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Heli Skottman, Alexandra Mikhailova, Tanja Ilmarinen, Goran Petrovski, Hannu M T Uusitalo, Anjula Ratnayake, Mehrdad Rafat; Bioengineered collagen matrices as carriers for human pluripotent stem cell derived limbal stem cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5637.
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Corneal epithelium is renewed by limbal stem cells (LSCs), a type of tissue-specific stem cells located in the basal layer of the limbus. Limbal stem cell deficiency (LSCD) is a major cause of corneal blindness worldwide, and its treatment remains a challenge. Human pluripotent stem cells (hPSCs) provide new opportunities for corneal regeneration. In this study, we investigated the use of bioengineered collagen matrices as ECM (extracellular matrix)-mimetic substrate carriers for hPSC-derived corneal LSC-like cells, aiming for clinical applications.
Differentiation of hPSCs towards LSC-like cells was directed using small-molecule induction followed by maturation in corneal epithelial medium. After 4 weeks of culture, differentiated cells were seeded onto the bioengineered matrices fabricated as transparent membranes of uniform thickness (100 µm). Standard cell culture plastic coated with human placental collagen IV was used as control. Cell attachment and morphology were monitored regularly using phase-contrast microscopy, and protein expression of several key markers, most importantly p63, CK15, Ki67, CK3 and CK12, was evaluated using immunofluorescence. Cell viability and proliferation activity were assessed after 15 days of culture on the bioengineered collagen matrices and the control, using the WST-1 Cell Proliferation Assay.
Differentiated LSC-like cells had the appropriate cuboidal morphology and were fairly small in size, expressing the LSC marker p63, but not CK3, a marker of terminally differentiated corneal epithelium. After two weeks of culture, cell proliferation was significantly higher on bioengineered collagen matrices than in control wells. Moreover, LSC markers CK15 and p63, along with proliferation marker Ki67 were highly expressed even after 30 days in culture. Overall, these cells retained their self-renewal capacity, but were also able to differentiate upon stimulation, as suggested by protein expression of CK3 and CK12.
Finding a scaffold suitable for transplantation of cells to the ocular surface is an important step towards clinical applications. Here we propose the use of a bioengineered collagen matrix as a scaffold for hPSC-derived LSC-like cells, applicable to cell replacement therapy.
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