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Methods for Cardiac Fibroblast Generation from iPSCs: Updates and Characterization

ABSTRACT: Since the initial isolation of human embryonic stem cells and subsequent discovery of reprogramming methods for somatic cells, thousands of protocols have been developed to create each of the hundreds of cell types found in vivo with significant focus on disease-prone systems, e.g. cardiovascular. Robust protocols exist for many of these cell types, save cardiac fibroblasts (CF). Very recently, several competing methods have been developed to generate these cells through a developmentally conserved epicardial pathway. Such methods generate epicardial cells, but here we report that prolonged exposure to growth factors such as bFGF induces a more native morphology and similar chromatin architecture to primary CFs. Media conditions for growth and assays are provided, as well as suggestions for seeding densities and timepoints for protein harvest of extracellular matrix. We demonstrate marker expression and matrix competency of resultant cells as shown next to primary human cardiac fibroblasts. These methods provide additional guidance to the original protocol and result in an increasingly stable phenotype

ORGANISM(S): Homo sapiens

PROVIDER: GSE167368 | GEO | 2021/12/01

REPOSITORIES: GEO

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Comparison of gene expression data between control and tissue specific epicardial PDGF receptor (a and b) knockout embryonic hearts

Project description:Epicardial cells undergo an epithelial-to-mesenchymal transtion (EMT) to generate coronary vascular smooth muscle cells (VSMC) and cardiac fibroblasts. Little is known about the mechanisms regulating EMT or the in vivo signals directing epicardial-derived cell (EPDC) fate. Here, we show that loss of PDGF signaling leads to a disruption in Sox9 expression, and when Sox9 expression was restored in mutant hearts, the EMT defect was rescued. Interestingly, mutants lacking only one of the PDGF genes exhibited a lineage specific requirement for the individual receptors. Loss of PDGFRα resulted in a deficit in cardiac fibroblast formation, while cVSMC development was unperturbed. Conversely, PDGFRβ was required for cVSMC development but not cardiac fibroblast development. Combined, our data demonstrate a novel role for PDGF receptors in epicardial EMT and EPDC development. GSM671723-GSM671724: Total RNA was isolated from E12.5 control and PDGF receptor epicardial knockout hearts using the Trizol reagent. RNA was processed as per manufacturer's instructions (Illumina Gene expression array, Illumina, inc. San Diego, CA, USA) GSM671877-GSM671882: Total RNA was isolated from E12.5 control and PDGF receptor epicardial knockout primary epicardial cultures using the Trizol reagent. RNA was processed as per manufacturer's instructions (Illumina Gene expression array, Illumina, inc. San Diego, CA, USA)

2011-02-28 | E-GEOD-27181 | biostudies-arrayexpress

Comparison of gene expression data between control and tissue specific epicardial PDGF receptor (a and b) knockout embryonic hearts

2011-02-28 | GSE27181 | GEO

RNA-seq of Pluripotent stem cell derived cardiac cells

Project description:Cardiac fibroblasts (CFBs) support heart function by secreting extracellular matrix and paracrine factors, respond to stress associated with injury and disease, and therefore are an increasingly important therapeutic target to treat heart disease. Here, we profile how developmental lineage of human pluripotent stem cell-derived CFBs, epicardial (EpiC-FB) and second heart field (SHF-FB) impacts their transcriptome. We compared this to human pluripotent stem cell derived cardiomyocytes, primary fetal cardiac fibroblasts, primary adult left ventricular fibroblasts, and primary adult dermal fibroblasts.

2021-08-11 | GSE168380 | GEO

PRMT1-Mdm4-p53 pathway controls epicardial invasion and cell fate transition

Project description:Epicardial cells are progenitors giving rise to the majority of cardiac fibroblasts, coronary smooth muscle cells, and pericytes during cardiac development, and critically modulating heart morphogenesis and coronary development. An integral phase of epicardial cell fate transition is epithelial-to-mesenchymal transition (EMT), which confers motility and facilitates cell fate transition. We identify a pathway involving protein arginine methyltransferase 1 (PRMT1) and its downstream p53 signaling that drives epicardial EMT and invasion. We show that PRMT1 determines the half-life of p53 through regulating alternative splicing of Mdm4, which is a key controller of p53 degradation. Loss of PRMT1 promotes the expression of Mdm4 short form, which inhibits p53 degradation. Accumulation of p53 subsequently enhances Slug degradation and blocks epicardial EMT. We further demonstrated that the PRMT1-Mdm4-p53 pathway drives epicardial cell fate transition into cardiac fibroblasts, coronary smooth muscle cells and pericytes in vivo, and modulates ventricular morphogenesis and coronary vessel formation. Together, our results establish critical functions of the PRMT1-Mdm4-p53 pathway in epicardial EMT, invasion and cell fate transition.

2020-08-18 | GSE122200 | GEO

scRNA-seq of E15.5 epicardium and epicardium-derived cells

Project description:The embryonic epicardium provides a source of multipotent progenitors for cardiac lineages, including pericytes, fibroblasts and smooth muscle cells. However, it remains unclear if the epicardium comprises of heterogeneous progenitors or if epicardial cells are multipotent. Using scRNA-seq, we analysed the expression profile of epicardial and epicardial-derived cells at E15.5 and found no evidence of discrete epicardial sub-compartments. Our findings also support the notion that epicardial fate is specified after epithelial-mesenchymal transition, not pre-determined.

2020-04-30 | GSE145832 | GEO

Gene expression of cardiac organoids at a single cell resolution in healthy and cardiac injury conditions

Project description:Cardiac fibroblast heterogeneity has been observed increasingly in data sets coming from model organisms during regeneration and cardiac injury. This works provides the first characterization of cardiac fibroblasts derived from hPSC epicardial cells in a healthy and injured state with single cell resolution.

2023-10-12 | GSE221500 | GEO

EPICARDIOID SINGLE-CELL GENOMICS UNCOVER PRINCIPLES OF HUMAN EPICARDIUM BIOLOGY IN HEART DEVELOPMENT AND DISEASE [scRNA-Seq]

Project description:Native human epicardial precursors are virtually inaccessible, as they appear in the embryo less than four weeks post-conception, at which point pregnancy may not yet be detected. Protocols have been established to generate epicardial cells and their progeny from human pluripotent stem cells in vitro. However, no current model is amenable to studying the many facets of human epicardial biology, notably epicardium formation, lineage heterogeneity, and functional crosstalk with other cardiac cell types during organ development and disease. Here, we generated human pluripotent stem cell-derived 3D heart organoids showing retinoic acid-dependent self-organization of the epicardium and myocardium, which we called epicardioids. Time course single-cell RNA sequencing revealed that epicardioids are formed through the specification of first heart field progenitors, a subset of which closely correspond to juxta-cardiac field (JCF) cells that have recently been identified in the embryonic mouse heart as novel common progenitors of myocardium and epicardium and have not yet been described and studied in humans. Analysis of chromatin accessibility by single-cell ATAC-seq additionally revealed key transcriptional programs guiding fate decisions along the epicardial lineage tree. Human epicardioids provide a unique model to gain fundamental insights into human epicardium biology and function during heart development, homeostasis, and disease.

2023-02-05 | GSE196513 | GEO

EPICARDIOID SINGLE-CELL GENOMICS UNCOVER PRINCIPLES OF HUMAN EPICARDIUM BIOLOGY IN HEART DEVELOPMENT AND DISEASE [scATAC-Seq]

2023-02-05 | GSE196512 | GEO

Genome-wide analysis of epicardial gene expression in E11.5 mouse hearts

Project description:Identification of epicardium-enriched genes in the embryonic heart. The epicardium encapsulates the heart and functions as a source of multipotent progenitor cells and paracrine factors essential for cardiac development and repair. Injury of the adult heart results in re-activation of a developmental gene program in the epicardium, but the transcriptional basis of epicardial gene expression has not been delineated. We established a mouse embryonic heart organ culture and gene expression system that facilitated the identification of epicardial enhancers activated during heart development and injury. Epicardial activation of these enhancers depends on a combinatorial transcriptional code centered on CCAAT/enhancer binding protein (C/EBP) transcription factors. Disruption of C/EBP signaling in the adult epicardium reduced injury-induced neutrophil infiltration and improved cardiac function. These findings reveal a transcriptional basis for epicardial activation and heart injury, providing a platform for enhancing cardiac regeneration. Total RNA obtained from lacZ-positive epicardial cells isolated from the E11.5 Tcf21lacZ hearts compared to total dissociated heart cells

2012-11-01 | E-GEOD-41959 | biostudies-arrayexpress

BNC1 is a master regulator of human epicardial cell heterogeneity and function

Project description:Single cell RNAseq of a model of epicardium derived from human pluripotent stem cell (hPSC-epi) reveals that distinct epicardial sub-populations are defined by high levels of expression for the transcription factors BNC1 and TCF21. Cells positive for the transcription factor WT1 are included in the BNC1 population, which confirms the heterogeneity observed in the mouse with TCF21 and WT1. Analysis of genes differentially expressed between the two populations suggests differences in their biological activities. We identified THY1 as a membrane marker of the TCF21 population and we show that THY1+ cells can differentiate into cardiac fibroblast and smooth muscle cells while THY1- cells mostly produce the latter. Knocking-down BNC1 during the establishment of the populations led to an homogeneous epicardium containing mostly the TCF21high populations and differentiating mainly into cardiac fibroblasts. Using network inference methods and transcriptomic data from the different epicardial lineages derived from the hPSC-epi, we identified a core transcriptional network organized around WT1, TCF21 and BNC1 and orchestrated by BNC1.

2019-12-03 | GSE122827 | GEO

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