Developmental changes in the in vitro activated regenerative activity of primitive mammary epithelial cells - PubMed (original) (raw)
Developmental changes in the in vitro activated regenerative activity of primitive mammary epithelial cells
Maisam Makarem et al. PLoS Biol. 2013.
Abstract
Many normal adult tissues contain rare stem cells with extensive self-maintaining regenerative potential. During development, the stem cells of the hematopoietic and neural systems undergo intrinsically specified changes in their self-renewal potential. In the mouse, mammary stem cells with transplantable regenerative activity are first detectable a few days before birth. They share some phenotypic properties with their adult counterparts but are enriched in a subpopulation that displays a distinct gene expression profile. Here we show that fetal mammary epithelial cells have a greater direct and inducible growth potential than their adult counterparts. The latter feature is revealed in a novel culture system that enables large numbers of in vitro clonogenic progenitors as well as mammary stem cells with serially transplantable activity to be produced within 7 days from single fetal or adult input cells. We further show that these responses are highly dependent on novel factors produced by fibroblasts. These findings provide new avenues for elucidating mechanisms that regulate normal mammary epithelial stem cell properties at the single-cell level, how these change during development, and how their perturbation may contribute to transformation.
Conflict of interest statement
The authors have declared that no competing interests exist.
Figures
Figure 1. Representative frequencies of phenotypes, CFCs, and MRUs in E18.5 fetal and adult mammary tissue.
(A) Representative gating and final FACS profiles of viable subsets of single cells isolated from dissociated E18.5 mammary glands after exclusion of hematopoietic (CD45+ and Ter119+) and endothelial (CD31+) cells. Embedded images are negative controls. (B) Frequency of total cells, CFCs, and MRUs measured in the indicated subsets and in suspended but not further separated fetal mammary (EpCAM+) cells. (C) Representative gating and final FACS profiles of viable subsets of single cells isolated from dissociated 8–12-wk-old adult mammary glands after exclusion of hematopoietic (CD45+ and Ter119+), endothelial (CD31+), and stromal (BP-1+) cells. A fluorescence-minus-one control was used to set the gate used to identify CD61+ cells. (D) Frequency of total cells, CFCs, and MRUs measured in the indicated subsets and in unseparated suspensions of adult mammary epithelial (EpCAM+) cells.
Figure 2. Quantification of the CFC and MRU content of fetal compared with adult mammary cells.
(A) Distribution of fetal MRUs and CFCs according to their expression of EpCAM. CFC values are the mean ± SEM for ≥4 experiments. MRU and 95% CI values were determined by LDA. The arrow indicates that MRUs, if present in the EpCAM− fraction, were below the limit of detection (indicated by the line; for details, see Table S4). (B) Comparison of CFC and MRU content of cells from >15 and 2 adult no. 4 glands, respectively, and >50 fetal glands. CFC values are geometric means. Error bars for MRU and CFC values show 95% CI.
Figure 3. Production of MRUs and CFCs in 7-d Matrigel cultures of adult and fetal mammary cells.
(A) General experimental design. Mammary cells were added to 50 µl of solidified Matrigel (±2.5×104 irradiated 3T3 fibroblasts) in 200 µl of medium and incubated for 7-d. Each well was then examined for the presence of one or more visible structures and the contents then fixed and stained or dissociated into a suspension of viable single cells to perform FACS, CFC, or MRU assays for comparison with corresponding starting (input) values. (B) CFC outputs from 7-d Matrigel cultures of unseparated adult mammary cells as a function of the input cell number (expressed as the number of input EpCAM+ cells) and the addition of irradiated fibroblasts. (C) CFC outputs from 7-d cultures of unseparated fetal cells as a function of the input cell number (expressed as the number of input EpCAM+ cells). Comparison of the corresponding relationship for adult cells (dotted line redrawn from (B)) shows a ∼5-fold higher CFC output by the fetal cells. (D) Comparison of increased numbers of MRUs obtained from 7-d Matrigel cultures of fetal and adult mammary cells (values determined by LDA as described in Tables S1, S2, S3, S4, S5, S6 and expressed relative to 100 EpCAM+ cells).
Figure 4. The effect of 3T3 cell CM and selected factors on fetal and adult basal and luminal mammary cell production of CFCs.
CFC assays were performed on 7-d cultures of 30 EpCAM++ fetal cells (green), 60 EpCAM+CD49f+ adult basal cells (blue), 100 EpCAM++CD49flow/−CD61+ adult luminal cells (red), and 300 adult EpCAM+ cells (purple) using various additives to determine their effects on CFC outputs. (A) Comparison of the effect of 80% 3T3 cell CM, 160 ng/ml Wnt3a±400 ng/ml R-Spondin 1 (R-Spo), or 16 ng/ml bFGF relative to added 3T3 cells (set = 100%). The concentrations shown are the final concentrations in the 250 µl cultures. Results are pooled from 3–6 experiments. The difference in CFC output between CM and no added 3T3 cells was significant for cultures initiated with all types of cells (p<0.05, one-way ANOVA with Bonferroni's multiple comparison test). (B) Effect of Wnt pathway inhibitors: XAV939 (0.8 µM for adult, 4 µM for fetal) or mDKK1 (160 ng/ml) in cultures with irradiated 3T3 cells (set = 100%). Results are pooled from 3–9 experiments. Only the effect of added XAV939 was significant, and only for basal cells (_p_ = 0.04, one-way ANOVA with Bonferroni's multiple comparison test). (C) Effect of 40 ng/ml HGF and 16 ng/ml CSF-1 on adult EpCAM+ cells. The difference in CFC output between added HGF or CSF-1 and no added 3T3 cells is not significant (_p_>0.99, one-way ANOVA with Bonferroni's multiple comparison test).
Figure 5. Morphology and cellular composition of structures generated in 7-d Matrigel cultures of single fetal and adult mammary cells.
(A) Gross appearance of representative structures from each of the three types of input cells tested. The scale shown indicates 30 µm. (B) Representative photomicrographs of sectioned structures stained with antibodies against the markers shown. Scale, 50 µm. (C) Flow cytometric analysis of dissociated PI− cells obtained from pooled harvests of cultures initiated with the same types of cells.
Figure 6. Comparison of CFC and MRU outputs in 7-d Matrigel cultures initiated with single fetal or adult mammary cells.
(A) Correlation between the total number of cells retrieved from individually assessed 7-d cultures with the corresponding number of CFCs detected. (B) Distribution of clonal CFC outputs in 7-d cultures initiated with different types of input cells. The dotted line indicates the median values of positive clones: 1,000 for clones derived from basal adult cells (n = 44), 140 for clones from CD61+ adult luminal cells (n = 35), and 13,000 for clones from fetal cells (n = 55). (C) Comparison of MRU outputs determined by LDA in 7-d Matrigel cultures initiated with single adult and fetal mammary cells. The value for fetal cells is significantly higher than either of the adult values (p<0.01, Chi-square p value, pair-wise comparison of stem cell frequencies, ELDA, Table S7).
Figure 7. Regenerated glands produced in vivo from MRU generated in vitro from structures generated from single fetal, basal, or luminal cells.
Whole mounts (upper panels) and sections (middle panels) of mammary glands produced in fat pads injected with cells from cultures initiated with single fetal cells (left panel), or single adult basal cells (middle panel) or single adult luminal cells (right panels). Scale bars, 100 µm (whole mounts) and 50 µm (sections). Lower panels show flow cytometry profiles of regenerated mammary glands from cultures initiated with single fetal cells (left panel), or single adult basal (middle panel) or six CD61+ luminal cells (right panels).
Figure 8. Microarray analysis of differentially expressed genes in MRU-enriched fetal and adult basal subsets of mammary cells.
(A) Comparison of transcripts detected in Agilent arrays of extracts of E18.5 fetal (CD31−CD45−Ter119−EpCAM++CD49f+) and adult basal (CD31−CD45−Ter119−BP-1−EpCAM+CD49f+) cells. Expression values of both datasets are shown as log2-normalized values. Shown in pink are the genes (probes) identified as significantly differentially expressed (≥2-fold, _p_≤0.05). (B) Heat map of the top 1% of differentially expressed genes (probes) that were higher in the fetal dataset and the top 1% of differentially expressed genes (probes) that were higher in the adult basal dataset (shown as log2 normalized gene expression values from panel A).
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