Results
Morphology and immunophenotype characteristics of cells from human fetal heart
Nucleated cells from human fetal heart plated at low-density formed individual colonies displaying fibroblast-like morphology. After subcultivation, nucleated cells proliferated with a population-doubling time of 23 hours and reached a confluent condition. Adherent cells could be readily expanded in vitro by means of successive cycles of trypsinization, seeding. And culture every 3 days for 20 passages displayed no visible changes in terms of their morphology under light microscopy. The immunophenotype of cells from adult and fetal bone marrow and fetal heart was determined by flow cytometry. The staining pattern of the cells was similar. As shown in Figure 1, adherent cells isolated from adult and fetal bone marrow and fetal heart were positive for CD73, CD105, HLA-ABC, CD44, CD29, CD166 and lacked of expression of CD45, CD34, CD86, HLA-DR. The phenotypic profile of adult, fetal bone marrow and fetal heart adherent cells did not change after 12 passages in culture.
Differentiation ability of cells from human fetal heart Adipogenic differentiation was apparent after 1 week of incubation; two weeks later intracellular lipid accumulation was visualized using Oil Red O staining (Figure 2. A, B, C). Deposition of mineralized matrix on the culture vessels was shown by von Kossa staining (Figure 2. E, F, G). The staining results indicated the differentiation of cultured cells into osteocytic lineage. The positive alcian blue staining (Figure 2. I, J, K) suggested the expression of type II collagen of chondrocyte.Control cells did not show spontaneous adipocyte, osteoblast or chondrocyte formation even after 3-4 weeks of cultivation (Figure 2. D, H, L)
Immunosuppressive effect of cells from human fetal heart in vitroThe MLR data suggested a nonspecific immunosuppressive effect of cells from bone marrow and fetal heart on human CD3+ T cell proliferation in a dose dependent manner (Figure 3).
Discussion
In this study we demonstrated that there were adherent cells with the characteristics of MPC resided in human fetal heart. They showed fibroblast like morphology and, like MPC from bone marrow, were positive for some mesenchymal markers, such as CD73 (SH2, SH3), CD105 (SH4) and negative for the endothelial/hematopoietic progenitor marker CD34 and the pan leukocyte marker CD45. This meant that there were not endothelial progenitor cells (EPC), which expressed CD45 and CD34. In addition, the adherent cells had similar ability to differentiate into adipocyte, osteoblast and chondrocyte. Finally and most importantly, as MPC, the adherent cells exerted an immunosuppressive effect on T cells that was beneficial for clinical application. The out-dated view was that the heart lacked a pool of stem cells capable of self-renewal and differentiation. But more and more evidences show that the adult heart, like the brain, is composed of mainly terminally differentiated parenchyma cells not reentering the cell cycle, is no doubt a terminally differentiated organ but containing adult stem cells supporting its regeneration. Exciting new evidence has emerged that the transplanted human heart harbors a population of primitive undifferentiated cells derived from both the recipient and the donor. These primitive cells may be cardiac stem cells and may play a pivotal role in the remodeling process following transplantation［10］. And recently, Beltrami AP et al［11］ have isolated cardiac stem cells from adult rat, which showed in vitro and in vivo self-renewing and multipotent. What is more, in vivo manipulation of these stem cells could regenerate large amounts of functional myocardium, shown to be one of the most extensive solid organ tissue regenerations by using stem cells reported so far. Maybe autologous cardiac-specific stem cells are more beneficial to clinical cell therapy for cardiac diseases. The adherent cells we isolated are not the same as the cardiac stem cell. Though they both are negative for CD34, CD45, cardiac stem cells do not express fibronectin and vimentin, which is different from the adherent cells (data no shown). The heart and the bone marrow, cardiomyocytes as well as bone marrow MPC, are of mesodermal origin, so we postulate that there will be MPC in adult heart. But it needs further study to make things clearer.
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