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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Cell Stem Cell. Author manuscript; available in PMC Jan 10, 2009.
Published in final edited form as:
PMCID: PMC2276651
NIHMSID: NIHMS39424

Lentiviral Transduction of Mammary Stem Cells for Analysis of Gene Function during Development and Cancer

SUMMARY

The mouse mammary gland is the only epithelial organ capable of complete regeneration upon orthotopic transplantation, making it ideally suited for in vivo gene function studies through viral mediated gene delivery. A hurdle that has challenged the widespread adoption of this technique has been the inability to transduce mammary stem cells effectively. We have overcome this limitation by infecting total primary mammary epithelial cells in suspension with high titer lentiviruses. Transduced cells gave rise to all major cell types of the mammary gland, and were capable of clonal outgrowth and functional differentiation in serial transplants. To demonstrate that this method is a valuable alternative to developing transgenic animals, we used lentiviral-mediated Wnt-1 overexpression to replicate MMTV-Wnt-1 mammary phenotypes and used a dominant-negative Xenopus Suppressor of Hairless to reveal a requirement for Notch signaling during ductal morphogenesis. Importantly, this method is also applicable to transduction of cells from other tissues.

Keywords: transduction, mammary stem cells, lentivirus, transplantation, reconstituted mammary gland, clonal outgrowth, functional differentiation, Wnt-1, inhibition of Notch signaling

INTRODUCTION

The mammary gland is a unique organ in that most of its development occurs postnatally (Wiseman and Werb, 2002). In mice, the mammary gland develops from a rudimentary branched epithelial structure to a highly ramified ductal network by ten weeks post-parturition (Sternlicht et al., 2006). This process is driven by specialized structures called terminal end buds (TEBs), which are located at the distal tip of developing ducts and mediate invasion into the surrounding fat pad (Hinck and Silberstein, 2005). The subtending ducts form a continuous epithelium consisting of an outer basal layer of contractile myoepithelial cells and an inner layer of luminal cells. During pregnancy, systemic hormones induce the development of lobulo-alveoli from ducts that functionally differentiate at parturition to produce milk. The lobulo-alveoli regress at the end of lactation and the mammary gland returns to a morphological state similar to that found in virgin mice (Richert et al., 2000). This process of functional differentiation and regression occurs during each round of pregnancy and is termed the “mammary cycle” (Clarkson, 2002).

Nearly half-a-century of research has established the existence of a rare, multipotent stem cell population that regulates postnatal growth and functional differentiation of the mammary gland (Daniel et al., 1968; Deome et al., 1959; Smith, 1996). Landmark studies demonstrated that ductal fragments from any region of the mammary gland have a capacity to regenerate a functional gland upon orthotopic transplantation that is independent of the age of donor mice (Deome et al., 1959; Young et al., 1971). More recently, two groups have isolated mammary stem cell (MaSC)-enriched mammary epithelial cell (MEC) populations by using antibodies against the cell surface proteins CD24 (heat stable antigen) in combination with either CD29 (β1-integrin) or CD49f (α6-integrin) (Shackleton et al., 2006; Stingl et al., 2006). While these MEC populations have significantly increased outgrowth capacity, the MaSCs still represent only a small proportion of the enriched population. Although mechanisms of MaSC differentiation are unknown, the progeny of these rare adult stem cells contribute to a hierarchy of progenitors that are committed to either ductal or lobulo-alveolar cell fates (Kordon and Smith, 1998; Smith, 1996).

Mammary gland research would benefit greatly from a rapid and convenient assay for testing gene function in vivo. Genetically engineered mice are costly and time consuming to develop. In addition, the hormonally sensitive promoters used to target genes to the mammary epithelium are most active during pregnancy and lactation, and drive expression in embryonic mammary glands as well as several other tissues (Wagner et al., 1997). Viral-mediated gene expression does not require tissue-specific promoters, but has thus far only been successful with genes that provide a selective advantage during outgrowth (Du et al., 2006; Edwards et al., 1992; Welm et al., 2005). Here, we describe the development of a simple, yet efficient method to transduce MaSCs ex vivo with high-titer lentiviral vectors that facilitates functional genetic studies on mammary development and tumorigenesis.

RESULTS

Efficient Transduction of Primary MECs in Suspension by Lentiviruses

We first tested a previously described monolayer viral infection and transplantation method (Welm et al., 2005) to express genes in mammary epithelium in vivo. We transplanted transduced primary MECs into epithelium-free (“cleared”) mammary fat pads (Daniel et al., 1968; Deome et al., 1959) of 3-week-old syngeneic mice and scored outgrowths for fluorescence after 8 weeks. When we used MSCV-Myc, a virus that co-expresses the oncogene c-myc with enhanced green fluorescent protein (EGFP) (Cormack et al., 1996), we observed outgrowths with almost the entire ductal network positive for EGFP fluorescence in most transplants, as reported previously (Welm et al., 2005) (Figures 1A and 1B, and Table 1). However, two retroviruses, based on either mouse stem cell virus (MSCV) (Van Parijs et al., 1999) or Moloney murine leukemia virus (MMLV) (Coffin and Varmus, 1996) that can only infect dividing cells, and an HIV-based lentivirus (Ventura et al., 2004) that can also infect non-dividing cells, were inefficient at producing transgenic outgrowths when expressing only EGFP (Figures 1C and 1D, and Table 1).

Figure 1
Comparison of the monolayer and suspension infection methods
Table 1
Efficiency of the monolayer and suspension infection methods

We next asked why there was poor EGFP expression in outgrowths from nononcogenic vectors. We observed that MEC colonies in monolayer cultures had two distinct cell populations: Cells located at the periphery of a colony had an elongated appearance and were preferentially infected, whereas cells in the center of a colony were cuboidal and poorly infected (Figure 1E). This difference in transduction efficiency occurred with all virus types and was not due to a difference in proliferation, since both populations exhibited significant bromodeoxyuridine (BrdU) incorporation (Figure S1). We transplanted the peripheral and central cells that were separated by differential trypsinization, and identified the central cells as the population with the highest MaSC content (Figure 1F). Thus, the MEC population with the greatest stem cell activity was poorly targeted by the monolayer infection method, regardless of the virus type used.

To improve transgenic outgrowth efficiency, we modified the protocol to infect primary MECs in suspension, rather than in monolayer. In addition to increasing the cell surface area accessible to virus, this method raises the effective viral titer by reducing the culture volume needed during the infection. We infected MaSC-enriched central cells from monolayer cultures or freshly prepared MECs. During the overnight infection in suspension, MECs formed large multicellular clusters (Figure 1G) composed of cells that expressed myoepithelial and luminal epithelial markers (Figure S1). Cells that failed to cluster were enriched for blood cell, stromal and apoptotic markers, and were depleted during washes prior to transplantation (Figure S1). Most of the transplants derived from HIV-EGFP infected aggregates gave rise to outgrowths that exhibited fluorescence throughout their ductal epithelium (Figures 1H and 1I, and Table 1). In contrast, few outgrowths from MMLV-EGFP infected MECs showed any fluorescence (Table 1). This reduced efficiency of MMLV-EGFP may result, in part, from the low proliferation rate observed in aggregated MECs (Figure S1). Collectively, these data show that infecting MECs in suspension with a lentivirus increases the representation of transduced cells in outgrowths. This suggests that combining lentiviral vectors with the suspension infection technique efficiently targets MaSCs.

MaSCs Are Transduced in Suspension

If MaSCs are transduced in suspension, we would expect EGFP to be expressed in all epithelial lineages of the mammary gland. In outgrowths derived from freshly prepared MECs infected with HIV-EGFP, we detected EGFP in myoepithelial cells, in both estrogen receptor (ER) positive and ER negative luminal epithelium, and in lobulo-alveoli of pregnant mammary glands (Figures 2A–2C and data not shown). Importantly, EGFP was not detected in periductal cells, indicating that transduced stroma does not contribute significantly to transgenic outgrowths (Figure 2A and 2B).

Figure 2
MaSCs are transduced by the suspension infection method

Since stem cells self-renew, transduced MaSCs should contribute to stem and progenitor populations in serially transplanted outgrowths. We transplanted small ductal fragments (<1.0 mm3) from lentiviral-infected primary outgrowths into secondary hosts and stained dissociated cells from both primary and secondary transplants with CD24 and CD49f antibodies (Stingl et al., 2006) to determine whether transduced MECs are represented in enriched stem and progenitor cell populations. FACS analysis of primary and secondary outgrowths derived from HIV-EGFP infected MECs revealed that transduced cells were present among mammary colony forming cells (MaCFC), myoepithelial cells (Myo), as well as the MaSC-containing mammary repopulating units (MRU) (Figure 2D).

The mammary gland contains at least three distinct progenitor populations: two have limited differentiation capacity and give rise to either ducts or alveoli upon transplantation, while only one is multipotent and capable of generating an entire functional mammary gland (Shackleton et al., 2006; Smith, 1996). We serially transplanted ductal fragments from a 7-month-old primary outgrowth, derived from freshly prepared MECs infected with a lentivirus expressing Zsgreen (Matz et al., 1999) (HIV-ZsGreen), into secondary and subsequently tertiary hosts to establish whether multipotent MaSCs were transduced. We observed Zsgreen fluorescence in each generation of transplants: throughout the ductal network of virgin recipients (Figure 2E and data not shown) and in lobulo-alveoli of pregnant recipients (Figure 2F and Figure 5M). These observations demonstrate that transduced MECs were capable of giving rise to both ducts and lobulo-alveoli in serial transplants, and imply that multipotent progenitors with regenerative capacity were targeted by the infection method.

Figure 5
HIV-dnXSu(H) outgrowths are reduced in size and display developmental defects

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 2G, lanes a–c) 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. Consistent with this notion, 16 secondary transplants derived from one primary outgrowth (Figure 2G, lanes d–h and data not shown) displayed a total of seven different integration patterns, each consisting of 1–4 distinct viral integrations. Interestingly, nine outgrowths exhibited the same doublet pattern (Figure 2G, lane f and data not shown). The reduction in the number of viral integrations per outgrowth and the duplication of these patterns in several secondary outgrowths suggest that these transplants were derived from one or few MaSCs. We then analyzed the tertiary transplants to determine whether these integration patterns would persist or segregate in serially related outgrowths. We observed the same doublet pattern in 6/6 tertiary outgrowths derived from one secondary transplant (Figure 2G, compare lane f to lane f’, and data not shown). Furthermore, two tertiary transplants, each derived from a separate secondary outgrowth, displayed distinct patterns that were also observed in Southern blots from the secondary transplants (Figure 2G, compare lane g to lane g’ and lane h to h’). These observations are consistent with earlier reports by Smith and colleagues demonstrating that retrovirally tagged epithelial fragments can give rise to clonally dominant outgrowths upon serial transplantation (Kordon and Smith 1998; Smith and Boulanger, 2002). Taken together, our data demonstrate that the 2×105 total primary MECs we routinely transplant contain multiple MaSCs, most of which are transduced with 1–4 viruses during the overnight infection in suspension.

Multiple Progenitors Contribute to Outgrowths

The previous results prompted us to examine the degree of cellular heterogeneity in outgrowths derived from MECs transplanted at non-limiting dilutions. Aliquots of freshly prepared MECs were separately infected with either HIV-Zsgreen or a lentivirus that expressed a monomeric red fluorescent protein (Campbell et al., 2002) fused to histone H2B (Kanda et al., 1998) (HIV-H2BmRFP). The separately infected populations were mixed at a 1:1 ratio and a total of 2×105 MECs were transplanted. The majority of outgrowths had both monochromatic and dichromatic regions, with the latter often displaying several patterns of fluorescence (Figure 3A and Movie S1). Most ductal segments in dichromatic regions were highly intermixed (Figure 3B and Movie S2) resulting in an amalgamation of red and green cells, while some ducts consisted of a dominant fluorescent cell type with focal cell clusters of the alternate color (Figure 3C and Movie S3). Interestingly, fluorescent patterns were not always maintained along an entire duct or its associated side branches. For example, a dichromatic duct could transition into a monochromatic duct or have side branches with a single dominant color (Figures 3A and 3D, and Movie S4). We then bred some transplant recipient mice and observed that lobulo-alveoli in late-pregnant outgrowths often displayed a dominant fluorescent color or were monochromatic rather than being highly intermixed (Figures 3F–3H). Thus, when MECs are transplanted at non-limiting dilutions both entire outgrowths as well as individual ducts can be derived from multiple progenitors, while lobulo-alveoli may originate from a single dominant alveolar progenitor.

Figure 3
Progenitor contribution in two-color outgrowths

We next hypothesized that these intermixed two-color outgrowths would transition to a predominantly monochromatic fluorescent phenotype if fewer MaSCs were transplanted. When we performed limiting dilution transplant analysis of freshly prepared primary MECs, we obtained a MaSC frequency of approximately 1/13,000 cells (data not shown). We therefore used a 100-fold dilution range and scored the limiting dilution outgrowths based on whether they were intermixed or composed of a dominant fluorescent color (Figures 3I and 3J, and Table 2). As we reduced the number of cells transplanted from 200,000 to 2,000, we observed a decrease in intermixed outgrowths with a concomitant increase in dominant color outgrowths (Figure 3J and Table 2). The success rate of these limiting dilution transplants was consistent with a MaSC frequency of 1/29,000 cells (range of 1/21,000–1/40,000), approximately half of that observed before the suspension infection. This lower frequency is likely due to culturing in suspension, which eliminates survival signals mediated through basement membrane attachment (Streuli and Gilmore, 1999), and to cytotoxicity associated with exposure to highly concentrated virus. Furthermore, we cannot rule out that expression of certain fluorescent markers may affect growth of transduced MECs, thereby reducing the apparent MaSC frequency. Consistent with this notion, the majority of dominant color outgrowths were Zsgreen positive (Table 2), which suggests that expression of the H2BmRFP fusion protein may have a negative effect on growth. Transplants derived from 10,000 MECs or less are likely to develop from a single MaSC. Accordingly, 12/14 outgrowths from such transplants were either monochromatic or exhibited a single dominant color (Figure 3I and Table 2). Therefore, the suspension infection method can be used to study the effect of genes in both polyclonal and monoclonal outgrowths by varying the number of MECs transplanted.

Table 2
Success rates and fluorescent phenotypes of limiting dilution two-color transplants

HIV-Wnt-1 Transplants Phenocopy MMTV-Wnt-1 Transgenic Mammary Glands

Our transduction technique would be most useful if it were a rapid alternative to producing transgenic mice. As a proof-of-concept, we asked if a lentivirus combined with the suspension infection method could phenocopy a transgenic mouse model of breast cancer. Mice that express Wnt-1 under the control of the mouse mammary tumor virus promoter (MMTV-Wnt-1) develop mammary hyperplasias that progress to adenocarcinoma with a median onset of 6 months (Tsukamoto et al., 1988). We transduced primary MECs with a lentivirus expressing Wnt-1 (HIV-Wnt-1) and analyzed outgrowths 1, 7 and 10 months after transplantation. By one month after transplantation, the HIV-Wnt-1 transplants exhibited increased ductal branching (compare Figure 4B to 4A), as has been observed previously with retroviral Wnt-1 infection (Edwards et al., 1992), and occupied at least twice as much of the fat pad as the contralateral HIV-Zsgreen control outgrowths (Figure 4C). By 7 months after transplantation, the HIV-Wnt-1 outgrowths displayed hyperplasias and adenomas similar to those observed in MMTV-Wnt1 glands (Tsukamoto et al., 1988) (Figures 4D and 4E). By 10 months, 1 of 16 transplants had a palpable adenocarcinoma (Figure 4F). Thus, we were able to replicate MMTV-Wnt-1 mammary phenotypes in outgrowths from lentiviral infected MECs.

Figure 4
HIV-Wnt-1 outgrowths phenocopy MMTV-Wnt-1 mammary glands

A Dominant-negative Xenopus Suppressor of Hairless (dnXSu[H]) Confers a Growth Disadvantage and Causes Developmental Defects

Efficient viral infection of MaSCs is necessary to avoid competition between transduced and untransduced progenitors during outgrowth (Edwards et al., 1996; Smith et al., 1991). This becomes especially important when studying genes that impede development or confer a growth disadvantage. We chose to manipulate the Notch pathway, which regulates stem cells in many systems (Bray, 2006). Notch proteins are transmembrane receptors that undergo a multi-step cleavage process to release the Notch intracellular domain (Nicd) upon ligand interaction. Nicd interacts with a RBP-J containing complex in the nucleus and subsequently activates genes implicated in cell fate decisions. To block Notch signaling we took advantage of dnXSu(H), a RBP-J homologue from Xenopus that interacts with Nicd and interferes with Notch signaling due to mutations that prevent binding to DNA (Duncan et al., 2005; Wettstein et al., 1997). We analyzed outgrowths from primary MECs infected in suspension with either HIV-dnXSu(H) or HIV-Zsgreen 5 weeks after transplantation, when TEBs are still present. HIV-dnXSu(H) outgrowths had severely dysplastic TEBs that were nearly four times larger than those from HIV-Zsgreen control outgrowths (Figures 5A–5E). Moreover, the HIV-dnXSu(H) outgrowths invaded less than half the fat pad area compared to HIV-Zsgreen controls (Figure 5F), and the overall ductal network was regionally hyper-branched, disordered and distended (Figures 5G–5I). TEBs from HIV-dnXSu(H) outgrowths exhibited expanded body-cell compartments and filled lumens, while mature ducts contained a bilayered epithelium with clear lumens (Figures 5J–5L). A previous study showed a requirement for Notch signaling during lobulo-alveolar development (Buono et al., 2006). Therefore, we transplanted ductal fragments from HIV-dnXSu(H) and HIV-Zsgreen outgrowths into contralateral glands of recipient mice that were impregnated after 6 weeks. Accordingly, the secondary dnXSu(H) expressing outgrowths exhibited markedly reduced lobulo-alveoli compared to contralateral HIV-Zsgreen control outgrowths (Figures 5M–5O). These data support an important role for Notch signaling during both ductal morphogenesis and pregnancy-induced differentiation. These results also demonstrate that our suspension infection method can overcome competition between untransduced progenitors and progenitors transduced with genes that confer growth disadvantages during outgrowth.

DISCUSSION

The mammary gland is a functionally regenerative organ (Deome et al., 1959) ideally suited for studying the in vivo effects of genes through viral-mediated gene delivery (Edwards et al., 1996). We have been able to transduce MaSCs efficiently by infecting primary MECs in suspension with high titer lentiviruses, and demonstrate the versatility of this technique with genes that have either positive or negative effects on mammary development. Importantly, we were able to do this without the need for enrichment (e.g., FACS isolation) of MRUs either before or after viral transduction.

Previously, two viral-mediated approaches have been used to study the effects of oncogenes on tumor development in the breast. However, both methods are inefficient at producing transgenic mammary glands when viruses with no selective advantage are used. The first approach involves ex vivo transduction of primary MECs grown in monolayer with either MMLV- (Edwards et al., 1992) or MSCV-based (Welm et al., 2005) retroviruses, followed by their transplantation into cleared inguinal fat pads. We show here that this method produces, at best, transgenic outgrowths in only a third of the transplants, with 15% of the ducts or less expressing EGFP (Table 1). The second approach involves in vivo transduction of only a few thousand cells per mammary gland with a modified avian retrovirus via intraductal injection of mice that express the cell surface receptor for this virus in their mammary epithelium (Du et al., 2006). Our approach of infecting primary MECs in suspension with lentiviruses is significantly more efficient than either method, producing transgenic outgrowths in up to 100% of the reconstituted glands with the majority of epithelium transduced (Table 2). The suspension infection technique is likely more effective due to superior targeting of MaSCs. This is supported by our observation that 37/38 limiting dilution transplants were fluorescent, suggesting that few MaSCs remain untransduced.

How does our in suspension infection technique enhance the transduction of MaSCs? Switching viral type was crucial for this approach to work, but does not necessarily explain the increase in efficiency, as lentivirus was equally incompetent as retrovirus at producing transgenic outgrowths with the monolayer infection method. The latter technique poorly targets cells within the center of MEC colonies, where we observe the greatest MaSC activity. In polarized epithelium, the efficiency of viral transduction depends on which epithelial surface is exposed to virus, possibly due to an unequal distribution of available VSV-G receptors in the apical and basolateral membranes (Borok et al., 2001; Johnson et al., 2000). Primary MECs grown in monolayer culture are associated through tight junctional complexes that confine viral access to the apical cell surface (Pickett et al., 1975; Shin et al., 2006). Our observation that these tightly associated central cells are refractory to infection unless they are detached by trypsinization, while loosely associated peripheral cells are infected preferentially, is consistent with MEC polarity affecting transduction. In addition to increasing the cell surface area accessible to virus, enzymatic digestion may also remove proteoglycans that can inhibit viral transduction (Batra et al., 1997). Important advantages of the suspension infection are that we can control the ratio of cells to virus more accurately and MECs can be infected with much higher viral titers than monolayer cultures. Thus, culturing and infecting MECs in suspension is likely to promote transduction of MaSCs through multiple mechanisms. Pertinent here, this infection technique is also applicable to human MECs isolated from reduction mammoplasty. Such cells can form ductal structures in fat pads of immunocompromised host mice, when transplanted together with human stromal cells (Kuperwasser et al., 2004). Furthermore, the utility of this approach is not restricted to the mammary gland, as we have observed robust transduction efficiencies in preliminary experiments with cell preparations from brain, lung and liver.

The two-color outgrowths shown here provide us with some insights into how MaSCs and their progenitors contribute to the generation of epithelial structures in reconstituted mammary glands. Mixed outgrowths from separately transduced MECs transplanted at non-limiting dilution contained heterogeneous ducts displaying complex fluorescent patterns. Our observation that neighboring cells in TEBs and mature ducts can express different fluorescent proteins is consistent with several ductal progenitors proliferating in close temporal and spatial proximity. In contrast, lobulo-alveoli were either monochromatic or exhibited a dominant fluorescent color. Taken together, these data support a scenario in which ducts originate from multiple progenitors, while lobulo-alveoli are derived from a dominant alveolar progenitor that may exist as a distinct cell population among the ductal epithelium (Smith, 1996). Further studies are required to determine whether similar patterns of cellular heterogeneity occur in intact mouse mammary glands.

Genetically engineered mice (GEM) are inherently limited by germ-line integration, which necessitates the use of tissue-specific promoters to restrict transgene expression. Viral-mediated transgene expression in the mammary gland can induce phenotypes that are different from those observed in GEM. For example, MSCV-mediated expression of the MET oncogene induces intraepithelial neoplasia in outgrowths, while MET-expressing transgenic mice have no discernible mammary phenotype (Welm et al., 2005). Similarly, avian retroviral-mediated expression of the polyoma middle T oncogene (PyMT) gives rise to tumors with different cellular characteristics and latency than tumors arising in MMTV-PyMT mice (Du et al., 2006). These studies imply that one should consider the potential cell types targeted when interpreting results. When we interfered with Notch signaling by expressing dnXSu(H) in outgrowths, we observed TEB dysplasia, branching defects and slow ductal development, whereas Buono et al. (2006) reported overtly normal ducts in transplants derived from MMTV-Cre/Rbp-jfl/fl MECs. It is possible that the disparity in virgin phenotypes results from targeting different cell types, since the MMTV promoter was used for Cre-mediated deletion of RBP-J, while dnXSu(H) was expressed from the ubiquitous EF1α promoter (Kim et al., 1990). However, we cannot rule out that dnXSu(H) interferes with other signaling pathways or that transduced non-epithelial cells contribute to the phenotype. The latter explanation is unlikely, since we have not observed transduced stroma outside the injection site and the ductal and alveolar phenotypes persisted in serial transplants. Transducing Rbp-jfl/fl MECs with a Cre-expressing virus could help elucidate Notch function during ductal morphogenesis.

There are several limitations of lentiviral-mediated gene delivery to develop transgenic outgrowths. Not surprisingly, we have observed that some genes are deleterious to outgrowth when expressed constitutively (data not shown). Hence, regulatable viral constructs will be necessary to elucidate the effects of such genes. Due to packaging constraints, lentiviral titers decrease significantly with large inserts, making some genes impractical for this application. In addition, it is possible that insertional mutagenesis could induce abnormal phenotypes in the mammary gland, but several independent rounds of infection and transplantation should reduce this risk.

Nevertheless, this technology provides an important alternative to GEM. Studies that were difficult to perform in GEM are now feasible with lentiviral-mediated transduction of MaSCs. Experiments that involve functional analysis of protein sub-domains, gene knockdown by siRNAs, or gene expression in distinct mammary lineages can be performed in months, at a fraction of the cost of developing GEM. At least 30 reconstituted mammary glands can be derived from transduced MECs isolated from a single mouse and these can be expanded further with serial transplantation. Partnering GEM donor MECs with viral transduction enables rapid analysis of genetic interactions and provides a powerful method to rescue mutant phenotypes. Thus, lentiviral-mediated gene delivery into MaSCs can be used in combination with, or in lieu of, GEM technology, depending on the question being asked.

EXPERIMENTAL PROCEDURES

Mice

FVB/N mice from Charles River Laboratories (Wilmington, MA) were maintained in a pathogen free facility and all mouse procedures were approved by the UCSF Institutional Animal Care and Use Committee (IACUC).

Viral Vectors and Virus Production

MSCV-EGFP and MSCV-Myc (formerly referred to as pMIG (Van Parijs et al., 1999) and pMIG-MYC (Welm et al., 2005), respectively) were previously described; all other viral vectors were constructed as outlined in the Supplemental Experimental Procedures. Virus was produced and titrated essentially as described (Zufferey and Trono, 2000), with modifications detailed in the Supplemental Experimental Procedures.

Preparation, Viral Transduction and Transplantation of Primary Mouse MECs

Primary mouse MEC cultures were generated from 8- to 12-week-old donor mice. Mammary glands number 3 (monolayer cultures only), 4 (without lymph node) and 5 were isolated and minced with a #10 scalpel for 5 min. For each gram of tissue, we used a digestion mixture of 5 ml collagenase buffer (RPMI-1640, 25 mM HEPES, 5% FBS, 100 µg/ml streptomycin, 100 U/ml penicillin G and 2 mg/ml collagenase IV [Sigma]) for 1 h at 37 °C with moderate shaking. Mammary organoids were subsequently separated from fat and single cells (such as fibroblasts and red blood cells) by centrifugation at 600 × g for 10 min followed by a series of four 2-sec pulses at 600 × g with resuspension in DMEM/F12. For monolayer infections, organoids were placed into 6-well plates (Welm et al., 2005) in primary growth medium (DMEM/F12, 5 µg/ml insulin, 1 µg/ml hydrocortisone, 10 ng/ml mouse EGF, 10% FBS, 100 µg/ml streptomycin, 100 U/ml penicillin G and 50 µg/ml gentamicin) and transduced 24 h and 48 h later by centrifuging the plates containing MECs for 1 h at 754 × g in the presence of 1×106 transducing units (TU)/ml virus and 1 µg/ml polybrene (Sigma). For suspension infections, organoids were trypsinized (0.5 mg/ml trypsin and 0.2 mg/ml EDTA in saline) for 20 min at 37 °C, washed with DMEM/F12, DNAse treated (0.1 mg/ml DNAse 1 [Sigma] in DMEM/F12 with 25 mM HEPES) for 3 min at 25 °C and filtered through a 70-µm cell strainer (BD Falcon). This single MEC suspension was transferred at 2×106 cells/well into 24-well ultra-low adhesion plates and transduced with 1×107 TU of virus (multiplicity of infection [MOI] is 5) for 16 h in a final volume of 0.8 ml primary growth medium. Transduced MECs were washed in Hank’s balanced salt solution (HANKS, with Ca2+ and Mg2+) and either 1×106 cells (monolayer infection) or aggregates equivalent to 2×105 cells (suspension infection) were injected in a 10 µl volume into cleared inguinal fat pads (Daniel et al., 1968; Deome et al., 1959). For the two-color limiting-dilution transplants, MECs were separately transduced as described above and the number of viable cells was determined again after infection by trypan blue exclusion to ensure 1:1 mixing of the two cell populations. The pooled MECs were then serially diluted in HANKS so that the 10 µl injection volume contained the appropriate cell number. For serial transplantation, small pieces of mammary gland tissue (< 1 mm3) containing a fluorescent ductal fragment were harvested from virgin hosts with either first or second generation outgrowths and transplanted into cleared fat pads.

Imaging of Reconstituted Mammary Glands

Reconstituted mammary glands were dissected and compressed between two glass slides to image fluorescent ducts with a MZ16 F fluorescent dissecting scope (Leica) and Spot Insight camera (Diagnostic Instruments). The glands were then fixed in 4% paraformaldehyde (PFA) for 4 h at 25 °C and either embedded in paraffin for histological analysis or processed as whole mounts, which were stained overnight with carmine-alum (0.2% carmine and 0.5% aluminum potassium sulfate) at 4 °C, dehydrated in a series of 70%, 95% and 100% ethanol and placed in Histoclear before imaging. The TEB, ductal outgrowth and fat pad areas were outlined in Adobe Photoshop and quantified in pixel numbers.

Outgrowth Potential of Peripheral and Central MEC populations

Four-day-old organoid cultures grown in monolayer were trypsinized for 2 min at 37 °C to release the ‘peripheral’ MECs. The ‘central’ MECs were collected after an additional 18 min of trypsin treatment and both cell populations were washed with HANKS prior to their transplantation into cleared inguinal fat pads. Cells (2–5×103 /10 µl) were injected and outgrowths were scored positive if ducts developed from the site of injection.

Fluorescent Imaging of Transplant Sections

Outgrowths were surgically removed, fixed in 4% PFA for 4 h at 25 °C, washed with phosphate buffered saline (PBS) and either embedded in paraffin or stored at −80 °C in Optimal Cutting Temperature compound (O.C.T., Tissue-Tek) after an additional overnight incubation in 30% sucrose at 4 °C. Deparaffinized 5-µm thick sections were blocked with IgG blocking reagent of a M.O.M. Kit (Vector Laboratories) for 1 h at 25 °C, washed in PBS and incubated overnight at 4 °C with rabbit anti-EGFP (Abcam, 1:50) and either Cy3-labeled mouse anti-α-smooth muscle actin (Sigma, 1:100) or mouse antiestrogen receptor (Dako Cytomation, 1:35) diluted in M.O.M diluent. The slides were then washed in PBS with 0.1% Tween-20 (Sigma) and incubated with Alexa-488-labeled goat anti-rabbit IgG and Alexa-568-labeled goat anti-mouse IgG (both Molecular Probes, both 1:500) in blocking buffer (3% bovine serum albumin in PBS) for 1 h at 25 °C. The slides were washed with PBS before being overlaid with Gel/Mount mounting media (Biomeda) to which 1 µg/ml 4′-6-diamidino-2-phenylindole (DAPI) was added. Cells were imaged on a Leica DMR microscope with a Leica DC500 camera. Frozen 30-µm thick sections were rinsed in PBS, incubated with 1 µg/ml DAPI for 1 h at 25 °C and rinsed once more with PBS before being cover-slipped. Fluorescent cells were imaged directly by spinning disk confocal microscopy (either a Zeiss Axiovert 200M with a Yokogawa CSU-10 or an Olympus IX81-ZDC microscope with an IX2-DSU).

Fluorescence Activated Cell Sorting

Single MEC suspensions (2×106 cells) from either primary and secondary transplants gated for EGFP fluorescence or wild type mouse mammary glands were incubated with biotin-conjugated rat anti-CD24 (BD Pharmingen, 1:100) and phycoerythrin-conjugated rat anti-CD49f (BD Pharmingen, 1:100) in HANKS with 2% FBS for 20 min at 4 °C. Cells were washed with HANKS containing 2% FBS, incubated with allophycocyanin-conjugated streptavidin (BD Biosciences, 1:200) for 10 min at 4 °C, washed again and analyzed on a FACSort flow cytometer (BD Bioscience). A forward and side scatter gate was used to exclude cell debris and clusters from the analysis.

DNA Isolation and Southern Blot Analysis

Virgin primary and late pregnant secondary or tertiary transplants were imaged as described, flash frozen in liquid nitrogen and stored at −80 °C until further processing. Individual glands were thawed on ice, minced with two #10 scalpels and their total DNA was purified with the DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer’s protocol. To determine viral integration patterns by Southern blot analysis (Kordon and Smith, 1998; Southern, 1975), 15 µg of DNA was digested overnight with BamHI and separated in a 0.8% agarose gel by electrophoresis. Afterwards, the DNA was blotted onto a positively charged nylon membrane that was hybridized overnight with a randomly primed 32P-labeled probe encoding ZsGreen. This 706 bp fragment was isolated from HIV-ZsGreen by digestion with NcoI and ClaI. Membranes were exposed for three days to storage phosphor screens, which were read by a Storm 860 phosphorimager (Molecular Dynamics).

Statistical Analysis

All values are shown as mean ± one standard deviation (s.d.). P-values were determined using the Student’s t-test with two-tailed distribution and unequal variance. A two-tailed Chi2 analysis was used to ascertain the P-value for the outgrowth potential of central and peripheral MECs. A two-tailed Fisher’s exact test was used to determine the P-value for limiting dilution outgrowths. MaSC frequency was determined using a complementary log-log binomial regression with 95% confidence levels.

Supplemental Data

Supplemental Data include one Figure, four Movies and Supplemental Experimental Procedures and References, and can be found with this article online at http://www.cellstemcell.com/.

Supplementary Material

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02

03

04

ACKNOWLEDGMENTS

We thank Michael McManus for providing pSICO, pMDLg/pRRE and RSV-Rev, Tannishtha Reya for dnXSu(H), Sean Megason for H2BmRFP, and Guangnan Li for EF1α and Wnt1-HA plasmids. We are grateful to Mikala Egeblad, Hanne Askautrud, Kathleen Karmel and Monica Mahler for help with spinning disk confocal microscopy and to Jennifer Lilla for Southern expertise. This study was supported by grants from the National Cancer Institute and the National Institute of Environmental Health Sciences (CA057621, CA084243 and ES012801), a UCSF Comprehensive Cancer Center Intramural Award from the Alexander and Margaret Stewart Trust, and by Postdoctoral Traineeship Awards from the Congressionally Directed Breast Cancer Research Program, US Department of Defense (B.E.W., DAMD-17-03-1-0498; G.J.P.D., DAMD–17-02-1-0333).

Footnotes

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