Re associated with mammary stem cells [19]. Isolation of a pure mammary stem cell population has not been possible thus far due to lack of definitive markers. However, a mammary stem cell-enriched population can be obtained using a combination of cell surface markers and fluorescence-activated cell sorting (FACS). The population of CD24+ CD49fhi cells contains basal cells, mammary stem cells and possibly luminal progenitors. Outgrowths arising from these cells are fully functional and able to produce milk when recipients are put through pregnancy [20,21]. Moreover, mammary stem cells express basal markers such as keratin (K) 5, K14, smooth muscle actin (SMA), smooth muscle myosin, vimentin and laminin [20,22]. Luminal cells are CD24+ CD49flo, express K18 and lack expression of these basal markers. Luminal progenitors can be distinguished by the expression of the CD61 surface molecule and have the ability to form colonies in vitro in both two-dimensional and three-dimensional Matrigel culture [23].Given the indispensable role of Stat3 in mESCs and intestinal crypt stem cells, and the essential role of Stat3 in mediating cell death during mammary gland involution, it was of interest to us to investigate the role of Stat3 in mammary gland-specific stem cells including both embryonic derived adult stem cells and those that are present following a full involution (PI-MECs).Materials and Methods Animal HusbandryMice bearing a Stat3 gene flanked by loxP sites (Stat3fl/fl) [24] 25837696 were crossed with a strain expressing Cre recombinase gene under either the b-lactoglobulin (BLG) promoter [25] or the K14 promoter. K14-Cre mice were kindly donated by Dr. Michaela Frye (Centre for Stem Cell Research, Cambridge, UK), were originally from The Jackson Laboratories (Bar Harbor, ME, USA), and have a mixed C57Bl/6 6 CBA background. All mice were maintained and bred in conventional cages within a specific pathogen free (SPF) animal facility. Immunodeficient CD1-Stat3 and Mammary Stem CellsFigure 3. Normal mammary gland development in Stat3 depleted glands. (A) Whole mount staining of mammary get 69056-38-8 glands of 5-week-old Stat3fl/fl;K14-Cre2 and Stat3fl/fl;K14-Cre+ females. (B) Flow cytometry analysis of luminal and basal cells isolated from mammary glands of Stat3fl/fl;K14-Cre2 and Stat3fl/fl;K14-Cre+ females four weeks after natural weaning. p value was determined using Student’s t test. ns: not significant. doi:10.1371/journal.pone.0052608.g003 Figure 2. BLG-Cre mediated deletion of Stat3 affects repopulating frequency of stem cells and outgrowth phenotype. (A) Whole mount staining of mammary outgrowths originating from CD24+ CD49fhi basal cells sorted from mammary glands of Stat3fl/fl,BLG-Cre2 and Stat3fl/fl;BLG-Cre+ 
females four weeks after natural weaning. (B) Limiting dilution analysis of the repopulating frequency of the mammary stem cell-enriched population sorted from mammary glands of Stat3fl/fl,BLG-Cre2 and Stat3fl/fl;BLG-Cre+ females four weeks after natural weaning. Number of outgrowths is shown per number of transplanted fat pads. CI: confidence interval. doi:10.1371/journal.pone.0052608.gATG CTA TTT GTA GG-39, Stat3 A 196 site deleted reverse 59-GCA GCA GAA TAC TCT ACA GCT C-39.Semi-quantitative RT-PCRRNA was extracted from sorted cells using TRIzol Reagent (Invitrogen) and cDNA was prepared using the Super Script FirstStrand Synthesis System for RT (Invitrogen) following the manufacturer’s instructions. Semi-quantitative RT-PCR was performed with the following pr.Re associated with mammary stem cells [19]. Isolation of a pure mammary stem cell population has not been possible thus far due to lack of definitive markers. However, a mammary stem cell-enriched population can be obtained using a combination of cell surface markers and fluorescence-activated cell sorting (FACS). The population of CD24+ CD49fhi cells contains basal cells, mammary stem cells and possibly luminal progenitors. Outgrowths arising from these cells are fully functional and able to produce milk when recipients are put through pregnancy [20,21]. Moreover, mammary stem cells express basal markers such as keratin (K) 5, K14, smooth muscle actin (SMA), smooth muscle myosin, vimentin and laminin [20,22]. Luminal cells are CD24+ CD49flo, express K18 and lack expression of these basal markers. Luminal progenitors can be distinguished by the expression of the CD61 surface molecule and have the ability to form colonies in vitro in both two-dimensional and three-dimensional Matrigel culture [23].Given the indispensable role of Stat3 in mESCs and intestinal crypt stem cells, and the essential role of Stat3 in mediating cell death during mammary gland involution, it was of interest to us to investigate the role of Stat3 in mammary gland-specific stem cells including both embryonic 
derived adult stem cells and those that are present following a full involution (PI-MECs).Materials and Methods Animal HusbandryMice bearing a Stat3 gene flanked by loxP sites (Stat3fl/fl) [24] 25837696 were crossed with a strain expressing Cre recombinase gene under either the b-lactoglobulin (BLG) promoter [25] or the K14 promoter. K14-Cre mice were kindly donated by Dr. Michaela Frye (Centre for Stem Cell Research, Cambridge, UK), were originally from The Jackson Laboratories (Bar Harbor, ME, USA), and have a mixed C57Bl/6 6 CBA background. All mice were maintained and bred in conventional cages within a specific pathogen free (SPF) animal facility. Immunodeficient CD1-Stat3 and Mammary Stem CellsFigure 3. Normal mammary gland development in Stat3 depleted glands. (A) Whole mount staining of mammary glands of 5-week-old Stat3fl/fl;K14-Cre2 and Stat3fl/fl;K14-Cre+ females. (B) Flow cytometry analysis of luminal and basal cells isolated from mammary glands of Stat3fl/fl;K14-Cre2 and Stat3fl/fl;K14-Cre+ females four weeks after natural weaning. p value was determined using Student’s t test. ns: not significant. doi:10.1371/journal.pone.0052608.g003 Figure 2. BLG-Cre mediated deletion of Stat3 affects repopulating frequency of stem cells and outgrowth phenotype. (A) Whole mount staining of mammary outgrowths originating from CD24+ CD49fhi basal cells sorted from mammary glands of Stat3fl/fl,BLG-Cre2 and Stat3fl/fl;BLG-Cre+ females four weeks after natural weaning. (B) Limiting dilution analysis of the repopulating frequency of the mammary stem cell-enriched population sorted from mammary glands of Stat3fl/fl,BLG-Cre2 and Stat3fl/fl;BLG-Cre+ females four weeks after natural weaning. Number of outgrowths is shown per number of transplanted fat pads. CI: confidence interval. doi:10.1371/journal.pone.0052608.gATG CTA TTT GTA GG-39, Stat3 deleted reverse 59-GCA GCA GAA TAC TCT ACA GCT C-39.Semi-quantitative RT-PCRRNA was extracted from sorted cells using TRIzol Reagent (Invitrogen) and cDNA was prepared using the Super Script FirstStrand Synthesis System for RT (Invitrogen) following the manufacturer’s instructions. Semi-quantitative RT-PCR was performed with the following pr.