A method for quantifying normal human mammary epithelial stem cells with in vivo regenerative ability (original) (raw)
Related papers
Nature Protocols, 2010
Mammary epithelial stem cells A number of studies in recent years have established that the mature cells in the mammary epithelium are continually generated by a multistep differentiation process from a pool of undifferentiated self-renewing mammary epithelial stem cells 1-4. The key to this progress has been the introduction of specific and quantitative functional assays that detect distinct subsets of cells within the hierarchy on the basis of the longevity and diversity of their particular regenerative abilities. The identification of a mouse mammary stem cell population was based on the development of a transplant assay that reveals the ability of a rare subpopulation of cells (termed 'mammary repopulating units' or MRUs) to individually regenerate an entire mammary tree in the epithelium-cleared mammary fat pad of an immune-compatible recipient mouse 1,2. In both mouse and human mammary tissue, a variety of lineage-restricted and bipotent populations of clonogenic mammary progenitor cells ('colony-forming cells' or CFCs) have also been recognized. The identification of these mammary CFCs is based on detecting their ability to form colonies of plastic-adherent daughter cells of particular lineages when cocultured at low density with irradiated fibroblasts in defined media containing epidermal growth factor 3,4. The finding that most mouse MRUs and CFCs can be prospectively isolated as distinct populations is the basis of the concept that mammary cell differentiation is organized as a highly regulated, multistep differentiation process. This concept, in turn, has set the stage for the development of breast cancer models in which tumorigenic 'cancer stem cells' are defined operationally by their ability to regenerate a tumor when transplanted into a suitable recipient 5 , although how these cancer stem cell populations relate developmentally to their normal counterparts remains a topic of intense interest. A key feature of all clonal assays is their restricted applicability to suspensions of viable single cells or to uniquely traceable single cells. A critical aspect of the assay design is therefore to optimize and define exogenous factors (e.g., growth factors, extracellular matrix, supportive stromal cells) that allow the cells of interest to demonstrate their maximal intrinsic regenerative potential. Xenotransplantation of human mammary epithelial stem cells Human mammary epithelial cells (HMECs) do not readily proliferate in the adipose environment of the mouse mammary fat pad 6. This has posed a significant challenge to the development of a transplantation assay protocol for human MRUs analogous to that used to assay mouse MRUs. One approach pioneered by Kuperwasser et al. has been to 'humanize' the fat pads of immunodeficient mice by colonizing with fibroblasts. This modification provides a supportive environment in which recognizable human mammary epithelial structures are regenerated when human mammary organoids 7,8 or dissociated human mammary cells 9 are subsequently transplanted. In this paper, we describe a different xenotransplant methodology 10,11 that we adapted from previous studies suggesting the ability of the subrenal capsule site to support the growth of implanted tissue fragments 12. These earlier studies include the important work of Cunha and colleagues 13 , who demonstrated the specific applicability of this site to support the propagation of intact fragments of mammary tissue. According to our protocol, dissociated suspensions of cells (or subsets thereof) isolated from normal human mammary tissue are combined with fibroblasts in collagen gels and these gels are then placed under the kidney capsule of highly immunedeficient hormone-supplemented mice. After 4 weeks, the gels are removed and evidence of a regenerated bilayered, normal-appearing human mammary gland structure can be identified (Fig. 1). Use of in vivo-regenerated mammary CFCs as an endpoint of mammary stem cell activity Histology can be used to detect evidence of regenerated mammary tissue in the implanted gels (Fig. 1a); however, discrimination of
Mammary Epithelial Stem Cells: Our Current Understanding
1999
It has recently been shown that the progeny froma single cell may comprise the epithelial population ofa fully developed lactating mammary outgrowth in mice.Serial transplantation of epithelial fragments from this clonally derived gland demonstratesthat the subsequently generated outgrowths are alsocomprised of progeny from the original antecedent.Similarly, genetic analysis of contiguous portions of individual human mammary ducts within the samebreast indicates their clonal derivation. Theseobservations support the concept that multipotenttissue-specific epithelial stem cells are present amongthe parenchymal cells of the mammary gland. Here,we present the developing evidence for the presence ofstem cells in virtually every renewing mammalian tissueas well as some classically considered to consist only of differentiated cells. Further, wereview the present morphologic and biologic evidence forstem cells and lineage-limited progenitor cells in humanand rodent mammary epithelium. Although a number of selective markers are known for variouslineage-limited hematopoietic cells and their progeny,our understanding of the biology of the precursor cellsfor mammary epithelium is just beginning. Our purpose here is to develop further interest in theclarification of these issues in the biology of themammary gland.
Growth and differentiation of progenitor/stem cells derived from the human mammary gland
Experimental Cell Research, 2004
Estrogen is necessary for the full development of the mammary gland and it is also involved in breast cancer development. We set out to identify and characterise progenitor/stem cells in the human mammary gland and to explore the role of estrogen in their proliferation and differentiation. Three candidate stem cell populations were isolated: double positive (DP) cells co-expressed the luminal and myoepithelial markers, EMA and CALLA, respectively, whereas double negative (DN) cells did not express these cell surface markers; side population (SP) cells were characterised by their differential ability to efflux the dye Hoechst 33342. The ABC transporter, breast cancer resistance protein (BCRP) was more highly expressed in SP cells than in non-SP cells and a specific BCRP inhibitor, Ko143, reduced SP formation, suggesting that BCRP confers the SP phenotype in mammary epithelial cells, as has been demonstrated in other tissues. Interestingly, SP cells were double negative for the EMA and CALLA antigens and therefore represent a separate and distinct population to DP cells. Single cell multiplex RT-PCR indicated that the SP and DN cells do not express detectable levels of ERa or ERh, suggesting that estrogen is not involved in their proliferation. DP cells expressed ERa but at a lower level than differentiated luminal cells. These findings invoke a potential strategy for the breast stem/progenitor cells to ignore the mitogenic effects of estrogen. All three cell populations generated mixed colonies containing both luminal and myoepithelial cells from a single cell and therefore represent candidate multipotent stem cells. However, DN cells predominately generated luminal colonies and exhibited a much higher cloning efficiency than differentiated luminal cells. Further characterisation of these candidate progenitor/stem cells should contribute to a better understanding of normal mammary gland development and breast tumorigenesis.
Use of Stem Cell Markers in Dissociated Mammary Populations
Methods in Molecular Biology, 2010
The regenerative potential of mammary epithelium facilitates assessment of the "stemness" of any epithelial subpopulation in transplantation assays. Thus, mammary tissue can be dissociated into single cells, stained for cell surface markers of interest and classified using fluorescence-activated cell sorting. The selected cells can then be transplanted into epithelium-devoided fat pads from recipient hosts. Recent publications have described markers that enrich for mammary repopulating potential. Here, we describe the materials and methods necessary to sort cells according to these markers. This approach can be used interchangeably with other cell surface markers with slight variation to the protocol.
PLoS ONE, 2010
Background: Mammary stem cells are maintained within specific microenvironments and recruited throughout lifetime to reconstitute de novo the mammary gland. Mammary stem cells have been isolated through the identification of specific cell surface markers and in vivo transplantation into cleared mammary fat pads. Accumulating evidence showed that during the reformation of mammary stem cell niches by dispersed epithelial cells in the context of the intact epithelium-free mammary stroma, non-mammary epithelial cells may be sequestered and reprogrammed to perform mammary epithelial cell functions and to adopt mammary epithelial characteristics during reconstruction of mammary epithelium in regenerating mammary tissue in vivo. Methodology/Principal Findings: To examine whether other types of progenitor cells are able to contribute to mammary branching morphogenesis, we examined the potential of murine embryonic stem (mES) cells, undergoing hematopoietic differentiation, to support mammary reconstitution in vivo. We observed that cells from day 14 embryoid bodies (EBs) under hematopoietic differentiation condition, but not supernatants derived from these cells, when transplanted into denuded mammary fat pads, were able to contribute to both the luminal and myoepithelial lineages in branching ductal structures resembling the ductal-alveolar architecture of the mammary tree. No teratomas were observed when these cells were transplanted in vivo. Conclusions/Significance: Our data provide evidence for the dominance of the tissue-specific mammary stem cell niche and its role in directing mES cells, undergoing hematopoietic differentiation, to reprogram into mammary epithelial cells and to promote mammary epithelial morphogenesis. These studies should also provide insights into regeneration of damaged mammary gland and the role of the mammary microenvironment in reprogramming cell fate.
Breast Cancer Research, 2011
Introduction: Utilizing single-cell cloning of the COMMA-D cell line engineered to express β-galactosidase (CDβ) cell line, which exhibits normal in vivo morphogenesis, distinct multipotent, ductal-limited, alveolar-limited and luminal-restricted progenitors, have been isolated and characterized. Methods: A single-cell suspension of CDβ cells was stained using Hoechst dye 33342, followed by analysis and sorting. Cells that effluxed the dye appeared on the left side of a FACS analysis panel and were referred to as side population (SP) cells. Cells that retained the dye appeared on the right side and were referred to as non-SP (NSP) cells. Cells from both SP and NSP regions were sorted and analyzed for outgrowth potential. Additionally, individual clones were derived from single cells sorted from each region.
Cancer research, 2007
The identification of mammary gland stem cells (MGSC) or progenitors is important for the study of normal breast development and tumorigenesis. Based on their immunophenotype, we have isolated a population of mouse mammary gland cells that are capable of forming ''mammospheres'' in vitro. Importantly, mammospheres are enriched for cells that regenerate an entire mammary gland on implantation into a mammary fat pad. We also undertook cytogenetic analyses of mammosphere-forming cells after prolonged culture, which provided preliminary insight into the genomic stability of these cells. Our identification of new cell surface markers for enriching mammosphere-initiating cells, including endoglin and prion protein, will facilitate the elucidation of the cell biology of MGSC. [Cancer Res 2007;67(17):8131-8]
Separation by cell size enriches for mammary stem cell repopulation activity
Stem cells translational medicine, 2013
Mammary gland reconstitution experiments, as well as lineage tracing experiments, have provided evidence for the existence of adult mammary stem cells (MaSCs). In addition, cell sorting techniques for specific cell surface markers (CD24(+)CD29(H)CD49f(H)Sca1(-)) have been used to prospectively isolate MaSC-enriched populations. Although these markers enrich for cell subpopulations that harbor MaSCs, they do not identify regenerative stem cells uniquely. Here, we report that MaSCs can be further defined by the property of cell size. Fluorescence-activated cell sorting was used to analyze sizing beads and further separate populations of cells with varying degrees of forward scatter (FSC). Cells with a low FSC that were approximately <10 μm in size lacked outgrowth potential and failed to reconstitute the mammary gland when transplanted into the cleared fat pads of syngeneic mice. In contrast, cells >10 μm in size with a higher FSC had increased outgrowth potential as compared wi...