人诱导多能干细胞和胚胎干细胞分化为血管周细胞和内皮细胞过程中差异表达基因分析

Analysis of differentially expressed genes during the differentiation of human induced pluripotent stem cells and embryonic stem cells into pericytes and endothelial cells

目的观察人诱导多能干细胞(hiPSC)、人胚胎干细胞(hESC)向血管周细胞和内皮细胞分化过程中差异表达基因(DEG)的情况,初步寻找可能参与调控分化的关键分子和信号通路。方法选取hiPSC、hESC,以mTeSR培养基进行扩增培养。“两步法”诱导hiPSC、hESC分化为血管周细胞和内皮细胞,免疫荧光染色鉴定周细胞,流式细胞仪分离鉴定内皮细胞。分化过程中,分别提取分化0、4、7、10 d的总RNA样本进行转录组测序,筛选持续显著DEG。对筛选得到的DEG进行基因本体(GO)富集分析、京都基因与基因组百科全书(KEGG)的信号通路富集分析。结果hiPSC、hESC在诱导培养条件下均可成功分化为血管周细胞和内皮细胞。转录组测序结果显示,hiPSC、hESC随分化时间延长,DEG显著上调或下调且变化趋势基本一致。分化过程中,血管周细胞和内皮细胞标志性基因显著上调。hiPSC、hESC共检测到持续DEG 491个。其中,hiPSC、hESC分别为164、335个;两个细胞系同时表达8个。其中,SLC30A3、LCK、TNFRSF8、PRDM14、GLB1L3持续表达下调;CLEC18C、CLEC18B、F2RL2持续表达上调。GO富集分析结果显示,持续上调表达的DEG主要富集在神经发生、分化、发育蛋白等条目中;持续下调表达的DEG主要富集在膜结构、磷脂代谢过程等条目中。KEGG通路分析结果显示,上调表达的基因主要富集于癌症相关通路、干细胞全能性调节通路、Wnt信号通路、Hippo信号通路;下调表达的基因主要富集于代谢相关通路。结论hiPSC、hESC向血管周细胞和内皮细胞分化程中,有8个DEG存在持续特异性表达变化;其可能通过激活Wnt通路、Hippo通路,抑制代谢通路,解除对干细胞多能性的维持以及影响细胞周期、抑制细胞增殖等作用,促进血管周细胞与内皮细胞的分化。

Objective To study the differentially expressed genes(DEG)during the differentiation of human induced pluripotent stem cells(hiPSC)and human embryonic stem cells(hESC)into pericytes and endothelial cells,and to identify key molecules and signaling pathways that may regulate this differentiation process.Methods hiPSC and hESC were selected and expanded using mTeSR medium.A"two-step method"was used to induce the differentiation of hiPSC and hESC into pericytes and endothelial cells.Pericytes were identified using immunofluorescence staining,while endothelial cells were isolated and identified using flow cytometry.Total RNA samples were extracted on days O,4,7,and 10 of differentiation and consistently significant DEGs were screened.Gene ontology(GO)enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)signal pathway enrichment analysis were performed on the screened DEGs.Results Both hiPSCs and hESCs successfully differentiated into pericytes and endothelial cells under induction conditions.Transcriptome sequencing results showed that with the extension of differentiation time,the DEGs in hiPSCs and hESCs were significantly upregulated or downregulated,following a generally consistent trend.During the differentiation process, marker genes for pericytes and endothelial cells were significantlyupregulated. A total of 491 persistent DEGs were detected in both hiPSC and hESC, with 164 unique to hiPSCsand 335 to hESCs, while 8 DEGs were co-expressed in both cell lines. Among these, SLC30A3, LCK, TNFRSF8,PRDM14, and GLBIL3 showed sustained downregulation, whereas CLEC18C, CLEC18B, and F2RL2 exhibitedsustained upregulation. GO enrichment analysis revealed that DEGs with sustained upregulation were primarilyenriched in terms related to neurogenesis, differentiation, and developmental proteins, while DEGs withsustained downregulation were enriched in terms related to membrane structure and phospholipid metabolicprocesses. KEGG pathway analysis showed that upregulated genes were primarily enriched in cancer-relatedpathways, pluripotency regulatory pathways, the Wnt signaling pathway, and the Hippo signalingpathway, whereas downregulated genes were predominantly enriched in metabolism-related pathways.Conclusions During the differentiation of hiPSC and hESC into pericytes and endothelial cells, 8 DEGs exhibitsustained specific expression changes. These changes may promote pericyte and endothelial cell differentiationby activating the Wnt and Hippo pathways, inhibiting metabolic pathways, releasing the maintenance of stemcell pluripotency, affecting the cell cycle, and inhibiting cell proliferation.