title: Human pluripotent stem cell-derived limbal epithelial stem cells on bioengineered matrices for corneal reconstruction.
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) - 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 hPSC-derived 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.
authors.affiliation: BioMediTech, University of Tampere, Tampere, Finland. Electronic address: alexandra.mikhailova@uta.fi.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Mikhailova
authors.affiliation: BioMediTech, University of Tampere, Tampere, Finland. Electronic address: tanja.ilmarinen@uta.fi.
authors.forename: Tanja
authors.initials: T
authors.lastname: Ilmarinen
authors.affiliation: LinkoCare Life Sciences AB, Linköping, Sweden. Electronic address: aratnayake@linkocare.com.
authors.forename: Anjula
authors.initials: A
authors.lastname: Ratnayake
authors.affiliation: Stem Cells and Eye Research Laboratory, Department of Ophthalmology, Faculty of Medicine, University of Szeged, Szeged, Hungary. Electronic address: petrovski.goran@med.u-szeged.hu.
authors.forename: Goran
authors.initials: G
authors.lastname: Petrovski
authors.affiliation: Department of Ophthalmology, SILK, University of Tampere and Tays Eye Center, Tampere, Finland. Electronic address: hannu.uusitalo@uta.fi.
authors.forename: Hannu
authors.initials: H
authors.lastname: Uusitalo
authors.affiliation: BioMediTech, University of Tampere, Tampere, Finland. Electronic address: heli.skottman@uta.fi.
authors.forename: Heli
authors.initials: H
authors.lastname: Skottman
authors.affiliation: LinkoCare Life Sciences AB, Linköping, Sweden; Department of Biomedical Engineering, Linköping University, Linköping, Sweden. Electronic address: mehrdad.rafat@liu.se.
authors.forename: Mehrdad
authors.initials: M
authors.lastname: Rafat
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D002452:Cell Count; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D003316:Corneal Diseases / Q000473:pathology / Q000601:surgery*; D016039:Corneal Transplantation / Q000379:methods*; D016895:Culture Media, Serum-Free; D019573:Epithelium, Corneal / Q000378:metabolism / Q000637:transplantation / Q000648:ultrastructure*; D006801:Humans; D016850:Limbus Corneae / Q000378:metabolism / Q000648:ultrastructure*; D008855:Microscopy, Electron, Scanning; D039904:Pluripotent Stem Cells / Q000378:metabolism / Q000648:ultrastructure*; D033581:Stem Cell Transplantation; D023822:Tissue Engineering / Q000379:methods*
chemical_list: D015415:Biomarkers; D016895:Culture Media, Serum-Free
keywords: Corneal epithelium; Human pluripotent stem cells; Limbal epithelial stem cells; Regenerative medicine; Serum-free culture conditions; Tissue engineering