Differentiation of Human-induced Pluripotent Stem Cell-derived Dental Stem Cells through Epithelial-Mesenchymal Interaction.

Research on tooth regeneration using human-induced pluripotent stem cells (hiPSCs) is valuable for autologous dental regeneration. Acquiring mesenchymal and epithelial cells as a resource for dental regeneration is necessary because mesenchymal-epithelial interactions play an essential role in dental development. We reported the establishment of hiPSCs-derived dental epithelial-like cell (EPI-iPSCs), but hiPSCs-derived dental mesenchymal stem cells (MSCs) have not yet been reported. This study was conducted to establish hiPSCs-derived MSCs and to differentiate them into dental cells with EPI-iPSCs. Considering that dental MSCs are derived from the neural crest, hiPSCs were induced to differentiate into MSCs through neural crest formation to acquire the properties of dental MSCs. To differentiate hiPSCs into MSCs through neural crest formation, established hiPSCs were cultured and differentiated with PA6 stromal cells and differentiated hiPSCs formed neurospheres on ultralow-attachment plates. Neurospheres were differentiated into MSCs in serum-supplemented medium. Neural crest-mediated MSCs (NC-MSCs) continuously showed typical MSC morphology and expressed MSC markers. After 8 days of odontogenic induction, the expression levels of odontogenic/mineralization-related genes and dentin sialophosphoprotein (DSPP) proteins were increased in the NC-MSCs alone group in the absence of coculturing with dental epithelial cells. The NC-MSCs and EPI-iPSCs coculture groups showed high expression levels of amelogenesis/odontogenic/mineralization-related genes and DSPP proteins. Furthermore, the NC-MSCs and EPI-iPSCs coculture group yielded calcium deposits earlier than the NC-MSCs alone group. These results indicated that established NC-MSCs from hiPSCs have dental differentiation capacity with dental epithelial cells. In addition, it was confirmed that hiPSCs-derived dental stem cells could be a novel cell source for autologous dental regeneration.

Differentiation and Establishment of Dental Epithelial-Like Stem Cells Derived from Human ESCs and iPSCs.

Tooth development and regeneration occur through reciprocal interactions between epithelial and ectodermal mesenchymal stem cells. However, the current studies on tooth development are limited, since epithelial stem cells are relatively difficult to obtain and maintain. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) may be alternative options for epithelial cell sources. To differentiate hESCs/hiPSCs into dental epithelial-like stem cells, this study investigated the hypothesis that direct interactions between pluripotent stem cells, such as hESCs or hiPSCs, and Hertwig's epithelial root sheath/epithelial rests of Malassez (HERS/ERM) cell line may induce epithelial differentiation. Epithelial-like stem cells derived from hES (EPI-ES) and hiPSC (EPI-iPSC) had morphological and immunophenotypic characteristics of HERS/ERM cells, as well as similar gene expression. To overcome a rare population and insufficient expansion of primary cells, EPI-iPSC 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.

Porcine PD-L1: cloning, characterization, and implications during xenotransplantation.

BACKGROUND: Effective intervention achieved by manipulating cell-mediated xenogeneic immune responses would critically increase the clinical feasibility of xenotransplantation as immediate hyperacute rejections become controllable through genetic modulations of donor organs. Endogenous negative regulatory signals like the programmed death 1 (PD-1)-programmed death ligand 1 (PD-L1) system are candidate targets for the control of cell-mediated xenogeneic immune response. METHODS: A porcine PD-L1 molecule was cloned using RACE (rapid amplification of cDNA ends) technology based on the human PD-L1 sequence. The functional effects of cloned porcine PD-L1 were tested on human CD4(+) T cell activation using porcine PD-L1-transfected bystander cells. Cellular proliferation was monitored by [3H] thymidine incorporation, and human T cell apoptosis was measured by flow cytometry. RESULTS: Porcine PD-L1 (GenBank accession number AY837780) was found to have 73.8% sequence homology with human PD-L1 and to contain two immunoglobulin domains in its extracellular region. Moreover, porcine PD-L1 expressed on Chinese hamster ovary (CHO) cells inhibited human CD4(+) T cell proliferation stimulated with anti-CD3 only or anti-CD3 plus anti-CD28. Percentages of apoptotic activated human T cells increased by over 30% in the presence of porcine PD-L1/CHO cells, and the addition of recombinant human PD-1-Fc fusion proteins during human T cell activation reversed the inhibitory effects of porcine PD-L1. CONCLUSIONS: Cloned porcine PD-L1 showed high sequence homology with human PD-L1 and a similar molecular structure. Moreover, porcine PD-L1 inhibited human CD4(+) T cell activation in human PD-1-dependent manner, and this involved activated T cell apoptosis. The authors suggest that PD-1-PD-L1 might play an important endogenous immune regulatory role during xenogeneic transplantation, and that the effective application of this system would improve transplanted xenogeneic organ survival.