Differentiation Of Immature Dental Pulp Stem Cells Into Corneal Epithelium Cultivated On Deepithelialized And Epithelialized Human Amniotic Membrane

Abstract

Purpose: : Evaluate the capacity of differentiation of human Immature Dental Pulp Stem Cells into corneal epithelium in different media and onto epithelialized and deepithelialized amniotic membrane.

Methods: : The amniotic membranes (AM) were provided by Obstetrics Department of Federal University of São Paulo, and were previously prepared in accordance to Covre et al. (2011). The culture of human Immature Dental Pulp Stem Cells (hIDPSC) were divided in two groups, that represent deepithelialized and epithelialized AM (AMD and AME, respectively), and subdivided into four groups in accordance to the culture media (Shem, KSFM, Knockout, DMEM and Epilife). After seven days of culture, the samples were fixed and prepared for immunocitochemistry analysis. The differentiation was comproved using K3/12 antibody. Antibodies against ABCG2, connexin 43 and p63 were also tested. The control groups were cultivated with medium that didn’t induce differentiation in both groups of AM. Histological and eletron microscopy (EM) analysis were performed. FISH was provided only in AME.

Results: : Immunocitochemistry analysis for differentiation showed positive reaction for K3/12 in the group of AMD with SHEM and Knockout DMEM media. Epilife and KSFM didn’t reacted. Control group of AMD presented positive reaction for ABCG2, p63 and connexin 43, which indicates that those cells remain undifferentiated, and negative reaction for K3/12. AME did not present reaction for any antibodies. In the histological and EM analysis of AMD with SHEM and Knockout DMEM media, we could observe similarities with corneal epithelium. These similarities couldn’t be observed in Epilife and KSFM media in AME and control groups. However, AME group presented cells like trophoblasts. These group was tested for FISH analysis. We could observe in all samples of these group four signals, two that represent AM (XX) and two that represent hIDPSC (XY). These data prove that hIDPSC onto the AME differentiated into another type of cell.

Conclusions: : Our results showed that hIDPSC onto AMD with Shem and Knockout media are able to differentiate into corneal epithelium cells. And AME are not indicated to be used as support for hIDPSC.

Keywords: cornea: epithelium • differentiation • proliferation