Prospective isolation and characterization of committed and multipotent progenitors from immortalized mouse mammary epithelial cells with morphogenic potential
© Kittrell et al.; licensee BioMed Central Ltd. 2011
Received: 14 September 2010
Accepted: 5 April 2011
Published: 5 April 2011
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.
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.
There was no difference in the outgrowth potential of the SP vs. NSP cells when 5,000 cells per fat pad were transplanted. However, individual clones derived from single cells sorted from either SP or NSP regions had varying growth potential. A total of nine clones were identified, four of which possessed in vivo mammary outgrowth potential and five of which lacked in vivo outgrowth potential. Two of the clones formed mammary lobuloalveolar structures that contained both ducts and alveoli and were termed multipotent. Two of the clones generated either ductal-only or alveolar-only structures and were referred to as ductal-limited or alveolar-limited progenitor clones, respectively. The ability to expand the clones in vitro allowed for the characterization of their unique molecular phenotypes. Among the mammary-specific markers tested, high cytokeratin 5 (CK5) expression was the only marker that correlated with the clones' outgrowth potential. Among the clones that did not show any in vivo outgrowth potential when transplanted alone, one clone showed in vivo growth and incorporated into the mammary lumen when mixed with normal mammary epithelial cells. This clone also showed the highest in vitro expression of CK8 and Elf5and may represent a luminal-restricted progenitor clone. In addition, six "biclones," each made from an SP cell plus an NSP cell, were analyzed. Of these six, three exhibited lobuloalveolar growth.
Distinct immortalized mammary progenitors have been isolated and characterized. Importantly, the results of this study provide further evidence for the existence of distinct ductal and alveolar mammary progenitors.
A mammary gland epithelial hierarchy is beginning to be defined. In 1996, Smith and colleagues  were the first to demonstrate, on the basis of limiting dilution transplantation studies, that the mouse mammary gland contained three distinct progenitors: lobular, ductal and lobuloalveolar. Later, Wagner and colleagues  discovered a parity-identified mammary epithelial subpopulation that was defined as a lobular-restricted progenitor cell. This cell type was also found in the luminal cell compartment of ducts, but was not a ductal progenitor cell. Although previous studies demonstrated the presence of these progenitors, their phenotypic characteristics were not known until now. Recently, flow cytometry (FACS) cell sorting followed by transplantation has allowed the phenotypic and functional characterization of progenitor and differentiated cells in mouse mammary glands and human breast cells. In the mouse mammary gland, cell surface markers have been used to define stem cells as lineage-negative (Lin-) CD24+/CD29high  and Lin-CD24+/CD49fhigh , luminal progenitors as Lin-CD29lowCD24+CD61+ , differentiated estrogen receptor-positive (ER+) luminal cells as CD24+/CD133+  and myoepithelial progenitors as CD29low/CD24low . In human breast cells, epithelial cell adhesion molecule-low (EPCAMlow/-)/CD49fhigh and EPCAM+/CD49f+/CD10+/Thy-1+ represent bipotent progenitors that generate both luminal and myoepithelial cells [7, 8], and EPCAM+/CD49f+/AC133+/MUC-1+ represent luminal progenitor cells . The differentiated luminal cells in human breast cells are characterized by EPCAM+CD49f-CD133+/MUC-1+ expression, while the differentiated myoepithelial cells are characterized by EPCAM+/CD49f-/CD10+/Thy-1+ expression . However, the distinct progenitors have not been prospectively isolated, expanded in vivo or in vitro or fully characterized. Using single-cell cloning, distinct mammary progenitor populations were isolated. The CD cell line was originally derived from midpregnant BALB/c mouse mammary glands . This cell line is unique in that transplantation of cells into the epithelium-free fat pads of syngeneic female mice generates mammary ductal and alveolar structures. The CD cell line harbors two distinct p53 mutations: (1) a G-to-C transversion resulting in substitution of tryptophan for cysteine at codon 138 and (2) a deletion of the first 21 nucleotides of exon 5 resulting in deletion of codons 123 to 129 . The experiments in this study were performed using COMMA-D cell line engineered to express β-galactosidase (CDβ) cells. CDβ cells were derived from the parental CD cells by transduction with Zeg+ retrovirus containing a bifunctional LacZ/Neomycin (β-geo) generated in Dr Soriano's laboratory [12, 13]. Detailed sequence analysis of the p53 gene in eight different clonal derivatives of the CDβ cell line showed that both mutant alleles were present in each clone, demonstrating that the CD cell line is indeed clonal with respect to the p53 gene and that each cell expresses two distinctive mutant alleles of p53 . As a result of these p53 mutations, the mammary outgrowths progress to mammary tumors after many months in vivo. Here we show that single-cell cloning of CDβ cells resulted in isolated ductal-limited, alveolar-limited and lobuloalveolar mammary progenitors. Molecular characterization of these clones using FACS- and immunofluorescence (IF)-defined markers specific for mammary progenitor activity. In summary, the progenitor clones derived from the CDβ cell line provide valuable tools for molecular characterization of multipotent and unipotent or committed mammary progenitors. Furthermore, the malignant potential of the CDβ clones will allow the study of the role of distinct progenitors in generating heterogeneous mammary tumors.
Materials and methods
CDβ cells grown in Dulbecco's modified Eagle's medium/Ham's F-12 nutrient mixture containing 2% adult bovine serum albumin, 5 ng/ml epidermal growth factor, 5 μg/ml insulin and 100 U/ml penicillin/streptomycin were trypsinized, and a single-cell suspension at 1 million cells/ml was prepared. The process for mammary cell staining using Hoechst 33342 dye (B2261, St. Louis, MO, USA) has been published previously . The Hoechst dye was excited at 350 nm and was fluorescence measured at two wavelength emissions of 450/20 band pass filter (Hoechst blue) and 675 EFLP optical filter (Hoechst red). Analysis and sorting were performed on a triple laser MoFlo (Cytomation, Fort Collins, CO, USA). After analysis, single cells from each side population (SP) and non-side population (NSP) region were sorted into individual wells of 96-well plates. For the biclones, one cell from the SP region and one cell from the NSP region were sorted into the same well.
Fat pad transplantation and pituitary isografts
Mammary epithelial clearance, transplantation procedures and pituitary isograft procedures have been described previously [15, 16]. These procedures were approved by the Institutional Review Board at Baylor College of Medicine, Houston, TX, USA. One hundred thousand cells from each clone were injected into the cleared fat pads of syngeneic (BALB/c) female mice. Eight weeks after cell transplantation fat pads were excised and prepared for the various staining procedures described in this section.
Whole mount, hematoxylin and eosin and X-gal staining of mammary outgrowths
Mammary glands were removed and fixed in 10% formalin for 24 hours. Fixative was removed, and tissue was placed in acetone for two incubations of 24 hours each. Glands were pretreated for 1 hour each in 100% and 95% alcohol, followed by staining overnight in hematoxylin. The next morning glands were immersed in running tap water for 1 hour, followed by dehydration in graded alcohol for 1 hour each at 70%, 95% and 100% (three times), as well as in xylene (three times). The tissues were kept in methylsalicylate for long-term storage. For X-gal staining, glands were fixed in 4% paraformaldehyde, followed by three washes using a buffer containing 0.2% NP-40, 0.01% NaDOC and 2 mM MgCl2 in 1× phosphate-buffered saline (PBS) and incubated overnight at 37°C in X-gal solution containing 1 mg/ml X-gal. Tissues were then dehydrated as described above, embedded in paraffin and sectioned.
Hematoxylin and eosin staining
Excised mammary outgrowths were fixed in 4% paraformaldehyde and then embedded in paraffin according to established protocols. Slides containing 5-μm sections were deparaffinized in xylene (8400 laboratory grade, Anapath brand; StatLab, Lewisville, TX, USA) and hydrated through graded ethanol series (100%, 95%, 80% and 70%), followed by hematoxylin staining (s212A Harris hematoxylin with glacial acetic acid; Poly Scientific (Bayshore, NY, USA) for 30 seconds and destaining in acid ethanol (1 ml of concentrated HCl + 400 ml of 70% ethanol), followed by eosin staining (176 Eosin Phloxine stain; Poly Scientific) for 45 seconds and dehydration in graded ethanol (95% and 100%) and xylene three times for 15 minutes each, followed by placement of a coverslip onto the slide by using xylene-based Permount (SP15-100 histological mounting medium, Fisher Scientific, Pittsburgh, PA, USA).
Immunofluorescence and antibodies
IF was performed following tissue deparaffinization by clearance in xylene and hydration through a graded ethanol series as described above. Microwave antigen retrieval (20 minutes) in 10 mM sodium citrate was performed for all the antibodies used for IF. A 5% solution of bovine serum albumin (BSA) in PBS + 0.5% Tween 20 was used as blocking buffer. For staining of cells grown in two dimensions (2D), cells were grown on coverslip slides and fixed in 2% paraformaldehyde for 15 minutes at room temperature without antigen retrieval. Sections were incubated with the following primary antibodies overnight at 4°C: ERα rabbit polyclonal antibody (SC-542, MC-20; Santa Cruz Biotechnology, Santa Cruz, CA, USA), smooth muscle actin (SMA) antibody (A14, A2547; Sigma, St. Louis, MO, USA), cytokeratin 5 (CK5) rabbit polyclonal antibody (PRB-160P; Covance, Richmond, CA, USA), CK6 rabbit polyclonal antibody (PRB-169P; Covance), CK8 rat monoclonal antibody (TROMA-1; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA), Na+, K+, 2Cl- type I cotransporter (NKCC1) rabbit polyclonal antibody (gift of Jim Turner, National Institutes of Health, Bethesda, MD, USA) and p63 mouse monoclonal antibody (MS-1081-P, Thermo Fisher Scientific, Fermont, CA, USA). Nuclei were counterstained with 4',6-diamidino-2-phenylindole (Dapi) (Vector Laboratories, Burlingame, CA, USA) and TO-PRO-3 iodide (T3605, Invitrogen, Carlsbad, CA, USA). Secondary antibodies used were anti-rabbit Alexa Fluor 563 antibody, anti-mouse Alexa Fluor 594 antibody and anti-rat Alexa Fluor 488 antibody (Invitrogen, Carlsbad, CA, USA). All primary antibodies were used at a 1:200 concentration, and secondary antibodies were used at a 1:500 concentration. Confocal microscopy was performed using a laser-scanning confocal microscope (model 510; Carl Zeiss MicroImaging, Inc., Thornwood, NY, USA). The acquisition software used was LSM Image Browser (Carl Zeiss MicroImaging, Inc.). Phase-contrast images were captured using an inverted microscope (CK40-SLP; Olympus, Southend-on-Sea, Essex SS2 5QH, UK). The acquisition software used was PhotoShop version 5.0 (Adobe, San Jose, CA, USA).
FACS and reagents
Primary antibodies used were anti-mouse CD29 fluorescein isothiocyanate (FITC)-conjugated antibody (102206; Biolegend, San Diego, CA, USA), anti-mouse CD24 R-phycoerythrin (PE)-conjugated antibody (553262; BD Biosciences, San Diego, CA, USA), anti-mouse CD24 FITC-conjugated (553261; BD Biosciences), anti-mouse CD49f FITC-conjugated antibody (557510; BD Biosciences), biotin-conjugated hamster anti-mouse CD61 antibody (553345; BD Biosciences), anti-stem cell antigen 1 (Sca-1)-PE antibody (553336; BD Biosciences) and anti-mouse prominin 1 antibody (AC133, 12-1331; eBioscience (San Diego, CA, USA). Biotin-conjugated CD61 was labeled using streptavidin-APC (554067; BD Biosciences). Isotype control staining was performed using PE-conjugated anti-rat immunoglobulin G2a (IgG2a) antibody (558067; BD Biosciences) and FITC-conjugated mouse anti-rat IgG1 antibody (553892; BD Biosciences). JSE3, 33A10, 50B8 and 44G3 antibodies were supernatants used at 1:1 concentration for 45 minutes at room temperature. All other antibody cells were stained at a final concentration of 1:200 for 30 minutes on ice, followed by washes in Hanks' balanced salt solution (Invitrogen, Carlsbad, CA, USA) containing 2% fetal bovine serum. FACS analysis and data acquisition were performed using the BD LSR II flow cytometer and BD FACSDiva-based software (BD Biosciences).
Intracellular staining and FACS analysis
Cells were harvested by trypsinization with 0.05% trypsin-ethylenediaminetetraacetic acid (Invitrogen). Cells were fixed for 15 minutes with Click-iT fixative (Invitrogen), permeabilized for 30 minutes with saponin-based permeabilization buffer (Invitrogen) and stained with primary CK5 antibody (1:50, PRB-160P; Covance) or with the rabbit IgG immunoglobulin isotype control antibody (BD Biosciences). Subsequently, samples were counterstained with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (1:100; Invitrogen) and resuspended in 300 μl of 1% BSA-PBS prior to analysis with the LSR II flow cytometer. Data analysis was performed using FlowJo software (Tree Star, Inc., Ashland, OR, USA).
RNA isolation and quantitative real-time polymerase chain reaction
Total RNA was isolated and reverse-transcribed (100 ng) using the TaqMan Gene Expression Cells-to-CT Kit (Ambion, Austin, TX, USA) following the manufacturer's instructions. The mRNA transcript levels of the housekeeping gene 18S and the target gene Elf5 were evaluated using commercially available TaqMan primers and probes (Hs03928990_g1, 18S; Mm00468732_m1 Elf5 (Applied Biosystems, Carlsbad, CA, USA)). The resulting cDNA was diluted 1:15 and subjected to quantitative real-time polymerase chain reaction (qRT-PCR) analysis in a 20-μl final reaction volume. Each sample was run in triplicate for both the target and the normalizer, and the average cycle threshold (Ct) was used in subsequent calculations. Each sample was also run as a minus reverse transcriptase control to confirm lack of DNA contamination. Gene expression was detected using the ABI 7900 fast real-time PCR system (Applied Biosystems). The 2-ΔΔCt method was used to calculate relative fold change values between samples, with one control sample set to 1 and all other samples compared to it.
Single-cell cloning of CDβ cells identified multipotent and committed progenitors
Transplantation results of the CDβ-derived clonesa
Number of fat pads filled, n(%)
Distinct progenitors generate outgrowths with appropriate luminal or basal orientation
As shown in Figures 3C, F, I and 3L, nuclear ER expression was undetectable in the CDβ-derived outgrowths, although it was readily detectable in the normal BALB/c mammary glands (Figure 3L). This was surprising, since many studies have reported the importance of ER expression for normal mammary development [18, 19]. However, studies using mouse models with complete germline deletion of ER (ER-/-) have shown that ER was not required for prepubertal mammary development, since at 3 weeks of age there was no difference between the mutant and wild-type littermates in the formation of a rudimentary mammary ductal tree . Nevertheless, there was a significant difference in ductal elongation postpuberty in female mice lacking ER . Furthermore, Medina and colleagues  characterized p53-null preneoplastic mammary outgrowth lines which lacked ER expression but retained normal ductal alveolar outgrowth potential. The outgrowths were immortal and progressed through IDH and carcinoma over time. Therefore, ER may not be required for mammary development in the CDβ-derived outgrowths, since they also lack wild-type p53 gene expression. The outgrowths were also examined for the expression of progesterone receptor (PR). PR expression was solely extranuclear in the outgrowths generated from all ductal, alveolar and multipotent progenitor clones (data not shown). Interestingly, extranuclear PR expression has been implicated in the activation of Src and mitogen-activated protein kinase (MAPK) that lead to cancer cell proliferation . These interactions may also mediate hyperplastic growth of CDβ-derived outgrowths and their progression to tumorigenesis.
High CK5 expression correlates with outgrowth potential
CDβ progenitor clones were examined for the expression of specific CKs and surface markers known to be expressed by mammary stem, progenitor and differentiated cells grown in 2D. These included markers of mouse mammary repopulating cells (CD24+/CD29high and CD24+/CD49fhigh), luminal ER-positive (CD24+/AC133+), Sca-1 (hematopoietic stem progenitor cell marker) and luminal progenitors (CD29low/CD24+CD61+) [3–6, 22]. Other surface markers analyzed included JSE3, 33A10, 50B8 and 44G3 (monoclonal antibodies developed in the laboratory of Dr Around Sonnenberg ) and aldehyde dehydrogenase (Aldefluor Kit, Stem Cell Technologies, Vancouver, BC, Canada). Additionally, the expression of CK5, CK8, CK6, SMA, NKCC1, total p63, TA p63 and ΔN p63 were examined by IF staining on cells grown in 2D culture, and additional staining was performed to analyze P63, TA p63, ΔN p63 and CK5 expression by FACS. P63 is a member of the p53 family. It is critical in the development of stratified epithelial tissues such as epidermis, breast and prostate. P63 is expressed in at least six isoforms that can be classified into two groups: those with a transactivating domain (TAp63) and those that lack this domain (ΔNp63) .
No correlation could be found between the expression of CK8, NKCC1, CK6, SMA, total and isoform-specific (TA and ΔN) p63 and Aldefluor and progenitor activity (data not shown). Our data show that Sca-1 and p63 expression levels change in vitro in cells grown in 2D, and therefore their expression could not be correlated with the clones' outgrowth potential (data not shown). Therefore, on the basis of our data, Sca-1 expression may not be correlated with mammary progenitor potential. The expression of individual p63 isoforms, TA and ΔN, by IF and FACS (ΔN and TA), also was not associated with in vivo progenitor potential (data not shown).
Restricted mammary luminal progenitor cells may exist in the mouse mammary gland
A role for stem cells or distinct progenitor cells as the cells of origin in many types of cancer has been proposed [29–32]. These studies have led to the hypothesis that in many types of cancers, including breast cancer, distinct progenitors and their abrogated self-renewal pathways may ultimately underlie the mechanisms of heterogeneity observed among subtypes of human breast cancer . The identification and further characterization of distinct mammary progenitors are necessary prerequisites for testing this hypothesis. Single-cell cloning of an immortalized mammary cell line, CDβ, was utilized to isolate mammary-specific multipotent, ductal and alveolar progenitor clones. Molecular phenotyping showed that mammary progenitors express significantly higher levels of CK5.
Sca-1 expression was not associated with outgrowth potential of the clones. The unique expression of Sca-1 in stem and progenitor subpopulations has been proposed for many cell types. Sca-1 (Ly-6A), a member of Ly6 gene family, is a glycosylphosphatidylinositol-anchored cell surface protein. Sca-1, when combined with other markers, c-kit and the hematopoietic lineage, identifies hematopoietic stem cells in the bone marrow . However, its role as a stem or progenitor cell marker in the mammary gland has been controversial .
CDβ clones that possess outgrowth potential contain significantly higher levels of CK5 expression. The expression of CK5 in mammary stem cells has been proposed previously [25–27]. In human breast tissue, there is a small population of CK5-expressing cells (5%) that lack the expression of other markers of glandular, CK8, CK18, CK19 or myoepithelial cells (SMA) . Additionally, the stem cell subpopulation in normal mouse mammary gland (Lin-CD24+CD29+) is enriched in the expression of CK5 and displays a basal phenotype [27, 36]. Dontu and colleagues  showed that mammospheres were enriched in undifferentiated cells and expressed the basal markers CK5 and CK14. These data demonstrate that high-level CK5-expressing cells may represent an enriched population of stem and progenitor cells. It will be interesting to explore whether CK5 serves only as a stem cell marker or perhaps is coregulated with a critical stem cell gene.
In light of the important role of estrogen signaling during normal mammary development, it was surprising that the CDβ-derived outgrowths lacked ER expression. This may be because the CDβ-derived outgrowths express mutated p53. It has been demonstrated that some of the p53-null preneoplastic mammary outgrowths lack ER expression, although they do develop normally . Therefore, the requirement for ER expression may apply only to wild-type p53-expressing mammary glands. Furthermore, ER-/- mammary glands develop normally until puberty. However, there is stunted ductal outgrowth after puberty. Therefore, the outgrowths generated by the CDβ-derived clones may develop without the expression of ER because the CDβ cells lack a functional p53 gene and/or the outgrowths grow in response to prepubertal growth hormones such as the parathyroid hormone signaling pathway . The progesterone signaling pathway has also been shown to play an important role in mammary ductal branching morphogenesis and alveolar development, as well as in mammary tumorigenesis [18, 21]. Interestingly, the expression of PR was extranuclear in the outgrowths derived from the progenitor clones. It has been demonstrated that PR may bind and activate Src-1 and MAPK signaling pathway and thus promote the proliferation of breast cancer cells. These extranuclear effects of PR may mediate mammary hyperplasia and tumor progression in the outgrowths derived from the CDβ cells.
The CDβ cell line is unique in that transplantation of cells into the epithelium-free fat pads of syngeneic female mice generates mammary outgrowths that eventually progress to tumors over time in vivo. Mammary tumors eventually form because the CDβ cell line harbors two distinct p53 mutations. Thus, the model provides a unique opportunity to study the cellular and molecular mechanisms underlying mammary tumor progression beginning at the normal stages.
The utilization of clones with distinct multipotent, ductal, alveolar and restricted luminal progenitor activity will provide valuable tools for studying the unique biology of these progenitor populations. The identification and molecular characterization of self-renewal pathways in stem and distinct progenitor cell subpopulations will not only reveal normal mechanisms for development but also help to elucidate the molecular pathways that are misregulated during tumorigenesis, ultimately leading to tumor heterogeneity.
COMMA-D cell line engineered to express β-galactosidase
hematoxylin and eosin
sodium potassium chloride cotransporter
stem cell antigen 1
smooth muscle actin
This work was supported by the National Cancer Institute grants F32 CA101560-03 (to FB), 5R00CA127462-05 (to FB), U01 CA84243 (to JMR and DM), and the Baylor College of Medicine Cytometry and Cell Sorting core National Center for Research Resources grant S10RR024574 from the National Institutes of Health. In addition, the authors thank Mike Cubbage, Chris Threeton, Tatiana Goltsova, Joel M Sederstrom and Candice Sellers for excellent flow cytometry assistance; Jeannie L Zhong for assistance with confocal microscopy; and David Edwards, Shirley Small and Maria Gonzalez-Rimbau for their superb technical support. The TROMA-1 (rat monoclonal anti-mouse CK8 antibody) developed by Philippe Brulet and Rolf Kemler was obtained from the Developmental Studies Hybridoma Bank, which was developed under the auspices of the National Institute of Child Health and Human Development and is maintained by the Department of Biology, University of Iowa, Iowa City, IA 52242.
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