The Odontogenic Differentiation Potentiality From Mouse Induced Pluripotent Stem (iPS)cell

Tooth tissue engineering that is generate functional bioengineeredreplacement teeth and dental tissue using stem cells. These stem cells can bedivided into2main types, dental stem cells, including dental pulp stem cells,dental follicle stem cells and periodontal ligament stem cells, andnonodnotogenic stem cells, for example embryonic stem cells and bonemarrow-derived cells. However, the sources of patients’ dental stem cells limitthe use of this approach, such as PDLSCs can be harvested from exfoliateddeciduous teeth, from extracted wisdom teeth or from extracted orthodonticteeth. Embryonic stem cells possess the ability of self-renewal and candifferentiate into all of cell types, but at least two important roadblocks to theiruse in humans must be overcome: ethical issues and post-transplantationimmune rejection. These concerns may be overcome if pluripotent stem cells can be directly derived from patients’ somatic cells. Studies have demonstratedthat iPS cells can differentiate into neurons, blood cells, islet cells, liver andkidney cells, however, few studies have been conducted on whether iPS cellscan differentiate into the odontogenic lineage. In this study, we discussed thepotentiality of induced pluripotent stem cells as candidate cells for teethregenerationPart one: Mouse iPS cells culture and characterization.Objective: To culture iPS cells （C5cell line）and confirm the multi-potency ofmouse iPS cells. Methods: The cryopreserved iPS cells were cultured onmitomycin-C-treated mouse embryo fibroblast (MEF) feeder cells. In vitrostudies：Cell morphological differences were observed under a phase-contrastmicroscope. Alkaline phosphatase(ALP) staining and immunofluorescencestaining analysis were performed. EB formation was achieved. In vivo study: toform,to characterization of mouse iPS cells, approximately2.0×106untreatediPS cells cultured on MEF were collected with fibrin gel as a carrier andimplanted into subcutaneous pockets of6-week-old nude mice (FMMU MedicalLaboratory Animal Center) for teratoma formation. Results: Mouse iPS cellsdisplayed the typical shapes: the colony with clear edge and smooth surface, andthe boundaries between cells were not clear. For ALP activity, enzyme assayresults revealed high ALP activity. EB was formed in vitro. Mouse iPS cellscultured in regular medium formed teratomas in vivo. Conclusion: Mouse iPScells possess the ability of self-renewal and can differentiate into all of celltypes.Part two: Differentiation of iPS cells into ameloblast-like cells cultured in ameloblasts serum-free conditioned medium (ASF-CM).Objective: To explore the possibility of mouse iPS cells differentiation intoameloblast-like cells induced by ASF-CM. Methods: ASF-CM induced mouseiPS cells for14days. Immunofluorescence staining analysis was performed atday14. Using immunofluorescence staining analysis detected the proteinexpression of AMBN, AMGN and CK14. Results: Cell morphologicaldifferences were observed. After treated with ASF-CM for14days, iPS cellswere differentiated into cobblestone-like cells, which resembled ameloblast-likecells. To further characterize the heterogeneous cell phenotypes present inmouse iPS-derived cells, we examined the expression of odontogenesis-relatedproteins AMBN, AMGN, CK14by immunofluorescence. We confirmed theexpression of AMBN, AMGN, and CK14at day14. In the group of untreated iPScell，the expression of AMBN, AMGN, and CK14was negative.Conclusion:ASF-CM provides a suitable microenvironment for inducing mouse iPS cellsdifferentiation into ameloblast-like cell lineage.Part three: Differentiation of BMP4-induced mouse iPS cells.Objective: To explore the possibility of mouse iPS cells differentiation intoodontogenic lineage induced by ameloblasts serum-free conditioned mediumsupplemented with BMP4(ASF-BMP4). Methods: In vitro studies：We culturedmouse iPS cells in ASF-CM or in ASF-BMP4for14days. The morphologicalchanges of differentiating mouse iPS cell cultured in ASF-BMP4were observed.Immunofluorescence of specific markers was performed in differentiating iPScells cultured in ASF-CM and in ASF-BMP4at day14. We examinedodontogenesis-related protein expression of AMBN, AMGN, CK14, Dmp1andDSP by immunofluorescence. DMP-1and DSP is odontoblast lineage marker.In vitro studies: To further investigate the in vivo differentiation capacity of vitro studies: To further investigate the in vivo differentiation capacity ofBMP4-induced mouse iPS cells, an in vivo transplantation assay was performed.Aggregates of iPS/CBB were implanted into the renal capsule of C57mice.Results: In vitro： The cells derived from ASF-BMP4-iPS cells showedmorphological characteristics of ameloblast-like cells and odontoblast-like cells.To further characterize the cell phenotypes of iPS-derived cells, we examinedthe expression of odontogenesis-related proteins AMBN, AMGN, CK14, DMP-1and DSP by immunofluorescence. The expressions of AMBN, AMGN, CK14were positive， and the expression of DMP-1and DSP was negative inASF-CM-treated iPS cells. The expression of AMBN, AMGN, CK14, DMP-1and DSP was all positive in BMP4-treated iPS cells. In vivo：ASF-CM-treatedmouse iPS cells only generated keratogenous tissues. ASF-BMP4-treated mouseiPS cells formed both ameloblast-like and odontoblast-like structures.Conclusion: ASF-BMP4-induced mouse iPS cells can differentiate intoameloblast-like and odontoblast-like cells.Part four: Noggin inhibited BMP4effect in the differentiation of iPS cells.Objective: To further determine whether the BMP growth factor signalingpathway in ASF-CM was essential to induce the differentiation of mouse iPScells into the odontogenic lineage. Methods: we cultured mouse iPS cells inASF-CM or in ASF-BMP4or in ASF-Noggin for14days. The morphologicalchanges of differentiating mouse iPS cells cultured in ASF-Noggin wereobserved. Immunofluorescence of specific markers was performed in3kinds ofconditioned medium induced iPS cells at day7and14. RT-PCR and westernblot examined the gene expression of Pax9, Msx2, AMBN, AMGN, Dmp1andDSP. Results: After treated with ASF-Noggin for14days, proliferation of these cells was slow. RT-PCR results showed that Pax9and Msx2were mainlyexpressed on day7, while AMBN, DMP-1, and DSPP were mainly expressed onday14. Compared to the cells cultured in ASF-CM, those cultured inASF-BMP4had markedly higher expression levels of Pax9and Msx2at day7and those of AMBN, DMP-1, and DSPP at day14s. Interestingly, the expressionof these genes in cultured in ASF-noggin was markedly lower than that inASF-CM. Western blot analysis consistent with those of RT-PCR. Conclusion:BMP4promoted the differentiation of mouse iPS cells whereas noggin reversedthis effect. BMP4is a key growth factor that is essential for the effectiveinduction of odontogenic differentiation.