Abstract
Purpose :
Limbal melanocytes (LMel) represent essential components of the corneal epithelial stem cell niche. Previous studies have shown that LMel exert various non-canonical functions in limbal niche regulation, e.g. by maintaining an undifferentiated phenotype of limbal epithelial stem/progenitor cells (LEPC) (Polisetti, Gießl et al. 2021). Because the molecular mechanisms underlying this intercellular communication have not been clarified, we explored the proteomic profile and effects of LMel-derived melanosomes, exosomes and soluble factors on LEPC phenotype.
Methods :
Primary cultures of LEPC, LMel, and limbal mesenchymal stromal cells (LMSC) were established following their isolation by enzymatic digestion from human corneoscleral rims. Contaminating cell types, such as epithelial cells or fibroblasts, were eliminated by magnetic activated cell sorting (MACS) using cell type-specific antibodies in stepwise purification protocols. LMel-derived melanosomes and exosomes were purified by several centrifugation steps, validated by electron microscopy and Western blotting, and analyzed by MALDI-TOF-MS. Proteins were identified by MASCOT peptide mass fingerprint search. LEPC were incubated with purified melanosomes, exosomes and exosome-depleted conditioned media and analyzed by qPCR, Western blotting and immunocytochemistry.
Results :
Typical melanosomes and exosomes could be purified from conditioned media of LMel. Approximately 600 proteins were identified in melanosomes and about 170 in exosomes relating to structural, metabolic and immunological functions. Melanosomes were phagocytosed by LEPC in vitro. LEPC incubated with melanosomes, exosomes and conditioned media showed an increased proliferation and significant upregulation of progenitor markers (e.g. K15, ABCG2, FZD7) compared to untreated controls, whereas differentiation markers (e.g. K3, IVL, DSG1) were downregulated on the mRNA and protein level.
Conclusions :
Based on their potent regulatory properties, LMel-derived melanosomes and exosomes may be a valuable novel tool in tissue engineering strategies and stem cell-based therapies for ocular surface reconstruction.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.