Extracellular matrix from decellularized mesenchymal stem cells improves cardiac gene expressions and oxidative resistance in cardiac C-kit cells

Wai Hoe Ng; Rajesh Ramasamy; Yoke Keong Yong; Siti Hawa Ngalim; Vuanghao Lim; Bakiah Shaharuddin; Jun Jie Tan

doi:10.1016/j.reth.2019.03.006

Extracellular matrix from decellularized mesenchymal stem cells improves cardiac gene expressions and oxidative resistance in cardiac C-kit cells

Regen Ther. 2019 May 10:11:8-16. doi: 10.1016/j.reth.2019.03.006. eCollection 2019 Dec.

Authors

Wai Hoe Ng¹, Rajesh Ramasamy², Yoke Keong Yong³, Siti Hawa Ngalim¹, Vuanghao Lim¹, Bakiah Shaharuddin¹, Jun Jie Tan¹

Affiliations

¹ Advanced Medical and Dental Institute, Universiti Sains Malaysia, Bertam, 13200 Kepala Batas, Penang, Malaysia.
² Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, 43400 Selangor Darul Ehsan, Malaysia.
³ Department of Human Anatomy, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, 43400 Selangor Darul Ehsan, Malaysia.

Abstract

Objective: Myocardial infarction remains the number one killer disease worldwide. Cellular therapy using cardiac c-kit cells (CCs) are capable of regenerating injured heart. Previous studies showed mesenchymal stem cell-derived (MSC) extracellular matrices can provide structural support and are capable of regulating stem cell functions and differentiation. This study aimed to evaluate the effects of human MSC-derived matrices for CC growth and differentiation.

Methods: Human Wharton's Jelly-derived MSCs were cultured in ascorbic acid supplemented medium for 14 days prior to decellularisation using two methods. 1% SDS/Triton X-100 (ST) or 20 mM ammonia/Triton X-100 (AT). CCs isolated from 4-week-old C57/BL6N mice were cultured on the decellularised MSC matrices, and induced to differentiate into cardiomyocytes in cardiogenic medium for 21 days. Cardiac differentiation was assessed by immunocytochemistry and qPCR. All data were analysed using ANOVA.

Results: In vitro decellularisation using ST method caused matrix delamination from the wells. In contrast, decellularisation using AT improved the matrix retention up to 30% (p < 0.05). This effect was further enhanced when MSCs were cultured in cardiogenic medium, with a matrix retention rate up to 90%. CCs cultured on cardiogenic MSC matrix (ECM^cardio), however, did not significantly improve its proliferation after 3 days (p < 0.05), but the viability of CCs was augmented to 67.2 ± 0.7% after 24-h exposure to H₂O₂ stress as compared to 42.9 ± 0.5% in control CCs (p < 0.05). Furthermore, CCs cultured on cardiogenic MSC matrices showed 1.7-fold up-regulation in cardiac troponin I (cTnI) gene expression after 21 days (p < 0.05).

Conclusion: Highest matrix retention can be obtained by decellularization using Ammonia/Triton-100 in 2-D culture. ECM^cardio could rescue CCs from exogenous hydrogen peroxide and further upregulated the cardiac gene expressions, offering an alternate in vitro priming strategy to precondition CCs which could potentially enhance its survival and function after in vivo transplantation.

Keywords: AT, ammonia/triton X-100; CC, cardiac c-kit cells; Cardiac c-kit cells; Cardiomyocyte differentiation; ECM, extracellular matrix; Extracellular matrices; LVEF, left ventricular ejection fraction; MI, myocardial infarction; MSC, mesenchymal stem cells; Mesenchymal stem cells; SMA, smooth muscle actinin; ST, SDS/Triton X-100; cTnI, cardiac troponin I; vWF, von Willibrand factor; αMHC, myosin heavy chain alpha; βMHC, myosin heavy chain beta.