Stat3 is required to maintain the full differentiation potential of mammary stem cells and the proliferative potential of mammary luminal progenitors - PubMed (original) (raw)

Stat3 is required to maintain the full differentiation potential of mammary stem cells and the proliferative potential of mammary luminal progenitors

Anna D Staniszewska et al. PLoS One. 2012.

Abstract

Stat3 has a defined role in mammary gland where it is a critical mediator of cell death during post-lactational regression. On the other hand, Stat3 is required for the self-renewal of embryonic stem cells and is sufficient for the induction of a naïve pluripotent state in epiblast stem cells. Mammary stem cells (MaSCs) have a high capacity for self-renewal and can grow robustly in transplantation experiments in vivo. However, a role for Stat3 in MaSCs has not been investigated. Here we show that depletion of Stat3 from basal cells results in reduced primary transplantation efficiency and diminishes the potential to generate ductal, but not alveolar, outgrowths. In addition, Stat3 is required for maximal proliferation of luminal progenitors.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Stat3fl/fl;BLG-Cre+ glands show incomplete involution and luminal progenitors have reduced proliferative capacity.

(A) RT-PCR analysis of Stat3 expression in FACS sorted populations of mammary epithelial cells. MRU: mammary repopulating units. (B, C) H&E staining of sections of Stat3fl/fl;BLG-Cre− and Stat3fl/fl;BLG-Cre+ mammary glands collected at day 5 of the second gestation (B) or four weeks after natural weaning (C). (D) Western blot analysis of four Stat3fl/fl;BLG-Cre− and five Stat3fl/fl;BLG-Cre+ mammary glands four weeks after natural weaning for the expression or activation of Stat5, Erk, Akt, β-casein and WAP. β-actin was used as a loading control. (E) Immunohistochemistry staining for pStat5 (red) and E-cadherin (green) in mammary gland sections from Stat3fl/fl;BLG-Cre− and Stat3fl/fl;BLG-Cre+ mice collected four weeks after natural weaning. Nuclei were stained with Hoechst 33342 (blue). (F) Flow cytometry analysis of luminal progenitors isolated from mammary glands of Stat3fl/fl;BLG-Cre− and Stat3fl/fl;BLG-Cre+ females four weeks after natural weaning. (G) In vitro colony forming analysis performed on CD24+ CD49fhi CD61+ luminal progenitor cells sorted from Stat3fl/fl;BLG-Cre− and Stat3fl/fl;BLG-Cre+ mammary glands. Points represent the value for each mouse and lines depict mean values for each group. p value was determined using Student’s t test, * p<0.05.

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-Cre− 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 Stat3 fl/fl,BLG-Cre_−_ and Stat3 fl/fl;BLG-Cre+ females four weeks after natural weaning. Number of outgrowths is shown per number of transplanted fat pads. CI: confidence interval.

Figure 3. Normal mammary gland development in Stat3 depleted glands.

(A) Whole mount staining of mammary glands of 5-week-old Stat3fl/fl;K14-Cre− and Stat3fl/fl;K14-Cre+ females. (B) Flow cytometry analysis of luminal and basal cells isolated from mammary glands of Stat3fl/fl;K14-Cre− and Stat3fl/fl;K14-Cre+ females four weeks after natural weaning. p value was determined using Student’s t test. ns: not significant.

Figure 4. Stat3 is required to maintain the multipotency of mammary stem cells and their proliferative potential.

(A) Whole mount staining of mammary outgrowths originating from CD24+ CD49fhi basal cells sorted from mammary glands of 5-week-old Stat3fl/fl,K14-Cre− and Stat3fl/fl;K14-Cre+ females. (B) Limiting dilution analysis to assess the repopulating frequency of the mammary stem cell-enriched population sorted from mammary glands of 5-week-old Stat3fl/fl,K14-Cre− and Stat3fl/fl;K14-Cre+ females. Number of outgrowths per number of transplanted fat pads and percentage of fat pad filled ± standard error of the mean are shown. CI: confidence interval. (C) H&E staining of mammary outgrowths originating from CD24+ CD49fhi basal cells sorted from mammary glands of 5-week-old Stat3fl/fl;K14-Cre− and Stat3fl/fl;K14-Cre+ females. (D, E) Immunohistochemistry staining for pStat5 (red, D), Ki67 (red, E) and E-cadherin (green) in mammary outgrowths originating from CD24+ CD49fhi cells from mammary glands of 5-week-old Stat3fl/fl,K14-Cre− and Stat3fl/fl;K14-Cre+ females. Nuclei were stained with Hoechst 33342 (blue).

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Stat3 is required to maintain the full differentiation potential of mammary stem cells and the proliferative potential of mammary luminal progenitors - PubMed (original) (raw)