Study On The Mechanism Of Telomere Mainteance In Human Bone Marrow-derived Mesenchymal Stem Cells

Because of their extensive proliferative potential, ability to self-renew and multipotential properties with regard to differentiation, human mesenchymal stem cells (MSCs) have attracted much attention. MSCs from bone marrow give rise to not only hematopoiesis-supporting stroma. but also multiple nonhematopoietic cell lineages, such as osteoblasts, chondrocytes, muscle cells, adipocytes, cardiomyocytes, neural precursors,neurogliocyte, epicytes, They can maintain their potentiality of multi-directional differentiation even after several population doublings of culture in vitro. In addition,MSCs possess in vivo immunomodulatory properties and they are not significantly immunogenic. For these reasons,MSCs were proposed for clinical applications include stem cell transplantation, stem cell strategies for the repair of damaged organs and gene therapy.MSCs in adult bone marrow (BM) are a very rare population (0.01% to 0.001% of BM mononuclear cells. As a result,it is necessary to culture and populate MSCs in vitro in order to obtain enough quantities of MSCs which is competent for clinical using.MSCs in vitro expansion of about 10 PDs (minimal expansion of MSCs used for transplantation), leads to an average loss of 1.0 kb of telomere sequence which is equivalent tothe loss of more than half their lifespan by the time they are reinflised. And their differentiation potential to adipocyte and osteocyte dropped in the late-passages. It is evident that the fundamental questions of maintaining the proliferative potential and lifespan still need to be resolved before MSCs can be used for safe and effective clinical applications in the context of both cell and gene therapies.Telomeres consist of TTAGGG repeats and associated proteins in human cells. They serve to protect the ends of chromosomes from end-to-end fusion, recombination and degradation. Telomeres have been associated with the molecular machinery critical for cell replicative lifespan.and their shortening is known to play an important part in the cell molecular aging process. In most normal somatic cells, telomere sequences are lost during replication in vitro and in vivo.When the telomere length is decreased to 4kbp-5kbp,the cells enter replicative senescence and lose their proliferative capacity. In contrast, immortal cells such as tumor and stem cells are capable of maintaining telomere function, in general, by activation of the reverse transcriptase telomerase or alternative lengthening of telomeres (ALT).Previous study reported that the telomere length of MSCs was shortening and their differentiation capacity compromised sharply associated with telomere shortening after 7-10 population doublings of expansion in vitro.These study hinted that the shorting of telomere in MSCs may be the mechanisms of reduction in proliferative, differentiative, and homing capacity of MSCs. However, the mechanisms of telomere maintenance of MSCs has not yet been addressed to date. With this regard, a series of experiments were designed to evaluate the underlying mechanism of telomere maintenance in MSCs.To this end, telomere length, extrachromosomal telomeric repeat DNA (ECTR DNA), ALT-associated PML bodies (APBs) and telomerase activity were detected in MSCs. This is the first report on telomerase expression in cycling MSCs, which had ever been believed to lack hTERT expression and telomerase activity.This paper was divided into three parts.In part I, bone marrow aspirates were collected and processed using density gradient centrifugation, from which light-density cells were pooled and cultured with low-glucose Dulbecco's Modified Eagle Medium(LG-DMED). Flow cytometry analysis demonstrated MSCs were uniformly positive for CD29, CD44 and CD166 while negative for CD14, CD34, CD45 and HLA-DR, which indicated no contamination of hematopoietic cells in these cells. The MSCs cultured here can successfully be induced to differentiate into osteogenic precursor cells, lipocytes and neuron cells.In part II, no telomerase activity was detected in MSCs by TRAP protocol. Iimmunofluorescence microscopy demonstrated that PML did not colocalize with TRF1 in MSCs and HeLa cells(a telomerase positive cell line) while it exclusively did in ALT cell line WI-38-2RA cells(a telomerase negative cell line). This results suggested no presence of APBs in MSCs. Furthermore, telomere lengths and ECTR DNA were determined by Southern blot analysis. The telomere in length seemed shorter and relatively more homogeneous in MSCs and HeLa cells than WI-38-2RA cells. The mTRF in MSCs was 8.0 kbp( TRF ranging from 2.7 , kbp~18.0 kbp);the mTRF in HeLa was 6.0 kbp( TRF ranging from 2.7 kbp~8.6 kbp);the mTRF in 293T was 5.0 kbp( TRF ranging from 2.7 kbp~8.6 kbp);the telomeres lengths in mTRF in ALT cell line WI-38-2RA was 21.2 kb (ranging from2.0 kbp~>21.2 kbp). As to ECTR DNA, it was only positive in WI-38-2RA cells, but not in MSCs and HeLa cells.In part III, TRAP protocol was employed to measure telomerase activity in passaged, cryopreserved and differentiated MSCs, respectively. The results indicated that MSCs were negative in telomerase activity and their passage and cryopreservation gave no impact on the level of its activity. Intriguingly, the telomerase activity was positive after their differentiation into adipocytes, which detected to be 11.80+2.52% in RTA. Morever, a cell cycle-dependent expression profile of telomerase was found in MSCs when they were synchronized by serum starvation and Aphidicolin treatment. Untreated MSCs expressed very low level of telomerase probed by Western blot with the 2C4 mAb, but the telomerase level had significantly increased when these cells were trapped in S/G2/M phase.Summary: 1. MSCs did not contain APBs and ECTR DNA which are characteristic of ALT mechanism and their telomere in length was shorter and relatively more homogeneous in length than ALT cell line(WI-38-2RA) 2. The telomerase activity of MSCs was very weakly positive detect by Western blot with the 2C4 mAb, but significantly increased when they were induced to differentiate into adipocytes or trapped in S/G2/M phase. Conclusion: The telomere of MSCs was maintained by telomerase pathway and the level of telomerase expression was associated with cell cycle stage.