The use of MaSCs from GFP mice

The use of MaSCs from GFP mice for in vivo transplant into wild type recipients enabled us to examine the purity of FACS-gated basal and luminal cell populations from the regenerated GFP glands. All epithelial amyloid (GFP positive) were converged in the CD24+ population, while the combination of CD24 and CD49f enabled the separation of luminal cells from basal cells efficiently (Fig. S5a). However, we also found 22% GFP negative cells within the basal gate and 5% GFP negative cells within the luminal gate (Fig. S5a). This indicates that a substantial portion of non-epithelial cells also share the same cell surface marker phenotype as the basal epithelial cells. Subsequent analysis showed that these non-epithelial cells alone cannot form spheres (Fig. S5b). The higher percentage of non-epithelial cells in the basal cell fraction may explain, in part, why basal cells had much lower SFE than luminal cells. Furthermore, differentiation in Matrigel culture showed predominantly solid or hollow structures for spheres derived from basal or luminal cells from the regenerated glands, similar to those of natural glands (Figs. S5c–f). We noticed uniform appearance of the complete hollow structure (Fig. S5f) in 3D culture of luminal derived spheres, suggesting a reduced complexity of luminal lineage from regenerated glands in comparison with the natural glands. This reduced complexity concept is consistent with the fact that unipotent basal MaSCs in adult glands (used in this study) are different from the multipotent MaSCs present in the fetus (Van Keymeulen et al., 2011). Future studies using multipotent MaSCs derived from fetal mammary glands for transplant may unravel the difference between basal MaSCs at different developmental stages. Based on our SFD assay, we estimated that 1 SFD-IC in 252 total basal cells or 1 SFD-IC in 1154 total epithelial cells (the sum of basal and luminal cells) was obtained from adult mammary glands from 2-month-old wild type virgin C57BL/6 mice at estrus phase. Our SFD-IC values are very similar to the MRU values (1 MRU in 51 to 160 basal cells) reported previously on the same mouse strain based on limited dilution IVT assay (Stingl et al., 2006). However, MRU frequency has been reported ranging from 1 MRU in 100 total cells to <1 MRU in 4900 cells in literature (Kordon and Smith, 1998; Moraes et al., 2007; Shackleton et al., 2006; Stingl et al., 2006). The large variation has been attributed to different methods used for mammary tissue dissociation and IVT (Stingl, 2009). The following are the supplementary data related to this article.
Acknowledgments This work was supported in part by funding from NIH Grants R01CA75253 and R01ES022057, the Bank of America Shelby Rae Tengg Foundation, the Mary Kay Foundation (#082-12), and the Cancer Therapy and Research Center at University of Texas Health Science Center at San Antonio through the NCI Cancer Center Support Grant 2P30CA054174-17 to the flow cytometry core facility. We thank Dr. John Stingl for his kind instruction in establishing the in vitro and in vivo assays in our laboratory. We also thank Dr. Rong Li for the use of the Nikon BioStation-IM.
Introduction Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have provided a platform for studying basic human development and disease mechanisms and hold great potential for future cell therapies (Murry and Keller, 2008). However, biomedical application of hESCs and iPSCs depends on the availability of robust cell expansion and differentiation protocols. Undifferentiated colonies of hESCs and iPSCs can be technically challenging to maintain and expand. For example, they thaw from frozen samples with low efficiency and then require co-culture with mouse embryonic fibroblasts (MEF), or expensive media and matrix proteins, in order to remain undifferentiated. Furthermore, they need daily medium changes, examination, and manual selection to ensure the cultures remain in an undifferentiated state. Finally, >10% of hESC and iPSC cultures develop karyotypic anomalies (Ben-David et al., 2011; Peterson et al., 2011; Taapken et al., 2011), which should be monitored as they could impact differentiation capabilities (Graf and Stadtfeld, 2008) and clinical applicability. Clearly, less time consuming and labor intensive culturing techniques would be advantageous.