Construction of human telomerase reverse transcriptase-immortalized rat bone marrow mesenchemal stem cell strains

doi:10.3969/j.issn.2095-4344.2015.23.003

Abstract

Abstract:

BACKGROUND: Because of convenient source, multi-lineage differentiation and low immunogenicity, bone marrow mesenchymal stem cells are the ideal cell type to serve as vectors of transgenic cells in pain management. However, the replicative senescence and small amount of cells obtained from the bone marrow restrict the application of bone marrow mesenchymal stem cells in pain research.
OBJECTIVE: To construct human telomerase reverse transcriptase (hTERT)-immortalized rat bone marrow mesenchymal stem cells as transgenic cellular vectors for pain therapy.
METHODS: Bone marrow mesenchymal stem cells were obtained from whole rat bone marrow, and then transfected with a lentivirus containing the hTERT (pLV-Puro-EF1α-hTERT) followed by puromycin selection. hTERT expression and telomerase activity in these transfected cells were determined by RT-PCR and TRAP. Morphological changes, capacity of cell growth and multi-lineage differentiation, chromosome karyotype and
tumorigenicity were observed in vitro. Moreover, the expression of cell surface molecule, Nestin, MHC-I and MHC-II in transfected cells were also detected by flow cytometry and immunocytochemistry.
RESULTS AND CONCLUSION: The bone marrow mesenchymal stem cells genetically modified by hTERT could be cultured and passaged through 30 generations in vitro. Compared to the primary and negative transfected cells, the hTERT-modified bone marrow mesenchymal stem cells showed higher expression of hTERT mRNA, telomerase activity and cell proliferation. Most of transfected cells stayed at G2/M and S stages. The proliferation index of the transfected cells were increased dramatically. The positive rates of CD29, CD44 and CD90 were over 70%, but the positive rates of CD34 and CD45 were less than 5%. Transfected cells were positive for Nestin in the cytoplasm, but negative for MHC-1 and MHC-11. In addition, this cell line continued to exhibit the characteristics of fibroblastic bone marrow mesenchymal stem cells, including phenotype, differentiation into osteoblasts, adipocytes and neuron-like cells. No chromosome abnormality and tumor formation were observed in this experiment. Taken together, these data suggests that the rat bone marrow mesenchymal stem cells immortalized by hTERT gene are constructed successfully and still maintain major stem cells characteristics, which provide safe and stable cell vectors as research base for pain therapy.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Bone Marrow, Mesenchymal Stem Cells, Telomerase, RNA-Directed DNA Polymerase

CLC Number:

R394.2

Liu Hui-ping, Zhong Xiao-long, Zhao Qing, Huang Wen-qi, An Ke. Construction of human telomerase reverse transcriptase-immortalized rat bone marrow mesenchemal stem cell strains [J]. Chinese Journal of Tissue Engineering Research, 2015, 19(23): 3621-3627.

Figures/Tables 8

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