Cobalt Chloride as a Hypoxia Mimicking Agent Induced HIF-1α and mTOR Expressions of Human Umbilical Cord Mesenchymal Stem Cells

Mefina Kuntjoro; Nike Hendrijantini; Eric Priyo Prasetyo; Bambang Agustono; Ratri Maya Sitalaksmi; Eryk Hendrianto; Aristika Dinaryanti; Marvin Rusli; Evelyn Tjendronegoro; Guang Hong

Authors

Mefina Kuntjoro
Nike Hendrijantini
Eric Priyo Prasetyo
Bambang Agustono
Ratri Maya Sitalaksmi
Eryk Hendrianto
Aristika Dinaryanti
Marvin Rusli
Evelyn Tjendronegoro
Guang Hong

Keywords:

Umbilical Cord, Mesenchymal Stem Cells, Stem Cell Research, Regeneration

Abstract

Objective: To assess the effects of cobalt chloride (CoCl₂) as a hypoxia mimicking agent on human umbilical cord mesenchymal stem cells (hUCMSCs) expression of HIF-1α and mTOR for use in regenerative dentistry. Material and Methods: Human umbilical cord mesenchymal stem cells were isolated and then cultured. The characteristics of stemness were screened and confirmed by flow cytometry. The experiment was conducted on hypoxia (H) and normoxia (N) groups. Each group was divided and incubated into 24-, 48-, and 72-hours observations. Hypoxic treatment was performed using 100 µM CoCl₂ on 5^th passage cells in a conventional incubator (37°C; 5% CO₂). Then, immunofluorescence of HIF-1α and mTOR was done. Data was analyzed statistically using One-way ANOVA and Tukey’s HSD. Results: Significant differences were found between normoxic and hypoxic groups on HIF-1α (p=0.015) and mTOR (p=0.000) expressions. The highest HIF-1α expression was found at 48 hours in the hypoxia group, while for mTOR at 24 hours in the hypoxia group. Conclusion: Hypoxia using cobalt chloride was able to increase human umbilical cord mesenchymal stem cells expression of HIF-1α and mTOR.

References

Mendes RT, Nguyen D, Stephens D, Pamuk F, Fernandes D, Hasturk H, et al. Hypoxia-induced endothelial cell responses-possible roles during periodontal disease. Clin Exp Dent Res 2018; 4(6):241-248. https://doi.org/10.1002/cre2.135

Devine DA, Marsh PD, Meade J. Modulation of host responses by oral comensal bacteria. J Oral Microbiol 2015; 7:26941. https://doi.org/10.3402/jom.v7.26941

Celik D, Kantarci A. Vascular changes and hypoxia in periodontal disease as a link to systemic complications. Pathogens 2021; 10(10):1280. https://doi.org/10.2290/pathogens10101280

Wu D, Yotnda P. Induction and testing of hypoxia in cell culture. J Vis Exp 2011; 54:2899. https://doi.org/10.3791/2899

Misawa MYO, Huynh-Ba G, Villar GM, Villar CC. Efficacy of stem cells on the healing of peri-implant defects: systematic review of preclinical studies. Clin Exp Dent Res 2016; 2(1):18-34. https://doi.org/10.1002/cre2.16

Chao YH, Wu HP, Chan CK, Tsai C, Peng CT, Wu KH. Umbilical cord-derived mesenchymal stem cells for hematopoietic stem cell transplantation. J Biomed Biotechnol 2012; 2012:759503. https://doi.org/10.1155/2012/759503

Kuntjoro M, Prasetyo EP, Cahyani F, Kamadjaja MJK, Hendrijantini N, Laksono H, et al. Lipopolysaccharide’s cytotoxicity on human umbilical cord mesenchymal stem cells. Pesqui Bras Odontopediatria Clín Integr 2020; 20:e0048. https://doi.org/10.1590/pboci.2020.153

Prasetyo EP, Kuntjoro M, Cahyani F, Goenharto S, Saraswati W, Juniarti DE, et al. Calcium hydroxide upregulates interleukin-10 expression in time dependent exposure and induces osteogenic differentiation of human umbilical cord mesenchymal stem cells. Int J Pharm Res 2021; 13(1):140-145. https://doi.org/10.31838/ijpr/2021.13.01.023

Kuntjoro M, Agustono B, Prasetyo EP, Salim S, Rantam FA, Hendrijantini N. The effect of advanced glycation endo products (AGEs) on human umbilical cord mesenchymal stem cells (hUCMSCs) with regard to osteogenesis and calcification. Res J Pharm Technol 2021; 14(8):4019-4024. https://doi.org/10.52711/0974-360X.2021.00696

Prasetyo EP, Kuntjoro M, Goenharto S, Juniarti DE, Cahyani F, Hendrijantini N, et al. Calcium hydroxide increases human umbilical cord mesenchymal stem cells expressions of apoptotic proteaseactivating factor-1, caspase-3 and caspase-9. Clin Cosmet Investig Dent 2021; 13:59-65. https://doi.org/10.2147/CCIDE.S284240

Li T, Xia M, Gao Y, Chen Y, Xu Y. Human umbilical cord mesenchymal stem cells: an overview of their potential in cell-based therapy. Expert Opin Biol Ther 2015; 15(9):1293-1306. https://doi.org/10.1517/14712598.2015.1051528

Ejtehadifar M, Shamsasenjan K, Movassaghpour A, Akbarzadehlaleh P, Dehdilani N, Abbasi P, et al. The effect of hypoxia on mesenchymal stem cell biology. Adv Pharm Bull 2015; 5(2):141-149. https://doi.org/10.15171/apb.2015.021

Bhandi S, Kahtani AA, Mashyakhy M, Alsofi L, Maganur PC, Vishwanathaiah S, et al. Modulation of the dental pulp stem cell secretory profile by hypoxia induction using cobalt chloride. J Pers Med 2021; 11(4):247. https://doi.org/10.3390/jpm11040247

Laksana K, Sooampon S, Pavasant P, Sriarj W. Cobalt chloride enhances the stemness of human dental pulp cells. J Endod 2017; 43(5):760-765. https://doi.org/10.1016/j.joen.2017.01.005

Nugraha AP, Prasetyo EP, Kuntjoro M, Ihsan IS, Dinaryanti A, Susilowati H, et al. The effect of cobalt (II) chloride in the viability percentage and the induced hypoxia inducible factor - 1 of human adipose mesenchymal stem cells (HAMSCs): An in vitro study. Syst Rev Pharm 2020; 11(6):308-314. https://doi.org/10.31838/srp.2020.6.49

Nugraha AP, Ihsan IS, Dinaryanti A, Hendrianto E, Susilowati H, Prasetyo EP, et al. Cobalt (II) chloride in enhancing hypoxia inducible factor-1a expression of gingival derived mesenchymal stem cells in vitro. Res J Pharm Technol 2021; 14(5):2639-2642. https://doi.org/10.52711/0974-360X.2021.00465

Chen Y, Zhao Q, Yang X, Yu X, Yu D, Zhao W. Effects of cobalt chloride on the stem cell marker expression and osteogenic differentiation of stem cells from human exfoliated deciduous teeth. Cell Stress Chaperones 2019; 24(3):527-538. https://doi.org/10.1007/s12192-019-00981-5

Moniz I, Ramalho-Santos J, Branco AF. Differential oxygen exposure modulates mesenchymal stem cell metabolism and proliferation through mTOR signaling. Int J Mol Sci 2022; 23(7):3749. https://doi.org/10.3390/ijms23073749

Vishwakarma A, Karp J. Biology and Engineering of Stem Cell Niches. New York: Elsevier; 2017.

Park IH, Kim KH, Choi HK, Shim JS, Whang SY, Hahn SJ, et al. Constitutive stabilization of hypoxia-inducible factor alpha selectively promotes the self-renewal of mesenchymal progenitors and maintains mesenchymal stromal cells in an undifferentiated state. Exp Mol Med 2013; 45(9):e44. https://doi.org/10.1038/emm.2013.87

Leontieva OV, Natarajan V, Demidenko ZN, Burdelya LG, Gudkov AV, Blagosklonny MV. Hypoxia suppresses conversion from proliferative arrest to cellular senescence. Proc Natl Acad Sci 2012; 109(33):13314-13318. https://doi.org/10.1073/pnas.1205690109

Lavrentieva A, Majore I, Kasper C, Hass R. Effects of hypoxic culture conditions on umbilical cord-derived human mesenchymal stem cells. Cell Commun Signal 2010; 8:18. https://doi.org/10.1186/1478-811X-8-18

Saxton RA, Sabatini DM. mTOR Signaling in growth, metabolism, and disease. Cell 2017; 168(6):960-976. https://doi.org/10.1016/j.cell.2017.02.004

Prasetyo EP, Widjiastuti I, Cahyani F, Kuntjoro M, Hariyani N, Winoto ER, et al. Cytotoxicity of calcium hydroxide on human umbilical cord mesenchymal stem cells. Pesqui Bras Odontopediatria Clín Integr 2020; 20:e0044. https://doi.org/10.1590/pboci.2020.141

Teti G, Focaroli S, Salvatore V, Mazzotti E, Ingra L, Mazzotti A, et al. The hypoxia-mimetic agent cobalt chloride differently affects human mesenchymal stem cells in their chondrogenic potential. Stem Cells Int 2018; 2018:3237253. https://doi.org/10.1155/2018/3237253

Ding DC, Chang YH, Shyu WC, Lin SZ. Human umbilical cord mesenchymal stem cells: A new era for stem cell therapy. Cell Transplant 2015; 24(3):339-347. https://doi.org/10.3727/096368915X686841

Cordero CB, Santander GM, Gonzalez DU, Quezada A, Silva CI, Vasquez C, et al. Allogenic cellular therapy in a mature tooth with apical periodontitis and accidental root perforation: A case report. J Endod 2020; 46(12):1920-1927. https://doi.org/10.1016/j.joen.2020.04.007

Wang L, Ott L, Seshareddy K, Weiss ML, Detamore MS. Musculoskeletal tissue engineering with human umbilical cord mesenchymal stromal cells. Regen Med 2011; 6(1):95-109. https://doi.org/10.2217/rme.10.98

Suda T, Takubo K, Semenza GL. Metabolic regulation of hematopoietic stem cells in the hypoxic niche. Cell Stem Cell 2011; 9(4):298-310. https://doi.org/10.1016/j.stem.2011.09.010

Liu Y, Ma T. Metabolic regulation of mesenchymal stem cell in expansion and therapeutic application. Biotechnol Prog 2015; 31(2):468-481. https://doi.org/10.1002/btpr.2034

Cimmino F, Avitabile M, Lasorsa VA, Montella A, Pezone L, Cantalupo S, et al. HIF-1 transcription activity: HIF1A driven response in normoxia and in hypoxia. BMC Med Genet 2019; 20:37. https://doi.org/10.1186/s12881-019-0767-1

Ho SS, Hung BP, Heyrani N, Lee MA, Leach JK. Hypoxic preconditioning of mesenchymal stem cells with subsequent spheroid formation accelerates repair of segmental bone defects. Stem Cells 2018; 36(9):1393-1403. https://doi.org/10.1002/stem.2853

Kierans SJ, Taylor CT. Regulation of glycolysis by the hypoxia-inducible factor (HIF): implications for cellular physiology. J Physiol 2021; 599(1):23-37. https://doi.org/10.1113/JP280572

Brugarolas J, Lei K, Hurley RL, Manning BD, Reiling JH, Hafen E, et al. Regulation of mTOR function in response to hypoxia by REDD1 and the TSC1/TSC2 tumor suppressor complex. Genes Dev 2004; 18(23):2893-2904. https://doi.org/10.1101/gad.1256804

Lee HJ, Ryu JM, Jung YH, Oh SY, Lee SJ, Han HJ. Novel pathway for hypoxia-induced proliferation and migration in human mesenchymal stem cells: Involvement of HIF-1α, FASN, and mTORC1. Stem Cells 2015; 33(7):2182-2195. https://doi.org/10.1002/stem.2020

Chu Y, Chen W, Peng W, Liu Y, Xu L, Zuo J, et al. Amnion-derived mesenchymal stem cell exosomes-mediated autophagy promotes the survival of trophoblasts under hypoxia through mTOR pathway by the downregulation of EZH2. Front Cell Dev Biol 2020; 8:545852. https://doi.org/10.3389/fcell.2020.545852

Blagosklonny MV. Hypoxia, MTOR and autophagy converging on senescence or quiescence. Autophagy 2013; 9(2):260-262. https://doi.org/10.4161/auto.22783

Lee SH, Lee YJ, Han HJ. Role of hypoxia-induced fibronectin-integrin β1 expression in embryonic stem cell proliferation and migration: Involvement of PI3K/Akt and FAK. J Cell Physiol 2011; 226(2):484-493. https://doi.org/10.1002/jcp.22358

Lee Y, Jung J, Cho KJ, Lee SK, Park JW, Oh IH, et al. Increased SCF/c-kit by hypoxia promotes autophagy of human placental chorionic plate-derived mesenchymal stem cells via regulating the phosphorylation of mTOR. J Cell Biochem 2013; 114(1):79-88. https://doi.org/10.1002/jcb.24303

Kuntjoro M, Hendrijantini N, Prasetyo EP, Legowo D, Sitalaksmi RM, Agustono B, et al. Human umbilical cord mesenchymal stem cells accelerate and increase implant osseointegration in diabetic rats. J Appl Oral Sci 2023; 31:e20220375. https://doi.org/10.1590/1678-7757-2022-0375