Objective Embryonic stem cells (ESCs) represent an extremely promising cell source for tissue-engineered myocardial tissue. But the role of extracellular matrix in control and guidance of ESCs differentiation and commitment into complex and viable three-dimensional (3D) myocardial tissue is still poorly understood. The purpose of this study was to identify and functionally characterise survive and differentiation of ESCs in 3D extracellular matrices isolated from myocardial tissues.
Methods Undifferentiated ESCs were cultured as previously described. Native myocardial tissues isolated from hearts of adult rabbit were treated with trypsin-EDTA and NaOH to remove cell components to gain acellular matrices. The acellularization of myocardial tissues was documented by haematoxylin-eosin staining (HE staining). Then ESCs were seeded onto the acellular matrices. Cell density and distribution were assessed by HE staining, cardiac transcription factors such as GATA4, NKX2.5, and atrial natriuretic factor (ANF) were evaluated by RT-PCR, and cardiac markers such as α-actinin, troponin I, and connexin43 were evaluated by immunocytochemistry. Ultrastructural analysis was examined under a transmission electron microscope.
Results The acellular matrices were incompletely covered by cells at 1 week, and the interstitiums of matrices were densely repopulated by cells at 2 weeks post seeding, indicating the rapid growth and expansion of these cells in the extracellular matrices and the formation of live constructs. Cardiac transcription factors GATA4 and NKX2.5 began to express on day10, but the expression of ANF was detected on day12. The constructs were positively immunostained with α-actinin, troponin I, and connexin43 at 2 weeks post seeding. Ultrastructural analysis indicates that cells in constructs exhibited the morphological features including sarcomeres, abundant glycogen and mitochondria and nascent junctional complexes.
Conclusions These results indicate that extracellular matrices have an important role to promote the differentiation of embryonic stem cells into cardiomyocytes. Such cell differentiation with extracellular matrices may be useful in forming tissue-engineered myocardial tissue to repair specific damaged hearts.