| Abstract: Cardiovascular diseases are a leading cause of morbidity and mortality. Currently, the valves, patches and conduits for replacement therapy or repair are imperfect and subject patients to one or more ongoing risks including thrombosis, limited durability, increased susceptibility to infection, and need for reoperations due to lack of growth in pediatric populations. A very promising approach to solve the above problems may be the use of tissue engineering with autologous cells. The successful use of tissue-engineered transplants is hampered by the need for vascularization. Recent advances have made possible the using of stem cells as cell sources for therapeutic angiogenesis, including the vascularization of engineered tissue grafts.Stem cells are undifferentiated cells capable of self-renewal and differentiation into multiple lineages of mature cells. Mesenchymal stem cells (MSCs) can be isolated from various adult tissues of human by their ability of adhering plastic culture plate wall. Despite the fact that bone marrow derived MSCs represent the main available source for cell therapies, the use of bone marrow derived MSCs is not always acceptable because of the significant decrease in cell number and proliferation/differentiation capacity with age. In addition, obtaining the therapeutic quantity of bone marrow requires general anesthesia and hospitalization. In this connection, most attention should be paid to tissues containing cells with higher proliferative potency, capability of differentiation, and low risk of contamination. Human umbilical cord is likely a feasible source of stem cells for its advantage over bone marrow such as vast abundance, lack of donor attrition, and low risk of viral transmission.In the first part of this paper, we described the isolation and characterization of stem cells from human umbilical cord tissue (UCDS cells). When initially plated, the UCDS cells appeared rounded in shape. After 72h of plating, the cells were adherent, elongated, and spindle-shaped. Flow cytometry results showed that UCDS cells were positive for CD13, CD29, CD44, CD90, CD166, and MHC-I, in addition, no expression of CD31, CD34, CD38, CD45, CD106, CD117 and CD144 or MHC-Ⅱwas observed. Most importantly, the cells were positive for Kossa staining and have the ability to accumulate different amounts of lipid vacuoles after cultivation in osteogenic and adipogenic medium. This results demonstrated that UCDS cells were a crowd of undifferentiated stem cells that were similar to bone marrow derived MSCs, have similar phenotype and differentiation ability.In the second part, we investigated the myocardial differentiation ability of UCDS cells. Two weeks after treatment. Some cells gradually increased in size and formed a ball-like or stick-like appearance, but we have not observed beating cells during the in vitro differentiation. RT-PCR results showed that differentiated cells express cardiomyocyte Specific cardiac troponin T gene. Immunocytochemistry showed that differentiated cells were strongly stained with cardiac sarcomericα-actin myosin and Troponin T at 1 month after 5-azacytidine treatment and more than 50% UCDS cells were positively stained. Longitudinal sections of UCDS cells which were directed toward cardiomyocytes were analyzed by transmission electron microscopy. After 4 weeks of induction with 5-azacytidine, some cells showed myofilaments, but their alignment are intricate.The goal of the third part of our study was to examine the endothelial potential of UCDS cells. UCDS cells were differentiated in an endothelial differentiation medium containing VEGF and bFGF. Differentiation into endothelial cells was determined by acetylated low-density lipoprotein (ac-LDL) incorporation and expression of endothelial-specific proteins. The uptake of DiI-labeled ac-LDL is a specific marker for endothelial cells in vitro. Immunofluorescence revealed that the induced cells were positive for ac-LDL uptake. Immunofluorescence studies confirmed their endothelial phenotype with expression of known endothelial cell markers including CD31, CD34 and 30-50% UCDS cells were positively stained. In addition, different staining from cells in the same area of culture showed the induced cells which were positive for Ac-LDL uptake were also positively stained with CD31 and CD34. In vivo, the transplanted UCDS cells were sprouting from local injection and differentiated into endothelial cells in a hindlimb ischemia mouse model. These findings indicate the presence of a cell population within the human umbilical cord that exhibits characteristics of endothelial progenitor cells. Therefore, human umbilical cord might represent a source of stem Cells useful for therapeutic angiogenesis and re-endothelialization of engineered tissue grafts.Umbilical cord derived stem cells can be easily extracted and cryopreserved, allowing for individuals to store their own samples for possible future autologous Use even if there was no immediate indication that stem cell therapy would be required. In the near future, cryopreserved autologous UCDS cells for therapeutic medicine may become available. |
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