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Aurelie Dos Santos, Alis Balayan, Martha L Funderburgh, Irona Khandaker, Elfren Baclagon, James Funderburgh, Sophie X. Deng; Potential of immortalized corneal stromal stem cells for corneal repair. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4358.
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© ARVO (1962-2015); The Authors (2016-present)
Human corneal stromal stem cells (CSSC) stimulate corneal regeneration during wound healing (Basu et al., 2014). However, primary CSSC exhibit a limited replicative life span. To overcome this limitation, we established an immortalized CSSC line to provide an unlimited source of potent CSSC.
CSSC were transduced by overexpression of cMYC or SV40 large T antigen (SV40T). Transduced cells were compared to primary CSSC for expression of mesenchymal stem cell markers and their potential for differentiation, using immunofluorescence and gene expression analysis. In addition, expression of anti-inflammatory gene TSG-6 in response to TNF-α was assessed by qRT-PCR.
Cell lines transduced by cMYC and SV40T showed morphology similar to the parental CSSC through >20 passages in culture. Furthermore, transduced cells overcame replicative senescence with 95.5±2.1% and 97.2±1.2% of Ki67 positive cells in cMYC and SV40T cells at passage 12, respectively. In contrast, only 2.1±5.4% of the parental cells were immunoreactive for Ki67 at passage 7. Compared to the parental CSSC, the expression of stem cell markers, Nanog and SOX2, declined in cMYC overexpressing cells but was retained in SV40T cells. In addition, the differentiation capacity towards keratocytes, after 7 days’ induction, was reduced in cMYC cells. Upregulation of TSG6 transcript (TNFAIP6) in response to TNF-alpha stimulation was maintained in cMYC cells with 4.4-, 3.0- and 1.4-fold expression in the parental CSSC (passage 4), cMYC (passage 12) and SV40T (passage 14) cells, respectively.
CSSC, transduced with SV40T, retained a phenotype close to that of the parental cells but had a diminished response to TNF-α treatment compared to cMYC overexpressing cells. Optimization of the immortalization is needed, as well as the selection of potent clones.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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