Immortalisation with hTERT Impacts on Sulphated Glycosaminoglycan Secretion and Immunophenotype in a Variable and Cell Specific Manner

Abstract

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.

Fig 1. hTERT expression and impact on cell morphology.

(A) Expression of the housekeeping gene ACTB and hTERT determined by RT-PCR in cells following recovery post transduction and with expansion in continuous cell culture. (B-I) Phase contrast micrographs of cells in monolayer culture pre- (B, D and F) and post- (C, E and G) transduction with hTERT or with empty vector (F and I) Scale = 150 μm, exact PD as indicated in image.

Fig 2. The effect of hTERT expression on senescence associated β Galactosidase (SA βGal) activity.

(A-M) SA βGal activity in early and late PD non-transduced and empty vector transduced cells, and late PD hTERT transduced cells. Scale = 100 μm, exact PD as indicated in image. (N-P) Percentage of cells stained positively for SA βGal activity, mean n = 4 ± SD. (** p≤0.01, **** p≤0.0001)

Fig 3. Flow cytometry analysis of cell surface marker expression.

(A) Expression of surface markers CD14, CD19, CD34, CD45, HLA-DR, CD73, CD90, CD105 (filled) with relevant isotype control (unfilled). (B) Quantification of the percentage of positive events compared to the relevant isotype control. Markers set to exclude 99% of isotype control events; ≥ 4x10⁴events were collected per sample. OK3 was tested at PD10, OK3H at PD50, BMA13 at PD6, BMA13H at PD18, 1C6 at PD44 and 1C6H at PD118.

Fig 4. Tri-lineage differentiation capacity of cells over 21 days.

Cells were cultured in monolayer for 21 days in the presence of the appropriate pro-differentiation supplements followed by fixation and staining. Osteogenic samples were stained with alizarin red for calcium deposits, scale = 150 μm. Chondrogenic samples were stained with alcian blue for SGAGs, images are from 1 well of a 24 well plate. Chondrogenic medium frequently caused cells to partially or fully separate from the substrate and contract into pellet-like structures. Adipogenic samples are stained with oil red O for lipid droplets, scale = 80 μm. Images of controls cells cultured in standard maintenance media and stained as appropriate are inset for comparison.

Fig 5. Quantification of sulphated glycosaminoglycan (sGAG) production and retention.

(A) DMMB quantification of sGAG in both the media fraction and monolayer fraction after culture in either MM (-) or PChM (+) for 20 days. (B) Normalisation of sGAG to DNA at Day 20 in MM (-) or PChM (+). (C) Fold change in sGAG at Day 20 compared to levels determined at Day 0 in monolayer (left), media (middle) and in total (right). (D) Fold change in DNA normalised sGAG at Day 20 compared to Day 0 in monolayer (left), media (middle) and in total (right). Bold line at a fold change of 1 indicates no change compared to Day 0 levels. # p<0.05, + p<0.01, *p<0.001 (mean n = 3 ±SD). Experiments were performed at OK3 PD6, OK3H PD50, BMA13 PD6, BMA13H PD16, 1C6 PD 42 and 1C6H PD84.