Epigenetic Mechanism For Spontaneous Transformation Of Mesenchymal Stem Cells

Mesenchymal stem cells (MSC) are attractive candidates for cell-based therapy and tissue engineering for injured organs or tissues. Although MSCs, as a most promising autologous cell source, show great potential in clinical applications, their fate after extensive in vitro expansion or in vivo transplantation remains inconclusive. Accumulating data have shown that MSCs from various animals, including human, frequently undergo spontaneous transformation during long-term culture. Transformed MSCs show some characteristics of malignant cells and are highly tumorigenic when injected into immunodeficient mice.The MSC transformation is spontaneous and occurs with a frequency much higher than that of natural mutations. Thus, it may be initiated by epigenetic changes rather than gene mutation. In addition, like normal somatic cells, MSCs undergo replicative senescence after a limited number of cell divisions, and the spontaneous transformation takes place after a period of substantial growth arrest, indicating senescence-escape is the key step during the cell’s transformation. How do MSCs escape cellular senescence? It is known that p16INK4a is one of key regulators for cellular senescence and its levels increase in senescent mammalian cells. However, p16INK4a is inactived in most immortalized cells and tumor cells, as well as transformed MSCs. What is the reason for silence of the p16INK4agene? The present research addressed this question by investigating the gene’s regulation during MSC spontaneous transformation at epigenetic level.Partl. Definition of key stages in spontaneous transformation of MSCs during long-term cultureObjective:Evidence from studies on embryonic development or stem cell differentiation confirmed that an epigenetic regulation of a gene’s expression usually takes place at the key points when a cell changes its fate, such as undergoing differentiation, phenotypic modulation, senescence or transformation etc. The objective of the present experiment was to define the key stages of the rat MSC spontaneous transformation for the followed epigenetic studies.Method:Bone marrow MSCs were isolated from femurs and tibias of male SD rats, and identified by checking their surface markers with flow cytometry and mesenchymal potential by induced differentiation assays. According to the cell’s growth profile (growth curve in each passage, accumulative population doublings (PD) with subcultures), and percentages of proliferating (positive for BrdU incorporation) and senescent (positive for SA-β-Gal staining) cells in total population, the MSCs at different stages during spontaneous transformation were defined as the pre-senescent, senescent, senescence-escaped and transformed MSCs (please see results).Result:When expanded to passage2, MSCs cultures were characterized by their colony-form growth, uniform fibroblast-like morphology, typical surface marker expression (97.5%±1.5CD29+,87.4%±2.5CD90+,0.7%±0.3CD34+and0.5%±0.2CD45+) and adiogenic and osteogenic potential. These cells were actively proliferating with more than75%cells incorporating BrdU and less than1%staining for SA-(3-gal. Accordingly, we defined these cells as "pre-senescent" MSCs. When expanded to passage6(24to25PDs), the MSCs ceased proliferating and showed an enlarged and flattened morphology with increased cytoplasmic granularity. These cells were less than1%incorporating BrdU while more than95%staining positive for SA-β-gal and were therefore defined "senescent" MSCs. The senescent MSCs usually maintained silence for about4weeks and after this period, a small number of re-proliferating cells appeared among senescent cells. By week5, the re-proliferating cells had formed small cellular colonies. We obtained cultures enriched in re-proliferating cells that lost the senescent morphology and marker (SA-β-gal positive staining), but had a BrdU incorporation rate of>75%. These cells were termed "senescence-escaped" MSCs. With a further4months of culture the "senescence-escaped" MSCs became highly proliferative, with no sign of growth arrest observed up to P80. Thus, the cells after P35were termed "transformed" MSCs and were identified by>90%BrdU incorporation and<0.1%SA-β-gal staining, and these cells had a similar surface marker pattern to their pre-senescent counterparts.Conclusion:1. The pontaneous transformation of rat MSCs can be divided into four key stages: the pre-senescent, senescent, senescence-escaped and transformed.2. Different from the human MSCs, rat MSCs have no crisis phase after senescen-escape. This may explain why the rat MSCs are much easier to undergo a pontaneous transformation.in vitro in comparison to their human counterparts. Part2. Changes in p16a expression and p16INK a gene-associated epigenetic modification during in vitro spontaneous transformation of rat MSCsObjective:To investigate the expression of p16INK4a, and p16INK4a gene-associated histone modification (H3K27me and H3K9me) and DNA methylation at different stages of MSC spontaneous transformation.Method:The pre-senescent, senescent, senescence-escaped and transformed MSCs were prepared according to criterion described in Part l. The expression p16INK4a in MSCs at four stages was evaluated with western blot and RT-qPCR. The p16INK4a gene-associated H3K27me and H3K9me were quantified with chromatin immunoprecipitation (ChIP). The DNA methylation of p16INK4a gene was analyzed with bisulfite DNA sequencing (BSP).Result:Compared with pre-senescent MSCs, the expression of p16INK4a was obviously up-regulated in senescent MSCs with a sharp decrease in p16INK4a gene associated H3K27me and CpG sites methylation. However, p16INK4a gene associated H3K9me was barely detected in either pre-senescent or senescent MSCs. In both senescence-escaped and transformed MSCs p16INK4a was barely detectable. H3K27me was reversed in senescence-escaped cells and completely restored to or slightly exceeded the pre-senescent levels in transformed MSCs, but the DNA methylation levels significantly increased to70-80%in senescence escaped MSCs and up to85-97%in transformed cells. This DNA hypermethylation in p16INK4a gene is accompanied by appearing and increasing of H3K9me.Conclusion:During in vitro spontaneous transformation of rat MSCs, Changes of p16INK4a expression was correlated with the levels of p16INK4a gene-associated histone modification and DNA methylation. In sensecent MSCs, up-regulation of p16INK4a is accompanied by decreased p16INK4a gene-associated H3K27me and DNA methylation. In senescence-escaped and transformed MSCs, the silence of p16INK4a gene due to DNA hypermethylaiton is associated not only with H3K27me but also with H3K9me. Part3. The effects of Ezh2on p16INK4a gene-associated epigenetic modificationsObjective:To investigate the effects of Ezh2on p16INK4a gene-associated histone modification and DNA methylation during MSC spontaneous transformation.Method:The expression of Ezh2in MSCs at four key stages of spontaneous transformation was evaluated with Western blot and RT-qPCR. The p16INKC4a gene-bound Ezh2was quantified with ChIP assay. The effects of Ezh2on p16INK4a gene-associated histone modification, DNA methylation and cellular senescence were checked by shRNA knockdown of Ezh2.Result:In comparison with pre-senescent cells, Ezh2in senescent MSCs was markedly down-regulated and disassociated from p16INK4a gene. In senescence-escaped and transformed cells, the expression of Ezh2reached levels more than2-fold higher than those in their pre-senescent counterparts, and the p16INK4a gene-bound Ezh2reappeared and restored to the pre-senescent levels. Although knockdown of Ezh2caused a sharp decrease in both global and p16INK4a gene-associated H3K27me in MSCs at all stages, the expected decrease in p16INK4a gene methylation and increase in p16INK4a expression were observed only in pre-senescent MSCs. Furthermore, Knockdown of Ezh2had no effect on p16INK4a gene-associated H3K9me and DNA hypermethylation in senescence-escaped and transformed MSCs. In addition, inhibition of Ezh2induced premature senescence in pre-senescent MSCs, but this effect was not observed in either senescence-escaped or transformed cells.Conclusion:The activity of p16INK4a gene during MSCs senecence is regulated epigenetically by Ezh2-mediated H3K27me and DNA methylation. But in senescence-escaped and transformed MSCs, the silence of the p16INK4a gene caused by a DNA hypermethylation is independent of Ezh2-mediated H3K27me mechanism, but associated with H3K9me, suggesting that an Ezh2/H3K27me-independent and H3K9me-enhanced DNA hypermethylation of the p16INK4a gene might be an epigenetic signature for MSC spontaneous transformation...