Promoting limbal stem cells proliferation and maintenance using post-thaw human amniotic membranes fortified by platelet lysate

In this study, we aimed to investigate the proliferation and maintenance rate of cultivated human limbal stem cells (hLSCs)-enriched after treating with post-thaw human amniotic membranes (HAMs) fortified by platelet lysate (PL). To prepare PL, three volunteers were selected and platelet rich plasma (PRP) were isolated from their peripheral blood. Thrombin solution was added afterwards at a 1:5 ratio and the produced PL was used to fortify HAMs after a freeze-thaw cycle. hLSCs obtained from corneo-scleral rings of donated cadaveric corneas and cultured on the post-thaw fortified HAMs as the test group. Cell viability as well as genotypic and phenotypic variations in hLSCs-enriched were compared between the test group and the hLSCs cultivated on post-thaw HAMs without PL (as the control group). Cell Viability assessment demonstrated a significant increase of the hLSCs-enriched viability in the test group on days 1 and 3 and a borderline increase after 1 week in comparison to the control group. Tp63 gene expression decreased remarkably on day 7. NGFR gene expression, however, showed a notable increase on days 1, 3, and 7. There was not a significant difference between the hLSCs-enriched in the test and control groups after 1 week, concerning Delta Np63 protein expression. Our findings strongly indicate that applying PL to fortify HAMs after thawing could potentiate hLSCs-enriched proliferation and differentiation to corneal epithelial precursor cells. In this respect, PL-fortified post-thaw HAMs may potentially improve ocular surface wound healing and re-epithelialization.