Methods of treating seizure activity

Abstract

Provided herein is a method of treating seizure activity by administering to a subject in need of treating seizure activity a therapeutically effective amount of a cellular composition that includes pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells. In some embodiments, at least 90% of the cells of the composition are post-mitotic cells. In some embodiments, at least 50% of the cells of the composition express LHX6, ERBB4, and GAD1.

Description

NTHER.014WO PATENT METHODS OF TREATING SEIZURE ACTIVITY CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application No. 63 / 587407, filed October 2, 2023, which is incorporated herein by reference in its entirety. BACKGROUND Field

[0002] The present disclosure relates to pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells and methods of use thereof in a cell therapy to treat seizure activity and / or neuronal hyperexcitability.

[0003] Mesial temporal lobe epilepsy (MTLE) is one of the most common types of focal epilepsy whereby seizures typically originate from a sclerotic hippocampus. Approximately one-third of people living with MTLE do not adequately respond to anti- seizure drugs and have few effective options. Surgery to remove or ablate the affected temporal lobe is one such option, but it is not indicated or effective for all and can result in adverse effects. SUMMARY

[0004] Provided herein are methods of treating seizure activity, comprising: identifying a subject in need of treating seizure activity; and administering to the subject a therapeutically effective amount of a cellular composition comprising pluripotent stem cell- derived, MGE-type inhibitory (e.g., GABAergic) neuron cells. In some embodiments, 90% or more of the cells of the cellular composition are post-mitotic cells. In some embodiments, the cellular composition includes pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells, where at least 50% of the cells of the composition express LHX6, ERBB4, and GAD1. In some embodiments, the cells of the cellular composition are substantially all pallial interneurons (e.g., are, are about, or are at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 90-100%, 90-98%, 92-95%, 92-97%, etc.) pallial interneurons). In some embodiments, a frequency of seizures may be reduced after the administering, thereby treating the seizure activity.

[0005] Also provided is use of a cellular composition for the treatment of seizure activity in a subject, the composition comprising pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells. In some embodiments, at least 90% of the cells of the composition are post-mitotic cells. In some embodiments, the cellular composition includes pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells, wherein at least 50% of the cells of the composition express LHX6, ERBB4, and GAD1.

[0006] Also provided is a cellular composition comprising pluripotent stem cell- derived, MGE-type inhibitory (e.g., GABAergic) neuron cells, for the preparation of a medicament for treatment of seizure activity in a subject. In some embodiments, at least 90% of the cells of the cellular composition are post-mitotic cells. In some embodiments, the cellular composition includes, or are enriched for, pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells, wherein at least 50% of the cells of the composition express LHX6, ERBB4, and GAD1.

[0007] Provided herein are therapeutic compositions of pluripotent stem cell- derived, MGE-type inhibitory (e.g., GABAergic) neuron cells. In some embodiments, at least 90% of the cells of the composition are post-mitotic cells. In some embodiments, the therapeutic composition includes pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells, wherein at least 50% of the cells of the composition express LHX6, ERBB4, and GAD1. In some embodiments, the composition may be used for the treatment of a focal onset seizure.

[0008] Also provided herein are therapeutic compositions comprising: a poloxamer; and pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells at a concentration of about 1 x 105cells / μL or greater. In some embodiments, the therapeutic composition includes pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells.

[0009] Provided herein are methods of preparing a therapeutic composition for administering to a subject, comprising: providing pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells; and preparing a composition comprising: a poloxamer; and the pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells at a concentration of about 1 x 105cells / μL or greater. In some embodiments,the cellular composition includes pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells.

[0010] In any method or composition herein, in some embodiments, a cellular composition of the present disclosure includes pluripotent stem cell-derived, subpallial, MGE-type GABAergic neuron cells, wherein at least 50% of the cells of the composition express LHX6, LHX8 or NKX2.1, and GAD1. In some embodiments, the composition is depleted for cells expressing ERBB4 or MAF / MAFB. In some embodiments, the subpallial, MGE-type GABAergic neuron cells include interneurons and / or projection neurons.

[0011] Provided herein are delivery systems for transplanting cells into a tissue, comprising: a delivery cannula that includes: a proximal portion comprising a cellular liquid composition comprising cells at a concentration of about 1 x 105cells / μL or greater; and a distal portion comprising a cell-free liquid chase vehicle, wherein the cellular liquid composition is stably held in the proximal portion by the liquid chase vehicle; and a displacement device connected to the distal end of the cannula and configured to cause the liquid chase vehicle to displace the cellular liquid composition to thereby expel the cellular liquid composition from the proximal end of the cannula.

[0012] Also provided is a delivery system for transplanting cells into a tissue, comprising: a delivery cannula that includes a cellular liquid composition comprising cells at a concentration of about 1 x 105cells / μL or greater; and a displacement device connected to a distal end of the cannula and configured to displace the cellular liquid composition in the cannula to thereby expel the cellular liquid composition from the proximal end of the cannula. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIGs. 1A and 1B are flow diagrams showing a method of treating a focal onset seizure, according to some non-limiting embodiments of the present disclosure.

[0014] FIGs. 2A-2G show post thaw in vitro characterization of end-of-process cells derived from human embryonic stem cells (hESCs), according to some non-limiting embodiments of the present disclosure. FIG. 2A is a schematic diagram showing neuronal subtypes derived from the MGE progenitor domain. FIGs. 2B and 2C are a collection of images showing ICC staining of the hESC-derived cells with pallial MGE and cholinergicmarkers. FIG. 2D is a graph showing marker expression profile of the hESC-derived cells, according to some non-limiting embodiments of the present disclosure, as determined by immunocytochemistry (ICC) analysis. FIG. 2E is a graph showing GABA release from hESC-derived pallial MGE-type interneuron lots, undifferentiated hESCs and spinal motor neuron cultures. FIG. 2F is a graph showing acetylcholine release from hESC-derived pallial MGE-type interneuron lots, undifferentiated hESCs and spinal motor neuron cultures. FIG. 2G is a graph showing quantification of live cells in unsorted / sorted cell lots after cryopreservation and thaw.

[0015] FIGs. 3A-3G show single cell RNA sequencing characterization of cell composition during in vitro differentiation, according to some non-limiting embodiments of the present disclosure. FIG. 3A is a plot showing UMAP (Uniform Manifold Approximation and Projection) visualization of cell clusters from samples combined. FIG. 3B is a collection of plots showing UMAP visualization of cell clusters from samples separately. FIG. 3C is a graph showing sample composition by cluster. FIG. 3D is a collection of plots visualizing gene expression across different clusters. FIG. 3E is a dot plot visualizing expression of key genes that define different cell categories. FIG. 3F is a dot plot showing projection of prediction scores between 0 and 1 onto day 14 and end-of-process clusters. FIG. 3G is a heatmap showing percentage of cells in each in vitro cluster that are assigned to different GE categories based on prediction scores.

[0016] FIGs.4A-4W show electrophysiological characterization of grafted human interneurons in the rodent pallium, according to some non-limiting embodiments of the present disclosure. FIG. 4A is a collection of fluorescence microscopy images showing examples of grafted cell morphologies. FIG. 4B is a collection of fluorescence microscopy images showing expression of human- and pallial interneuron specific markers. FIG. 4C is a collection of fluorescence microscopy images showing HNA staining of recorded human cells in cortical slices. FIGs. 4D and 4E are electrophysiology traces showing action potential recorded from transplanted human interneurons. FIGS. 4F-4M are graphs showing physiological properties of grafted human cells. FIG. 4N is a collection of traces showing current recordings before and after treatment with NBQX. FIGs. 4O-4Q are graphs showing properties of glutamate–mediated spontaneous excitatory postsynaptic currents (sEPSCs) in transplanted human interneurons. FIGs. 4R and 4S are electrophysiology traces showingexamples of human interneurons firing evoked APs. FIG. 4T is a collection of fluorescence microscopy images showing IHC staining of SYN-ChR2-YFP labeled human interneurons. FIGs. 4U-4W are graphs showing properties of sEPSCs in transplanted human interneurons. FIG. 4X is an electrophysiology trace showing recorded currents in light-stimulated ChR2- expressing human interneurons. FIG. 4Y is an electrophysiology trace showing recorded currents in endogenous mouse neurons after light stimulation of the human interneurons. FIG. 4Z is a graph showing the amplitude of light-evoked inhibitory postsynaptic currents in mouse neurons.

[0017] FIGs. 5A-5H show an overview of the chronic MTLE mouse model and seizure suppression after cell transplantation. FIG. 5A is a schematic diagram showing the experimental timeline. FIGs. 5B and 5C are a collection of EEG traces at various time points post-transplant. FIG. 5D is a graph showing electrographic mesiotemporal seizure frequency for epileptic animals treated with hESC-derived interneurons. FIG. 5E is a graph showing cumulative duration of seizures for epileptic animals treated with hESC-derived interneurons. FIG. 5F is a graph showing electrographic mesiotemporal seizure frequency for epileptic animals treated with hESC-derived interneurons. FIG. 5G is a fluorescence microscopy image showing staining of the hippocampus for markers for human cells and interneurons. FIG. 5H is a fluorescence microscopy image showing staining of the hippocampus for markers for human cells and neurons.

[0018] FIGS. 6A-6G show histological characterization of interneuron in the epileptic hippocampus. FIG. 6A is a collection of fluorescence microscopy images showing staining for markers of immature neurons and human cells. FIG. 6B is a collection of fluorescence microscopy images showing staining for markers of MGE interneurons and human cells. FIG. 6C is a collection of fluorescence microscopy images showing staining for a human-specific neuronal marker. FIG. 6D is a collection of fluorescence microscopy images showing staining for a human housekeeping gene and GABAergic neuronal marker. FIG. 6E is a collection of fluorescence microscopy images showing staining for human cells, MGE interneuron markers, and a perineuronal net marker, as well as lack of expression of off-target markers. FIG. 6F is a graph showing the rate of persistence of human cells. FIG. 6G is graph showing marker expression in persisting human cells.

[0019] FIGs. 7A-7J show human interneuron dose-response activity in the MTLE model, according to some non-limiting embodiments of the present disclosure. FIG. 7A is a graph showing seizure frequency in epileptic animals treated with different doses of hESC- derived MGE-type pallial interneurons, according to some non-limiting embodiments of the present disclosure. FIG. 7B is a collection of images showing immunohistochemical staining of the hippocampus for HNA and DAPI. FIG. 7C is a collection of images showing immunohistochemical staining of the hippocampus for HNA and LHX6. FIG. 7D is a collection of images showing immunohistochemical staining of the hippocampus for HNA and SST. FIGs. 7E-7J show quantification of human cell persistence and fate. FIGs. 7E and 7H are graphs showing, respectively, the total number of persisting human cells at 8.5 MPT and rate of persistence as a percentage of the initial dose. FIGs. 7F and 7I are graphs showing quantification of the total number of LHX6 positive human cells and the percentage of human cells expressing LHX6, respectively. FIGs. 7G and 7J are graphs showing the total number of SST positive human cells and the percentage human cells expressing SST, respectively.

[0020] FIGs. 8A-8Q show epileptic hippocampal pathology, behavioral outcome and animal survival after human interneuron transplantation, according to some non-limiting embodiments of the present disclosure. FIG. 8A is an image showing DAPI labeling of the granule cell (GC) layer in a naïve animal. FIG. 8B is an image showing DAPI labeling of the GC layer in an epileptic animal treated with vehicle. FIGs. 8C, 8D, 8E, and 8F are each an image showing DAPI labeling of the GC layer in an epileptic animal treated with the indicated dose of hESC-derived interneurons (per hippocampus). FIGs. 8G and 8H are graphs showing measurements of the GCL width and area, respectively. FIGs. 8I and 8J are images showing immunohistochemical staining of the hippocampus for caspase3 with NEUN and HNA. FIGs. 8K and 8L are images showing immunohistochemical staining of the hippocampus for calbindin and HNA. FIG. 8M is a collection of bar graphs showing results of a modified Irwin screen. FIG. 8N is a collection of bar graphs showing open field test results. FIG. 8O is a bar graph showing Y maze results. FIG. 8P is a bar graph showing Barnes Maze results. FIG. 8Q is survival curve for epileptic animals treated with either vehicle, or transplanted with cells.

[0021] FIGs. 9A-9G show in vitro characterization of hESC-derived VZ-like MGE progenitors after the two-week patterning phase, according to some non-limiting embodiments of the present disclosure. FIG. 9A is a collection of images showing FOXG1 expression in hESC-derived MGE-like progenitors by ICC (middle and bottom panels) and in the developing mouse brain at E13.5 by in situ hybridization (top panel). FIG. 9B is a collection of images showing NKX2-1 expression in hESC-derived MGE-like progenitors by ICC (middle and bottom panels) and in the developing mouse brain at E13.5 by in situ hybridization (top panel). FIG. 9C is a collection of images showing OTX-2 expression in hESC-derived MGE-like progenitors by ICC (middle and bottom panels) and in the developing mouse brain at E13.5 by in situ hybridization (top panel). FIG. 9D is a collection of images showing LHX6 staining in hESC-derived MGE-like progenitors by ICC (bottom panel) and in the developing mouse brain at E13.5 by in situ hybridization (top panel). FIG. 9E is a collection of images showing PAX6 staining in hESC-derived MGE-like progenitors by ICC (bottom panel) and in the developing mouse brain at E13.5 by in situ hybridization (top panel). FIG. 9F is a collection of images showing NKX2-2 staining in hESC-derived MGE-like progenitors by ICC (bottom panel) and in the developing mouse brain at E13.5 by in situ hybridization (top panel). FIG. 9G is a graph quantifying expression of different markers in hESC-derived MGE-like progenitors across different lots. FIG. 9H is a graph quantifying expression of different markers in hESC-derived MGE-like progenitors in lots 1 and 2.

[0022] FIGs. 10A-10J show efficient and reproducible hESC differentiation into MGE-type pallial interneurons, according to some non-limiting embodiments of the present disclosure. FIG. 10A is a schematic showing MGE pallial-type interneuron differentiation from hESCs and key markers used to characterize each stage. FIG. 10B is a collection of images showing expression of the indicated markers by ICC at different time points. FIG. 10C is a graph quantifying expression of the indicated markers over the different time points. FIG. 10D is a collection of images showing expression of the indicated markers by ICC at different time points. FIG. 10E is a graph quantifying expression of the indicated markers over the different time points. FIG. 10F is a collection of images showing expression of the indicated markers by ICC at different time points. FIG. 10G is a graph quantifying expression of the indicated markers over the different time points. FIG. 10H is a graphshowing the percentage of cells staining positive for the indicated markers, with or without MEK pathway inhibition. FIGs. 10I and 10J are a collection of images showing LHX8 expression in end-of-process pallial interneuron, with or without MEK pathway inhibition.

[0023] FIGs. 11A-11E show that no residual proliferative progenitors or pluripotent stem cells were identified in the end-of-process (week 6) samples, according to some non-limiting embodiments of the present disclosure. FIG. 11A is a graph quantifying Ki67 staining of cells derived from hESC with or without NOTCH and CDK pathway inhibition. FIG. 11B is a graph quantifying 5-ethynyl-2’-deoxyuridine (EdU) staining of cells derived from hESC with or without NOTCH and CDK pathway inhibition. FIG. 11C is a collection of images showing Edu staining in cells derived from hESC with (bottom panels) or without (top panels) NOTCH and CDK pathway inhibition. FIG. 11D is a collection of images showing Ki67 staining in cells derived from hESC with or without NOTCH and CDK pathway inhibition. FIGs. 11E and 11F are graphs quantifying expression of the indicated markers in cells derived from hESC with or without NOTCH and CDK pathway inhibition. FIGs. 11G-11I are a collection of flow cytometry plots showing distribution of cells expressing the indicated pluripotency markers for in-process (Week 4, FIG. 11G) and end-of- process (FIG. 11H) samples. FIG. 11I is a table showing the limits of detection (LOD) for residual TRA-1-60+ / OCT4+ cells for in-process and end-of-process samples.

[0024] FIGs. 12A-12E show in vitro migration phenotypes of hESC-derived pallial MGE-type interneurons, according to some non-limiting embodiments of the present disclosure, compared to human endogenous MGE interneurons and commercially available hPSC-derived GABAergic cells. FIG. 12A is a collection of phase contrast images showing a cell aggregate before (left most panel) and after 3 days of migration (remaining panels). FIG. 12B is a collection of images showing staining for LHX6, MAFB, and ERBB4 by immunocytochemistry (ICC). FIG. 12C is a graph quantifying percent migrating cells after 3 days. FIG. 12D is a collection of images comparing marker expression in hESC-derived pallial GABAergic MGE-type interneurons, according to some non-limiting embodiments of the present disclosure, and two commercial sources of hPSC-derived GABAergic neurons. FIG. 12E is a table comparing qualitative analysis of marker expression in pallial GABAergic interneurons derived from hESC, according to some non-limiting embodiments of the present disclosure, and two commercial sources of hPSC-derived GABAergic neurons.

[0025] FIGs. 13A-13C show single cell RNA sequencing gene expression patterns in vitro and in the developing human GE. FIG. 13A is a dot plot showing expression level of genes in each cluster. FIGs. 13B and 13C are a collection of violin plots showing expression of genes associated with different cell stages and cell types, by sample (FIG. 13B) and by cluster (FIG. 13C). FIG. 13D is plot visualizing gene expression across clusters.

[0026] FIGs. 14A-14H show reproducible seizure suppression achieved with additional independent cell production lots. FIG. 14A is an image showing the right hippocampus and the kainate injection site (CA1). FIG. 14B is a collection of images showing the right hippocampus overlayed with the sites of administration of the hESC- derived interneurons (dots) and the site of depth electrode implantation (arrow). FIG. 14C is a graph showing seizure frequency based on EEG recordings made at the indicated time points. FIG. 14D and 14E are graphs showing seizure frequency and cumulative seizure duration based on EEG recordings made at the indicated time points. FIG. 14F is a collection of images showing expression of SST and HNA in transplanted human cells. FIG. 14G is a collection of images showing fluorescence in situ hybridization (FISH) staining of the distribution of transplanted human cells in the hippocampus. FIG. 14H is a collection of images showing immunohistochemistry staining of transplanted human cells in the hippocampus.

[0027] FIG. 15 is a collection of graphs showing stability of cryopreserved hPSC- derived, pallial, MGE-type GABAergic interneurons after thawing.

[0028] FIGs. 16A and 16B are collection of graphs showing consistent delivery of hPSC-derived, pallial, MGE-type GABAergic interneurons held in a cannula configured to hold and deliver the cells, according to some non-limiting embodiments of the present disclosure.

[0029] FIG. 17 is a collection of schematics and plots showing stability of cells post-thaw and after a hold period in a cannula, according to some non-limiting embodiments of the present disclosure.

[0030] FIG. 18 is a collection of images showing migration potential of cells post-thaw and after a hold period in a cannula, according to some non-limiting embodiments of the present disclosure.

[0031] FIGs. 19A-19I show molecular characterization of human pallial interneurons 1-18 months post transplantation (MPT) using single nuclei RNA sequencing (snRNAseq). FIG. 19A is a plot showing UMAP (Uniform Manifold Approximation and Projection) visualization of cell clusters. FIG. 19B is a collection of plots showing prediction scores of transplanted hESC-derived pallial interneuron (pIN) gene expression compared with endogenous human pINs from adult M1 cortex. FIG. 19C is a collection of dot plots showing expression level of key genes that define different cell categories by the transplanted hESC-derived pINs. FIGs. 19D-19F are a collection of plots showing expression level of different genes across cells among the transplanted hESC-derived pINs. FIG. 19G is a plot showing subclass categories of transplanted human cells based on prediction and cluster marker analyses. FIG. 19H is a graph showing quantification of graft composition by subclass categories across 5 time points post-transplant. FIG. 19I is plot showing projection of hESC-derived MGE pIN subclasses onto human prenatal-adult prefrontal cortex interneuron dataset.

[0032] FIG. 20 is a collection of graphs showing the effect of single-dose administration of NRTX-1001 in subjects with drug-resistant MTLE, according to some non- limiting embodiments of the present disclosure.

[0033] FIG. 21 is a collection of graphs showing levels of metabolites in the hippocampus of a subject treated with NRTX-1001, according to some non-limiting embodiments of the present disclosure.

[0034] FIG. 22 depicts data showing the results of neurocognitive tests in subjects treated with NRTX-1001, according to some non-limiting embodiments of the present disclosure.

[0035] FIG. 23A is a collection of graphs showing the effect of transplanting hESC-derived interneurons on urethane-induced theta oscillations in a 5xFAD Alzheimer’s disease model.

[0036] FIG. 23B is a collection of graphs showing the effect of transplanting hESC-derived interneurons on 5xFAD AD mice treated with riluzole.

[0037] FIG. 24 is a schematic diagram showing a cell delivery system and use thereof, according to some non-limiting embodiments of the present disclosure.

[0038] FIGs. 25A-25N shows stable graft composition from 1 to 18 MPT comprised of PVALB and SST subclasses, according to some non-limiting embodiments of the present disclosure. FIG. 25A shows the experimental design: Cell lots (N=5) were transplanted into the neonate cortex in 3 deposits per hemisphere (75,000 cells / deposit). Cortical tissues (region of interest, ROI) were dissected at 1 MPT (N=3 lots), 3-4 MPT (N=3 lots), 6-7 MPT (N=4 lots), 12 MPT (N=2 lots) and 18 MPT (N=2 lots). Tissues from 2-3 animals were dissected for each lot and each time point and pooled for nuclei isolation. Nuclei were stained with an antibody against human nuclear antigen (HNA), followed by fluorescence activated nuclear sorting (FANS) to enrich rare human cells. The entire sorted samples were captured on microfluidic chips using the 10X controller (10X Genomics), followed by cDNA and library preparation for sequencing. Cells were aligned to both human and mouse transcriptomes, filtering out any captured mouse nuclei from downstream analyses. FIG. 25B shows integrated UMAP clustering of all the human cells across time points. FIG. 25C shows feature plots of GABAergic (GAD1, GAD2) and MGE (LHX6, SOX6) markers. FIG. 25D shows expression by cluster of genes that characterize different cell categories, including general markers of neurons, GABAergic and MGE neurons, preoptic area (POA), caudal and lateral GE (CGE / LGE), neuronal progenitors (NPC), cycling cells, ESC, as well as genes associated with glial cells, glutamatergic neurons (Glu), dopaminergic neurons (DA), serotonergic neurons (5HT) and cholinergic neurons (Ach). FIG. 25E shows population prediction scores comparing transplanted cells with human adult brain superclusters corresponding to diverse cell types42. FIG. 25F shows MGE interneuron prediction scores overlaid onto grafted cell UMAP. FIG. 25G shows graft composition over time based on transcriptional similarity to adult brain superclusters. FIG. 25H shows MGE- and CGE-derived interneuron subclass prediction scores overlaid onto grafted cell UMAP. For reference, the middle temporal gyrus (MTG) interneuron dataset was used48. FIG. 25I shows expression of markers associated with pIN subclasses listed at the top of each column. FIG. 25J shows UMAP visualization of the interneuron subclasses identified in the transplanted cells based on clustering and prediction analyses. FIGs. 25K and 25L show graft composition by subclass over time. FIG. 25M shows repeated measurements of a single lot composition transplanted from five different vials (n=5 technical replicates) at 1 and 4 MPT.FIG. 25N shows expression of subclass-enriched genes. For FIGs. 25G, 25K, 25L, data shown are mean ± SEM.

[0039] FIGs. 26A-26L show additional characterization of post-transplant cell identity. FIG. 26A shows quality metrics of post-transplant cell clusters, including number of unique genes detected (nFeature), number of total RNA molecules (nCount), percentage of mitochondrial and ribosomal genes, according to some non-limiting embodiments of the present disclosure. FIGs. 26B and 26C show UMAP colored by Age (FIG. 26B) and by batch ID (FIG. 26C). FIG. 26D shows expression of key genes over time. FIGs. 26E and 26F show prediction scores projected onto the integrated UMAPs based on comparisons to the listed human adult brain cell types (FIG. 26E) and brain regions (FIG. 26F)42. The ridge plot on the right panel in FIG. 26F shows distribution of prediction scores for the transplanted population compared to each reference brain region. FIG. 26G shows prediction scores projected onto the integrated UMAPs based on comparisons to endogenous human PVALB, SST and SST NPY subclasses, separated by age. The ridge plots show distribution of prediction scores for each subclass over time compared to adult human MTG (middle panels) 48 and M1 cortex (right panels)49reference datasets. FIG. 26H shows expression of several conserved markers (ERBB4, SLIT2, SHISA9, RUNX2) enriched in endogenous PVALB INs compared to SST INs in the adult human M1 cortex and mouse cortex + hippocampus (adapted from the Allen Brain Map: Cell Types Database). FIGs. 26I-26L show grafted human cells analyzed by fluorescence in situ hybridization (FISH) using RNAscope probes against the GABAergic marker glutamate decarboxylase 1 (GAD1), PVALB-enriched marker SLIT2, SST and NPY to assess the main cell populations. FIG. 26I shows MALAT1 was chosen as a FISH probe target to identify all human cells due to its strong and ubiquitous expression, regardless of subclass or MPT (99.99% of the cells are positive). FIG. 26J shows expression of GAD1, SLIT2, SST, and NPY transcripts by subclass over time. FIG. 26K shows representative FISH images showing expression of listed targets in transplanted human cells in the mouse cortex. Note that the GAD1 probe, although designed against human mRNA sequence, also binds to endogenous mouse GAD1. FIG. 26L shows quantification of MALAT1+ cells co-expressing listed targets. Each dot is from a different hemisphere, after averaging technical replicates (4 sections each). 1 MPT (n=3 hemi, 2 cell lots), 4-18 MPT (n=8 hemi, 3 cell lots). Lines show mean ± SEM.

[0040] FIGs. 27A-27J show transplantation environment triggers expression of synaptic genes enabling rapid cell fate identification, according to some non-limiting embodiments of the present disclosure. FIGs. 27A, and 27B show integrated clustering analysis of one lot (010519S1) sequenced prior to transplantation (whole cells = PRE cell, and nuclei = PRE nuc), after one month of in vitro (30 DIV), 14 days post-transplantation (14 DPT) and 1 MPT, colored by cluster (FIG. 27A) and by subclass (FIG. 27B). FIG. 27C shows alluvial plots showing transcriptomic relationship between pre- and post-transplant cell populations. FIG. 27D shows graft composition by subclass at 14 DPT and 1 MPT. FIGs. 27E and 27F show PVALB, SST and SST NPY prediction scores across samples shown as feature plots (FIG. 27E) and ridge plots (FIG. 27F). FIG. 27G shows expression of cell-type- specific genes upregulated in 1 MPT vs 30 DIV samples. FIGs. 27H-27J show evaluation of graft development post-transplantation compared with human cortical development spanning from prenatal to postnatal stages. The quantification is based on predicted cell stage analysis using cortical developmental datasets as a reference51. FIG. 27H shows distribution of grafted cell maturation states over time on the top relative to broad human stages, corresponding to developmental changes in neuronal physiology shown on the bottom (adapted from19). FIG. 27I shows grafted cell maturation states over time separated by the three main subclasses. FIG. 27J shows on the left, the re-clustering of endogenous human interneurons from prefrontal cortex, PFC (original dataset from51) colored by age, with the CGE- and MGE-derived interneuron populations indicated. On the right, projection of grafted cells at different stages colored by subclass onto human PFC interneurons.

[0041] FIGs. 28A-28G show gene expression patterns under different conditions and at different stages post-transplant. FIGs. 28A and 28B show integrated clustering analysis of lot 010519S1 sequenced prior to transplantation (PRE cells and PRE nuclei), after 30 DIV (nuclei), 14 DPT (nuclei) and 1 MPT (nuclei), colored by cluster (FIG. 28A) and by subclass (FIG. 28B). FIG. 28C shows expression of ERBB4 (enriched in PVALB subclass) and RELN (enriched in SST subclass) in PRE, 30 DIV and 1 MPT nuclei samples. FIG. 28D shows GO term analysis for biological processes enriched in subclass-specific genes that are upregulated in 1 MPT vs 30 DIV samples. FIGs. 28E-28G show GO term analysis for biological processes (FIG. 28E), cellular components (FIG. 28F), and molecular functions (FIG. 28G) enriched at each of the main transcriptional states: PRE, 1 MPT, and 3+ MPT.

[0042] FIGs. 29A-29L show cell development is characterized by major transcriptional changes during the first 3 MPT followed by gradual unfolding of gene modules involved in regulation of synaptic transmission and membrane potential. FIGs. 29A- 29C show clustering analysis of all the cells pooled together (without integration), colored by MPT (FIG. 29A), by subclass with all the stages combined (FIG. 29B), or separated by MPT (FIG. 29C). FIG. 29D shows expression of neuronal, GABAergic and MGE markers across transcriptional states. FIG. 29E shows stage-specific expression pattern corresponding to pre- transplant (PRE), 1 MPT and 3-18 MPT transcriptional states. FIG. 29F shows gene networks of top 4 GO biological processes enriched at each of the main transcriptional states. FIG. 29G shows identification of genes through DGE analysis that continue to be upregulated over time. FIG. 29H shows GO term analysis for biological processes that are enriched after 3 MPT and 6 MPT. FIGs. 29I and 29J show expression of gene modules that regulate chemical synaptic transmission (FIG. 29I) and membrane potential (FIG. 29J) in the grafts over time (top plots). In the bottom plots, expression of the same modules is plotted for 18 MPT grafts in the context of human cortical development. FIGs. 29K and 29L show expression of genes identified in continuously upregulated modules involved in vesicle- mediated transport in synapse, modulation of chemical synaptic transmission and synapse organization (FIG. 29K), as well as regulation of presynaptic membrane and action potential (FIG. 29L).

[0043] FIGs. 30A-30G show hMGE-pIN grafts are comprised of multiple PVALB and SST subtypes, according to some non-limiting embodiments of the present disclosure. FIG. 30A shows the cluster correlation to each other. FIG. 30B shows the proportion of nuclei from the 18 MPT ESC-derived clusters (columns) that correspond to endogenous adult interneuron subtypes (rows) defined in the cell census atlas of the mammalian primary motor cortex10based on prediction analysis. Of the 72 transcriptionally defined CGE- and MGE-derived interneuron subtypes, only the ones which transplanted hMGE-pINs match by >10% are included in this heatmap. To the right, human cluster cellular taxonomy classification is shown, based on mouse / human cross-species overlap analysis, where multimodal data (morphology, physiology and transcriptomics) were applied to define the mouse interneuron subtypes10. FIG. 30C shows UMAP of all the transplanted cells, colored by transcriptionally distinct populations based on data-driven marker analyses,cluster correlations and overlap with human interneuron subtypes. FIG. 30D shows graft composition by subtype over time. Bar graphs shown mean ± SEM. FIG. 30E shows top subtype markers. FIG. 30F shows expression of transcription factors, sodium, calcium and potassium voltage gated ion channels and regulatory subunits, as well as cell adhesion molecules involved in cell recognition43, in pooled 12-18 MPT data across subtypes. FIG. 30G shows expression of several voltage gated ion channels over time and across subtypes.

[0044] FIGs. 31A-31S shows grafted hMGE-pINs acquire histological features of known PVALB and SST subtypes, according to some non-limiting embodiments of the present disclosure. FIG. 31A shows a diagram showing transplantation sites and dosing in the neonatal cortex for histological characterization. FIG. 31B shows distribution of hMGE-pINs labeled with human-specific nuclear antigen (HNA), the immature neuronal marker DCX, and the MGE-lineage marker LHX6 at 1 MPT (coronal view). FIG. 31C shows the percent of HNA+ cells persisting at 1, 4, and 17 MPT out of the number of cells that were injected. FIG. 31D shows the relative rostrocaudal distribution of HNA+ cells at 1 and 4 MPT. FIG. 31E shows the relative positioning of hMGE-pINs in the cortex based on expression of ERBB4 / SST (upper panel, n=199 cells from 3 transplanted animals; 22.6% ERBB4+, 41.2% SST+) and SST / NOS1 (lower panel, n=396 cells from 3 transplanted animals; 49.4% SST+, 9.6% NOS1+). Dashed lines indicate the surface, layers I / II, III / IV, and V / VI transitions (from top to bottom). FIG. 31F shows an example of hMGE-pIN with Martinotti morphology. Martinotti cells are characterized by mid-to-low SST and CNTNAP4 expression and long ascending axon collaterals that reach layer I to profusely ramify. FIGs. 31G and 31H show human SST-positive cells co-expressing Martinotti markers CALB1 (FIG. 31G) and ETV1 (FIG. 31H). FIG. 31I and 31J show human SST-positive cells co-expressing SST1 (predicted non-Martinotti subtype) cluster markers GULP1 (FIG. 31I) and NPAS3 (FIG. 31J). FIG. 31K shows an example of a GFP-labeled long-range projection (LRP) neuron in layer VI with a long axonal collateral projecting parallel to the corpus callosum (inset shows SST+ expression in its soma and higher magnifications of the long projection). FIGs. 31L and 31M show human cells with LRP characteristics frequently express SST, NPY, and NOS1. FIG. 31N-31P show representative images of NPY+SST+ axonal projections, which are frequently found in the caudal hippocampus (medial DG-CA3 (FIG. 31N)), midline corpus callosum (FIG. 31O), and caudate putamen (next to the nucleus accumbens (FIG. 31P)). FIG. 31Qshows an example of a ERBB4+ (SST negative) hIN with basket cell features in layer IV. FIG. 31R shows these cells form axonal arborizations (marked by human-specific SYP) around the somata of pyramidal neurons and interneurons. FIG. 31S shows nuclear expression of the PV2 (predicted Basket subtype) cluster marker BCL6. Inset shows BCL6 nuclear expression and lack of SST expression. All images and quantifications were taken from animals at 7-15 MPT.

[0045] FIGs. 32A-32N’ show hMGE-pINs exhibit subclass-specific synaptic connectivity features in the host mouse cortex, according to some non-limiting embodiments of the present disclosure. FIGs. 32A-32B show increased cortical expression of the human- specific synaptophysin (SYP) in layers I-II (FIG. 32A) and IV (FIG. 32B) at 1, 7, and 13 MPT. FIG. 32C show Diagram showing a pyramidal projection neuron with dendritic (Pex5L) and somatic (NeuN), as well as presynaptic (Gephyrin and GabaR1) marker expression. Postsynaptic and axonal markers associated with SST (green) and PVALB (red) subclasses are illustrated collocalizing with human-specific TAU and SYP. FIG. 32D and 32E show examples of inhibitory synapses found in layer I composed of presynaptic human SYP and SST and postsynaptic Gephyrin (arrows). FIG. 32F shows SST-expressing human axonal terminals are intimately intertwined with Pex5L+ distal dendrites of pyramidal cells (FIG. 32F’, 32F” show higher magnifications with SST expression). FIG. 32G, 32G’ show representative images showing co-expression of human-specific SYP and synaptoporin (SYPR) in axonal terminals in layers I / II. FIGs. 32H and 32I show examples of human axonal processes in layer I co-expressing SST, the glutamate ionotropic receptor, GRIN3A (FIG. 32H), and the SST1-cluster marker, ARHGAP6 (arrows in 32I; additional examples are shown in 32I’ and I”). FIGs. 32J-32L show Human axonal terminals show perisomatic synaptic patterns associated with PVALB cells in layer IV. Examples of perisomatic synapses formed between presynaptic human SYP (FIG. 32J, 32K) and LGI2 (FIG. 32L), and host cell postsynaptic GabaR1 (arrows). Bottom panels (FIGs. 32J’-32L”) correspond to additional examples for each marker combination at higher magnification. NeuN labels neuronal cell bodies (FIG. 32K). FIG. 32M and 32N show representative images showing perisomatic human axonal terminals co-expressing the PVALB-enriched potassium channels, KIR3.1 (encoded by KCNJ3) and KV3.2 (encoded by KCNC2) (FIG. 32M and 32N,respectively). Insets (32M’ and 32N’) correspond to higher magnification of their respective images. Samples were obtained at 7-18 MPT.

[0046] FIGs. 33A-33D show stability of cells post-thaw and following a hold period in a cannula, according to some non-limiting embodiments of the present disclosure. The data demonstrate that the viability (FIG. 33A), concentration (FIG. 33B), metabolic activity (FIG. 33C), and migratory potential (FIG. 33D) of cells held for 48 hours at 2-18°C are comparable to the overnight hold reference control after 7 hours of cannula retention at room temperature. The study includes N = 5-10 independent experiments using 6 different cell batches. Error bars represent the standard error of the mean.

[0047] FIGs. 34A-34C show persistence, migration, and distribution of hPSC- derived GABAergic interneurons at 1 month-post-transplantation (1 MPT) into neonate P1-2 rodent brains, according to some non-limiting embodiments of the present disclosure. Results show that cell persistence (FIG. 34A), migration (FIG. 34B) and distribution of cells (FIG. 34C) following hypothermic storage at 2-18C for 48-72 hrs are comparable to the overnight reference control dose preparation method.

[0048] FIGs. 35A-35B show single-cell RNA sequencing analysis of hPSC- derived GABAergic interneurons after 1 month and 4 months post-transplantation, according to some non-limiting embodiments of the present disclosure. Transplantation into rodent brains revealed no off-target cell types, off-target gene expression, or significant differences in gene expression patterns (FIG. 35A). The gene expression profiles and composition were consistent between cells stored at 2-18°C for 48-72 hours and the overnight reference control, and were comparable between the 1-month and 4-month post-transplantation time points (FIG. 35B).

[0049] FIGs. 36A-36B show effects of neurotransmitter receptor antagonists on spontaneous calcium activity from in vitro human pallial interneuron (hpIN) cultures, according to some non-limiting embodiments of the present disclosure. FIG. 36A shows calcium traces show activity of 30 neurons before (baseline) and after the addition of small molecule antagonists (dashed red line). After 17 days in vitro, samples were treated using control media (vehicle), two sets of two compounds (Picrotoxin + Saclofen or NBQX + AP- 5), or TTX, all diluted in vehicle media. FIG. 36B shows fold change in firing rate after application of each set of compounds shows no significant changes in calcium activity beforeand after addition of vehicle (control media), picrotoxin and saclofen (GABAA and GABAB receptor antagonists, respectively), or NBQX and AP-5 (AMPA- and NMDA-receptor antagonists, respectively). However, the addition of TTX (Na+ channel blocker) almost completely abolished calcium activity, indicating that the calcium activity is mediated by action potentials. Statistics: One way ANOVA with Bonferroni post-hoc analysis (ns-no significance, *p<0.05, **p<0.01, ***p<0.001).

[0050] FIGs. 37A-37T show multi-electrode array analysis of co-cultured hiPSC derived glutamatergic neurons, mouse primary astrocytes with or without human GABAergic pallial interneurons (hpIN) , according to some non-limiting embodiments of the present disclosure. FIG. 37A shows representative immunostaining images demonstrating survival of glutamatergic neurons (EMX2, TBR1), astrocytes (GFAP) and interneurons (LHX6, SST) after 40 days of co-culture in vitro on the MEA plate (FIGs. 37B-37C). Analysis of synchrony index and mean firing rate for Cell Dynamics Incorporated (CDI) iPSC-derived glutamatergic neurons with only primary mouse astrocytes (blue, CDI Gluta alone) or with human pallial interneurons (green, CDI Gluta + hpIN). FIGs. 37D-37E shows analysis of synchrony index and mean firing rate for Bit.bio (ioGluta) iPSC-derived glutamatergic neurons with only primary mouse astrocytes (purple, BitBio Gluta alone) or with human pallial interneurons (red, BitBio Gluta + hpIN). FIG. 37F-37K show connectivity (synchronicity) and neuron network dynamics (network) measurements for 24 days in vitro (DIV) (highlighted dashed box in FIGs. 37D-37E) show a clear difference + / - hpIN. FIG. 37L shows ioGluta only cultures show no changes in neural acivity after the addition of the GABAA receptor antagonist, picrotoxin. However, ioGluta + hpIN show an obvious change in network activity after the addition of picrotoxin. FIG. 37M shows PCA analysis revealing that ioGluta-only cultures cluster together before and after picrotoxin addition, while ioGluta + hpIN cultures cluster separately at baseline but move closer to ioGluta-only wells, post- picrotoxin treatment. FIG. 37N-37Q show UMAP analysis of all network events detected at DIV 40 in the MEA system across 4 samples (n=6 wells per sample): (FIG. 37N) ioGluta alone at baseline, (FIG. 37O) ioGluta alone post 20 minutes treatment with picrotoxin, (FIG. 37P) ioGluta co-cultured with hpIN at baseline and (FIG. 37Q) ioGluat+hpIN after a 20 minutes picrotoxin treatment. FIG. 37R shows Louvain clustering analysis identified 6 main clusters in the UMAP representation of all network event properties. FIG. 37S shows aspecific color has been attributed to each cluster. FIG. 37T shows quantification of network event distribution across the 6 identified clusters demonstrate ioGluta alone cultures show similar distribution of the network events before and after picrotoxin addition. However, ioGluta + hpIN clustering shows a very different distribution with a large portion of network events in cluster#2 not shared with ioGluta alone cultures. The addition of picrotoxin to ioGluta + hpIN leads to a distribution of network events in these clusters that resembles ioGluta alone before and after picrotoxin. Acronyms: CC – Cross-correlegram, NB – network burst, NBpC – network burst per electrode.

[0051] FIGs. 38A-38B shows epileptic MTLE mouse model behavioral outcome after human pIN transplantation, according to some non-limiting embodiments of the present disclosure. (Open Field test (FIG. 38A, general anxiety, spatial locomotion and travel velocity) and Light-Dark Emergence (FIG. 38B, Anxiety). Also shown are percent of animals emerged and non-emerged. Data are shown for the middle dose (200K). Kruskal Wallis test for differences between groups; significant differences are indicated by asterisk (P<0.05).

[0052] FIGs. 39A-39E show an epileptic MTLE mouse model hippocampal pathology analysis showing that the progression of dentate granule cell dispersion from before transplant to 8.5 months post-human pIN transplantation is halted, with similar dispersion observed across time points post-transplant, according to some non-limiting embodiments of the present disclosure. FIG. 39A shows granule cell dispersion analysis at 1 MPSE (age and time-matched to efficacy study mice at time of pIN transplantation. FIGs. 39A-39D show granule cell dispersion analysis at 8.5 MPT in age-matched mice: Prox1 labeling shows representative granule cell (GC) layer in early epileptic mice (FIG. 39A), naive mouse age-matched to end of study mice (FIG. 39B), epileptic cell-treated mice at end of study (FIG. 39C) and epileptic vehicle-treated mice at end of study (FIG. 39D). FIG. 39E shows Average GC layer area. All data are mean ± SEM. The Kruskal Wallis statistic, followed by Dunn’s test, was significant between all three groups at the end of the study (P<0.05). GC area in epileptic mice at time of transplant was not significantly different from cell-treated mice at the end of study.

[0053] FIGs. 40A-40D show percent change from baseline in neurocognitive and quality of life test performance after low dose administration of NRTX-1001 to humanMTLE subjects in the ongoing Phase 1 / 2 study, according to some non-limiting embodiments of the present disclosure. FIG. 40A shows BNT = Boston Naming Test total score. FIG. 40B shows BVMT: Brief Visuospatial Memory Test delayed recall score. FIG. 40C shows RAVLT: Ray Auditory Verbal Learning Test delayed recall score. FIG. 40D shows QOLIE- 31P: Quality of Life total score. T-score measurements with reliable change index noted.

[0054] FIGs. 41A-41B show percent change from baseline in monthly seizure frequency after low dose administration of NRTX-1001 to human MTLE subjects in the ongoing Phase 1 / 2 study, according to some non-limiting embodiments of the present disclosure. FIG. 41A shows all seizures plotted, including focal aware, impaired awareness, and focal to bilateral tonic clonic seizures. FIG. 41B shows only disabling seizures plotted, including focal aware, impaired awareness, and focal to bilateral tonic clonic seizures as above but excluding aware seizures without objective manifestation (auras).

[0055] FIGs. 42A-42B show histological characterization of amyloid plaques in two Alzheimer’s disease (AD) mouse models: 5XFAD and TG2576, according to some non- limiting embodiments of the present disclosure. FIG. 41A shows amyloid plaque deposits are observed in the cortex and hippocampus in two models of AD, TG2576 and 5XFAD at 21 and 16 months of age. FIG. 41B shows higher magnification images of the amyloid plaques (cyan) surrounded by activated microglia marked by CD68 (red) in the TG2576 and 5XFAD models.

[0056] FIGs. 43A-43B show human interneuron transplantation in two mouse models of Alzheimer’s disease, according to some non-limiting embodiments of the present disclosure. FIG. 43A shows images of human ESC-derived interneurons transplanted into the rostro-dorsal and caudal-ventral hippocampus of 5XFAD mice at 11 MPT. The transplanted cells (marked by human-specific nuclear antigen (HNA) and the interneuron subtype marker, SST) persist and migrate throughout the hippocampus. In addition, the transplanted cells project extensively to surrounding areas as marked by the human-synaptophysin (SYP) marker. FIG. 43B shows images of human ESC-derived interneurons transplanted into the rostro-dorsal and caudal-ventral hippocampus of TG1576 mice at 16 MPT. The transplanted cells (marked by human-specific nuclear antigen (HNA) and the interneuron subtype marker, SST) persist and migrate throughout the hippocampus. In addition, the transplanted cells project extensively to surrounding areas as marked by the human-TAU marker.

[0057] FIGs. 44A-44B show human interneuron transplantation reduces the number of amyloid plaques in the TG2576 AD mouse model, according to some non-limiting embodiments of the present disclosure. FIG. 44A shows quantification of plaque densities in the hippocampus at 3 different levels (rostral, middle and caudal) in vehicle-injected and human interneuron transplanted animals. FIG. 44B shows the effect of human cell transplantation in the average plaque size in the hippocampus of TG2576 mice.

[0058] FIG. 45 shows human interneuron transplantation rescues animal survival in the TG2576 AD mouse model, according to some non-limiting embodiments of the present disclosure. Percentages of surviving mice since the time of cell transplantation in naive (NCAR) and TG2576 transgenic mice injected with vehicle (VEH) or human interneurons (CELL).

[0059] FIGs. 46A-46B show human interneuron transplantation increases the power of gamma oscillations in the TG2576 AD mouse model during the resting phase, according to some non-limiting embodiments of the present disclosure. FIG. 46A shows relative power with respect to the resting phase of theta (4-12 Hz, left) and gamma (20-60 Hz) oscillations in non-carrier (NCAR, gray), vehicle injected transgenic (VEH, red), and human cell transplanted transgenic (CELL, blue) in 24 hr EEG recording. FIG. 46B shows absolute power of theta (4-12 Hz, left) and gamma (20-60 Hz) oscillations in NCAR (gray), vehicle injected (red), and human cell transplanted (blue).

[0060] FIGs. 47A-47C show embryonic induction of mTor, according to some non-limiting embodiments of the present disclosure. In utero electroporation of a constitutively active form of Rheb, a main transducer of the mTor pathway. Expression plasmid of Rheb-CA and GFP is shown in FIG. 47A, and the electroporation method is illustrated in FIG. 47B (at E15.5). FIG. 47C shows GFP and phospho-S6 (a mTor indicator) expression in the medial prefrontal cortex (mPFC) at E18.5 (left). GFP can be used to screen for GFP+ pups at P1 (middle and right panels).

[0061] FIGs. 48A-48B show histological validation of embryonic induction of mTor, according to some non-limiting embodiments of the present disclosure. FIG. 48A shows GFP, GFPA, and pS6 expression in the mPFC of two different animals at P30. GFAP is a marker of active gliosis, a hallmark of FCD. FIG. 48B shows higher magnificationimages of the examples in FIG. 48A. This illustrates differences in RhebCA-GFP expression in different animals, and the activation of pS6 and GFAP.

[0062] FIGs. 49A-49D show neonatal induction of mTor, according to some non- limiting embodiments of the present disclosure. FIGs. 49A-49B show neonatal delivery of a constitutively active form of Rheb by AAVs. Diagram of an AAV backbone with Rheb-CA and GFP driven by the projection neuron CamKII promoter. AAVs were delivered into the mPFC in P0-P1 CD1 pups at several different doses. FIG. 49C shows RhebCA / GFP- expressing cells correspond to cortical projection neurons (marked by NeuroD2), but not to GABAergic interneurons (marked by Lhx6). FIG. 49D shows co-expression of GFP and phospho-S6 in transduced cells and induction of GFAP in the mPFC at P30.

[0063] FIGs. 50A-50B show histological validation of the neonatal induction of mTor in immunosuppressed mice, according to some non-limiting embodiments of the present disclosure. FIG. 50A shows co-expression of GFP, Rheb, and phospho-S6 in transduced cells and induction of GFAP in the mPFC of Scid-beige mice at P30. FIG. 50B shows high magnification images showing co-expression of GFP and phospho-S6 (marked by arrows) in transduced cells in the medial mPFC.

[0064] FIGs. 51A-51C show electrographic validation of the embryonic induction of mTor, according to some non-limiting embodiments of the present disclosure. FIG. 51A shows examples of generalized seizures recorded with superficial electrodes in animals embryonically induced with Rheb-CA. FIG. 51B shows average seizure rates from a cohort of 6 embryonically-induced animals (3 weeks recording). FIG. 51C shows daily seizure recording of 3 animals with generalized seizures.

[0065] FIGs. 52A-52B shows electrographic validation of the neonatal induction of mTor, according to some non-limiting embodiments of the present disclosure. FIGs. 52A- 52B shows examples of epileptiform activity recorded at 2 months post-implant with cortical electrodes in CD1 (FIG. 52A) and Scid-beige (FIG. 52B) animals induced with Rheb-CA AAVs neonatally. DETAILED DESCRIPTION

[0066] Provided herein are methods of treating seizure activity, e.g., mesial temporal lobe epilepsy (MTLE), using a cellular composition of pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells. In some embodiments, a substantial portion, e.g., at least 90%, of the cells of the cellular composition are post-mitotic. In general terms, a method of the present disclosure includes identifying a subject in need of treating seizure activity; and administering a therapeutically effective amount of a cellular composition enriched for pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells. In some embodiments, the cellular composition administered to the subject includes (or is enriched for) pallial, MGE-type GABAergic interneuron cells derived from pluripotent stem cells (e.g., hESCs). In some embodiments, at least 90% of the cells of the cellular composition administered to the subject are pallial MGE-type GABAergic interneuron cells derived from pluripotent stem cells. In some embodiments, the method reduces the frequency of seizures in the subject, e.g., by at least about 50% in some embodiments. In some embodiments, administering the pluripotent stem cell-derived, MGE- type inhibitory (e.g., GABAergic) neuron cells (e.g., pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells) can restore local inhibitory tone to reduce the frequency of seizures in the subject. In any method of the present disclosure, in some embodiments, the MGE-type inhibitory (e.g., GABAergic) neuron cells include MGE-type inhibitory (e.g., GABAergic) interneuron cells. In some embodiments, the MGE-type inhibitory (e.g., GABAergic) neuron cells are of the pallial lineage. In any method of the present disclosure, in some embodiments, the pluripotent stem cell-derived, MGE-type GABAergic neuron cells are pallial, MGE-type GABAergic interneuron cells. In some embodiments, the MGE-type inhibitory (e.g., GABAergic) neuron cells are of the subpallial lineage. In any method of the present disclosure, in some embodiments, the pluripotent stem cell-derived, MGE-type GABAergic neuron cells are subpallial, MGE-type GABAergic neuron cells.

[0067] In some embodiments, the administered cells locally disperse, mature into cortical / hippocampal-type interneurons, and persist in the subject’s temporal lobe, e.g. hippocampus, for an extended duration. In some embodiments, the high proportion of post- mitotic cells in the administered cellular composition promotes a stable cell fate of the pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells) after administration to the subject. In some embodiments, administering the pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pluripotent stem cell- derived, pallial, MGE-type GABAergic interneuron cells) reduces granule cell dispersion and / or neuronal degeneration in the subject’s temporal lobe. In some embodiments, administering the pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells) treats seizure activity, e.g., MTLE, such as MTLE that does not respond to anti-seizure drugs. In some embodiments, methods of the present disclosure provide a cell therapy alternative to resection / ablation surgery for drug-resistant MTLE. In some embodiments, administration of the pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells) to the subject’s brain (e.g., hippocampus, cortex, and / or amygdala) provides a targeted treatment for the seizure activity that minimizes side effects such as behavioral abnormalities and / or adverse immunological reactions by the subject. In some embodiments, the pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells) are administered to one or more regions of the temporal lobe: hippocampus, subiculum, entorhinal cortex, and / or parahippocampal gyrus. In some embodiments, the interneurons, precursors, and / or pluripotent stem cells, are genetically modified to evade the recipient's immune system (e.g., immune-cloaking). Terms

[0068] Unless indicated otherwise, terms have their customary and ordinary meaning as understood by one of ordinary skill in the art in view of the present disclosure.

[0069] “Seizure” or “seizure activity” as used herein has its customary and ordinary meaning as understood by one of ordinary skill in the art in view of the present disclosure. A seizure can include an uncontrolled, abnormal and / or synchronous brain activity, and / or one or more symptoms thereof. Unless indicated otherwise, reference to a seizure includes epileptic seizure-like discharges or epileptiform activity in the brain. A seizure may cause one or more of changes in the level of consciousness, behavior, memory, or emotional states. The seizure can be convulsive or non-convulsive. The seizure can be provoked or unprovoked. A seizure can include a focal seizure (e.g., focal aware seizure and / or focal impaired awareness seizure) or a focal to bilateral tonic-clonic seizure.

[0070] “Focal onset seizure” or “focal seizure” as used herein has its customary and ordinary meaning as understood by one of ordinary skill in the art in view of the present disclosure, and denotes a seizure that occurs in or originates from one hemisphere or a subpart thereof of a subject’s brain. “Generalized seizure” as used herein has its customary and ordinary meaning to one of ordinary skill in the art in view of the present disclosure, and denotes a seizure that affects the whole brain or that is not limited to one hemisphere. A focal onset seizure can become generalized in some cases. A focal onset seizure can include a focal aware seizure and / or focal impaired awareness seizure.

[0071] “Epilepsy” as used herein has its customary and ordinary meaning as understood by one of ordinary skill in the art in view of the present disclosure. An epilepsy refers to a disorder characterized by recurring seizures. “Focal epilepsy” refers to recurring seizures that affect one hemisphere or a subpart thereof of a subject’s brain.

[0072] “Cellular composition” as used herein refers to an isolated collection or population of cells. The composition can include, without limitation, primary cells, stem cells, cells differentiated from stem cells, and cell lines. The cells of the composition can be suspended in, adhered to and / or embedded in any suitable medium or substrate.

[0073] A “progenitor” or “progenitor cell” as used herein refers to a mitotic cell that is differentiating from a pluripotent stem cell, and is capable of further differentiating into a terminally-differentiated cell (e.g., as determined by changes in marker expression). A progenitor cell in some embodiments can differentiate into a desired terminally-differentiated cell (“on target”), or terminally-differentiated cell that is not desired (“off target”).

[0074] “MGE-type” as used herein with reference to progenitors, lineages, neurons, and interneurons, refers to cells that express markers expressed by cells in the MGE region of the developing brain, or by neurons differentiated from cells of the MGE region. In general, MGE progenitor cells express markers such as, Foxg1, homeobox gene Nkx2.1, LIM-homeobox genes Lhx6, Lhx7, or Lhx8. MGE progenitor cells are capable of differentiating into interneurons and / or projection neurons under suitable differentiation conditions.

[0075] “Pluripotent stem cell” or “pluripotent cell” refers to a cell that has the ability, under appropriate conditions, of producing progeny of several different cell types that are derivatives of any one of the three germinal layers (endoderm, mesoderm, and ectoderm).Pluripotent stem (PS) cells are capable of forming teratomas. Examples of pluripotent stem cells are embryonic stem (ES) cells, embryonic germ stem (EG) cells, embryonal carcinoma stem (EC) cells, and induced pluripotent stem (iPS) cells. PS cells may be from any one of a variety of organisms, including, e.g., human; primate; non-human primate; canine; feline; murine; equine; porcine; avian; camel; bovine; ovine, and so on.

[0076] “Express” as used herein refers to the presence of a gene product (e.g., mRNA and / or protein) in or associated with a cell or a group of cells at a level above a relevant reference level (e.g., a predetermined level for detecting the gene product, or a level of the gene product that effectively differentiates between populations of cells known to either express or not express the gene product). In some cases, the reference level is a background level of expression.

[0077] As used herein, “post-mitotic” denotes a cell in a state after it has exited the cell cycle. Cell cycle exit may be reversible or irreversible. In some embodiments, a post-mitotic cell is terminally differentiated. A post-mitotic cell can be characterized by expression, or lack thereof, of one or more markers (e.g., Ki67, ASPM), and / or measuring incorporation, or lack thereof, of a nucleoside analog.

[0078] “Effective amount” as used herein refers to an amount sufficient to bring about a desired result. “Therapeutically effective amount” as used herein refers to an amount sufficient for a therapeutic agent to bring about a therapeutically desired result.

[0079] As used herein, “treat” or “treatment” refer to curing, preventing occurrence of, ameliorating, preventing deterioration of, reducing one or more symptoms of, and / or slowing the progress of a condition or disease.

[0080] “Subject” or “individual” as used herein can be any one of a variety of animals, such as a mammal. Mammals include, but are not limited to, humans, primates, horses, pigs, cows, dogs, cats, mice and rats, etc. In some embodiments, a subject can be male or female. “Subject” and “patient” are used interchangeably herein.

[0081] “Transforming growth factor betas”, “TGF-βs”, and “TGFBs” are used interchangeably herein to refer to the TGFB secreted proteins belonging to the subfamily of the transforming growth factor β (TGFβ) superfamily. TGFBs (TGFB1, TGFB2, TGFB3) are multifunctional peptides that regulate proliferation, differentiation, adhesion, and migration and in many cell types. The mature peptides may be found as homodimers or asheterodimers with other TGFB family members. TGFBs interact with transforming growth factor beta receptors (TGF-βRs, or TGFBRs) on the cell surface, which binding activates MAP kinase-, Akt-, Rho- and Rac / cdc42-directed signal transduction pathways, the reorganization of the cellular architecture and nuclear localization of SMAD proteins, and the modulation of target gene transcription. Inhibitors of TGFB signaling, can be readily be identified by any of a number of suitable assays, for example competitive binding assays for binding to TGFB or TGFB receptors, or functional assays, e.g. measuring suppression of activity of downstream signaling proteins such as MAPK, Akt, Rho, Rac, and SMADs, e.g., AR-Smad, etc.

[0082] “Bone morphogenic proteins” or “BMPs” as used herein refers to the family of growth factors that is a subfamily of the transforming growth factor β (TGF β) superfamily. BMPs (e.g. BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9 / GDF, BMP10, BMP11 / GDF11, BMP12 / GDF7, BMP13 / GDF6, BMP14 / GDF5, BMP15 / GDF9B) were first discovered by their ability to induce the formation of bone and cartilage. BMPs interact with specific receptors on the cell surface, referred to as bone morphogenetic protein receptors (BMPRs). Signal transduction through BMPRs results in mobilization of members of the SMAD family of proteins, which in turn modulate transcription of target genes. Inhibitors of BMP signaling, can readily be identified by any of a number of suitable assays, for example competitive binding assays for binding to BMP or BMP receptors, or functional assays, e.g., measuring enhancement of activity of downstream signaling proteins such as relocalization of SMADs, such as, BR-Smad to the nucleus and transcriptional activation of downstream gene targets.

[0083] “Wnts” as used herein refers to the family of highly conserved secreted signaling molecules which play key roles in both embryogenesis and mature tissues. The human Wnt gene family has at least 19 members (Wnt-1, Wnt-2, Wnt-2B / Wnt-13, Wnt-3, Wnt3a, Wnt-4, Wnt-5A, Wnt-5B, Wnt-6, Wnt-7A, Wnt-7B, Wnt-8A, Wnt-8B, Wnt-9A / Wnt- 14, Wnt-9B / Wnt-15, Wnt-10A, Wnt-10B, Wnt-11 Wnt-16). Wnt proteins modulate cell activity by binding to Wnt receptor complexes that include a polypeptide from the Frizzled (Fz) family of proteins and a polypeptide of the low-density lipoprotein receptor (LDLR)- related protein (LRP) family of proteins. Once activated by Wnt binding, the Wnt receptor complex will activate one or more intracellular signaling cascades. These include thecanonical Wnt signaling pathway; the Wnt / planar cell polarity (Wnt / PCP) pathway; and the Wnt-calcium (Wnt / Ca2+) pathway.

[0084] “Sonic hedgehog” and “shh” as used herein refers to a member of the Hedgehog (Hh) protein family, that is a secreted glycoprotein that undergoes post- translational modifications, including auto-catalytic cleavage and cholesterol coupling to amino-terminal peptide to form the fragment that possesses the signaling activity. Hh binds to the 12-step transmembrane protein Ptch (Ptch1 and Ptch2), thereby alleviating Ptch- mediated suppression of Smoothened (Smo). Activation Smo triggers a series of intracellular events that culminate in the stabilization of Gli transcription factors (Gli1, Gli2, and Gli3) and the expression of Gli-dependent genes that are responsible for cell proliferation, cell survival, angiogenesis, and invasion.

[0085] “Notch” as used herein refers to a signaling receptor in the Notch signaling pathway that controls cell fate during development, cell survival and cell proliferation. The Notch receptor is a single-pass transmembrane receptor containing numerous tandem epidermal growth factor (EGF)-like repeats and three cysteine-rich Notch / LIN-12 repeats within a large extracellular domain. Notch receptors are activated by single-pass transmembrane ligands of the Delta, Serrated, Lag-2 (DSL) family. Mammalian Notch receptors undergo cleavage to form the mature receptor and also following ligand binding to activate downstream signaling. A furin-like protease cleaves the Notch receptors during maturation to generate juxtamembrane heterodimers that comprise a non-covalently associated extracellular subunit and a transmembrane subunit held together in an auto- inhibitory state. Ligand binding relieves this inhibition and induces cleavage of the Notch receptor by an ADAM-type metalloprotease and a gamma-secretase, the latter of which releases the intracellular domain (ICD) into the cytoplasm, allowing it to translocate into the nucleus to activate gene transcription. Cleavage by ADAM occurs within the non-ligand binding cleavage domain within the membrane proximal negative regulatory region.

[0086] “CDK” or “cyclin dependent kinase” as used herein refers to a family of serine-threonine kinase proteins that regulate progression through the various phases of the cell cycle. CDKs use ATP as a substrate to phosphorylate diverse polypeptides in a sequence dependent manner. Cyclins are a family of proteins characterized by a homology region,containing approximately 100 amino acids, termed the “cyclin box” which is used in binding to, and defining selectivity for, specific CDK partner proteins.

[0087] “MEK” as used herein refers to one of three kinases that are part of the MAP (mitogen-activated protein) kinase (MAPK) pathway that regulates cell proliferation and / or differentiation. The MAPK pathway encompasses a cascade of phosphorylation events involving three key kinases, namely Raf, MEK (MAP kinase kinase) and ERK (MAP kinase). Active GTP-bound Ras results in the activation and indirect phosphorylation of Raf kinase. Raf then phosphorylates MEK1 and 2. Activated MEK then phosphorylates its only known substrates, the MAP kinases, ERK1 and 2. Phosphorylated ERK dimerizes and then translocates to the nucleus where it accumulates. In the nucleus, ERK is involved in several important cellular functions, including but not limited to nuclear transport, signal transduction, DNA repair, nucleosome assembly and translocation, and mRNA processing and translation. Overall, treatment of cells with growth factors leads to the activation of ERK1 and 2 which results in proliferation and, in some cases, differentiation.

[0088] As used herein with reference to a numerical value, “K” indicates units of a thousand, and “M” indicates units of a million. For examples “200K” indicates “200,000”, and “1.5M” indicates “1,500,000.”

[0089] The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. The abbreviation, “e.g.” is used herein to indicate a non- limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.” The term “about” as used herein to, for example, define the values and ranges of molecular weights means that the indicated values and / or range limits can vary within ±20%, e.g., within ±10%, including within ±5%. The use of “about” before a number includes the number itself. For example, “about 5” provides express support for “5.” METHODS

[0090] Provided herein are methods of treating seizure activity, e.g., mesial temporal lobe epilepsy (MTLE), in a subject in need thereof. With reference to FIG. 1A, a method of treating seizure activity is provided. The method 100a can include at block 110a identifying a subject in need of treating seizure activity. The method can further include atblock 120a administering (e.g., intracranially administering) a therapeutically effective amount of a cellular composition comprising pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells, as described herein. The pluripotent stem cell- derived, MGE-type inhibitory (e.g., GABAergic) neuron cells can in some embodiments be post-mitotic cells, where for example, at least 90% of the cells are post-mitotic cells. In some embodiments, at least 50% of the cells of the composition express markers for MGE- type GABAergic neuron cells (e.g., express at least LHX6 and GAD1). In some embodiments, at least 50% of the cells of the composition express markers for pallial, MGE- type GABAergic interneuron cells (e.g., express LHX6, ERBB4, and GAD1). In any method of the present disclosure, in some embodiments, a cellular composition includes pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells. In some embodiments, at least 50% of the cells of the composition express markers for subpallial, MGE-type GABAergic neuron cells (e.g., at least 50% of the cells express LHX6, LHX8 or NKX2.1, and GAD1, and do not express ERBB4). With reference to FIG. 1B, a method 100b of the present disclosure can include: at block 110b identifying a subject in need of treating seizure activity. The method can further include at block 120b administering (e.g., intracranially administering) a therapeutically effective amount of a cellular composition comprising pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells, as described herein.

[0091] A subject treated by the methods of the present disclosure can have hyperexcitability and / or hyperactivity in neurons or a network thereof. In some embodiments, the subject is suffering from hyperexcitability and / or hyperactivity in neurons or a network thereof, where the hyperexcitability and / or hyperactivity is associated with a neurological condition. In some embodiments, the subject has a neurological condition that is associated with neuronal hyperexcitability and / or hyperactivity in one or more brain regions. In some embodiments, the subject has a neurodegenerative disease, such as but not limited to Alzheimer’s disease (AD) or Parkinson’s disease (PD) that is associated with the neuronal hyperexcitability and / or hyperactivity. In some embodiments, the subject suffers from epileptic seizure-like discharges in the brain associated with Alzheimer’s disease, focal cortical dysplasia (FCD), or amnesic mild cognitive impairment (aMCI). In some embodiments, subject has AD and exhibits one or more symptoms of AD. In someembodiments, the AD subject has or exhibits symptoms of tauopathy. In some embodiments, the subject has schizophrenia, spasticity, and / or neuropathic pain that is associated with the neuronal hyperexcitability and / or hyperactivity. In some embodiments, the subject has a neuropsychiatric disorder associated with the neuronal hyperexcitability and / or hyperactivity.

[0092] In some embodiments, the subject is suffering from hyperexcitability and / or hyperactivity in one or more brain regions, e.g., in the temporal lobe. In some embodiments, the subject is suffering from hyperexcitability and / or hyperactivity in the hippocampus. In some embodiments, the subject is suffering from hyperexcitability and / or hyperactivity in one or more of the following regions of the temporal lobe: hippocampus, subiculum, entorhinal cortex, and / or parahippocampal gyrus.

[0093] In some embodiments, the subject is in need of treatment of seizures and / or symptoms thereof. In some embodiments, the seizure is convulsive. In some embodiments, the seizure is non-convulsive (e.g., is an absence seizure). In some embodiments, the seizure is a focal onset seizure, or a focal epilepsy. In some embodiments, the seizure is localized to and / or originates from the temporal lobe of the left or right hemisphere of the subject’s brain. In some embodiments, the seizure is localized to and / or originates from one or more subregions of the temporal lobe. In some embodiments, the seizure is localized to and / or originates from the subiculum, entorhinal cortex, and / or parahippocampal gyrus. In some embodiments, the seizure is localized to and / or originates from the hippocampus (e.g., left and / or right hippocampus) and / or from the amygdala (e.g., left or right amygdala). In some embodiments, the subject suffers from a focal onset seizure that develops into a generalized seizure. In some embodiments, the subject does not have a generalized seizure. In some embodiments, the subject has a chronic seizure (e.g., a recurring seizure). In some embodiments, the subject has an epilepsy, e.g., a focal epilepsy or a temporal lobe epilepsy. In some embodiments, the subject has temporal lobe epilepsy (TLE) (e.g., drug-resistant TLE). In some embodiments, the subject suffers from a neocortical onset focal epilepsy. In some embodiments, the subject suffers from focal seizure activity. In some embodiments, the subject has mesial temporal lobe epilepsy (MTLE). In some embodiments, the subject has AD with epileptic activity. In some embodiments, the subject has AD and has, or suffers from symptoms of, tauopathy.

[0094] In some embodiments, the subject has been diagnosed as having or suffering from seizure activity. In some embodiments, the subject has been diagnosed to have one or more conditions underlying the seizures (e.g., an infection, disease, stroke, trauma, neurological disorder, metabolic disorder, developmental disorder, drug exposure, or stress). The subject can be identified as in need of treating seizure activity by any suitable option. In some embodiments, the subject is identified as having or suffering from seizure activity based on a clinical manifestation of symptoms of seizure. In some embodiments, the subject is experiencing or has experienced one or more symptoms of the seizure. In some embodiments, the subject is identified as suffering from seizure activity based on a subjective assessment of one or more symptoms of the seizure. In some embodiments, the subject is identified as suffering from seizure activity based on brain monitoring. In some embodiments, the subject is identified as suffering from seizure activity after undergoing one or more brain monitoring such as, without limitation, computed tomography (CT) scan, magnetic resonance imaging (MRI), MR spectroscopy (MRS), functional MRI (fMRI), electroencephalography (EEG), intracranial EEG, positron emission tomography (PET), and single photon emission computed tomography (SPECT).

[0095] In some embodiments, the seizure activity is an electrographic seizure. In some embodiments, the subject is identified as having or suffering from electrographic seizures. In some embodiments, the electrographic seizure is detected using an EEG. In some embodiments, an electrographic seizure includes a spike train of about 1 Hz or more, e.g., about 2 Hz, about 3 Hz, about 4Hz, about 5 Hz or more, or at a frequency in a range defined by any two of the preceding values (e.g., 1-5 Hz, 2-5 Hz, 3-4 Hz, 3-5 Hz, etc.), for example, as detected by EEG. In some embodiments, an electrographic seizure includes a spike train having a duration of about 5 seconds or longer, e.g., about 10 seconds, about 15 seconds, about 20 seconds, about 30 seconds or longer, or a period of time in a range defined by any two of the preceding values (e.g., 5-30 seconds, 10-20 seconds, 15-30 seconds, 5-20 seconds, etc.). In some embodiments, an electrographic seizure includes an inter-event (or inter-spike train) interval of about 1 second or more, e.g., about 2 seconds, about 3 seconds, about 5 seconds, about 10 seconds, about 15 seconds, about 20 seconds, about 30 seconds or more, or a period of time in a range defined by any two of the preceding values (e.g., 1-30 seconds, 2-20 seconds, 3-30 seconds, 5-15 seconds, 5-30 seconds, etc.).

[0096] In some embodiments, a chronic or recurring seizure can occur at one or more frequencies. In some embodiments, the seizure occurs at a frequency of once per 30 minutes or more, e.g., 2 times per 30 minutes or more, about 5 times per 30 minutes or more, e.g., about 10 times per 30 minutes or more, about 15 times per 30 minutes or more, about 20 times per 30 minutes or more, about 30 times per 30 minutes or more, about 50 times per 30 minutes or more, or at a frequency in a range defined by any two of the preceding values (e.g., 1-50 times / 30 minutes, 2-30 times / 30 minutes, 3-20 times / 30 minutes, 5-15 times / 30 minutes, 10-15 times / 30 minutes, etc.). In some embodiments, the seizure occurs at a frequency of once per week or more, e.g., 2 times per week or more, about 5 times per week or more, about 10 times per week or more, about 15 times per week or more, about 20 times per week or more, about 30 times per week or more, about 50 per week times or more, or at a frequency in a range defined by any two of the preceding values (e.g., 1-50 times / week, 2-30 times / week, 3-20 times / week, 5-15 times / week, 10-15 times / week, etc.). In some embodiments, the seizure occurs at a frequency of once per month or more, e.g., 2 times per month or more, about 5 times per month or more, about 10 times per month or more, about 15 times per month or more, about 20 times per month or more, about 30 times per month or more, about 50 times per month or more, or at a frequency in a range defined by any two of the preceding values (e.g., 1-50 times / month, 2-30 times / month, 3-20 times / month, 5-15 times / month, 10-15 times / month, etc.). In some embodiments, the frequency at which the seizure occurs (without administering the therapeutic compositions of the present disclosure) does not vary substantially over a month, two months, 3 months, 4 months, 6 months, or over 12 months or more. In some embodiments, the frequency at which the seizure occurs (without administering the therapeutic compositions of the present disclosure) increases over a month, two months, 3 months, 4 months, 6 months, or over 12 months or more.

[0097] The length of an episode of a seizure from which the subject is suffering can vary. In some embodiments, an episode of the seizure lasts for about 5 second or longer, e.g., about 10 seconds or longer, about 30 seconds or longer, about 60 seconds or longer, about 90 seconds or longer, about 120 seconds or longer, about 180 seconds or longer, about 4 minutes or longer, about 5 minutes or longer, about 10 minutes or longer, about 15 minutes or longer, about 20 minutes or longer, or about 30 minutes or longer, or a period of time in a range defined by any two of the preceding values. In some embodiments, an episode of theseizure lasts from about 5 second to about 30 minutes. In some embodiments, an episode of the seizure lasts from about 5-10 seconds, 10-30 seconds, 30-60 seconds, 60-90 seconds, 90- 120 seconds, 120-180 seconds, 3-4 minutes, 4-5 minutes, 5-10 minutes, 10-15 minutes, 15-20 minutes, or 20-30 minutes.

[0098] In some embodiments, the seizure activity is a chronic condition. In some embodiments, the seizure activity is a provoked seizure, or a non-provoked seizure. In some embodiments, the seizure activity is associated with an infection, disease (e.g., a tumor), stroke, trauma, neurological disorder, metabolic disorder, developmental disorder, drug exposure (e.g., drug overdose), focal cortical dysplasia, tuberous sclerosis, or stress. In some embodiments, the seizure activity is due to a traumatic brain injury (e.g., to the temporal lobe), focal cortical dysplasia, tuberous sclerosis, or due to a neurological disorder. In some embodiments, the seizure activity is due to a stroke, a tumor, or a developmental disorder. In some embodiments, the subject has mesial temporal sclerosis. In some embodiments, the subject has hippocampal sclerosis, and / or an extrahippocampal sclerosis (e.g., sclerosis of the amygdala). In some embodiments, the subject has one or more lesions, e.g., epileptogenic lesions, in the brain. In some embodiments, the subject has one or more lesions, e.g., epileptogenic lesions, in the temporal lobe. In some embodiments, the subject has 1, 2, 3, 4, 5 or more lesions, e.g., epileptogenic lesions, in the brain (e.g., in the temporal lobe). In some embodiments, the subject has increased granule cell dispersion in the temporal lobe, e.g., in the hippocampus. In some embodiments, the cause of the seizure is unknown.

[0099] In some embodiments, the seizure activity is resistant to a conventional anti-seizure therapy. In some embodiments, the subject’s seizure activity is not adequately treated after treatment with one, two or more anti-seizure therapies. In some embodiments, the subject has drug-resistant TLE, e.g., drug-resistant MTLE. Drug-resistant seizure or epilepsy as used herein has its ordinary and customary meaning as understood by one of ordinary skill in the art in view of the present disclosure. In some embodiments, drug- resistant TLE is resistant to treatment with carbamazepine or levetiracetam.

[0100] In some embodiments, the method includes identifying mesial temporal sclerosis in the subject’s brain. In some embodiments, the method includes identifying hippocampal sclerosis, and / or an extrahippocampal sclerosis (e.g., sclerosis of the amygdala). In some embodiments, the method includes identifying a lesion, e.g., an epileptogenic legion,in the subject’s brain (such as in the temporal lobe). Any suitable option can be used to identify a lesion or mesial temporal sclerosis in the subject’s brain. In some embodiments, the method includes using one or more suitable imaging techniques (e.g., computed tomography (CT) scan, magnetic resonance imaging (MRI), MR spectroscopy (MRS), functional MRI (fMRI), electroencephalography (EEG), intracranial EEG, positron emission tomography (PET), and single photon emission computed tomography (SPECT), etc.) to identify mesial temporal sclerosis or a lesion, e.g., an epileptogenic legion, in the subject’s brain, before, concurrent to, and / or after administering the cellular composition. In some embodiments, the method includes using one or more suitable imaging techniques, as described herein, to determine that the mesial temporal sclerosis is reduced or eliminated after administering the cellular composition. In some embodiments, the method includes using one or more suitable imaging techniques, as described herein, to determine that the lesion is reduced or eliminated after administering the cellular composition.

[0101] In some embodiments, the method includes using one or more suitable brain activity monitoring techniques to determine whether the subject exhibits seizure activity, e.g., before and / or after administering the cellular composition. In some embodiments, the method includes using one or more suitable brain activity monitoring techniques, as described herein, to determine that the frequency of seizures is reduced after administering the therapeutically effective amount of the cellular composition containing the pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells). In some embodiments, the method includes performing one or more of computed tomography (CT) scan, magnetic resonance imaging (MRI), MR spectroscopy (MRS), functional MRI (fMRI), electroencephalography (EEG), intracranial EEG, positron emission tomography (PET), and single photon emission computed tomography (SPECT) on the subject.

[0102] In some embodiments, seizure activity is measured by self-assessment of the frequency, severity and / or type of seizure experienced by the subject over a suitable time period. A subject can make a self-assessment of the frequency, severity and / or type of the seizure using any suitable option. In some embodiments, the subject has described the frequency, severity and / or type of seizure experienced over a suitable time period (e.g., daily, weekly, monthly, etc.) in a seizure diary. In some embodiments, the subject has orallyconveyed the frequency, severity and / or type of seizure experienced over a suitable time period (e.g., daily, weekly, monthly, etc.). In some embodiments, the method includes comparing the frequency, severity and / or type of seizure experienced by the subject before and after administration of the cellular composition as recorded by the subject in a seizure diary.

[0103] In some embodiments, the method includes administering the therapeutically effective amount of the cellular composition to the temporal lobe of the subject. In some embodiments, the method includes administering the therapeutically effective amount of the cellular composition to the temporal lobe ipsilateral to seizure activity in the subject’s brain (e.g., ipsilateral to a focal onset seizure). In some embodiments, where the focal onset seizure is in or originates from the left hemisphere (e.g., left hippocampus, left amygdala) of the subject’s brain, the method includes administering the therapeutically effective amount of the cellular composition to the left temporal lobe. In some embodiments, where the focal onset seizure is in or originates from the right hemisphere (e.g., right hippocampus, right amygdala) of the subject’s brain, the method includes administering the therapeutically effective amount of the cellular composition to the right temporal lobe. In some embodiments, the method includes administering the cellular composition only to the temporal lobe ipsilateral to the focal onset seizure. In some embodiments, the method includes administering the therapeutically effective amount of the cellular composition to the temporal lobe bilaterally. In some embodiments, the cellular composition comprises genetically modified or gene edited cells, e.g., to evade the recipient's immune system, as described herein.

[0104] In some embodiments, the cellular composition is administered as one or more deposits in the subject’s brain. In some embodiments, the cellular composition is administered as 1, 2, 3, 4, 5, 6, 7, 8 or more deposits in the subject’s brain. In some embodiments, the method include administering the cellular composition to one or more sites, e.g., 2, 3, 4, 5, 6, 7, 8, or more sites, in the hippocampus, cortex, subiculum, entorhinal cortex, parahippocampal gyrus, and / or amygdala. In some embodiments the cellular composition is administered to one or more distinct sites (e.g., target sites) within the temporal lobe (e.g., the hippocampus, subiculum, entorhinal cortex, parahippocampal gyrus, and / or amygdala) ipsilateral to the focal onset seizure. Suitable sites within the temporallobe include, without limitation, the hippocampus, cortex, amygdala, subiculum, entorhinal cortex, and / or the parahippocampal gyrus. In some embodiments, the cellular composition is administered to one or more sites in the temporal lobe ipsilateral to the focal onset seizure. In some embodiments, the cells are administered to at least 1, 2, 3, 4, 5, 6, 7, or 8 or more different sites within the subject’s temporal lobe ipsilateral to the focal onset seizure. In some embodiments, administration of the pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells) to the subject’s brain (e.g., temporal lobe: hippocampus, subiculum, entorhinal cortex, and parahippocampal gyrus) provides a targeted treatment for the seizure activity that minimizes side effects such as behavioral abnormalities.

[0105] In some embodiments, the cells of the composition are administered to, around, or distal to, a site of mesial temporal sclerosis in the subject’s brain. In some embodiments, the number of cells administered (per hemisphere or per deposit) depends on the size and / or extent of mesial temporal sclerosis in the subject’s brain.

[0106] In some embodiments, the cells of the composition are administered to, around, or distal to, one or more sites of a lesion, e.g., epileptogenic lesion, in the subject’s brain (e.g., temporal lobe). In some embodiments, the number of cells administered (per hemisphere or per deposit) depends on the size and / or extent of a lesion (e.g., epileptogenic lesion).

[0107] In some embodiments, the cells of the composition are administered to, around, or distal to, one or more sites within the subject’s brain identified by functional neuroimaging. In some embodiments, the cells of the composition are administered to, around, or distal to, one or more sites identified as a seizure onset zone using functional neuroimaging. In some embodiments, the cells of the composition are administered to, around, or distal to, one or more sites identified as an epileptogenic zone using functional neuroimaging. Any suitable option for functional neuroimaging can be used. In some embodiments, functional neuroimaging comprises one or more of PET, SPECT, magnetoencephalogram / magnetic source imaging (MEG / MSI), diffusion tensor imaging (DTI), fMRI, fMRI-EEG, MR spectroscopy (MRS), arterial spin labeling (ASL). In some embodiments, the subject does not or is not known to have a mesial temporal sclerosis or a lesion.

[0108] Any suitable volume of the cellular composition can be administered per deposit. In some embodiments, the cellular composition is delivered in a volume of, of about, or of at most 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 8, 6, 5, 4, 3, 2 μL per deposit, or of, of about, or of at least 0.1, 0.5, 0.8, 1, 1.2, 1.5, 2, 2.5, 3 μL per deposit, or a volume per deposit in a range defined by any two of the preceding values (e.g., 0.1-100 μL per deposit, 0.1-50 μL per deposit, 0.1-20 μL per deposit, 0.5-10 μL per deposit, 1.0-5 μL per deposit, 1.5 -4 μL per deposit, 1-2 μL per deposit, 2-4 μL per deposit, etc.). In some embodiments, the cellular composition is delivered in a volume of about 50 μL or less (e.g., 1-50 μL) per deposit. In some embodiments, the cellular composition is delivered in a volume of about 20 μL or less (e.g., 1-20 μL) per deposit. In some embodiments, the cellular composition is delivered in a volume of about 10 μL or less (e.g., 1-10 μL) per deposit.

[0109] Any suitable number of the cells can be administered to the subject. In some embodiments, the cellular composition administered to the subject includes, per hemisphere, about 1 x 104cells or more, about 2 x 104cells or more, about 3 x 104cells or more, about 5 x 104cells or more, about 1 x 105cells or more, about 2 x 105cells or more, about 5 x 105cells or more, about 1 x 106cells or more, about 2 x 106cells or more, about 5 x 106cells or more, about 1 x 107cells or more, about 2 x 107cells or more, about 5 x 107cells or more, about 1 x 108cells or more, about 1 x 109cells or more, about 2 x 109cells or more, about 5 x 109cells or more, about 1 x 1010cells or more, about 2 x 1010cells or more, about 5 x 1010cells or more, about 1 x 1011cells or more, about 2 x 1011cells or more, about 5 x 1011cells or more, about 1 x 1012cells or more, or a number of cells within a range defined by any two of the preceding values (e.g., 3 x 104- 1 x 1012cells, 1 x 105- 5 x 1011cells, 3 x 105- 1 x 1011cells, 1 x 106- 1 x 1010cells, 3 x 104– 2 x 106, or 1 x 107- 1 x 109cells, etc.). In some embodiments, the cellular composition administered to the subject includes, per hemisphere, from about 1 x 104to about 1 x 108cells, from about 2 x 104to about 5 x 107cells, from about 5 x 104to about 1 x 107cells, from about 1 x 105to about 1 x 107cells, from about 2 x 105to about 5 x 106cells, from about 5 x 105to about 5 x 106cells, from about 3 x 104to about 1 x 1012cells, from about 3 x 104to about 1 x 1011cells, from about 1 x 106to about 1 x 1010cells, or from about 3 x 104to about 2 x 106cells. In some embodiments, the cellular composition administered to the subject includes, per hemisphere, from about 1 x 105to about 1 x 109cells, from about 2 x 105to about 5 x 108cells, fromabout 5 x 105to about 1 x 108cells, from about 1 x 106to about 1 x 108cells, from about 2 x 106to about 5 x 107cells, from about 5 x 106to about 5 x 107cells, from about 3 x 105to about 1 x 1012cells, from about 3 x 105to about 1 x 1011cells, from about 1 x 106to about 1 x 1010cells, or from about 3 x 106to about 5 x 107cells. In some embodiments, the cellular composition administered to the subject includes, per hemisphere, about 1 x 107cells to about 5 x 107cells.

[0110] In some embodiments, the number of cells administered to the subject per deposit is about 1 x 103cells or more, about 2 x 103cells or more, about 5 x 103cells or more, about 1 x 104cells or more, about 2 x 104cells or more, about 5 x 104cells or more, about 1 x 105cells or more, about 2 x 105cells or more, about 5 x 105cells or more, about 1 x 106cells or more, about 2 x 106cells or more, about 5 x 106cells or more, about 1 x 107cells or more, about 1 x 107cells or more, about 2 x 107cells or more, about 5 x 107cells or more, about 1 x 108cells or more, about 1 x 109cells or more cells, or a number of cells within a range defined by any two of the preceding values (e.g., 1 x 103- 1 x 109, 1 x 104- 1 x 108, 1 x 104- 2 x 108, 1 x 105- 1 x 107, 1 x 105- 1 x 106, or 1 x 103- 1 x 107cells, etc.).

[0111] In some embodiments, the cellular composition is divided equally for administering to multiple sites, such that each deposit includes approximately the same number of cells of the composition. In some embodiments, at least two different deposits include substantially different numbers of cells of the composition. In some embodiments, the number of cells administered in two different deposits differs by about 50% or less, e.g., about 40% or less, about 30% or less, about 20% or less, about 10% or less, about 5% or less, about 2% or less, or at most by a percentage within a range defined by any two of the preceding values (e.g., at most 2-50%, 2-40%, 5-30%, 5-20%, or 10-20%, etc.).

[0112] In some embodiments, the number of cells administered to the subject is based on a scaling factor applied to the number of cells administered in preclinical studies using a species that is different from the subject. In some embodiments, the scaling factor takes into account a ratio of the volume of a brain region (e.g., hippocampus) between the species used in the preclinical studies and the species to which the subject belongs. In some embodiments, the species used in preclinical studies is a mouse and the subject is a human subject, where the ratio of the volume of a brain region (e.g., hippocampus) between the two species is between about 100 and about 200, e.g., between about 120 and about 190, betweenabout 130 and about 180, between about 140 and about 180, between about 150 and 170. In some embodiments, the species used in preclinical studies is a mouse and the subject is a human subject, where the ratio of the volume of a brain region (e.g., hippocampus) between the two species is between about 150 and 170. In some embodiments, the species used in preclinical studies is a mouse and the subject is a human subject, where the scaling factor is at least about 5 fold, e.g., at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 12 fold, at least about 14 fold, at least about 16 fold, at least about 18 fold, at least about 20 fold, at least about 25 fold, at least about 30 fold, at least about 50 fold, at least about 75 fold, at least about 100 fold, or a fold difference in a range defined by any two of the preceding values (e.g., 5-100 fold, 5-75 fold, 5-50 fold, 6-30 fold, 6-25 fold, 7-20 fold, 8-12 fold, 18-25 fold, etc.). In some embodiments, the species used in preclinical studies is a mouse and the subject is a human subject, where the scaling factor is about 10 fold. In some embodiments, the species used in preclinical studies is a mouse and the subject is a human subject, where the scaling factor is about 20 fold.

[0113] The methods can employ any suitable option for administering the cellular composition to the subject’s brain. In general, the cells are administered intracranially. In some embodiments, the cells are administered using a stereotactic system. In some embodiments, administering includes injecting or implanting the cellular composition of the present disclosure into one or more target sites in the subject. In some embodiments, administering includes injecting or implanting the cellular composition of the present disclosure as one or more deposits in the subject’s brain, as described herein. In some embodiments, the cells of the present disclosure can be inserted into a delivery device, which facilitates introduction by injection or implantation of the cells into the subject’s brain. In some embodiments, such delivery devices include a tubular structure with an internal compartment, e.g., cannula, or catheters, for holding the cells and fluids and / or for injecting cells and fluids into the body of a subject. In some embodiments, the tubular structure additionally have a needle tip through which the cells can be introduced into the subject at a desired location. In some embodiments, the method includes using a cannula or catheter, or similar device, to administer the cellular composition to a subject (e.g., to the subject’s brain). In some embodiments, the method includes using a delivery device, as describedherein, to administer the cellular composition to the subject (e.g., to the subject’s brain). In some embodiments, the method includes detecting the position of the delivery device (for example, the proximal end (e.g., the subject-facing end) of the delivery device) holding the cellular composition in the subject’s brain while, or for at least some portion of, administering the cellular composition to a subject. In some embodiments, the cellular composition includes a contrast agent that facilitates visualizing the position of the delivery device (for example, the proximal end (e.g., the subject-facing end) of the delivery device) in the subject’s brain during administration of the cellular composition. In some embodiments, the method includes using MRI to visualize the administration or delivery of the cellular composition (e.g., where the delivery vehicle includes an MRI contrast agent).

[0114] The cellular composition of the present disclosure, or a portion thereof, can be in any suitable form for inserting into the delivery device. In some embodiments, the method includes formulating the cellular composition of the present disclosure, or a portion thereof, in a suitable form for inserting into the delivery device. For example, without limitation, the cells can be suspended in a solution or embedded in a support matrix when contained in such a delivery device. As used herein, the term “solution” includes a pharmaceutically acceptable vehicle, carrier, or diluent in which the cells remain viable. Pharmaceutically acceptable vehicles, carriers, and diluents can include, without limitation, saline, aqueous buffer solutions, solvents and / or dispersion media. In some embodiments, the solution is sterile and fluid to facilitate delivery. In some embodiments, the solution is stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi through the use of, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In some embodiments, solutions of the present disclosure are prepared in a pharmaceutically acceptable carrier or diluent and, as required, other ingredients enumerated above, followed by filter sterilization.

[0115] In some embodiments, the cellular composition is provided in a suitable delivery vehicle. In some embodiments, the delivery vehicle includes pharmaceutically acceptable salts, supplements, lipid polymers, non-ionic surfactants, and / or MRI contrast agents, such as, without limitation, gadolinium. In some embodiments, the delivery vehicle includes a non-ionic surfactant, e.g., a poloxamer. Suitable poloxamers include, withoutlimitation, P124, P188, P237, P338, and / or P407. Any suitable amount of the poloxamer can be included in the delivery vehicle. In some embodiments, the delivery vehicle includes, v / v, about 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, or a percentage in a range defined by any two of the preceding values (e.g., 0.01%-2%, 0.05%-1%, 0.1%-0.5%, 0.2%-1.5%, etc.) of the poloxamer. In some embodiments, the delivery vehicle includes a poloxamer at 0.01%-2%, v / v. In some embodiments, the delivery vehicle includes a poloxamer at 0.1%-1%, v / v.

[0116] In some embodiments, the delivery vehicle includes a preservation supplement (e.g., neural supplements, B27, N2, buffers, CSB, trehalose, sugars, salts, lipids, proteins, reducing agents, and / or Hypothermosol®). Any suitable amount of the preservation supplement can be included in the delivery vehicle. In some embodiments, the delivery vehicle includes, v / v, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or a percentage in a range defined by any two of the preceding values (e.g., 1-20%, 2-15%, 5-15%, 5-20%, etc.) of the preservation supplement.

[0117] In some embodiments, the delivery vehicle includes a base medium. Any suitable based medium can be used. In some embodiments, the base medium includes, without limitation, aCSF, HBSS, MEM, DMEM, L-15, neurobasal medium, and combinations thereof. In some embodiments, the delivery vehicle includes a contrast agent. Any suitable contrast agent for imaging applications can be used. In some embodiments, the contrast agent is a paramagnetic contrast agent. In some embodiments, the contrast agent is an MRI contrast agent, including, without limitation, a gadolinium- or manganese-based contrast agent. In some embodiments, the contrast agent is an iodine-based or barium-based contrast agent. In some embodiments, the delivery vehicle includes one or more salts, e.g., sodium chloride, potassium chloride, etc. In some embodiments, the delivery vehicle includes a suitable buffer at physiological pH.

[0118] In some embodiments, the cellular composition includes one or more salts, e.g., sodium chloride, potassium chloride, etc. In some embodiments, the cellular composition includes a salt (e.g., sodium chloride) at a concentration of, of about, or of at least 100, 120, 140, 160, 180, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, or 400 mM, or an osmolality of, of about, or of at most 600, 590, 580, 570, 560, 550, 540, 530, 520, 510, 500, 490, 480, 470, 460, 450, 440,430, 420, 410, or 400 mM, or at a concentration in a range defined by any two of the preceding values (e.g., 100-600 mM, 200-500 mM, 150-450 mM, 250-500 mM, 300-450 mM, etc.). In some embodiments, the cellular composition includes sodium chloride at a concentration of 100-600 mM. In some embodiments, the cellular composition includes sodium chloride at a concentration of 100-600 mM. In some embodiments, the cellular composition has an osmolality of, of about, or of at least 100, 120, 140, 160, 180, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 mOsm / kg, or an osmolality of, of about, or of at most 1,000, 900, 800, 750, 700, 690, 680, 670, 660, 650, 640, 630, 620, 610, 600, 590, 580, 570, 560, 550, 540, 530, 520, 510, or 500 mOsm / kg, or an osmolality in a range defined by any two of the preceding values (e.g., 100-1,000 mOsm / kg, 100-900 mOsm / kg, 200-800 mOsm / kg, 200-600 mOsm / kg, 200-500 mOsm / kg, 250-500 mOsm / kg, 300-450 mOsm / kg, etc.). In some embodiments, the cellular composition has an osmolality of at least 200 mOsm / kg. In some embodiments, the cellular composition has an osmolality of at least 300 mOsm / kg. In some embodiments, the cellular composition has an osmolality of 100-900 mOsm / kg. In some embodiments, the cellular composition has an osmolality of 300-500 mOsm / kg.

[0119] In some embodiments, the cellular composition includes a high concentration of cells (e.g., pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells) in suspension, e.g., when administered to the subject. Without being bound by theory, a sufficiently high concentration of cells in the cellular composition effectively raises the viscosity such that the cellular composition can be held stably in a defined volume at the tip of a cannula, or a catheter, or similar delivery device, where the rest of the internal compartment of the cannula can be filled with a cell-free liquid chase vehicle or inner stylet. In some embodiments, the cellular composition includes cells at a concentration of about 0.1 x 106cells per microliter or more, e.g., about 0.2 x 106cells per microliter, about 0.3 x 106cells per microliter, about 0.5 x 106cells per microliter, about 0.75 x 106cells per microliter, about 1 x 106cells per microliter, about 1.5 x 106cells per microliter, about 2 x 106cells per microliter, about 3 x 106cells per microliter, about 4 x 106cells per microliter or more, or at a concentration in a range defined by any two of the preceding values (e.g., 0.1 x 106-4 x 106cells per microliter, 0.2 x 106-2 x 106cells permicroliter, 0.2 x 106-1.5 x 106cells per microliter, 0.1 x 106-3 x 106cells per microliter, etc.). In some embodiments, the cellular composition includes cells at a concentration of about 1 x 105cells / μL or greater, and up to about 2 x 106cells per microliter. In some embodiments, the cellular composition includes cells at a concentration of, of about, or of at least 0.1 x 106, 0.2 x 106, 0.3 x 106, 0.4 x 106, 0.5 x 106, 0.6 x 106, 0.7 x 106, 0.8 x 106, 0.9 x 106, 1 x 106, 1.1 x 106, 1.2 x 106, 1.3 x 106, 1.4 x 106, 1.5 x 106, 1.6 x 106, 1.7 x 106, 1.8 x 106, 1.9 x 106, or 2 x 106cells per microliter, or at a concentration in a range defined by any two of the preceding values (e.g., 0.1 x 106– 2 x 106cells per microliter, 0.5 x 106– 2 x 106cells per microliter, 0.6 x 106– 1.2 x 106cells per microliter, 0.6 x 106– 1 x 106cells per microliter, 0.8 x 106– 1.2 x 106cells per microliter, etc.). In some embodiments, the cellular composition for administering to the subject includes cells at a concentration of about 0.8 x 106cells per microliter or more (e.g., about 0.9 x 106cells per microliter, about 1.0 x 106cells per microliter, about 1.1 x 106cells per microliter, about 1.2 x 106cells per microliter, about 1.5 x 106cells per microliter, including up to about 2.0 x 106cells per microliter). In some embodiments, the cellular composition includes cells at a concentration of about 0.5 x 106cells per microliter to about 1.5 x 106cells per microliter. In some embodiments, the cellular composition includes cells at a concentration of about 0.6 x 106cells per microliter to about 1.2 x 106cells per microliter. In some embodiments, the cellular composition includes cells at a concentration of about 0.9 x 106cells per microliter to about 1.2 x 106cells per microliter. In some embodiments, the cellular composition includes cells at a concentration of about 1 x 106cells per microliter.

[0120] In some embodiments, the therapeutically effective amount of the cellular composition is administered at a concentration of about 1 x 105cells / μL or greater. In some embodiments, the therapeutically effective amount of the cellular composition is administered at a concentration of about 1 x 105cells / μL or greater, and up to about 2 x 106cells per microliter. In some embodiments, the therapeutically effective amount of the cellular composition is administered at a concentration of, of about, or of at least 0.1 x 106, 0.2 x 106, 0.3 x 106, 0.4 x 106, 0.5 x 106, 0.6 x 106, 0.7 x 106, 0.8 x 106, 0.9 x 106, 1 x 106, 1.1 x 106, 1.2 x 106, 1.3 x 106, 1.4 x 106, 1.5 x 106, 1.6 x 106, 1.7 x 106, 1.8 x 106, 1.9 x 106, or 2 x 106cells per microliter, or at a concentration in a range defined by any two of the preceding values (e.g., 0.1 x 106– 2 x 106cells per microliter, 0.5 x 106– 2 x 106cells per microliter,0.6 x 106– 1.2 x 106cells per microliter, 0.6 x 106– 1 x 106cells per microliter, 0.8 x 106– 1.2 x 106cells per microliter, etc.). In some embodiments, the therapeutically effective amount of the cellular composition is administered at a concentration of about 0.5 x 106cells per microliter to about 2 x 106cells per microliter. In some embodiments, the therapeutically effective amount of the cellular composition is administered at a concentration of about 0.6 x 106cells per microliter to about 1.2 x 106cells per microliter. In some embodiments, the therapeutically effective amount of the cellular composition is administered at a concentration of about 0.9 x 106cells per microliter to about 1.2 x 106cells per microliter. In some embodiments, the therapeutically effective amount of the cellular composition is administered at a concentration of about 1 x 106cells per microliter.

[0121] In some embodiments, a cellular composition, such as the cellular compositions of pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells described herein (e.g., pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells), having a high concentration of cells is delivered to a subject using a delivery device, such as, but not limited to, a cannula or catheter. In some embodiments, delivering a cellular composition, such as the cellular compositions of pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells), to the subject involves loading the cellular composition from the proximal end of the delivery device (e.g., cannula) and displacing the cellular composition from the proximal end of the delivery device with a chase vehicle solution (e.g., a cell-free liquid chase vehicle), or inner stylet, such that the desired volume of the cellular composition is expelled from the delivery device outlet at the proximal end. As used herein, “proximal” refers to a position along the delivery device (e.g., cannula) that is closer to the subject to which the cellular composition is being delivered than a position that is “distal”. In some embodiments, a delivery device (e.g., cannula) is configured to deliver the cellular composition, where the delivery device includes the cellular composition at the proximal portion, and a chase vehicle solution that fills the rest of the internal compartment of the delivery device. In some embodiments, the cellular composition is loaded at the proximal portion of the delivery device (e.g., cannula) and a chase vehicle solution fills the rest of the internal compartment of the delivery device. In some embodiments, the chase vehicle solution includes the same pharmaceuticallyacceptable carrier as the cellular composition. In some embodiments, the chase vehicle solution is a cell-free liquid composition that includes the same pharmaceutically acceptable carrier as the cellular composition. In some embodiments, the chase vehicle solution (or liquid chase vehicle) is the same as the delivery vehicle in which the cellular composition is provided for administration to a subject, but in a cell-free format. In some embodiments, the chase vehicle solution has a lower viscosity than the cellular composition. In some embodiments, the delivery device (e.g., cannula) is connected at the distal end to a displacement device (e.g., syringe or stylet traversing the inner lumen of the cannula) configured to expel the cellular composition from the cannula, where the syringe volume is filled with the chase vehicle solution. In some embodiments, the chase vehicle is not required (e.g., where the cellular composition is displaced by a stylet positioned in the cannula and configured to push the cellular composition out of the proximal end of the cannula). In some embodiments, the displacement device is attached to a pump configured to drive the syringe plunger and thereby expel the cellular composition from the delivery device (e.g., cannula) in a controlled manner. In some embodiments, the cellular composition is stably held in the delivery device (e.g., cannula) for a sufficient period of time to allow delivery to the subject. For example, substantially all of the cells remain in the volume at the end or tip of the delivery device (e.g., cannula) at which the composition is initially placed for a suitable period of time before administering to a subject. In some embodiments, the cellular composition is stably held in the cannula, before administering to a subject, for about 0.5 hours or more, e.g., about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 6 hours, about 8 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours or longer, or for a length of time within a range defined by any two of the preceding values (e.g., 0.5-96 hours, 1-72 hour, 2-48 hours, 6-96 hours, etc.).

[0122] In some embodiments, the cellular composition of the present disclosure has been cryopreserved before use. In some embodiments, the method includes obtaining cryopreserved cells that include the pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells) before administering. In some embodiments, the method includes thawing a cryopreserved cellular composition that contains the pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells) before administering. The cellular composition may be cryopreserved for any suitable length of time, for example, at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 6 months, 9 months, 12 months, 18 months, 2 years, 5 years or more, or a length of time defined by any two of the preceding values (e.g., 1 week – 5 years, 2 weeks – 2 years, 1 month – 1 year, or 6 months to 2 years, etc.), before the administering.

[0123] The pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells) can be preserved in any suitable cryopreservation formulation. In some embodiments, a cryopreservation formulation includes a cryoprotective agent, an energy substrate, and supplements, that prevent intracellular icicle formation (e.g., 0.5–20% DMSO (e.g., 0.5-1%, 1-2%, 2-5%, 5-10%, 10-15%, or 15-20% DMSO)), protect cell membranes by association with the cell surface, scavenge free radicals while providing pH buffering, oncotic / osmotic support, and stable ionic concentrations at low temperatures. In some embodiments, the pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells) are cryopreserved before use. In some embodiments, cryopreserving the cellular composition includes, before placing the cells in a cryoformulation that contains DMSO, incubating the cells in a buffer containing high molecular weight disaccharides, amino acids, and proteins such that cellular membrane structures are physically and chemically stable across a range of temperatures (e.g., 0-37°C, such as 4°C) and over a range of time periods in the buffer (e.g., 1 minute to 4 hours or more, such as overnight, including 30 minutes). In some embodiments, the cells are incubated in the buffer at about 0°C or higher, e.g., about 2 °C, about 4°C, about 6°C, about 10°C, about 12°C, about 15°C, about 18°C, about 20°C, about 25°C, about 30°C, or about 37°C, or at a temperature in a range defined by any two of the preceding values (e.g., 0-37°C, 2-30°C, 2-6°C, 10-30°C, 2-10°C, etc.). In some embodiments, the cells are incubated in the buffer for about 1 minute or longer, e.g., about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 1.5 hours, about 2 hours, about 3 hours, about 4 hours or longer, or overnight, or for an amount of time within a range defined by any two of the preceding values (e.g., 1minute-4 hours, 1-60 minutes, 20-45 minutes, 1-4 hours, 10 minutes-1.5 hours, etc.). In some embodiments, cryopreserving the cells includes exposing the cells in the cryoformulation to an ambient temperature that is decreasing at a controlled rate, past the point of ice crystal nucleation, to about -20 to -80°C. In some embodiments, exposing the cells in the cryoformulation to an ambient temperature that is decreasing includes cooling the ambient temperature at a specified rate, such as about 0.5°C / min or faster, e.g., about 1°C / min, about 2°C / min, about 3°C / min, about 4°C / min, or about 5°C / min, or at a rate in a range defined by any two of the preceding values (e.g., 0.5-5°C / min, 0.5-2°C / min, 1- 4°C / min, 1-3°C / min, etc.). Exposing the cells in the cryoformulation to an ambient temperature that is decreasing at a controlled rate can be done using any suitable option, for example, without limitation, by placing the cells in a controlled rate freezer under conditions such that the ambient temperature is decreased at the controlled rate. In some embodiments, after freezing, the cells are stored at <-80°C, such as, without limitation, in liquid nitrogen vapor or liquid phase, e.g., at <-150°C.

[0124] In some embodiments, the cryopreserved pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells) are thawed by incubating the cryopreserved cells at 30-40°C (e.g., at, or at about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40°C) for 0.5-5 minutes (e.g., for, or for about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 minutes), and further rehydrating the cells for 0.5-2 minutes / mL (e.g., for, or for about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.4, 1.6, 1.8, or 2 minutes / mL) in thaw medium. In some embodiments, the thaw medium contains a nuclease (e.g., DNase), supplements and sodium ions, to scavenge free radicals while maintaining high osmolarity in order to reduce the risk of rapid cell expansion and cellular membrane damage. Any suitable nuclease can be used. Suitable nucleases include, without limitation, DNase I, Benzonase®, Micrococcal nuclease. In some embodiments, the thaw medium contains a nuclease (e.g., DNase) at about 500 units / mL or more, e.g., about 750 units / mL, about 1000 units / mL, about 1500 units / mL, about 2000 units / mL, or an amount in a range defined by any two of the preceding values (e.g., 500-2000 units / mL, 750- 1500 units / mL, 500-1000 units / mL, etc.). In some embodiments, the thaw medium contains sodium ions at a concentration of, of about, or of at least 100, 150, 200, 250, 300, 350, 400,450, 500, or 600 mM, or a concentration in a range defined by any two of the preceding values (e.g., 100-600 mM, 150-500 mM, 200-600 mM, 200-400 mM, 100-200 mM, etc.).

[0125] In some embodiments, the thawed and rehydrated cells are further incubated before formulating in a delivery formulation. In some embodiments, the cells (e.g., after thawing and rehydrating) are incubated in a buffered solution that contains poloxamers, ions, and nutrients to facilitate cell membrane repair, restore intracellular electrochemical equilibrium, and maintain physiological pH balance. Suitable poloxamers include, without limitation, P124, P188, P237, P338, and / or P407. In some embodiments, the buffered solution includes sodium chloride at a concentration of, of about, or of at least 100, 150, 200, 250, 300, 350, 400, 450, 500, or 600 mM, or a concentration in a range defined by any two of the preceding values (e.g., 100-600 mM, 150-500 mM, 200-600 mM, 200-400 mM, 100-200 mM, etc.). In some embodiments, the buffered solution includes sodium chloride at a concentration in a range of 100-600 mM. In some embodiments, the buffered solution includes sodium chloride at a concentration in a range of 200-600 mM. In some embodiments, the cells (e.g., with or without prior cryopreservation) are incubated in a buffered solution that contains poloxamers, ions, and nutrients before administering to a subject, as described herein. The cells can be incubated in the buffered solution at any suitable concentration, including at a high concentration while minimizing shear stress and reducing cell aggregation. In some embodiments, the cells are incubated in the buffered solution at a concentration of about 0.1 x 106cells / μL or higher, e.g., about 0.5 x 106cells / mL, about 1 x 106cells / mL, about 2 x 106cells / mL, about 5 x 106cells / mL, about 10 x 106cells / mL, about 20 x 106cells / mL, about 50 x 106cells / mL, about 100 x 106cells / mL, about 200 x 106cells / mL, or about 300 x 106cells / mL, or at a concentration in a range defined by any two of the preceding values (e.g., 0.1 x 106cells / mL-300 x 106cells / mL, 0.2 x 106cells / mL-200 x 106cells / mL, 0.5 x 106cells / mL-100 x 106cells / mL, 1 x 106cells / mL- 300 x 106cells / mL, 2 x 106cells / mL-200 x 106cells / mL, etc.). The cells can be incubated in the buffered solution for any suitable time period (e.g., after thawing and rehydrating). In some embodiments, the cells are incubated in the buffered solution for about 0.5 hours or more, e.g., about 1 hour, about 2 hours, about 5 hours, about 10 hours, about 15 hours, about 20 hours, about 30 hours, or for a time period in a range defined by any two of the preceding values (e.g., 0.5-30 hours, 1-30 hours, 1-20 hours, 2-20 hours, 1-10 hours, etc.). The cellscan be incubated in the buffered solution at any suitable temperature. In some embodiments, the cells are incubated in the buffered solution at about 0°C or higher, e.g., about 2 °C, about 4°C, about 6°C, about 10°C, about 12°C, about 15°C, about 18°C, about 20°C, about 25°C, or at a temperature in a range defined by any two of the preceding values (e.g., 0-25°C, 2- 25°C, 2-6°C, 10-20°C, 2-10°C, etc.).

[0126] In some embodiments, the cells (with or without prior cryopreservation) can be further held or stored for a second time period before administering to a subject. In some embodiments, the cells (with or without prior cryopreservation) can be further held or stored for a second time period, for example, for up to 1-7 days (e.g., 1, 2, 3, 4, 5, 6, 7 days, etc.) at 0-37°C (e.g., 4°C, 12°C, 20°C, 25°C, 37°C, etc.) before administering to a subject. In some embodiments, the cellular composition (e.g., thawed cellular composition) can be further stored with high viability and recovery at 0-37°C for up to 1-7 days when held in the buffered solution to allow long-term stability of the cells. In some embodiments, the method includes holding (or storing) the cellular composition (e.g., thawed cellular composition) for 1-7 days (e.g., about 1, 2, 3, 4, 5, 6, or about 7 days, etc.) at 0-37°C (e.g., about 4°C, 12°C, 20°C, 25°C, or about 37°C, etc.) before administering to a subject. In some embodiments, the method includes holding (or storing) the cellular composition (e.g., thawed cellular composition) for up to about 5 days before administering to a subject. In some embodiments, the method includes holding (or storing) the cellular composition (e.g., thawed cellular composition) for up to about 5 days at about 4°C before administering to a subject. In some embodiments, the method includes holding (or storing) the cellular composition (e.g., thawed cellular composition) for up to about 7 days before administering to a subject. In some embodiments, the method includes holding (or storing) the cellular composition (e.g., thawed cellular composition) for up to about 7 days at about 4°C before administering to a subject. In some embodiments, the lower the temperature at which the cells (e.g., thawed cells) are held or stored, the longer the cells can be held or stored before administering to a subject. In some embodiments, the method incudes holding the cells (e.g., thawed cells) at a temperature of, of about, or of at most 2, 4, 6, 8, 10, 12, 15, 17, 18, 19, 20, 21, 22, 25°C, or at a temperature in a range defined by any two of the preceding values (e.g., 4-25 °C, 4-20°C, 10- 20°C, 8-15°C, 4-12°C, 2-18°C, etc.), or at room temperature before administering to a subject. The thawed cellular composition can be held (or stored) at any suitable stagebetween after thawing and delivering to the subject. In some embodiments, the cellular composition (e.g., thawed cellular composition) is held (or stored) before loading into a delivery device (e.g., a cannula), in a suitable compartment (e.g., a tube, a syringe for attaching to a cannula, etc.). In some embodiments, the cellular composition (e.g., thawed cellular composition) is held (or stored) after loading into a delivery device (e.g., a cannula) and before delivering to the subject. In some embodiments, the cells (e.g., thawed and incubated cells) are further concentrated (e.g., before loading the cells into a delivery device, as described herein) to, to about, or to at least 0.1 x 106cells per microliter (e.g., 0.1 x 106-4 x 106cells per microliter). In some embodiments, the cells (with or without prior cryopreservation) are further concentrated and held or stored for the second time period in a suitable delivery vehicle, as described herein, before administering to the subject.

[0127] In some embodiments, the pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pluripotent stem cell-derived, pallial, MGE- type GABAergic interneuron cells) are an unsorted population of cells differentiated from pluripotent stem cells. In some embodiments, the pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pluripotent stem cell-derived, pallial, MGE- type GABAergic interneuron cells) are a sorted population of cells differentiated from pluripotent stem cells. In some embodiments, the pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells have been sorted (e.g., to enrich for the pallial or subpallial lineage) using one or more of flow cytometry, fluorescence activated cell sorting (FACS), or magnetic-activated cell sorting (MACS).

[0128] In some embodiments, administration of the cellular composition reduces seizure frequency. In some embodiments, a method of the present disclosure provides for a reduction in the frequency of seizures by about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, about 99% or more, by about 100%, or by a percentage in a range defined by any two of the preceding values (e.g., 50-100%. 55-99%, 60-95%, 70-90%, or 75-95%, etc.), after administering the cellular composition. In some embodiments, administration of the cellular composition reduces the duration of seizure episodes. In some embodiments, a method of the present disclosure provides for a reduction in the duration of seizure episodes by about 50% or more,about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, about 99% or more, or about 100%, or by a percentage in a range defined by any two of the preceding values (e.g., 50-100%. 55-99%, 60-95%, 70-90%, or 75- 95%, etc.), after administering the cellular composition. In some embodiments, administration of the cellular composition reduces seizure intensity (or severity). In some embodiments, the intensity (or severity) of seizures is reduced by about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, about 99% or more, or about 100%, or by a percentage in a range defined by any two of the preceding values (e.g., 50-100%. 55-99%, 60-95%, 70-90%, or 75-95%, etc.), after administering the cellular composition. In some embodiments, seizure frequency, duration of seizure episodes, or seizure intensity (or severity) is determined based on a subjective assessment of the frequency of seizures, duration of seizure episodes, or intensity (or severity) of a seizure (e.g., the most severe seizure experienced in the last week, last month, etc.), respectively. In some embodiments, the seizure frequency, duration of seizure episodes, or seizure intensity (or severity) is determined based on a measurement of brain activity using, e.g., EEG, CT scan, MRI, fMRI, MRS, PET or SPECT before and / or after the administering.

[0129] In some embodiments, administration of the cellular composition reduces spontaneous electrographic seizure activity in the subject. In some embodiments, co- administration of the cellular composition of the present disclosure with an anti-seizure medication may have a synergistic therapeutic effect. For example, without being bound by theory, a benzodiazepine anti-seizure medication may increase the affinity of the subject’s GABA receptors for GABA produced by the pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pluripotent stem cell-derived, pallial, MGE- type GABAergic interneuron cells). As used herein, co-administering two or more therapeutic agents to a subject includes administering a therapeutic agent such that the therapeutic agent effectively exhibits its therapeutic effect during any time interval in which one or more other therapeutic agent effectively exhibits their therapeutic effect. Forexample, co-administering can include administering one therapeutic agent before, at the same time as, or after administering a second therapeutic agent. In some embodiments, administration of the cellular composition reduces the dose of an anti-seizure medication, e.g., antiepileptic drug, required to treat the seizure in the subject. In some embodiments, administration of the cellular composition reduces the dose of an anti-seizure medication required to treat the seizure by about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, about 99% or more, or about 100%, or by a percentage in a range defined by any two of the preceding values (e.g., 20-100%, 25-99%, 30-95%, 30-90%, 40-90%, 50-80%, 60- 95%, 70-99%, etc.), after administering the cellular composition. In some embodiments, the patient does not require any further treatment with an anti-seizure medication, e.g., antiepileptic drug, for the seizure activity after administering the cellular composition to the subject. In some embodiments, a subject that is administered the cellular composition has about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or greater chance of reducing seizure frequency by 75% or more. In some embodiments, the therapeutic effect (e.g., reduction in seizure frequency, reduction in sclerosis, and / or reduction in lesion size) of administering the cellular composition to the subject is observed at least 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 12 months, 15 months, 18 months 21 months, 24 months or longer after the administration. In some embodiments, the method includes measuring a therapeutic effect (e.g., reduction in seizure frequency, reduction in sclerosis, and / or reduction in lesion size) of administering the cellular composition to the subject. In some embodiments, administration of the cellular composition reduces tauopathy or one or more symptoms thereof (e.g., in a subject having AD).

[0130] In some embodiments, the method includes detecting or measuring, e.g., non-invasively detecting or measuring, one or more therapeutic effects of administering the cellular composition to the subject. In some embodiments, the method includes measuring a reduction in seizure activity (e.g., seizure frequency and / or intensity) in the subject, as described herein. In some embodiments, the method includes using EEG to measuretherapeutic effects of administering the cellular composition to the subject. In some embodiments, the method includes using one or more brain imaging techniques to measure one or more structural and / or metabolic changes in the subject’s brain indicative of a therapeutic effect of administering the cellular composition. In some embodiments, the method includes using one or more biomarkers of seizure activity to monitor, e.g., non- invasively monitor, the therapeutic effect. In some embodiments, the biomarkers of seizure activity include one or more blood-based biomarkers. In some embodiments, the method includes measuring the change in level of one or more of N-acetylaspartate (NAA), myoinositol, glutamate, γ-Aminobutyric acid (GABA) in the subject’s brain or a region thereof. In some embodiments, the method includes measuring the level of one or more of N-acetylaspartate (NAA), myoinositol, glutamate, γ-Aminobutyric acid (GABA) before and / or after administering the cellular composition. The metabolites in the brain can be detected using any suitable option. In some embodiments, the level of one or more of N- acetylaspartate (NAA), myoinositol, glutamate, γ-Aminobutyric acid (GABA) are detected in the subject’s brain using magnetic resonance spectroscopy (MRS). In some embodiments, the method includes measuring the change in one or more of volume, edema, blood flow, oxygen metabolism and / or glucose levels in the subject’s brain or a region thereof. The volume, edema, blood flow, oxygen metabolism and / or glucose levels can be measured using any suitable option. In some embodiments, the method includes using magnetic resonance imaging (MRI) and / or positron emission tomography (PET) to detect volume, edema, blood flow, oxygen metabolism and / or glucose levels in the subject’s brain or a region thereof. In some embodiments, the method includes measuring (e.g., non-invasively measuring) the therapeutic effect using one or more biomarkers of seizure activity. In some embodiments, the one or more biomarkers comprises N-acetylaspartate (NAA), myoinositol, glutamate, GABA, volume, edema, blood flow, oxygen metabolism, and glucose.

[0131] In some embodiments, administration of the cellular composition reduces the extent of sclerosis, and / or the size of an epileptogenic lesion in the subject’s brain. In some embodiments, ministration of the cellular composition reduces the extent of sclerosis or size of an epileptogenic lesion in the subject’s brain by about 5% or more, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 100%, or by a percentage in range defined by any two of the preceding values (e.g., 5-100%, 10-90%, 20-50%, 50-90%, 40-70%, 80-100%, etc.) compared to the extent of sclerosis or size of the lesion before administering. The extent of sclerosis and / or size of the epileptogenic lesion can be determined using any suitable option. Suitable options include, without limitation, CT scan, MRI, MRS, EEG, PET, SPECT, or fMRI. In some embodiments, the method includes using one or more of a CT scan, MRI, MRS, EEG, PET, SPECT, or fMRI to detect the presence and / or measure the size of an epileptogenic lesion in the subject’s brain, before and / or after administering the cellular composition. In some embodiments, the method includes using one or more of a CT scan, MRI, MRS, EEG, PET, SPECT, or fMRI before administering the cellular composition, e.g., for diagnosis and / or localizing an onset zone (e.g., a seizure or epileptogenic onset zone). In some embodiments, the method includes using one or more of a CT scan, MRI, MRS, EEG, PET, SPECT, or fMRI after administering the cellular composition, e.g., to monitor therapeutic efficacy. In some embodiments, the method includes using one or more of a CT scan, MRI, MRS, EEG, PET, SPECT, or fMRI to determine a reduction in extent of sclerosis or size of an epileptogenic lesion in the subject’s brain after administering the cellular composition.

[0132] In some embodiments, administration of the cellular composition increases or restores viable neurons in the subject’s brain or a region thereof. In some embodiments, the level of NAA correlates with viable neurons in the subject’s brain or a region thereof. In some embodiments, administration of the cellular composition increases the level of NAA in the subject’s brain or a region thereof. In some embodiments, administration of the cellular composition increases the level of NAA in the subject’s brain or a region thereof by about 5% or more, e.g., 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more, or by a percentage in a range defined by any two of the preceding values (e.g., 5-100%, 10-90%, 15-80%, 20-100%, 5-50%, 50-100%, etc.) relative to the level before administering. In some embodiments, administration of the cellular composition restores the level of NAA in the subject’s brain or a region thereof to a level comparable to (e.g., within 30%, 20%, 10%, 5% of, or substantially the same as) the level in a healthy subject or a subject that is not suffering from seizure activity.

[0133] In some embodiments, administration of the cellular composition reduces inflammation in the subject’s brain or a region thereof. In some embodiments, the level of myoinositol correlates with inflammation in the subject’s brain or a region thereof. In someembodiments, administration of the cellular composition reduces the level of myoinositol in the subject’s brain or a region thereof. In some embodiments, administration of the cellular composition reduces the level of myoinositol in the subject’s brain or a region thereof by about 5% or more, e.g., 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 100%, or by a percentage in a range defined by any two of the preceding values (e.g., 5-100%, 10-90%, 15-80%, 20-100%, 5-50%, 5-20%, etc.) relative to the level before administering. In some embodiments, administration of the cellular composition restores the level of myoinositol in the subject’s brain or a region thereof to a level comparable to (e.g., within 30%, 20%, 10%, 5% of, or substantially the same as) the level in a healthy subject or a subject that is not suffering from seizure activity.

[0134] In some embodiments, administration of the cellular composition reduces or restores neural excitation in the subject’s brain or a region thereof. In some embodiments, the level of glutamate correlates with inflammation in the subject’s brain or a region thereof. In some embodiments, administration of the cellular composition reduces the level of glutamate in the subject’s brain or a region thereof. In some embodiments, administration of the cellular composition reduces the level of glutamate in the subject’s brain or a region thereof by about 5% or more, e.g., 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 100%, or by a percentage in a range defined by any two of the preceding values (e.g., 5-100%, 10-90%, 15-80%, 20-100%, 5-50%, 5-20%, etc.) relative to the level before administering. In some embodiments, administration of the cellular composition restores the level of glutamate in the subject’s brain or a region thereof to a level comparable to (e.g., within 30%, 20%, 10%, 5% of, or substantially the same as) the level in a healthy subject or a subject that is not suffering from seizure activity.

[0135] In some embodiments, administration of the cellular composition restores GABAergic tone in the subject’s brain or a region thereof. In some embodiments, the level of GABA correlates with GABAergic tone in the subject’s brain or a region thereof. In some embodiments, administration of the cellular composition restores the level of GABA in the subject’s brain or a region thereof to a level comparable to (e.g., within 30%, 20%, 10%, 5% of, or substantially the same as) the level in a healthy subject or a subject that is not suffering from seizure activity.

[0136] In some embodiments, the subject has a cognitive (or neurocognitive) impairment associated with the seizure activity. In some embodiments, the cognitive (or neurocognitive) impairment includes a memory impairment, including, without limitation, impairment in short-term memory, long-term memory, or working memory. In some embodiments, the memory impairment includes, without limitation, impairment in verbal and / or visuospatial memory. In some embodiments, the method includes performing one or more neurocognitive assessments of the subject before and / or after the administration. Any suitable neurocognitive assessment can be performed, including without limitation, tests for word retrieval, verbal memory, and / or visuospatial memory.

[0137] In some embodiments, administration of the cellular composition ameliorates, reduces, or restores cognitive (or neurocognitive) impairment associated with the seizure activity in the subject. In some embodiments, administration of the cellular composition slows or prevents further deterioration of the neurocognitive capacity of the subject. In some embodiments, administration of the cellular composition at least partially restores the neurocognitive capacity of the subject to a level within a range of a healthy individual. In some embodiments, administration of the cellular composition at least partially improves the neurocognitive capacity of the subject compared to the level before administration.

[0138] In some embodiments, administration of the cellular composition improves the quality of life of the subject. The quality of life can be measured using any suitable option. In some embodiments, the subject’s self-assessment of quality of life is used to measure improvement before and after administration of the cellular composition. In some embodiments, administration of the cellular composition reduces the risk of death associated with the seizure activity and / or symptoms thereof.

[0139] Methods of the present disclosure can provide for delivery of, or transplanting, pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells) to a host brain, where the cells are capable of functionally integrating into the endogenous host brain tissue. In some embodiments, about or at least 0.1, 0.2, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15%, or a percentage in a range defined by any two of the preceding values (e.g., 0.1-15%, 0.5-10%, 1-5%, 3-15%, 3-12%, 4-10%, or 4-8%, etc.) of the administered cellspersist in the host brain for, for about, or for at least 3, 6, 9, 12, 15, 18, 21, 24, 36, 48, 60, 72, months, or a period of time in a range defined by any two of the preceding values (e.g., 3-72 months, 6-60 months, 12-48 months, 6-12 months, etc.) after administration. In some embodiments, persistence or persistence rate denotes the percentage of transplanted cells that remain in the brain of the subject to whom the cellular composition has been administered, relative to the total number of cells administered. In some embodiments, the persistence rate of the administered cells in the host brain at 6 months or later is, is about, or is at least 0.1, 0.2, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15% , or a percentage in a range defined by any two of the preceding values (e.g., 0.1-15%, 0.5-10%, 1-5%, 3-15%, 3-12%, 4-10%, or 4- 8%, etc.). In some embodiments, the persistence rate of the administered cells in the host brain at 8 months or later is, is about, or is at least 0.1, 0.2, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15%, or a percentage in a range defined by any two of the preceding values (e.g., 0.1-15%, 0.5-10%, 1-5%, 3-15%, 3-12%, 4-10%, or 4-8%, etc.). In some embodiments, the persistence rate of the administered cells in the host brain at 10 months or later is, is about, or is at least 0.1, 0.2, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15%, or a percentage in a range defined by any two of the preceding values (e.g., 0.1-15%, 0.5-10%, 1-5%, 3-15%, 3-12%, 4- 10%, or 4-8%, etc.). In some embodiments, the persistence rate of the administered cells in the host brain at 12 months or later is, is about, or is at least 0.1, 0.2, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15%, or a percentage in a range defined by any two of the preceding values (e.g., 0.1-15%, 0.5-10%, 1-5%, 3-15%, 3-12%, 4-10%, or 4-8%, etc.). In some embodiments, the persistence rate of the administered cells in the host brain at 15 months or later is, is about, or is at least 0.1, 0.2, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15%%, or a percentage in a range defined by any two of the preceding values (e.g., 0.1-15%, 0.5-10%, 1- 5%, 3-15%, 3-12%, 4-10%, or 4-8%, etc.). In some embodiments, the persistence rate of the administered cells in the host brain at 18 months or later is, is about, or is at least 0.1, 0.2, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15%, or a percentage in a range defined by any two of the preceding values (e.g., 0.1-15%, 0.5-10%, 1-5%, 3-15%, 3-12%, 4-10%, or 4-8%, etc.). In some embodiments, the persistence rate of the administered cells in the host brain at 24 months or later is, is about, or is at least 0.1, 0.2, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15%, or a percentage in a range defined by any two of the preceding values (e.g., 0.1- 15%, 0.5-10%, 1-5%, 3-15%, 3-12%, 4-10%, or 4-8%, etc.). In some embodiments, thepersistence rate of the administered cells in the host brain at 36 months or later is, is about, or is at least 0.1, 0.2, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15%, or a percentage in a range defined by any two of the preceding values (e.g., 0.1-15%, 0.5-10%, 1-5%, 3-15%, 3-12%, 4- 10%, or 4-8%, etc.). In some embodiments, the persistence rate of the administered cells in the host brain at 48 months or later is, is about, or is at least 0.1, 0.2, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15%, or a percentage in a range defined by any two of the preceding values (e.g., 0.1-15%, 0.5-10%, 1-5%, 3-15%, 3-12%, 4-10%, or 4-8%, etc.). In some embodiments, about, or at least 0.1, 0.2, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15%, or a percentage in a range defined by any two of the preceding values (e.g., 0.1-15%, 0.5-10%, 1- 5%, 3-15%, 3-12%, 4-10%, or 4-8%, etc.) of the administered cells functionally integrate into the subject’s brain as GABAergic neurons. In some embodiments, about or at least 0.1, 0.2, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15%, or a percentage in a range defined by any two of the preceding values (e.g., 0.1-15%, 0.5-10%, 1-5%, 3-15%, 3-12%, 4-10%, or 4- 8%, etc.) of the administered cells functionally integrate into the subject’s brain as GABAergic interneurons, e.g., where the administered composition includes pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells. In some embodiments, 3-15% of the administered cells functionally integrate into the subject’s brain as GABAergic interneurons, e.g., where the administered composition includes pluripotent stem cell- derived, pallial, MGE-type GABAergic interneuron cells. In some embodiments, about 3 % or more, 4% or more, 5% or more, 6% or more, 8% or more, 9% or more, 10% or more, or about 12% or more, or a percentage in a range defined by any two of the preceding values (e.g., 3-15%, 3-12%, 4-10%, or 4-8%, etc.) of the administered cells functionally integrate into the subject’s brain as GABAergic projection neurons, e.g., where the administered composition includes pluripotent stem cell-derived, subpallial, MGE-type GABAergic neuron cells.

[0140] In some embodiments, the percentage of post-mitotic cells in the administered cellular composition is sufficiently high to result in a high percentage of the cells persisting in the transplanted brain retaining the MGE-type GABAergic neuron cell fate. In some embodiments, the cells of the cellular composition retain the pallial, MGE-type GABAergic interneuron cell fate after transplantation to the host brain. In some embodiments, the cells of the cellular composition retain the subpallial, MGE-typeGABAergic neuron cell fate after transplantation to the host brain. In some embodiments, the high-percentage of post-mitotic cells in the administered cellular composition promotes a higher percentage of cells persisting in the transplanted brain that retain the MGE-type GABAergic neuron cell fate (e.g., pallial, MGE-type GABAergic interneuron cell fate), for example, after, after about, or after at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, 48, months, or a length of time in a range defined by any two of the preceding values (e.g., 3-48 months, 4-36 months, 6-24 months, 9-12 months, 12-48 months, etc.). In some embodiments, the percentage of cells retaining a MGE-type GABAergic neuron cell fate (e.g., pallial, MGE- type GABAergic interneuron cell fate) among the cells persisting in the transplanted brain is, is about, or is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 50-100%, 55-95%, 60-85%, 90-98%, etc.) about 3 months or more after administration. In some embodiments, the percentage of cells retaining a MGE-type GABAergic neuron cell fate (e.g., pallial, MGE-type GABAergic interneuron cell fate) among the cells persisting in the transplanted brain is, is about, or is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 50-100%, 55-95%, 60-85%, 90-98%, etc.) about 6 months or more after administration. In some embodiments, the percentage of cells retaining a MGE-type GABAergic neuron cell fate (e.g., pallial, MGE-type GABAergic interneuron cell fate) among the cells persisting in the transplanted brain is, is about, or is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 50-100%, 55-95%, 60-85%, 90-98%, etc.) about 9 months or more after administration. In some embodiments, the percentage of cells retaining a MGE-type GABAergic neuron cell fate (e.g., pallial, MGE-type GABAergic interneuron cell fate) among the cells persisting in the transplanted brain is, is about, or is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 50-100%, 55-95%, 60-85%, 90-98%, etc.) about 12 months or more after administration. In some embodiments, the percentage of cells retaining a MGE-type GABAergic neuron cell fate (e.g., pallial, MGE-type GABAergic interneuron cell fate) among the cells persisting in the transplanted brain is, is about, or is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 50-100%, 55-95%,60-85%, 90-98%, etc.) about 24 months or more after administration. In some embodiments, the percentage of cells retaining a MGE-type GABAergic neuron cell fate (e.g., pallial, MGE-type GABAergic interneuron cell fate) among the cells persisting in the transplanted brain is, is about, or is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 50-100%, 55-95%, 60-85%, 90-98%, etc.) about 36 months or more after administration. In some embodiments, the percentage of cells retaining a MGE-type GABAergic neuron cell fate (e.g., pallial, MGE-type GABAergic interneuron cell fate) among the cells persisting in the transplanted brain is, is about, or is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 50-100%, 55-95%, 60-85%, 90-98%, etc.) about 48 months or more after administration. In some embodiments, the percentage of cells retaining a MGE-type GABAergic neuron cell fate (e.g., pallial, MGE-type GABAergic interneuron cell fate) among the cells persisting in the transplanted brain is, is about, or is at least 50%, about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years or more after administration. In some embodiments, the percentage of cells retaining a MGE-type GABAergic neuron cell fate (e.g., pallial, MGE-type GABAergic interneuron cell fate) among the cells persisting in the transplanted brain is in the range of 50-90%, about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years or more after administration. In some embodiments, the cells persisting in the subject and that retain a pallial, MGE-type GABAergic interneuron cell fate express one or more of GAD1, GAD2, LHX6, SOX6, NXPH1, ERBB4, SST, NPY, MEF2c, and ARX. In some embodiments, expression of one or more markers of a pallial interneuron lineage (e.g., ERBB4, SST, NPY, MAF, and / or MAFB) indicates retention of a pallial interneuron cell fate in the persisting cells. In some embodiments, expression of one or more markers of a MGE-type lineage (e.g., SOX6, and / or LHX6) indicates retention of a MGE- type GABAergic neuron cell fate in the persisting cells. In some embodiments, expression of one or more markers of a GABAergic neuron lineage (e.g., GAD1 and / or GAD2) indicates retention of a GABAergic neuron cell fate in the persisting cells. In some embodiments, expression of one or more markers of a subpallial interneuron lineage (e.g., LHX8 and / or NKX2.1) indicates retention of a subpallial neuron cell fate in the persisting cells. In some embodiments, detecting electrophysiological properties of a GABAergic neuron (e.g.,inhibitory post-synaptic potential) indicates retention of a GABAergic neuron cell fate in the persisting cells.

[0141] In some embodiments, the method includes administering one or more additional doses of the cellular composition to the subject after an initial administration of the therapeutically effective amount of the cellular composition. In some embodiments, the one or more additional doses is administered at about the same dose (e.g., same number of cells) as the initial dose. In some embodiments, the one or more additional doses is administered at a different dose (e.g., different number of cells) than the initial dose. In some embodiments, the therapeutically effective amount of the cellular composition in the one or more additional doses includes fewer cells than the number of cells in the therapeutically effective amount of the cellular composition in the initial dose. In some embodiments, the therapeutically effective amount of the cellular composition of the one or more additional doses includes at most about 100%, 95%, 90%, 80%, 70%. 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.1% or less, or a percentage in between any two of the preceding values (e.g., 0.1-100%, 1-95%, 5-90%, 10-80%, 10-70%, 20-60%, 30-50%, 10-50%, 10-30%, etc.), of the number of cells as the therapeutically effective amount of the cellular composition in the initial dose. In some embodiments, the therapeutically effective amount of the cellular composition of the one or more additional doses includes more cells than the number of cells in the therapeutically effective amount of the cellular composition in the initial dose. In some embodiments, the patient does not require any further administration of the cellular composition to treat the seizure activity after the initial dose. In some embodiments, the patient does not require any further treatment for the seizure activity after administering the cellular composition to the subject.

[0142] In some embodiments, the method further includes co-administering an anti-seizure medication to the subject. The co-administered anti-seizure medication can be administered before, after, or concurrently with the administration of the cellular composition. In some embodiments, the co-administered anti-seizure medication is one suitable for treating generalized seizure. The anti-seizure medication can be administered using any suitable option. In some embodiments, administration is oral or parenteral. In some embodiments, administration is local or systemic. In some embodiments, administration is intravenous, intracranial, subcutaneous, intramuscular, intraperitoneal. Insome embodiments, the anti-seizure medication includes one or more benzodiazepines. Suitable anti-seizure medications include, without limitation, Carbamazepine, Cenobamate, Clobozam, Clonazepam, Diazepam, Ethosuximide, Felbamate, Fenfluramine, Gabapentin, Lacosamide, Lamotrigine, Levetiracetam, Lorazepam, Midazolam, Oxcarbazepine, Phenobarbital, Phenytoin, Pregabalin, Primidone, Tiagabine, Topiramate, Valproic acid, Vigabatrin, Zonisamide.

[0143] In some embodiments, the method includes any suitable option to reduce or minimize adverse immunological reactions by the subject (e.g., immune-cloaking). In some embodiments, one or more immunosuppressants are co-administered to the subject. In some embodiments, immunosuppressants promote long-term engraftment of allogeneic or autologous cells. Any suitable immunosuppressant can be administered to a subject, e.g., to promote engraftment of the transplanted cells herein. Suitable immunosuppressants include, without limitation, glucocorticosteroids (e.g., prednisolone, cortisone, prednisone, dexamethasone, etc.), calcineurin inhibitors (e.g., cyclosporin A, tacrolimus), and cytostatic agents (e.g., azathioprine, and mycophenolate mofetil (MMF)). In some embodiments, one or more immunosuppressants are administered to the subject before, at the same time as, or after administering the cellular composition to the subject. In some embodiments, the method includes administering an immunosuppressant to the subject at an initial dose before and / or after administering the cellular composition to the subject. In some embodiments, the method includes gradually reducing the dose of the immunosuppressant after a time period post-administration. The immunosuppressant can be administered to the subject at any suitable time period before administering the cellular composition to the subject. In some embodiments, the method includes administering an immunosuppressant to the subject for a time period of, of about, or of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 17, 21, 24, or 28 days, or a number of days in a range defined by any two of the preceding values (e.g., 1-28 days, 2-24 days, 3-14 days, 4-10 days, etc.) before administering the cellular composition to the subject. The immunosuppressant can be administered to the subject for any suitable time period after administering the cellular composition to the subject. In some embodiments, the method includes administering an immunosuppressant to the subject for a time period of, of about, or of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 17, 21, 24, or 28 months, or a number of days in a range defined by any two of the preceding values (e.g., 1-28 months, 2-24 months, 3-14months, 4-12 months, etc.) after administering the cellular composition to the subject. In some embodiments, the method includes administering an initial dose of an immunosuppressant to the subject for an initial time period of, of about, or of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 17, 21, 24, or 28 months, or a number of days in a range defined by any two of the preceding values (e.g., 1-28 months, 2-24 months, 3-14 months, 4-12 months, etc.) after administering the cellular composition to the subject, and then reducing the dose after the initial time period. In some embodiments, the method includes use of genetically modified pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pallial, MGE-type GABAergic interneuron cells), as described herein, for example, to reduce an immunological response against the administered cellular composition by the subject. In some embodiments, the method includes immunosuppression by genetically modifying the interneurons, precursors, or pluripotent stem cells of the present disclosure. In some embodiments, the genetic modification contributes to evasion of the recipient's immune system by the transplanted or administered cells. Any suitable gene that inhibits the expression or activity of immune-activating genes can be mutated to reduce or abolish activity, or knocked out, and / or any suitable genetic modification that activates expression or activity of immune-inhibiting genes can be made in the pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells (or a precursor thereof). In some embodiments, the method includes genetically modifying one or more genes to reduce or abolish activity, and / or knocking out one or more immune-activating genes (e.g., B2M, a HLA class I gene, and / or a HLA class II gene, etc.) in the pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells (or a precursor thereof). In some embodiments, the method includes genetically modifying the pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells (or a precursor thereof) to activate expression or activity of immune-inhibiting genes. In some embodiments, the pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells (or a precursor thereof) are genetically modified to reduce or abolish expression of one or more of: B2M, a HLA class I gene, and / or a HLA class II gene. In some embodiments, the pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells are genetically modified to knock out one or more of: B2M, a HLA class I gene, and / or a HLA class II gene. Genetically modifying the pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells (or a precursor thereof) can bedone using any suitable option for genetic modification and / or gene editing. Suitable options include, without limitation, viral infection (e.g., adeno-associated virus, adenovirus, herpes simplex virus, lentivirus, retrovirus, etc.), zinc finger nuclease, TALEN, CRISPR-Cas nuclease, etc.

[0144] Also provided is a method of treating seizure activity, comprising: identifying a subject in need of treating seizure activity; and intracranially administering to the subject a cellular composition comprising: about 5 x 104to about 1 x 1012pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells at a concentration in a range of about 1 x 105to about 4 x 106cells / μL, wherein at least 50% of the cells of the composition express LHX6, ERBB4, and GAD1, wherein 90% or more of the cells of the composition are post-mitotic cells; a poloxamer at 0.1-1%, v / v; and sodium chloride at 100- 600 mM. PLURIPOTENT STEM CELL-DERIVED, MGE-TYPE GABAERGIC NEURON CELLS

[0145] The cellular composition for use in the present disclosure generally includes pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells). In some embodiments, the cellular composition is sufficiently enriched in the pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells) so as to provide a therapeutic result when administered in a therapeutically effective amount to a subject in need of treating seizure activity. In some embodiments, the cellular composition includes NRTX-1001 prepared by Neurona Therapeutics Inc.

[0146] In some embodiments, the cellular composition is a composition enriched for MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pallial, MGE-type GABAergic interneuron cells). In some embodiments, the combination of markers characteristic of MGE-type inhibitory (e.g., GABAergic) neuron cells includes at least LHX6 and GAD1. In some embodiments, about 50% or more, e.g., 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 50-100%, 60-99%, 70-95%, 80-100%, 85-95%, etc.) of the cells of the composition are MGE-type inhibitory (e.g., GABAergic) neuron cells. In someembodiments, about 50% or more, e.g., 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 50- 100%, 60-99%, 70-95%, 80-100%, 85-95%, etc.) of the cells of the composition are pallial, MGE-type inhibitory (e.g., GABAergic) interneuron cells. In some embodiments, at least 80% of the cells of the composition are pallial, MGE-type inhibitory (e.g., GABAergic) interneuron cells. In some embodiments, about 85% of the cells of the composition are pallial, MGE-type inhibitory (e.g., GABAergic) interneuron cells. In some embodiments, about 50% or more, e.g., 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 50-100%, 60-99%, 70-95%, 80-100%, 85-95%, etc.) of the cells of the composition express a combination of markers characteristic of pallial, MGE-type inhibitory (e.g., GABAergic) interneuron cells. In some embodiments, the combination of markers characteristic of pallial, MGE-type inhibitory (e.g., GABAergic) interneuron cells includes at least (i) LHX6, (ii) ERBB4, and (iii) GAD1. In some embodiments, the combination of markers characteristic of pallial, MGE-type inhibitory (e.g., GABAergic) interneuron cells includes at least two or more (e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or all) of: DLX6-AS1, GAD1, GAD2, LHX6, SST, SLC32A1, ARX, DLX1, DLX2, DLX5, MEF2C, ZEB2, SOX6, NXPH1, CXCR4, ACKR3, MAF, MAFB, and ERBB4. In some embodiments, the combination of markers characteristic of pallial, MGE-type inhibitory (e.g., GABAergic) interneuron cells includes at least (i) LHX6, (ii) ERBB4, (iii) GAD1, and optionally (iv) one or more of DLX6-AS1, GAD2, SST, SLC32A1, ARX, DLX1, DLX2, DLX5, MEF2C, ZEB2, SOX6, NXPH1, CXCR4, MAF, MAFB, and ACKR3. In some embodiments, at least 80% (e.g., about 80, 85, 90, 95% or more) of cells of the cellular composition express (i) LHX6, (ii) ERBB4, (iii) GAD1. In some embodiments, at least 50%, e.g., 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or more, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 50-100%, 60-99%, 70-95%, 80-100%, 85-95%, etc.) of the cells of the cellular composition further express at least one of MAF and MAFB. In some embodiments, 20-55% or 20-30% of cells of the cellular composition express SST.

[0147] In some embodiments, the cells in the composition are characterized by expression (or lack thereof) of one or more markers in a manner consistent with the cell type of interest, such as pallial, MGE-type inhibitory (e.g., GABAergic) interneuron cells capableof integrating into the host brain upon transplantation. Expression of a marker can refer to mRNA and / or protein expression. In some embodiments, the cellular composition is enriched for cells expressing two or more, or three or more, markers of a pallial, MGE-type inhibitory (e.g., GABAergic) interneuron lineage, as noted above. In some embodiments, about, or at least 45%, 50%, 52%, 54%, 56%, 57%, 58%, 60%, 62%, 64%, 66%, 68%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 85%, 90%, 95%, 97%, or about 100%, or a percentage within a range defined by any two of the preceding values (e.g., 45-100%, 50-100%, 50-80%, 60-90%, 75-100%, 80-100%, 85-98%, 90-95%, 90-100%, etc.), of the cells of the composition express LHX6. In some embodiments, about, or at least 45%, 50%, 52%, 54%, 56%, 57%, 58%, 60%, 62%, 64%, 66%, 68%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 85%, 90%, 95%, 97%, or about 100%, or a percentage within a range defined by any two of the preceding values (e.g., 45-100%, 50-100%, 50-80%, 60-90%, 75-100%, 80-100%, 85-98%, 90-95%, 90-100%, etc.), of the cells of the composition express GAD1. In some embodiments, about, or at least 45%, 50%, 52%, 54%, 56%, 57%, 58%, 60%, 62%, 64%, 66%, 68%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 85%, 90%, 95%, 97%, or about 100%, or a percentage within a range defined by any two of the preceding values (e.g., 45-100%, 50-100%, 60-90%, 75-97%, 80-100%, 85-98%, 90-95%, 90-100%, etc.), of the cells of the composition express ERBB4. In some embodiments, about, or at least 45%, 50%, 52%, 54%, 56%, 57%, 58%, 60%, 62%, 64%, 66%, 68%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 85%, 90%, 95%, 97%, or about 100%, or a percentage within a range defined by any two of the preceding values (e.g., 45-100%, 50-100%, 50-80%, 60-90%, 75-100%, 80-100%, 85- 98%, 90-95%, 90-100%, etc.), of the cells of the composition express LHX6 and ERBB4. In some embodiments, about, or at least 45%, 50%, 52%, 54%, 56%, 57%, 58%, 60%, 62%, 64%, 66%, 68%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 85%, 90%, 95%, 97%, or about 100%, or a percentage within a range defined by any two of the preceding values (e.g., 45- 100%, 50-100%, 50-80%, 60-90%, 75-100%, 80-100%, 85-98%, 90-95%, 90-100%, etc.), of the cells of the composition express LHX6 and GAD1. In some embodiments, about, or at least 45%, 50%, 52%, 54%, 56%, 57%, 58%, 60%, 62%, 64%, 66%, 68%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 85%, 90%, 95%, 97%, or about 100%, or a percentage within a range defined by any two of the preceding values (e.g., 45-100%, 50-100%, 50-80%, 60-90%, 75- 100%, 80-100%, 85-98%, 90-95%, 90-100%, etc.), of the cells of the composition expressERBB4 and GAD1. In some embodiments, about, or at least 45%, 50%, 52%, 54%, 56%, 57%, 58%, 60%, 62%, 64%, 66%, 68%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 85%, 90%, 95%, 97%, or about 100%, or a percentage within a range defined by any two of the preceding values (e.g., 45-100%, 50-100%, 50-80%, 60-90%, 75-100%, 80-100%, 85-98%, 90-95%, 90-100%, etc.), of the cells of the composition express LHX6, ERBB4, and GAD1. In some embodiments, the cellular composition is enriched for cells expressing LHX6, ERBB4, and / or GAD1, compared to before being differentiated in vitro (e.g., embryonic stem cells) or compared to during the differentiation in vitro (e.g., MGE-type progenitor cells).

[0148] In some embodiments, about, or at least 45%, 50%, 52%, 54%, 56%, 57%, 58%, 60%, 62%, 64%, 66%, 68%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 85%, 90%, 95%, 97%, or about 100%, or a percentage within a range defined by any two of the preceding values (e.g., 45-100%, 50-100%, 60-90%, 75-97%, 80-100%, 85-98%, 90-95%, 90-100%, etc.), of the cells of the composition express at least one of MAF and MAFB. In some embodiments, about, or at least 45%, 50%, 52%, 54%, 56%, 57%, 58%, 60%, 62%, 64%, 66%, 68%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 85%, 90%, 95%, 97%, or about 100%, or a percentage within a range defined by any two of the preceding values (e.g., 45-100%, 50-100%, 60-90%, 75-97%, 80-100%, 85-98%, 90-95%, 90-100%, etc.), of the cells of the composition express MAF. In some embodiments, about, or at least 45%, 50%, 52%, 54%, 56%, 57%, 58%, 60%, 62%, 64%, 66%, 68%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 85%, 90%, 95%, 97%, or about 100%, or a percentage within a range defined by any two of the preceding values (e.g., 45-100%, 50-100%, 60-90%, 75-97%, 80-100%, 85-98%, 90-95%, 90-100%, etc.), of the cells of the composition express MAFB.

[0149] In some embodiments, the cells of the cellular composition express two or more (e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or all) markers for pallial, MGE-type inhibitory (e.g., GABAergic) interneurons selected from: DLX6-AS1, GAD1, GAD2, LHX6, SST, SLC32A1, ARX, DLX1, DLX2, DLX5, MEF2C, ZEB2, SOX6, NXPH1, CXCR4, ACKR3, MAF, MAFB, and ERBB4. In some embodiments, the cellular composition is enriched for cells expressing two or more (e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or all) markers for GABAergic interneurons selected from: DLX6- AS1, GAD1, GAD2, LHX6, SST, SLC32A1, ARX, DLX1, DLX2, DLX5, MEF2C, ZEB2,SOX6, NXPH1, CXCR4, ACKR3, MAF, MAFB, and ERBB4, compared to before being differentiated in vitro (e.g., embryonic stem cells) or compared to during the differentiation in vitro (e.g., MGE-type progenitor cells). In some embodiments, about, at least, or at least about 45%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or about 100%, or a percentage within a range defined by any two of the preceding values (e.g., 45- 100%, 50-100%, 60-90%, 75-97%, 80-100%, 85-97%, 90-95%, 90-100%, etc.), of the cells of the cellular composition express two or more (e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or all) markers for pallial, MGE-type inhibitory (e.g., GABAergic) interneurons selected from: DLX6-AS1, GAD1, GAD2, LHX6, SST, SLC32A1, ARX, DLX1, DLX2, DLX5, MEF2C, ZEB2, SOX6, NXPH1, CXCR4, ACKR3, MAF, MAFB, and ERBB4.

[0150] In some embodiments, the cells of the cellular composition express one or more pan-neuronal markers selected from: DCX, MAPT, STMN2, C1orf61, and MAP2. In some embodiments, about, at least, or at least about at least about 45%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or about 100%, or a percentage within a range defined by any two of the preceding values (e.g., 45-100%, 50-100%, 60-90%, 75-97%, 80- 100%, 85-97%, 90-95%, 90-100%, etc.), of the cells of the cellular composition express one or more pan-neuronal markers selected from: DCX, MAPT, STMN2, C1orf61, and MAP2.

[0151] In some embodiments, the cellular composition is depleted for cells expressing one or more markers that are indicative of an off-target cell type (e.g., a non- pallial, MGE-type inhibitory interneuron lineage). In some embodiments, at most 20% of the cells of the composition are subpallial neurons. In some embodiments, at most 15% of the cells of the composition are subpallial neurons. In some embodiments, the cellular composition is depleted for cells expressing one or more off-target markers selected from LHX8, OTX2, SP8, ISL1, NKX2-1, VIM, KI67, ASPM, POU5F1, NANOG, OLIG1, OLIG2, PDGFRA, GFAP, SLC17A6, SLC17A7, NEUROD2. In some embodiments, the cellular composition is depleted for cells expressing any of the off-target markers selected from LHX8, OTX2, SP8, ISL1, NKX2-1, VIM, KI67, ASPM, POU5F1, NANOG, OLIG1, OLIG2, PDGFRA, GFAP, SLC17A6, SLC17A7, NEUROD2. In some embodiments, about 50% or less, e.g., 45%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 3%, 1%, or about 0%, or a percentage within a range defined by any two of the preceding values (e.g., 50-0%, 45-1%,45-5%, 35-10%, 50-3%, etc.), of the cells of the cellular composition express one or more off-target markers selected from LHX8, OTX2, SP8, ISL1, NKX2-1, VIM, KI67, ASPM, POU5F1, NANOG, OLIG1, OLIG2, PDGFRA, GFAP, SLC17A6, SLC17A7, NEUROD2. In some embodiments, about 50% or less, e.g., 45%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 3%, 1%, or about 0%, or a percentage within a range defined by any two of the preceding values (e.g., 50-0%, 45-1%, 45-5%, 35-10%, 50-3%, etc.), of the cells of the cellular composition express any of the off-target markers selected from LHX8, OTX2, SP8, ISL1, NKX2-1, VIM, KI67, ASPM, POU5F1, NANOG, OLIG1, OLIG2, PDGFRA, GFAP, SLC17A6, SLC17A7, NEUROD2.

[0152] In some embodiments, the cellular composition is depleted for cells expressing one or more markers for MGE-type progenitor cells selected from: LHX8, NKX2-1, and VIM, compared to during the differentiation in vitro (e.g., compared to MGE- type progenitor cells). In some embodiments, about or at most 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 2%, 1% or less, or a percentage in a range define by any two of the preceding values (e.g., 80-1%, 60-5%, 50- 10%, 80-20%, 75-20%, 70-25%, 65-30%, etc.) of cells of the cellular composition express LHX8. In some embodiments, about, or at least 45%, 50%, 52%, 54%, 56%, 57%, 58%, 60%, 62%, 64%, 66%, 68%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 85%, 90%, 95%, 97%, or about 100%, or a percentage within a range defined by any two of the preceding values (e.g., 45-100%, 50-100%, 50-80%, 60-90%, 75-100%, 80-100%, 85-98%, 90-95%, 90- 100%, etc.), of the cells of the composition express LHX6, ERBB4, and GAD1, and the composition is further depleted for cells expressing LHX8. In some embodiments, about or at most 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 2%, 1% or less, or a percentage in a range define by any two of the preceding values (e.g., 80-1%, 60-5%, 50-10%, 80-20%, 75-20%, 70-25%, 65-30%, etc.) of cells of the cellular composition express NKX2-1.

[0153] In some embodiments, the cellular composition is depleted for cells expressing one or more markers for glial cells selected from: SLC1A3, GFAP, OLIG1 and OLIG2, compared to before being differentiated in vitro (e.g., embryonic stem cells) or compared to during the differentiation in vitro (e.g., MGE-type progenitor cells). In some embodiments, about 5% or less, e.g., about 4%, about 3%, about 2%, about 1%, or about 0%,or a percentage in a range define by any two of the preceding values (e.g., 5-0%, 5-1%, 4- 1%, 3-1%, 5-2%, 4-2%, etc.) of cells of the cellular composition express OLIG2.

[0154] In some embodiments, about 50% or more, e.g., 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 50-100%, 60-99%, 70-95%, 80-100%, 85-95%, etc.) of the cells of the composition are subpallial, MGE-type inhibitory (e.g., GABAergic) neuron cells. In some embodiments, the subpallial, MGE-type GABAergic neuron cells include interneurons and / or projection neurons. In some embodiments, the combination of markers characteristic of a subpallial, MGE-type inhibitory (e.g., GABAergic) neuron cell fate includes at least (i) LHX6, (ii) LHX8 and / or NKX2.1, and (iii) GAD1. In some embodiments, about 50% or more, e.g., 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 50-100%, 60-99%, 70-95%, 80- 100%, 85-95%, etc.) of the cells of the composition express at least LHX8 and / or NKX2.1. In some embodiments, about 50% or more, e.g., 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 50-100%, 60-99%, 70-95%, 80-100%, 85-95%, etc.) of the cells of the composition express at least (i) LHX6, (ii) LHX8 and / or NKX2.1, and (iii) GAD1. In some embodiments, a subpallial, MGE-type inhibitory (e.g., GABAergic) neuron cell fate is characterized by a lack of ERBB4 expression. In some embodiments, the cellular composition is enriched for cells expressing two or more, or three or more, markers of a subpallial, MGE-type inhibitory (e.g., GABAergic) neuron lineage, as noted above. In some embodiments, the cellular composition is enriched for cells expressing LHX6, LHX8 and / or NKX2.1, and / or GAD1, compared to before being differentiated in vitro (e.g., embryonic stem cells) or compared to during the differentiation in vitro (e.g., MGE-type progenitor cells). In some embodiments, the cellular composition is depleted for cells expressing one or more markers that are indicative of an off-target cell type (e.g., a non-subpallial, MGE-type inhibitory neuron lineage). In some embodiments, at most 20% of the cells of the composition are pallial interneurons. In some embodiments, at most 15% of the cells of the composition are pallial interneurons. In some embodiments, the cellular composition is depleted for cells expressing one or more off-target markers selected from ERBB4, MAF, MAFB, OTX2, SP8, ISL1, VIM, KI67, ASPM, POU5F1, NANOG, OLIG1, OLIG2, PDGFRA, GFAP, SLC17A6,SLC17A7, NEUROD2. In some embodiments, about 50% or less, e.g., 45%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 3%, 1%, or about 0%, or a percentage within a range defined by any two of the preceding values (e.g., 50-0%, 45-1%, 45-5%, 35-10%, 50-3%, etc.), of the cells of the cellular composition express one or more off-target markers selected from ERBB4, MAF, MAFB, OTX2, SP8, ISL1, VIM, KI67, ASPM, POU5F1, NANOG, OLIG1, OLIG2, PDGFRA, GFAP, SLC17A6, SLC17A7, NEUROD2. In some embodiments, about 50% or less, e.g., 45%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 3%, 1%, or about 0%, or a percentage within a range defined by any two of the preceding values (e.g., 50-0%, 45-1%, 45-5%, 35-10%, 50-3%, etc.), of the cells of the cellular composition express any of the off- target markers selected from ERBB4, MAF, MAFB, OTX2, SP8, ISL1, VIM, KI67, ASPM, POU5F1, NANOG, OLIG1, OLIG2, PDGFRA, GFAP, SLC17A6, SLC17A7, NEUROD2. In some embodiments, about or at most 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 2%, 1% or less, or a percentage in a range define by any two of the preceding values (e.g., 80-1%, 60-5%, 50-10%, 80-20%, 75-20%, 70-25%, 65-30%, etc.) of cells of the cellular composition express ERBB4. In some embodiments, about or at most 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 2%, 1% or less, or a percentage in a range define by any two of the preceding values (e.g., 80-1%, 60-5%, 50-10%, 80-20%, 75-20%, 70-25%, 65-30%, etc.) of cells of the cellular composition express MAF and / or MAFB. In some embodiments, about 50% or more, e.g., 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 50-100%, 60-99%, 70-95%, 80- 100%, 85-95%, etc.) of the cells of the composition express at least (i) LHX6, (ii) LHX8 and / or NKX2.1, and (iii) GAD1, and the composition is further depleted for cells expressing ERBB4. In some embodiments, about 50% or more, e.g., 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 50-100%, 60-99%, 70-95%, 80-100%, 85-95%, etc.) of the cells of the composition express at least (i) LHX6, (ii) LHX8 and / or NKX2.1, and (iii) GAD1, and do not express ERBB4. In some embodiments, about 50% or more, e.g., 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 50-100%, 60-99%, 70-95%, 80-100%, 85-95%, etc.) of the cells of the composition express at least (i) LHX6, (ii) LHX8 and / or NKX2.1, and (iii) GAD1, andthe composition is further depleted for cells expressing MAF or MAFB. In some embodiments, about 50% or more, e.g., 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 50- 100%, 60-99%, 70-95%, 80-100%, 85-95%, etc.) of the cells of the composition express at least (i) LHX6, (ii) LHX8 and / or NKX2.1, and (iii) GAD1, and do not express MAF or MAFB.

[0155] The percentage of cells expressing markers, e.g., pallial, MGE-type interneuron markers (such as, without limitation, DLX6-AS1, GAD1, LHX6, SST, SLC32A1, ARX, DLX5, MEF2C, NXPH1, MAF, MAFB, and ERBB4), or non-pallial, MGE-type neuron markers in the cellular composition, can be determined, if desired, using any suitable option. In some embodiments, expression is determined by detecting a level of protein in the cells. Suitable options for measuring expression level of a protein include, without limitation, ELISA, Western blotting, radioimmunoassay, immunoprecipitation, immunostaining (including, e.g., immunohistochemistry and immunocytochemistry), flow cytometry or fluorescence activated cell sorting (“FACS”) analysis, and homogeneous time- resolved fluorescence (HTRF) assays. In some embodiments, expression is determined by detecting a level of mRNA. Suitable options for measuring expression level of a mRNA include, without limitation, Northern blot hybridization, ribonuclease RNA protection, in situ hybridization (ISH) (e.g., fluorescence in situ hybridization (FISH)), microarray analysis, reverse-transcription polymerase chain reaction (RT-PCR), high throughput or next generation sequencing. In some embodiments, the expression level of one marker is measured using an option that is different from that used to measure the expression level of another marker. For example, without limitation, one marker can be measured using immunohistochemistry, and another marker can be measured using FACS. As another example, without limitation, one marker can be measured using immunohistochemistry, and another marker can be measured using ISH or FISH. In some embodiments, the expression level of one marker is measured using an option that is the same as that used to measure the expression level of another marker. In some embodiments, any one or more characteristics (e.g., marker expression, mitotic state, migratory capacity, etc.) of the cells of the composition can be determined, if desired, for at least a subset of the cells of the composition. In some embodiments, a representative portion or subset of the cells of thecomposition are assayed to determine one or more characteristics (e.g., marker expression, mitotic state, migratory capacity, etc.) of the cells of the composition.

[0156] In some embodiments, substantially all cells of the composition do not contain or do not express an exogenous MGE-type progenitor cell marker. The exogenous MGE-type progenitor cell marker can be any marker for MGE-type progenitor cells that is not native to the cells of the composition. In some embodiments, an exogenous MGE-type progenitor cell marker is a transgenic marker. In some embodiments, an exogenous MGE- type progenitor cell marker is an enhancer-promoter reporter gene, e.g., a fluorescent protein, such as a green fluorescent protein (GFP), expressed under an enhancer-promoter specific for MGE-type progenitor cell. In some embodiments, at most 30%, at most 20%, at most 15%, at most 10%, at most 5%, at most 3%, at most 2%, at most 1%, or about 0%, or at most a percentage in a range define by any two of the preceding values (e.g., at most 30-0%, at most 20-1%, at most 15-5%, at most 30-3%, etc.) of cells of the pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells) have an exogenous MGE-type progenitor cell marker. In some embodiments, substantially none of the pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells) has an exogenous MGE-type progenitor cell marker.

[0157] In some embodiments, substantially all cells of the composition do not contain or express an exogenous marker. The exogenous marker can be any marker that is not native to the cells of the composition. In some embodiments, an exogenous marker is a transgenic marker. In some embodiments, an exogenous marker is an enhancer-promoter reporter gene, e.g., a fluorescent protein, such as a green fluorescent protein (GFP), expressed under an enhancer-promoter. In some embodiments, at most 30%, at most 20%, at most 15%, at most 10%, at most 5%, at most 3%, at most 2%, at most 1%, or about 0%, or at most a percentage in a range define by any two of the preceding values (e.g., at most 30-0%, at most 20-1%, at most 15-5%, at most 30-3%, etc.) of cells of the pluripotent stem cell- derived, MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pluripotent stem cell- derived, pallial, MGE-type GABAergic interneuron cells) have an exogenous marker. In some embodiments, substantially none of the pluripotent stem cell-derived, MGE-typeinhibitory (e.g., GABAergic) neuron cells (e.g., pluripotent stem cell-derived, pallial, MGE- type GABAergic interneuron cells) has an exogenous marker.

[0158] In some embodiments, about 90% or more, e.g., about 95% or more, about 97% or more, about 98% or more, about 98.5% or more, about 99% or more, about 99.5% or more, about 99.7% or more, about 99.8% or more, about 99.9% or more, about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 90-100%, 95-99%, 98-99.9%, 99-100%, etc.) of the cells of the composition are post-mitotic cells. If desired, the percentage of post-mitotic cells in the composition (or a representative portion thereof) can be determined using any suitable option, such as measuring incorporation of a nucleoside analog (e.g., BrdU or EdU), or measuring expression of a proliferation marker (e.g., KI67 and / or ASPM). In some embodiments, about 10% or less, e.g., about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1% or less, about 0.5% or less, about 0.1% or less, about 0%, or a percentage in a range defined by any two of the preceding values (e.g., 10-0%, 8-0.1%, 5- 0.1%, 3-0.1%, 1-0.1%, 3-1%, 2-0.1%, 2-0%, etc.) of the cells of the cellular composition express KI67 and / or ASPM. In some embodiments, the cellular composition substantially lacks cells expressing one or more markers for cell cycling selected from: KI67 and ASPM. In some embodiments, 10% or less of the cells of the cellular composition express KI67 and / or ASPM. In some embodiments, 5% or less of the cells of the cellular composition express KI67 and / or ASPM. In some embodiments, 2% or less of the cells of the cellular composition express KI67 and / or ASPM. In some embodiments, 1% or less of the cells of the cellular composition express KI67 and / or ASPM. In some embodiments, 0.5% or less of the cells of the cellular composition express KI67 and / or ASPM. In some embodiments, about 10% or less, e.g., about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1% or less, about 0.5% or less, about 0.1% or less, about 0%, or a percentage in a range defined by any two of the preceding values (e.g., 10-0%, 8-0.1%, 5-0.1%, 3-0.1%, 1-0.1%, 3-1%, 2-0.1%, 2- 0%, etc.) of the cells of the cellular composition incorporate a nucleoside analog (e.g., BrdU or EdU). In some embodiments, the cellular composition substantially lacks cells that incorporate a nucleoside analog (e.g., BrdU or EdU).

[0159] Any suitable option can be used to increase the percentage of post-mitotic cells in the cellular composition. In some embodiments, an agent that promotes cell cycle exit and differentiation is used to increase the percentage of post-mitotic cells in the cellular composition. A suitable agent that promotes cell cycle exit and differentiation includes, without limitation, an inhibitor of Notch signaling. Suitable inhibitors of Notch signaling include, without limitation, a γ-secretase inhibitor. Suitable γ-secretase inhibitors include, without limitation, DAPT, BMS906024, BMS986115, E-2012, avagacestat, nirogacestat, begacestat, semagacestat, MK-0752, tarenflurbil, RG-4733, PF 3084014, hydrobromid, CultureOne, Compound E, and itanapraced. In some embodiments, the Notch inhibitor is DAPT. In some embodiments, the percentage of post-mitotic cells among the cells of the cellular composition is increased by providing the inhibitor of Notch signaling to the culture for at least about the last week, e.g., the last 1.5 weeks, the last 2 weeks, or the last 2.5 weeks, of differentiation of the pluripotent stem cells into the MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells).

[0160] In some embodiments, cells of the cellular composition are migratory cells. In some embodiments, about 5% or more, e.g., about 7% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, or a percentage in a range defined by any two of the preceding values (e.g., 5-30%, 7-25%, 10- 30%, 15-25%, etc.) of the cells of the composition are migratory cells, e.g., as determined by an in vitro migration assay. In some embodiments, the proportion of migratory cells among cells of the cellular composition is at least 50%, e.g., 60%, 70%, 80%, 90%, 100%, 120%, 150%, 175%, 200%, 250%, 300% or more, or a percentage in a range defined by any two of the preceding values (e.g., 50-300%, 60-250%, 70-200%, 100-200%, 120-175%, 100-300%, etc.) of the proportion of migratory cells among primary human MGE interneurons, e.g., as determined by an in vitro migration assay.

[0161] In some embodiments, the cellular composition comprises GABAergic cells. In some embodiments, the cellular composition comprises GABA-secreting cells. If desired, GABA secretion and / or amount of GABA secretion can be detected using any suitable assay, such as an in vitro GABA release assay. In some embodiments, cells of the cellular composition secrete GABA in an in vitro GABA release assay. In someembodiments, cells of the cellular composition secrete GABA constitutively in an in vitro GABA release assay. In some embodiments, cells of the cellular composition increase GABA secretion upon stimulation, e.g., by potassium chloride, sodium chloride or other salt solution, in an in vitro GABA release assay.

[0162] The MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells) of the present disclosure are generally derived from pluripotent stem (PS) cells. The MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells) can be cells derived from any suitable PS cells. Suitable PS cells include, without limitation, embryonic stem cells (ESC) and induced pluripotent stem cells (iPS cells). In some embodiments, the MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells) are derived from neural stem cells. In some embodiments, the MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells) are obtained through reprogramming or transdifferentiation of cells, e.g., somatic cells, neural cells obtained from the MGE, Cortex, Sub-Cortex, other regions of the brain, or non- neural cells.

[0163] The cellular composition for use in the present methods can be obtained using any suitable option. In some embodiments, a cellular composition is obtained by differentiating pluripotent stem cells, e.g., from ESCs or iPS cells, into the MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pallial, MGE-type GABAergic interneuron cells). In some embodiments, the MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pallial, MGE-type GABAergic interneuron cells) are derived from embryonic stem (ES) cells (e.g., hESCs) or induced pluripotent stem (iPS) cells. In some embodiments, the pluripotent stem cells are expanded before differentiating. In some embodiments, the pluripotent stem cells (e.g., hESCs) are expanded for, for about, or for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or at least 3, 4, 5, 6, 7, or 8 weeks, or for a length of time in a range defined by any two of the preceding values (e.g., 1-8 weeks, 1-14 days, 2-10 days, 3-10 days, 3-7 days, etc.). In some embodiments, the pluripotent stem cells (e.g., hESCs) are expanded for about 5 days. In some embodiments, the pluripotent stem cells after expanding retain or substantially retain their pluripotency. In some embodiments, the pluripotent stem cellsmaintain expression of one or more markers of pluripotent stem cells after expanding. In some embodiments, the pluripotent stem cells exhibit a similar or substantially the same pattern of expression of one or more markers of pluripotent stem cells after expanding compared to the pattern of expression before expanding. In some embodiments, the markers of pluripotent stem cells include one or more of OCT4, NANOG, TRA1-60, and SSEA3 / 4. In some embodiments, the pluripotent stem cells maintain expression of OCT4, NANOG, TRA1-60, and SSEA3 / 4 after expanding. In some embodiments, the expanded pluripotent stem cells are OCT4+, NANOG+, TRA1-60+, and SSEA3 / 4+. Any suitable option for expanding pluripotent stem cells (e.g., hESCs) can be used. In some embodiments, expanding the pluripotent stem cells (e.g., hESCs) includes culturing the cells in the presence of an FGF2 and / or TGFβ signaling activator. In some embodiments, the TGFβ signaling activator includes a SMAD signaling activator. In some embodiments the SMAD signaling activator includes, without limitation, TGFs (e.g., TGFβ3), BMPs (e.g., BMP2, BMP4, BMP8), Activin, Nodal, GDF, and IDE1. In some embodiments, the FGF2 signaling activator includes FGF2.

[0164] Any suitable option for differentiating a population of pluripotent stem cells in vitro into a MGE-type neuron lineage (e.g., pallial, MGE-type interneuron lineage) can be used, e.g., as provided herein. In some embodiments, differentiating the population of pluripotent stem cells into the MGE-type neuron lineage includes culturing a population, or a differentiating population, of pluripotent stem cells in the presence of one or more of a sonic hedgehog (shh) signaling activator, a SMAD signaling inhibitor, a wnt signaling inhibitor, a bone morphogenetic protein (BMP) signaling inhibitor, a neural inducing supplement, a Rho- associated kinase (ROCK) signaling inhibitor, MEK signaling inhibitor, CDK signaling inhibitor, and a Notch signaling inhibitor. As used herein, “a differentiating population of pluripotent stem cells” refers to cells that are in the process of developing features of a cell lineage of interest, starting from the pluripotent stem cells. Thus, in some embodiments, cells of the differentiating population may have one or more features characteristic of the pluripotent stem cells and one or more features characteristic of the cell lineage of interest. In some embodiments, a population of pluripotent stem cells that are differentiating into a MGE-type neuron lineage (e.g., pallial, MGE-type interneuron lineage) includes MGE-type progenitor cells.

[0165] In some embodiments, differentiating the population of pluripotent stem cells includes supplementing a culture of pluripotent stem cells with one or more of a sonic hedgehog (shh) signaling activator, a SMAD signaling inhibitor, a wnt signaling inhibitor, a bone morphogenetic protein (BMP) signaling inhibitor, a neural inducing supplement, a Rho- associated kinase (ROCK) signaling inhibitor, MEK signaling inhibitor, CDK signaling inhibitor, and a Notch signaling inhibitor. In some embodiments, each of the one or more of a sonic hedgehog (shh) signaling activator, a SMAD signaling inhibitor, a wnt signaling inhibitor, a BMP signaling inhibitor, a neural inducing supplement, a ROCK signaling inhibitor, a MEK signaling inhibitor, a CDK signaling inhibitor, and a Notch signaling inhibitor is present in the culture during a time period that spans at least a portion of the differentiation process. In some embodiments, the culture of pluripotent stem cells is a serum-free culture. In some embodiments, the culture of pluripotent stem cells is a feeder cell-free culture. In some embodiments, the culture of pluripotent stem cells includes a feeder cell, e.g., a human feeder cell. Any suitable feeder cell can be used to culture the pluripotent stem cells in the presence of feeder cells. In some embodiments, the culture of pluripotent stem cells is a xeno-free culture.

[0166] In some embodiments, differentiating the population of pluripotent stem cells includes a first time period (e.g., to promote definitive neuroectoderm specification and MGE patterning), a second time period following the first time period (e.g., to promote MGE progenitor expansion and pallial interneuron (or subpallial neuron) commitment), and a third time period following the second time period (e.g., to promote cell cycle exit and differentiation). In some embodiments, differentiating the population of pluripotent stem cells includes culturing the cells in a suitable base medium during the differentiation time course. Any suitable based medium can be used. In some embodiments, the base medium includes without limitation, MEM, DMEM, DMEM-F12, L-15, neurobasal medium, and combinations thereof. In some embodiments, the base medium includes a supplement for neuronal cell culture. Any suitable supplement for neuronal cell culture can be used for differentiating pluripotent stem cells. In some embodiments, the neural inducing supplement is, without limitation, B-27TM(with or without vitamin A), NS21, N2 supplement-B, or an equivalent supplement, and combinations thereof. In some embodiments, the base medium includes amino acids.

[0167] In some embodiments, differentiating the population of pluripotent stem cells includes culturing the pluripotent stem cells (e.g., hESCs) in the presence of one or more MGE patterning factors, including a ROCK signaling inhibitor, BMP signaling inhibitor, a TGF-β signaling inhibitor, a wnt signaling inhibitor, and a shh signaling activator during at least a portion of the first time period. In some embodiments, the first time period is, is about, or is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days, or a length of time in a range defined by any two of the preceding values (e.g., 7-21 days, 10-18 days, 12-16 days, etc.). In some embodiments, the first time period is or is about 2 weeks. In some embodiments, the one or more MGE patterning factors are removed from the culture at the end of the first time period. In some embodiments, each of the MGE patterning factors are added to the culture for any suitable portion of the first time period to promote definitive neuroectoderm specification and MGE patterning. In some embodiments, one or more of the MGE patterning factors are added to the culture at the start of the first time period (e.g., D0 of the differentiation time course). As used herein, the differentiation time course denotes the number of days of the differentiation process starting from the pluripotent stem cells (after and excluding any stem cell expansion stage noted above). In some embodiments, one or more of the MGE patterning factors are added to the culture 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days after the start of the first time period (e.g., D1, D2, D3, D5, D6, D7, D8, D9, D10, D11, D12, D13, D14 or later of the differentiation time course). In some embodiments, the cells are cultured in the presence of one or more of the MGE patterning factors for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days, or a length of time in a range defined by any two of the preceding values (e.g., 1-14 days, 2-10 days, 2-7 days, 4-10 days, 7-14 days, etc.), during the first time period. In some embodiments, the cells are cultured in the presence of one or more of the MGE patterning factors between D0-D14, D0- D7, D1-D7, D1-D14, D2-D5, D2-D7, D7-D12, or D7-D14 of the differentiation time course. Any suitable amount of the MGE patterning factors can be used to promote definitive neuroectoderm specification and MGE patterning. In some embodiments, each of the one or more MGE patterning factors is provided in the culture medium independently at a concentration of, of about, or of at least 1, 5, 10, 20, 50, 100, 150, 200, 250, 500, or 750 nM, or of, of about, or of at least 1, 5, 10, 20, 50, 100, or 200 μM, or a concentration in a rangedefined by any two of the preceding values (e.g., 1 nM-200 μM, 50 nM-100 μM, 100 nM- 100 μM, or 100 nM-50 μM).

[0168] In some embodiments, differentiating the population of pluripotent stem cells further includes culturing the pluripotent stem cells (e.g., hESCs) in the presence of a FGF / VEGF / PDGF inhibitor (e.g., a receptor tyrosine kinase (RTK) inhibitor) during at least a portion of the first time period. Any suitable FGF / VEGF / PDGF inhibitor can be used. In some embodiments, the FGF / VEGF / PDGF inhibitor is, without limitation, SU5402, SU6668, BIBF1120, ZM323881, SU11652, or XL999.

[0169] Without being bound by theory, culturing the pluripotent stem cells during the first time period can promote definitive neuroectoderm specification and MGE patterning. For convenience and without limitation, the first time period may be referred to as the “MGE patterning” phase). In some embodiments, the population of cells (e.g., differentiating cells) that have been cultured for the first time period (e.g., MGE patterned cells) is enriched for cells expressing one or more of SOX1, FOXG1, NKX2-1. In some embodiments, the population of cells (e.g., differentiating cells) that have been cultured for the first time period (e.g., MGE patterned cells) is depleted for cells expressing PAX6. In some embodiments, the cells (e.g., differentiating cells) that have been cultured for the first time period (e.g., MGE patterned cells) are SOX1+, FOXG1+, NKX2-1+, and PAX6-.

[0170] The shh signaling activator can be any suitable activator of shh signaling. In some embodiments, the shh signaling activator includes, without limitation, shh or a derivative thereof, purmorphamine, SAG smoothened agonist, Hh-Ag1.5, or derivatives and analogs thereof. In some embodiments, the shh signaling activator includes SAG. In some embodiments, the shh signaling activator is added to the culture at the start of the first time period (e.g., D0 of the differentiation time course). In some embodiments, the shh signaling activator is added to the culture 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days after the start of the first time period (e.g., D1, D2, D3, D5, D6, D7, D8, D9, D10, D11, D12, D13, D14 or later of the differentiation time course). In some embodiments, the cells are cultured in the presence of the shh signaling activator for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days, or a length of time in a range defined by any two of the preceding values (e.g., 1- 14 days, 2-10 days, 2-7 days, 4-10 days, 7-14 days, etc.), during the first time period. In some embodiments, the cells are cultured in the presence of the shh signaling activatorbetween D0-D14, D0-D7, D1-D7, D1-D14, D2-D5, D2-D7, D7-D12, or D7-D14 of the differentiation time course. Any suitable amount of the shh signaling activator can be used to promote definitive neuroectoderm specification and MGE patterning. In some embodiments, the shh signaling activator is provided in the culture medium at a concentration of, of about, or of at least 1, 5, 10, 20, 50, 100, 150, 200, 250, 500, or 750 nM, or of, of about, or of at least 1, 5, 10, 20, 50, 100, or 200 μM, or a concentration in a range defined by any two of the preceding values (e.g., 1 nM-200 μM, 50 nM-100 μM, 100 nM-100 μM, or 100 nM-50 μM). In some embodiments, the shh signaling activator is provided in the culture medium at a concentration of or of about 100 nM.

[0171] Any suitable inhibitor or activator of SMAD signaling can be used for differentiating pluripotent stem cells. In some embodiments, the SMAD signaling modulator is a SMAD signaling inhibitor, such as, without limitation, TGF-β inhibitor, BMP inhibitor, Activin inhibitor, Nodal inhibitor, or GDF signaling pathway inhibitor. In some embodiments, the SMAD signaling inhibitor includes a TGF-β inhibitor. In some embodiments, the TGF-β inhibitor includes, without limitation, SB431542, Galunisertib, LDN-193189, and / or K02288. In some embodiments, the TGF-β inhibitor includes SB431542. In some embodiments, the TGF-β inhibitor is added to the culture at the start of the first time period (e.g., D0 of the differentiation time course). In some embodiments, the TGF-β inhibitor is added to the culture 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days after the start of the first time period (e.g., D1, D2, D3, D5, D6, D7, D8, D9, D10, D11, D12, D13, D14 or later of the differentiation time course). In some embodiments, the cells are cultured in the presence of the TGF-β inhibitor for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days, or a length of time in a range defined by any two of the preceding values (e.g., 1-14 days, 2-10 days, 2-7 days, 4-10 days, 7-14 days, etc.), during the first time period. In some embodiments, the cells are cultured in the presence of the TGF-β inhibitor between D0- D14, D0-D7, D1-D7, D1-D14, D2-D5, D2-D7, D7-D12, or D7-D14 of the differentiation time course. Any suitable amount of the TGF-β inhibitor can be used to promote definitive neuroectoderm specification and MGE patterning. In some embodiments, the TGF-β inhibitor is provided in the culture medium at a concentration of, of about, or of at least 1, 5, 10, 20, 50, 100, 150, 200, 250, 500, or 750 nM, or of, of about, or of at least 1, 5, 10, 20, 50, 100, or 200 μM, or a concentration in a range defined by any two of the preceding values(e.g., 1 nM-200 μM, 50 nM-100 μM, 100 nM-100 μM, or 100 nM-50 μM). In some embodiments, the TGF-β inhibitor is provided in the culture medium at a concentration of or of about 10 μM.

[0172] Any suitable inhibitor of BMP signaling can be used to for differentiating pluripotent stem cells. In some embodiments, the BMP signaling inhibitor includes, without limitation, LDN193189, M4K2163, Dorsomorphin, DMH-1, and / or ML 347. In some embodiments, the BMP signaling inhibitor includes LDN193189. In some embodiments, the BMP signaling inhibitor is added to the culture at the start of the first time period (e.g., D0 of the differentiation time course). In some embodiments, the BMP signaling inhibitor is added to the culture 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days after the start of the first time period (e.g., D1, D2, D3, D5, D6, D7, D8, D9, D10, D11, D12, D13, D14 or later of the differentiation time course). In some embodiments, the cells are cultured in the presence of the BMP signaling inhibitor for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days, or a length of time in a range defined by any two of the preceding values (e.g., 1-14 days, 2-10 days, 2-7 days, 4-10 days, 7-14 days, etc.), during the first time period. In some embodiments, the cells are cultured in the presence of the SMAD signaling inhibitor between D0-D14, D0-D7, D1-D7, D1-D14, D2-D5, D2-D7, D7-D12, or D7-D14 of the differentiation time course. Any suitable amount of the BMP signaling inhibitor can be used to promote definitive neuroectoderm specification and MGE patterning. In some embodiments, the BMP signaling inhibitor is provided in the culture medium at a concentration of, of about, or of at least 1, 5, 10, 20, 50, 100, 150, 200, 250, 500, or 750 nM, or of, of about, or of at least 1, 5, 10, 20, 50, 100, or 200 μM, or a concentration in a range defined by any two of the preceding values (e.g., 1 nM-200 μM, 50 nM-100 μM, 100 nM-100 μM, or 100 nM-50 μM). In some embodiments, the BMP signaling inhibitor is provided in the culture medium at a concentration of or of about 10 μM.

[0173] Any suitable inhibitor of wnt signaling can be used for differentiating pluripotent stem cells. In some embodiments, the wnt signaling inhibitor includes, without limitation, soluble frizzled polypeptides comprising the wnt binding domains; soluble frizzled related polypeptides; wnt specific antibodies; frizzled specific antibodies; and other molecules capable of blocking extracellular wnt signaling; Dkk gene family such as, but not limited to, Dkk-1, Dkk-2, Dkk-3, and Dkk-4, and the Dkk-3 related protein Soggy (Sgy);Wise; or a small molecule inhibitor, such as, but not limited to CKI-7, IWP analogs, IWR analogs, XAV939, 53AH, Wnt-C59. In some embodiments, the wnt signaling inhibitor includes XAV939. In some embodiments, the wnt signaling inhibitor is added to the culture at the start of the first time period (e.g., D0 of the differentiation time course). In some embodiments, the wnt signaling inhibitor is added to the culture 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days after the start of the first time period (e.g., D1, D2, D3, D5, D6, D7, D8, D9, D10, D11, D12, D13, D14 or later of the differentiation time course). In some embodiments, the cells are cultured in the presence of the wnt signaling inhibitor for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days, or a length of time in a range defined by any two of the preceding values (e.g., 1-14 days, 2-10 days, 2-7 days, 4-10 days, 7-14 days, etc.), during the first time period. In some embodiments, the cells are cultured in the presence of the wnt signaling inhibitor between D0-D14, D0-D7, D1-D7, D1-D14, D2-D5, D2-D7, D7- D12, or D7-D14 of the differentiation time course. Any suitable amount of the wnt signaling inhibitor can be used to promote definitive neuroectoderm specification and MGE patterning. In some embodiments, the wnt signaling inhibitor is provided in the culture medium at a concentration of, of about, or of at least 1, 5, 10, 20, 50, 100, 150, 200, 250, 500, or 750 nM, or of, of about, or of at least 1, 5, 10, 20, 50, 100, or 200 μM, or a concentration in a range defined by any two of the preceding values (e.g., 1 nM-200 μM, 50 nM-100 μM, 100 nM- 100 μM, or 100 nM-50 μM). In some embodiments, the wnt signaling inhibitor is provided in the culture medium at a concentration of or of about 10 μM.

[0174] Any suitable inhibitor of ROCK signaling can be used for differentiating pluripotent stem cells. In some embodiments, the ROCK signaling inhibitor includes, without limitation, Y27623, thiazovivin, fasudil, ripasudil, or KD025. In some embodiments, the ROCK signaling inhibitor includes Y27623. In some embodiments, the ROCK signaling inhibitor is added to the culture at the start of the first time period (e.g., D0 of the differentiation time course). In some embodiments, the ROCK signaling inhibitor is added to the culture 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days after the start of the first time period (e.g., D1, D2, D3, D5, D6, D7, D8, D9, D10, D11, D12, D13, D14 or later of the differentiation time course). In some embodiments, the cells are cultured in the presence of the ROCK signaling inhibitor for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days, or a length of time in a range defined by any two of the preceding values (e.g., 1-14 days, 2-10days, 2-7 days, 4-10 days, 7-14 days, etc.), during the first time period. In some embodiments, the cells are cultured in the presence of the ROCK signaling inhibitor between D0-D14, D0-D7, D1-D7, D1-D14, D2-D5, D2-D7, D7-D12, or D7-D14 of the differentiation time course. Any suitable amount of the ROCK signaling inhibitor can be used to promote definitive neuroectoderm specification and MGE patterning. In some embodiments, the ROCK signaling inhibitor is provided in the culture medium at a concentration of, of about, or of at least 1, 5, 10, 20, 50, 100, 150, 200, 250, 500, or 750 nM, or of, of about, or of at least 1, 5, 10, 20, 50, 100, or 200 μM, or a concentration in a range defined by any two of the preceding values (e.g., 1 nM-200 μM, 50 nM-100 μM, 100 nM-100 μM, or 100 nM-50 μM). In some embodiments, the ROCK signaling inhibitor is provided in the culture medium at a concentration of or of about 10 μM.

[0175] In some embodiments, differentiating the population of pluripotent stem cells includes further culturing the cells (e.g., differentiating cells) that have been cultured for the first time period (e.g., MGE patterned cells), in the presence of a MEK signaling inhibitor during at least a portion of the second time period. In some embodiments, the second time period is, is about, or is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 days, or a length of time in a range defined by any two of the preceding values (e.g., 7-28 days, 7-21 days, 10-18 days, 12-16 days, etc.). In some embodiments, the second time period is or is about 2 weeks. In some embodiments, the cells (e.g., differentiating cells) are cultured without the signaling inhibitors (e.g., MEK signaling inhibitor) or activators as described herein during a portion (e.g., at the start) of the second time period. In some embodiments, the cells (e.g., differentiating cells) are cultured without the MEK signaling inhibitor during a portion (e.g., at the start) of the second time period. In some embodiments, the MEK signaling inhibitor is removed from the culture at the end of the second time period. In some embodiments, culturing the cells (e.g., differentiating cells) during the second time period can promote MGE progenitor expansion and pallial interneuron commitment. For convenience and without limitation, the second time period may be referred to as the “expansion and commitment” phase). In some embodiments, the population of cells (e.g., differentiating cells) that have been cultured for the second time period (e.g., expanded and committed cells) is enriched for cells expressing LHX8 and / or LHX6. In some embodiments, the population of cells (e.g., differentiating cells) that havebeen cultured for the second time period (e.g., expanded and committed cells) is depleted for cells expressing high levels of NKX2-1, e.g., NKX2-1 expressed at levels expressed by MGE precursor cells. In some embodiments, the cells (e.g., differentiating cells) that have been cultured for the second time period (e.g., expanded and committed cells) are LHX8+, and have declining NKX2-1 expression and increasing LHX6 expression. In some embodiments, NKX2.1 continues to be expressed. In some embodiments, culturing the cells (e.g., differentiating cells) during the second time period can promote MGE progenitor expansion and subpallial neuron commitment (e.g., by omitting the MEK signaling inhibitor).

[0176] Any suitable inhibitor of MEK signaling can be used for differentiating pluripotent stem cells into a pallial interneuron lineage. In some embodiments, the MEK signaling inhibitor includes, without limitation, PD0325901. In some embodiments, the MEK signaling inhibitor includes PD0325901. In some embodiments, the MEK signaling inhibitor is added to the culture at the start of the second time period (e.g., around D14 of the differentiation time course). In some embodiments, the MEK signaling inhibitor is added to the culture 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days after the start of the second time period (e.g., D14, D15, D16, D17, D18, D19, D20, D21, D22, D23, D24, D25, D26, D27, D28 or later of the differentiation time course). In some embodiments, the cells are cultured in the presence of the MEK signaling inhibitor for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days, or a length of time in a range defined by any two of the preceding values (e.g., 1-14 days, 2-10 days, 2-7 days, 4-10 days, 7-14 days, etc.), during the second time period. In some embodiments, the cells are cultured in the presence of the MEK signaling inhibitor between D14-D28, D14-D21, D15-D21, D15-D28, or D21-D28 of the differentiation time course. Any suitable amount of the MEK signaling inhibitor can be used to promote MGE progenitor expansion and pallial interneuron commitment. In some embodiments, the MEK signaling inhibitor is provided in the culture medium at a concentration of, of about, or of at least 1, 5, 10, 20, 50, 100, 150, 200, 250, 500, or 750 nM, or of, of about, or of at least 1, 2, 5, 10, 20, 50, 100, or 200 μM, or a concentration in a range defined by any two of the preceding values (e.g., 1 nM-200 μM, 50 nM-100 μM, 100 nM- 100 μM, or 100 nM-50 μM). In some embodiments, the MEK signaling inhibitor is provided in the culture medium at a concentration of, or of about 2 μM.

[0177] In some embodiments, differentiating the population of pluripotent stem cells includes further culturing the cells (e.g., differentiating cells) that have been cultured for the second time period (e.g., expanded and committed cells), in the presence of one or more cell cycle exit and differentiation factors, including a MEK signaling inhibitor, a CDK signaling inhibitor, and a Notch signaling inhibitor during at least a portion of the third time period. In some embodiments, the third time period is, is about, or is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days, or a length of time in a range defined by any two of the preceding values (e.g., 7-21 days, 10-18 days, 12-16 days, etc.). In some embodiments, the third time period is or is about 2 weeks. In some embodiments, the one or more cell cycle exit and differentiation factors are removed from the culture at the end of the third time period. In some embodiments, each of the cell cycle exit and differentiation factors are added to the culture for any suitable portion of the third time period to promote cell cycle exit and differentiation. In some embodiments, one or more of the cell cycle exit and differentiation factors are added to the culture at the start of the third time period (e.g., around D28 of the differentiation time course). In some embodiments, one or more of the cell cycle exit and differentiation factors are added to the culture 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days after the start of the third time period (e.g., D28, D29, D30, D31, D32, D33, D34, D35, D36, D37, D38, D39, D40 or later of the differentiation time course). In some embodiments, the cells are cultured in the presence of one or more of the cell cycle exit and differentiation factors for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days, or a length of time in a range defined by any two of the preceding values (e.g., 1-14 days, 2-10 days, 2-7 days, 4-10 days, 7-14 days, etc.), during the third time period. In some embodiments, the cells are cultured in the presence of one or more of the cell cycle exit and differentiation factors between D28-D42, D28-D35, D29-D35, D29-D42, D30-D33, D30- D35, D35-D42, or D35-D40 of the differentiation time course. Any suitable amount of the cell cycle exit and differentiation factors can be used to promote cell cycle exit and differentiation. In some embodiments, each of the one or more cell cycle exit and differentiation factors is provided in the culture medium independently at a concentration of, of about, or of at least 1, 5, 10, 20, 50, 100, 150, 200, 250, 500, or 750 nM, or of, of about, or of at least 1, 5, 10, 20, 50, 100, or 200 μM, or a concentration in a range defined by any twoof the preceding values (e.g., 1 nM-200 μM, 50 nM-100 μM, 100 nM-100 μM, or 100 nM-50 μM).

[0178] In some embodiments, culturing the cells (e.g., differentiating cells) during the third time period promotes cell cycle exit and differentiation. For convenience and without limitation, the third time period may be referred to as “cell cycle exit” phase). For convenience and without limitation, the differentiated cells after culturing in the first, second and third time periods may be referred to as end-of-process (EOP) neurons. In some embodiments, the population of EOP neurons is enriched for cells expressing one or more of LHX6, GAD1, and ERBB4 / CXCR4. In some embodiments, obtaining the cellular composition for use in the present methods includes enriching the differentiated cells for cells expressing one or more of LHX6, GAD1, and ERBB4 / CXCR4 / CXCR7. In some embodiments, enriching the differentiated cells includes using a binding moiety (e.g., antibody) that specifically binds to ERBB4 or CXCR4 or CXCR7, e.g., using FACS or MACS. In some embodiments, the population of EOP neurons is depleted for cells expressing LHX8 and / or NKX2-1. In some embodiments, the EOP neurons are LHX6+, GAD1+, ERBB4+ / CXCR4+ / CXCR7+, LHX8- and NKX2-1-.

[0179] Any suitable agent that inhibits Notch signaling can be used during the differentiation process for obtaining the MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pallial, MGE-type GABAergic interneuron cells). In some embodiments, the Notch signaling inhibitor includes, a γ-secretase inhibitor. In some embodiments, the Notch signaling inhibitor includes, without limitation, DAPT, BMS906024, BMS986115, E-2012, avagacestat, nirogacestat, begacestat, semagacestat, MK-0752, tarenflurbil, RG-4733, itanapraced, PF3084014, and / or Culture One. In some embodiments, the Notch signaling inhibitor is added to the culture at the start of the third time period (e.g., around D28 of the differentiation time course). In some embodiments, the Notch signaling inhibitor is added to the culture 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days after the start of the third time period (e.g., D28, D29, D30, D31, D32, D33, D34, D35, D36, D37, D38, D39, D40 or later of the differentiation time course). In some embodiments, the cells are cultured in the presence of the Notch signaling inhibitor for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days, or a length of time in a range defined by any two of the preceding values (e.g., 1-14 days, 2-10 days, 2-7 days, 4-10 days, 7-14 days, etc.), during the third time period. In someembodiments, the cells are cultured in the presence of the Notch signaling inhibitor between D28-D42, D28-D35, D29-D35, D29-D42, D30-D33, D30-D35, D35-D42, or D35-D40 of the differentiation time course. Any suitable amount of the Notch signaling inhibitor can be used to promote cell cycle exit and differentiation. In some embodiments, the Notch signaling inhibitor is provided in the culture medium at a concentration of, of about, or of at least 1, 5, 10, 20, 50, 100, 150, 200, 250, 500, or 750 nM, or of, of about, or of at least 1, 5, 10, 20, 50, 100, or 200 μM, or a concentration in a range defined by any two of the preceding values (e.g., 1 nM-200 μM, 50 nM-100 μM, 100 nM-100 μM, or 100 nM-50 μM). In some embodiments, the Notch signaling inhibitor is provided in the culture medium at a concentration of or of about 10 μM.

[0180] In some embodiments, differentiating the population of pluripotent stem cells into the MGE-type neuron lineage (e.g., pallial, MGE-type interneuron lineage) includes culturing the differentiating population of pluripotent stem cells in the presence of a NOTCH signaling inhibitor for a time period after obtaining MGE-like progenitor cells. In some embodiments, differentiating pluripotent stem cells to obtain the pallial, MGE-type inhibitory (e.g., GABAergic) interneuron cells includes culturing MGE-like progenitor cells derived from pluripotent stem cells in the presence of a Notch signaling inhibitor. In some embodiments, differentiating pluripotent stem cells includes culturing the population of pluripotent stem cells in the presence of a sonic hedgehog (shh) signaling activator, a TGFbeta signaling inhibitor, and a wnt signaling inhibitor to obtain MGE-like progenitor cells, and further culturing the MGE-like progenitor cells in the presence of a Notch signaling inhibitor, to obtain pallial, MGE-type inhibitory (e.g., GABAergic) interneuron cells.

[0181] Any suitable inhibitor of CDK signaling can be used for differentiating pluripotent stem cells. In some embodiments, the CDK signaling inhibitor includes, without limitation, PD0332991, ribociclib, abemaciclib, flavopiridol, or AT7519. In some embodiments, the CDK signaling inhibitor includes PD0332991. In some embodiments, the CDK signaling inhibitor is added to the culture at the start of the third time period (e.g., around D28 of the differentiation time course). In some embodiments, the CDK signaling inhibitor is added to the culture 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days after the start of the third time period (e.g., D28, D29, D30, D31, D32, D33, D34, D35, D36, D37,D38, D39, D40 or later of the differentiation time course). In some embodiments, the cells are cultured in the presence of the CDK signaling inhibitor for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days, or a length of time in a range defined by any two of the preceding values (e.g., 1-14 days, 2-10 days, 2-7 days, 4-10 days, 7-14 days, etc.), during the third time period. In some embodiments, the cells are cultured in the presence of the CDK signaling inhibitor between D28-D42, D28-D35, D29-D35, D29-D42, D30-D33, D30-D35, D35-D42, or D35-D40 of the differentiation time course. Any suitable amount of the CDK signaling inhibitor can be used to promote cell cycle exit and differentiation. In some embodiments, the CDK signaling inhibitor is provided in the culture medium at a concentration of, of about, or of at least 1, 5, 10, 20, 50, 100, 150, 200, 250, 500, or 750 nM, or of, of about, or of at least 1, 5, 10, 20, 50, 100, or 200 μM, or a concentration in a range defined by any two of the preceding values (e.g., 1 nM-200 μM, 50 nM-100 μM, 100 nM-100 μM, or 100 nM-50 μM). In some embodiments, the CDK signaling inhibitor is provided in the culture medium at a concentration of or of about 10 μM.

[0182] Any suitable inhibitor of MEK signaling can be used for differentiating pluripotent stem cells. In some embodiments, the MEK signaling inhibitor includes, without limitation, PD0325901. In some embodiments, the MEK signaling inhibitor includes PD0325901. In some embodiments, the MEK signaling inhibitor is added to the culture at the start of the third time period (e.g., around D28 of the differentiation time course). In some embodiments, the MEK signaling inhibitor is not added to the culture during the second time period, and is added at the start of the third time period (e.g., at D28 of the differentiation time course). In some embodiments, the MEK signaling inhibitor is added to the culture 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days after the start of the third time period (e.g., D28, D29, D30, D31, D32, D33, D34, D35, D36, D37, D38, D39, D40 or later of the differentiation time course). In some embodiments, the cells are cultured in the presence of the MEK signaling inhibitor for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days, or a length of time in a range defined by any two of the preceding values (e.g., 1-14 days, 2-10 days, 2-7 days, 4-10 days, 7-14 days, etc.), during the third time period. In some embodiments, the cells are cultured in the presence of the MEK signaling inhibitor between D28-D42, D28-D35, D29-D35, D29-D42, D30-D33, D30-D35, D35-D42, or D35-D40 of the differentiation time course. Any suitable amount of the MEK signaling inhibitor can be usedto promote cell cycle exit and differentiation. In some embodiments, the MEK signaling inhibitor is provided in the culture medium at a concentration of, of about, or of at least 1, 5, 10, 20, 50, 100, 150, 200, 250, 500, or 750 nM, or of, of about, or of at least 1, 2, 5, 10, 20, 50, 100, or 200 μM, or a concentration in a range defined by any two of the preceding values (e.g., 1 nM-200 μM, 50 nM-100 μM, 100 nM-100 μM, or 100 nM-50 μM). In some embodiments, the MEK signaling inhibitor is provided in the culture medium at a concentration of or of about 2 μM.

[0183] In some embodiments, differentiating the population of pluripotent stem cells includes further culturing the cells (e.g., differentiating cells) that have been cultured for the second time period (e.g., expanded and committed cells), in the presence of a wnt signaling activator (e.g., a wnt, a GSK3 inhibitor, beta-catenin activator, etc.) during at least a portion of the third time period. Any suitable wnt signaling activator can be used. In some embodiments, the wnt signaling activator is, without limitation, BIO(6-bromoindirubin-3'- oxime), LY2090314, SB-216763, CHIR99021, WAY 262611, LP 922056, or DCA. In some embodiments, the wnt signaling activator is added to the culture at the start of the third time period (e.g., around D28 of the differentiation time course). In some embodiments, the wnt signaling activator is added to the culture 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days after the start of the third time period (e.g., D28, D29, D30, D31, D32, D33, D34, D35, D36, D37, D38, D39, D40 or later of the differentiation time course). In some embodiments, the cells are cultured in the presence of the wnt signaling activator for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days, or a length of time in a range defined by any two of the preceding values (e.g., 1-14 days, 2-10 days, 2-7 days, 4-10 days, 7-14 days, etc.), during the third time period. In some embodiments, the cells are cultured in the presence of the wnt signaling activator between D28-D42, D28-D35, D29-D35, D29-D42, D30-D33, D30-D35, D35-D42, or D35-D40 of the differentiation time course. Any suitable amount of the wnt signaling activator can be used to promote cell cycle exit and differentiation. In some embodiments, the wnt signaling activator is provided in the culture medium at a concentration of, of about, or of at least 1, 5, 10, 20, 50, 100, 150, 200, 250, 500, or 750 nM, or of, of about, or of at least 1, 5, 10, 20, 50, 100, or 200 μM, or a concentration in a range defined by any two of the preceding values (e.g., 1 nM-200 μM, 50 nM-100 μM, 100 nM- 100 μM, or 100 nM-50 μM).

[0184] In some embodiments, deriving the MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pallial, MGE-type GABAergic interneuron cells) does not include sorting the differentiating population of pluripotent stem cells, e.g., using FACS or MACS, based on expression of an exogenous marker for MGE-type progenitors. In some embodiment, the pluripotent stem cells do not have or express an exogenous marker for MGE-type lineage cells (e.g., a transgenically-expressed marker for MGE-type progenitors or GABAergic cortical interneurons). In some embodiments, the MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pallial, MGE-type GABAergic interneuron cells) are derived from pluripotent cells without using sorting techniques, e.g., FACS or MACS, based on expression of an exogenous MGE-type progenitor marker.

[0185] In some embodiments, deriving the MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pallial, MGE-type GABAergic interneuron cells) includes enriching for cells expressing one or more MGE-type neuron-specific cell-surface markers (e.g., one or more pallial, MGE-type interneuron-specific cell-surface markers). Any suitable option can be used to enrich the differentiating population for cells based on expression of one or more MGE-type neuron-specific cell-surface markers (e.g., one or more pallial, MGE- type interneuron-specific cell-surface markers). Suitable options include, without limitation, flow cytometry, FACS, MACS, etc. In some embodiments, deriving the MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pallial, MGE-type GABAergic interneuron cells) does not include sorting for cells expressing one or more MGE-type interneuron- specific cell-surface markers. In some embodiments, the population of differentiated cells (e.g., MGE-type GABAergic interneuron cells) includes a mix of pallial, MGE-type GABAergic interneuron cells (e.g., cells expressing markers for the pallial, MGE-type GABAergic interneuron lineage) and subpallial, MGE-type GABAergic neuron cells (e.g., cells expressing markers for the subpallial, MGE-type GABAergic neuron lineage), without sorting. In some embodiments, differentiating the pluripotent stem cells according to options described herein generates, without sorting, a population of MGE-type GABAergic neuron cells that is or is about 80%, 85%, or 90%, or a percentage in a range defined by any two of the preceding values (e.g., 80-90%, 85-90%, 80-85%) of the pallial interneuron lineage. In some embodiments, differentiating the pluripotent stem cells according to options described herein generates, without sorting, a population of MGE-type GABAergic neuron cells that isor is about 10%, 15%, or 20%, or a percentage in a range defined by any two of the preceding values (e.g., 10-20%, 15-20%, 10-15%) of the subpallial neuron lineage.

[0186] In some embodiments, differentiating pluripotent stem cells to obtain subpallial, MGE-type inhibitory (e.g., GABAergic) neuron cells is carried out using any suitable option. In some embodiments, subpallial, MGE-type inhibitory (e.g., GABAergic) neuron cells are obtained by differentiating pluripotent stem cells as described herein, and enriching for LHX6+, ERBB4-, LHX8+ and / or NKX2.1+, GAD1+ cells. In some embodiments, subpallial, MGE-type inhibitory (e.g., GABAergic) neuron cells are obtained by differentiating pluripotent stem cells as described herein, but omitting the MEK inhibitor during the differentiation process. In some embodiments, subpallial, MGE-type inhibitory (e.g., GABAergic) neuron cells are enriched by sorting for cells, for example, by FACS or MACS. In some embodiments, subpallial, MGE-type inhibitory (e.g., GABAergic) neuron cells are enriched by sorting for cells using binding agents to one or more surface markers (e.g., NRP2+) expressed on cells of the subpallial lineage. In some embodiments, subpallial, MGE-type inhibitory (e.g., GABAergic) neuron cells are enriched by depleting non-pallial cells using binding agents to one or more surface markers (e.g., ERBB4+) not expressed on cells of the subpallial lineage. In some embodiments, the subpallial, MGE-type GABAergic neuron cells include interneurons and / or projection neurons.

[0187] In some embodiments, the method includes cryopreserving the MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pallial, MGE-type GABAergic interneuron cells) after deriving the cells from the pluripotent stem cells. Any suitable option for cryopreserving mammalian cells can be used. In some embodiments, the method includes thawing a cryopreserved cellular composition that includes the MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pallial, MGE-type GABAergic interneuron cells) before administering the cells.

[0188] In some embodiments, the pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pallial, MGE-type GABAergic interneuron cells) are autologous to the subject. In some embodiments, the pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pallial, MGE-type GABAergic interneuron cells) are heterologous to the subject. In some embodiments, the interneurons, precursors, and / or pluripotent stem cells of the present disclosure, are genetically modified.In some embodiments, the interneurons, precursors, and / or pluripotent stem cells of the present disclosure include one or more mutations (e.g., nucleic acid substitutions, deletions, insertions, chromosomal rearrangements, etc.) relative to a native genome. In some embodiments, the pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pallial, MGE-type GABAergic interneuron cells) are genetically modified. In some embodiments, the genetic modification alters (e.g., reduces, abolishes, enhances, etc.) an expression and / or function of a gene product (e.g., protein) regulated or encoded by the modified genetic locus. Any suitable genetically-encoded function can be altered through genetic modification of the pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells (and / or precursor thereof). In some embodiments, the pluripotent stem cell- derived, MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pallial, MGE-type GABAergic interneuron cells) are genetically modified to reduce an immunological response against the administered cellular composition by the subject. Any suitable gene immune- activating genes can be mutated to reduce or abolish activity, or can be knocked out, and / or any suitable genetic modification that activates expression or activity of immune-inhibiting genes can be present in the pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells. In some embodiments, the pluripotent stem cell-derived, pallial, MGE- type GABAergic interneuron cells are genetically modified to reduce or abolish expression of one or more of: B2M, a HLA class I gene, and / or a HLA class II gene. In some embodiments, the pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells are genetically modified to knock out one or more of: B2M, a HLA class I gene, and / or a HLA class II gene. SYSTEMS

[0189] Also provided is a delivery system for transplanting cells, such as but not limited to pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pallial, MGE-type GABAergic interneuron cells) as provided herein, into a tissue, such as but not limited to one or more regions of the brain, as provided herein. The system can include a delivery cannula (or catheter, or similar device) that includes: a proximal portion comprising a cellular liquid composition having cells (e.g., pluripotent stem cell-derived, MGE-type inhibitory (e.g., GABAergic) neuron cells (e.g., pallial, MGE-type GABAergicinterneuron cells), or NRTX-1001) at a concentration of about 1 x 105cells / μL or greater; and a distal portion comprising a liquid chase vehicle, wherein the cellular liquid composition is stably held in the proximal portion by the liquid chase vehicle; and a displacement device (e.g., a syringe with optional syringe pump) connected to the distal end of the cannula and configured to cause the liquid chase vehicle to displace the cellular liquid composition to thereby expel the cellular liquid composition from the proximal end of the cannula. In some embodiments, a stylet that traverses the inner lumen of the cannula is used to displace the cellular liquid composition, with or without the liquid chase vehicle, to thereby expel the cellular liquid composition from the proximal end of the cannula. The cellular liquid composition can include cells at any suitable concentration, as described herein, such as at about 1 x 105cells / μL or greater (e.g., about 0.5 x 106cells per microliter to about 1.5 x 106cells per microliter, about 0.9 x 106cells per microliter to about 1.2 x 106cells per microliter, or about 1 x 106cells per microliter). In some embodiments, the cellular liquid composition includes cells at a concentration of about 1 x 105cells / μL or greater, and up to about 2 x 106cells per microliter. In some embodiments, the cellular liquid composition includes cells at a concentration of, of about, or of at least 0.1 x 106, 0.2 x 106, 0.3 x 106, 0.4 x 106, 0.5 x 106, 0.6 x 106, 0.7 x 106, 0.8 x 106, 0.9 x 106, 1 x 106, 1.1 x 106, 1.2 x 106, 1.3 x 106, 1.4 x 106, 1.5 x 106, 1.6 x 106, 1.7 x 106, 1.8 x 106, 1.9 x 106, or 2 x 106cells per microliter, or at a concentration in a range defined by any two of the preceding values (e.g., 0.1 x 106– 2 x 106cells per microliter, 0.5 x 106– 2 x 106cells per microliter, 0.6 x 106– 1.2 x 106cells per microliter, 0.6 x 106– 1 x 106cells per microliter, 0.8 x 106– 1.2 x 106cells per microliter, etc.). In some embodiments, the cellular liquid composition includes cells at a concentration of about 0.6 x 106cells per microliter to about 1.2 x 106cells per microliter. In some embodiments, the system includes a pump (e.g., syringe pump) connected to the displacement device.

[0190] Provided herein is a delivery system for transplanting cells. The system can include a delivery cannula containing a cellular liquid composition comprising cells at a concentration of about 1 x 105cells / μL or greater; and a displacement device connected to a distal end of the cannula and configured to displace the cellular liquid composition in the cannula to thereby expel the cellular liquid composition from the proximal end of the cannula. In some embodiments, the displacement device includes a stylet configured totraverse the inner lumen of the delivery cannula to thereby expel the cellular liquid composition from the proximal end of the cannula.

[0191] In some embodiments, the system includes a monitoring device configured to monitor the delivery site in the subject’s brain during administration of the cellular composition. In some embodiments, the monitoring device is configured to detect the position of the proximal end of the cannula at a site of delivery in a subject. Any suitable monitoring device can be used. In some embodiments, the monitoring device includes, without limitation, a CT scanner, MRI scanner, PET scanner, or a SPECT scanner.

[0192] The system of the present disclosure can be used to carry out any of the methods of treating seizure activity as described herein.

[0193] Further non-limiting embodiments of the present disclosure are provided in the following numbered arrangements. 1. A method of treating seizure activity, comprising: identifying a subject in need of treating seizure activity; and administering to the subject a therapeutically effective amount of a cellular composition comprising pluripotent stem cell-derived, pallial, MGE-type inhibitory (e.g., GABAergic) interneuron cells, wherein at least 50% of the cells of the composition express LHX6, ERBB4, and GAD1, and wherein a frequency of seizures is reduced after the administering, thereby treating the seizure activity. 2. The method of arrangement 1, wherein the subject has a focal epilepsy. 3. The method of arrangement 1, wherein the subject has a chronic seizure activity. 4. The method of any one of the preceding arrangements, wherein the subject has mesial temporal sclerosis. 5. The method of arrangement 4, wherein the mesial temporal sclerosis is reduced after the administering 6. The method of any one of the preceding arrangements, wherein the subject has an epileptogenic lesion in the brain. 7. The method of arrangement 6, wherein the epileptogenic lesion is reduced after the administering.8. The method of any one of the preceding arrangements, wherein at most 50% of the cells of the composition express NKX2-1. 9. The method of any one of the preceding arrangements, wherein at most 40% of the cells of the composition express LHX8. 10. The method of any one of the preceding arrangements, wherein the at least 50% of the cells of the composition further express at least one of MAF and MAFB. 11. The method of any one of the preceding arrangements, wherein the therapeutically effective amount comprises about 5 x 104cells or more. 12. The method of arrangement 11, wherein the therapeutically effective amount comprises between about 5 x 104to about 1 x 1012cells. 13. The method of any one of the preceding arrangements, wherein the therapeutically effective amount of the cellular composition is administered at a concentration of about 1 x 105cells / μL or greater. 14. The method of any one of the preceding arrangements, wherein the cellular composition comprises sodium chloride at 100-600 mM. 15. The method of any one of the preceding arrangements, wherein the cellular composition has an osmolality of at least 200 mOsm / kg. 16. The method of any one of the preceding arrangements, wherein at most 40% of the cells express an MGE progenitor cell marker or a non-pallial MGE-type neural cell marker. 17. The method of any one of the preceding arrangements, wherein at most 10% of the cells of the composition express KI67. 18. The method of any one of the preceding arrangements, wherein at most 10% of the cells of the composition express OLIG2. 19. The method of any one of the preceding arrangements, wherein cells of the composition are GABA-secreting cells. 20. The method of any one of the preceding arrangements, wherein cells of the composition are migratory cells. 21. The method of any one of the preceding arrangements, wherein at least 50% of the cells of the composition are pallial MGE-type inhibitory (e.g., GABAergic) interneuron cells.22. The method of any one of the preceding arrangements, wherein the subject has temporal lobe epilepsy (TLE). 23. The method of arrangement 22, wherein the TLE is drug-resistant TLE. 24. The method of any one of the preceding arrangements, wherein the subject suffers from a neocortical onset focal epilepsy. 25. The method of any one of the preceding arrangements, wherein the subject suffers from focal seizure activity. 26. The method of any one of the preceding arrangements, comprising administering the therapeutically effective amount of the cellular composition to the subject’s brain. 27. The method of any one of the preceding arrangements, further comprising performing one or more of computed tomography (CT) scan, magnetic resonance imaging (MRI), MR spectroscopy (MRS), functional MRI (fMRI), electroencephalography (EEG), intracranial EEG, positron emission tomography (PET), and single photon emission computed tomography (SPECT) on the subject. 28. The method of any one of the preceding arrangements, further comprising monitoring brain activity in the subject. 29. The method of any one of the preceding arrangements, further comprising identifying a mesial temporal sclerosis in the subject, or one or more lesions in the brain. 30. The method of arrangement 29, wherein identifying the mesial temporal sclerosis in the subject or the one or more lesions comprises performing one or more of a CT scan, MRI, MRS, EEG, PET, SPECT, or fMRI. 31. The method of any one of the preceding arrangements, wherein the frequency of seizures is reduced by about 50% or more after the administering. 32. The method of any one of the preceding arrangements, further comprising performing one or more neurocognitive assessments of the subject. 33. The method of any one of the preceding arrangements, wherein the cellular composition is a thawed cellular composition. 34. The method of arrangement 33, comprising holding the thawed cellular composition for up to about 5 days before administering to the subject.35. The method of arrangement 33 or 34, comprising holding the thawed cellular composition at an ambient temperature of 4-25°C, or 2-18°C, or at room temperature, before administering to the subject. 36. The method of any one of the preceding arrangements, further comprising thawing a cryopreserved cellular composition comprising the pluripotent stem cell-derived, pallial, MGE-type, inhibitory (e.g., GABAergic) interneuron cells before administering. 37. The method of any one of the preceding arrangements, wherein the administering comprises administering the cellular composition as one or more deposits in the subject’s brain. 38. The method of arrangement 37, wherein the therapeutically effective amount of the cellular composition is delivered in a volume of about 50 μL or less per deposit. 39. The method of any one of the preceding arrangements, wherein the administering comprises administering the cellular composition to one or more sites in the temporal lobe, optionally wherein the one or more sites in the temporal lobe include the hippocampus, cortex, and / or amygdala, optionally wherein the one or more sites in the temporal lobe include the hippocampus, subiculum, entorhinal cortex, and / or parahippocampal gyrus. 40. The method of any one of the preceding arrangements, wherein the cells are human cells. 41. The method of any one of the preceding arrangements, wherein the subject is a human subject. 42. The method of any one of the preceding arrangements, further comprising administering an immunosuppressant before and / or after administering the therapeutically effective amount of the cellular composition. 43. The method of any one of the preceding arrangements, wherein the seizure activity is due to a traumatic brain injury, stroke, tumor, focal cortical dysplasia, tuberous sclerosis, developmental disorder, or a neurological disorder. 44. The method of any one of the preceding arrangements, wherein the subject suffers from epileptic seizure-like discharges in the brain associated with Alzheimer’s disease, focal cortical dysplasia (FCD), or amnesic mild cognitive impairment (aMCI).45. The method of any one of the preceding arrangements, further comprising monitoring a therapeutic effect of administering the cellular composition in the subject. 46. The method of arrangement 45, comprising non-invasively monitoring the therapeutic effect using one or more biomarkers of seizure activity. 47. The method of arrangement 46, wherein the one or more biomarkers comprises N-acetylaspartate (NAA), myoinositol, glutamate, GABA, volume, edema, blood flow, oxygen metabolism, and glucose. 48. The method of any one of arrangements 45-47, comprising using EEG, imaging, and / or one or more blood-based biomarkers to monitor the therapeutic effect. 49. A method of treating seizure activity, comprising: identifying a subject in need of treating seizure activity; and intracranially administering to the subject a cellular composition comprising: about 5 x 104to about 1 x 1012pluripotent stem cell-derived, pallial, MGE- type GABAergic interneuron cells at a concentration in a range of about 1 x 105to about 4 x 106cells / μL, wherein at least 50% of the cells of the composition express LHX6, ERBB4, and GAD1, wherein 90% or more of the cells of the composition are post-mitotic cells; a poloxamer at 0.1-1%, v / v; and sodium chloride at 100-600 mM. 50. Use of a cellular composition for the treatment of seizure activity in a subject, the composition comprising pluripotent stem cell-derived, pallial, MGE-type inhibitory (e.g., GABAergic) interneuron cells, wherein at least 50% of the cells of the composition express LHX6, ERBB4, and at least one of MAF and MAFB, optionally wherein at least 90% of the cells are post-mitotic cells. 51. A cellular composition of pluripotent stem cell-derived, pallial MGE-type inhibitory (e.g., GABAergic) interneuron cells for the preparation of a medicament for treatment of seizure activity in a subject, wherein at least 50% of the cells of the composition express LHX6, ERBB4, and GAD1. 52. A therapeutic composition of pluripotent stem cell-derived, pallial MGE-type inhibitory (e.g., GABAergic) interneuron cells for the treatment of focal onset seizure, wherein at least 50% of the cells of the composition express LHX6, ERBB4, and GAD1.53. A therapeutic composition comprising: a poloxamer; and pluripotent stem cell-derived, pallial, MGE-type inhibitory (e.g., GABAergic) interneuron cells at a concentration of about 1 x 105cells / μL or greater. 54. A method of preparing a therapeutic composition for administering to a subject, comprising: providing pluripotent stem cell-derived, pallial, MGE-type inhibitory (e.g., GABAergic) interneuron cells; and preparing a cellular composition comprising: a poloxamer; and the pluripotent stem cell-derived, pallial, MGE-type inhibitory (e.g., GABAergic) interneuron cells at a concentration of about 1 x 105cells / μL or greater. 55. A delivery system for transplanting cells into a tissue, comprising: a delivery cannula comprising: a proximal portion comprising a cellular liquid composition comprising cells at a concentration of about 1 x 105cells / μL or greater; and a distal portion comprising a cell-free liquid chase vehicle, wherein the cellular liquid composition is stably held in the proximal portion by the liquid chase vehicle; and a displacement device connected to a distal end of the cannula and configured to cause the liquid chase vehicle to displace the cellular liquid composition to thereby expel the cellular liquid composition from a proximal end of the cannula. 56. A delivery system for transplanting cells into a tissue, comprising: a delivery cannula comprising a cellular liquid composition comprising cells at a concentration of about 1 x 105cells / μL or greater; and a displacement device connected to a distal end of the cannula and configured to displace the cellular liquid composition in the cannula to thereby expel the cellular liquid composition from a proximal end of the cannula. 57. The delivery system of arrangement 55 or 56, wherein the displacement device comprises a syringe, or a stylet traversing the inner lumen of the delivery cannula.58. The delivery system of any one of arrangements 55-57, further comprising a pump connected to the displacement device. 59. The delivery system of any one of arrangements 55-58, wherein the cellular liquid composition comprises a contrast agent. 60. The delivery system of arrangement 59, wherein the contrast agent comprises an MRI contrast agent. 61. The delivery system of arrangement 59 or 60, wherein the contrast agent comprises a gadolinium-based contrast agent. 62. The delivery system of any one of arrangements 55-59, further comprising a monitoring device configured to detect the position of the proximal end of the cannula at a site of delivery in a subject. 63. The delivery system of arrangement 62, wherein the monitoring device comprises a CT scanner, MRI scanner, PET scanner, or a SPECT scanner. 64. The delivery system of any one of arrangements 55-63, wherein the cells are neuron cells. 65. The delivery system of arrangement 64, wherein the cells comprise pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells. 66. A method of treating seizure activity, comprising: identifying a subject in need of treating seizure activity; and administering to the subject a therapeutically effective amount of a cellular liquid composition comprising pluripotent stem cell-derived, pallial, MGE-type inhibitory (e.g., GABAergic) interneuron cells using the system of any one of arrangements 55-65, wherein the cellular liquid composition comprises the pluripotent stem cell-derived, pallial, MGE-type inhibitory (e.g., GABAergic) interneuron cells. 67. The method of arrangement 66, comprising detecting the position of the delivery cannula in the subject’s brain during the administering. 68. The method of arrangement 66 or 67, wherein the detecting comprises using MRI to visualize the delivery cannula in the subject’s brain.69. The method, therapeutic composition, or delivery system of any one of the preceding arrangements, wherein the cellular composition or the cellular liquid composition comprises NRTX-1001. 70. The method, therapeutic composition, or delivery system of any one of the preceding arrangements, wherein at least 50% of the cells of the composition express LHX6, ERBB4, and GAD1, and wherein at least 90% of the cells are post-mitotic cells. 71. The method, therapeutic composition, or delivery system of any one of the preceding arrangements, wherein at least 50% of the cells persisting in the subject retain a pallial, MGE-type GABAergic interneuron cell fate at least 6 months after administration. 72. The method, therapeutic composition, or delivery system of arrangement 71, wherein the cells persisting in the subject and that retain the pallial, MGE-type GABAergic interneuron cell fate express one or more of GAD1, GAD2, LHX6, SOX6, NXPH1, ERBB4, SST, NPY, MEF2c, and ARX. 73. The method, therapeutic composition, or delivery system of any one of the preceding arrangements, wherein the pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells are genetically modified. 74. The method, therapeutic composition, or delivery system of arrangement 73, wherein the pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells are genetically modified to reduce an immunological response against the administered cellular composition by the subject, optionally wherein the pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells comprises: a mutation in one or more immune-activating genes that reduces or abolishes expression or activity thereof; and / or a genetic modification that activates expression or activity of one or more immune-inhibiting genes. 75. The method, therapeutic composition, or delivery system of arrangement 74, wherein pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells are genetically modified to reduce or abolish expression of one or more of: B2M, a HLA class I gene, and / or a HLA class II gene.EXAMPLES Example 1

[0194] This non-limiting example introduces Examples 2-9.

[0195] Epilepsy, one of the most common central nervous system disorders, is characterized by abnormal, excessive, and / or synchronous electrical activity in the brain. Imbalanced neural excitatory and inhibitory activity results in hyperactive neuronal networks that precipitate and propagate seizures. Consequently, anti-seizure drugs (ASDs) have been designed to decrease neuronal activity. However, one-third of individuals with epilepsy have drug-resistant seizures. ASDs that potentiate the inhibitory neurotransmitter GABA are effective acute seizure suppressants. Still, these drugs are typically not first-line medications for chronic epilepsy due to systemic and neurotoxic side effects, tolerance, and addiction. There are few effective options for people living with drug-resistant chronic epilepsy. Surgical resection or laser ablation of the seizure focus can be effective options for some with drug-resistant MTLE, resulting in seizure freedom in 50-75% of cases. However, these surgeries are tissue-destructive, can cause serious adverse effects, including increased risk of verbal memory impairment in the dominant hemisphere, and cannot be performed on both temporal lobes for those with bilateral MTLE. Thus, the development of non-destructive therapeutic strategies that target regions of the brain where seizures begin, spare surrounding tissue, and avoid the toxicities associated with ASDs, could provide a safer and more effective option.

[0196] Inhibitory cell therapy represents a strategy that could locally restore normal GABAergic tone to seizure-onset foci and thereby repair underlying pathophysiology. Pallial (commonly referred to as “cortical”) GABAergic local circuit interneurons are the primary source of inhibition in the neocortex and hippocampus. Pallial interneurons are born in subcortical germinal zones and migrate tangentially to the cortex and hippocampus, where they disperse to their final positions and gradually acquire mature neurochemical and physiological characteristics. Different types of pallial interneurons are generated by distinct germinal domains in the developing subpallium: the MGE and the nearby preoptic area (POA) generate somatostatin (SST)- and parvalbumin (PV)-expressing pallial interneuron classes, while the caudal ganglionic eminence (CGE) generates serotonin receptor 3A (HTR3A)-expressing interneuron classes. A loss of MGE-derived interneurons,including SST, neuropeptide Y (NPY), and PV-expressing subtypes, has been observed in hippocampal tissue resected from MTLE patients or post-autopsy. Furthermore, mutations in genes required for interneuron generation and function have been identified in various developmental epileptic disorders. Consistent with these findings, SST and PV interneuron subtypes are lost or dysfunctional in animal models of epilepsy. Additionally, seizures can be induced by selective MGE-type pallial interneuron ablation and reduced by selective MGE- type interneuron activation.

[0197] Proof-of-concept efficacy of inhibitory interneuron cell therapy has been demonstrated after transplantation of primary embryonic rodent MGE cells into experimental models of epilepsy. The transplanted primary MGE cells locally dispersed, stably persisted, matured into pallial interneuron subtypes, and suppressed seizures. A renewable cell source of human MGE-type interneurons would encourage widespread clinical use.

[0198] Human pluripotent stem cells (hPSCs) represent an expandable cell source from which inhibitory interneurons can be derived. Multiple derivation methods for generating human GABAergic neurons from MGE-like progenitors have been published using either embryonic or induced pluripotent stem cells. Subsequent transplantation studies of hPSC-derived GABAergic neurons into experimental epilepsy models reported promising results, including seizure reduction. However, despite the widespread use of the term “cortical interneurons”, these cell preparations were not specifically enriched for cortical or pallial-type MGE-derived interneurons. Rather, each consisted of a mixture of different GABAergic populations, including CGE-derived interneurons and likely non-migratory GABAergic projection neurons, as evidenced by marker expression and graft phenotypes in the above reports. This distinction is important because MGE progenitors produce several cell lineages in addition to pallial GABAergic interneurons, including GABAergic and cholinergic striatal interneurons, GABAergic and cholinergic subpallial projection neurons, oligodendrocytes, and astrocytes. Thus, while pallial MGE interneurons are specifically implicated in the pathophysiology of MTLE and represent the most anatomically and physiologically relevant population for cell replacement therapy, a clinically appropriate source of these cells has not been described previously in the literature, and the potential of human pallial interneuron cell therapy to suppress seizures has remained unexplored.

[0199] A novel pallial MGE-type GABAergic interneuron cell therapy derived from a clinical-grade human embryonic stem cell (hESC) line was developed, as shown in Examples 2-9. Significant suppression of mesiotemporal seizures and histopathology improvement following cell transplantation into a preclinical MTLE model was demonstrated. The human pallial MGE-type interneurons were harvested and cryopreserved at a committed post-mitotic, migratory stage of development to ensure dispersion and integration within local circuits, mitigating the risks of proliferation and differentiation into undesired cell fates. Extensive molecular and functional characterization of the cell therapy candidate was made, including single-cell RNA sequencing (scRNA-seq), in vitro migration, and GABA secretion analyses. Following intrahippocampal transplantation into a chronic mouse model of MTLE, hESC-derived pallial MGE-type interneurons were analyzed for cellular migration and persistence, graft composition, synaptic connectivity, and dose- dependent reduction of seizure activity.

[0200] In some embodiments, human pallial MGE-type GABAergic interneuron cell therapy can provide treatment of drug-resistant focal epilepsies. Example 2

[0201] This non-limiting example provides materials and methods used in Examples 3-10. A. In vitro hESC line and banks

[0202] The pallial MGE-type GABAergic interneurons described in this study were differentiated from research-grade working cell banks derived from a human embryonic stem cell (hESC) line. The cell line is listed in the NIH hESC Registry as an “Approved Line” (eligible for use in NIH-supported research). Donor consent was obtained for use of the cells in research, clinical and commercial development. Donor screening was performed using FDA’s Donor Eligibility Guidelines for HCT / Ps. The cell line was derived, expanded, and banked under cGMP conditions using qualified raw materials to generate a GMP seed cell bank (SCB), and research-grade master cell banks (rMCB) and working cell banks (rWCB). Karyotypic analysis and pluripotency marker expression were assessed across all hESC cell banks, demonstrating genomic stability and high purity. The pluripotent stem cellbanks were tested for sterility and found negative for relevant viruses, mycoplasma, and adventitious agents. Pluripotency of the undifferentiated cells was demonstrated by expression of markers such as TRA-1-60, OCT-4, and NANOG; the in vitro differentiation of the cells to endoderm, ectoderm, and mesoderm lineage; and the demonstration of teratoma formation when engrafted in vivo. hESC stability

[0203] The hESC working cell banks (WCB) were tested for genetic stability per ICH Q5D (ICH_Q5D 1998) by evaluating the cell substrate for consistent production of the intended cell fate. MGE-type patterning and differentiation to pallial interneurons using the manufacturing process was initiated from rWCB ESCs after up to five additional passages of ESC expansion to reflect the intended limit of cultivation, and genetic stability was characterized using the complementary orthogonal methods of karyotype analysis by G- banding and single nucleotide polymorphism (SNP) arrays. Since the product being manufactured is post-mitotic and karyotype analysis by G-banding requires mitotic cells, karyotype analysis was performed during the ESC expansion and genome-wide genomic copy number variants (CNVs) and regions of homozygosity analysis (ROH) were assessed during both ESC expansion and end-of-process (EOP) interneuron differentiation using SNP arrays.

[0204] In addition, exon capture, next generation sequencing and variant analysis of the TP53 gene was performed using gDNA isolated from ESCs and EOP interneurons, with at least 100X on target coverage. Libraries were prepared using CleanPlex® TP53 Panel (Paragon Genomics) and sequenced on the Illumina platform and analyzed by ACGT Inc for TP53 mutations recurrently acquired in human pluripotent stem cells during in vitro culture (Merkle et al, 2017). The characterizations demonstrated:

[0205] a. no genetic abnormality occurs during ESC expansion or following the interneuron production process from ESCs at the maximum limit of in vitro cultivation; and

[0206] b. consistent production of the intended pallial interneuron cell product at the maximum limit of in vitro cultivation. hESC expansion

[0207] A cGMP grade hESC line was adapted to a feeder-free, xeno-free culture system using human recombinant vitronectin (A27940, Life Technologies) coated dishes,Essential 8 medium kit (A2656101, Life Technologies) and dissociation reagent ReLeSR (100-0484, STEMCELL Technologies). Media was changed daily, and cells were passaged every 5 days, for a total of 5 passages, before being cryopreserved to sequentially generate a GMP SCB, and research-grade rMCBs and rWCBs. MGE pallial-type interneuron differentiation

[0208] For each differentiation experiment, one vial of rWCB was thawed. The thawed hESCs were seeded into adherent culture and expanded for 5 days post-thaw using conditions described above. To initiate differentiation, the expanded hESCs were dissociated into a single cell suspension using StemPro Accutase (A1110501, Life Technologies) and resuspended in differentiation media consisting of Neurobasal-A (50%; 10888, Life Technologies) and DMEM / F12 (50%; 21331, Life Technologies), supplemented with GlutaMAX (1X; 35050-061, Life Technologies), B-27 without Vitamin A (1X; 12587-010 Life Technologies), N2 supplement-B (1X; 07156, Stem Cell Technologies), Penicillin- Streptomycin (1X; 15140-122, Life Technologies), L-Ascorbic Acid (200μM; A5960, Sigma), β-Mercaptoethanol (55M; 21985-023 Life Technologies), and MEM non-essential amino acids (1 / 2X; 11140-050, Life Technologies). The resuspended hESCs were exposed to patterning and differentiation cues from day 0, adjusting small molecule combinations over time and changing media every 2 to 3 days, using only chemically-defined reagents and small molecules, in the absence of knockout serum replacement (KSR).

[0209] During the three main protocol phases (Fig 10A), small molecules modulating the listed cell signaling pathways were introduced to promote robust MGE patterning and differentiation into pallial-type GABAergic interneurons. During the neuroectoderm specification and MGE patterning phase, ROCK inhibitor (Y27623 10μM; 1254 Tocris) with TGFβ and BMP pathway inhibition induced neuroectoderm (SB431542 10μM; 1614 Tocris / LDN193189250nM; 6053 Tocris). WNT pathway inhibition conferred forebrain identity (XAV939 10μM; 3748 Tocris) and SHH pathway activation induced ventral forebrain MGE-like progenitors (SAG 100nM; 4366 Tocris). During the second MGE progenitor expansion and pallial interneuron commitment phase, a MEK pathway inhibitor was applied to further improve the efficiency, kinetics, and specificity of pallial-type GABAergic interneuron lineage (PD0325901 2μM; 4192 Tocris). Finally, during the third phase, inhibitors of the MEK (PD0325901 2μM; 4192 Tocris), CDK (PD0332991 2μM;S1116 Selleck Chemicals) and NOTCH (DAPT 10μM; 2634 Tocris) pathways were added to induce cell cycle exit and differentiation into post-mitotic MGE pallial-type GABAergic interneurons. Cell batch processing and cryopreservation

[0210] At the end of the differentiation process, cells were harvested and dissociated to single cells using TrypLE™ Select Enzyme and Benzonase (1 / 10000; Sigma Aldrich). A portion of the harvested cell suspension was subsequently processed using magnetic-activated cell sorting (MACS) for ERBB4. Single cells were incubated with a biotinylated primary antibody against human ERBB4 (BAF1131 R&D Systems), followed by incubation with Anti-Biotin MicroBeads (130-105- 637, Miltenyi Biotec), and magnetic sorting was performed on the CliniMACS according to manufacturer instructions (Miltenyi Biotec). Unsorted (pre-sort) and sorted (post-positive selection) cell populations were cryopreserved using the CryoMed controlled-rate freezer (Fisher Scientific), before further storage in vapor-phase liquid nitrogen. Immunocytochemistry

[0211] Thawed cells were counted using the NC-200 cell counter (Chemometec) and seeded into 384-well plates coated with Poly-L-Ornithine / Fibronectin / Laminin (PO / Fib / Lam) at ~4.0E5-4.3E5 cells / cm2. Cells were fixed with 4% Paraformaldehyde (PFA) for 10 minutes 3-16 hours after plating and processed for staining within two weeks. Briefly, cells were incubated with a blocking buffer (10% Goat Serum, 0.1% Triton X-100 in DPBS (Dulbecco’s phosphate-buffered saline)) for 30 minutes at ambient temperature. Primary and secondary antibody dilutions were prepared in blocking buffer and kept on ice. Primary antibody incubation was done at 4°C overnight, followed by two 30-minute washes with PBS-T (DPBS, 0.1% Triton X-100). Secondary antibodies (Alexa Fluor conjugated, Life Technologies) incubation was done at ambient temperature in the dark for 2 hours, followed by two 30-minute washes with PBS-T. The second wash was replaced with DPBS for imaging and storage. Aspiration and washes in 384-well plates were automated using the plate washer ELX406 (Biotek).

[0212] Images were acquired using the Leica Dmi8 microscope with a 20X objective. A minimum of three-five fields of view were captured (typically 250-350 cells perfield) for each sample and antibody combination. Analysis was performed manually using ImageJ software.

[0213] For higher throughput sample images acquisition, analysis and quantification, the high content screening imaging system CX5 (Life Technologies) was used. Independent combinations of antibodies (up to 4 independent channels for the detection of Hoechst, Alexa Fluor AF 488, AF547 and AF650) were acquired independently with specific exposure times. Adjusting background correction and segmentation parameters, using “spot detector” bio application, nuclei were identified as primary objects to define regions of interest on subsequent channels, and determine percent of positive cells expressing a given marker, based on morphological and intensity thresholding. Assessment of residual pluripotent cells by flow cytometry

[0214] Cells were stained on day 28 and at the end of process (post-thaw) with PE / Cy7 anti-human-TRA-1-60 (Biolegend) for surface TRA-1-60, Fixable Far Red dye (ThermoFisher Scientific) for viability and AF488 anti-human-OCT4 (BD Bioscience) for intracellular OCT4. The cells were analyzed using an Attune-NxT Acoustic Focusing Flow Cytometer and data were analyzed using FlowJo (BD Biosciences) software. To assess the presence of residual pluripotency markers in the final cryopreserved product, an extensive qualification was performed to determine the assay repeatability, accuracy, linearity and limits of detection (LOD) per ICH Q2 guidelines. Samples were stained in triplicate (1E7 cells per sample) for better population statistics. From this study the method was determined to be accurate in detecting pluripotent cells at a LOD of 0.002%. When the method was employed to analyze samples at the end of the process, the percentage of pluripotent cells detected was always below the LOD. Primeflow

[0215] Cells were thawed, counted using the NC-200 cell counter, stained using Fixable Aqua Dead Cell Stain Kit (Life Technologies, cat# L34957), and processed for RNA Primeflow assay (Thermo Fisher, cat# 88-18005-210) according to the manufacturer’s instructions. Reactions were run in triplicate. Data were acquired using the Attune NXT Flow Cytometer (Life Technologies). All events in the sample were recorded (in the range of 6E4-3E5). Results were analyzed using the FlowJo software. Fixable Aqua and FSC were used to gate live cells. Single cells were then gated on forward scatter area and height (FSC-A versus FSC-H) and side scatter area and height (SSC-A versus SSC-H). Using dapB as the negative control, gates were set in histogram plots for AF488 and AF647 probes. The same gates were then applied to all the test samples accordingly to determine the percentage of expression in cell lots. Quantitative results were reported as the average ± standard deviation of 3 technical replicates. GABA release

[0216] To perform the GABA release assay, cryopreserved cells were thawed, and the viability and cell concentration were determined using the NC-250 cell counter (Chemotec). Cells were seeded onto a polyornithine / fibronectin / laminin coated 96-well plate at a density of 0.65E6 cells / cm2in culture media. Each cell lot was seeded in six wells, and cells were cultured at 37°C, 5% CO2 for 8 days, with media changes every 2-3 days. On day 8, spent medium was replaced with fresh pre-warmed medium either alone or supplemented with 90 mM potassium chloride (in triplicate wells per condition). After a 30-minute incubation at 37°C, 5% CO2, cell-free supernatants were collected and stored at -80°C. The samples were submitted for liquid chromatography and mass spectrometry (LC / MS-MS) analysis of GABA and Acetylcholine (Ach) neurotransmitter content (Charles Rivers Laboratories).

[0217] To confirm functional specificity, the GABA release assay was performed using hESCs and spinal motor neurons (SMNs, BrainXcell), neither of which normally secrete GABA. hESCs were seeded at a density of 25E3 cells / cm2in E8 media containing ROCK inhibitor seventy-two hours prior to sample collection. One day post-seeding, the media was changed to the same neuronal culture media as hESC-derived interneurons, and the cells were fed daily until sample collection. SMNs were thawed and seeded into a polyornithine / fibronectin / laminin coated 96-well plate at a density of 0.65E6 cells / cm2and cultured for 8 days as described by the manufacturer. The sample collection was performed the same way for ESCs and SMNs as for the hESC-derived interneurons on day 8. In vitro cell migration

[0218] To assess the migration potential of hESC-derived pallial MGE-type interneurons compared to human fetal MGE-derived interneurons and other in vitro-derived GABAergic cell preparations, a migration assay was developed, in which cells were aggregated and embedded into a MatrigelTM (CB-40230, Fisher Scientific) drop coveredwith culture medium, and then incubated for three days to allow for the visualization and quantification of cells migrating away from the main cell aggregate. Primary human MGE was dissected from mid-gestational age tissue (GW18-20) followed by dissociation and sorting for ERBB4. All hPSC-derived interneurons were thawed prior to aggregation. The commercial sources of hPSC-derived GABAergic cells were from Cellular Dynamics International (vendor “X”: iCell GABA Neurons, cat #C1008) and BrainXell (vendor “Y”: GABAergic Neurons, cat #BX-0400). Cells from different sources were prepared by one operator, assigned a random ID and handed to a different operator de-identified for assays and data analyses.

[0219] To perform the assay, 5E3 cells in 100 μl of culture medium were spun down in 96-well V bottom plates (S-bio, MS-9096VZ) and allowed to aggregate at 37°C, 5% CO2 over a period of three days. The aggregates were then embedded into a MatrigelTMdrop in the center of a 96-well flat bottom assay plate with the aid of a dissecting microscope inside the biosafety cabinet (BSC) (one aggregate per well, twelve aggregates per sample). After a 15-minute incubation at ambient temperature, 200 μl of culture medium was added to the wells, followed by incubation at 37°C, 5% CO2 for three days.

[0220] On day 3 of migration, samples were fixed for 30 minutes with a 2% PFA and 1X Hoechst solution at ambient temperature, protected from light. The fixation solution was washed away three times using DBPS. Migration images were acquired using the CX5 instrument with a 4X objective, projecting the maximum intensity across 375 µm (15 z-plans, 25 µm steps). The number of Hoechst positive cells that migrated away from the aggregate was quantified automatically using “spot detector” bio application. Percentage of migrating cells was determined based on the number of Hoechst positive spots detected divided by 5E3 (input cells that were aggregated). Single cell RNA sequencing

[0221] To objectively assess cell composition, scRNA-seq analysis was performed using the 10X Genomics platform on the hESCs prior to the start of differentiation, hESC-derived MGE-type VZ-like progenitors on day 14 of differentiation, and six pairs of unsorted / sorted cell lots at the end of the differentiation process. All samples were thawed the day of capture and transported on ice to SeqMatic (Fremont, CA) for downstream processing and data generation.

[0222] Individual cells were captured and barcoded using the chromium controller, cDNA libraries were prepared using next GEM 3’ V3.1 kits (10x Genomics), cDNA libraries were quantified using the Agilent Tapestation 4200 and pooled for sequencing. Pooled libraries were quantified using Lightcycler 96 qPCR and sequenced by the Illumina NovaSeq 6000. Typically, ~8E3-12E3 cells were captured per sample. About 200 million reads were obtained for each sample, with an average read depth of ~20,000 reads per cell.

[0223] Cell Ranger v3.1.0 (10x Genomics) was used to demultiplex FASTQ files for each sample, align reads to the human GRCh38 genome downloaded from 10x Genomics, and quantify the expression levels for each gene in each cell from each sample. R version 4.0.3 and Seurat v3.2.2 were used to quality control, normalize, cluster, and visualize the cells from all samples. Cells were filtered for quality by having at least 1000 expressed genes, at least 1000 unique reads, no more than 20% reads of mitochondrial genes and no more than 40% reads of ribosomal genes. Cells were normalized by the ‘LogNormalize’ method. The top 3000 genes were identified by ‘FindVariableFeatures’ with ‘vst’ selection method for downstream integration analysis. Seurat CCA (Canonical Correlation Analysis) integration workflow was applied to remove the batch effects between different cell lots. hESCs and Day 14 progenitors were merged with the integrated differentiated lots for clustering. PCA (Principal component analysis) was applied, 30 PCs (principal components) were selected, and cells were clustered with Seurat standard workflow. Using a dimension reduction technique called UMAP (Uniform Manifold Approximation and Projection), cells are visualized in a 2-dimensional space and colored by groups, where each dot represents one cell, and groups of cells with similar gene expression form into clusters. Top markers for each cluster were identified by using the Seurat ‘FindAllMarkers’ function with ROC method and the cell type of each cluster were determined by the expression of typical cell markers of each type. Gene expression was visualized with violin-plot, dot-plot and feature-plot built in Seurat package.

[0224] The sequencing data discussed herein have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE208672.

[0225] To compare and classify in vitro hESC-derived interneurons with published data, human developing GE snRNAseq data were downloaded from GEO dbs (Shi et al., 2021), and processed to identify MGE, CGE, LGE and other clusters as described in the original report. PCA and UMAP models of cell types were computed and the human developing GE reference was constructed by following the Seurat reference mapping procedure. The cell type classification of hESC-derived interneurons was assigned based on prediction scores using the human GE cells as a reference. Each in vitro cell was assigned a score between 0 and 1 for each cell type based on the transcriptomic similarity between the query and the reference (human developing GE cells). The prediction scores of each cell for each cell type category were projected onto UMAP clusters and the predicted-cell-type compositions (percentage) for each cluster were visualized with the heatmap. B. In vivo Animals

[0226] Up to 85 NOG (NOD.Cg-PrkdcscidIl2rgtm1Sug / JicTac) male mice per study were obtained from Taconic in the range of approximately 5-6 weeks of age for comparisons of different cell lots or doses and vehicle groups. Male mice were chosen because they display more consistent seizures in this model. Mice were assigned a unique identifier number on arrival, which was marked on the mouse by ear-notches and tail tattoos. Animals were maintained in an animal facility under standard temperature and humidity on a 12:12 light / dark cycle. Bottled water and standard chow were provided ad libitum. Single-use cages were used with irradiated Alpha-Dri bedding. Prior to induction, animals were co-housed when possible. Colony health was monitored through monthly diagnostic sample submission for microbiology. All described surgical procedures were accompanied by s.c. meloxicam (Metacam® 10 mg / kg in 2ml / kg i.p.) as a non-steroidal anti-inflammatory drug, antibiotic enrofloxacin (Baytril® 10 mg / kg in 4.4 ml / kg s.c.), and local anesthesia on the scalp with bupivacaine (2.5 mg / kg in 5 ml / kg s.c.). After every surgery, animals received 1 ml warmed Ringer’s solution s.c. to rehydrate, and their cages were placed on a heating pad until full recovery from anesthesia.Epilepsy induction

[0227] After acclimation of 2 weeks, animals received 0.21 µg kainic acid (KA; Tocris) dissolved in 50 nL NaCl into the right dorsal hippocampus to induce epilepsy (coordinates in relation to Bregma: AP -2.0; ML +1.6; DV -1.65), using a pulled glass needle with 70-90 µm diameter at the tip and a connected microinjection system (Narishige). At this first surgery, the body weight of the animals was 23-24 g on average. After injection of KA, mice were kept under isoflurane anesthesia for a total of 1 hour to avoid the more severe seizures associated with recovery from inhaled anesthesia. After recovery from anesthesia, animals were closely monitored for occurrence of generalized seizures. All mice began seizing within 90 min post-anesthesia and were then returned to the animal housing room in single-housed cages. Status epilepticus (SE) was not interrupted in this model. Mice were injected with 1 mL lactated Ringer’s solution to ensure rehydration during / after SE. Additionally, food pellets and water as a gel were provided at the bottom of the cage. If necessary, mice were placed on heating pads and manually provided with Ensure® liquid diet supplement on the day after SE. If failure to thrive was observed, the mouse was euthanized. Mortality during and within the first days after SE was <10%. Cell transplantation for focal KA model

[0228] Transplantation of hESC-derived MGE-type pallial interneurons or vehicle (serum-free base medium) was performed four to five weeks after SE induction to ensure that the mice displayed spontaneous seizures and were chronically epileptic prior to treatment. Cells were transplanted using the same system as for the KA injections, a pulled glass needle with 70-90 um diameter at the tip and a connected microinjection system (Narishige). Cells were injected in 4-6 different deposits per brain hemisphere in a dose of 5E3 (5K) – 6E5 (600K) cells per deposit depending on the target dose from 2.5E4 (25K) to 1.5E6 (1.5M) per hemisphere. After delivery of the hESC-derived interneurons, the needle remained in the tissue for another 30 seconds to prevent backflow of cell suspension along the needle tract. Control mice received similar amounts of vehicle solution into the same deposit coordinates. Electrode implantation and EEG recording

[0229] For recording an electroencephalogram (EEG), transmitters from Data Science International (DSI) were implanted and connected in-house to a custom-madebipolar electrode (PlasticsOne). The DSI transmitter was implanted subcutaneously over the left flank of the mouse and the electrode was lowered into the right dorsal CA1 of the hippocampus into the same site as the previous KA injection (coordinates in relation to Bregma: AP -2.0; ML +1.6; DV -1.65). The electrode was secured in the skull by implanting three holding screws (2 rostral, 1 caudal) and Loctite glue. Implantation happened in the same surgery as cell / vehicle transplantation or within a few weeks of transplantation surgery. After DSI transmitter implantation, EEG and video recordings (Ponemah; DSI) were acquired in multi-day 24-hour sessions for each animal enrolled in the study; the data were saved to a server approximately daily and archived at least monthly on backup storage hard drives. EEG was recorded 24 / 7 for 1-3 weeks at various time points post-SE (Fig. 4A) using Ponemah software. Recording time points for most studies were between 1-2 MPT, 5, and 7 MPT. Some studies (lot 3S & 4S) were recorded also at earlier and later time points (Figs. 14D, 14E). Brain slice electrophysiology

[0230] For electrophysiology studies, cells were seeded onto PO / Fib / Lam coated multiwell plates in culture media. Following overnight incubation at 37°C, 5% CO2, culture medium was replaced with fresh medium containing lentivirus supernatants (Lenti-UbC-GFP or Lenti-Syn-ChR2-YFP) based on the optimal titration that was determined empirically in a separate experiment to achieve 95% GFP+ or YFP+ expression. After a three-day incubation, culture medium was removed followed by three washes with PBS to remove any residual viral particles. Cells were harvested using 0.25% trypsin solution, quenched with FBS (20% final).

[0231] Upon harvesting, cells were concentrated by centrifugation (150g, 4 minutes at room temperature). Concentrated cell suspension was loaded into beveled glass micropipettes (internal diameter ≈80-90 μm, Wiretrol 5 μl, Drummond Scientific Company) prefilled with mineral oil and mounted on a microinjector. Concentration of the cell suspension within the needle was determined using a hemocytometer just prior to the injection. Postnatal day (P)0-2 Scid-beige (CRL) mice were anesthetized by hypothermia for 1-2 minutes and subsequently positioned in a clay mold to stabilize the head. Recipient mice were injected 3 times bilaterally with 50E3 cells / injection site (total = 300E3 cells per pup). Injections were performed 0.75, 1.25, 2.5 mm anterior from Lambda, 0.75, 1.0, 1.0 mm awayfrom the midline (respectively) and at a depth of 0.35 mm from the surface of the skin. After the injections were completed, the needle was slowly removed and transplant recipients were placed on a warm surface to recover from hypothermia. The mice were then returned to their mothers until they were weaned (P21).

[0232] Adult (6-8 weeks old) NOG male mice were transplanted as described above. A total dose of 200E3 cells / hippocampus was delivered into the mouse hippocampus at 4 different sites (50E3 cells / injection) with respect to bregma (AP: -1.85; ML: +1.8; DV: - 1.7 mm; AP: -2.5; ML: +2.65; DV: -1.9 mm; AP: -3.15; ML: +2.8; DV: -2.4 mm and AP: - 3.15; ML: +3.25; DV: -3.45 mm). Mice up to one year of age were deeply anesthetized with intraperitoneal injection of freshly prepared Avertin (250 mg / Kg) and perfused with ice-cold sucrose-based solution (210 mM Sucrose, 2.5 mM KCl, 10 mM MgSO4, 2 mM CaCl2, 1.25 mM NaH2PO4, 24 mM NaHCO3, 11 mM glucose). The brain was removed and sectioned into 300 μm thick slices, which were transferred to 34℃ ACSF (126 mM NaCl, 3 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 1.2 mM NaH2PO4, 26 mM NaHCO3, 10 mM glucose) for 30 minutes at room temperature for no more than 6 hours for later use.

[0233] For older animals, a previously published “protective recovery method” was adapted. Mice were deeply anesthetized and transcardially perfused with ice-cold “NMDG-HEPES aCSF” (92 mM NMDG, 92 mM HCl, 2.5 mM KCl, 1.2 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 5 mM sodium ascorbate, 2 mM thiourea, 3 mM sodium pyruvate, 10 mM MgSO4, 0.5 mM CaCl2). The brain was dissected and sectioned into 300 μm coronal slices. Slices were transferred to 34℃ “NMDG-HEPES aCSF” for 40 minutes to recover. During the recovery phase, Na+ spike-in solution (2 M NaCl in NMDG-HEPES aCSF) was added at the following time points: 15 min: 0.25 mL; 20 min: 0.25 mL; 25 min: 0.5 mL; 30 min: 1 mL; 35 min: 2 mL. Slices were then stored in “HEPES holding aCSF” (92 mM NaCl, 2.5 mM KCl, 1.2 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 5 mM sodium ascorbate, 2 mM thiourea, 3 mM sodium pyruvate, 2 mM MgSO4, 2 mM CaCl2) at room temperature for no more than 6 hours for later measurements in “recording aCSF” (124 mM NaCl, 2.5 mM KCl, 1.2 mM NaH2PO4, 24 mM NaHCO3, 5 mM HEPES, 12.5 mM glucose, 2 mM MgSO4, 2 mM CaCl2). All solutions were balanced for 30 minutes with carbogen (95% O2, 5% CO2) before use.

[0234] Borosilicate glass pipettes (4-8 Mohm) were backfilled with a solution containing: 140 mM K-gluconate, 2 mM MgCl2, 10 mM HEPES, 0.2 mM EGTA, 4 mM MgATP, 0.3 mM NaGTP, 10 mM Phosphocreatine d-tris, and 0.25% (g / 100mL) biocytin. Signals were collected at room temperature at a sampling rate of 50K, filtered using a 10K bessel filter (MultiClamp 700B, Axon Instruments, Molecular Devices) and digitized (Digidata 1320A, Axon Instruments, Molecular Devices). Recording data were further filtered using Clampfit (v10.7), “Lowpass” function with Gaussian type and 2000 Hz -3 dB cutoff. Stimfit (0.15.8, github(dot)com / neurodroid / stimfit) and python were used to calculate and plot the parameters. Behavior

[0235] For all assays, experimenters were masked to animal treatment group, and test order was based on the experimenter randomly selecting a cage.

[0236] Modified Irwin’s Test: ...

Claims

WHAT IS CLAIMED IS:

1. A method of treating seizure activity, comprising: identifying a subject in need of treating seizure activity; and administering to the subject a therapeutically effective amount of a cellular composition comprising pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells, wherein at least 50% of the cells of the composition express LHX6, ERBB4, and GAD1, and wherein a frequency of seizures is reduced after the administering, thereby treating the seizure activity.

2. The method of claim 1, wherein the subject has a focal epilepsy.

3. The method of claim 1, wherein the subject has a chronic seizure activity.

4. The method of claim 1, wherein the subject has mesial temporal sclerosis.

5. The method of claim 4, wherein the mesial temporal sclerosis is reduced after the administering 6. The method of claim 1, wherein the subject has an epileptogenic lesion in the brain.

7. The method of claim 6, wherein the epileptogenic lesion is reduced after the administering.

8. The method of claim 1, wherein at most 50% of the cells of the composition express NKX2-1.

9. The method of claim 8, wherein at most 40% of the cells of the composition express LHX8.

10. The method of claim 1, wherein the at least 50% of the cells of the composition further express at least one of MAF and MAFB.

11. The method of claim 1, wherein the therapeutically effective amount comprises about 5 x 104cells or more.

12. The method of claim 11, wherein the therapeutically effective amount comprises between about 5 x 104to about 1 x 1012cells.

13. The method of claim 1, wherein the therapeutically effective amount of the cellular composition is administered at a concentration of about 1 x 105cells / μL or greater.

14. The method of claim 1, wherein the cellular composition comprises sodium chloride at 100-600 mM.

15. The method of claim 14, wherein the cellular composition has an osmolality of at least 200 mOsm / kg.

16. The method of claim 1, wherein at most 40% of the cells express an MGE progenitor cell marker or a non-pallial MGE-type neural cell marker.

17. The method of claim 16, wherein at most 10% of the cells of the composition express KI67.

18. The method of claim 16, wherein at most 10% of the cells of the composition express OLIG2.

19. The method of claim 1, wherein cells of the composition are GABA-secreting cells.

20. The method of claim 1, wherein cells of the composition are migratory cells.

21. The method of claim 1, wherein at least 50% of the cells of the composition are pallial MGE-type GABAergic interneuron cells.

22. The method of claim 1, wherein the subject has temporal lobe epilepsy (TLE).

23. The method of claim 22, wherein the TLE is drug-resistant TLE.

24. The method of claim 1, wherein the subject suffers from a neocortical onset focal epilepsy.

25. The method of claim 1, wherein the subject suffers from focal seizure activity.

26. The method of claim 1, comprising administering the therapeutically effective amount of the cellular composition to the subject’s brain.

27. The method of claim 1, further comprising performing one or more of computed tomography (CT) scan, magnetic resonance imaging (MRI), MR spectroscopy (MRS), functional MRI (fMRI), electroencephalography (EEG), intracranial EEG, positron emission tomography (PET), and single photon emission computed tomography (SPECT) on the subject.

28. The method of claim 27, further comprising monitoring brain activity in the subject.

29. The method of claim 1, further comprising identifying a mesial temporal sclerosis in the subject, or one or more lesions in the brain.

30. The method of claim 29, wherein identifying the mesial temporal sclerosis in the subject or the one or more lesions comprises performing one or more of a CT scan, MRI, MRS, EEG, PET, SPECT, or fMRI.

31. The method of claim , wherein the frequency of seizures is reduced by about 50% or more after the administering.

32. The method of claim 1, further comprising performing one or more neurocognitive assessments of the subject.

33. The method of claim 1, wherein the cellular composition is a thawed cellular composition.

34. The method of claim 33, comprising holding the thawed cellular composition for up to about 5 days before administering to the subject.

35. The method of claim 34, comprising holding the thawed cellular composition at an ambient temperature of 2-18°C before administering to the subject.

36. The method of claim 33, further comprising thawing a cryopreserved cellular composition comprising the pluripotent stem cell-derived, pallial, MGE-type, GABAergic interneuron cells before administering.

37. The method of claim 1, wherein the administering comprises administering the cellular composition as one or more deposits in the subject’s brain.

38. The method of claim 37, wherein the therapeutically effective amount of the cellular composition is delivered in a volume of about 50 μL or less per deposit.

39. The method of claim 37, wherein the administering comprises administering the cellular composition to one or more sites in the temporal lobe, optionally wherein the one or more sites in the temporal lobe include the hippocampus, cortex, and / or amygdala, optionally wherein the one or more sites in the temporal lobe include the hippocampus, subiculum, entorhinal cortex, and / or parahippocampal gyrus.

40. The method of claim 1, wherein the cells are human cells.

41. The method of claim 1, wherein the subject is a human subject.

42. The method of claim 1, further comprising administering an immunosuppressant to the subject before and / or after administering the therapeutically effective amount of the cellular composition.

43. The method of claim 1, wherein the seizure activity is due to a traumatic brain injury, stroke, tumor, focal cortical dysplasia, tuberous sclerosis, developmental disorder, or a neurological disorder.

44. The method of claim 1, wherein the subject suffers from epileptic seizure-like discharges in the brain associated with Alzheimer’s disease, focal cortical dysplasia (FCD), or amnesic mild cognitive impairment (aMCI).

45. The method of claim 1, further comprising monitoring a therapeutic effect of administering the cellular composition in the subject.

46. The method of claim 45, comprising non-invasively monitoring the therapeutic effect using one or more biomarkers of seizure activity.

47. The method of claim 46, wherein the one or more biomarkers comprises N- acetylaspartate (NAA), myoinositol, glutamate, GABA, volume, edema, blood flow, oxygen metabolism, and glucose.

48. The method of claim 45, comprising using EEG, imaging, and / or one or more blood-based biomarkers to monitor the therapeutic effect.

49. A method of treating seizure activity, comprising: identifying a subject in need of treating seizure activity; and intracranially administering to the subject a cellular composition comprising: about 5 x 104to about 1 x 1012pluripotent stem cell-derived, pallial, MGE- type GABAergic interneuron cells at a concentration in a range of about 1 x 105to about 4 x 106cells / μL, wherein at least 50% of the cells of the composition express LHX6, ERBB4, and GAD1, wherein 90% or more of the cells of the composition are post-mitotic cells; a poloxamer at 0.1-1%, v / v; and sodium chloride at 100-600 mM.

50. Use of a cellular composition for the treatment of seizure activity in a subject, the composition comprising pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells, wherein at least 50% of the cells of the composition express LHX6, ERBB4, and at least one of MAF and MAFB, optionally wherein at least 90% of the cells are post-mitotic.

51. A cellular composition comprising pluripotent stem cell-derived, pallial MGE-type GABAergic interneuron cells, for the preparation of a medicament for treatment of seizure activity in a subject, wherein at least 50% of the cells of the composition express LHX6, ERBB4, and GAD1.

52. A therapeutic composition of pluripotent stem cell-derived pallial MGE-type GABAergic interneuron cells for the treatment of focal onset seizure, wherein at least 50% of the cells of the composition express LHX6, ERBB4, and GAD1.

53. A therapeutic composition comprising: a poloxamer; and pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells at a concentration of about 1 x 105cells / μL or greater.

54. A method of preparing a therapeutic composition for administering to a subject, comprising: providing pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells; and preparing a cellular composition comprising: a poloxamer; and the pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells at a concentration of about 1 x 105cells / μL or greater.

55. A delivery system for transplanting cells into a tissue, comprising: a delivery cannula comprising: a proximal portion comprising a cellular liquid composition comprising cells at a concentration of about 1 x 105cells / μL or greater; and a distal portion comprising a cell-free liquid chase vehicle, wherein the cellular liquid composition is stably held in the proximal portion by the liquid chase vehicle; and a displacement device connected to a distal end of the cannula and configured to cause the liquid chase vehicle to displace the cellular liquid composition to thereby expel the cellular liquid composition from a proximal end of the cannula.

56. A delivery system for transplanting cells into a tissue, comprising:a delivery cannula comprising a cellular liquid composition comprising cells at a concentration of about 1 x 105cells / μL or greater; and a displacement device connected to a distal end of the cannula and configured to displace the cellular liquid composition in the cannula to thereby expel the cellular liquid composition from a proximal end of the cannula.

57. The delivery system of claim 56, wherein the displacement device comprises a syringe, or a stylet traversing the inner lumen of the delivery cannula.

58. The delivery system of claim 57, further comprising a pump connected to the displacement device.

59. The delivery system of claim 58, wherein the cellular liquid composition comprises a contrast agent.

60. The delivery system of claim 59, wherein the contrast agent comprises an MRI contrast agent.

61. The delivery system of claim 60, wherein the contrast agent comprises a gadolinium-based contrast agent.

62. The delivery system of claim 59, further comprising a monitoring device configured to detect the position of the proximal end of the cannula at a site of delivery in a subject.

63. The delivery system of claim 62, wherein the monitoring device comprises a CT scanner, MRI scanner, PET scanner, or a SPECT scanner.

64. The delivery system of claim 56, wherein the cells are neuron cells.

65. The delivery system of claim 64, wherein the cells comprise pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells.

66. A method of treating seizure activity, comprising: identifying a subject in need of treating seizure activity; and administering to the subject a therapeutically effective amount of a cellular liquid composition comprising pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells using the system of any one of claims 55-65, wherein the cellular liquid composition comprises the pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells.

67. The method of claim 66, comprising detecting the position of the delivery cannula in the subject’s brain during the administering.

68. The method of claim 67, wherein the detecting comprises using MRI to visualize the delivery cannula in the subject’s brain.

69. The method, therapeutic composition, or delivery system of any one of the preceding claims, wherein the cellular composition or the cellular liquid composition comprises NRTX-1001.

70. The method, therapeutic composition, or delivery system of any one of the preceding claims, wherein at least 50% of the cells of the composition express LHX6, ERBB4, and GAD1, and wherein at least 90% of the cells of the composition are post-mitotic cells.

71. The method, therapeutic composition, or delivery system of any one of the preceding claims, wherein at least 50% of the cells persisting in the subject retain a pallial, MGE-type GABAergic interneuron cell fate about 6 months or more after administration.

72. The method, therapeutic composition, or delivery system of claim 71, wherein the cells persisting in the subject and that retain the pallial, MGE-type GABAergic interneuron cell fate express one or more of GAD1, GAD2, LHX6, SOX6, NXPH1, ERBB4, SST, NPY, MEF2c, and ARX.

73. The method of claim 1, wherein the pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells are genetically modified.

74. The method of claim 73, wherein the pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells are genetically modified to reduce an immunological response against the administered cellular composition by the subject, optionally wherein the pluripotent stem cell-derived, pallial, MGE-type GABAergic interneuron cells comprises: a mutation in one or more immune-activating genes that reduces or abolishes expression or activity thereof; and / or a genetic modification that activates expression or activity of one or more immune-inhibiting genes.

75. The method of claim 74, wherein the pluripotent stem cell-derived, pallial, MGE- type GABAergic interneuron cells are genetically modified to reduce or abolish expression of one or more of: B2M, a HLA class I gene, and / or a HLA class II gene.

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