Project description:To systematically examine the cellular identity of INS+ cells derived after H1152 treatment, INSw/GFP HES3 hESCs were differentiated to a pancreatic progenitor population and treated with 10 µM H1152 for 8 days in sphere culture. The INS-GFP+ cells of H1152 or DMSO treated spheres were purified by cell sorting and transcripts profiled using RNA-seq.
Project description:Differentiation of INSGFP/w hESCs using published protocols demonstrated that all GFP+ cells co-expressed insulin, confirming the fidelity of the reporter gene. INS-GFP+ cells also co-expressed glucagon and somatostatin, confirming prior studies regarding the polyhormonal nature of early hESC derived insulin-expressing cells. INSGFP/w hESCs were employed to develop a 96 well format spin Embryoid Body (EB) differentiation protocol that utilized the recombinant protein based fully defined medium, APEL. Like INS-GFP+ cells generated with other methods, those derived using the spin EB protocol expressed a collection of pancreatic related transcription factors including ISL1, PAX6 and NKX2.2. However, in contrast to previous methods, the spin EB protocol yielded INS-GFP+ cells that also co-expressed the beta-cell transcription factor, NKX6.1 and comprised a substantial proportion of monohormonal insulin+ cells.
Project description:Human pluripotent stem cells (hPSCs) have the potential to generate any human cell type, and one widely recognized goal is to make pancreatic β cells. To this end, comparisons between differentiated cell types produced in vitro and their in vivo counterparts are essential to validate hPSC-derived cells. Genome-wide transcriptional analysis of sorted insulin-expressing (INS(+)) cells derived from three independent hPSC lines, human fetal pancreata, and adult human islets points to two major conclusions: (i) Different hPSC lines produce highly similar INS(+) cells and (ii) hPSC-derived INS(+) (hPSC-INS(+)) cells more closely resemble human fetal β cells than adult β cells. This study provides a direct comparison of transcriptional programs between pure hPSC-INS(+) cells and true β cells and provides a catalog of genes whose manipulation may convert hPSC-INS(+) cells into functional β cells RNA is isolated and processed using MARIS from the following samples: H1 human embryonic stem cells (hESCs) in duplicate, HUES8 hESCs in duplicate, human induced pluripotent stem cells (hiPSCs) in duplicate, H1 cells differentiated to a stage in which insulin-expressing cells are present (stage 6) in duplicate, HUES8 cells differentiated to stage 6 in duplicate, hiPSCs differentiated to stage 6, insulin-expressing cells sorted from H1 cells differentiated to stage 6 in duplicate, insulin-expressing cells sorted from HUES8 cells differentiated to stage 6 in duplicate, insulin-expressing cells sorted from hiPSCs differentiated to stage 6 in duplicate, human week 16 fetal pancreata in duplicate, insulin-expressing cells sorted from human week 16 fetal pancreata in triplicate, adult human pancreatic islets in triplicate, and insulin-expressing cells sorted from adult human pancreatic islets in triplicate.
Project description:Human embryonic stem cell-reporter line hESC-NKX2.5(eGFP/w) were differentiated to cardiomyocytes (CMs) by utilizing the spin embryoid body method. During differentiation the cells were treated with DMSO or retinoic acid (RA) from day 4-7. At day 31, cells were sorted based on GFP prior to RNA isolation. The results of this microarray demonstrate that CMs treated with RA during differentiation exhibit atrial-like gene expression profile, while DMSO-treated cells show ventricular-like gene profile.
Project description:Human embryonic stem cell-reporter line hESC-NKX2.5(eGFP/w) were differentiated to cardiomyocytes (CMs) by utilizing the spin embryoid body method. During differentiation the cells were treated with DMSO or retinoic acid (RA) from day 4-7. At day 31, cells were sorted based on GFP prior to RNA isolation. The results of this microarray demonstrate that CMs treated with RA during differentiation exhibit atrial-like gene expression profile, while DMSO-treated cells show ventricular-like gene profile. CMs treated with DMSO or RA during differentiation were sorted for GFP and analyzed for differential gene expression.
Project description:The experiment was designed to unravel how endogenous signaling in differentiating hESCs is affected by exogenous factors. RNA-seq of human ES cell lines HUES4 and H1, differentiated for 5 or 7 days towards pancreatic endoderm. The cells were treated with IWP-L6, TGFb1 or both from day 3 and onwards. Vehicle controls were done with 0.1% DMSO.
Project description:n=3 Ctrl and n=3 HD iPSC lines differentiated into cortical neurons were treated with DMSO or 10nM Branaplam for 72h and RNA-seq was performed.
Project description:Aiming to identify insulin-independent modulators of glucose homeostasis, we performed a drug screen on zebrafish insulin (ins) mutants and identified androgen receptor (AR) antagonists. To investigate how AR antagonism mediates glucose level reduction in ins mutants, we evaluated the effects of antagonist treatment using transcriptomic studies. RNA-Seq analyses were performed on 120 hours post fertilization (hpf) ins mutants treated with Flutamide or Cyproterone starting at 84 hpf compared to vehicle (DMSO) treated mutants.
