| Dental pulp stem cells(DPSCs) are mesenchymal-derived cells residing within the perivascular niche of dental pulp and are considered to originate from migrating cranial neural crest cells. These cells are self-renewal and multipotent stem cells which can be induced to differentiate in-vitro into the classic mesenchymal stem cell lineage cell types of osteoblasts/odontoblasts, adipocytes and chondrocytes. Additionally, a number of studies have demonstrated that it also has the potential for endothelial, myogenic, hepatocytic and melanocytic differentiation. Therefore, recent studies on DPSCs promote the development of tissue engineering and regenerative medicine. The pulp tissue is heterogeneous, so DPSCs from pulp tissues are heterogeneous cells which are expected to produce unwanted biological activity. It has been reported that different clones of DPSCs exhibit different behaviour(including the capacities of proliferation and differentiation), according to the age of the donor, the method of isolation, as well as the condition of the pulp tissue. DPSCs provide a potential source of progenitor cells for tissue engineering. However, the abilities of expansion and differentiation in vitro should be considered before use. Therefore, we plan to isolate different clones from different patients and then compare their proliferation rates and potential to differentiate into three mesenchymal lineages(including osteogenic, adipogenic and chondrogenic), in order to find the best clone as the candidate for further tissue engineering research.Smooth muscle is an involuntary nonstriated muscle which can be found in most parts of human organs. The nervous system mainly including the peripheral and central nervous systems traditionally lacks the capacity for endogenous repair and regeneration after injury. Loss of smooth muscle and nervous function caused by system diseases and trauma profoundly hampers the ability to perform many routine activities of human being, even threaten their life. The rapid development of tissue engineering and regenerative medicine provides a promising alternative treatment for smooth muscle and nervous repair/regeneration. As one of the promising sources for tissue engineering and regenerative medicine, it is still unclear whether DPSCs could be induced to differentiate into neuron and smooth muscle cells(SMCs) for nervous and smooth muscle regeneration.In this study, we isolated one of DPSCs clones with the best capacity of proliferation and differentiation and then further investigated the differentiation protocols and molecular mechanisms for SMCs and neuronal differentiation, in order to provide a promising alterative source of cells for further in smooth muscle and nervous tissue engineering.The results are as follows: 1. Isolation and selection of DPSCs clones from different patients.We isolated four clones of DPSCs from three different patients by using fibronectin based selection protocol. The level of population doublings(PDs) during expansion culture and RT-PCR were monitored to analyse the proliferation rate and the gene expression of MSCs markers of different clones, respectively. Then three clones were induced to differentiate into three mesenchymal lineages(including osteogenic, adipogenic and chondrogenic) in appropriate differentiation condition in vitro to compare their capacity of differentiation. The DPSCs clone A32 demonstrated the highest proliferation capacity extending beyond 80PDs(about 300 days) and the best differentiation capacity into three mesenchymal lineages including ostengenic, adipogenic and chondrogenic of four clones, which make it to be the best clone as the candidate for further tissue engineering research. 2. The study for the differentiation of SMCs-like cells from DPSCs clone A32The SMCs conditional medium(CM) was obtained from SMCs. The various concentration of CM and the growth factor of transforming growth factor beta 1(TGF-β1) were evaluated to ascertain which were the most potent inducers of differentiation. Here, we demonstrate the ability of DPSCs clone A32 to differentiate into SMCs in growth environment containing 20% CM and 2.5ng/ml TGF-β1. After 14 days of exposure to this medium, the morphology is demonstrated to change from the spindle shape of the A32 to the "hill and valley" morphology of SMCs over time. The gene and protein expression of SMCs-specific markers(α-SMA, desmin, myosin, and calponin) increased over time. We then further found the induced A32 formed monolayer structure and generated the smooth muscle-like phenotype after an extended differentiation duration. Additionally, we further in-depth investigate the molecular mechanism of A32 differentiation into SMCs. We found the canonical Wnt signaling(also known as Wnt/β-catenin signaling) play a vital role in the regulation of A32 differentiation into SMCs. It, on the one hand, promoted the process of SMCs-differentiation from A32 by regulating the expression of growth factors, including TGF-β1, hepatocyte growth factor(HGF), platelet-derived growth factor-BB(PDGF-BB) and vascular endothelial growth factor(VEGF). On the other hand, it maintained the pluripotency of A32 in the process of SMCs-differentiation by regulating the expression of basic fibroblast growth factor(bFGF) and epidermal growth factor(EGF). 3. The study for the differentiation of neuronal-like cells from DPSCs clone A32.In this study, two-steps of neuronal differentiation protocol was used to induce the A32 differentiation into neuronal-like cells in vitro. In the initial step, the attached fibroblast-like A32 was changed into the floating neurosphere-like bodies by culturing in the neural stem cells(NSCs) medium for 10 days on low-attachment culture surfaces. The gene and protein expression of NSCs specific markers were increased, which demonstrated A32 has the potential to differentiate into NSCs-like phenotype. A further maturation step, with the addition of neurotrophic factors to the neurobasal medium, the NSCs-like cells from A32 further differentiated into the neuronal-like cells demonstrated by a more complex neuronal morphology change and the expression of later stage markers, such as Neurofilament(NF) and microtubule-associated protein 2(MAP2). Additionally, we investigated the expression of different stages of neuronal markers, growth factors(including TGF-β1, bFGF, EGF), as well as neurotrophic factors(including Brain-Derived Nurotrophic Factor(BDNF), Nerve Growth Factor(NGF), Neurotrophin-3(NT-3) in the process of neuronal differentiation from A32. We found different growth factors and neurotrophic factors play different roles during the process of neuronal differentiation from A32.In conclusion, we investigated the characterization of different clones of DPSCs,and isolated one of DPSCs clones A32 with the best proliferation and differentiation capacity, and further demonstrated that this clone have the potential to be induced to differentiate into SMCs-like and neuron-like cells. The data in this study about the differentiation protocols as well as the molecular mechanisms represents a step forward in providing a better understanding for further research in smooth muscle and neuronal tissue engineering. |
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