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% O₂) and hypoxic (5% O₂) groups, and those in adherent culture were divided into normoxic, hypoxic and hypoxia + HIF-1α 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-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 of β-catenin and VEGFA (P < 0.05) and protein expressions of HIF-1 α, 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.
Echinomycin/metabolism* ; Embryoid Bodies/metabolism* ; Humans ; Hypoxia/metabolism* ; Induced Pluripotent Stem Cells/metabolism* ; beta Catenin/metabolism*

Hypoxia-inducible factor-1 (HIF-1), as a transcription factor, plays an important role in the adaptation to hypoxic microenvironment within tumors. It can induce a series of genes transcription that participate in angiogenesis, glucose metabolism, cell proliferation, and cell migration/invasion. Thus HIF-1 not only allows cancer cells to survive in hypoxic microenvironment, but also makes the tumor more aggressive. Moreover, HIF-1 also induces tumors to acquire resistance to chemo-/radio-therapy, and is related to poor prognosis. HIF-1 emerges gradually as a potential target to develop new antitumor drugs. This paper reviews recent progress in this field.
Amphotericin B ; pharmacology ; Animals ; Antineoplastic Agents ; pharmacology ; Echinomycin ; pharmacology ; Humans ; Hypoxia-Inducible Factor 1 ; antagonists & inhibitors ; genetics ; metabolism ; Indazoles ; pharmacology ; Sirolimus ; analogs & derivatives ; pharmacology ; Topotecan ; pharmacology ; Transcription, Genetic

Echinomycin is a small-molecule inhibitor of hypoxia-inducible factor-1 DNA-binding activity, which plays a crucial role in ovarian ovulation in mammalians. The present study was designed to test the hypothesis that hypoxia-inducible factor (HIF)-1alpha-mediated endothelin (ET)-2 expressions contributed to ovarian ovulation in response to human chorionic gonadotropin (hCG) during gonadotropin-induced superuvulation. By real-time RT-PCR analysis, ET-2 mRNA level was found to significantly decrease in the ovaries after echinomycin treatment, while HIF-1alpha mRNA and protein expression was not obviously changed. Further analysis also showed that these changes of ET-2 mRNA were consistent with HIF-1 activity in the ovaires, which is similar with HIF-1alpha and ET-2 expression in the granulosa cells with gonadotropin and echinomycin treatments. The results of HIF-1alpha and ET-2 expression in the granulosa cells transfected with cis-element oligodeoxynucleotide (dsODN) under gonadotropin treatment further indicated HIF-1alpha directly mediated the transcriptional activation of ET-2 during gonadotropin-induced superuvulation. Taken together, these results demonstrated that HIF-1alpha-mediated ET-2 transcriptional activation is one of the important mechanisms regulating gonadotropin-induced mammalian ovulatory precess in vivo.
Animals ; Cells, Cultured ; Chorionic Gonadotropin/*pharmacology ; Echinomycin/*pharmacology ; Endothelin-2/genetics/*metabolism ; Female ; Gonadotropins, Equine/*pharmacology ; Granulosa Cells/drug effects/metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/*antagonists & inhibitors/genetics/metabolism/physiology ; Oligonucleotides/genetics ; Ovary/cytology/drug effects/physiology ; Rats ; Rats, Sprague-Dawley ; Superovulation/*drug effects ; Transcriptional Activation