Insight in Hypoxia-Mimetic Agents as Potential Tools for Mesenchymal Stem Cell Priming in Regenerative Medicine

Table 2

Effect of hypoxia on MSC viability, proliferation, and clonogenicity.

Treatment conditions		Types of stem cells	The effect compared to normoxia (methods of analysis)	Ref.
O2 concentration	Time/passage
1%	2 d	hBM-MSC	Proliferation (DNA Quant-iT Picrogreen assay), clonogenicity (Giemsa staining), and metabolic activity (Vybrant assay) increased; HIF-1α downregulated (qRT-PCR), the proapoptotic genes: BAX, BCL-2, and CASP-3 downregulated (qRT-PCR)	[57]
1%	2 d	rBM-MSC	The proliferation decreased (Trypan Blue staining, cell count)	[80]
1%	7 d	hBM-MSC	Proliferation significantly reduced (MTS proliferation assay)	[58]
1%	7 d	hBM-MSC	HIF-1α upregulated three-folds (qRT-PCR)	[58]
1%	9 d	hAD-MSC	Proliferation increased 1.7-folds (Trypan Blue staining, cell count)	[62]
1%	10 d	hBM-MSC	Proliferation (DNA Quant-iT Picrogreen assay) decreased, and metabolic activity increased (Vybrant assay), HIF-1α downregulated (qRT-PCR), the proapoptotic genes BCL-2 and CASP-3 downregulated, BAX upregulated (qRT-PCR)	[57]
1%	14 d	mBM-MSC	Viability (MTT viability assay) and proliferation (BrdU cell proliferation assay) increased, the main metabolic regulators like Hk2 upregulated (sqRT-PCR), shift to anaerobic glycolysis, the Slc16a3 (MCT-4) gene upregulated under prolonged hypoxia (qRT-PCT), the MCT-4 level increased under prolonged hypoxia (WB)	[68]
1%	14 d	rBM-MSC	Clonogenicity increased (crystal violet staining)	[80]
1%	21 d	hAD-MSC	Cell aging reduced, telomeres longer 1.5-folds (qPCR)	[62]
1%	21 d	hBM-MSC	A slowdown of cell cycle progression, accumulation in G1 phase under prolonged hypoxia (flow cytometry)	[58]
1-3%	16 h	hBM-MSC	Viability and proliferation (flow cytometry) maintained, Akt signaling pathway activated (WB)	[81]
1.5%	1 d	hBM-MSC; hUCB-MSC	Proliferation increased (Trypan Blue staining, cell count) and the cell cycle faster progression (flow cytometry), HIF-1α increased (WB)	[82]
1.5%	3 d	hUC-MSC	Proliferation decreased (Trypan Blue staining, cell count), LDHA, GLUT-1, and PDK-1 upregulated (RT-PCR), glutamate production decreased (HPLC), glucose consumption significantly increased (YSI 2700 analyzer)	[69]
2%	2 d	hBM-MSC	Proliferation (DNA Quant-iT Picrogreen assay), clonogenicity (Giemsa staining), and viability (flow cytometry) increased	[57]
2%	2 d	hWJ-MSC	Expression of the genes HIF1-α, HIF-2α, Notch2, and JAGGED1 increased (RT-PCR)	[64]
2%	7 d	hBM-MSC	A high growth rate maintained even after confluency–multilayer formation (cell count, growth curve), HIF-2α upregulated (RT-PCR)	[38]
2%	7 d	hBM-MSC	Clonogenicity increased (crystal violet staining)	[63]
2%	12 d	hBM-MSC	Higher proliferation rate (Trypan Blue staining, cell count), the number of actively dividing cells significantly increased (PKH26 Red Fluorescent Cell Linker kit), the cellular division started earlier in the cell cycle (PKH26 staining, flow cytometry)	[63]
2%	20 d	hBM-MSC	Clonogenicity (colony count from microscopic images) and doubling time (cell count and growth curve) maintained, cellular senescence reduced (β-galactosidase staining, histochemistry)	[71]
2%	Passages 2-7	hBM-MSC	Higher cell number in each passage from 2 to 7 (Trypan Blue staining, cell count)	[38]
2%	10 passages	hWJ-MSC	Faster growth rates and higher total cell number yielded (cell area count, image analysis), normal karyotype maintained (Giemsa staining)	[64]
2%	64 d	hBM-MSC	Homogenous morphology of rapidly self-renewing cells maintained up to 52 d (microscopy analysis)	[83]
2.5%	3 d	hUC-MSC	Proliferation increased (cell counting under a microscope), HIF-1α increased (WB), PDK-1, GLUT-1, and LDHA upregulated (RT-PCR), glutamate production diminished (HPLC), glucose consumption significantly increased (YSI 2700 SELECT analyzer)	[69]
2.5%	>3 d	hUCB-MSC	Cell viability (at 24 h and 2 d) increased (Trypan Blue staining, cell count, and MTT); proliferation (at 3 d) increased (Trypan Blue staining, cell count), CFU-F number in vitro significantly enhanced (Giemsa staining)	[60]
2.5%	>3 d	hUCB-MSCs	Cell metabolic activity (MTT), CFU-F number (Giemsa staining), and proliferation (at 2 and 3 d) (Trypan Blue staining, cell count) increased, doubling time reduced (at 2 and 3 d) (Trypan Blue staining, cell count), cell death inhibited (at 2 and 3 d) (microscope analysis)	[84]
3%	~100 d; Passage 1	hBM-MSC	Proliferative lifespan with additional 10 PD improved (flow cytometry), transcription of hypoxia-related the genes encoding VHL, HIF-1, PH-4, HYOU1, HIF1AN, HIG, and HIG unaltered (qPCR)	[33]
3%	Over 25 passages	hBM-MSC	Cell growth improved (Trypan Blue staining, cell count), population doublings increased (Trypan Blue staining, cell count), oxidative stress reactions (DHE, flow cytometry) and nuclear alterations such as damage of DNA, telomere shortening, and chromosomal abnormalities (DAPI, Q-FISH, Breast Aneusomy Multicolor Probe kit) limited, glycolysis increased (OCR/ECAR, F96 Flux analyzer)	[32]
5%	2 d	hBM-MSC	Proliferation rate lowered (DNA Quant-iT Picrogreen assay), clonogenicity (Giemsa staining), and metabolic activity elevated (Vybrant assay)	[57]
5%	3 d	hUC-MSC	Proliferation increased (Trypan Blue staining, cell count), LDHA, PDK-1, and GLUT-1 upregulation (RT-PCR)	[69]
5%	4 d	rBM-MSC	Proliferation rate increased (flow cytometry)	[85]
5%	4 d	hBM-MSC	Clonogenicity (crystal violet staining), proliferation (EDU Proliferation kit), and metabolic activity (Alamar Blue staining) increased	[65]
5%	14 d	hBM-MSC	Clonogenicity decreased at primary cells and the passage 1 but increased at the passages 2 and 3 (crystal violet staining)	[66]
5%	20 d	hBM-MSC	Colony formation significantly reduced (colony count from microscopic images), doubling time maintained (Trypan Blue staining, cell count, growth curve), cellular senescence reduced (β-galactosidase staining, histochemistry, blue stained cell count)	[71]
5%	Passage 1-10	hBM-MSC	The number of population doublings increased (Trypan Blue staining, cell count), cellular senescence reduced (β-galactosidase staining, histochemistry, blue stained cell count)	[72]

Hypoxic preconditioning in 2.5% O₂ for 15 minutes, then reoxygenation at 21% O₂ for 30 minutes, and again hypoxia preconditioning at 2.5% O₂ for 3 days; d: day/days; h: human; m: mouse; r: rat; PD: population doublings; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; BrdU: 5-bromo tetrazolium inner salt-20-deoxyuridine; MTS: tetrazolium inner salt; WB: Western Blotting.