Immortalisation with hTERT impacts on sulphated glycosaminoglycan secretion and chondrogenic potential in a variable and cell specific manner

Dale, T. P.; De Castro, A.; Parkinson, E. K.; Kuiper, N. J.; Forsyth, N. R.

Abstract

Introduction: Limited therapeutic options for the treatment of cartilage damage have driven the development of cell therapy alternatives. How-ever, many of the relevant cell types are difficult to study due to the rapid onset of replicative senescence; this may be ameliorated by for cedreexpression of telomerase reverse transcriptase (hTERT). We have quantified sulphated glycosaminoglycans (sGAGs), essential for maintaining cartilage biomechanical properties, from human bone marrow mesenchymal stem cells (BMA13), chondrocytes (OK3) embryonic stem cell derived cells (1C6) and their hTERT transduced counterparts(BMA13H, OK3H, 1C6H).

Materials and methods: Pellets of~2.59105cells were formed by centrifugation at 300 g for 3 minutes. Pellets were maintained in a 2% O2,5% CO2, 73% N2incubator for 20 days in maintenance media (MM) orpro-chondrogenic media (PCM) supplemented with TGF-b3. SGAG content of proteinase K digested pellets and spent culture media at day0 and day 20 were determined using the dimethyl methylene blueassay and DNA content by Picogreenâassay. Pellets were fixed in 4%paraformaldehyde and paraffin embedded for sectioning and histology.

Results: Centrifuged cells formed free floating pellets within 24 hours that were larger in PCM than in MM and stained abundantly for extra-cellular matrix sGAGs (Fig. 1). In the presence of PCM, DNA normalized (lgsGAG/lg DNA), pellet associated, sGAG levels increased in all three primary cell types compared to day 0 levels (OK3>BMA13>1C6) as did 1C6H transduced cells. However in contrast to this,BMA13H and OK3H transduced cell pellets in PCM had reduced levels of sGAG production in comparison to their non- transduced counter-parts (Fig. 2). In addition to pellet associated sGAG, spent culture media also contained sGAG. Levels in MM were 8.4, 46.2 and 92.8% oftotal quantified sGAG from pellet and media in BMA13, 1C6, and OK3and 62.4, 77.4 and 92.5% in transduced counterparts. Media sGAGwas always highest in MM, and in the presence of PCM was reduced to2.4, 34.8 and 70.8%, and 27.9, 41.8 and 65.5% in primary and transduced cells respectively.

Discussion and conclusions: All three primary cell types had a promising response to pro-chondrogenic conditions with OK3 chondrocytes producing the largest quantities of sGAG/cell. We have determined that hTERT immortalization has a variable and cell type specific outcome and that whilst this can be a successful approach in some cases, there can also be significant impairment of chondrogenic potential.

Disclosure: Authors have no conflicts of interest to disclose

Citation

Dale, T. P., De Castro, A., Parkinson, E. K., Kuiper, N. J., & Forsyth, N. R. (2014, June). Immortalisation with hTERT impacts on sulphated glycosaminoglycan secretion and chondrogenic potential in a variable and cell specific manner. Poster presented at Tissue Engineering & Regenerative Medicine International Society, European Chapter Meeting, Magazzini del Cotone Congress Center, Genova, Italy

• Presentation Conference Type: Poster
• Conference Name: Tissue Engineering & Regenerative Medicine International Society, European Chapter Meeting
• Conference Location: Magazzini del Cotone Congress Center, Genova, Italy
• Start Date: Jun 10, 2014
• End Date: Jun 13, 2014
• Deposit Date: Jun 12, 2023
• Publisher URL: https://onlinelibrary.wiley.com/doi/epdf/10.1002/term.1932