| Objective:Through observation the biological characteristics of human bone marrow stromal cells (hBMSCs) during in vitro expansion, We intended to understand the role of 20S proteasome in replicative senescence. Based on these results, we explored the mechanism underlying hBMSCs senescence that would have impact for the quality control of hBMSCs preparations used for therapeutic application.Method:hBMSCs were cultured in DMEM containing 10%FBS and passaged about 90% confluence. hBMSCs from both early (passage 1 to 3),middle (passage7 to 9),and late (passage 12 to 14) passages were used to study. (1) The biological characteristics of hBMSCs were observed under the phase contrast micoscpore at all stages and senescence-associatedβ-galactosidase (SA-p-gal) staining was performed to detect senescence cells. Meanwhile, the MTT assay and the bromodeoxyuridine (BrdU) incorporation assay were used to investigate cell proliferation at different passages.(2) Expression of 20S proteasome was analyzed by immunostaing in hBMSCs during in vitro expansion. (3) Early passage cells were exposed to 10μM MG132 (proteasomal inhibition) for 2 h, then left to recover for 22 h and treatment was repeated 3 additional times. Subsequently SA-P-gal staining, MTT and BrdU assay were performed to investigate the effects of proteasomal inhibition on early passage hBMSCs.Results:(1) Early hBMSCs were thinner and spindle-shaped, displayed fibroblast-like morphology. After long-term culture, cells became much larger with irregular and flat shape, nuclei became more circumscribed, and the cytoplasm began to be granular with many inclusions and aggradations in phase contrast microscopy. Proliferation potential of hBMSCs gradually decreased in the course of long-term cultivation.The percentage of SA-β-gal-positive cells in Early, middle and late passage cultures were 9%±7%,54%±8%and 90%±8% respectively, indicated that senescence cells were increased in vitro expansion. MTT analysis demonstrated that the viability of hBMSCs (early,0.61±0.04; middle,0.53±0.05; late, O.37±0.03; p<0.05) were decreased with replicative senescence. Furthermore, BrdU incoperation in late passages (8%±2%) was significantly decreased, compared to early passages (73%±11%; p<0.05).These results suggested that morphology and proliferation capacity of hBMSCs is affected by in vitro expansion. (2) Express of 20S in hBMSCs was detected by immunostaining, the percentage of 20S positive cells was reduced to 31% in late passages, suggesting loss of proteasome function may be related to replicative senescence of hBMSCs. (3) Inhibition of proteasome activity by MG132, the percentage of SA-β-gal-positive cells (87%±13.2%; p<0.05) was dramatically increased in early passage cells as compared to DMSO vehicle control (19%±6.8%), whereas decreased proliferation capacity was observed in early passage cells treatment with MG132 by MTT assay(MG132:0.36±0.06; DMSO:0.47±0.08; p<0.05) as well as BrdU incoperation (MG132,7%±2%; DMSO,54%±4%; p<0.05). These observations demonstrated that Partial inhibition of proteasomes in young cells induced a senescence-like phenotype, thus strongly suggesting a central role of the proteasome during cellular senescence and maintenance in hBMSCs.Conclusions:(1) hBMSCs exhibit a senescence phenotype upon prolonged in vitro culture. (2) Decreased proteasome activity is observed in replicative senescence of hBMSCs.(3) Partial inhibition of proteasomes in young cells caused by treatment with specific inhibitors induce a senescence-like phenotype, thus demonstrating proteasome malfunction may contribute to the cellular senescence of hBMSCs.
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