Molecular and Cellular Features of Murine Craniofacial and Trunk Neural Crest Cells as Stem Cell-Like Cells

et al. (2014) Molecular and Cellular Features of Murine Craniofacial and Trunk Neural Crest Cells as Stem Cell-Like Cells. PLoS ONE 9(1): e84072. doi:10.1371/journal.pone.0084072 Molecular and Cellular Features of Murine Craniofacial and Trunk Neural Crest Cells as Stem Cell-Like Cells Kunie Hagiwara 0 Takeshi Obayashi 0 Nobuyuki Sakayori 0 Emiko Yamanishi 0 Ryuhei Hayashi 0 Noriko Osumi 0 Toru Nakazawa 0 Kohji Nishida 0 Hitoshi Okazawa, Tokyo Medical and Dental University, Japan 0 1 Department of Ophthalmology, Tohoku University Graduate School of Medicine, Seiryo-cho, Aoba-ku, Sendai, Japan, 2 Division of Applied Informatics for Human and Life Science, Tohoku University Graduate School of Information Science, Aramaki-Aza-Aoba, Aoba-ku, Sendai, Japan, 3 Division of Developmental Neuroscience, Tohoku University Graduate School of Medicine, Seiryo-cho, Aoba-ku, Sendai, Japan, 4 Department of Ophthalmology, Osaka University Graduate School of Medicine , Yamadaoka, Suita , Japan 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. - Funding: This study was supported by a project for the realization of regenerative medicine from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan given to K.N. and by KAKENHI from MEXT to N.O. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Neural crest cells (NCCs) are cell populations that originate in the early stage of the vertebrate embryo from the dorsal region of the neural tube. They delaminate from the border of neural and non-neural areas of the ectoderm. After delamination, NCCs vigorously proliferate during migration towards various locations within the embryonic body, and differentiate into a wide range of cell types and tissues, including neurons and glial cells of the peripheral nervous systems (PNS), smooth muscles of the heart and great vessels, bone, cartilage, connective tissue of the face, and melanocytes in the skin. The migration patterns and differentiation fates of NCCs have been well characterized in avian and rodent embryos [1]. Trunk NCCs (tNCCs) emerge from the trunk region of the neural epithelium, and those migrating just beneath the ectoderm will form pigment cells in the skin and others taking a ventral pathway via the somites will differentiate into neurons and glia of the PNS as well as chromaffin cells in the adrenal gland [2]. Craniofacial NCCs (cNCCs) emerge from the forebrain, midbrain and hindbrain regions of the neural epithelium, and populate the frontonasal area or the pharyngeal arches depending on their original positions [3]. These cNCCs produce not only neurons, glia and melanocytes, but also the majority of the connective and skeletal tissue of the head [1]. Therefore, cNCCs show wider variation in their differentiated cell types than tNCCs during normal development. Another feature characteristic to NCCs is its relation to tumor formation. Melanoma is a common skin cancer derived from pigment cells of NC-origin [4]. It is also believed that neuroblastoma, one of the most frequent child cancers occurred in the sympathetic nervous systems and adrenal gland, is originated from the NCCs [5]. Another example of a cancer thought to be NCorigin is Ewing sarcoma, an aggressive bone and soft tissue tumors [6]. Considering a recent idea of cancer stem cells [7,8], NCCs may share molecular features common to malignant tumors. In the present study, we performed transcriptome analyses of cNCCs and tNCCs using genetically engineered mice that specifically label NCCs. We also clarified difference in expression profiles of cNCCs and tNCCs from those of inducible pluriopotent stem cells (iPSCs) and embryonic stem cells (ESCs). Furthermore, we also carried out sphere-forming and differentiation assays to know proliferation and differentiation potentials of cNCCs and tNCCs in vitro. Both of approaches consistently revealed differential characters of NCCs as multipotent stem cells, and possibly as cancer stem cells. These results not only provide useful information for NCC application in regenerative medicine but also contribute to develop specific therapeutics for preventing metastatic cascades of NC-derived tumors. Materials and Methods Animals Transgenic (TG) mice expressing the Cre enzyme induced by the myelin protein zero (P0) promoter [9] were crossed with the CAG-CAT-EGFP TG line [10]. In P0-Cre/floxed-EGFP double TG (P0-Cre; EGFP) mice, NCCs were identified by evaluating the expression of EGFP after P0-Cre-mediated DNA recombination [11]. To eliminate pigmentation in the embryonic tissue of double TG lines, mice originally of the C57/BL6J background were crossed with mice of an ICR background (Japan Charles River, Tokyo, Japan) for 56 generations as described previously [12]. At mid-day of identifying a vaginal plug was considered as E0.5. P0Cre recombinase TG mice were kindly provided by Dr. K. Yamamura (Kumamoto University, Kumamoto, Japan). CAGCAT-EGFP TG mice were kindly provided by Dr. J. Miyazaki (Osaka University, Osaka, Japan) and maintained at Tohoku University. All experimental animal procedures described in this study were approved by the Ethics Committee for Animal Experiments of Tohoku University Graduate School of Medicine (#2012-134). Preparation of cells from mouse embryos P0-Cre; EGFP embryos were resected into Hanks (...truncated)