Studies from other glandular tissues like mammary gland reve

Studies from other glandular tissues, like mammary gland, revealed CD24hi/CD29lo- (Shackleton et al., 2006) expressing NVP-TNKS656 as potential stem cells. In mice, we reported that intraglandular transplantation of primary-sphere-derived CD24+/CD29+ cells could ameliorate radiation-induced hyposalivation to some extent (Nanduri et al., 2011). Therefore, we next tested whether subpopulations exist in primary-sphere-derived single cells (Figure 1A) that express CD24 and/or CD29 using flow cytometry. Unlike the situation in the mammary gland (Shackleton et al., 2006), we did not detect a clear separation between subsets. Therefore, the cells were arbitrarily separated into four subsets (CD24hi/CD29lo, CD24hi/CD29hi, CD24med/CD29hi, and CD24lo/CD29hi) using the gating strategy shown in Figure 3A. Sorted single cells were plated into MM culture medium. After 5–7 days, secondary spheres were formed from all subsets (Figure 3B), albeit with different efficiencies. Clearly, the CD24hi/CD29hi cells showed the highest secondary-sphere-formation capacity (Figure 3C). Next, these secondary spheres were induced to form organoids. Although all four subsets formed organoids, these were morphologically very distinct (Figure 3D). Detailed analysis of the different types of organoids showed that CD24hi/CD29hi and CD24med/CD29hi cells have the ability to form both ductal and lobular organoids whereas CD24hi/CD29lo and CD24lo/CD29hi cells form predominantly lobular organoids (Figure 3E), most likely implying a more committed cell state in the latter two populations. To assess their self-renewal potential, cells from all populations were cultured under MM conditions. Most cells from CD24hi/CD29lo and CD24lo/CD29hi subsets do not proliferate or die during culture as observed with trypan-blue-based cell counting. Very few form secondary spheres that did not yield enough cells to allow passaging. CD24med/CD29hi- and CD24hi/CD29hi-derived cells, however, were able to self-renew for four passages and greater than five passages, respectively (Figure S1A), indicating the higher potential of these populations. During self-renewal, spheres are dissociated to single cells at every passage. To minimize dissociation-induced stress, the Rho-inhibitor Y-27632, known to protect against dissociation-induced cell stress, was added (Zhang et al., 2011; enriched medium [EM]). It enhanced the initial secondary sphere-formation percentage of CD24hi/CD29hi cells (Figure S1B) from 2.5% ± 0.68% (MM) to 10.9% ± 3.9% (EM). Next, CD24hi/CD29hi cells were cultured under EM conditions, which resulted in a more-rapid and pronounced expansion of cells capable of forming secondary spheres. In general, the percentage of cells capable of forming spheres was increased with increasing passages (Figure 4A) from 11.54% ± 5.02% at passage 1 (P1) to 25.51% ± 1.86% at P12 (p < 0.05; Figure S1C), indicative of enrichment for sphere-forming cells. This is further emphasized by the significant increase (Figure 4B) in cells expressing CD24hi/CD29hi from P1 to P9 (Figure S1D). Compared to expansion from unselected cells, a striking 4-fold increased (5,734 ± 1,148 cells) number of CD24hi/CD29hi cells was generated after seven passages (Figure 4C). Thus, a pronounced expansion of apparent SG stem cells can be obtained with these optimized in vitro cultures. Such expansion may be a key element in the future therapeutic use of these cells to restore SG function in patients included in prolonged (multiple weeks) radiotherapy regimens (Vergeer et al., 2009). Karyotyping of passaged sphere cells showed doubling of the number of chromosomes in about 50% of the cells that were cultured beyond passage 3 (data not shown), a phenomenon known to occur in mice (Tolar et al., 2007). However, we have so far not seen any tumor formation after transplantation of these cells (see below). To identify differentially regulated genes in long-term self-renewing SG cells, genome-wide gene expression analysis (GEO accession number GSE59559) was performed on primary-sphere-derived CD24hi/CD29hi cells (P0) and CD24hi/CD29hi cells that were passaged ten (P10) times. Gene-expression analysis with a stringent threshold of p < 0.0001 has identified 229 probes that are differentially expressed between P0 and P10 CD24hi/CD29hi cells (Figure S1E). Among these, 13 genes were upregulated (>logFc 4.0) in P10 compared to P0 cells, indicating their enrichment during later passages (Table S3). These include CCD3, PORCN, CAV1, SNCG, TRP63, CCND2, KRT5, LGALS7, SNAI2, NTRK2, GPR87, VSNL1, and DST (Figure S1F).