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Kishore Reddy Katikireddy, Reza Dana, Ula Jurkunas; Differentiation Potential of Limbal Fibroblasts and Bone Marrow Mesenchymal Stem Cells to Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):981.
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© ARVO (1962-2015); The Authors (2016-present)
It has been shown that human limbal fibroblasts (LFs) and bone marrow mesenchymal stem cells (BM MSCs) are multipotent. Specifically, a side population of stage-specific embryonic antigen-4 (SSEA4)-positive LFs differentiates into a variety of cell types. The present study compared the stem cell characteristics of SSEA4+ and SSEA4- LFs to those of BM MSCs, and determined their potential to differentiate into the corneal epithelial phenotype.
Human cadaveric limbal tissue (n=6) was treated with dispase to remove the epithelium and endothelium. Single cell suspensions were obtained by digesting the stroma with 0.025% trypsin. Stem cell enrichment was performed by exposure of BM MSCs and LF cells in KnockOut ESC/iPSC medium for 12-15 days. LF and BM MSCs were sorted for SSEA4+ and SSEA4- cells by Magnetic-Activated Cell Sorting. Cell doubling time (CDT), stem cell (SC) marker analysis, and colony forming efficacy (CFE) were performed on sorted LF and BM MSCs. Epithelial phenotype was achieved using induction and differentiation media. Differentiated cells were characterized for corneal cytokeratins (CKs).
After separation, enriched LFs and BM MSCs, 97.4 ± 0.6% and 93.5 ± 0.7% SSEA4+ subgroups were achieved, respectively. The CDT of SSEA4+ LFs was 102 ± 1 hr and SSEA4- LFs was 58.2 ± 1.5 hrs (p=0.002). CDT of SSEA4+ BM MSCs was 105 ± 1 hr. and SSEA4- BM MSC was 56.3 ± 2 hrs (p=0.002). LF and BM MSC subgroups were negative for pan-cytokeratin. After enrichment, SSEA4+ cells showed the ability to form cell aggregates, while SSEA4- cells were mostly adherent to the culture plates. The transcript levels of SC markers OCT4, SOX2, Nanog and Rexo1 were higher in SSEA4+ than SSEA4- groups of LF and BM MSCs (p<0.05). Upon induction and differentiation, both SSEA4+ LFs and BM MSCs exhibited an epithelial morphology and positivity for CK3, CK12, and CK8, with high CFE (p<0.02), whereas the SSEA4- cells exhibited a fibroblast morphology and were negative for corneal epithelial markers.
Although both LFs and BM MSCs express stem cell markers and have some transdifferentiation potential, only SSEA4+ LFs clearly demonstrate the ability to differentiate into corneal epithelial cell phenotype. These findings establish a potential alternative source of corneal epithelial cells that can be harvested for ocular surface reconstruction.
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