Dale, TP and de Castro, A and Kuiper, NJ and Parkinson, EK and Forsyth, NR (2015) Immortalisation with hTERT Impacts on Sulphated Glycosaminoglycan Secretion and Immunophenotype in a Variable and Cell Specific Manner. PLoS One, 10 (7). e0133745 -?. ISSN 1932-6203
This is the latest version of this item.
Immortalisation with hTERT Impacts on Sulphated Glycosaminoglycan Secretion and Immunophenotype in a Variable and Cell Specific Manner..pdf - Published Version
Available under License Creative Commons Attribution.
Download (10MB) | Preview
BACKGROUND: Limited options for the treatment of cartilage damage have driven the development of tissue engineered or cell therapy alternatives reliant on ex vivo cell expansion. The study of chondrogenesis in primary cells is difficult due to progressive cellular aging and senescence. Immortalisation via the reintroduction of the catalytic component of telomerase, hTERT, could allow repeated, longitudinal studies to be performed while bypassing senescent phenotypes. METHODS: Three human cell types: bone marrow-derived stromal cells (BMA13), embryonic stem cell-derived (1C6) and chondrocytes (OK3) were transduced with hTERT (BMA13H, 1C6H and OK3H) and proliferation, surface marker expression and tri-lineage differentiation capacity determined. The sulphated glycosaminoglycan (sGAG) content of the monolayer and spent media was quantified in maintenance media (MM) and pro-chondrogenic media (PChM) and normalised to DNA. RESULTS: hTERT expression was confirmed in transduced cells with proliferation enhancement in 1C6H and OK3H cells but not BMA13H. All cells were negative for leukocyte markers (CD19, CD34, CD45) and CD73 positive. CD14 was expressed at low levels on OK3 and OK3H and HLA-DR on BMA13 (84.8%). CD90 was high for BMA13 (84.9%) and OK3 (97.3%) and moderate for 1C6 (56.7%), expression was reduced in BMA13H (33.7%) and 1C6H (1.6%). CD105 levels varied (BMA13 87.7%, 1C6 8.2%, OK3 43.3%) and underwent reduction in OK3H (25.1%). 1C6 and BMA13 demonstrated osteogenic and adipogenic differentiation but mineralised matrix and lipid accumulation appeared reduced post hTERT transduction. Chondrogenic differentiation resulted in increased monolayer-associated sGAG in all primary cells and 1C6H (p<0.001), and BMA13H (p<0.05). In contrast OK3H demonstrated reduced monolayer-associated sGAG in PChM (p<0.001). Media-associated sGAG accounted for ≥55% (PChM-1C6) and ≥74% (MM-1C6H). CONCLUSION: In conclusion, hTERT transduction could, but did not always, prevent senescence and cell phenotype, including differentiation potential, was affected in a variable manner. As such, these cells are not a direct substitute for primary cells in cartilage regeneration research.
|Uncontrolled Keywords:||Chondrocytes, Cell staining, Cell differentiation, Senescence, Cell immortalization, Alizarin staining, Cartilage, Mesenchymal stem cells|
|Subjects:||R Medicine > R Medicine (General)|
|Divisions:||Faculty of Medicine and Health Sciences > Institute for Science and Technology in Medicine|
|Date Deposited:||20 Aug 2015 11:38|
|Last Modified:||30 Nov 2016 14:18|
Available Versions of this Item
Immortalisation with hTERT Impacts on Sulphated Glycosaminoglycan Secretion and Immunophenotype in a Variable and Cell Specific Manner. (deposited 29 Jul 2015 11:56)
- Immortalisation with hTERT Impacts on Sulphated Glycosaminoglycan Secretion and Immunophenotype in a Variable and Cell Specific Manner. (deposited 20 Aug 2015 11:38) [Currently Displayed]
Actions (login required)