| Dental pulp stem cells (DPSCs) were first isolated and characterized from human teeth and most studies have focused on using human DPSCs for dentin regeneration. However, mouse DPSCs have not been well characterized and their origin(s) have not yet been elucidated. I examined if murine DPSCs are neural crest derived and determined their in vitro and in vivo capacity. DPSCs from neonatal mice expressed embryonic stem cell and neural crest genes, but lacked expression of mesodermal genes. Cells isolated from the Wnt1-Cre/R26R-LacZ mouse, a reporter of neural crest-derived tissues, indicated that DPSCs were Wnt1-marked and therefore of neural crest origin. Clonal DPSCs showed multi-differentiation in neural crest lineage for odontoblasts, chondrocytes, adipocytes, neurons, and smooth muscles. In vivo subcutaneous transplantation with hydroxyapatite/tricalcium phosphate, based on tissue/cell morphology and specific antibody staining, revealed that the clones differentiated into odontoblast-like cells and produced dentin/pulp-like structure. Conversely, femur-derived bone marrow stromal cells (BMSCs) gave rise to osteoblast-like cells and generated bone-like structure. Interestingly, the capillary distribution in the DPSC transplants showed close proximity to odontoblasts whereas in the BMSC transplants bone condensations were distant to capillaries resembling dentinogenesis in the former vs. osteogenesis in the latter.;Loss of functional salivary gland causes patients' moribidities from difficulties in swallowing and speech, as well as oral diseases. Stem cell therapy is considered a potential therapeutic alternative. However, combinatory approaches including not only salivary gland stem cells but also supportive cells and appropriate extracellular matrix are necessary to form a functional salivary gland. Like tooth formation, the development of salivary gland requires epithelium interacting with neural crest-derived mesenchyme. I used the human salivary gland (HSG) cell line as a model to study the effects of DPSCs on salivary gland differentiation. In vitro differentiation on matrigel showed that HSG alone and HSG co-cultured with Wnt1-Cre/R26R-LacZ derived DPSCs (HSG+DPSC) differentiated into acinar-like structures. However, HSG formed more mature (higher expression of LAMP-1 and CD44), larger and increased numbers of acini in HSG+DPSC. Subcutaneous co-transplantation of HSG and DPSCs with hyaluronic acid (HA) hydrogels after 2 weeks was evaluated by Q-RT-PCR, morphology and immunohistology. Compared to HSG transplants which only showed undifferentiated tumor-like cells, HSG+DPSC demonstrated (1) higher expression of murine mesenchymal marker Fgf-7, (2) higher expression of mature human salivary gland differentiation marker alpha-amylase-1 (AMY-1), (3) higher expression of murine endothelial, vWF, neuronal, NF-200, and angiogenic markers, Vegfr-3 and Vegf-c, (4) mucin-secreting acinar- and duct-like structures with abundant blood vessels at the interface with DPSCs, and (5) more mature glandular structures double-positive for salivary gland differentiation markers CD44 and LAMP-1. These results indicate that DPSCs supported and enhanced HSG differentiation into functional salivary gland tissue.;In addition, DPSCs have previously demonstrated potential pericyte-like topography and function. However, the mechanisms regulating their pericyte function are still yet to be elucidated. DPSC angiogenic and pericyte function were investigated Tie2-GFP derived dental pulp cells were negative for GFP driven by the endothelial Tie2 transgene, indicating an absence of endothelial cells. Endothelial cells co-cultured with DPSCs formed more mature in vitro tube-like structures as compared to those co-cultured with BMSCs. Many DPSCs were located adjacent to vascular tubes, suggesting a pericyte location and function. In vivo DPSCs subcutaneously transplanted in matrigel (MG) (DPSC-MG) induced more vessel formation than BMSC-MG. DPSCs expressed higher Vegfd, Vegfr3, EphrinB2 levels. Soluble Flt (sFlt), an angiogenic inhibitor that binds VEGF-A, significantly decreased the amount of blood vessels in DPSC-MG, but not in BMSC-MG. sFlt inhibited VEGFR2 and downstream ERK signaling and down-regulated Vegfa, Vegf receptors and EphrinB2 expression in DPSCs. Therefore, DPSC-induced angiogenesis is VEGF-dependent. DPSCs enhance angiogenesis by secreting VEGF-A, -C, -D and forming tight associations with vessels, resembling pericyte-like cells. Taken together, I demonstrate the existence of neural crest-derived DPSCs with differentiation capacity into cranial mesenchymal tissues and other neural crest-derived tissues. I also illustrate the potential of DPSCs as inductive mesenchyme for salivary gland regeneration, repair, and tissue engineering, and provide first insights into the mechanism(s) of DPSC angiogenic capacity and their function as pericytes. DPSCs hold promise as a stem cell source for regenerating neural crest derived tissues, and the trophic and angiogenic properties of DPSCs also highlight this stem cell source useful for tissue regeneration. |
