The Underlying Mechanism of Telomere Maintenance in Human Bone Marrow-Derived Mesenchymal Stem Cells.

Objective: Human bone marrow-derived mesenchymal stem cells(MSCs) are thought to be promising tools in cell and gene therapy. Unfortunately, the low frequency of MSCs in bone marrow and rapid aging in in vitro expansion, which profoundly compromise their proliferative capacity, give rise to a huge hindrance for their clinical use. Previous study indicated that MSCs would undergo quick telomere shortening as well as reduced replicative capacity during in vitro expansion. These findings suggested that MSCs’ telomere loss might be associated with their decreased proliferative and differentiative potentials. However, the mechanisms by which MSCs maintain their telomere homeostasis have not yet been fully addressed to date. In the present study, we compared the telomere length, the distribution pattern of telomeric repeat binding factor 1(TRF1) between MSCs and other telomerase-positive cells or telomerase-negative cells, detected extrachromosomal telomeric repeat DNA (ECTR DNA) in MSCs and the variation of telomerase activity during cell cycle progression in order to unveil the mystery of telomere regulation in MSCs.

METHODS: MSCs were isolated from healthy human bone marrow (n=34) by the plastic adherence protocols and identified by flow cytometry with markers of CD14, CD45, CD44, HLA-DR, CD34, CD29 and CD166. Telomere length and ECTR DNA were detected with Southern hybridization. The TRF1 distribution were probed with immunofluorescence staining. Telomeric repeat amplification protocol (TRAP ) and/or semi-quantitive Western blot assay were performed to determine the telomerase activity in MSCs, MSCs-derived adipocytes and telomerase levels during cell cycle progression. MSCs were synchronized by serum starvation and Aphidicolin treatment for the aforementioned assay.

RESULTS: The mean telomere restriction fragment (mTRF) in MSCs was 8.0 kbp( range, 2.7 kbp-18.0 kbp), similar to telomerase-positive HeLa cells 6.0 kbp (range, 2.7 kbp-8.6 kbp) and 293T cells 5.0 kbp(range, 2.7 kbp-8.6 kbp); while the mTRF in telomerase-negative cells WI-38–2RA was 21.2 kb (range 2.0 kbp->21.2 kbp). The results indicated that telomere length in MSCs and HeLa cells were shorter and relatively more homogeneous than WI-38–2RA cells. TRF1 did not coincide with promyelocytic leukemia (PML) nuclear body in MSCs and HeLa cells while it exclusively did in WI-38–2RA cells. ECTR DNA was negative in MSCs and HeLa cells but positive in WI-38–2RA cells. Detected by TRAP, telomerase activity in MSCs(n=34) was negative with relative telomerase activity (RTA) of 1.44%±0.77%, but it was positive in MSCs-derived adipocytes (n=3) with RTA of 11.80±2.52%(P<0.001). Moreover, 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 extremely 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 phase.

CONCLUSION: Since MSCs possessed similar features to telomerase-positive cells in telomere length, TRF1 localization pattern and ECTR DNA which were distinct from telomerase-negative ALT cells, and they had increased telomerase activity following differentiation into adipocytes and entrance into S phase, We postulated that the telomere in MSCs was maintained by telomerase pathway other than ALT pathway. The telomerase expression level of MSCs was tightly regulated with cell cycle progression.