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Mary M. Mann, James L. Funderburgh, Yiqin Du, Martha L. Funderburgh, Nancy B. Zurowski; Assessing Potential of Corneal Stromal Stem Cells to Differentiate to Keratocytes. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5153.
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To be able to use stem cells in bioengineering applications it is necessary to reliably determine their stem cell potential. This study examined methods for assessing the potential of human corneal stromal stem cells (CSSC) to express keratocyte phenotype when cultured on rigid or collagen gel substrata.
Human corneal stromal stem cells harvested from adult donor corneas were expanded clonally in a stem cell growth media and then these cells were plated on substrata of tissue culture (TC) plastic, dry collagen, or collagen gels in a culture medium which induces keratocyte differentiation. Upregulation of known keratocyte markers (AQP1, ALDH3A1, CHST6, PTDGS and PDK4) was examined by qPCR, flow cytometry, western blot, and immunostaining. CSSC grown on plastic in a maintenance medium for stem cells, was used as the control.
Up-regulation of mRNA for all the markers was observed by 2-4 days, with TC-plastic eliciting the greatest response. However, flow cytometry repeatedly showed a greater proportion of cells staining for keratocyte proteins in CSSC cultured on collagen gels. After 6 days, proportions of cells staining for keratocyte proteins reached near 100% in the gel cultures.
Stem cell phenotype is usually assessed by expression of ‘stem cell marker’ genes, often of unknown function. In this study we developed a quality control assay of stem cells based on their ability to adopt a differentiated phenotype. Flow cytometry proved to be more informative than PCR because it provided the proportion of differentiated cells as well as a quantitative assessment of protein expression levels. The study also demonstrated that differentiation of CSSC was markedly influenced by the substratum on which the cells are cultured. The study therefore suggests that characterization of stem cells by their progenitor potential and not their stem cell marker expression may be a preferable means of quality control for populations of these highly plastic cells.
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