Dedifferentiation Effect of hTERT on hMSCs Through Epigenetic Mechanism and Thus Enhances Osteogenesis

陳俊利(Chun-Li Chen) ； Shih-Chieh Hung ； Chien-Mei Liu ； Lein-Tuan Hou

《植牙界》 3期 (2009/01) Pp. 41-43

https://doi.org/10.6373/CYC.200901.0041

摘要

雖然無限制的細胞複製與絕對的多能性分化能力仍為胚胎幹細胞所獨有，然而倫理道德上的考量徹底阻礙了胚胎幹細胞在臨床上的應用可能性，也因此，人類骨髓間葉幹細胞似乎成為重要的特屬於個人或特屬於特定疾病的幹細胞來源。然而，細胞老化與自然分化嚴重阻礙了人類骨髓幹細胞培養時的擴增與其應用，在目前的實驗中，我們發現外在表現人類終端不但能阻止自然分化，並且還能夠增進人類骨髓幹細胞分化的效率，然而，最令人感到興趣的，是我們成功的誘導了這些基因轉殖後的人類骨髓幹細胞朝向生殖及胎盤種系細胞分化。我們想更進一步嘗試證明人類終端轉錄?能夠透過表觀基因的重整，尤其是DNA的去甲基化，來引發人類骨髓幹細胞去分化的現象。我們假設廣泛性的針對CpG island之甲基化情形進行分析，可能得以顯示經由轉殖人類終端基因的骨髓幹細胞，其整體甲基化程度的下降，並且去甲基化的現象，可能高度集中於與發育或分化高度相關的基因，並因此使得這些基因在適當的刺激之下得以表現。而經由主成分分析，我們預期經由轉殖人類終端基因的骨髓幹細胞，其基因表達特徵可能因此趨近於胚胎幹細胞。這些資料將得以指出人類終端在去分化上的重要性，因為其不但能夠回復骨髓幹細胞的多能性分化，並且可能經由表觀基因的機制抑制自然分化。而這些結果將對建立人類骨髓幹細胞在未來臨床應用上的方便性及可及性往前邁進一大步。

並列摘要

Purpose: Cellular senescence and spontaneous differentiation are critical phenomena that impair long-term study of human mesenchymal stem cells (hMSCs) in vitro, despite its high proliferation rate and multipotency comparing to those of differentiated cells derived from different tissues. In addition, bone formation capacity, which might be the most critical factor in implantology, would also be severely influenced by cellular senescence and spontaneous differentiation of hMSCs. The present study was designed to overcome these problems and also explored to drive hMSCs toward stemness and thus to enhance osteogenesis.
Methods and Results: Ectopic expression of telomerase in a previously developed hMSCline was found to enhance pluripotency and block spontaneous differentiation of the cells.Surprisingly, the telomerase-transfected hMSCs (3A6) had a differentiation potential farbeyond the normal hMSCs. They expressed trophoectoderm and germline specific markersin vitro, similar to those of embryonic stem cells upon perturbations with BMP4 and retinoicacid, respectively. Furthermore, the telomerase-transfected hMSCs (3A6) displayed higherosteogenic and neural differentiation efficiency than their parental cells while there was adecrease in DNA methylation level as proved by a global CpG island methylation profileanalysis. Possible underlying mechanisms were assayed by DNA methylation and its regulationenzymes. Notably, the demethylated CpG islands were found to be highly associated withdevelopment and differentiation associated genes. Principal component analysis further pointedout the expression profile of the cells converged toward embryonic stem cells.
Conclusions: These data demonstrate the reversion of pluripotency, blockage ofspontaneous differentiation and improvement of osteogenesis capacity of hMSCs by ectopicexpression of telomerase through epigenetic mechanism.