Analysis Of DNA Methylation Profile During Erythroid Differentiation From HESCs/hiPSCs And Differentiation Of HHFMSCs Into RBCs By OCT4

Erythropoiesis may be controlled by both genetics and epigenetics programs.Genome expression dynamics may be regulated by DNA methylation for promotingerythropoiesis, and the transcription factors may induce the differentiationof erythrocytes by regulating transcription of target genes. Here, we investigate theeffects of DNA methylation during differentiation of human embryonic stem cells(hESCs) and iPSCs toward erythroid cells based on combination of human wholegenome DNA methylation array and gene expression array, and research the role ofOCT4to induce the human hair follicle mesenchymal stem cells (hHFMSCs) fortransdifferentiation into red blood cells.1. Effects of DNA methylation driving erythroid differentiation from hESCs.Analysis of differentially methylated CpG (DMC) sites and gene expression wererespectively performed. The three cell samples (hESCs, n=3; hES-EBs, n=3; andhES-BLs, n=3or n=4) were randomly distributed across the arrays. The DNAmethylation portrait was described according to the distribution of CpG content and theneighborhood context, functional genomic distribution, and associated RNAtranscription. Correlation between gene expression and DNA methylation was detected,and then the functions of genes with anti-correlation were enriched by GO and KEGGpathway following by constructing network of regulation by GeneSpring software. Atlast, the results from arrays were verified by RT-qPCR and real-time MSP. The resultsare shown below:(1) Profiling of DNA methylation at CpG sites during erythroid differentiationfrom hESCsWe identified639DMC sites occurred in hES-EBs in comparison to hESCs, and100991DMC sites as well as134736DMC sites occurred in hES-BLs in comparison to hES-EBs and hESCs respectively. The hypermethylated sites enriched in CpGislands and shores and promoters, and hypomethylated enriched in CpG shelves andopen sea and gene body, however, the percentages of association with coding RNAwere not shown substantial difference.（2）Profiling of gene expression changes during erythroid differentiation fromhESCs①The expression of810genes were changed in hES-EBs versus hESCs, and5264genes as well as5843genes expressed differently in hES-BLs as compared tohES-EBs and hESCs respectively.②Upon DAVID analysis for GO term enrichment in hES-EBs versus hESCs,up-regulated genes were involved in development of the circulatory system, whiledown-regulated genes were involved in the biological adhesion and nervous systemdevelopment; upon differentiation of hES-EBs into hES-BLs, up-regulated geneswere found to be involved in hematopoiesis, particularly in erythrocyte developmentand maturation, while the majority of the down-regulated genes clustered into thecategory of nervous system development and embryonic development.③KEGG pathway analysis showed that up-regulated genes in hES-EBs ascompared to hESCs were mainly involved in TGF-beta, pathways in cancer, andcomplement and coagulation cascades. None of pathway was enriched fordown-regulated genes. Upon differentiation of hES-EBs into hES-BLs, up-regulatedgenes were mainly involved in hematopoietic cell lineage, chemokine signalingpathway, and JAK-STATs signaling pathway, etc., while the down-regulated genesclustered into the pathways of tight junction and cell cycle.(3) Analysis of genes with anti-correlation between DNA methylation and geneexpression.①Selection of genes with anti-correlation and profiling the genomic distributonof DMC sites: we identified15genes with anti-correlation upon differentiation ofhESCs into hES-EBs, and2624genes with anti-correlation were selected in hES-BLsas compared to hES-EBs. DMC sites were mainly enriched in promoters and genebodys.②GO analysis and genomic distribution of DMC sites in genes involved in specific biological processes: upon DAVID analysis for GO term enrichment inhES-BLs versus hES-EBs, up-regulated genes were involved in erythrocyte specificcellular components, molecular functions, and also the biological processes; whiledown-regulated genes were involved in the cell junctions, embryonic development,and nervous system development; hematopoiesis genes were hypomethylated andup-regulated, and their hypomethylated sites were enriched in CpG shelves and opensea, hypomethylation of gene body was involved in enhancers and DHSs;pluripotency genes and neurogenesis genes were hypermethylated anddown-regualted, and their DMC sites were mainly enriched in CpG islands andshores.③KEGG analysis and gene regulatory networks: a total of53signalingpathways were enriched, upregulated genes involved in38pathways, down-regulatedgenes involved in15signaling pathways. Among these pathways, chemokine andhematopoietic cell lineage have highest enrichment degree,.in the process of erythroiddifferentiation, OCT4was hyermethylated and down-regulated, and the expression of101OCT4target genes have changed. Erythropoiesis related genes were upregulated,and embryonic and nervous development genes were down-regulated.(6) Verification of DNA methylation and gene expression microarray results①Real-time MSP showed that when hES-EBs and hES-BLs were compared tohESCs respectively, partly demethylation occurred in promoters of GATA2andGYPB, and the promoter of SOX2were partly methylated, which validated ourmicroarray-based genome-wide measurement of DNA methylation.②RT-qPCR showed that when hES-EBs and hES-BLs were compared tohESCs respectively, the hematopoiesis genes (GATA2, TAL1, LMO2, CD34, andGYPB) were up-regulated and the pluripotency genes OCT4and SOX2weredown-regulated, which validated the microarray-based genome-wide measurement ofgene expression.2. Effects of DNA methylation upon erythroid differentiation from hiPSCsWe performed the analysis of gene expression and DNA methylation in three cellsamples (iPSC，n=3；iPS-EB，n=3；iPS-BL，n=3) using the same method mentionedabove, the results are shown below: (1) Profiling of DNA methylation at CpG sites during erythroid differentiationfrom hiPSCsWe identified759DMC sites occurred in iPS-EBs in comparison to iPSCs, thehypermethylated sites were more than hypomethylated ones; and66922DMC sites aswell as59003DMC sites occurred in iPS-BLs in comparison to iPS-EBs and iPSCsrespectively. The hypomethylated sites were more than hypermethylated ones. Thegenes with DMC sites were mainly involved in metabolic process, biologicalregulation, and development process.(2) Profiling of Gene expression changes during erythroid differentiation fromiPSCsThe expression of1473genes were changed in iPS-EBs versus iPSCs, and2776genes as well as5137genes expressed differently in iPS-BLs as compared to iPS-EBsand iPSCs respectively. In iPS-EBs versus iPSCs, up-regulated genes were involvedin vasculature and heart development; while down-regulated genes were involved inresponds to stimulations and process of the circulatory system. Upon differentiation ofiPS-EBs into iPS-BLs, up-regulated genes were found to be involved mainly inhematopoiesis and erythrocyte differentiation, whtile the down-regulated genes wereinvolved in nervous system development; In iPS-BLs versus iPSCs up-regulatedgenes were found to be involved mainly in heme metabolism and regulation ofcell apoptosis; while the majority of the down-regulated genes clustered into thecategories of cell cycle and cell-cell junctions.(3) Analysis of genes with anti-correlation between DNA methylation and geneexpression.①Selection of genes with anti-correlation and profiling the genomic distributonof DMC sites: we identified15genes with anti-correlation upon differentiation ofiPSCs into iPS-EBs, and2185genes with anti-correlation were selected in iPS-BLs ascompared to iPS-EBs. DMC sites were mainly enriched in promoters and gene bodys.②GO analysis: upon DAVID analysis for GO term enrichment in iPS-BLs ascompared to iPS-EBs, genes with anti-correlation were involved in regulationof vascular size, blood circulation, and cell adhesion, etc., but the biological processesof erythrocyte were not found. (4) The expression changes in target genes of OCT4during erythrocytedifferentiation from iPSCsExpression of OCT4was up-regulated upon differentiation of iPSCs intoiPS-EBs, and total36of target genes were differentially expressed; while expressionof OCT4was not changed upon differentiation of iPS-EBs into iPS-BLs, and total52of target genes were differently exressed. Both of up-and down-regulated genes wereinvovled in embryonic development, nervous development, and mesodermdevelopment, and few of hematopoiesis genes were up-regulated.3. Transdifferentiation of human hair follicle mesenchymal stem cells into redblood cells by OCT4hHFMSCs were infected with lentivirus expressing OCT4following byobersivation of celluar morphology and expression of hematopoiesis genes. FloatinghHFMSCsOCT4were cultured in hematopoietic medium supplemented with acombination of hematopoietic cytokines, then cells were cultured in erythroid cellexpansion medium, and finally, late stage erythroid precursors were cultured inerythroid enucleation medium to induce enucleation. During trans-differentiation oferythrocyte, we observed celluar morphology using Wright-Giemsa staining, detectedexpression of erythrocyte specific genes using immunofluorescence cytochemistryand flow cytometry, finally, detected the expression of target genes of OCT4usingRT-qPCR, the results are shown below:（1） Enforced expression of OCT4in hHFMSCs leads to the changes incellular morphology and expression of hematopoiesis genes①About88.92%of OCT4lentivirus transfected hHFMSCs (hHFMSCsOCT4)were EGFP+as shown by flow cytometric analysis. Expression of OCT4gene wasconfirmed at both mRNA and protein levels by RT-PCR and Western analyses.②After transduction, cell size gradually decreased，and spindle-shaped cellschanged to round-like cells. After14days, a subset of cells emerged fromOCT4-transfected hHFMSCsOCT4, which were easily floating and formed clustersafter re-platting.③Flow cytometry analysis showed that1.5%of CD45+cells were emergedafter transduction, and a population of CD34+cells (up to2.19%) was increased inXIII floating hHFMSCsOCT4.（2） Genenration of erythroid cells from floating hHFMSCsOCT4with thestimulation of hematopoietic cytokines①Hematopoietic clusters were observed at day7, from day10to day20, wesuccesively obtained large classic BFU-E and late progenitors CFU-E.Morphologically, Wright-Giemsa staining showed that the first macroblast wasobserved at day7, and then basophilic normoblast and acidophilic normoblasts weregenerated between days10and15. By day20, the number of acidophilic normoblastswas no longer increased.②Both CD71+and CD235a+erythroid cells could be detected between day7and day20, and hHFMSC-derived erythrocytes were AB blood type for expressions ofblood group A and B antigens.③After enucleation at day23more than80%of erythrocytes expressed adultglobin β-chain.④Progressive decrease of cell size and N/C ratio, and accumulation ofhemoglobin were observed during transdifferentiation.（3） Expression of OCT4turns on erythropoiesis program in hHFMSCsEnforced expression of OCT4significantly increased expression of pluripotentgene (OCT4, NANOG, SEMA3A and LEFTY2) and hematopoietic genes (FLI1,TAL1, HBG1, and CA2); at day21after hematopoietic induction, expression levelsof SOX2, LEFTY2, and OCT4were decreased, and the expression levels of TAL1,HBG1, and RHD were increased.In summary, our data emphasize that erythroid differentiation requiresdevelopment stage dependent genome-wide expression dynamics, which may beregulated by DNA methylation. Therefore, the current study provides a potentialepigenetics strategy for in vitro erythroid differentiation of pluripotent stem cells.Moreover, we have developed a robust trans-differentiation strategy in vitro whichdirectly converse hHFMSCs into enucleated mature RBCs, that will provide analternative way to generate adult RBCs for patient-specific transfusion.