Enhancement of neuronal differentiation in neural progenitor cells

Abstract

The present invention relates to a method for obtaining a cell population containing nerve cells and directing the differentiation of nerve cells into neurons by contacting the cell population with a NOTCH signaling inhibitor, the NOTCH signaling inhibitor being selected from LY411575, abagacestat, dibenzazepine, and PF-03084014.

Description

[Technical Field]

[0001] This invention generally relates to the field of stem cells, such as human embryonic stem cells. Methods for obtaining stem cell-derived nerve cells are provided. Specifically, methods for directing the differentiation of stem cell-derived nerve cells into neurons in the forebrain, midbrain, and hindbrain / spinal cord regions are provided. [Background technology]

[0002] The prospects for using human pluripotent stem cells (hPSCs) to treat a variety of pathological conditions are considered very promising. These therapies include cell replacement therapy for neurological conditions such as Parkinson's disease, stroke, paralysis, epilepsy, and other such disorders. However, for these therapies to be viable, the development of in vitro methods for artificially producing stem cell-derived products for delivery to the central nervous system (CNS) is necessary. Differentiation of hPSCs into defined cell types is a difficult process to control, and often the offspring produced from in vitro protocols are heterogeneous. Typically, when differentiating into nerve cells, the resulting mixture of cell types includes neurons (and various neuronal subtypes within these, such as glutamatergic and dopaminergic neurons), glial cells, neural stem cells (NSCs), and other non-neuronal cells (e.g., meningeal stromal cells). Such heterogeneous cultures are suboptimal for disease modeling studies or many cell replacement therapies.

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

[0004] In cases of stroke or paralysis resulting from spinal cord injury, only specific neuronal focal identity cell types and specific neuronal subtypes are likely required to restore lost function in both cases, while glial cells, stromal cells, or other types of neurons are likely not necessary. Thus, for these adaptations, it is also beneficial to have maximum control over the differentiation process in order to have refined cell products for maximum efficacy and safety.

[0005] Therefore, an object of the present invention is to overcome the aforementioned problems, and in particular to provide a method that can direct the differentiation of nerve cells into neurons. [Overview of the Initiative]

[0006] The objectives outlined above are achieved by embodiments of the present invention. In addition, the present invention may also solve further problems that become apparent from the disclosure of exemplary embodiments.

[0007] In a first aspect, the present invention provides a method comprising obtaining a cell population including nerve cells and contacting the cell population with a NOTCH signaling inhibitor, wherein the NOTCH signaling inhibitor is selected from LY411575, abagacestat, dibenzazepine, and PF-03084014, preferably LY411575. Specifically, the method is for directing the differentiation of nerve cells into neurons, meaning that the developmental fate of nerve cells is influenced toward a particular outcome, namely, neuron. However, differentiation into neurons is not necessarily completed by this method, and cells do not necessarily develop to their final fate in the method disclosed herein. Preferably, cells are directed toward differentiation toward neurons in vitro only if it is suitable for late administration to a patient and further development in vivo results in a neuronal fate. The inventors have found that the results of protocols for differentiating cell populations such as human PSCs toward or into neurons can be improved by inhibiting the NOTCH signaling pathway using alternative compounds rather than those commonly used in the field of neuronal differentiation, namely the small molecule DAPT. Surprisingly, the inventors have found that compound LY411575 demonstrates improved upregulation of neuronal genes and downregulation of NPC genes during differentiation compared to DAPT. The inventors have further identified alternative compounds abagacestat, dibenzazepine, and PF-03084014 as having similar capabilities. Specifically, as neurons further differentiate toward terminal phase fate, this inhibition thus further reduces the proportion of late-proliferating cells (representing off-target lines), reduces the proportion of stromal cells such as VLMCs and glial cells such as astrocytes, and simultaneously increases the proportion of neurons. The inventors have found that the above-mentioned compounds are superior to, for example, the use of DAPT. 24 hours after administration, cells treated with either of the compounds produced more neurons (HuC / D or INA-positive cells) and had lower proliferative capacity (SOX2 and / or Ki67-positive cells) compared to similar treatment with DAPT.

[0008] Importantly, the inventors have found that the timing of inhibiting NOTCH signaling relative to the developmental stage of the cells influences the outcome. In one embodiment, the cell population is obtained by differentiating pluripotent stem cells (PSCs) into neurons, such as by neural induction. Those skilled in the art will recognize that cells undergo a series of developmental stages, initially becoming neural stem cells, and then progressing toward neuron identity as neuroblast intermediate progenitor cells. Specifically, the inventors have identified a window of opportunity during neuronal development, during which the neuron is particularly susceptible to influence so as to be directed toward neuronal fate. In a preferred embodiment, more than 2% (e.g., more than 5%, more than 10%, more than 15%, more than 20%, or more than 25%) of the cell population are INA+ / SOX2- at the time the cell population is exposed to the NOTCH signaling inhibitor.

[0009] In preferred embodiments, a population of PSCs is neurogenically induced according to well-known methods, such as exposing the PSCs to an inhibitor of SMAD protein signaling, and selectively targeting both the TGF-beta and BMP pathways. The inventors also recognize that culturing the neurogenically induced cell population for an excessively long period closes the window of opportunity. In the aforementioned methods for neurogenically induced PSCs, it has been found that the cultured cell population may be difficult to harvest due to exceeding a certain threshold of INA+ / SOX2- resulting from the formation of an inseparable mesh of neurites.

[0010] The in vitro cell populations obtained according to this method may be used for the treatment of neurological conditions such as Parkinson's disease, and the higher purity of the cell product may provide a reduction in surgical procedure time and cranial injection. In addition, improved product purity and reduced impurities offer enhanced safety and potential efficacy profiles. [Brief explanation of the drawing]

[0011] [Figure 1]Figure 1 shows a schematic diagram of the developing brain in sagittal section. The dorsal forebrain region generated using the differentiation protocols described in Figures 2 and 20 is highlighted in the viewfinder. [Figure 2] Figure 2 shows a simplified schematic illustrating the stages of dorsal forebrain differentiation. The procedure begins with hPSCs (DIV0) differentiated into dorsal FB NSC cells after neural induction, as well as the absence of cues for ventral and caudal differentiation. Cells were profiled via flow cytometry analysis in DIV18 (Figure 3) and DIV30 (Figure 4) (indicated by asterisks) of the differentiation protocol. Administration of the compound of the present invention, the NOTCH inhibitor LY411575, occurred in DIV30 of differentiation (black arrow). [Figure 3] Figure 3 shows a bar graph of the results of intracellular flow cytometry protein analysis of dorsal FB cell cultures in DIV18. It shows the expression of the NSC marker SOX2, FB lineage markers OTX2 and PAX6, as well as the FB IPC marker TBR2, and the neuronal marker INA, described as the INA+ / SOX2- population. The graph shows the percentage of cells out of total viable cells. The levels of these markers indicate that these cells are dorsal forebrain NSCs. [Figure 4] Figure 4 shows a bar graph of the results of intracellular flow cytometry protein analysis of cell cultures in DIV30. It shows the expression of the NSC marker SOX2 and the proliferation marker KI67. This also shows the expression of the neuronal marker INA, described as an INA+ / SOX2- population, as well as the FB lineage markers OTX2 and PAX6. The graph shows the percentage of cells out of total viable cells. The levels of these markers indicate that these cells are dorsal forebrain neurons. [Figure 5]Figure 5 shows a bar graph of the results of ICC analysis of cell cultures on day 35 in vitro after contacting the cell cultures with the NOTCH inhibitor LY411575 for 24 hours from DIV30 to DIV31. Results of cultures without intervention (control, white bars), and cultures with administration of LY411575 for 24 hours (black bars) are shown. Protein expression is shown for the NSC marker SOX2 and the proliferation marker KI67, as well as the neuron marker HuC / D. The graph displays the percentage of cells out of the total cells. [Figure 6] Figure 6 shows a bar graph of the results of ICC analysis of cell cultures on day 35 in vitro after contacting the cell cultures with the NOTCH inhibitor LY411575 for 72 hours from DIV30 to DIV33. Results of cultures without intervention (control, white bars), and cultures with administration of LY411575 for 72 hours (black bars) are shown, and protein expression is shown for the NSC marker SOX2 and the proliferation marker KI67, as well as the neuron marker HuC / D. The graph displays the percentage of cells out of the total cells. [Figure 7] Figure 7 shows a schematic diagram of the developing brain in a sagittal section. The ventral midbrain region generated using the differentiation protocol shown in Figures 8 and 23 is highlighted within the viewfinder. [Figure 8] Figure 8 shows a simplified schematic diagram illustrating the stages of ventral MB differentiation. The procedure starts with hPSCs (DIV0) that are differentiated into ventral MB NSCs after neural induction, ventralization, and caudalization. Cell profiles via flow cytometry analysis were performed at DIV16 (Figure 9) and DIV22 (Figure 10) (represented by asterisks) of the differentiation protocol. The compound of the present invention, the NOTCH inhibitor LY411575, is administered at DIV22 of the differentiation protocol (black arrow). [Figure 9]Figure 9 shows a bar graph of the results of intracellular flow cytometry protein analysis of cell cultures in DIV16. It shows the expression of the NSC marker SOX2 and the proliferation marker KI67. It also shows the expression of ventral MB as well as the floor plate lineage markers FOXA2, LMX1A, EN1, and OTX2, and also the expression of the MB IPC marker ASCL1 and the neuron marker INA shown as INA and as the INA+ / SOX2- population. The graph shows the percentage of cells out of all viable cells. [Figure 10] Figure 10 shows a bar graph of the results of intracellular flow cytometry protein analysis of ventral MB cell cultures in DIV22 when administration of the NOTCH inhibitor LY411575 was initiated. It shows the expression of the NSC marker SOX2 and the proliferation marker KI67. It also shows the expression of not only the floor plate lineage marker FOXA2, but also the MB IPC marker ASCL1 and the neuron marker INA shown as INA and as the INA+ / SOX2- population. The graph shows the percentage of cells out of all viable cells. [Figure 11] Figure 11 shows a bar graph of the results of ICC analysis of cell cultures on days 29 - 30 in vitro after administration of LY411575 for 24 hours from DIV22 to DIV23. It shows the results of cultures without intervention (control, white bars), and cultures with administration of the NOTCH inhibitor LY411575 (black bars). Protein expression for the NSC marker SOX2, the proliferation marker KI67, the neuron marker HuC / D, and the VM floor plate lineage marker FOXA2 is shown. The graph shows the percentage of cells out of all cells. [Figure 12]Figure 12 shows bar graphs of ICC analysis results for cell cultures at in vitro days 29–30 after administration of LY411575 between 72–96 hours. Results are shown for cultures without intervention (control, white bars) and cultures administered with the NOTCH inhibitor LY411575 (black bars). Protein expression for the NSC marker SOX2, the proliferation marker KI67, the neuron marker HuC / D, and the floorplate lineage marker FOXA2 is shown. The graphs show the percentage of cells out of total cells. [Figure 13] Figure 13 shows bar graphs of ICC analysis results for ventralized MB cell cultures on day 29 in vitro after administration of different NOTCH inhibitors over a 24-hour period (DIV22 to DIV23). Results are shown for unintervention cultures (control, white bars), DAPT administration (dotted bars), abagacestat (thin diagonal striped bars), PF-03084014 (thick diagonal striped bars), and LY411575 (black bars). Protein expression for the NSC marker SOX2 and proliferation marker KI67, the neuron marker HuC / D, and the floorplate lineage marker FOXA2 is shown. The graphs show the percentage of cells out of total cells. [Figure 14] Figure 14 shows a schematic diagram of the developing brain in sagittal section. The hindbrain / spinal cord region generated using the differentiation protocol described in Figure 15 is highlighted in the viewfinder. [Figure 15] Figure 15 shows a simplified schematic diagram illustrating the stages of hindbrain / spinal cord differentiation and an overview of the experiment. The procedure begins with hPSCs (DIV0) that differentiate into HB / SCord NSCs after neural induction, caudalization, and ventralization. Cells were profiled via flow cytometry analysis at DIV15 (Figure 16) (asterisk) of the differentiation protocol. The compound of the present invention, the NOTCH inhibitor LY411575, is administered at DIV15, as indicated by the black arrow in the schematic diagram. [Figure 16]Figure 16 shows a bar graph of the results of intracellular flow cytometry protein analysis of HB / SCord cell cultures in DIV15 at the start of administration of the NOTCH inhibitor LY411575. It shows the expression of off-target markers that identify not only the NSC marker SOX2, the HB / SCord precursor domain markers NKX6.1, OLIG2, NKX2.2, and PAX6, but also the ventral MB and floor plate lineage markers OTX2 and FOXA2, which indicate that these cells together belong to the ventromedial posterior brain / spinal cord lineage. [Figure 17] Figure 17 shows a bar graph of the results of ICC analysis of cell cultures at in vitro day 20 after administration of LY411575 over a 24-hour period from DIV15 to DIV16. Results are shown for cultures without intervention (control, white bars) and those administered with LY411575 (black bars). Protein expression is shown for the NSC marker SOX2 and the proliferation marker KI67, as well as the neuron marker HuC / D. The graph displays the percentage of cells out of total cells. [Figure 18] Figure 18 shows a bar graph of the results of ICC analysis of cell cultures at in vitro day 20 after administration of LY411575 over 72 hours from DIV15 to DIV18. Results are shown for cultures without intervention (control, white bars) and those administered with LY411575 (black bars). Protein expression is shown for the NSC marker SOX2 and the proliferation marker KI67, as well as the neuron marker HuC / D. The graph shows the percentage of cells out of total cells. [Figure 19]Figure 19 shows bar graphs of ICC analysis results for ventralized MB cultures at in vitro day 29 after administration of the NOTCH inhibitor LY411575 at different concentrations over a 24-hour period from DIV22 to DIV23. Results are shown for cultures without intervention (control, white bars) and for administration of 0.2 μM LY411575 (dotted bars), 0.5 μM LY411575 (thin striped bars), 2 μM LY411575 (thick diagonal striped bars), 10 μM LY411575 (horizontal striped bars), 40 μM LY411575 (thick diagonal striped bars), and 100 μM LY411575 (black bars). Protein expression for the NSC marker SOX2 and proliferation marker KI67, neuron marker HuC / D, and VM floorplate marker FOXA2 are shown. The graph displays the percentage of cells out of the total number of cells. [Figure 20] Figure 20 shows a simplified schematic illustrating the stages of dorsal forebrain differentiation. The procedure begins with hPSCs (DIV0) differentiated into dorsal FB NSC cells after neural induction, and without cues for ventral and caudal differentiation. Cells were profiled via flow cytometry analysis in DIV18, DIV25, and DIV27 of the differentiation protocol, and immunocytochemistry in DIV30 (represented by graph icons). Administration of the compound of the present invention, the NOTCH inhibitor LY411575, occurred in DIV25 of differentiation (black arrow). Cells were cryopreserved in DIV27 (represented by asterisks). [Figure 21] Figure 21 shows a bar graph of the results of intracellular flow cytometry protein analysis of dorsal FB cell cultures in DIV25. It shows the expression of the NSC marker SOX2, the proliferation marker Ki67, the FB lineage markers OTX2, SOX1, and PAX6, as well as the cortical / dorsal FB IPC marker TBR2, and the neuronal marker INA, which is described as an INA+ / SOX2- population. The graph shows the percentage of cells out of total viable cells. The levels of these markers indicate that these cells are dorsal forebrain neurons. [Figure 22]Figure 22 shows bar graphs of flow cytometry analysis results for dorsal FB cell cultures at in vitro day 27 after administration of the NOTCH inhibitor LY411575 for 24 hours or 48 hours (DIV25 / DIV26 to DIV27). Results are shown for cultures without intervention (control, white bars), LY411575 administration for 24 hours (diagonal striped bars), and LY411575 administration for 48 hours (black bars). Protein expression is shown for the NSC marker SOX2 and the proliferation marker KI67, and the neuronal marker HuC / D is shown as HuC / D or HuCD+ / SOX2- and the cortical IPC marker TBR2. The graphs show the percentage of cells among all viable cells. [Figure 23] Figure 23 shows a simplified schematic diagram illustrating the stages of ventral MB differentiation. The procedure begins with hPSCs (DIV0) differentiated into ventral MB NSCs after neural induction, ventralization, and caudalization. Cell profiling via flow cytometry analysis was performed at DIV16, DIV22, and DIV24, and immunocytochemistry was performed at DIV36 of the differentiation protocol (represented by graph icons). The compound of the present invention, the NOTCH inhibitor LY411575, is administered at DIV22 of the differentiation protocol (black arrow). Cells were cryopreserved at DIV24 (represented by asterisks). [Figure 24] Figure 24 shows bar graphs of flow cytometry analysis results for ventral MB cell cultures at in vitro day 24 after administration of the NOTCH inhibitor LY411575 for 24 or 48 hours (DIV22 / DIV23 to DIV24). Results are shown for cultures without intervention (control, white bars), LY411575 administration for 24 hours (diagonal striped bars), and LY411575 administration for 48 hours (black bars). Protein expression is shown for the NSC marker SOX2 and the proliferation marker KI67, the neuronal marker HuC / D is shown as HuC / D or HuCD+ / SOX2-, the cortical IPC marker TBR2, and the floorplate marker FOXA2. The graphs show the percentage of cells out of total viable cells. [Modes for carrying out the invention]

[0012] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. The practice of the present invention will, unless otherwise suggested, utilize conventional methods of chemistry, biochemistry, biophysics, molecular biology, cell biology, genetics, immunology, and pharmacology known to those skilled in the art.

[0013] It should be noted that all headings and subheadings in this specification are for convenience only and should not be construed as limiting the invention.

[0014] The use of any examples or illustrative language provided herein (e.g., "etc.") is solely intended to make the invention clearer and, unless otherwise claimed, does not limit the scope of the invention.

[0015] definition General definition As used herein, “a,” “an,” or “the” can mean one or more. Unless otherwise indicated herein, terms presented in the singular form also include plural situations.

[0016] Furthermore, as used herein, “and / or” means and encompasses all possible combinations of one or more of the associated enumerated items, as well as the absence of any combinations when interpreted as an alternative ("or"). Moreover, the present invention also intends that in some embodiments of the present invention, any feature or combination of features described herein may be excluded or omitted.

[0017] stem cells Stem cells should be understood as undifferentiated cells that possess differentiation and proliferative capabilities (especially self-renewal), but maintain their differentiation potential. Stem cells are classified according to their differentiation potential into pluripotent stem cells, multipotent stem cells, unipotent stem cells, and similar types.

[0018] As used herein, the term “pluripotent stem cell” (PSC) refers to any cell lineage that can be cultured in vitro and that belongs to any of the three germ layers (ectoderm, mesoderm, or endoderm), as well as stem cells that have the ability to differentiate into lineage-limited undifferentiated stem cells that have typically lost the ability to form certain cell types, lineages, or developmental regions through gene editing.

[0019] 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, and similar sources. Examples of pluripotent stem cells (PSCs) include embryonic stem cells (ESCs), embryonic germ cells (EG cells), induced pluripotent stem cells (iPSCs), and similar sources.

[0020] As used herein, the term “induced pluripotent stem cells” (also known as iPS cells or iPSCs) means one type of pluripotent stem cell that can be generated directly from cells that have a nucleus, and not from PSCs. Non-pluripotent cells can be converted into pluripotent stem cells by introducing the products of a specific set of pluripotency-related genes.

[0021] As used herein, the term “embryonic stem cells” means pluripotent stem cells derived from the inner cell mass of a blastocyst. Pluripotent embryonic stem cells may also be derived from parthenogenetic organisms, for example, as described in International Publication No. 2003 / 046141. In addition, embryonic stem cells can be produced from a single blastomeres or by culturing the inner cell mass obtained without destroying the embryo. Embryonic stem cells are available from given tissues and are also commercially available. Preferably, the methods and products of the present invention are based on stem cells derived from either human PSCs, i.e., human induced pluripotent stem cells, or human embryonic stem cells including parthenogenetic organisms.

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

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

[0024] As used herein, the term “in vitro” means that the cells are provided and maintained outside the body of a human or animal, such as in a flask, multiwell, or petri dish. Therefore, the cells are cultured in a cell culture medium.

[0025] As used herein, the term “non-natural” means that cells derived from pluripotent stem cells, but which may be of human origin, are artificial constructs that do not exist in nature. Generally, the goal in the field of stem cell therapy is to provide cells that are as similar as possible to the cells of the human body. However, it is never possible to mimic the development that pluripotent stem cells undergo during the embryonic and fetal stages to such an extent that mature cells become indistinguishable from natural cells of the human body. Essentially, in one embodiment of the present invention, the cells are artificial.

[0026] As used herein, the term “artificial” with respect to cells may include materials that are naturally occurring in nature but have been modified into non-naturally occurring structures. This includes human stem cells that differentiate into non-naturally occurring cells that mimic human cells.

[0027] protocol Throughout this application, the terms “method” and “protocol” may be used interchangeably when referring to the process of culturing or differentiating cells.

[0028] As used herein, the terms “days” in relation to the protocol and similarly “in vitro days (DIV)” refer to the specific time required to perform a particular step in the differentiation procedure.

[0029] Generally, and unless otherwise specified, "Day 0" refers to the start of the protocol, which includes, for example, plating the stem cells, transferring the stem cells to an incubator, or contacting the stem cells in the current cell culture medium with a compound before transplantation. Typically, the start of the protocol is by transferring the undifferentiated stem cells to another cell culture medium and / or container, for example by plating or incubation, and / or by first contacting the undifferentiated stem cells with a compound that affects the undifferentiated stem cells in such a way that the differentiation process is initiated.

[0030] In the context of the method, "cells" refers to all cells in a cell population, regardless of cell type.

[0031] When referring to "Day X," such as Day 1, Day 2, etc., this refers to the start of the protocol on Day 0. Those skilled in the art will recognize that, unless otherwise specified, the exact time for the execution of the process may vary. Therefore, "Day X" means encompassing a time range such as ±10 hours, ±8 hours, ±6 hours, ±4 hours, ±2 hours, or ±1 hour.

[0032] Stem cell culture As used herein, the term “culture” refers to a continuous procedure, which is performed throughout the method to maintain the viability of the cells at various stages. After the cells of interest have been isolated from, for example, living tissue or embryo, they are subsequently maintained under carefully controlled conditions. These conditions vary depending on the cell type but generally consist of suitable containers equipped with substrates and / or culture media that supply essential nutrients (amino acids, carbohydrates, vitamins, minerals), growth factors, hormones, and gases (CO2, O2) and regulate the physicochemical environment (pH buffer, osmotic pressure, temperature).

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

[0034] As used herein, the term “incubator” refers to any suitable incubator capable of supporting cell culture. Non-limiting examples include culture dishes, petri dishes, and plates (microtiter plates, microplates, deep-well plates, etc. of 6-well, 24-well, 48-well, 96-well, 384-well, 9600-well and similar sizes), flasks, chamber slides, tubes, cell factories, roller bottles, spinner flasks, hollow fibers, microcarriers, or beads.

[0035] Where used herein and referred to in a protocol, the term “provide stem cells” means obtaining a batch of cells by the methods described above and, optionally, transferring the cells to a different environment, such as by seeding them onto a new substrate. Those skilled in the art will readily recognize that stem cells are vulnerable to such migration, that the procedure requires care, and that maintaining the stem cells in the original cell culture medium may facilitate a more sustainable migration of cells before replacing the cell culture medium with another cell culture medium more suitable for further differentiation processes.

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

[0037] As used herein, the term “marker” refers to a spontaneously occurring, identifiable expression produced by a cell that can be correlated with a particular characteristic of the cell. In preferred embodiments, the marker is a gene expression or proteomics expression that can be detected and also correlated with the identity of the cell. The marker may be referred to as a gene expression, which can be readily translated into the expression of the corresponding mRNA and protein.

[0038] When used herein, in reference to any marker such as surface proteins or transcription factors disclosed herein, the terms “negative” or “-” refer to a marker that is not expressed in a cell or cell population, while the terms “weak” or “low” refer to a marker that is expressed in a cell at a reduced level compared to the average expression of the marker in the cell population or compared to a reference sample.

[0039] As used herein, the terms “positive” or “+” used with respect to any marker such as surface proteins or transcription factors disclosed herein refer to the marker expressed in a cell or cell population, while the terms “high” or “strong” refer to the marker expressed at an increased level in a cell compared to the mean expression of the marker in the cell population or compared to a reference sample.

[0040] As used herein, annotation INA+ / SOX2- refers to a situation in which a given cell is identified by two or more markers, i.e., a cell that is both INA+ and SOX2-.

[0041] As used herein, the term “differentiation” broadly refers to the process by which a cell progresses from an undifferentiated state or a state different from the intended differentiated state to a specific differentiated state, such as from an immature state to a less immature state, or from an immature state to a mature state, and this may occur continuously during the implementation of the method. In relation to pluripotent stem cells, the term “differentiation” refers to the process by which a cell progresses from an undifferentiated state to a specific differentiated state, i.e., from an immature state to a less immature state or a terminal state. Changes in cell interactions and maturation occur when a cell loses markers for undifferentiated cells or gains markers for differentiated cells. The loss or gain of a single marker may indicate that a cell has “fully differentiated” or “terminally differentiated.” A “terminally differentiated” cell is the final stage of a developmental lineage and cannot differentiate further.

[0042] As used herein, the term “contact” in relation to culturing or differentiating cells means exposing cells to a particular compound, for example, by placing the compound in a location that allows it to come into contact with the cells, in order to produce “contacted” cells. Contact may be achieved by any preferred means. A non-limiting example of contact is by adding the compound to the cell culture medium of the cells. Cell contact is assumed to occur as long as the cells and the particular compound are in close proximity, for example, as long as the compound is present in the cell culture medium at a suitable concentration.

[0043] As used herein, the term “inhibitor” refers to a compound that reduces, suppresses, or downregulates processes such as signaling pathways that can promote cell differentiation.

[0044] As used herein, the term “activator” refers to a compound that induces, stimulates, or upregulates processes such as signaling pathways that can promote cell differentiation.

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

[0046] As used herein, the term “cell population” refers to a group of cells in the same culture. A cell population may be, for example, a mixture of different types of cells, or cells at various stages of development, such as cells at different stages of maturation toward identical or similar specialized characteristics, or a more homogeneous composition of cells having common markers.

[0047] As used herein, the term “neuron population” refers to a population of cells that include nerve cells.

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

[0049] Neuroectoderm cells As used herein, the term “nerve” refers to the nervous system.

[0050] As used herein, the term “neuron” (unless otherwise specified) means cells whose natural corresponding portion naturally forms a part of the ectoderm, more specifically a part of the neuroectoderm, encompassing cells at any developmental stage within this germ layer, from NSCs to neurons and other terminally differentiated cell types (e.g., glial cells), i.e., including neural stem cell stages and neuroblast stages. Thus, neurons and their precursors are considered a specific type of nerve cell.

[0051] As used herein, the terms “neuron,” “neuronal,” and “nerve cell” may be used interchangeably to refer to nerve cells that have undergone postmittal differentiation into specialized cells. Neurons are characterized by the expression of the marker INA, or other equivalent markers such as ELAVL3, ELAVL4 (typically detected with the antibody HuC / D (also known as HuCD)), RFBOX3 (typically detected with the antibody NeuN), STMN2, NCMA1, or other such broad neuronal markers.

[0052] As used herein, the terms “neural stem cells” or “NSCs” and “neural progenitor cells” or “NPCs” are interchangeable terms used to refer to the self-regenerating (though typically limited) pluripotent cells of the nervous system that can give rise to countless more specialized cells of the CNS and PNS. NSCs and NPCs typically express transcription factors such as SOX2, NES, PAX6, SOX1, OTX2, OTX1, NKX6.1, OLIG2, NKX2.2, FOXG1, FOXA2, or LMX1A, as well as proliferation / cell cycle genes such as KI67 (also known as MKI67) and TOP2A, CDK1, MCM2, MCM4, and PCNA.

[0053] As used herein, the term “intermediate progenitor cell” or “IPC” refers to a cell that is typically asymmetrically divided, sometimes pluripotent or typically amphipotent or unipotent, and capable of self-renewal to a limited extent. Intermediate progenitor cells ultimately give rise to terminally differentiated cell types, such as neurons. The terms “neuroblast,” “intermediate precursor cell,” “intermediate progenitor cell,” and “radial glial cell” may be used interchangeably.

[0054] As used herein, the terms “neuroblast” or “intermediate progenitor cell” mean a cell expressing a gene associated with this stage, such as ASCL1 (also known as MASH1), SOX4, EOMES (also known as TBR2), NHLH1, EMX1, EMX2, DLX1, DLX5, DLX6, NFIA, NFIB, NFIX, MATH1 (also known as ATOH1), one of the Neurod or Neurog gene families, or other such genes. These cells are destined to become neurons.

[0055] As used herein, the terms “neuron progenitor,” “neuron precursor,” and “neuron precursor” may be used interchangeably and refer to nerve cells that have the potential or tendency to further specialize into neurons. The terms “neuron precursor” and “non-native neuron precursor” may be used interchangeably.

[0056] The nerve cells according to the present invention may have specific local identities, such as cells specific to the forebrain, midbrain, hindbrain, spinal cord, etc.

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

[0058] As used herein, the term “dorsal forebrain” in relation to cells means neurons that possess certain characteristics of neurons that spontaneously arise in the dorsal forebrain. Typically, dorsal forebrain neurons are characterized by the expression of certain markers, such as PAX6 and OTX2. Specifically, as used herein, the term “dorsal forebrain neural stem cell” refers to neural stem cells that possess the characteristics of neural stem cells that spontaneously arise in the dorsal forebrain. Dorsal forebrain neural progenitor cells may be characterized by the co-expression of two or more markers from SOX1, PAX6, OTX2, FOXG1, EMX1, EMX2, and SOX2.

[0059] As used herein, the term “midbrain” refers to the medial region of the neural tube (on the rostral-caudal axis) and the CNS that gives rise to the structures including the substantia nigra.

[0060] As used herein, the term “ventral midbrain” in relation to cells means neural cells that possess certain characteristics of neural cells that spontaneously arise in the ventral midbrain. Typically, ventral midbrain neurons 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 neural stem cells that possess the characteristics of neural stem cells that spontaneously arise in the ventral midbrain. Ventral midbrain neural progenitor cells may be characterized by the co-expression of two or more markers from FOXA1, FOXA2, LMX1A, LMX1B, EN1, OTX2, and SOX2.

[0061] As used herein, the terms “hindbrain” and “spinal cord” refer to the caudal region of the neural tube that is caudal to the isthmus organizer.

[0062] As used herein, the terms “glutamatergic cell” or “glutamatergic neuron” or “glutamatergic neuron” refer to cells that have the ability to synthesize the neurotransmitter glutamate.

[0063] As used herein, the terms “dopaminergic (DA) cell” or “dopaminergic neuron” or “dopamine neuron” refer to cells that have the ability to synthesize the neurotransmitter dopamine.

[0064] As used herein, the term “stromal cell” refers to a cell that has the ability to become a connective tissue cell or a fibroblast.

[0065] As used herein, the term "VLMC" refers to vascular peala cells and is considered a type of stromal cell present in the central nervous system (CNS).

[0066] As used herein, the term “glial cell” refers to non-neuronal cells of the central nervous system (brain and spinal cord) and the peripheral nervous system that do not produce electrical impulses, which provide support and protection to neurons. Examples include astrocytes and oligodendrocytes and their precursors, glial progenitor cells, or glial blasts.

[0067] Direction of differentiation into neurons A general embodiment of the present invention provides a method comprising obtaining a cell population containing nerve cells and contacting the cell population with a NOTCH signaling inhibitor, wherein the NOTCH signaling inhibitor is selected from LY411575, abagacestat, dibenzazepine, and PF-03084014. As used herein, the term "NOTCH" refers to the Notch receptor signaling pathway. As used herein, "LY411575" refers to the compound having CAS number 209984-57-6. The molecular formula of LY411575 is C 26 H 23 It is F2N3O4, while its chemical structure is sometimes shown as follows (Chemical Formula 1). [ka]

[0068] The chemical name for LY411575 is sometimes N-2((2S)-2-(3,5-difluorophenyl)-2-hydroxyethanol)-N1-((7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepine-7-yL)-l-alaninamide, or alternatively (2S)-2-(3,5-difluorophenyl)-2-hydroxyacetyl]amino]-N-[(7S)-5-methyl-6-oxo-7Hbenzo[d][1]benzazepine-7-yl]propanamide.

[0069] As used herein, the term "PF-03084014" refers to the compound having CAS number 1962925-29-6. The molecular formula of PF-03084014 is C 27 H 41 Its chemical name is F2N5O.HBr. Its chemical name is (2S)-2-[[(2S)-6,8-difluoro-1,2,3,4-tetrahydro-2-naphthalenyl]amino]-N-[1-[2-[(2,2-dimethylpropyl)amino]-1,1-dimethylethyl]-1H-imidazole-4-yl]pentanamide dihydrobromide, while its chemical structure is as follows: Chemical Formula 2: [ka]

[0070] As used herein, “dibenzazepine,” “DBZ,” or “XX” refers to the compound having CAS number 209984-56-5. The molecular formula of dibenzazepine is C 26 H 23 It is F2N3O3. Its chemical name is N-[(1S)-2-[[(7S)-6,7-dihydro-5-methyl-6-oxo-5H-dibenz[b,d]azepine-7-yl]amino]-1-methyl-2-oxoethyl]-3,5-difluorobenzeneacetamide, while its chemical structure is as follows: Chemical formula 3: [ka]

[0071] As used herein, "avagacestat" refers to the compound having CAS number 1146699-66-2. The molecular formula of abagacestat is C 20 H 17 It is ClF4N4O4S. Its chemical name is (2R)-2-[[(4-chlorophenyl)sulfonyl][[2-fluoro-4-(1,2,4-oxadiazole-3-yl)phenyl]methyl]amino]-5,5,5-trifluoropentanamide, while its chemical structure is as follows: Chemical formula 4: [ka]

[0072] The method is intended to increase the number of cells in a population of cells that have neuronal destiny. As used herein, the term “neuronal destiny” with respect to a cell means that, given that it is permitted to mature under favorable conditions, the developmental destiny of that cell will be restricted to developing into a neuron.

[0073] In one embodiment, the method is for directing the differentiation of nerve cells into neurons. As used herein, the term “directing differentiation” means influencing the developmental fate of a cell toward a particular outcome. Directing cell differentiation is not necessarily a continuous process, and cells do not necessarily have to develop to their final fate during the methods disclosed herein. In a preferred embodiment, cell differentiation is directed in vitro so that cells may later develop toward a particular fate, in particular, a neuronal fate, in vivo. Thus, in one embodiment, the method is in vitro.

[0074] In one embodiment, the method is for increasing the percentage of nerve cells that differentiate into neurons. In one embodiment, the method is for decreasing the percentage of nerve cells that have the potential to differentiate into stromal cells such as vascular leptomeningeal cells (VLMCs) and / or glial cells such as astrocytes. In further embodiments, the increase or decrease in the percentage of cells having a particular outcome occurs when the cell population is further cultured under favorable conditions or after administration of the cell population to a subject.

[0075] The inventors have found that the results of protocols for differentiating cell populations such as human PSCs toward or into neurons can be improved by inhibiting the NOTCH signaling pathway using alternative compounds rather than those commonly used in the field of neuronal differentiation, namely the small molecule DAPT. Surprisingly, the inventors have found that compound LY411575 demonstrates improved upregulation of neuronal genes and downregulation of NPC genes during differentiation compared to DAPT. The inventors have further identified alternative compounds abagacestat, dibenzazepine, and PF-03084014 as having similar capabilities. Specifically, as neurons further differentiate toward terminal phase fate, this inhibition thus further reduces the proportion of late-proliferating cells (representing off-target lines), reduces the proportion of stromal cells such as VLMCs and glial cells such as astrocytes, and simultaneously increases the proportion of neurons. The inventors have found that the above-mentioned compounds are superior to, for example, the use of DAPT. 24 hours after administration, cells treated with either of the compounds produced more neurons (HuCD or INA-positive cells) and had lower proliferative capacity (SOX2 and / or Ki67-positive cells) compared to similar treatment with DAPT (Figure 13).

[0076] The cell population obtained by this method includes nerve cells. In one embodiment, at least 80%, 85%, 90%, 95%, or 99% of the cell population are nerve cells at the time the cell population is exposed to the NOTCH signaling inhibitor. Ideally, the cell population consists of nerve cells. The nerve cells may be obtained by any preferred method. In one embodiment, the cell population containing nerve cells is obtained by differentiating pluripotent stem cells (PSCs) into nerve cells. In one embodiment, the cell population containing nerve cells is obtained by neural induction. As used herein, the term “neural induction” means the process by which pluripotent cells are exposed to signals that direct the cells to become neural stem or progenitor cells, unless they are exposed to signals that cause them to deviate to an alternative fate. Thus, in one embodiment, the cell population is cultured to induce differentiation into nerve cells before being exposed to the NOTCH signaling inhibitor. The exposure of the cell population to the NOTCH signaling inhibitor is performed before the cryopreservation of the cell population. In a particularly preferred embodiment, contact of the cell population with a NOTCH signaling inhibitor is performed 24–48 hours before cryopreservation of the cell population.

[0077] In a cell population used for neural induction, individual cells differentiate and progress at different rates, and therefore the cell population contains a composition of neurons at different developmental stages. Thus, in one embodiment, the cell population includes neural stem cells, neuroblast intermediate progenitor cells, and neurons at the time the cell population is brought into contact with a NOTCH signaling inhibitor. The inventors have identified a preferred timing for when the composition of neurons is optimal for directing the differentiation of cells into neurons, i.e., when to bring the cell population containing neurons into contact with a NOTCH signaling inhibitor. In one embodiment, at least 1% (e.g., at least 2%) of the cell population is INA+ / SOX2- at the time the cell population is brought into contact with the NOTCH signaling inhibitor (see Figures 21 and 10). Thus, this means that in order to effectively direct the differentiation of neurons toward neurons, a certain percentage of individual cells need to be both positive for INA expression and negative for SOX2 expression.

[0078] In another embodiment, the preferred timing for when the composition of ventral midbrain nerve cells is optimal for directing the differentiation of cells into neurons, i.e., when to expose the cell population containing nerve cells to a NOTCH signaling inhibitor, was determined based on the total number of cells positive for INA expression. In one embodiment, the level of cells positive for ASCL1 expression is also evaluated. In one embodiment, at least 5% of the cell population is INA+ at the time the cell population is exposed to a NOTCH signaling inhibitor at a rate of at least 35%, such as 10%, 15%, 20%, 25%, 30%, etc. In another embodiment, 5–35% (10–25%, 10–20%, more preferably 10–15%, etc.) of the cell population is INA+ at the time the cell population is exposed to a NOTCH signaling inhibitor.

[0079] In one embodiment, at least 20% (25%, 30%, 35%, 40%, 50%, 60%, 70%, at least 75%, etc.) of the cell population are ASCL1+ at the time the cell population is brought into contact with the NOTCH signaling inhibitor. In another embodiment, 20-75% (20-70%, 25-70%, more preferably 35-65%, etc.) of the cell population are ASCL1+ at the time the cell population is brought into contact with the NOTCH signaling inhibitor.

[0080] In another embodiment, the composition of dorsal forebrain neurons was determined based on the total number of cells positive for INA expression, which determined the preferred timing for directing the differentiation of the cells into neurons, i.e., when to contact the cell population containing neurons with a NOTCH signaling inhibitor. In one embodiment, the level of cells positive for TBR2 expression is also evaluated. In one embodiment, at least 5% of the cell population is INA+ at the time the cell population is contacted with a NOTCH signaling inhibitor at a rate of at least 35%, such as 10%, 15%, 20%, 25%, 30%, etc. In another embodiment, 5–35% (5–25%, 5–20%, more preferably 10–20%, etc.) of the cell population is INA+ at the time the cell population is contacted with a NOTCH signaling inhibitor.

[0081] In one embodiment, at least 5% (10%, 15%, 20%, 30%, etc., at least 40%) of the cell population are TBR2+ at the time the cell population is brought into contact with the NOTCH signaling inhibitor. In another embodiment, 5-40% (5-30%, 5-20%, more preferably 5-15%, etc.) of the cell population are TBR2+ at the time the cell population is brought into contact with the NOTCH signaling inhibitor.

[0082] Neurons exhibiting either of these expression profiles specifically indicate the progression of the cell to the terminal phase fate of a neuron and are considered unable to further differentiate or divide. Neurons are cells that possess neurites (axons and dendrites) and transmit information using electrical signals. Those skilled in the art will readily know how to determine this by single-cell expression analysis, such as using scRNAseq. In one embodiment, marker expression by cells is measured using scRNAseq, flow cytometry, ICC, qPCR, or any other such method. In one embodiment, marker expression by cells is measured according to Examples 9, 13, 14, and 15. In one embodiment, 2-60%, 2-50%, 2-40%, 2-30%, or 2-25%, preferably 2-25%, of the cell population is INA+ / SOX2- at the time the cell population is exposed to the NOTCH signaling inhibitor. In one embodiment, the cell population is contacted with a NOTCH signaling inhibitor when at least 1% of the cell population is INA+ / SOX2-, or within 30 days thereafter (e.g., within 25, 20, 15, 10, or 5 days). In a preferred embodiment, the cell population is contacted with a NOTCH signaling inhibitor when at least 2% of the cell population is INA+ / SOX2-, or within 1 to 5 days thereafter. In a preferred embodiment, the cell population is contacted with a NOTCH signaling inhibitor when at least 5% of the cell population is INA+, or within 1 to 5 days thereof. In a preferred embodiment, the cell population is contacted with a NOTCH signaling inhibitor when at least 30% of the cell population is ASCL1+, or within 1 to 5 days thereof. In a preferred embodiment, the cell population is contacted with a NOTCH signaling inhibitor when at least 5% of the cell population is TBR2+, or within 1 to 5 days thereof.

[0083] In one embodiment, at the time of contact with the NOTCH signaling inhibitor, at least 1.5% (preferably at least 2.5%, more preferably at least 5%) of the cell population are neuroblast intermediate progenitor cells. In one embodiment, 1.5–75% (e.g., 1.5–60%) (preferably 2.5–50%, more preferably 5–60%) of the cell population are neuroblast intermediate progenitor cells at the time of contact with the NOTCH signaling inhibitor. In one embodiment, the cell population is contacted with the NOTCH signaling inhibitor when 1.5% of the cell population are neuroblast intermediate progenitor cells, or within 30 days thereafter (e.g., within 25, 20, 15, 10, 5, or 1 day). In one embodiment, neuroblast intermediate progenitor cells express markers selected from ASCL1, Neurod1, Neurod2, Neurod4, Neurod6, Neurog1, Neurog2, Neurog3, TBR2, NHLH1, NFIA, NFIB, NFIX, and SOX4. Those skilled in the art will recognize that the markers indicating neuroblast intermediate progenitor cells are distinct from the specific brain region to which the nerve cells belong. In one embodiment, the nerve cells are specific to a region selected from the forebrain, midbrain, and hindbrain / spinal cord regions. In one embodiment, the forebrain region is the dorsal forebrain region. In one embodiment, the midbrain region is the ventral midbrain region.

[0084] In one embodiment, the concentration of the NOTCH signaling inhibitor is at least 0.1 μM (preferably at least 0.2 μM, 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, or at least 10 μM). In one embodiment, the concentration of the NOTCH signaling inhibitor is 0.2 μM to 1000 μM (10 μM to 100 μM, preferably 50 μM to 100 μM). In one embodiment, the concentration of the NOTCH signaling inhibitor is less than 1000 μM (900 μM, 800 μM, 700 μM, 600 μM, 500 μM, 400 μM, 300 μM, 200 μM, or less than 100 μM).

[0085] In one embodiment, the cell population is in contact with the NOTCH signaling inhibitor for at least 1 / 2 hour (such as at least 3, 6, or 12 hours), or for at least 1, 2, 3, 4, 5, or 6 days, preferably 1 to 2 days. In one embodiment, the NOTCH signaling inhibitor is replaced at least every 12 to 36 hours, preferably at least every 18 to 30 hours, more preferably at least every 24 hours. In one embodiment, a cell population is in contact with a NOTCH signaling inhibitor for 1 / 2 hour to 10 days, 1 / 2 hour to 9 days, 1 / 2 hour to 8 days, 1 / 2 hour to 7 days, 1 / 2 hour to 6 days, 1 / 2 hour to 5 days, 1 / 2 hour to 4 days, 1 / 2 hour to 3 days, 1 / 2 hour to 2 days, 12 hours to 9 days, 12 hours to 8 days, 12 hours to 7 days, 12 hours to 6 days, 12 hours to 5 days, 12 hours to 4 days, 12 hours to 3 days, 12 hours to 2 days, 1 day to 10 days, 1 day to 9 days, 1 day to 8 days, 1 day to 7 days, 1 day to 6 days, 1 day to 5 days, 1 day to 4 days, 1 day to 3 days, or 1 day to 2 days, preferably 12 hours to 2 days.

[0086] In one embodiment, the cell population has not been exposed to any other NOTCH inhibitor prior to the time at which it is brought into contact with the NOTCH signaling inhibitor.

[0087] In one embodiment, the cell population is harvested after inhibition of NOTCH signaling. As used herein, the term “harvesting” means that the cells are collected and transferred to an environment in which they will not further develop into neurons. In one embodiment, the cell population is harvested before the point in time when 60–100% of the cell population is INA+ / SOX2-. In one embodiment, the cell population is harvested before the point in time when 60–95% of the cell population is INA+ / SOX2-. In one embodiment, the cell population is harvested within 10 days after the end of contact of the cell population with the NOTCH signaling inhibitor (e.g., 9, 8, 7, 6, 5, 4, 3, 2, or 1 day after the end of contact of the cell population with the NOTCH signaling inhibitor), preferably within 1 or 2 days. In one embodiment, the cell population is harvested after inhibition of NOTCH signaling and cryopreserved. In one embodiment, the cell population is cryopreserved immediately after harvesting. In one embodiment, the cell population is cryopreserved within 10 days (9, 8, 7, 6, 5, 4, 3, 2, or 1 day, for example) after the end of contact with the NOTCH signaling inhibitor, preferably within 1 or 2 days. In embodiments in which the cell population is cryopreserved, the cell population is typically cryopreserved in a cryoprotective agent containing DMSO.

[0088] In a preferred embodiment, the cell population is cultured under culture conditions suitable for maintaining nerve cells. In one embodiment, the cell population is cultured in a culture medium suitable for maintaining nerve cells, and the culture medium is basal neuronal medium. In one embodiment, the culture medium contains one or more selected from B27 supplement, ascorbic acid, BDNF, GDNF, dcAMP, and DAPT. Preferably, the culture medium does not contain DAPT. In one embodiment, the culture medium is replaced at least every 12 to 36 hours, preferably at least every 18 to 30 hours, and more preferably at least every 24 hours.

[0089] Differentiation of PSCs into nerve cells In the initial step of the method of the present invention, a cell population containing nerve cells is obtained. The cell population may be obtained by any preferred means. However, in a preferred embodiment, the cell population containing nerve cells is obtained by differentiating PSCs into nerve cells. Thus, in one embodiment, the cell population is derived from PSCs. In a preferred embodiment, the cell population is derived from human cells. In one embodiment, the PSCs are human embryonic stem cells or human induced pluripotent stem cells.

[0090] In further embodiments, cell populations containing neurons are obtained by differentiating PSCs into neurons specific to regions selected from the forebrain, midbrain, and hindbrain / spinal cord. Protocols for differentiating PSCs into different cell types of neuroectoderm are well known and apply to all protocols in which PSCs are first neurogenically induced, and the cells then undergo a series of developmental stages, initially becoming neural stem cells, then progressing to neuroblast intermediate progenitor cells, and then optionally proceeding to differentiation into neurons toward neuronal identity. Depending on the specific region, development may take longer or shorter periods. However, we have found that regardless of the time it takes for a particular region and cells to progress through different developmental stages, the preferred timing for contacting the cell population with a NOTCH signaling inhibitor can be established based on the percentage of cells that are both positive for marker INA expression and negative for marker SOX2 expression, total expression of marker INA and / or total expression of TBR2 or ASCL1. At this stage, the cell population typically includes neural stem cells, neuroblast intermediate progenitor cells, and neurons.

[0091] In a typical embodiment, a cell population containing neurons is obtained by differentiating pluripotent stem cells (PSCs) into neurons for 14–35 days. By differentiating the cells, the cell population is exposed to signals that induce the cells to become neurons. Depending on the specific protocol, the cell population may be exposed to different signals at different times, such as by contacting the cell population with compounds that activate and / or inhibit certain signaling pathways, and once the cell population has been exposed to different signals, further differentiation is permitted by simply culturing the cell population in a culture medium suitable for further differentiation of the cells, without further exposure to these or any other signals. Regardless of whether the cells are permitted to further differentiate for a period of time without exposure to differentiation-directing signals, in a preferred embodiment, the cell population is cultured in a culture medium suitable for maintaining neurons, and the culture medium is a neural basal medium.

[0092] In one embodiment, a cell population containing neurons is obtained by differentiating pluripotent stem cells (PSCs) into neurons over a period of 18–35 days, and the neurons are then differentiated into neurons specific to the forebrain region. In this embodiment, the cell population is neurogenically induced, not ventrally or caudally induced, and obtains dorsal forebrain neurons. In one embodiment, the neurons are differentiated into neurons specific to the forebrain region, preferably the dorsal forebrain region, and the cell population is not exposed to a NOTCH signaling inhibitor until at least 24 days after the initiation of differentiation of the cell population into forebrain-specific neurons (e.g., at least 24, 26, 28, 30, 32, 33, or 35 days). Preferably, the cell population is not exposed to a NOTCH signaling inhibitor until 25–28 days after the initiation of differentiation of the cell population into neurons (Figure 20).

[0093] In a preferred embodiment, the cell population is not exposed to an inhibitor of NOTCH signaling until at least 24 days after the onset of differentiation of the cell population into forebrain-specific neurons (for example, at least 24, 26, 28, 30, 32, 33, or 35 days later, and at the point when at least 5% of the cell population is INA+).

[0094] In a preferred embodiment, the cell population is not exposed to the NOTCH signaling inhibitor until at least 24 days after the onset of differentiation of the cell population into forebrain-specific neurons (e.g., at least 24, 26, 28, 30, 32, 33, or 35 days) and at least 5% of the cell population is TBR2+.

[0095] In another embodiment, a cell population containing neurons is obtained by differentiating pluripotent stem cells (PSCs) into neurons over a period of 14–26 days, and the neurons are then differentiated into neurons specific to the midbrain region, preferably the ventral midbrain region. In such embodiments, the cell population is neurogenically induced, ventralized, and caudalized to obtain ventral midbrain neurons. Furthermore, in one embodiment, less than 60% (e.g., less than 50%, less than 40%, less than 30%, less than 20%, less than 10%) of the cell population expresses KI67 at the time of contact with a NOTCH signaling inhibitor. Specifically, for the midbrain region, the aforementioned number of KI67-positive cells may represent a cell population containing enough neuroblast intermediate progenitor cells to be treated with a NOTCH signaling inhibitor. In a further embodiment, 20–80% (preferably 30–70%, more preferably 40–60%) of the cell population expresses KI67 at the time of contact with a NOTCH signaling inhibitor. In embodiments in which nerve cells differentiate into nerve cells specific to the midbrain region, preferably the ventral midbrain region, the cell population is not in contact with the NOTCH signaling inhibitor until at least 20 days after the onset of differentiation of the cell population into nerve cells (at least 21, 22, 23, 24, 25, or 26 days, preferably at least 22 days, etc.). Preferably, the cell population is not in contact with the NOTCH signaling inhibitor until 22–25 days after the onset of differentiation of the cell population into nerve cells (Figure 23).

[0096] In a preferred embodiment, the cell population is not exposed to the NOTCH signaling inhibitor until at least 20 days after the initiation of differentiation of the cell population into midbrain region-specific neurons (such as at least 21, 22, 23, 24, 25, or 26 days), preferably at least 22 days, and at least 10% of the cell population is INA+. In a preferred embodiment, the cell population is not exposed to an inhibitor of NOTCH signaling until at least 20 days after the initiation of differentiation of the cell population into midbrain region-specific neurons (such as at least 21, 22, 23, 24, 25, or 26 days), preferably at least 22 days, and at least 30% of the cell population is ASCL1+.

[0097] In another embodiment, a cell population containing nerve cells is obtained by differentiating pluripotent stem cells (PSCs) into nerve cells over a period of 14–26 days, and the nerve cells are then differentiated into nerve cells specific to the hindbrain / spinal cord region.

[0098] In a preferred embodiment, a cell population containing neurons is obtained by inducing pluripotent cells, such as PSCs, into neurons. The cells may be induced by any preferred method. However, in a preferred embodiment, a cell population containing neurons is obtained by contacting the cell population with an inhibitor of Small Mothers Against Decapentaplegic (SMAD) protein signaling. In one embodiment, a cell population containing neurons is obtained by contacting the cell population with at least two inhibitors of SMAD protein signaling. 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 PSCs with one or more inhibitors of the SMAD signaling pathway is a robust technique for differentiating cells into the nervous system. In one embodiment, the inhibitors of the SMAD signaling pathway are selected from Noggin, LY364947, SB431542, RepSox, DMH1, DMH2, LND-212854, and LDN-193189.

[0099] In a preferred embodiment, the cell population is cultured in a culture medium suitable for maintaining nerve cells, and the culture medium is a basal neuronal medium. In one embodiment, the culture medium contains one or more components selected from B27 supplement, ascorbic acid, BDNF, GDNF, dcAMP, and DAPT. Preferably, the culture medium does not contain DAPT.

[0100] Protocols specific to the forebrain region In one embodiment, the cell population is differentiated into forebrain-specific neurons. In one embodiment, the cell population is differentiated into forebrain-specific neurons, and the cell population is exposed to an inhibitory pathway of Small Mothers Against Decapentaplegic (SMAD) protein signaling for about 0–11 days, followed by exposure to FGF2 for about 7–9 days, and then the cell population is continued to be cultured, typically for a further 9–12 days, in a basal medium free of culture growth factors, morphogens, or small molecules.

[0101] Examples of differentiation into dorsal forebrain / mantle cells can be found in the publications Shi et al., Livesey “Human cerebral cortex development from pluripotent stem cells to functional excitatory synapses” 2011, and Espuny-Camacho et al., Vanderhaegen “Pyramidal Neurons Derived from Human Pluripotent Stem Cells Integrate Efficiently into Mouse Brain Circuits In Vivo” 2012.

[0102] A protocol specific to the ventral midbrain region. In one embodiment, the cell population is differentiated into ventral midbrain neurons. In one 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, optionally, an activator of fibroblast growth factor (FGF) signaling, and optionally, ascorbic acid and / or optionally, brain-derived neurotrophic factor (BDNF). In one embodiment, a 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 optionally, an activator of fibroblast growth factor (FGF) signaling, as well as optionally ascorbic acid and / or optionally brain-derived neurotrophic factor (BDNF). The cell population is then cultured in a culture medium suitable for maintaining neurons without further contact with the SMAD protein signaling inhibitor, SHH signaling activator, Wnt signaling activator, or FGF signaling activator. Thus, neurons are allowed to further differentiate without continuous exposure to differentiation-directing signals.

[0103] In one embodiment for differentiating PSCs into ventral midbrain neurons, 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” may be used interchangeably and refer to the process by which pluripotent cells thereby assume ventral gene expression identity equivalent to that of embryonic or embryonic structure cells, i.e., neural tube cells. In one embodiment, PSCs are ventralized by contacting the cells with an activator of the SHH signaling pathway. As used herein, the term “activator of the Sonic Hedgehog signaling pathway” refers to any molecule or compound having the ability to activate the SHH signaling pathway. Activation of the SHH pathway is well known to be involved in the induction and maintenance of ventral neural tube structure. In one embodiment, the activator of the SHH signaling pathway is selected from SHH, purmorphamine, and SAG.

[0104] In one embodiment for differentiating PSCs into ventral midbrain neurons, the cell population is differentiated into ventral midbrain neurons by inducing caudalization of the cell population. As used herein, the term “caudalization” refers to the process by which pluripotent cells assume caudal gene expression identity equivalent to that of embryonic or embryonic structure cells, i.e., neural tube cells. In one embodiment, PSCs are caudalized by contacting the cells with a Wnt signaling pathway activator. As used herein, the term “Wnt signaling activator” refers to any molecule or compound having the ability to activate the Wnt signaling pathway. Wnt signaling inhibitors are well known to be involved in the caudalization of neurons. In one embodiment, the Wnt signaling activator reduces GSK3-beta for activation of Wnt signaling. Therefore, in certain embodiments, the Wnt activator is a GSK3-beta inhibitor. In one embodiment, the Wnt signaling activator is selected from CHIR99021 and recombinant Wnt protein.

[0105] In one embodiment for differentiating PSCs into ventral midbrain neurons, the cell population is differentiated into ventral midbrain neurons by further inducing caudalization of the cell population. Therefore, in one embodiment, the cell population is brought into contact with a fibroblast growth factor (FGF) activator. In one embodiment, the FGF signaling activator is selected from FGF8b.

[0106] In a particular embodiment, the concentration of the SMAD protein signaling inhibitor is 1 μM to 50 μM, the SHH signaling activator is 200 ng / ml to 800 ng / ml, the Wnt signaling inhibitor is 0.1 μM to 1 μM, the FGF signaling activator is 10 ng / ml to 200 ng / ml, the ascorbic acid is 50 μM to 500 μM, and / or the BDNF is 1 ng / ml to 50 ng / ml.

[0107] In one embodiment, a population of pluripotent stem cells is exposed to a SMAD protein signaling inhibitor for 5 to 9 days. In a specific embodiment, the cell population is exposed to a SMAD protein signaling inhibitor from day 0 to day 5 to 9. In one embodiment, a population of PSCs is exposed to an SHH signaling activator for 5 to 9 days (e.g., 7 to 9 days), preferably for 9 days. In a preferred embodiment, the cell population is exposed to a Wnt signaling inhibitor from day 0 to day 5 to 9. In one embodiment, the concentration of the SHH signaling activator is 200 ng / ml to 800 ng / ml. In one embodiment, a population of pluripotent stem cells is exposed to a Wnt signaling inhibitor for 5 to 9 days (e.g., 7 to 9 days), preferably for 9 days. In a preferred embodiment, the cell population is exposed to a Wnt signaling inhibitor from day 0 to day 5 to 9. In one embodiment, the concentration of the Wnt signaling activator is 0.1 μM to 1 μM. In one embodiment, a population of PSCs is exposed to an FGF signaling activator for 7–12 days after discontinuing 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 exposed to an FGF signaling activator from days 5–9 to 7–12, or at the end of contact with an inhibitor of SMAD protein signaling, an inhibitor of Wnt signaling, and / or an activator of SHH signaling. In a particular embodiment, the cell population is exposed to an inhibitor of SMAD protein signaling, an activator of SHH signaling, and an inhibitor of Wnt signaling from days 0–9, and then exposed to an FGF signaling activator from days 9–16. In one embodiment, the FGF signaling activator is FGF8b. In one embodiment, the concentration of the FGF signaling activator is 10 ng / ml to 200 ng / ml. In one embodiment, the cell population is exposed to ascorbic acid for 5 to 7 days starting from day 10 or day 11. In one embodiment, the concentration of ascorbic acid is 10 μM to 400 μM, preferably 100 μM to 300 μM, preferably 150 μM to 250 μM, and more preferably about 200 μM.In one embodiment, the cell population is exposed to BDNF for 5 to 7 days starting from day 10 or day 11. In one embodiment, the concentration of BDNF is 1 ng / ml to 40 ng / ml, preferably 10 ng / ml to 40 ng / ml, preferably 15 ng / ml to 30 ng / ml, and more preferably about 20 ng / ml.

[0108] An example of differentiation into ventral midbrain neurons can be found in Example 3.

[0109] Protocols specific to the hindbrain / spinal cord region In one embodiment, the cell population is differentiated into neurons specific to the hindbrain / spinal cord region. In one embodiment, the cell population is differentiated into neurons specific to the hindbrain / spinal cord region, and the cell population is also contacted with a protein signaling pathway inhibitor (SMAD) for about 0–6 days and a WNT agonist such as CHIR for about 0–15 days, and the cell population is also differentiated by contacting a ventralizing molecule such as SHH or SAG for about 6–15 days, and by contacting a cell population with retinoic acid, which is a major factor for this region.

[0110] Examples of differentiation into hindbrain / spinal cord cells can be found in publications such as Du et al., Zhang “Generation and expansion of highly pure motor neuron progenitors from human pluripotent stem cells” 2014, Amaroso et al., Wichterle “Accelerated High-Yield Generation of Limb-Innervating Motor Neurons from Human Stem Cells” 2013, and Butts et al., McDevitt “V2a interneuron differentiation from mouse and human pluripotent stem cells” 2019.

[0111] A group of nerve cells Aspects of the present invention relate to cell populations comprising nerve cells that can be obtained by any of the methods described herein. In certain embodiments, cell populations comprising nerve cells are obtained by any one of the methods described herein. In particular, the inventors were able to identify parameters that distinguish cell populations harvested immediately after inhibition of NOTCH signaling from cell populations not subjected to the method of directing differentiation toward neurons according to the present invention. However, it is evident that cells are stimulated by signaling and that the proportion of cells retaining neuronal fate increases, which becomes even more evident when the cell populations can be further cultured. Accordingly, aspects of the present invention relate to cell populations comprising nerve cells that, when cultured in vitro for 5 to 7 days in a culture medium suitable for maintaining nerve cells, result in a cell population in which at least 35%, preferably 50%, is HuC / D positive. In one embodiment, marker expression by cells is measured using scRNAseq. In one embodiment, marker expression by cells is measured according to Examples 9, 13, 14, and 15. In one embodiment, the cell population is cultured according to Examples 10, 11, and 12, depending on the region to which the nerve cells are specific. In one embodiment, the cell population is in vitro. In one embodiment, the nerve cells are non-natural. In one embodiment, the nerve cells are artificial. In one embodiment, the cell population is derived from human cells. In a preferred embodiment, the cell population is derived from stem cells. In a particular embodiment, the cell population is derived from pluripotent stem cells. In a further embodiment, the cell population is derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs). In one embodiment, the nerve cells are specific to a region selected from the forebrain, midbrain, and hindbrain / spinal cord regions. In a further embodiment, the nerve cells are ventral midbrain nerve cells. In a further embodiment, the nerve cells are dorsal forebrain nerve cells.

[0112] In one embodiment, the cells of a cell population are genetically modified. In one embodiment, pluripotent stem cells are genetically modified, and the genetic modification persists in nerve cells obtained according to one of the methods described herein. In a particular embodiment, the cells of a cell population are genetically modified to be hypoimmunogenic. As used herein, the terms “hypoimmunogenic” and “immune-evading” with respect to cells may be used interchangeably and refer to characteristics of cells that reduce the tendency of such cells to cause immune rejection by the recipient to which they are transplanted. Typically, certain surface markers are overexpressed or silenced. In one embodiment, the genetically modified cells have reduced expression of MHC-I and / or MHC-II. In one embodiment, cells are genetically modified to express one or more tolerance-genic factors or analogs such as HLA-E, HLA-G, CD46, CD47, CD55, CD59, and PD-L1. Examples and methods for making genetically modified cells immune-evading are described in International Publication Nos. 2012 / 145384, 2013 / 158292, 2016 / 142532, 2016 / 183041, 2018 / 132783, 2018 / 175390, 2019 / 161271, 2020 / 018615, 2020 / 018620, 2020 / 049535, 2020 / 168317, 2021 / 195426, 2022 / 012591, and 2020 / 260563. In some embodiments, genome editing technologies (e.g., CRISPR / Cas or TALEN systems) are used to regulate the expression of specific genes (e.g., reduce, eliminate, and / or increase their expression).

[0113] In one embodiment, the recombination for low immunogenicity includes reduced expression of MHC-I human leukocyte antigens compared to wild-type stem cells, reduced expression of MHC-II human leukocyte antigens compared to wild-type stem cells, and / or increased expression of tolerogenic factors compared to wild-type stem cells. In one embodiment, the MHC-I human leukocyte antigens are HLA-A, HLA-B, and HLA-C. In one embodiment, the MHC-II human leukocyte antigens are HLA-DP, HLA-DQ, and HLA-DR. In one embodiment, the tolerogenic factors are selected from CD46, CD47, CD55, CD59, PD-L1, HLA-E, and HLA-G.

[0114] In one embodiment, cells in a cell population are genetically modified to be lineage-restricted. As used herein, the term “lineage-restricted” with respect to cells means that the cells are functionally and / or structurally restricted to differentiate into a particular cell type.

[0115] In one embodiment, the cell population includes at least 1,000 cells (such as 10,000 cells, 100,000 cells, 1,000,000 cells, or 10,000,000 cells). In one embodiment, the cell population is cryopreserved.

[0116] Therefore, another aspect of the present invention relates to a composition comprising a cell population and a cryoprotectant. In embodiments in which the cell population is cryopreserved, the cell population is cryopreserved in a cryoprotectant typically comprising DMSO.

[0117] Use as a medicine Another aspect of the present invention relates to in vitro cell populations or compositions thereof described herein for use as pharmaceuticals. In one embodiment, the cell population for use as a pharmaceutical is for the treatment of neurological conditions. In one embodiment, the neurological conditions are selected from Parkinson's disease, stroke, traumatic brain injury, spinal cord injury, Huntington's disease, dementia, Alzheimer's disease, and other neurological conditions in which neurons are lost or become dysfunctional. In one embodiment, the cell population comprises nerve cells contacted with an inhibitor of NOTCH signaling. In one embodiment, the nerve cells are forebrain nerve cells. In one embodiment, the cell population comprising forebrain nerve cells is for the treatment of stroke. In one embodiment, the nerve cells are ventral midbrain nerve cells. In one embodiment, the cell population comprising ventral midbrain nerve cells is for the treatment of Parkinson's disease. In one embodiment, the nerve cells are hindbrain / spinal cord nerve cells. In one embodiment, the cell population comprising hindbrain / spinal cord nerve cells is for the treatment of paralysis, spinal cord injury, diabetic neuropathy, or chronic pain.

[0118] Another embodiment relates to a method for treating a neurological condition, comprising administering an effective amount of a cell population to a patient according to the present invention. In one embodiment, the neurological condition is selected from Parkinson's disease, stroke, traumatic brain injury, spinal cord injury, Huntington's disease, dementia, Alzheimer's disease, and other neurological conditions in which neurons are lost or become dysfunctional. In a particular embodiment, the neurological condition is Parkinson's disease.

[0119] As used herein, the term “subject” refers to a human patient suffering from Parkinson’s disease or stroke. In one embodiment, “therapeutic dose” with respect to the treatment of these disorders using cell products means a dose of 200,000 to 40,000,000 cells. Administration of nerve cells to the subject is intended by surgery.

[0120] In this embodiment, the in vitro cell population described herein is differentiated by the addition of a NOTCH inhibitor, the NOTCH signaling inhibitor being LY411575, and at the time described herein, and after the cell population is cryopreserved, vials of cryopreserved cells that can be used for administration to a subject are obtained.

[0121] In vivo orientation and differentiation of nerve cells into neurons Another embodiment relates to a method for treating neurological conditions, comprising administering a therapeutically effective dose of a cell population including nerve cells and a NOTCH signaling inhibitor to a subject, wherein the NOTCH signaling inhibitor is LY411575. The inventors predict that a similar effect in directing differentiation toward neurons can be achieved in vivo by co-administering the NOTCH signaling inhibitor together with nerve cells. Thus, in one embodiment, nerve cells are not the subject of the method of directing differentiation toward neurons as disclosed herein. As used herein, the term “subject” refers to a human patient suffering from Parkinson’s disease or stroke. In one embodiment, “therapeutic dose” in relation to the treatment of these disorders with cell products means a dose of 200,000 to 40,000,000 cells. Administration of ventral midbrain nerve cells to a subject is intended by surgery. In one embodiment, ventral midbrain neurons co-express three or more markers selected from the list of FOXA2, LMX1A, EN1, OTX2, ASCL1, Neurod1, PITX3, and SOX2. In one embodiment, forebrain neurons co-express three or more markers selected from the list of SOX2, OTX2, PAX6, FOXG1, TBR2, EMX2, NFIA, and SOX1.

[0122] One aspect of the present invention relates to a composition comprising a cell population including nerve cells and a NOTCH signaling inhibitor, wherein the NOTCH signaling inhibitor is LY411575.

[0123] Accordingly, aspects of the present invention relate to LY411575 for use in the treatment of neurological conditions. Specifically, one embodiment relates to LY411575 for use in the treatment of Parkinson's disease in subjects to whom a therapeutically effective dose of ventral midbrain nerve cells is administered.

[0124] Those skilled in the art will understand that the embodiments described in this section with respect to therapeutic methods, drug use, and secondary medical use are equally applicable to all the embodiments described.

[0125] Specific Embodiments Herein, aspects of the present invention will be further described by the following non-limiting embodiments. 1. A method comprising obtaining a cell population containing nerve cells and contacting the cell population with a NOTCH signaling inhibitor, wherein the NOTCH signaling inhibitor is selected from LY411575, abagacestat, dibenzazepine, and PF-03084014. 2. The method according to the prior embodiment, wherein the NOTCH signaling inhibitor is LY411575. 3. A method according to any one of the prior embodiments, wherein the method is for directing the differentiation of nerve cells into neurons. 4. A method according to any one of the prior embodiments, wherein the method is for increasing the percentage of nerve cells that have the potential to differentiate into neurons. 5. The method according to any one of the prior embodiments, wherein the method is for increasing the percentage of nerve cells that have the potential to differentiate into neurons after the completion of contacting the cell population with a NOTCH signaling inhibitor when the cell population is further cultured under suitable conditions or after administration of the cell population to a subject. 6. The method according to any one of the prior embodiments, wherein the method is for reducing the proportion of nerve cells that have the potential to differentiate into stromal cells such as vascular leptomeningeal cells (VLMCs) and / or glial cells such as astrocytes. 7. The method according to any one of the preceding embodiments, wherein the method is for reducing the proportion of neurons with the potential to differentiate into stromal cells such as vascular leptomeningeal cells (VLMCs) and / or glial cells such as astrocytes, after the completion of contacting the cell population with a NOTCH signaling inhibitor when the cell population is further cultured under suitable conditions or after administration of the cell population to a subject. 8. The method according to any one of the prior embodiments, wherein a cell population containing nerve cells is obtained by differentiating pluripotent stem cells (PSCs) into nerve cells. 9. A method according to any one of the prior embodiments, wherein a population of cells including nerve cells is obtained by neural induction. 10. The method according to any one of the preceding embodiments, wherein the cell population is cultured to induce differentiation into nerve cells before contacting the cell population with a NOTCH signaling inhibitor. 11. The method according to any one of the preceding embodiments, wherein at least 80%, 85%, 90%, 95%, or 99% of the cell population are neurons at the time the cell population is brought into contact with a NOTCH signaling inhibitor. 12. The method of any one of the prior embodiments, wherein at least 1%, such as at least 2%, of the cell population is INA+ / SOX2- at the time the cell population is brought into contact with the NOTCH signaling inhibitor. 13. The method according to any one of the preceding embodiments, wherein 1-60% or 2-60% of the cell population are INA+ / SOX2- at the time the cell population is brought into contact with the NOTCH signaling inhibitor. 14. The method of any one of the prior embodiments, wherein the cell population comes into contact with a NOTCH signaling inhibitor at a time when at least 1% of the cell population is INA+ / SOX2-, or within 30 days thereof (such as within 25, 20, 15, 10, or 5 days thereof). 15. The method according to any one of the preceding embodiments, wherein 60-100%, or 65-95%, preferably 70-90%, more preferably 75-85%, of the cell population is SOX2-positive at the time the cell population is brought into contact with a NOTCH signaling inhibitor. 16. The method of any one of the preceding embodiments, wherein less than 90% of the cell population is SOX2-positive at the time the cell population is exposed to a NOTCH signaling inhibitor. 17. The method according to any one of the preceding embodiments, wherein at least 1.5% of the cell population are neuroblast intermediate progenitor cells, preferably at least 2.5%, more preferably at least 5%, at the time the cells are brought into contact with the NOTCH signaling inhibitor. 18. The method according to any one of the preceding embodiments, wherein at least 1.5 to 75% (e.g., 1.5 to 60%) of the cell population are neuroblast intermediate progenitor cells, preferably 2.5 to 50%, more preferably 5 to 60%, at the time the cells are brought into contact with the NOTCH signaling inhibitor. 19. The method according to any one of the preceding embodiments, wherein the cell population comes into contact with a NOTCH signaling inhibitor at a time when at least 1.5% of the cell population are neuroblast intermediate progenitor cells, or within 30 days thereof (e.g., within 25, 20, 15, 10, or 5 days thereof). 20. The method according to any one of Embodiments 17 to 19, wherein the neuroblast intermediate progenitor cells express a marker selected from ASCL1, Neurod1, Neurod2, Neurod4, Neurod6, Neurog1, Neurog2, Neurog3, TBR2, NHLH1, NFIA, NFIB, NFIX, and SOX4. 21. The method according to any one of the preceding embodiments, wherein the cell population includes neural stem cells, neuroblast intermediate progenitor cells, and neurons at the point in contact with the NOTCH signaling inhibitor. 22. The method according to any one of the prior embodiments, wherein the nerve cells are specific to a region selected from the forebrain, midbrain, and hindbrain / spinal cord. 23. The method according to a prior embodiment, wherein the nerve cells are specific to the hindbrain / spinal cord, specifically the medial ventrocaudal NPC of the hindbrain or spinal cord region, more specifically, the ventral 0-2 region, referred to as the V0, V1, or V2 subtype. 24. The method according to any one of the prior embodiments, wherein a population of cells including nerve cells is obtained by differentiating pluripotent stem cells into nerve cells specific to a region selected from the forebrain, midbrain, and hindbrain / spinal cord. 25. The method according to any one of the preceding embodiments, wherein the neurons are specific to the forebrain region, and 2–25% of the cell population are INA+ / SOX2- at the time the cell population is brought into contact with a NOTCH signaling inhibitor. 26. The method according to any one of the prior embodiments, wherein the neurons are specific to the forebrain region, and at least 5% of the neurons (e.g., 10%, 15%, 20%, 25%, 30%, at least 35%) are INA+ at the time the neurons are brought into contact with the NOTCH signaling inhibitor. 27. The method according to any one of the preceding embodiments, wherein the neurons are specific to the forebrain region, and 5-35% (5-25%, 5-20%, more preferably 10-20%, etc.) of the neurons are INA+ at the time the neurons are brought into contact with the NOTCH signaling inhibitor. 28. The method according to any one of the preceding embodiments, wherein the neurons are specific to the forebrain region, and at least 5% (e.g., 10%, 15%, 20%, 30%, or at least 40%) of the neurons are TBR2+ at the time the neurons are brought into contact with a NOTCH signaling inhibitor. 29. The method according to any one of the preceding embodiments, wherein the neurons are specific to the forebrain region, and 5-40% (5-30%, 5-20%, more preferably 5-15%, etc.) of the neurons are TBR2+ at the time the neurons are brought into contact with a NOTCH signaling inhibitor. 30. The method according to any one of the preceding embodiments 1 to 24, wherein the neurons are specific to the premidbrain region, and 0-10%, 0-5%, 1-5%, or 2-5%, preferably 2-5%, of the cell population are INA+ / SOX2- at the time the cell population is brought into contact with the NOTCH signaling inhibitor. 31. The method according to Embodiment 30, wherein the nerve cells are specific to the midbrain region, and at least 5% of the nerve cells (e.g., 10%, 15%, 20%, 25%, 30%, at least 35%) are INA+ at the time the nerve cells are brought into contact with the NOTCH signaling inhibitor. 32. The method according to any one of embodiments 30 to 31, wherein the nerve cells are specific to the midbrain region, and 5 to 35% (10 to 25%, 10 to 20%, more preferably 10 to 15%, etc.) of the nerve cells are INA+ at the time of contact with the NOTCH signaling inhibitor. 33. The method according to any one of embodiments 30 to 32, wherein the nerve cells are specific to the midbrain region, and at least 20% of the nerve cells (25%, 30%, 35%, 40%, 50%, 60%, 70%, at least 75%, etc.) are ASCL1+ at the time the nerve cells are brought into contact with the NOTCH signaling inhibitor. 34. The method according to any one of embodiments 30 to 33, wherein the nerve cells are specific to the midbrain region, and 20 to 75% (20 to 70%, 25 to 70%, more preferably 35 to 65%, etc.) of the nerve cells are ASCL1+ at the time the nerve cells are brought into contact with the NOTCH signaling inhibitor. 35. The method according to any one of Embodiments 1 to 24, wherein the nerve cells are specific to the hindbrain / spinal cord region, and 0-15% of the cell population are INA+ / SOX2- at the time the cell population is brought into contact with a NOTCH signaling inhibitor. 36. The method according to any one of the preceding embodiments, wherein the concentration of the NOTCH signaling inhibitor is at least 1 μM (preferably at least 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, or at least 10 μM, etc.). 37. The method according to any one of the preceding embodiments, wherein the concentration of the NOTCH signaling inhibitor is 0.2 μM to 1000 μM (10 μM to 100 μM, preferably 50 μM to 100 μM, etc.). 38. The method according to any one of the preceding embodiments, wherein the concentration of the inhibitor of NOTCH signaling is less than 1000 μM (such as less than 900 μM, 800 μM, 700 μM, 600 μM, 500 μM, 400 μM, 300 μM, 200 μM, or 100 μM). 39. The method according to any one of the preceding embodiments, wherein the cell population is contacted with an inhibitor of NOTCH signaling for at least 1 / 2 hour (such as for 3, 6, or 12 hours, or for at least 1, 2, 3, 4, 5, or 6 days), preferably for 1 to 2 days. 40. The method according to any one of the preceding embodiments, wherein the cell population is contacted with an inhibitor of NOTCH signaling for less than 10 days (such as less than 9 days, 8 days, or 7 days). 41. The method according to any one of the preceding embodiments, wherein the cell population is contacted with an inhibitor of NOTCH signaling for 1 / 2 hour to 10 days, 1 / 2 hour to 9 days, 1 / 2 hour to 8 days, 1 / 2 hour to 7 days, 1 / 2 hour to 6 days, 1 / 2 hour to 5 days, 1 / 2 hour to 4 days, 1 / 2 hour to 3 days, 1 / 2 hour to 2 days, 12 hours to 9 days, 12 hours to 8 days, 12 hours to 7 days, 12 hours to 6 days, 12 hours to 5 days, 12 hours to 4 days, 12 hours to 3 days, 12 hours to 2 days, 1 day to 10 days, 1 day to 9 days, 1 day to 8 days, 1 day to 7 days, 1 day to 6 days, 1 day to 5 days, 1 day to 4 days, 1 day to 3 days, or 1 day to 2 days, preferably for 12 hours to 2 days. 42. The method according to any one of the preceding embodiments, wherein the cell population is not contacted with DAPT. 43. The cell population has a density of 0.5×10 6 cells / cm 2 ~2.0×10 6 cells / cm 2 (0.8×10 6 cells / cm 2 ~1.5×10 6 cells / cm 2 etc.) at the time when the cell population is contacted with an inhibitor of NOTCH signaling. The method according to any of the preceding embodiments. 44. The method according to any one of the preceding embodiments, wherein the cell population is harvested after inhibition of NOTCH signaling. 45. The method according to any one of the preceding embodiments, wherein the cell population is harvested before more than 60-100% of the cell population is INA+ / SOX2-. 46. ​​The method according to any one of embodiments 43 and 44, wherein the cell population is harvested within 10 days, preferably within 1 to 2 days, after the completion of contact with the NOTCH signaling inhibitor of the cell population, such as 9, 8, 7, 6, 5, 4, 3, 2, or 1 day after the completion of contact with the NOTCH signaling inhibitor of the cell population. 47. The method according to any one of the preceding embodiments, wherein a cell population is collected and cryopreserved after inhibition of NOTCH signaling. 48. The method according to the preceding embodiment, wherein the cell population is cryopreserved immediately after collection. 49. The method according to any one of embodiments 46 and 47, wherein the cell population is cryopreserved within 10 days, preferably within 1 to 2 days, after the completion of contact with the NOTCH signaling inhibitor of the cell population, such as 9, 8, 7, 6, 5, 4, 3, 2, or 1 day after the completion of contact with the NOTCH signaling inhibitor of the cell population. 50. The method according to any one of embodiments 46 to 48, wherein the cell population is cryopreserved in a cryoprotective agent typically containing DMSO. 51. The method according to any one of the preceding embodiments, wherein the NOTCH signaling inhibitor is replaced at least every 12 to 36 hours, preferably at least every 18 to 30 hours, and more preferably at least every 24 hours. 52. The method according to any one of the prior embodiments, wherein the cell population is derived from PSCs. 53. The method according to any one of the prior embodiments, wherein the cell population is derived from human cells. 54. The method according to the preceding embodiment, wherein the PSC is a human embryonic stem cell or a human induced pluripotent stem cell. 55. The method according to any one of the prior embodiments, wherein a cell population containing nerve cells is obtained by differentiating pluripotent stem cells (PSCs) into nerve cells over a period of 14 to 35 days. 56. The method according to any one of the preceding embodiments, wherein a cell population including nerve cells is obtained by differentiating pluripotent stem cells (PSCs) into nerve cells over a period of 18 to 35 days, and the nerve cells are differentiated into nerve cells specific to the forebrain region. 57. The method according to any one of embodiments 55 to 56, wherein the cell population does not come into contact with a NOTCH signaling inhibitor until at least 24 days after the initiation of differentiation of the cell population into forebrain-specific neurons (for example, at least 24, 26, 28, 30, 32, 33, or 35 days later, and at the point when 5% of the cell population is INA+). 58. The method according to any one of embodiments 55 to 57, wherein the cell population does not come into contact with a NOTCH signaling inhibitor until at least 24 days after the onset of differentiation of the cell population into forebrain-specific neurons (for example, at least 24, 26, 28, 30, 32, 33, or 35 days later, and at least 5% of the cell population is TBR2+). 59. The method according to Embodiment 55, wherein a population of cells including nerve cells is obtained by differentiating pluripotent stem cells (PSCs) into nerve cells over a period of 14 to 26 days, and the nerve cells are differentiated into nerve cells specific to the midbrain region, preferably the ventral midbrain region. 60. The method according to Embodiment 59, wherein the cell population does not come into contact with a NOTCH signaling inhibitor until at least 20 days after the initiation of differentiation of the cell population into neurons specific to the midbrain region (at least 21, 22, 23, 24, 25, or 26 days later, preferably at least 22 days, etc.) and at the point when at least 10% of the cell population is INA+. 61. The method according to any one of embodiments 59 to 60, wherein the cell population does not come into contact with a NOTCH signaling inhibitor until at least 20 days after the initiation of differentiation of the cell population into midbrain region-specific neurons (at least 21, 22, 23, 24, 25, or 26 days, preferably at least 22 days, etc.) and at the point when at least 30% of the cell population is ASCL1+. 62. The method according to the preceding embodiment, wherein less than 30% (preferably less than 20%, more preferably less than 10%) of the cell population express KI67 at the time the cells are brought into contact with a NOTCH signaling inhibitor. 63. The method according to Embodiment 62, wherein 5-30% (preferably 29-70%) of the cell population expresses KI67 at the time of contact with the NOTCH signaling inhibitor. 64. The method according to any one of embodiments 52 to 63, wherein the cell population does not come into contact with the NOTCH signaling inhibitor until at least 20 days after the initiation of differentiation of the cell population into nerve cells (such as at least 21, 22, 23, 24, 25, or 26 days), preferably until at least 22 days. 65. The method according to any one of Embodiments 1 to 55, wherein a population of cells including nerve cells is obtained by differentiating pluripotent stem cells (PSCs) into nerve cells over a period of 14 to 26 days, and the nerve cells are then differentiated into nerve cells specific to the hindbrain / spinal cord region. 66. The method according to any one of the preceding embodiments, wherein a cell population is cultured in a culture medium suitable for maintaining nerve cells, and the culture medium is a basal neuronal medium. 67. The method according to Embodiment 66, wherein the culture medium comprises one or more components selected from B27 supplement, ascorbic acid, BDNF, GDNF, dcAMP, and DAPT. 68. The method according to Embodiment 67, wherein the culture medium does not contain DAPT. 69. The method according to any one of the prior embodiments, wherein a population of cells including nerve cells is obtained by contacting the population of cells with an inhibitor of Small Mothers Against Decapentaplegic (SMAD) protein signaling. 70. The method according to any one of the prior embodiments, wherein a population of cells including nerve cells is obtained by contacting the cell population with at least two inhibitors of SMAD protein signaling. 71. The method according to any one of Embodiments 69 and 70, wherein the SMAD signaling inhibitor is selected from Noggin, LY364947, SB431542, RepSox, DMH1, DMH2, LND-212854, and LDN-193189. 72. The method according to any one of Embodiments 1-58 and 66-71, wherein a cell population is differentiated into forebrain region-specific neurons, comprising contacting the cell population with an inhibitor of the (SMAD) protein signaling pathway for about 0-10 days, followed by contacting the cell population with FGF2 for about 7-9 days, and then culturing the cell population in basal neuronal medium for an additional 9-12 days. 73. The method according to any one of Embodiments 1-55, 59-64, and 66-71, wherein a cell population is differentiated into ventral midbrain neurons, and the method involves contacting the cell population 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 optionally an activator of fibroblast growth factor (FGF) signaling, as well as optionally ascorbic acid and / or optionally brain-derived neurotrophic factor (BDNF). 74. The method according to a prior embodiment, wherein a 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 optionally an activator of fibroblast growth factor (FGF) signaling, and optionally ascorbic acid and / or optionally brain-derived neurotrophic factor (BDNF), and then the cell population is further cultured in a culture medium suitable for maintaining neurons without contacting the cell population with an inhibitor of SMAD protein signaling, an activator of SHH signaling, an activator of Wnt signaling, or an activator of FGF signaling. 75. The method according to any one of embodiments 73 and 74, wherein the activator of SHH signaling is selected from SHH, prumorphamine, and SAG. 76. The method according to any one of embodiments 73 to 75, wherein the Wnt signaling activator is selected from CHIR99021. 77. The method according to any one of embodiments 73 to 76, wherein the activator of FGF signaling is selected from FGF8b. 78. a) The concentration of the SMAD protein signaling inhibitor is 1 μM to 50 μM. b) The concentration of the SHH signaling activator is 200 ng / ml to 800 ng / ml. c) The concentration of the Wnt signaling inhibitor is 0.1 μM to 1 μM. d) The concentration of the FGF signaling activator is 10 ng / ml to 200 ng / ml. e) The concentration of ascorbic acid is 50 μM to 500 μM, and / or f) The method according to any one of embodiments 73 to 77, wherein the concentration of BDNF is 1 ng / ml to 50 ng / ml. 79. The method according to any one of embodiments 73 to 78, wherein a cell population is exposed to a SMAD protein signaling inhibitor for 5 to 9 days. 80. The method according to any one of embodiments 73 to 79, wherein a cell population is exposed to a Wnt signaling inhibitor for 5 to 9 days. 81. The method according to any one of embodiments 73 to 80, wherein a cell population is brought into contact with an SHH signaling activator for 5 to 9 days. 82. The method according to any one of embodiments 73 to 81, wherein a cell population is contacted with an FGF signaling activator for 7 to 12 days after the completion of contact with an inhibitor of SMAD protein signaling, an inhibitor of Wnt signaling, and / or an activator of SHH signaling. 83. The method according to any one of embodiments 73 to 82, wherein a cell population is brought into contact with a SMAD protein signaling inhibitor for a period from day 0 to day 9, or from day 0 to day 5 to day 9. 84. The method according to any one of Embodiments 73 to 83, wherein a cell population is brought into contact with a Wnt signaling inhibitor for a period from day 0 to day 9, or from day 0 to day 5 to day 9. 85. The method according to any one of embodiments 73 to 84, wherein a cell population is brought into contact with an SHH signaling activator for a period from day 0 to day 5 to day 9, such as from day 0 to day 9. 86. The method according to any one of embodiments 73 to 85, wherein the cell population is exposed to an FGF signaling activator for 7 to 12 days, starting from day 5 to 9, or at the end of contact with an inhibitor of SMAD protein signaling, an inhibitor of Wnt signaling, and / or an activator of SHH signaling. 87. The method according to the preceding embodiment, wherein a cell population is contacted with an FGF signaling activator between days 9 and 16. 88. The method according to any one of embodiments 73 to 87, wherein the cell population is brought into contact with ascorbic acid for 5 to 7 days starting from the 10th or 11th day. 89. The method according to any one of embodiments 73 to 88, wherein the concentration of ascorbic acid is 10 μM to 400 μM. 90. The method according to any one of embodiments 73 to 89, wherein a cell population is brought into contact with BDNF for 5 to 7 days starting from day 10 or day 11. 91. The method according to any one of embodiments 73 to 90, wherein the concentration of BDNF is 1 ng / ml to 40 ng / ml. 92. The method according to any one of Embodiments 73 to 91, wherein a cell population including nerve cells is brought into contact with an inhibitor of SMAD protein signaling, an activator of SHH signaling, and an inhibitor of Wnt signaling on days 0 to 9, and optionally brought into contact with an activator of FGF signaling on days 9 to 16 to induce differentiation of PSCs into nerve cells. 93. The method according to the prior embodiment, wherein a cell population is contacted with an FGF signaling activator from day 9 to day 16. 94. The method according to any one of Embodiments 1 to 55 and 65 to 71, comprising differentiating a cell population into neurons specific to the hindbrain / spinal cord region, and contacting the cell population with a caudal agent such as CHIR99021 on days 0 to 12, a ventral agent such as SHH, SAG, or purmorphamine on days 6 to 15, and a hindbrain / spinal cord promoting compound retinoic acid on days 6 to 15. 95. A method according to any one of the prior embodiments, which is performed in vitro. 96. The method according to any one of the preceding embodiments, wherein the expression of a marker by cells is measured using scRNAseq, qPCR, ICC, or flow cytometry. 97. The method according to any one of the prior embodiments, wherein the expression of a marker by cells is measured according to any of the methods described in Example 9, Example 13, Example 14, and Example 15. 98. The method according to any one of the prior embodiments, wherein the expression of a marker by cells is measured according to one of the methods described in Example 14. 99. A cell population including nerve cells that can be obtained by the method of any one of Embodiments 1 to 98. 100. A cell population comprising nerve cells, wherein when cultured in vitro for 5 to 7 days in a culture medium suitable for maintaining nerve cells, the resulting cell population is at least 35%, preferably 50%, HuC / D positive. 101. A cell population according to a prior embodiment, wherein the expression of a marker by cells is measured using scRNAseq, qPCR, flow cytometry, or ICC. 102. A cell population according to any one of Embodiments 100 and 101, wherein the expression of a marker by cells is measured according to any of the methods described in Example 9, Example 13, Example 14, and Example 15. 103. A cell population according to any one of Embodiments 100 to 102, wherein the expression of a marker by cells is measured according to the method described in Example 14. 104. A cell population according to any one of Embodiments 100 to 103, wherein the cells are ventral midbrain NPCs and the cell population is cultured according to Example 11. 105. A cell population according to any one of Embodiments 100 to 103, wherein the cells are dorsal forebrain NPCs of the cerebral cortex or LGE region, and the cell population is cultured according to Example 10. 106. A cell population according to any one of Embodiments 100 to 103, wherein the cells are from the hindbrain / spinal cord region and the cell population is cultured according to Example 12. 107. A cell population according to any one of embodiments 99 to 106, wherein the cell population is in vitro. 108. A cell population according to any one of embodiments 99 to 107, wherein the nerve cells are non-natural. 109. A cell population according to any one of embodiments 99 to 108, wherein the nerve cells are artificial. 110. A cell population according to any one of embodiments 99 to 109, wherein the cell population is derived from stem cells. 111. A cell population according to any one of embodiments 99 to 110, wherein the cell population is derived from pluripotent stem cells. 112. A cell population according to a prior embodiment, wherein pluripotent stem cells are genetically modified, and the genetic modification is sustained within nerve cells. 113. A cell population according to any one of embodiments 111 to 112, wherein the nerve cells are derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs). 114. A cell population according to any one of embodiments 99 to 113, wherein the nerve cells are specific to a region selected from the forebrain, midbrain, and hindbrain / spinal cord. 115. The cell population according to the prior embodiment, wherein the nerve cells are ventral midbrain nerve cells. 116. A cell population according to any one of embodiments 99 to 115, wherein the cells of the cell population are genetically modified. 117. A cell population according to a prior embodiment, wherein the cells of the cell population are genetically modified to be low immunogenic and / or lineage-limited. 118. Cell populations according to a prior embodiment, wherein genetic modification for low immunogenicity includes reduced expression of MHC-I human leukocyte antigen in wild-type stem cells, reduced expression of MHC-II human leukocyte antigen in wild-type stem cells, and / or increased expression of tolerance factors in wild-type stem cells. 119. The cell population according to the prior embodiment, wherein the MHC-I human leukocyte antigens are HLA-A, HLA-B, and HLA-C. 120. A cell population according to any one of embodiments 118 to 119, wherein the MHC-II human leukocyte antigens are HLA-DP, HLA-DQ, and HLA-DR. 121. A cell population according to any one of embodiments 118 to 120, wherein the tolerance factor is selected from CD46, CD47, CD55, CD59, PD-L1, HLA-E, and HLA-G. 122. A cell population according to any one of embodiments 99 to 121, wherein the cell population comprises at least 1,000 cells (such as 10,000 cells, 100,000 cells, 1,000,000 cells, or 10,000,000 cells). 123. A cell population according to any one of embodiments 99 to 122, wherein the cell population is cryopreserved. 124. A composition comprising a cell population and a cryoprotectant as described in any one of the prior embodiments. 125. The composition according to the prior embodiment, wherein the cryoprotectant comprises DMSO. 126. An in vitro cell population according to any one of the prior embodiments, or a composition according to any one of the prior embodiments, for use as a pharmaceutical. 127. An in vitro cell population as described in a prior embodiment for the treatment of neurological pathologies. 128. An in vitro cell population according to a prior embodiment, wherein the neurological condition is selected from the group including Parkinson's disease, stroke, traumatic brain injury, spinal cord injury, Huntington's disease, dementia, Alzheimer's disease, and other neurological conditions resulting in loss or dysfunction of neurons. 129. An in vitro cell population according to Embodiment 126 for the treatment of Parkinson's disease, wherein the nerve cells are ventral midbrain nerve cells. 130. An in vitro cell population according to Embodiment 126 for the treatment of stroke, wherein the nerve cells are forebrain nerve cells. 131. An in vitro cell population according to Embodiment 126 for the treatment of paralysis, spinal cord injury, diabetic neuropathy, or chronic pain, wherein the nerve cells are posterior brain / spinal cord nerve cells. 132. A method for treating a neurological condition, comprising administering to a patient an effective amount of a cell population described in any one of the preceding embodiments. 133. The method according to a prior embodiment, wherein the neurological condition is selected from Parkinson's disease, stroke, traumatic brain injury, spinal cord injury, Huntington's disease, dementia, Alzheimer's disease, and other neurological conditions resulting in loss or dysfunction of neurons. 134. The method according to Embodiment 132, wherein the neurological condition is Parkinson's disease. 135. A method for treating a neurological condition, comprising administering to a subject in need thereof a therapeutically effective amount of a population of cells including nerve cells and a NOTCH signaling inhibitor, wherein the NOTCH signaling inhibitor is selected from LY411575, abagacestat, dibenzazepine, and PF-03084014. 136. The method according to a prior embodiment, wherein the inhibitor of NOTCH signaling is LY411575. 137. A composition comprising a cell population including nerve cells and a NOTCH signaling inhibitor, wherein the NOTCH signaling inhibitor is selected from LY411575, abagacestat, dibenzazepine, and PF-03084014. 138. The composition according to the prior embodiment, wherein the inhibitor of NOTCH signaling is LY411575. 139. LY411575 for use in the treatment of neurological conditions. 140. LY411575 for use in the treatment of Parkinson's disease in patients receiving a therapeutically effective dose of ventral midbrain nerve cells. 141. LY411575 for use in the treatment of stroke in patients receiving a therapeutically effective dose of dorsal anterior cranial nerve cells. 142. Abagacestat for use in the treatment of neurological conditions. 143. Abagacestat for use in the treatment of Parkinson's disease in patients receiving a therapeutically effective dose of ventral midbrain nerve cells. 144. Abagacestat for use in the treatment of stroke in patients receiving a therapeutically effective dose of dorsal anterior cranial nerve cells. 145. Dibenzazepine for use in the treatment of neurological conditions. 146. Dibenzazepine for use in the treatment of Parkinson's disease in patients receiving a therapeutically effective dose of ventral midbrain nerve cells. 147. Dibenzazepine for use in the treatment of stroke in patients receiving a therapeutically effective dose of dorsal anterior cranial nerve cells. 148. PF-03084014 for use in the treatment of neurological conditions. 149. PF-03084014 for use in the treatment of Parkinson's disease in patients receiving a therapeutically effective dose of ventral midbrain nerve cells. 150. PF-03084014 for use in the treatment of stroke in patients receiving a therapeutically effective dose of dorsal anterior cranial nerve cells. [Examples]

[0126] The following are non-limiting embodiments for carrying out the present invention.

[0127] Example 1: Differentiation of human pluripotent stem cells into dorsal forebrain neurons Human embryonic stem cell (hESC) lines RC17 (Roslin CT) and 3053 (Novo Nordisk A / S) were subjected to a human laminin-521 matrix (0.7~1.2 μg / cm³). 2Cells were cultured in iPS Brew XF medium (Miltenyi Biotec) supplemented with 60 U / mL penicillin-streptomycin (PS, Thermo Fisher Scientific) on culture devices coated with Biolamina. The medium was changed daily, and cells were subcultured every 4–6 days in 0.5 mM EDTA (Thermo Fisher Scientific). The cultures were maintained at 37°C, 95% humidity, and 5% CO2 level.

[0128] hESCs were differentiated into dorsal forebrain neurons according to established protocols (Shi et al. 2011; Shi et al. 2012). The hESCs were seeded and then administered laminin-521 (1.2 μg / cm³). 2 Cells were cultured in culture devices coated with BioLamina and, once a 95-100% confluent monolayer was formed, were exposed to differentiation medium. From DIV0-19, cells were cultured in CPN medium: 50% DMEM / F12+Glutamax (Gibco), 50% Neurobasal (Gibco), 2% B27 supplement, vitamin A-containing CTS (Thermo Fisher), 1% N2 supplement CTS (Thermo Fisher), 5% GlutaMAX (Thermo Fisher), 0.2% penicillin streptomycin (P / S, Thermo Fisher), 1% NEAA (Gibco), and 0.089% β-mercaptoethanol (Gibco), supplemented with BMP pathway inhibitor SB431542 (10 μM; Miltenyi Biotec) and noggin (100 ng / mL, Miltenyi Biotec). In DIV11, cells were dissociated using 0.5 mM EDTA and then passaged in a 1:2 ratio. N2 / B27 medium was supplemented in DIV11-18 with fibroblast growth factor b (20 ng / mL, R&D). In DIV18, cells were dissociated using 0.5 mM EDTA and then either cryopreserved or passaged in a 1:2 ratio. From DIV19 onward, cells were cultured in CPN basal medium (Figure 2).

[0129] Example 2: Administration of NOTCH inhibitor LY411575 to dorsal anterior cranial nerve cultures To generate hPSC-derived cell cultures or cell products rich in neurons and dorsal forebrain neurons, and deficient in other cell types, a novel NOCTH pathway-targeting inhibitor, LY411575, was administered to dorsal forebrain neuronal cell cultures.

[0130] FB NPCs were differentiated from hPSCs by patterning factors that induce neuronal / ectoderm specification, and the cells were cultured until they acquired dorsal forebrain identity (resulting from DIV18). DIV18 cultures were then profiled by flow cytometry to confirm correct patterning (Figure 3, Table 1). Specifically, the cells were FB NPCs because they expressed high levels of PAX6 (>86.2%) and OTX2 (>88.6%) (Figure 3, Table 1). These cultures were left without neuronal / ectoderm patterning factors for further differentiation in vitro for a period of 12 days (DIV18-30). During this period and at all prior time points (in vitro days 0-30), the cell cultures were not exposed to any NOTCH inhibitors (Figure 2). However, since they are mostly in the precursor stage (as indicated by high precursor marker expression and low IPC and neuron marker expression), we also anticipate that these cells may be a population that can be treated with NOTCH inhibitor compounds and converted into neurons.

[0131] Cells at 30 days in vitro were profiled by flow cytometry before exposure to the NOTCH inhibitor LY411575 (Figure 4, Table 2), and the protein expression levels of dorsal forebrain focal identity transcription factors (PAX6 and OTX2) and cell stage markers (NSC and proliferation markers SOX2 and KI67; TBR2, which identifies IPC; and INA+ / SOX2-, which identifies neurons) were assessed. These cultures were observed to be dorsal forebrain in their focal identity and to express the dorsal forebrain marker PAX6 (Figure 4, Table 2). These cells were still in the NSC stage, as indicated by the high expression of the NSC marker SOX2 (Figure 4, Table 2). These cultures also showed several levels of the intermediate precursor marker TBR2 and low levels of postmittal neurons, which were distinguishable by INA+ / SOX2- staining (Figure 4, Table 2).

[0132] After administration of the compound (LY411575), all groups were differentiated in vitro for a further 5 days in the absence of any NOTCH inhibitors, then fixed in 4% paraformaldehyde and stained with DAPI to identify all cell nuclei and primary antibodies against SOX2 (1:300), Ki-67 (1:250), and HuC / D (1:100), followed by the identification of fluorescently labeled secondary antibodies. Images were acquired using a Zeiss Axio Observer microscope with Zen 3.2 (Zen Pro) software, and cells were counted using ImageJ software.

[0133] The IF counts showed comparable results for both 24-hour and 72-hour administration (Figures 5 and 6, Tables 3 and 4). All groups treated with LY411575 were found to have higher expression of the neuronal marker HuC / D (in total cells) in both conditions (92.9% and 94.4%, black bars) compared with cultures not exposed to any NOTCH inhibitor at any time point (64.1% and 61.3%, white bars). Cultures exposed to the NOTCH inhibitor LY411575 were also observed to have reduced levels of the NSC marker SOX2 and the proliferation marker Ki67 in both conditions (Figures 5 and 6, black bars) compared with the control (Figures 5 and 6, white bars), indicating that these cells differentiated into neurons at a higher rate compared to the control in response to these inhibitors.

[0134] These results support the hypothesis that the addition of the NOTCH inhibitor LY411575 can promote the differentiation of dorsal forebrain NSCs into dorsal forebrain neurons while dramatically reducing the amount of proliferative and undesirable cell types (Figures 5 and 6, Tables 3 and 4). [Table 1] [Table 2] [Table 3] [Table 4]

[0135] Example 3: Differentiation of human pluripotent stem cells into ventral midbrain nerve cells Human embryonic stem cell (hESC) lines RC17 (Roslin CT) and 3053 (Novo Nordisk A / S) were subjected to a human laminin-521 matrix (0.7~1.2 μg / cm³). 2Cells were cultured in iPS Brew XF medium (Miltenyi Biotec) supplemented with 60 U / mL penicillin-streptomycin (PS, Thermo Fisher Scientific) on culture devices coated with Biolamina. The medium was changed daily, and cells were subcultured every 4–6 days in 0.5 mM EDTA (Thermo Fisher Scientific). The cultures were maintained at 37°C, 95% humidity, and 5% CO2 level.

[0136] hESCs were differentiated into ventral midbrain neurons according to established protocols (e.g., Nolbrant et al., 2017, Kirkeby et al., 2017). Briefly, hESCs were proliferated to 70-90% confluence and then dissociated using 0.5 mM EDTA. The cells were then treated with human laminin-111 (1.2 μg / cm³). 2 , 10 cell culture flasks or plates coated with BioLamina 4 cells / cm 2Cells were seeded and immediately brought into contact with differentiation medium. Cells were exposed to N2-based medium for in vitro days (DIV) 0-8, supplemented with 50% DMEM / F12+Glutamax (Gibco), 50% Neurobasal (Gibco), 1% N2 supplement CTS (Thermo Fisher Scientific), 5% GlutaMAX (Thermo Fisher Scientific), 0.2% PS (Thermo Fisher Scientific), and SMAD inhibitor SB431542 (10 μM, Miltenyi Biotec), noggin (100 ng / mL, Miltenyi Biotec) for neural induction, Sonic Hedgehog C24II (SHH, 500 ng / mL, Miltenyi Biotec) for ventral fate, and GSK3β inhibitor CHIR99021 (CHIR, 0.5-0.6 μM, Miltenyi Biotec) to promote caudalization. In N2-based medium, fibroblast growth factor 8b (FGF8b, 100 ng / mL, Miltenyi Biotec) was supplemented in DIV9-11. In DIV11, cells were dissociated with accutase (Thermo Fisher Scientific), and human laminin-111 (1.2 μg / cm³) was added to DIV11-16 medium (Neurobasal, 2% B27 supplement, vitamin A-free CTS (Thermo Fisher Scientific)), 5% GlutaMAX, 0.2% PS, and 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). 2 ) in a cell culture flask or plate coated with 0.8 x 10 6 individual cells / m 2The cells were seeded. At 16 days in vitro (DIV), the cells were dissociated with acutase, and during the extended in vitro culture, poly-L-ornithine (0.002%) and laminin-521 (1.5 μg / cm³) were added to B27 medium supplemented with BDNF (20 ng / mL), GDNF (20 ng / mL), L-ascorbic acid (200 μM), dcAMP (500 μM), and Y-27632 (10 μM). 2 Cell culture flasks / plates coated with ) were either cryopreserved or reseeded to allow further differentiation and maturation of ventral midbrain neural stem cells into neurons (Figures 7 and 8). To confirm correct patterning, flow cytometry analysis of DIV16 was performed using regional markers expressed in ventral midbrain FOXA2, OTX2, LMX1A, and EN1 (Figure 9).

[0137] [References] Nolbrant S,Heuer A,Parmar M,Kirkeby A.Generation of high-purity human ventral midbrain dopaminergic progenitors for in vitro maturation and intracerebral transplantation.Nat Protoc.2017 Sep;12(9):1962-1979.doi: 10.1038 / nprot.2017.078.Epub 2017 Aug 31.PMID:28858290.

[0138] Kirkeby A, Nolbrant S, Tiklova K, Heuer A, Kee N, Cardoso T, Ottosson DR, Lelos MJ, Rifes P, Dunnett SB, Grealish S, Perlmann T, Parmar M. Predictive Markers Guide Differentiation to Improve Graft Outcome in Clinical Translation of hESC-Based Therapy for Parkinson's Disease.Cell Stem Cell.2017 Jan 5;20(1):135-148.doi:10.1016 / j.stem.2016.09.004.Epub 2016 Oct 27.PMID:28094017;PMCID: PMC5222722.

[0139] Example 4: Administration of NOTCH inhibitor LY411575 to ventral midbrain nerve cultures To generate hPSC-derived cell cultures or cell products rich in neurons and ventral midbrain dopaminergic neurons, and deficient in other cell types (i.e., glial precursors, glial cells, stromal cells, and proliferating cells), the NOCTH pathway-targeting inhibitor LY411575 was administered to ventral midbrain neuronal cell cultures.

[0140] VM NPC cultures were first differentiated from hPSCs by patterning factors that induce neuronal / ectoderm specification, ventralization, and caudalization (induced by DIV16), and these were profiled by flow cytometry to confirm VM NPC identity (Figure 9). Specifically, the cells were VM NPCs because they expressed high levels of FOXA2 (>79.9%), OTX2 (>83.2%), EN1 (>66.3%), and LMX1A (>61.8%) (Figure 9, Table 5). These cultures were left without neuronal / ectoderm, ventralization, and caudalization patterning factors and further differentiated in vitro (DIV 16-22) for a period of 6 days, and the cell cultures were not exposed to any NOTCH inhibitors at any of the preceding time points (in vitro days 0-22) (Figure 8).

[0141] Cells at day 22 in vitro were profiled by flow cytometry before exposure to the NOTCH inhibitor LY411575 (Figure 10, Table 6), and the protein expression levels of ventral midbrain floorplate local identity transcription factor (FOXA2) and cell stage markers (NSC and proliferation markers SOX2 and KI67; ASCL1, which identifies IPCs; and INA, which identifies neurons) were assessed. These analyzed cultures were observed to be ventral midbrain in terms of their local identity and expressed the floorplate marker FOXA2 (>82% of all cells) (Figure 10). These cells were still in the NSC stage, as indicated by the high expression of NSC markers SOX2 and KI67 (>86% and >51%; Figure 10, Table 6). These cultures also showed high levels of the intermediate precursor marker ASCL1 (48%, Figure 10, Table 6) and low levels of the neuronal marker INA (12.8%, Figure 10, Table 6). Postmitotic neurons (3.3%) were also distinguishable by INA+ / SOX2- staining (Figure 10 and Table 6).

[0142] After evaluating the correct patterning and maturation stages of the cultures, DIV22 cells were exposed to the NOTCH inhibitor LY411575 for either 24 or 72 hours. Following compound administration, all groups were differentiated in vitro for a further 7 days in the absence of any NOTCH inhibitor, then fixed in 4% paraformaldehyde and stained with DAPI to identify all cell nuclei and primary antibodies against SOX2 (1:300), Ki-67 (1:250), HuC / D (1:100), and FOXA2 (1:200), followed by fluorescently labeled secondary antibodies. Images were acquired using a Zeiss Axio Observer microscope with Zen 3.2 (Zen Pro) software, and cells were counted using ImageJ software.

[0143] IF counts showed similar results for both 24-hour and 72-96 hour administrations (Figures 11 and 12, Tables 7 and 8). All groups were found to maintain high expression (of all cells) of the ventral midbrain floorplate lineage marker FOXA2 (>80%, Figures 11 and 12). Cultures exposed to the NOTCH inhibitor LY411575 had increased levels of the neuronal marker HuC / D at both 24-hour and 72-96 hour administrations (54.8% and 67.3%, Figures 11 and 12, black bars) compared to control cells not exposed to NOTCH inhibition (35.2% and 29.7%, Figures 11 and 12, white bars). Treated cells were also observed to have decreased levels of the NSC marker SOX2 and the proliferation marker Ki67 under both conditions (Figures 11 and 12, black bars) compared to controls (Figures 11 and 12, white bars).

[0144] These results support the hypothesis that the addition of the NOTCH inhibitor LY411575 can promote the differentiation of ventral midbrain floorplate NSCs into ventral midbrain floorplate neurons and reduce the amount of proliferative and undesirable cell types without altering the expression of floorplate lineage transcription factors (Figures 11 and 12, Tables 7 and 8). [Table 5] [Table 6] [Table 7] [Table 8]

[0145] Example 5: Administration of different NOTCH inhibitors to ventral midbrain nerve cell cultures Novel NOTCH pathway-targeting inhibitors, LY411575, abagacestat, and PF-03084014, were administered to ventral midbrain neuronal cell cultures to generate hPSC-derived cell cultures or cell products rich in neurons and ventral midbrain dopaminergic neurons, and deficient in other cell types (i.e., glial precursors, glial cells, stromal cells, and proliferating cells). The effects were then compared to those obtained by administering NOTCH inhibitors commonly used in in-situ DAPT.

[0146] VM NPC cultures were first differentiated from hPSCs by patterning factors that induce neuronal / ectoderm specification, ventralization, and caudalization, as described in Example 4. DIV16 cells were then profiled via flow cytometry (Figure 9, Table 5), further differentiated in vitro for a period of 6 days (DIV16-22) without any NOTCH inhibitors (Figure 8), and their protein expression levels were analyzed using flow cytometry as shown in Example 4 (Figure 10, Table 6).

[0147] After evaluating the correct patterning and maturation stages of the cultures, DIV22 NPC cells were treated for 24 hours with novel NOTCH inhibitors LY411575, abagacestat, and PF-03084014.

[0148] After administration of the compound (LY411575), all groups were differentiated in vitro for a further 7 days in the absence of any NOTCH inhibitors, then fixed in 4% paraformaldehyde and stained with DAPI to identify all cell nuclei and primary antibodies against SOX2 (1:300), Ki-67 (1:250), HuC / D (1:100), and FOXA2 (1:200), followed by the identification of fluorescently labeled secondary antibodies. Images were acquired using a Zeiss Axio Observer microscope with Zen 3.2 (Zen Pro) software, and cells were counted using ImageJ software.

[0149] All groups were found to maintain high expression (in all cells) of the ventral midbrain floorplate lineage marker FOXA2 (>60%, Figure 13, Table 9). Cultures exposed to the novel NOTCH inhibitors LY411575 (black bars), abagacestat (thin striped bars), and PF-03084014 (thick striped bars) showed increased expression levels of the neuronal marker HuC / D at all time points compared to control cells (white bars) that were not exposed to NOTCH inhibition (Figure 13, Table 9). Treated cells were also observed to have decreased levels of the NSC marker SOX2 and the proliferation marker Ki67 compared to controls (Figure 13, Table 9).

[0150] Interestingly, the protein expression levels of the neuronal marker HuC / D were higher in cultures treated with the novel NOTCH inhibitors LY411575, abagacestat, and PF-03084014 compared to cultures treated with the NOTCH inhibitors commonly used in in-situ DAPT (dotted bars) (Figure 13, Table 9). Furthermore, all groups exposed to the novel NOTCH inhibitors LY411575, abagacestat, and PF-03084014 had lower expression levels of the NSC marker SOX2 and the proliferation marker Ki67 compared to cells treated with DAPT (Figure 16, Table 11).

[0151] These results summarize that novel compounds targeting the NOTCH pathway LY411575, abagacestat, and PF-03084014 had the effect of increasing neurons and decreasing NSC and proliferating cell percentages. A comparison between the results of cultures treated with novel NOTCH inhibitors (LY411575, abagacestat, and PF-03084014) and those treated with the well-known NOTCH inhibitor DAPT shows that administration of the novel compounds resulted in a higher number of HuC / D neurons, and lower levels of NSCs and proliferation markers SOX2 and Ki67 (Figure 13, Table 9) compared to DAPT administration, indicating that novel NOTCH inhibitors are better suited to solving the problem of obtaining purified cell products, which have been well-received in the field of stem cell-derived neuronal generation. [Table 9]

[0152] Example 6: Titration of LY411575 in ventral midbrain nerve cultures To test the extent to which the NOTCH inhibitor LY411575 promotes differentiation of ventral midbrain dopaminergic neurons and its toxicity can be examined, titrations of the compound were performed.

[0153] VM NPC cultures were differentiated from hPSCs according to Example 3. After cell culture profiling at DIV16, further differentiation was performed in the absence of any NOTCH inhibitor up to DIV22, and after re-analysis of protein levels in the DIV22 culture as described in Example 3 (Figures 9 and 10, Table 5, and Table 6), VM NPCs were contacted with the NOTCH inhibitor LY411575 at different concentrations for 24 hours. Specifically, LY411575 was administered at 0.2 μM, 0.5 μM, 2 μM, 10 μM, 40 μM, and 100 μM. After administration of the compound (LY411575), all groups were differentiated in vitro for a further 7 days without any NOTCH inhibitors, then fixed in 4% paraformaldehyde and stained with DAPI to identify all cell nuclei and primary antibodies against SOX2 (1:300), Ki-67 (1:250), HuC / D (1:100), and FOXA2 (1:200), followed by the identification of fluorescently labeled secondary antibodies. Images were acquired using a Zeiss Axio Observer microscope with Zen 3.2 (Zen Pro) software, and cells were counted using ImageJ software.

[0154] The results show that floorplate marker FOXA2 levels remained high across all groups (>62.7%, Figure 19, Table 10), indicating that the cultures maintained their local identity. Furthermore, all concentrations tested are thought to promote a higher level of differentiation of ventral midbrain NPCs into ventral midbrain neurons compared to cultures not in contact with any NOTCH inhibitor (CONTROL: white bars), as indicated by increased levels of the neuronal marker HuC / D, as well as decreased levels of the NSC marker SOX2 and the proliferation marker Ki67 (Figure 19, Table 10).

[0155] These results indicate that the NOTCH inhibitor LY411575 acts across a wide range of concentrations, promoting neuronal differentiation and reducing the number of proliferating cells in culture (Figure 19, Table 10). Furthermore, higher concentrations of LY411575 (black bars) appear to be non-toxic to cells and further help reduce the amount of undesirable proliferating cells in the culture (Figure 19, Table 10). [Table 10]

[0156] Example 7: Differentiation of human pluripotent stem cells into hindbrain / spinal cord nerve cells Undifferentiated hESCs at 80-90% confluence were dissociated with 0.5 mM EDTA (Thermo Fisher) for 5-7 minutes at room temperature and separated from the flask for reseeding; EDTA was used because it generates small aggregates, thus allowing for uniform distribution of cells before the start of the differentiation procedure. The hESC aggregates were seeded and evenly distributed into 24-well plates or T25 flasks (Sarstedt) pre-coated with 10 μg / mL laminin-521. Cells were maintained for the first 24 hours using iPS Brew XF (Miltenyi) and supplemented with 10 μM of the wax-associated kinase inhibitor Y27632 (ROCKi; Sigma) to promote cell survival. Differentiation was initiated when the cells reached 90-100% confluence (approximately 24 hours later). For the first six days of differentiation, cells were maintained in "Panneuron Basal Medium" - 40% basal neural fluid (Gibco), 40% DMEM / F12+GlutaMAX (Gibco), 5% B27 supplement (Thermo Fisher), 1% N2 supplement (Thermo Fisher), 1% MEM non-essential amino acids (NEAA, Gibco), 0.5% GlutaMAX (Gibco), 0.2% penicillin / streptomycin (P / S, Thermo Fisher) - supplemented with SMAD pathway inhibitors SB431542 (10 nM, Miltenyi Biotec) and noggin (100 ng / mL, Miltenyi Biotec) for neural induction, and 3 μM CHIR99021 (CHIR, Miltenyi Biotec) for caudal differentiation. The medium was changed daily, and cells were washed with DPBS- / -(Invitrogen) before medium changes to remove accumulated cell death. On day 6, the cells were dissociated using acutase (Innovative Cell Technologies) and coated with 10 μg / mL of LN-521 in a 1:6 ratio (cells / cm³). 2Cells were subcultured using 10 μM ROCKi Y27632 by reseeding. From days 6–12, cells were grown in panneuronal basal medium supplemented with the SMAD pathway inhibitor SB431542 (10 nM; Miltenyi Biotec) and noggin (100 ng / mL; Miltenyi Biotec), as well as 1 μM CHIR, 40–100 nM Smoothened agonist (SAG; Millipore), and 500 nM retinoic acid (RA; Sigma). The medium was changed daily, and cells were washed with DPBS- / - before the medium change to remove cell death. From days 12–15, cells were grown in panneuronal basal medium (-vitamin A)-5% B27 supplement-vitamin A (Thermo Fisher)-40–100 nM SAG and 500 nM RA. On day 15, the cells were dissociated using acutase at 37°C for 6-8 minutes, and then coated with 10 μg / mL LN-521 in a 1:6 ratio (cells / cm³). 2 The cells were subculturized using 10 μM ROCKi Y27632 by reseeding. Hindbrain / spinal cord NPCs were collected on day 12 or 15 of differentiation and often cryopreserved, and the cells were exposed to NOTCH inhibitors for neuronal conversion at DIV15 or DIV15–22. From day 18 onward, the cultures were maintained in panneuronal basal medium (-VitA) for neuronal maturation. The medium was changed daily, and cells were washed with DPBS- / - before the medium change to remove cell death. On day 24 of differentiation, the cultures were changed every 3–4 days for the remainder of the culture duration.

[0157] Example 8: Administration of different NOTCH inhibitors to hindbrain / spinal cord nerve cell cultures To generate hPSC-derived cell cultures or cell products rich in neurons and hindbrain / spinal cord neurons, and deficient in other cell types, a novel NOTCH pathway-targeting inhibitor, LY411575, was administered to hindbrain / spinal cord neuronal cell cultures.

[0158] First, HB / SCord NPC cultures were differentiated from hPSCs by patterning factors that induce neuronal / ectoderm differentiation and ventralization (occurring by DIV15). These were profiled by flow cytometry to confirm HB / SCord NPC identity (Figure 16, Table 11). Specifically, the cells did not express OTX2 at varying levels, but expressed NKX6.1, PAX6, and OLIG2, thus confirming they were HB / SCord NPCs (Figure 16, Table 11).

[0159] After the cells acquired HB / SCord NPC identity, they were exposed to the NOTCH inhibitor LY411575 for either 24 or 72 hours (Figure 15) to promote neuronal maturation.

[0160] After administration of the compound (LY411575), all groups were differentiated in vitro for a further 5 days in the absence of any NOTCH inhibitors, then fixed in 4% paraformaldehyde and stained with DAPI to identify all cell nuclei and primary antibodies against SOX2 (1:300), Ki-67 (1:250), and HuC / D (1:100), followed by the identification of fluorescently labeled secondary antibodies. Images were acquired using a Zeiss Axio Observer microscope with Zen 3.2 (Zen Pro) software, and cells were counted using ImageJ Fiji software.

[0161] The IF counts showed similar results for both 24-hour and 72-hour administrations (Figures 17 and 18, Tables 12 and 13). Specifically, all groups treated with LY411575 were found to have higher expression of the neuronal marker HuC / D (total cells) after both 24-hour and 72-hour administrations (77% and 80%, black bars) compared to cultures not exposed to any NOTCH inhibitor at any time point (36.3% and 21.6%, white bars). Cultures exposed to the NOTCH inhibitor LY411575 were also observed to have reduced levels of the NSC marker SOX2 and the proliferation marker Ki67 under both conditions compared to the control. NSC marker SOX2 and proliferation marker Ki67 decreased from 69.9% and 41.1% in the control group (white bars) to 55.6% and 8.6% in the 24-hour LY411575-treated group (black bars). Similar results were achieved by administering LY411575 for 72 hours, which reduced SOX2 and Ki67 levels from 78% and 59.6% (white bars) to 32.3% and 9.6% (black bars) (Figures 17 and 18, Tables 12 and 13).

[0162] These results support the hypothesis that the addition of the NOTCH inhibitor LY411575 can promote the differentiation of hindbrain / spinal cord NSCs into hindbrain / spinal cord neurons while dramatically reducing the amount of proliferative and undesirable cell types (Figures 17 and 18, Tables 12 and 13). [Table 11] [Table 12] [Table 13]

[0163] Example 9: Single-cell protein marker expression assay by flow cytometry analysis Ventral midbrain dopaminergic (vmDA) progenitor cells were generated from hESCs in 2D in vitro cultures using the reagents described in Example 1 and Table 14. At various times after the start of differentiation, the cell cultures were dissociated into single-cell suspensions using accutase, counted on a NucleoCounter NC-200, and collected in N2 medium (CTS® Neurobasal® medium supplemented with 1% CTS® N-2 supplement). Dead cells were labeled using the LIVE / DEAD® Fixable Near-IR Dead Cell Stain Kit. The cells were then resuspended in B27 medium (CTS® Neurobasal® medium supplemented with 1% B-27® supplement, no vitamin A, 2 mM GlutaMAX®, 60 U / mL penicillin-streptomycin, and 10 μM ROCK inhibitor). Next, the cells were fixed and permeabilized using the BD Transcription Factor Buffer Set (BD Biosciences) according to the manufacturer's instructions. The fixed cells were then stained with a fluorescently conjugated antibody, and the samples were acquired on a BD LSR Fortessa (BD Biosciences) or CytoFLEX S (Beckman Coulter). The fcs files were exported and analyzed with FlowJo 10.8.1.

[0164] To determine whether cells were considered positive for the protein expression of their marker, a gate was set at the edge of the fluorescence signal of the negative control sample, as routinely done in situ. Examples of negative control samples used included unstained control, fluorescence minus 1 (FMO) control, and most preferably biological negative control sample. All cells present above these threshold gates when the experimental sample was run were considered positive.

[0165] A two-parameter dot plot was generated to measure the expression of two related proteins. Co-expression of the two proteins is represented by the percentage of cells in the upper right quadrant (Q2), while single-positive cells are represented by the number of cells in Q1 and Q3. [Table 14-1] [Table 14-2]

[0166] Example 10: After NOTCH inhibition, the dorsal forebrain neuronal population was further cultured and its expression profile was evaluated. After administration of NOTCH inhibitors, cells were conditioned in panneuron medium or other similar support media supplemented with BDNF (40 ng / mL), GDNF (40 ng / mL), L-ascorbic acid (200 μM), dcAMP (50 μM), and laminin (1 ug / mL) with poly-L-ornithine (0.002%) and laminin-521 (1.5 μg / cm³). 2 The cells are cultured for a further 5 days in 2D culture in wells coated with ), and at this point, time is allowed for the migration of neural precursors and intermediates to allow for final differentiation into dorsal forebrain neurons. After 5 days of culture, the expression profile of the cell population can be evaluated according to Example 14.

[0167] Example 11: After NOTCH inhibition, the ventral midbrain neuron population was further cultured and its expression profile was evaluated. After administration of NOTCH inhibitors, cells were subjected to a neuronal support medium supplemented with BDNF (20 ng / mL), GDNF (20 ng / mL), L-ascorbic acid (200 μM), and dcAMP (500 μM), in which poly-L-ornithine (0.002%) and laminin-521 (1.5 μg / cm³) were added. 2 The cells are cultured for a further 7 days in 2D culture in wells coated with ), and at this point, time is allowed for the migration of neural precursors and intermediates to allow for final differentiation into ventral midbrain neurons. After 5 days of culture, the expression profile of the cell population can be evaluated according to Example 14.

[0168] Example 12: Further culture of hindbrain / spinal cord cell populations after NOTCH inhibition and evaluate the expression profile. After administration of NOTCH inhibitors, cells were conditioned in panneuronal basal medium supplemented with B27 (-vitamin A) with poly-L-ornithine (0.002%) and laminin-521 (1.5 μg / cm³). 2Cultured for an additional 5 days in 2D culture in wells coated with and at this point, allow time for the migration of neural precursors and intermediates and finally differentiate them into hindbrain neurons. Five days after the culture, the expression profile of the cell population can be evaluated according to Example 14.

[0169] Example 13: RNA sequencing method To perform single-cell RNA sequencing (scRNA-seq) or single-nucleus RNA sequencing, not only undifferentiated PSCs but also those of differentiated cells were dissociated into single-cell suspensions with Accutase, Tryple Select, or other such reagents, and 3000 - 10000 cells were processed using the 10X Genomics Chromium Platform and sequenced on the NextSeq550. The data were processed in the R programming language using the 10X Cellranger and Seurat analysis packages. Samples were analyzed, filtered for low-quality or multiplet cells, analyzed separately for each individual experiment, and then combined into one dataset not only for the selected differentiated cell lineages but also for hPSC cells, and then this was analyzed using the standard Seurat workflow as outlined for Seurat version 3, i.e., normalization using SCTransform and finally using the first 29 major components for the integrated tSNE plot.

[0170] Example 14: Immunocytochemistry (ICC) Cells were fixed in 4% paraformaldehyde (Alfa Aesar) for 10 minutes at room temperature. Nonspecific antibody binding was blocked by incubating cells for 30 minutes in phosphate-buffered saline (PBS) containing 0.02% sodium azide solution (Ampliqon) without PADT buffer; 0.5% Triton X-100 (Sigma); and 5% donkey serum (Jackson Labs), followed by overnight incubation at 4°C with primary antibody (see Table 14). Cells were washed three times with PBS without Ca2+ and Mg2+, blocked with PADT buffer for 15 minutes, protected from light, and incubated for 2 hours at room temperature with fluorophore-conjugated secondary antibody. (See Table 14) Next, cells were counterstained with DAPI (10 μg / mL) at room temperature for 5 minutes, washed three times with Ca2+ and Mg2+-free PBS, and stored at 4°C in Ca2+ and Mg2+-free PBS supplemented with 0.02% sodium azide. Images were acquired using a Zeiss Axio Observer microscope equipped with an Axiocam 512 camera and ZEN 3.2(Pro) software (Zeiss). Automated or manual counting of each cell relative to the DAPI nucleus can also be performed for quantification of ICC staining.

[0171] Example 15: Quantitative Real-Time PCR (qPCR, or qRT-PCR) To perform qPCR, RNA was extracted from cells using Trizol and converted to cDNA, and then analyzed using quantitative real-time polymerase chain reaction (qPCR) for target genes such as SOX2, KI67, HuCD, NeuN, INA, ASCL1, FOXA2, TH, LMX1A, EN1, or other relevant markers for ventral midbrain neurons. qPCR was typically performed over three or more independent biological replicates, each with three consecutive technical replicates, and normalized for housekeeping genes such as GAPDH or HPRT1.

[0172] While certain features of the present invention are illustrated and described herein, many modifications, substitutions, alterations, and equivalents will be conceivable to those skilled in the art. It should therefore be understood that the appended claims are intended to encompass all such modifications and alterations that fall within the true spirit of the invention.

[0173] Example 16: Administration of NOTCH inhibitor LY411575 to dorsal anterior cranial nerve cultures Experiments were conducted to further investigate the timing of NOTCH inhibitor addition. The experiments were carried out along the line in Example 2. DIV18 cultures were left without neuronal / ectoderm patterning factors and were left at a high cell density (0.5 × 10⁶). 6 cells / cm 2 ~2.0×10 6 cells / cm 2 Further differentiation occurred in vitro for a period of 7 days (DIV18-25). The DIV25 culture was profiled by flow cytometry to confirm correct patterning for dorsal forebrain identity (Figure 21, Table 15). In DIV25, a NOTCH inhibitor was added for 1-2 days prior to cryopreservation (Figure 20). At any point prior to this (in vitro days 0-25), the cell culture was not exposed to any NOTCH inhibitor. Cells in DIV27 were later thawed and analyzed using flow cytometry to confirm that the addition of a NOTCH inhibitor promoted the maturation of dorsal FB neurons into neurons (Figure 22 and Table 16). This experiment confirms that the addition of a NOTCH inhibitor in DIV25 is as good as the addition of a NOTCH inhibitor in DIV30. [Table 15] [Table 16]

[0174] Example 17: Administration of NOTCH inhibitor LY411575 to ventral midbrain nerve cultures Experiments were conducted to further investigate the timing of NOTCH inhibitor addition. The experiments were carried out along the line in Example 4. DIV16 cultures were left without neuronal / ectoderm patterning factors and were left at a high cell density (0.5 × 10⁶). 6 cells / cm 2 ~2.0×10 6 cells / cm 2 Further differentiation occurred in vitro for a period of 6 days (DIV16-22). DIV22 cultures were profiled by flow cytometry to confirm correct patterning for dorsal forebrain identity (Figure 10 and Table 6). In DIV22, a NOTCH inhibitor was added for 1-2 days prior to cryopreservation (Figure 23). At any point prior to this (in vitro days 0-22), the cell cultures were not exposed to any NOTCH inhibitor. Cells in DIV24 were later thawed and analyzed using flow cytometry to confirm that the addition of a NOTCH inhibitor promoted the maturation of ventral MB neurons into neurons (Figure 24 and Table 17). This experiment confirms that the NOTCH inhibitor of the present invention can be added in high-density cell cultures. [Table 17]

Claims

1. A method for directing the differentiation of nerve cells into neurons, comprising obtaining a cell population containing nerve cells and contacting the cell population with a NOTCH signaling inhibitor, wherein the NOTCH signaling inhibitor is selected from LY411575, abagacestat, dibenzazepine, and PF-03084014.

2. The method according to claim 1, wherein the NOTCH signaling inhibitor is LY411575.

3. The method according to any one of claims 1 to 2, wherein the cell population including nerve cells is obtained by differentiating pluripotent stem cells (PSCs) into nerve cells.

4. The method according to any one of claims 1 to 3, wherein the cell population, including nerve cells, is obtained by differentiating pluripotent stem cells (PSCs) into nerve cells over a period of 14 to 35 days.

5. The method according to any one of claims 1 to 4, wherein the nerve cells are specific to a region selected from the forebrain, midbrain, and hindbrain / spinal cord.

6. The method according to claim 5, wherein the cell population, including nerve cells, is obtained by differentiating pluripotent stem cells (PSCs) into nerve cells over a period of 14 to 26 days, and the nerve cells are differentiated into nerve cells specific to the midbrain region, preferably the ventral midbrain region, and the cell population is not in contact with the NOTCH signaling inhibitor for at least 20 days after the cell population begins differentiation into nerve cells.

7. The method according to claim 6, wherein the cell population, including nerve cells, is obtained by differentiating pluripotent stem cells (PSCs) into nerve cells for 18 to 35 days, and the nerve cells are differentiated into nerve cells specific to the forebrain region, preferably the dorsal forebrain region, and the cell population is not in contact with the NOTCH signaling inhibitor for at least 24 days after the cell population begins to differentiate into nerve cells.

8. The method according to any one of claims 1 to 7, wherein at least 70% of the cell population express SOX2, and 5 to 35% of the cell population are INA+ at the time the cell population is brought into contact with the NOTCH signaling inhibitor.

9. The method according to any one of claims 1 to 8, wherein the nerve cells are specific to the ventral midbrain region, and 20 to 75% of the cell population are ASCL1+ at the time the cell population is brought into contact with the NOTCH signaling inhibitor.

10. The method according to any one of claims 1 to 7, wherein the nerve cells are specific to the forebrain region, and 5 to 40% of the cell population are TBR2+ at the time the cell population is brought into contact with the NOTCH signaling inhibitor.

11. The method according to any one of claims 1 to 10, wherein the cell population is contacted with the NOTCH signaling inhibitor for 12 hours to 2 days.

12. The method according to any one of claims 1 to 11, wherein the concentration of the NOTCH signaling inhibitor is 0.2 μM to 1000 μM.

13. The method according to any one of claims 1 to 12, wherein the cell population is brought into contact with the NOTCH inhibitor 48 hours before cryopreservation.

14. At the point when the cell population is brought into contact with the NOTCH signaling inhibitor, 0.5 × 10 ６ cells / cm ２ ~2.0 x 10 ６ cells / cm ２ The method according to any one of claims 1 to 13, having the density of

15. An in vitro cell population comprising nerve cells obtained by the method according to any one of claims 1 to 14, or a composition comprising the in vitro cell population comprising nerve cells, wherein the nerve cells are specific to a region selected from the forebrain, midbrain, and hindbrain / spinal cord.

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