Project description:Develop a new guided induced pluripotent stem cell differentiation protocol that rapidly generates diverse range of cardiac-relevant cell types.
Project description:We apply short-read RNA sequencing technology to identify transcripts expressed during four time points of a human induced pluripotent stem cell derived cardiomyocyte differentiation protocol, corresponding to pluripotent, mesoderm, early cardiomyocyte, and cardiomyocyte cell stages. The RNA-seq reads are used to generate custom protein sequence database for proteogenomic applications and downstream mass spectrometry analysis. We demonstrate that this custom RNA-seq-guided proteomics approach can be used to identify protein isoforms that are differentially regulated during cardiac differentiation.
Project description:microRNAs are post transcriptional regulators involved in several biological processes. The main objective of this project is to identify all the microRNAs involved in the regulation of the cardiac differentiation from human pluripotent stem cells. The final goal is to understand RNA networks related to cardiac lineage commitment during human embryonic development.
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:Human induced pluripotent stem cells (hiPSCs) hold significant potential for advancing our understanding of heart development. Differentiating hiPSCs into cardiac cells allows for modeling early cardiac developmental processes and exploring key signaling pathways. However, variations in differentiation efficiency and poor reproducibility of hiPSC-derived cardiomyocytes (hiPSC-CMs) production have remained a challenge. Here, we report a unique metabolic method to promote hiPSC-CM differentiation that involves marked suppression of the mitochondrial oxidative phosphorylation from the mesendoderm to the cardiac mesoderm, which is regulated by PHGDH, a rate-limiting enzyme in the serine synthesis pathway (SSP). Mechanistically, the analysis of scRNA-seq revealed that SSP inhibition promotes CM lineage differentiation by disrupting the cardiopharyngeal mesoderm lineage differentiation. Our findings show that SSP can regulate cardiac differentiation and have implications in elucidating the potential mechanisms of heart development and pathogenesis of heart disease.
Project description:The mouse embryonic stem cell’s differentiation was guided by several treatments, and each stage of differentiation was examined. Keywords: development stage
Project description:Pathological variants in NOTCH1 have been implicated in multiple types of congenital heart defects including bicuspid aortic valve and hypoplastic left heart syndrome (HLHS). To probe how NOTCH1 deficiency affects cardiac development, we generated homozygous NOTCH1 knockout (N1KO) human induced pluripotent stem cells (iPSCs). We then ran single-cell RNA-seq to temporally profile transcriptomic changes during cardiac differentiation in wild type (WT) and N1KO iPSCs. We collected differentiating cells at multiple time points corresponding to different development stages, i.e., Day 0 (D0: pluripotent stem cell), D2 (mesoderm), D5 (cardiac mesoderm), D10 (cardiac progenitor), D14 (early cardiomyocyte), and D30 (fetal cardiomyocyte). Single-cell transcriptomics analysis reveals that NOTCH1 disruption impairs human ventricular cardiomyocyte differentiation and proliferation through balancing cell fate determination of cardiac mesoderm toward the first heart field, second heart field, and epicardial lineages.
Project description:Cardiovascular diseases are a major cause of life-threatening burden around the world. The heart has a very low regeneration capacity and donor organs for transplantation are scarce. Therefore regeneration of lost myocardium with stem cell-derived cardiomyocytes (CMs) provides an attractive strategy for heart repair. Human pluripotent stem cells (hPSCs) can be efficiently differentiated in vitro into CMs but the molecular mechanisms behind this process are still not fully understood. In particular identification of secreted autocrine and/or paracrine factors that function as important extrinsic signals remained elusive because the mass spectrometry (MS)-based identification of secreted proteins from cell culture supernatants is impeded by high levels of albumin present in common differentiation media. Thus we established an albumin-free cardiomyogenic differentiation medium and performed secretomics at seven different time points during in vitro differentiation. This analysis led to the identification of 4832 proteins with 1802 being significantly altered during differentiation and 431 of these were annotated as secreted according to gene ontology. Bioinformatics revealed enrichment of extrinsic Wnt pathway-related proteins 3 days upon induction of differentiation and of extracellular matrix proteins in the resulting CMs. Numerous extrinsic components of Wnt, Activin A, Nodal, TGFβ, BMP or FGF signaling pathways were quantitatively assessed during differentiation. Notably, the abundance of pathway agonists was generally lower compared to the respective antagonists but their curves of progression over timer were rather similar. We hypothesize that Activin A, Nodal and TGFβ signaling are turned down shortly upon initiation of cardiac differentiation whereas BMP signaling is switched on. Wnt and FGF signaling peaks between d0 and d3 of differentiation and interestingly, Activin A and TGFβ signaling seem to be reactivated at the cardiac progenitor stages and/or in early CMs.