Mammary stem cell repertoire: new insights in aging epithelial populations
Introduction
The action of mammary stem cells and their mitotic progeny is fundamental to normal mammary growth, differentiation and regeneration in successive cycles of pregnancy, lactation and involution. An important feature of the mammary gland is the regenerative capacity of its epithelium, which is demonstrated upon successive reproductive cycles. During pregnancy in the mouse, a massive increase (25- to 27-fold) in the number of mammary epithelial cells occurs within the mammary fat pad (Nicoll and Tucker, 1965, Kordon and Smith, 1998). Following lactation, this massive epithelial population is subsequently reduced to a number very close to that in the nulliparous fat pad through apoptotic cell death during involution. Mammary outgrowths in transplanted mammary fat pads also grow massively in impregnated hosts and involute at the end of lactation. A typical mammary implant contains between 5 and 7×103 epithelial cells (Smith and Medina, 1988), after ductal growth, the implanted gland contains roughly 2.5×106 epithelial cells an increase of ∼500-fold. During pregnancy the number of epithelial cells in a typical outgrowth increases an additional 25-fold resulting in a ∼10 000-fold increase over the implanted epithelial population (Kordon and Smith, 1998).
Limiting dilution transplantation studies have identified three distinct multipotent epithelial cells within the mouse mammary gland (Smith, 1996). These cells are characterized by their distinctive ability to produce secretory lobules, generate branching mammary ducts or recreate the entire functional (lactating) mammary epithelium upon transplantation into a breeding host. Each appears to have the capacity for self-renewal, but the lobule-limited and ductal-restricted progenitors appear to have a smaller reproductive capacity than the fully competent progenitor. Cap cells and terminal bud formation seem to be within the province of the ductal-restricted progenitor, whereas lobule development and expansion is absent. The opposite is true for the lobule-limited progenitor. Similar experiments with dispersed mammary cells in the rat have also demonstrated lobule-limited and ductal-limited epithelial progenitors, within the mammary population (Kamiya et al., 1998, Kamiya et al., 1999).
Our earlier studies indicate that clonally derived, multipotent stem cells, positioned throughout the mature fully developed mammary gland, have the capacity to produce sufficient differentiated progeny to recapitulate an entire functional gland (Kordon and Smith, 1998). We demonstrated that second generation outgrowths derived by transplanting random fragments from the original implant after involution produced mammary populations exhibiting the identical MMTV proviral insertions. These second generation populations were shown by dissociation and limiting dilution transplantation studies to contain all three multipotent mammary cell types described above. We therefore proposed that all three types arose from a single pluripotent precursor and that this precursor was capable of self-renewal and existed in all epithelial portions of the mouse mammary gland.
Normal mammary transplants to the epithelium-divested mammary fat pad in nulliparous hosts show growth senescence during serial propagation. It was also shown that growth senescent ductal implants in situ were able to respond to pregnancy and produce secretory lobules and milk protein indicating that growth senescence for ductal morphogenesis was distinct from that for secretory lobulogenesis (Daniel et al., 1971). From this observation, it seemed to us that both lobule-committed and ductal-committed progenitors might exist at any one time within an implant and possess different reproductive capacities. Alternatively, a primary antecedent responsible for the generation of both of these lineage-committed progenitors loses its capacity to produce one type independent of the other. Therefore, two alternative interpretations could be applied to our results indicating that individual mammary outgrowths may be clonally derived based upon the identity provided by proviral tagging. Originally, we postulated that the entire original outgrowth was generated from the progeny of one or a few lineally related stem cells, whose predecessor had acquired MMTV proviral insertions. During ductal growth and extension these cells were self-renewed and distributed throughout the ductal tree. Therefore upon subsequent transplantation the process was repeated and the same proviral tagging pattern was maintained in the subsequent generation. However, because of the existence of multipotent lineage-limited progenitors within the outgrowths, the pattern of proviral tagging observed in the original outgrowth might result from the sum of several different provirally tagged cellular clones that are maintained at a similar relative fraction throughout the gland. Further, these sub-populations might possess similar or identical growth potentials such that they were maintained in the second outgrowth generation. In an attempt to distinguish between these possibilities, we have serially transplanted apparent clonal mammary populations through several generations in breeding recipients (where both ductal and lobular development are supported) to growth senescence. All subsequent generations were evaluated regarding their proviral content. In their recent review (Weissman et al., 2001) of stem and progenitor cells, Weissman, Anderson and Gage wrote, ‘We believe that a claim of stem cell differentiation should include marking or isolating prospectively the cell from the donor, and demonstrating, at the clonal level, that this is a cell from the donor, and that such cells have the capacity to repopulate and regenerate stem cells, oligolineage progenitors and differentiated and functional progeny’. In the present study an individual mammary epithelial stem cell has not been prospectively isolated. Nevertheless, we have demonstrated that MMTV proviral insertions can mark a pluripotent mammary epithelial cell and have shown at the clonal level that all subsequent progeny arise from that antecedent including premalignant and malignant populations. Further, growth senescence is a direct result of the aging of local mammary stem cells.
Section snippets
Mice
CzechII mice infected with MMTV (Czech) (Callahan et al., 1982) were used as donors and hosts of the mammary epithelial transplants. The mice were held in a closed colony, maintained on a 12-h light/dark cycle under controlled temperature and humidity, and were given laboratory chow supplemented with birdseed and water ad libitum. Bedding was hardwood chip and was autoclaved prior to use. All animal care and treatment was conducted strictly according to the rules and procedures defined by the
Establishment of the clonal derivation of outgrowth, R12
The experimental design is depicted in Fig. 1. Typically, the implant is placed in the center of the cleared fat pad. The implant grows and expands by generating multiple growing termini that radiate from the implant site. Lobular development occurs behind these advancing end buds on the subtending ducts and is seen as lateral budding. Ductal growth ceases as the termini approach the margins of the fat pad whereas lobular development continues in the pregnant host until parturition. All
Discussion
Mammary epithelium shows ductal growth senescence upon serial transplantation in non-bred mice. Our study used breeding hosts to evaluate this aging process and we found that the population aged at a different rate with respect to producing secretory lobule development as compared to ductal morphogenesis. Further, the capacity to produce ductal growth generally resulted in an outgrowth, which filled or nearly filled the fat pad while secretory lobule development in a duct-senescent outgrowth
Acknowledgements
We would like to thank Drs Robert Callahan and Susan Rasmussen for helpful suggestions, scientific discussion and critical reading of the manuscript. Amanda Kalmanowicz and Katy Bernhard provided excellent animal care and husbandry over the extended duration of our transplantation analysis.
References (39)
- et al.
Three division-competent, structurally-distinct cell populations contribute to murine mammary epithelial renewal
Tissue Cell
(1997) - et al.
Influence of cell division on an aging process. Life span of mouse mammary epithelium during serial propagation in vivo
Exp. Cell Res.
(1971) - et al.
The influence of mammogenic hormones on serially transplanted mouse mammary gland
Exp. Gerontol.
(1971) - et al.
Expression of mouse mammary tumor virus envelope protein does not prevent superinfection in vivo or in vitro
Virology
(1999) - et al.
Ectopic TGF beta 1 expression in the secretory mammary epithelium induces early senescence of the epithelial stem cell population
Dev. Biol.
(1995) - et al.
Estimates of parenchymal, stromal, and lymph node deoxyribonucleic acid in mammary glands of C3H/Crgl-2 mice
Life Sci.
(1965) - et al.
Microsatellite alterations indicating monoclonality in a typical hyperplasias associated with breast cancer
Hum. Pathol.
(1997) Stem cell fate and patterning in mammalian epidermis
Curr. Opin. Genet. Dev.
(2001)- et al.
The influence of host and tissue age on life span and growth rate of serially transplanted mouse mammary gland
Exp. Gerontol.
(1971) - et al.
Reducing mammary cancer risk through premature stem cell senescence
Oncogene
(2001)
Impairment of mammary lobular development induced by the expression of TGFb1 under the control of WAP promoter does not suppress tumorigenesis in MMTV-infected transgenic mice
Int. J. Cancer
Novel class of mouse mammary tumor virus-related DNA sequences found in all species of Mus, including mice lacking the virus proviral genome
Proc. Natl. Acad. Sci. USA
MMTV-induced mammary tumorigenesis: gene discovery, progression to malignancy and cellular pathways
Oncogene
The in vivo life span of normal and preneoplastic mouse mammary glands: a serial transplantation study
Proc. Natl. Acad. Sci. USA
Loss of heterozygosity in normal tissue adjacent to breast carcinomas
Science
Mammary tumorigenesis in feral mice: identification of a new int locus in mouse mammary tumor virus (Czech II)-induced mammary tumors
J. Virol.
Quantitative studies of ductal versus alveolar differentiation from rat mammary clonogens
Proc. Soc. Exp. Biol. Med.
Kinetics of mammary clonogenic cells and rat mammary cancer induction by X-rays or fission neutrons
J. Radiat. Res. (Tokyo)
Cited by (36)
Long-label-retaining mammary epithelial cells are created early in ductal development and distributed throughout the branching ducts
2019, Mechanisms of DevelopmentCitation Excerpt :After a prolonged chase during which much of the branching duct morphogenesis was completed, 3HTdR-label retaining epithelial cells (LREC) were detected among the epithelium of the maturing glands. Labeling newly synthesized DNA in these glands with a different marker, 5-bromodeoxyuridine (5BrdU), resulted in the appearance of doubly labeled nuclei in a large percentage of the LREC (Smith, 2005; Smith and Boulanger, 2002). In contrast, label-retaining cells within the associated lymph nodes did not incorporate 5BrdU during the pulse, indicating that they were not traversing the cell cycle.
Lentiviral Transduction of Mammary Stem Cells for Analysis of Gene Function during Development and Cancer
2008, Cell Stem CellCitation Excerpt :We next performed Southern blot analysis on DNA from the serially transplanted HIV-Zsgreen outgrowths to determine the viral integration patterns during each generation of outgrowth. A recurrence of these patterns in successive outgrowth generations would confirm that MaSCs were transduced (Kordon and Smith, 1998; Smith and Boulanger, 2002). Southern blots from primary outgrowths (Figure 2Ga–2Gc) often showed many bands together with a broad background smear that was absent in the wild-type negative control (Figure 2G, WT), suggesting that these transplants were derived from several transduced MaSCs and progenitors.
Mammary stem cells come of age, prospectively
2006, Trends in Molecular MedicineRetinoids and steroids in bovine mammary gland immunobiology
2005, Livestock Production Science
- 1
These authors contributed equally in the development of the experimental data presented in this paper.