Enhancing neuronal differentiation of ventral midbrain neural progenitor cells

Abstract

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

Description

Technical Field

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

[0002] The prospect of using human pluripotent stem cells (hPSC) to treat various conditions seems very bright. Treatment includes cell replacement therapy for neurological conditions such as Parkinson's disease and stroke. However, in order to make such treatment feasible, it is necessary to develop in vitro methods to artificially produce stem cell-derived products so that they are delivered to the central nervous system (CNS). The differentiation of hPSC to a defined cell type is an uncontrollable process; in many cases, the offspring produced by in vitro schemes are heterogeneous. Typically, when differentiating to neural cells, the mixture of cell types produced includes neurons (and including a variety of neuronal subtypes, such as glutamatergic neurons and dopaminergic neurons), glial cells, neural stem cells (NSC) and other non-neuronal cells (e.g., meningeal matrix cells). This type of heterogeneous culture is not optimal for disease modeling studies or many cell replacement therapies.

[0003] Cell replacement therapy for Parkinson's disease is a classic example. In Parkinson's disease, A9 ventral midbrain dopaminergic neurons (vmDA) are lost and are arguably the only cell type that must be transplanted to restore the lost function. However, all current academic scientific publications and all ongoing human clinical trials transplant multipotent progenitor cell populations, which results in a mixed cell population in addition to vmDA in the body. This mixed population includes cell types such as proliferating NSCs, vascular leptomeningeal cells (VLMCs), non-vmDA neurons, and astrocytes. Non-dopaminergic neurons cannot restore Parkinson's disease function, while some neurons (i.e., 5-hydroxytryptaminergic neurons) produce negative gain-of-function behaviors in patients, as seen in clinical trials transplanting human fetal cells, and more generally, these non-vmDAs bring unknown safety and efficacy risks. Therefore, there is a need to provide methods to ensure that patients are treated with ventral midbrain neurons or their progenitors.

[0004] It is therefore an object of the present invention to overcome the above challenges and in particular to provide methods that can direct the differentiation of cells into ventral midbrain neurons. Summary of the invention

[0005] The objects outlined above are achieved by aspects of the present invention. In addition, the present invention can also solve other problems, which will be 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 NSC with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. Specifically, the method is used to guide the differentiation of ventral midbrain NSC into neurons, which means that the developmental fate of ventral midbrain neural cells is affected towards a certain result, namely neurons. However, in the process of the method disclosed herein, differentiation into neurons is not necessarily completed, and cells may not necessarily develop into the final fate. Preferably, cells in the progenitor cell stage are guided toward differentiation into neurons in vitro and are suitable for administration to patients, wherein further development from the progenitor cell stage in vivo produces a neuronal fate. The inventors have found that by jointly inhibiting the MEK signaling pathway and the NOTCH signaling pathway, differentiation and preparation of ventral midbrain neurons from cell populations such as human PSCs can be improved. In particular, the inventors were surprised to find that the effect of MEK pathway inhibition can be enhanced by jointly adding NOTCH pathway antagonists / inhibitors. Specifically, this combined inhibition reduces the proportion of late proliferating cells (which represent off-target lineages), reduces the proportion of non-neural cells (such as matrix cells, such as VLMC), and increases the proportion of neurons. Importantly, the inventors have found that the timing of inhibiting MEK and NOTCH signaling relative to the developmental stage of the cell has an effect on the outcome. In a preferred embodiment, the neural cells at the time of inhibiting MEK and NOTCH signaling are at a stage where the neural cells contain a mixture of neural stem cells, intermediate precursor cells of neuroblasts, and a small amount of neurons. In a preferred embodiment, the ventral midbrain NSCs before inhibiting MEK signaling are neurally induced, ventralized, and caudalized. In a preferred embodiment, at least 5% of the cell populations comprising ventral midbrain NSCs co-express markers FOXA2, LMX1A, EN1, OTX2, and SOX2. A cell population comprising such a mixture of ventral midbrain neural cells can be produced by any method. However, in an embodiment, the cell population is neurally induced, caudalized, and ventralized before inhibiting MEK and NOTCH signaling. The cell population can be derived from pluripotent cells, such as PSCs. Therefore, in a preferred embodiment, a cell population of PSCs is subjected to neural induction, ventralization and caudalization according to well-known methods, such as exposing PSCs to inhibitors of SMAD protein signaling, activators of SHH signaling and inhibitors of Wnt signaling, and further contacting the cell population with an activator of FGF signaling. A cell population of neural induction cells such as ventral midbrain NSCs can be obtained according to a differentiation method that lasts, for example, 16 days from the start of neural induction, wherein the resulting cell population mainly comprises neural stem cells.Further culturing the cell population, for example, for up to 8 days, allows more neural stem cells to further develop into neuroblast intermediate progenitor cells, but still retains a window of opportunity to guide the ventral midbrain neural cells to further differentiate into neurons by contacting the cells with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. In the above-mentioned method of neural induction of PSCs, it has been found that cells cultured for too long (e.g., more than 28 days) may be difficult to harvest, for example, due to the formation of neurite networks that cannot be separated.

[0007] Those skilled in the art will recognize that the optimal time for contacting a cell population with an inhibitor may vary depending on the specific protocol used to differentiate a PSC into a ventral midbrain NSC. However, the inventors have determined the expression profile of a cell population comprising a ventral midbrain NSC that provides an improved neuronal outcome. Therefore, in a preferred embodiment, when a cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, 40-60% of the cell population comprising the ventral midbrain NSC express ASCL1, 45-65% of the cell population comprising the ventral midbrain NSC express KI67, less than 10-15% of the cell population comprising the ventral midbrain NSC express INA, 2-5% of the cell population comprising the ventral midbrain NSC is INA+ / SOX2-, and 80-95% of the cell population comprising the ventral midbrain NSC express SOX2. Thus, in embodiments where a cell population is differentiated into a cell population comprising ventral midbrain NSCs, the cell population is allowed to differentiate until the cell population acquires the aforementioned expression profile, at which time the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling.

[0008] The in vitro cell population obtained according to the method can be used to treat Parkinson's disease, and due to the higher purity of the cell product, the operation time can be shortened and the intracranial injection can be reduced. In addition, the improvement of product purity and the reduction of impurities are expected to provide enhanced safety and potential efficacy profiles.

[0009] Another aspect of the present invention relates to an inhibitor of MEK signaling for treating Parkinson's disease in a subject to whom a therapeutically effective amount of ventral midbrain NSC has been administered. Specifically, the ventral midbrain NSC co-expresses markers FOXA2, LMX1A, EN1, OTX2, and SOX2. The inventors have thought that the effects of MEK and NOTCH inhibition confirmed in vitro can be directly converted into in vivo effects, guiding the administered ventral midbrain NSC to differentiate into neurons. Therefore, the inventors believe that after a cell population comprising ventral midbrain NSC is administered to a subject, for example, by surgery, the administered cells can be further guided to differentiate into neurons, thereby reducing the proportion of late proliferating cells and non-neuronal cells such as VLMC, and increasing the proportion of neurons. In an embodiment, the inhibitor of MEK signaling is Mirdametinib, a clinically tested small molecule that acts as a MEK inhibitor and crosses the blood-brain barrier. In an embodiment, the ventral midbrain NSC is co-administered with an inhibitor of MEK signaling or treated with an inhibitor before transplantation.

[0010] Similar aspects of the invention relate to a combination of an inhibitor of MEK signaling, an inhibitor of NOTCH signaling, and a cell population comprising ventral midbrain NSCs for treating 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 treating Parkinson's disease. Here, the inventors contemplate co-administering ventral midbrain NSCs with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. Prior to co-administration, 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. It has been demonstrated that even a short exposure to an inhibitor of MEK and NOTCH signaling directs a cell population comprising ventral midbrain NSCs toward a neuronal fate, and a single co-administration of cells and inhibitors may be sufficient to direct further cell maturation in vivo, thereby reducing the dose administration required for the patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 A simplified schematic diagram illustrating the stages of VM differentiation is shown. The process begins with hPSCs (shown in white graphics), which differentiate into VM NSC cells (shown in vertical striped graphics) after neural induction, ventralization, and caudalization. After these steps, and when VM IPCs (shown in horizontal striped graphics) and VM neurons (shown in black graphics) have low expression, a compound of the invention, MEK and / or NOTCH inhibitors, is administered. Adding inhibitors immediately after neural induction, ventralization, and caudalization is referred to as "early administration."

[0012] Figure 2 Bar graphs of intracellular flow cytometry protein analysis results of cell cultures at the start of early compound administration are shown. Expression of NSC marker SOX2 and proliferation marker KI67 are shown. Expression of VM and floor plate lineage markers FOXA2, LMX1A and OTX2 and VM floor plate IPC marker ASCL1 and neuronal marker INA are also shown. The figure shows the percentage of cells in total viable cells.

[0013] Figure 3 Shown is a bar graph of the intracellular flow cytometry protein analysis results of cell culture on the 22nd day in vitro after early application.Show the results of the following culture: no intervention (control, black bars), use of MEK inhibitors (+MEKi, white bars), use of NOTCH inhibitors (+NOTCHi, narrow striped bars), and use of MEK and NOTCH inhibitors (+MEKi+NOTCHi, thick striped bars).Show the protein expression of NSC marker SOX2 and proliferation marker KI67, neuron marker INA and VM floor plate lineage markers FOXA2 and LMX1A.The figure shows the percentage of cells in total living cells.

[0014] Figure 4 Representative immunofluorescence images of cultures that received early administration and differentiated for an extended period of time to day 35 in vitro are shown. Results are shown for control conditions (AB), after administration of a NOTCH inhibitor (CD), after administration of a MEK inhibitor (EF), and after administration of both a MEK and NOTCH inhibitor (GH). Staining of all nuclei with DAPI (A, C, E, G) and staining of SOX2 cells (B, D, F, H).

[0015] Figure 5 Representative immunofluorescence images of cultures that received early administration and differentiated for an extended period of time to day 35 in vitro are shown. Results are shown for control conditions (AB), after administration of a NOTCH inhibitor (CD), after administration of a MEK inhibitor (EF), and after administration of both a MEK and NOTCH inhibitor (GH). Staining of all neuronal fibers with the marker INA (A, C, E, G) and staining of all proliferating cells with KI67 (B, D, F, H).

[0016] Figure 6Representative immunofluorescence images of cultures that received early administration and differentiated for an extended period of time to day 35 in vitro are shown. Results are shown for control conditions (AB), after administration of a NOTCH inhibitor (CD), after administration of a MEK inhibitor (EF), and after administration of both a MEK and NOTCH inhibitor (GH). Staining of all nuclei with DAPI (A, C, E, G) and staining of all dopamine neurons with tyrosine hydroxylase (B, D, F, H).

[0017] Figure 7 Representative immunofluorescence images of cultures that received early administration and differentiated for an extended period of time to day 35 in vitro are shown. Results are shown for control conditions (AB), after administration of a NOTCH inhibitor (CD), after administration of a MEK inhibitor (EF), and after administration of both a MEK and NOTCH inhibitor (GH). Staining of all ventral midbrain floor plate lineage cells with the marker LMX1A (A, C, E, G) and staining of non-neuronal stromal cells with the marker COL1A1 (B, D, F, H).

[0018] Figure 8 A simplified schematic diagram illustrating the stages of VM differentiation is shown. The process begins with hPSCs (represented by white graphics), which differentiate into VM NSC cells (represented by vertical stripes graphics) after neural induction, ventralization, and caudalization. After these steps, and in the absence of patterning factors, after a period of time, when the ratio of VM IPCs (represented by horizontal stripes graphics) and VM neurons (represented by black graphics) increases and VM NSCs decrease, a compound of the present invention, MEK and / or NOTCH inhibitors, is administered. Adding inhibitors after a period of neural induction, ventralization, and caudalization in the absence of patterning factors is referred to as "late administration."

[0019] Fig. 9 Bar graphs of intracellular flow cytometry protein analysis of cell cultures are shown when cells are subjected to late compound administration (MEKi, NOTCHi, or MEKi+NOTCHi); note that these cells were not previously exposed to any NOTCH or MEK inhibitors. Expression of NSC marker SOX2 and proliferation marker KI67 is shown. Expression of VM and floor plate lineage markers FOXA2, LMX1A, and OTX2 as well as VM floor plate IPC marker ASCL1 and neuronal marker INA are also shown. The figure shows the percentage of cells in total viable cells.

[0020] Fig.10Bar graphs of intracellular flow cytometry protein analysis results of cell cultures at the start of late compound administration are shown. Expression of NSC marker SOX2 and proliferation marker KI67 are shown. Expression of VM and floor plate lineage markers FOXA2, LMX1A and OTX2 and VM floor plate IPC marker ASCL1 and neuronal marker INA are also shown. The figure shows the percentage of cells in total viable cells.

[0021] Fig.11 Bar graphs of intracellular flow cytometry protein analysis of cell cultures are shown while late compound administration (MEKi, NOTCHi or MEKi+NOTCHi) is performed, however, these cells have been previously exposed to NOTCH inhibitors, as is conventionally done in the art, and this is not ideal and not preferred for administration of MEKi and / or NOTCHi treatments. Expression of NSC marker SOX2 and proliferation marker KI67 is shown. Expression of VM and floor plate lineage markers FOXA2, LMX1A and OTX2 as well as VM floor plate IPC marker ASCL1 and neuronal marker INA are also shown. The figure shows the percentage of cells as a percentage of total viable cells.

[0022] Fig.12 Representative immunofluorescence images of cultures that received late administration and differentiated for an extended period of time to day 40 in vitro are shown. Results are shown for control conditions (AB), after administration of a NOTCH inhibitor (CD), after administration of a MEK inhibitor (EF), and after administration of both a MEK and NOTCH inhibitor (GH). Staining of all nuclei with DAPI (A, C, E, G) and staining of SOX2 cells (B, D, F, H).

[0023] Fig.13 Representative immunofluorescence images of cultures that received late administration and differentiated for an extended period of time to day 40 in vitro are shown. Results are shown for control conditions (AB), after administration of a NOTCH inhibitor (CD), after administration of a MEK inhibitor (EF), and after administration of both a MEK and NOTCH inhibitor (GH). Staining of all neuronal fibers with the marker INA (A, C, E, G) and staining of all proliferating cells with KI67 (B, D, F, H).

[0024] Fig.14Representative immunofluorescence images of cultures that received late administration and differentiated for an extended period of time to day 40 in vitro are shown. Results are shown for control conditions (AB), after administration of a NOTCH inhibitor (CD), after administration of a MEK inhibitor (EF), and after administration of both a MEK and NOTCH inhibitor (GH). Staining of all nuclei with DAPI (A, C, E, G) and staining of all dopamine neurons with tyrosine hydroxylase (B, D, F, H).

[0025] Fig.15 Representative immunofluorescence images of cultures that received late administration and differentiated for an extended period of time to day 40 in vitro are shown. Results are shown for control conditions (AB), after administration of a NOTCH inhibitor (CD), after administration of a MEK inhibitor (EF), and after administration of both a MEK and NOTCH inhibitor (GH). Staining of all ventral midbrain floor plate lineage cells with the marker LMX1A (A, C, E, G) and staining of non-neuronal stromal cells with the marker COL1A1 (B, D, F, H).

[0026] Fig.16 A scheme for neural induction, ventralization and caudalization of a cell population of PSCs to become ventral midbrain NSCs and further directing cell differentiation toward neurons is shown. In (A), a cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling at day 16. In (B), after removal of patterning factors (i.e., SMAD inhibitors, SHH agonists, WNT agonists, and FGF8), the cell population is allowed to further differentiate for a period of time in the intentional absence of NOTCHi, and then contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling at day 22.

[0027] Fig.17 A simplified schematic diagram illustrating the stages of VM differentiation is shown. The process begins with hPSCs (represented by white graphics), which differentiate into VM NSC cells (represented by vertical stripes graphics) after neural induction, ventralization, and caudalization. After these steps, and after a period of time in the absence of patterning factors, when the ratio of VM IPCs (represented by horizontal stripes graphics) and VM neurons (represented by black graphics) increases and VM NSCs decrease, a compound of the invention, a NOTCH inhibitor, is administered. Adding a NOTCH inhibitor after a period of neural induction, ventralization, and caudalization in the absence of patterning factors is referred to as "late administration of a single NOTCHi". DETAILED DESCRIPTION

[0028] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which the invention belongs. Unless otherwise specified, conventional methods of chemistry, biochemistry, biophysics, molecular biology, cell biology, genetics, immunology and pharmacology known to those skilled in the art are used in the practice of the present invention.

[0029] It should be noted that all headings and subheadings used herein are for convenience only and should not be construed as limiting the invention in any way.

[0030] The use of any and all examples, or exemplary language (eg, "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0031] definition

[0032] General Definition

[0033] As used herein, "a" or "an" or "the" may mean one or more than one. Unless otherwise specified in the specification, terms presented in the singular form also include plural cases.

[0034] As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the relevant listed items, and when interpreted in an alternative manner ("or"), refers to the lack of a combination. In addition, the present invention also contemplates that in some embodiments of the present invention, any feature or combination of features set forth herein may be excluded or omitted.

[0035] Stem Cells

[0036] "Stem cells" should be understood as undifferentiated cells that have differentiation potential and proliferation ability (especially self-renewal ability) but retain differentiation potential. According to their differentiation potential, stem cells include categories such as pluripotent stem cells, multipotent stem cells, unipotent stem cells, etc.

[0037] As used herein, the term "pluripotent stem cell" (PSC) refers to stem cells that can be cultured in vitro and have the potential to differentiate into any cell lineage belonging to the three germ layers (ectoderm, mesoderm, endoderm), as well as lineage-restricted undifferentiated stem cells that have lost the ability to form certain cell types, lineages, or developmental regions, usually through gene editing.

[0038] Pluripotent stem cells can be induced or isolated from fertilized eggs, somatic cell nuclear transfer embryos, germ stem cells, stem cells in tissues, somatic cells, etc. Examples of pluripotent stem cells (PSCs) include embryonic stem cells (ESCs), embryonic germ cells (EG cells), induced pluripotent stem cells (iPSCs), etc.

[0039] As used herein, the term "induced pluripotent stem cells" (also referred to as iPS cells or iPSCs) refers to a type of pluripotent stem cell that can be generated directly from a cell that is not a PSC and has a nucleus. Non-pluripotent cells can be converted into pluripotent stem cells by introducing the products of a specific set of pluripotency-related genes.

[0040] 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 can also be derived from parthenogenetic bodies (parthenotes), as described in, for example, WO 2003 / 046141. In addition, embryonic stem cells can be produced from a single blastomere or produced by culturing an inner cell mass obtained without destroying an embryo. Embryonic stem cells can be obtained from a given organization or can be commercially available. Preferably, the method and product of the present invention are based on human PSC, i.e. stem cells derived from human induced pluripotent stem cells or human embryonic stem cells (including parthenogenetic bodies).

[0041] As used herein, the term "multipotent stem cell" means a stem cell that has the potential to differentiate into multiple types of tissues or cells (but not all kinds), and is usually restricted to one germ layer. Neural stem cells are an example of multipotent stem cells restricted to the neural lineage.

[0042] As used herein, the term "unipotent stem cell" means a stem cell that has the potential to differentiate into only one specific cell type.

[0043] As used herein, the term "in vitro" means that cells are provided and maintained outside the human or animal body, for example in a container such as a flask, multiwell or culture dish. It can be seen that the cells are cultured in a cell culture medium.

[0044] As used herein, the term "non-natural" refers to an artificial construct that does not exist in nature, although the cell is derived from a pluripotent stem cell that may have a human origin. In general, providing cells that are as similar as possible to human cells is a goal in the field of stem cell therapy. However, it may never be possible to simulate the development that pluripotent stem cells experience in the embryonic and fetal stages to the extent that mature cells are indistinguishable from natural human cells. In essence, in embodiments of the present invention, the cell is artificial.

[0045] As used herein, the term "artificial" in relation to cells can include materials that occur naturally in nature but are modified into non-naturally occurring constructs. This includes human stem cells that differentiate into non-naturally occurring cells that mimic human body cells.

[0046] plan

[0047] Throughout this application, the terms "method" and "protocol" are used interchangeably when referring to cell culture or differentiation processes.

[0048] As used herein, the term "day" and the similar term day in vitro (DIV) in relation to a protocol refer to the specific time during which certain steps are performed during a differentiation procedure.

[0049] In general, unless otherwise indicated, "Day 0" refers to the start of the protocol, which is performed by, for example, but not limited to, plating or transferring the stem cells to a culture vessel, or contacting the stem cells with a compound in their current cell culture medium prior to transferring the stem cells. Typically, the protocol is initiated by transferring undifferentiated stem cells to a different cell culture medium and / or container, for example, but not limited to, by plating or incubation, and / or first contacting the undifferentiated stem cells with one or more compounds that affect the undifferentiated stem cells in a manner that initiates the differentiation process.

[0050] When referring to "cells" in the methods, all cells of a cell population are meant, regardless of cell type.

[0051] When reference is made to "Day X," such as Day 1, Day 2, etc., this is relative to the start of the protocol on Day 0. One of ordinary skill in the art will recognize that the exact time of day that the step is performed may vary unless otherwise specified. Thus, "Day X" is intended to encompass time spans such as + / -10 hours, + / -8 hours, + / -6 hours, + / -4 hours, + / -2 hours, or + / -1 hour.

[0052] Cultivating stem cells

[0053] As used herein, the term "culture" refers to a continuous procedure that is used throughout the method to maintain the viability of the cells at their various stages. After the cells of interest are isolated from, for example, but not limited to, living tissue or embryos, they are maintained under carefully controlled conditions. These conditions vary for each cell type, but are generally composed of a suitable container with a substrate and / or culture medium that provides essential nutrients (amino acids, carbohydrates, vitamins, minerals), growth factors, hormones, and gases (CO2, O2) and regulates the physicochemical environment (pH buffer, osmotic pressure, temperature).

[0054] As used herein, the term "cell culture medium" refers to a liquid or gel designed to support cell growth. Cell culture medium typically contains an appropriate energy source and compounds that regulate the cell cycle.

[0055] As used herein, the term "culture vessel" refers to any suitable culture vessel that can support cell culture. Non-limiting examples include culture dishes, culture plates and plates (microtiter plates, microtiter plates, deep well plates, etc., with 6 holes, 24 holes, 48 ​​holes, 96 holes, 384 holes, 9600 holes, etc.), flasks, compartment slides, tubes, cell factory systems (Cell Factory), roller bottles, spinner bottles, hollow fibers, microcarriers or beads.

[0056] As used herein, the term "providing stem cells" when referred to in the protocol means obtaining a batch of cells by a method such as described above, and optionally transferring the cells to a different environment, such as by seeding onto a new substrate. One of ordinary skill in the art will readily recognize that stem cells are fragile to such transfer, and thus the procedure requires caution, and maintaining the stem cells in the original cell culture medium before replacing the cell culture medium with another cell culture medium more suitable for the further differentiation process can promote more sustainable cell transfer.

[0057] Differentiating stem cells

[0058] As used herein, the term "expression" in relation to a gene or protein refers to the presence of an RNA molecule, which can be detected using assays such as reverse transcription quantitative polymerase chain reaction (RT-qPCR), RNA sequencing, and / or the presence of a protein, which can be detected, for example, 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 can be considered expressed when at least one molecule is detected (e.g., in RNA sequencing), or a limit of detection above the background / noise level can be defined relative to a control sample, such as in flow cytometry. One skilled in the art will readily understand that when referring to the "expression" of a cell population, such as "a cell population that expresses X% of marker Y," it means that X% of the cells in the cell population express marker Y.

[0059] As used herein, the term "co-expression" refers to the expression of multiple markers by a single cell.

[0060] As used herein, the term "marker" refers to a naturally occurring identifiable expression by a cell that can be associated with certain properties of the cell. In preferred embodiments, the marker is a genetic expression or a proteomic expression that can be detected and associated with the identity of the cell. A marker can be referred to as a gene. This can be easily translated into expression of the corresponding mRNA and protein.

[0061] As used herein, when used in conjunction with any marker disclosed herein, such as a surface protein or transcription factor, the term "negative" or "-" means that the marker is not expressed in a cell or cell population, while the term "weak" or "low" means that the marker is expressed at a reduced level in a cell compared to the average expression of the marker in a cell population or compared to a reference sample.

[0062] As used herein, when used in conjunction with any marker disclosed herein, such as a surface protein or transcription factor, the term "positive" or "+" means that the marker is expressed in a cell or cell population, while the term "high" or "strong" means that the marker is expressed at an increased level in a cell compared to the average expression of the marker in a cell population or compared to a reference sample.

[0063] As used herein, the term "differentiation" refers generally to the process by which a cell progresses from an undifferentiated state or a state different from an expected differentiation state to a specific differentiation state, such as a process by which an immature state progresses to a more mature state or from an immature state to a mature state, which can occur continuously when the method is performed. The term "differentiation" associated with pluripotent stem cells refers to the process by which a cell progresses from an undifferentiated state to a specific differentiation state, i.e., a process by which an immature state progresses to a more mature state or a terminal state. When a cell loses a marker of an undifferentiated cell or gains a marker of a differentiated cell, changes in cell interaction and cell maturation occur. The loss or gain of a single marker can indicate that a cell has been "completely differentiated" or "terminally differentiated." Terminally differentiated" cells are the final stage of a developmental lineage and cannot differentiate further.

[0064] As used herein, the term "contacting" in relation to culturing or differentiating cells refers to exposing cells to a particular compound by, for example, placing the particular compound in a position that allows it to reach the cell, so as to produce a "contacted" cell. Contacting can be accomplished using any suitable means. A non-limiting example of contacting is by adding the compound to the cell culture medium of the cell. Contact of the cell is assumed to occur as long as the cell and the particular compound are in proximity, for example, the compound is present in the cell culture medium at a suitable concentration.

[0065] As used herein, the term "inhibitor" refers to a compound that decreases or inhibits or downregulates a process, such as a signaling pathway that promotes cell differentiation.

[0066] As used herein, the term "activator" refers to a compound that induces or stimulates or upregulates a process, such as a signaling pathway that promotes cell differentiation.

[0067] Stem Cell Derived Products

[0068] As used herein, the term "differentiated cell" refers to a cell that has progressed from an undifferentiated state to a more mature state, such as a pluripotent stem cell. A differentiated cell can be, for example, a more mature specialized cell, such as a progenitor cell, or fully mature into a specialized / terminal cell type.

[0069] As used herein, the term "cell population" refers to a plurality of cells in the same culture. A cell population can be, for example, a mixture of cells of different types or cells at different stages of development (e.g., cells at different stages of maturation leading to the same or similar specialized characteristics), or it can be a more homogeneous composition of cells with common markers.

[0070] As used herein, the term "neural cell population" refers to a cell population comprising neural cells.

[0071] As used herein, the terms "genetically modified" and "genetically engineered" related to cells are used interchangeably and refer to cells that have undergone artificial manipulation, modification, or recombination of DNA or other nucleic acid molecules to change the characteristics (phenotype) of the cell. Such cells are no longer considered to be naturally occurring cells. For genetically modified stem cells, even if the stem cells are further differentiated into specialized cells, the traits produced by gene editing still exist, thus making the specialized cells genetically modified and artificial, i.e., non-naturally occurring. 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 transplant rejection. A method for obtaining HLA-deficient stem cells is disclosed in WO / 2020 / 260563.

[0072] Neuroectodermal cells

[0073] As used herein, the term "neural" refers to the nervous system.

[0074] As used herein, the term "neural cell" (unless otherwise indicated) refers to cells whose native counterparts naturally constitute a portion of the ectoderm, more specifically the neuroectoderm, and is intended to encompass cells within this germ layer at any stage of development, such as NSCs all the way up 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 specific types of neural cells.

[0075] As used herein, the terms "neuron" and "neuronal cell" are used interchangeably and refer to a neural cell that is postmitotic and eventually differentiates into specialized cells. Neurons are characterized by the expression of the marker INA or other equivalent markers such as ELAVL3, ELAVL4 (usually detected with antibody HuC / D), RBFOX3 (usually detected with antibody NeuN), STMN2, NCMA1 or other such broad neuronal markers.

[0076] As used herein, the terms "neural stem cells" or "NSCs" and "neural precursor cells" or "NPCs" are interchangeable terms that refer to self-renewing, multipotent cells of the nervous system that are capable of generating large numbers of more specialized CNS and PNS cells. NSCs and NPCs typically express transcription factors such as SOX2, NES, PAX6, SOX1, OTX2, OTX1, NKX6.1, OLIG2, NKX2.2, FOXG1, FOXA2, or LMX1A.

[0077] As used herein, the term "neuroblast" refers to an intermediate precursor cell that is usually multipotent or only unipotent and can only self-renew 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" are used interchangeably.

[0078] As used herein, the term "neuroblast" or "intermediate progenitor cell" refers to a cell that has expressed a gene associated with this stage, such as any member of the ASCL1, SOX4, MASH1, EOMES, NHLH1, NEUROD or NEUROG gene family or other such genes. These cells are destined to become neurons.

[0079] As used herein, the terms "neuronal progenitor cell", "neuronal precursor" and "neuronal precursor" are used interchangeably and refer to neural cells that have the potential or tendency to further specialize into neurons. The terms "neuronal precursor" and "non-natural neuronal precursor" are used interchangeably.

[0080] Neural cells according to the present invention may have a specific regional identity, such as midbrain-specific cells.

[0081] As used herein, the term "forebrain" refers to the neural tube and rostral region of the CNS, which gives rise to structures including the cerebral cortex and striatum.

[0082] As used herein, the term "midbrain" refers to the region intermediate to the neural tube and the CNS (on the rostral-caudal axis), which gives rise to structures including the substantia nigra.

[0083] As used herein, the term "ventral midbrain" in relation to cells refers to a neural cell having certain properties of a neural cell naturally present in the ventral midbrain. Typically, ventral midbrain neural cells are characterized by the expression of certain markers such as FOXA2 and LMX1A. Specifically, as used herein, the term "ventral midbrain neural stem cell" refers to a neural stem cell having the characteristics of a neural stem cell naturally present in the ventral midbrain. The ventral midbrain NSC or NPC may be characterized by the co-expression of the markers FOXA2, LMX1A, EN1, OTX2, and SOX2.

[0084] As used herein, the terms "hindbrain" and "spinal cord" refer to the caudal region of the neural tube, which is located caudal to the isthmic organoid.

[0085] As used herein, the term "dopaminergic (DA) cell" or "dopaminergic neuron" or "dopamine neuron" refers to a cell that is capable of synthesizing the neurotransmitter dopamine.

[0086] As used herein, the term "stromal cells" refers to cells that have the ability to become connective tissue cells or fibroblast identity cells.

[0087] As used herein, the term "VLMC" refers to vascular leptomeningeal cells, and are considered a type of stromal cell located in the CNS.

[0088] As used herein, the term "glial cell" refers to non-neuronal cells in the central nervous system (brain and spinal cord) and peripheral nervous system that do not generate electrical impulses and provide support and protection for neurons. Examples include astrocytes and oligodendrocytes and their precursors, glial precursor cells or glial cells.

[0089] Directs differentiation into ventral midbrain neurons

[0090] In a general aspect of the invention, a method is provided, comprising contacting a cell population comprising ventral midbrain NSCs with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. Specifically, in embodiments, the method is used to direct the differentiation of ventral midbrain NSCs into neurons. In further embodiments, the method is used to differentiate ventral midbrain NSCs into ventral midbrain neurons. In preferred embodiments, ventral midbrain NSCs are neurally induced, ventralized, and caudalized. In embodiments, the cell population is neurally induced, ventralized, and caudalized to obtain ventral midbrain NSCs. In embodiments, the method is in vitro.

[0091] The method aims to increase the number of cells with a neuronal fate in a cell population.As used herein, the term "neuronal fate" in relation to a cell means that the developmental fate of the cell is restricted so that it will develop into a neuron.

[0092] As used herein, the term "directed differentiation" refers to influencing the developmental fate of a cell toward a certain outcome. In the process of the methods disclosed herein, directing cell differentiation is not necessarily a continuous process, and the cell may not necessarily develop into a final fate. In a preferred embodiment, the differentiation of cells is directed in vitro so that the cells can subsequently develop toward a specific fate, particularly a neuronal fate, in vivo.

[0093] Thus, in general embodiments, the methods are used to increase the proportion of neural cells that differentiate into neurons. In embodiments, the methods are used to decrease the proportion of neural cells that differentiate into stromal cells such as vascular leptomeningeal cells (VLMCs), non-vmDA neurons and / or astrocytes.

[0094] As used herein, the term "MEK" refers to MAPK / ERK kinase, and the term "MEK signaling" refers to 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 embodiments, inhibitors of MEK signaling inhibit MKK1 (MEK1) and MKK2 (MEK2). In embodiments, inhibitors of MEK signaling inhibit activation and downstream signaling of MEK. In embodiments, inhibitors of MEK signaling are effective inhibitors that inhibit ERK phosphorylation. This applies to all aspects of the invention.

[0095] 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 an embodiment, the inhibitor of NOTCH signaling achieves this by targeting γ-secretase or γ-secretase complex. This applies to all aspects of the present invention. The inventors further discovered that in order to guide the cell population to the fate of ventral midbrain neurons, contacting the cell population with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling provides a synergistic effect.

[0096] In embodiments, 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 the cell population comprising ventral midbrain NSCs co-express the markers FOXA2, LMX1A, EN1, OTX2 and SOX2. The inventors have found that inhibition of MEK signaling is particularly effective in increasing neuronal differentiation for NSCs that are neurally induced, ventralized and caudalized to the extent that they co-express the markers FOXA2, LMX1A, EN1, OTX2 and SOX2.

[0097] The inventors have determined the expression profile of a cell population comprising ventral midbrain NSCs that provides improved neuronal outcomes. In embodiments, when a cell population comprising ventral midbrain NSCs is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, 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 express ASCL1. In embodiments, when a cell population comprising ventral midbrain NSCs is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, 25-65%, 30-65%, 35-65%, 40-65% or 45-65%, preferably 45-65% of the cell population express KI67. In embodiments, when a cell population comprising ventral midbrain NSCs is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, less than 20%, preferably less than 15%, of the cell population express INA. In embodiments, when a cell population comprising ventral midbrain NSCs is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, 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 express INA. In embodiments, when a cell population comprising ventral midbrain NSCs is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, less than 10%, preferably less than 5%, of the cell population is INA+ / SOX2-. In embodiments, when a cell population comprising ventral midbrain NSCs is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, 0-10%, 0-5%, 1-5%, or 2-5%, preferably 2-5%, of the cell population are INA+ / SOX2-. In embodiments, when a cell population comprising ventral midbrain NSCs is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, 75-95%, preferably 80-95% of the cell population express SOX2. In a preferred embodiment, all of the above apply, i.e., when the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, 40-60% of the cell population comprising ventral midbrain NSCs express ASCL1, 45-65% of the cell population comprising ventral midbrain NSCs express KI67, 10-15% of the cell population comprising ventral midbrain NSCs express INA, 2-5% of the cell population comprising ventral midbrain NSCs are INA+ / SOX2-, and 80-95% of the cell population comprising ventral midbrain NSCs express SOX2.In a preferred embodiment, the cell population has not been contacted with an inhibitor of NOTCH signaling, such as DAPT, prior to having the preferred expression profile described above.

[0098] In an embodiment, before more than 70% or 60% of the cell population comprising the ventral midbrain NSC further develops into an intermediate neural precursor, the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. In another embodiment, before more than 20%, 15%, 10% or 5% of the cell population comprising the ventral midbrain NSC further develops into an intermediate neural precursor or a neuron, the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. Specifically, in another embodiment, before more than 20%, 15%, 10% or 5% of the cell population comprising the ventral midbrain NSC express one of the markers ASCL1 and INA, the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling.

[0099] As used herein, the term "intermediate neural precursor" refers to a cell at a developmental stage between a neural stem cell and a neuron. The ventral midbrain neural cells at this stage are characterized by the expression of the marker ASCL1. The inventors have found that the window of opportunity for effectively guiding the cell population to develop into neurons is before the cells mature to become neurons or other terminal neural cells such as astrocytes, matrix cells such as VLMCs, etc.

[0100] In an 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 whose chemical name is N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide, CAS number 391210-10-9, and is a potent MKK1 (MEK1) and MKK2 (MEK2) inhibitor.

[0101] In embodiments, the concentration of the inhibitor of MEK signaling is at least 5 μM, preferably at least 10 μM. In embodiments, 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.

[0102] In an 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 whose chemical name is (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 with a CAS number of 1146699-66-2. As used herein, the term "PF-03084014" refers to a compound with a CAS number of 1290543-63-3. As used herein, the terms "LY450139" and "Semagacestat" can be used interchangeably and refer to a compound with a CAS number of 425386-60-3.

[0103] In embodiments, the concentration of the inhibitor of NOTCH signaling is at least 1 μM, preferably at least 10 μM. In further embodiments, the concentration of the inhibitor of NOTCH signaling is 1 μM to 100 μM, 1 μM to 50 μM, 1 μM to 40 μM, or 1 μM to 30 μM.

[0104] In embodiments, 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 embodiments, the cell population is contacted with an inhibitor of MEK 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 days to about 15 days. In embodiments, 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 embodiments, a population of cells is contacted with 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 days to about 15 days.

[0105] In embodiments, the inhibitor of MEK signaling and the inhibitor of NOTCH signaling are administered to the cells at least every 48 hours, 36 hours, preferably at least every 30 hours, and more preferably at least every 24 hours. In embodiments, the inhibitor of MEK signaling and the inhibitor of NOTCH signaling are replaced at least every 12 to 36 hours, preferably at least every 18 to 30 hours, and more preferably at least every 24 hours. In preferred embodiments, during the contact of the cell population with the inhibitor, it is ensured that the cells are fully exposed to the inhibitor. In embodiments, the cell population is continuously exposed by sufficient culture medium replacement, for example, once every 24 hours. Alternatively, the cell population can be fully exposed to the inhibitor by a biomaterial or substance that ensures the sustained release of the inhibitor.

[0106] In embodiments, the cell population is at least partially contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling simultaneously. In embodiments, the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling simultaneously.

[0107] According to the present invention, cells are guided in vitro toward differentiation into neurons so as to be suitable for subsequent administration to a patient, wherein further development in the body leads to a neuronal fate. After the method of guiding differentiation toward neuronal development, it may not be immediately administered to the patient. Therefore, in an embodiment, a cell colony is cryopreserved after the inhibition of MEK and NOTCH signaling. In an embodiment, within 4 days after the inhibition of MEK and NOTCH signaling ends, such as within 3 days, 2 days, 1 day, 12 hours, 6 hours or 3 hours, a cell colony is cryopreserved. In an embodiment, a cell colony is cryopreserved immediately after the inhibition of MEK and NOTCH signaling ends. In an embodiment, a cell colony is cryopreserved in DMSO or using a cryoprotectant that does not contain DMSO. Those skilled in the art will know that such cryoprotectants are commercially available, and the technology for cryopreserving a cell colony such as a cell colony comprising a neuron is well known.

[0108] According to the present invention, a cell population comprising ventral midbrain NSCs is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling to guide the cell population toward the fate of ventral midbrain neurons. Therefore, when at least a portion of the cells have differentiated into ventral midbrain NSCs, the cell population is contacted with one or both of the inhibitors. Nevertheless, the cells may be contacted with one or both of the inhibitors before this stage to achieve similar or other effects. However, in a preferred embodiment, the cell population is not contacted with an inhibitor of MEK signaling or an inhibitor of NOTCH signaling before the cell population is neurally induced, ventralized, and caudalized. In a preferred embodiment, the cell population is not contacted with an inhibitor of NOTCH signaling, such as DAPT, before the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling according to the method.

[0109] The inventors have determined that the expression profile of a cell population comprising NSCs is particularly suitable for directing differentiation toward neurons. Therefore, aspects of the present invention also relate to an in vitro method for directing ventral midbrain NSCs to differentiate into neurons, comprising contacting a cell population comprising ventral midbrain NSCs with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, wherein 0.5-66%, preferably 40-60%, of the cell population comprising ventral midbrain NSCs expresses ASCL1, 25-65%, preferably 45-65% of the cell population comprising ventral midbrain NSCs expresses KI67, 0-20%, preferably 10-15% of the cell population comprising ventral midbrain NSCs expresses INA, 0-10%, preferably 2-5% of the cell population comprising ventral midbrain NSCs are INA+ / SOX2-, and 75-95%, preferably 80-95% of the cell population comprising ventral midbrain NSCs express SOX2. It will be appreciated by those skilled in the art that the above-mentioned embodiments are equally applicable to this specific aspect.

[0110] Differentiation of PSCs into ventral midbrain NSCs

[0111] General aspects of methods related to guiding ventral midbrain NSC to neuronal differentiation require a cell population comprising ventral midbrain NSC as a starting material. The cell population comprising ventral midbrain NSC can be obtained by any suitable method. Therefore, in an embodiment, the method includes an initial step of differentiating the cell population into ventral midbrain NSC. In an embodiment, the cell population is derived from PSC. In such an embodiment, the cell population is subjected to neural induction, ventralization and caudalization to obtain ventral midbrain NSC. Therefore, in an embodiment, before contacting the cell population with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, the cell population is cultured to induce differentiation into ventral midbrain NSC.

[0112] In an embodiment, 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), prior to contacting the cell population with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling.

[0113] In an embodiment of differentiating PSC into ventral midbrain NSC, the cell population is differentiated into ventral midbrain neural cells by contacting the cell population with an inhibitor of SMAD protein signaling, such as at least two inhibitors of SMAD protein signaling, thereby performing neural induction on the cell population. As used herein, the term "SMAD protein signaling" refers to the Small Mothers Against Decapentaplegic (SMAD) protein signaling pathway. Those skilled in the art will recognize that contacting PSC with one or more inhibitors of the SMAD signaling pathway is a reliable technique for differentiating cells into neural lineages. In an embodiment, an inhibitor of the SMAD signaling pathway is selected from Noggin, LY364947, SB431542, RepSox, and LDN-193189, or a similar compound.

[0114] In an embodiment of differentiating PSCs into ventral midbrain NSCs, a 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" are used interchangeably and refer to the process in which pluripotent cells present a ventral gene expression identity equivalent to that of an embryo or embryonic structure, i.e., a cell of a neural tube. In an embodiment, 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 capable of activating the SHH signaling pathway. It is well known that activation of the SHH pathway is responsible for the induction and maintenance of the ventral neural tube structure. In an embodiment, an activator of SHH signaling is selected from SHH, purmorphamine, and SAG, or similar compounds.

[0115] In embodiments where PSC is differentiated into ventral midbrain NSC, the cell population is differentiated into ventral midbrain neural cells by inducing caudal lateralization of the cell population. As used herein, the term "caudal lateralization" refers to the process in which pluripotent cells present a caudal gene expression identity equivalent to that of cells of an embryo or embryonic structure, i.e., a neural tube. In embodiments, PSC is caudal lateralized by contacting the cell 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. It is well known that inhibitors of Wnt signaling are responsible for the caudal lateralization of neural cells. In embodiments, an activator of Wnt signaling reduces GSK3-β for activation of Wnt signaling. Therefore, in certain embodiments, the Wnt activator is an inhibitor of GSK3-β. In embodiments, the activator of Wnt signaling is selected from CHIR99021 and recombinant Wnt protein.

[0116] In embodiments where PSCs are differentiated into ventral midbrain NSCs, the cell population is differentiated into ventral midbrain neural cells by further inducing caudal lateralization of the cell population. Thus, in embodiments, the cell population is contacted with an activator of fibroblast growth factor (FGF). In embodiments, the activator of FGF signaling is FGF8b.

[0117] In certain embodiments, the concentration of the inhibitor of SMAD protein signaling is 1 μM to 50 μM, the concentration of the activator of SHH signaling is 200 ng / ml to 800 ng / ml, the concentration of the inhibitor of Wnt signaling is 0.1 μM to 1 μM, the concentration of the activator of FGF signaling is 10 ng / ml to 200 ng / ml, the concentration of ascorbic acid is 50 μM to 500 μM, and / or the concentration of BDNF is 1 ng / ml to 50 ng / ml.

[0118] In preferred embodiments, the PSCs are human embryonic stem cells or human induced pluripotent stem cells.

[0119] A further aspect of the present invention relates to an in vitro method for directing the differentiation of a PSC cell population into ventral midbrain neurons, comprising culturing a cell population of PSCs, 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, optional ascorbic acid, and optional BDNF to obtain a cell population comprising ventral midbrain NSCs, wherein the cell population comprising ventral midbrain NSCs is further contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling to direct differentiation into ventral midbrain neurons.

[0120] In an embodiment, the cell population of pluripotent stem cells is contacted with an inhibitor of SMAD protein signaling for 5 to 9 days. In a specific embodiment, the cell population is contacted with an inhibitor of SMAD protein signaling for 5 to 9 days starting from day 0.

[0121] In an embodiment, the cell population of PSCs is contacted with an activator of SHH signaling for 5 to 9 days, such as 7 to 9 days, preferably 9 days. In a preferred embodiment, the cell population is contacted with an inhibitor of Wnt signaling for 5 to 9 days starting from day 0. In an embodiment, the concentration of the activator of SHH signaling is 200 ng / ml to 800 ng / ml.

[0122] In an embodiment, the cell population of pluripotent stem cells is contacted with the inhibitor of Wnt signaling for 5 to 9 days, such as 7 to 9 days, preferably 9 days. In a preferred embodiment, the cell population is contacted with the inhibitor of Wnt signaling for 5 to 9 days from day 0. In an embodiment, the concentration of the activator of Wnt signaling is 0.1 μM to 1 μM.

[0123] In an embodiment, the cell population of PSC is contacted with an activator of FGF signaling for 7 to 12 days after contact with an inhibitor of SMAD protein signaling, an inhibitor of Wnt signaling, and / or an activator of SHH signaling. In another embodiment, the cell population is contacted with an activator of FGF signaling for 7 to 12 days from day 5 to 9, or when contact with an inhibitor of SMAD protein signaling, an inhibitor of Wnt signaling, and / or an activator of SHH signaling is completed. In a specific 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 day 9, and then the cell population is contacted with an activator of FGF signaling from day 9 to day 16. In an embodiment, the activator of FGF signaling is FGF8b. In an embodiment, the concentration of the activator of FGF signaling is 10 ng / ml to 200 ng / ml.

[0124] In embodiments, the cell population is contacted with ascorbic acid for 5 to 7 days from day 10 or 11. In embodiments, 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.

[0125] In embodiments, the cell population is contacted with BDNF for 5 to 7 days, starting from day 10 or 11. In embodiments, 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.

[0126] In embodiments, the cell population is allowed to differentiate into ventral midbrain NSCs for 14 to 24 days, such as 15 to 20 days, before contacting the cell population with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. In embodiments, 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 days to about 10 days. In embodiments, the cell population is contacted with an inhibitor of MEK and NOTCH 0 to 10 days after the cell population is no longer in contact with an activator of FGF signaling.

[0127] In preferred embodiments, the cell population is not contacted with an inhibitor of NOTCH signaling, such as DAPT, while differentiating the cell population toward ventral midbrain NSCs.

[0128] In an embodiment, the cell population comprising ventral midbrain NSCs is cultured in a suitable culture medium for 6 to 8 days from day 16, 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, prior to contacting the cell population with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. Those skilled in the art will recognize that when the cell population is further cultured in the absence of such components, the cells will continue to differentiate.

[0129] In alternative embodiments, the cell population is contacted with an inhibitor of Wnt signaling for at least 14 days, rather than contacting the cell population with an activator of FGF. In such embodiments, 0 to 10 days after the cell population is no longer in contact with the inhibitor of Wnt signaling, the cells are contacted with an inhibitor of MEK and NOTCH.

[0130] In an embodiment, the cell population is harvested before 28 days, such as before 27 days, 26 days or 25 days, preferably before 26 days, from the start of differentiation of the cell population towards ventral midbrain NSC.

[0131] In embodiments, the cell population is harvested on day 28, day 27, day 26, or day 25, preferably day 25, from the start of differentiation of the cell population toward ventral midbrain NSCs.

[0132] As used herein, the term "harvest" refers to collecting cells and transferring them to a new environment. This can be to re-inoculate the cells into a new in vitro culture system. This can also be a situation where the cells cannot further develop into neurons, such as in an embodiment, the cell colony is cryopreserved at the time of harvest. Therefore, in an embodiment, the cell colony is cryopreserved after the inhibition of MEK signaling and the inhibition of NOTCH signaling. In an embodiment, the cell colony is cryopreserved within 4 days, such as 3 days, 2 days, 1 day, 12 hours, 6 hours, 3 hours or 1 hour after the inhibition of MEK signaling and the inhibition of NOTCH signaling ends. In an embodiment, the cell colony is cryopreserved within 4 days after harvest, such as 3 days, 2 days, 1 day, 12 hours, 6 hours or 3 hours.

[0133] In embodiments, the cell population is at least partially contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling simultaneously. In specific embodiments, the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling simultaneously.

[0134] In embodiments, the cell colony is cultured in two-dimensional culture. In further embodiments, the cell colony is initially seeded on a substrate. In embodiments, the substrate comprises an extracellular matrix. In further embodiments, the substrate comprises poly-L-lysine, poly-D-lysine, poly-ornithine, laminin, fibronectin and / or collagen, and / or fragments thereof. In more specific embodiments, the laminin or its fragment is selected from laminin-111, laminin-521 and laminin-511. In embodiments, the cell colony is cultured on a laminin-111 substrate. In embodiments, the concentration of the laminin substrate is about 10 μg / ml.

[0135] In embodiments, when a cell population comprising ventral midbrain NSCs is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, 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 express ASCL1. In embodiments, when a cell population comprising ventral midbrain NSCs is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, 25-65%, 30-65%, 35-65%, 40-65%, or 45-65%, preferably 45-65%, of the cell population express KI67. In embodiments, when a cell population comprising ventral midbrain NSCs is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, less than 20%, preferably less than 15%, of the cell population express INA. In embodiments, when a cell population comprising ventral midbrain NSCs is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, 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 express INA. In embodiments, when a cell population comprising ventral midbrain NSCs is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, less than 10%, preferably less than 5%, of the cell population is INA+ / SOX2-. In embodiments, when a cell population comprising ventral midbrain NSCs is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, 0-10%, 0-5%, 1-5%, or 2-5%, preferably 2-5%, of the cell population are INA+ / SOX2-. In embodiments, when a cell population comprising ventral midbrain NSCs is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, 75-95%, preferably 80-95% of the cell population express SOX2. In a preferred embodiment, all of the above apply, i.e., when the cell population is contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, 40-60% of the cell population comprising ventral midbrain NSCs express ASCL1, 45-65% of the cell population comprising ventral midbrain NSCs express KI67, 10-15% of the cell population comprising ventral midbrain NSCs express INA, 2-5% of the cell population comprising ventral midbrain NSCs are INA+ / SOX2-, and 80-95% of the cell population comprising ventral midbrain NSCs express SOX2.

[0136] In an embodiment, a cell population is cultured by obtaining a neural microsphere, which comprises the following steps: obtaining neural stem precursor progenitor cells, aggregating neural stem precursor progenitor cells to form neural microspheres, and further maturing the neural stem precursor progenitor cells of the neural microspheres. In an embodiment, the method comprises an additional step of seeding the neural stem precursor progenitor cells in a well suitable for maintaining the neural microspheres in static non-adherent culture before the step of aggregating the neural stem precursor progenitor cells. The method of culturing a cell population in a neural microsphere is described in detail in WO / 2021 / 099532, which is incorporated herein by reference.

[0137] Late-stage neural differentiation using inhibitors of NOTCH signaling

[0138] Another aspect of the present invention relates to an in vitro method for directing the differentiation of a cell population into ventral midbrain neurons, comprising the steps of culturing a cell population comprising PSCs, inducing the cell population to differentiate into ventral midbrain NSCs, allowing the ventral midbrain NSCs to mature, and contacting the cell population comprising the ventral midbrain NSCs with an inhibitor of NOTCH signaling, wherein the ventral midbrain NSCs express the markers FOXA2, LMX1A, EN1, OTX2, and SOX2.

[0139] In embodiments, the cell population is contacted with the inhibitor of NOTCH signaling at least 20 days, preferably 21 days, more preferably 22 days, after initially inducing the cell population to differentiate into ventral midbrain NSC.

[0140] In embodiments, the ventral midbrain NSCs are allowed to mature for at least 4 days prior to contacting the population of cells comprising ventral midbrain NSCs with the inhibitor of NOTCH signaling.

[0141] In embodiments, the ventral midbrain NSCs are contacted with an inhibitor of NOTCH signaling no earlier than 4 days after at least 70% of the cells in the population express the markers FOXA2, LMX1A, EN1, OTX2, and SOX2.

[0142] Cell populations of ventral midbrain neurons

[0143] One aspect of the present invention relates to a cell population comprising ventral midbrain neural cells that can be obtained by any method described herein. In a specific embodiment, a cell population comprising ventral midbrain neural cells is obtained by a method according to any of the foregoing embodiments. It is noteworthy that the inventors have not yet been able to determine the parameters that can distinguish between a cell population harvested immediately after inhibiting MEK signaling and inhibiting NOTCH signaling and a cell population that has not undergone a method for guiding differentiation toward neurons according to the present invention. However, it is clear that the cells have been stimulated by signaling, and the proportion of cells with a neuronal fate has increased, which will become immediately apparent once the cell population is further cultured. Therefore, one aspect of the present invention relates to a cell population comprising ventral midbrain NSCs, wherein when the cell population is cultured in vitro for 5 days in a culture medium suitable for maintaining neural cells, a cell population in which at least 50% are neurons (determined according to HuCD, INA or related markers) and <15% of the cells are proliferating (determined according to KI67, MKI67 or other such proliferation markers) is produced (Example 5). In an embodiment, scRNAseq is used to measure the expression of cell markers according to Example 7. In an embodiment, the expression of cell markers is measured according to immunocytochemistry (ICC) as described in Example 8. In embodiments, the expression of a marker of the cell is measured according to flow cytometry as described in Example 2. In embodiments, the cell population is cultured according to Example 6.

[0144] Thus, one aspect of the invention relates to a cell population comprising ventral midbrain NSCs, wherein when the cell population is cultured in vitro for 5 days in a medium suitable for maintaining neural cells, a cell population of at least 25% is HuCD+ / SOX2- or HUCD+ or NEUN+ / SOX2- is produced. In embodiments, the expression of markers of the cells is measured using scRNAseq according to Example 7. In embodiments, the expression of markers of the cells is measured according to immunocytochemistry (ICC) as described in Example 8. In embodiments, the expression of markers of the cells is measured according to flow cytometry as described in Example 2. In embodiments, the cell population is cultured according to Example 6.

[0145] In embodiments, the cell colony is in vitro. In embodiments, the ventral midbrain neural cells are non-natural. In embodiments, the ventral midbrain neural cells are artificial. In preferred embodiments, the ventral midbrain neural cells are stem cell derived. In specific embodiments, the ventral midbrain neural cells are stem cell derived from pluripotent stem cells. In further embodiments, the ventral midbrain neural cells are stem cell derived from human embryonic stem cells (hESC) or human induced pluripotent stem cells (hiPSC).

[0146] In embodiments, the cells of the cell colony are genetically modified. In embodiments, pluripotent stem cells are genetically modified, and the genetic modification persists in the ventral midbrain neural cells obtained according to any method described herein. In certain embodiments, the cells of the cell colony are genetically modified to become low immunogenic. As used herein, the terms "low immunogenicity" and "immune escape" related to cells can be used interchangeably, referring to the cell characteristics that make cells less susceptible to immune rejection of the subject transplanted with such cells. Typically, specific surface markers are overexpressed or silenced. In embodiments, genetically modified cells have reduced MHC-I and / or MHC-II expression. In embodiments, cells are genetically modified to express one or more tolerogenic factors or their analogs, such as HLA-E, HLA-G, CD46, CD47, CD55, CD59 and PD-L1. WO2012145384, WO2013158292, WO2016142532, WO2016183041, WO2018132783, WO2018175390, WO2019161271, WO2020018615, WO2020018620, WO2020049535, WO2020168317, WO2021195426, WO2022012591 and WO2020260563 describe examples and methods of genetically modifying cells to make them have immune evasion. In some aspects, genome editing technology (e.g., CRISPR / Cas or TALEN systems) is used to regulate (e.g., reduce, eliminate and / or increase) the expression of specific genes.

[0147] In embodiments, genetic modifications for low immunogenicity include: reduced expression of MHC-I human leukocyte antigens relative to wild-type stem cells; reduced expression of MHC-II human leukocyte antigens relative to wild-type stem cells; and / or increased expression of tolerogenic factors relative to wild-type stem cells. In embodiments, MHC-I human leukocyte antigens are HLA-A, HLA-B, and HLA-C. In embodiments, MHC-II human leukocyte antigens are HLA-DP, HLA-DQ, and HLA-DR. In embodiments, tolerogenic factors are selected from CD46, CD47, D55, CD59, PD-L1, HLA-E, and HLA-G.

[0148] In embodiments, cells of the cell population are genetically modified to be lineage restricted. As used herein, the term "lineage restricted" in relation to a cell means that the cell is functionally and / or structurally restricted to differentiate into certain cell types.

[0149] In embodiments, the cell population is cryopreserved. In embodiments, the cell population is cryopreserved in DMSO, or using a cryoprotectant that does not contain DMSO.

[0150] In embodiments, the population of cells comprises at least 1,000 cells, 10,000 cells, 100,000 cells, 1,000,000 cells, or 10,000,000 cells.

[0151] Cell populations for use as drugs

[0152] Another aspect of the invention relates to an in vitro cell population or composition thereof according to any embodiment described herein for use as a medicament. In an embodiment, the cell population comprises ventral midbrain neural cells that have been contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling. In a preferred embodiment, the cell population is used to treat Parkinson's disease.

[0153] Another aspect relates to a method of treating or preventing a nervous system condition comprising administering to a patient an effective amount of a cell population according to the invention. In a preferred embodiment, the nervous system condition is Parkinson's disease.

[0154] On the other hand, it is related to a composition comprising a cell population containing ventral midbrain NSC, an inhibitor of MEK signaling, and an inhibitor of NOTCH signaling. In embodiments, the inhibitor of MEK signaling is Mirdametinib. In embodiments, the composition is used to treat Parkinson's disease. In embodiments, the cell population comprising ventral midbrain NSC is characterized in that when cultured in vitro for 5 days in a culture medium suitable for maintaining neural cells, a cell population in which at least 50% is INA+ or HuCD+ and <15% is KI67+ is produced. In embodiments, the cell population is cultured according to Example 6. In embodiments, a cell population comprising ventral midbrain NSC, wherein when the cell population is cultured in vitro for 5 days in a culture medium suitable for maintaining neural cells, a cell population in which at least 25% is HuCD+ / SOX2- or HUCD or NEUN+ / SOX2- is produced. In embodiments, the cell population is cultured according to Example 6.

[0155] In an embodiment, the composition further comprises a cryoprotectant. In an embodiment, the cryoprotectant is DMSO. In an embodiment, the cryoprotectant does not contain DMSO. In vivo guidance of ventral midbrain neural cell differentiation into neurons

[0156] Another aspect relates to a method for treating Parkinson's disease, comprising administering to a subject a therapeutically effective amount of an inhibitor of ventral midbrain NSC and MEK signaling. In embodiments, 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 embodiments, a "therapeutically effective amount" related to the use of a cell product to treat Parkinson's disease refers to a dose of 200,000 to 10,000,000 cells. It is envisioned that a cell population comprising ventral midbrain NSC is administered to a subject by surgery. In embodiments, ventral midbrain NSC co-expresses markers FOXA2, LMX1A, EN1, OTX2, and SOX2. In embodiments, ventral midbrain NSC is obtained according to the methods disclosed herein. The administration of an inhibitor of MEK signaling and / or an inhibitor of NOTCH signaling can be performed by any suitable means, such as oral or subcutaneous injection. In embodiments, the administration of an inhibitor of MEK signaling and / or an 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 Mirdametinib. In an embodiment, the inhibitor of NOTCH signaling is selected from DAPT, MRK-560, MRK-003, LY900009, AL-101, Crenigacestat (LY3039478), MK0752, Nirogacestat (PF-03084017, RO4929097 (RG473), CT16, PTG12, anti-NRR1, anti-NRR2, Brontictuzumab (Brontictuzumab)

[0157] (OMP-52M51), Tarextumab (OMP-59R5), 15D11, Anti-Jag1 / 2, Anti-DII1, YW152F, MMGZ01, mABL001, HMD4-2, Demcizumab (OMP-21M18), Enoticumab (REGN421), MEDI0639, Navicixizumab

[0158] (OMP-305B83), ABT-165, NOV1501 (ABL001; HD105), IMR-1, RIN1, SAHM1 and CB-103. In the embodiment, an inhibitor of NOTCH signaling that has been clinically tested is selected. Detailed information on the listed NOTCH inhibitors can be found in Front.Cell Dev.Biol., May 28, 2021, Vol.9, 2021 (Table 1). In the embodiment, an inhibitor of MEK signaling and / or an inhibitor of NOTCH signaling is administered to the patient on the same day that the ventral midbrain NSC is administered. In the embodiment, an inhibitor of MEK signaling and / or an inhibitor of NOTCH signaling is administered before the cells are transplanted so that a steady state is achieved in the subject when the cells are administered. In the embodiment, an inhibitor of MEK signaling and / or an inhibitor of NOTCH signaling is administered from 6 days before cell transplantation to 6 days after cell transplantation. In embodiments, the subject is administered an inhibitor of MEK signaling and / or an inhibitor of NOTCH signaling for at least 2 days, such as at least 3, 4, 5, 6, 7, 8, 9 or 10 days. In embodiments, the subject is administered an inhibitor of MEK signaling and / or an inhibitor of NOTCH signaling for 1 to 12 days. The dosage regimen may be any suitable dose so that the plasma concentration of the inhibitor of MEK signaling and / or the inhibitor of NOTCH signaling is within an effective window for guiding the administered ventral midbrain NSC to differentiate into neurons. In embodiments, the inhibitor of MEK signaling is Mirdametinib, and the dosage regimen is 1 mg to 30 mg bid (twice a day), 1 mg to 20 mg bid, 1 mg to 10 mg bid, 1 to 5 mg bid or 2 to 4 mg bid.

[0159] In an embodiment, an inhibitor of MEK signaling and / or an inhibitor of NOTCH signaling is co-administered with a cell population comprising ventral midbrain NSCs, e.g., a cell population that has been differentiated for 14 to 24 days, preferably at least 16 days, optionally wherein the cell population has not been contacted with an inhibitor of MEK signaling and / or an inhibitor of NOTCH signaling prior to administration to a patient.

[0160] Thus, aspects of the invention relate to inhibitors of MEK signaling for use in treating Parkinson's disease in a subject to which a therapeutically effective amount of ventral midbrain NSCs has been administered. Another aspect relates to inhibitors of MEK signaling for use in treating Parkinson's disease in a subject by co-administering with a therapeutically effective amount of ventral midbrain NSCs. In an embodiment, an inhibitor of NOTCH signaling is further co-administered to the subject. Specifically, one aspect relates to Mirdametinib for use in treating Parkinson's disease. More specifically, one aspect relates to Mirdametinib for use in treating Parkinson's disease in a subject to which a therapeutically effective amount of ventral midbrain NSCs has been administered, or to Mirdametinib for use in treating Parkinson's disease in a subject by co-administering 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.

[0161] Another aspect relates to an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, which are used in combination to treat Parkinson's disease in a subject to whom a therapeutically effective amount of ventral midbrain NSC has been administered. Similarly, one aspect relates to an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, which are used in combination in a method of directing ventral midbrain NSC to differentiate into ventral midbrain neurons, wherein the method comprises administering an inhibitor of MEK signaling and an inhibitor of NOTCH signaling to a subject to whom ventral midbrain NSC has been administered. In embodiments, an inhibitor of MEK signaling and an inhibitor of NOTCH signaling are administered within 6 days after administration of ventral midbrain NSC. In embodiments, an inhibitor of MEK signaling and an inhibitor of NOTCH signaling are administered for at least 2 days, such as at least 4, 5 or 6 days.

[0162] Another aspect relates to an inhibitor of MEK signaling, an inhibitor of NOTCH signaling, and a cell population comprising ventral midbrain NSCs, which are used in combination to treat Parkinson's disease. In an embodiment, the inhibitor of MEK signaling and the inhibitor of NOTCH signaling are co-administered with the cell population comprising ventral midbrain NSCs. In an embodiment, the inhibitor of MEK signaling is Mirdametinib.

[0163] Another aspect of the invention relates to a composition comprising a cell population containing ventral midbrain NSCs and an inhibitor of MEK signaling. In embodiments, the inhibitor of MEK signaling is Mirdametinib. In embodiments, the composition further comprises an inhibitor of NOTCH signaling. In specific embodiments, the inhibitor of NOTCH signaling is DAPT. Another aspect relates to a composition comprising a cell population containing ventral midbrain NSCs, an inhibitor of MEK signaling, and an inhibitor of NOTCH signaling. In embodiments, the composition is in vitro.

[0164] It will be appreciated by those skilled in the art that the embodiments mentioned in this section relating to methods of treatment, use as a medicament and second medical use are equally applicable to all mentioned aspects.

[0165] Cell populations for the treatment of Parkinson's disease

[0166] The inventors have realized that obtaining a cell population comprising ventral midbrain neurons without contacting the cell population with an inhibitor of NOTCH, such as DAPT, better reflects the natural development of neurons. Although the inventors still prefer to further contact the cell population with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, a cell population having a preferred expression profile when contacting the cells with these inhibitors may itself be more suitable for treating Parkinson's disease compared to prior art methods. Therefore, another aspect of the present invention relates to a cell population for treating Parkinson's disease, wherein 80-95% of the cell population express SOX2, 40-60% of the cell population express ASCL1, 30-65% of the cell population express KI67, 10-20% of the cell population express INA, and 2-5% of the cell population are INA+ / SOX2-( Fig. 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 both the MEK inhibitor and the NOTCH inhibitor. These cells are obtained according to the methods disclosed herein prior to contacting the cell population with the inhibitor, wherein the cell population is allowed to differentiate into ventral midbrain NSCs for at least 20 days, such as 22-24 days.

[0167] The cell population may be administered to a patient alone, or may be co-delivered with an inhibitor of MEK signaling and / or an inhibitor of NOTCH signaling. Accordingly, one aspect relates to a composition comprising the above cell population and an inhibitor of MEK signaling and / or an inhibitor of NOTCH signaling.

[0168] Specific implementation plan

[0169] Aspects of the present invention will now be further described by the following non-limiting embodiments:

[0170] 1. A method comprising contacting a cell population comprising ventral midbrain NSCs with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling.

[0171] 2. The method according to the preceding embodiment, wherein the method is used to guide the differentiation of ventral midbrain NSCs into neurons.

[0172] 3. The method according to any one of the preceding embodiments, wherein the method is used to differentiate ventral midbrain NSCs into ventral midbrain neurons.

[0173] 4. The method according to any one of the preceding embodiments, wherein the ventral midbrain NSCs are neurally induced, ventralized and caudalized.

[0174] 5. The method according to any one of the preceding embodiments, wherein the cell population is subjected to neural induction, ventralization and caudalization to obtain ventral midbrain NSCs.

[0175] 6. The method according to any one of the preceding embodiments, wherein the inhibitor of MEK signaling inhibits MEK1 and MEK2.

[0176] 7. A method according to any one of the preceding embodiments, wherein the inhibitor of NOTCH signaling targets y-secretase.

[0177] 8. The method according to any one of the preceding embodiments, wherein at least 5% of the cell population comprising ventral midbrain NSCs co-express the markers FOXA2, LMX1A, EN1, OTX2, and SOX2.

[0178] 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 prior to contacting the cell population with the inhibitor of MEK signaling and the inhibitor of NOTCH signaling.

[0179] 10. A method according to any one of embodiments 5 to 9, wherein before more than 70% or 60% of the cell population comprising ventral midbrain NSCs further develop into intermediate neural precursors, the cell population is contacted with the inhibitor of MEK signaling and the inhibitor of NOTCH signaling.

[0180] 11. The method according to any one of the preceding embodiments, wherein when the cell population comprising ventral midbrain NSCs is contacted with the inhibitor of MEK signaling and the inhibitor of NOTCH signaling, 0.5-65%, preferably 30-65%, more preferably 40-60% of the cell population express ASCL1.

[0181] 12. The method according to any one of the preceding embodiments, wherein when the cell population comprising ventral midbrain NSCs is contacted with the inhibitor of MEK signaling and the inhibitor of NOTCH signaling, 25-65%, preferably 45-65%, of the cell population express KI67.

[0182] 13. The method according to any one of the preceding embodiments, wherein when the cell population comprising ventral midbrain NSCs is contacted with the inhibitor of MEK signaling and the inhibitor of NOTCH signaling, less than 20%, preferably less than 15%, of the cell population express INA.

[0183] 14. The method according to any one of the preceding embodiments, wherein when the cell population comprising ventral midbrain NSCs is contacted with the inhibitor of MEK signaling and the inhibitor of NOTCH signaling, 0-20%, preferably 10-15% of the cell population express INA.

[0184] 15. A method according to any of the preceding embodiments, wherein when the cell population comprising ventral midbrain NSCs is contacted with the inhibitor of MEK signaling and the inhibitor of NOTCH signaling, less than 10%, preferably less than 5%, of the cell population are INA+ / SOX2-.

[0185] 16. A method according to any one of the preceding embodiments, wherein when the cell population comprising ventral midbrain NSCs is contacted with the inhibitor of MEK signaling and the inhibitor of NOTCH signaling, 0-10%, preferably 2-5%, of the cell population are INA+ / SOX2-.

[0186] 17. The method according to any one of the preceding embodiments, wherein when the cell population comprising ventral midbrain NSCs is contacted with the inhibitor of MEK signaling and the inhibitor of NOTCH signaling, 75-95%, preferably 80-95% of the cell population express SOX2.

[0187] 18. A method according to any one of the preceding embodiments 5 to 10, wherein the cell population is contacted with the inhibitor of MEK signaling and the inhibitor of NOTCH signaling before more than 20% of the cell population comprising ventral midbrain NSCs further develop into intermediate neural precursors or neurons.

[0188] 19. The method of any one of embodiments 5 to 10 and 18, wherein the cell population is contacted with the inhibitor of MEK signaling and the inhibitor of NOTCH signaling before more than 20% of the cell population comprising ventral midbrain NSCs express one of the markers ASCL1 and INA.

[0189] 20. A method according to any one of the preceding embodiments, wherein the inhibitor of MEK signaling is selected from PD0325901, trametinib (GSK1120212), selumetinib (AZD6244), pimacetinib (AS703026), MEK162, cobimetinib, PD184352, PD173074, BIX02189, AZD8330, PD318088, rivatinib and PD98059, preferably PD0325901.

[0190] 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.

[0191] 22. The method according to any one of the preceding embodiments, wherein the concentration of the inhibitor of MEK signaling is 5 μM to 100 μM.

[0192] 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.

[0193] 24. The method according to any of the preceding embodiments, wherein the inhibitor of NOTCH signaling is selected from DAPT, MRK-560, MRK-003, LY900009, AL-101, Crenigacestat (LY3039478), MK0752, Nirogacestat (PF-03084017, RO4929097 (RG473), CT16, PTG12, anti-NRR1, anti-NRR2, Brontictuzumab (OMP-52M51), Tarex (Tarex tumab) (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.

[0194] 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.

[0195] 26. The method according to any one of the preceding embodiments, wherein the concentration of the inhibitor of NOTCH signaling is 1 μM to 100 μM.

[0196] 27. A method according to any of the preceding embodiments, wherein the cell population is contacted with the 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.

[0197] 28. A method according to any of the preceding embodiments, wherein the cell population is contacted with the inhibitor of MEK 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 days to about 15 days.

[0198] 29. A method according to any of the preceding embodiments, wherein the cell population is contacted with the 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.

[0199] 30. A method according to any of the preceding embodiments, wherein the cell population is contacted with the 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 days to about 15 days.

[0200] 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.

[0201] 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 to 36 hours, preferably at least every 18 to 30 hours, more preferably at least every 24 hours.

[0202] 33. A method according to any one of the preceding embodiments, wherein the cell population is at least partially contacted with the inhibitor of MEK signaling and the inhibitor of NOTCH signaling simultaneously.

[0203] 34. A method according to any one of the preceding embodiments, wherein the cell population is contacted with the inhibitor of MEK signaling and the inhibitor of NOTCH signaling simultaneously.

[0204] 35. A method according to any one of the preceding embodiments, wherein the cell population is not contacted with the inhibitor of NOTCH signaling before contacting the cell population with the inhibitor of MEK signaling and the inhibitor of NOTCH signaling according to the method.

[0205] 36. A method according to any of the preceding embodiments, wherein the cell population is cryopreserved following inhibition of MEK and NOTCH signaling.

[0206] 37. A method according to the preceding embodiment, wherein the cell population is cryopreserved within 4 days, such as 3 days, 2 days, 1 day, 12 hours, 6 hours or 3 hours after the end of inhibition of MEK and NOTCH signaling.

[0207] 38. A method according to the preceding embodiment, wherein the cell population is cryopreserved immediately after the inhibition of MEK and NOTCH signaling is completed.

[0208] 39. A method according to any one of embodiments 36 to 38, wherein the cell population is cryopreserved in DMSO, or using a cryoprotectant that does not contain DMSO.

[0209] 40. The method of any one of the preceding embodiments, wherein the cell population is cultured to induce differentiation into ventral midbrain NSCs prior to contacting the cell population with the inhibitor of MEK signaling and the inhibitor of NOTCH signaling.

[0210] 41. A method according to any one of the preceding embodiments, wherein before contacting the cell population with the inhibitor of MEK signaling and the 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).

[0211] 42. A method according to the preceding embodiment, wherein the cell population is contacted with at least two SMAD protein signaling inhibitors.

[0212] 43. A method according to any one of embodiments 41 and 42, wherein the inhibitor of SMAD signaling is selected from Noggin, LY364947, SB431542, RepSox and LDN-193189, or a similar compound.

[0213] 44. A method according to any one of embodiments 41 to 43, wherein the activator of SHH signaling is selected from SHH, purmorphamine, SAG or a similar compound.

[0214] 45. A method according to any one of embodiments 41 to 44, wherein the activator of Wnt signaling is CHIR99021.

[0215] 46. ​​A method according to any one of embodiments 41 to 45, wherein the activator of FGF signaling is FGF8b.

[0216] 47. The method of any one of embodiments 41 to 46, wherein:

[0217] a) the concentration of the inhibitor of SMAD protein signaling is 1 μM to 50 μM,

[0218] b) the concentration of the SHH signal transduction activator is 200 ng / ml to 800 ng / ml,

[0219] c) the concentration of the Wnt signaling inhibitor is 0.1 μM to 1 μM,

[0220] d) the concentration of the FGF signaling activator is 10 ng / ml to 200 ng / ml,

[0221] e) ascorbic acid at a concentration of 50 μM to 500 μM, and / or

[0222] f) The concentration of BDNF is 1 ng / ml to 50 ng / ml.

[0223] 48. A method according to any of the preceding embodiments, wherein the cell population is derived from PSC.

[0224] 49. The method according to the preceding embodiment, wherein the PSC is a human embryonic stem cell or a human induced pluripotent stem cell.

[0225] 50. The method according to any one of the preceding embodiments, wherein the method is in vitro.

[0226] 51. An in vitro method for guiding the differentiation of a PSC cell population into ventral midbrain neurons, comprising culturing a PSC cell population, contacting the PSC cell population with an inhibitor of SMAD protein signaling, an inhibitor of Wnt signaling, an activator of SHH signaling, an activator of FGF signaling, optional ascorbic acid and optional BDNF to obtain a cell population containing ventral midbrain NSCs, wherein the cell population containing ventral midbrain NSCs is further contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling to guide differentiation into ventral midbrain neurons.

[0227] 52. The method according to the preceding embodiment, wherein the cell population is differentiated into ventral midbrain NSCs for 14 to 24 days prior to contacting the cell population with the inhibitor of MEK signaling and the inhibitor of NOTCH signaling.

[0228] 53. A method according to any one of embodiments 51 and 52, wherein the cell population is harvested before 28 days, such as 27 days, 26 days or 25 days, preferably before 26 days, from the start of differentiation of the cell population into ventral midbrain NSCs.

[0229] 54. A method according to any one of embodiments 51 to 53, wherein the cell population is harvested on day 28, day 27, day 26 or day 25, preferably day 25, from the start of differentiation of the cell population into ventral midbrain NSCs.

[0230] 55. A method according to any one of embodiments 51 to 54, wherein the cell population is contacted with the inhibitor of MEK signaling and the 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 days to about 10 days.

[0231] 56. A method according to any one of embodiments 51 to 55, wherein the cell population is contacted with the inhibitor of MEK signaling and the inhibitor of NOTCH signaling starting 0 to 10 days after the cell population is no longer in contact with the activator of FGF signaling.

[0232] 57. A method according to any one of embodiments 51 to 56, wherein the cell population is neurally induced, ventralized and caudalized for 16 days prior to contacting the cell population with the inhibitor of MEK signaling and the inhibitor of NOTCH signaling, and then cultured in a suitable culture medium for 6 to 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.

[0233] 58. A method according to any one of the preceding embodiments 51 to 57, wherein the cell population is at least partially contacted with the inhibitor of MEK signaling and the inhibitor of NOTCH signaling simultaneously.

[0234] 59. A method according to any one of embodiments 51 to 58, wherein the cell population is contacted with the inhibitor of MEK signaling and the inhibitor of NOTCH signaling simultaneously.

[0235] 60. A method according to any one of embodiments 51 to 59, wherein the cell population of pluripotent stem cells is contacted with an inhibitor of SMAD protein signaling for 5 to 9 days.

[0236] 61. A method according to any one of embodiments 51 to 60, wherein the cell population of pluripotent stem cells is contacted with an inhibitor of Wnt signaling for 5 to 9 days.

[0237] 62. A method according to any one of embodiments 51 to 61, wherein the cell population of pluripotent stem cells is contacted with an activator of SHH signaling for 5 to 9 days.

[0238] 63. A 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 contact with an inhibitor of SMAD protein signaling, an inhibitor of Wnt signaling and / or an activator of SHH signaling is completed.

[0239] 64. A method according to any one of embodiments 51 to 63, wherein the cell population is contacted with the inhibitor of SMAD protein signaling for 5 to 9 days starting from day 0.

[0240] 65. A method according to any one of embodiments 51 to 64, wherein the cell population is contacted with the inhibitor of Wnt signaling for 5 to 9 days starting from day 0.

[0241] 66. A method according to any one of embodiments 51 to 65, wherein the cell population is contacted with the activator of SHH signaling for 5 to 9 days starting from day 0.

[0242] 67. A method according to any one of embodiments 51 to 66, wherein the cell population is contacted with an activator of FGF signaling for 7 to 12 days, starting from day 5 to 9, or when contact with the inhibitor of SMAD protein signaling, the inhibitor of Wnt signaling and / or the activator of SHH signaling ends.

[0243] 68. The method of any one of embodiments 51 to 67, wherein the cell population is contacted with ascorbic acid for 5 to 7 days starting from day 10 or 11.

[0244] 69. The method of any one of embodiments 51 to 68, wherein the concentration of ascorbic acid is 10 μM to 400 μM.

[0245] 70. The method of any one of embodiments 51 to 69, wherein the cell population is contacted with BDNF for 5 to 7 days starting from day 10 or 11.

[0246] 71. The method of any one of embodiments 51 to 70, wherein the concentration of BDNF is from 1 ng / ml to 40 ng / ml.

[0247] 72. The method according to any one of embodiments 51 to 71, wherein the cell population is cryopreserved after MEK signaling inhibition and NOTCH signaling inhibition.

[0248] 73. A method according to the above embodiment, wherein the cell population is cryopreserved within 4 days, such as 3 days, 2 days, 1 day, 12 hours, 6 hours, 3 hours or 1 hour after the end of MEK signaling inhibition and NOTCH signaling inhibition.

[0249] 74. A method according to any one of embodiments 72 and 73, wherein the cell population is cryopreserved upon harvest.

[0250] 75. A method according to the preceding embodiments, wherein 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 harvesting.

[0251] 76. A method according to any one of embodiments 51 to 75, wherein when the cell population comprising ventral midbrain NSCs is contacted with the inhibitor of MEK signaling and the inhibitor of NOTCH signaling, 0.5-65%, preferably 30-65%, more preferably 40-60% of the cell population express ASCL1.

[0252] 77. A method according to any one of embodiments 51 to 76, wherein when the cell population comprising ventral midbrain NSCs is contacted with the inhibitor of MEK signaling and the inhibitor of NOTCH signaling, 25-65%, preferably 45-65%, of the cell population express KI67.

[0253] 78. A method according to any one of embodiments 51 to 77, wherein when the cell population comprising ventral midbrain NSCs is contacted with the inhibitor of MEK signaling and the inhibitor of NOTCH signaling, less than 20%, preferably less than 15%, of the cell population express INA.

[0254] 79. A method according to any one of embodiments 51 to 78, wherein when the cell population comprising ventral midbrain NSCs is contacted with the inhibitor of MEK signaling and the inhibitor of NOTCH signaling, 0-20%, preferably 10-15% of the cell population express INA.

[0255] 80. A method according to any one of embodiments 51 to 79, wherein when the cell population comprising ventral midbrain NSCs is contacted with the inhibitor of MEK signaling and the inhibitor of NOTCH signaling, less than 10%, preferably less than 5%, of the cell population are INA+ / SOX2-.

[0256] 81. A method according to any one of embodiments 51 to 80, wherein when the cell population comprising ventral midbrain NSCs is contacted with the inhibitor of MEK signaling and the inhibitor of NOTCH signaling, 0-10%, preferably 2-5%, of the cell population are INA+ / SOX2-.

[0257] 82. A method according to any one of embodiments 51 to 81, wherein when the cell population comprising ventral midbrain NSCs is contacted with the inhibitor of MEK signaling and the inhibitor of NOTCH signaling, 75-95%, preferably 80-95%, of the cell population express SOX2.

[0258] 83. An in vitro method for directing a cell population to differentiate into ventral midbrain neurons, comprising the following steps:

[0259] - culturing a cell population comprising PSCs,

[0260] - inducing the cell population to differentiate into ventral midbrain NSCs,

[0261] - maturing the ventral midbrain NSCs, and

[0262] - contacting a cell population comprising ventral midbrain NSCs with an inhibitor of NOTCH signaling,

[0263] The ventral midbrain NSCs express the markers FOXA2, LMX1A, EN1, OTX2 and SOX2.

[0264] 84. A method according to the preceding embodiment, wherein the cell population is contacted with the inhibitor of NOTCH signaling at least 20 days, preferably 21 days, and more preferably 22 days after initially inducing the cell population to differentiate into ventral midbrain NSCs.

[0265] 85. A cell population comprising ventral midbrain neural cells obtainable by the method according to any one of embodiments 1 to 84.

[0266] 86. A cell population comprising ventral midbrain neural cells obtained by the method according to any one of embodiments 1 to 84.

[0267] 87. A cell population comprising ventral midbrain NSCs, wherein when said cell population is cultured in vitro for 5 days in a medium suitable for maintaining neural cells, a cell population is produced in which at least 50% are INA+ or HUCD+ and less than 15% are KI67+.

[0268] 88. A cell population comprising ventral midbrain NSCs, wherein when said cell population is cultured in vitro for 5 days in a medium suitable for maintaining neural cells, a cell population is produced in which at least 25% are HuCD+ / SOX2- or HUCD+ or NEUN+ / SOX2-.

[0269] 89. A cell population according to any one of embodiments 87 and 88, wherein the expression of markers of the cells is measured using single-cell RNA sequencing, nucleic acid RNA sequencing, immunocytochemistry (ICC), qPCR or FACS.

[0270] 90. A cell population according to any one of embodiments 87 to 89, wherein the expression of markers of the cells is measured according to Example 7.

[0271] 91. A cell population according to any one of embodiments 87 to 89, wherein the expression of markers of the cells is measured according to Example 8.

[0272] 92. A cell population according to any one of embodiments 87 to 89, wherein the expression of markers of the cells is measured according to Example 9.

[0273] 93. A cell population according to any one of embodiments 87 to 92, wherein the cell population is cultured according to Example 6.

[0274] 94. A cell population according to any one of embodiments 85 to 93, wherein the cell population is in vitro.

[0275] 95. A cell population according to any one of embodiments 85 to 94, wherein the ventral midbrain neurons are non-natural.

[0276] 96. A cell population according to any one of embodiments 85 to 95, wherein the ventral midbrain neurons are artificial.

[0277] 97. A cell population according to any one of embodiments 85 to 96, wherein the ventral midbrain neural cells are stem cell derived.

[0278] 98. A cell population according to any one of embodiments 85 to 97, wherein the ventral midbrain neural cells are stem cell derived from pluripotent stem cells.

[0279] 99. A cell population according to the preceding embodiment, wherein the pluripotent stem cells are genetically modified, and wherein the genetic modification persists in the ventral midbrain neural cells.

[0280] 100. A cell population according to any one of embodiments 98 and 99, wherein the ventral midbrain neural cells are stem cell derived from human embryonic stem cells (hESC) or human induced pluripotent stem cells (hiPSC).

[0281] 101. A cell population according to any one of embodiments 85 to 100, wherein the cells of the cell population are genetically modified.

[0282] 102. A cell population according to the preceding embodiments, wherein the cells of the cell population are genetically modified to be low immunogenic and / or lineage restricted.

[0283] 103. A cell population according to the preceding embodiments, wherein the genetic modification for low immunogenicity includes: reduced expression of MHC-I human leukocyte antigen relative to wild-type stem cells; reduced expression of MHC-II human leukocyte antigen relative to wild-type stem cells; and / or increased expression of tolerogenic factors relative to wild-type stem cells.

[0284] 104. A cell population according to the preceding embodiment, wherein the MHC-I human leukocyte antigens are HLA-A, HLA-B and HLA-C.

[0285] 105. A cell population according to any one of embodiments 103 and 104, wherein the MHC-II human leukocyte antigens are HLA-DP, HLA-DQ and HLA-DR.

[0286] 106. The cell population of any one of embodiments 103 to 105, wherein the tolerogenic factors are selected from CD46, CD47, CD55, CD59, PD-L1, HLA-E and HLA-G.

[0287] 107. A 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.

[0288] 108. A cell population according to any one of embodiments 85 to 107, wherein the cell population is cryopreserved.

[0289] 109. A composition comprising a cell population according to any one of embodiments 85 to 108.

[0290] 110. The composition according to the preceding embodiment, further comprising a cryoprotectant.

[0291] 111. A composition according to the preceding embodiment, wherein the cryoprotectant is DMSO, or wherein the cryoprotectant does not contain DMSO.

[0292] 112. An in vitro cell population according to any one of embodiments 85 to 108 for use as a medicament.

[0293] 113. An in vitro cell population according to the preceding embodiments, for use in treating Parkinson's disease.

[0294] 114. A method of treating a nervous system condition comprising administering to a patient an effective amount of the cell population of any one of embodiments 85 to 108.

[0295] 115. The method according to the preceding embodiment, wherein the neurological condition is Parkinson's disease.

[0296] 116. A method of treating Parkinson's disease comprising administering to a subject a therapeutically effective amount of a ventral midbrain NSC and an inhibitor of MEK signaling.

[0297] 117. The method according to the preceding embodiment, wherein the inhibitor of MEK signaling is PD0325901.

[0298] 118. The method of any one of embodiments 116 and 117, wherein the ventral midbrain NSCs co-express the markers FOXA2, LMX1A, EN1, OTX2, and SOX2.

[0299] 119. The method of any one of embodiments 116 to 118, further comprising administering an inhibitor of NOTCH signaling.

[0300] 120. A method according to the preceding embodiments, wherein the inhibitor of NOTCH signaling is selected from DAPT, MRK-560, MRK-003, LY900009, AL-101, Crenigacestat (LY3039478), MK0752, Nirogacestat (PF-03084017, RO4929097 (RG473), CT16, PTG12, anti-NRR1, anti-NRR2, Brontictuzumab (OMP-52M51), Tarextuzumab (Tarextuzumab umab) (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.

[0301] 121. The method according to the preceding embodiments, wherein the inhibitor of NOTCH signaling is DAPT.

[0302] 122. An inhibitor of MEK signaling and an inhibitor of NOTCH signaling for use in combination in treating Parkinson's disease in a subject to whom a therapeutically effective amount of ventral midbrain NSC has been administered.

[0303] 123. The inhibitor of MEK signaling and the inhibitor of NOTCH signaling according to the preceding embodiments, wherein the inhibitor of MEK signaling and the inhibitor of NOTCH signaling are administered at least within 6 days after administration of the ventral midbrain NSCs.

[0304] 124. An inhibitor of MEK signaling and an inhibitor of NOTCH signaling used in combination according to 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.

[0305] 125. An inhibitor of MEK signaling, an inhibitor of NOTCH signaling, and a cell population comprising ventral midbrain NSCs, for use in combination in treating Parkinson's disease.

[0306] 126. The inhibitor of MEK signaling, the inhibitor of NOTCH signaling, and the cell population according to 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.

[0307] 127. An inhibitor of MEK signaling, an inhibitor of NOTCH signaling, and a cell population according to the preceding embodiments, wherein the inhibitor of MEK signaling is Mirdametinib.

[0308] 128. An inhibitor of MEK signaling for use in treating Parkinson's disease in a subject to whom a therapeutically effective amount of ventral midbrain NSC has been administered.

[0309] 129. An inhibitor of MEK signaling according to the preceding embodiment, wherein the ventral midbrain NSCs have been administered surgically.

[0310] 130.Mirdametinib for the treatment of Parkinson's disease.

[0311] 131. Mirdametinib for use in treating Parkinson's disease in a subject to whom a therapeutically effective amount of ventral midbrain NSC has been administered.

[0312] 132. A composition comprising a cell population comprising ventral midbrain NSCs, an inhibitor of MEK signaling, and an inhibitor of NOTCH signaling.

[0313] 133. A composition according to the preceding embodiment, wherein the inhibitor of MEK signaling is Mirdametinib.

[0314] 134. A composition according to any one of embodiments 132 and 133, for use in treating Parkinson's disease.

[0315] 135. A composition according to any one of embodiments 132 to 134, wherein the cell population comprising ventral midbrain NSCs is according to embodiments 87 and / or 88.

[0316] 136. The composition of any one of embodiments 132 to 135, wherein the composition is in vitro.

[0317] 137. An in vitro cell population for treating Parkinson's disease, wherein 80-95% of the cell population express SOX2, 40-60% of the cell population express ASCL1, 30-65% of the cell population express KI67, 10-20% of the cell population express INA, and 2-5% of the cell population are INA+ / SOX2-.

[0318] 138. The cell population according to the preceding embodiment, wherein the cell population has not been contacted with DAPT.

[0319] 139. A composition comprising the cell population according to embodiment 137 or 138 and an inhibitor of MEK signaling and / or an inhibitor of NOTCH signaling.

[0320] Example

[0321] The following are non-limiting examples for practicing the present invention.

[0322] Example 1: Differentiation of human pluripotent stem cells into ventral midbrain neurons

[0323] hPSCs can be differentiated into ventral midbrain neurons according to several protocols that have been published since the seminal paper by Lorenz Studer's group in 2011 entitled "Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease". Shown below is one way to perform the differentiation procedure, which follows the publications described in the references in this section (Nolbrant et al., 2017 and Kirkeby et al., 2017).

[0324] Human embryonic stem cell (hESC) lines RC17 (Roslin CT) and 3053 (Novo Nordisk A / S) and other hESC lines developed by Novo Nordisk were cultured on human laminin-521 matrix (0.7-1.2 μg / cm 2 The cells were cultured in iPSBrew XF medium (Miltenyi Biotec) supplemented with 60 U / mL penicillin-streptomycin (PS; Thermo Fisher Scientific) on culture vessels coated with 1% EDTA (Thermo Fisher Scientific). The medium was changed every day, and cells were passaged every 4-6 days with EDTA 0.5 mM (Thermo Fisher Scientific). The cultures were maintained at 37°C, 95% humidity, and 5% CO2.

[0325] 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. Cells were plated at 10 4 cells / cm 2 Inoculated with human laminin-111 (1.2 μg / cm 2 From day 0-8 in vitro (DIV 0-8), cells were exposed to N2-based medium: 50% DMEM / F12+Glutamax (Gibco), 50% Neurobasal (Gibco), 1% N2 supplement CTS (ThermoFisher Scientific), 5% GlutaMAX (Thermo Fisher Scientific), 0.2% PS (ThermoFisher Scientific) and supplemented with 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, GSK3β inhibitor CHIR99021 (CHIR; 0.5-0.6 μM; Miltenyi Biotec) to promote caudal lateralization. From DIV9-11, N2-based medium was supplemented with fibroblast growth factor 8b (FGF8b; 100 ng / mL; Miltenyi Biotec). At DIV11, cells were dissociated with accutase (Thermo Fisher Scientific) and cultured at 0.8 × 10 6 Cells / m 2 Inoculated with human laminin-111 (1.2 μg / cm 2)-coated cell culture flasks or culture plates in DIV11-16 medium (Neurobasal, 2% B27 supplement CTS without vitamin A (Thermo Fisher Scientific), 5% GlutaMAX, 0.2% PS, and supplemented with FGF8b (100 ng / mL), L-ascorbic acid (AA; 200 μM; Sigma), and human brain-derived neurotrophic factor (BDNF; 20 ng / mL; Miltenyi Biotec)) supplemented with 10 μM Y-27632 (Miltenyi Biotec). At day 16 in vitro (DIV 16), cells were dissociated with accutase and either cryopreserved or replated in culture medium supplemented with poly-L-ornithine (0.002%) and laminin-521 (1.5 μg / cm 2 )-coated cell culture flasks / plates in B27 medium supplemented 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) for prolonged in vitro culture, thereby allowing the ventral midbrain neural stem cells to further differentiate and mature into neurons.

[0326] References:

[0327] · 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 September; 12(9): 1962-1979. doi: 10.1038 / nprot.2017.078. Epub August 31, 2017.PMID:28858290.

[0328] ·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 MarkersGuide 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.

[0329] Example 2: Flow cytometry analysis of single cell protein marker expression

[0330] Ventral midbrain dopaminergic (vmDA) progenitors were generated from hESCs in 2D in vitro culture as described in Example 1 and using the reagents described in Table 1. At various days after the start of differentiation, cell cultures were dissociated into single cell suspensions using Accutase, counted on a NucleoCounter NC-200, and collected in N2 medium (supplemented with 1% CTS TM CTS of N-2 Supplements TM Neurobasal TM Use LIVE / DEAD TM The dead cells can be marked with a fixable near-infrared dead cell staining kit. The cells are then resuspended in B27 medium (supplemented with 1% B-27 without vitamin A). TM Supplements, 2mMGlutaMAX TM , 60U / mL penicillin-streptomycin, 10μM ROCK inhibitor CTS TM Neurobasal TM The cells were then fixed and permeabilized using the BD Transcription Factor Buffer Kit (BD Biosciences) according to the manufacturer's instructions. The fixed cells were then stained with fluorescently conjugated antibodies and samples were acquired on a BD LSR Fortessa or BD FACSymphony (BD Biosciences). The fcs files were exported and analyzed in FlowJo 10.5.03.

[0331] To determine whether a cell is considered positive for protein expression of a marker, a gate is set in the field at the edge of the fluorescent signal of the negative control sample, as is routinely done. Examples of negative control samples used include unstained controls, fluorescence minus one (FMO) controls, and most preferably biological negative control samples; all cells present above these threshold gates when the experimental samples are run are considered positive. Antibodies: FOXA2 (1:320, Miltenyi), OTX2 (1:320, Miltenyi), SOX2 (1:40, BD), LMX1A (1:2500, manufactured by Novo Nordisk), EN1 (1:40, AtlasAntibodies), ASCL1 (1:2500, Mitlenyi), INA (1:3000, AtlasAntibodies).

[0332] Table 1: List of reagents used for FACS, ICC and cell culture

[0333]

[0334]

[0335] Example 3: Early in vitro administration of MEKi and / or NOTCHi

[0336] In order to generate hPSC-derived cell cultures or cell products that are rich in ventral midbrain dopaminergic neurons and depleted of other cell types (i.e., other neurons, glial precursors, glial cells, stromal cells, proliferating cells), novel inhibitors and inhibitor combinations are applied to ventral midbrain neural cell cultures. Immediately after the hPSC neural / ectoderm specification (with small molecule SMAD inhibitors, adding head protein), ventralization (with SHH pathway agonists such as SHH, purmorphamine or SAG) and caudal lateralization (with WNT agonists such as WNT proteins, small molecules CHIR999021), the culture of VM NPCs is exposed to MEK inhibitors and / or NOTCH inhibitors. This exposure immediately after neural ectoderm patterning, ventralization and caudal lateralization is referred to as "early administration" ( Figure 1 Early administration refers to the time when the culture is mainly composed of VM NSCs and minimal expression of VM IPC and / or VM neuronal markers corresponding to DIV16 of the protocol in Example 1 is observed ( Figure 1 ).

[0337] Cells treated with MEK inhibitors and / or NOTCH inhibitors early were profiled by flow cytometry ( Figure 2) to assess the protein expression levels of ventral midbrain floor plate regional identity transcription factors (FOXA2, LMX1A, OTX2, EN1) and cell stage markers (KI67, which identifies highly proliferative cells such as NSCs; SOX2, which identifies NSCs; ASCL1, which identifies IPCs; INA, which identifies neurons).

[0338] Table 2: FCM Overview VM DIV16

[0339]

[0340] It was observed that the cultures analyzed belonged to the ventral midbrain in their regional identity, as evidenced by the expression of their relevant ventral midbrain floor plate markers FOXA2 (>50% of total cells), LMX1A (>45% of total cells), and OTX2 (>70% of total cells), as well as EN1 (>50% of total cells) ( Figure 2 and Table 2). These cells were still in the NSC stage, as indicated by their high expression of the NSC marker SOX2 (>70%) and the proliferation marker KI67 (>30%). These cultures began to express low levels of markers of ventral midbrain IPCs (ASCL1, 0.48-2.86%) and markers of neurons as determined by total INA expression (1.43-3.12%) by the time of exposure, but did not yet express INA+ / SOX2- (0%) ( Figure 2 and Table 2 ).

[0341] After 5 days of exposure to MEKi alone, NOTCHi alone, or both MEKi and NOTCHi, cultures were reanalyzed using flow cytometry to assess protein expression levels of key genes and compared to untreated controls ( Figure 3 and Table 3).

[0342] Table 3: FCM results, early treatment (DIV16-22)

[0343]

[0344] Table 3 (continued)

[0345]

[0346] All groups showed high expression of ventral midbrain floor plate lineage markers FOXA2 and LMX1A (>72.4 of total cells) ( Figure 3 , Table 3). It was observed that control cells maintained high expression of the NSC marker SOX2 (78.2-93.7%) and high levels of the proliferation marker KI67 (46.1-73.3%), accompanied by low levels of the neuronal marker INA ( Figure 3, black bars). Taken together, this suggests that control cultures are primarily ventral midbrain floor plate neural stem cells. In contrast, cultures treated with MEKi alone ( Figure 3 , open bars, and Table 3) and cultures treated with NOTCHi only ( Figure 3 Downregulation of the expression of NSC and proliferation markers SOX2 and KI67 was observed in both 1:1 and 2:1 inhibitors (bars, narrow stripes); indicating that these cells are differentiating toward neurons at a higher rate in response to these inhibitors compared to controls. The most striking results were observed for the combined administration of MEKi and NOTCHi to the cultures ( Figure 3 , thick striped bars, and Table 3). Combined administration reduced the expression of the NSC marker SOX2 to a greater extent (32.4-53%) and significantly reduced the level of the proliferation marker KI67 (4.24-5.55% of all cells), accompanied by a significant increase in the expression level of the neuronal marker INA (42%) ( Figure 3 and Table 3). Taken together, these results suggest that dual administration of MEKi and NOTCHi has a synergistic effect in increasing the proportion of neurons and decreasing the proportion of NSCs and proliferating cells ( Figure 3 and Table 3).

[0347] These results support the hypothesis that early addition of MEKi, NOTCHi, and especially 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 , Figure 2 and Figure 3 , Tables 2 and 3).

[0348] In the case of administration of the compound and Figure 2 and Figure 3 After flow cytometry analysis as indicated, all groups were differentiated in vitro for 11 days and then fixed in 4% paraformaldehyde and stained with DAPI to recognize all 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 fluorescently labeled secondary antibodies. Images were acquired on an Olympus IX81 microscope using CellSens software. Immunofluorescence (IF) staining confirmed Figure 3 Flow cytometry results of Figure 2, where prolonged differentiation showed that treatment with NOTCHi alone reduced the amount of SOX2-labeled NSCs compared to untreated controls ( Figure 4 AD) and the amount of proliferating cells labeled by KI67 ( Figure 5B, D), while the amount of neurons labeled by INA was maintained ( Figure 5 A, C).

[0349] In addition, consistent with flow cytometry results from earlier time points, prolonged differentiation demonstrated that MEKi treatment alone reduced the amount of SOX2-labeled NSCs compared to untreated controls ( Figure 4 AB, EF) and the amount of proliferating cells labeled by KI67 ( Figure 5 B, F), while the amount of neurons labeled by INA was maintained ( Figure 5 A, E). Further matching the flow cytometry results at earlier time points, prolonged differentiation confirmed that treatment with MEKi and NOTCHi reduced the amount of NSCs labeled by SOX2 compared to untreated controls ( Figure 4 AB, GH) and the amount of proliferating cells labeled by KI67 ( Figure 5 B, H), while the amount of neurons labeled by INA was maintained ( Figure 5 A, G).

[0350] Prolonged differentiation allowed analysis of markers of subtype lineages of interest, specifically tyrosine hydroxylase (TH), a rate-limiting enzyme that identifies dopamine neurons, and COL1A1, a gene expressed by non-neuronal stromal cells. IF staining results of prolonged differentiation showed that treatment with NOTCHi alone, MEKi alone, or dual treatment with MEKi and NOTCHi did not prevent the formation of dopamine neurons ( Figure 6 B, D, F, H), and comparison with the total number of cells labeled with DAPI showed that the proportion of TH neurons increased under inhibitor treatment conditions ( Figure 6 AH). The results of IF staining of prolonged differentiation also showed that treatment with NOTCHi alone, MEKi alone, or dual treatment with MEKi and NOTCHi did not prevent the expression of the ventral midbrain lineage marker LMX1A ( Figure 7 Most notably, treatment with either MEKi alone or NOTCHi inhibitor alone significantly reduced the proportion of stromal COL1A1 cells compared with untreated control cultures ( Figure 7 B, DF), while the dual administration of MEKi and NOTCHi further reduced their ratios ( Figure 7 H).

[0351] These results support the hypothesis that early addition of MEKi, NOTCHi, and especially combined administration of MEKi and NOTCHi, can promote the differentiation of ventral midbrain floor plate NSCs into ventral midbrain floor plate neurons, especially dopamine 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 ( Figure 4-Figure 7 ).

[0352] Example 4: Late in vitro administration of MEKi and / or NOTCHi

[0353] To assess the extent and ability of MEKi and / or NOTCHi to promote differentiation of ventral midbrain floor plate NSCs into ventral midbrain floor plate neurons, particularly dopamine neurons, at the expense of non-neuronal cells such as stromal cells, further studies were performed on cultures that had differentiated to later developmental stages at later in vitro time points ( Figure 8 and Fig.11 and summarized in Tables 4 and 5).

[0354] VM NPC cultures that had completed the hPSC neural / ectodermal specification, ventralization, and caudalization stages were allowed to further differentiate in vitro for 6 days in the absence of these patterning factors. During this time (days 16-22 in vitro) and at all time points prior to this (days 0-22 in vitro), cell cultures were not exposed to MEK inhibitors and / or NOTCH inhibitors ( Figure 8 ). During the period from day 16 to day 22, Figure 1-Figure 2 Compared to the early administration in Table 2, cells acquired a greater proportion of cells with more mature developmental identities, such as IPCs and neurons, because the level of SOX2 decreased, and the percentage of cells expressing the intermediate progenitor and neuronal markers ASCL1 and INA and INA+ / SOX2- increased ( Fig. 9 , Table 4). The cells were then exposed to MEK inhibitors and / or NOTCH inhibitors ( Figure 8 ), which is called "late administration".

[0355] We envision that this population could be transplanted into animal models of Parkinson's disease for the purpose of restoring function and into human patients for therapeutic purposes.

[0356] Table 4: FCM profile VM DIV22 (no early NOTCHi)

[0357] FOXA2 SOX2 Ki67 ASCL1 INA INA+ / SOX2- #1 83.8 90.1 52.4 42.3 13.9 2.61 #2 90.6 91.2 60.8 61.6 13.1 3.71 #3 89.5 92.2 56.2 58.9 12.4 3.53 #4 63.4 79.6 50.9 44.4 13 3.3 #5 88.4 84.9 29.1 39.1 11.8 average value 82.1 86.2 51.0 48.0 12.8 3.29 SEM 4.27 2.36 4.61 3.95 0.35 0.24

[0358] Prior to late administration, cells were profiled by flow cytometry ( Fig. 9 and Table 4) to assess the protein expression levels of ventral midbrain floor plate regional identity transcription factors (FOXA2) and cell stage markers (NSC and proliferation markers SOX2 and KI67; ASCL1, which identifies IPCs; INA, which identifies neurons). It was observed that these analyzed cultures belonged to the ventral midbrain in terms of their regional identity and expressed the ventral midbrain floor plate marker FOXA2 (>60% of all cells) ( Fig. 9These cells are still in the NSC stage, as evidenced by their high expression of NSC markers SOX2 and KI67 (>79% and >50%, Fig. 9 , Table 4). It is worth noting that these cultures Figure 1-Figure 2 The early-administered cultures treated with medium were more mature, as the levels of ASCL1 increased dramatically to 48%, total INA expression increased to 12.8%, and some cells were now INA+ / SOX2-, accounting for 3.28%, ( Fig. 9 and Table 4).

[0359] After 5 days of exposure to MEKi alone, NOTCHi alone, or both MEKi and NOTCHi, cultures were reanalyzed using flow cytometry to assess protein expression levels of key genes and compared to untreated controls ( Fig.10 , Table 5). In addition, the cultures were differentiated for another 7 days in the absence of any NOTCH inhibitors, and ICC was performed using DAPI that recognizes all nuclei and primary antibodies against FOXA2 (1:200), LMX1A (1:1000), SOX2 (1:300), Ki-67 (1:250), and HuC / D (1:100).

[0360] Table 5: FCM / ICC results, late administration (DIV22-29)

[0361]

[0362] Table 5 (continued)

[0363]

[0364] It was found that all groups maintained high expression (as a percentage of total cells) of the ventral midbrain floor plate lineage markers FOXA2 (>68%) and LMX1A (>80%) ( Fig.10 and Table 5). It was observed that control cells maintained high expression of the NSC marker SOX2 (average 74%) and high levels of the proliferation marker KI67 (average 34.6%), accompanied by moderate levels of the neuronal marker INA (average 30.5%) ( Fig.10 , open bars, and Table 5). Taken together, this suggests that at this late in vitro time point, control cultures are primarily ventral midbrain floor plate neural stem cells, with a subset of neurons. In contrast, cultures treated with MEKi alone ( Fig.10 , narrow striped bars, and Table 5) and cultures treated with NOTCHi only ( Fig.10, thick striped bars, and Table 5) all observed downregulation of NSC and proliferation markers SOX2 and KI67; indicating that these cells are differentiating into neurons at a higher rate in response to these inhibitors compared to controls. Fig.10 , black / solid bars, Table 5) also reduced the expression of the NSC marker SOX2 (average 45.4%) and significantly reduced the level of the proliferation marker KI67 (average 3.7% for all cells), accompanied by a significant increase in the expression level of the neuronal marker INA (average 68.8%) ( Fig.10 , Table 5). Taken together, these results indicate that MEKi, NOTCHi, and especially the dual administration of these two pathway inhibitors, have the effect of increasing the proportion of neurons and decreasing the proportion of NSCs and proliferating cells. Comparison between the results of early and late treatment of VM neural cells with MEKi and NOTCHi showed that late administration produced a greater number of INA neurons (average 68.8%) compared to early administration (average 42%).

[0365] 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 altering the expression of floor plate lineage transcription factors. Taken together, the data suggest that late administration of MEKi and NOTCHi is preferred and superior for converting VM cells into VM neurons.

[0366] After compound administration and flow cytometry analysis as indicated, all groups were differentiated in vitro for another 11 days, then fixed in 4% paraformaldehyde and stained with DAPI, which recognizes all 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 fluorescently labeled secondary antibodies. Images were acquired on an Olympus IX81 microscope using CellSens software. Immunofluorescence (IF) staining confirmed the flow cytometry results, where prolonged differentiation showed that treatment with NOTCHi alone reduced the amount of NSCs labeled by SOX2 compared to untreated controls ( Fig.12 AD) and the amount of proliferating cells labeled by KI67 ( Fig.13 B, D), while the amount of neurons labeled by INA was maintained ( Fig.13 A, C).

[0367] In addition, consistent with flow cytometry results from earlier time points, prolonged differentiation demonstrated that MEKi treatment alone reduced the amount of SOX2-labeled NSCs compared to untreated controls ( Fig.12 AB, EF) and the amount of proliferating cells labeled by KI67 ( Fig.13 B, F), while the amount of neurons labeled by INA was maintained ( Fig.13 A, E). Further matching the flow cytometry results at earlier time points, prolonged differentiation confirmed that treatment with MEKi and NOTCHi reduced the amount of NSCs labeled by SOX2 compared to untreated controls ( Fig.12 AB, GH) and the amount of proliferating cells labeled by KI67 ( Fig.13 B, H), while the amount of neurons labeled by INA was maintained ( Fig.13 A, G).

[0368] Prolonged differentiation allowed analysis of markers of subtype lineages of interest, specifically tyrosine hydroxylase (TH), a rate-limiting enzyme that identifies dopamine neurons, and COL1A1, a gene expressed by non-neuronal stromal cells. IF staining results of prolonged differentiation showed that treatment with NOTCHi alone, MEKi alone, or dual treatment with MEKi and NOTCHi did not prevent the formation of dopamine neurons ( Fig.14 B, D, F, H), and comparison with the total number of cells labeled with DAPI showed that the proportion of TH neurons increased under inhibitor treatment conditions ( Fig.14 AH). The results of IF staining of prolonged differentiation also showed that treatment with NOTCHi alone, MEKi alone, or dual treatment with MEKi and NOTCHi did not prevent the expression of the ventral midbrain lineage marker LMX1A ( Fig.15 Most notably, treatment with either MEKi alone or NOTCHi inhibitor alone significantly reduced the proportion of stromal COL1A1 cells compared with untreated control cultures ( Fig.15 B, D, F), while the dual administration of MEKi and NOTCHi further reduced their ratios ( Fig.15 H).

[0369] These results support the hypothesis that late addition of MEKi, NOTCHi, and especially combined administration of MEKi and NOTCHi, can promote the differentiation of ventral midbrain floor plate NSCs into ventral midbrain floor plate neurons, especially dopamine 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 ( Figure 12-Figure 15 ).

[0370] Example 5: Overview of late VM neurons treated with NOTCHi at DIV16-22

[0371] In this embodiment, VM NPC cultures that have completed the hPSC neural / ectoderm specification, ventralization and caudalization stages are further differentiated in vitro for 6 days from day 16 without these patterning factors, and exposed to NOTCH inhibitors (DAPT) every 48-72 hours between DIV16-22. In the art, cultures are usually exposed to NOTCH inhibitors immediately after stopping the use of one or more or all patterning factors; for example, patterning factors are factors used to induce the neural ectoderm of the embryo (i.e., head protein (NOGGIN), SMAD inhibitors) and / or dorsal-ventral patterning factors (i.e., BMP, SHH, SAG, purmorphamine) and / or rostral-caudal patterning factors (i.e., WNT proteins, CHIR, FGF8), as described in publications Nolbrant et al., 2017, Kriks et al., 2011, Nishimura et al., 2023. During this time (DIV16-22) and at all time points prior to this (days in vitro 0-16), cell cultures were not exposed to MEK inhibitors.

[0372] At DIV16, when the cultures were predominantly NPCs and contained no or <2% INA+ / SOX2- cells ( Figure 2 , Table 1), adding NOTCH inhibition immediately after removal of patterning factors until DIV22 resulted in a profound effect on cell stage / maturation, meaning a profound effect on the proportion of IPCs and neurons at DIV22. This can be observed at the protein level by flow cytometry profiles, see Fig. 9 and Table 4, which shows the profile at DIV22 in the absence of any early NOTCH inhibition and compared with that with early NOTCHi Fig.11 and Table 6. Of note, early (DIV16-22) treatment with NOTCH inhibition resulted in a higher proportion of neurons that were INA+ cells (24.7% with NOTCHi and 12.8% without NOTCHi) or INA+ / SOX2- cells (8.4% with NOTCHi and 3.3% without NOTCHi). Therefore, we expected that this early treatment with NOTCHi, immediately after withdrawal of patterning factors, would result in cultures that were not optimal for exposure to dual MEKi / NOTCHi treatment and conversion to neurons, as more cells were already at the neuronal stage. Furthermore, we expected that such a population with more neurons would not be optimal for transplantation (as neurons are fragile and sensitive to transplantation stress), and we expected that Fig. 9and Table 4. Populations containing many ASCL1+ IPCs (51.8%) and with few neuronal (INA+ or INA+ / SOX2-) cells were superior for transplantation and / or addition of dual MEK and NOTCH inhibition.

[0373] References:

[0374] Kaneyasu Nishimura, Shanzheng Yang, Ka Wai Lee, Emilia Sif Asgrimsdottir, 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 hESCdifferentiation.2023.Stem Cell Reports. https: / / doi.org / 10.1016 / j.stemcr.2022.10.016

[0375] ·Sonja Kriks,Jae-Won Shim,Jinghua Piao,Yosif M Ganat,Dustin RWakeman,Zhong Xie,Luis Carrillo-Reid,Gordon Auyeung,Chris Antonacci,AmandaBuch,Lichuan Yang,M Flint Beal,D James Surmeier,Jeffrey H Kordower,Viviane Tabar,Lorenz Studer.Dopamine derived neurons from human ES cells efficiently engraft in animal models of Parkinson's disease.2011.Nature.10.1038 / nature10648

[0376] Table 6: FCM profile VM DIV22 (+ early NOTCHi)

[0377] FOXA2 SOX2 Ki67 ASCL1 INA INA+ / SOX2- #1 85.4 72 5.9 50.4 23.6 7.3 #2 98.2 70.5 4.8 57.1 34.2 13.6 #3 82.1 64.7 6.0 40.5 19 5.7 #4 94.9 65.7 6.6 51.8 45.6 20.1 #5 67.6 69.5 6.1 36.0 18.3 7.0 #6 69.7 70.9 6.4 41.1 30.5 13.8 #7 76.2 86.3 8.1 41.7 19.2 4.7 #8 82.7 88.3 9.7 45.8 22.4 6.2 #9 78.0 77.2 7.8 16.2 13.8 2.6 #10 75.5 78.5 5.2 17.3 13.3 5.0 #11 35.2 76.4 19.8 25.6 32.0 6.0 average value 76.9 74.5 7.9 38.5 24.7 8.4 SEM 5.04 2.32 1.27 4.11 2.97 1.58

[0378] Example 6: Further culturing of cell populations after MEK / NOTCH inhibition to assess expression profiles

[0379] After administration of MEK and NOTCH inhibitors, cells were cultured in neural support medium supplemented with BDNF (20 ng / ml), GDNF (20 ng / ml), L-ascorbic acid (200 μM), dcAMP (500 μM) or other such support medium in the presence of poly-L-ornithine (0.002%) and laminin-521 (1.5 μg / cm 2 ) coated wells in two-dimensional culture for another 5 days, which allows time for the transformation of neural precursors and intermediates and final differentiation into ventral midbrain neurons. After 5 days of culture, the expression profile of the cell population can be evaluated according to any of the methods described in Examples 7, 8 and 9.

[0380] Example 7: RNA sequencing method

[0381] For single-cell RNA sequencing (scRNA-seq) or single-cell nuclear RNA sequencing, undifferentiated PSCs as well as differentiated cells were dissociated into single-cell suspensions using accutase, trypleselect or other such reagents, and 3,000-10,000 cells were processed using the 10XGenomics Chromium Platform and sequenced on the NextSeq550. Data were processed using 10X cellranger and the Seurat analysis package in the R programming language. Samples were analyzed, low-quality or multi-state cells were filtered, and analyzed separately for each individual experiment, and then cells of selected differentiated cell lineages as well as hPSCs were combined into one dataset and then analyzed using the standard Seurat workflow as outlined for Seurat version 3, i.e., normalization using SCTransform, and finally analysis of the unified tSNE plot using the first 29 principal components.

[0382] Example 8: Immunocytochemistry (ICC)

[0383] Cells were fixed in 4% paraformaldehyde (Alfa Aesar) for 10 min at room temperature. 2+ and Mg 2+The sections were incubated with 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 min, followed by incubation with the primary antibody (see Table X) overnight at 4°C to block nonspecific antibody binding. 2+ and Mg 2 + The cells were washed three times with PBS, blocked with PADT buffer for 15 minutes, and incubated with fluorophore-conjugated secondary antibodies (see Table X) for 2 hours at room temperature in the dark. The cells were then counterstained with DAPI (10 μg / mL) for 5 minutes at room temperature and incubated with Ca-free 2+ and Mg 2+ Wash 3 times with PBS without Ca 2+ and Mg 2+ But stored in PBS supplemented with 0.02% sodium azide at 4° C. Images were captured using a Zeiss Axio Observer microscope equipped with an Axiocam 512 camera and ZEN 3.2 (Pro) software (Zeiss).

[0384] Example 9: Quantitative real-time PCR (qPCR or qRT-PCR)

[0385] For qPCR, RNA is extracted from cells using Trizol and converted into cDNA, which is then analyzed using quantitative real-time polymerase chain reaction (qPCR) for genes of interest such as SOX2, KI67, HuCD, NeuN, INA, ASCL1, FOXA2, TH, LMX1A, EN1, or other relevant markers of ventral midbrain neurons. qPCR is typically performed on three technical replicates of each of three or more independent biological replicates and normalized to housekeeping genes such as GAPDH or HPRT1.

[0386] Although certain features of the present invention have been illustrated and described herein, many modifications, substitutions, changes and equivalents will now occur to those skilled in the art. Therefore, it should be understood that the appended claims are intended to cover all such modifications and changes that fall within the true scope of the invention.

Claims

1. An in vitro method for directing the differentiation of a PSC cell population into ventral midbrain neurons, comprising culturing a PSC cell population, contacting the PSC cell population with an inhibitor of SMAD protein signaling, an inhibitor of Wnt signaling, an activator of SHH signaling, an activator of FGF signaling, optional ascorbic acid and optional BDNF to obtain a cell population containing ventral midbrain NSCs, wherein the cell population containing ventral midbrain NSCs is further contacted with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling to direct differentiation into ventral midbrain neurons, and wherein the cell population is allowed to differentiate into ventral midbrain NSCs for 14 to 24 days before contacting the cell population with the inhibitor of MEK signaling and the inhibitor of NOTCH signaling.

2. An in vitro method for directing the differentiation of ventral midbrain NSCs into neurons, comprising contacting a cell population comprising ventral midbrain NSCs with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, wherein at least 5% of the cell population comprising ventral midbrain NSCs co-expresses the markers FOXA2, LMX1A, EN1, OTX2, and SOX2, and wherein the cell population is differentiated into ventral midbrain NSCs for 14 to 24 days prior to contacting the cell population with the inhibitor of MEK signaling and the inhibitor of NOTCH signaling.

3. An in vitro method for directing the differentiation of ventral midbrain NSCs into neurons, comprising contacting a cell population comprising ventral midbrain NSCs with an inhibitor of MEK signaling and an inhibitor of NOTCH signaling, wherein 40-60% of the cell population comprising ventral midbrain NSCs express ASCL1, 45-65% of the cell population comprising ventral midbrain NSCs express KI67, 10-15% of the cell population comprising ventral midbrain NSCs express INA, 2-5% of the cell population comprising ventral midbrain NSCs are INA+ / SOX2-, and 80-95% of the cell population comprising ventral midbrain NSCs express SOX2.

4. The method according to any one of the preceding claims, comprising initially differentiating the cell population towards ventral midbrain NSCs, wherein the cell population is harvested on day 28, day 27, day 26 or day 25, preferably day 25, from the initial differentiation of the cell population towards ventral midbrain NSCs.

5. The method according to any one of the preceding claims, wherein the inhibitor of MEK signaling is selected from PD0325901, trametinib (GSK1120212), selumetinib (AZD6244), pimacetinib (AS703026), MEK162, cobimetinib, PD184352, PD173074, BIX02189, AZD8330, PD318088, refactinib and PD98059, preferably PD0325901.

6. The method according to any one of the preceding claims, wherein the inhibitor of NOTCH signaling is selected from DAPT, Avagacestat, PF-03084014 and LY450139.

7. The method of any of the preceding claims, wherein the cell population is contacted with the inhibitor of MEK signaling and the 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.

8. The method according to any one of the preceding claims, wherein the cell population is contacted at least partially simultaneously with the inhibitor of MEK signaling and the inhibitor of NOTCH signaling.

9. The method of any one of the preceding claims, wherein the cell population is contacted simultaneously with the inhibitor of MEK signaling and an inhibitor of NOTCH signaling.

10. The method of the preceding claim, wherein the cell population is cryopreserved within 4 days after the end of MEK signaling inhibition and NOTCH signaling inhibition.

11. An in vitro cell population comprising ventral midbrain NSCs, wherein when said cell population is cultured in vitro for 5 days in a medium suitable for maintaining neural cells, a cell population is produced in which at least 50% are INA+ or HuCD+ and less than 15% are KI67+.

12. A cell population according to the preceding claim, wherein the cell population is cultured according to Example 6.

13. The cell population according to any one of claims 11 and 12, wherein the expression of markers of the cells is measured according to the method described in Example 8.

14. A composition comprising a cell population comprising ventral midbrain NSCs, an inhibitor of MEK signaling, and an inhibitor of NOTCH signaling.

15. A composition according to the preceding claim for use in the treatment of Parkinson's disease.

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