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Gary Hin-Fai Yam, Ericia PW Teo, Matthew J Lovatt, Bee-Tin Goh, Jodhbir S Mehta; Directing human adult periodontal ligament stem cells to corneal stromal keratocytes. Invest. Ophthalmol. Vis. Sci. 2016;57(12):886. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Dental stem cells (SCs) have drawn much attention due to their accessibility, plasticity and applicability for regenerative medicine. Periodontal ligament SC (PDLSC) shares similar cranial neural crest (NC) origin with corneal stromal keratoctye (CSK). This study investigated the differentiation potential of postnatal PDLSC to CSK in vitro.
Human PDLSCs from the third molars extracted with informed consent were propagated to passage 3~6 in serum culture. They were grown as spheroids with B27, N2 and basic FGF to enrich NC progenitors. The cells were then treated with CSK-inducing medium containing chick embryo extract, L-ascorbate-2-phosphate, basic FGF and TGFβ3 under conditions: (1) inside human amnion stroma or (2) on collagen-I coated surface with soluble human amnion stromal extract (ASE>3kDa). Marker expression for NC, CSK and other developmental lineages was examined by immunofluorescence, quantitative PCR and flow cytometry. Controls were naïve PDLSCs and normal human corneal stroma.
Primary PDLSCs displayed mesenchymal properties with proliferation, plastic adherence and expression of CD44 and CD90, as well as NC genes (nestin, Snail, Slug). Spheroids positively expressing nestin and p75/NGFR were generated in 5 days when PDLSCs were plated in low attachment condition and the spheroid-forming efficiency was 4.1% (NC enrichment). The dissociated spheroid cells expressed keratocan after cultured inside human amnion stroma with CSK-inducing medium for 14 days. When spheroid cells were plated on collagen-I surface with inducing medium and ASE>3kDa for 14 days, they exhibited CSK-like dendritic morphology and expressed stromal crystallins (ALDH1A1, ALDH3A1), lumican, B3GNT7 and CHST6 (keratan sulfate-synthesizing enzymes). Flow cytometry revealed >80% cells expressing keratocan. They had negligible expression of markers related to other lineages, including myogenesis, neurogenesis, vasculogenesis and fibrogenesis.
Our study established a potential of adult human PDLSC as an autologous cell source for CSK in corneal cell therapy and stromal reconstruction.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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