Cardiac derived ECM enhances cardiogenic properties of Human Cardiac Progenitor Cells

摘要

Introduction: During the last few years, cardiac tissue engineering has emerged as a new therapeutic approach for the regeneration of the infarcted heart. Different factors can influence the success of a tissue engineering application, the most important of which are the choice of cells and matrix. The ideal matrix should promote cell engraftment, survival and differentiation of the transplanted cells as well as mimic the properties of the endogenous cardiac ECM. A porcine derived myocardial matrix hydrogel has been shown to improve cardiac function upon injection into an infarcted heart and to promote cardiogenesis in murine cardiac progenitor cells (mCPCs). However, its effect on clinically applicable human CPCs (hCPCs) still needs to be addressed. Materials, Methods and Results: In this study, we investigated the in vitro influence of the myocardial matrix hydrogel on the cardiogenic phenotype of Sca-1-like human derived CPCs. Human fetal and adult CPCs (hfCPCs and haCPCs) were isolated from heart biopsies and encapsulated in the myocardial matrix and compared to collagen type Ⅰ hydrogels. RT-PCR analysis of encapsulated hfCPCs displayed a significant increase in the cardiac transcription marker Gata-4 as well as in the sarcomeric protein MLC2v and the vascular marker VEGF receptor 2 (VEGF-R2) after 4 days in culture (n=6; p＜0.05). Similarly increased levels were observed for early cardiac transcription factors Nkx2.5 and mef-2c as well as vascular markers VEGF-R2 and CD31 when haCPCs were cultured in the matrix compared to collagen (n=5; p＜0.05). Cell viability was sustained in both hydrogels up to 1 week in culture, as showed by a cell viability assay. Hematoxylin and Eosin staining showed that the cells remain homogenously dispersed in the hydrogels, with increasing nuclear densities upon culturing. Interestingly, the myocardial matrix cultured human fetal and adult CPCs showed a significant increase in the proliferation marker Ki67 after 4 days in culture when compared to collagen group (n=6 and n=4 respectively; p＜0.05). Cellular protein expression of Nkx2.5 and Tnl was also confirmed by immunofluorescence at day 4 and 7. Finally, to evaluate if the matrix is able to preserve CPCs viability, encapsulated CPCs were treated with 500 μM (hfCPCs) or 750 μM (haCPCs) H_2O_2 for 16 hours. Evaluation via Alamar blue assay showed an improved survival of encapsulated cells in the myocardial matrix when compared to the collagen matrix (n=9 and n=8 respectively; p＜0.05). Conclusion: In summary, we showed that the myocardial matrix hydrogel is advantageous for hCPCs culture and encapsulation, enhancing CPCs proliferation, survival and cardiogenic potential compared to collagen. This work demonstrates the potential of the myocardial matrix as an in vitro platform and warrants its further investigation for cell transplantation in the diseased myocardium.