Amanda J. Kedaigle

Vestibular hair cells are mechanoreceptors critical for detecting head position and motion. In mammals, hair cell loss causes vestibular dysfunction as spontaneous regeneration is nearly absent. Constitutive expression of exogenous ATOH1, a hair cell transcription factor, increases hair cell regeneration, however, these cells fail to fully mature. Here, we profiled mouse utricles at 14 time points, and defined transcriptomes of developing and mature vestibular hair cells. To mimic native hair cells which downregulate endogenous ATOH1 as they mature, we engineered viral vectors carrying the supporting cell promoters GFAP and RLBP1. In utricles damaged ex vivo, both CMV-ATOH1 and GFAP-ATOH1 increased regeneration more effectively than RLBP1-ATOH1, while GFAP-ATOH1 and RLBP1-ATOH1 induced hair cells with more mature transcriptomes. In utricles damaged in vivo, GFAP-ATOH1 induced regeneration of hair cells expressing genes indicative of maturing type II hair cells, and more hair cells with bundles and synapses than untreated organs. Together our results demonstrate the efficacy of spatiotemporal control of ATOH1 overexpression in inner ear hair cell regeneration.

Vestibular hair cells are mechanoreceptors critical for detecting head position and motion. In mammals, hair cell loss causes vestibular dysfunction as spontaneous regeneration is nearly absent. Constitutive expression of exogenous ATOH1, a hair cell transcription factor, increases regeneration of hair cells, but these cells fail to mature. To mimic native hair cells which downregulate endogenous ATOH1 as they mature, we engineered viral vectors carrying the supporting cell promoters GFAP and RLBP1. In utricles damaged ex vivo, both CMV-ATOH1 and GFAP-ATOH1 increased regeneration more effectively than RLBP1-ATOH1, while GFAP-ATOH1 and RLBP1-ATOH1 induced hair cells exhibiting more mature transcriptomes. In utricles damaged in vivo, GFAP-ATOH1 induced regeneration of hair cells expressing genes representing maturing type II hair cells, and more hair cells with bundles and synapses than untreated organs. Together our results demonstrate the efficacy of spatiotemporal control of ATOH1 overexpression in inner ear regeneration.

De novo heterozygous loss-of-function mutations in PTEN are strongly associated with Autism spectrum disorders (ASD); however, it is unclear how heterozygous mutations in this gene affects different cell types during human brain development, and how these effects vary across individuals. Here, we used human cortical organoids from different donors to identify cell-type-specific developmental events that are affected by heterozygous mutations in PTEN. We profiled individual organoids by single-cell RNA-seq, proteomics, and spatial transcriptomics, and revealed abnormalities in developmental timing in human outer radial glia progenitors and deep layer cortical projection neurons, which varied with the donor genetic background. Calcium imaging in intact organoids showed that both accelerated and delayed neuronal development phenotypes resulted in similar abnormal activity of local circuits, irrespective of genetic background. The work reveals donor-dependent, cell-type specific developmental phenotypes of PTEN heterozygosity that later converge on disrupted neuronal activity.

De novo heterozygous loss-of-function mutations in PTEN are strongly associated with Autism spectrum disorders (ASD); however, it is unclear how heterozygous mutations in this gene affects different cell types during human brain development, and how these effects vary across individuals. Here, we used human cortical organoids from different donors to identify cell-type-specific developmental events that are affected by heterozygous mutations in PTEN . We profiled individual organoids by single-cell RNA-seq, proteomics and spatial transcriptomics, and revealed abnormalities in developmental timing in human outer radial glia progenitors and deep layer cortical projection neurons, which varied with the donor genetic background. Calcium imaging in intact organoids showed that both accelerated and delayed neuronal development phenotypes resulted in similar abnormal activity of local circuits, irrespective of genetic background. The work reveals donor-dependent, cell-type specific developmental phenotypes of PTEN heterozygosity that later converge on disrupted neuronal activity.

Realizing the full utility of brain organoids as experimental systems to study human cortical development requires understanding whether organoids replicate the cellular and molecular events of this complex process precisely, reproducibly, and with fidelity to the embryo. Here we present a comprehensive single-cell transcriptomic, epigenetic, and spatial atlas of human cortical organoid development, comprising over 610,000 cells, spanning initial generation of neural progenitors through production of differentiated neuronal and glial subtypes. We define the lineage relationships and longitudinal molecular trajectories of cortical cell types during development in organoids, and show that developmental processes of cellular diversification in organoids correlate closely to endogenous ones, irrespective of metabolic state. Using this data, we identify genes with predicted human-specific roles in lineage establishment, and discover a developmental origin for the transcriptional diversity of human callosal projection neurons, a population that has undergone dramatic expansion and diversification during human evolution. Our work provides a comprehensive, single-cell molecular map of human corticogenesis in vitro , identifying developmental trajectories and molecular mechanisms associated with human cellular diversification.

Genetic risk for autism spectrum disorder (ASD) is associated with hundreds of genes spanning a wide range of biological functions^1,2,3,4,5,6. The alterations in the human brain resulting from mutations in these genes remain unclear. Furthermore, their phenotypic manifestation varies across individuals^7,8. Here we used organoid models of the human cerebral cortex to identify cell-type-specific developmental abnormalities that result from haploinsufficiency in three ASD risk genes—SUV420H1 (also known as KMT5B), ARID1B and CHD8—in multiple cell lines from different donors, using single-cell RNA-sequencing (scRNA-seq) analysis of more than 745,000 cells and proteomic analysis of individual organoids, to identify phenotypic convergence. Each of the three mutations confers asynchronous development of two main cortical neuronal lineages—γ-aminobutyric-acid-releasing (GABAergic) neurons and deep-layer excitatory projection neurons—but acts through largely distinct molecular pathways. Although these phenotypes are consistent across cell lines, their expressivity is influenced by the individual genomic context, in a manner that is dependent on both the risk gene and the developmental defect. Calcium imaging in intact organoids shows that these early-stage developmental changes are followed by abnormal circuit activity. This research uncovers cell-type-specific neurodevelopmental abnormalities that are shared across ASD risk genes and are finely modulated by human genomic context, finding convergence in the neurobiological basis of how different risk genes contribute to ASD pathology.

We have previously established induced pluripotent stem cell (iPSC) models of Huntington’s disease (HD), demonstrating CAG-repeat-expansion-dependent cell biological changes and toxicity. However, the current differentiation protocols are cumbersome and time consuming, making preparation of large quantities of cells for biochemical or screening assays difficult. Here, we report the generation of immortalized striatal precursor neurons (ISPNs) with normal (33) and expanded (180) CAG repeats from HD iPSCs, differentiated to a phenotype resembling medium spiny neurons (MSN), as a proof of principle for a more tractable patient-derived cell model. For immortalization, we used co-expression of the enzymatic component of telomerase hTERT and conditional expression of c-Myc. ISPNs can be propagated as stable adherent cell lines, and rapidly differentiated into highly homogeneous MSN-like cultures within 2 weeks, as demonstrated by immunocytochemical criteria. Differentiated ISPNs recapitulate major HD-related phenotypes of the parental iPSC model, including brain-derived neurotrophic factor (BDNF)-withdrawal-induced cell death that can be rescued by small molecules previously validated in the parental iPSC model. Proteome and RNA-seq analyses demonstrate separation of HD versus control samples by principal component analysis. We identified several networks, pathways, and upstream regulators, also found altered in HD iPSCs, other HD models, and HD patient samples. HD ISPN lines may be useful for studying HD-related cellular pathogenesis, and for use as a platform for HD target identification and screening experimental therapeutics. The described approach for generation of ISPNs from differentiated patient-derived iPSCs could be applied to a larger allelic series of HD cell lines, and to comparable modeling of other genetic disorders.

Tissue-resident innate lymphoid cells (ILCs) help sustain barrier function and respond to local signals. ILCs are traditionally classified as ILC1, ILC2 or ILC3 on the basis of their expression of specific transcription factors and cytokines¹. In the skin, disease-specific production of ILC3-associated cytokines interleukin (IL)-17 and IL-22 in response to IL-23 signalling contributes to dermal inflammation in psoriasis. However, it is not known whether this response is initiated by pre-committed ILCs or by cell-state transitions. Here we show that the induction of psoriasis in mice by IL-23 or imiquimod reconfigures a spectrum of skin ILCs, which converge on a pathogenic ILC3-like state. Tissue-resident ILCs were necessary and sufficient, in the absence of circulatory ILCs, to drive pathology. Single-cell RNA-sequencing (scRNA-seq) profiles of skin ILCs along a time course of psoriatic inflammation formed a dense transcriptional continuum—even at steady state—reflecting fluid ILC states, including a naive or quiescent-like state and an ILC2 effector state. Upon disease induction, the continuum shifted rapidly to span a mixed, ILC3-like subset also expressing cytokines characteristic of ILC2s, which we inferred as arising through multiple trajectories. We confirmed the transition potential of quiescent-like and ILC2 states using in vitro experiments, single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) and in vivo fate mapping. Our results highlight the range and flexibility of skin ILC responses, suggesting that immune activities primed in healthy tissues dynamically adapt to provocations and, left unchecked, drive pathological remodelling.

Genetic risk for autism spectrum disorder (ASD) has been associated with hundreds of genes spanning a wide range of biological functions. The phenotypic alterations in the human brain resulting from mutations in ASD risk genes remain unclear, and the level at which these alterations converge on shared disease pathology is poorly understood. Here, we leveraged reproducible organoid models of the human cerebral cortex to identify cell type-specific developmental abnormalities associated with haploinsufficiency in three ASD risk genes, SUV420H1 ( KMT5B ), PTEN , and CHD8 . We performed comprehensive single-cell RNA-sequencing (scRNA-seq) of over 400,000 cells, and proteomic analysis on individual organoids sampled at different developmental stages to investigate phenotypic convergence among these genes. We find that within a defined period of early cortical development, each of the three mutations demonstrates accelerated development of cortical neurons. Notably, they do so by affecting different neuronal populations: excitatory deep layer ( SUV420H1 ) and callosal ( PTEN ) neurons, and inhibitory interneurons ( CHD8 ). This work shows that haploinsufficiency in ASD risk genes converge on early developmental defects in the generation of neurons of the cortical microcircuit.