s-PRGF Supports the Expansion of Human Limbal Epithelial Stem/Progenitor Cells

Abstract

Purpose: To investigate if serum derived from plasma rich in growth factors (s-PRGF) have the capacity to support the expansion of human limbal epithelial stem/progenitor cells (LSCs).

Methods: Limbal explants were cultured on denuded amniotic membrane in Supplemented Hormonal Epithelial Medium (SHEM) supplemented with 5% fetal bovine serum (FBS), 10% human serum (HS) or 10% s-PRGF. Single LSCs cultured on a growth-arrested 3T3-J2 monolayer with SHEM with 5% FBS were used as a control. Cell morphology, cell growth rate and expression level of putative stem cell and differentiation markers were examined.

Results: Cell growth rate based on the population doubling time was similar for LSCs in the form of tissue explants cultured with either serum preparation or FBS, and similar to that of the control. LSCs cultured with s-PRGF had a higher Ki67 mRNA expression level compared to control (p>0.001). The expression level of the putative stem cell markers ABCG2, ΔNp63 and K14 was comparable in all the conditions tested in the tissue explants form (p>0.05). LSCs cultured with HS had a significantly lower mRNA expression of the differentiation marker K12 compared to control (p=0.000) while there were no significant differences in the K12 expression level for LSCs cultured with s-PRGF compared to control (p=0.14). No significant differences were found between LSCs cultured with HS, s-PRGF or FBS in the percentage of K12⁺ (1.1% to 3.1%) and K14⁺ (93.9% to 96.0%) cells (p>0.05). The percentage of K14+ cells of LSCs cultured with any of the three serum in the tissue explants form (around 95%) was higher than that of the control (86.4%, p>0.01). The percentage of p63α bright cells in LSCs cultured with s-PRGF (18.5%) and HS (14.4%) was significantly higher than in cells cultured with FBS (11.7%) and in the control (12.4%) (p>0.001 for s-PRGF versus FBS and control, and for HS versus FBS).

Conclusions: s-PRGF appears to have the same capability as HS to expand the LSC population.