Abstract 1415: Tumorgenicity of Long-Term Cultured Mouse Bone Marrow-Derived Mesenchymal Stem Cells
Bone marrow-derived mesenchymal stem cells (MSC) are considered an ideal source for cell-based therapy for myocardial infarction, since these cells are multipotent and can secrete a variety of growth factors and cytokines that can prevent the death of cardiomyocytes post-infarction, induce angiogenesis, attract endogenous stem cell homing and attenuate inflammation. However, the effect of in vitro expansion, which is necessary to generate a sufficient number of cells for injection in vivo, on MSCs remains unclear. We tested the hypothesis that in vitro culture could modify the properties of MSCs. Methods: Mouse MSCs were harvested from tibias and femurs using the plastic adherent method and cultured to passage 50. Cell growth rate was monitored by cell counting. Expression of cell cycle inhibitors, p16 and p19, and proliferation cell nuclear antigen (PCNA) were analyzed by western blot. The tumorgenicity of MSCs at passages 10 and 50 were tested by culturing in soft agar and inoculation to immunodeficient mice. Chromosome numbers and DNA content were also analyzed. Results: We found that throughout the passages the cell growth rate progressively increased; the cell doubling time decreased from 4.1 ± 0.6 days for passage 3 to 1.2 ± 0.2 for passage 45 and the expression of p16, p19 and PCNA gradually increased, which suggested immortalization of MSCs in long-term culture. Cells at passage 50 had a higher DNA content than cells at passage 2. Many later-passage cells had abnormal chromosome numbers. Passage 50, but not passage 10, MSCs could grow in soft agar and generated subcutaneous tumor after subcutaneous injection into severe combined immunodeficient mice.
Conclusions: Common belief is that adult stem cells, especially autologous stem cells, are safe and will not generate tumors in vivo. However, we found that prolonged in vitro culture altered MSC properties and resulted in their immortalization. Thus, in vitro expanded MSCs need to be carefully characterized before clinical application to patients.
This research has received full or partial funding support from the American Heart Association, AHA National Center.