Immortalization Of Mesenchymal Stem Cells From Bone Marrow Of Rhesus Monkey

ObjectiveBone marrow mesenchymal stem cell (BMSC) is a good choice for cell transplantation and tissue engineering due to its multilineage differentiation and low immunogenicity. Our previous experiments indicated that normal Rhesus monkey BMSCs (RhBMSCs) would undergo growth arrest when the PD (population doubling) number exceeded 20. The limited proliferative capacity of RhBMSCs will hamper their application in cell transplantation and tissue engineering. Establishing an immortalized RhBMSCs lineage might solve the problem.MethodsRhBMSCs were isolated from bone marrow of Rhesus monkey (3 years old) by different method, cultured in different medium, and identified in morphology, phenotype, differentiation and karyotype. Then the cells were steadily transfected by plasmid containing human telomerase reverse transcriptase gene (pCI-neo-hTERT) at PD2. Expression of hTERT, proliferation, phenotype (SH-2, SH-3, SB-10, CD29, CD34, CD45 and HLA-DR), differentiation toward osteogenic lineages, and karyotype of transfected cells were analyzed and compared with that of untransfected RhBMSCs.ResultsAfter transfected with plasmid containing hTERT, the RhBMSCs proliferated vigorously and have undergone more than 50 PDs till now. Apoptosis of transfected RhBMSCs at PD40 was only 4.5%, versus untransfected RhBMSCs at PD15 which more than 33.5%. Compared with normal RhBMSCs, the life span of transfected RhBMSCs had been prolonged, and remained similar morphology, clone and karyotype as normal RhBMCs. More than 95% transfected RhBMSCs were positive for stem cell markers including SH-2, SH-3, SB-10 and CD29, and negative for CD34, CD45 and HLA-DR. Transfected RhBMSCs possessed the ability to differentiate into osteogenic lineages, as same as that of normal RhBMSCs.ConclusionOur results demonstrate that hTERT gene has been transfected into RhBMSCs. After transfection, the life span of RhBMSCs has been prolonged, and the cells show vigorous proliferation activity. Phenotype (SH-2, SH-3, SB-10, CD29, CD34, CD45 and HLA-DR), differentiation, and karyotype of transfected RhBMSCs have no significant difference with untransfected cells. The transfected RhBMSCs are potential as cell source for cell transplantation as well as tissue engineering.