Activin A and BMP4 Modulation of Wnt/?-catenin Signaling Directs Fate Specification of Mesoderm Derivatives from Human Embryonic Stem Cells
Ontology highlight
ABSTRACT: We report the derivation of 2 different methods of generating cardiac myocytes from human ESCs. The traditional route is via cardiac progenitor cells and the second, new approach is through re-directing hemogenic endothelium into the cardiac lineage using inhibition of Wnt/b-catenin signaling Examination of 2 different cardiac populations using RNA-seq
Project description:This project compares Baf60c homozygous deletions to WT at two time points during cardiac differentiation. This data is used in two separate projects: first to assess the role of Baf60c in regulating cardiac gene expression, and second to assess the repeatability of results from different RNA-seq analysis tools. There are 12 samples total; 3 replicates for each group, times 2 genetic backgrounds (WT and Baf60c homozygous KO), times two timepoints (cardiomyocyte (D10) and cardiac precusor (D5.3)).
Project description:Human heart development is governed by transcription factor (TF) networks controlling dynamic and temporal gene expression alterations. Therefore, to comprehensively characterize these transcriptional regulations, day-to-day transcriptomic profiles were generated throughout the directed cardiac differentiation, starting from three distinct human induced pluripotent stem cell lines from healthy donors (32 days).
Project description:We have developed a protocol to generate cardiopharyngeal mesoderm (CPM) in vitro by Mesp1 induction in ES cells. The goal of this study is to compare the transcriptome of CPM-derived cardiac and skeletal myogenic progenitors to identify novel lineage-specific markers. mRNA profiles of CPM-derived D6 (early) and D12 (late), cardiac (BMP) and skeletal myogenic (control) progenitors were generated
Project description:Human pluripotent stem cell-derived cardiomyocytes (CMs) are a promising tool for cardiac cell therapy. To optimize graft cells for cardiac reconstruction, we compared the engraftment efficiency of intramyocardially-injected undifferentiated-induced pluripotent stem cells (iPSCs), day4 mesodermal cells, and day8, day20, and day30 purified iPSC-CMs after initial differentiation by tracing the engraftment ratio (ER) using in vivo bioluminescence imaging. This analysis revealed the ER of day20 CMs was significantly higher compared to other cells. Transplantation of day20 CMs into the infarcted hearts of immunodeficient mice showed significant functional improvement. Moreover, the imaging signal and ratio of Ki67-positive CMs at 3 months post injection indicated engrafted CMs proliferated in the host heart. Although this graft growth reached a plateau at 3 months, histological analysis confirmed progressive maturation from 3 to 6 months. These results suggested that day20 CMs had very high engraftment, proliferation, and therapeutic potential in host mouse hearts. Differentiated cells, N=10 Undifferentiated pluripotent stem cells, N=1 Heart samples, N=6
Project description:Polycomb complexes are essential regulators of stem cell identity, yet very little is known about their molecular mechanisms during cell differentiation. Pcgf proteins (Pcgf1/2/3/4/5/6) are core subunits of the Polycomb repressive complex 1 (PRC1). It has been recently proposed that specific Pcgf proteins are associated to particular PRC1 complexes, yet the molecular and biological functions of different Pcgf proteins remains largely elusive. Using specific differentiation protocols, we have elucidated a role for Pcgf2/Mel18 in specifically regulating mesoderm differentiation. Mechanistically, during early cardiac mesoderm differentiation, Pcgf2/Mel18 functions as a classical Polycomb protein by repressing pluripotency, lineage specification, late cardiac differentiation and negative regulators of the BMP pathway, yet Pcgf2/Mel18 also positively regulates the expression of key mesoderm transcription factors, revealing a novel function of Pcgf2/Mel18 in gene activation during cardiac differentiation. Mel18 depletion results in an unbalance of pathways that positively and negatively regulate cardiac differentiation. We propose that Mel18 is a novel epigenetic factor that controls mesoderm differentiation by opposing molecular mechanisms. List of ChIPseq samples: Mel18 in ESCs and MES, Ring1b, RYBP and Cbx2 in MES, IgG in MESs. List of RNAseq experiments: Mel18 KD, Ring1b KO and CTR in ESCs, Mel18 KD and CTR in MES, Mel18 KD and CTR in CMs.
Project description:Human pluripotent stem cells (hPSCs) such as embryonic stem cells and induced pluripotent stem cells are promising materials for cell-based regenerative therapies to heart diseases. However, until realization there are many hurdles such as high efficiency of cardiac differentiation of hPSCs and production of clinical-grade cardiac cells derived from hPSCs. Here, we show that a novel small molecule KY02111 robustly enhances differentiation to functional cardiomyocytes from hPSCs. To reveal how KY02111 function on promoting cardiac differentiation of hPSCs, we analyzed the gene expression profiles in KY02111-treated IMR90-1 hiPSCs using the microarray technique. At Day3 of cardiac differentiation from hiPSCs, KY02111 or DMSO was added in the culture and then the cell population was harvested after 12 or 24 hours for RNA extraction.
Project description:We discovered induction of circular RNA in human fetal tissues, including the heart. In this study, we were able to recapitulate this induction by in vitro directed differentiation of hESCs to cardiomyocytes, paving the way for future studies into circular RNA regulation. We harvested hESCs at sequential stages of differentiation: undifferentiated (day 0), mesoderm (day 2), cardiac progenitor (day 5) and definitive cardiomyocyte (day 14). We performed RNA sequencing in biological triplicate, with 3-8 technical replicates each.
Project description:Translational regulation is of paramount importance for proteome remodeling during stem cell differentiation both at the global and transcript-specific levels. In this study, we characterized translational remodeling during hepatogenic differentiation of induced pluripotent stem cells (iPSCs) by polysome profiling. We demonstrate that protein synthesis increases during exit from pluripotency, and is then globally repressed during later steps of hepatogenic maturation. This global downregulation of translation is accompanied by a decrease in the protein abundance of components of the translation machinery, which involves a global reduction in translational efficiency of terminal oligopyrimidine tract (TOP) mRNA encoding translation-related factors. Despite global translational repression during hepatogenic differentiation, key hepatogenic genes remain efficiently translated, and the translation of several transcripts involved in hepato-specific functions and metabolic maturation are even induced. We conclude that, during hepatogenic differentiation, a global decrease in protein synthesis is accompanied by a specific translational rewiring of hepato-specific transcripts.
Project description:We monitored 9 pluripotent stem cell lines across three time points of hepatic directed differentiation, representing 3 developmental stages: undifferentiated (T0), definitive endoderm (T5), and early hepatocyte (T24). ESCs (n=3) and patient-derived normal (n=3) or PiZZ (n=3) iPSCs were analyzed in the undifferentiated state (T0), after differentiation to definitive endoderm (T5), and upon reaching hepatic stage (T24) for a total of 27 samples. We sought to test the hypothesis that a single transgene-free iPSC clone from each donor could be used to detect disease-specific differences between the normal cohort and the PiZZ cohort, anticipating that this difference would emerge only at a developmental stage in which the mutant AAT gene is expressed. Cells were sorted before analysis at T0 and T5 after antibody staining for TRA1-80+/SSEA3+ (T0) or C-kit+/CXCR4+ (T5) cells. ESCs (n=3) and patient-derived normal (n=3) or PiZZ (n=3) iPSCs were analyzed in the undifferentiated state (T0), after differentiation to definitive endoderm (T5), and upon reaching hepatic stage (T24) for a total of 27 samples. Cells were sorted before analysis at T0 and T5 after antibody staining for TRA1-80+/SSEA3+ (T0) or C-kit+/CXCR4+ (T5) cells. RNA was extracted at each stage, biotin labeled, and hybridized to either Affymetrix GeneChip Human Gene 1.0 ST or miRNA 2.0 arrays. For methylation analysis, gDNA was sorted from the same 27 samples before undergoing bisulfite conversion. Bisulfite converted DNA was then amplified and purified prior to overnight hybridization to Illumina’s Infinium HumanMethylation 450 BeadChips. Next day staining of hybridized arrays produced methylation-dependent differential fluorescence that was detected via an Illumina iScan array scanner.
Project description:We encapsulated common marmoset pluripotent stem cells (cmPSCs) in agarose microgels and optimised culture conditions to generate epiblast and amnion spheroids. Using this system, we investigate the effects of signalling perturbations on epiblast and amnion spheroids as a model for early lineage specification