AGING OF MESENCHYMAL STEM CELLS IN VITRO: IMPLICATIONS IN PHENOTYPE AND DIFFERENTIATION CAPACITY

Background & Aim

Human mesenchymal stem cells (hMSC) have been used in various clinical protocols; these cells are multipotent and can be isolated from various sources. They are able to adhere to plastic, differentiate into mesodermal lineage and express specific markers (CD90⁺/CD73⁺/CD105⁺/CD45^-/CD34^-/CD19^-/CD11b^-/HLA-DR^-). Despite these features and the easily culture method, hMSC present a limited growing time that can affect its use in clinical protocols. In order to stablish the upper limit by which hMSC could be cultivated, hMSC proliferating rate was analyzed from P0 to P16; hMSC were also analyzed regarding its capacity to differentiate into mesodermal cells, imunophenotyping profile, capacity to decrease mononuclear cells proliferation in vitro and its telomere lenght. hMSC was isolated from bone marrow from healthy donors.

Methods, Results & Conclusion

Population doubling time was observed during 16 passages; the cumulative doubling time increased during the first 5 passages and seems to be maintained from P5 to P8. After P8, proliferation rate slowed down. Differentiation capacity, imunophenotyping, MNC proliferation assay and telomere lenght were analyzed in P5, P7, P10 and P14 or P15. Differentiation capacity was decreased by P10 when was observed a less differentiation into adipocytes (very few cells were observed with lipid droplets in the cytoplasm), chondrocytes (a less presence of proteolycans) and osteoblasts (less calcium deposits). Related to the markers expression., its results corroborate the ones observed in differentiation assay where there was a decrease in CD73 (55%) and CD105 (73%) expression on P10; CD90 presented a expression higher than 85% in all passages analyzed. The negative markers such as CD19, HLA-DR and CD34 presented a higher expression on P10 (7,53%, 7,73% and 3,11%, respectivelly). The telomere lenght showed a decrease on P10 when compared to the other passages analyzed (8.9 versus 9.5 on P0, 9.4 on P3, 9.6 on P5 and 9.5 on P7). The immunomodulatory analysis also showed a decrease in modulate the MNCs proliferation on passage 10; when analyzing 1 MSC:1 MNC, the number of proliferating cells increased from 12% (P5), 16% (P7) to 57% (P10) and 66% (P15). All these data suggest that hMSC is changing its proliferative into a senescent profile when cells reach the P10. In conclusion, hMSC could be used for clinical purposes until passage 7-8, since passage 10 already showed great modificiations related to potency, imunophenotyping, proliferation and telomere length.