Int J Stem Cells.  2021 May;14(2):221-228. 10.15283/ijsc20096.

Optimal Hypoxic Preconditioning of Human Embryonic Stem Cell-Derived Mesenchymal Stem Cells (hES-MSCs) and Their Characteristics

Affiliations
  • 1Department of Translational Medicine, Graduate School of Biomedical Science & Engineering, Hanyang University, Seoul, Korea
  • 2Department of Internal Medicine, Hanyang University School of Medicine, Seoul, Korea
  • 3Department of Biomedical Sciences, Pak-Austria Fachhochschule: Institute of Applied Sciences and Technology, Mang, Haripur, Pakistan
  • 4Department of Internal Medicine, Eulji University School of Medicine, Seoul, Korea

Abstract

Background and Objectives
Hypoxia is frequently used to enhance stem cell function. However, the optimal level of hypoxia for growth and function of human embryonic stem cell-derived mesenchymal stem cells (hES-MSCs) is yet to be determined. The purpose of this study was to find the optimal level of hypoxia for hES-MSCs and characteristics of hES-MSCs cultured under these optimal hypoxic conditions.
Methods and Results
Cell viability and changes in the morphology of hES-MSCs were determined through cell proliferation and CCK-8 assay. The hES-MSCs were preconditioned under various hypoxic conditions (0.5∼5% O2 and 24∼72 h). The expression of cytokines in each culture condition was compared using cytokine array analysis. The morphology of hES-MSCs did not change under various hypoxic culture conditions. hES-MSCs viability after 48 h incubation in 2% O2condition was higher than that in normoxic condition. HIF1α expression was increased up to six folds after 48 h of hypoxic preconditioning. HIF1α expression in hES-MSCs peaked after 48 h of incubation in 1% O2 condition. The expressions of PDGF-BB, IGFBP-6, VEGF-A, and angiogenin were increased after hES-MSCs were incubated for 48 h in 2% O2 condition.
Conclusions
The hES-MSCs viability and expressions of PDGF-BB, IGFBP-6, VEGF-A, and angiogenin increased after 48 h incubation in 2% O2 condition.

Keyword

Hypoxia; Stem cell; Function; VEGF

Figure

  • Fig. 1 Morphology and viability of hES-MSCs under normal and hypoxic conditions. (A) Morphological analysis of hES-MSCs cultured under normal (21% O2) and various hypoxic incubation conditions (0.5%, 1%, 2%, and 5% O2) for 24, 48 and 72 h. The morphology was observed using an optical microscope; Magni-fication: 400×. (B) Cell viability was assessed using CCK-8 cell viability assay; (n=6, mean±SEM; ns, *p< 0.05, **p<0.01, ***p<0.001 and ****p<0.0001 vs. normal condition using One-way ANOVA-Dunnett’s multiple comparison test).

  • Fig. 2 Correlation between HIF1α protein expression and cell viability of hES-MSCs under various hypoxic conditions. (A) HIF1α protein expression under normal and hypoxic conditions. (B) HIF1α protein quantification using densitometry. (C) Correlation between cell viability and HIF1α relative expression. (D) HIF1α protein expression at various low O2 partial pressures. (E) Cell viability comparison with or without HIF1α inhibitor in hypoxia; (n=6, mean±SEM; ns, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 vs. normal condition using One-way ANOVA-Dunnett’s multiple comparison test).

  • Fig. 3 Comparison of hES-MSCs cytokines expressions under various hypoxic conditions. (A) Membrane after Ray Bio human cytokine antibody array. (B) Heat map demonstrating relative expressions of biomarkers from screening cytokine antibody array in normal vs. various hypoxic conditions. (C, D) Comparison of cytokines expressions in various hypoxic culture conditions.


Reference

References

1. Harris DT. 2009; Non-haematological uses of cord blood stem cells. Br J Haematol. 147:177–184. DOI: 10.1111/j.1365-2141.2009.07767.x. PMID: 19796266.
Article
2. Haque N, Rahman MT, Abu Kasim NH, Alabsi AM. 2013; Hypoxic culture conditions as a solution for mesenchymal stem cell based regenerative therapy. ScientificWorldJournal. 2013:632972. DOI: 10.1155/2013/632972. PMID: 24068884. PMCID: PMC3771429.
Article
3. Li O, Tormin A, Sundberg B, Hyllner J, Le Blanc K, Scheding S. 2013; Human embryonic stem cell-derived mesenchymal stroma cells (hES-MSCs) engraft in vivo and support hematopoiesis without suppressing immune function: implications for off-the shelf ES-MSC therapies. PLoS One. 8:e55319. DOI: 10.1371/journal.pone.0055319. PMID: 23383153. PMCID: PMC3558469.
Article
4. Wang X, Kimbrel EA, Ijichi K, Paul D, Lazorchak AS, Chu J, Kouris NA, Yavanian GJ, Lu SJ, Pachter JS, Crocker SJ, Lanza R, Xu RH. 2014; Human ESC-derived MSCs outperform bone marrow MSCs in the treatment of an EAE model of multiple sclerosis. Stem Cell Reports. 3:115–130. DOI: 10.1016/j.stemcr.2014.04.020. PMID: 25068126. PMCID: PMC4110787.
Article
5. Kern S, Eichler H, Stoeve J, Klüter H, Bieback K. 2006; Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells. 24:1294–1301. DOI: 10.1634/stemcells.2005-0342. PMID: 16410387.
Article
6. Kim DS, Kim JH, Lee JK, Choi SJ, Kim JS, Jeun SS, Oh W, Yang YS, Chang JW. 2009; Overexpression of CXC chemokine receptors is required for the superior glioma-tracking property of umbilical cord blood-derived mesenchymal stem cells. Stem Cells Dev. 18:511–519. DOI: 10.1089/scd.2008.0050. PMID: 18624673.
Article
7. Kim SW, Han H, Chae GT, Lee SH, Bo S, Yoon JH, Lee YS, Lee KS, Park HK, Kang KS. 2006; Successful stem cell therapy using umbilical cord blood-derived multipotent stem cells for Buerger's disease and ischemic limb disease animal model. Stem Cells. 24:1620–1626. DOI: 10.1634/stemcells.2005-0365. PMID: 16497946.
Article
8. Grayson WL, Zhao F, Bunnell B, Ma T. 2007; Hypoxia enhances proliferation and tissue formation of human mesenchymal stem cells. Biochem Biophys Res Commun. 358:948–953. DOI: 10.1016/j.bbrc.2007.05.054. PMID: 17521616.
Article
9. Hung SP, Ho JH, Shih YR, Lo T, Lee OK. 2012; Hypoxia promotes proliferation and osteogenic differentiation potentials of human mesenchymal stem cells. J Orthop Res. 30:260–266. DOI: 10.1002/jor.21517. PMID: 21809383.
Article
10. Razban V, Lotfi AS, Soleimani M, Ahmadi H, Massumi M, Khajeh S, Ghaedi M, Arjmand S, Najavand S, Khoshdel A. 2012; HIF-1α overexpression induces angiogenesis in mesenchymal stem cells. Biores Open Access. 1:174–183. DOI: 10.1089/biores.2012.9905. PMID: 23514846. PMCID: PMC3559201.
Article
11. Das R, Jahr H, van Osch GJ, Farrell E. 2010; The role of hypoxia in bone marrow-derived mesenchymal stem cells: considerations for regenerative medicine approaches. Tissue Eng Part B Rev. 16:159–168. DOI: 10.1089/ten.teb.2009.0296. PMID: 19698058.
Article
12. Hu X, Yu SP, Fraser JL, Lu Z, Ogle ME, Wang JA, Wei L. 2008; Transplantation of hypoxia-preconditioned mesenchymal stem cells improves infarcted heart function via enhanced survival of implanted cells and angiogenesis. J Thorac Cardiovasc Surg. 135:799–808. DOI: 10.1016/j.jtcvs.2007.07.071. PMID: 18374759.
Article
13. Wang JA, Chen TL, Jiang J, Shi H, Gui C, Luo RH, Xie XJ, Xiang MX, Zhang X. 2008; Hypoxic preconditioning attenuates hypoxia/reoxygenation-induced apoptosis in mesenchymal stem cells. Acta Pharmacol Sin. 29:74–82. DOI: 10.1111/j.1745-7254.2008.00716.x. PMID: 18158868.
Article
14. Luo Z, Wu F, Xue E, Huang L, Yan P, Pan X, Zhou Y. 2019; Hypoxia preconditioning promotes bone marrow mesenchymal stem cells survival by inducing HIF-1α in injured neuronal cells derived exosomes culture system. Cell Death Dis. 10:134. DOI: 10.1038/s41419-019-1410-y. PMID: 30755595. PMCID: PMC6372680.
Article
15. Liu J, Hao H, Xia L, Ti D, Huang H, Dong L, Tong C, Hou Q, Zhao Y, Liu H, Fu X, Han W. 2015; Hypoxia pretreatment of bone marrow mesenchymal stem cells facilitates angiogenesis by improving the function of endothelial cells in diabetic rats with lower ischemia. PLoS One. 10:e0126715. DOI: 10.1371/journal.pone.0126715. PMID: 25996677. PMCID: PMC4440823.
Article
16. Xiong X, Sun Y, Wang X. 2020; HIF1A/miR-20a-5p/TGFβ1 axis modulates adipose-derived stem cells in a paracrine manner to affect the angiogenesis of human dermal microvascular endothelial cells. J Cell Physiol. 235:2091–2101. DOI: 10.1002/jcp.29111. PMID: 31368162.
Article
17. van Es MA, Schelhaas HJ, van Vught PW, Ticozzi N, Andersen PM, Groen EJ, Schulte C, Blauw HM, Koppers M, Diekstra FP, Fumoto K, LeClerc AL, Keagle P, Bloem BR, Scheffer H, van Nuenen BF, van Blitterswijk M, van Rheenen W, Wills AM, Lowe PP, Hu GF, Yu W, Kishikawa H, Wu D, Folkerth RD, Mariani C, Goldwurm S, Pezzoli G, Van Damme P, Lemmens R, Dahlberg C, Birve A, Fernández-Santiago R, Waibel S, Klein C, Weber M, van der Kooi AJ, de Visser M, Verbaan D, van Hilten JJ, Heutink P, Hennekam EA, Cuppen E, Berg D, Brown RH Jr, Silani V, Gasser T, Ludolph AC, Robberecht W, Ophoff RA, Veldink JH, Pasterkamp RJ, de Bakker PI, Landers JE, van de Warrenburg BP, van den Berg LH. 2011; Angiogenin variants in Parkinson disease and amyotrophic lateral sclerosis. Ann Neurol. 70:964–973. DOI: 10.1002/ana.22611. PMID: 22190368. PMCID: PMC5560057.
Article
18. Whitney KE, Liebowitz A, Bolia IK, Chahla J, Ravuri S, Evans TA, Philippon MJ, Huard J. 2017; Current perspectives on biological approaches for osteoarthritis. Ann N Y Acad Sci. 1410:26–43. DOI: 10.1111/nyas.13554. PMID: 29265418.
Article
19. Koay EJ, Athanasiou KA. 2008; Hypoxic chondrogenic differentiation of human embryonic stem cells enhances cartilage protein synthesis and biomechanical functionality. Osteoar-thritis Cartilage. 16:1450–1456. DOI: 10.1016/j.joca.2008.04.007. PMID: 18541445.
Article
20. Khan WS, Adesida AB, Hardingham TE. 2007; Hypoxic conditions increase hypoxia-inducible transcription factor 2alpha and enhance chondrogenesis in stem cells from the infrapatellar fat pad of osteoarthritis patients. Arthritis Res Ther. 9:R55. DOI: 10.1186/ar2211. PMID: 17537234. PMCID: PMC2206341.
21. Scherer K, Schünke M, Sellckau R, Hassenpflug J, Kurz B. 2004; The influence of oxygen and hydrostatic pressure on articular chondrocytes and adherent bone marrow cells in vitro. Biorheology. 41:323–333. PMID: 15299265.
22. Lennon DP, Edmison JM, Caplan AI. 2001; Cultivation of rat marrow-derived mesenchymal stem cells in reduced oxygen tension: effects on in vitro and in vivo osteochondrogenesis. J Cell Physiol. 187:345–355. DOI: 10.1002/jcp.1081. PMID: 11319758.
Article
23. Abdollahi H, Harris LJ, Zhang P, McIlhenny S, Srinivas V, Tulenko T, DiMuzio PJ. 2011; The role of hypoxia in stem cell differentiation and therapeutics. J Surg Res. 165:112–117. DOI: 10.1016/j.jss.2009.09.057. PMID: 20080246. PMCID: PMC2891942.
Article
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