In Vitro Culture And Identification Of Cardiac Stem Cells From Adult Mini-pigs

Objectives : Cardiac stem cell regenerative therapy is a new treatment of myocardial infarction. However, there is no uniform in vitro culture standard due to the limited cell number and the difficulty in amplification of cardiac stem cells. The cells obtained from many domestic laboratories fail to achieve their multipotent differentiation, which is not enough to be strong evidence of cardiac stem cell existence. This study aimed to explore an efficient and stable method for culture and systemic identification of cardiac stem cells from mini-pigs, to lay a solid foundation for cell biological therapy of myocardial infarction. Methods: Adult female Bama mini-pigs weighing from 25 to 30 kilograms were selected for this study. The right atrial appendage tissue was resected through heart surgery from a small incision on the right second intercostal. Primary cardiac stem cells obtained by explants method were cultured in cardiosphere-growth medium and passaged according to cell growth. Purified third generation cell lines were selected to detect cell phenotype such as c-kit,CD34, CD45, CD31, CD90 and Lineage by flow cytometry; to measure the expression of early caridac transcription factors Nkx2.5 and GATA-4 through Western Blot; and to observe the positive expression of CX43、cTnT、α-SMA、vWF after induced by 5-AZA, rh-PDGF-BB or bFGF through immunofluorescence to clarify their pluripotent capacity. Results: 1.The primary cells isolated from adult mini-pigs’ right appendage tissue appeared to be small, phase-bright under inverted microscope and grew slowly. While the passaged cells were spindle in morphology and grew adherently. They grew much easier than the primary cells. 2.Flow cytometric analyses confirmed that purified cells mainly express c-kit and did not express CD31, CD34, CD45, CD90 and Lineage. 3.Western blot results showed positive expression of cardiac early specifictranscripti- on factor Nkx2.5 and GATA-4. 4.Purified cells successfully expressed cardiomyocyte markers(cTnT, CX43),smooth muscle marker(α-SMA) and endothelial marker(vWF) after induced by 5-AZA, rhPDGF-BB, and bFGF. Conlusion: 1.Cells with the phenotype of c-kit+ CD31- CD34- CD45- CD90- Lineage- could be separated from adult mini-pigs’ right atrial heart specimens successfully. They can express stem cell surface marker positively. 2.The cells isolated in this study are capable of expressing cardiac early specific transcription factor Nkx2.5 and GATA-4, suggesting that the cells can regulate their early differentiation to cardiomyocyte. 3.The cells can express specific marker protein such as CX43, cTnT, α-SMA, vWF after induced in vitro, suggesting their multiple differentiation potential towards myocardial cells, smooth muscle cells and endothelial cells. 4.The cells isolated and purified from primary tissue explant were identified to be cardiac stem cells by flow cytometry, Western Blot and immunofluo- rescence assay. This study established an efficient and standard method for expansion and identification of cardiac stem cells from adult porcine heart in vitro, which provi- des instructions for clinical application.