Human pluripotent stem (hPS) cells can, in theory, be differentiated into any cell type, making them a powerful in vitro model for human biology. Recent technological advances have facilitated large-scale hPS cell studies that allow investigation of the genetic regulation of molecular phenotypes and their contribution to high-order phenotypes such as human disease. Integrating hPS cells with single-cell sequencing makes identifying context-dependent genetic effects during cell development or upon experimental manipulation possible.
View Article and Find Full Text PDFThe mechanisms by which DNA alleles contribute to disease risk, drug response, and other human phenotypes are highly context-specific, varying across cell types and different conditions. Human induced pluripotent stem cells are uniquely suited to study these context-dependent effects but cell lines from hundreds or thousands of individuals are required. Village cultures, where multiple induced pluripotent stem lines are cultured and differentiated in a single dish, provide an elegant solution for scaling induced pluripotent stem experiments to the necessary sample sizes required for population-scale studies.
View Article and Find Full Text PDFTo assess the transcriptomic profile of disease-specific cell populations, fibroblasts from patients with primary open-angle glaucoma (POAG) were reprogrammed into induced pluripotent stem cells (iPSCs) before being differentiated into retinal organoids and compared with those from healthy individuals. We performed single-cell RNA sequencing of a total of 247,520 cells and identified cluster-specific molecular signatures. Comparing the gene expression profile between cases and controls, we identified novel genetic associations for this blinding disease.
View Article and Find Full Text PDFPrecursors of the adult hematopoietic system arise from the aorta-gonad-mesonephros (AGM) region shortly after the embryonic circulation is established. Here, we develop a microfluidic culture system to mimic the primitive embryonic circulation and address the hypothesis that circulatory flow and shear stress enhance embryonic blood development. Embryonic (HOXA) hematopoiesis was derived from human pluripotent stem cells and induced from mesoderm by small-molecule manipulation of TGF-β and WNT signaling (SB/CHIR).
View Article and Find Full Text PDFThere are currently no treatments for geographic atrophy, the advanced form of age-related macular degeneration. Hence, innovative studies are needed to model this condition and prevent or delay its progression. Induced pluripotent stem cells generated from patients with geographic atrophy and healthy individuals were differentiated to retinal pigment epithelium.
View Article and Find Full Text PDFWe report that cardiac fibroblasts (CFs) and mesenchymal progenitors are more hypoxic than other cardiac interstitial populations, express more hypoxia-inducible factor 1α (HIF-1α), and exhibit increased glycolytic metabolism. CF-specific deletion of Hif-1a resulted in decreased HIF-1 target gene expression and increased mesenchymal progenitors in uninjured hearts and increased CF activation without proliferation following sham injury, as demonstrated using single-cell RNA sequencing (scRNA-seq). After myocardial infarction (MI), however, there was ∼50% increased CF proliferation and excessive scarring and contractile dysfunction, a scenario replicated in 3D engineered cardiac microtissues.
View Article and Find Full Text PDFFront Cell Dev Biol
August 2021
Platelet-derived growth factors (PDGFs) are powerful inducers of cellular mitosis, migration, angiogenesis, and matrix modulation that play pivotal roles in the development, homeostasis, and healing of cardiac tissues. PDGFs are key signaling molecules and important drug targets in the treatment of cardiovascular disease as multiple researchers have shown that delivery of recombinant PDGF ligands during or after myocardial infarction can reduce mortality and improve cardiac function in both rodents and porcine models. The mechanism involved cannot be easily elucidated due to the complexity of PDGF regulatory activities, crosstalk with other protein tyrosine kinase activators, and diversity of the pathological milieu.
View Article and Find Full Text PDFBesides cardiomyocytes, the heart contains numerous interstitial cell types, including cardiac fibroblasts, endothelial cells, immune (myeloid and lymphoid) cells, and mural cells (pericytes and vascular smooth muscle cells), which play key roles in heart repair, regeneration, and disease. We recently published a comprehensive map of cardiac stromal cell heterogeneity and flux in healthy and infarcted hearts using single-cell RNA sequencing (scRNA-seq) ( Farbehi , 2019 ). Here, we describe the FACS (Fluorescent Activated Cell Sorting)-based method used in that study for isolation of two cardiac cell fractions from adult mouse ventricles: the total interstitial cell population (TIP; non-cardiomyocytes) and enriched (-GFP) cardiac fibroblasts.
View Article and Find Full Text PDFBasal breast cancer is associated with younger age, early relapse, and a high mortality rate. Here, we use unbiased droplet-based single-cell RNA sequencing (RNA-seq) to elucidate the cellular basis of tumor progression during the specification of the basal breast cancer subtype from the luminal progenitor population in the MMTV-PyMT (mouse mammary tumor virus-polyoma middle tumor-antigen) mammary tumor model. We find that basal-like cancer cells resemble the alveolar lineage that is specified upon pregnancy and encompass the acquisition of an aberrant post-lactation developmental program of involution that triggers remodeling of the tumor microenvironment and metastatic dissemination.
View Article and Find Full Text PDFDroplet based scRNA-seq systems such as Drop-seq, inDrop and Chromium 10X have been the catalyst for the wide adoption of high-throughput scRNA-seq technologies in the research laboratory. In order to understand the capabilities of these systems to deeply interrogate biology; here we provide a practical guide through all the steps involved in a typical scRNA-seq experiment. Through comparing and contrasting these three main droplet based systems (and their derivatives), we provide an overview of all critical considerations in obtaining high quality and biologically relevant data.
View Article and Find Full Text PDFBesides cardiomyocytes (CM), the heart contains numerous interstitial cell types which play key roles in heart repair, regeneration and disease, including fibroblast, vascular and immune cells. However, a comprehensive understanding of this interactive cell community is lacking. We performed single-cell RNA-sequencing of the total non-CM fraction and enriched (-GFP) fibroblast lineage cells from murine hearts at days 3 and 7 post-sham or myocardial infarction (MI) surgery.
View Article and Find Full Text PDFRegulation of tissue development and repair depends on communication between neighbouring cells. Recent advances in cell micro-contact printing and microfluidics have facilitated the in-vitro study of homotypic and heterotypic cell-cell interaction. Nonetheless, these techniques are still complicated to perform and as a result, are seldom used by biologists.
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