P-Cadherin Is Expressed by Epithelial Progenitor Cells and Melanocytes in the Human Corneal Limbus

Abstract

Interactions between limbal epithelial progenitor cells (LEPC) and surrounding niche cells, which include limbal mesenchymal stromal cells (LMSC) and melanocytes (LM), are essential for the maintenance of the limbal stem cell niche required for a transparent corneal surface. P-cadherin (P-cad) is a critical stem cell niche adhesion molecule at various epithelial stem cell niches; however, conflicting observations were reported on the presence of P-cad in the limbal region. To explore this issue, we assessed the location and phenotype of P-cad+ cells by confocal microscopy of human corneoscleral tissue. In subsequent fluorescence-activated cell sorting (FACS) experiments, we used antibodies against P-cad along with CD90 and CD117 for the enrichment of LEPC, LMSC and LM, respectively. The sorted cells were characterized by immunophenotyping and the repopulation of decellularized limbal scaffolds was evaluated. Our findings demonstrate that P-cad is expressed by epithelial progenitor cells as well as melanocytes in the human limbal epithelial stem cell niche. The modified flow sorting addressing P-cad as well as CD90 and CD117 yielded enriched LEPC (CD90−CD117−P-cad+) and pure populations of LMSC (CD90+CD117−P-cad−) and LM (CD90−CD117+P-cad+). The enriched LEPC showed the expression of epithelial progenitor markers and better colony-forming ability than their P-cad− counterparts. The cultured LEPC and LM exhibited P-cad expression at intercellular junctions and successfully repopulated decellularized limbal scaffolds. These data suggest that P-cad is a critical cell–cell adhesion molecule, connecting LEPC and LM, which may play an important role in the long-term maintenance of LEPC at the limbal stem cell niche; moreover, these findings led to further improvement of cell enrichment protocols to enhance the yield of LEPC.

Transcriptomic Landscape and Functional Characterization of Human Induced Pluripotent Stem Cell-Derived Limbal Epithelial Progenitor Cells

“…Differentiated hiPSC in CEM were dissociated using Accutase (Life Technologies, Karlsruhe, Germany) and resuspended in ice-cold staining buffer (Keratinocyte serum-free medium supplemented with 2% fetal bovine serum (FBS) and IX insulin transferrin selenium solution (Life Technologies, Karlsruhe, Germany). The harvested cells were filtered with a cell strainer (40 µm, BD Biosciences, Heidelberg, Germany) and flow sorting was performed according to our previously reported method [ 14 , 29 ]. Briefly, single-cell suspensions were incubated with conjugated antibodies ( Supplementary Table S2 ) on ice for 1 h. After three washes, cells were resuspended in ice-cold PBS, and flow cytometry was performed on a FACSCanto II (BD Biosciences, Heidelberg, Germany ) using FACS Diva Software (BD FACSDiva 8.0.1, BD Pharmingen; BD Biosciences, Heidelberg, Germany).…”

“…T-LEPC (CD90 − CD117 − P-cadherin + ) and LM (CD90 − CD117 + P-cadherin + ) from cadaveric limbal tissues were isolated and cultured as described previously [ 14 ].…”

“…Clonal expansion of hiPSC-LEPC (P0, 300 cells/cm [ 2 ]) were studied on feeder layers using mitomycin C-treated 3T3 fibroblasts as described previously [ 14 ] (n = 4).…”

“…To study the possible interactions of LM and hiPSC-LEPC, the co-culture experiments were performed as demonstrated in our earlier publication [ 14 ] (n = 3).…”

“…A different approach promotes autogenic eye-like differentiation of human iPSC in self-forming ectodermal autonomous multi-zones (SEAM) giving rise to several ocular cell types [ 13 ] including corneal epithelial cells, which enabled functional recovery in an animal model of LSCD [ 13 ]. The gene expression profile of hiPSC-derived corneal epithelial cells has been analyzed in comparison with native differentiated limbal epithelial cells (paired box (PAX)6 + cytokeratin(CK)12 + ) [ 13 ] but not with LEPC (PAX6 + CK12 − ), which are present in the basal layer of limbal epithelium and can be isolated by flow sorting (P-cadherin + CD90 − CD117 − ) [ 14 ]. Moreover, it is important to evaluate the angiogenic and immunogenic potential of hiPSC-LEPCs before applying them clinically, as many factors influence the immunological and angiogenic properties of hiPSC-LEPC, such as long differentiation times and culture medium composition [ 15 ].…”