Mesenchymal stem cells (MSCs) are the valuable sources of tissue regeneration, especially for the cellular cardiomyoplasty[9-11]. However, the best source of these cells has not been determined. The most important thing to obtain MSCs is the proliferation and differentiation capacities, however, these capacities decrease with aging[12-13]. The UCB-MSCs are the most young donors, so these cells could be an excellent source of MSCs. In the present study, we further confirmed that whether UCB-MSCs could be a new valuable source of MSCs. Results showed that compared with bone marrow-derived MSCs, UCB-derived mononuclear cells (Figure 1a) are more easily obtained, and furthermore, the afflication of bone marrow puncture could be avoided. In addition, following induction with 5-azaserine, the successful differentiation rate of cardiomyocytes (Figure 1b) from UCB-MSCs is higher and differentiation process is easier compared with bone marrow-derived MSCs[14], this may be related to young donor of the UCB-MSCs[15-16].
Present results demonstrated that there was no difference in the telomerase activity (Figure 2) between the UCB-MSCs lineages with and without 5-azaserine induction. Telomerase activity has been shown to be specifically expressed in immortal cells, such as cancer and germ cells, where it compensates for telomere shortening during DNA replication and thus stabilizes telomere length. Therefore, expression of telomerase activity in pluripotent stem cells may be a necessary and essential step in the maintenance and differentiation of those cells[17]. Following 5-azaserine induction, USB-MSCs showed apparent telomerase activity, implying their pluripotency and ability for further amplification; but there was no significant difference compared with no 5-azaserine induction, which suggests the safety of 5-azaserine induction.
The cardiomyocytes from UCB-MSCs have been obtained, but whether they are safe for clinical transplantation treatment[18-19], especially in terms of cellular cardiomyoplasty. We further detected the presence of pro-oncogenes of MSCs with and without 5-azaserine introduction, including p53, cyclinA, cdk2, C-fos, β-actin, h-TERT and c-myc. The chromosomal karyotypes and tumor formation in nude mice were also investigated.
The present results also showed that there was no significant difference in the presence of pro-oncogenes of MSCs between with and without 5-azaserine induction (Figure 3), and that no abnormal chromosomal karyotypes (Figure 4) in UCB-MSCs were observed after 5-azaserine induction. Chromosomes are visible during active cell division when chromosomal materials are condensed. Normally, the number, shape, and structure of chromosomes are stable, however, various factors can influence karyotype changes, with most tumor cells having abnormal chromosome karyotypes. Additionally, transplantation of 5-azaserine treated MSCs did not result in tumor formation in nude mice (Figure 5). All these findings suggest that UCB-MSCs are safe for transplantation treatment.
We determined the surface molecular markers of UCB-MSCs in pervious study[20]. Flow cytometry was used to further purify these MSCs. Flow cytometry applying ?uoresceinated antibodies can be used to determine surface molecules present in the expanded cell population. Flow cytometric analysis showed that these cells were negative for CD34 human cardiomyocyte marker, but were positive for human MSC markers CD44 and CD90, which is consistent with that reported in the literature for the bone marrow counterpart, indicating the property of UCB-MSCs. UCB-MSCs have been shown to lead to new tissue formation after homing and engrafting to the heart, potentially capable of replacing the lost myocardium and inhibiting the apoptosis of cardiomyocytes, and ultimately improving the functional performance of damaged heart[5-8].
Interestingly, the present results demonstrated that the apoptosis of cardiomyocytes could be inhibited when it was co-cultured with UCB-MSCs (Figure 6). This could be a paracrine mechanism whereby cardiomyocytes-released factors are quenched by MSCs, thereby inhibiting the formation of apoptotic signals[21-22]. Enhancing survival signaling cascades can lead to inhibition of apoptosis[23-24] and control the loss of cardiomyocytes. These results could prove a new mechanism and provide a valuable new therapeutic tool in the treatment of debilitating illnesses, such as myocardial infarction, and a useful strategy for slowing the development of heart failure. Since apoptosis has been known to contribute to the formation of cardiovascular disease, therefore, UCB-MSCs may be another viable source of somatic cells that can potentially be used to treat cardiovascular disease. However, the data is inconclusive and this area warrants further study to characterize the ability of these cells to actively differentiate into other cell types, and the apoptosis inhibiting mechanisms.
