Neural Crest-Derived Dental Pulp Stem Cells Function as Ectomesenchyme to Support Salivary Gland Tissue Formation

Abstract

Xerostomia, dry mouth due to loss of functional salivary gland, is caused by Sjogren’s syndrome, radiotherapy for head and neck cancer, medications and aging, leading to patients’ suffer 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. Dental pulp stem cells (DPSC) isolated from murine dental pulp is neural crest-derived. Herein, we used the human salivary gland (HSG) cell line as a model to study the effects of DPSC on salivary gland differentiation. Upon in vitro differentiation on Matrigel, HSG alone and HSG cocultured with Wnt1-Cre/R26R-LacZ derived DPSC (HSG+DPSC) differentiated into acinar-like structures. However, HSG formed more mature (higher expression of LAMP-1 and CD44), larger and increased numbers of acinar structures in HSG+DPSC. In vivo subcutaneous co-transplantation of HSG and DPSC with hyaluronic acid (HA) hydrogel after 2 weeks was evaluated by Q-RT- PCR, morphological and immunohistological assessment. 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 alphaamylase- 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 DPSC; and (5) more mature glandular structures double-positive for salivary gland differentiation markers CD44 and LAMP-1. These results indicate that DPSC supported and enhanced HSG differentiation into functional salivary gland tissue. This study illustrates the potential of DPSC as inductive mesenchyme for salivary gland regeneration, repair, and tissue engineering.