Search

The outstanding differentiation capacities and easier access from adult tissues, cells derived from neural crest cells (NCCs) have fascinated scientists in developmental biology and regenerative medicine. Differentiation potentials of NCCs are known to depend on their originating regions. Here, we report differential molecular features between craniofacial (cNCCs) and trunk (tNCCs) NCCs by analyzing transcription profiles and sphere forming assays of NCCs from P0-Cre/floxed-EGFP mouse embryos. We identified up-regulation of genes linked to carcinogenesis in cNCCs that were not previously reported to be related to NCCs, which was considered to be, an interesting feature in regard with carcinogenic potentials of NCCs such as melanoma and neuroblastoma. Wnt signal related genes were statistically up-regulated in cNCCs, also suggesting potential involvement of cNCCs in carcinogenesis. We also noticed intense expression of mesenchymal and neuronal markers in cNCCs and tNCCs, respectively. Consistent results were obtained from in vitro sphere-forming and differentiation assays. These results were in accordance with previous notion about differential potentials of cNCCs and tNCCs. We thus propose that sorting NCCs from P0-Cre/floxed-EGFP mice might be useful for the basic and translational research of NCCs. Furthermore, these newly-identified genes up-regulated in cNCC would provide helpful information on NC-originating tumors, developmental disorders in NCC derivatives, and potential applications of NCCs in regenerative medicine.

We assessed the maintenance and distribution of epithelial stem/progenitor cells after corneal reconstruction using tissue-engineered oral mucosal cell sheets in a rat model. Oral mucosal biopsy specimens were excised from green fluorescent protein (GFP) rats and enzymatically treated with Dispase II. These cells were cultured on inserts with mitomycin C-treated NIH/3T3 cells, and the resulting cell sheets were harvested. These tissue-engineered cell sheets from GFP rats were transplanted onto the eyes of a nude rat limbal stem cell deficiency model. Eight weeks after surgery, ocular surfaces were completely covered by the epithelium with GFP-positive cells. Transplanted corneas expressed p63 in the basal layers and K14 in all epithelial layers. Epithelial cells harvested from the central and peripheral areas of reconstructed corneas were isolated for a colony-forming assay, which showed that the colony-forming efficiency of the peripheral epithelial cells was significantly higher than that of the central epithelial cells 8 weeks after corneal reconstruction. Thus, in this rat model, the peripheral cornea could maintain more stem/progenitor cells than the central cornea after corneal reconstruction using oral mucosal epithelial cell sheets.

Induced pluripotent stem (iPS) cells can be established from somatic cells. However, there is currently no established strategy to generate corneal epithelial cells from iPS cells. In this study, we investigated whether corneal epithelial cells could be differentiated from iPS cells. We tested 2 distinct sources: human adult dermal fibroblast (HDF)-derived iPS cells (253G1) and human adult corneal limbal epithelial cells (HLEC)-derived iPS cells (L1B41). We first established iPS cells from HLEC by introducing the Yamanaka 4 factors. Corneal epithelial cells were successfully induced from the iPS cells by the stromal cell-derived inducing activity (SDIA) differentiation method, as Pax6(+)/K12(+) corneal epithelial colonies were observed after prolonged differentiation culture (12 weeks or later) in both the L1B41 and 253G1 iPS cells following retinal pigment epithelial and lens cell induction. Interestingly, the corneal epithelial differentiation efficiency was higher in L1B41 than in 253G1. DNA methylation analysis revealed that a small proportion of differentially methylated regions still existed between L1B41 and 253G1 iPS cells even though no significant difference in methylation status was detected in the specific corneal epithelium-related genes such as K12, K3, and Pax6. The present study is the first to demonstrate a strategy for corneal epithelial cell differentiation from human iPS cells, and further suggests that the epigenomic status is associated with the propensity of iPS cells to differentiate into corneal epithelial cells.