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So-Hyang Chung, Hyun Jung Lee, Soojung Shin; Ex vivo limbal epithelial sheet generation on amniotic membrane slide scaffold. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1378.
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© ARVO (1962-2015); The Authors (2016-present)
In patients with limbal stem cell deficiency (LSCD), transplantation of ex vivo cultured limbal epithelial sheets has achieved an appreciable success rate in terms of ocular surface reconstruction and visual outcomes. Here we compared the stemness and proliferation potential of limbal epithelial sheets in parallel without substrate, with sutured HAM onto transwell, and with new HAM slide scaffold.
Human limbal explants from limbal biopsy were set in either control medium (SHEM), SHEM without cholera toxin (w/o CT), or human AB serum 5% w/o CT, and on either the transwell (TW), HAM onto transwell (HAMTW) or HAM slide scaffold (HAMS). We determined outgrowth size from limbal biopsy explants, protein and mRNA expression of limbal stem/progenitor cell markers p63α and ABCG2, and colony forming efficiency (CFE). Cultures limbal epithelial sheets in HAMS were transplanted onto corneas of LSCD rabbit models.
Human AB serum 5% w/o CT caused a very large increase in CFE, ABCG2 efflux activity and p63α and ABCG2 expression. Limbal explant on the HAMS showed increased outgrowth area, cell yield and ki67 expression (p < 0.05) compared to those on TW or HAMTW. Increased ABCG2 efflux activity, stem/progenitor cell markers expression JC1low, and CFE was confirmed in the HAMTW and HAMS compared to TW, respectively (p < 0.05). The percent of corneal epithelial differentiation marker CK12 positive cells was statistically lower in HAMTW and HAMS. At 4 weeks after transplantation in LSCD rabbit models, the basal cell layer intensively expressed p63α and superficial layers expressed CK12 in rabbit cornea. All corneal epithelial layers were intensively stained by anti-human nuclei antibody, indicating that regenerated corneal epithelial cells were originated from transplanted human limbal epithelial sheets grown onto HAMS rather than zonal repopulation by rabbit limbal stem cells.
In conclusion, limbal explant culture onto HAM slide scaffold enhances the growth of expanded limbal epithelial sheet and stemness with the sheet.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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