Identification of gene expression profile of neural crest-derived cells isolated from submandibular glands of adult mice.

Neural crest cells in the embryo migrate to reach target sites as neural crest-derived cells (NCDCs) where they differentiate into a variety of derivatives. Some NCDCs are maintained in an undifferentiated state throughout the life of the animal and are considered to be a useful cell source for regenerative medicine. However, no established method to obtain NCDCs sufficient for regenerative medicine from adults with high purity has been presented, since their distribution in adult tissues is not fully understood. It is critical to identify reliable markers for NCDCs in adults, as the expressions of P0 and Wnt1, the most reliable NCDC markers, are shut off in the embryonic stage. To analyze the characteristics of NCDCs in adult tissues, we utilized a double transgenic mouse strain, P0-Cre/CAG-CAT-EGFP transgenic mice (P0 mice), in which NCDCs were shown to express EGFP and we were able to recognize GFP-positive cells in those. We focused on the submandibular glands (SMGs), which are known to be derived from the neural crest. GFP-positive cells were shown to be scattered like islands in the SMGs of adult P0 mice. We surgically removed SMGs from adult mice and digested samples into single cell suspensions. GFP-positive cells separated using flow cytometry expressed a high level of Sox10, a marker of embryonic neural crest cells, suggesting successful isolation of NCDCs. To identify candidate marker genes in isolated NCDCs, we performed DNA microarray analyses and real-time PCR analysis of GFP-positive and -negative cells isolated from P0 mice, then selected genes showing differential gene expression patterns. As compared to GFP-negative cells, GFP-positive cells expressed Gpr4 and Ednrb at higher levels, whereas Pdgfra and Pdgfrb were expressed at lower levels. Furthermore, DNA microarray analysis showed that GFP-positive cells were positive for aquaporin 5, a marker for acinar cells. Together, our results indicate that NCDCs in adult SMGs have characteristic gene expression profiles specially their cell surface molecules. Cell sorting using a combination of these specific cell surface proteins would be a useful strategy for isolation of NCDCs from SMGs with high purity.