RNA-seq of definitive endoderm and pancreatic progenitor cells derived in vitro from human pluripotent stem cells in order to study the role of transcription factor GATA6 in human pancreas development
ABSTRACT: Mutant GATA6 hPSCs were obtained by TALEN genome editing or re-programmed from patient fibroblasts. Along with wild-type H9 cells, these GATA6 mutant cell lines were differentiated into SOX17+/CXCR4+ endodermal cells (day 3) and PDX1+ pancreatic progenitor cells (day 12). The purpose of this work was to study the role of GATA6 in the development of the human pancreas at a molecular level.
Project description:Mutant GATA6 hPSCs were obtained by TALEN genome editing or re-programmed from patient fibroblasts. Along with wild-type H9 cells, these GATA6 mutant cell lines were differentiated into SOX17+/CXCR4+ endodermal cells (day 3). The purpose of this work was to study the role of GATA6 in the development of the human pancreas at a molecular level.
Project description:Cardiomyocytes can be differentiated from human pluripotent stem cells (hPSCs) in defined conditions, but efficient and consistent cardiomyocyte differentiation often requires expensive reagents such as B27 supplement or recombinant albumin. Using a chemically defined albumin-free (E8 basal) medium, we identified heparin as a novel factor that significantly promotes cardiomyocyte differentiation efficiency, and developed an efficient method to differentiate hPSCs into cardiomyocytes. The treatment of heparin helped cardiomyocyte differentiation consistently reach at least 80% purity (up to 95%) from more than 10 different hPSC lines in chemically defined DMEM/F-12 based medium on either Matrigel or defined matrices like Vitronectin and Synthemax. One of heparin’s main functions was to act as a WNT modulator that helped promote robust and consistent cardiomyocyte production. Our study provides an efficient, reliable, and cost-effective method for cardiomyocyte derivation from hPSCs that can be used for potential large-scale drug screening, disease modeling, and future cellular therapies. 12 human pluripotent stem cells (hPSCs) at three different cardiac differentiation times (0 Days, 3 Days, 6 Days, 10 Days) under different culture conditions (+/- Heparin, +/- IWP2).
Project description:Single-cell RNA-seq (scRNA-seq) on nocodazole and DMSO treated cells before and after differentiation into endoderm. hPSC colonies were treated with DMSO or 100ng/ml nocodazole for 16 hours and induced to differentiate into definitive endoderm for three days. Single cells were subsequently collected in either undifferentiated conditions (Und) or after 3 days of endoderm differentiation (Endoderm) and then sorted onto 384 well plates for Smart-Seq2 processing.
Project description:This study aims to profile the transcriptomes of single naive and primed human embryonic stem cells. Cells from the H9 line were cultured to select for naive or primed phenotypes, and a sequencing library was generated from each single cell using the Smart-seq2 method. This was repeated for multiple experimental batches, i.e., independent cultures. Batch 1 consists of sequencing runs 2383 and 2384; batch 2 consists of runs 2678, 2679, 2739 and 2740; and batch 3 consists of runs 2780 and 2781. Transcriptional profiles for all cells were obtained from the sequencing data and used to explore substructure and heterogeneity in the population for each phenotype.
Project description:Reliable, scalable and time-efficient culture methods are required to fully realize the clinical and industrial applications of human pluripotent stem (hPS) cells. Here we present a completely defined, xeno-free medium that supports long-term propagation of hPS cells on uncoated tissue-culture plastic. The medium consists of the Essential 8 (E8) formulation supplemented with Inter-α-inhibitor (IαI), a human serum-derived protein, recently demonstrated to activate key pluripotency pathways in mouse PS cells. IαI efficiently induces attachment and long-term growth of both embryonic and induced hPS cell lines when added as a soluble protein to the medium at seeding. IαI-supplementation efficiently supports adaptation of feeder-dependent hPS cells to xeno-free conditions, clonal growth as well as single-cell survival in the absence of Rho-associated kinase inhibitor (ROCKi). This time-efficient and simplified culture method paves the way for large-scale, high-throughput hPS cell culture, and will be valuable for both basic research and commercial applications. SNP genotyping with Illumina HumanOmniExpressExome-array version 8v1-2_A, encompassing >964.000 single nucleotide polymorphism (SNP)-markers, were performed by the SNP&SEQ technology Platform in Uppsala (www.genotyping.se), according to the manufacturer’s instructions. Each of the four hPS cell lines (i.e. H181, H207, HUES1 and K2C) were analyzed in three separate SNP-array experiments for the 12 samples described here. A first analysis was performed after 2-5 passages of growth for initial reference (4 samples), and subsequently, two analyses were performed per cell line after 16-21 passages in E8:VN or E8:IαI culture (8 samples). The CN-calls of the autosomal chromosomes were illustrated using the Nexus-software.
Project description:Differentiation into diverse cell lineages requires orchestration of gene regulatory networks guiding cell fate choices. Genetic factors acting through changes in transcriptional levels can contribute to cardiovascular disease risk by impacting early stages of development and have cell type-specific effects. We set out to characterize lineage trajectory progression of subpopulations and identify potential disease-related genes by examining their expression changes in single cells during early stages of cardiac lineage specification. Using 43,168 single-cell transcriptomes, we developed novel classification and trajectory analysis methods to dissect cellular composition and gene networks across five discrete time points underlying lineage derivation of mesoderm, definitive endoderm, vascular endothelium, cardiac precursors, and definitive cell types that comprise cardiomyocytes and a previously unrecognized cardiac outflow tract population.
Project description:Murine ES cell gene expression before RA induction are used to compare gene expression for time-points of 8, 12, 16, 24, 36, 48, 60 and 72 hours post-induction. KH2 ES Cell RA Differentiation Time-course
Project description:Transcription factor-mediated reprogramming is a powerful method to study cell fate changes. In this work, we demonstrate that the transcription factor Gata6 can initiate reprograming of multiple cell types to induced extraembryonic endoderm (iXEN) cells. Intriguingly, Gata6 is sufficient to drive iXEN cells from mouse pluripotent cells and differentiated neural cells. Furthermore, GATA6 induction in human ES (hES) cells also downregulates pluripotency gene expression and upregulates extraembryonic endoderm genes, revealing a conserved function in mediating this cell fate switch. Profiling transcriptional changes following Gata6 induction in mES cells reveals step-wise pluripotency factor disengagement, with initial repression of Nanog and Esrrb, then Sox2 and finally Oct4, alongside step-wise activation of extraembryonic endoderm genes. Chromatin immunoprecipitation and subsequent high-throughput sequencing analysis shows Gata6 enrichment near both pluripotency and endoderm genes, suggesting that Gata6 functions as both a direct repressor and activator. Together this demonstrates that Gata6 is a versatile and potent reprogramming factor that can act alone to drive a cell fate switch from diverse cell types. (1) Microarray analysis of Gata6 overexpressing cells from 12 to 144 hours of doxycycline treatment in mouse embryonic stem (mES) cells compared to uninduced mES cells, embryo-derived XEN cells and Sox7 overexpressing mES cells after 144 hours of doxycycline treatment. (2) ChIP-seq analysis of Gata6 binding 36 hours following doxycycline treatment. (3) ChIP-seq analysis of Gata6 binding in embryo-derived XEN cells. (4) RNA-seq analysis of GATA6 overexpressing cells following 144 hours of induction in hES cells.