A Quiescent Bcl11b High Stem Cell Population Is Required for Maintenance of the Mammary Gland

Abstract

Stem cells in many tissues sustain themselves by entering a quiescent state to avoid genomic insults and to prevent exhaustion caused by excessive proliferation. In the mammary gland, the identity and characteristics of quiescent epithelial stem cells are not clear. Here, we identify a quiescent mammary epithelial cell population expressing high levels of Bcl11b and located at the interface between luminal and basal cells. Bcl11b^high cells are enriched for cells that can regenerate mammary glands in secondary transplants. Loss of Bcl11b leads to a Cdkn2a-dependent exhaustion of ductal epithelium and loss of epithelial cell regenerative capacity. Gain- and loss-of-function studies show that Bcl11b induces cells to enter the G₀ phase of the cell cycle and become quiescent. Taken together, these results suggest that Bcl11b acts as a central intrinsic regulator of mammary epithelial stem cell quiescence and exhaustion and is necessary for long-term maintenance of the mammary gland.

Keywords: quiescence; stem cell exhaustion.

Fig 1. Bcl11b expression is restricted to CD49f^highCD24^medLineage⁻ cells and shows sporadic localization in the basal layer

(A) FACS plot of a mammary gland from an 8–12 week old adult mouse with surface markers CD24 and CD49f. Red circles show gates for Basal1(CD49f^highCD24^medLineage⁻), Basal2(CD49f^highCD24^lowLineage⁻), LUM1(CD49f^lowCD24^highLineage⁻) and LUM2(CD49f^lowCD24^lowLineage⁻) populations. (B,C) Single cell gene expression analysis of Bcl11b in Basal1, Basal2 and Luminal cells. Cell and gene clustering are superimposed with the expression panel in (B). Red color indicates high expression, green indicates low expression and gray indicates no expression. The Ct value of Bcl11b in the original gates is shown in (C). In this scale bar, blue color indicates low Ct value (high expression), while red color indicates high Ct value (low expression). (D) Real time PCR quantification of Bcl11b mRNA levels in Basal1, Basal2 and Luminal cells with ActB as an internal control. Data were presented as Mean±SEM. N=3 (E) Immunofluorescence of the mammary gland from an 8 week old mouse. Red: Krt14; Green: Bcl11b; Blue: DAPI. Scale bar, 20μm (F) Schematic diagram of Bcl11b tdTomato reporter mouse. (G) A representative FACS plot of a mammary gland from a Bcl11b^tdtomato/wt reporter mouse. The TdTomato expression level is shown for EpCAM+ mammary cells. (H) Tdtomato expression level is shown for CD49f^highCD24^medLineage⁻ population. (I) Real time PCR confirming Bcl11b^high population isolated from Bcl11b tdtomato reporter mice enriched for Bcl11b mRNA expression. N=3, p<0.01. (J) Mammary bifurcating mammary duct of a 12-week-old Bcl11b^tdtomato/wt mouse. Stacked images were shown on the left panel. Optical sections were shown on the right panel. Yellow arrowheads point to individual tdTomato positive cells. Scale bar, 100μm. See also Figure S1

Fig. 2. Deletion of Bcl11b causes mammary gland hypoplasia

(A) Whole mount image of Bcl11b^flox/flox and Krt14-cre Bcl11b^flox/flox inguinal mammary gland at week 9. (B) Quantification of mammary gland size from littermates (control and Bcl11b mutant) at adulthood (10–14 weeks). Data are plotted as Mean±S.D. (p<0.0001, n=16). (C) Image of collagenase digested mammary gland fragments from Krt14-cre mTmG Bcl11b^wt/wt and Krt14-cre mTmG Bcl11b^flox/flox mice showing GFP and tdTomato. (D) FACS analysis of Krt14-cre mTmG Bcl11b^wt/wt and Krt14-cre mTmG Bcl11b^flox/flox mice with CD49f and EpCAM. EpCAM positive mammary epithelia were gated and plotted using GFP. (E) Quantification of GFP positive cells of whole mammary epithelia, basal cells and luminal cells. Data were presented as mean±SD. (n=5) (F) Total MRU number quantified by limiting dilution transplantation of lineage negative mammary cells from control and Krt14-cre Bcl11b^flox/flox mice. Data were normalized to the control MRU number. n=3, p<0.01 (G) Images and pie chart of the secondary transplant of the mammary outgrowths from control and Krt14-cre Bcl11b^flox/flox mice. (H) Whole mount images of control (adeno-cre Bcl11b^wt/wt) and mutant (adeno-cre Bcl11b^flox/flox) transplants. (I) Pie chart of limiting dilution transplant of adenovirus-cre mediated Bcl11b knockout mammary epithelia. (J) ELDA plot of limiting dilution transplant of adenovirus-cre mediated Bcl11b knockout mammary epithelia. N=3, p<0.01. See also figure S2.

Fig. 3. Cells expressing high levels of Bcl11b are enriched for CD49f^highCD24^medLineage⁻ cells

(A) Colony formation assay for CD49f^highCD24^medLin⁻ expressing high versus low levels of Bcl11b. 1K cells/well were seeded for each condition and cultured for a week. Number of colonies were counted and plotted in the bar graph. The data are presented as mean ±S.E.M (N=3, p<0.01) (B) ELDA plot of Limiting dilution transplant of the Bcl11b^high and Bcl11b^low CD49f^highCD24^medLineage⁻ populations (C,D,E) Single cell transplants of Bcl11b^high CD49f^highCD24^medLineage⁻ cells. A schematic diagram of single cell sorting and a representative Bcl11b^high single cell image in a Terasaki plate is shown in (C,D). A whole mount image of a representative singe cell transplant outgrowth is shown in (E) with the table showing the numbers of total injections and outgrowths (E). (F) The mammary outgrowths from Bcl11b^highCD49f ^highCD24^medlin⁻ cells were dissected and dissociated to single cells. Single cell suspensions from two distinct mammary outgrowths were passaged to 6 recipient mice for secondary transplant. Pie chart shows the tree coverage of the mammary fat pad. See also Figure S3.

Fig. 4. Bcl11b^high cells in mammary gland are quiescent

(A) Flow plot showing Bcl11b^high cells are distinct from Procr+ cells within CD49f^highCD24^medLin⁻gate. DNA content analysis of Bcl11b^high and procr+ CD49f^highCD24^medLin⁻. Procr+ CD49f^highCD24^medLin⁻contained more proliferating cells (11%) than Bcl11b^high CD49f^highCD24^medLin⁻ (0.67%). (B) Single cell gene expression analysis of Procr+ and Procr- basal cells. (C) EdU incorporation assay for Bcl11b^high cells. Adult mice (8–12weeks) were injected via IP with 1.25mg/20g EdU for one day and analyzed for Edu incorporation (red), Bcl11b (green), Krt14 (blue) 24 hrs later. (D,E) Immunofluorescence co-staining of Bcl11b and Ki67 in mammary gland frozen sections from a pubescent mouse showing mammary ducts and terminal end bud. Green: Bcl11b. Red: Ki67. Blue: DAPI (F) Single cell gene expression analysis showing that the mRNA expressions of Bcl11b and Ki67 are mutually exclusive. (G) Left panel: CD49f^highCD24^medLineage⁻ cells were sorted and stained with Pyronin Y and Hoechst 33342. The G₀, G1 and SG₂M cells were sorted and subjected to real time PCR. Right panel: real time PCR analysis of Bcl11b expression level in various cell populations. N=3 (H) Images showing day1 and day11 culture of PKH26 stained CD49f^highCD24^medLineage⁻ cells, and sorting gates of PKH26 retaining and non-retaining cells. (I) Real time quantification of Bcl11b expression in the PKH26 retaining (PKH26^high) and PKH26 low (PKH26⁻) populations. Data are presented as mean ±S.E.M (N=4, p<0.05). See also Figure S4

Fig. 5. Bcl11b functionally regulates mammary epithelial cells

(A) Schematic diagram showing the strategy of the competition growth assay. Sorted CD49f^highCD24^medLineage⁻ cells were infected with a Bcl11b inducible construct. Infected cells (GFP+) and uninfected cells (GFP−) were mixed in a 1:1 ratio and cultured in the absence (Dox−) or presence (Dox+) of Doxycycline. Colonies were digested to single cells and analyzed by flow cytometry. (B) Competition growth assay with induced Bcl11b or Nr2f1 in the presence or absence of 100ng/ml Doxycycline for 3 days (C) Quantification of GFP percentage in Bcl11b or Nr2f1 competition growth assay. Data are presented as mean ±S.D. (N=3). (D) Images of the colonies arising from CD49f^highCD24^medLineage⁻ cells in a 3D culture from Krt14-cre Bcl11b^wt/wt and Krt14-cre Bcl11b^flox/flox mice. Images on day3 and day5 are shown. (E) The colony size of each individual colony was quantified and plotted over time. (F,G) Quantification of the sizes of colonies in a single cell culture assay from Krt14-cre Bcl11b^wt/wt and Krt14-cre Bcl11b^flox/flox mice. Single CD49f^highCD24^medLineage⁻ cells were sorted to 96-well plates (1cell/well) and cultured for colony formation. Data are presented as mean ±S.D. (p<0.05) (H) Induced deletion of Bcl11b with a Rosa26-creERT2 Bcl11b^flox/flox mTmG mouse. 8 week-old Rosa26-creERT2 Bcl11b^flox/flox mTmG mouse was administered with one dose 1mg/10g tamoxifen and left in husbandry for 3 weeks. Mammary glands were dissected and analyzed by flow cytometry. (I) Gene expression analysis of mouse basal cells at different stages of mouse pregnancy. Mammary glands from mice at different pregnancy stages p0, p6.5, p12.5, p18.5, lactation L1, were harvested and dissociated into single cells. Sorted basal cells were subjected to real time PCR analysis. (J) Hormone regulation of Bcl11b expression in mammary gland. Adult mice were ovariectomized and treated with vehicle (corn oil) or Estrogen (10ug)+Progesterone (1mg). Bcl11b expression was quantified by real time PCR, compared with pregnant mammary gland at stage p12.5. N=3 See also Figure S5,6.

Fig. 6. Bcl11b regulation of CD49f^highCD24^medLineage⁻ proliferation and engraftment via Cdkn1a(p21) and Cdkn2a(p16)

(A) Schematic Diagram showing the process of real time PCR analysis of Bcl11b induced and uninduced populations in colonies formed by CD49f^highCD24^medLin⁻. (B) mRNA expression levels of selected CDKIs (p21, p16, p19, p27, p57) after induced Bcl11b expression in CD49f^highCD24^medLineage⁻ cells in 3D culture. GFP+ cells from Dox-(0ng/ml) and Dox+(50ng/ml) samples were sorted for real time gene expression analysis. (C) Competition growth assay with Bcl11b induction in p21 knockout mice. Note: p21 knockout partially rescues Bcl11b-mediated repression of proliferation. N=3 (D) Gene expression plot of Cdkn2a(p16) versus Bcl11b from the currently available 10,000 mouse microarrays. Each dot represents one microarray dataset. (E) Gene expression plot of p53 versus Bcl11b from the currently available 10,000 mouse microarrays. Each dot represents one microarray dataset. (F) Schematic diagram showing the design of the p16 shRNA and p53 shRNA rescue assays. Basal1 cells from Bcl11b^flox/flox mice were sorted and transduced with p16 or p53 shRNA lentivirus and cultured for a week on 3D matrigel. Mammary colonies were then dissociated and transduced with adeno-cre-RFP and cultured overnight. The GFP and RFP double positive cells were then sorted and transplanted into NSG recipient mice. (G) Limiting dilution transplants of adeno-cre mediated Bcl11b knockout basal cells infected with p16 shRNA or p53 shRNA vectors. In contrast to p53 shRNA, which did not show significant difference in repopulation frequency, p16 shRNA did rescue the engraftment by ~3 fold. (N=3, P_pSIH vs p16 shRNA<0.05, P_{pSIH vs p53shRNA}=0.63).