低氧环境可体外促进人诱导多能干细胞分化为拟胚体

Hypoxia promotes differentiation of human induced pluripotent stem cells into embryoid bodies in vitro

目的探讨人诱导多能干细胞向拟胚体分化过程中,生理性低氧环境在拟胚体悬浮和贴壁阶段的影响及其相关机制。方法本研究中拟胚体悬浮阶段分为悬浮常氧组(21%O_(2))和悬浮低氧组(5%O_(2)),贴壁阶段分为贴壁常氧组(21%O_(2))、贴壁低氧组(5%O_(2))、贴壁低氧组+HIF-1α抑制剂Echinomycin。首先采用免疫荧光法鉴定人诱导多能干细胞的多能性,人诱导多能干细胞消化后分别在21%氧气和5%氧气条件下悬浮培养5 d,利用显微镜观察悬浮阶段拟胚体的形成和形态变化,5 d后检测拟胚体中三胚层标记基因的表达情况。接着分别取等量的拟胚体接种到明胶包被的培养皿,继续在21%和5%氧气条件下培养2 d,观察贴壁阶段拟胚体的粘附性能和粘附后向外周扩增速度的差异,检测低氧信号通路相关的HIF-1α、β-catenin和VEGFA的表达情况,随后在5%氧气条件下使用HIF-1α特异性抑制剂Echinomycin后,再次检测低氧信号通路相关的HIF-1α、β-catenin和VEGFA的表达情况。结果常氧组和低氧组的拟胚体均具有三胚层分化的潜力。与常氧培养组相比,低氧组悬浮拟胚体周围的凋亡碎片明显减少,且较早出现囊性拟胚体,获得的拟胚体大小更加均匀一致。此外,低氧组的拟胚体贴壁数量明显高于常氧组(P<0.05),拟胚体贴壁后向外生长的速度明显快于常氧组(P<0.05)。低氧组HIF-1α的mRNA未显著上调(P>0.05),而β-catenin和VEGFA的mRNA显著上调(P<0.05);在蛋白质水平,低氧信号通路相关的HIF-1α、β-catenin和VEGFA的表达均上调(P<0.05)。低氧组使用HIF-1α特异性抑制剂Echinomycin后,低氧信号通路相关的HIF-1α、β-catenin和VEGFA的蛋白质表达均下调(P<0.05)。结论低氧条件不影响拟胚体的三胚层分化潜力。生理性低氧可以促进悬浮拟胚体的形成和成熟,并且能增强拟胚体的贴壁和贴壁后增殖性能,初步证实通过激活HIF-1α/β-catenin/VEGFA信号通路,提升人诱导多能干细胞的分化能力。

Objective To investigate effects of physiological hypoxic conditions on suspension and adherence of embryoid bodies(EBs)during differentiation of human induced pluripotent stem cells(hiPSCs)and explore the underlying mechanisms.Methods EBs in suspension culture were divided into normoxic(21%O_(2))and hypoxic(5%O_(2))groups,and those in adherent culture were divided into normoxic,hypoxic and hypoxia+HIF-1αinhibitor(echinomycin)groups.After characterization of the pluripotency with immunofluorescence assay,the hiPSCs were digested and suspended under normoxic and hypoxic conditions for 5 days,and the formation and morphological changes of the EBs were observed microscopically;the expressions of the markers genes of the 3 germ layers in the EBs were detected.The EBs were then inoculated into petri dishes for further culture in normoxic and hypoxic conditions for another 2 days,after which the adhesion and peripheral expansion rate of the adherent EBs were observed;the changes in the expressions of HIF-1α,β-catenin and VEGFA were detected in response to hypoxic culture and echinomycin treatment.Results The EBs cultured in normoxic and hypoxic conditions were all capable of differentiation into the 3 germ layers.The EBs cultured in hypoxic conditions showed reduced apoptotic debris around them with earlier appearance of cystic EBs and more uniform sizes as compared with those in normoxic culture.Hypoxic culture induced more adherent EBs than normoxic culture(P<0.05)with also a greater outgrowth rate of the adherent EBs(P<0.05).The EBs in hypoxic culture showed significantly up-regulated mRNA expressions ofβ-catenin and VEGFA(P<0.05)and protein expressions of HIF-1α,β-catenin and VEGFA(P<0.05),and their protein expresisons levels were significantly lowered after treatment with echinomycin(P<0.05).Conclusion Hypoxia can promote the formation and maturation of suspended EBs and enhance their adherence and post-adherent proliferation without affecting their pluripotency for differentiation into all the 3 germ layers.Our results provide preliminary evidence that activation of HIF-1α/β-catenin/VEGFA signaling pathway can enhance the differentiation potential of hiPSCs.