Isolation and Characterization of Neural Crest-Derived Stem Cells from Dental Pulp of Neonatal Mice

Dental pulp stem cells (DPSCs) are shown to reside within the tooth and play an important role in dentin regeneration. DPSCs were first isolated and characterized from human teeth and most studies have focused on using this adult stem cell for clinical applications. However, mouse DPSCs have not been well characterized and their origin(s) have not yet been elucidated. Herein we examined if murine DPSCs are neural crest derived and determined their in vitro and in vivo capacity. DPSCs from neonatal murine tooth pulp expressed embryonic stem cell and neural crest related genes, but lacked expression of mesodermal genes. Cells isolated from the Wnt1-Cre/R26R-LacZ model, 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. Following in vivo subcutaneous transplantation with hydroxyapatite/tricalcium phosphate, based on tissue/cell morphology and specific antibody staining, the clones differentiated into odontoblast-like cells and produced dentin-like structure. Conversely, 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. Thus we demonstrate the existence of neural crest-derived DPSCs with differentiation capacity into cranial mesenchymal tissues and other neural crest-derived tissues. In turn, DPSCs hold promise as a source for regenerating cranial mesenchyme and other neural crest derived tissues

Characterization of Dental Pulp Stem Cells and Their Potential Clinical Applications

Thesis (Ph.D.)--University of Washington, 2013Dental 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

The Prevalence of Xerostomia in Older Thai Individuals with Type II Diabetes Mellitus and Its Association with Type of Toothpaste and Oral Functions: A Cross-Sectional Study Using Questionnaires

Aim: To investigate the prevalence of xerostomia in older people with diabetes mellitus and its impacts on oral functions, as well as to determine potential risk factors for xerostomia. Methods: An analytical cross-sectional study was conducted on 623 older type 2 diabetes mellitus (T2DM) Thai people using valid structural questionnaires. Patients were interviewed, and data were recorded. Xerostomia was assessed using subjective symptom questionnaires. Risk factors for xerostomia were analyzed using bivariate and multiple logistic regression analyses. Results: Among the study participants, 38.4% of the older T2DM people had xerostomia, which is associated with sex, age, type of toothpaste, years of diabetes, hemoglobin A1c level, other systemic diseases, medication, smoking, alcohol consumption, and denture wearing. It was significant that xerostomia was associated with toothpaste containing spicy herbal extracts (OR: 9.32 [3.46 to 15.25]), while toothpaste containing artificial sweeteners tended to lower the risk of xerostomia. In addition, older T2DM adults with xerostomia had greater impaired oral functions, which include difficulties in speaking (OR: 3.31 [1.11 to 9.80]), tasting (OR: 5.12 [3.26 to 8.06]), swallowing (OR: 3.59 [2.32 to 5.53]), and chewing (OR: 3.34 [1.15 to 5.82]). Conclusions: Xerostomia is prevalent in older Thai people with T2DM. The results suggest that toothpaste containing spicy herbal extracts might increase the risk of xerostomia, resulting in various oral function problems. Therefore, greater awareness of xerostomia in this group should be raised to monitor dental health, and professionals should work in parallel with other aspects of oral health promotion

Isolation, Characterization, and Transplantation of Cardiac Endothelial Cells

Isolation and ex vivo expansion of cardiac endothelial cells have been a recurrent challenge due to difficulties in isolation, cell heterogeneity, lack of specific markers to identify myocardial endothelial cells, and inadequate conditions to maintain long-term cultures. Herein, we developed a method for isolation, characterization, and expansion of cardiac endothelial cells applicable to study endothelial cell biology and clinical applications such as neoangiogenesis. First, we dissociated the cells from murine heart by mechanical disaggregation and enzymatic digestion. Then, we used flow cytometry coupled with specific markers to isolate endothelial cells from murine hearts. CD45+ cells were gated out to eliminate the hematopoietic cells. CD31+/Sca-1+ cells were isolated as endothelial cells. Cells isolated from atrium grew faster than those from ventricle. Cardiac endothelial cells maintain endothelial cell function such as vascular tube formation and acetylated-LDL uptake in vitro. Finally, cardiac endothelial cells formed microvessels in dorsal matrigel plug and engrafted in cardiac microvessels following intravenous and intra-arterial injections. In conclusion, our multicolor flow cytometry method is an effective method to analyze and purify endothelial cells from murine heart, which in turn can be ex vivo expanded to study the biology of endothelial cells or for clinical applications such as therapeutic angiogenesis

An In Vitro Culture System for Long-Term Expansion of Epithelial and Mesenchymal Salivary Gland Cells: Role of TGF-β1 in Salivary Gland Epithelial and Mesenchymal Differentiation

Despite a pivotal role in salivary gland development, homeostasis, and disease, the role of salivary gland mesenchyme is not well understood. In this study, we used the Col1a1-GFP mouse model to characterize the salivary gland mesenchyme in vitro and in vivo. The Col1a1-GFP transgene was exclusively expressed in the salivary gland mesenchyme. Ex vivo culture of mixed salivary gland cells in DMEM plus serum medium allowed long-term expansion of salivary gland epithelial and mesenchymal cells. The role of TGF-β1 in salivary gland development and disease is complex. Therefore, we used this in vitro culture system to study the effects of TGF-β1 on salivary gland cell differentiation. TGF-β1 induced the expression of collagen, and inhibited the formation of acini-like structures in close proximity to mesenchymal cells, which adapted a fibroblastic phenotype. In contrast, TGF-βR1 inhibition increased acini genes and fibroblast growth factors (Fgf-7 and Fgf-10), decreased collagen and induced formation of larger, mature acini-like structures. Thus, inhibition of TGF-β signaling may be beneficial for salivary gland differentiation; however, due to differential effects of TGF-β1 in salivary gland epithelial versus mesenchymal cells, selective inhibition is desirable. In conclusion, this mixed salivary gland cell culture system can be used to study epithelial-mesenchymal interactions and the effects of differentiating inducers and inhibitors

A γ-Secretase Inhibitor Attenuates Cell Cycle Progression and Invasion in Human Oral Squamous Cell Carcinoma: An In Vitro Study

Notch signaling is associated with many human malignancies, including oral squamous cell carcinoma (OSCC). However, the exact function of Notch signaling in OSCC remains unclear. Here, we investigated the effect of Notch signaling inhibition using a γ-secretase inhibitor (DAPT) on OSCC behaviours in vitro. Bioinformatic analysis of public-available gene expression profiles revealed the dysregulation of the Notch signaling pathway in OSCC compared with normal tissues, indicating the role of Notch signaling in OSCC regulation. RNA sequencing analysis of DAPT-treated human OSCC cells revealed the dysregulation of genes related to cell cycle-related pathways. Blocking Notch signaling significantly inhibited cell proliferation. DAPT-induced G0/G1 cell cycle arrest induced cell apoptosis. Furthermore, cell migration and invasion were also reduced in DAPT-treated cells. These findings indicate that Notch signaling activation participates in OSCC regulation by promoting cell growth, cell cycle progression, cell migration, and invasion. These mechanisms could facilitate OSCC progression. These results imply the potential use of Notch signaling inhibitors as a candidate adjuvant treatment in OSCC patients

Harnessing Sphingosine-1-Phosphate Signaling and Nanotopographical Cues To Regulate Skeletal Muscle Maturation and Vascularization

Despite possessing substantial regenerative capacity, skeletal muscle can suffer from loss of function due to catastrophic traumatic injury or degenerative disease. In such cases, engineered tissue grafts hold the potential to restore function and improve patient quality of life. Requirements for successful integration of engineered tissue grafts with the host musculature include cell alignment that mimics host tissue architecture and directional functionality, as well as vascularization to ensure tissue survival. Here, we have developed biomimetic nanopatterned poly­(lactic-<i>co</i>-glycolic acid) substrates conjugated with sphingosine-1-phosphate (S1P), a potent angiogenic and myogenic factor, to enhance myoblast and endothelial maturation. Primary muscle cells cultured on these functionalized S1P nanopatterned substrates developed a highly aligned and elongated morphology and exhibited higher expression levels of myosin heavy chain, in addition to genes characteristic of mature skeletal muscle. We also found that S1P enhanced angiogenic potential in these cultures, as evidenced by elevated expression of endothelial-related genes. Computational analyses of live-cell videos showed a significantly improved functionality of tissues cultured on S1P-functionalized nanopatterns as indicated by greater myotube contraction displacements and velocities. In summary, our study demonstrates that biomimetic nanotopography and S1P can be combined to synergistically regulate the maturation and vascularization of engineered skeletal muscles