Reversibly Immortalized Mouse Articular Chondrocytes Acquire Long-Term Proliferative Capability While Retaining Chondrogenic Phenotype

Abstract

Cartilage tissue engineering holds great promise for treating cartilaginous pathologies including degenerative disorders and traumatic injuries. Effective cartilage regeneration requires an optimal combination of biomaterial scaffolds, chondrogenic seed cells, and biofactors. Obtaining sufficient chondrocytes remains a major challenge due to the limited proliferative capability of primary chondrocytes. Here we investigate if reversibly immortalized mouse articular chondrocytes (iMACs) acquire long-term proliferative capability while retaining the chondrogenic phenotype. Primary mouse articular chondrocytes (MACs) can be efficiently immortalized with a retroviral vector-expressing SV40 large T antigen flanked with Cre/loxP sites. iMACs exhibit long-term proliferation in culture, although the immortalization phenotype can be reversed by Cre recombinase. iMACs express the chondrocyte markers Col2a1 and aggrecan and produce chondroid matrix in micromass culture. iMACs form subcutaneous cartilaginous masses in athymic mice. Histologic analysis and chondroid matrix staining demonstrate that iMACs can survive, proliferate, and produce chondroid matrix. The chondrogenic growth factor BMP2 promotes iMACs to produce more mature chondroid matrix resembling mature articular cartilage. Taken together, our results demonstrate that iMACs acquire long-term proliferative capability without losing the intrinsic chondrogenic features of MACs. Thus, iMACs provide a valuable cellular platform to optimize biomaterial scaffolds for cartilage regeneration, to identify biofactors that promote the proliferation and differentiation of chondrogenic progenitors, and to elucidate the molecular mechanisms underlying chondrogenesis.

Functional Characteristics of Reversibly Immortalized Hepatic Progenitor Cells Derived from Mouse Embryonic Liver

“…3B) [49]. We have successfully used this system and immortalized primary progenitors from various sources [50,51,52,54,62]. Accordingly, we successfully generated immortalized hepatic progenitor (iHP) cells isolated from different stages of embryonic liver tissues (Fig.…”

“…More recently, we demonstrate that piggyBac transposon-mediated stable expression of SV40 T antigen is a more efficient approach to the immortalizing primary cells [60]. The most noteworthy features shared by all of the SV40 T antigen-immortalized cells include that 1) they are reversible and become mortal upon the removal of SV40 T antigen from the immortalized cells; 2) stable expression of SV40 T antigen does not completely inhibit the differentiation potential although the removal of SV40 T antigen has been shown to improve the terminal differentiation; and 3) the immortalized cells are non-tumorigenic in immune compromised mice, even after an extended observation period [50,51,52,54]. …”

“…The ability of SV40 large T antigen to immortalize primary cells may be largely dependent on its ability to complex with p53 [104]. Using the reversible immortalization system expressing SV40 T antigen [49], we have immortalized several types of progenitors and adult cells, including MEFs, mouse cardiomyogenic progenitor cells, mouse articular chondrocytes, stem cells of dental apical papilla, and mouse melanoblastic progenitor cells [50,51,52,53,54]. More recently, we demonstrate that piggyBac transposon-mediated stable expression of SV40 T antigen is a more efficient approach to the immortalizing primary cells [60].…”

“…The retroviral vector SSR#69 expressing SV40 large T antigen flanked with LoxP sites and pCLAmpho packaging vector were co-transfected into HEK-293 cells to produce packaged retrovirus as described [49,50,51,52,53,54]. Subconfluent primary fetal liver cells isolated above were infected with retrovirus and selected with hygromycin B (0.3mg/mL, Invitrogen) for 7∼10 days.…”

“…PCR products were approximately 100∼150 bps. SYBR Green-based qPCR analysis was carried out by using the thermocycler Opticon II DNA Engine (Bio-Rad, CA) with a standard pUC19 plasmid as described elsewhere [48,54]. The qPCR reactions were done in triplicate.…”