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

Table 4

Effect of hypoxia pre/treatment on MSC differentiation.

Treatment conditions		Type of stem cells	The effect compared to normoxia (methods of analysis)	Ref.
O2 concentration	Time/passage
1%	Pretreatment for 2 d	rBM-MSC	Osteogenesis increased (ALP activity, 7 d; Alizarin Red S, 21 d)	[80]
1%	Pretreatment for 3 d	hBM-MSC	Osteogenic potential of MSC maintained, for high-density spheroid osteogenic potential enhanced; increase in the ALP activity related to the spheroid cell density (ALP staining after 14 d), osteocalcin level maintained (ELISA)	[89]
1%	Pretreatment for 14 d	hBM-MSC	Adipogenic differentiation impaired (Oil Red staining, 14-20 d), osteogenic potential reduced (Alizarin Red staining, 21 d, calcium precipitates detected)	[58]
1%	Pretreatment for 21 d	hAD-MSC	Chondrogenic potential increased (Alcian Blue staining, 21 d); high expression of the SOX9 and COL2A1 genes (RT-PCR), osteogenic potential slightly reduced (Von Kossa staining, 21 d, manual counting of calcified areas); lower expression of ALP and unchanged OPN (RT-PCR), adipogenic potential slightly reduced (Oil Red staining, 21 d); lower expression of the ADPN and LPL genes (RT-PCR)	[62]
1%	Treatment for 9 d	Coculture; hBM-MSC; HUVEC	Osteogenic potential maintained (Alizarin Red stain, 9 d), expression of the osteogenic RUNX2 and ALP genes retained and upregulation COLIA1 (qRT-PCR)	[52]
1.5%	Pretreatment for 1 d	BM-MSC; UCB-MSC	Osteogenic potential (Von Kossa staining, 14 d) and expression of the RUNX2 gene retained (RT-PCR), adipogenic differentiation potential retained (Oil Red staining, 21Coll type IId) and its corresponding marker gene PPAR-α (RT-PCR), chondrogenesis (Coll type II detection, 21d), and expression of the SOX9 gene increased (RT-PCR)	[82]
2%	Pretreatment for 7 d	hAD-MSC	Osteogenesis enhanced (Von Kossa staining, 22Coll type II d), adipogenesis enhanced (Oil Red staining, 21Coll type II d)	[114]
2%	Pretreatment for 8 d	mAD-MSC	Early chondrogenesis increased (Alcian Blue staining, sGAGs assay kit, 6 d), osteogenesis after 7 d maintained (ALP activity, colorimetric assay), after 21 d—decreased (Alizarin Red staining)	[116]
2%	Pretreatment at passage 2 and/or treatment for 21 d	hBM-MSC	Osteogenic potential reduced (Alizarin Red staining, 21 d), osteogenic potential reduced due to hypoxia pretreatment in cells grown in normoxia and hypoxia conditions	[71]
2%	Treatment for 14 d	hBM-MSC	Osteogenic potential maintained (Von Kossa staining and ALP activity, 14 d), adipogenic potential maintained (Oil Red staining, 14 d)	[63]
2%	Treatment for 14 d	hBM-MSC	Osteogenic potential preserved (Von Kossa staining, 14 d), the above capacities preserved up to the seventh passage	[38]
2%	Treatment for 14-17 d	hWJ-MSC	Chondrogenic potential maintained (Alcian Blue staining, 14-17 d)	[64]
2%	Treatment for 18 days	hWJ-MSC	Adipogenic potential increased (Oil Red staining, 18 d)	[63]
2%	Treatment for 21 days	hBM-MSC	Adipogenic potential preserved (Nile Red staining, 21 d) up to the seventh passage	[38]
2%	Treatment for 21 days	hWJ-MSC	Osteogenic potential increased (Von Kossa staining, 21 d)	[64]
2.5%	Pretreatment for more than 3 days	hUC-MSC	Osteogenic (Alizarin Red staining, 21 d), adipogenic (Oil Red staining, 21 d), and chondrogenic (Alcian Blue staining, 21 d) potential preserved	[84]
3%	Pretreatment for 7-10 d and/or treatment for 21 d	hBM-MSC	Both hypoxia pretreatment and hypoxic treatment during differentiation preserve osteogenic (Alizarin Red staining, 21 d) and adipogenic (Oil Red staining, 21 d) potential for primary cells only, diminished adipogenesis and inhibited osteogenesis with increase in the passage number from 1 to 4, the genes ALPL, IBSP, FABP4, and LPL downregulated (qRT-PCR) with increase in the passage number from 1 to 4, osteogenic differentiation (passage 1) stimulated upon transition from 3 to 20% O2	[33]
1-3%	Pretreatment for 16 h	hBM-MSC	Osteogenic (Alizarin Red staining), adipogenic (Oil Red staining), and chondrogenic (Alcian Blue staining) differential potential maintained	[81]
1-5%	Treatment for 21 d	hBM-MSC	Osteogenic differentiation reduced (Alizarin Red assay, 7, 14, and 21 d), ALP and OPN expressed at low levels below 5% O2 (WB)	[125]
5%	Pretreatment at passages 2-4 and/or treatment for 21 d	hWJ-MSC	Osteogenesis (Von Kossa staining, 21 d) and cartilage differentiation (Masson’s trichrome staining, 21 d) maintained at the same level, hypoxic/normoxic pretreatment and treatment did not affect MSC differentiation potential	[126]
5%	Pretreatment up to passage 2 and/or treatment for 28 d	hBM-MSC	Osteogenic potential (Alizarin Red staining, 28 d), and the expression of the ALPL and RUNX2 genes increased (RT-PCR), adipogenic potential (Oil Red staining, 28 d) increased; the expression level of the LPL and PPARγ genes maintained (RT-PCR), both hypoxia pretreatment and hypoxic treatment increased differentiation potential	[66]
5%	Treatment for 14 d	hBM-MSC	Osteogenic potential (ALP activity, 14 d) and the BGLAP, RUNX2, and COLL2 gene expression increased (RT-PCR)	[65]
5%	Treatment for 21 d	hBM-MSC	Adipogenesis reduced (Oil Red staining, 21 d)	[65]
5%	Treatment for 28 d	hBM-MSC	Chondrogenesis reduced (Alcian Blue staining, 28 d)	[65]
8%	Treatment for 8 d	mBM-MSC	Adipogenesis increased after 8 d (Sudan Black staining)	[123]
8%	Treatment for 14 d	mBM-MSC	Oct4 inhibited (qRT-PCR)	[123]

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.