Treatment options for xerostomia include administration of s

Treatment options for xerostomia include administration of saliva substitutes or stimulants (Fox, 2004). Saliva substitutes might improve some, but not all, problems associated with SG dysfunction, whereas stimulants are only useful for people with some remaining SG function. Alternative approaches to restore SG function have been pursued, for instance, the development of bioengineered glands (Ogawa et al., 2013). Although this may be a good model to study SG regeneration, it might not be clinically translatable due to its origin from embryonic SGs. Another potential option is to rescue these patients using autologous stem cell transplantation that may regenerate the damaged tissue and thus provide long-term recovery. It has been shown that ductal ligation induced damage to the SG-stimulated proliferation of CD29- and CD49f-expressing btk inhibitor (Matsumoto et al., 2007), indicating the existence of regenerative cells in this area of the SG. We reported earlier that murine (Lombaert et al., 2008) and human (Feng et al., 2009) stem/progenitor cells can be cultured into salispheres (primary spheres) via an enrichment culture in vitro. In preclinical models, we demonstrated the potential of autologous adult stem cell transplantation to restore radiation-damaged SG function (Lombaert et al., 2008; Nanduri et al., 2011) and tissue homeostasis (Nanduri et al., 2013). Murine SG primary-sphere-derived c-KIT+ cells were able to restore SG function in hyposalivation mouse model. Unfortunately, scarce adult human biopsy material contains very low numbers of c-KIT+ cells (Feng et al., 2009; Pringle et al., 2013), limiting their clinical potential. An alternative strategy is therefore necessary to generate sufficient stem/progenitor cells numbers to enable translation of this therapy to the clinic. Expanding the number of stem cells ex vivo represents a way to circumvent this problem. In contrast to induced pluripotent stem cells and embryonic stem cells, adult stem cells are not easily propagated and expanded. Self-renewal/expansion has been reported for only a few types of adult stem cells, including neural (Kalani et al., 2008), intestinal (Barker et al., 2007), and liver stem cells (Huch et al., 2013), but the long-term functional activity of these cultured cells remains to be assessed.
Results First, in vitro assays were used to test self-renewal and differentiation properties of primary spheres, being a putative stem or progenitor cell population. To test their self-renewal ability, murine primary-sphere-derived single cells were fluorescence-activated cell sorting sorted and seeded into a Matrigel-based matrix (10,000 cells/gel) supplemented with minimal culture medium (MM) (see the Experimental Procedures; Figure 1A). Within 5–7 days 0.44% ± 0.03% of the single cells formed secondary spheres (Figure 1B, MM). When primary-sphere-derived single cells from DsRed and enhanced GFP (EGFP) transgenic mice were mixed and coseeded, more than 99% of all secondary spheres were single colored (Figure 1C), indicating that the spheres are not formed by clumping of cells, but rather through the outgrowth from single cells. To test the ability of putative SG stem cells to differentiate into mature cell lineages, single-cell-derived spheres were plated into a 3D matrix mixture of collagen and Matrigel, supplemented with minimal medium with 10% fetal calf serum (Figure 1A, Differentiation). Within 1–3 weeks, the seeded spheres changed morphologically into diverse organoid-like structures. Two types of organoids, ductal and lobular, could be discerned. Ductal organoids contained a lumen (arrows) and were surrounded by cells that stained positive for CK7 and CK18 (Figure 1D), which is consistently expressed in SG duct cells. The lobular organoids were devoid of tubular extensions but rather contained compact, round, lobule-like structures, which expressed the acinar cell protein aquaporin-5 (Figure 1E, AQP5). To confirm genome-wide signs of differentiation in the organoids, a gene expression analysis was performed (details in the Experimental Procedures; Gene Expression Omnibus [GEO] accession number GSE59559). Data from organoids were compared to that of primary spheres (representing a relatively undifferentiated state) using differential gene expression analysis. Results identified 302 differentially expressed probes in organoids when compared to primary spheres (Figure 2A) with a stringent threshold (p < 0.0001). Among these, 102 probes were upregulated (>logFc2.0) in organoids (Figure 2B) compared to primary spheres (Table S1 available online). They were tested for gene ontology classifiers that revealed significant enrichment in biological processes including gland morphogenesis (GO: 0022612), gland development (GO: 0048732), and many others listed in Table S2. At the individual gene level, these 102 genes include calcium-channel-related proteins (CPNE8), secretin (role in pancreatic duct secretion; Lee et al., 2012); cranio-facial-development-related RBMS3 gene (Jayasena and Bronner, 2012), vitamin-K-dependent protein (PROS1), vesicle-associated membrane protein (VAMP4; Sramkova et al., 2012), STRA6 (Petrakova et al., 2014) known to be expressed in SG, SG-branching morphogenesis-related (TGM2 and NTN4), and embryonic SG end bud differentiation marker (fibroblast growth factor 1; Patel et al., 2006), indicating SG-specific differentiation. These findings show that single-cell-derived spheres are capable of secondary sphere formation, which can form differentiated SG lineages in vitro.