Effect of Cryopreserved Amniotic Membrane Orientation on the Expression of Limbal Mesenchymal and Epithelial Stem Cell Markers in Prolonged Limbal Explant Cultures

Abstract

PurposeTo evaluate the effect of prolonged limbal explants cultured without any scaffolds or on amniotic membrane (AM) on the viability, proliferation and differentiation potential of putative phenotypically defined cultured limbal mesenchymal (LMSC) and epithelial stem cells (LESC).MethodsLimbal explants were cultivated on cryopreserved intact AM or plastic plates using medium supplemented with only human serum. AM was positioned with either the epithelial or stromal side up. The outgrowing cells were immunophenotyped for the co-expression of mesenchymal stem cell markers (CD73/CD90/CD105 positive and CD45 negative), proliferation and putative progenitor markers (CXCR4, CD117), epithelial markers and antigen presenting cell markers (CD80, CD83, CD86) by flow cytometry. Immunohistochemistry on limbal cultures cultivated on AM was carried out with antibodies against pan-cytokeratin, p63, Ki67.ResultsMorphological and immunostaining analyses revealed two distinct stem cell population types, which could be identified over prolonged culturing time periods. Expression of LMSC markers and CXCR4 was significantly higher (p < 0.05) in cultures cultivated without AM. However, no statistically significant difference was observed in CD117 expression. The cells cultivated on AM retained an epithelial cell structure, which was further confirmed by histology examination. Histology revealed limbal epithelial growth and p63, Ki67 positive cells on both sides of AM.ConclusionLimbal cells cultivated on AM exhibited a lower expression profile of LMSC and CXCR4 markers as limbal cells cultivated on plastic culture plates. However, CD117 expression was similar. Histology confirmed limbal epithelial cell growth on both sides of AM, with no morphological differences, or positivity of cells for p63 and Ki67.

CD200 Expression Marks a Population of Quiescent Limbal Epithelial Stem Cells with Holoclone Forming Ability

“…LEGEND Screen analysis confirmed high expression of the commonly cited limbal epithelial cell markers: EGFR (88.81 ± 6.02) , SSEA‐4 (54.02% ± 5.93%) , CD71 (88.76 ± 5.92) , integrin β5 (91.45 ± 1.24) , integrin α6 (92.54 ± 6.41) , E‐cadherin (88.48 ± 6.06) as well as many other general markers of corneal epithelium. The presence of other markers previously related to limbal epithelial cells was also confirmed: CD40 (26.00 ± 6.94) , CD117 (c‐kit) (8.22 ± 2.56) , CD146 (67.04 ± 2.87), and CD166 (95.08 ± 0.97) as well as the presence of putative LSC marker integrin α9/β1 (4.85 ± 1.98) . The expression of the autophagy marker LAMP1 (84.82 ± 11.89) was also high, corroborating with previously reported data on limbal epithelial cultures .…”

Explant culture: An advantageous method for isolation of mesenchymal stem cells from human tissues

“…Different populations of stem cells have been detected in human corneal tissue . Recently, MSC was also isolated from human cornea and found to play role in injury/wound healing process within the tissue . Luznic et al.…”

“…explanted limbal rim pieces including the epithelium as well as some of the superficial limbal stromal tissue. They detected two distinct population of stem cells after long‐term culture (when explants were cultivated for over 30 days) and found that after a second 30‐day explantation of same pieces into another dish, MSC population became 2‐fold in number . In another study, Vereb et al.…”

Identification and characterization of dendritic cell subtypes in preserved and cultured cadaveric human corneolimbal tissue on amniotic membrane

“…) and were performed as recently described (Lužnik et al. ). In brief, the remaining limbal rim was cut into approximately 1 × 2 × 0.25 mm equal pieces of limbal explants (biopsy), obtaining 5–6 limbal explants from each donor.…”