Abstract
Dissecting the gene expression programs which control the early stage cardiovascular development is essential for understanding the molecular mechanisms of human heart development and heart disease. Here, we performed transcriptome sequencing (RNA-seq) of highly purified human Embryonic Stem Cells (hESCs), hESC-derived Multipotential Cardiovascular Progenitors (MCPs) and MCP-specified three cardiovascular lineages. A novel algorithm, named as Gene Expression Pattern Analyzer (GEPA), was developed to obtain a refined lineage-specificity map of all sequenced genes, which reveals dynamic changes of transcriptional factor networks underlying early human cardiovascular development. Moreover, our GEPA predictions captured ~90% of top-ranked regulatory cardiac genes that were previously predicted based on chromatin signature changes in hESCs, and further defined their cardiovascular lineage-specificities, indicating that our multi-fate comparison analysis could predict novel regulatory genes. Furthermore, GEPA analysis revealed the MCP-specific expressions of genes in ephrin signaling pathway, positive role of which in cardiomyocyte differentiation was further validated experimentally. By using RNA-seq plus GEPA workflow, we also identified stage-specific RNA splicing switch and lineage-enriched long non-coding RNAs during human cardiovascular differentiation. Overall, our study utilized multi-cell-fate transcriptomic comparison analysis to establish a lineage-specific gene expression map for predicting and validating novel regulatory mechanisms underlying early human cardiovascular development.
Highlights
Heart formation is a stepwise process, including the consecutive differentiation of mesoderm, cardiac progenitor, and the terminal specification of cardiovascular lineage cells[1,2,3]
Pluripotency marker genes, such as Nanog Homeobox (NANOG), POU5F1 (OCT4), and SOX2 were exclusively expressed in human Embryonic Stem Cells (hESCs)
principle component analysis (PCA) revealed that the whole gene expression signature of multipotential cardiovascular progenitor cells (MCPs) was close to that of hESCs and far from those of the specified cardiovascular lineages, indicating a temporal change of whole transcriptional signatures during cardiovascular differentiation
Summary
Heart formation is a stepwise process, including the consecutive differentiation of mesoderm, cardiac progenitor, and the terminal specification of cardiovascular lineage cells[1,2,3]. With the recent advances in stem cell biology, the majority of human somatic cell types could be differentiated from human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs)[2] This provides a unique access to the purified populations of cell lineages of interest at different differentiation stages. A recent study showed that during cardiac differentiation in human ESCs, cardiac regulatory genes, most of which are transcriptional factors, have distinct dynamic patterns of histone modifications from the CM-specific structural sarcomeric genes, indicating that combined analysis of histone modification dynamics plus gene expression profiles could be used to predict regulatory genes in early human CM development[13]. We performed deep-transcriptome sequencing (RNA-seq) of hESCs, MCPs, CMs, SMs and ECs, which represent pluripotency, multipotency and lineage-specification stages of early human heart formation, respectively. All the results demonstrate that the cardiovascular cell resources and multi-cell-fate comparison algorithm could allow us to uncover and validate novel regulatory mechanisms during early human cardiovascular development
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