Methods for generating cortical excitatory neurons

Abstract

The present disclosure provides a method for generating cortical excitatory neurons, a cortical excitatory neuron generated by such a method, and a composition comprising such a cell. Specifically, the present disclosure provides an in vitro method for inducing differentiation of stem cells, comprising contacting the stem cells with at least one inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling, at least one inhibitor of Wingless (Wnt) signaling, and at least one inhibitor of Notch signaling to obtain a cell population of differentiated cells, wherein at least about 50% of the differentiated cells express at least one cortical excitatory neuron marker. The present disclosure also provides the use of the cortical excitatory neurons and compositions comprising the same for preventing and / or treating neurodegenerative disorders.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 134,651, filed January 7, 2021, the contents of which are incorporated by reference in their entirety and to which priority is claimed.

[0002] 1. Introduction The present disclosure provides methods for generating cortical excitatory neurons, cortical excitatory neurons generated by such methods, and compositions comprising such cortical excitatory neurons. The present disclosure also provides uses of the cortical excitatory neurons and compositions comprising same for preventing and / or treating neurodegenerative, neurodevelopmental, and / or neuropsychiatric disorders. [Background technology]

[0003] 2.Background Previously, embryonic and somatic stem cells have been used as therapeutic agents and model systems for neurodegenerative, neurodevelopmental, and / or neuropsychiatric disorders. Research and technological developments on directed differentiation of embryonic and somatic stem cells have been conducted in the field of diseases of the central nervous system (CNS), such as Alzheimer's disease, frontotemporal dementia, Parkinson's disease, schizophrenia, autism, depression, intellectual disability, amyotrophic lateral sclerosis, and stroke. However, the results of these studies showed little in vivo ability to restore neuronal function and often led to undesirable tumor growth in patients.

[0004] Thus, there remains a need for improved methods for generating cortical neurons suitable for treating neurodegenerative, neurodevelopmental, and / or neuropsychiatric disorders. Summary of the Invention

[0005] 3. Overview of the Invention The present disclosure provides methods for generating cortical excitatory neurons, cortical excitatory neurons generated by such methods, and compositions comprising such cells. The present disclosure also provides uses of the cortical excitatory neurons and compositions comprising same for preventing and / or treating neurodegenerative, neurodevelopmental, and / or neuropsychiatric disorders.

[0006] In certain embodiments, the present disclosure provides an in vitro method for inducing differentiation of stem cells, comprising contacting the stem cells with at least one inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling and at least one inhibitor of Wingless (Wnt) signaling, and contacting the cells with at least one inhibitor of Notch signaling to obtain a cell population of differentiated cells, wherein at least about 50% of the differentiated cells express at least one cortical excitatory neuron marker, and the initial contact of the cells with the at least one inhibitor of Notch signaling is at least about 10 days after the initial contact of the cells with the at least one inhibitor of SMAD signaling. In certain embodiments, at least about 75% of the differentiated cells express at least one cortical excitatory neuron marker. In certain embodiments, at least about 95% of the differentiated cells express at least one cortical excitatory neuron marker.

[0007] In certain embodiments, the cells are cultured such that initial contacting of the cells with the at least one inhibitor of Notch signaling is about 20 days after initial contacting of the cells with the at least one inhibitor of SMAD signaling.

[0008] In certain embodiments, the cells are contacted with at least one inhibitor of Notch signaling for at least about 1 day.

[0009] In certain embodiments, the cells are contacted with at least one inhibitor of Notch signaling for up to about 20 days.

[0010] In certain embodiments, the cells are contacted with at least one inhibitor of Notch signaling for about 10 days.

[0011] In certain embodiments, the cells are contacted with at least one inhibitor of SMAD signaling for about 10 days.

[0012] In certain embodiments, the cells are contacted with at least one inhibitor of Wnt signaling for up to about 3 days.

[0013] In certain embodiments, the at least one inhibitor of Notch signaling comprises a gamma-secretase inhibitor.

[0014] In certain embodiments, the gamma-secretase inhibitor comprises DAPT, a derivative thereof, or a mixture thereof.

[0015] In certain embodiments, the at least one inhibitor of SMAD signaling is selected from an inhibitor of TGFβ / Activin-Nodal signaling, an inhibitor of bone morphogenetic protein (BMP) signaling, and combinations thereof.

[0016] In certain embodiments, the at least one inhibitor of TGFβ / activin-nodal signaling comprises an inhibitor of ALK5.

[0017] In certain embodiments, the at least one inhibitor of TGFβ / activin-nodal signaling comprises SB431542 or a derivative thereof, or a mixture thereof.

[0018] In certain embodiments, the at least one inhibitor of TGFβ / activin-nodal signaling comprises SB431542.

[0019] In certain embodiments, the at least one inhibitor of BMP signaling comprises LDN193189, Noggin, dorsomorphin, derivatives thereof, or mixtures thereof.

[0020] In certain embodiments, the inhibitor of at least one BMP comprises LDN-193189.

[0021] In certain embodiments, the at least one inhibitor of Wnt signaling comprises a compound selected from the group consisting of XAV939, IWP2, DKK1, IWR1, IWP L6, Wnt-C59, JW 55, derivatives thereof, and combinations thereof.

[0022] In certain embodiments, the at least one inhibitor of Wnt signaling comprises XAV939.

[0023] In certain embodiments, the at least one cortical excitatory neuron marker is selected from TBR1 (T-Box Brain 1), MAP2 (microtubule associated protein 2), FOXG1, CTIP2, DCX, TUBB3, FOXP2, vGlut1 / 2, TLE4, and combinations thereof.

[0024] In certain embodiments, the differentiated cells do not express at least one marker selected from KI67, CTIP2 (chicken ovalbumin upstream promoter transcription factor interacting protein 2), SATB2 (special AT-rich sequence binding protein 2), and combinations thereof.

[0025] In certain embodiments, the stem cells are selected from human, non-human primate, or rodent non-embryonic stem cells; human, non-human primate, or rodent embryonic stem cells; human, non-human primate, or rodent induced pluripotent stem cells; and human, non-human primate, or rodent recombinant pluripotent cells.

[0026] In certain embodiments, the stem cells are human stem cells.

[0027] In certain embodiments, the stem cells are pluripotent or multipotent stem cells.

[0028] In certain embodiments, the stem cells are pluripotent stem cells.

[0029] In certain embodiments, the pluripotent stem cells are selected from embryonic stem cells, induced pluripotent stem cells, and combinations thereof.

[0030] In certain embodiments, the present disclosure provides a cell population of in vitro differentiated cells, wherein the in vitro differentiated cells are obtained by the methods of the present disclosure.

[0031] In certain embodiments, the present disclosure provides cell populations of in vitro differentiated cells, wherein at least about 50%, at least about 75%, or at least about 95% of the cells express at least one cortical excitatory neuron marker.

[0032] In certain embodiments, the at least one cortical excitatory neuron marker is selected from TBR1, MAP2, CTIP2, FOXG1, DCX, TUBB3, FOXP2, vGlut1 / 2, TLE4, and combinations thereof.

[0033] In certain embodiments, less than about 30% of the differentiated cells express at least one marker selected from KI67, CTIP2, SATB2, and combinations thereof.

[0034] In certain embodiments, the present disclosure provides a composition comprising a cell population of the present disclosure.

[0035] In certain embodiments, the composition is a pharmaceutical composition further comprising a pharma- ceutically acceptable carrier.

[0036] In certain embodiments, the present disclosure provides a kit for inducing differentiation of stem cells into cortical excitatory neurons, comprising: (a) at least one inhibitor of SMAD signaling; (b) at least one inhibitor of Wnt signaling; (c) at least one inhibitor of Notch signaling.

[0037] In certain embodiments, the kit further comprises (d) instructions for inducing differentiation of the stem cells into a population of differentiated cells expressing at least one cortical excitatory neuron marker.

[0038] In certain embodiments, the present disclosure provides a method of preventing and / or treating a neurodegenerative, neurodevelopmental and / or neuropsychiatric disorder in a subject, the method comprising administering to the subject an effective amount of a cell population disclosed herein or a composition disclosed herein.

[0039] In certain embodiments, the neurodegenerative or neurodevelopmental and / or neuropsychiatric disorder is selected from Alzheimer's disease, frontotemporal dementia, Parkinson's disease, schizophrenia, autism, depression, intellectual disability, amyotrophic lateral sclerosis, and stroke.

[0040] In certain embodiments, the present disclosure provides a cell population of the present disclosure or a composition of the present disclosure for use in preventing and / or treating a neurodegenerative, neurodevelopmental, and / or neuropsychiatric disorder in a subject.

[0041] In certain embodiments, the neurodegenerative, neurodevelopmental, and / or neuropsychiatric disorder is selected from Alzheimer's disease, frontotemporal dementia, Parkinson's disease, schizophrenia, autism, depression, intellectual disability, amyotrophic lateral sclerosis, and stroke. 4. Brief description of the drawings [Brief description of the drawings]

[0042] [Figure 1A]1A-1C show robust induction of cortical precursor cells from hPSCs. FIG. 1A shows a method of generating cortical excitatory neurons according to certain embodiments of the presently disclosed subject matter. FIG. 1B shows qRT-PCR analysis data, which showed progressive and robust induction of telencephalic cortical markers and efficient downregulation of pluripotency-related genes. FIG. 1C shows uniform induction of cortical specific genes FOXG1 and PAX6, and minimal contamination of ventral telencephalic markers. [Figure 1B] 1A-1C show robust induction of cortical precursor cells from hPSCs. FIG. 1A shows a method of generating cortical excitatory neurons according to certain embodiments of the presently disclosed subject matter. FIG. 1B shows qRT-PCR analysis data, which showed progressive and robust induction of telencephalic cortical markers and efficient downregulation of pluripotency-related genes. FIG. 1C shows uniform induction of cortical specific genes FOXG1 and PAX6, and minimal contamination of ventral telencephalic markers. [Figure 1C] 1A-1C show robust induction of cortical precursor cells from hPSCs. FIG. 1A shows a method of generating cortical excitatory neurons according to certain embodiments of the presently disclosed subject matter. FIG. 1B shows qRT-PCR analysis data, which showed progressive and robust induction of telencephalic cortical markers and efficient downregulation of pluripotency-related genes. FIG. 1C shows uniform induction of cortical specific genes FOXG1 and PAX6, and minimal contamination of ventral telencephalic markers. [Figure 2A] Figures 2A-2D show rapid depletion of precursor cells and synchronized neurogenesis. Figure 2A shows that Ki67+ precursor cells were rapidly depleted and a pure population of MAP2+ neurons was generated by day 25 of differentiation. Figure 2B provides EdU dating studies that confirmed the generation of approximately age-matched neurons. Figure 2C shows quantification. Figure 2D shows the generation of a highly pure population of neurons with TBR1+ identity (>80%) by synchronized neurogenesis. [Figure 2B]Figures 2A-2D show rapid depletion of precursor cells and synchronized neurogenesis. Figure 2A shows that Ki67+ precursor cells were rapidly depleted and a pure population of MAP2+ neurons was generated by day 25 of differentiation. Figure 2B provides EdU dating studies that confirmed the generation of approximately age-matched neurons. Figure 2C shows quantification. Figure 2D shows the generation of a highly pure population of neurons with TBR1+ identity (>80%) by synchronized neurogenesis. [Figure 2C] Figures 2A-2D show rapid depletion of precursor cells and synchronized neurogenesis. Figure 2A shows that Ki67+ precursor cells were rapidly depleted and a pure population of MAP2+ neurons was generated by day 25 of differentiation. Figure 2B provides EdU dating studies that confirmed the generation of approximately age-matched neurons. Figure 2C shows quantification. Figure 2D shows the generation of a highly pure population of neurons with TBR1+ identity (>80%) by synchronized neurogenesis. [Figure 2D] Figures 2A-2D show rapid depletion of precursor cells and synchronized neurogenesis. Figure 2A shows that Ki67+ precursor cells were rapidly depleted and a pure population of MAP2+ neurons was generated by day 25 of differentiation. Figure 2B provides EdU dating studies that confirmed the generation of approximately age-matched neurons. Figure 2C shows quantification. Figure 2D shows the generation of a highly pure population of neurons with TBR1+ identity (>80%) by synchronized neurogenesis. [Figure 3A] Figures 3A-3B show the maintenance of synchronized pure hPSC-derived cortical neurons with high viability in long-term culture. Figure 3A provides representative images of hPSC-derived cortical neurons in long-term culture. Figures 3B and 3C provide qRT-PCR analysis for KI67 / MAP2 marker expression, which confirmed the maintenance of synchrony and purity conditions in long-term culture. [Figure 3B]Figures 3A-3B show the maintenance of synchronized pure hPSC-derived cortical neurons with high viability in long-term culture. Figure 3A provides representative images of hPSC-derived cortical neurons in long-term culture. Figures 3B and 3C provide qRT-PCR analysis for KI67 / MAP2 marker expression, which confirmed the maintenance of synchrony and purity conditions in long-term culture. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] 5. Detailed Description The present disclosure provides methods for generating cortical excitatory neurons, cortical excitatory neurons generated by such methods, and compositions comprising such cells. The present disclosure also provides uses of the cortical excitatory neurons and compositions comprising same for preventing and / or treating neurodegenerative disorders.

[0044] The present disclosure is based at least on the discovery that the methods of the present disclosure generate populations of highly pure synchronized excitatory cortical neurons, which can further be maintained in vivo for extended periods of time with high viability and purity.

[0045] Non-limiting embodiments of the presently disclosed subject matter are illustrated in the specification and by way of examples.

[0046] For purposes of clarity of disclosure, and not by way of limitation, the detailed description is divided into the following subsections.

[0047] 5.1. Definition; 5.2. Methods for differentiating stem cells; 5.3. Cell populations and compositions; 5.4. Methods of treating neurodegenerative disorders; and 5.5.Kit

[0048] 5.1.Definition The terms used herein generally have their ordinary meanings in the art, within the context of this disclosure and within the specific context in which each term is used. Certain terms are discussed below or elsewhere in the specification to provide additional guidance to the practitioner in describing the compositions and methods of the present disclosure and how to make and use them.

[0049] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 3 or more than 3 standard deviations, as is customary in the art. Alternatively, "about" can mean within a range of up to 20%, e.g., up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, e.g., within 5-fold or within 2-fold, of a value.

[0050] As used herein, the term "signal transduction" with respect to "signal transduction proteins" refers to proteins that are activated or otherwise affected by ligand binding to membrane receptor proteins or some other stimulus. Examples of signal transduction proteins include, but are not limited to, SMADs, and Wingless (Wnt) complex proteins, including beta-catenin, NOTCH, transforming growth factor beta (TGFP), activin, nodal, and glycogen synthase kinase 3 beta (GSK3β) proteins, and bone morphogenetic proteins (BMPs). For many cell surface receptors or internal receptor proteins, the ligand-receptor interaction is not directly related to the cell's response. A ligand-activated receptor can first interact with other proteins within the cell before the ultimate physiological effect of the ligand on the cell's behavior occurs. In many cases, the behavior of a series of several interacting cellular proteins changes after receptor activation or inhibition. The entire series of cellular changes induced by receptor activation is called a signal transduction mechanism or signal transduction pathway.

[0051] As used herein, the term "signals" refers to internal and external factors that control changes in the structure and function of cells. They can be chemical or physical in nature.

[0052] As used herein, the term "ligand" refers to molecules and proteins that bind to receptors, such as transforming growth factor beta (TFGP), activin, nodal, bone morphogenetic protein (BMP), and the like.

[0053] As used herein, an "inhibitor" refers to a compound or molecule (e.g., a small molecule, peptide, peptidomimetic, natural compound, siRNA, antisense nucleic acid, aptamer, or antibody) that interferes with (e.g., reduces, decreases, suppresses, eliminates, or blocks) the signaling function of a molecule or pathway. An inhibitor can be any compound or molecule that alters any activity of a specified protein (signaling molecule, any molecule involved in a specified signaling molecule, a specified associated molecule, such as glycogen synthase kinase 3β (GSK3β)) (e.g., including, but not limited to, a signaling molecule described herein) through, for example, directly contacting SMAD signaling, contacting SMAD mRNA, causing a conformational change in SMAD, reducing SMAD protein levels, or preventing SMAD interaction with a signaling partner (e.g., including those described herein), and affecting the expression of a SMAD target gene (e.g., those described herein).

[0054] Inhibitors also include molecules that indirectly regulate biological activities, such as those of SMADs, by interfering with upstream signaling molecules. (For example, within the extracellular domain, examples of signaling molecules and effects include Noggin, which sequesters bone morphogenetic proteins and inhibits activation of ALK receptors 1, 2, 3 and 6, thus preventing downstream SMAD activation. Similarly, Chordin, Cerberus, and Follistatin similarly sequester extracellular activators of SMAD signaling. Bambi, a transmembrane protein, also acts as a pseudoreceptor to sequestrate extracellular TGFβ signaling molecules.) Antibodies that block upstream or downstream proteins are contemplated for use in neutralizing extracellular activators of protein signaling, etc. Inhibitors are described in terms of competitive inhibition (binding to the active site in such a way as to preclude or reduce binding of another known binding compound) and allosteric inhibition (binding to the protein in such a way that alters the conformation of the protein in a manner that prevents binding of the compound to the active site of the protein) in addition to inhibition induced by binding to and affecting a molecule upstream of a specified signaling molecule, which in turn causes inhibition of the specified molecule. Inhibitors can be "direct inhibitors" that inhibit the signaling target or a signaling target pathway by actually contacting the signaling target.

[0055] As used herein, the term "WNT" or "Wingless" with respect to a ligand refers to a group of secreted proteins that can interact with WNT receptors, such as those of the Frizzled and LRPDerailed / RYK receptor families (e.g., integration 1 in humans). As used herein, the term "WNT or Wingless signaling pathway" refers to a signaling pathway composed of Wnt family ligands and Wnt family receptors, such as Frizzled and LRPDerailed / RYK receptors, mediated with or without β-catenin. In certain embodiments, the WNT signaling pathway includes mediation by β-catenin, such as WNT / -catenin.

[0056] As used herein, the term "derivative" refers to a compound having a similar core structure.

[0057] As used herein, the term "cell population" or "cell population" refers to a group of at least two cells. In non-limiting examples, a cell population can include at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000 cells. A population can be a pure population that includes one cell type, for example, a population of cortical excitatory neurons, or a population of undifferentiated stem cells. Alternatively, a population can include more than one cell type, for example, a mixed cell population.

[0058] As used herein, the term "stem cell" refers to a cell that has the capacity to divide indefinitely in culture and give rise to specialized cells.

[0059] As used herein, the terms "embryonic stem cells" and "ESCs" refer to primitive (undifferentiated) cells derived from preimplantation stage embryos that can divide in culture without differentiation for extended periods of time and are known to develop into cells and tissues of the three primary germ layers. Human embryonic stem cells refer to embryonic stem cells derived from human embryos. As used herein, the terms "human embryonic stem cells" or "hESCs" refer to a type of pluripotent stem cell derived from early stage human embryos up to and including the blastocyst stage that can divide in culture without differentiation for extended periods of time and are known to develop into cells and tissues of the three primary germ layers.

[0060] As used herein, the term "embryonic stem cell line" refers to a population of embryonic stem cells cultured under in vitro conditions that allow proliferation without differentiation for up to several days, months to years.

[0061] As used herein, the term "totipotent" refers to the ability to give rise to all cell types of the body, as well as all of the cell types that make up extraembryonic tissues such as the placenta.

[0062] As used herein, the term "multipotent" refers to the ability to develop into more than one cell type of the body.

[0063] As used herein, the term "pluripotent" refers to the capacity to develop into an organism's three developmental germ layers, including endoderm, mesoderm and ectoderm.

[0064] As used herein, the term "induced pluripotent stem cells" or "iPSCs" refers to a type of pluripotent stem cell that is formed by introducing certain embryonic genes (e.g., but not limited to, OCT4, SOX2 and KLF4 transgenes) (see, e.g., Takahashi and Yamanaka Cell 126, 663-676 (2006), incorporated herein by reference) into somatic cells.

[0065] As used herein, the term "somatic cell" refers to any cell in the body other than a gamete (egg or sperm), and is sometimes referred to as an "adult" cell.

[0066] As used herein, the term "somatic (adult) stem cells" refers to relatively rare undifferentiated cells found in many organs and differentiated tissues, which have limited capacity for both self-renewal (in the laboratory) and differentiation.

[0067] As used herein, the term "neuron" refers to a nerve cell, which is the primary functional unit of the nervous system. A neuron consists of a cell body and its processes--an axon and at least one dendrite. Neurons transmit information to other neurons or cells by releasing neurotransmitters at synapses.

[0068] As used herein, the term "proliferation" refers to an increase in cell number.

[0069] As used herein, the term "undifferentiated" refers to cells that have not yet developed into a specialized cell type.

[0070] As used herein, the term "differentiation" refers to the process by which unspecialized embryonic cells acquire the characteristics of specialized cells, such as neurons, cardiac, hepatic, or muscle cells. Differentiation is usually controlled by the interaction of the cell's genes with the physical and chemical conditions outside the cell, via signaling pathways that involve proteins embedded in the cell surface.

[0071] As used herein, the term "directed differentiation" refers to the manipulation of stem cell culture conditions to induce differentiation into specific (e.g., desired) cell types, such as neural precursors, neural crest precursors, head placode precursors, and non-neural ectodermal precursors. In the context of stem cells, "directed differentiation" refers to the use of small molecules, growth factor proteins, and other growth conditions to promote the transition of stem cells from a pluripotent state to a more mature or specialized cell fate.

[0072] As used herein, the term "inducing differentiation" with respect to a cell refers to changing a default cell type (genotype and / or phenotype) to a non-default cell type (genotype and / or phenotype). Thus, "inducing differentiation in a stem cell" refers to inducing a stem cell (e.g., a human stem cell) to divide into progeny cells that have characteristics distinct from the stem cell, such as genotype (e.g., changes in gene expression determined by genetic analysis such as microarrays) and / or phenotype (e.g., changes in expression of protein markers of cortical excitatory neurons such as TBR1, MAP2, CTIP2, FOXG1, DCX, TUBB3, FOXP2, vGlut1 / 2, and TLE4).

[0073] As used herein, the term "cell culture" refers to the growth of cells in vitro in an artificial medium for research or medical treatment.

[0074] As used herein, the term "culture medium" refers to a liquid containing nutrients to cover, nourish and support cells in a culture vessel such as a petri dish, multi-well plate, etc. Culture medium may also contain growth factors that are added to produce desired changes in the cells.

[0075] As used herein, the term "contacting" a cell(s) with a compound (e.g., at least one inhibitor, activator and / or inducer) refers to providing the compound in a location that allows the cell(s) to access the compound. Contacting can be achieved using any suitable method. For example, contacting can be achieved by adding a concentrated form of the compound to a cell or cell population, e.g., in the context of a cell culture, to achieve a desired concentration. Contacting can also be achieved by including the compound as a component of a formulated culture medium.

[0076] As used herein, the term "in vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments include, but are not limited to, test tubes and cell cultures.

[0077] As used herein, the term "in vivo" refers to the natural environment (eg, an animal or a cell) and to processes or reactions that occur within a natural environment, such as embryonic development, cell differentiation, neurulation, etc.

[0078] As used herein, the term "express" with respect to a gene or protein refers to producing mRNA or protein that can be observed using an assay such as a microarray assay, an antibody staining assay, or the like.

[0079] As used herein, the term "marker" or "cell marker" refers to a gene or protein that identifies a particular cell or cell type. A cell marker may not be limited to one marker, but may refer to a "pattern" of markers such that a specified group of markers can distinguish a cell or cell type from another cell or cell type.

[0080] As used herein, the terms "derived from" or "established from" or "differentiated from" when made with reference to any cell disclosed herein refer to cells obtained (e.g., isolated, purified, etc.) from the ultimate parent cell of the cell line, tissue (such as a dissociated embryo), or fluid using any manipulation, such as, but not limited to, single cell isolation, in vitro culture, treatment and / or mutagenesis, such as with proteins, chemicals, radiation, infection with viruses, transfection with DNA sequences (e.g., with morphogens), etc., selection (such as by continuous culture) of any cells contained within the cultured parent cell. Derived cells may be selected from a mixed population by response to growth factors, cytokines, a selected course of cytokine treatment, adherence, lack of adherence, sorting procedures, etc.

[0081] An "individual" or "subject" herein is a vertebrate, such as a human or a non-human animal, such as a mammal. Mammals include, but are not limited to, humans, non-human primates, farm animals, sports animals, rodents, and pets. Non-limiting examples of non-human animal subjects include rodents, such as mice, rats, hamsters, and guinea pigs; rabbits; dogs; cats; sheep; pigs; goats; cows; horses; and non-human primates, such as apes and monkeys.

[0082] As used herein, the term "disease" refers to any condition or disorder that impairs or interferes with the normal function of a cell, tissue, or organ.

[0083] As used herein, the term "treat" or "treatment" refers to clinical intervention in an attempt to change the disease course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. The therapeutic effect of treatment includes, but is not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing the rate of disease progression, amelioration or alleviation of the disease state, and remission or improvement of prognosis. By preventing the progression of a disease or disorder, treatment can prevent deterioration by the disorder in an affected or diagnosed subject, or a subject suspected of having the disorder, but treatment can also prevent the onset of the disorder or symptoms of the disorder in a subject at risk of or suspected of having the disorder.

[0084] As used herein, the term "cortical excitatory neurons" refers to a type of cerebral cortical neuron that can release neurotransmitters, such as glutamate, that can increase the likelihood that a connected postsynaptic neuron will fire. "Cortical excitatory neurons" also refer to "cortical projection neurons." Non-limiting examples of cortical excitatory neurons include corticofugal projection neurons and cortico-cortical projection neurons.

[0085] 5.2. Methods for Differentiating Stem Cells The present disclosure provides in vitro methods for inducing differentiation of stem cells (e.g., human stem cells). The subject matter of the present disclosure provides in vitro methods for inducing differentiation of stem cells to produce cortical neurons, e.g., cortical excitatory neurons. In certain embodiments, the stem cells are pluripotent stem cells. In certain embodiments, the pluripotent stem cells are selected from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and combinations thereof. In certain embodiments, the stem cells are multipotent stem cells. Non-limiting examples of stem cells that can be used with the methods of the present disclosure include human, non-human primate, or rodent non-embryonic stem cells, embryonic stem cells, induced non-embryonic pluripotent cells, and induced pluripotent cells. In certain embodiments, the stem cells are human stem cells. Non-limiting examples of human stem cells include human pluripotent stem cells (hPSCs) (including but not limited to human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)), human parthenogenetic stem cells, primordial germ cell-like pluripotent stem cells, epiblast stem cells, F class pluripotent stem cells, somatic stem cells, cancer stem cells, or any other cells capable of lineage-specific differentiation. In certain embodiments, the stem cells are embryonic stem cells (ESCs). In certain embodiments, the stem cells are human embryonic stem cells (hESCs). In certain embodiments, the stem cells are induced pluripotent stem cells (iPSCs). In certain embodiments, the stem cells are human induced pluripotent stem cells (hiPSCs).

[0086] The present disclosure relates to stem cell derived cortical neurons, e.g., cortical excitatory neurons. In certain embodiments, differentiation of stem cells into cortical excitatory neurons includes in vitro differentiation of stem cells into cells expressing at least one cortical progenitor marker (also called "cortical progenitor cells"), and in vitro differentiation of cells expressing at least one cortical progenitor marker into cells expressing at least one cortical excitatory neuron marker (also called "cortical excitatory neurons").

[0087] In certain embodiments, the present disclosure provides methods for inducing differentiation of stem cells comprising contacting the stem cells with at least one inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling (referred to as a "SMAD inhibitor") and at least one inhibitor of Wingless (Wnt) signaling (referred to as a "Wnt inhibitor"), and contacting the cells with at least one inhibitor of Notch signaling (referred to as a "Notch inhibitor") to obtain a cell population comprising differentiated cells expressing at least one cortical excitatory neuron marker.

[0088] 5.2.1. Differentiation of stem cells into cortical progenitor cells In certain embodiments, cells expressing at least one cortical progenitor cell marker are differentiated in vitro from stem cells (e.g., human stem cells) by contacting the stem cells with at least one inhibitor of SMAD signaling and at least one inhibitor of Wnt signaling.

[0089] Non-limiting examples of cortical progenitor cell markers include Forkhead Box G1 (FOXG1), Paired Box 6 (PAX6), Empty Spiracles Homeobox 2 (EMX2), FEZ Family Zinc Finger 2 (FEZF2), OTX2, NEUROG1, NEUROG2, HES5, and combinations thereof.

[0090] Non-limiting examples of SMAD inhibitors include inhibitors of transforming growth factor beta (TGFβ) / activin-nodal signaling (referred to as "TGFβ / activin-nodal inhibitors"), and inhibitors of bone morphogenetic protein (BMP) signaling. In certain embodiments, TGFβ / activin-nodal inhibitors can neutralize ligands including TGFβ, BMP, Nodal, and activin, and / or block their signaling pathways by blocking receptors and downstream effectors. Non-limiting examples of TGFβ / activin-nodal inhibitors include those disclosed in WO 2010 / 096496, WO 2011 / 149762, WO 2013 / 067362, WO 2014 / 176606, WO 2015 / 077648, Chambers et al., Nat Biotechnol. 2009 Mar;27(3):275-80, Kriks et al., Nature. 2011 Nov 6;480(7378):547-51, and Chambers et al., Nat Biotechnol. 2012 Jul 1;30(7):715-20(2012), all of which are incorporated by reference in their entirety for all purposes. In certain embodiments, the at least one TGFβ / activin-nodal inhibitor is selected from an inhibitor of ALK5, an inhibitor of ALK4, an inhibitor of ALK7, and combinations thereof. In certain embodiments, the TGFβ / activin-nodal inhibitor comprises an inhibitor of ALK5. In certain embodiments, the TGFβ / activin-nodal inhibitor is a small molecule selected from SB431542, derivatives thereof, and mixtures thereof. "SB431542" has the CAS number 301836-41-9, 22 H 18 N 4 O 3 and the name 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide, see, for example, the following structure: [ka]

[0091] In certain embodiments, the TGFβ / activin-nodal inhibitor comprises SB431542. In certain embodiments, the TGFβ / activin-nodal inhibitor comprises a derivative of SB431542. In certain embodiments, the derivative of SB431542 is A83-01.

[0092] In certain embodiments, the at least one SMAD inhibitor comprises an inhibitor of BMP signaling (referred to as a "BMP inhibitor"). Non-limiting examples of BMP inhibitors include those disclosed in WO 2011 / 149762, Chambers et al., Nat Biotechnol. 2009 Mar;27(3):275-80, Kriks et al., Nature. 2011 Nov 6;480(7378):547-51, and Chambers et al., Nat Biotechnol. 2012 Jul 1;30(7):715-20, all of which are incorporated by reference in their entirety. In certain embodiments, the BMP inhibitor is a small molecule selected from LDN193189, noggin, dorsomorphin, derivatives thereof, and mixtures thereof. "LDN193189" refers to a C 25 H 22 N 6 It refers to the small molecule DM-3189, which has the chemical formula and IUPAC name 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline. [ka]

[0093] LDN193189 can function as a SMAD signaling inhibitor. LDN193189 is also a highly potent small molecule inhibitor of ALK2, ALK3 and ALK6, protein tyrosine kinase (PTK), and inhibits the signaling of members of the ALK1 and ALK3 family of type I TGFβ receptors, leading to the inhibition of the transmission of multiple biological signals, including bone morphogenetic protein (BMP) BMP2, BMP4, BMP6, BMP7 and activin cytokine signals, and the subsequent SMAD phosphorylation of Smad1, Smad5 and Smad8 (Yu et al. (2008) Nat Med 14:1363-1369; Cuny et al. (2008) Bioorg.Med.Chem.Lett.18:4388-4392, incorporated herein by reference).

[0094] In certain embodiments, the BMP inhibitor comprises LDN 193189. In certain embodiments, the BMP inhibitor comprises Noggin.

[0095] In certain embodiments, the stem cells are exposed to one SMAD inhibitor, for example, one TGFβ / activin-nodal inhibitor. In certain embodiments, the one TGFβ / activin-nodal inhibitor is SB431542 or A83-01. In certain embodiments, the stem cells are exposed to two SMAD inhibitors. In certain embodiments, the two SMAD inhibitors are a TGFβ / activin-nodal inhibitor and a BMP inhibitor. In certain embodiments, the stem cells are exposed to SB431542 or A83-01, and LDN193189 or Noggin. In certain embodiments, the stem cells are exposed to SB431542 and LDN193189.

[0096] In certain embodiments, the stem cells are exposed to or contacted with at least one SMAD inhibitor for at least about 3 days, at least about 5 days, at least about 10 days, or at least about 15 days. In certain embodiments, the stem cells are exposed to or contacted with at least one SMAD inhibitor for at least about 3 days. In certain embodiments, the stem cells are contacted to or exposed to at least one SMAD inhibitor for up to about 5 days, up to about 10 days, up to about 15 days, or up to about 20 days. In certain embodiments, the stem cells are contacted to or exposed to at least one SMAD inhibitor for up to about 20 days. In certain embodiments, the stem cells are contacted or exposed to at least one SMAD inhibitor for about 3 days to about 20 days, about 3 days to about 15 days, about 3 days to about 10 days, about 10 days to about 20 days, or about 10 days to about 15 days. In certain embodiments, the stem cells are contacted or exposed to at least one SMAD inhibitor for about 3 days to about 15 days. In certain embodiments, the stem cells are contacted or exposed to at least one SMAD inhibitor for about 5 days, about 10 days, about 15 days, or about 20 days. In certain embodiments, the stem cells are contacted or exposed to at least one SMAD inhibitor for about 10 days. In certain embodiments, the stem cells are contacted or exposed to at least one SMAD inhibitor for about 9 days or 10 days. In certain embodiments, the stem cells are contacted with or exposed to at least one SMAD inhibitor from day 0 to day 9. In certain embodiments, the at least one SMAD inhibitor is added daily or every other day to the cell culture medium comprising the stem cells from day 0 to day 9. In certain embodiments, the at least one SMAD inhibitor is added daily (every day) to the cell culture medium comprising the stem cells from day 0 to day 9.

[0097] In certain embodiments, the at least one SMAD inhibitor comprises a TGFβ / activin-nodal inhibitor and the cells are contacted with or exposed to the TGFβ / activin-nodal inhibitor. In certain embodiments, the concentration of the TGFβ / activin-nodal inhibitor contacted with or exposed to the cells is between about 1 μM and about 20 μM, between about 1 μM and about 10 μM, between about 1 μM and about 15 μM, between about 10 μM and about 15 μM, between about 5 μM and about 10 μM, between about 5 μM and about 15 μM, between about 5 μM and about 20 μM, or between about 15 μM and about 20 μM. In certain embodiments, the concentration of the TGFβ / activin-nodal inhibitor contacted with or exposed to the cells is between about 5 μM and about 10 μM. In certain embodiments, the concentration of TGFβ / activin-nodal inhibitor contacted with or exposed to the cells is between about 10 μM. In certain embodiments, the TGFβ / activin-nodal inhibitor comprises SB431542 or a derivative thereof (e.g., A83-01). In certain embodiments, the TGFβ / activin-nodal inhibitor comprises SB431542.

[0098] In certain embodiments, the at least one SMAD inhibitor comprises a BMP inhibitor. In certain embodiments, the cells are contacted with or exposed to a BMP inhibitor. In certain embodiments, the concentration of the BMP inhibitor contacted with or exposed to the cells is between about 10 nM and about 200 nM, or between about 10 nM and about 50 nM, or between about 10 nM and about 150 nM, or between about 10 nM and about 100 nM, or between about 50 nM and about 200 nM, or between about 50 nM and about 150 nM, or between about 50 nM and about 100 nM, or between about 100 nM and about 200 nM, or between about 100 nM and about 150 nM. In certain embodiments, the concentration of the BMP inhibitor contacted with or exposed to the cells is between about 50 nM and about 150 nM. In certain embodiments, the concentration of the BMP inhibitor contacted or exposed to the cells is about 50 nM to about 100 nM. In certain embodiments, the concentration of the BMP inhibitor contacted or exposed to the cells is about 100 nM. In certain embodiments, the BMP inhibitor comprises LDN193189 or a derivative thereof. In certain embodiments, the BMP inhibitor comprises LDN193189.

[0099] In certain embodiments, the at least one SMAD inhibitor comprises a TGFβ / activin-nodal inhibitor and a BMP inhibitor. In certain embodiments, the cells are contacted or exposed to the TGFβ / activin-nodal inhibitor and the BMP inhibitor simultaneously. In certain embodiments, the stem cells are contacted or exposed to the TGFβ / activin-nodal inhibitor and the BMP inhibitor for about 10 days. In certain embodiments, the stem cells are contacted or exposed to the TGFβ / activin-nodal inhibitor and the BMP inhibitor for about 9 or 10 days. In certain embodiments, the cells are contacted or exposed to the TGFβ / activin-nodal inhibitor and the BMP inhibitor from day 0 to day 9. In certain embodiments, the cells are exposed or contacted to the TGFβ / activin-nodal inhibitor and the BMP inhibitor every day (e.g., every day) or every other day. In certain embodiments, the TGFβ / activin-nodal inhibitor and the BMP inhibitor are added to the cell culture medium containing the stem cells every day or every other day from day 0 to day 9. In certain embodiments, the TGFβ / activin-nodal inhibitor and the BMP inhibitor are added to the cell culture medium containing the stem cells every day (daily) from day 0 to day 9.

[0100] "Wingless" or "Wnt" refers to a signaling pathway composed of Wnt family ligands and Wnt family receptors, such as Frizzled and LRPDerailed / RYK receptors, mediated with or without β-catenin. Wnt proteins are involved in several developmental processes, including tumorigenesis, and cell fate regulation and patterning during embryogenesis. The Wnt pathway includes proteins either downstream or upstream of Wnt protein activity. For example, this can include LRPS, LRP6, Dkk, GSK-3, Wnt10B, Wnt6, Wnt3 (e.g., Wnt 3A), Wnt1, or any of the other proteins discussed herein, and the genes encoding these proteins.

[0101] The Wnt pathway also includes pathways downstream of Wnt, such as the LRPS pathway or HBM pathway, the Dkk pathway, the p-catenin pathway, the MAPKAPK2 pathway, and the OPG / RANK pathway. "LRP5 pathway" and "IBM pathway" refer to any protein / gene including LRP5 or HBM mutant, and a protein downstream of LRPS or HBM mutant. "β-catenin pathway" refers to any protein / gene including β-catenin and a protein downstream of β-catenin. "MAPKAPK2 pathway" refers to any protein / gene including MAPKAPK2 and a protein downstream of MAPKAPK2. "OPG / RANKL pathway" refers to any protein / gene including OPG / RANKL, and a protein downstream of OPG and RANKL. "Dkk pathway" refers to any protein / gene involved in the interaction of Dkk-1, which is part of the Wnt pathway, with LRP5 and / or LRP6. Dkk-I inhibits LRP5 activity.

[0102] As used herein, the term "Wnt signaling inhibitor" or "Wnt inhibitor" refers to any agent that can act by directly inhibiting the normal function of Wnt protein, as well as any agent that inhibits Wnt signaling pathway and thus reproduces the function of Wnt. Wnt inhibitors include, for example, XAV939 (Hauang et al. Nature 461:614-620 (2009)), vitamin A (retinoic acid), lithium, flavonoids, Dickkopf1 (Dkk1), insulin-like growth factor binding protein (IGFBP) (WO 2009 / 131166), and siRNA against β-catenin.

[0103] Non-limiting examples of inhibitors of Wnt signaling include XAV939, IWP2, DKK1, IWR1, IWP L6, Wnt-C59, JW 55, derivatives thereof, and combinations thereof.

[0104] Additional Wnt inhibitors include, but are not limited to, IWR compounds, IWP compounds and other Wnt inhibitors described in WO 09155001 and Chen et al., Nat Chem Biol 5:100-7 (2009).

[0105] XAV939 is a potent small molecule inhibitor of tankyrase (TNKS) 1 and 2, with IC 50 The values ​​are 11 nM and 4 nM, respectively. Huang et al., Nature 461:614-620 (2009). By inhibiting TNKS activity, XAV939 increases the protein levels of the Axin-GSK3β complex and promotes the degradation of β-catenin in SW480 cells. Known Wnt inhibitors also include Dickkopf proteins, secreted Frizzled-related proteins (sFRPs), Wnt inhibitory factor 1 (WIF-1), and Soggy. Members of the Dickkopf-related protein family (Dkk-1 to Dkk-4) are secreted proteins with two cysteine-rich domains separated by a linker region. Dkk-3 and Dkk-4 also have one prokineticin domain. Dkk-1, Dkk-2, Dkk-3, and Dkk-4 function as antagonists of canonical Wnt signaling by binding to LRP5 / 6 and preventing LRP5 / 6 interaction with the Wnt-Frizzled complex. Dkk-1, Dkk-2, Dkk-3, and Dkk-4 also bind to cell surface Kremen-1 or Kremen-2 and promote LRP5 / 6 internalization. Antagonistic activity of Dkk-3 has not been demonstrated. Dkk proteins have distinct expression patterns in adult and embryonic tissues and have a wide range of effects on tissue development and morphogenesis.

[0106] The Dkk family also includes Soggy, which is homologous to Dkk-3 but not to other family members. sFRPs are a family of five Wnt-binding glycoproteins that are similar to membrane-bound Frizzled. The largest family of Wnt inhibitors includes two groups, the first consisting of sFRP1, sFRP2, and sFRP5, and the second group including sFRP3 and sFRP4. All are secreted and derived from unique genes, and there are no alternative splice forms of the Frizzled family. Each sFRP contains an N-terminal cysteine-rich domain (CRO). Other Wnt inhibitors include WIF-1 (Wnt inhibitory factor 1), a secreted protein that binds to Wnt proteins and inhibits their activity.

[0107] "IWP2" or "Inhibitor of WNT Production-2" refers to the IUPAC name N-(6-methyl-1,3-benzothiazol-2-yl)-2-[(4-oxo-3-phenyl-6,7-dihydrothieno[3,2-d]pyrimidin-2-yl)thio]acetamide, which has the following formula: [ka]

[0108] IWP-2 inhibits the WNT pathway at the level of the pathway activator porcupine (IC 50 = 27 nM). Porcupin is a membrane-bound acyltransferase that palmitoylates WNT proteins, conferring WNT secretion and signaling capacity.

[0109] In certain embodiments, the stem cells are contacted or exposed to at least one Wnt inhibitor for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, or at least about 5 days. In certain embodiments, the stem cells are contacted with at least one Wnt inhibitor for up to about 1 day, up to about 5 days, or up to about 10 days. In certain embodiments, the stem cells are contacted with at least one Wnt inhibitor for up to about 5 days. In certain embodiments, the stem cells are contacted with at least one Wnt inhibitor for up to about 3 days. In certain embodiments, the stem cells are contacted with at least one Wnt inhibitor for about 1 day to about 5 days. In certain embodiments, the stem cells are contacted with at least one Wnt inhibitor for about 2 days. In certain embodiments, the stem cells are contacted with at least one Wnt inhibitor for about 3 days. In certain embodiments, the stem cells are exposed or contacted with at least one Wnt inhibitor from day 0 to day 2. In certain embodiments, the stem cells are exposed to or contacted with at least one Wnt inhibitor every day (daily) or every other day. In certain embodiments, at least one Wnt inhibitor is added to the cell culture medium containing the stem cells every day or every other day from day 0 to day 2. In certain embodiments, at least one Wnt inhibitor is added to the cell culture medium containing the stem cells every day (daily) from day 0 to day 2.

[0110] In certain embodiments, the concentration of the at least one Wnt inhibitor contacted with or exposed to the cells is about 1 μM to about 20 μM, about 1 μM to about 15 μM, about 1 μM to about 10 μM, about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 5 μM to about 15 μM, about 15 μM to about 20 μM, about 5 μM to about 20 μM, or about 10 μM to about 15 μM. In certain embodiments, the concentration of the at least one Wnt inhibitor contacted with or exposed to the cells is about 1 μM to about 5 μM. In certain embodiments, the cells are contacted with the at least one Wnt inhibitor at a concentration of about 2 μM. In certain embodiments, the Wnt inhibitor comprises XAV939 or a derivative thereof. In certain embodiments, the Wnt inhibitor comprises XAV939.

[0111] 5.2.2. Differentiation of cortical progenitor cells into cortical excitatory neurons In certain embodiments, cells expressing at least one cortical excitatory neuron marker are differentiated in vitro from cells expressing at least one cortical progenitor cell marker (e.g., obtained by the methods described in Section 5.2.1) by contacting the cells expressing at least one cortical progenitor cell marker (e.g., obtained by the methods described in Section 5.2.1) with at least one inhibitor of Notch signaling (referred to as a "Notch inhibitor"). Exposure of the cells to at least one Notch inhibitor can promote rapid cell cycle exit and induce synchronized neurogenesis. In certain embodiments, the cells are passaged at low density to obtain synchronized and / or pure cortical excitatory neurons.

[0112] Non-limiting examples of cortical excitatory neuron markers include TBR1 (T-Box Brain 1), MAP2 (microtubule-associated protein 2), FOXG1, CTIP2, DCX, TUBB3, FOXP2, vGlut1 / 2, and TLE4.

[0113] Non-limiting examples of Notch inhibitors include DAPT (Dovey et al., Journal of neurochemistry 76, 173-181 (2001)), Begacestat (5-chloro-N-[(1S)-3,3,3 trifluoro-1-(hydroxymethyl)-2-(trifluoromethyl)propyl]-2-thiophenesulfonamide) (Mayer et al., J. Med. Chem. 51:7348 (2008)), DBZ (N-[(1S)-2-[[(7S)-6,7-dihydro-5-methyl-6-oxo-5H-dibenz[b,d]azepin-7-yl]amino]-1-methyl-2-oxoethyl]-3,5-difluorobenzeneacetamide) (van Es et al., Nature 435:959 (2005)), BMS 299897 (2-[(1R)-1-[[(4-chlorophenyl)sulfonyl](2,5-difluorophenyl)amino]ethyl-5-fluorobenzenebutanoate] (Goldstein et al., J. Pharmacol. Exp. Ther. 323:102(2007)), Compound W (3,5-bis(4-nitrophenoxy)benzoic acid) (Okochi et al., J. Biol. Chem. 281:7890(2006)), Flurizan ((R)-2-fluoro-α-methyl[1,1'-biphenyl]-4-acetic acid (Eriksen et al., J. Pharmacol. Exp. Ther. 323:102(2007)), al., J. Clin. Invest. 112:440(2003)), L-685,458 ((5S)-(tert-butoxycarbonylamino)-6-phenyl-(4R)-hydroxy-(2R)-benzylhexanoyl)-L-leucine-L-phenylalaninamide) (Shearman et al., Biochemistry 39:8698(2000)), JLK 6 (7-amino-4-chloro-3-methoxy-1H-2-benzopyran) (Petit et al., Nat. Cell. Biol. 3:507(2001)), MRK 560 (N-[cis-4-[(4-chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]-1,1,1-trifluoromethanesulfonamide) (Best et al., J. Clin. Invest. 112:440(2003)), L-685,458 ((5S)-(tert-butoxycarbonylamino)-6-phenyl-(4R)-hydroxy-(2R)-benzylhexanoyl)-L-leucine-L-phenylalaninamide) (Shearman et al., Biochemistry 39:8698(2000)), JLK 6 (7-amino-4-chloro-3-methoxy-1H-2-benzopyran) (Petit et al., Nat. Cell. Biol. 3:507(2001)), al., J. Pharm. Exp. Ther.317:786(2006)), PF 3084014 hydrobromide ((2S)-2-[[(2S)-6,8-difluoro-1,2,3,4-tetrahydro-2-naphthalenyl]amino]-N-[1-[2-[(2,2-dimethylpropyl)amino]-1,1-dimethylethyl]-1H-imidazol-4-yl]pentanamide dihydrobromide) (Lanz et al., J. Pharmacol. Exp. Ther. 334:269(2010)), or derivatives thereof.

[0114] In certain embodiments, the term "DAPT" refers to an example of a gamma-secretase inhibitor that inhibits NOTCH, which is N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester; LY-374973, N--[N-(3,5-difluorophenylacetyl)-L-alanyl]-S-phenylglycine t-butyl ester; N--[N-(3,5-difluorophenylacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (C 23 H 26 F 2 N 2 O 4 DAPT derivatives have been described as dipeptidic γ-secretase specific inhibitors, also known as DAPT derivatives having the chemical formula: ... [ka]

[0115] In certain embodiments, the at least one Notch inhibitor is selected from DAPT, BMS 299897, Compound E, Compound W, DBZ, L-685,458, PF 3084014 hydrobromide, derivatives thereof, and combinations thereof.

[0116] The present inventors have discovered that initial contact or exposure of cells to at least one Notch inhibitor can affect the purity of cortical neurons (e.g., cortical excitatory neurons), for example, to obtain a pure and synchronized population of cortical neurons (e.g., cortical excitatory neurons), which can further be maintained in long-term culture.

[0117] In certain embodiments, the initial contact or exposure of the cells (e.g., cortical progenitor cells) with at least one Notch inhibitor is at least about 10 days, at least about 15 days, or at least about 20 days from the initial contact or exposure of the cells with at least one SMAD inhibitor. In certain embodiments, the initial contact or exposure of the cells with at least one Notch inhibitor is within about 15 days, within about 20 days, or within about 25 days from the initial exposure of the stem cells to at least one SMAD inhibitor. In certain embodiments, the initial contact or exposure of the cells with at least one Notch inhibitor is between about 10 days and about 25 days, between about 10 days and about 20 days, between about 10 days and about 15 days, or between about 15 days and about 20 days from the initial contact of the cells with at least one SMAD inhibitor. In certain embodiments, the initial contacting of the cells with the at least one Notch inhibitor is about 20 days after the initial contacting of the cells with the at least one SMAD inhibitor.

[0118] In certain embodiments, cells (e.g., cortical progenitor cells) are exposed to or contacted with at least one Notch inhibitor for at least about 1 day, at least about 5 days, at least about 10 days, at least about 15 days, at least about 20 days, at least about 25 days, or at least about 30 days. In certain embodiments, cells (e.g., cortical progenitor cells) are exposed to or contacted with at least one Notch inhibitor for at least about 1 day. In certain embodiments, cells are contacted with or exposed to at least one Notch inhibitor for up to about 5 days, about 10 days, up to about 15 days, up to about 20 days, up to about 25 days, or up to about 30 days. In certain embodiments, cells are contacted with or exposed to at least one Notch inhibitor for up to about 10 days. In certain embodiments, the cells are contacted with or exposed to at least one Notch inhibitor for about 1 day to about 30 days, about 1 day to about 20 days, about 1 day to about 15 days, about 1 day to about 10 days, about 1 day to about 5 days, about 10 days to about 15 days, about 10 days to about 20 days, about 20 days to about 30 days, about 10 days to about 30 days, or about 15 days to about 20 days. In certain embodiments, the cells are contacted with or exposed to at least one Notch inhibitor for about 1 day to about 10 days. In certain embodiments, the cells are contacted with or exposed to at least one Notch inhibitor for about 10 days. In certain embodiments, the stem cells are contacted with or exposed to at least one Notch inhibitor from about day 20 to about day 30, with day 0 being the day the cells are first exposed to at least one SMAD inhibitor. In certain embodiments, the cells are exposed to at least one Notch inhibitor every day, every other day, or about every 5 days. In certain embodiments, the at least one Notch inhibitor is added to the cell culture medium containing the cells (e.g., cortical progenitor cells) every day, every other day, or about every 5 days from about day 20 to about day 30.In certain embodiments, at least one Notch inhibitor is added to cell culture medium containing cells (eg, cortical progenitor cells) about every 5 days from about day 20 to about day 30.

[0119] In certain embodiments, the concentration of the Notch inhibitor contacted with or exposed to a cell (e.g., a cortical progenitor cell) is between about 1 μM and about 20 μM, between about 1 μM and about 10 μM, between about 1 μM and about 15 μM, between about 10 μM and about 15 μM, between about 5 μM and about 10 μM, between about 5 μM and about 15 μM, between about 5 μM and about 20 μM, or between about 15 μM and about 20 μM. In certain embodiments, the concentration of the Notch inhibitor contacted with or exposed to a cell is about 5 μM to about 10 μM. In certain embodiments, the concentration of the Notch inhibitor contacted with or exposed to a cell is about 10 μM. In certain embodiments, the Notch inhibitor comprises DAPT or a derivative thereof. In certain embodiments, the Notch inhibitor comprises DAPT.

[0120] In certain embodiments, the cells are dissociated and replated prior to initial contacting of the cells with at least one Notch inhibitor.

[0121] 5.2.3. Cell Culture Media In certain embodiments, the inhibitor is added to the cell culture medium containing the cells. Suitable cell culture media include, but are not limited to, Knockout Serum Replacement ("KSR") medium, Neurobasal® medium (NB), N2 medium, B-27 medium, and Essential 8® / Essential 6® ("E8 / E6") medium, and combinations thereof. KSR medium, NB medium, N2 medium, B-27 medium, E8 / E6 medium are commercially available. KSR medium is a defined serum-free formulation optimized for the growth and maintenance of undifferentiated hESCs in culture.

[0122] In certain embodiments, the cell culture medium is E8 / E6 medium. E8 / E6 medium is a feeder-free and xeno-free medium that supports the growth and proliferation of human pluripotent stem cells. E8 / E6 medium has been shown to support somatic cell reprogramming. Furthermore, E8 / E6 medium can be used as a base for the formulation of custom media for the culture of PSCs. An example of E8 / E6 medium is described in Chen et al., Nat Methods 2011 May;8(5):424-9, which is incorporated by reference in its entirety. An example of E8 / E6 medium is disclosed in WO 15 / 077648, which is incorporated by reference in its entirety. In certain embodiments, the E8 / E6 cell culture medium is composed of DMEM / F12, ascorbic acid, selenium, insulin, NaHCO 3 , transferrin, FGF2, and TGFβ. E8 / E6 medium differs from KSR medium in that E8 / E6 medium does not contain active BMP or Wnt components. Thus, in certain embodiments, when E8 / E6 medium is used to culture and differentiate a stem cell population of the present disclosure into a cortical progenitor cell population, it is not necessary to add at least one inhibitor of SMAD signaling (e.g., one that inhibits BMP) to the E8 / E6 medium. In certain embodiments, the cell culture medium for differentiating stem cells into cortical progenitor cells (e.g., as described in Section 5.2.1) is E8 / E6 medium containing at least one SMAD inhibitor (e.g., SB431542, optionally LDN19318). In certain embodiments, the cell culture medium for differentiating stem cells into cortical progenitor cells from stem cells (e.g., as described in Section 5.2.1) is E8 / E6 medium containing at least one SMAD inhibitor (e.g., SB431542, optionally LDN19318) and at least one Wnt inhibitor. In certain embodiments, the cell culture medium from days 0 to 2 is E8 / E6 medium containing at least one SMAD inhibitor (e.g., SB431542, optionally LDN19318) and at least one Wnt inhibitor. In certain embodiments, the cell culture medium from days 3 to 9 is E8 / E6 medium containing at least one SMAD inhibitor (e.g., SB431542, optionally LDN19318).

[0123] In certain embodiments, the cell culture medium for differentiating stem cells into cortical progenitor cells (e.g., as described in Section 5.2.1) is N2 / B27 medium. In certain embodiments, the cell culture medium from day 10 to day 20 is N2 medium supplemented with B27.

[0124] In certain embodiments, the cell culture medium for differentiating cortical progenitor cells into cortical excitatory neurons (e.g., as described in Section 5.2.2) is NB medium containing at least one Notch inhibitor (e.g., DAPT). In certain embodiments, the cell culture medium from day 20 to day 29 is N2 medium containing at least one Notch inhibitor (e.g., DAPT). In certain embodiments, the N2 medium is further supplemented with B27, L-glutamine, and Y-27632.

[0125] In certain embodiments, the cortical excitatory neurons (e.g., obtained according to the method described in Section 5.2.2) are cultured in a maturation medium. In certain embodiments, the maturation medium is NB medium. In certain embodiments, the NB medium comprises one or more molecules selected from L-glutamine, brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), cyclic adenosine monophosphate (cAMP), and ascorbic acid (AA).

[0126] In certain embodiments, the cortical excitatory neurons can be maintained in the maturation medium for an extended period of time. In certain embodiments, the cortical excitatory neurons can be maintained in the maturation medium for at least about 10 days, at least about 50, at least about 10 weeks, at least about 15 weeks, at least about 20 weeks, at least about 25 weeks, at least about 30 weeks, or at least about 40 weeks. In certain embodiments, the cortical excitatory neurons can be maintained in the maturation medium for about 10 weeks. In certain embodiments, the cell culture medium is KSR medium. The components of KSR medium are disclosed in WO 2011 / 149762. In certain embodiments, the KSR medium comprises Knockout DMEM, Knockout serum replacement, L-glutamine, Pen / Strep, MEM, and 13-mercaptoethanol. In a specific embodiment, 1 liter of KSR medium contains 820 mL of Knockout DMEM, 150 mL of Knockout Serum Replacement, 10 mL of 200 mM L-glutamine, 10 mL of Pen / Strep, 10 mL of 10 mM MEM, and 55 μM 13-mercaptoethanol.

[0127] 5.3. Cell Populations and Compositions The present disclosure provides cell populations of in vitro differentiated cells, in which at least about 50% (e.g., at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%) of the differentiated cells express at least one cortical excitatory neuron marker. In certain embodiments, at least about 75% of the differentiated cells express at least one cortical excitatory neuron marker. In certain embodiments, at least about 95% of the differentiated cells express at least one cortical excitatory neuron marker.

[0128] The cortical excitatory neuronal cell populations of the present disclosure can be maintained in vitro for extended periods of time, in certain embodiments, the cortical excitatory neuronal cell populations of the present disclosure can be maintained in vitro for at least about 10 days, at least about 50, at least about 10 weeks, at least about 15 weeks, at least about 20 weeks, at least about 25 weeks, at least about 30 weeks, or at least about 40 weeks.

[0129] Non-limiting examples of cortical excitatory neuron markers include TBR1, MAP2, CTIP2, FOXG1, DCX, TUBB3, FOXP2, vGlut1 / 2 and TLE4.

[0130] The present disclosure also provides compositions comprising such a population of cortical excitatory neuronal cells. In certain embodiments, the in vitro differentiated cells are obtained by the differentiation methods described herein, for example, in Section 5.2.

[0131] In certain embodiments, less than about 30% (e.g., less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, or less than about 0.1%) of the differentiated cells express at least one marker selected from KI67, CTIP2 (chicken ovalbumin upstream promoter transcription factor interacting protein 2), SATB2 (special AT-rich sequence binding protein 2), and combinations thereof.

[0132] In certain embodiments, the cells are included in a composition that further comprises a biocompatible scaffold or matrix, such as a biocompatible three-dimensional scaffold that promotes tissue regeneration when the cells are placed or implanted in a subject. In certain embodiments, the biocompatible scaffold comprises an extracellular matrix material, a synthetic polymer, a cytokine, a collagen, a polypeptide or protein, a polysaccharide including fibronectin, laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparin sulfate, chondroitin sulfate, agarose or gelatin, and / or a hydrogel. (See, e.g., U.S. Patent Application Publication Nos. 2015 / 0159135, 2011 / 0296542, 2009 / 0123433, and 2008 / 0268019, the contents of each of which are incorporated by reference in their entirety.)

[0133] In certain embodiments, the composition comprises about 1×10 4 ~Approx. 1×10 10 , about 1×10 4 ~Approx. 1×10 5 , about 1×10 5 ~Approx. 1×10 9 , about 1×10 5 ~Approx. 1×10 6 , about 1×10 5 ~Approx. 1×10 7 , about 1×10 6 ~Approx. 1×10 7 , about 1×10 6 ~Approx. 1×10 8 , about 1×10 7 ~Approx. 1×10 8 , about 1×10 8 ~Approx. 1×10 9 , about 1×10 8 ~Approx. 1×10 10 , or about 1 × 10 9 ~Approx. 1×10 10 of in vitro differentiated cortical excitatory neurons.

[0134] In certain embodiments, the composition is frozen. In certain embodiments, the composition further comprises at least one cryoprotectant, such as, but not limited to, dimethylsulfoxide (DMSO), glycerol, polyethylene glycol, sucrose, trehalose, dextrose, or a combination thereof.

[0135] In certain embodiments, the composition further comprises a biocompatible scaffold or matrix, for example, a biocompatible three-dimensional scaffold that promotes tissue regeneration when the cells are placed or implanted in a subject. In certain embodiments, the biocompatible scaffold comprises an extracellular matrix material, a synthetic polymer, a cytokine, a collagen, a polypeptide or protein, a polysaccharide including fibronectin, laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparin sulfate, chondroitin sulfate, agarose or gelatin, and / or a hydrogel. (See, e.g., U.S. Patent Application Publication Nos. 2015 / 0159135, 2011 / 0296542, 2009 / 0123433, and 2008 / 0268019, the contents of each of which are incorporated by reference in their entirety.)

[0136] In certain embodiments, the composition is a pharmaceutical composition comprising a pharma- ceutical acceptable carrier.The composition can be used to prevent and / or treat neurodegenerative disease, neurodevelopmental disease, and / or neuropsychiatric disorder.Non-limiting examples of neurodegenerative disease, neurodevelopmental disease, and / or neuropsychiatric disorder include Alzheimer's disease, frontotemporal dementia, Parkinson's disease, schizophrenia, autism, depression, intellectual disability, amyotrophic lateral sclerosis, and stroke.

[0137] The presently disclosed subject matter also provides a device comprising differentiated cortical excitatory neurons or a composition comprising the same, as disclosed herein. Non-limiting examples of devices include syringes, fine glass tubes, stereotactic needles and cannulas.

[0138] 5.4. Methods for Treating Neurodegenerative Disorders The cell populations and compositions disclosed herein (e.g., those disclosed in Section 5.3) can be used to prevent and / or treat neurodegenerative, neurodevelopmental, and / or neuropsychiatric disorders. The subject matter of the present disclosure provides methods of preventing and / or treating neurodegenerative, neurodevelopmental, and / or neuropsychiatric disorders. In certain embodiments, the methods include administering a stem cell-derived cortical excitatory neuron of the present disclosure or a composition thereof to a subject suffering from a neurodegenerative, neurodevelopmental, and / or neuropsychiatric disorder. In certain embodiments, the composition is a pharmaceutical composition further comprising a pharma- ceutically acceptable carrier.

[0139] Non-limiting examples of neurodegenerative disorders include Alzheimer's disease, frontotemporal dementia, Parkinson's disease, schizophrenia, autism, depression, intellectual disability, amyotrophic lateral sclerosis, and stroke.

[0140] In certain embodiments, the neurodegenerative, neurodevelopmental, and / or neuropsychiatric disorder is Parkinson's disease, whose primary motor symptoms include, but are not limited to, tremors in the hands, arms, legs, jaw, and face, bradykinesia or slowness of movement, stiffness or rigidity of the limbs and trunk, and postural instability or impaired balance and coordination.

[0141] In certain embodiments, Parkinson's disease refers to a disease associated with a lack of dopamine in the basal ganglia, the part of the brain that controls movement.Symptoms include tremor, bradykinesia (extremely slow movement), flexed posture, postural instability, and rigidity.Non-limiting examples of Parkinsonism disease include corticobasal degeneration, dementia with Lewy bodies, multiple system atrophy, and progressive supranuclear palsy.

[0142] The cells or compositions can be administered or provided systemically or directly to a subject to treat or prevent a neurodegenerative, neurodevelopmental, and / or neuropsychiatric disorder. In certain embodiments, the cells or compositions are directly injected into an organ of interest (e.g., the central nervous system (CNS)). In certain embodiments, the cells or compositions are directly injected into the cerebral cortex.

[0143] The cells or compositions can be administered in any physiologically acceptable vehicle. The cells or compositions can be administered by local intracranial injection, orthotopic (OT) injection, systemic injection, intravenous injection, or parenteral administration. In certain embodiments, the cells or compositions are administered to a subject suffering from a neurodegenerative disorder via intracranial injection, for example, local intracranial injection into the CNS.

[0144] The cells or compositions may be conveniently provided as a sterile liquid preparation, for example, an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition that may be buffered to a selected pH. Liquid preparations are usually easier to prepare than gels, other viscous compositions, and solid compositions. Furthermore, liquid compositions are somewhat more convenient to administer, particularly by injection. Viscous compositions, on the other hand, may be formulated within an appropriate viscosity range to provide a longer contact period with a particular tissue. A liquid or viscous composition may include a carrier, which may be, for example, a solvent or dispersion medium containing water, saline, phosphate buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof. A sterile injectable solution may be prepared by incorporating the subject composition of the present disclosure, for example, a composition comprising the stem cell-derived cortical excitatory neurons of the present disclosure, in a required amount of an appropriate solvent, with various amounts of other ingredients as required. Such compositions may be mixed with suitable carriers, diluents, or excipients, such as sterile water, saline, glucose, dextrose, and the like. The compositions may also be lyophilized. Depending on the route of administration and the desired preparation, the composition may contain auxiliary substances such as wetting agents, dispersing or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or thickening additives, preservatives, flavoring agents, coloring agents, etc. Suitable preparations can be prepared without undue experimentation by reference to standard texts such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985, which is incorporated herein by reference.

[0145] Various additives that enhance the stability and sterility of the compositions can be added, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, etc. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, alum monostearate and gelatin.

[0146] The viscosity of the composition can be maintained at a selected level using a pharma- ceutically acceptable thickening agent if desired. Methylcellulose can be used because it is readily and economically available and easy to handle. Other suitable thickening agents include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, and the like. The concentration of the thickening agent can depend on the drug selected. The important point is to use an amount that achieves the selected viscosity. The selection of suitable carriers and other additives depends on the exact route of administration and the nature of the particular dosage form, for example, a liquid dosage form (e.g., whether the composition is formulated into a solution, suspension, gel, or another liquid form, for example, a time-release form or a liquid-filled form).

[0147] Those skilled in the art will recognize that the components of the composition should be selected to be chemically inert and not affect the viability or efficacy of the stem cell-derived precursors of the present disclosure. This does not present a problem to those skilled in the art of chemical and pharmaceutical principles, or can be easily avoided from this disclosure and the documents cited herein, by reference to standard textbooks, or by simple experimentation (without undue experimentation).

[0148] One consideration regarding therapeutic use of cells is the amount of cells required to achieve an optimal effect, including but not limited to, repopulation of CNS regions in a subject suffering from a neurodegenerative disorder and / or improvement in the function of the CNS in a subject.

[0149] In certain embodiments, the composition comprises an effective amount of stem cell-derived cortical excitatory neurons. As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount sufficient to affect a beneficial or desired clinical outcome during treatment. An effective amount can be administered to a subject in at least one dose. In terms of treatment, an effective amount is an amount sufficient to alleviate, improve, stabilize, reverse or delay the progression of a neurodegenerative or pituitary disorder, or to reduce the pathological consequences of a neurodegenerative disorder. An effective amount is generally determined by a physician on a case-by-case basis and is within the skill of a person of ordinary skill in the art. Several factors are usually taken into consideration when determining the appropriate dosage to achieve an effective amount. These factors include the age, sex and weight of the subject, the condition being treated, the severity of the condition, and the form and effective concentration of the cells administered.

[0150] In certain embodiments, the effective amount of cells is sufficient to repopulate CNS regions in a subject suffering from a neurodegenerative, neurodevelopmental, and / or neuropsychiatric disorder. In certain embodiments, the effective amount of cells is sufficient to improve the function of the CNS in a subject suffering from a neurodegenerative, neurodevelopmental, and / or neuropsychiatric disorder, for example, the improved function can be about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, or about 100% of the function of a normal CNS.

[0151] The amount of cells administered will vary depending on the subject being treated. In certain embodiments, about 1×10 4 ~Approx. 1×10 10 , about 1×10 4 ~Approx. 1×10 5 , about 1×10 5 ~Approx. 1×10 9 , about 1×10 5 ~Approx. 1×10 6 , about 1×10 5 ~Approx. 1×10 7 , about 1×10 6 ~Approx. 1×10 7 , about 1×10 6 ~Approx. 1×108 , about 1×10 7 ~Approx. 1×10 8 , about 1×10 8 ~Approx. 1×10 9 , about 1×10 8 ~Approx. 1×10 10 , or about 1 × 10 9 ~Approx. 1×10 10 In certain embodiments, about 1×10 cells are administered to the subject. 5 pieces~approx. 1×10 7 In certain embodiments, about 1×10 cells are administered to a subject suffering from a neurodegenerative disorder. 6 pieces~approx. 1×10 7 In certain embodiments, about 1×10 cells are administered to a subject suffering from a neurodegenerative disorder. 6 pieces~approx. 4×10 6 The cells are administered to a subject suffering from a neurodegenerative disorder. The exact determination of what will be considered an effective dose may be based on factors individual to each subject, including the size, age, sex, weight and condition of the particular subject. Dosage amounts can be readily ascertained by those skilled in the art from this disclosure and the knowledge of those skilled in the art.

[0152] 5.5.Kit The subject matter of the present disclosure provides a kit for inducing differentiation of stem cells into cortical excitatory neurons. In certain embodiments, the kit comprises (a) at least one inhibitor of SMAD signaling, (b) at least one inhibitor of Wnt signaling, and (c) at least one inhibitor of Notch signaling. In certain embodiments, the kit further comprises (d) instructions for inducing differentiation of the stem cells into a population of differentiated cells expressing at least one cortical excitatory neuron marker.

[0153] In certain embodiments, the instructions include contacting the stem cells with the inhibitors in a particular order.

[0154] In certain embodiments, the instructions include contacting the stem cells with the inhibitor according to the methods described in Section 5.2.

[0155] In certain embodiments, the present disclosure provides a kit comprising an effective amount of a unit dosage form of a cell population or composition disclosed herein. In certain embodiments, the kit comprises a sterile container containing the therapeutic composition, which may be a box, an ampoule, a bottle, a vial, a tube, a bag, a pouch, a blister pack, or other suitable container form known in the art. Such a container may be made of plastic, glass, laminated paper, metal foil, or other material suitable for holding pharmaceutical agents.

[0156] In certain embodiments, the kit includes instructions for administering the cell population or composition to a subject suffering from a neurodegenerative, neurodevelopmental, and / or neuropsychiatric disorder. The instructions may include information regarding the use of the cells or composition to treat and / or prevent a neurodegenerative, neurodevelopmental, and / or neuropsychiatric disorder. In certain embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosing schedule and administration for treating and / or preventing a neurodegenerative, neurodevelopmental, and / or neuropsychiatric disorder, or a symptom thereof; cautions; warnings; instructions; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions may be printed directly on the container (if present), or as a label affixed to the container, or as a separate sheet, pamphlet, card, or folder provided in or with the container. EXAMPLES

[0157] 6. Working Example The presently disclosed subject matter will be better understood by reference to the following examples, which are provided by way of illustration of the presently disclosed subject matter, and not by way of limitation. Example 1: Generation of synchronized pure cortical excitatory neurons from hPSCs

[0158] The examples disclosed herein describe the directed differentiation of hPSCs into synchronized populations of cortical excitatory neurons with very high purity in two steps.

[0159] Step 1: hPSCs were robustly differentiated towards a homogenous population of dorsal cortical progenitor cells expressing specific markers including FOXG1 and PAX6 (Figures 1A-1C).

[0160] Step 2: Synchronized TBR1 cells are then cultured to promote rapid cell cycle exit and can be maintained in long-term culture. + Cortical precursor cells were passaged under conditions that induce the generation of pure populations of cortical excitatory neurons (Figures 2A-2D, Figures 3A-3B).

[0161] Step 1 involved patterning hPSCs towards a homogenous population of cortical progenitor cells: Day -1: hPSCs were dissociated as single cells and plated on Matrigel-coated plates at 3 × 10 5 cells / cm 2 The cells were seeded at a density of 10 μM and cultured in E8 medium containing 10 μM Y-27632. □ Days 0-2: Cells were fed daily with E6 medium supplemented with 10 μM SB431542, 100 nM LDN193189 and 2 μM XAV939. □ Days 3-9: Cells were fed daily with E6 supplemented with 10 μM SB431542 and 100 nM LDN19318. □ Days 10-20: Cells were fed daily with N2 / B27 medium. Step 2 involved passaging and synchronized neurogenesis: Days 20-29: Precursor cells are dissociated into single cell suspensions and plated at 1-1.5 × 10 5 cells / cm 2 Cells were cultured in NB medium supplemented with B27, L-glutamine, 10 uM Y-27632, and 10 uM DAPT, and half of the medium in each plate was replaced every 5 days until day 30. Day 30 - endpoint: Cells were maintained in NB supplemented with B27, L-glutamine, 20 μg / ml BDNF, 20 μg / ml GDNF, 200 μM dbcAMP, 200 μM ascorbic acid (AA). Half of the medium was changed approximately every 4-5 days.

[0162] Step 1 of the disclosed method robustly induced the differentiation of hPSCs toward a homogenous population of cortical precursor cells. The mRNA levels of pluripotency-associated genes NANOG and OCT4, and telencephalic cortex markers FOXG1, Emx2, Pax6, and Fezf2 were determined. As shown in FIG. 1B, telencephalic cortex markers were progressively and robustly induced, and pluripotency-associated genes were efficiently reduced. The cortex-specific markers FOXG1 and PAX6, and the ventral telencephalic markers Gsx2 and Nkx2.1 were also evaluated. Immunofluorescence staining revealed homogenous induction of cortex-specific genes FOXG1 and PAX6, and minimal contamination of ventral telencephalic markers (FIG. 1C).

[0163] Step 2 of the disclosed method rapidly depleted precursor cells and synchronized neurogenesis. Ki67+ precursor cells were rapidly depleted, generating a pure population of MAP2+ neurons by day 25 of differentiation (Figures 2A and 2C). 5-ethynyl-2'-deoxyuridine (EdU) developmental date determination confirmed the generation of neurons of approximately the same age (Figures 2B and 2C). Notably, synchronized neurogenesis generated a highly pure population of neurons of TBR1+ identity (>80%) (Figure 2D).

[0164] The disclosed method was able to maintain synchronized and pure hPSC-derived cortical neurons with high viability in long-term culture. Representative images of hPSC-derived cortical neurons in long-term culture are shown in Figure 3A. qRT-PCR analysis of KI67 / MAP2 marker expression confirmed the maintenance of synchrony and purity conditions in long-term culture (Figure 3B).

[0165] Although the subject matter of the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the present disclosure. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the processes, machines, manufacture, and compositions of matter, means, methods and steps described herein. Those skilled in the art will readily appreciate from this disclosure of the presently disclosed subject matter, processes, machines, manufacture, compositions of matter, means, methods, or steps, now existing or later developed, which perform substantially the same function or achieve substantially the same result, as corresponding embodiments described herein can be utilized in accordance with the presently disclosed subject matter. It is therefore intended that the appended claims include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.

[0166] Various patents, patent applications, publications, product descriptions, protocols, and sequence accession numbers are cited throughout this application, the disclosures of which are hereby incorporated by reference in their entireties for all purposes.

Claims

1. 1. An in vitro method for inducing differentiation of a stem cell, comprising:

20. The method of claim 19, further comprising contacting the stem cells with at least one inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling and at least one inhibitor of Wingless (Wnt) signaling, and contacting the cells with at least one inhibitor of Notch signaling to obtain a cell population of differentiated cells, wherein at least about 50%, at least about 75%, or at least about 95% of the differentiated cells express at least one cortical excitatory neuron marker, and wherein initial contacting of the cells with the at least one inhibitor of Notch signaling is at least about 10 days after initial contacting of the cells with the at least one inhibitor of SMAD signaling.

2. 2. The method of claim 1, wherein the initial contacting of the cells with the at least one inhibitor of Notch signaling is about 20 days after the initial contacting of the cells with the at least one inhibitor of SMAD signaling.

3. The method of claim 1 or 2, wherein the cells are contacted with the at least one inhibitor of Notch signaling for at least about 1 day.

4. The method according to any one of claims 1 to 3, wherein the cells are contacted with the at least one inhibitor of Notch signalling for up to about 20 days.

5. The method according to any one of claims 1 to 4, wherein said cells are contacted with said at least one inhibitor of Notch signalling for about 10 days.

6. The method of any one of claims 1 to 5, wherein said cells are contacted with said at least one inhibitor of SMAD signaling for about 10 days.

7. The method of any one of claims 1 to 6, wherein said cells are contacted with said at least one inhibitor of Wnt signaling for up to about 3 days.

8. The method of any one of claims 1 to 7, wherein the at least one inhibitor of Notch signalling comprises a γ-secretase inhibitor.

9. The method of claim 8, wherein the gamma-secretase inhibitor comprises DAPT, a derivative thereof, or a mixture thereof.

10. 10. The method of any one of claims 1 to 9, wherein the at least one inhibitor of SMAD signaling is selected from an inhibitor of TGFβ / Activin-Nodal signaling, an inhibitor of bone morphogenetic protein (BMP) signaling, and combinations thereof.

11. The method of claim 10, wherein the at least one inhibitor of TGFβ / activin-nodal signaling comprises an inhibitor of ALK5.

12. The method of claim 10 or 11, wherein the at least one inhibitor of TGFβ / activin-nodal signaling comprises SB431542 or a derivative thereof, or a mixture thereof.

13. The method of any one of claims 10 to 12, wherein the at least one inhibitor of TGFβ / activin-nodal signaling comprises SB431542.

14. 14. The method of claim 13, wherein the at least one inhibitor of BMP signaling comprises LDN193189, Noggin, dorsomorphin, derivatives thereof, or mixtures thereof.

15. The method of claim 10 or 14, wherein the inhibitor of at least one BMP comprises LDN-193189.

16. 16. The method of any one of claims 1 to 15, wherein the at least one inhibitor of Wnt signaling comprises a compound selected from the group consisting of XAV939, IWP2, DKK1, IWR1, IWP L6, Wnt-C59, JW 55, derivatives thereof, and combinations thereof.

17. The method of any one of claims 1 to 16, wherein the at least one inhibitor of Wnt signaling comprises XAV939.

18. 18. The method of any one of claims 1 to 17, wherein the at least one cortical excitatory neuron marker is selected from TBR1 (T-Box Brain 1), MAP2 (Microtubule Associated Protein 2), FOXG1, CTIP2, DCX, TUBB3, FOXP2, vGlut1 / 2, TLE4, and combinations thereof.

19. 19. The method of any one of claims 1 to 18, wherein the differentiated cells do not express at least one marker selected from KI67, CTIP2 (chicken ovalbumin upstream promoter transcription factor interacting protein 2), SATB2 (special AT-rich sequence binding protein 2), and combinations thereof.

20. 20. The method of any one of claims 1 to 19, wherein the stem cells are selected from human, non-human primate or rodent non-embryonic stem cells; human, non-human primate or rodent embryonic stem cells; human, non-human primate or rodent induced pluripotent stem cells; and human, non-human primate or rodent recombinant pluripotent cells.

21. The method according to any one of claims 1 to 20, wherein the stem cells are human stem cells.

22. The method according to any one of claims 1 to 21, wherein the stem cells are pluripotent or multipotent stem cells.

23. The method according to any one of claims 1 to 22, wherein the stem cells are pluripotent stem cells.

24. The method according to any one of claims 1 to 12, wherein the pluripotent stem cells are selected from embryonic stem cells, induced pluripotent stem cells, and combinations thereof.

25. A cell population of in vitro differentiated cells, said in vitro differentiated cells being obtainable according to the method of any one of claims 1 to 24.

26. A cell population of in vitro differentiated cells, wherein at least about 50%, or at least about 75%, or at least about 95% of the cells express at least one cortical excitatory neuron marker.

27. 27. The cell population of claim 26, wherein the at least one cortical excitatory neuron marker is selected from TBR1, MAP2, FOXG1, CTIP2, DCX, TUBB3, FOXP2, vGlut1 / 2, TLE4, and combinations thereof.

28. 28. The cell population of claim 26 or 27, wherein less than about 30% of the differentiated cells express at least one marker selected from KI67, CTIP2, SATB2, and combinations thereof.

29. A composition comprising the cell population according to any one of claims 25 to 28.

30. 30. The composition of claim 29, which is a pharmaceutical composition further comprising a pharma- ceutically acceptable carrier.

31. A kit for inducing differentiation of stem cells into cortical excitatory neurons, comprising: (a) at least one inhibitor of SMAD signaling; (b) at least one inhibitor of Wnt signaling; (c) at least one inhibitor of Notch signaling.

32. 32. The kit of claim 31, further comprising: (d) instructions for inducing differentiation of the stem cells into a population of differentiated cells expressing at least one cortical excitatory neuron marker.

33. A composition comprising the cell population of any one of claims 25 to 28, or the composition of claim 29 or 30, for preventing and / or treating a neurodegenerative, neurodevelopmental and / or neuropsychiatric disorder in a subject.

34. 34. The composition of claim 33, wherein the neurodegenerative disorder, the neurodevelopmental disorder, and / or the neuropsychiatric disorder is selected from Alzheimer's disease, frontotemporal dementia, Parkinson's disease, schizophrenia, autism, depression, intellectual disability, amyotrophic lateral sclerosis, and stroke.

35. 31. The composition of any one of claims 29 or 30 for use in the prevention and / or treatment of a neurodegenerative, neurodevelopmental and / or neuropsychiatric disorder in a subject.

36. 36. The composition for use according to claim 35, wherein the neurodegenerative disorder, the neurodevelopmental disorder and / or the neuropsychiatric disorder is Alzheimer's disease, frontotemporal dementia, Parkinson's disease, schizophrenia, autism, depression, intellectual disability, amyotrophic lateral sclerosis, and stroke.

Images