Characteristic Of Cranial Neural Crest Cells And Their Differentiation Into Neural Cells And Osteoblasts

Neural crest cells detach from dorsal neural tube. They migrate to diverse location in the developing embryo where they generate a number of different cell types. These include neurons, glia, and many other non-neural cells. Neural crest cells are one of the most multipotent embryonic cells in body. Mostly cells in dental papilla, dental sac, periodontium, bone and muscles are derived from cranial neural crest cells (CNCC). Research on the development and differentiation of CNCC can help us understand the formation of tissue and organ in orofacial region. In addition, the pluripotent CNCC can also be one of candidate seed cells in the research of tissue engineering. Therefore, it is of great import to study the culture and differentiation condition of CNCC in vitro.It is well known that CNCC can differentiate into neural cells, osteoblasts and odontoblasts in vivo, however, it is still not clear how CNCC give rise to a multitude of cell types in vitro. The present work has some limitations because of the following two reasons: (1) CNCC is difficult to obtain, and (2) CNCC is easy to differentiate.To elucidate the characteristic and differentiation of CNCC, the present study focused on the CNCC culture condition and how they differentiate into neural cells and osteoblasts. The present work showed the following:(1) The neural tubes of somite 6 before were removed from E8.5 Balb/C mice and cultured in serum-free DMEM/F12 medium. The neural tubes were detached and discarded after 2 days. CNCC were typsinized and cultured. Primary CNCC appeared to be fibroblast-like. The doubling population time of CNCC was 34.4 hours. Results of immunohistochemistry showed that the specificantibodyi"'/iDneurotrophin receptor p75 was positively stained, while cytokeratin, vimentin, NF and GFAP were negative.(2) To observe the roles of serum on CNCC. CNCC became long and thin after treated with 10% PCS DMEM medium. GFAP-immunoreactive cells were seen in serum-treated group. No positive signals were found in normal serum-free medium.(3) To evaluate the effects of different doses of rhBMP2 on CNCC. After cultured in 50ng/ml rhBMP-2 medium, it shapes like neuron with different length of neurites. However, CNCC maintained its fibroblast-like shape. Immunocytochemical examination also revealed that the cells exhibiting a neuronal morphology expressed NF in rhBMP-2 treated group. TEM results showed that the number of endoplasmic reticulum and mitochondrial decreased and neuroendocrine particles were found in rhBMP-2 treated group. So rhBMP-2 can induce the differentiation of CNCC into neurons. The appropriate dose of rhBMP-2 was 50mg/ml and the percents of neuron cells were 47.1% after calculating the NF positive cells.(4) Expression of Tyrosine hydroxylase (TH) and Gamma-aminobutyric acid-alpha (GABAA) receptor y subunit mRNA changed significantly before and after the addition of rhBMP-2 into the medium. The results of RT-PCR showed that mRNA of TH and GABAA receptor y subunit was expressed in rhBMP-2 treated group. Thissuggests that rhBMP2 induced neurons were of adrenal phenotype.(5) pBK-BMP-2 was transfected into CNCC by using Fugene 6. Positive cell clones were selected with G-418. The stable transfection and expression of BMP-2 of CNCC were determined by immunohistochemistry and in situ hybridization. The typical neurite-like processes were observed in the BMP2 transected cells. NF positive staining further proved that BMP-2 gene transfected cells were of neuronal phenotype.(6) Mineralization induction medium was used to assess if CNCC can differentiate into osteoblasts in vitro. 20 days after culturing in mineralization medium, the shapes of CNCC changed from fibroblasts-like to polygonal. The doubling population time of CNCC prolonged to 57.8 hours in treated group. Treated cells showed decreased proliferative ability but with enhanced Alkaline phosphatase activity and Osteocalcin quantity (P<0.05). These results suggest that serum-free medium can maintain the status of CNCC and serum medium canno...