Immortalization of Cementoblast Progenitor Cells With Bmi-1 and TERT

Abstract

A cementoblast progenitor cell line designated BCPb8 was successfully isolated from dental follicle cells immortalized with Bmi-1 and hTERT. BCPb8 showed the potential to differentiate into cementoblasts on implantation into immunodeficient mice. BCPb8 was confirmed to be the first established cementoblast progenitor cell line and will provide a useful model for investigating cementogenesis. Introduction:The dental follicle is the mesenchymal tissue surrounding the developing tooth germ. During tooth root development, progenitor cells present in the dental follicle are believed to play a central role in the formation of periodontal components (cementum, periodontal ligament, and alveolar bone). However, little more is known about the biology of these progenitors. Previously, we observed that cultured bovine dental follicle cells (BDFCs) contained putative cementoblast progenitors. To further analyze the biology of these cells, we attempted to isolate cementoblast progenitors from immortalized BDFC through expression of the polycomb group protein, Bmi-1, and human telomerase reverse transcriptase (hTERT). Materials and Methods: BDFCs were transduced with replication-deficient retroviruses carrying human Bmi-1(LXSN-Bmi-1), and hTERT (LXSH-hTERT) for immortalization. Single cell clones were established from immortalized BDFC, and differentiation into cementoblasts was assessed by implantation into immunodeficient mice. Results and Conclusion: BDFCs expressing Bmi-1 and hTERT showed an extended life span-90 population doublings more than normal BDFCs-and still contained cells with the potential to differentiate into cementoblasts on implantation into immunodeficient mice. From these cells, we established a clonal cell line, designated BCPb8, which formed cementum-like tissue that was reactive to the anti-cementum-specific monoclonal antibody 3G9 and expressed mRNA for bone sialoprotein, osteocalcin, osteopontin, and type I collagen on implantation. Thus, by using Bmi-1 and hTERT, we succeeded in immortalizing cementoblast progenitor cells from BDFC without affecting differentiation potential. The BCPb8 cell line is the first immortalized clonal cell line of cementoblast progenitors and could be a useful tool not only to study cementogenesis but also to develop regeneration therapy for patients with periodontitis.

Characteristic phenotype of immortalized periodontal cells isolated from a Marfan syndrome type I patient

“…Recently, attempts have been made to establish immortalized PDL cells by introducing hTERT (Berry et al 2003;Kamata et al 2004;Fujita et al 2005;Saito et al 2005;Fujii et al 2006;Zhang et al 2006). Cyclin-dependent kinase inhibitors p16 Ink4a and p21 WAF1 induce premature senescence in human cells by telomere-independent mechanisms (Ramirez et al 2001).…”

“…The production and infection of LXSN-Bmi-1 and MSCVpuro-hTERT retroviruses were performed as described previously (Kyo et al 2003;Saito et al 2005). The infected cells were selected in the presence of geneticin (125 μg/ml) or puromycin (0.5 μg/ml).…”

“…After electrophoretic resolution of the cell lysate (20 μg each protein) on 12.5% SDS-polyacrylamide gels, the proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Amersham, N.J.). The subsequent detection procedure was performed as described previously (Saito et al 2005).…”

“…Fiber formation in the HPDL2 and M-HPL1 cells was assessed as described previously (Handa et al 2002;Saito et al 2005;Yokoi et al 2007). Briefly, 1.5×10 6 cells were incubated with 40 mg hydroxyapatite particles and fibrin clot (mixture of mouse fibrinogen and thrombin; Sigma).…”

Periodontal Ligament Stem Cells

“…As a consequence, clonal PDL cell lines derived from mice (Saito et al, 2002), swine (Ibi et al, 2007) and humans (Kamata et al, 2004;Fujita et al, 2005;Pi et al, 2007;Mi et al, 2011) have been developed. In addition, clonal cell lines from mouse dental follicle (DF) cells as a source of PDLSCs have also been reported (Saito et al, 2005;Luan et al, 2006). However, the multipotency in these cell lines has not been analyzed in detail.…”