| Cell transplantation, as a revolutionary idea, has opened a new exciting perspective for the treatment of infarcted myocardium. Recently, bone marrow stromal cells (MSCs) have been considered as a promising candidate because of their some advantages such as easiness to obtain by a simple routine bone marrow aspiration, ability to self-renew and recently reported novel potential to differentiate into cardiomyogenic cells with the induction of 5-azacytidine or when exposed to a cardiac milieu. However, the precise understanding of the molecular control on this differentiation awaits some further experimental work, and the claim that adult stem cells can be converted into multiple unexpected cell types is also questioned by some experts. To give a further investigation on the potential of cardiomyogenic differentiation of MSCs and explore a new strategy for the therapy of myocardial infarction with cellular transplantation and gene engineering, the present study was performed in several parts as following:1. Morphology and Growth Properties of MSCs in vitro from Wistar RatsTo investigate the morphologic characteristics and proliferation kinetics of MSCs in culture ex vivo, the full marrow was isolated by centrifugation in vitro from wistar rats. After a primary culcure of 24 h, MSCs were seen to attach to culture dishes sparsely after discarding the nonadherent cells by the first medium change and by washing with Hank's balanced salt solution (HBSS) three times. And the majority of the cells displayed a spindle-like shape. These cells began to proliferate at about day 3 or day 4, and gradually grew to form small colonies. By day 7, the number of cellular colonies with different size had obviously increased (about 8-12 colonies /60-mm dish). As growth of cells continued, colonies gradually expanded in size with the adjacent ones interconnected with each other. However, the cultures failed to reach confluency by day 12, although the cells in thecenter of the colonies had formed several overcrowded layers and nearly ceased proliferation at this time. Passaged MSCs were larger in size and more heterogeneous in morphology and in growth properties. Grossly, the MSCs in subcultures could be divided into two types: small spindle- or triangle-like, and broad flattened cells. The flattened cells seldom proliferated and it seemed that the spindle- and triangle-like MSCs gradually transformed into broad flattened cells with passages. At the end of the first passage, about 15-18% of cells were flattened, but at the end of passage 4, at least more than 57% broad flattened cells were observed.When seeded at a density of 2 845 cells/cm2 and cultured under common conditions, rat MSCs could be expanded up to 21.94 cell doublings in 30 days of successive subcultures. This was accompanied by a gradual loss of their replication ability with passages. For primary culture, the cells remained quiescent during the first 4 days of culture, and then quickly replicated until day 10 when the average cell number reached (15.37 + 0.10) X 104. The number of cells did not significantly increase during the following 4 days of culture (days 11 ~ 14). The patterns of the growth curves of MSCs in passages 1 and 2 were similar to those of primary cells. However, the cells showed a shortened quiescent period (days 1 ~ 3) before proliferation and the cell number kept increasing significantly for a shorter period (days 4 ~ 7 in PI and days 4 - 6 in P2). The increase of average cell numbers was considerably slowed in passage 3, and the cells almost ceased proliferation in passage 4.The present study proved that rat MSCs could not be extensively expanded in vitro and they were easy to become senescent with passaging; and the multidifferentiation potential, the application values for cellular transplantation as target cells and vectors of gene delivery shoud be questioned and investigated furtherly.2. The Investigation on the Effects of 5-Azacytine as a Cytosine Analogue on the Cardiomyogenic Differentiation of MSCsTo investigate whether a...
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