Impaired odontogenic differentiation of senescent dental mesenchymal stem cells is associated with loss of Bmi-1 expression

Abstract

Introduction: Dental mesenchymal stem cells (dMSCs) might differentiate into odontoblast-like cells and form mineralized nodules. In the current study, we investigated the effects of senescence on odontogenic differentiation of dMSCs.

Methods: dMSCs were serially subcultured until senescence. Telomere lengths and telomerase activities were determined by quantitative polymerase chain reaction. Expression of genes involved in cell proliferation and differentiation, eg, Bmi-1, p16(INK4A), osteocalcin (OC), dentin sialoprotein (DSP), bone sialoprotein (BSP), and dentin matrix protein-1 (DMP-1) were assayed by Western blotting and quantitative reverse transcription polymerase chain reaction. Exogenous Bmi-1 was expressed in dMSCs by using retroviral vectors. Odontogenic differentiation was assayed by alkaline phosphatase activity.

Results: Subculture-induced replicative senescence of dMSCs led to reduced expression of Bmi-1, OC, DSP, and BSP compared with rapidly proliferating cells, whereas p16(INK4A) level increased. The cells exhibited progressive loss of telomeric DNA during subculture, presumably as a result of lack of telomerase activity. Bmi-1 transduction did not affect proliferation of cells but enhanced the expression of OC and DSP in the late passage cultures. Bmi-1-transduced cells also demonstrated enhanced alkaline phosphatase activity and mineralized nodule formation.

Conclusions: These results indicate that dMSCs lose their odontogenic differentiation potential during senescence, in part by reduced Bmi-1 expression.

Figure 1. DPSC undergo limited lifespan and replicative senescence upon serial subculture

(A) Replication kinetics of DPSC and SCAP were determined and plotted against time in culture. DPSC reached a maximum of PD 64 and SCAP reached a maximum of PD 46. (B) Relative telomere length was determined by q-PCR analysis of 30 ng DNA of SCC4, 16B, NHK, NHF, DPSC (PD 17, 29, 54, >65), SCAP (PD 18, 28, 41, 44). Average telomere versus single copy gene (T/S) ratio was used to determine telomere length. (C) Telomerase activities in SCC4, NHK, DPSC and SCAP were determined in dose dependent manner using SYBR Green Q-TRAP assay. (D) Protein extracts of DPSC at PDs 16, 32, and 54 were analyzed by Western blotting for expression of Bmi-1 and p16^INK4A. β-actin was used as a loading control. (E) DPSC at the indicated PDs were assessed for mRNA expression of Bmi-1 and INK4A by RT-qPCR. (F) Presenescent (rapidly proliferating) and senescent DPSC were stained for SA β-Gal activity. Original magnification, 100x. Green signal represents the positive SA β-Gal staining.

Figure 2. DPSC and SCAP exhibit abrogation of odontogenic differentiation upon senescence

(A) DPSC cultures at varying PD levels were assessed for mRNA expression of OC, DSPP, and BSP by RT-qPCR. (B) Protein extract collected from DPSC at PDs 13 and 48 were assayed seven days post-confluence for the ALP activity and the protein expression level. Protein extracts were analyzed by Western blotting in which β-actin was used as a loading control. (C) ALP activity and the protein expression were assayed in SCAP at PDs 14 and 44 using the same method as shown in panel B. DPSC and SCAP at the indicated PDs were stained for the ALP activity seven days post-confluence. (D) Photographs of the ALP staining are shown for SCAP and DPSC at varying PD levels.

Figure 3. Bmi-1 transduction in DPSC enhances odontogenic differentiation and mineralization capacity

(A) Protein extracts from induced DPSC/B0 and DPSC/Bmi-1 were collected 7 days post-confluence and ALP activity for odontogenic differentiation was measured. Protein extracts from these cultures were also analyzed by Western Blotting for ALP and Bmi-1 protein expression. β-actin was used as a loading control. (B) Infected DPSC were cultured under calcifying conditions for 28 days post-confluence, stained for Alizarin Red and subsequently destained for 1 hour for quantification. (C) ALP activity was determined in SCAP/B0 and SCAP/Bmi-1 cells after 7 days of culture in calcifying condition post-confluence. (D) Odontoblastic markers OC, DSPP, BSP, and DMP-1 mRNA expression of early (PD 11) and late passage DPSC/B0 (PD 27) and DPSC/Bmi-1 (PD 28) were quantified by RT-qPCR.