As expected, typical ameloblast-related gene markers (and and and < 0.05. Abbreviations HERS/ERMHertwigs epithelial root sheath/epithelial rests of MalassezhESCsHuman embryonic stem cellshiPSCsHuman induced pluripotent stem cellshdDPSCDental pulp stem cells from human exfoliated Calcitetrol deciduous teethEPI-ES
EPI-iPSCDental epithelial-like stem cells derived from hESCsDental epithelial-like stem cells derived from hiPSCs Author Contributions Conceptualization, M.J.; data curation, M.J.; formal analysis, G.-H.K., J.Y., D.-H.J., and G.Y.C.; funding acquisition, G.L. was immortalized with the SV40 large T antigen. The immortalized EPI-iPSC cell line had a normal karyotype, and a short tandem repeat (STR) analysis verified that it was derived from hiPSCs. The EPI-iPSC cell line co-cultured with dental pulp stem cells displayed increased amelogenic and odontogenic gene expression, exhibited higher dentin sialoprotein (DSPP) protein expression, and promoted mineralized nodule formation. These results indicated that the direct co-culture of hESCs/hiPSCs with HERS/ERM successfully established dental epithelial-like stem cells. Moreover, this differentiation protocol could help with understanding the functional roles of cell-to-cell communication and tissue engineering of teeth. and and and which are stemness-related markers. (c) Fluorescence-activated cell sorting (FACS) analysis of EPI-iPSC. EPI-iPSC was positive for mesenchymal markers (CD29) and HLA type I, but negative for hematopoietic cell markers (CD10, CD45, and HLA-DR) and an endothelial cell marker (CD31). All data were replicated three times. Open in a separate window Figure 4 Characterization of dental epithelial-like stem cell lines derived from hiPSC. (a) Immunofluorescence staining for the expression of SV40 in the EPI-iPSC cell line. Primary HERS/ERM cells did not express SV40, whereas the established EPI-iPSC cell line expressed SV40. (b) Morphology and passaging of the EPI-iPSC cell line. EPI-iPSC-SV40 showed the typical epithelial cell-like shape and clonal expansion until passage 15. The morphology was maintained through subculture. Magnifications are at 400. (c) Growth of three EPI-iPSC-SV40 lines. Cumulative cell numbers of EPI-iPSC showed that they maintained stable proliferation for 40 days. (d) Expression of epithelial stem cell and stemness-related genes in the EPI-iPSC cell line (passage 10). EPI-iPSC cell line was positive for which are stemness-related markers. (e) FACS analysis of the EPI-iPSC cell line (passage 10). EPI-iPSC was positive for CD29 and HLA-I, and negative for CD10, CD45, HLA-DR, and CD31. (f) Karyotype of the EPI-iPSC cell line. The EPI-iPSC cell line at passage 10 showed a normal karyotype with 46, XY. (g) Origin Calcitetrol of the EPI-iPSC cell line. Microsatellite (STR) analysis, which is a PCR-based microsatellite method, showed that the differentiated EPI-iPSC cell line was derived from hiPSC. All data were obtained from three replicates. Table 1 STR analysis showed that the EPI-iPSC cell line matched human iPSCs. was examined. After EMT induction, the EPI-iPSC cell line demonstrated a down-regulated expression of E-cadherin. On the other hand, expressions of N-cadherin and Vimentin were significantly up-regulated. (Figure 5b). These data suggested that the EPI-iPSC cell line could acquire mesenchymal phenotypes through EMT. Open in a separate window Figure 5 Epithelial-mesenchymal transition (EMT) of HERS/ERM cells and the EPI-iPSC cell line. The EMT was induced by TGF-1 for 48 h. (a) Morphology of the EPI-iPSC cell line after 48 h of TGF-1 treatment. All of these cells lost epithelial cell polarity and cell-to-cell contact. (b) EMT-related gene expression of the EPI-iPSC cell line after EMT induction. When all cell types were treated with TGF-1, the gene expression of N-cadherin and Vimentin was increased in primary HERS/ERM and epithelial-like cells. However, the levels of E-cadherin were decreased. All data shown are the mean S.D. from the levels of three replicates. Data are presented as the mean SD, = 6 per group. ** < 0.01, * Calcitetrol < 0.05. N/I: no induction. 2.4. Differentiation Potential of Differentiated Dental Epithelial-Like Stem Cell Lines Derived Calcitetrol From hiPSC To observe the synergetic effect of EPI-iPSC and hdDPSC, co-culture was performed with or without osteogenic medium for 20 days. The expression of ameloblast/odontoblast markers was measured with qRT-PCR and a western blot. Amelogenin, the major structural protein of the enamel organic matrix, was notably increased in EPI-iPSC alone RUNX2 or the co-culture group when odontogenic differentiation was induced. Ameloblast matrix protein expression, including enamelin and the proteinase KLK4, was only upregulated in the co-culture group with the induction medium compared with hdDPSC or EPI-iPSC alone. An early and proliferative stage marker of mineralization, Calcitetrol such as Runx2 and BSP of the matrix maturation marker, was increased in the co-culture group with a5OB. OCN, which is an even later stage marker than matrix.