Fostering cell-cell interactions and integrating angiocrine factors to promote the development of salivary microtissues in 3D.

Salivary gland development requires vascular input signals. However, whether endothelial cell-secreted angiocrine factors have any inductive effects on adult human salivary gland stem/progenitor cells (hS/PCs) is unknown. To advance the engineering of functional salivary glands, we probed the effects of epithelial and endothelial crosstalk on the growth and differentiation of hS/PCs. Culture of hS/PCs in agarose microwells led to the formation of multicellular spheroids with close cell-cell contacts. Compared with the day 3 culture maintained in the hepatocyte media (HEP) typically used for hS/PCs, 7-day culture in the endothelial cell growth media (EMG2), significantly increased the expression of genes encoding the E-cadherin (CDH1), stem/progenitor markers (KRT5, KRT14, MYC), acinar markers (AQP5, AMY, SLC12A1), and extracellular matrix proteins (LAMA1, FN1). Subsequent cultivation of hS/PC spheroids in a hyaluronic acid (HA)-derived, cell-adhesive, and proteolytically degradable hydrogel yielded hydrogel-encapsulated microtissues with complex, multilobulated structures, consisting of fibronectin-encased lobules connected by F-actin structures. Addition of a CD31+/vWF+ endothelial cell monolayer on top of the hS/PC-laden gel construct led to the development of salivary gland microtissues containing differentiated cells expressing key acinar and ductal cell markers. In vivo work showed that the cell-free HA gels implanted in the partially resected rat parotid gland were degraded in 21 days and did not adversely affect the native tissue structure. Collectively, fostering epithelial cell-cell interaction and integrating endothelial cell-secreted angiocrine signals led to the development of pro-acinar salivary gland mimetics from adult salivary gland stem/progenitor cells.