Abstract
Purpose :
To isolate putative conjunctival epithelial stem cells by low-affinity neurotrophin receptor P75 and identify the stemness of P75++ cells.
Methods :
Human conjunctival epithelial cells were divided into three cell subsets: P75++, P75+ and P75- by P75-conjugated immunomagnetic beads, and the separation efficiency was verified. To identify the characteristics of different cell subpopulations, the expression of conjunctival stem cell markers ABCG2 and P63, as well as differentiation-associated cytokeratin CK4 and CK13 at both mRNA and protein levels were detected by RT-PCR , western blot and immunofluorescence. The ability of clonal formation of each isolated cell fractions was demonstrated by colony-forming efficiency (CFE) assay. Immunofluorescence staining of Ki67 was used to evaluate the different cell cycle status. The proliferation potential of different cell subsets was observed by CCK8 and long-term cultivation in vitro. To compare their ability of tissue formation, cells of P75++ and P75- were cultured on amniotic acellular matrix for 10 days and then the tissue of conjunctiva epithelium formed by each other were examined by histomorphology and immunohistochemistry.
Results :
After analysing the characteristics of P75++ cells by RT-PCR and western blot , we found that these cells were in a relatively immature state in vivo, as they least expressed the differentiation markers CK4 and CK13, but expressed ABCG2 and P63 at a relatively high level. CFE showed strong colony-forming ability of P75++ cells and immunofluorescence staining of ki67 demonstrated that these cells also retained a relatively slow-cycling phenotype, which have been both considered as important characteristics of stem cells. During the long-term cultivation in vitro, P75++ cells showed the greatest proliferation potential and tissue formation ability on amniotic membrane than all the other cell subsets.
Conclusions :
Here we draw the conclusion that P75 can be used as a proper stem cell marker to isolate conjunctival epithelial stem cells, and thus provide a satisfying source of stem cells for tissue engineering conjunctival epithelium reconstruction.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.