The Underlying Mechanisms For Differentiation Of Human Pluripotent Stem Cells Into Cardiac Lineage

Background and objective:Cardiac differentiation from human pluripotent stem cells provides a unique opportunity to study human heart development in vitro and offers a potential cell source for cardiac regeneration.Compared to the large body of studies investigating cardiac matu ration and cardiomyocyte subtype-specific induction,molecular events underlying cardiac lineage commitment from pluripotent stem cells at early stage remain poorly characterized.On the other hand,enzymes that can catalyze changes in the structure of chromatin,chromatin remodeling complexes,are usually assembled into multi-subunits complexes to function.They are useful for gene transcription,cell cycle development,DNA replication and damage repair in eukaryotes.Despite the importance of chromatin remodeling complexes for normal heart development,little is known about how chromatin remodeling complexes are programmed for cardiac differentiation.Research reports that the chromatin remodeling complex INO80 participates in many biological processes,including DNA repair.transcription,and replication,that plays an important regulatory role.The INO80 complex can bind to the promoter region of the major transcription factor gene of embryonic stem cells to keep the chromatin region open.Mutations of the INO80 complex have been found to be related to cardiovascular disease,but the role of the INO80 complex in cardiomyocyte differentiation in vitro has not been explored.The purpose of this study was to use single-cell sequencing technology to explore the mechanism of early cardiac cell fate commitment from the single cell level and quest chromatin remodeling complex INO80 in regulating cardiomyocyte fate determination.Methods:The six key time points(TO,T2,T5,T9,T14,T60)in the differentiation process were sequenced by single cell sequencing;the expression of the representative proteins of the four cell subsets of T5 was further verified by immunofluorescence technology;the expression of ETS1 and INO80 was constructed by shRNA knockdown,and the change of cardiac-specific gene expression was detected by quantitative RT-PCR;the differential expression genes of the after knocking down INO80 were analyzed by RNA-seq.The mechanism was studied by ChIP-seq and Mnase-seqResults:In order to uncover key molecular events and regulators controlling cardiac lineage commitment from a pluripotent state during differentiation,On the one hand,we performed single-cell RNA-Seq sequencing and obtained high-quality data for 6879 cells collected from 6 stages during cardiac differentiation from human embryonic stem cells and identified multiple cell subpopulations with distinct molecular features,including pluripotent stem cell subpopulation,early meso-endoderm cell subpopulation,late meso-endoderm cell subpopulation,cardiac progenitor cell subpopulation,endoderm like cell subpopulation,cardiomyocyte subpopulation,etc.By constructing the developmental locus map of myocardial differentiation and the hypothetical ligand receptor interaction analysis,we found that there was a potential paracrine pathway regulating cardiac precursor cells in the endodermal cells of T5,which may provide a cell microenvironment for the fate determination of cardiac lineage on the 5th day.Through constructing developmental trajectory of cardiac differentiation and putative ligand-receptor interactions,we revealed crosstalk between cardiac progenitor cells and endoderm cells,which could potentially provide a cellular microenvironment supporting cardiac lineage commitment at day 5.In addition,computational analyses of single-cell RNA-Seq data unveiled ETS1(ETS Proto-Oncogene 1)activation as an important downstream event induced by crosstalk between cardiac progenitor cells and endoderm cells.Consistent with the findings from single-cell analysis,chromatin immunoprecipitation followed by high-throughput sequencing(ChIP-Seq)against ETS1 revealed genomic occupancy of ETS 1 at cardiac structural genes at day 9 and day 14,whereas ETS1 depletion by shRNA dramatically compromised cardiac differentiation.Western blot analysis revealed that the chromatin remodeling complex INO80 was significantly upregulated at cardiac progenitor stage during cardiac differentiation and regulated transcription of key regulatory genes for cardiac lineage commitment.Through the chromatin immunoprecipitation sequencing(ChIP-seq)of INO80 at each key stage of cardiac differentiation,five INO80 genome-wide binding patterns were identitied,of which cluster 4 showed a gradual combination increase from the pluripotent stem cell stage to the cardiomyocyte stage,this cluster genes are significantly enriched in the cardiac tissue development pathway.Proteomic analysis uncovered distinct combinatorial assembly of the INO80 subunits during cardiac differentiation.Our data identified BRG1,SWI/SNF chromatin-remodeling core subunit,which physically interacts and functionally cooperates with INO80 that are required for transcriptional regulation of the target genes.Cross-analysis with epigenetic dynamics elucidated unidentified interactions between INO80 and H3K4me3 in cardiac differentiation.Finally,INO80 loss by shRNA resulted in growth retard of terminally differentiated cardiomyocytes.Conclusion:Together,our study not only characterized the molecular features of different cell types during cardiac differentiation in vitro and identified ETS1 as a crucial factor induced by cell-cell crosstalk contributing to cardiac lineage commitment from a pluripotent state,but also identified INO80 as an essential factors for transcriptional activation of critical cardiac specific genes during cardiac progenitor induction.Our results also establish the relevance of different ATP-dependent chromatin remodelers in the cardiac differentiation of human embryonic stem cell.Our work has important implications for understanding human heart development at early embryonic stage,as well as directed manipulation of cardiac differentiation in regenerative medicine.