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

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.

Fig. 1

Isolation and characterisation of cardiac c-kit cells. (A) Immunocytochemistry staining of cardiac c-kit cells. CCs expressed c-kit, cardiac transcription factors (GATA4, NKX2.5), pluripotent marker (SOX2) but negative for mast cell marker (Tryptase). (B) Dexamethasone-directed cardiac differentiation of CCs for 21 days showed positive expression of cardiac markers (cTnI), smooth muscle actinin (SMA) and endothelial marker (vWF). Nuclei were stained with DAPI (Blue). Scale bar = 100 μm.

Fig. 2

Extracellular matrix generation. (A) Schematic diagram illustrated the process of ECM generation. (B) MSCs cultured on day 14 were decellularised using SDS/Triton X-100 (ST) or ammonia/Triton X-100 (AT). (C) The number of well-retaining ECM following treatments with ST or AT. (D) Representative images observed under phase contrast microscopy following decellularisation and recellularization of ascorbic acid- and cardiogenic-treated MSCs. (E) The number of well-retaining ECM following treatment with AT. (F) Protein quantification of ECM^AA and ECM^Cardio. *p < 0.05 vs. ST, #p < 0.05 vs. ECM^AA.

Fig. 3

The effect of decellularised MSC-ECM on CC proliferation and resistance to oxidative stress. (A) The cell number of CC cultured on surface with or without ECM for 3 days were assessed using Presto Blue ®. (B) Viability of CCs post-H₂O₂ treatment. *p < 0.05 vs. No ECM; #p < 0.05 vs. ECM^AA

Fig. 4

The effect of decellularised MSC-ECM on CC cardiac differentiation. (A) Immunofluorescence staining of differentiated CCs on ECM (cTnI: cardiac troponin I); SMA: smooth muscle actinin; vWF: von Willibrand factor. (B) Cardiac gene expressions were assessed by qPCR. *p < 0.05 vs. No ECM.