| Background and Objective:Heart is the first organ function during mammalian embryogenesis formed,and cardiac abnormalities lead to various heart diseases.Elucidation of the mechanisms of human heart development,will help to reveal the etiology and pathogenesis of heart disease,provide clues for the treatment of cardiac regeneration.For decades,rodent models have made important contributions to the exploration of human heart developmental biology and diseases.However,the essential differences between species make the results based on rodents unable to accurately reflect the results of humans,and thus cannot be translated into clinical applications.Human pluripotent stem cells(hPSCs),especially induced pluripotent stem cells(iPSCs)derived from somatic cell reprogramming technology,provide an inexhaustible source of cells for the study of human heart development.The pathogenesis of cardiovascular disease as well as the field of cardiac regenerative cell-based therapy offers an unprecedented opportunity.hPSCs can differentiate into various cardiac lineages and reproduce some important steps of in vivo heart development in vitro.However,most of the current program directed differentiation of cardiac lineage is the use of adherent monolayer cell differentiation,the formation of a single cell type and does not reflect the complexity of real three-dimensional organs and tissues,ignoring other non-cardiac cells(such as epicardial,Cardiac fibroblasts,etc.)contribution to heart development.Even 3D suspension culture,also failed to form a structure in cardiac tissue,it is difficult to process morphology occurs during heart development simulation.Organoids are 3D complexes derived from stem cells or organ-specific progenitor cells and formed through self-assembly.They can simulate real organizational structure and functional characteristics of the organ to a certain extent,it is in understanding human organ development,disease modeling,and regenerative medicine with unprecedented advantages.So far,using organoids as developmental models has decoded many key events in the development of organs,however,previous studies on human heart organoids have not formed a structure similar to the heart in the body.Therefore,this study intends to use human pluripotent stem cells to construct heart organoids in vitro,and establish a new model that can not only cross species differences,but also effectively simulate human early embryonic heart development in vitro.This study provides a powerful platform for studying human heart development and heart disease modeling.Methods:Scientific issues of this study are how to use human pluripotent stem cells to construct heart organoids in vitro.Around this scientific question,three key questions need to be solved.First,clear cell lineages of early heart development constitute,secondly,to establish a stable cardiac lineage cells in vitro system,and finally,to build from heart organ culture system.Therefore,this research will carry out research from the following aspects.The first part:Collect samples of 4 aborted fetuses to obtain complete heart tissue,single cells were obtained after digestion of 3 samples,database construction and 10x genomics single cell sequencing were performed,and the sequencing results were analyzed by bioinformatics to clarify the cell lineage composition in the early stage of heart development.The remaining one case was stained with HE and immunofluorescence.The second part:Collect the discarded foreskin tissue after circumcision,obtain skin fibroblasts by tissue block attachment method,and use reprogramming technology to generate iPSCs.Alkaline phosphatase staining,immunofluorescence staining and flow cytometry were used to detect the pluripotency of iPSC,the G-banding method was used to analyze the karyotype of iPSCs,and teratoma formation assay was used to detect the multi differentiation potential of iPSCs.Then adopt the modified "GiWi" scheme to establish a stable iPSC-CM differentiation system,use qRT-PCR and other methods to detect gene expression during the differentiation process,use immunofluorescence staining and flow cytometry to evaluate the differentiation efficiency of cardiomyocytes,and The cardiomyocytes were identified,and the electrophysiological function of the cardiomyocytes was detected by patch clamp technique.Then adopt the modified "GiWi" scheme to establish a stable iPSC-CM differentiation system,qRT-PCR and other methods were used to detect the gene expression in the process of differentiation.Immunofluorescence staining and flow cytometry were used to evaluate the differentiation efficiency of cardiomyocytes,and the cardiomyocytes were identified.Patch clamp technique was used to detect the electrophysiological function of cardiomyocytes.The third part:Use immunofluorescence staining to identify the expression of cardiovascular precursor cells NKX2-5 and ISL1 during cardiomyocyte differentiation.Select the stage where NKX2-5 and ISL1 are highly expressed,and use CHIR99021 to activate the Wnt signaling pathway again to induce partial differentiation of cells into epicardial lineage cells.Subsequently,cells expressing NKX2-5,ISL1,and WT1 were mixed to construct self-assembled outer membrane heart organoids,and the culture system was optimized,and the cell composition of the organoids was detected by immunofluorescence staining.Results:In the first part,the transcription map of the intact heart of the embryo in the middle stage of development was successfully drawn for the first time:3 heart samples were captured 8075,9095,13992 cell numbers,corresponding to the average number of reads per cell is 40833,36359,21346,the median of the gene per cell 1565,1702,1213.After filtration strict quality control,we were retained 4916,5719,8311 cells for subsequent transcriptome analysis,the average in each single cell were detected to 2751,2821,2065 genes.Eventually we defined the embryonic heart eight major cell types and 15 sub-cell types,for the first time identified a fetal heart lymphocyte population,but also to correct errors identified in previous studies of endocardial cell population.Our research has provided a relatively complete single-cell transcriptional map of the human embryonic heart so far.The second part successfully established iPSC directed cardiomyocyte differentiation system:The skin fibroblasts with better activity were obtained by the tissue block adhesion method,and then the cells were transfected with Sendai virus for reprogramming.Alkaline phosphatase staining confirmed that embryonic stem cells(ESCs)-like cell clones were formed during the reprogramming process.By selecting clones,we finally selected a cell line with better growth for identification and follow-up research(named B12-6).Using immunofluorescence staining and flow cytometry to detect pluripotency genes or proteins OCT4,SOX-2,NANOG,SSEA4 and Tra160 are all highly expressed in B12-6 cells.After 20 generations of subculture,the karyotype is normal,and the teratoma formation experiment confirmed that B12-6 has a good ability to differentiate into three germ layers.Therefore,we used it to construct a cardiomyocyte differentiation system.Adopting the modified "GiWi"scheme,by regulating the Wnt signaling pathway,B12-6 was successfully induced to differentiate into beating cardiomyocytes with a differentiation efficiency close to 90%.The samples collected during the differentiation process were tested by qRT-PCR,and it was confirmed that the gene expression during the differentiation process was similar to the development of the heart in vivo.The immunofluorescence of the differentiated cardiomyocytes indicated that the cardiomyocyte-specific proteins cTnT and a-actinin-4 were highly expressed.The third part successfully constructed an outer-membrane heart organoid:By performing immunofluorescence staining on cells of different days during the differentiation process,we determined that the cardiovascular precursor cells NKX2-5 and ISL1 were highly expressed on the 9th day of differentiation.By reactivating the Wnt signaling pathway,immunofluorescence staining and qRT-PCR results confirmed that NKX2-5+and ISL1+cells were partially induced to express WT1 and TBX18 epicardial lineage cells.Harvest the cells on the 11th day of differentiation,and then let the cells self-assemble into a beating heart organoid in a 96-well U-shaped plate.Immunofluorescence staining confirmed that this organoid has epicardial and myocardial structures,similar to the epicardial and myocardial layers of a real heart.In order to further explore whether the epicardial organoids we constructed can simulate the epithelial to mesenchy-mal transition(EMT)process in vitro,we cultured them adherently.During the culture process,a large number of cells emigrating around the organoids can be seen.Immunofluorescence staining confirmed that these cells include vascular smooth muscle cells,vascular endothelial cells and cardiac fibroblasts,which are highly similar to epicardial-derived cells.Conclusions:1.Using the whole embryonic heart digestion method,we defined 8 main cell types and 15 subcellular types of embryonic heart by using scRNA-seq technology,identified the lymphocyte population of fetal heart for the first time,and corrected the wrong identification of endocardial cell group in previous studies.Our study has provided a relatively complete single-cell transcription map of the human embryonic heart so far,and provided guidance for the subsequent construction of cardiac organoids.2.Through the improved "GiWi protocol",we have successfully established a cell line B12-6 CM directed differentiation system.The system can efficiently produce cardiomyocytes without adding any cytokines and growth factors,and the differentiation process is similar to the in vivo heart development process.Therefore,it can be used to simulate heart development in vitro and also lay the foundation for subsequent experiments.3.We have identified the stage of high expression of cardiovascular precursor cells in the differentiation of cardiomyocytes,and selected the cardiovascular precursor cells at this stage to construct human epicardial heart organoids.This model may be a valuable tool for studying human heart development and the etiology of CHD.In view of the important role of the epicardium in the field of heart regeneration,this model may also be used to simulate heart regeneration in vitro. |
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