. 2020 Nov 29;10(12):1614. doi: 10.3390/biom10121614

Table 1.

The summary of studies investigating the impact of hypoxia on angiogenesis-related markers and properties of pluripotent stem cells.

Cell Type	Hypoxia Level	Duration ^#	Scheme of the Experiment	Angiogenesis-Related Markers and/or Properties Increased vs. Normoxia (20% or 21% O₂)	References
mESCs	1% O₂	7 days	Undifferentiated mESCs plated on collagen IV-coated plate and differentiated in ECDM containing Iscove’s Modified Dulbecco’s Medium (50%), 50% Ham’s F-12 medium (50%), supplemented with N2, B27 retinoic acid, 0.05% lipid-rich BSA, ascorbic acid (50 ng/mL), MTG (50 ng/mL), human VEGF (50 ng/mL), human FGF2 (10 ng/mL), and BMP4 (2 ng/mL)	CD144, PECAM1 mRNA and protein Percentage of CD144⁺PECAM1⁺ cell population HIF-1α protein Glut1, Pdk1, Pdk3, Pdk4, Ldha, Etv2, Ephb2, Notch1, Dll4 mRNA	[76]
hESCs	1% O₂	7 days	Undifferentiated hESCs plated on Matrigel-coated plate in Essential 8 medium. The next day, medium replaced with ECDM containing Iscove’s Modified Dulbecco’s Medium (50%), Ham’s F-12 medium (50%), insulin-transferrin-selenium-X, chemically defined lipid concentrate (1%), BSA (5 mg/mL), ascorbic acid (50 ng/mL), and 1-Thioglycerol (200 μM) supplemented with activin A (25ng/mL), BMP4 (10 ng/mL), VEGF (50 ng/mL), CHIR (1.5 μM) and incubated for 3 days. At days 3, 5, 7 medium replaced with serum-free differentiation medium supplemented with VEGF (50 ng/mL) and SB431542 (10 μM)	Percentage of CD144⁺PECAM1⁺ cell population
hESCs	1% and/or 5% O₂	up to 15 days	Undifferentiated hESCs cultured on ECM from human foreskin fibroblasts under hypoxic conditions in a foreskin-fibroblast conditioned medium composed of DMEM/F12, BME (100 µM), L-glutamine (1 mM), NEAA (100 mM), serum replacement (20%) without changing the media	Endothelial-like morphology of the cells VEGF, VEGFB, VEGFR2 mRNA ANGPTL4 mRNA and protein CD34 mRNA and protein, CD34⁺ cell population ECs phenotype: * CD34⁺ population: 57.7% VEGF⁺ population: 53.4% ANGPTL4⁺ population: 80.8% PECAM1⁺ population: 11.6% vWF⁺ population: 31.9%	[79]
hESCs	1% and/or 5% O₂	up to 15 days	As above but after 5–7 days cells plated on Matrigel and further cultured for 1–7 days	Sprouting of cellular aggregates expressing CD34 (24 h) Formation of cordlike structures expressing CD34 and VEGFR2 (3 days) More dense cordlike structures expressing PECAM1 and vWF (7 days)	[79]
mEBs Derived from mESCs	1% O₂	3 days	After 3 days from mEBs formation, mEBs were kept under hypoxic conditions for 3 days for spontaneous differentiation	Vegf, Vegfr2, Pecam1, Acta2, Cd144, Vegfr1, Fgf2 mRNA HIF-1α, HIF-2α protein	[77]
mEBs Derived from mESCs	1% O₂	2 days	After 3 days from mEBs formation, mEBs were kept under hypoxic conditions for 2 days and seeded on a plate coated with 0.3% gelatin in DMEM/10% FBS. Next day, the culture medium was replaced with EGM2 medium supplemented with FBS (5%) and cells were incubated up to 14 days	Spreading of mEBs PECAM1⁺, CD144⁺ cells HIF-1α protein Vegf, Pecam1, Vegfr2, Cd144 mRNA Junctional distribution of PECAM1 exhibiting tubular and branching structure especially in the central region on 7 and 14 days after reattachment α-SMA, CD144 positive cells in the outgrowth region on later time points, 14 days after reattachment	[77]
mEBs Derived from mESCs	3% O₂	5 to 10 days	mEBs differentiated under hypoxic conditions in methylcellulose or in suspension for 5 to 9 days without exogenous VEGF	PECAM1⁺ cells	[78]
			mEBs differentiated for 7 and 10 days	Adm, Ang1, Ang2, Vegfr2, Tie2, Tie1 mRNA (by day 7) Epo, Tie2, Tie1 mRNA (at day 10)
			Differentiation of mESCs into 10-day EBs in methylcellulose containing VEGF (25 ng/mL) and FGF2 (100 ng/mL). EBs were then replated in collagen-type-I gel matrix for 4 days	Increased percentage of highly angiogenic, sprouting cells
			As above but with a lower concentration of FGF2 (25 ng/mL) and VEGF (5 ng/mL or without this factor)	Elevated number of highly angiogenic, sprouting cells
			EBs differentiated in suspension or methylcellulose cultures in the absence of exogenous VEGF under hypoxic conditions up to 9 days	VEGFR2⁺mVEGFR1⁺ cells sVEGFR1 protein
hEBs Derived from hESCs	3% O₂	7 days	hEBs transferred to hypoxic conditions 3 days after hEBs formation (cultured in DMEM/F12 medium supplemented with serum replacement (10%), L-glutamine (1 mM), NEAA (1%), BME (100 mM) without FGF2 treatment	VEGFR2, PECAM1, CD144, TIE2, FGFR1, PDGFBR mRNA HIF-1α, VEGF, FGF2, ANG1, PDGFB/PDGF-BB on mRNA and protein VEGF, FGF2, PDGF-BB and, to a lesser extent ANG1 on protein (secreted) Percentage of VEGFR1⁺, TIE2⁺, VEGFR2⁺, CD144⁺ and PECAM1⁺ cell populations	[80]
hEBs Derived from hESCs	3% O₂	7 days	Cells plated on Matrigel after 7 days for additional 3 days	PECAM1⁺ and vWF⁺ cells spontaneously forming vessel-like structures Increased number of sprouts and, to a lesser extent, their length	[80]
hEBs Derived from hESCs	1% and 5% O₂	7 days	Differentiating hEBs exposed to hypoxic conditions in a sealed 6.2-L modulator incubator; half-media changes occurred every 3 to 4 days as needed	HIF-1α protein VEGF, GLUT1 mRNA	[81]
hESCs and/or hiPSCs	5% O₂	6 days (primed) or 12 days (continuous)	hPSCs plated onto collagen IV-coated plates and cultured in a differentiation medium composed of α-MEM, FBS (10%) and BME (0.1 mM) Primed: cells attached in normoxic conditions for 4 hours and then subjected to hypoxia. On day 6, differentiated cells were collected, seeded on collagen-type-IV-coated plates in ECGM supplemented with FBS (2%) VEGF (50 ng/mL) and SB431542 (10 µM) for an additional 6 days Continuous: cells attached for 4 hours in normoxic conditions, and then subjected to continuous 5% O₂ conditions	After 6 days: CD34, VEGFR2, CD56 mRNA Primed and continuous: CD144, PECAM1 mRNA; PECAM1⁺ cells; lectin binding, uptake of acLDL, tube formation on Matrigel Continuous: endothelial-like morphology with bundles of elongated cells and cobblestone area-forming cells; CD144⁺ cells; CD144 and PDGFRβ localized with CD144⁺ clusters surrounded by PDGFRβ⁺ cells	[82]
hESCs and/or hiPSCs	1% and 5% O₂	up to 3 days	hESC and hiPSC cells grown on an inactivated mouse embryonic feeder layer in a growth medium consisting of 80% ES-DMEM/F12 supplemented with 20% KSR and FGF2 (4 and 10 ng/mL for hESCs and hiPSCs, respectively). For the experiment, cells were seeded on Matrigel-coated plates for feeder-free culturing in a conditioned medium supplemented with the same FGF2 concentrations above. Cells were allowed to attach in atmospheric oxygen for 24 h before the culture under low oxygen tension.	HIF-1α protein VEGF, ANG1, ANG2, GLUT1 mRNA	[88]

List of abbreviations: acLDL—acetylated low-density lipoprotein; ACTA2—actin alpha 2, also known as alpha smooth muscle actin, α-SMA; ADM—adrenomedullin; ANG1—angiopoietin 1; ANG2—angiopoietin 2; ANGPTL4—angiopoietin-like 4; α-MEM—Minimum Essential Medium Eagle-alpha modification; B27—serum-free supplement used to support the growth and viability; BME—β-mercaptoethanol; BMP4—bone morphogenetic protein 4; BSA—bovine serum albumin; CD144—cluster of differentiation 144, also known as vascular endothelial cadherin, VE-cad; CD34—cluster of differentiation 34, transmembrane phosphoglycoprotein; CD56—cluster of differentiation 56, also known as neural cell adhesion molecule, NCAM; CHIR—specific inhibitor of glycogen synthase-3β; DLL4—delta-like 4; DMEM—Dulbecco’s Modified Eagle Medium; DMEM/F-12—Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12; ECDM—Endothelial Cell Differentiation Medium; ECGM—endothelial cell growth media; ECM—extracellular matrix; ECs—endothelial cells; EGM2—Endothelial Cell Growth Medium 2; EPHB2—ephrin receptor B2; EPO—erythropoietin; FBS—fetal bovine serum; FGF2—basic fibroblast growth factor, bGFG; FGFR1—fibroblast growth factor receptor 1; GLUT1—glucose transporter 1; hEBs—human embryoid bodies; hESCs—human embryonic stem cells; HIF-1α—hypoxia-inducible factor-1α; HIF-2α—hypoxia-inducible factor-2α; hiPSCs—human induced pluripotent stem cells; hPSCs—human pluripotent stem cells; IGF-1—insulin-like growth factor-1; KSR—knockout serum replacement; LDHA—lactate dehydrogenase A; mEBs—mouse embryoid bodies; mESCs—mouse embryonic stem cells; MTG—monothioglycerol; mVEGFR1—membrane-bound vascular endothelial growth factor receptor 1; N2—serum-free supplement based on Bottenstein’s N-1 formulation; NEAA—nonessential amino acids; NOTCH1—Notch homolog 1, translocation-associated; PDGFB/PDGF-BB—platelet-derived growth-factor beta/platelet-derived growth factor beta polypeptide; PDGFBR—platelet-derived growth factor receptor beta; PDK1/3/4—pyruvate dehydrogenase kinase 1/3/4; PECAM1—platelet endothelial cell adhesion molecule, also known as cluster of differentiation 31, CD31; SB431542—inhibitor of the activin receptor-like kinase (ALK) receptors, ALK5, ALK4 and ALK; SDF-1—stromal cell-derived factor-1; sVEGFR1—soluble vascular endothelial growth factor receptor 1; TIE1—tyrosine kinase with immunoglobulin-like and EGF-like domains 1; TIE2—tyrosine kinase with immunoglobulin-like and EGF-like domains 2; VCAM-1—vascular cell adhesion protein-1; VEGF—vascular endothelial growth factor A; VEGFB—vascular endothelial growth factor B; VEGFR1—vascular endothelial growth factor receptor 1; VEGFR2—vascular endothelial growth factor receptor 2; vWF—von Willebrand factor. ^#, not all analyses were performed at the endpoint of the experiment; * not directly compared to normoxia.