RESUMEN
The liver has the remarkable capacity to regenerate. In the clinic, regeneration is induced by portal vein embolization, which redirects portal blood flow, resulting in liver hypertrophy in locations with increased blood supply, and atrophy of embolized segments. Here, we apply single-cell and single-nucleus transcriptomics on healthy, hypertrophied, and atrophied patient-derived liver samples to explore cell states in the regenerating liver. Our data unveils pervasive upregulation of genes associated with developmental processes, cellular adhesion, and inflammation in post-portal vein embolization liver, disrupted portal-central hepatocyte zonation, and altered cell subtype composition of endothelial and immune cells. Interlineage crosstalk analysis reveals mesenchymal cells as an interaction hub between immune and endothelial cells, and highlights the importance of extracellular matrix proteins in liver regeneration. Moreover, we establish tissue-scale iterative indirect immunofluorescence imaging for high-dimensional spatial analysis of perivascular microenvironments, uncovering changes to tissue architecture in regenerating liver lobules. Altogether, our data is a rich resource revealing cellular and histological changes in human liver regeneration.
Asunto(s)
Embolización Terapéutica , Regeneración Hepática , Hígado , Vena Porta , Humanos , Regeneración Hepática/fisiología , Embolización Terapéutica/métodos , Hepatocitos/metabolismo , Análisis de la Célula Individual , Transcriptoma , Masculino , Células Endoteliales/metabolismo , Femenino , Hipertrofia , Persona de Mediana EdadRESUMEN
Primary human hepatocytes are widely used to evaluate liver toxicity of drugs, but they are scarce and demanding to culture. Stem cell-derived hepatocytes are increasingly discussed as alternatives. To obtain a better appreciation of the molecular processes during the differentiation of induced pluripotent stem cells into hepatocytes, we employ a quantitative proteomic approach to follow the expression of 9,000 proteins, 12,000 phosphorylation sites, and 800 acetylation sites over time. The analysis reveals stage-specific markers, a major molecular switch between hepatic endoderm versus immature hepatocyte-like cells impacting, e.g., metabolism, the cell cycle, kinase activity, and the expression of drug transporters. Comparing the proteomes of two- (2D) and three-dimensional (3D)-derived hepatocytes with fetal and adult liver indicates a fetal-like status of the in vitro models and lower expression of important ADME/Tox proteins. The collective data enable constructing a molecular roadmap of hepatocyte development that serves as a valuable resource for future research.
Asunto(s)
Células Madre Pluripotentes Inducidas , Proteoma , Adulto , Diferenciación Celular , Hepatocitos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Proteoma/metabolismo , ProteómicaRESUMEN
Basal progenitors (BPs), including intermediate progenitors and basal radial glia, are generated from apical radial glia and are enriched in gyrencephalic species like humans, contributing to neuronal expansion. Shortly after generation, BPs delaminate towards the subventricular zone, where they further proliferate before differentiation. Gene expression alterations involved in BP delamination and function in humans are poorly understood. Here, we study the role of LGALS3BP, so far known as a cancer biomarker, which is a secreted protein enriched in human neural progenitors (NPCs). We show that individuals with LGALS3BP de novo variants exhibit altered local gyrification, sulcal depth, surface area and thickness in their cortex. Additionally, using cerebral organoids, human fetal tissues and mice, we show that LGALS3BP regulates the position of NPCs. Single-cell RNA-sequencing and proteomics reveal that LGALS3BP-mediated mechanisms involve the extracellular matrix in NPCs' anchoring and migration within the human brain. We propose that its temporal expression influences NPCs' delamination, corticogenesis and gyrification extrinsically.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Biomarcadores de Tumor/metabolismo , Corteza Cerebral/citología , Vesículas Extracelulares/metabolismo , Células Madre Pluripotentes Inducidas/citología , Neocórtex/citología , Células-Madre Neurales/citología , Neuroglía/metabolismo , Animales , Diferenciación Celular , Corteza Cerebral/metabolismo , Femenino , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Ventrículos Laterales/citología , Ventrículos Laterales/metabolismo , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Neocórtex/metabolismo , Células-Madre Neurales/metabolismoRESUMEN
It is a major challenge to integrate single-cell sequencing data across experiments, conditions, batches, time points, and other technical considerations. New computational methods are required that can integrate samples while simultaneously preserving biological information. Here, we propose an unsupervised reference-free data representation, cluster similarity spectrum (CSS), where each cell is represented by its similarities to clusters independently identified across samples. We show that CSS can be used to assess cellular heterogeneity and enable reconstruction of differentiation trajectories from cerebral organoid and other single-cell transcriptomic data, and to integrate data across experimental conditions and human individuals.
Asunto(s)
Genómica/métodos , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Humanos , Aprendizaje Automático no SupervisadoRESUMEN
Three-dimensional (3D) tissues grown in culture from human stem cells offer the incredible opportunity to analyze and manipulate human development, and to generate patient-specific models of disease. Methods to sequence DNA and RNA in single cells are being used to analyze these so-called 'organoid' systems in high-resolution. Single-cell transcriptomics has been used to quantitate the similarity of organoid cells to primary tissue counterparts in the brain, intestine, liver, and kidney, as well as identify cell-specific responses to environmental variables and disease conditions. The merging of these two technologies, single-cell genomics and organoids, will have profound impact on personalized medicine in the near future.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Organoides/metabolismo , Análisis de la Célula Individual/métodos , Transcriptoma/genética , Humanos , Especificidad de Órganos/genética , Medicina de PrecisiónRESUMEN
Ectopic expression of defined transcription factors can force direct cell-fate conversion from one lineage to another in the absence of cell division. Several transcription factor cocktails have enabled successful reprogramming of various somatic cell types into induced neurons (iNs) of distinct neurotransmitter phenotype. However, the nature of the intermediate states that drive the reprogramming trajectory toward distinct iN types is largely unknown. Here we show that successful direct reprogramming of adult human brain pericytes into functional iNs by Ascl1 and Sox2 encompasses transient activation of a neural stem cell-like gene expression program that precedes bifurcation into distinct neuronal lineages. During this transient state, key signaling components relevant for neural induction and neural stem cell maintenance are regulated by and functionally contribute to iN reprogramming and maturation. Thus, Ascl1- and Sox2-mediated reprogramming into a broad spectrum of iN types involves the unfolding of a developmental program via neural stem cell-like intermediates.