Bone marrow derived human mesenchymal stem cells (hMSCs) are considered one of the most promising and prospective resources for cell and gene therapy in mesenchymal and non-mesenchymal applications because of their great self-renewal and versatile plasticity in vitro and in vivo . However, there are still two major hindrances, loss of stem-like properties, namely self-renewal and multipotency, and spontaneous differentiation, encountered during in vitro expansion of MSCs . Loss of stem-like properties could be defined as diminished replication, altered functionality , and deteriorated potential for differentiation . Spontaneous differentiation, known as the emergence of lineage-specific markers without any directed differentiation, would diminish the proportion of undifferentiated stem cells, and therefore compromised the benefit of hMSCs for clinical application. Thus, identifying methods for inhibiting loss of stem-like properties and spontaneous differentiation, and reversing hMSCs to a more primitive state has attracted great research interest.
In a previous attempt to immortalize hMSCs with increased life span, we have established a cell line-KP by transferring HPV16 E6E7 genes into hMSCs . Though KP successfully overcomes the drawback of cellular senescence and could be passaged over 100 population doublings (PDs), the phenomenon of spontaneous differentiation could not be avoided . Telomerase, known to maintain the telomere length, has been indicated to play a role in self-renewal and pluripotency of embryonic stem cells (ESCs) . However, hMSCs express no telomerase activity with telomere shortening in a rate similar to non-stem cells (30-120 bp/population doubling), and cease to divide when the telomere length is less than 10 kb . Besides, ectopic expression of human telomerase reverse transcriptase (hTERT), the catalytic component of telomerase, has been proven not only to bypass cellular senescence and extend life span , but also to influence differentiation potential . Notably, a recent report has unraveled a fascinating fact that TERT might play a crucial role in gene regulation directly or indirectly, which finally caused profound changes in gene expressions of mouse skin . What's most important, the authors further demonstrated that the effect of TERT on gene regulation is irrelevant to its catalytic enzyme action at telomere ends .
In mammals, DNA methylation of cytosines in cytosine guanine dinucleotide (CpG) islands, known to mediate epigenetic gene silencing [12, 13], plays pivotal roles in embryonic development [14–16] and ESC differentiation . For example, treating ESCs or somatic cells with demethylation agent such as 5-azacytidine (5-AzaC) resulted in dedifferentiation, thereby pointing out the association of DNA methylation with the differentiation state [18–20]. These results also imply methods that reverse the differentiation state of stem or progenitor cells will induce changes in DNA methylation patterns .
In this study, we hypothesized, after ectopic expression of HPV16 E6E7 and hTERT, hMSCs would bypass loss of stem-like properties and block spontaneous differentiation with changes in DNA methylation patterns. Meanwhile, we also tried to demonstrate the heightened differentiation potential of HPV16 E6E7 and hTERT-transfected hMSCs by directing germline and trophoectoderm differentiation. Finally, the roles of DNA methylation-modification factors, such as DNA methyltransferases (DNMTs) in the reversion of hMSCs to a more primitive state would be explored.