Hypoxia Influences the Vascular Expansion and Differentiation of Embryonic Stem Cell Cultures Through the Temporal Expression of Vascular Endothelial Growth Factor Receptors in an ARNT-Dependent Manner

Abstract

Adaptive responses to low oxygen (O 2 ) tension (hypoxia) are mediated by the heterodimeric transcription factor hypoxia inducible factor (HIF). When stabilized by hypoxia, bHLH-PAS a-and b-(HIF-1b or ARNT) HIF complex regulate the expression of multiple genes, including vascular endothelial growth factor (VEGF). To investigate the mechanism(s) through which hypoxia contributes to blood vessel development, we used embryonic stem cell (ESC) differentiation cultures that develop into embryoid bodies (EBs) mimicking early embryonic development. Significantly, low O 2 levels promote vascular development and maturation in wild-type (WT) ESC cultures measured by an increase in the numbers of CD31 1 endothelial cells (ECs) and sprouting angiogenic EBs, but refractory in Arnt 2/2 and Vegf 2/2 ESC cultures. Thus, we propose that hypoxia promotes the production of ECs and contributes to the development and maturation of vessels. Our findings further demonstrate that hypoxia alters the temporal expression of VEGF receptors Flk-1 (VEGFR-2) and the membrane and soluble forms of the antagonistic receptor Flt-1 (VEGFR-1). Moreover, these receptors are distinctly expressed in differentiating Arnt 2/2 and Vegf 2/2 EBs. These results support existing models in which VEGF signaling is tightly regulated during specific biologic events, but also provide important novel evidence that, in response to physiologic hypoxia, HIF mediates a distinct stoichiometric pattern of VEGF receptors throughout EB differentiation analogous to the formation of vascular networks during embryogenesis. STEM CELLS 2010;28:799-809 Disclosure of potential conflicts of interest is found at the end of this article.

Cited2 Gene Controls Pluripotency and Cardiomyocyte Differentiation of Murine Embryonic Stem Cells through Oct4 Gene

“…To generate gelatin-adapted ESCs, cells were weaned off feeder cells by serial passages on 6-well plates coated with 1% gelatin (Sigma) as described elsewhere (31)(32)(33)(34).…”

“…For hematopoietic differentiation, a two-step differentiation method was used as described (31). For primary differentiation, gelatin-adapted ESC (3,000 cells) were seeded on methylcellulose-based semi-solid media containing Iscove's modified Dulbecco's medium, 15% FBS (STEMCELL Technologies), IL-3, and stem cell factor (R&D Systems) as described previously (31).…”

“…For primary differentiation, gelatin-adapted ESC (3,000 cells) were seeded on methylcellulose-based semi-solid media containing Iscove's modified Dulbecco's medium, 15% FBS (STEMCELL Technologies), IL-3, and stem cell factor (R&D Systems) as described previously (31). Medium was replenished on EB day 6.…”

Cited2, a Transcriptional Modulator Protein, Regulates Metabolism in Murine Embryonic Stem Cells

“…ESCs were induced to differentiate via a methylcellulose-based method as described previously (22). For hypoxia experiments, ESCs were cultured or induced to differentiate in an incubator with an adjustable oxygen concentration, as described elsewhere (24).…”

Oxygen-Controlled Automated Neural Differentiation of Mouse Embryonic Stem Cells

“…Physiological oxygen levels that occur during early embryo development have been shown to maintain ESC in a pluripotent state [29][30][31] and to improve their directed differentiation towards cardiac [15,20], vascular [32], neural [33][34][35][36][37] and retinal lineages [38]. Studies from our laboratory revealed that the yield of neural cells from mouse ESC (mESC) can be greatly enhanced by manually operating at 2% O 2 [33].…”