WO2022020798A1 - Methods of differentiating neurons and identification of disease phenotypes thereof - Google Patents

Abstract

The present disclosure provides methods of lineage specific differentiation of pluripotent stem cells, including induced pluripotent stem cells, into inhibitory and excitatory neurons. Also provided are methods of identifying disease phenotypes using the method of differentiation, including disease phenotypes related to neurodevelopmental diseases or disorders.

Description

METHODS OF DIFFERENTIATING NEURONS AND IDENTIFICATION OF DISEASE PHENOTYPES THEREOF

Cross-Reference to Related Applications

[0001] The subject patent application claims the benefit of priority to U.S. Provisional Patent Application Number 63/056,487 (filed July 24, 2020; now pending). The full disclosure of the priority application is incorporated herein by reference in its entirety and for all purposes.

Field

[0002] The present disclosure relates to methods of lineage specific differentiation of pluripotent stem cells, including induced pluripotent stem cells, into inhibitory and excitatory neurons. Also provided are methods of identifying disease phenotypes using the methods of differentiation, including disease phenotypes related to neurodevelopmental diseases or disorders.

Background

[0003] Various methods for differentiating pluripotent stem cells into lineage specific cell populations and the resulting cellular compositions are contemplated for studying disease phenotypes. However, in some cases, such methods are limited in their ability to produce particular types of cells consistently, such as inhibitory GABAergic neurons or excitatory neurons, or in their ability to accurately model a disease or disorder. Improved methods and cellular compositions thereof are needed, including to provide for improved methods for differentiating cells, such as for use in identifying phenotypes associated with a neurodevelopmental disease or disorder.

Brief Description of the Drawings

[0004] Figure 1 shows a schematic of GABAergic differentiation, including duration of Wnt inhibition and FGF2 treatment as variables. [0005] Figure 2 shows a directed GABAergic differentiation protocol, including Wnt inhibition during neural induction.

[0006] Figure 3 shows miniature inhibitory postsynaptic current (mIPSC) traces, recorded in the presence in tetrodotoxin (TTX) or bicuculline.

[0007] Figure 4 shows representative mIPSC traces of control and FXS neurons on Day 52 and Day 62 of the directed differentiation protocol.

[0008] Figure 5 shows superimposed mIPSC events obtained by averaging individual events of control and FXS neurons.

[0009] Figure 6 shows measurements of mIPSC frequency, rise time and amplitudes (Wilcoxon signed-rank tests; * p < 0.05, ** p < 0.01, *** p < 0.001).

[0010] Figure 7 shows spontaneous firing activity as measured from Day 47 to Day 77 of the directed differentiation protocol in control and FXS cultures (Wilcoxon signed-rank tests; p < 0.05, ** p < 0.01, *** p < 0.001).

[0011] Figure 8 shows burst frequency, frequency in bust, and number of total spikes within burst as measured from Day 47 to Day 77 of the directed differentiation protocol in control and FXS cultures (Wilcoxon signed-rank tests; p < 0.05, ** p < 0.01, *** p < 0.001).

[0012] Figure 9 shows quantification of expression of KCC1, KCC2 and MAP2 on Day 52, Day 62 and Day 72 of the directed differentiation protocol by immunohistochemistry (* p <

0.05, ** p < 0.01, *** p < 0.001).

[0013] Figure 10 shows mosaic FXS-iPSC lines subcloned into cell lines with larger or smaller size CGG trinucleotide expansions.

[0014] Figure 11 shows a schematic of the excitatory neuronal differentiation protocol.

[0015] Figure 12 shows expression of FOXG1 on Day 15 of the differentiation protocol.

[0016] Figure 13 shows action potentials upon depolarization in control and FXS-derived cells, as measured by whole-cell patch clamp.

[0017] Figure 14 shows that differentially expressed genes are enriched with autism- associated genes, especially at later time points. (A) Overall, 27 differentially expressed genes overlap with 144 Category I genes from the SFARI database. The over-representation test was carried out by hypergeometric test, and p-value of overlap by chance is 2.56e-05. (B) List of overlapping genes. (C) Over-representation test using the list of overlapping genes suggests these genes play a role in the histone lysine methylation process. (D) Overlap between SFARI genes and DEGs by cell type and time point. Dotted line indicates statistical significance (p < 0.05). Detailed Description

[0018] The present disclosure relates to methods of lineage specific differentiation of pluripotent stem cells (PSCs), such as embryonic stem (ES) cells or induced pluripotent stem cells (iPSCs). Specifically described are methods of directing lineage specific differentiation of PSCs, including isogenic PSC lines, into inhibitory GABAergic neurons and excitatory neurons, and use of such methods to identify disease or disorder phenotypes associated therewith. As exemplified herein (e.g., Example 1), effectiveness and reliability of the methods of the invention are demonstrated by consistent expression of key marker genes. Also encompassed by the invention are inhibitory neurons and excitatory neurons that can be obtained with the differentiation methods of the invention. The cells, including those at any stage of the differentiation methods provided herein, are further contemplated for various uses including, but not limited to, identification of disease phenotypes.

[0019] In some aspects, PSCs are differentiated into inhibitory GABAergic neurons or inhibitory GABAergic neural precursors. In some aspects, PSCs are differentiated into inhibitory GABAergic neurons. In some aspects, PSCs are differentiated into inhibitory GABAergic neural precursors. In some aspects, iPSCs are differentiated into inhibitory GABAergic neurons or inhibitory GABAergic neural precursors. In some aspects, iPSCs are differentiated into inhibitory GABAergic neurons. In some aspects, iPSCs are differentiated into inhibitory GABAergic neural precursors. In some aspects, embryonic stem cells (ESCs) are differentiated into inhibitory GABAergic neurons or inhibitory GABAergic neural precursors. In some aspects, ESCs are differentiated into inhibitory GABAergic neurons. In some aspects, ESCs are differentiated into inhibitory GABAergic neural precursors.

[0020] In some aspects, PSCs are differentiated into excitatory neurons or excitatory neural precursors. In some aspects, PSCs are differentiated into excitatory neurons. In some aspects, PSCs are differentiated into excitatory neural precursors. In some aspects, iPSCs are differentiated into excitatory neurons or excitatory neural precursors. In some aspects, iPSCs are differentiated into excitatory neurons. In some aspects, iPSCs are differentiated into excitatory neural precursors. In some aspects, embryonic stem cells (ESCs) are differentiated into excitatory neurons or excitatory neural precursors. In some aspects, ESCs are differentiated into excitatory neurons. In some aspects, ESCs are differentiated into excitatory neural precursors. [0021] The provided embodiments address problems related to the recapitulation and identification of disease or disorder phenotypes in cellular models, such as those associated with neurodevelopmental disorders.

[0022] FXS (Fragile X syndrome) is the leading monogenic cause of intellectual disability and autism spectrum disorder, affecting as many as 1 in 3,600 in the general population (Cornish, Turk et al. 2008). It is an X-linked disease caused by the expansion of a trinucleotide repeat in the 5’UTR region of the FMR1 (Fragile X Mental Retardation- 1) gene. This results in the hypermethylation of its locus, silencing the transcription and translation of FMR1. Fragile X Mental Retardation Protein (FMRP, encoded by FMR1 ) has numerous roles in regulating RNA metabolism and translation; it directly binds to both mRNA and translational machinery (Pasciuto and Bagni 2014). FMRP recognizes the quadruplex RNA structure (Suhl, Chopra et al. 2014), regulates RNA stability (Zalfa, Eleuteri et al. 2007, La Fata, Gartner et al. 2014, Li, Stockton et al. 2016) binds to ribosomal proteins and stall ribosomal translocation. FMRP -null cells in general have upregulated global protein synthesis (Jacquemont, Pacini et al. 2018). The study of FXS is particularly interesting because of its relationship with autism spectrum disorder (ASD). 60% of males and 20% of females who have been diagnosed with FXS meet the diagnostic criteria of ASD. Notably, many risk variants associated with ASD are on genes regulated by FMRP. Expression of ASD associated genes peaks during the third quarter of prenatal development (Schork, Won et al. 2019), towards the end of neurogenesis (Malik, Vinukonda et al. 2013).

[0023] Previous studies have focused largely on synaptogenesis and circuit malformation caused by loss of FMRP in FMR1 knockout (KO) mice, especially in the excitatory neurons (Hou, Antion et al. 2006, Napoli, Mercaldo et al. 2008, Edbauer, Neilson et al. 2010, Darnell, Van Driesche et al. 2011, He, Nomura et al. 2014). Much less is known about deficits in inhibitory neurons in FXS. Studies in fetal GABAergic neuron development in FXS are also lacking.

[0024] While current studies of disease mechanisms underlying FXS have focused largely on the effect of FMRP loss in excitatory neurons, emerging evidence suggests that GABAergic inhibitory networks are also affected in FXS and such perturbations appear early in development. However, research in embryonic development of GABAergic neurons in FXS is relatively sparse, and the potential aberrancies in the neurogenesis of GABAergic lineage neurons in FXS is not well characterized. [0025] Findings described herein indicate a delay in the GABAA reversal potential switching in the maturing FXS inhibitory neural network. Such phenotypic alterations indicate a maturation delay in FXS GABAergic neurogenesis that may have profound implications for excitatory-inhibitory circuit development.

[0026] Further, there is a great need for models of human disease that accurately reproduce detailed characteristics of disease pathology. Genetically modified and humanized rodent models are not optimal for investigation of diseases such as autism and Fragile X Syndrome, which are caused by dysfunction during fetal development of the human brain. Use of such models has led to drug targets failing in clinical trials. As described herein, the mosaicism of the trinucleotide repeat lengths in the FMR1 gene in cells derived from individuals with FXS was exploited to establish an isogenic culture system that allows for the identification of phenotypes underlying neurodevelopmental disease, and the screening of potential disease-modifying factors. Development of isogenic pairs of cell lines that do and do not express disease characteristics (e.g., expression of any of the various marker genes or differentially expressed genes detailed in the Examples herein) allows discrimination of the phenotypic characteristics that are relevant to the disease, and not confounded by genetic background. By identifying the specific underlying causes of a disease phenotype and a relevant cellular model of the phenotype, screening for phenoytype-reversing compounds will similarly be more relevant.

[0027] To this end, isogenic cell lines with different CGG repeat lengths were isolated from the same patient using single cell subcloning, and the cells derived therefrom were investigated throughout neuronal differentiation and maturation to identify disease-associated phenotypes. In some cases, isogenic FXS cell lines were differentiated into excitatory cortical progenitors and neurons. The isogenic cell line and culture system used herein indicates that excitatory cortical neurons derived from FXS cell lines exhibit reduced expression of FOXG1+, increased BMP signaling, aberrant methylation patterns, and reduced synaptic activity. The identification of these phenotypes by the culture and differentiation methods described herein allows for the identification of new targets for the reversal or treatment of neurodevelopmental diseases, including FXS.

[0028] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0029] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

I. DEFINITIONS

[0030] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0031] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or more.” It is understood that aspects and variations described herein include “consisting of’ and/or “consisting essentially of’ aspects and variations.

[0032] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.

[0033] The term “about” as used herein refers to the usual error range for the respective value readily known. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”. [0034] As used herein, a statement that a cell or population of cells is “positive” for a particular marker refers to the detectable presence on or in the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the presence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is detectable by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions and/or at a level substantially similar to that for cell known to be positive for the marker, and/or at a level substantially higher than that for a cell known to be negative for the marker.

[0035] As used herein, a statement that a cell or population of cells is “negative” for a particular marker refers to the absence of substantial detectable presence on or in the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the absence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is not detected by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions, and/or at a level substantially lower than that for cell known to be positive for the marker, and/or at a level substantially similar as compared to that for a cell known to be negative for the marker.

[0036] The term "expression" or "expressed" as used herein in reference to a gene refers to the transcriptional and/or translational product of that gene. The level of expression of a DNA molecule in a cell may be determined on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell (Sambrook et ak, 1989, Molecular Cloning: A Laboratory Manual, 18.1-18.88).

[0037] As used herein, the term "stem cell" refers to a cell characterized by the ability of self-renewal through mitotic cell division and the potential to differentiate into a tissue or an organ. Among mammalian stem cells, embryonic and somatic stem cells can be distinguished. Embryonic stem cells reside in the blastocyst and give rise to embryonic tissues, whereas somatic stem cells reside in adult tissues for the purpose of tissue regeneration and repair.

[0038] As used herein, the term “adult stem cell” refers to an undifferentiated cell found in an individual after embryonic development. Adult stem cells multiply by cell division to replenish dying cells and regenerate damaged tissue. An adult stem cell has the ability to divide and create another cell like itself or to create a more differentiated cell. Even though adult stem cells are associated with the expression of pluripotency markers such as Rexl, Nanog, Oct4 or Sox2, they do not have the ability of pluripotent stem cells to differentiate into the cell types of all three germ layers.

[0039] As used herein, the terms "induced pluripotent stem cell," "iPS" and "iPSC" refer to a pluripotent stem cell artificially derived (e.g., through man-made manipulation) from anon- pluripotent cell. A "non-pluripotent cell" can be a cell of lesser potency to self-renew and differentiate than a pluripotent stem cell. Cells of lesser potency can be, but are not limited to adult stem cells, tissue specific progenitor cells, primary or secondary cells.

[0040] As used herein, the term "pluripotent" or "pluripotency" refers to cells with the ability to give rise to progeny that can undergo differentiation, under appropriate conditions, into cell types that collectively exhibit characteristics associated with cell lineages from the three germ layers (endoderm, mesoderm, and ectoderm). Pluripotent stem cells can contribute to tissues of a prenatal, postnatal or adult organism.

[0041] As used herein, the term "pluripotent stem cell characteristics" refer to characteristics of a cell that distinguish pluripotent stem cells from other cells. Expression or non-expression of certain combinations of molecular markers are examples of characteristics of pluripotent stem cells. More specifically, human pluripotent stem cells may express at least some, and optionally all, of the markers from the following non-limiting list: SSEA-3, SSEA-4, TRA-1-60, TRA-1- 81, TRA-2-49/6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Lin28, Rexl, and Nanog. Cell morphologies associated with pluripotent stem cells are also pluripotent stem cell characteristics.

[0042] As used herein, the term "reprogramming" refers to the process of dedifferentiating a non-pluripotent cell into a cell exhibiting pluripotent stem cell characteristics.

[0043] As used herein, the term "cell culture" may refer to an in vitro population of cells residing outside of an organism. The cell culture can be established from primary cells isolated from a cell bank or animal, or secondary cells that are derived from one of these sources and immortalized for long-term in vitro cultures.

[0044] As used herein, the terms "culture," "culturing," "grow," "growing," "maintain," "maintaining," "expand," "expanding," etc., when referring to cell culture itself or the process of culturing, can be used interchangeably to mean that a cell is maintained outside the body (e.g., ex vivo) under conditions suitable for survival. Cultured cells are allowed to survive, and culturing can result in cell growth, differentiation, or division. [0045] As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.

[0046] The term “pharmaceutical composition” refers to a composition suitable for pharmaceutical use, such as in a mammalian subject (e.g., a human). A pharmaceutical composition typically comprises an effective amount of an active agent (e.g., cells) and a carrier, excipient, or diluent. The carrier, excipient, or diluent is typically a pharmaceutically acceptable carrier, excipient or diluent, respectively.

[0047] A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0048] The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.

[0049] As used herein, a “subject” is a mammal, such as a human or other animal, and typically is human.

II. METHODS FOR DIFFERENTIATING CELLS

[0050] Provided herein are methods for differentiating pluripotent stem cells into inhibitory (GABAergic) neurons. In some embodiments, the method includes (a) performing a first incubation that is initiated on day -1, the first incubation includes culturing pluripotent stem cells in a culture vessel under conditions to produce neural progenitors, wherein during the first incubation, the cells are exposed to (i) an inhibitor of ROCK; (ii) an inhibitor of TGF- /activing- Nodal signaling; (iii) an inhibitor of bone morphogenetic protein (BMP) signaling; and (iv) an inhibitor of Wnt/p-catenin signaling; and (b) performing a second incubation comprising culturing the neural progenitors under conditions to differentiate the neural progenitors into inhibitory neurons. In some embodiments, culturing the neural progenitors under conditions to differentiate the neural progenitors into inhibitory neurons includes exposing the cells to (i) brain-derived neurotrophic factor (BDNF); (ii) ascorbic acid; (iii) glial cell-derived neurotrophic factor (GDNF); and (iv) cyclic AMP (cAMP) (collectively “BAGC”); and (v) an inhibitor of ROCK. In some aspects, the pluripotent stem cells are derived from a subject having a neurodevelopmental disease or condition. In some aspects, the neurodevelopmental disease or condition is Fragile X Syndrome or autism.

[0051] Also provided herein are methods of identifying a neurodevelopmental disease or condition phenotype, comprising use of any of the methods described herein. In some aspects, the method includes (a) performing a first incubation that is initiated on day -1, the first incubation including culturing pluripotent stem cells in a culture vessel under conditions to produce neural progenitors, wherein during the first incubation, the cells are exposed to (i) an inhibitor of ROCK; (ii) an inhibitor of TGF- /activing-Nodal signaling; (iii) an inhibitor of bone morphogenetic protein (BMP) signaling; and (iv) an inhibitor of Wnt/p-catenin signaling, wherein the pluripotent stem cells are derived from a subject having a neurodevelopmental disease or condition and/or the pluripotent stem cells have a mutation in the FMR1 gene; (b) performing a second incubation including culturing the neural progenitors under conditions to differentiate the neural progenitors into inhibitory neurons; and (c) comparing a phenotype of the pluripotent stem cells, neural progenitors, and/or inhibitory neurons produced by the method to the phenotype of reference pluripotent stem cells, neural progenitors, and/or inhibitory neurons produced by the method that are not derived from a subject having a neurodevelopmental disease or condition and/or do not have a mutation in the FMR1 gene. In some embodiments, culturing the neural progenitors under conditions to differentiate the neural progenitors into inhibitory neurons includes exposing the cells to (i) brain-derived neurotrophic factor (BDNF); (ii) ascorbic acid; (iii) glial cell-derived neurotrophic factor (GDNF); and (iv) cyclic AMP (cAMP) (collectively “BAGC”); and (v) an inhibitor of ROCK. In some embodiments, the method includes harvesting the inhibitory neurons. In some embodiments, the inhibitory neurons are harvested between about day 23 and about day 80. In some embodiment, the inhibitory neurons are harvested on day 80. In some aspects, the neurodevelopmental disease or condition is Fragile X Syndrome or autism.

[0052] Also provided herein are methods for differentiating pluripotent stem cells into excitatory (glutamatergic) neurons.

[0053] Also provided are cells produced by any of the methods described herein, and compositions thereof.

III. ARTICLES OF MANUFACTURE

[0054] Also provided are articles of manufacture, systems, apparatuses, and kits useful in performing the provided methods. Also provided are articles of manufacture, including: (i) one or more reagents for differentiation of pluripotent stem cells into inhibitory neurons; and (ii) instructions for use of the one or more reagents for performing any methods described herein.

[0055] In some embodiments, the reagent for differentiation is or includes a small molecule, capable of inhibiting Rho-associated, coiled-coil containing protein kinase (ROCK). In some of any such embodiments, the reagent for inhibiting ROCK is Y-27632. In some of any such embodiments, the reagent for differentiation is or includes a small molecule, capable of inhibiting TGF- /activing-Nodal signaling. In some of any such embodiments, the reagent for differentiation is or includes SB431542. In some of any such embodiments, the reagent for differentiation is or includes a small molecule, capable of inhibiting BMP signaling. In some of any such embodiments, the reagent for inhibiting BMP signaling is LDN193189. In some of any such embodiments, the reagent for differentiation is or includes a small molecule, capable of inhibiting Wnt/p-catenin signaling. In some of any such embodiments, the reagent for inhibiting Wnt/p-catenin signaling is XAV9390. In some embodiments, the reagents for differentiation include one or more of Y-27632, SB431542, LDN193189, and XAV9390. In some embodiments, the reagents for differentiation include each of Y-27632, SB431542, LDN193189, and XAV9390.

[0056] In some of any of such embodiments, the reagent for differentiation is or includes one or more of BDNF (e.g., rhBDNF), GDNF (e.g., rhGDNF), cyclic AMP (cAMP), and ascorbic acid. In some of any of such embodiments, the reagent for differentiation is or includes each of BDNF (e.g., rhBDNF), GDNF (e.g., rhGDNF), cAMP, and ascorbic acid.

[0057] The reagents in the kit in one embodiment may be in solution, may be frozen, or may be lyophilized.

[0058] Also provided are articles of manufacture, including (i) any composition described herein; and (ii) instructions for administering the composition to a subject.

[0059] In some embodiments, the articles of manufacture or kits include one or more containers, typically a plurality of containers, packaging material, and a label or package insert on or associated with the container or containers and/or packaging, generally including instructions for use, e.g., instructions for reagents for differentiation of pluripotent cells into inhibitory (GABAergic) neurons, and instructions to carry out any of the methods provided herein. In some aspects, the provided articles of manufacture contain reagents for differentiation and/or maturation of cells, for example, at one or more steps of the manufacturing process, such as any reagents described herein. [0060] The articles of manufacture provided herein contain packaging materials. Packaging materials for use in packaging the provided materials are well known to those of skill in the art. See, for example, U.S. Patent Nos. 5,323,907, 5,052,558 and 5,033,252, each of which is incorporated herein in its entirety. Examples of packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, disposable laboratory supplies, e.g., pipette tips and/or plastic plates, or bottles. The articles of manufacture or kits can include a device so as to facilitate dispensing of the materials or to facilitate use in a high-throughput or large-scale manner, e.g., to facilitate use in robotic equipment. Typically, the packaging is non-reactive with the compositions contained therein.

[0061] In some embodiments, the reagents and/or cell compositions are packaged separately. In some embodiments, each container can have a single compartment. In some embodiments, other components of the articles of manufacture or kits are packaged separately, or together in a single compartment

IV. EXAMPLES

[0062] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1: Neuronal differentiation of inhibitory neurons from pluripotent stem cells

A. Differentiation of Inhibitory (GABAergic) Neurons from Pluripotent Stem Cells [0063] A total of six pluripotent stem cells lines were differentiated into inhibitory (“GABAergic”) neurons, as set forth in Table 1. Two cell lines (cell lines “1” and “2”) were derived from induced pluripotent stem cell (iPSC) lines from two different individuals with fragile X syndrome (FXS). Two of the cell lines (cell lines “3” and “4”) were an isogenic pair of human embryonic stem cell (hESC) lines, in which one line had a targeted loss of fragile X mental retardation protein (FMRP) expression (“ FMR1 knockout”), and the other did not.

Fragile X mental retardation 1 (FMR1) knockout was achieved through CRISPR/Cas9 editing via a guide ribonucleic acid (RNA) targeting the junction of intron and exon 3, and confirmed by Western blotting. The final two cell lines (cell lines “5” and “6”) served as control pluripotent stem cells (PSCs): a non-disease human embryonic stem cell (hESC) line and an induced pluripotent stem cell (iPSC) line derived from a healthy individual.

Table 1. List of Cell Lines

[0064] All cell lines were cultured with mTeSR™l -based on Geltrex™ substrate. Before reaching 80% confluence, cells were passaged routinely at a 1:3 - 1:5 ratio with dispase. During passage, cell aggregates were gently dissociated 3-5 times using Pasteur pipets 0.55 cm in diameter (Coming).

[0065] Once the cells reached 80% confluence, they were dissociated with Accutase™ and plated on Geltrex™-coated 12-well plates at 210,000 cells/cm² in TeSR™-E6 media, supplemented with 10 mM Y-27632, an inhibitor of rho-associated, coiled-coil containing protein kinase (ROCK). The next day (Day 0), Y-27632 was withdrawn, and cortical induction was initiated TeSR™-E6 media in the presence of 10 pM SB421542, a small molecule inhibitor of the transforming growth factor-beta 1 (TGF-bI) activin receptor-like kinases (ALKs), and 100 nM LDN193189, a small molecule inhibitor of the bone morphogenetic (BMP) pathway (“neural induction media”).

[0066] On Day 10 of differentiation, neural progenitors were passaged as aggregates at a 1:3 ratio, and were re-plated on poly-d-lysine- and laminin-coated 12-well plates in a progenitor expansion media supplemented with Y-27632. The progenitor expansion media was composed with 1-to-l ratio of Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) and Neurobasal™ media, lx N-2 Supplement, lx B-27™ Supplement minus vitamin A, 0.5 mg/ml of bovine serum albumin (BSA), lx Penicillin-Streptomycin, and 100 pM beta- mercaptoethanol. Progenitors were allowed to expand from Days 10-25, during which cells were re-plated weekly as aggregates. Fibroblast growth factor 2 (FGF2) was optionally provided in the progenitor expansion media at 20 ng/mL on Days 10-24.

[0067] On Day 25 of differentiation, cells were dissociated with Accutase™, and seeded on 24-well plates at 130,000 cells/cm² in neuronal maturation media supplemented with 10 pM Y- 27632. Neuronal maturation media was composed of Neurobasal™ Plus medium, lx B-27™ Plus Supplement, lx N-2 Supplement, 100 pM cyclic adenosine monophosphate (cAMP), 20 ng/ml brain-derived neurotrophic factor (BDNF), 20 ng/mL glial cell line-derived neurotrophic factor (GDNF), and 200 nM ascorbic acid (vitamin C). The small molecule Wingless-INT (Wnt) inhibitor XAV939 was optionally provided in the neural induction media at 2 mM on Days 0-4 or Days 0-9. A schematic of the differentiation scheme and checkpoints is shown in Fig. 1.

[0068] On Day 30 of differentiation, when areal identity of neural progenitors is largely determined, RNA was isolated from cells from each condition and the expression of canonical markers for the dorsal telencephalon (cortex), medial ganglionic eminence (MGE), as well as lateral ganglionic eminence (LGE)/caudal ganglionic eminence (CGE) was quantified by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR).

[0069] The relative expression of these genes is examined. Forebrain marker forkhead box G1 (FOXG1) was not observed to be significantly changed among the different conditions. Excitatory lineage genes Dorsal genes homeobox protein (EMX1), eomesodermin (EOMES), solute carrier family 17 member 7 (SLC17A7), and T-box brain transcription factor 1 (TBR1) were upregulated with FGF2 addition during the progenitor proliferation phase. By contrast, inhibitory lineage genes GE markers glutamate decarboxylase 2 (GAD2), GS homeobox 2 (GSX2) and NK2 homeobox 1 (NKX2-1) also showed increased expression were downregulated with FGF2 addition during the progenitor proliferation phase.

[0070] It was found that Wnt inhibition and not adding FGF2 promoted GE fate determination. When FGF2 was added during progenitor proliferation phase, the excitatory lineage genes (EMX1, EOMES, SLC17A7, and TBR1) were upregulated, and the inhibitory lineages genes (GAD2, GSX2, NKX2-1) were downregulated.

[0071] The duration of Wnt inhibition by XAV939 also shifted the dorsal-ventral identity. While XAV939 treatment from Days 0-4 showed modest differences from untreated cells, treatment from Days 0-9 shifted the cells to a more ventral identity, even without additional SHH, as evidenced by the upregulation of NKX2-1 while maintaining the expression of LGE/CGE marker GSX2. Along the same trend, excitatory lineage markers were reduced with this treatment. These results indicate that Wnt inhibition throughout the neural induction phase during differentiation Days 0-9 is sufficient to generate a differentiated cell culture that dominated by a mixture of GE identity cells, without additional SHH and FGF2 treatments.

[0072] Differentiation was performed on five batches of all six cell lines to test for reproducibility. Expressions of key marker genes were measured by qPCR from the five batches of differentiated cell lines collected on Days 9, 17, 24, 32, 42, 52, 62 and 72. Reproducibility was demonstrated by consistent expression of key marker genes. [0073] Control and FXS cell lines consistently showed expression of GE markers GSX2, NKX2-1 at progenitor stage (day 24). There was an increase of inhibitory neuron lineage markers GAD2, solute carrier family 6 member 1 (SLC6A1), calbindin 2 (CALB2), reelin (RELN), somatostatin (SST) and neuropeptide Y (NPY) over time. The expression peaked at Day 42 and stayed at the plateau, indicating neurogenesis likely proceeded until Day 42. Excitatory neuron lineage markers, calcium/calmodulin dependent protein kinase II alpha (CAMK2A), SLC17A7, EOMES, neurogenin-2 (NEUROG2), EMX1 and TBR1 showed low expression overall (relative to inhibitory markers). For EMX1 and TBR1, expression levels became lower over time, peaking at Day 17 but slowly returning to near 0.

[0074] FMR1 was never expressed in FXS-iPSCs derived-cells but had high expression in control cells throughout the culture period ( FMR1 transcripts were also detected in the FMR1 KO line but FMRP is not made in these cell lines). FOXG1 and LHX2 were highly expressed throughout the duration of culture, validating the telencephalon identity of the cells. Expression of PAX6 remained high throughout the course of differentiation of the various cell lines.

[0075] Principal component analysis (PCA) was carried out on this qPCR dataset to analyze variance explained by disease group, differentiation day, and batch of differentiation. 42.48% of the variance was explained by the first principal component (PCI). PCI was mostly explained by the Day of differentiation. Disease status and batch of differentiation did not show clear clustering on the PCA plots, indicating they likely did not contribute to major variance in this dataset. These results indicate that a mixed population of GE-derived inhibitory neurons, resistant to cell line and batch-to-batch differences, can be generated from the inhibitory neuron derivation protocol described in this Example.

B. Defined Differentiation Protocol for Inhibitory (GABAergic Neurons)

[0076] In a related experiment, pluripotent stem cells were differentiated into inhibitory (GABAergic) neurons based on the described differentiation protocol, and cell identity was characterized by immunocytochemistry. A schematic in Fig. 2 illustrates the directed differentiation protocol. As before, when cells reached 80% confluence, they were dissociated with Accutase™ and plated on Geltrex™-coated 12-well plates at 210,000 cells/cm² in TeSR™- E6 media, supplemented with 10 mM Y-27632. The next day (Day 0), Y-27632 was withdrawn, and cortical induction was initiated TeSR™-E6 media in the presence of 10 pM SB421542, 100 nM LDN193189. In this experiment, XAV939 was provided at 2 pM on Days 0-9 for all cells. On Day 10, small molecule treatment was withdrawn. In contrast to the initial experiment that allowed progenitors to expand from Day 10 to Day 25, progenitors were allowed to expand from Day 10 to Day 22. On Day 22, cells were disassociated and seeded as single cells for terminal differentiation and maturation in the presence of neurotrophic factors BDNF, GDNF, cAMP and ascorbic acid (vitamin C).

[0077] Telencephalon markers FOXG1 and LHX2 were expressed in almost all cells on Day 22. On Day 22, this protocol was also observed to yield ventral forebrain progenitor cells. Cells positive for GE markers NKX2-1, GSX2, COUPTFII (counterstained with DAPI (4', 6- diamidino-2-phenylindole) for the nuclei), and PAX6 accounted for the majority of the cells in the differentiated culture. No significant difference was observed in expression of these markers between FXS and control cells.

[0078] On Day 62, the majority of the neurons were gamma aminobutyric acid (GABA) positive. Neuronal soma were identified by expression of microtubule associated protein 2 (MAP2). By this time, inhibitory neuron subtypes such as SST-expressing or CALB2-expressing inhibitory neurons were abundant. No significant difference was observed in expression of these markers between FXS and control.

[0079] Together, these data indicate that the directed differentiation protocol described in the second experiment and represented by Fig. 2 is capable of producing GABAergic neurons derived from either control or FXS pluripotent stem cells. Delay in FXS GABAergic inhibitory network maturation

[0080] The six human pluripotent stem cell lines described in Example 1 A were differentiated into GABAergic neurons using the protocol described in Example IB to compare the formation of functional synapses between the cell lines.

[0081] On Day 52 and Day 62 of the differentiation protocol, miniature postsynaptic currents were measured using voltage clamp mode with intracellular recording. Miniature inhibitory postsynaptic current (mIPSC) was recorded in the presence of tetrodotoxin (TTX) at the end of each recording to confirm inhibitory postsynaptic currents, as shown in Fig. 3. Upon administration of bicuculline, a GABAA receptor blocker, no postsynaptic events were observed, indicating the previously observed currents were miniature inhibitory postsynaptic events. Representative mIPSC traces of control and FXS neurons on Day 52 and Day 62 are shown in Fig. 4. No excitatory events were detected, indicating that the protocol described in Example IB yielded a predominantly inhibitory neuron cell culture. [0082] Superimposed mIPSC events were obtained by averaging individual events of control and FXS neurons (Fig. 5). Measurements of mIPSC frequency, rise time, and amplitudes between FXS and control GABAergic neurons on Day 52 and Day 62 are shown in Fig. 6. Differences in mIPSC frequency and mIPSC amplitudes between FXS- and control-derived inhibitory neurons were not observed. The mIPSC rise time was, however, observed to be significantly faster in FXS neurons on Day 62, likely due to a difference in GABAA receptor channel dynamic between FXS and control neurons. Overall, both FXS and control neurons formed functional GABAergic synapses in culture.

[0083] To test if a delay in GABAA reversal potential switch occurs in FXS PSC-derived inhibitory neurons, spontaneous activities from maturing FXS and control cultures were recorded every five days from Days 47-77 using a micro-electrode array (MEA). Both control and FXS neurons on Day 62 showed an increase in activity initially but transitioned to a steady decrease in activities later.

[0084] Altered network activity was detected in FXS neuronal culture in vitro. Spontaneous firing activity was measured from Day 47 to 77 in FXS and control cultures. During peak activity periods, however, FXS neurons fired more frequently and had more network activities, as shown in Fig. 7. Mean firing rate of active electrodes and mean ISI (inter-spike intervals) were inversely correlated. FXS neurons also exhibited higher mean burst frequencies during this period and a greater number of spikes in bursts as shown in Fig. 8. The mean frequency in burst, however, did not show differences between control and FXS, also shown in Fig. 8. This indicated longer burst durations in FXS. Importantly, both the peaks in mean firing rate and bursting rate occurred earlier in control neurons, consistent with a finding that the GABAA reversal potential switch is delayed in the maturing FXS inhibitory neuron culture. That is, the control culture had a steady decrease in activity through day 62 and 77 while FXS culture had a sharp decrease in activity on day 72. This indicated the transition of GABA was continuous in control cells but was more abrupt in FXS cells.

[0085] The expression of two key chloride transporters that regulate GABAAreversal potential switch was also investigated. Immunocytochemistry against K+/C1- cotransporter 1 (KCC1) and K+/C1- cotransporter 2 (KCC2) antigens was performed on Day 52, Day 62, and Day 72 control and FXS iPSCs derived inhibitory neurons to determine if a defective transition from KCC1 to KCC2 expression was also true in FXS iPSC-derived GABAergic neurons. Immature neurons have an upregulation of KCC1. As neurons mature, KCC1 is downregulated and replaced by KCC2. The upregulation of KCC2 leads to a negative shift of reversal potential of the GABBA receptor.

[0086] FXS neurons showed reduced KCC2+ expression during maturation (from Days 52, 62, and 72) compared to that of control neurons, as quantified in Fig. 9. On Day 52, there were comparable percentages of KCC1+ neurons and mean KCC1 intensity in both FXS and control and by Day 62 and 72, the number of KCC1+ neurons decreased sharply to almost zero. Overall, there was little difference in KCC1 expression between control and FXS neurons derived from iPSCs.

[0087] A steady increase of KCC2 expression and number of KCC2 expressing cells from Day 52 to Day 72 was also observed in both control and FXS cultures. However, the magnitude of increase was much larger in control cells compared to in FXS cells. At each timepoint, there were significantly more KCC2+ neurons in control culture compared to FXS culture.

[0088] An aberrant transition from KCC1 to KCC2 protein expression in the FXS maturing GABAergic neuronal culture was observed. These observations were consistent with delayed KCC2 protein expression in the maturing FXS GABAergic neuronal cultures, similar to the Fmrl knockout mouse developmental phenotype.

Example 3: Differentiation of Isogenic Cells into Excitatory Cortical Progenitors and

Neurons

[0089] Isogenic cell lines were generated by isolating and subcloning single cells from the same human subject with mosaic Fragile X Syndrome, such that the isogenic cells lines had different CGG repeat lengths. Ten to fifteen subclones were produced from two different human subjects, each having FXS, to produce four iPSC cell lines (two isogenic cell lines from each patient). CGG repeat size length in each clone was determined using a capillary electrophoresis method. Clones with more than 200 repeats (full mutation; “FXS cell lines”) and clones with shorter repeat lengths (“control cell lines”) were identified. Neither transcripts of the FMR1 gene or the FMRP protein were detected in clones with full mutation CGG repeat length. Fig. 10 shows mosaic FXS-iPSC lines subcloned into cell lines with larger or smaller size CGG trinucleotide expansions.

[0090] The four iPSC lines were differentiated into excitatory cortical progenitors and neurons using a dual SMAD and Wnt inhibition protocol, to produce deep and upper layer excitatory neurons. The Wnt inhibitor was used to further direct neural progenitors to a dorsal forebrain identity. [0091] Briefly, the iPSCs were cultured as a monolayer in the presence of SB431542, dorsomorphin (DM), and XAV939 from Day 0 through Day 9 of culture to produce cortical progenitors. SB431542, DM, and XAV939 were withdrawn on Day 10 and cortical progenitors were allowed to expand. On Day 22, neuronal differentiation was induced by exposure to BDNF, GDNF, ascorbic acid (vitamin C), and cyclic AMP (cAMP). A schematic of the differentiation protocol is shown in Fig. 11. Neuroepithelial cells were detected at about Day 10, as evidenced by a uniform layer of PAX6+FOXG1+ cells. Intermediate progenitors (TBR2+) began to emerge at Day 22 at the periphery of the neural rosettes. Around Day 30, layer VI-V neurons (CTIP2+TBR1+) could be identified in culture. Finally, upper neurons expressing CUX1, POU3F2, and SATB2 appeared at Day 60.

[0092] To allow for the determination of differences between neurons derived from the FXS cell lines and neurons derived from the isogenic control cell lines, neurons resulting from culture were separated from progenitor cells by sorting with a neuronal fluorescent dye (NeuO; Stem Cell Technologies). FMR1 and FMRP expression in sorted progenitors and neurons was analyzed. A 2-fold increase in Beta III Tubulin expression in NeuO+ cells was observed by immunoblotting, indicating an enrichment of neurons after cell sorting. In addition, RT-qPCR revealed a 2.8-fold higher level of FMR1 gene expression in NeuO+ cells, as compared to unsorted or NeuO- cells, underscoring the importance of separating neurons and progenitors for downstream analyses. The selected neurons were examined for expression of FOXG1+, BMP signaling, DNA methylation patterns, and synaptic activity.

[0093] Expression of telencephalic markers in cells derived from FXS and control cell lines was analyzed by immunocytochemistry (ICC). Differentiation of three control cell lines demonstrated robust rosette formation from day 8 to 22. In contrast, FXS-derived cells failed to form neural rosettes from Day 8 to Day 22. High expression of the early dorsal forebrain marker

PAX6 was observed in both control and FXS cells at Day 15. However, expression of the telencephalic marker FOXG1 was observed to be reduced in FXS cells at Day 15 (Fig. 12).

Approximately 80% of control cells were FOXG1 -positive, whereas only 2-10% of FXS cells expressed FOXG1 at Day 15. When results were compared to /'M/N-KO-hESC cell lines, a reduction of FOXG1+ cells was also observed, but compared to a 70-80% reduction in FOXG1+ cells in FXS-iPSCs, /'M/N-KO-hESC lines showed a less severe phenotype, with 20-30% reduction of FOXG1+ cells at Day 15. These data indicate that the silencing of FMR1 by DNA methylation in FXS iPSCs has more profound phenotypic effects than simply silencing of the gene by knockout, suggesting a simple gene knockout is insufficient to model the disease in human cells, and epigenetic effects are necessary to include in a model of neurodevelopmental disease.

[0094] Expression of genes involved in early forebrain development, including Notch signaling, Wnt signaling, and BMP signaling, was analyzed by gene expression microarray.

Both positive and negative changes in the downstream target genes of Wnt and Notch signaling were found. In particular, BMP signaling was significantly upregulated in FXS cells, as evidenced by upregulation of expression of both BMP ligands, BMP4 and BMP7, and downstream transcription factors, FOX01 and SMAD3. Phosphorylated levels of SMAD 1, 5, and 8 (pSMAD 1, 5, 8) were also examined to validate an overactive BMP signaling. Immunostaining against pSMAD 1, 5, 8 was observed to be increased in FXS untreated cells on Day 15. To determine whether hyperactivation of BMP signaling causes FOXG1 inhibition in FXS cells, FXS cells were treated with Noggin, a BMP inhibitor, from Day 10 to Day 15, and FOXG1 expression was assessed. A dose-dependent increase in the percentage of FOXG1+ cells was observed in both FXS cell lines. In addition, Noggin treatment reduced immunoreactivity of antibodies to pSMAD 1, 5, 8 in FXS cells. The highest concentration of Noggin only increased the percentage of FOXG1+ cells from 2-10% to 40% in FXS cells at Day 15, as compared to -80% in control cells, indicating other factors also contribute to the FOXG1 phenotype.

[0095] Methylation patterns between FXS cell lines and their isogenic control cell lines was assessed by DNA methylation profiling using the Infmium HumanMethylation450 BeadChip. Specifically, global DNA methylation patterns were profiled in non-disease, FXS FMR1+ and FXS FMR1- cells at Days 0, 3, 6, 9, and 12 of the excitatory neuronal differentiation method. 1,603 differentially methylated cytosines (DMCs) specific to FXS cells were identified. Both gains and losses of methylation were observed in FXS FMR1- lines relative to non-disease lines. Methylation patterns were observed to be shared by both types of FXS cells ( FMR1+ and FMR1-), which differed from non-disease cells. At some loci, FXS FMR1+ cells exhibited intermediate global methylation pattern compared to non-disease and FXS FMR1- cells. Both DNA hypomethylation and hypermethylation specific to FXS FMR1- cells were observed. Only 5.6% (90 of 1,603) of candidate DMCs occurred on the X chromosome, while 93.9% (1,506 of 1,603) were located on autosomes, indicating DNA methylation abnormalities in FXS FMR1- cells are not restricted to the X chromosome.

[0096] The electrophysiology of iPSC-derived Day 80 neurons from control (n=45) and FXS

(n=31) was characterized by whole-cell patch clamp. Voltage responses were recorded under a current step stimulation starting at -50 pA, which was incremented by 2.5 pA until intense firing was observed. Both control and FXS cells exhibited healthy action potentials upon depolarization and no voltage sag under hyperpolarizing current (Fig. 13).

[0097] Resting membrane potential, estimated membrane capacitance, input resistance at resting stage and rheobase, and the threshold current sufficient to elicit firing were measured. No significant differences between control and FXS-derived neurons were observed. Spontaneous excitatory post-synaptic currents (sEPSCs) from control and FXS-derived cells were also recorded. FXS cells exhibited significantly reduced frequency of sEPSC (p<0.05).

Example 4: Shared and cluster-specific differentially expressed genes (DEGs) in FXS

[0098] To understand cell type-specific differences between FXS and control, we performed differential gene expression analysis at the two time points representing early (day 22) and late (day 42-48) stages of neurogenesis. For each time point, cells from each cluster were compared separately. Gene expression profiles were fihed with a hurdle model that accounts for the probability of gene dropout, cellular gene detection rate, and disease effect using MAST. A likelihood ratio test was performed by dropping the disease variable in the hurdle model to detect genes that were differentially expressed between clusters. Genes were considered differentially expressed relative to control if there was at least a 10% change in gene expression with a false discovery rate less than 0.05.

[0099] On day 22, only four differentially expressed genes (DEGs) were shared between progenitor and neuronal clusters: EIF5A, a translation initiation factor, was broadly upregulated in FXS; RPS4Y1, RPS26, and CHCHD2 were broadly downregulated in FXS. CHCHD2 is associated with neuronal migration and was identified as an FXS candidate gene in our previous study as well as a downregulated gene in multiple intemeuron clusters in autism brain. TAGLN3, upregulated in multiple neuronal clusters, is a neuron-specific protein that regulates neurite growth. Most DEGs in FXS cells were cell cluster-specific on day 22, and many have functions in protein translation ( EIF5A , RPL13A, RPS10 and RPLP0 ) and cytoskeleton remodeling (TAGLN3, MAP IB, ACTG1, ACTB, TUBA1B, TMSB10, and DYNLL1). However, no gene ontology (GO) terms were significantly enriched in these DEGs. Overall, the difference between FXS and control was minor on day 22.

[0100] At the later stage of neurogenesis, the differences between FXS and control became more pronounced. At this stage, many DEGs in FXS were shared between progenitor and neuronal clusters. Notably, among the most significant differentially expressed genes was a group of ribosome genes ( RPS29 , RPS27, RPLP1, RPL41, RPL39, RPL36A, RPL17, EIF3F, EEF2, and EEF1D) that were universally upregulated in FXS across clusters. This was consistent with our previous factor loading analysis that indicated that translation was broadly upregulated in FXS cells across cell types. Apparent differential expression of ribosomal genes sometimes can be caused by artificial sampling of outlier samples that have more ribosomal reads than expected. To test if this were the case in our dataset, ribosomal protein read percentages were plotted across samples at each time point. We found the percentage of reads belonging to ribosome genes was similar across different samples for each time point, which suggests that the differential expression of ribosomal genes on day 42-48 represents a biological difference between FXS and control cells and not an artifact.

[0101] Many of the FXS-associated downregulated genes were also shared across clusters. Among them, many ( IJQCRH , IJQCR10, COX6A1, UQCR11, and CCDC90B) are nuclear- encoded mitochondrial genes that have important roles in electron transfer through the respiratory chain.

[0102] Burden analyses demonstrated that not only did the number of DEGs increase dramatically from day 22 (< 30 genes) to day 42-48 (300-1000 genes), but that certain cell types were more affected than others. For example, progenitors had an enrichment of DEGs at day 22 whereas the most affected cell types at day 42-48 were the cholinergic cells, followed by the GABAergic intemeurons. Gene set enrichment analysis (GSEA) on the day 42-48 DEGs revealed that the most significant upregulated GO biological processes in FXS (day 42-48) were cytoplasmic translation, neuroblast proliferation, and WNT and BMP signaling, while downregulated processes included neuronal functions such as action potential and synaptic processes, as well as mitochondrial functions (hydrogen peroxide and electron transport).

Example 5: Overrep resen tation of autism risk genes among FXS DEGs

[0103] Because of the significant overlap between manifestations of FXS and ASD (autism spectrum disorder), we asked whether genes that are securely implicated in autism (SFARI category I) were enriched in the FXS DEGs. We found a significant overlap between FXS DEGs and ASD risk genes (p = 1.04e-03, hypergeometric test): among the 1793 DEGs in FXS, 27 overlap with SFARI (category I) genes (Fig. 14A-B). Enrichment here was also cluster-specific. ASD risk genes were significantly enriched in the GABAergic projection neurons and the cholinergic clusters on day 22 as well as the progenitor and the GABAergic CGE/ LGE intemeuron and the cholinergic clusters on day 42-48 (Fig. 14C). [0104] The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.

Claims

We claim:

1. A method of differentiating inhibitory neurons, the method comprising:

(a) performing a first incubation that is initiated on day -1, the first incubation comprising culturing pluripotent stem cells in a culture vessel under conditions to produce neural progenitors, wherein during the first incubation, the cells are exposed to (i) an inhibitor of ROCK; (ii) an inhibitor of TGF- /activing-Nodal signaling; (iii) an inhibitor of bone morphogenetic protein (BMP) signaling; and (iv) an inhibitor of Wnt/p-catenin signaling; and

(b) performing a second incubation comprising culturing the neural progenitors under conditions to differentiate the neural progenitors into inhibitory neurons.

2. The method of claim 1, wherein the cells are exposed to the inhibitor of TGF- b/activing-Nodal signaling beginning at day 0 and through day 9, inclusive of each day.

3. The method of claim 1 or claim 2, wherein the cells are exposed to the inhibitor of bone morphogenetic protein (BMP) signaling beginning at day 0 and through day 9, inclusive of each day.

4. The method of any of claims 1-3, wherein the cells are exposed to the inhibitor of Wnt/p-catenin signaling beginning at day 0 and through day 9, inclusive of each day.

5. The method of any of claims 1-4, wherein the cells: are exposed to the inhibitor of ROCK on day -1; and are not exposed to the inhibitor of ROCK beginning on day 0 and through day 9, inclusive of each day.

6. The method of any of claims 1-5, wherein the cells are exposed to the inhibitor of ROCK beginning on day 10.

7. The method of any of claims 1-6, wherein culturing the neural progenitors under conditions to differentiate the neural progenitors into inhibitory neurons comprises exposing the cells to (i) brain-derived neurotrophic factor (BDNF); (ii) ascorbic acid; (iii) glial cell-derived neurotrophic factor (GDNF); and (iv) cyclic AMP (cAMP) (collectively “BAGC”); and (v) an inhibitor of ROCK.

8. The method of any of claims 1-7, wherein the cells are exposed to BAGC and the inhibitor of ROCK beginning on day 22.

9. The method of any of claims 1-8, wherein the cells are exposed to BAGC and the inhibitor of ROCK beginning at day 22 and until harvest of the differentiated inhibitory neurons, optionally until about day 80.

10. The method of any of claims 1-9, wherein the pluripotent stem cells are derived from a subject having a neurodevelopmental disease or condition, optionally Fragile X Syndrome.

11. The method of any of claims 1-10, wherein the pluripotent stem cells comprise pluripotent stem cells having a mutation in the FMR1 gene, optionally an expansion of a cysteine-glycine-glycine (“CGG”) trinucleotide repeat in the FMR1 gene.

12. The method of any of claims 1-11, wherein the pluripotent stem cells are derived from a subject having Fragile X Syndrome.

13. The method of any of claims 10-12, wherein GABAA reversal potential switch is delayed in the inhibitory neurons produced by the method, compared to reference inhibitory neurons produced by the same method.

14. The method of claim 12 or claim 13, wherein the reference neural progenitors and/or the reference inhibitory neurons produced by the same method are not derived from a subject having a neurodevelopmental disease or condition and/or do not have a mutation in the FMR1 gene.

15. The method of any one of claims 1-14, further comprising harvesting the inhibitory neurons.

16. The method of claim 15, wherein the inhibitory neurons are harvested between about day 23 and about day 80.

17. A cell produced by the method of any of claims 1-16.

18. The cell of claim 17 , wherein the cell is derived from a subject having a neurodevelopmental disease or condition and/or has a mutation in the FMR1 gene.

19. The cell of claim 17 or claim 18, wherein the cell is derived from a subject having a neurodevelopmental disease or condition and/or has a mutation in the FMR1 gene and exhibits delayed GABAA reversal potential switch, compared to a reference cell.

20. The cell of any of claims 17-19, wherein the cell is derived from a subject having Fragile X Syndrome.

21. The cell of any of claims 17-20, wherein the reference cell is not derived from a subject having a neurodevelopmental disease or condition and/or does not have a mutation in the FMR1 gene.

22. A method of identifying a neurodevelopmental disease or condition phenotype, comprising use of the method of any of claims 1-16 and/or the cell of any of claims 17-21.

23. A method of identifying a neurodevelopmental disease or condition phenotype, the method comprising:

(a) performing a first incubation that is initiated on day -1, the first incubation comprising culturing pluripotent stem cells in a culture vessel under conditions to produce neural progenitors, wherein during the first incubation, the cells are exposed to (i) an inhibitor of ROCK; (ii) an inhibitor of TGF- /activing-Nodal signaling; (iii) an inhibitor of bone morphogenetic protein (BMP) signaling; and (iv) an inhibitor of Wnt/p-catenin signaling, wherein the pluripotent stem cells are derived from a subject having a neurodevelopmental disease or condition and/or the pluripotent stem cells have a mutation in the FMR1 gene; (b) performing a second incubation comprising culturing the neural progenitors under conditions to differentiate the neural progenitors into inhibitory neurons; and

(c) comparing a phenotype of the pluripotent stem cells, neural progenitors, and/or inhibitory neurons produced by the method to the phenotype of reference pluripotent stem cells, neural progenitors, and/or inhibitory neurons produced by the method that are not derived from a subject having a neurodevelopmental disease or condition and/or do not have a mutation in the FMR1 gene.

24. The method of claim 23, wherein the cells are exposed to the inhibitor of TGF- b/activing-Nodal signaling beginning at day 0 and through day 9, inclusive of each day.

25. The method of claim 23 or claim 24, wherein the cells are exposed to the inhibitor of bone morphogenetic protein (BMP) signaling beginning at day 0 and through day 9, inclusive of each day.

26. The method of any of claims 23-25, wherein the cells are exposed to the inhibitor of Wnt/p-catenin signaling beginning at day 0 and through day 9, inclusive of each day.

27. The method of any of claims 23-26, wherein the cells: are exposed to the inhibitor of ROCK on day -1; and are not exposed to the inhibitor of ROCK beginning on day 0 and through day 9, inclusive of each day.

28. The method of any of claims 23-27, wherein the cells are exposed to the inhibitor of ROCK beginning on day 10.

29. The method of any of claims 23-28, wherein culturing the neural progenitors under conditions to differentiate the neural progenitors into inhibitory neurons comprises exposing the cells to (i) brain-derived neurotrophic factor (BDNF); (ii) ascorbic acid; (iii) glial cell-derived neurotrophic factor (GDNF); and (iv) cyclic AMP (cAMP) (collectively “BAGC”); and (v) an inhibitor of ROCK.

30. The method of any of claims 23-29, wherein the cells are exposed to BAGC and the inhibitor of ROCK beginning on day 22.

31. The method of any of claims 23-30, wherein the cells are exposed to BAGC and the inhibitor of ROCK beginning at day 22 and until harvest of the differentiated inhibitory neurons, optionally until about day 80.

32. The method of any of claims 10-16 and 22-31 or the cell of any one of claims 17- 21, wherein the inhibitor of ROCK is Y-27632.

33. The method of any of claims 10-16 and 22-32 or the cell of any one of claims 17- 21, wherein SB421542 is provided in culture at a concentration of about 10 mM.

34. The method of any of claims 10-16 and 22-33 or the cell of any one of claims 17- 21, wherein the inhibitor of TGF- /activing-Nodal signaling is SB421542.

35. The method of any of claims 10-16 and 22-34 or the cell of any one of claims 17- 21, wherein SB421542 is provided in culture at a concentration of about 10 mM.

36. The method of any of claims 10-16 and 22-35 or the cell of any one of claims 17- 21, wherein the inhibitor of bone morphogenetic protein (BMP) signaling is LDN193189.

37. The method of any of claims 10-16 and 22-36 or the cell of any one of claims 17- 21, wherein LDN193189 is provided in culture at a concentration of about 100 nM.

38. The method of any of claims 10-16 and 22-37 or the cell of any one of claims 17- 21, wherein the inhibitor of Wnt/p-catenin signaling is XAV939.

39. The method of any of claims 10-16 and 22-38 or the cell of any one of claims 17- 21, wherein XAV939 is provided in the culture at a concentration of about 2 mM.

40. The method of any of claims 10-16 and 22-39 or the cell of any one of claims 17- 21, wherein BDNF is provided in the culture at a concentration of about 20 ng/mL, ascorbic acid is provided in the culture at a concentration of about 200 nM, GDNF is provided in the culture at a concentration of about 20 ng/mL, and/or cAMP is provided in the culture at a concentration of about 100 mM.

41. The method of any of claims 10-16 and 22-40 or the cell of any of claims 17-21, wherein the neurodevelopmental disease or condition is Fragile X Syndrome or autism.

42. The method of any of claims 10-16 and 22-41 or the cell of any of claims 17-21, wherein the subject is a human.