate. In one embodiment, the substrate comprises an extracellular matrix. In a further embodiment, the substrate comprises poly-L-lysine, poly-D-lysine, poly-ornithine, laminin, fibronectin, and / or collagen, and / or fragments thereof. In a more specific embodiment, the laminin or fragment thereof is selected from the group consisting of laminin-111, laminin 521, and laminin 511. In one embodiment, the cell population is cultured on a laminin-111 substrate. In one embodiment, the concentration of the laminin substrate is about 10 μg / ml.

[0109] In one embodiment, 0.5 to 65%, 5 to 65%, 10 to 65%, 15 to 65%, 20 to 65%, 25 to 65%, 30 to 65%, 35 to 65%, or 40 to 65% of the cell population containing ventral midbrain NSCs, preferably 30 to 65%, more preferably 40 to 65%, express ASCL1 when the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. In one embodiment, 25 to 65%, 30 to 65%, 35 to 65%, 40 to 65%, or 45 to 65% of the cell population containing ventral midbrain NSCs, preferably 45 to 65%, express KI67 when the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. In one embodiment, less than 20%, preferably less than 15% of the cell population containing ventral midbrain NSCs express INA when the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. In one embodiment, 0 to 20%, 0.1 to 20%, 0 to 15%, 0.1 to 15%, 5 to 20%, 10 to 20%, 5 to 15%, or 10 to 15% of the cell population containing ventral midbrain NSCs, preferably 10 to 15%, express INA when the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. In one embodiment, less than 10%, preferably less than 5% of the cell population containing ventral midbrain NSCs are INA+ / SOX2- when the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. In one embodiment, 0 to 10%, 0 to 5%, 1 to 5%, or 2 to 5% of the cell population containing ventral midbrain NSCs, preferably 2 to 5%, are INA+ / SOX2- when the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. In one embodiment, 75 to 95%, preferably 80 to 95% of the cell population containing ventral midbrain NSCs express SOX2 when the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling.In a preferred embodiment, all of the above are applied, i.e., when the cell population contacts an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, 40-60% of the cell population containing ventral midbrain NSCs expresses ASCL1, 45-65% of the cell population containing ventral midbrain NSCs expresses KI67, 10-15% of the cell population containing ventral midbrain NSCs expresses INA, 2-5% of the cell population containing ventral midbrain NSCs is INA+ / SOX2-, and 80-95% of the cell population containing ventral midbrain NSCs expresses SOX2.

[0110] In one embodiment, the cell population is cultured by obtaining neural stem progenitor cells, aggregating the neural stem progenitor cells to form nervous system microspheres, and further maturing the neural stem progenitor cells of the nervous system microspheres. In one embodiment, the method includes an additional step of seeding the neural stem progenitor cells in a well suitable for maintaining the nervous system microspheres in static non-adherent culture before the step of aggregating the neural stem progenitor cells. The method for culturing the cell population in the nervous system microspheres is detailed in International Publication No. WO 2021 / 099532, which is incorporated herein by reference.

[0111] Late neural differentiation by using an inhibitor of NOTCH signaling Another aspect of the present invention is an in vitro method for directing the differentiation of a cell population into ventral midbrain neurons, which includes culturing a cell population containing PSCs, inducing the differentiation of the cell population into ventral midbrain NSCs, maturing the ventral midbrain NSCs, and contacting the cell population containing the ventral midbrain NSCs with an inhibitor of NOTCH signaling, wherein the ventral midbrain NSCs express markers FOXA2, LMX1A, EN1, OTX2, and SOX2.

[0112] In one embodiment, the cell population contacts the inhibitor of NOTCH signaling at least 20 days, preferably 21 days, more preferably 22 days after initially inducing the differentiation of the cell population into ventral midbrain NSCs.

[0113] In one embodiment, the ventral midbrain NSCs are matured for at least 4 days before contacting the cell population containing the ventral midbrain NSCs with an inhibitor of NOTCH signaling.

[0114] In one embodiment, the ventral midbrain NSCs are contacted with an inhibitor of NOTCH signaling after day 4 following the expression of at least 70% of the cell population of the marker FOXA2, LMX1A, EN1, OTX2, and SOX2.

[0115] Cell population of ventral midbrain nervous system cells Aspects of the invention relate to a cell population comprising ventral midbrain nervous system cells obtainable by any of the methods described herein. In certain embodiments, the cell population comprising ventral midbrain nervous system cells is obtained by the method according to any one of the previous embodiments. In particular, the inventors were unable to identify parameters that distinguish the cell population taken immediately after inhibition of MEK signaling and inhibition of NOTCH signaling as compared to cell populations not subjected to methods that direct differentiation towards neurons according to the invention. However, it is clear that the cells are stimulated by signaling and that the proportion of cells maintaining a neuronal fate is increasing, which becomes apparent immediately upon further culturing of the cell population. Accordingly, aspects of the invention are cell populations comprising ventral midbrain NSCs that, when cultured in vitro for 5 days in a culture medium suitable for maintaining nervous system cells, are at least 50% neurons as determined by HuCD, INA, or related markers and <15% of the cells are proliferating as determined by KI67, MKI67, or other such proliferation markers (Example 5). In one embodiment, the expression of markers by the cells is measured using scRNAseq according to Example 7. In one embodiment, the expression of markers by the cells is measured according to immunocytochemistry (ICC) as described in Example 8. In one embodiment, the expression of markers by the cells is measured according to flow cytometry as described in Example 2. In one embodiment, the cell population is cultured according to Example 6.

[0116] Accordingly, an aspect of the present invention relates to a cell population comprising ventral midbrain NSCs, which, when cultured in vitro for 5 days in a culture medium suitable for maintaining nervous system cells, results in a cell population in which at least 25% are HuCD+ / SOX2- or HUCD or NEUN+ / SOX2-. In one embodiment, the expression of the marker by the cells is measured using scRNAseq according to Example 7. In one embodiment, the expression of the marker by the cells is measured according to immunocytochemistry (ICC) as described in Example 8. In one embodiment, the expression of the marker by the cells is measured according to flow cytometry as described in Example 2. In one embodiment, the cell population is cultured according to Example 6.

[0117] In one embodiment, the cell population is in vitro. In one embodiment, the ventral midbrain nervous system cells are non-natural. In one embodiment, the ventral midbrain nervous system cells are artificial. In a preferred embodiment, the ventral midbrain nervous system cells are stem cell-derived. In certain embodiments, the ventral midbrain nervous system cells are stem cells derived from pluripotent stem cells. In a further embodiment, the ventral midbrain nervous system cells are stem cells derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs).

[0118] In one embodiment, the cells of the cell population are genetically engineered. In one embodiment, the pluripotent stem cells are genetically engineered and the genetic engineering persists in the ventral midbrain nervous system cells obtained according to any of the methods described herein. In certain embodiments, the cells of the cell population are genetically engineered to be hypoimmunogenic. As used herein, the terms "hypoimmunogenic" and "immune evasive" with respect to cells may be used interchangeably and refer to the property of a cell that reduces the tendency of immune rejection by the subject into which such cells are transplanted. Typically, certain surface markers are overexpressed or silenced. In one embodiment, the genetically engineered cells have reduced expression of MHC-I and / or MHC-II. In one embodiment, the cells are genetically engineered to express one or more tolerogenic factors or analogs thereof such as HLA-E, HLA-G, CD46, CD47, CD55, CD59, and PD-L1. Examples and methods for genetically engineering cells to be immune evasive are described in International Publication Nos. WO 2012 / 145384, WO 2013 / 158292, WO 2016 / 142532, WO 2016 / 183041, WO 2018 / 132783, WO 2018 / 175390, WO 2019 / 161271, WO 2020 / 018615, WO 2020 / 018620, WO 2020 / 049535, WO 2020 / 168317, WO 2021 / 195426, WO 2022 / 012591, and WO 2020 / 260563. In some aspects, genome editing techniques (e.g., CRISPR / Cas or TALEN systems) are used to regulate (e.g., reduce, eliminate, and / or increase) the expression of specific genes.

[0119] In one embodiment, genetic recombination for low immunogenicity includes reduced expression of MHC-I human leukocyte antigen in wild-type stem cells, reduced expression of MHC-II human leukocyte antigen in wild-type stem cells, and / or increased expression of tolerogenic factors in wild-type stem cells. In one embodiment, the MHC-I human leukocyte antigen is HLA-A, HLA-B, and HLA-C. In one embodiment, the MHC-II human leukocyte antigen is HLA-DP, HLA-DQ, and HLA-DR. In one embodiment, the tolerogenic factors are selected from CD46, CD47, CD55, CD59, PD-L1, HLA-E, and HLA-G.

[0120] In one embodiment, the cells of the cell population are genetically recombined to be lineage-restricted. As used herein, the term "lineage-restricted" with respect to a cell means that the cell is functionally and / or structurally restricted to differentiate into a particular cell type.

[0121] In one embodiment, the cell population is cryopreserved. In one embodiment, the cell population is cryopreserved in DMSO or using a cryoprotectant that does not contain DMSO.

[0122] In one embodiment, the cell population comprises at least 1,000 cells, 10,000 cells, 100,000 cells, 1,000,000 cells, or 10,000,000 cells.

[0123] A cell population for use as a medicament Another aspect of the invention relates to a cell population or a composition thereof in vitro according to any one of the embodiments described herein for use as a medicament. In one embodiment, the cell population comprises ventral midbrain nervous system cells contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. In a preferred embodiment, the cell population is for the treatment of Parkinson's disease.

[0124] Another aspect relates to a method of treating a neurological condition, comprising administering to a patient an effective amount of a cell population according to the present invention. In a preferred embodiment, the neurological condition is Parkinson's disease.

[0125] Another aspect relates to a composition comprising a cell population comprising ventral midbrain NSCs, an inhibitor of MEK signaling, and an inhibitor of NOTCH signaling. In one embodiment, the inhibitor of MEK signaling is midostaurin. In one embodiment, the composition is for the treatment of Parkinson's disease. In one embodiment, the cell population comprising ventral midbrain NSCs is characterized by yielding a cell population that is at least 50% INA+ or HuCD+ and <15% KI67+ when cultured in vitro for 5 days in a culture medium suitable for maintaining nervous system cells. In one embodiment, the cell population is cultured according to Example 6. In one embodiment, the cell population comprising ventral midbrain NSCs yields a cell population that is at least 25% HuCD+ / SOX2- or HUCD or NEUN+ / SOX2- when cultured in vitro for 5 days in a culture medium suitable for maintaining nervous system cells. In one embodiment, the cell population is cultured according to Example 6.

[0126] In one embodiment, the composition further comprises a cryoprotectant. In one embodiment, the cryoprotectant is DMSO. In one embodiment, the cryoprotectant does not contain DMSO.

[0127] Directing the differentiation of ventral midbrain nervous system cells into neurons in vivo Another aspect relates to a method for the treatment of Parkinson's disease, the method comprising administering to a subject a therapeutically effective amount of ventral midbrain NSCs and an inhibitor of MEK signaling. In one embodiment, the method further comprises administering an inhibitor of NOTCH signaling. As used herein, the term "subject" refers to a human patient suffering from Parkinson's disease. In one embodiment, a "therapeutically effective amount" for the treatment of Parkinson's disease using cell products means a dose of 200,000 to 10,000,000 cells. Administration of a cell population comprising ventral midbrain NSCs to a subject is contemplated by surgery. In one embodiment, the ventral midbrain NSCs co-express the markers FOXA2, LMX1A, EN1, OTX2, and SOX2. In one embodiment, the ventral midbrain NSCs are obtained according to the methods disclosed herein. Administration of the inhibitor of MEK signaling and / or the inhibitor of NOTCH signaling may be by any suitable means such as oral or subcutaneous injection. In one embodiment, the administration of the inhibitor of MEK signaling and / or the inhibitor of NOTCH signaling is oral. In a preferred embodiment, the inhibitor of MEK signaling is PD0325901. PD0325901 may also be referred to by the generic name of midostaurin. In one embodiment, the inhibitor of NOTCH signaling is selected from DAPT, MRK-560, MRK-003, LY900009, AL-101, lenigastat (LY3039478), MK0752, nilogacestat (PF-03084017, RO4929097 (RG473), CT16, PTG12, anti-NRR1, anti-NRR2, bronchiquibizumab (OMP-52M51), taletrectinib (OMP-59R5), 15D11, anti-Jag1 / 2, anti-DII1, YW152F, MMGZ01, mABL001, HMD4-2, demcizumab (OMP-21M18), enoticumab (REGN421), MEDI0639, navicixizumab (OMP-305B83), ABT-165, NOV1501 (ABL001; HD105), IMR-1, RIN1, SAHM1, and CB-103. In one embodiment, an inhibitor of NOTCH signaling that has been clinically tested is selected.Details of the Notch inhibitors listed can be found in Front. Cell Dev. Biol., 28 May 2021, Vol. 9, 2021 (Table 1). In one embodiment, an inhibitor of MEK signaling and / or an inhibitor of Notch signaling is administered starting on the same day as the day on which the ventral midbrain NSCs are administered to the patient. In one embodiment, administration of the inhibitor of MEK signaling and / or the inhibitor of Notch signaling is initiated prior to transplantation of the cells such that steady state is reached in the subject at the time of administration of the cells. In one embodiment, administration of the inhibitor of MEK signaling and / or the inhibitor of Notch signaling is initiated from 6 days before to 6 days after transplantation of the cells. In one embodiment, the inhibitor of MEK signaling and / or the inhibitor of Notch signaling is administered to the subject for at least 2 days, for example, at least 3, 4, 5, 6, 7, 8, 9, or 10 days. In one embodiment, the inhibitor of MEK signaling and / or the inhibitor of Notch signaling is administered to the subject for 1 to 12 days. The dosing regimen may be any suitable dosage to provide a plasma concentration of the inhibitor of MEK signaling and / or the inhibitor of Notch signaling within a range effective to direct the differentiation of the administered ventral midbrain NSCs into neurons. In one embodiment, the inhibitor of MEK signaling is midostaurin, and the dosing regimen is 1 mg to 30 mg bid (twice daily), 1 mg to 20 mg bid, 1 mg to 10 mg bid, 1 to 5 mg bid, or 2 to 4 mg bid.

[0128] In one embodiment, an inhibitor of MEK signaling and / or an inhibitor of Notch signaling is co-administered with a cell population comprising ventral midbrain NSCs, for example, a cell population differentiated for 14 to 24 days, preferably at least 16 days, and optionally, the cell population has not been contacted with the inhibitor of MEK signaling and / or the inhibitor of Notch signaling prior to administration to the patient.

[0129] Accordingly, aspects of the present invention relate to inhibitors of MEK signaling for use in the treatment of Parkinson's disease in a subject administered a therapeutically effective amount of ventral midbrain NSCs. Further aspects relate to inhibitors of MEK signaling for use in the treatment of Parkinson's disease in a subject by co - administration with a therapeutically effective amount of ventral midbrain NSCs. In one embodiment, the subject is further co - administered an inhibitor of NOTCH signaling. Specifically, one aspect relates to midostaurin for use in the treatment of Parkinson's disease. More specifically, one aspect relates to midostaurin for use in the treatment of Parkinson's disease in a subject administered a therapeutically effective amount of ventral midbrain NSCs, or for use in the treatment of Parkinson's disease in a subject by co - administration with a therapeutically effective amount of ventral midbrain NSCs. In a preferred embodiment, the ventral midbrain NSCs co - express the markers FOXA2, LMX1A, EN1, OTX2, and SOX2.

[0130] Another aspect relates to inhibitors of MEK signaling and inhibitors of NOTCH signaling for combined use in the treatment of Parkinson's disease in a subject administered a therapeutically effective amount of ventral midbrain NSCs. Similarly, one aspect relates to inhibitors of MEK signaling and inhibitors of NOTCH signaling for combined use in a method of directing the differentiation of ventral midbrain NSCs into ventral midbrain neurons, the method comprising administering to a subject administered ventral midbrain NSCs a combination of an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. In one embodiment, the inhibitor of MEK signaling and the inhibitor of NOTCH signaling are administered within at least 6 days after administration of the ventral midbrain NSCs. In one embodiment, the inhibitor of MEK signaling and the inhibitor of NOTCH signaling are administered for at least 2 days, for example at least 4, 5, or 6 days.

[0131] A further aspect relates to an inhibitor of MEK signaling, an inhibitor of NOTCH signaling, and a cell population comprising ventral midbrain NSCs for use in combination in the treatment of Parkinson's disease. In one embodiment, the inhibitor of MEK signaling and the inhibitor of NOTCH signaling are co-administered together with a cell population comprising ventral midbrain NSCs. In one embodiment, the inhibitor of MEK signaling is midostaurin.

[0132] Another aspect of the invention relates to a composition comprising a cell population comprising ventral midbrain NSCs and an inhibitor of MEK signaling. In one embodiment, the inhibitor of MEK signaling is midostaurin. In one embodiment, the composition further comprises an inhibitor of NOTCH signaling. In certain specific embodiments, the inhibitor of NOTCH signaling is DAPT. Another aspect relates to a composition comprising a cell population comprising ventral midbrain NSCs, an inhibitor of MEK signaling, and an inhibitor of NOTCH signaling. In one embodiment, the composition is in vitro.

[0133] Those skilled in the art will understand that the embodiments referred to in this section with respect to methods of treatment, use as medicaments, and second medical uses may be equally applicable to all of the aspects described.

[0134] Cell populations for use in the treatment of Parkinson's disease The inventors recognized that obtaining a cell population containing ventral midbrain nervous system cells without contacting the cell population with an inhibitor of NOTCH, such as DAPT, better reflects the natural development of nervous system cells. The inventors still prefer to further contact the cell population with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, while a cell population having a preferred expression profile at the time of contacting the cells with these inhibitors may itself be more suitable for the treatment of Parkinson's disease compared to prior art methods. Accordingly, another aspect of the present invention relates to a cell population for use in the treatment of Parkinson's disease, wherein 80 - 95% of the cell population expresses SOX2, 40 - 60% of the cell population expresses ASCL1, 30 - 65% of the cell population expresses KI67, 10 - 20% of the cell population expresses INA, and 2 - 5% of the cell population is INA+ / SOX2- (Figure 9, Table 3). In a preferred embodiment, the cell population has not been contacted with DAPT or other inhibitors of NOTCH signaling prior to contacting with both an MEK inhibitor and a NOTCH inhibitor. These cells are obtained according to the methods disclosed herein prior to contacting the cell population with the inhibitors, and the cell population is differentiated into ventral midbrain NSCs for at least 20 days, for example 22 - 24 days.

[0135] The cell population may be administered to a patient by itself or may be co-delivered together with an inhibitor of MEK signaling and / or an inhibitor of NOTCH signaling. Accordingly, one aspect relates to a composition comprising the aforementioned cell population and an inhibitor of MEK signaling and / or an inhibitor of NOTCH signaling.

[0136] Specific embodiments Here, aspects of the present invention are further described by the following non-limiting embodiments.

[0137] 1. A method comprising contacting a cell population comprising ventral midbrain NSCs with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. 2. The method according to the preceding embodiment for directing the differentiation of ventral midbrain NSCs into neurons. 3. The method according to any one of the preceding embodiments for differentiating ventral midbrain NSCs into ventral midbrain neurons. 4. The method according to any one of the preceding embodiments, wherein the ventral midbrain NSCs are neuroinduced, ventralized, and caudalized. 5. The method according to any one of the preceding embodiments, wherein a cell population is neuroinduced, ventralized, and caudalized to obtain ventral midbrain NSCs. 6. The method according to any one of the preceding embodiments, wherein an inhibitor of MEK signaling inhibits MEK1 and MEK2. 7. The method according to any one of the preceding embodiments, wherein an inhibitor of NOTCH signaling targets γ-secretase. 8. The method according to any one of the preceding embodiments, wherein at least 5% of the cell population comprising ventral midbrain NSCs co-expresses the markers FOXA2, LMX1A, EN1, OTX2, and SOX2. 9. The method according to any one of the preceding embodiments, wherein the cell population is differentiated into ventral midbrain NSCs for 14 to 24 days before contacting the cell population with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. 10. The method according to any one of embodiments 5 to 9, wherein a cell population comprising ventral midbrain NSCs is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling before more than 70% or more than 60% of the cell population further develops into intermediate nervous system precursors. 11. The method according to any one of the preceding embodiments, wherein 0.5 to 65%, preferably 30 to 65%, more preferably 40 to 60% of the cell population comprising ventral midbrain NSCs expresses ASCL1 at the time when the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. 12. The method according to any one of the preceding embodiments, wherein 25 to 65%, preferably 45 to 65% of the cell population comprising ventral midbrain NSCs expresses KI67 at the time when the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. 13. The method according to any one of the preceding embodiments, wherein less than 20%, preferably less than 15% of the cell population comprising ventral midbrain NSCs expresses INA when the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. 14. The method according to any one of the preceding embodiments, wherein 0 to 20%, preferably 10 to 15% of the cell population comprising ventral midbrain NSCs expresses INA when the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. 15. The method according to any one of the preceding embodiments, wherein less than 10%, preferably less than 5% of the cell population comprising ventral midbrain NSCs is INA+ / SOX2- when the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. 16. The method according to any one of the preceding embodiments, wherein 0 to 10%, preferably 2 to 5% of the cell population comprising ventral midbrain NSCs is INA+ / SOX2- when the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. 17. The method according to any one of the preceding embodiments, wherein 75 to 95%, preferably 80 to 95% of the cell population comprising ventral midbrain NSCs expresses SOX2 when the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. 18. The method according to any one of embodiments 5 to 10 of the preceding, wherein the cell population comprising ventral midbrain NSCs is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling before more than 20% of the cell population further develops into intermediate nervous system precursors or neurons. 19. The method according to any one of embodiments 5 to 10 and 18, wherein the cell population comprising ventral midbrain NSCs is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling before more than 20% of the cell population expresses one of the markers ASCL1 and INA. 20. The method according to any one of the preceding embodiments, wherein the inhibitor of MEK signaling is selected from PD0325901, trametinib (GSK1120212), selumetinib (AZD6244), pimasertib (AS703026), MEK162, cobimetinib, PD184352, PD173074, BIX02189, AZD8330, PD318088, refametinib, and PD98059, preferably PD0325901. 21. The method according to any one of the preceding embodiments, wherein the concentration of the inhibitor of MEK signaling is at least 5 μM, preferably at least 10 μM. 22. The method according to any one of the preceding embodiments, wherein the concentration of the inhibitor of MEK signaling is from 5 μM to 100 μM. 23. The method according to any one of the preceding embodiments, wherein the inhibitor of NOTCH signaling is selected from DAPT, avagacestat, PF-03084014, and LY450139. 24. The method according to any one of the preceding embodiments, wherein the inhibitor of NOTCH signaling is selected from DAPT, MRK-560, MRK-003, LY900009, AL-101, clenigacestat (LY3039478), MK0752, nilogacestat (PF-03084017, RO4929097 (RG473), CT16, PTG12, anti-NRR1, anti-NRR2, bronchiquizumab (OMP-52M51), talexizumab (OMP-59R5), 15D11, anti-Jag1 / 2, anti-DII1, YW152F, MMGZ01, mABL001, HMD4-2, demcizumab (OMP-21M18), enoticumab (REGN421), MEDI0639, navicixizumab (OMP-305B83), ABT-165, NOV1501 (ABL001; HD105), IMR-1, RIN1, SAHM1, and CB-103. 25. The method according to any one of the preceding embodiments, wherein the concentration of the inhibitor of NOTCH signaling is at least 1 μM, preferably at least 10 μM. 26. The method according to any one of the preceding embodiments, wherein the concentration of the inhibitor of Notch signaling is from 1 μM to 100 μM. 27. The method according to any one of the preceding embodiments, wherein the cell population is contacted with an inhibitor of MEK signaling for at least 1 / 2 hour, 1 hour, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours, or 18 hours, or 24 hours, or at least 2 days, 3 days, 4 days, 5 days, or 6 days. 28. The method according to any one of the preceding embodiments, wherein the cell population is contacted with an inhibitor of MEK signaling for from 1 / 2 hour to 15 days, from 1 / 2 hour to 10 days, from 1 / 2 hour to 5 days, from 1 / 2 hour to 2 days, from 1 hour to 2 days, from 3 hours to 2 days, from 6 hours to 2 days, from 12 hours to 2 days, from 18 hours to 2 days, or from 1 to 2 days, or from about 2 to about 15 days. 29. The method according to any one of the preceding embodiments, wherein the cell population is contacted with an inhibitor of Notch signaling for at least 1 / 2 hour, 1 hour, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours, or 18 hours, or 24 hours, or at least 2 days, 3 days, 4 days, 5 days, or 6 days. 30. The method according to any one of the preceding embodiments, wherein the cell population is contacted with an inhibitor of Notch signaling for from 1 / 2 hour to 15 days, from 1 / 2 hour to 10 days, from 1 / 2 hour to 5 days, from 1 / 2 hour to 2 days, from 1 hour to 2 days, from 3 hours to 2 days, from 6 hours to 2 days, from 12 hours to 2 days, from 18 hours to 2 days, or from 1 to 2 days, or from about 2 to about 15 days. 31. The method according to any one of the preceding embodiments, wherein the inhibitor of MEK signaling and the inhibitor of Notch signaling are replaced at least every 36 hours, preferably at least every 30 hours, more preferably at least every 24 hours. 32. The method according to any one of the preceding embodiments, wherein the inhibitor of MEK signaling and the inhibitor of Notch signaling are replaced at least every 12 - 36 hours, preferably at least every 18 - 30 hours, more preferably at least every 24 hours. 33. The method according to any one of the preceding embodiments, wherein the cell population is contacted at least partially and simultaneously with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. 34. The method according to any one of the preceding embodiments, wherein the cell population is contacted simultaneously with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. 35. The method according to any one of the preceding embodiments, wherein the cell population is not contacted with an inhibitor of NOTCH signaling before contacting the cell population with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling according to the method. 36. The method according to any one of the preceding embodiments, wherein the cell population is cryopreserved after inhibition of MEK and NOTCH signaling. 37. The method according to the preceding embodiment, wherein the cell population is cryopreserved within 4 days, such as within 3 days, within 2 days, within 1 day, within 12 hours, within 6 hours, or within 3 hours after the end of inhibition of MEK and NOTCH signaling. 38. The method according to the preceding embodiment, wherein the cell population is cryopreserved immediately after the end of inhibition of MEK and NOTCH signaling. 39. The method according to any one of embodiments 36 to 38, wherein the cell population is cryopreserved in DMSO or using a cryoprotectant without DMSO. 40. The method according to any one of the preceding embodiments, wherein the cell population is cultured to induce differentiation into ventral midbrain NSCs before contacting the cell population with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. 41. Before contacting the cell population with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, the cell population is contacted with an inhibitor of Small Mothers Against Decapentaplegic (SMAD) protein signaling, an activator of sonic hedgehog (SHH) signaling, an activator of wingless (Wnt) signaling, and / or an activator of fibroblast growth factor (FGF) signaling, and optionally ascorbic acid and / or optionally brain-derived neurotrophic factor (BDNF), according to any one of the preceding embodiments. 42. The method according to the preceding embodiment, wherein the cell population is contacted with at least two inhibitors of SMAD protein signaling. 43. The method according to any one of embodiments 41 and 42, wherein the inhibitor(s) of SMAD signaling is selected from noggin, LY364947, SB431542, RepSox, and LDN-193189, or similar compounds. 44. The method according to any one of embodiments 41 to 43, wherein the activator of SHH signaling is selected from SHH, purmorphamine, SAG, or similar compounds. 45. The method according to any one of embodiments 41 to 44, wherein the activator of Wnt signaling is CHIR99021. 46. The method according to any one of embodiments 41 to 45, wherein the activator of FGF signaling is FGF8b. 47. a) The concentration of the inhibitor of SMAD protein signaling is from 1 μM to 50 μM, b) The concentration of the activator of SHH signaling is from 200 ng / ml to 800 ng / ml, c) The concentration of the inhibitor of Wnt signaling is from 0.1 μM to 1 μM, d) The concentration of the activator of FGF signaling is from 10 ng / ml to 200 ng / ml, e) The concentration of ascorbic acid is from 50 μM to 500 μM and / or f) The method according to any one of Embodiments 41 to 46, wherein the concentration of BDNF is from 1 ng / ml to 50 ng / ml. 48. The method according to any one of the preceding embodiments, wherein the cell population is derived from PSC. 49. The method according to the preceding embodiment, wherein the PSC is a human embryonic stem cell or a human induced pluripotent stem cell. 50. The method according to any one of the preceding embodiments, which is in vitro. 51. An in vitro method for directing the differentiation of a cell population of PSC into ventral midbrain neurons, comprising culturing the cell population of PSC and contacting the cell population of PSC with an inhibitor of SMAD protein signaling, an inhibitor of Wnt signaling, an activator of SHH signaling, an activator of FGF signaling, optionally ascorbic acid, and optionally BDNF to obtain a cell population comprising ventral midbrain NSC, and further contacting the cell population comprising ventral midbrain NSC with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling to direct the differentiation into ventral midbrain neurons. 52. The method according to the preceding embodiment, wherein the cell population is differentiated into ventral midbrain NSC for 14 to 24 days before contacting the cell population with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. 53. The method according to any one of Embodiments 51 and 52, wherein the cell population is collected, for example, 27 days, 26 days, or 25 days before the start of differentiation of the cell population into ventral midbrain NSC, preferably 26 days before. 54. The method according to any one of Embodiments 51 to 53, wherein the cell population is collected on the 28th, 27th, 26th, or 25th day, preferably the 25th day, from the start of differentiation of the cell population into ventral midbrain NSC. 55. The method according to any one of embodiments 51 - 54, wherein the cell population is contacted with a cell population having an inhibitor of MEK signaling and an inhibitor of NOTCH signaling for 1 / 2 hour to 15 days, 1 / 2 hour to 10 days, 1 / 2 hour to 5 days, 1 / 2 hour to 2 days, 1 hour to 2 days, 3 hours to 2 days, 6 hours to 2 days, 12 hours to 2 days, 18 hours to 2 days, or 1 - 2 days, or about 2 - about 10 days. 56. The method according to any one of embodiments 51 - 55, wherein the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling 0 - 10 days after the cell population has ceased to be in contact with an activator of FGF signaling. 57. Before contacting the cell population with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, the cell population is subjected to neural induction, ventralization, and caudalization for 16 days, and subsequently the cell population is cultured in a suitable culture medium for 6 - 8 days without contacting the cell population with an inhibitor of SMAD protein signaling, an inhibitor of Wnt signaling, an activator of SHH signaling, an activator of FGF signaling, or an inhibitor of NOTCH signaling such as DAPT. The method according to any one of embodiments 51 - 56. 58. The method according to any one of the preceding embodiments 51 - 57, wherein the cell population is contacted at least partially simultaneously with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. 59. The method according to any one of embodiments 51 - 58, wherein the cell population is contacted simultaneously with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. 60. The method according to any one of embodiments 51 - 59, wherein a cell population of pluripotent stem cells is contacted with an inhibitor of SMAD protein signaling for 5 - 9 days. 61. The method according to any one of embodiments 51 - 60, wherein a cell population of pluripotent stem cells is contacted with an inhibitor of Wnt signaling for 5 - 9 days. 62. The method according to any one of embodiments 51 - 61, wherein a cell population of pluripotent stem cells is contacted with an activator of SHH signaling for 5 - 9 days. 63. The method according to any one of embodiments 51 to 62, wherein the cell population of pluripotent stem cells is contacted with an activator of FGF signaling for 7 to 12 days after the end of contact with an inhibitor of SMAD protein signaling, an inhibitor of Wnt signaling, and / or an activator of SHH signaling. 64. The method according to any one of embodiments 51 to 63, wherein the cell population is contacted with an inhibitor of SMAD protein signaling from about day 0 to 5 to 9 days. 65. The method according to any one of embodiments 51 to 64, wherein the cell population is contacted with an inhibitor of Wnt signaling from day 0 to 5 to 9 days. 66. The method according to any one of embodiments 51 to 65, wherein the cell population is contacted with an activator of SHH signaling from day 0 to 5 to 9 days. 67. The method according to any one of embodiments 51 to 66, wherein the cell population is contacted with an activator of FGF signaling from days 5 to 9 to 7 to 12 days or at the end of contact with an inhibitor of SMAD protein signaling, an inhibitor of Wnt signaling, and / or an activator of SHH signaling. 68. The method according to any one of embodiments 51 to 67, wherein the cell population is contacted with ascorbic acid from day 10 or 11 to 5 to 7 days. 69. The method according to any one of embodiments 51 to 68, wherein the concentration of ascorbic acid is 10 μM to 400 μM. 70. The method according to any one of embodiments 51 to 69, wherein the cell population is contacted with BDNF from day 10 or 11 to 5 to 7 days. 71. The method according to any one of embodiments 51 to 70, wherein the concentration of BDNF is 1 ng / ml to 40 ng / ml. 72. The method according to any one of embodiments 51 to 71, wherein the cell population is cryopreserved after inhibition of MEK signaling and inhibition of NOTCH signaling. 73. The method according to the preceding embodiments, wherein the cell population is cryopreserved within 4 days, such as within 3 days, within 2 days, within 1 day, within 12 hours, within 6 hours, within 3 hours, or within 1 hour after the inhibition of MEK signaling and the inhibition of NOTCH signaling are terminated. 74. The method according to any one of embodiments 72 and 73, wherein the cell population is cryopreserved at the time of collection. 75. The method according to the preceding embodiments, wherein the cell population is cryopreserved within 4 days, such as within 3 days, within 2 days, within 1 day, within 12 hours, within 6 hours, or within 3 hours after collection. 76. The method according to any one of embodiments 51 - 75, wherein 0.5 - 65%, preferably 30 - 65%, more preferably 40 - 60% of the cell population containing ventral midbrain NSCs expresses ASCL1 at the time when the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. 77. The method according to any one of embodiments 51 - 76, wherein 25 - 65%, preferably 45 - 65% of the cell population containing ventral midbrain NSCs expresses KI67 at the time when the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. 78. The method according to any one of embodiments 51 - 77, wherein less than 20%, preferably less than 15% of the cell population containing ventral midbrain NSCs expresses INA at the time when the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. 79. The method according to any one of embodiments 51 - 78, wherein 0 - 20%, preferably 10 - 15% of the cell population containing ventral midbrain NSCs expresses INA at the time when the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. 80. The method according to any one of embodiments 51 - 79, wherein less than 10%, preferably less than 5% of the cell population containing ventral midbrain NSCs is INA+ / SOX2- at the time when the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. 81. The method according to any one of embodiments 51 to 80, wherein when the cell population is brought into contact with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, 0 to 10%, preferably 2 to 5% of the cell population containing ventral midbrain NSCs is INA+ / SOX2-. 82. The method according to any one of embodiments 51 to 81, wherein when the cell population is brought into contact with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, 75 to 95%, preferably 80 to 95% of the cell population containing ventral midbrain NSCs expresses SOX2. 83. An in vitro method for directing the differentiation of a cell population into ventral midbrain neurons, comprising: - culturing a cell population containing PSCs; - inducing the differentiation of the cell population into ventral midbrain NSCs; - maturing the ventral midbrain NSCs; - contacting the cell population containing ventral midbrain NSCs with an inhibitor of NOTCH signaling, wherein the ventral midbrain NSCs express the markers FOXA2, LMX1A, EN1, OTX2, and SOX2. 84. The method according to the preceding embodiments, wherein the cell population is brought into contact with an inhibitor of NOTCH signaling at least 20 days, preferably 21 days, more preferably 22 days after initially inducing the differentiation of the cell population into ventral midbrain NSCs. 85. A cell population containing ventral midbrain nervous system cells obtainable by the method according to any one of embodiments 1 to 84. 86. A cell population containing ventral midbrain nervous system cells obtained by the method according to any one of embodiments 1 to 84. 87. A cell population containing ventral midbrain NSCs, which when cultured in vitro for 5 days in a culture medium suitable for maintaining nervous system cells, results in a cell population in which at least 50% is INA+ or HUCD+ and less than 15% is KI67+. A cell population comprising ventral midbrain NSCs, which, when cultured in vitro for 5 days in a culture medium suitable for maintaining nervous system cells, results in a cell population in which at least 25% are HuCD+ / SOX2- or HUCD+ or NEUN+ / SOX2-. 89. The cell population according to any one of embodiments 87 and 88, wherein the expression of the marker by the cells is measured using single cell RNA sequencing, nucleic acid RNA sequencing, immunocytochemistry (ICC), qPCR, or FACS. 90. The cell population according to any one of embodiments 87 to 89, wherein the expression of the marker by the cells is measured according to Example 7. 91. The cell population according to any one of embodiments 87 to 89, wherein the expression of the marker by the cells is measured according to Example 8. 92. The cell population according to any one of embodiments 87 to 89, wherein the expression of the marker by the cells is measured according to Example 9. 93. The cell population according to any one of embodiments 87 to 92, wherein the cell population is cultured according to Example 6. 94. The cell population according to any one of embodiments 85 to 93, wherein the cell population is in vitro. 95. The cell population according to any one of embodiments 85 to 94, wherein the ventral midbrain nervous system cells are non-natural. 96. The cell population according to any one of embodiments 85 to 95, wherein the ventral midbrain nervous system cells are artificial. 97. The cell population according to any one of embodiments 85 to 96, wherein the ventral midbrain nervous system cells are derived from stem cells. 98. The nerve cells according to any one of embodiments 85 to 97, wherein the ventral midbrain nervous system cells are stem cells derived from pluripotent stem cells. 99. The cell population according to the preceding embodiment, wherein the pluripotent stem cells are genetically modified and the genetic modification persists in the ventral midbrain nervous system cells. 100. The cell population according to any one of embodiments 98 and 99, wherein the ventral midbrain nervous system cells are stem cells derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs). 101. The cell population according to any one of embodiments 85 to 100, wherein the cells of the cell population are genetically modified. 102. The cell population according to the preceding embodiment, wherein the cells of the cell population are genetically modified so as to be hypoimmunogenic and / or lineage-restricted. 103. The cell population according to the preceding embodiment, wherein the genetic modification for hypoimmunogenicity comprises reduced expression of MHC-I human leukocyte antigen, reduced expression of MHC-II human leukocyte antigen, and / or increased expression of a tolerogenic factor, relative to wild-type stem cells. 104. The cell population according to the preceding embodiment, wherein the MHC-I human leukocyte antigen is HLA-A, HLA-B, and HLA-C. 105. The cell population according to any one of embodiments 103 and 104, wherein the MHC-II human leukocyte antigen is HLA-DP, HLA-DQ, and HLA-DR. 106. The cell population according to any one of embodiments 103 to 105, wherein the tolerogenic factor is selected from CD46, CD47, CD55, CD59, PD-L1, HLA-E, and HLA-G. 107. The cell population according to any one of embodiments 85 to 106, wherein the cell population comprises at least 1,000 cells, 10,000 cells, 100,000 cells, 1,000,000 cells, or 10,000,000 cells. 108. The cell population according to any one of embodiments 85 to 107, wherein the cell population is cryopreserved. 109. A composition comprising the cell population according to any one of embodiments 85 to 108. 110. The composition according to the preceding embodiment, further comprising a cryoprotectant. 111. The composition according to the preceding embodiment, wherein the cryoprotectant is DMSO or the cryoprotectant does not contain DMSO. 112. An in vitro cell population according to any one of embodiments 85 to 108 for use as a medicament. 113. An in vitro cell population according to the preceding embodiments for the treatment of Parkinson's disease. 114. A method of treating a neurological condition comprising administering to a patient an effective amount of the cell population according to any one of embodiments 85 - 108. 115. The method according to the preceding embodiments, wherein the neurological condition is Parkinson's disease. 116. A method of treating Parkinson's disease, the method comprising administering to a subject a therapeutically effective amount of ventral midbrain NSCs and an inhibitor of MEK signaling. 117. The method according to the preceding embodiments, wherein the inhibitor of MEK signaling is PD0325901. 118. The method according to any one of embodiments 116 and 117, wherein the ventral midbrain NSCs co - express the markers FOXA2, LMX1A, EN1, OTX2, and SOX2. 119. The method according to any one of embodiments 116 - 118, further comprising administering an inhibitor of NOTCH signaling. 120. The inhibitor of NOTCH signaling is selected from DAPT, MRK - 560, MRK - 003, LY900009, AL - 101, Crenigacestat (LY3039478), MK0752, Nilotacestat (PF - 03084017, RO4929097 (RG473), CT16, PTG12, anti - NRR1, anti - NRR2, Bronchiticuzumab (OMP - 52M51), Talexizumab (OMP - 59R5), 15D11, anti - Jag1 / 2, anti - DII1, YW152F, MMGZ01, mABL001, HMD4 - 2, Demcizumab (OMP - 21M18), Enoticumab (REGN421), MEDI0639, Navicixizumab (OMP - 305B83), ABT - 165, NOV1501 (ABL001; HD105), IMR - 1, RIN1, SAHM1, and CB - 103, according to the preceding embodiments. 121. The method according to the preceding embodiments, wherein the inhibitor of NOTCH signaling is DAPT. An inhibitor of MEK signaling and an inhibitor of NOTCH signaling for use in combination in the treatment of Parkinson's disease in a subject administered a therapeutically effective amount of ventral midbrain NSCs. 123. An inhibitor of MEK signaling and an inhibitor of NOTCH signaling, as described in the preceding embodiments, wherein the inhibitor of MEK signaling and the inhibitor of NOTCH signaling are administered within at least 6 days after administration of ventral midbrain NSCs. 124. A combination of an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, as described in any one of embodiments 122 and 123, wherein the inhibitor of MEK signaling and the inhibitor of NOTCH signaling are administered for at least 2 days, such as at least 4, 5, or 6 days. 125. A cell population comprising an inhibitor of MEK signaling, an inhibitor of NOTCH signaling, and ventral midbrain NSCs for use in combination in the treatment of Parkinson's disease. 126. An inhibitor of MEK signaling, an inhibitor of NOTCH signaling, and a cell population, as described in the preceding embodiments, wherein the inhibitor of MEK signaling and the inhibitor of NOTCH signaling are co-administered together with the cell population comprising ventral midbrain NSCs. 127. An inhibitor of MEK signaling, an inhibitor of NOTCH signaling, and a cell population, as described in the preceding embodiments, wherein the inhibitor of MEK signaling is midostaurin. 128. An inhibitor of MEK signaling for use in the treatment of Parkinson's disease in a subject administered a therapeutically effective amount of ventral midbrain NSCs. 129. An inhibitor of MEK signaling, as described in the preceding embodiments, wherein the ventral midbrain NSCs are administered surgically. 130. Midostaurin for use in the treatment of Parkinson's disease. 131. Midostaurin for use in the treatment of Parkinson's disease in a subject administered a therapeutically effective amount of ventral midbrain NSCs. 132. A composition comprising a cell population comprising ventral midbrain NSCs, an inhibitor of MEK signaling, and an inhibitor of NOTCH signaling. 133. The composition according to the preceding embodiment, wherein the inhibitor of MEK signal transduction is midostaurin. 134. The composition according to any one of embodiments 132 and 133 for the treatment of Parkinson's disease. 135. The composition according to any one of embodiments 132 to 134, wherein the cell population comprising ventral midbrain NSCs is as described in embodiments 87 and / or 88. 136. The composition according to any one of embodiments 132 to 135, wherein the composition is in vitro. 137. An in vitro cell population for use in the treatment of Parkinson's disease, wherein 80-95% of the cell population expresses SOX2, 40-60% of the cell population expresses ASCL1, 30-65% of the cell population expresses KI67, 10-20% of the cell population expresses INA, and 2-5% of the cell population is INA+ / SOX2-. 138. The cell population according to the preceding embodiment, wherein the cell population has not been contacted with DAPT. 139. A composition comprising the cell population according to embodiment 137 or 138, an inhibitor of MEK signal transduction, and / or an inhibitor of NOTCH signal transduction.

Examples

[0138] The following are non-limiting examples for practicing the present invention.

[0139] Example 1: Differentiation of human pluripotent stem cells into ventral midbrain nervous system cells hPSCs can be differentiated into ventral midbrain neural cells according to several protocols that have been published since 2011, when an important paper titled "Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease" by the research group of Lorenz Studer was published. One method of performing the differentiation procedure is shown below according to the published content described by the references in this section (Nolbrant et al., 2017 and Kirkeby et al., 2017).

[0140] Human embryonic stem cell (hESC) lines RC17 (Roslin CT) and 3053 (Novo Nordisk A / S), as well as other hESC lines developed by Novo Nordisk, were cultured in iPS Brew XF medium (Miltenyi Biotec) supplemented with 60 U / mL penicillin-streptomycin (P-S, Thermo Fisher Scientific) on culture vessels coated with human laminin-521 (0.7 - 1.2 μg / cm 2 , Biolamina). The medium was changed daily, and the cells were passaged every 4 - 6 days with 0.5 mM EDTA (Thermo Fisher Scientific). The cultures were maintained at 37 °C, 95% humidity, and 5% CO2 level.

[0141] hESCs were differentiated into ventral midbrain neurons according to established protocols (Nolbrant et al., 2017, Kirkeby et al., 2017). Briefly, hESCs were grown to 70 - 90% confluence and then dissociated with 0.5 mM EDTA. The cells were seeded at 10 2 cells / cm 4 onto cell culture flasks or plates coated with human laminin-111 (1.2 μg / cm 2It was seeded and immediately contacted with the differentiation medium. The cells were exposed to an N2-based medium; 50% DMEM / F12 + Glutamax (Gibco), 50% Neurobasal (Gibco), 1% N2 supplement CTS (Thermo Fisher Scientific), 5% GlutaMAX (Thermo Fisher Scientific), 0.2% P-S (Thermo Fisher Scientific), and the SMAD inhibitor SB431542 (10 μM, Miltenyi Biotec), Noggin for neural induction (100 ng / mL, Miltenyi Biotec), Sonic hedgehog C24II (SHH, 500 ng / mL, Miltenyi Biotec) for ventral fate, and the GSK3β inhibitor CHIR99021 (CHIR, 0.5 - 0.6 μM, Miltenyi Biotec) to promote caudalization, from in vitro days (DIV) 0 to 8. The N2-based medium was supplemented with fibroblast growth factor 8b (FGF8b, 100 ng / mL, Miltenyi Biotec) at DIV9 - 11. At DIV11, the cells were dissociated with Accutase (Thermo Fisher Scientific) and seeded onto cell culture flasks or plates coated with human laminin-111 (1.2 μg / cm 2 ) at a density of 0.8×10 6 cells / m 2Cells were seeded. On day 16 in vitro (DIV), cells were dissociated with Accutase and further differentiated and matured into neurons from ventral midbrain neural stem cells by either cryopreservation or reseeding in cell culture flasks / plates coated with poly-L-ornithine (0.002%) and laminin-521 (1.5 μg / cm 2 ) while supplementing B27 medium with BDNF (20 ng / mL), GDNF (20 ng / mL), L-ascorbic acid (200 μM), dcAMP (500 μM), DAPT (10 μM), and Y-27632 (10 μM) to extend in vitro culture.

[0142] References: · Nolbrant S, Heuer A, Parmar M, Kirkeby A. Generation of high-purity human ventral midbrain dopaminergic progenitors for in vitro maturation and intracerebral transplantation. Nat Protoc. 2017 Sep;12(9):1962 - 1979. doi:10.1038 / nprot.2017.078. Epub 2017 Aug 31. PMID:28858290. · Kirkeby A, Nolbrant S, Tiklova K, Heuer A, Kee N, Cardoso T, Ottosson DR, Lelos MJ, Rifes P, Dunnett SB, Grealish S, Perlmann T, Parmar M. Predictive Markers Guide Differentiation to Improve Graft Outcome in Clinical Translation of hESC-Based Therapy for Parkinson’s Disease. Cell Stem Cell. 2017 Jan 5;20(1):135 - 148. doi:10.1016 / j.stem.2016.09.004. Epub 2016 Oct 27. PMID:28094017; PMCID:PMC5222722.

[0143] Example 2: Single-Cell Protein Marker Expression Assay by Flow Cytometry Analysis Ventral midbrain dopaminergic (vmDA) progenitor cells were generated from hESCs in 2D in vitro cultures as described in Example 1 and using the reagents described in Table 1. After various days from the start of differentiation, the cell cultures were dissociated into single-cell suspensions using Accutase, counted on a NucleoCounter NC-200, and collected in N2 medium (CTS(™) Neurobasal(™) medium supplemented with 1% CTS(™) N-2 supplement). Dead cells were labeled using the LIVE / DEAD(™) Fixable Near-IR Dead Cell Stain Kit. The cells were then resuspended in B27 medium (CTS(™) Neurobasal(™) medium supplemented with 1% B-27(™) supplement, without vitamin A, 2 mM GlutaMAX(™), 60 U / mL penicillin-streptomycin, 10 μM ROCK inhibitor). The cells were then fixed and permeabilized using the BD Transcription Factor Buffer Set (BD Biosciences) according to the manufacturer's instructions. The fixed cells were then stained with fluorescent-conjugated antibodies and the samples were acquired on a BD LSR Fortessa or BD FACSymphony (BD Biosciences). The fcs files were exported and analyzed with FlowJo 10.5.03.

[0144] To determine whether a cell was considered positive in the protein expression of the marker, the gates were set at the edge of the fluorescence signal of the negative control sample as routinely done in the art. Examples of negative control samples used included unstained controls, fluorescence minus one (FMO) controls, and most preferably biological negative control samples, and all cells that were present above these threshold gates when the experimental samples were run were considered positive. Antibodies: FOXA2 (1:320, Miltenyi), OTX2 (1:320, Miltenyi), SOX2 (1:40, BD), LMX1A (1:2500, Novo Nordisk production), EN1 (1:40, AtlasAntibodies), ASCL1 (1:2500, Mitlenyi), INA (1:3000, AtlasAntibodies).

Table 1

[0145] Example 3: Early in vitro administration of MEKi and / or NOTCHi To generate hPSC-derived cell cultures or cell products rich in ventral midbrain dopaminergic neurons and lacking other cell types (i.e., other neurons, glial precursors, glial cells, stromal cells, proliferative cells), novel inhibitors and combinations of inhibitors were administered to ventral midbrain nervous system cell cultures. Cultures of VM NPCs were exposed to MEK inhibitors and / or NOTCH inhibitors immediately after the stage of hPSC neural / ectodermal specification (performed with a small molecule SMAD inhibitor (addition of noggin)), after ventralization (performed with an SHH pathway agonist such as SHH, purmorphamine, or SAG), and after caudalization (performed with a WNT agonist such as WNT protein, the small molecule CHIR999021). This exposure immediately after neural ectodermal patterning, ventralization, and caudalization is referred to as "early administration" (Figure 1). Early administration was when the cultures consisted mainly of VM NSCs and minimal expression of VM IPC and / or VM neuron markers was observed (Figure 1), corresponding to DIV16 of the protocol of Example 1.

[0146] Cells exposed to early administration of MEK inhibitor and / or NOTCH inhibitor were profiled by flow cytometry (Figure 2), and the protein expression levels of ventral midbrain floor plate regional identity transcription factors (FOXA2, LMX1A, OTX2, EN1) and cell stage markers (KI67 that identifies highly proliferative cells such as NSCs, SOX2 that identifies NSCs, ASCL1 that identifies IP Cs, INA that identifies neurons) were evaluated. [Table 2]

[0147] The cultures analyzed were observed to be ventral midbrain in their regional identity, as shown by the expression of the relevant ventral midbrain floor plate markers FOXA2 (>50% of all cells), LMX1A (>45% of all cells), and OTX2 (>70% of all cells), as well as EN1 (>50% of all cells) (Figure 2 and Table 2). These cells were still at the NSC stage, as indicated by the high expression of the NSC marker SOX2 (>70%) and the proliferation marker KI67 (>30%). These cultures had initiated by the time of exposure and expressed low levels of the ventral midbrain IP C (ASCL1, 0.48 - 2.86%) and neurons (determined by INA total expression (1.43 - 3.12%)), and INA+ / SOX2- (0%) had not yet expressed (Figure 2 and Table 2).

[0148] After 5 days of exposure to MEKi alone, NOTCHi alone, or both MEKi and NOTCHi cultures, the cultures were re - analyzed using flow cytometry to evaluate the protein expression levels of key genes and compared to untreated controls (Figure 3 and Table 3). [Table 3]

[0149] All groups were found to maintain high expression of the ventral midbrain floor plate lineage markers FOXA2 and LMX1A (>72.4 of all cells) (Figure 3, Table 3). Control cells were observed to simultaneously maintain high expression of the NSC marker SOX2 (78.2 - 93.7%) and high levels of the proliferation marker KI67 (46.1 - 73.3%), and low levels of the neuronal marker INA (Figure 3, black bars). Collectively, this indicates that the control cultures were predominantly ventral midbrain floor plate neural stem cells. In contrast, both the MEKi alone-treated cultures (Figure 3, white bars, and Table 3) and the NOTCHi alone-treated cultures (Figure 3, thin striped bars) were observed to have downregulated expression of NSC as well as the proliferation markers SOX2 and KI67, indicating that these cells were differentiating into neurons at a higher rate compared to the control in response to these inhibitors. The most significant observations were seen with the combined administration of MEKi and NOTCHi to the cultures (Figure 3, thick striped bars, and Table 3). The combined administration more significantly decreased the expression of the NSC marker SOX2 (32.4 - 53%) and dramatically decreased the level of the proliferation marker KI67 (4.24 - 5.55 of all cells), which was accompanied by a dramatic increase in the expression level of the neuronal marker INA (42%) (Figure 3 and Table 3). Collectively, these results indicate that dual administration of both MEKi and NOTCHi had a synergistic effect in increasing the proportion of neurons and decreasing the proportions of NSCs and proliferating cells (Figure 3 and Table 3).

[0150] These results support the hypothesis that early addition of either MEKi, NOTCHi, and furthermore the combined administration of MEKi and NOTCHi, can promote the differentiation of ventral midbrain floor plate NSCs into ventral midbrain floor plate neurons without altering the expression of floor plate lineage transcription factors and cell types (Figure 1 and Figure 2 and Figure 3, Table 2 and Table 3).

[0151] After administration of the compounds and flow cytometry analysis performed in FIGS. 2 and 3, all groups were further differentiated in vitro for 11 days, then fixed in 4% paraformaldehyde and stained with DAPI to identify all cell nuclei and primary antibodies against SOX2 (1:300), Ki-67 (1:250), LMX1A (1:3000), INA (1:200), tyrosine hydroxylase (TH; 1:500) and COL1A1 (1:300), followed by identification of fluorescently labeled secondary antibodies. Images were obtained with an Olympus IX81 microscope using CellSens software. Immunofluorescence (IF) staining confirmed the results of flow cytometry in FIG. 3 with extended differentiation, showing that treatment with NOTCHi alone decreased the amounts of NSCs identified by SOX2 (FIGS. 4A - D) and proliferating cells identified by KI67 (FIGS. 5B, D) while maintaining the amount of neurons identified by INA compared to untreated controls (FIGS. 5A, C).

[0152] Also, consistent with the results of flow cytometry at earlier time points, extended differentiation ensured that treatment with MEKi alone decreased the amounts of NSCs identified by SOX2 (FIGS. 4A - B, E - F) and proliferating cells identified by KI67 (FIGS. 5B, F) while maintaining the amount of neurons identified by INA compared to untreated controls (FIGS. 5A, E). Further consistent with the results of flow cytometry at earlier time points, extended differentiation ensured that treatment with MEKi and NOTCHi decreased the amounts of NSCs identified by SOX2 (FIGS. 4A - B, G - H) and proliferating cells identified by KI67 (FIGS. 5B, H) while maintaining the amount of neurons identified by INA compared to untreated controls (FIGS. 5A, G).

[0153] By extending the differentiation, it became possible to analyze markers of the target subtype lineage, particularly tyrosine hydroxylase (TH), which is the rate-limiting enzyme that identifies dopaminergic neurons, and COL1A1, a gene expressed by non-neural stromal cells. The IF staining results of the extended differentiation showed that treatment with NOTCHi alone, treatment with MEKi alone, or dual treatment with both MEKi and NOTCHi did not prevent the formation of dopaminergic neurons (Figs. 6B, D, F, H), and comparison with the total number of cells identified by DAPI showed an increase in the proportion of TH neurons in the inhibitor-treated state (Figs. 6A-H). The IF staining results of the extended differentiation also showed that treatment with NOTCHi alone, treatment with MEKi alone, or dual treatment with both MEKi and NOTCHi did not prevent the expression of the ventral midbrain lineage marker LMX1A (Figs. 7A, C, E, G). Most notably, treatment with either the MEKi alone inhibitor or the NOTCHi alone inhibitor dramatically reduced the proportion of stromal COL1A1 cells compared to the control untreated cultures (Figs. 7B, D-F), while dual administration of MEKi and NOTCHi further reduced that proportion (Fig. 7H).

[0154] These results support the hypothesis that early addition of either MEKi, NOTCHi, or even the combined administration of MEKi and NOTCHi can promote the differentiation of ventral midbrain floor plate NSCs into ventral midbrain floor plate neurons, particularly dopaminergic neurons, at the expense of non-neural cells such as stromal cells without modifying the expression of floor plate lineage transcription factors such as LMX1A (Figs. 4-7).

[0155] Example 4: Late in vitro administration of MEKi and / or NOTCHi To evaluate the scope and ability of MEKi and / or NOTCHi to promote the differentiation of ventral midbrain floor plate NSCs into ventral midbrain floor plate neurons, particularly dopaminergic neurons, at the expense of non-neuronal cells such as stromal cells, additional studies were also performed in vitro on cultures differentiated to late developmental stages at a late time point (summarized in FIGS. 8 and 11 and Tables 4 and 5).

[0156] Cultures of VM NPCs that had completed the stages of hPSC neural / ectodermal specification, ventralization, and caudalization were left without these patterning factors and allowed to further differentiate in vitro for a period of 6 days. During this period (days 16 - 22 in vitro) and all previous time points (days 0 - 22 in vitro), the cell cultures were not exposed to MEK inhibitor and / or NOTCH inhibitor (FIG. 8). During this 16 - 22 day period, compared to the early administration time points in FIGS. 1 - 2 and Table 2, the level of SOX2 decreased and there was an increase in the proportion of cells expressing the intermediate progenitor and neuron markers ASCL1 and INA and INA+ / SOX2− (FIG. 9, Table 4), so the cells acquired a higher proportion of cells with more mature developmental identities such as IPCs and neurons. The cells were then exposed to MEK inhibitor and / or NOTCH inhibitor (FIG. 8), which is referred to as "late administration".

[0157] The inventors contemplate that this population can be used for the purpose of restoring function in animal models of Parkinson's disease and for transplantation into human patients for therapeutic purposes. [Table 4]

[0158] Before late administration, cells were profiled by flow cytometry (Figure 9 and Table 4) to evaluate the protein expression levels of ventral midbrain floor plate homeobox transcription factor (FOXA2) and cell stage markers (NSC and proliferation markers SOX2 and KI67; ASCL1 to identify IPCs; INA to identify neurons). These analyzed cultures expressed the ventral midbrain floor plate marker FOXA2 (>60% of all cells) and were observed to be ventral midbrain in their regional identity (Figure 9). These cells were still at the NSC stage, as indicated by high expression of the NSC markers SOX2 and KI67 (>79% and >50%; Figure 9, 4). Notably, these cultures were more mature than the early administration cultures treated in Figures 1 - 2, as the levels of ASCL1 increased dramatically up to 48% and total expression of INA increased to 12.8%, where some cells were 3.28% INA+ / SOX2- (Figure 9 and Figure 4).

[0159] After 5 days of exposure to MEKi alone, NOTCHi alone, or both MEKi and NOTCHi, the cultures were re - analyzed using flow cytometry to evaluate the protein expression levels of key genes and compared to untreated controls (Figure 10, Table 5). Additionally, the cultures were further differentiated for 7 days in the absence of any NOTCH inhibitor and ICC was performed using DAPI to identify all cell nuclei and primary antibodies against FOXA2 (1:200), LMX1A (1:1000), SOX2 (1:300), Ki - 67 (1:250), and HuC / D (1:100)

Table 5

[0160] All groups were found to maintain high expression (of total cells) of the ventral midbrain floor plate lineage markers FOXA2 (>68%) and LMX1A (>80%) (Figure 10 and Table 5). Control cells were observed to maintain high expression of the NSC marker SOX2 (average 74%) and high levels of the proliferation marker KI67 (average 34.6%), along with moderate levels of the neuronal marker INA (average 30.5%) (Figure 10, white bars and Table 5). Collectively, this indicates that the control cultures were mainly ventral midbrain floor plate neural stem cells, and a subset were neurons at this late time point in vitro. In contrast, both MEKi-only treated cultures (Figure 10, thin striped bars, and Table 5) and NOTCHi-only treated cultures (Figure 10, thick striped bars, and Table 5) were observed to have downregulated expression of the NSC as well as the proliferation markers SOX2 and KI67, indicating that in response to these inhibitors, their cells were differentiating into neurons at a higher rate compared to the control. Co-administration of MEKi and NOTCHi to the cultures (Figure 10, black / filled bars, Table 5) also decreased the expression of the NSC marker SOX2 (average 45.4%) and dramatically decreased the level of the proliferation marker KI67 (average 3.7% of all cells), which was accompanied by a dramatic increase in the expression level of the neuronal marker INA (average 68.8%) (Figure 10, Table 5). Collectively, these results indicate that dual administration of MEKi, NOTCHi, or even inhibitors of both pathways had the effect of increasing neurons and decreasing the proportion of NSCs and proliferating cells. Comparison of the results of early vs. late treatment of VM neural cells with MEKi and NOTCHi indicates that late administration resulted in a greater amount of INA neurons (average 68.8%) compared to early administration (average 42%).

[0161] These results support the hypothesis that late addition of MEKi or NOTCHi, or combined administration of MEKi and NOTCHi, can promote the differentiation of ventral midbrain floor plate NSCs into ventral midbrain floor plate neurons without modifying the expression of floor plate lineage transcription factors. Overall, the data indicate that late administration of MEKi and NOTCHi is favorable and superior for shifting VM cells into VM neurons.

[0162] After administration of the compounds and flow cytometry analysis performed in the figures, all groups were further differentiated in vitro for 11 days, then fixed in 4% paraformaldehyde and stained with DAPI to identify all cell nuclei and primary antibodies against SOX2 (1:300), Ki-67 (1:250), LMX1A (1:1000), tyrosine hydroxylase (TH; 1:500) and COL1A1 (1:300), followed by identification of fluorescently labeled secondary antibodies. Images were obtained with an Olympus IX81 microscope using CellSens software. Immunofluorescence (IF) staining confirmed the results of flow cytometry of extended differentiation, showing that treatment with NOTCHi alone decreased the amount of NSCs identified by SOX2 (Figure 12A - D) and proliferating cells identified by KI67 (Figure 13B, D), while maintaining the amount of neurons identified by INA compared to untreated controls (Figure 13A, C).

[0163] Also in line with the flow cytometry results at earlier time points, by extending differentiation, treatment with MEKi alone, while maintaining the amount of neurons identified by INA compared to untreated controls, decreased the amount of NSCs identified by SOX2 (Figures 12A - B, E - F) and the amount of proliferating cells identified by KI67 (Figures 13B, F) (Figure 13A, E). Even more in line with the flow cytometry results at earlier time points, by extending differentiation, treatment with MEKi and NOTCHi, while maintaining the amount of neurons identified by INA compared to untreated controls, decreased the amount of NSCs identified by SOX2 (Figures 12A - B, G - H) and the amount of proliferating cells identified by KI67 (Figures 13B, H) (Figure 13A, G).

[0164] By extending differentiation, it became possible to analyze markers of the target subtype lineage, particularly tyrosine hydroxylase (TH), the rate - limiting enzyme that identifies dopaminergic neurons, and COL1A1, a gene expressed by non - neural stromal cells. The IF staining results of extended differentiation showed that treatment with NOTCHi alone, MEKi alone, or dual treatment with both MEKi and NOTCHi did not prevent the formation of dopaminergic neurons (Figures 14B, D, F, H), and comparison with the total number of cells identified by DAPI showed an increase in the proportion of TH neurons in the inhibitor - treated state (Figures 14A - H). The IF staining results of extended differentiation also showed that treatment with NOTCHi alone, MEKi alone, or dual treatment with both MEKi and NOTCHi did not prevent the expression of the ventral midbrain lineage marker LMX1A (Figures 15A, C, E, G). Most notably, treatment with either the MEKi inhibitor alone or the NOTCHi inhibitor alone dramatically decreased the proportion of stromal COL1A1 cells compared to control untreated cultures (Figures 15B, D, F), while dual administration of MEKi and NOTCHi further decreased that proportion (Figure 15H).

[0165] These results support the hypothesis that late addition of either MEKi, NOTCHi, or even the combination of MEKi and NOTCHi can promote the differentiation of ventral midbrain floor plate NSCs into ventral midbrain floor plate neurons, particularly dopaminergic neurons, at the expense of non-neuronal cells such as stromal cells without altering the expression of floor plate lineage transcription factors such as LMX1A (Figs. 12-15).

[0166] Example 5: Profile of late-stage VM nervous system cells after early treatment with NOTCHi from DIV16-22 In this example, cultures of VM NPCs that had completed the stages of hPSC neural / ectodermal specification, ventralization, and caudalization were left without these patterning factors and allowed to further differentiate in vitro for a period of 6 days starting from day 16 and were exposed to the NOTCH inhibitor (DAPT) every 48-72 hours between DIV16-22. Exposing the cultures to the NOTCH inhibitor immediately after cessation of the use of one or more or all of the patterning factors is typically carried out in the art, and the patterning factors are provided, for example, as described in the publications Nolbrant et al., 2017, Kriks et al., 2011, Nishimura et al., 2023, those provided to induce the embryonic neuroectoderm (i.e., noggin, SMAD inhibitor) and / or dorsal-ventral patterning factors (i.e., BMP, SHH, SAG, purmorphamine) and / or rostral-caudal patterning factors (i.e., WNT protein, CHIR, FGF8). During this period (DIV16-22) and all previous time points (days 0-16 in vitro), the cell cultures were not exposed to the MEK inhibitor.

[0167] Immediately after the patterning factor is removed by DIV16, when the culture is mainly NPC and does not contain or contains <2% of INA+ / SOX2- cells (Figure 2, Table 1), and by DIV22, the addition of this NOTCH inhibition has a significant impact on the cell stage / maturation that means the proportion of IPCs and neurons at DIV22. This is observable at the protein level via the flow cytometry profile. Refer to Figure 9 and Table 4 showing the profile at DIV22 where no early NOTCH inhibition was performed, and compare with Figure 11 and Table 6 where early NOTCHi was performed. In particular, early (DIV16 - 22) treatment with NOTCH inhibition results in a higher proportion of neurons, such as INA+ cells (24.7% with NOTCHi compared to 12.8% without NOTCHi) or INA+ / SOX2- cells (8.4% with NOTCHi compared to 3.3% with NOTCHi). Therefore, the inventors contemplate that this early treatment with NOTCHi immediately after the removal of the patterning factor is not an optimal culture for exposure to the dual treatment of MEKi / NOTCHi and conversion to neurons because there are already more cells in the neuronal stage. Furthermore, the inventors expect that this population with more neurons is not optimal for transplantation (because neurons are vulnerable and sensitive to transplantation stress), and they expect that the population in Figure 9 and Table 4, which contains many ASCL1+ IPCs (51.8%) with minimal neurons (INA+ or INA+ / SOX2-) cells, is more optimal for transplantation and / or for the additional dual inhibition of MEK and NOTCH.

[0168] Reference: ·Kaneyasu Nishimura,Shanzheng Yang,Ka Wai Lee,Emila Sif Asgrmsdottir,Kasra Nikouei,Wojciech Paslawski,Sabine Gnodde,Guochang Lyu,Lijuan Hu,Carmen Salto,Per Svenningsson,Jens Hjerling-Leffler,Sten Linnarsson,and Ernest Arenas.Single-cell transcriptomics reveals correct developmental dynamics and high quality midbrain cell types by improved hESC differentiation.2023.Stem Cell Reports.https: / / doi.org / 10.1016 / j.stemcr.2022.10.016 ·Sonja Kriks,Jae-Won Shim,Jinghua Piao,Yosif M Ganat,Dustin R Wakeman,Zhong Xie,Luis Carrillo-Reid,Gordon Auyeung,Chris Antonacci,Amanda Buch,Lichuan Yang,M Flint Beal,D James Surmeier,Jeffrey H Kordower,Viviane Tabar,Lorenz Studer.Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease.2011.Nature. 10.1038 / nature10648

Table 6

[0169] Example 6: Further culture the cell population after MEK / NOTCH inhibition to evaluate the expression profile After administration of the MEK inhibitor and the NOTCH inhibitor, the cells are further cultured for 5 more days in a 2D culture in wells coated with poly-L-ornithine (0.002%) and laminin-521 (1.5 μg / cm2) in a neural support medium supplemented with BDNF (20 ng / mL), GDNF (20 ng / mL), L-ascorbic acid (200 μM), and dcAMP (500 μM) or other such support media. This time allows for the migration of neural precursors and intermediates and final differentiation into ventral midbrain neurons. Five days after the culture, the expression profile of the cell population can be evaluated according to any one of the methods described in Example 7, Example 8, and Example 9.

[0170] Example 7: RNA Sequencing Method To perform single-cell RNA sequencing (scRNA-seq) or single-nucleus RNA sequencing, those of undifferentiated PSCs as well as differentiated cells were dissociated into single-cell suspensions with Accutase, Tryple Select, or other such reagents, 3000 - 10000 cells were processed using the 10X Genomics Chromium Platform, and sequenced with NextSeq550. The data was processed in the R programming language using the 10X Cellranger and Seurat analysis packages. After analyzing the samples, filtering for low-quality cells or multiplet cells, and analyzing each individual experiment separately, the selected differentiated cell lineages as well as the cells of hPSCs were combined into one dataset, which was then analyzed using the standard Seurat workflow as outlined for Seurat version 3, i.e., normalization using SCTransform and finally using the first 29 principal components of the integrated tSNE plot.

[0171] Example 8: Immunocytochemistry (ICC) Cells were fixed in 4% paraformaldehyde (Alfa Aesar) for 10 minutes at room temperature. Nonspecific antibody binding was blocked by incubating the cells with PADT buffer, calcium- and magnesium-free phosphate-buffered saline (PBS) (Gibco) containing 0.02% sodium azide solution (Ampliqon), 0.5% Triton X-100 (Sigma), and 5% donkey serum (Jackson Labs) for 30 minutes, followed by incubation overnight at 4°C with the primary antibody (see Table X). Cells were washed three times with calcium- and magnesium-free PBS, blocked with PADT buffer for 15 minutes, and incubated for 2 hours at room temperature with a fluorophore-conjugated secondary antibody (see Table X), protected from light. Next, cells were counterstained with DAPI (10 μg / mL) for 5 minutes at room temperature, washed three times with calcium- and magnesium-free PBS, and stored at 4°C in calcium- and magnesium-free PBS supplemented with 0.02% sodium azide. Images were captured using a Zeiss Axio Observer microscope equipped with an Axiocam 512 camera and ZEN 3.2 (Pro) software (Zeiss).

[0172] Example 9: Quantitative real-time PCR (qPCR, or qRT-PCR) To perform qPCR, RNA was extracted from cells using Trizol, converted to cDNA, and subsequently analyzed for the genes of interest, such as SOX2, KI67, HuCD, NeuN, INA, ASCL1, FOXA2, TH, LMX1A, EN1, or other relevant markers for ventral midbrain CNS cells, using quantitative real-time polymerase chain reaction (qPCR). qPCR was typically performed in triplicate technical replicates for each of three or more independent biological replicates and normalized to a housekeeping gene such as GAPDH or HPRT1.

[0173] While certain features of the present invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will occur to those skilled in the art. Accordingly, it is to be understood that the appended claims are intended to cover all such modifications and changes that fall within the true spirit of the invention.

Claims

[Claim 1] The invention described in the specification.

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