Primary Cilia as a Biomarker in Mesenchymal Stem Cells Senescence: Influencing Osteoblastic Differentiation Potency Associated with Hedgehog Signaling Regulation

Figure 2

Osteogenic differentiation of P0 and P4 MSCs. Control or senescent cells were cultured with osteogenic differentiation medium for one week and subjected to ALP staining or detection of the gene and protein expression. (a) Representative bright-field images showing the changes in MSCs during differentiation induction and ALP staining, which was stronger in P0 MSCs than in P4 MSCs. . (b) The expression of osteogenic genes (RUNX2, ALP, and Col I) was measured by qPCR with GAPDH as the reference gene. Student’s -test. versus the corresponding P0 MSCs cultured under the same differentiation conditions; ^# versus MSCs cultured in basic medium without induction of osteogenic differentiation. (c) The protein blots for RUNX2, ALP, Col I, and CDKN2A (p16) as well as (d) the quantification of the expression of these proteins relative to that of GAPDH showed that aged P4 MSCs exhibited decreased potency and increased expression of the senescence marker CDKN2A. All these results showed that the inhibition of osteogenic differentiation by expansion was associated with senescence. versus the corresponding P0 MSCs.

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