RESUMEN
Human organoids recapitulating the cell-type diversity and function of their target organ are valuable for basic and translational research. We developed light-sensitive human retinal organoids with multiple nuclear and synaptic layers and functional synapses. We sequenced the RNA of 285,441 single cells from these organoids at seven developmental time points and from the periphery, fovea, pigment epithelium and choroid of light-responsive adult human retinas, and performed histochemistry. Cell types in organoids matured in vitro to a stable "developed" state at a rate similar to human retina development in vivo. Transcriptomes of organoid cell types converged toward the transcriptomes of adult peripheral retinal cell types. Expression of disease-associated genes was cell-type-specific in adult retina, and cell-type specificity was retained in organoids. We implicate unexpected cell types in diseases such as macular degeneration. This resource identifies cellular targets for studying disease mechanisms in organoids and for targeted repair in human retinas.
Asunto(s)
Diferenciación Celular/genética , Organoides/citología , Organoides/metabolismo , Retina/citología , Retina/metabolismo , Análisis de la Célula Individual/métodos , Sinapsis/fisiología , Transcriptoma/genética , Técnicas de Cultivo de Célula/métodos , Línea Celular , Electrofisiología , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Predisposición Genética a la Enfermedad/genética , Humanos , Hibridación in Situ , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Microscopía Electrónica , Familia de Multigenes , Naftoquinonas , Organoides/efectos de la radiación , Organoides/ultraestructura , Retina/patología , Retina/efectos de la radiaciónRESUMEN
Human organoids allow the study of proliferation, lineage specification, and 3D tissue development. Here we present a genome-wide CRISPR screen in induced pluripotent stem cell (iPSC)-derived kidney organoids. The combination of inducible genome editing, longitudinal sampling, and endpoint sorting of tubular and stromal cells generated a complex, high-quality dataset uncovering a broad spectrum of insightful biology from early development to "adult" epithelial morphogenesis. Our functional dataset allows improving mesoderm induction by ROCK inhibition, contains monogenetic and complex trait kidney disease genes, confirms two additional congenital anomalies of the kidney and urinary tract (CAKUT) genes (CCDC170 and MYH7B), and provides a large candidate list of ciliopathy-related genes. Finally, identification of a cis-inhibitory effect of Jagged1 controlling epithelial proliferation shows how mosaic knockouts in pooled CRISPR screening can reveal ways of communication between heterogeneous cell populations in complex tissues. These data serve as a rich resource for the kidney research community and as a benchmark for future iPSC-derived organoid CRISPR screens.
Asunto(s)
Células Madre Pluripotentes Inducidas , Organoides , Edición Génica , Humanos , Riñón , OrganogénesisRESUMEN
TREM2 is a transmembrane protein expressed exclusively in microglia in the brain that regulates inflammatory responses to pathological conditions. Proteolytic cleavage of membrane TREM2 affects microglial function and is associated with Alzheimer's disease, but the consequence of reduced TREM2 proteolytic cleavage has not been determined. Here, we generate a transgenic mouse model of reduced Trem2 shedding (Trem2-Ile-Pro-Asp [IPD]) through amino-acid substitution of an ADAM-protease recognition site. We show that Trem2-IPD mice display increased Trem2 cell-surface-receptor load, survival, and function in myeloid cells. Using single-cell transcriptomic profiling of mouse cortex, we show that sustained Trem2 stabilization induces a shift of fate in microglial maturation and accelerates microglial responses to Aß pathology in a mouse model of Alzheimer's disease. Our data indicate that reduction of Trem2 proteolytic cleavage aggravates neuroinflammation during the course of Alzheimer's disease pathology, suggesting that TREM2 shedding is a critical regulator of microglial activity in pathological states.
Asunto(s)
Enfermedad de Alzheimer , Glicoproteínas de Membrana , Microglía , Receptores Inmunológicos , Enfermedad de Alzheimer/metabolismo , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Transgénicos , Microglía/metabolismo , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismoRESUMEN
Fibrosis is characterized by the excessive production of collagen and other extracellular matrix (ECM) components and represents a leading cause of morbidity and mortality worldwide. Previous studies of nonalcoholic steatohepatitis (NASH) with fibrosis were largely restricted to bulk transcriptome profiles. Thus, our understanding of this disease is limited by an incomplete characterization of liver cell types in general and hepatic stellate cells (HSCs) in particular, given that activated HSCs are the major hepatic fibrogenic cell population. To help fill this gap, we profiled 17,810 non-parenchymal cells derived from six healthy human livers. In conjunction with public single-cell data of fibrotic/cirrhotic human livers, these profiles enable the identification of potential intercellular signaling axes (e.g., ITGAV-LAMC1, TNFRSF11B-VWF and NOTCH2-DLL4) and master regulators (e.g., RUNX1 and CREB3L1) responsible for the activation of HSCs during fibrogenesis. Bulk RNA-seq data of NASH patient livers and rodent models for liver fibrosis of diverse etiologies allowed us to evaluate the translatability of candidate therapeutic targets for NASH-related fibrosis. We identified 61 liver fibrosis-associated genes (e.g., AEBP1, PRRX1 and LARP6) that may serve as a repertoire of translatable drug target candidates. Consistent with the above regulon results, gene regulatory network analysis allowed the identification of CREB3L1 as a master regulator of many of the 61 genes. Together, this study highlights potential cell-cell interactions and master regulators that underlie HSC activation and reveals genes that may represent prospective hallmark signatures for liver fibrosis.
Asunto(s)
Células Estrelladas Hepáticas , Enfermedad del Hígado Graso no Alcohólico , Transcriptoma , Animales , Voluntarios Sanos , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/metabolismo , Células Estrelladas Hepáticas/patología , Humanos , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Ratas , Análisis de la Célula IndividualRESUMEN
OBJECTIVE: To identify an MS-specific immune cell population by deep immune phenotyping and relate it to soluble signaling molecules in CSF. METHODS: We analyzed surface expression of 22 markers in paired blood/CSF samples from 39 patients using mass cytometry (cytometry by time of flight). We also measured the concentrations of 296 signaling molecules in CSF using proximity extension assay. Results were analyzed using highly automated unsupervised algorithmic informatics. RESULTS: Mass cytometry objectively identified a B-cell population characterized by the expression of CD49d, CD69, CD27, CXCR3, and human leukocyte antigen (HLA)-DR as clearly associated with MS. Concentrations of the B cell-related factors, notably FCRL2, were increased in MS CSF, especially in early stages of the disease. The B-cell trophic factor B cell activating factor (BAFF) was decreased in MS. Proteins involved in neural plasticity were also reduced in MS. CONCLUSION: When analyzed without a priori assumptions, both the soluble and the cellular compartments of the CSF in MS were characterized by markers related to B cells, and the strongest candidate for an MS-specific cell type has a B-cell phenotype.
Asunto(s)
Factor Activador de Células B/líquido cefalorraquídeo , Linfocitos B/citología , Biomarcadores/líquido cefalorraquídeo , Esclerosis Múltiple/inmunología , Adulto , Linfocitos B/inmunología , Biomarcadores/análisis , Femenino , Humanos , Masculino , Persona de Mediana Edad , FenotipoRESUMEN
Millions of putative transcriptional regulatory elements (TREs) have been cataloged in the human genome, yet their functional relevance in specific pathophysiological settings remains to be determined. This is critical to understand how oncogenic transcription factors (TFs) engage specific TREs to impose transcriptional programs underlying malignant phenotypes. Here, we combine cutting edge CRISPR screens and epigenomic profiling to functionally survey ≈15,000 TREs engaged by estrogen receptor (ER). We show that ER exerts its oncogenic role in breast cancer by engaging TREs enriched in GATA3, TFAP2C, and H3K27Ac signal. These TREs control critical downstream TFs, among which TFAP2C plays an essential role in ER-driven cell proliferation. Together, our work reveals novel insights into a critical oncogenic transcription program and provides a framework to map regulatory networks, enabling to dissect the function of the noncoding genome of cancer cells.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Redes Reguladoras de Genes , Carcinogénesis/genética , Epigenómica , Genoma Humano , Humanos , Elementos Reguladores de la TranscripciónRESUMEN
Acute kidney injury (AKI) and chronic kidney diseases are associated with high mortality and morbidity. Although the underlying mechanisms determining the transition from acute to chronic injury are not completely understood, immune-mediated processes are critical in renal injury. We have performed a comparison of 2 mouse models leading to either kidney regeneration or fibrosis. Using global gene expression profiling we could identify immune-related pathways accounting for the majority of the observed transcriptional changes during fibrosis. Unbiased examination of the immune cell composition, using single-cell RNA sequencing, revealed major changes in tissue-resident macrophages and T cells. Following injury, there was a marked increase in tissue-resident IL-33R+ and IL-2Ra+ regulatory T cells (Tregs). Expansion of this population before injury protected the kidney from injury and fibrosis. Transcriptional profiling of Tregs showed a differential upregulation of regenerative and proangiogenic pathways during regeneration, whereas in the fibrotic environment they expressed markers of hyperactivation and fibrosis. Our data point to a hitherto underappreciated plasticity in Treg function within the same tissue, dictated by environmental cues. Overall, we provide a detailed cellular and molecular characterization of the immunological changes during kidney injury, regeneration, and fibrosis.
Asunto(s)
Lesión Renal Aguda/inmunología , Linfocitos T Reguladores/inmunología , Lesión Renal Aguda/etiología , Lesión Renal Aguda/genética , Lesión Renal Aguda/prevención & control , Animales , Biopsia , Modelos Animales de Enfermedad , Fibrosis/genética , Fibrosis/inmunología , Fibrosis/prevención & control , Perfilación de la Expresión Génica , Interleucina-2/inmunología , Interleucina-33/inmunología , Riñón/patología , Riñón/fisiopatología , Ratones , Regeneración , Daño por Reperfusión/complicacionesRESUMEN
We develop CellSIUS (Cell Subtype Identification from Upregulated gene Sets) to fill a methodology gap for rare cell population identification for scRNA-seq data. CellSIUS outperforms existing algorithms for specificity and selectivity for rare cell types and their transcriptomic signature identification in synthetic and complex biological data. Characterization of a human pluripotent cell differentiation protocol recapitulating deep-layer corticogenesis using CellSIUS reveals unrecognized complexity in human stem cell-derived cellular populations. CellSIUS enables identification of novel rare cell populations and their signature genes providing the means to study those populations in vitro in light of their role in health and disease.
Asunto(s)
Análisis de la Célula Individual/métodos , Transcriptoma , Algoritmos , Línea Celular , Humanos , Neuronas/citologíaRESUMEN
Biliary epithelial cells (BECs) form bile ducts in the liver and are facultative liver stem cells that establish a ductular reaction (DR) to support liver regeneration following injury. Liver damage induces periportal LGR5+ putative liver stem cells that can form BEC-like organoids, suggesting that RSPO-LGR4/5-mediated WNT/ß-catenin activity is important for a DR. We addressed the roles of this and other signaling pathways in a DR by performing a focused CRISPR-based loss-of-function screen in BEC-like organoids, followed by in vivo validation and single-cell RNA sequencing. We found that BECs lack and do not require LGR4/5-mediated WNT/ß-catenin signaling during a DR, whereas YAP and mTORC1 signaling are required for this process. Upregulation of AXIN2 and LGR5 is required in hepatocytes to enable their regenerative capacity in response to injury. Together, these data highlight heterogeneity within the BEC pool, delineate signaling pathways involved in a DR, and clarify the identity and roles of injury-induced periportal LGR5+ cells.
Asunto(s)
Lesión Pulmonar Aguda/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Conductos Biliares/patología , Proteínas de Ciclo Celular/metabolismo , Células Epiteliales/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteína Axina/genética , Proteína Axina/metabolismo , Proteínas de Ciclo Celular/genética , Células Cultivadas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Modelos Animales de Enfermedad , Humanos , Regeneración Hepática , Masculino , Ratones , Ratones Endogámicos C57BL , Piridinas/toxicidad , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Trombospondinas/genética , Trombospondinas/metabolismo , Vía de Señalización Wnt , Proteínas Señalizadoras YAPRESUMEN
Chemogenomic profiling is a powerful and unbiased approach to elucidate pharmacological targets and the mechanism of bioactive compounds. Until recently, genome-wide, high-resolution experiments of this nature have been limited to fungal systems due to lack of mammalian genome-wide deletion collections. With the example of a novel nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, we demonstrate that the CRISPR/Cas9 system enables the generation of transient homo- and heterozygous deletion libraries and allows for the identification of efficacy targets and pathways mediating hypersensitivity and resistance relevant to the compound mechanism of action.