Fang L, Feng Z, Mei J, Zhou J, Lin Z
Guangdong Cardiovascular Institute, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou 510080, China.
Ji Hua Institute of Biomedical Engineering Technology, Ji Hua Laboratory, Foshan 528200, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2022 Jun 20;42(6):929-936. doi: 10.12122/j.issn.1673-4254.2022.06.18.
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.
EBs in suspension culture were divided into normoxic (21% O) and hypoxic (5% O) groups, and those in adherent culture were divided into normoxic, hypoxic and hypoxia + HIF-1 inhibitor (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-1, β-catenin and VEGFA were detected in response to hypoxic culture and echinomycin treatment.
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 ( < 0.05) with also a greater outgrowth rate of the adherent EBs ( < 0.05). The EBs in hypoxic culture showed significantly up-regulated mRNA expressions of β-catenin and VEGFA ( < 0.05) and protein expressions of HIF-1 , β-catenin and VEGFA ( < 0.05), and their protein expresisons levels were significantly lowered after treatment with echinomycin ( < 0.05).
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-1/β-catenin/VEGFA signaling pathway can enhance the differentiation potential of hiPSCs.
研究生理性缺氧条件对人诱导多能干细胞（hiPSCs）分化过程中拟胚体（EBs）悬浮和贴壁的影响，并探讨其潜在机制。
将悬浮培养的EBs分为常氧（21% O₂）组和缺氧（5% O₂）组，将贴壁培养的EBs分为常氧组、缺氧组和缺氧 + HIF-1抑制剂（棘霉素）组。用免疫荧光法鉴定多能性后，将hiPSCs在常氧和缺氧条件下消化并悬浮5天，显微镜下观察EBs的形成及形态变化；检测EBs中三个胚层标志基因的表达。然后将EBs接种到培养皿中，在常氧和缺氧条件下进一步培养2天，之后观察贴壁EBs的贴壁及周边扩展率；检测缺氧培养和棘霉素处理后HIF-1、β-连环蛋白和VEGFA表达的变化。
在常氧和缺氧条件下培养的EBs均能分化为三个胚层。与常氧培养相比，缺氧条件下培养的EBs周围凋亡碎片减少，囊状EBs出现更早且大小更均匀。缺氧培养诱导产生的贴壁EBs比常氧培养更多（P < 0.05），贴壁EBs的扩展率也更高（P < 0.05）。缺氧培养的EBs中β-连环蛋白和VEGFA的mRNA表达显著上调（P < 0.05），HIF-1、β-连环蛋白和VEGFA的蛋白表达也显著上调（P < 0.05），用棘霉素处理后其蛋白表达水平显著降低（P < 0.05）。
缺氧可促进悬浮EBs的形成和成熟，增强其贴壁及贴壁后增殖能力，且不影响其向三个胚层分化的多能性。我们的结果提供了初步证据，表明HIF-1/β-连环蛋白/VEGFA信号通路的激活可增强hiPSCs的分化潜能。
