<b>Induction of neural crest cells from human dental pulp-derived induced pluripotent stem </b><b>cells </b>

Abstract

We previously generated induced pluripotent stem (iPS) cells from human dental pulp cells of deciduous teeth. Neural crest cells (NCCs) play a vital role in the development of the oral and maxillofacial region. Therefore, NCCs represent a cell source for bone, cartilage, and tooth-related tissue engineering. In this study, we examined whether iPS cells are capable of differentiating into NCCs through modification of the human embryonic stem cell protocol. First, iPS cells were dissociated into single cells and then reaggregated in low-cell-adhesion plates with neural induction medium for 8 days in suspension culture to form neurospheres. The neurospheres were transferred to fibronectin-coated dishes and formed rosette structures. The migrated cells from the rosettes abundantly expressed NCC markers, as evidenced by real-time polymerase chain reaction, immunofluorescence, and flow cytometric analysis. Furthermore, the migrated cells exhibited the ability to differentiate into neural crest lineage cells in vitro. They also exhibited tissue-forming potential in vivo, differentiating into bone and cartilage. Collectively, the migrated cells had similar characteristics to those of NCCs. These results suggest that human dental pulp cell-derived iPS cells are capable of differentiating into NCCs. Therefore, iPS cell-derived NCCs represent cell sources for bone and cartilage tissue engineering.

Odontogenic Tissue Generation Derived from Human Induced Pluripotent Stem Cells Using Tissue Engineering Application

“…3 -and cementum using tissue engineering application when the iPS cells are combined with tooth germ cells isolated from porcine third molar teeth in the late bell stage. This potential was confi rmed by the detection of human nuclei by the immunohistochemistry in consistent with other studies 22,24,25) . Recently, the ability of mouse iPS cells to form toothrelated structures has also been confirmed by recombination of mice incisor cells at the cap stage and tooth germ cells from ED14.5 mice in vivo 17) , although the fi nding that iPS cells alone do not form tooth tissues is consistent with our observations.…”

“…How did we recognize whether the transplanted iPS cells differentiated into ameloblasts, odontoblasts, and cementoblasts? We used immunohistochemical staining with human antibody, a wellestablished technique to recognize human cells 22,24,25) . Furthermore, porcine dental papilla cells and rat bone marrow cells were negatively stained with human nuclei in this study.…”

“…First, we stained human dental pulp cells, porcine dental papilla cells and rat bone marrow cells with mouse anti-human nuclei antibody (1:250; MAB1281; Merck Millipore, Darmstadt, Germany) for immunocytochemistry to validate the specificity of this antibody as a primary antibody against human origin cells. Alexa Flour 488 goat anti-mouse IgG (1:500; Thermo Fisher Scientific) was then used as a secondary antibody 22) .…”

ALX1-related Frontonasal Dysplasia Results From Defective Neural Crest Cell Development and Migration

“…The central challenge of the work with iPSC models of human disease remains the lack of available human transcriptomic cell data to allow for an understanding which stage of development is modeled by the cellular lineages derived. NCC are characterized in vitro by the expression of markers identified to be specific to this cell population, namely P75, CD57, CD90, CD73 and CD105 (Billon, Iannarelli et al, 2007, Kawano, Toriumi et al, 2017, Minarcik & Golden, 2003 as well as their multi-lineage differentiation ability. NCC formation is a stepwise process coordinated by a spatiotemporally specific gene expression pattern.…”

A new alternative for corneal endothelial regeneration using autologous dental pulp stem cells

“…After initially comparing the traditional iPSC differentiation method with our novel protocol, we then compared our adhesion culture differentiation protocol of DPSC with a suspension differentiation method. The suspension method consisted of a modi cation from a previously described protocol based on neurospheres (NS) formation [25]. For this purpose, DPSC were cultured on ultra-low adhesion 24-well plates (Corning) in the previously described NC induction medium at a cell density of 2 x 10 4 cells/well.…”