Enhancing neuronal differentiation of neural progenitor cells

Abstract

The present invention relates to a method for directing differentiation of neural cells into neurons by obtaining a cell population comprising neural cells and contacting the cell population with an inhibitor of NOTCH signaling, wherein the inhibitor of NOTCH signaling is selected from LY411575, Avagacestat, Dibenzazepine, and PF-03084014.

Description

[0001] ENHANCING NEURONAL DIFFERENTIATION OF NEURAL PROGENITOR CELLS

[0002] TECHNICAL FIELD

[0003] The present invention relates generally to the field of stem cells, such as human embryonic stem cells. Methods are provided for obtaining stem cell-derived neural cells. Specifically, methods are provided for directing differentiation of stem cell-derived neural cells into neurons of the forebrain, midbrain and hindbrain / spinal cord region.

[0004] BACKGROUND

[0005] The prospect of using human pluripotent stem cells (hPSCs) in the treatment of various conditions seems very promising. Treatments include cell-replacement therapy of neurological conditions such as Parkinson’s disease, stroke, paralysis, epilepsy and other such disorders. For such treatments to become viable, however, it requires the development of in vitro methods to artificially produce the stem cell-derived products for their delivery to the central nervous system (CNS). The differentiation of hPSCs into defined cell types is a difficult process to control; in many cases the progeny generated from in vitro protocols are heterogenous. Typically, when differentiating into neural cells, the mixture of cell types produced includes neurons (and within this a variety of neuronal subtypes such as glutamatergic and dopaminergic neurons), glia, neural stem cells (NSCs) as well as other non-neuronal cells (e.g., meningeal stromal cells). Such heterogenous cultures are suboptimal for disease modelling studies or many cell replacement therapies.

[0006] Cellular replacement therapy for Parkinson’s disease is a prime example. In Parkinson’s disease, the A9 ventral midbrain dopaminergic neurons (vmDAs) are lost and arguably the sole cell type that must be transplanted to restore lost function. Presently, however, all academic scientific publications and all human clinical trials underway are transplanting a multipotent progenitor population that results in mixed populations of cells in vivo, in addition to vmDAs. 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 behaviors in patients as seen in clinical trials transplanting human fetal cells, and more generally these non-vmDAs carry unknown safety and efficacy risks. Consequently, there is a need to provide methods which ensure that patients are treated with ventral midbrain neurons or progenitors thereof.

[0007] In the case of stroke or paralysis due to damage of the spinal cord, it is likely that only specific neuronal regional identity cell types and specific subtypes of neurons are required to restore lost function in both cases, while glial cells, stromal cells or other types of neurons are not necessary. As such for these indications it is also valuable to have maximal control over a differentiation process to have a refined cell product for maximal efficacy and safety.

[0008] It is therefore an object of the present invention to overcome the aforementioned challenges, in particular to provide methods which can direct the differentiation of neural cells into neurons.

[0009] SUMMARY

[0010] The object as outlined above is achieved by the aspects of the present invention. In addition, the present invention may also solve further problems, which will be apparent from the disclosure of the exemplary embodiments.

[0011] In a first aspect the present invention has provided a method comprising obtaining a cell population comprising neural cells and contacting the cell population with an inhibitor of NOTCH signaling, wherein the inhibitor of NOTCH signaling is selected from LY411575, Avagacestat, Dibenzazepine, and PF-03084014, preferably LY411575. Specifically, the method is for directing differentiation of neural cells into neurons, meaning that the developmental fate of the neural cells is affected towards a certain outcome, i.e., neurons. However, according to the method the differentiation into neurons is not necessarily completed and the cells may not necessarily develop into the final fate during the method as disclosed herein. Preferably, the cells are directed towards differentiation towards neurons in vitro, only to be suitable for later administration into a patient wherein the further development in vivo results in a neuronal fate. The present inventors have found that the outcome of protocol for differentiating a cell population, such as human PSCs, towards or into neurons can be improved with the inhibition of the NOTCH signaling pathway using alternative compounds than usually used in the field of neural differentiation, namely the small molecule DAPT. The present invention has surprisingly found that the compound LY411575 demonstrates improved upregulation of neuronal genes and downregulation of NPC genes during the differentiation compared to DAPT. The present inventors have further identified the alternative compounds Avagacestat, Dibenzazepine, and PF-03084014 as having similar abilities. Specifically, this inhibition thus further reduces the proportion of latestage proliferative cells (which represents off-target lineages), reduces the proportion of stromal cells, such as VLMCs, and glial cells, such as astrocytes, and at the same time increases the proportion of neurons, when the neural cells are further differentiated towards a terminal fate. The present inventors have found the above-mentioned compounds are superior compared to the use of e.g., DAPT. After 24 hours of administration cells treated with any of the compounds produce more neurons (HuC / D or INA positive cells) and are less proliferative (SOX2 and / or Ki67 positive cells) as compared to similar treatment with DAPT.

[0012] Importantly, the present inventors have found that the timing of inhibiting the NOTCH signaling relative to the developmental stage of the cells is influential on the outcome. In an embodiment, the cell population is obtained by differentiating pluripotent stem cells (PSCs) into neural cells, such as by neural induction. A person skilled in the art will recognize that the cells undergo a series of developmental stages, initially becoming neural stem cells, then progressing into neuroblast intermediate precursor cells, towards neuronal identity. Specifically, the present inventors have found a window of opportunity during the neural cell development, in which timeframe during development the neural cells are particularly susceptible for being directed into a neuronal fate. In a preferred embodiment, more than 2%, such as more than 5%, 10%, 15%, 20%, or 25%, of the cell population are INA+ / SOX2-, at the time of contacting the cell population with the inhibitor of NOTCH signaling.

[0013] In a preferred embodiment, the cell population of PSCs is neurally induced according to well-known methods, such as exposing the PSCs to an inhibitor of SMAD protein signaling, optionally targeting both the TGF-beta and BMP pathways. The present inventors also recognize that culturing the cell population of neurally induced cells for too long closes this window of opportunity. In the aforementioned method of neurally inducing PSCs, it has been found that a cell population cultured for beyond a certain threshold of INA+ / SOX2- makes it difficult to harvest, i.e., due to formation of an inseparable mesh of neurites.

[0014] An in vitro cell population obtained according to the method may be used for the treatment of neurological conditions, such as Parkinson’s disease, and may provide reduced surgical procedure time and cranial injections due to a higher purity cell product. In addition, improved product purity and reduced impurities provide an enhanced safety and potentially efficacy profile.

[0015] BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 : shows a schematic representation of the developing brain in a sagittal section. Highlighted in the viewfinder is the dorsal forebrain region that is generated with the differentiation protocol described in Figure 2 and Figure 20.

[0017] Figure 2: shows a simplified schematic illustrating the stages of dorsal forebrain differentiation. The procedure begins with hPSCs (DIV0) that are differentiated to dorsal FB NSCs cells following neural induction, and the absence of ventralization and caudalization cues. Cells were profiled via flow cytometry analysis at DIV18 (Figure 3) and DIV30 (Figure 4) of the differentiation protocol (represented by the asterisks). Administration of compound of the invention, NOTCH inhibitor LY411575, occurred at DIV30 of the differentiation (black arrow).

[0018] Figure 3: shows a bar graph of the results of intracellular flow cytometry protein analysis of Dorsal FB cell cultures at DIV18. Expression is shown for the NSC marker SOX2, for FB lineage markers OTX2 and PAX6, as well as FB I PC marker TBR2 and neuronal marker INA, described as INA+ / SOX2- population. The graph displays the percentage of cells of the total viable cells. The level of these markers shows these cells are NSCs of the dorsal forebrain.

[0019] Figure 4: shows a bar graph of the results of intracellular flow cytometry protein analysis of cell cultures at DIV30. Expression is shown for the NSC marker SOX2 and proliferative marker KI67. Expression is also shown for FB lineage markers OTX2 and PAX6, as well as neuronal marker INA, described as INA+ / SOX2- population. The graph displays the percentage of cells of the total viable cells. The level of these markers shows these cells are neural cells of the dorsal forebrain.

[0020] Figure 5: shows a bar graph of the results of ICC analysis of cell cultures at day in vitro 35 after they have been contacted with NOTCH inhibitor LY411575 for 24h from DIV30 to DIV31. Results are shown of cultures with no intervention (Control, white bars) and with administration of LY411575 for 24h (black bars). Protein expression is shown for the NSC marker SOX2 and proliferative marker KI67, and neuronal marker HuC / D. The graph displays the percentage of cells of the total cells.

[0021] Figure 6: shows a bar graph of the results of ICC analysis of cell cultures at day in vitro 35 after they have being contacted with NOTCH inhibitor LY411575 for 72h from DIV30 to DIV33. Results are shown of cultures with no intervention (Control, white bars) and with administration of LY411575 for 72h (black bars), Protein expression is shown for the NSC marker SOX2 and proliferative marker KI67, and neuronal marker HuC / D. The graph displays the percentage of cells of the total cells.

[0022] Figure 7: shows a schematic representation of the developing brain in a sagittal section. Highlighted in the viewfinder is the ventral midbrain region that is generated with the differentiation protocol shown in Figure 8 and Figure 23.

[0023] Figure 8: shows a simplified schematic illustrating the stages of ventral MB differentiation. The procedure begins with hPSCs (DIVO) that are differentiated to ventral MB NSCs following neural induction, ventralization and caudalization. Profile of the cells via flow cytometry analysis was performed at DIV16 (Figure 9) and DIV22 (Figure 10) of the differentiation protocol (represented by the asterisks). Compound of the invention, NOTCH inhibitor LY411575, is administered at DIV22 of the differentiation protocol (black arrow).

[0024] Figure 9: shows a bar graph of the results of intracellular flow cytometry protein analysis of cell cultures at DIV16. Expression is shown for the NSC marker SOX2 and proliferative marker KI67. Expression is also shown for ventral MB and floor plate lineage markers FOXA2, LMX1A, EN1 and OTX2, as well as the MB IPC marker ASCL1 and neuronal marker INA, shown as INA and as INA+ / SOX2- population. The graph displays the percentage of cells of the total viable cells.

[0025] Figure 10: shows a bar graph of the results of intracellular flow cytometry protein analysis of ventral MB cell cultures at DIV22, when the administration of NOTCH inhibitor LY411575 begins. Expression is shown for the NSC marker SOX2 and proliferative marker KI67. Expression is also shown for floor plate lineage marker FOXA2 as well as the MB IPC marker ASCL1 and neuronal marker INA, shown as INA and as INA+ / SOX2- population. The graph displays the percentage of cells of the total viable cells.

[0026] Figure 11 : shows a bar graph of the results of ICC analysis of cell cultures at day in vitro 29-30 after LY411575 administration for 24h from DIV22 to DIV23. Results are shown of cultures with no intervention (Control, white bars) and administration of NOTCH inhibitor LY411575 (black bars). Protein expression is shown for the NSC marker SOX2 and proliferative marker KI67, neuronal marker HuC / D and VM floor plate lineage marker FOXA2. The graph displays the percentage of cells of the total cells.

[0027] Figure 12: shows a bar graph of the results of ICC analysis of cell cultures at day in vitro 29-30 after LY411575 administration for 72-96h. Results are shown of cultures with no intervention (Control, white bars) and the administration of NOTCH inhibitor LY411575 (black bars). Protein expression is shown for the NSC marker SOX2 and proliferative marker KI67, neuronal marker HuC / D and floor plate lineage marker FOXA2. The graph displays the percentage of cells of the total cells.

[0028] Figure 13: shows a bar graph of the results of ICC analysis of ventral MB cell cultures at day in vitro 29 after administration of different NOTCH inhibitors for 24h (from DIV22 to DIV23). Results are shown of cultures with no intervention (Control, white bars), administration of DAPT (dotted bar), Avagacestat (narrow diagonal striped bars), PF-03084014 (bold diagonal striped bars), and LY411575 (black bars). Protein expression is shown for the NSC marker SOX2 and proliferative marker KI67, neuronal marker HuC / D and floor plate lineage marker FOXA2. The graph displays the percentage of cells of the total cells. Figure 14: shows a schematic representation of the developing brain in a sagittal section. Highlighted in the viewfinder is the Hindbrain / Spinal Cord region that is generated with the differentiation protocol described in Figure 15.

[0029] Figure 15: shows a simplified schematic illustrating the stages of hindbrain / spinal cord differentiation and the experiment overview. The procedure begins with hPSCs (DIVO) that are differentiated to HB / SCord NSCs following neural induction, caudalization and ventralization. Cells were profiled via flow cytometry analysis at DIV15 (Figure 16) of the differentiation protocol (asterisk). Compound of the invention, NOTCH inhibitor LY411575, is administered at DIV15 as indicated by the black arrow in the schematic.

[0030] Figure 16: shows a bar graph of the results of intracellular flow cytometry protein analysis of HB / SCord cell cultures at DIV15, when the administration of NOTCH inhibitor LY411575 begins. Expression is shown for the NSC marker SOX2, HB / SCord progenitor domain markers NKX6.1 , OLIG2, NKX2.2 and PAX6, as well as off target markers identifying ventral MB and floor plate lineage markers OTX2 and FOXA2 that together shows these cells are of the ventral-medial hindbrain / spinal cord lineage.

[0031] Figure 17: shows a bar graph of the results of ICC analysis of cell cultures at day in vitro 20 after LY411575 administration for 24h from DIV15 to DIV16. Results are shown of cultures with no intervention (Control, white bars) and administration of LY411575 (black bars). Protein expression is shown for the NSC marker SOX2 and proliferative marker KI67, and neuronal marker HuC / D. The graph displays the percentage of cells of the total cells.

[0032] Figure 18: shows a bar graph of the results of ICC analysis of cell cultures at day in vitro 20 after LY411575 administration for 72h from DIV15 to DIV18. Results are shown of cultures with no intervention (Control, white bars) and administration of LY411575 (black bars). Protein expression is shown for the NSC marker SOX2 and proliferative marker KI67, and neuronal marker HuC / D. The graph displays the percentage of cells of the total cells.

[0033] Figure 19: shows a bar graph of the results of ICC analysis of ventral MB cultures at day in vitro 29 after administration of NOTCH inhibitor LY411575 at different concentrations for 24h from DIV22 to DIV23. Results are shown of cultures with no intervention (Control, white bars) and administration of 0.2uM LY411575 (dotted bars), 0.5uM LY411575 (narrow striped bars), 2uM LY411575 (bold angled striped bars), 10uM LY411575 (horizontal striped bars), 40uM LY411575 (bold angled striped bars) and 100uM LY411575 (black bars). Protein expression is shown for the NSC marker SOX2 and proliferative marker KI67, Neuronal marker HuC / D and VM floor plate marker FOXA2. The graph displays the percentage of cells of the total cells. Figure 20: shows a simplified schematic illustrating the stages of dorsal forebrain differentiation. The procedure begins with hPSCs (DIVO) that are differentiated to dorsal FB NSCs cells following neural induction, and the absence of ventralization and caudalization cues. Cells were profiled via flow cytometry analysis at DIV18, DIV25, and DIV27, and immunocytochemistry at DIV30 of the differentiation protocol (represented by the graph icon). Administration of compound of the invention, NOTCH inhibitor LY411575, occurred at DIV25 of the differentiation (black arrow). Cells were cryopreserved at DIV27 (represented by asterix).

[0034] Figure 21 : shows a bar graph of the results of intracellular flow cytometry protein analysis of dorsal FB cell cultures at DIV25. Expression is shown for the NSC marker SOX2, the proliferative marker Ki67, for FB lineage markers OTX2, SOX1 and PAX6, as well as cortical / dorsal FB I PC marker TBR2 and neuronal marker INA, described as INA and INA+ / SOX2- population. The graph displays the percentage of cells of the total viable cells. The level of these markers shows these cells are neural cells of the dorsal forebrain.

[0035] Figure 22: shows a bar graph of the results of flow cytometry analysis of dorsal FB cell cultures at day in vitro 27 after administration of NOTCH inhibitor LY411575 for 24h or 48h (from DIV25 / DIV26 to DIV27). Results are shown of cultures with no intervention (Control, white bars), administration of LY411575 for 24 hours (diagonal striped bars) and administration of LY411575 for 48 hours (black bars). Protein expression is shown for the NSC marker SOX2 and proliferative marker KI67, neuronal marker HuC / D, shown as HuC / D or HuCD+ / SOX2- and cortical I PC marker TBR2. The graph displays the percentage of cells of the total viable cells.

[0036] Figure 23: shows a simplified schematic illustrating the stages of ventral MB differentiation. The procedure begins with hPSCs (DIVO) that are differentiated to ventral MB NSCs following neural induction, ventralization and caudalization. Profile of the cells via flow cytometry analysis was performed at DIV16, DIV22 and DIV24, and immunocytochemistry at DI 36 of the differentiation protocol (represented by the graph icon). Compound of the invention, NOTCH inhibitor LY411575, is administered at DIV22 of the differentiation protocol (black arrow). Cells were cryopreserved at DIV24 (represented by asterix).

[0037] Figure 24: shows a bar graph of the results of flow cytometry analysis of ventral MB cell cultures at day in vitro 24 after administration of NOTCH inhibitor LY411575 for 24h or 48h (from DIV22 / DIV23 to DIV24). Results are shown of cultures with no intervention (Control, white bars), administration of LY411575 for 24 hours (diagonal striped bars) and administration of LY411575 for 48 hours (black bars). Protein expression is shown for the NSC marker SOX2 and proliferative marker KI67, neuronal marker HuC / D, shown as HuC / D or HuCD+ / SOX2- and cortical I PC marker TBR2, and floor plate marker FOXA2. The graph displays the percentage of cells of the total viable cells.

[0038] DESCRIPTION

[0039] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The practice of the present invention employs, unless otherwise indicated, conventional methods of chemistry, biochemistry, biophysics, molecular biology, cell biology, genetics, immunology and pharmacology, known to those skilled in the art.

[0040] It is noted that all headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way.

[0041] The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0042] Definitions

[0043] General definitions

[0044] As used herein, “a” or “an” or “the” can mean one or more than one. Unless otherwise indicated in the specification, terms presented in singular form also include the plural situation.

[0045] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”). Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted.

[0046] Stem cells

[0047] By “stem cell” is to be understood an undifferentiated cell having differentiation potency and proliferative capacity (particularly self-renewal competence) but maintaining differentiation potency. The stem cell includes categories such as pluripotent stem cell, multipotent stem cell, unipotent stem cell and the like according to their differentiation potentiality.

[0048] As used herein, the term “pluripotent stem cell” (PSC) refers to a stem cell capable of being cultured in vitro and having a potency to differentiate into any cell lineage belonging to the three germ layers (ectoderm, mesoderm, endoderm), as well as lineage restricted undifferentiated stem cells that have lost the capacity to form some cell type(s), lineage(s) or developmental region(s) typically through genetic editing.

[0049] A pluripotent stem cell can be induced or isolated from a fertilized egg, somatic nuclear transfer embryo, germ stem cell, stem cell in a tissue, somatic cell and the like. Examples of the pluripotent stem cell (PSC) include embryonic stem cell (ESC), embryonic germ cell (EG cell), induced pluripotent stem cell (iPSC) and the like.

[0050] As used herein, the term “induced pluripotent stem cell” (also known as iPS cells or iPSCs) means a type of pluripotent stem cell that can be generated directly from cells that are not PSCs and have a nucleus. By the introduction of products of specific sets of pluripotency-associated genes non-pluripotent cells can be converted into pluripotent stem cells.

[0051] As used herein, the term “embryonic stem cell” means a pluripotent stem cell derived from the inner cell mass of a blastocyst. Pluripotent embryonic stem cells may also be derived from parthenotes as described in e.g., WO 2003 / 046141. Additionally, embryonic stem cells can be produced from a single blastomere or by culturing an inner cell mass obtained without the destruction of the embryo. Embryonic stem cells are available from given organizations and are also commercially available. Preferably, the methods and products of the present invention are based on human PSCs, i.e., stem cells derived from either human induced pluripotent stem cells or human embryonic stem cells, including parthenotes.

[0052] As used herein, the term “multipotent stem cell” means a stem cell having a potency to differentiate into plural types of tissues or cells, though not all kinds and is typically restricted to one germ layer. A neural stem cell is an example of a multipotent stem cell restricted to the neural lineage.

[0053] As used herein, the term “unipotent stem cell” means a stem cell having a potency to differentiate into only one particular cell type.

[0054] As used herein, the term “in vitro" means that the cells are provided and maintained outside of the human or animal body, such as in a vessel like a flask, multiwell or petri dish. It follows that the cells are cultured in a cell culturing medium.

[0055] As used herein, the term “non-native” means that the cells although derived from pluripotent stem cells, which may have human origin, is an artificial construct, that does not exist in nature. In general, it is an object within the field of stem cell therapy to provide cells, which resemble the cells of the human body as much as possible. However, it may never become possible to mimic the development which the pluripotent stem cells undergo during the embryonic and fetal stage to such an extent that the mature cells are indistinguishable from native cells of the human body. Inherently, in an embodiment of the present invention, the cells are artificial.

[0056] As used herein, the term “artificial” in reference to cells may comprise material naturally occurring in nature but modified to a construct not naturally occurring. This includes human stem cells, which are differentiated into non-naturally occurring cells mimicking the cells of the human body.

[0057] Protocol

[0058] Throughout this application the terms “method” and “protocol”, when referring to processes for culturing or differentiating cells, may be used interchangeably.

[0059] As used herein, the term “day” and similarly day in vitro (DIV) in reference to the protocols refers to a specific time for carrying out certain steps during the differentiation procedure.

[0060] In general, and unless otherwise stated, “day 0” refers to the initiation of the protocol, this is by for example, but not limited to, plating the stem cells or transferring the stem cells to an incubator or contacting the stem cells in their current cell culture medium with a compound prior to transfer of the stem cells. Typically, the initiation of the protocol will be by transferring undifferentiated stem cells to a different cell culture medium and / or container such as, but not limited to, by plating or incubating, and / or with the first contacting of the undifferentiated stem cells with a compound or compounds that affects the undifferentiated stem cells in such a way that a differentiation process is initiated.

[0061] When referring to “the cells” in a method is meant all cells of the cell population, regardless of cell type.

[0062] When referring to “day X”, such as day 1 , day 2 etc., it is relative to the initiation of the protocol at day 0. One of ordinary skill in the art will recognize that unless otherwise specified the exact time of the day for carrying out the step may vary. Accordingly, “day X” is meant to encompass a time span such as of + / -10 hours, + / -8 hours, + / -6 hours, + / -4 hours, +1-2 hours, or + / -1 hours.

[0063] Culturing stem cells

[0064] As used herein, the term “culturing” refers to a continuous procedure, which is employed throughout the method in order to maintain the viability of the cells at their various stages. After the cells of interest have been isolated from, for example but not limited to, living tissue or embryo, they are subsequently maintained under carefully controlled conditions. These conditions vary for each cell type, but generally consist of a suitable vessel with a substrate and / or medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals), growth factors, hormones, and gases (CO2, O2), and regulates the physio-chemical environment (pH buffer, osmotic pressure, temperature).

[0065] As used herein, the term “cell culture medium” refers to a liquid or gel designed to support the growth of cells. Cell culture media generally comprise an appropriate source of energy and compounds which regulate the cell cycle.

[0066] As used herein, the term “incubator” refers to any suitable incubator that may support a cell culture. Non-limiting examples include culture dish, petri dish and plate (microtiter plate, microplate, deep well plate etc. of 6 well, 24 well, 48 well, 96 well, 384 well, 9600 well and the like), flask, chamber slide, tube, Cell Factory, roller bottle, spinner flask, hollow fiber, microcarrier, or bead.

[0067] As used herein, the term “providing stem cells” when referred to in a protocol means obtaining a batch of cells by methods such as described above and optionally transferring the cells into a different environment such as by seeding onto a new substrate. One of ordinary skill in the art will readily recognize that stem cells are fragile to such transfer and the procedure requires diligence and that maintaining the stem cells in the origin cell culture medium may facilitate a more sustainable transfer of the cells before replacing a cell culture medium with another cell culture medium more suitable for a further differentiation process.

[0068] Differentiating stem cells

[0069] As used herein, the term “expressing” in relation to a gene or protein refers to the presence of an RNA molecule, which can be detected using assays such as reverse transcription quantitative polymerase chain reaction (RT-qPCR), RNA sequencing and the like, and / or a protein, which can be detected for example using antibody-based assays such as flow cytometry, immunocytochemistry / immunofluorescence, and the like. Depending on the sensitivity and specificity of the assay, a gene or protein may be considered expressed when a minimum of one molecule is detected such as in RNA sequencing, or the limit of detection above background / noise levels may be defined in relation to control samples such as in flow cytometry. A person skilled in the art will readily understand that when referring to the “expression” of a cell population, such as “the cell population expressing X% of a marker Y”, is meant that X% of cells in said cell population express the marker Y.

[0070] As used herein, the term “marker” refers to a naturally occurring identifiable expression made by a cell, which can be correlated with certain properties of the cell. In a preferred embodiment the marker is a genetic or proteomic expression, which can be detected and correlated with the identity of the cell. The markers may be referred to by gene. This can readily be translated into the expression of the corresponding mRNA and proteins.

[0071] As used herein, the term “negative” or when used in reference to any marker such as a surface protein or transcription factor disclosed herein refers to the marker not being expressed in a cell or a population of cells, while the term “weak” or “low” refers to the marker being expressed at a reduced level in a cell as compared to the mean expression of the marker in a population of cells or as compared to a reference sample.

[0072] As used herein, the term “positive” or “+“ when used in reference to any marker such as a surface protein or transcription factor disclosed herein refers to the marker being expressed in a cell or a population of cells, while the term “high” or “strong” refers to the marker being expressed at an increased level in a cell as compared to the mean expression of the marker in a population of cells or as compared to a reference sample.

[0073] As used herein, the annotation INA+ / SOX2- refers to the situation where a given cell is identified by more than one marker, such as namely a cell being both INA+ and SOX2-.

[0074] As used herein, the term “differentiation” refers broadly to the process wherein cells progress from an undifferentiated state or a state different from the intended differentiated state to a specific differentiated state, e.g., from an immature state to a less immature state or from an immature state to a mature state, which may occur continuously as the method is performed. The term “differentiation” in respect to pluripotent stem cells refers to the process wherein cells progress from an undifferentiated state to a specific differentiated state, i.e. , from an immature state to a less immature state or to a terminal state. Changes in cell interaction and maturation occur as cells lose markers of undifferentiated cells or gain markers of differentiated cells. Loss or gain of a single marker can indicate that a cell has “fully differentiated” or “terminally differentiated”. “Terminally differentiated” cells are the final stage of a developmental lineage and cannot further differentiate.

[0075] As used herein, by the term “contacting” in reference to culturing or differentiating cells is meant exposing the cells to e.g., a specific compound by placing the specific compound in a location that will allow it to touch the cell in order to produce “contacted” cells. The contacting may be accomplished using any suitable means. A non-limiting example of contacting is by adding the compound to a cell culture medium of the cells. The contacting of the cells is assumed to occur as long as the cells and specific compound are in proximity, e.g., the compound is present in a suitable concentration in the cell culture medium.

[0076] As used herein, the term “inhibitor” refers to a compound that reduces or suppresses or down-regulates a process, such as a signaling pathway which can promote cell differentiation. As used herein, the term “activator” refers to a compound that induces or stimulates or up-regulates a process, such as a signaling pathway which can promote cell differentiation.

[0077] Stem cell-derived products

[0078] As used herein, the term “differentiated cells” refers to cells such as pluripotent stem cells which have progressed from an undifferentiated state to a less immature state. Differentiated cells may be e.g., less immature specialized cell such as progenitor cells or matured fully into a specialized / terminal cell type.

[0079] As used herein, the term “cell population” refers to a plurality of cells in the same culture. The cell population may be e.g., a mixture of cells of different types, or cells at various developmental stages such as cells at various maturity stages towards the same or similar specialized feature or it may be a more homogeneous composition of cells with common markers.

[0080] As used herein, the term “neural cell population” refers to a cell population comprising neural cells.

[0081] As used herein, the terms “genetically modified” and “genetically engineered” in reference to a cell may be used interchangeably and refer to a cell which has been subjected to an artificial manipulation, modification, or recombination of DNA or other nucleic acid molecules in order to alter the characteristics (phenotype) of that cell. Such a cell can no longer be considered a naturally occurring cell. In case of genetically modified stem cells the traits resulting from the gene editing persist even as the stem cell is further differentiated into a specialized cell, thus rendering the specialized cell genetically modified and artificial, i.e. , non-naturally occurring. An example of genetically modified stem cells are HLA-deficient stem cells, which are also referred to as universal donor cells and intended to overcome the problem of graft rejection. A method for obtaining HLA-deficient stem cells is disclosed in WO / 2020 / 260563.

[0082] Neuroectodermal cells

[0083] As used herein, the term “neural” refers to the nervous system.

[0084] As used herein, the term “neural cell” (unless otherwise stated) refers to a cell, where the native counterpart naturally forms part of the ectoderm germ layer, more specifically the neuroectoderm and is meant to encompass cells at any stage of development within this germ layer, such as NSCs all the way through to neurons and other terminally differentiated cell types (e.g., glial cells), i.e., cell stages such as neural stem cell stage and neuroblast stage. Accordingly, neurons and precursors thereof are considered specific types of neural cells.

[0085] As used herein, the terms “neuron”, “neuronal” and “nerve cell” may be used interchangeably referring to neural cells which are post-mitotic and have terminally differentiated into a specialized cell. A neuron is characterized by expression of the marker INA, or other equivalent markers such as ELAVL3, ELAVL4 (typically detected with the antibody HuC / D, also known as HuCD), RBFOX3 (typically detected with the antibody NeuN), STMN2, NCMA1 or other such broad neuronal markers.

[0086] As used herein, the term “neural stem cell” or “NSC” and “neural precursor cell” or “NPC” are terms that are used interchangeably to refer to a self-renewing (although typically with limits) multipotent cell of the nervous system capable of giving rise to a vast array of 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 and proliferative / cell cycle genes such as KI67 (also known as MKI67) and TOP2A, CDK1 , MCM2, MCM4 and PCNA.

[0087] As used herein, the term “intermediate precursor cell” or “I PC” refers to a cell which is sometimes multipotent or typically bi-potent or unipotent and can self-renew only to a limited extent, typically dividing asymmetrically. An intermediate precursor cell finally gives rise to terminally differentiated cell types such as neurons. The terms “neuroblast cell” and “intermediate precursor cell” and “intermediate progenitor cell” and “radial glial cell” may be used interchangeably.

[0088] As used herein, the term “neuroblast” or “intermediate precursor cell” means a cell that has expressed 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), any of the NEUROD or NEUROG gene family or other such genes. These cells are those which are destined to become neurons.

[0089] As used herein, the terms “neuron progenitor”, “precursor of a neuron” and “neuron precursor” may be used interchangeably and refer to a neural cell with the potential or propensity to further specialize into a neuron. The terms “neuron precursor” and “non-native neuron precursor” may be used interchangeably.

[0090] The neural cells according to the present invention may have a specific regional identity, such as cells specific to the forebrain, midbrain, hindbrain, spinal cord etc.

[0091] 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 the striatum. As used herein, the term “dorsal forebrain” in reference to a cell means a neural cell having certain properties of a neural cell naturally occurring in the dorsal forebrain. Typically, dorsal forebrain neural cells 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 a neural stem cell having the characteristics of a neural stem cell naturally occurring in the dorsal forebrain. A dorsal forebrain neural precursor cell may be characterized by the co-expression of two or more markers from SOX1, PAX6, OTX2, FOXG1 , EMX1, EMX2 and SOX2.

[0092] As used herein, the term “midbrain” refers to the medial region of the neural tube and CNS (on the rostro-caudal axis) that gives rise to structures including the substantia nigra.

[0093] As used herein, the term “ventral midbrain” in reference to a cell means a neural cell having certain properties of a neural cell naturally occurring in the ventral midbrain. Typically, ventral midbrain neural cells are characterized by the expression of certain markers such as FOXA2 and LMX1A. Specifically, as used herein the term “ventral midbrain neural stem cell” refers to a neural stem cell having the characteristics of a neural stem cell naturally occurring in the ventral midbrain. A ventral midbrain neural precursor cell may be characterized by the co-expression of two or more markers from FOXA1, FOXA2, LMX1A, LMX1B, EN1, OTX2, and SOX2.

[0094] As used herein, the terms “hindbrain” and “spinal cord” refer to the caudal regions of the neural tube that are caudal to the isthmus organizer.

[0095] As used herein, the term “glutamatergic cell” or “glutamatergic neuron” or “glutamate neuron” refers to a cell that is capable of synthesizing the neurotransmitter glutamate.

[0096] As used herein, the term “dopaminergic (DA) cell” or “dopaminergic neuron” or “dopamine neuron” refers to a cell that is capable of synthesizing the neurotransmitter dopamine.

[0097] As used herein, the term “stromal cell” refers to cells having the capacity to become connective tissue cells or cells of a fibroblast identity.

[0098] As used herein, the term “VLMC” means vascular leptomeningeal cells and is consider a type of stromal cell resident in the CNS.

[0099] As used herein, the term “glial cell” refers to cells that are non-neuronal cells in the central nervous system (brain and spinal cord) and the peripheral nervous system that do not produce electrical impulses, that perform support and protection for neurons. Examples include astrocytes and oligodendrocytes and their precursors, glial precursor cells or glioblasts.

[0100] Directing differentiation into neurons

[0101] In a general aspect of the present invention is provided a method comprising obtaining a cell population comprising neural cells and contacting the cell population with an inhibitor of NOTCH signaling, wherein the inhibitor of NOTCH signaling is selected from LY411575, Avagacestat, Dibenzazepine, and PF-03084014. As used herein, the term “NOTCH” refers to the signaling pathway of the notch receptors. As used herein, “LY411575” refers to a compound having CAS No. 209984-57-6. The molecular formula of LY411575 is C26H23F2N3O4 while the chemical structure is sometimes as shown below (Chem. 1).

[0102] Chem. 1

[0103] The chemical name for LY411575 is sometimes, N-2((2S)-2-(3,5-difluorophenyl)-2- hydroxyethanoyl)-N1-((7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d] azepin-7-yL)-l- alaninamid or alternatively (2S)-2-(3,5-difluorophenyl)-2-hydroxyacetyl]amino]-N-[(7S)-5- methyl-6-oxo-7Hbenzo[d][1]benzazepin-7-yl]propanamide.

[0104] As used herein, “PF-03084014” refers to a compound having CAS No. 1962925-29- 6. The molecular formula of PF-03084014 is C27H4iF2NsO.HBr. The 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]-1 H-imidazol-4-yl]pentanamide dihydrobromide, while the chemical structure is according to Chem. 2:

[0105] Chem. 2

[0106] As used herein, “Dibenzazepine” or “DBZ” or “XX” refers to a compound having CAS No. 209984-56-5. The molecular formula of Dibenzazepine is C26H23F2N3O3. The chemical name is / V-[(1 S)-2-[[(7S)-6,7-Dihydro-5-methyl-6-oxo-5 / 7-dibenz[b,d]azepin-7-yl]amino]-1- methyl-2-oxoethyl]-3,5-difluorobenzeneacetamide, while the chemical structure is according to Chem. 3:

[0107] Chem. 3 As used herein, “Avagacestat” refers to a compound having CAS No. 1146699-66-2.

[0108] The molecular formula of Avagacestat is C20H17CIF4N4O4S. The chemical name is (2R)-2- [[(4-Chlorophenyl)sulfonyl][[2-fluoro-4-(1,2,4-oxadiazol-3-yl)phenyl]methyl]amino]-5,5,5- trifluoropentanamide, while the chemical structure is according to Chem. 4:

[0109] Chem. 4 The method is intended to increase the number of cells in the cell population having a neuronal fate. As used herein, the term "neuronal fate" in reference to a cell means the developmental fate of that cell becomes restricted such that it will develop into a neuron if allowed to mature under suitable conditions.

[0110] In an embodiment, the method is for directing differentiation of neural cells into neurons. As used herein, the term “directing differentiation” means affecting the developmental fate of a cell towards a certain outcome. Directing the differentiation of a cell is not necessarily a continuous process and the cell may not necessarily develop into the final fate during the method as disclosed herein. In a preferred embodiment, the differentiation of the cells is directed in vitro so that the cells may later develop in vivo towards a certain fate, specifically a neuronal fate. Accordingly, in an embodiment the method is in vitro.

[0111] In an embodiment, the method is for increasing the proportion of neural cells differentiating into neurons. In an embodiment, the method is for decreasing the proportion of neural cells having the potential to differentiate into stromal cells, such as vascular leptomeningeal cells (VLMCs), and / or glial cells, such as astrocytes. In a further embodiment, the increase or decrease in proportion of cells with a specific outcome is when the cell population is further cultured under suitable conditions or following administration of the cell population into a subject.

[0112] The present inventors have found that the outcome of protocol for differentiating a cell population, such as human PSCs, towards or into neurons can be improved with the inhibition of the NOTCH signaling pathway using alternative compounds than usually used in the field of neural differentiation, namely the small molecule DAPT. The present invention has surprisingly found that the compound LY411575 demonstrates improved upregulation of neuronal genes and downregulation of NPC genes during the differentiation compared to DAPT. The present inventors have further identified the alternative compounds Avagacestat, Dibenzazepine, and PF-03084014 as having similar abilities. Specifically, this inhibition thus further reduces the proportion of late-stage proliferative cells (which represents off-target lineages), reduces the proportion of stromal cells, such as VLMCs, and glial cells, such as astrocytes, and at the same time increases the proportion of neurons, when the neural cells are further differentiated towards a terminal fate. The present inventors have found the above-mentioned compounds are superior compared to the use of e.g., DAPT. After 24 hours of administration cells treated with any of the compounds produce more neurons (HuCD or INA positive cells) and are less proliferative (SOX2 and / or Ki67 positive cells) as compared to similar treatment with DAPT (figure 13).

[0113] The cell population according to the method comprises neural cells. In an embodiment, at least 80%, 85%, 90%, 95%, or 99% of the cell population are neural cells at the time of contacting the cell population with the inhibitor of NOTCH signaling. Ideally, the cell population consists of neural cells. The neural cells may be obtained according to any suitable method. In an embodiment, the cell population comprising neural cells is obtained by differentiating pluripotent stem cells (PSCs) into neural cells. In an embodiment, the cell population comprising neural cells is obtained by neural induction. As used herein, by the term “neural induction” is meant a process by which pluripotent cells are exposed to signals that will instruct the cells to become neural stem or precursor cells unless exposed to signals that divert them to alternative fates. Accordingly, in an embodiment, the cell population is cultured to induce differentiation into neural cells prior to contacting the cell population with the inhibitor of NOTCH signaling. The contacting of the cell population with the inhibitor of NOTCH signaling is done prior to cryopreservation of the cell population. In a particularly preferred embodiment, the contacting of the cell population with the inhibitor of NOTCH signaling is done 24-48 hours prior to cryopreservation of the cell population.

[0114] In a cell population subject to neural induction, the individual cells will differentiate and progress at different pace and the cell population will therefore comprise a composition of neural cells at different developmental stages. Accordingly, in an embodiment, the cell population comprises neural stem cells, neuroblast intermediate precursor cells, and neurons at the time of contacting the cell population with the inhibitor of NOTCH signaling. The present inventors have identified a preferred timing wherein the composition of neural cells is optimal for directing differentiation of the cells into neurons, i.e., the timing for when to contact the cell population comprising neural cells with the inhibitor of NOTCH signaling. In an embodiment, at least 1%, such as at least 2%, of the cell population are INA+ / SOX2- at the time of contacting the cell population with the inhibitor of NOTCH signaling (see figure 21 and 10). Accordingly, this means that for effectively directing differentiation of the neural cells towards neurons a certain proportion of individual cells needs to be both positive for the expression of INA and negative for the expression of SOX2.

[0115] In another embodiment, the preferred timing wherein the composition of ventral midbrain neural cells is optimal for directing differentiation of the cells into neurons, i.e., the timing for when to contact the cell population comprising neural cells with the inhibitor of NOTCH signaling, is determined based on the total number of cells positive for the expression of INA. In one embodiment the level of cells positive for the expression of ASCL1 is also evaluated. In one embodiment at least 5% of the cell population is INA+ at the time of contacting the cell population with the inhibitor of NOTCH signaling, such as 10%, 15%, 20%, 25%, 30%, such as at least 35%. In another embodiment 5-35% of the cell population is INA+ at the time of contacting the cell population with the inhibitor of NOTCH signaling, such as 10-25%, 10-20%, more preferably 10-15%.

[0116] In one embodiment at least 20% of the cell population is ASCL1+ at the time of contacting the cell population with the inhibitor of NOTCH signaling, such as 25%, 30%, 35%, 40%, 50%, 60%, 70% such as at least 75%. In another embodiment 20-75% of the cell population is ASCL1+ at the time of contacting the cell population with the inhibitor of NOTCH signaling, such as 20-70%, 25-70%, more preferably 35-65%.

[0117] In another embodiment, the preferred timing wherein the composition of dorsal forebrain neural cells is optimal for directing differentiation of the cells into neurons, i.e., the timing for when to contact the cell population comprising neural cells with the inhibitor of NOTCH signaling, is determined based on the total number of cells positive for the expression of INA. In one embodiment the level of cells positive for the expression of TBR2 is also evaluated. In one embodiment at least 5% of the cell population is INA+ at the time of contacting the cell population with the inhibitor of NOTCH signaling, such as 10%, 15%, 20%, 25%, 30%, such as at least 35%. In another embodiment 5-35% of the cell population is INA+ at the time of contacting the cell population with the inhibitor of NOTCH signaling, such as 5-25%, 5-20%, more preferably 10-20%.

[0118] In one embodiment at least 5% of the cell population is TBR2+ at the time of contacting the cell population with the inhibitor of NOTCH signaling, such as 10%, 15%, 20%, 30%, such as at least 40%. In another embodiment 5-40% of the cell population is TBR2+ at the time of contacting the cell population with the inhibitor of NOTCH signaling, such as 5-30%, 5-20%, more preferably 5-15%.

[0119] A neural cell featuring either of such expression profiles is considered to be indicative of the cell’s progression to a terminal fate specifically that of a neuron and cannot differentiate further or divide; neurons being cells that have neurites (axons and dendrites) and transmit information with electrical signals. A person skilled in the art will readily know how to determine this by single cell expression analysis, such as using scRNAseq. In an embodiment, the expression of markers by the cells is measured using scRNAseq, flow cytometry, ICC, qPCR or some other such method. In an embodiment, the expression of markers by the cells is measured according to Example 9, Example 13, Example 14, and Example 15. In an embodiment, 2-60%, 2-50%, 2-40%, 2-30%, or 2-25%, preferably 2-25%, of the cell population are INA+ / SOX2- at the time of contacting the cell population with the inhibitor of NOTCH signaling. In an embodiment, the cell population is contacted with the inhibitor of NOTCH signaling at the time when at least 1% of the cell population are INA+ / SOX2- or within 30 days thereof, such as within 25, 20, 15, 10, or 5 days thereof. In a preferred embodiment the cell population is contacted with inhibitor of NOTCH signaling at the time when at least 2% of the cell population are INA+ / SOX2- or within 1 to 5 days thereof.

[0120] In a preferred embodiment the cell population is contacted with inhibitor of NOTCH signaling at the time when at least 5% of the cell population are INA+ or within 1 to 5 days thereof.

[0121] In a preferred embodiment the cell population is contacted with inhibitor of NOTCH signaling at the time when at least 30% of the cell population are ASCL1+ or within 1 to 5 days thereof. In a preferred embodiment the cell population is contacted with inhibitor of NOTCH signaling at the time when at least 5% of the cell population are TBR2+ or within 1 to 5 days thereof.

[0122] In an embodiment, at least 1.5% of the cell population are neuroblast intermediate precursor cells, preferably at least 2.5%, more preferably at least 5% at the time of contacting the cells with the inhibitor of NOTCH signaling. In an embodiment, 1.5-75%, such as 1.5-60% of the cell population are neuroblast intermediate precursor cells, preferably 2.5- 50%, more preferably 5-60% at the time of contacting the cells with the inhibitor of NOTCH signaling. In an embodiment, the cell population is contacted with the inhibitor of NOTCH signaling at the time when 1.5% of the cell population are neuroblast intermediate precursor cells or within 30 days thereof, such as within 25, 20, 15, 10, 5 or 1 day(s) thereof. In an embodiment, the neuroblast intermediate precursor cells express a marker selected from ASCL1 , NEUROD1, NEUROD2, NEUROD4, NEUROD6, NEUROG1 , NEUROG2, NEUROG3, TBR2, NHLH1 , NFIA, NFIB, NFIX and SOX4. A person skilled in the art will recognize that markers indicative of neuroblast intermediate precursor cells are distinct to the specific region of the brain of which the neural cells belong. In an embodiment, the neural cells are specific to a region selected from forebrain, midbrain, and hindbrain / spinal cord. In an embodiment, the forebrain region is dorsal forebrain region. In an embodiment, the midbrain region is ventral midbrain region.

[0123] In an embodiment, the concentration of the inhibitor of NOTCH signaling is at least 0.1 pM, such as preferably at least 0.2 pM, 0.5 pM, 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, or at least 10 pM. In an embodiment, the concentration of the inhibitor of NOTCH signaling is from 0.2 pM to 1000 pM, such as 10 pM to 100 pM, preferably 50 pM to 100 pM. In an embodiment, the concentration of the inhibitor of NOTCH signaling is less than 1000 pM, such as less than 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM or 100 pM.

[0124] In an embodiment, the cell population is contacted with the inhibitor of NOTCH signaling for at least 14 hour, such as at least 3, 6, or 12 hours, or for at least 1 , 2, 3, 4, 5, or 6 days, preferably for 1-2 days. In an embodiment, the inhibitor of NOTCH signaling 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 an embodiment, the cell population is contacted with the inhibitor of NOTCH signaling from 14 hour to 10 days, 14 hour to 9 days, 14 hour to 8 days, 14 hour to 7 days, 14 hour to 6 days, 14 hour to 5 days, 14 hour to 4 days, 14 hour to 3 days, 14 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 from 12 hours to 2 days.

[0125] In an embodiment, the cell population has not before the timepoint for contacting it with the inhibitor of NOTCH signaling, been exposed to any other NOTCH inhibitor.

[0126] In an embodiment, the cell population is harvested following inhibition of NOTCH signaling. As used herein, the term “harvesting” means that the cells are collected and transferred to an environment, wherein the cells do not further develop into neurons. In an embodiment, the cell population is harvested prior to the timepoint where more than 60- 100% of the cell population are INA+ / SOX2-. In an embodiment, the cell population is harvested prior to the timepoint where more than 60-95% of the cell population are INA+ / SOX2-. In an embodiment, the cell population is harvested within 10 days following ending contacting the cell population with the inhibitor of NOTCH signaling, such as 9, 8, 7, 6, 5, 4, 3, 2, or 1 day(s) following ending contacting the cell population with the inhibitor of NOTCH signaling, preferably within 1 day or 2 days. In an embodiment, the cell population is harvested and cryopreserved following the inhibition of NOTCH signaling. In an embodiment, the cell population is cryopreserved immediately following harvesting. In an embodiment, the cell population is cryopreserved within 10 days following ending contacting the cell population with the inhibitor of NOTCH signaling, such as 9, 8, 7, 6, 5, 4, 3, 2, or 1 day(s) following ending contacting the cell population with the inhibitor of NOTCH signaling, preferably within 1 day or 2 days. In an embodiment wherein the cell population is cryopreserved, the cell population is cryopreserved in cryoprotective agents, typically comprising DMSO. In a preferred embodiment, the cell population is cultured under suitable culturing conditions for maintaining neural cells. In an embodiment, the cell population is cultured in a culture medium suitable for maintaining neural cells, wherein the culture medium is a neurobasal medium. In an embodiment, the culture medium comprises one or more selected from B27 supplement, ascorbic acid, BDNF, GDNF, dcAMP, and DAPT. Preferably, the culture medium does not comprise DAPT. In an embodiment, the culture medium is replaced at least every 12 to 36 hours, preferably at least every 18 to 30 hours, more preferably at least every 24 hours.

[0127] Differentiation of PSCs into neural cells

[0128] In an initial step according to the method of the present invention a cell population comprising neural cells is obtained. The cell population may be obtained by any suitable means. However, in a preferred embodiment, the cell population comprising neural cells is obtained by differentiating PSCs into neural cells. Accordingly, in an embodiment, the cell population is derived from PSCs. In a preferred embodiment, the cell population is derived from human cells. In an embodiment, the PSCs are human embryonic stem cells or human induced pluripotent stem cells.

[0129] In a further embodiment, the cell population comprising neural cells is obtained by differentiating PSCs into neural cells specific to a region selected from forebrain, midbrain, and hindbrain / spinal cord. Protocols for differentiating PSCs into the different cell types of the neuroectoderm are well-known, and it applies to all protocols that the PSCs are initially neurally induced whereafter the cells progress in differentiation into neural cells by undergoing a series of developmental stages, initially becoming neural stem cells, then progressing into neuroblast intermediate precursor cells, and optionally towards neuronal identity. Depending on the specific region the development may take a longer or shorter time. However, the present inventors have found that regardless of the specific region and time it takes for the cells to progress through the different developmental stages, the preferred timing of contacting the cell population with the inhibitor of NOTCH signaling can be established based on the proportion of cells being both positive for the expression of the marker INA and negative for the expression of the marker SOX2, the total expression of the marker INA and / or the total expression of TBR2 or ASCL1. At this stage the cell population typically comprises neural stem cells, neuroblast intermediate precursor cells, and neurons.

[0130] In a general embodiment, the cell population comprising neural cells is obtained by differentiating pluripotent stem cells (PSCs) into neural cells for 14 to 35 days. By differentiating the cells, the cell population is exposed to signals that will induce the cells to become neural cells. 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 activates and / or inhibits certain signaling pathways, and it may be that once the cell population has been exposed to the different signals then further differentiation is allowed to occur without further exposure to these or any signals by merely culturing the cell population in a culture medium suitable for the cells to further differentiate. Whether or not the cells are allowed to further differentiate for a period of time without exposure to signals directing differentiation then in a preferred embodiment, the cell population is cultured in a culture medium suitable for maintaining neural cells, wherein the culture medium is a neurobasal medium.

[0131] In an embodiment, the cell population comprising neural cells is obtained by differentiating pluripotent stem cells (PSCs) into neural cells for 18 to 35 days, and wherein the neural cells are differentiated into neural cells specific to the forebrain region. In such an embodiment, the cell population is neurally induced, not ventralized, and not caudalized to obtain dorsal forebrain neural cells. In an embodiment wherein the neural cells are differentiated into neural cells specific to the forebrain region, preferably dorsal forebrain region, the cell population is not contacted with the inhibitor of NOTCH signaling until at least 24 days after initiating differentiation of the cell population into neural cells specific to the forebrain region, such as at least 24 days, 26 days, 28 days, 30 days, 32 days, 33 days, or 35 days after. Preferably the cell population is not contacted with the inhibitor of NOTCH signaling until 25-28 days after initiating differentiation of the cell population into neural cells (Figure 20).

[0132] In a preferred embodiment the cell population is not contacted with inhibitor of NOTCH signaling until at least 24 days after initiating differentiation of the cell population into neural cells specific to the forebrain region, such as at least 24 days, 26 days, 28 days, 30 days, 32 days, 33 days, or 35 days after and at the time when at least 5% of the cell population are INA+.

[0133] In a preferred embodiment the cell population is not contacted with inhibitor of NOTCH signaling until at least 24 days after initiating differentiation of the cell population into neural cells specific to the forebrain region, such as at least 24 days, 26 days, 28 days, 30 days, 32 days, 33 days, or 35 days after and at the time when at least 5% of the cell population are TBR2+ In another embodiment, the cell population comprising neural cells is obtained by differentiating pluripotent stem cells (PSCs) into neural cells for 14 to 26 days, and wherein the neural cells are differentiated into neural cells specific to the midbrain region, preferably ventral midbrain region. In such an embodiment, the cell population is neurally induced, ventralized, and caudalized to obtain ventral midbrain neural cells. Further to this, in an embodiment, less than 60% of the cell population express KI67, such as less than 50%, such as less than 40%, such as less than 30%, such as less than 20%, such as less than 10%, at the time of contacting the cells with the inhibitor of NOTCH signaling. Specifically for the midbrain region, the abovementioned number of KI67 positive cells may be indicative of a cell population comprising sufficient neuroblast intermediate precursor cells to be treated with the inhibitor of NOTCH signaling. In a further embodiment, 20-80% of the cell population express KI67, preferably 30-70%, more preferably 40-60%, at the time of contacting the cells with the inhibitor of NOTCH signaling. In an embodiment wherein the neural cells are differentiated into neural cells specific to the midbrain region, preferably ventral midbrain region, the cell population is not contacted with the inhibitor of NOTCH signaling until at least

[0134] 20 days after initiating differentiation of the cell population into neural cells, such as at least

[0135] 21 days, 22 days, 23 days, 24 days, 25 days, or 26 days after, preferably at least 22 days after. Preferably the cell population is not contacted with the inhibitor of NOTCH signaling until 22-25 days after initiating differentiation of the cell population into neural cells (Figure 23).

[0136] In a preferred embodiment the cell population is not contacted with inhibitor of NOTCH signaling until at least 20 days after initiating differentiation of the cell population into neural cells specific to the midbrain region, such as at least 21 days, 22 days, 23 days, 24 days, 25 days, or 26 days after, preferably at least 22 days and at the time when at least 10% of the cell population are INA+.

[0137] In a preferred embodiment the cell population is not contacted with inhibitor of NOTCH signaling until at least 20 days after initiating differentiation of the cell population into neural cells specific to the midbrain region, such as at least 21 days, 22 days, 23 days, 24 days, 25 days, or 26 days after, preferably at least 22 days and at the time when at least 30% of the cell population are ASCL1+.

[0138] In another embodiment, the cell population comprising neural cells is obtained by differentiating pluripotent stem cells (PSCs) into neural cells for 14 to 26 days, and wherein the neural cells are differentiated into neural cells specific to the hindbrain / spinal cord region. In a preferred embodiment, the cell population comprising neural cells is obtained by neurally inducing pluripotent cell, such as PSCs. The cells may be neurally induced by any suitable method. However, in a preferred embodiment, the cell population comprising neural cells is obtained by contacting the cell population with an inhibitor of Small Mothers Against Decapentaplegic (SMAD) protein signaling. In an embodiment, the cell population comprising neural cells 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. A person 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 neural lineage. In an embodiment, the inhibitor of the SMAD signaling pathway is selected from Noggin, LY364947, SB431542, RepSox, DMH1 , DMH2, LND-212854, and LDN-193189.

[0139] In a preferred embodiment, the cell population is cultured in a culture medium suitable for maintaining neural cells, wherein the culture medium is a neurobasal medium. In an embodiment, the culture medium comprises one or more components selected from B27 supplement, ascorbic acid, BDNF, GDNF, dcAMP, and DAPT. Preferably, the culture medium does not comprise DAPT.

[0140] Protocol specific to the forebrain region

[0141] In an embodiment, the cell population is differentiated into neural cells specific to the forebrain region. In an embodiment, the cell population is differentiated into neural cells specific to the forebrain region comprising contacting the cell population with an inhibitor of Small Mothers Against Decapentaplegic (SMAD) protein signaling pathway for about 0-11 days, followed by contacting the cell population with FGF2 for about 7-9 days, and following this can be continued to culture the cell population and typically in basal media without growth factors or morphogens or small molecules for an additional 9-12 days.

[0142] Examples of differentiation to dorsal forebrain / pallial cells are found in the publications by 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. Protocol specific to the ventral midbrain region

[0143] In an embodiment, the cell population is differentiated into ventral midbrain neural cells. In an embodiment, the cell population is contacted with an inhibitor of Small Mothers Against Decapentaplegic (SMAD) protein signaling, an activator of Sonic Hedgehog (SHH) signaling, an activator of wingless (Wnt) signaling, and / or, optionally, an activator of fibroblast growth factor (FGF) signaling, and, optionally, ascorbic acid and / or, optionally, Brain-derived neurotrophic factor (BDNF). In an embodiment, following 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, and, optionally, ascorbic acid and / or, optionally, Brain-derived neurotrophic factor (BDNF), the cell population is further cultured in a culture medium suitable for maintaining neural cells 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. Accordingly, the neural cells are allowed to further differentiate without continuous exposure to signals directing the differentiation.

[0144] In an embodiment for differentiating PSCs into ventral midbrain neural cells, the cell population is differentiated into ventral midbrain neural stem cells by inducing ventralization of the cell population. As used herein, the terms “ventralization” and “ventral patterning” may be used interchangeably and refer to the process whereby pluripotent cells assume a ventral gene expression identity equivalent to cells of an embryo or embryonic structure, i.e., neural tube. In an embodiment, the PSCs are ventralized by contacting the cells with an activator of the SHH signaling pathway. As used herein, the term “activator of sonic hedgehog signaling” refers to any molecule or compound that is capable of activating a SHH signaling pathway. Activation of the SHH pathway is well known as being responsible for the induction and maintenance of ventral neural tube structures. In an embodiment, the activator of SHH signaling is selected from SHH, purmorphamine, and SAG.

[0145] In an embodiment for differentiating PSCs into ventral midbrain neural cells, the cell population is differentiated into ventral midbrain neural cells by inducing caudalization of the cell population. As used herein, the term “caudalization” refers to the process whereby pluripotent cells assume a caudal gene expression identity equivalent to cells of an embryo or embryonic structure, i.e., neural tube. In an embodiment, the PSCs are caudalized by contacting the cells with an activator of the Wnt signaling pathway. As used herein, the term “activator of Wnt signaling” refers to any molecule or compound that is capable of activating a Wnt signaling pathway. Inhibitors of Wnt signaling is well known as being responsible for caudalizing of neural cells. In an embodiment, the activator of Wnt signaling lowers GSK3- beta for activation of Wnt signaling. Accordingly, in certain embodiments, the Wnt activator is an inhibitor of GSK3-beta. In an embodiment, the activator of Wnt signaling is selected from CHIR99021 and a recombinant Wnt protein.

[0146] In an embodiment for differentiating PSCs into ventral midbrain neural cells, the cell population is differentiated into ventral midbrain neural cells by further inducing caudalization of the cell population. Accordingly, in an embodiment, the cell population is contacted with an activator of fibroblast growth factor (FGF). In an embodiment, the activator of FGF signaling is selected from FGF8b.

[0147] In a certain embodiment, the concentration of the inhibitor(s) of SMAD protein signaling is from 1 pM to 50 pM, the activator of SHH signaling is from 200 ng / ml to 800 ng / ml, the inhibitor of Wnt signaling is from 0.1 pM to 1 pM, the activator of FGF signaling is from 10 ng / ml to 200 ng / ml, ascorbic acid is from 50 pM to 500 pM, and / or BDNF is from 1 ng / ml to 50 ng / ml.

[0148] In an embodiment, the cell population of pluripotent stem cells is contacted with the inhibitor of SMAD protein signaling for 5 to 9 days. In a certain embodiment, the cell population is contacted with the inhibitor of SMAD protein signaling for 5 to 9 days from day 0. In an embodiment, the cell population of PSCs is contacted with the activator of SHH signaling for 5 to 9 days, such as 7 to 9 days, preferably for 9 days. In a preferred embodiment, the cell population is contacted with the inhibitor of Wnt signaling for 5 to 9 days from day 0. In an embodiment, the concentration of the activator of SHH signaling is from 200 ng / ml to 800 ng / ml. In an embodiment, the cell population of pluripotent stem cells is contacted with the inhibitor of Wnt signaling for 5 to 9 days, such as 7 to 9 days, preferably for 9 days. In a preferred embodiment, the cell population is contacted with the inhibitor of Wnt signaling for 5 to 9 days from day 0. In an embodiment, the concentration of the activator of Wnt signaling is from 0.1 pM to 1 pM. In an embodiment, the cell population of PSCs is contacted with the activator of FGF signaling for 7 to 12 days following ended contacting with the inhibitor of SMAD protein signaling, inhibitor of Wnt signaling, and / or activator of SHH signaling. In another embodiment, the cell population is contacted with the activator of FGF signaling for 7 to 12 days from day 5 to 9, or when ending contacting with the inhibitor of SMAD protein signaling, inhibitor of Wnt signaling, and / or activator of SHH signaling. In a certain embodiment, the cell population is contacted with the inhibitor of SMAD protein signaling, the activator of SHH signaling, and the inhibitor of Wnt signaling from day 0 to day 9, subsequently, the cell population is contacted with the activator of FGF signaling from day 9 to day 16. In an embodiment, the activator of FGF signaling is FGF8b. In an embodiment, the concentration of the activator of FGF signaling is from 10 ng / ml to 200 ng / ml. In an embodiment, the cell population is contacted with ascorbic acid for 5 to 7 days from day 10 or 11. In an embodiment, the concentration of ascorbic acid is from 10 pM to 400 pM, preferably from 100 pM to 300 pM, preferably from 150 pM to 250 pM, more preferably about 200 pM. In an embodiment, the cell population is contacted with BDNF for 5 to 7 days from day 10 or 11. In an embodiment, the concentration of BDNF is from 1 ng / ml to 40 ng / ml, preferably from 10 ng / ml to 40 ng / ml, preferably 15 ng / ml to 30 ng / ml, more preferably about 20 ng / ml.

[0149] Examples of differentiation to ventral midbrain neural cells are found in the Example 3.

[0150] Protocol specific to the hindbrain / spinal cord region

[0151] In an embodiment, the cell population is differentiated into neural cells specific to the hindbrain / spinal cord region. In an embodiment, the cell population is differentiated into neural cells specific to the hindbrain / spinal cord region comprising contacting the cell population with an inhibitor of (SMAD) protein signaling pathway from about 0-6 days, and also WNT agonist such as CHIR from about 0-15 days and also by contacting the cell population with a ventralizing molecule such as SHH or SAG from about 6-15 days and also by contacting the cell population with Retinoic Acid a key factor for this region.

[0152] Examples of differentiation to hindbrain / spinal cord cells are found in the publications by 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; Butts et al., McDevitt “V2a interneuron differentiation from mouse and human pluripotent stem cells” 2019.

[0153] Cell population of neural cells

[0154] An aspect of the present invention relates to a cell population comprising neural cells obtainable by any of the methods described herein. In a certain embodiment, the cell population comprising neural cells is obtained by the method according to any one of the methods described herein. Notably, the present inventors have been able to identify parameters which distinguish the cell population harvested immediately following the inhibition of NOTCH signaling, as compared to a cell population which have not been subject to the method of directing differentiation towards neurons according to the present invention. However, it is also evident that the cells are stimulated by the signaling and that the proportion of cells holding neuronal fate has been increased, which becomes even more apparent once the cell population is allowed to culture further. Accordingly, an aspect of the present invention relates to a cell population comprising neural cells, wherein the cell population when cultured in vitro for 5-7 days in a culture medium suitable for maintaining neural cells results in a cell population wherein at least 35% are HuC / D positive, preferably at least 50%. In an embodiment, the expression of markers by the cells is measured using scRNAseq. In an embodiment, the expression of markers by the cells is measured according to Example 9, Example 13, Example 14, and Example 15. In an embodiment, the cell population is cultured according to Example 10, Example 11, and Example 12, depending on the region that the neural cells are specific to. In an embodiment, the cell population is in vitro. In an embodiment, the neural cells are non-native. In an embodiment, the neural cells are artificial. In an embodiment, the cell population is derived from human cells. In a preferred embodiment, the cell population is derived from stem cells. In a certain 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 an embodiment, the neural cells are specific to a region selected from forebrain, midbrain, and hindbrain / spinal cord. In a further embodiment, the neural cells are ventral midbrain neural cells. In a further embodiment, the neural cells are dorsal forebrain neural cells.

[0155] In an embodiment, the cells of the cell population are genetically modified. In an embodiment, the pluripotent stem cells are genetically modified, and the genetic modifications persist in the neural cells obtained according to the any of the methods described herein. In certain embodiments the cells of the cell population are genetically modified to become hypoimmunogenic. As used herein, the terms “hypoimmunogenic” and “immune evasive” in reference to a cell may be used interchangeably and refer to properties of the cell making it less prone to immune rejection by a subject into which such cells are transplanted. Typically, specific surface markers are overexpressed or silenced. In an embodiment, the genetically modified cells have reduced expression of MHC-I and / or MHC- II. In an embodiment, the cells are genetically modified to express one or more tolerogenic factors or analogues thereof, such as HLA-E, HLA-G, CD46, CD47, CD55, CD59, and PD- L1. Examples and methods for genetically modifying cells to be immune evasive are described in WO2012145384, WO2013158292, WO2016142532, W02016183041 , WO2018132783, WO2018175390, WO2019161271 , W02020018615, W02020018620, W02020049535, WO2020168317, WO2021195426, WO2022012591 , and W02020260563. In some aspects, genome editing technologies (e.g., the CRISPR / Cas or TALEN systems) are used to modulate (e.g., reduce, eliminate and / or increase) the expression of specific genes.

[0156] In an embodiment, the genetic modifications for hypoimmunogenicity comprise reduced expression of MHC-I human leukocyte antigens relative to a wild-type stem cell, reduced expression of MHC-II human leukocyte antigens relative to a wild-type stem cell, and / or increased expression of a tolerogenic factor relative to wild-type stem cell. In an embodiment, the MHC-I human leukocyte antigens are HLA-A, HLA-B, and HLA-C. In an embodiment, the MHC-II human leukocyte antigens are HLA-DP, HLA-DQ, and HLA-DR. In an embodiment, the tolerogenic factor is selected from CD46, CD47, CD55, CD59, PD-L1 , HLA-E, and HLA-G.

[0157] In an embodiment, the cells of the cell population are genetically modified to be lineage restricted. As used herein, the term “lineage restricted” in reference to a cell means that the cell is functionally and / or structurally limited to differentiate into certain cell types.

[0158] In an embodiment, the cell population comprises at least 1,000 cells, such as at least 10,000 cells, 100,000 cells, 1 ,000,000 cells, or 10,000,000 cells. In an embodiment, the cell population is cryopreserved.

[0159] Accordingly, another aspect of the present invention relates to a composition comprising the cell population and a cryoprotectant. In an embodiment wherein the cell population is cryopreserved, the cell population is cryopreserved in cryoprotective agents, typically comprising DMSO.

[0160] Use as a medicament

[0161] Another aspect of the present invention relates to an in vitro cell population described herein or a composition thereof for use as a medicament. In an embodiment, the cell population for use as a medicament is for the treatment of a neurological condition. In an 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 wherein neurons are lost or dysfunctional. In an embodiment, the cell population comprises neural cells having been contacted with an inhibitor of NOTCH signaling. In an embodiment, the neural cells are forebrain neural cells. In an embodiment, the cell population comprising forebrain neural cells is for the treatment of stroke. In an embodiment, the neural cells are ventral midbrain neural cells. In an embodiment, the cell population comprising ventral midbrain neural cells is for the treatment of Parkinson’s disease. In an embodiment, the neural cells are hindbrain / spinal cord neural cells. In an embodiment, the cell population comprising hindbrain / spinal cord neural cells is for the treatment of paralysis, spinal cord injury, diabetic neuropathy, or chronic pain.

[0162] Another aspect relates to a method of treatment of a neurological condition comprising the administration to a patient of an effective amount of a cell population according to the present invention. In an 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 wherein neurons are lost or dysfunctional. In a specific embodiment, the neurological condition is Parkinson’s disease.

[0163] As used herein the term “subject” refers to a human patient suffering from Parkinson’s disease or Stroke. In an embodiment, a “therapeutically effective amount” in reference to a treatment of these disorders with a cell product means a dose of 200,000 to 40,000,000 cells. Administration to a subject of the neural cells is contemplated by surgery.

[0164] In this embodiment an in vitro cell population described herein is differentiated with the addition of a NOTCH inhibitor, wherein the inhibitor of NOTCH signaling is LY411575, at the timepoints described herein and following that cryopreserving the cell population, thereby obtaining a vial of cryopreserved cells which can be used for administration to a subject.

[0165] In vivo directing differentiation of neural cells into neurons

[0166] Another aspect relates to a method for treatment of a neurological condition, comprising administering to a subject a therapeutically effective amount of a cell population comprising neural cells, and an inhibitor of NOTCH signaling, wherein the inhibitor of NOTCH signaling is LY411575. The present inventors envisage that a similar effect in directing differentiation towards neurons can be achieved in vivo by co-administering the inhibitor of NOTCH signaling together with the neural cells. In an embodiment, the neural cells have therefore not been subject to the method of directing differentiation towards neurons as disclosed herein. As used herein the term “subject” refers to a human patient suffering from Parkinson’s disease or Stroke. In an embodiment, a “therapeutically effective amount” in reference to a treatment of these disorders with a cell product means a dose of 200,000 to 40,000,000 cells. Administration to a subject of the ventral midbrain neural cells is contemplated by surgery. In an embodiment, the ventral midbrain neural cells co-express 3 or more of the markers selected from the list of FOXA2, LMX1A, EN1 , OTX2, ASCL1 , NEUROD1 , PITX3 and SOX2. In an embodiment, the forebrain neural cells co-express 3 or more of the markers selected from the list of SOX2, OTX2, PAX6, FOXG1, TBR2, EMX2, NFIA and SOX1. It follows that an aspect of the present invention relates to a composition comprising the cell population comprising neural cells, and an inhibitor of NOTCH signaling, wherein the inhibitor of NOTCH signaling is LY411575.

[0167] Accordingly, an aspect of the present invention relates to LY411575 for use in the treatment of a neurological condition. Specifically, one embodiment relates to LY411575 for use in the treatment of Parkinson’s disease in a subject being administered with a therapeutically effective amount of ventral midbrain neural cells.

[0168] A person skilled in the art will appreciate that the embodiments referred to in this section relating to methods of treatment, use as a medicament and second medical uses may equally apply to all aspects mentioned.

[0169] Particular embodiments

[0170] The aspects of the present invention are now further described by the following nonlimiting embodiments:

[0171] 1. A method comprising obtaining a cell population comprising neural cells and contacting the cell population with an inhibitor of NOTCH signaling, wherein the inhibitor of NOTCH signaling is selected from LY411575, Avagacestat, Dibenzazepine, and PF-03084014.

[0172] 2. The method according to the preceding embodiment, wherein the inhibitor of NOTCH signaling is LY411575.

[0173] 3. The method according to any one of the preceding embodiments, wherein the method is for directing differentiation of neural cells into neurons.

[0174] 4. The method according to any one of the preceding embodiments, wherein the method is for increasing the proportion of neural cells having the potential to differentiate into neurons.

[0175] 5. The method according to any one of the preceding embodiments, wherein the method is for increasing the proportion of neural cells having the potential to differentiate into neurons following end of contacting the cell population with the inhibitor of NOTCH signaling, when the cell population is further cultured under suitable conditions or following administration of the cell population into a subject. 6. The method according to any one of the preceding embodiments, wherein the method is for decreasing the proportion of neural cells having the potential to differentiate into stromal cells, such as vascular leptomeningeal cells (VLMCs), and / or glial cells, such as astrocytes.

[0176] 7. The method according to any one of the preceding embodiments, wherein the method is for decreasing the proportion of neural cells having the potential to differentiate into stromal cells, such as vascular leptomeningeal cells (VLMCs), and / or glial cells, such as astrocytes following end of contacting the cell population with the inhibitor of NOTCH signaling, when the cell population is further cultured under suitable conditions or following administration of the cell population into a subject.

[0177] 8. The method according to any one of the preceding embodiments, wherein the cell population comprising neural cells is obtained by differentiating pluripotent stem cells (PSCs) into neural cells.

[0178] 9. The method according to any one of the preceding embodiments, wherein the cell population comprising neural cells is obtained by neural induction.

[0179] 10. The method according to any one of the preceding embodiments, wherein the cell population is cultured to induce differentiation into neural cells prior to contacting the cell population with the inhibitor of NOTCH signaling.

[0180] 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 neural cells at the time of contacting the cell population with the inhibitor of NOTCH signaling.

[0181] 12. The method according to any one of the preceding embodiments, wherein at least 1%, such at least 2%, of the cell population are INA+ / SOX2- at the time of contacting the cell population with the inhibitor of NOTCH signaling. 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 of contacting the cell population with the inhibitor of NOTCH signaling.

[0182] 14. The method according to any one of the preceding embodiments, wherein the cell population is contacted with the inhibitor of NOTCH signaling at the time when at least 1% of the cell population are INA+ / SOX2- or within 30 days thereof, such as within 25, 20, 15, 10, or 5 days thereof.

[0183] 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 are SOX2 positive at the time of contacting the cell population with the inhibitor of NOTCH signaling.

[0184] 16. The method according to any one of the preceding embodiments, wherein less than 90% of the cell population is SOX2 positive at the time of contacting the cell population with the inhibitor of NOTCH signaling.

[0185] 17. The method according to any one of the preceding embodiments, wherein at least 1.5% of the cell population are neuroblast intermediate precursor cells, preferably at least 2.5%, more preferably at least 5% at the time of contacting the cells with the inhibitor of NOTCH signaling.

[0186] 18. The method according to any one of the preceding embodiments, wherein 1.5-75%, such as 1.5-60% of the cell population are neuroblast intermediate precursor cells, preferably 2.5-50%, more preferably 5-60% at the time of contacting the cells with the inhibitor of NOTCH signaling.

[0187] 19. The method according to any one of the preceding embodiments, wherein the cell population is contacted with the inhibitor of NOTCH signaling at the time when at least 1.5% of the cell population are neuroblast intermediate precursor cells or within 30 days thereof, such as within 25, 20, 15, 10, or 5 days thereof.

[0188] 20. The method according to any one of the embodiments 17 to 19, wherein the neuroblast intermediate precursor cells express a marker selected from ASCL1, NEUROD1 , NEUROD2, NEUROD4, NEUROD6, NEUROG1 , NEUROG2, NEUROG3, TBR2, NHLH1 , NFIA, NFIB, NFIX and SOX4. The method according to any one of the preceding embodiments, wherein the cell population comprises neural stem cells, neuroblast intermediate precursor cells, and neurons at the time of contacting the cell population with the inhibitor of NOTCH signaling. The method according to any one of the preceding embodiments, wherein the neural cells are specific to a region selected from forebrain, midbrain, and hindbrain / spinal cord. The method according to the preceding embodiment, wherein the neural cells are specific to the hindbrain / spinal cord, specifically medial-ventral caudal NPCs of the hindbrain or spinal cord region, more specifically including ventral 0-2 regions referred to as VO, V1 or V2 subtypes. The method according to any one of the preceding embodiments, wherein the cell population comprising neural cells is obtained by differentiating pluripotent stem cells into neural cells specific to a region selected from forebrain, midbrain, and hindbrain / spinal cord. The method according to any one of the preceding embodiments, wherein the neural cells are specific to the forebrain region, and wherein 2-25%, of the cell population are INA+ / SOX2- at the time of contacting the cell population with the inhibitor of NOTCH signaling. The method according to any one of the preceding embodiments, wherein the neural cells are specific to the forebrain region, and wherein at least 5% of the neural cells is INA+ at the time of contacting the neural cells with the inhibitor of NOTCH signaling, such as 10%, 15%, 20%, 25%, 30%, such as at least 35%. The method according to any one of the preceding embodiments, wherein the neural cells are specific to the forebrain region, and wherein 5-35% of the neural cells is INA+ at the time of contacting the neural cells with the inhibitor of NOTCH signaling, such as 5-25%, 5-20%, more preferably 10-20%. The method according to any one of the preceding embodiments, wherein the neural cells are specific to the forebrain region, and wherein at least 5% of the neural cells is TBR2+ at the time of contacting the neural cells with the inhibitor of NOTCH signaling, such as 10%, 15%, 20%, 30%, such as at least 40%. The method according to any one of the preceding embodiments, wherein the neural cells are specific to the forebrain region, and wherein 5-40% of the neural cells is TBR2+ at the time of contacting the neural cells with the inhibitor of NOTCH signaling, such as 5-30%, 5-20%, more preferably 5-15%. The method according to any one of embodiments 1 to 24, wherein the neural cells are specific to the midbrain region, and wherein 0-10%, 0-5%, 1-5%, or 2-5%, preferably 2-5%, of the cell population are INA+ / SOX2- at the time of contacting the cell population with the inhibitor of NOTCH signaling. The method according to embodiment 30, wherein the neural cells are specific to the midbrain region, and wherein at least 5% of the neural cells is INA+ at the time of contacting the neural cells with the inhibitor of NOTCH signaling, such as 10%, 15%, 20%, 25%, 30%, such as at least 35%. The method according to any one of embodiments 30 to 31 , wherein the neural cells are specific to the midbrain region, and wherein 5-35% of the neural cells is INA+ at the time of contacting the neural cells with the inhibitor of NOTCH signaling, such as 10-25%, 10-20%, more preferably 10-15%. The method according to any one of embodiments 30 to 32, wherein the neural cells are specific to the midbrain region, and wherein at least 20% of the neural cells is ASCL1+ at the time of contacting the neural cells with the inhibitor of NOTCH signaling, such as 25%, 30%, 35%, 40%, 50%, 60%, 70% such as at least 75%. The method according to any one of embodiments 30 to 33, wherein the neural cells are specific to the midbrain region, and wherein 20-75% of the neural cells is ASCL1+ at the time of contacting the neural cells with the inhibitor of NOTCH signaling, such as 20-70%, 25-70%, more preferably 35-65%. The method according to any one of embodiments 1 to 24, wherein the neural cells are specific to the hindbrain / spinal cord region, and wherein 0-15% of the cell population are INA+ / SOX2- at the time of contacting the cell population with the inhibitor of NOTCH signaling. The method according to any one of the preceding embodiments, wherein the concentration of the inhibitor of NOTCH signaling is at least 1 pM, such as preferably at least 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, or at least 10 pM. The method according to any one of the preceding embodiments, wherein the concentration of the inhibitor of NOTCH signaling is from 0.2 pM to 1000 pM, such as 10 pM to 100 pM, preferably 50 pM to 100 pM. The method according to any one of the preceding embodiments, wherein the concentration of the inhibitor of NOTCH signaling is less than 1000 pM, such as less than 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM or 100 pM. The method according to any one of the preceding embodiments, wherein the cell population is contacted with the inhibitor of NOTCH signaling for at least 14 hour, such as at least 3, 6, or 12 hours, or for at least 1 , 2, 3, 4, 5, or 6 days, preferably for 1-2 days. The method according to any one of the preceding embodiments, wherein the cell population is contacted with the inhibitor of NOTCH signaling for less than 10 days, such as less than 9 days, 8 days, or 7 days. The method according to any one of the preceding embodiments, wherein the cell population is contacted with the inhibitor of NOTCH signaling from 14 hour to 10 days, 14 hour to 9 days, 14 hour to 8 days, 14 hour to 7 days, 14 hour to 6 days, 14 hour to 5 days, 14 hour to 4 days, 14 hour to 3 days, 14 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 from 12 hours to 2 days.

[0189] 42. The method according to any one of the preceding embodiments, wherein the cell population is not contacted with DAPT.

[0190] 43. The method according to any of the preceding embodiments, wherein the cell population has a density of 0.5 x 106cells / cm2- 2.0 x 106cells / cm2, such as 0.8 x 106cells / cm2- 1.5 x 106cells / cm2at the time of contacting the cell population with the inhibitor of NOTCH signaling.

[0191] 44. The method according to any one of the preceding embodiments, wherein the cell population is harvested following inhibition of NOTCH signaling.

[0192] 45. The method according to any one of the preceding embodiments, wherein the cell population is harvested prior to more than 60-100% of the cell population are INA+ / SOX2-.

[0193] 46. The method according to any one of the embodiments 44 and 45, wherein the cell population is harvested within 10 days following ending contacting the cell population with the inhibitor of NOTCH signaling, such as 9, 8, 7, 6, 5, 4, 3, 2, or 1 day(s) following ending contacting the cell population with the inhibitor of NOTCH signaling, preferably within 1-2 days.

[0194] 47. The method according to any one of the preceding embodiments, wherein the cell population is harvested and cryopreserved following the inhibition of NOTCH signaling.

[0195] 48. The method according to the preceding embodiment, wherein the cell population is cryopreserved immediately following harvesting. 49. The method according to any one of the embodiments 47 and 48, wherein the cell population is cryopreserved within 10 days following ending contacting the cell population with the inhibitor of NOTCH signaling, such as 9, 8, 7, 6, 5, 4, 3, 2, or 1 day(s) following ending contacting the cell population with the inhibitor of NOTCH signaling, preferably within 1-2 days.

[0196] 50. The method according to any one of the embodiments 47 to 49, wherein the cell population is cryopreserved in cryoprotective agents, typically comprising DMSO.

[0197] 51. The method according to any one of the preceding embodiments, wherein the inhibitor of NOTCH signaling is replaced at least every 12 to 36 hours, preferably at least every 18 to 30 hours, more preferably at least every 24 hours.

[0198] 52. The method according to any one of the preceding embodiments, wherein the cell population is derived from PSCs.

[0199] 53. The method according to any one of the preceding embodiments, wherein the cell population is derived from human cells.

[0200] 54. The method according to the preceding embodiment, wherein the PSCs are human embryonic stem cells or human induced pluripotent stem cells.

[0201] 55. The method according to any one of the preceding embodiments, wherein the cell population comprising neural cells is obtained by differentiating pluripotent stem cells (PSCs) into neural cells for 14 to 35 days.

[0202] 56. The method according to any one of the preceding embodiments, wherein the cell population comprising neural cells is obtained by differentiating pluripotent stem cells (PSCs) into neural cells for 18 to 35 days, and wherein the neural cells are differentiated into neural cells specific to the forebrain region.

[0203] 57. The method according to any one of embodiments 55 to 56, wherein the cell population is not contacted with inhibitor of NOTCH signaling until at least 24 days after initiating differentiation of the cell population into neural cells specific to the forebrain region, such as at least 24 days, 26 days, 28 days, 30 days, 32 days, 33 days, or 35 days after and at the time when 5% of the cell population are INA+.

[0204] 58. The method according to any one of embodiments 55 to 57, wherein the cell population is not contacted with inhibitor of NOTCH signaling until at least 24 days after initiating differentiation of the cell population into neural cells specific to the forebrain region, such as at least 24 days, 26 days, 28 days, 30 days, 32 days, 33 days, or 35 days after and at the time when at least 5% of the cell population are TBR2+.

[0205] 59. The method according to embodiment 55, wherein the cell population comprising neural cells is obtained by differentiating pluripotent stem cells (PSCs) into neural cells for 14 to 26 days, and wherein the neural cells are differentiated into neural cells specific to the midbrain region, preferably ventral midbrain region.

[0206] 60. The method according to embodiment 59, wherein the cell population is not contacted with inhibitor of NOTCH signaling until at least 20 days after initiating differentiation of the cell population into neural cells specific to the midbrain region, such as at least 21 days, 22 days, 23 days, 24 days, 25 days, or 26 days after, preferably at least 22 days and at the time when at least 10% of the cell population are INA+.

[0207] 61 . The method according to any one of embodiments 59 to 60, wherein the cell population is not contacted with inhibitor of NOTCH signaling until at least 20 days after initiating differentiation of the cell population into neural cells specific to the midbrain region, such as at least 21 days, 22 days, 23 days, 24 days, 25 days, or 26 days after, preferably at least 22 days and at the time when at least 30% of the cell population are ASCL1+.

[0208] 62. The method according to the preceding embodiment, wherein less than 30% of the cell population express KI67, preferably less than 20%, more preferably less than 10%, at the time of contacting the cells with the inhibitor of NOTCH signaling. 63. The method according to embodiment 62, wherein 5-30% of the cell population express KI67, preferably 29-70%%, at the time of contacting the cells with the inhibitor of NOTCH signaling.

[0209] 64. The method according to any one of the embodiments 52 to 63, wherein the cell population is not contacted with the inhibitor of NOTCH signaling until at least 20 days after initiating differentiation of the cell population into neural cells, such as at least 21 days, 22 days, 23 days, 24 days, 25 days, or 26 days after, preferably at least 22 days after.

[0210] 65. The method according to any one of the embodiments 1 to 55, wherein the cell population comprising neural cells is obtained by differentiating pluripotent stem cells (PSCs) into neural cells for 14 to 26 days, and wherein the neural cells are differentiated into neural cells specific to the hindbrain / spinal cord region.

[0211] 66. The method according to any one of the preceding embodiments, wherein the cell population is cultured in a culture medium suitable for maintaining neural cells, wherein the culture medium is a neurobasal medium.

[0212] 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.

[0213] 68. The method according to embodiment 67, wherein the culture medium does not comprise DAPT.

[0214] 69. The method according to any one of the preceding embodiments, wherein the cell population comprising neural cells is obtained by contacting the cell population with an inhibitor of Small Mothers Against Decapentaplegic (SMAD) protein signaling.

[0215] 70. The method according to any one of the preceding embodiments, wherein the cell population comprising neural 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 the embodiments 69 and 70, wherein the inhibitor(s) of SMAD signaling is selected from Noggin, LY364947, SB431542, RepSox, DMH1, DMH2, LND-212854 and LDN-193189.

[0216] 72. The method according to any one of the embodiments 1 to 56 and 66 to 71, wherein the cell population is differentiated into neural cells specific to the forebrain region comprising contacting the cell population with an inhibitor of (SMAD) protein signaling pathway for about 0-10 days, followed by contacting the cell population with FGF2 for about 7-9 days, and followed by culturing the cell population in basal neural media for an additional 9-12 days.

[0217] 73. The method according to any one of the embodiments 1 to 55 and 59 to 64 and 66 to 71 , wherein the cell population is differentiated into ventral midbrain neural cells, the method comprises 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, and, optionally, ascorbic acid and / or, optionally, Brain-derived neurotrophic factor (BDNF).

[0218] 74. The method according to the preceding embodiment, wherein following 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, and, optionally, ascorbic acid and / or, optionally, Brain-derived neurotrophic factor (BDNF), the cell population is further cultured in a culture medium suitable for maintaining neural cells 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.

[0219] 75. The method according to any one of the embodiments 73 and 74, wherein the activator of SHH signaling is selected from SHH, purmorphamine, SAG.

[0220] 76. The method according to any one of the embodiments 73 to 75, wherein the activator of Wnt signaling is selected from CHIR99021. The method according to any one of the embodiments 73 to 76, wherein the activator of FGF signaling is selected from FGF8b. The method according to any one of the embodiments 73 to 77, wherein the concentration of: a) the inhibitor(s) of SMAD protein signaling is from 1 pM to 50 pM, b) the activator of SHH signaling is from 200 ng / ml to 800 ng / ml, c) the inhibitor of Wnt signaling is from 0.1 pM to 1 pM, d) the activator of FGF signaling is from 10 ng / ml to 200 ng / ml, e) ascorbic acid is from 50 pM to 500 pM, and / or f) BDNF is from 1 ng / ml to 50 ng / ml. The method according to any one of the embodiments 73 to 78, wherein the cell population is contacted with the inhibitor of SMAD protein signaling for 5 to 9 days. The method according to any one of the embodiments 73 to 79, wherein the cell population is contacted with the inhibitor of Wnt signaling for 5 to 9 days. The method according to any one of the embodiments 73 to 80, wherein the cell population is contacted with the activator of SHH signaling for 5 to 9 days. The method according to any one of the embodiments 73 to 81 , wherein the cell population is contacted with the activator of FGF signaling for 7 to 12 days following ended contacting with the inhibitor of SMAD protein signaling, inhibitor of Wnt signaling, and / or activator of SHH signaling. The method according to any one of the embodiments 73 to 82, wherein the cell population is contacted with the inhibitor of SMAD protein signaling for 5 to 9 days from day 0, such as from day 0 to day 9. The method according to any one of the embodiments 73 to 83, wherein the cell population is contacted with the inhibitor of Wnt signaling for 5 to 9 days from day 0, such as from day 0 to day 9. 85. The method according to any one of the embodiments 73 to 84, wherein the cell population is contacted with the activator of SHH signaling for 5 to 9 days from day 0, such as from day 0 to day 9.

[0221] 86. The method according to any one of the embodiments 73 to 85, wherein the cell population is contacted with the activator of FGF signaling for 7 to 12 days from day 5 to day 9, or at the time of ending contacting with the inhibitor of SMAD protein signaling, inhibitor of Wnt signaling, and / or activator of SHH signaling.

[0222] 87. The method according to the preceding embodiment, wherein the cell population is contacted with the activator of FGF signaling from day 9 to day 16.

[0223] 88. The method according to any one of the embodiments 73 to 87, wherein the cell population is contacted with ascorbic acid for 5 to 7 days from day 10 or day 11.

[0224] 89. The method according to any one of the embodiments 73 to 88, wherein the concentration of ascorbic acid is from 10 pM to 400 pM.

[0225] 90. The method according to any one of the embodiments 73 to 89, wherein the cell population is contacted with BDNF for 5 to 7 days from day 10 or day 11.

[0226] 91. The method according to any one of the embodiments 73 to 90, wherein the concentration of BDNF is from 1 ng / ml to 40 ng / ml.

[0227] 92. The method according to any one of the embodiments 73 to 91 , wherein the cell population comprising neural cells is obtained by inducing differentiation of PSCs into neural cells by contacting the cell population with an inhibitor of SMAD protein signaling, an activator of SHH signaling, and an inhibitor of Wnt signaling, from day 0 to day 9, and, optionally, contacting the cell population with an activator of FGF signaling from day 9 to day 16.

[0228] 93. The method according to the preceding embodiment, wherein the cell population is contacted an activator of FGF signaling from day 9 to day 16. 94. The method according to any one of the embodiments 1 to 55 and 65 to 71, wherein the cell population is differentiated into neural cells specific to the hindbrain / spinal cord region comprising contacting the cell population with a caudalizing agent such as CHIR99021 from day 0-12, and a ventralizing agent such as SHH or SAG or Purmorphamine from day 6-15, and the hindbrain / spinal cord promoting compound retinoic acid from day 6-15.

[0229] 95. The method according to any one of the preceding embodiments, wherein the method is in vitro.

[0230] 96. The method according to any one of the preceding embodiments, wherein the expression of markers by the cells is measured using scRNAseq, qPCR, ICC, or flow cytometry.

[0231] 97. The method according to any one of the preceding embodiments, wherein the expression of markers by the cells is measured according to any of the methods described in Example 9, Example 13, Example 14, and Example 15.

[0232] 98. The method according to any one of the preceding embodiments, wherein the expression of markers by the cells is measured according to any of the method described in Example 14.

[0233] 99. A cell population comprising neural cells obtainable by the method according to any one of embodiments 1 to 98.

[0234] 100. A cell population comprising neural cells, wherein the cell population when cultured in vitro for 5-7 days in a culture medium suitable for maintaining neural cells results in a cell population wherein at least 35% are HuC / D positive, preferably at least 50%.

[0235] 101. The cell population according to the preceding embodiment, wherein the expression of markers by the cells is measured using scRNAseq, qPCR, flow cytometry, or ICC. 102. The cell population according to any one of the embodiments 100 and 101, wherein the expression of markers by the cells is measured according to any of the methods described in Example 9, Example 13, Example 14, and Example 15.

[0236] 103. The cell population according to any one of the embodiments 100 to 102, wherein the expression of markers by the cells is measured according to the method described in Example 14.

[0237] 104. The cell population according to any one of the embodiments 100 to 103, wherein the cells are ventral midbrain NPCs, and wherein the cell population is cultured according to Example 11.

[0238] 105. The cell population according to any one of the embodiments 1000 to 103, wherein the cells are dorsal forebrain NPCs of the pallium or LGE region, and wherein the cell population is cultured according to Example 10.

[0239] 106. The cell population according to any one of the embodiments 100 to 103, wherein the cells are of the hindbrain / spinal cord region, and wherein the cell population is cultured according to Example 12

[0240] 107. The cell population according to any one of the embodiments 99 to 106, wherein the cell population is in vitro.

[0241] 108. The cell population according to any one of the embodiments 99 to 107, wherein the neural cells are non-native.

[0242] 109. The cell population according to any one of the embodiments 99 to 108, wherein the neural cells are artificial.

[0243] 110. The cell population according to any one of the embodiments 99 to 109, wherein the cell population is derived from stem cells.

[0244] 111. The cell population according to any one of the embodiments 99 to 110, wherein the cell population is derived from pluripotent stem cells. 112. The cell population according to the preceding embodiment, wherein the pluripotent stem cells are genetically modified and wherein the genetic modifications persist in the neural cells.

[0245] 113. The cell population according to any one of the embodiments 111 to 112, wherein the neural cells are derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs).

[0246] 114. The cell population according to any one of the embodiments 99 to 113, wherein the neural cells are specific to a region selected from forebrain, midbrain, and hindbrain / spinal cord.

[0247] 115. The cell population according to the preceding embodiment, wherein the neural cells are ventral midbrain neural cells.

[0248] 116. The cell population according to any one of the embodiments 99 to 115, wherein the cells of the cell population are genetically modified.

[0249] 117. The cell population according to the preceding embodiment, wherein the cells of the cell population are genetically modified to be hypoimmunogenic and / or lineage restricted.

[0250] 118. The cell population according to the preceding embodiment, wherein the genetic modifications for hypoimmunogenicity comprise reduced expression of MHC-I human leukocyte antigens relative to a wild-type stem cell, reduced expression of MHC-II human leukocyte antigens relative to a wild-type stem cell, and / or increased expression of a tolerogenic factor relative to wild-type stem cell.

[0251] 119. The cell population according to the preceding embodiment, wherein the MHC-I human leukocyte antigens are HLA-A, HLA-B, and HLA-C.

[0252] 120. The 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. The cell population according to any one of embodiments 118 to 120, wherein the tolerogenic factor is selected from CD46, CD47, CD55, CD59, PD-L1, HLA-E, and HLA-G.

[0253] 122. The cell population according to any one of the embodiments 99 to 121 , wherein the cell population comprises at least 1,000 cells, such as at least 10,000 cells, 100,000 cells, 1 ,000,000 cells, or 10,000,000 cells.

[0254] 123. The cell population according to any one of the embodiments 99 to 122, wherein the cell population is cryopreserved.

[0255] 124. A composition comprising the cell population according to any one of the preceding embodiments and a cryoprotectant.

[0256] 125. The composition according to the preceding embodiment, wherein the cryoprotectant comprises DMSO.

[0257] 126. An in vitro cell population according to any one of the preceding embodiments or a composition according to any one of the preceding embodiments for use as a medicament.

[0258] 127. The in vitro cell population according to the preceding embodiment for the treatment of a neurological condition.

[0259] 128. The in vitro cell population according to the preceding 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 wherein neurons are lost or dysfunctional.

[0260] 129. The in vitro cell population according to embodiment 126 for the treatment of Parkinson’s disease, wherein the neural cells are ventral midbrain neural cells.

[0261] 130. The in vitro cell population according to embodiment 126 for the treatment of stroke, wherein the neural cells are forebrain neural cells. 131. The in vitro cell population according to embodiment 126 for the treatment of paralysis, spinal cord injury, diabetic neuropathy, or chronic pain, wherein the neural cells are hindbrain / spinal cord neural cells.

[0262] 132. A method of treatment of a neurological condition comprising the administration to a patient of an effective amount of a cell population according to any one of the preceding embodiments.

[0263] 133. The method according to the preceding 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 wherein neurons are lost or dysfunctional.

[0264] 134. The method according to embodiment 132, wherein the neurological condition is Parkinson’s disease.

[0265] 135. A method for treatment of a neurological condition, comprising administering to a subject in need thereof a therapeutically effective amount of a cell population comprising neural cells, and an inhibitor of NOTCH signaling, wherein the inhibitor of NOTCH signaling is selected from LY411575, Avagacestat, Dibenzazepine, and PF- 03084014.

[0266] 136. The method according to the preceding embodiment, wherein the inhibitor of NOTCH signaling is LY411575.

[0267] 137. A composition comprising the cell population comprising neural cells, and an inhibitor of NOTCH signaling, wherein the inhibitor of NOTCH signaling is selected from LY411575, Avagacestat, Dibenzazepine, and PF-03084014.

[0268] 138. The composition according to the preceding embodiment, wherein the inhibitor of NOTCH signaling is LY411575.

[0269] 139. LY411575 for use in the treatment of a neurological condition. 140. LY411575 for use in the treatment of Parkinson’s disease in a subject being administered with a therapeutically effective amount of ventral midbrain neural cells.

[0270] 141. LY411575 for use in the treatment of stroke in a subject being administered with a therapeutically effective amount of dorsal forebrain neural cells.

[0271] 142. Avagacestat for use in the treatment of a neurological condition.

[0272] 143. Avagacestat for use in the treatment of Parkinson’s disease in a subject being administered with a therapeutically effective amount of ventral midbrain neural cells.

[0273] 144. Avagacestat for use in the treatment of stroke in a subject being administered with a therapeutically effective amount of dorsal forebrain neural cells.

[0274] 145. Dibenzazepine for use in the treatment of a neurological condition.

[0275] 146. Dibenzazepine for use in the treatment of Parkinson’s disease in a subject being administered with a therapeutically effective amount of ventral midbrain neural cells.

[0276] 147. Dibenzazepine for use in the treatment of stroke in a subject being administered with a therapeutically effective amount of dorsal forebrain neural cells

[0277] 148. PF-03084014 for use in the treatment of a neurological condition.

[0278] 149. PF-03084014 for use in the treatment of Parkinson’s disease in a subject being administered with a therapeutically effective amount of ventral midbrain neural cells.

[0279] 150. PF-03084014 for use in the treatment of stroke in a subject being administered with a therapeutically effective amount of dorsal forebrain neural cells

[0280] Examples

[0281] The following are non-limiting examples for carrying out the invention. Example 1 : Differentiation of human pluripotent stem cells to dorsal forebrain neural cells

[0282] Human embryonic stem cell (hESCs) lines RC17 (Roslin CT) and 3053 (Novo Nordisk A / S) were cultured in iPS Brew XF media (Miltenyi Biotec) supplemented with 60 U / rnL Penicillin-Streptomycin (P-S; Thermo Fisher Scientific) on human laminin-521 matrix (0.7-1.2 pg / cm2; Biolamina) coated culture ware. Media was changed daily, and cells passaged with EDTA 0.5mM (Thermo Fisher Scientific) every 4-6 days. Cultures were maintained at 37°C, humidity 95% and a 5% CO2 level. hESCs were differentiated to dorsal forebrain neurons according to an established protocol (Shi et al. 2011 ; Shi et al 2012). hESCs were seeded and cultured on laminin-521 (1.2 pg / cm2) (BioLamina) coated culture ware, and once forming a 95-100% confluent monolayer exposed to differentiation media. From DIVO-19, the cells were cultured in an CPN-based media: 50% DMEM / F12+Glutamax (Gibco) 50% Neurobasal (Gibco), 2% B27 supplement with vitamin A CTS (Thermo Fisher), 1 % N2 supplement CTS (Thermo Fisher), 5% GlutaMAX (Thermo Fisher), 0.2% Penicillin streptomycin (P / S; Thermo Fisher), 1% NEAA (Gibco), 0,089% B-Mercaptoethanol (Gibco), supplemented with BMP pathway inhibitors SB431542 (10 pM; Miltenyi Biotec) and Noggin (100 ng / mL; Miltenyi Biotec) for neural induction. On DIV11 , the cells were dissociated with 0.5mM EDTA and passaged at a 1 :2 ratio. N2 / B27-based media was supplemented with fibroblast growth factor b (20 ng / mL; R&D) from DIV11-18. On DIV18, the cells were dissociated with 0.5mM EDTA and either cryopreserved or passaged at a 1 :2 ratio. From DIV19 onwards, the cells were cultured in CPN base media (Figure 2).

[0283] Example 2: Administration of NOTCH inhibitor LY411575 to dorsal forebrain neural cultures

[0284] To generate hPSC derived cell cultures or cell products which are enriched in neurons and dorsal forebrain neurons and depleted in other cell types, a novel inhibitor targeting the NOCTH pathway, LY411575, was administered to dorsal forebrain neural cell cultures.

[0285] FB NPCs were differentiated from hPSCs by patterning factors that induce neural / ectodermal specification and cultured until the cells acquired a dorsal forebrain identity (which occurred by DIV18). DIV 18 cultures were then profiled by flow cytometry to confirm correct patterning (Figure 3, Table 1). Specifically, cells were FB NPCs as they expressed high levels of PAX6 (>86,2%) and OTX2 (>88,6%) (Figure 3, Table 1). These cultures were left without neural / ectodermal patterning factors to further differentiate for a period of time of 12 days in vitro (DIV 18-30). For this period and at all points before this (day in vitro 0-30) the cell cultures were not exposed to any NOTCH inhibition (Figure 2). However, as they are largely at the precursor stage (as shown by high precursor marker expression and low I PC and neuronal marker expression) we anticipate that these cells could also be a cell population that can be treated with NOTCH inhibitor compounds and converted to neurons.

[0286] Cells at day in vitro 30 were profiled by flow cytometry before being exposed to NOTCH inhibitor LY411575 (Figure 4, Table 2) to assess the protein expression levels of dorsal forebrain regional identity transcription factor (PAX6 and OTX2) and cell stage markers (NSC and proliferative markers SOX2 and KI67; TBR2 which identifies I PCs; INA+ / SOX2-which identifies neurons). These cultures were observed to be dorsal forebrain in their regional identity, expressing dorsal forebrain marker PAX6 (Figure 4, Table 2). These cells were still at the NSC stage as shown by their high expression of the NSC marker SOX2 (Figure 4, Table 2). These cultures showed also some levels of intermediate precursor marker TBR2 and low levels of post-mitotic neurons identifiable by INA+ / SOX2- staining (Figure 4, Table 2).

[0287] Following compound (LY411575) administration, all groups were differentiated for a further 5 days in vitro with the absence of any NOTCH inhibitors and 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 fluorescently labelled secondary antibodies. Images were acquired on a Zeiss Axio Observer microscope using Zen 3.2 (Zen Pro) software and cells were counted using Imaged software.

[0288] IF counts show comparable results for both 24 hours administration and 72 hours administration (Figure 5 and 6, Table 3 and Table 4). All groups treated with LY411575 were found to have higher expression (of total cells) of neuronal markers HuC / D at both (92,9% and 94,4%, black bars) compared to cultures there were not contacted with any NOTCH inhibition at any point (64,1% and 61.3%, white bars). Cultures contacted with NOTCH inhibitor LY411575 were also observed to have decreased levels of NSC marker SOX2 and proliferative marker Ki67 in both conditions (Figure 5 and 6, black bars) compared to the control (Figure 5 and 6, white bars); indicating these cells are differentiating to neurons at a higher rate compared to controls in response to these inhibitors.

[0289] These results support the hypothesis that addition of NOTCH inhibitor LY411575 is able to promote the differentiation of dorsal forebrain NSCs into dorsal forebrain neurons while dramatically decreasing the amount of proliferative and unwanted cell types (Figure 5 and 6, Table 3 and Table 4). Table 1 : profile of FB cells DIV18

[0290] Table 2: profile of FB cells DIV30 Table 3: FB results of LY411575 administration for 24h

[0291] Table 4: FB results of LY411575 administration for 72h

[0292] Example 3: Differentiation of human pluripotent stem cells to ventral midbrain neural cells Human embryonic stem cell (hESCs) lines RC17 (Roslin CT) and 3053 (Novo Nordisk A / S) were cultured in iPS Brew XF media (Miltenyi Biotec) supplemented with 60 U / rnL Penicillinstreptomycin (P-S; Thermo Fisher Scientific) on human laminin-521 matrix (0.7-1.2 pg / cm2; Biolamina) coated culture ware. Media was changed daily, and cells passaged with EDTA 0.5mM (Thermo Fisher Scientific) every 4-6 days. Cultures were maintained at 37°C, humidity 95% and a 5% CO2 level. hESCs were differentiated to ventral midbrain neurons according to an established protocol (e.g. Nolbrant et al., 2017; Kirkeby et al., 2017). In brief, hESC were grown to 70-90% confluency, then disassociated with 0.5mM EDTA. The cells were seeded at 104cells / cm2in cell culture flasks or plates coated with human laminin-111 (1.2 pg / cm2; BioLamina) and immediately put into contact with differentiation media. The cells were exposed to N2-based media from days in vitro (DIV) 0-8; 50% DMEM / F12+Glutamax (Gibco), 50% Neurobasal (Gibco), 1% N2 supplement CTS (Thermo Fisher Scientific), 5% GlutaMAX (Thermo Fisher Scientific), 0.2% P-S (Thermo Fisher Scientific) and supplemented with SMAD inhibitors SB431542 (10 pM; Miltenyi Biotec), Noggin (100 ng / mL; Miltenyi Biotec) for neural induction, Sonic Hedgehog C24II (SHH; 500 ng / mL; Miltenyi Biotec) for ventral fate, GSK3p inhibitor CHIR99021 (CHIR; 0.5-0.6 pM; Miltenyi Biotec) to promote caudalization. N2-based media was supplemented with fibroblast growth factor 8b (FGF8b; 100 ng / mL; Miltenyi Biotec) from DIV9-11. At DIV11 , the cells were dissociated with accutase (Thermo Fisher Scientific) and seeded at 0.8x106cells / m2in a cell culture flask or plate coated with human laminin-111 (1.2 pg / cm2) in DIV11-16 media (Neurobasal, 2% B27 supplement without vitamin A CTS (Thermo Fisher Scientific)), 5% GlutaMAX, 0.2% P-S and supplemented with FGF8b (100 ng / mL), L- ascorbic acid (AA; 200 pM; Sigma), human Brain Derived Neurotrophic Factor (BDNF; 20 ng / mL; Miltenyi Biotec)) supplemented with Y-27632 (Miltenyi Biotec) at 10 pM. At day in vitro (DIV) 16, the cells were dissociated with accutase and either cryopreserved, or re-seeded in cell culture flasks / plates coated with poly-L-ornithine (0.002%) and Laminin-521 (1.5 pg / cm2) in B27 media supplemented with BDNF (20 ng / mL), GDNF (20 ng / mL), L-ascorbic acid (200 pM), dcAMP (500 pM), and Y-27632 (10 pM) for extended in vitro culture allowing further differentiation and maturation of ventral midbrain neural stem cells into neurons (Figure 7 and Figure 8). To prove the correct patterning, flow cytometry analysis at DIV16 is performed using regional markers expressed in the ventral midbrain FOXA2, OTX2, LMX1A and EN1 (Figure 9)

[0293] References:

[0294] • 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.

[0295] • Kirkeby A, Nolbrant S, Tikiova 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.

[0296] Example 4: Administration of NOTCH inhibitor LY411575 to ventral midbrain neural cultures To generate hPSC derived cell cultures or cell products which are enriched in neurons and ventral midbrain dopaminergic neurons and depleted in other cell types (i.e. , glial precursors, glial cells, stromal cells, proliferative cells) a novel inhibitor targeting the NOCTH pathway, LY411575, was administered to ventral midbrain neural cell cultures.

[0297] Cultures of VM NPCs first were differentiated from hPSCs by patterning factors that induce neural / ectodermal specification, ventralization and caudalization (which occurred by DIV16) and these were profiled by flow cytometry to confirm VM NPC identity (Figure 9). Specifically, cells were VM NPCs as 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 neural / ectodermal, ventralization and caudilization patterning factors to further differentiate for a period of time of 6 days in vitro (DIV 16-22) for this period and at all points before this (day in vitro 0-22) the cell cultures were not exposed to any NOTCH inhibitors (Figure 8).

[0298] Cells at day in vitro 22 were profiled by flow cytometry before being exposed to NOTCH inhibitor LY411575 (Figure 10, Table 6) to assess the protein expression levels of ventral midbrain floor plate regional identity transcription factor (FOXA2) and cell stage markers (NSC and proliferative markers SOX2 and KI67; ASCL1 which identifies I PCs; INA which identifies neurons). These analyzed cultures were observed to be ventral midbrain in their regional identity, expressing the floor plate marker FOXA2 (>82% of all cells) (Figure 10). These cells were still at the NSC stage as shown by their high expression of the NSC marker SOX2 and KI67 (>86% and >51%; Figure 10, Table 6). These cultures showed also high levels of intermediate precursor marker ASCL1 (48%, Figure 10, Table 6) and low levels of the neuron marker INA (12.8%; Figure 10, Table 6) post-mitotic neurons identifiable by INA+ / SOX2- staining (3.3%) (Figure 10 and Table 6).

[0299] After assessing the correct patterning and the maturation stage of the culture, DIV22 cells were exposed to NOTCH inhibitor LY411575 for either 24 hours or 72 hours. Following compound administration, all groups were differentiated for a further 7 days in vitro with the absence of any NOTCH inhibitors and 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 labelled secondary antibodies. Images were acquired on a Zeiss Axio Observer microscope using Zen 3.2 (Zen Pro) software and cells were counted using Imaged software.

[0300] IF counts show similar results for both 24 hours administration and 72-96 hours administration (Figure 11 and 12, Table 7 and Table 8). All groups were found to maintain high expression (of total cells) of the ventral midbrain floor plate lineage markers FOXA2 (>80%, Figure 11 and 12). Cultures contacted with NOTCH inhibitor LY411575 have an increased level of neuronal marker HuC / D after both 24 hours and 72-96 hours administration (54.8% and 67.3%; Figure 11 and 12, black bars) compared to control cells that were not contacted with NOTCH inhibition at any time (35.2% and 29.7%, Figure 11 and 12, white bars). Treated cells were also observed to have decreased levels of NSC marker SOX2 and proliferative marker Ki67 in both conditions (Figure 11 and 12, black bars) compared to the control (Figure 11 and 12, white bars).

[0301] These results support the hypothesis that addition of NOTCH inhibitor LY411575 is able to promote the differentiation of ventral midbrain floor plate NSCs into ventral midbrain floor plate neurons without altering the expression of floor plate lineage transcription factors and decrease the amount of proliferative and unwanted cell types (Figure 11 and 12, Table 7 and Table 8).

[0302] Table 5: Profile of VM cells DIV16

[0303] Table 6: Profile of VM cells DIV22

[0304] Table 7: Results after LY411575 administration for 24h

[0305] Table 8: Results after LY411575 administration for 72h-96h

[0306] Example 5: Administration of different NOTCH inhibitors to ventral midbrain neural cell culture To generate hPSC derived cell cultures or cell products which are enriched in neurons and ventral midbrain dopaminergic neurons and depleted in other cell types (i.e. , glial precursors, glial cells, stromal cells, proliferative cells) novel inhibitors targeting the NOTCH pathway, LY411575, Avagacestat and PF-03084014 were administered to ventral midbrain neural cell cultures and the effect was compared to the effect obtained by administering the NOTCH inhibitor commonly used in the field DAPT.

[0307] Cultures of VM NPCs first were differentiated from hPSCs by patterning factors that induce neural / ectodermal specification, ventralization and caudalization as described in Example 4. DIV16 cells were then profiled via flow cytometry (Figure 9, Table 5), further differentiated for a period of time of 6 days in vitro (DIV 16-22) without any NOTCH inhibitors (Figure 8) and their protein expression level was analyzed using flow cytometry (Figure 10, Table 6) as indicated in Example 4.

[0308] After assessing the correct patterning and maturation stage of the culture, DI 22 NPC cells were treated with novel NOTCH inhibitors LY411575, Avagacestat and PF-03084014 for 24 hours.

[0309] Following compound (LY411575) administration, all groups were differentiated for a further 7 days in vitro with the absence of any NOTCH inhibitors and 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 labelled secondary antibodies. Images were acquired on a Zeiss Axio Observer microscope using Zen 3.2 (Zen Pro) software and cells were counted using Imaged software.

[0310] All groups were found to maintain high expression (of total cells) of the ventral midbrain floor plate lineage markers FOXA2 (>60%, Figure 13, Table 9). Cultures contacted with novels NOTCH inhibitors LY411575 (black bars), Avagacestat (narrow striped bar) and PF-03084014 (bold striped bar) show an increased expression levels of neuron marker HuC / D compared to control cells that were not contacted with NOTCH inhibition at any time (white bars). (Figure 13, Table 9). Treated cells were also observed to have decreased levels of NSC marker SOX2 and proliferative marker Ki67 compared to the control (Figure 13, Table 9).

[0311] Interestingly, protein expression levels of neuronal marker HuC / D are higher in cultures treated with novel NOTCH inhibitors LY411575, Avagacestat and PF-03084014 compared to NOTCH inhibitor commonly used in the field DAPT (dotted bar) (Figure 13, Table 9). Additionally, all groups contacted with novel NOTCH inhibitors LY411575, Avagacestat and PF-03084014 have a lower expression level of NSC marker SOX2 and proliferative marker Ki67 compared to cells treated with DAPT (Figure 13, Table 9).

[0312] Together these results indicate that novel compounds targeting the NOTCH pathway LY411575, Avagacestat and PF-03084014 had the effect of increasing neuronal and decreasing NSCs and proliferative cell proportions. Comparisons between the results of cultures treated with novel NOTCH inhibitors (LY411575, Avagacestat and PF-03084014) and well-known NOTCH inhibitor DAPT show that novel compounds administration resulted in a higher amount of HuC / D neurons, and lower levels of NSC and proliferative markers SOX2 and Ki67 compared to DAPT administration (Figure 13, Table 9) indicating that the novel NOTCH inhibitors are better suited to solve the problem of obtaining a refined cell product which is sought after in the field of generation of stem cell-derived neural cells.

[0313] Table 9: Ventral MB NOTCHi inhibitors 24 hours

[0314] Example 6: Titration of LY411575 titration in ventral midbrain neural cultures

[0315] In order to test the range at which NOTCH inhibitor LY411575 was able to promote differentiation of ventral midbrain dopaminergic neurons and test its toxicity, a titration of the compound was performed.

[0316] VM NPC culture were differentiated from hPSCs according to Example 3. Following cell culture profile at DI 16, further differentiation in the absence of any NOTCH inhibitors until DIV22 and re-analysis of protein levels of DIV22 culture as described in Example 3 (Figure 9 and 10, Table 5 and Table 6), VM NPC were contacted with NOTCH inhibitor LY411575 at different concentrations for 24 hours. Specifically, LY411575 was administered at 0.2 pM, 0.5 pM, 2 pM, 10 pM, 40 pM and 100 pM.

[0317] Following compound (LY411575) administration, all groups were differentiated for a further 7 days in vitro without any NOTCH inhibitors and 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 labelled secondary antibodies. Images were acquired on a Zeiss Axio Observer microscope using Zen 3.2 (Zen Pro) software and cells were counted using Imaged software.

[0318] Results show that levels of floor plate marker FOXA2 remain high throughout all groups (>62.7%, Figure 19, Table 10) indicating that the cultures maintain their regional identity. Additionally, all concentration tested seem to promote the differentiation of ventral midbrain NPCs to ventral midbrain neurons to higher levels compared to cultures that have not been contacted with any NOTCH inhibitor (CONTROL: white bars) as indicated by the increased amount of neuronal marker HuC / D and the decreased levels of NSC marker S0X2 and proliferative marker Ki67 (Figure 19, Table 10).

[0319] These results show that NOTCH inhibitor LY411575 works at a wide range of concentration, promoting neuronal differentiation and decreasing the number of proliferative cells in the culture (Figure 19, Table 10). Additionally, it appears that LY411575 at higher concentrations (black bars) is not toxic for the cells and helps to further reduce the amount of unwanted and proliferative cells in the culture (Figure 19, Table 10).

[0320] Table 10: LY411575 titration

[0321] Example 7: Differentiation of human pluripotent stem cells to hindbrain / spinal cord neural cells

[0322] Undifferentiated hESCs at 80-90% confluence were disassociated with EDTA 0.5mM (Thermo Fisher) for 5-7 min at RT to detach from flasks for re-seeding; EDTA was utilized as it generates small aggregates thus allowing even distribution of cells before initiation of the differentiation procedure. Aggregates of hPSCs were seeded into 10pg / mL Laminin-521 precoated 24w / plates or T25 flasks (Sarstedt) and distributed evenly. The first 24 hours, cells were maintained with iPS-Brew (Miltenyi) and supplemented with 10pM Rho- associated kinase inhibitor Y27632 (ROCKi; Sigma) to enhance cell survival. When cells reached 90-100% confluency (after approximately 24 hours) the differentiation was initiated. For the first 6 days of differentiation, cells were maintained in “Pan Neuronal Base Media” - 40% Neurobasal (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 pM; Miltenyi Biotec) and Noggin (100 ng / mL; Miltenyi Biotec) for neural induction. 3pM CHIR99021 (CHIR; Miltenyi) was added for caudalization. Media was changed daily, and cells were washed with DPBS- / - (Invitrogen) prior to media change to remove cell death that had accumulated. At day 6, cells were dissociated using Accutase (Innovative Cell Technologies) and passaged with 10pM ROCKi Y27632 by re-seeding to 1 :6 ratio cells per cm2 coated with 10pg / mL LN-521. From days 6-12, cells were grown in Pan Neuronal Base Media supplemented with SMAD pathway inhibitors SB431542 (10 pM; Miltenyi Biotec) and Noggin (100 ng / mL; Miltenyi Biotec) and also 1 M CHIR, 40-100 nM Smoothened Agonist (SAG; Millipore) and 500 nM Retinoic acid (RA; Sigma). Media was changed daily, and cells were washed with DPBS- / -prior to media change to remove cell death. From days 12 to 15, cells were grown in Pan Neuronal Base Media (-Vitamin A) - 5% B27 supplement -Vitamin A (Thermo Fisher) - supplemented with 40-100 nM SAG and 500nM RA. At day 15, cells were dissociated using Accutase for 6-8 min at 37°C and passaged with 10pM ROCKi Y27632 by re-seeding to 1:6 ratio cells per cm2 coated with 10pg / mL LN-521. Hindbrain / spinal cord NPCs were collected at day 12 or 15 of differentiation and frequently cryopreserved, at DIV15 or DIV15-22 cells were exposed to NOTCH inhibitors for neuronal conversion. From day 18 onwards cultures were maintained in pan neuronal base media (-VitA) for neuronal maturation. Media was changed daily, and cells were washed with DPBS- / -prior to media change to remove cell death. On day 24 of differentiation, cultures were media changed every 3-4 days for the remainder of the culture duration.

[0323] Example 8: Administration of different NOTCH inhibitors to hindbrain / spinal cord neural cell culture

[0324] To generate hPSC derived cell cultures or cell products which are enriched in neurons and hindbrain / spinal cord neurons and depleted in other cell types, a novel inhibitor targeting the NOTCH pathway, LY411575, was administered to hindbrain / spinal cord neural cell cultures.

[0325] Cultures of HB / SCord NPCs first were differentiated from hPSCs by patterning factors that induce neural / ectodermal specification and ventralization (which occurred by DIV15). These were profiled by flow cytometry to confirm HB / SCord NPC identity (Figure 16, Table 11). Specifically, cells were HB / SCord NPCs as they did not express OTX2, expressed NKX6.1 , PAX6 and OLIG2 in varying amounts (Figure 16, Table 11).

[0326] After the cells acquired a HB / SCord NPC identity, they were contacted with NOTCH inhibitor LY411575 for either 24 hours or 72 hours (Figure 15) to promote neuronal maturation.

[0327] Following compound (LY411575) administration, all groups were differentiated for a further 5 days in vitro in the absence of any NOTCH inhibition and 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 fluorescently labelled secondary antibodies. Images were acquired on a Zeiss Axio Observer microscope using Zen 3.2 (Zen Pro) software and cells were counted using ImagJ Fiji software.

[0328] IF counts show similar results for both 24 hours administration and 72 hours administration (Figure 17 and 18, Table 12 and Table 13). Specifically, all groups treated with LY411575 were found to have higher expression (of total cells) of neuronal markers HuC / D after both 24 hours and 72 hours administration (77% and 80%, black bars) compared to cultures there were not contacted with any NOTCH inhibition at any point (36,3% and 21 ,6%, white bars). Cultures contacted with NOTCH inhibitor LY411575 were also observed to have decreased levels of NSC marker SOX2 and proliferative marker Ki67 in both conditions compared to the control. NSC marker SOX2 and proliferative marker Ki67 decreased from 69,9% and 41 ,1% of the control group (white bars) to 55,6% and 8.6% of the 24 hours LY411575 treated group (black bar). Similar results were achieved with the administration of LY411575 for 72 hours where levels of SOX2 and Ki67 decreased from 78% and 59,6% (white bars) to 32,3% and 9.6% (black bars) (Figure 17 and 18, Table 12 and Table 13).

[0329] These results support the hypothesis that addition of NOTCH inhibitor LY411575 is able to promote the differentiation of hindbrain / spinal cord NSCs into hindbrain / spinal cord neurons while dramatically decreasing the amount of proliferative and unwanted cell types (Figure 17 and 18, Table 12 and Table 13).

[0330] Table 11 : HB / SCord profile DIV15

[0331] Table 12: results after LY411575 administration for 24h Table 13: results after LY411575 administration for 72h

[0332] Example 9: Flow Cytometry analytical single cell protein marker expression assay

[0333] Ventral midbrain dopaminergic (vmDA) progenitor cells were generated from hESCs in 2D in vitro culture as described in Example 1 and using reagents described in Table 14. At various days after initiating the differentiation the cell culture was dissociated into a single cell suspension using Accutase, counted on a NucleoCounter NC-200 and collected in N2 media (CTS™ Neurobasal™ medium supplemented with 1 % CTS™ N-2 supplement). Dead cells were labelled using a LIVE / DEAD™ Fixable Near-IR Dead Cell Stain Kit. The cells were then resuspended in B27 media (CTS™ Neurobasal™ medium supplemented with 1% B-27™ supplement without vitamin A, 2 mM GlutaMAX™, 60 U / rnL Penicillin-Streptomycin, 10 pM ROCK inhibitor). Cells were then fixed and permeabilized using the BD Transcription Factor Buffer Set (BD Biosciences) according to the manufacturer’s instructions. The fixed cells were then stained with fluorescently conjugated antibodies, and the samples acquired on a BD LSR Fortessa (BD Biosciences) or CytoFLEX S (Beckman Coulter). The fcs files were exported and analyzed in FlowJo 10.8.1.

[0334] To determine whether a cell was considered positive in its protein expression of a marker, gates were set as routinely done in the field at the edge of the fluorescent signal of a negative control sample. Examples of negative control samples used included unstained controls, fluorescence minus one (FMO) controls and most preferably biological negative control samples; all cells present above these threshold gates when experimental samples were run were considered positive.

[0335] To measure the expression of two proteins in relation to one another, two-parameter dot plots were generated. Co-expression of the two proteins is represented by the percentage of cells in the top right quadrant (Q2), while single positive cells are represented by the amount of cells in Q1 and Q3. Table 14: List of reagents used for FACS experiment

[0336]

[0337] Example 10: Further culturing of dorsal forebrain neural cell population after NOTCH inhibition to assess expression profile

[0338] After administration with NOTCH inhibitors, cells are cultured for a further 5 days in a 2D culture in wells coated with Poly-L- ornithine (0.002%) and Laminin-521 (1.5 pg / cm2) in panneuronal media supplemented with BDNF (40 ng / mL), GDNF (40 ng / mL), L-ascorbic acid (200 pM), dcAMP (50 pM), Laminin (1ug / mL) and this time allows time for the transition of neural precursors and intermediates to terminally differentiate into dorsal forebrain neurons. After 5 days of culture the expression profile of the cell population can be assessed according to Example 14.

[0339] Example 11 : Further culturing of ventral midbrain neural cell population after NOTCH inhibition to assess expression profile

[0340] After administration with NOTCH inhibitors, cells are cultured for a further 7 days in a 2D culture in wells coated with poly-L-ornithine (0.002%) and Laminin-521 (1.5 pg / cm2) in neural supportive media supplemented with BDNF (20 ng / mL), GDNF (20 ng / mL), L-ascorbic acid (200 pM), dcAMP (500 pM), and this time allows time for the transition of neural precursors and intermediates to terminally differentiate into ventral midbrain neurons. After 5 days of culture the expression profile of the cell population can be assessed according to Example 14. Example 12: Further culturinq of hindbrain / spinal cord cell population after NOTCH inhibition to assess expression profile

[0341] After administration with NOTCH inhibitors, cells are cultured for a further 5 days in a 2D culture in wells coated with poly-L-ornithine (0.002%) and Laminin-521 (1.5 pg / cm2) in pan neuronal base media supplemented with B27 (-Vitamin A) and this time allows time for the transition of neural precursors and intermediates to terminally differentiate into hindbrain neurons. After 5 days of culture the expression profile of the cell population can be assessed according to Example 14.

[0342] Example 13: RNA sequencing methodoloqy

[0343] To perform single cell RNA sequencing (scRNA-seq) or single nuclei RNA sequencing, undifferentiated PSCs as well as 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 a NextSeq550. Data was processed using 10X cellranger and the Seurat analysis package in R programming language. Samples were analyzed, filtered for low quality or multiplet cells and analyzed separately for each individual experiment before combining the cells of the selected differentiated cell lineages of choice as well as the hPSCs into one dataset that were then analyzed using the standard Seurat workflow as outlined for Seurat version 3, i.e., normalizing using SCTransform and finally using the first 29 principal components for the unified tSNE plots.

[0344] Example 14: Immunocytochemistry (ICC)

[0345] Cells were fixed in 4% paraformaldehyde (Alfa Aesar) for 10 minutes at room temperature. Unspecific antibody binding was blocked by incubating cells with PADT buffer; phosphate-buffered saline (PBS) without Ca2+ and Mg2+ (Gibco) with 0.02% sodium azide solution (Ampliqon), 0.5% Triton X-100 (Sigma), and 5% Donkey serum (Jackson Labs) for 30 minutes, followed by overnight incubation with primary antibodies (see Table 14) at 4°C. The cells were washed 3 times with PBS without Ca2+ and Mg2+, blocked with PADT buffer for 15 minutes, and incubated with fluorophore-conjugated secondary antibodies (see Table 14) for 2 hours at room temperature, protected from light. The cells were then counterstained with DAPI (10 g / mL) for 5 minutes at room temperature, washed 3 times with PBS without Ca2+ and Mg2+, and stored at 4°C in PBS without Ca2+ and Mg2+ supplemented with 0.02% sodium azide. Images were captured with 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 DAPI nuclei can also be performed for quantification of ICC staining.

[0346] Example 15: Quantitative real time PCR (qPCR, or qRT-PCR)

[0347] To perform qPCR RNA is extracted from cells with Trizol and converted to cDNA and subsequently analyzed using quantitative real-time polymerase chain reaction (qPCR) for genes of interest such as S0X2, KI67, HuCD, NeuN, INA, ASCL1, F0XA2, TH, LMX1A, EN1 or other relevant markers for ventral midbrain neural cells. qPCR is typically performed across triplicate technical replicates for each of 3 or more independent biological replicates and normalized against housekeeping genes such as GAPDH or HPRT1.

[0348] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

[0349] Example 16: Administration of NOTCH inhibitor LY411575 to dorsal forebrain neural cultures An experiment was performed to further investigate the time point at which to add the NOTCH inhibitor. The experiment was performed along the lines of example 2. DIV18 cultures were left without neural / ectodermal patterning factors to further differentiate for a period of time of 7 days in vitro (DIV18-25) at high cell density (0.5 x 106cells / cm2- 2.0 x 106cells / cm2). DIV25 cultures were profiled by flow cytometry to confirm correct patterning for dorsal forebrain identity (Figure 21 and Table 15). At DIV25 NOTCH inhibitor was added for 1 to 2 days before cryopreservation (Figure 20). At no time point before this (day in vitro 0- 25) were the cell cultures exposed to any NOTCH inhibition. The cells at DIV27 were later thawed and analyzed with flow cytometry to confirm that addition of the NOTCH inhibitor promotes the maturation of the dorsal FB neural cells into neurons (Figure 22 and Table 16). This experiment confirms that adding the NOTCH inhibitor at DIV25 is equally good as adding the NOTCH inhibitor at DIV30.

[0350] Table 15: profile of FB cells DIV25

[0351]

[0352] Table 16: results of NOTCHi administration in dorsal FB cells (DIV27) Example 17: Administration of NOTCH inhibitor LY411575 to ventral midbrain neural cultures An experiment was performed to further investigate the time point at which to add the NOTCH inhibitor. The experiment was performed along the lines of example 4. DIV16 cultures were left without neural / ectodermal patterning factors to further differentiate for a period of time of 6 days in vitro (DIV16-22) at high cell density (0.5 x 106cells / cm2- 2.0 x 106cells / cm2). DIV22 cultures were profiled by flow cytometry to confirm correct patterning for dorsal forebrain identity (Figure 10 and Table 6). At DIV22 NOTCH inhibitor was added for 1 to 2 days before cryopreservation (Figure 23). At no time point before this (day in vitro 0-22) were the cell cultures exposed to any NOTCH inhibition. The cells at DIV24 were later thawed and analyzed with flow cytometry to confirm that addition of the NOTCH inhibitor promotes the maturation of the ventral MB neural cells into neurons (Figure 24 and Table 17). This experiment confirms that it is possible to add the NOTCH inhibitor of the invention at high density cell cultures.

[0353] Table 17: results of NOTCH inhibitor LY411575 administration in ventral MB cells (DIV24)

Claims

CLAIMS1. A method for directing differentiation of neural cells into neurons comprising obtaining a cell population comprising neural cells and contacting said cell population with an inhibitor of NOTCH signaling, wherein the inhibitor of NOTCH signaling is selected from LY411575, Avagacestat, Dibenzazepine, and PF- 03084014.

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

3. The method according to any one of the preceding claims, wherein the cell population comprising neural cells is obtained by differentiating pluripotent stem cells (PSCs) into neural cells.

4. The method according to any one of the preceding claims, wherein the cell population comprising neural cells is obtained by differentiating pluripotent stem cells (PSCs) into neural cells for 14 to 35 days.

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

6. The method according to the preceding claim, wherein the cell population comprising neural cells is obtained by differentiating pluripotent stem cells (PSCs) into neural cells for 14 to 26 days, and wherein the neural cells are differentiated into neural cells specific to the midbrain region, preferably ventral midbrain region, and wherein the cell population is not contacted with the inhibitor of NOTCH signaling until at least 20 days after initiating differentiation of the cell population into neural cells.

7. The method according to the preceding claim, wherein the cell population comprising neural cells is obtained by differentiating pluripotent stem cells (PSCs) into neural cells for 18 to 35 days, and wherein the neural cells are differentiated into neural cells specific to the forebrain region, preferably dorsal forebrain region, and wherein the cell population is not contacted with the inhibitor of NOTCH signalinguntil at least 24 days after initiating differentiation of the cell population into neural cells.

8. The method according to any one of the preceding claims, wherein at least 70% of the cell population express SOX2 and wherein 5-35% of the cell population are INA+ at the time of contacting the cell population with the inhibitor of NOTCH signaling.

9. The method according to any one of the preceding claims, wherein the neural cells are specific to the ventral midbrain region, and wherein 20-75% of the cell population are ASCL1+ at the time of contacting the cell population with the inhibitor of NOTCH signaling.

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

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

12. The method according to any one of the preceding claims, wherein the concentration of the inhibitor of NOTCH signaling is from 0.2 pM to 1000 pM.

13. The method according to any one of the preceding claims, wherein the cell population is contacted with the NOTCH inhibitor 48h prior to cryopreservation.

14. The method according to any of the preceding embodiments, wherein the cell population has a density of 0.5 x 106cells / cm2- 2.0 x 106cells / cm2at the time of contacting the cell population with the inhibitor of NOTCH signaling.

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

Images