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

Abstract

BackgroundLimited 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.MethodsThree 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).ConclusionIn 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.

Usefulness of Mesenchymal Cell Lines for Bone and Cartilage Regeneration Research

“…Positive for ARS, APS, and Runx2 upregulation [71] Positive for ABS, TBS, and GAG assay in pellet culture [72]; low chondrogenic potential but stimulation of chondrocyte differentiation Positive for OROS, adipogenesis-related genes upregulation [71,73], and NRS [73] BMA13H Positive for ARS (reduced compared with primary cells) [74] Positive for ABS and GAG assay in 2D culture [74]; also positive for TBS, PSR and aggrecan and ColII immunostaining in 3D culture [75]; chondrogenic potential reduced compared with primary cells [74] Positive for OROS (reduced compared with primary cells) [74] SCP-1…”

“…It has been stated that hTERT transduction allows senescence evasion while maintaining in vitro and in vivo osteogenic ability of MSCs [63,91]. Transduction of hTERT alone has been employed to generate iMSC lines, but, since hTERT has no effect over non-replicative senescence, it has also been reported to fail to immortalize MSCs derived from bone marrow [13,18,58,74,104] and adipose tissue [85]. Skårn et al (2014) described that only one out of nine hTERT-transduced bone marrow-derived MSC clones was able to proliferate over 40 PDs, and even this clone showed a slow proliferation rate similar to that of primary MSCs [71].…”

“…On the other hand, there were four iMSC lines that showed reduced osteogenic potential in comparison with primary MSCs, all of them transduced with hTERT alone: BMA13H, hASCs-T, GB/hTERT MSCs, and Pelt cells. Two of these cell lines, BMA13H and hASCs-T, were incompletely immortalized [74,85], and so in these cases the reduction of osteogenic potential could be a result of the progressive loss of differentiation potential that occurs in primary MSCs as well. Also, nonreplicative senescence effects cannot be discarded in the reduction of osteogenic potential observed in GB/hTERT MSCs (derived from umbilical cord) and Pelt cells (derived from the periodontal ligament) [86,90].…”

“…Two iMSC lines did not show any chondrogenic potential when assessed in two-dimensional culture: UE6E7T-2, derived from bone marrow and transduced with E6/E7 and hTERT [99]; and iSuPS, derived from coronal sutures and transduced with SV40LT [50]. Three other iMSC lines (SCP-1, BMA13H, and 3 Hits hMPC), all derived from bone marrow-MSCs, showed reduced chondrogenic potential in comparison to primary cells; all these iMSC lines were chondrogenically induced in three-dimensional culture and contained hTERT as immortalization gene, and two of them were incompletely immortalized [14,74,101].…”

Molecular Phenotyping of Telomerized Human Bone Marrow Skeletal Stem Cells Reveals a Genetic Program of Enhanced Proliferation and Maintenance of Differentiation Responses

“…We observed similar differentiation capacity in vitro between telomerized hMSC and primary hMSC. Whereas Dale and colleagues showed that hTERT transduction of HMSCs affected differentiation potential of the cells to varying degrees, several published studies reported that telomerization in a number of stromal cell populations maintain biological characteristics of the cells. Balducci and colleagues generated an adipose‐derived stromal cell line using transfection with hTERT and sv40 or HPV E6/E7 and the cells retained osteogenic and adipogenic differentiation potential.…”