Human pluripotent stem cell-derived limbal epithelial stem cells on
Authors
Alexandra Mikhailova, Tanja Ilmarinen, Anjula Ratnayake, Goran Petrovski, Hannu Uusitalo, Heli Skottman, Mehrdad Rafat
Institution
University of Tampere,
Country
Finland
Year
2015
Journal
Experimental Eye Research
Abstract
Corneal epithelium is renewed by limbal epithelial stem cells (LESCs), a type of tissue-specific stem cells
located in the limbal palisades of Vogt at the corneo-scleral junction. Acute trauma or inflammatory
disorders of the ocular surface can destroy these stem cells, leading to limbal stem cell deficiency (LSCD)
e a painful and vision-threatening condition. Treating these disorders is often challenging and complex,
especially in bilateral cases with extensive damage. Human pluripotent stem cells (hPSCs) provide new
opportunities for corneal reconstruction using cell-based therapy. Here, we investigated the use of hPSCderived
LESC-like cells on bioengineered collagen matrices in serum-free conditions, aiming for clinical
applications to reconstruct the corneal epithelium and partially replace the damaged stroma. Differentiation
of hPSCs towards LESC-like cells was directed using small-molecule induction followed by
maturation in corneal epithelium culture medium. After four to five weeks of culture, differentiated cells
were seeded onto bioengineered matrices fabricated as transparent membranes of uniform thickness,
using medical-grade porcine collagen type I and a hybrid cross-linking technology. The bioengineered
matrices were fully transparent, with high water content and swelling capacity, and parallel lamellar
microstructure. Cell proliferation of hPSC-LESCs was significantly higher on bioengineered matrices than
on collagen-coated control wells after two weeks of culture, and LESC markers p63 and cytokeratin 15,
along with proliferation marker Ki67 were expressed even after 30 days in culture. Overall, hPSC-LESCs
retained their capacity to self-renew and proliferate, but were also able to terminally differentiate upon
stimulation, as suggested by protein expression of cytokeratins 3 and 12. We propose the use of bioengineered
collagen matrices as carriers for the clinically-relevant hPSC-derived LESC-like cells, as a
novel tissue engineering approach for corneal reconstruction.