Descemet Membrane Versus Amniotic Membrane for Limbal Stem Cell Therapy.

PURPOSE: To compare biomarker expression and proliferation rates of limbal epithelial stem cells (LESCs) cultured on Descemet membrane (DM) versus amniotic membrane (AM). To investigate the suitability of DM as a potential long-term scaffold for transplantation of cultivated limbal stem cells on the ocular surface.

METHODS

Donor limbal epithelial cells were pooled, reseeded, and expanded on decellularized DM and AM. LESC biomarker expression was assessed by In-Cell Western and immunocytochemical staining. Cell proliferation was evaluated by BrdU incorporation. Airlift organ cultures were performed on DM-limbal tissue constructs and analyzed with 3D immunofluorescence microscopy to determine if DM could support stratified epithelial growth. Suitability of DM as a potential long-term scaffold for cultured LESCs on the cornea was assessed by evaluating transparency and resistance to collagenase digestion versus AM.

RESULTS

Cultured cells exhibited higher expression of putative LESC markers (ABCG2, ABCB5), lower expression of transient amplifying cell marker (p63α), and lower cell proliferation rates on DM versus AM, indicating DM maintained LESC stemness better than AM. Under airlifting conditions, cells on DM stratified with differentiation and expression of corneal epithelial cell biomarkers in superficial layers, while maintaining LESC biomarker expression in basal layers. DM was more transparent and resistant to collagenase digestion than AM.

CONCLUSIONS

DM promotes LESC stemness better than AM in ex vivo culture and can support stratified corneal epithelium. DM is also a more transparent and potentially durable epithelial scaffold than AM. DM is a promising new alternative to AM for limbal stem cell therapy.