Effect of Long-Term Cultures on Proliferative Capacity and Genome Stability of Stem Cells Derived from Adipose Tissue and Bone Marrow in Pigs.

Abstract

Mesenchymal stem cells (MSCs) are multipotent adult stem cells that have been isolated from various tissues. Bone marrow (BM)-derived MSCs (BMSCs) and Adipose tissue-derived MSCs (AMSCs) are main MSCs. Although they all derive from the mesoderm, there maybe some differences between them. Here, we collected BMSCs and AMSCs, maintained them in identical culture conditions and compared basic biological characteristics between them including immunophenotype, differentiation potential, cell growth characteristics, senescence and Pluripotency-specific marker gene (Nanog, Oct4, Sox2, c-Myc and Klf4) expressions. Adipose tissue and bone marrow were obtained from one-week old piglets, enzymatically dissociated using 1.0mg/mL collagenase type I in low-glucose DMEM supplemented with 10% bovine serum albumin. Digestion was carried out continuously for 45 min at 37°C and followed by a 15 min centrifugation (1000 x g). These initial plates represented the passage 0 (P0). At 24 h intervals, cells were washed with phosphate-buffered saline (PBS) to remove floating erythrocytes and other unattached cells. This procedure was repeated for 3 days once a day. The plating and expansion medium consisted of low-glucose DMEM with 10% FBS and 1% penicillin/streptomycin. Cells were maintained at a condition of 37°C with 5% CO2 in tissue culture dishes or flasks and medium was changed twice each week. Cells reached confluence within 10-14 days for initial plating (P0). Once 80% confluence was reached, the adherent cells were detached with 0.25% trypsin-ethylenediaminetetra-acetic acid and then replated at 1.0 x 104 cells/cm2 and passaged every 3-5 days until they were ready for analysis (up to passage 20). For detecting protein level, western blotting was used to check out gene's protein content of Nanog, Oct4, Sox2, c-Myc and Klf4 in different passages MSCs. Our results showed that the mean population doubling time (97.14 ± 8.04 h) of AMSC was obviously longer than that (60.35 ± 5.13 h) of BMSC. The frequency of nuclear aberrations in BMSC was noticeably lower than that in AMSCs regardless of the passage number increasing. Moreover, as the cells went through several passages, both two MSCs showed the change in content of pluripotency-specific marker gene. As the passages increased, all the marker gene's content became lower and lower. Additionally, gene array analysis of BMSCs showed substantially different gene expression compared to AMSCs. In passage 1, BMSCs expressed c-Myc gene's content obviously more than AMSCs, but other genes content expressed similarly. Moreover, AMSCs have been influenced seriously as passage passed down than BMSCs. Western blotting results showed the same change as gene expressions. Collectively, these results indicate that BMSCs display higher proliferative capacity and are more stable than AMSCs in long-term cultures. Because MSCs are being explored to regenerate damaged tissue and treat cardiovascular disease, brain and diabetes, so our results underscore the need to consider specificities among model systems that may influence outcomes when designing potential human applications. This work was supported by the National Basic Research Program of China (2009CB941001). (poster)