Ectopic Telomerase Expression Fails to Maintain Chondrogenic Capacity in Three-Dimensional Cultures of Clinically Relevant Cell Types

Abstract

The poor healing capacity of cartilage and lack of effective treatment for associated disease and trauma makes it a strong candidate for a regenerative medicine approach. Potential therapies tested to date, although effective, have met with a number of intrinsic difficulties possibly related to limited autologous chondrocyte cell yield and quality of cartilage produced. A potential mechanism to bypass limited cell yields and improve quality of differentiation is to immortalize relevant cell types through the ectopic expression of telomerase. Pellet cultures of human chondrocytes (OK3), bone marrow mesenchymal stem cells (BMA13), and embryonic stem cell (H1 line)-derived cells (1C6) and their human telomerase reverse transcriptase (hTERT) transduced counterparts were maintained for 20 days in standard maintenance medium (MM) or transforming growth factor-β3-supplemented prochondrogenic medium (PChM). Pellets were assessed for volume and density by microcomputed tomography. Quantitative gene expression (COL1A2, COL2A1, COL3A1, COL6A3, COL10A1, ACAN, COMP, SOX9); sulfated glycosaminoglycans (sGAGs), and DNA quantification were performed. Histology and immunohistochemistry were used to determine matrix constituent distribution. Pellet culture in PChM resulted in significantly larger pellets with an overall increased density when compared with MM culture. Gene expression analysis revealed similarities in expression patterns between telomerase-transduced and parental cells in both MM and PChM. Of the three parental cell types examined OK3 and BMA13 produced similar amounts of pellet-associated sGAG in PChM (4.62 ± 1.20 and 4.91 ± 1.37 μg, respectively) with lower amounts in 1C6 (2.89 ± 0.52 μg), corresponding to 3.1, 2.3, and 1.6-fold increases from day 0. In comparison, telomerase-transduced cells all had much lower sGAG with OK3H at 2.74 ± 0.11 μg, BMA13H 1.29 ± 0.34 μg, and 1C6H 0.52 ± 0.01 μg corresponding to 1.2, 0.87, and 0.34-fold changes compared with day 0. Histology of day 20 pellets displayed reduced staining overall for collagens and sGAG in telomerase-transduced cells, most notably with alterations in aggrecan and collagen VI; all cells stained positively for collagen II. We conclude that while telomerase transduction may be an effective technique to extend cellular proliferative capacity, it is not sufficient in isolation to sustain a naive chondrogenic phenotype across multiple cell types.