Telomerase Activity During Differentiation Of Mesenchymal Stem Cells Derived From Human Umbilical Cord Blood Into Neuron-like Cells With EGF And BFGF

Neural stem cells (NSCs) are the self-renewing, multipotent cells that generate neurons, astrocytes, and oligodendrocytes. Althrough there are multipotent NSCs in the brain, they could not generate enough differentiated progeny when brain damaged because of the small amount of NSCs and the lack of enough positive stimulating signals in the brain. And it is difficult to acquire NSCs from the adult brain. The clinical utility of NSCs from Embryonic stem cells and fetal brain is restricted because of ethical and immune rejection problems. So, it has great sense to discover a new source of NSCs for basic and clinical studies in neuroscience field.Mesenchymal stem cells (MSCs) can transdifferentiate into multiple cell lineages and be isolated, cultured and proliferated easily. These characteristics of MSCs make them ideal engineering cells in cell and gene therapies. Not only bone marrow but also unbilical cord blood and peripheral blood harbor MSCs. MSCs from human unbinical cord blood have been shown to give rise to neural cells the same as bone MSCs in recent studies.It is one of the necessaries for MSCs to maintain poliferation ability that they have modest telomerase activity, which reflect indirectly the ability of poliferation and security for transplantation of MSCs. To evaluate the telomerase activity during differentiation of MSCs derived from human umbilical cord blood into neuron-like cells with EGF and bFGF in vitro for potential safe clinical application of MSCs.Methods:Mononuclear cells (MNCs) were prepared from heparinized human umbilical cord blood (5 0-1 00ml) samples (donated from volunteers) by centrifugation over Lymphoprep (density 1.077/ml) and divided into three parts. One part was cryopreserved in liquid nitrogen, the other two parts were resuspended in DMEM/F12 medium containing 20 % fetal bovine serum and allowed to adhere to T-75 tissue culture flasks in humidified atmosphere with 5% CO2 at 37 . After 24 hours, the non-adherent cells were removed, and adherent cells were subsequently cultured for 1, 4, 7, 10, 14 and 21 day(s) with or without recombinant human EGF (10 ng/ml) and bFGF (10 ng/ml). Phenotypic changes were monitored by light microscopy. The cryopreserved MNCs and harvested cells on day 1, 4, 7 ,10,14 and 21 respectively were used to detected the expression of nestin, neurofilament subunit M (NF-M) and hTERT by reverse-transcript polymerase chain reaction (RT-PCR) and telomerase activity by TRAP(PCR)-ELISA during differentiation of mesenchymal stem cells derived from human umbilical cord blood into neuron-like cells.Results:1 .The results showed that compared to the cryopreserved MNCs, higher expression of NF-M mRNA were found in the cultured cells along with culture time, especially in the presence of EGF and bFGF; but low levels of nestin and hTERT mRNA were detected and downregulated in the course of culture, especially in the cultured cells without addition of EGF and bFGF. On day 7, no detectable nestin and hTERT mRNA expression were found.2. Compared to the cryopreserved MNCs, low levels of telomerase activity were detected by TRAP (PCR) -ELISA and downregulated in the course of differentiation ofMSCs into neuron-like cells, especially in the cultured cells without addition of EGF and bFGF (PO.01). On day 21, no detectable telomerase activity was found.Conclusions:1. It is concluded that the combination of human recombinant EGF and bFGF could promote the differentiation of MSCs derived from human umbilical cord blood into neuron-like cells.2. It is concluded that the human recombinant EGF and bFGF could downregulate the expression of nestin and hTERT mRNA in the process of the differentiation of MSCs derived from human umbilical cord blood into neuron-like cells.3.The combination of human recombinant EGF and bFGF could downregulate the telomerase activity in the process of the differentiation of MSCs derived from human umbilical cord blood into neuron-like cells.4.The research would provide the theory basis of potential safe clinical application o...