| Dental pulp stem cells (DPSCs) was firstly isolated and termed by Shi S and Gronthos S in 2000. It is thought that they are progenitors exist in dental pulp. They could be activated by dental damage and terminally differentiated into functional odontoblasts. The founding of DPSCs made tooth regeneration becoming a realistic possibility. Using DPSCs as seeding cells, the bioengineer tooth project funded by NIH has succeeded and patented in 2000.Just like other adult stem cells, DPSCs could not maintain in an undifferentiated state for a long time when cultured in vitro, then will lose self-renewal capabilities. Moreover, DPSCs still cannot be purified because of a lack of specific markers, which made it more difficult to obtain enough stem cells as seeding cells. One of the solutions is to improve the self-renewal capabilities while maintaining pluripotent properties by gene transfer.Notch signaling defines an evolutionary conserved cell interaction mechanism, which play an important role in theproliferation, differentiation and apoptosis of many cells. Activation of Notch signaling pathway leads to transcriptional suppression of lineage-specific genes that cause inhibition of differentiation, resulting in the maintenance of cells in an uncommitted state. This made Notch ligand Delta a potential ideal candidates for the self-renewal and expansion of stem cells. The tentative plan was firstly applied in hematopoietic stem cells, and obtained encouraging results. It turned out that hematopoietic stem cells be immortalized by constitutive Notchl and Delta has the function as the mitogenic regulators of hematopoietic stem cells. It is speculated that Delta may play a similar role to other tissue-specific stem cells, including DPSCs. Notchl is expressed by stem cells resided in the cervical loop epithelium, and Notch signaling pathway play a central role in fate decision of those stem cells. Notch receptors and their ligands are involved in both patterning and cell fate determination during odontogenesis. Moreover, many cell factors, which are closely related with odontogenesis, could regulate expression of Notch receptors or their ligands. On the basis of all those studies, we intend to transfer Deltal gene into cultured DPSCs by retrovirus-mediated gene transduction to elucidate the effects of Deltal on the self-renewal and differentiation of DPSCs. Furthermore, we preliminarily explore the feasibility of dental pulp regeneration using Deltal -gene-transduced-DPSCs as seeding cells. The main methods and results were as follows:1. Isolation, culture and identification of human DPSCs: DPSCs were isolated by collagenase type I and dispase digestion of dental pulp derived from third molars as reported, then cultured and subcultured.Identification of human dental pulp stem cells: (1) Colony-forming efficiency of cells derived from dental pulp tissue was 2~15clones/103 cells plated.(2) Characterization of the immunophenotype of DPSCs in vitro: DPSCs were found to express many different markers, including vimentin , collagen type I , GFAP, nestin and osteocalcin by immunohistochemical and RT-PCR assay, while they failed to react with MyoD, PPARy and DSPP.(3) Differentiation potential of DPSCs:Grown in an adipogenic cocktail medium(100ug/ml indomethacin + 10ug/ml insulin + 0.5mM IBMX+1uM Dex )for three weeks, some DPSCs expressed fat cell markers of PPARy and LPL, and formed oil red O- positive lipid clusters in five weeks.After culture with a myogenic-inductive medium containing 10uM 5-Aza, DPSCs were found to express MyoD, desmin and myosin, markers of myocyte in 24~48h, 15th and 21th day respectively. In addition, fusion were found between two to three cells and myotube like cells were observed at about 18th day.Long-term cultures of DPSCs grown in differentiation inductive medium demonstrated the capacity to form Von kossa-positive condensed nodules with high levels of calcium.The results of this part imply that cells we isolated from adult hum...
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