| Summary: | 碩士 === 國立臺灣大學 === 臨床牙醫學研究所 === 96 === Background: Cellular senescence and spontaneous differentiation are critical phenomena that impair long-tern study of cells in vitro. The same problems are similarly encountered in the investigation of human mesenchymal stem cells (hMSCs), despite its high proliferation rate and multipotency comparing to those of differentiated cells derived from different tissues. The present study was desigmed to overcome these problems and also explored to drive hBMSCs toward stemness.
Materials and Methods: Primary hBMSCs, E6E7-transfected hBMSCs (KP cells) and Htert transfected cells (3A6) were used in the present study under regular culture condition. Flow cytometry using many stem cell markers was applied to screen the similarity of studied cell types. Major targeted cells, 3A6, were generated by insertion of phTERT-IRES2-EGFP gene fragment using Nucleofector technology. Functional analysis on differentiation and dedifferentiation of all investigationed hBMSCs, KP and 3A6 were evaluated by RT-PCR of osteogenic, neural and adipogenic gene messages cytochemical staining and in vitro mineralization. Mater embryonic transcription gene markers, such as Oct4 and Nanog etc, and germline differentiation were further examined by RT-PCR, in unstimulated and stimulated BMP4 and RA condition.
Stemness related evidence and possible mechanism were explored by measurements of demethylation level of genomic DNA with CpG island microarray and level of various demethylation enzymes, such as DNMT1, 3A, 3B and EZH2 by real-time PCR between 3A6 and KP cells. Further relative relashinship between 3A6 and ESC was investigated by microarray expression data sets and principal component analysis in AffymetrixTM U133 and principal compared with public accessible array databasesat Gene Expression Omnibus (GEO) (http: //www.ncbi.nlm.nih.gov/geo/).
Results: Ectopic expression of telomerase in a previously developed hMSC line was found to enhance pluripotency and block spontaneous differentiation of the cells. Surprisingly, the telomerase-transfected hMSCs (3A6) had a differentiation potential far beyond the normal hMSCs. They expressed trophoectoderm and germline specific markers in vitro, similar to those of embryonic stem cells, upon perturbations with BMP4 and retinoic acid, respectively. Furthermore, the telomerase-transfected hMSCs (3A6) displayed higher osteogenic and neural differentiation efficiency than their parental cells did, while there was a decrease in DNA methylation level as proved by a global CpG island methylation profile analysis. Possible underlying mechanisms were assayed by DNA methylation and its regulation enzymes. Notably, the demethylated CpG islands were found to be highly associated with development and differentiation associated genes. Principal component analysis further pointed out the expression profile of the cells converged toward embryonic stem cells.
Conclusions: In addition to the preservation of stem cells’ characteristics in hTERT-transduced hMSCs, our present data also demonstrated the first pilot evidence that the reversion of pluripotency and blockage of spontaneous differentiation of hMSCs could be initiated in immortalized hBMSCs cell line by ectopic expression of telomerase.
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