The varying pathways of mammary gland development across species and evolutionary history are underexplored, largely due to a lack of model systems. Recent progress in organoid technology holds the promise of enabling in-depth studies of the developmental adaptations that have occurred throughout the evolution of different species, fostering beneficial phenotypes. The practical application of this technology for mammary glands has been mostly confined to rodents and humans. In the current study, we have successfully created next-generation 3D mammary gland organoids from eight eutherian mammals and the first branched organoid of a marsupial mammary gland. Using mammary organoids, we identified a role for ROCK protein in regulating branching morphogenesis, a role that manifests differently in organoids from different mammals. This finding demonstrates the utility of the 3D organoid model for understanding the evolution and adaptations of signaling pathways. These achievements highlight the potential for organoid models to expand our understanding of mammary gland biology and evolution, and their potential utility in studies of lactation or breast cancer.

The emergence of mammals in evolution is marked by the appearance of a mammary gland: a secretory gland able to provide nutrition to offspring. The mammary gland evolved from a cutaneous, likely hair-associated, gland, with evidence pointing to the apocrine sweat gland as the precursor of the mammary gland (Oftedal, 2002), and has undergone remarkable evolutionary transformations, adapting to diverse ecological pressures and reproductive strategies.

The earliest extant mammals are the monotremes, consisting of the platypus and four species of echidna. Monotremes are egg-laying mammals that diverged from therian mammals about 190 million years ago (Oftedal and Dhouailly, 2013) (Fig. 1A). The monotreme mammary gland is a patch of special hairs, where milk is secreted from a gland at the base of each hair, of which there are ∼100-150 in the echidna (Griffiths, 1978).

Fig. 1.

Generation of marsupial mammary gland organoids. (A) The timeline of mammalian group divergence in evolution. (B) Illustration of mammary gland structure in each of the three mammalian groups, compared with an apocrine gland. (C) Bright-field images depicting the development of opossum mammary gland organoids during 3 weeks in culture. Arrows indicate ductal tip enlargements. (D) Whole-mount in situ hybridization detection of keratin 14 in 3D opossum organoids. Image on the right is an enlargement of the area outlined in the image on the left.

Fig. 1.

Generation of marsupial mammary gland organoids. (A) The timeline of mammalian group divergence in evolution. (B) Illustration of mammary gland structure in each of the three mammalian groups, compared with an apocrine gland. (C) Bright-field images depicting the development of opossum mammary gland organoids during 3 weeks in culture. Arrows indicate ductal tip enlargements. (D) Whole-mount in situ hybridization detection of keratin 14 in 3D opossum organoids. Image on the right is an enlargement of the area outlined in the image on the left.

The next mammalian group to diverge was the marsupials. Marsupials, also known as Metatheria, diverged from eutherians approximately 166 million years ago (Nilsson et al., 2010; Deakin et al., 2012; Oftedal and Dhouailly, 2013). The marsupial mammary gland develops in association with a hair follicle and represents a more ancient version of a gland when compared with eutherian mammary glands (Oftedal, 2002). Marsupial young are born altricial, relying less on placentation and completing their development postnatally, with an extended period of lactation. Marsupials comprise about 6% of mammals, while ∼94% of mammals are eutherians.

Eutherian mammals are the most taxonomically diverse group of mammals, with over 4000 species representing a large variety of mammary gland morphologies, milk compositions and lactation strategies that evolved throughout mammalian evolution. The morphological distinction between the monotreme, marsupial and eutherian mammary glands, and how it relates to the evolutionary precursor of the gland is illustrated in Fig. 1B.

Investigating the evolution of the mammary gland can offer insights into fundamental biological processes, including tissue development, hormonal regulation and lactation. For example, certain species have evolved unique adaptations, which can serve as valuable models for understanding the regulation of milk composition and the cyclical regeneration of mammary glands (Sharp et al., 2007, 2017). The marsupial mammary gland is of interest due to the uniquely enriched composition of its milk, particularly during early lactation, that compensates for short gestation periods (Green et al., 1980). In addition, some marsupials can simultaneously produce different milk compositions to feed offspring of different ages, a phenomenon known as asynchronous concurrent lactation (ACL), which provides an opportunity to explore the local versus systemic regulation of milk composition (Nicholas, 1988). In some species, mammary cancer rarely occurs, whereas in others, including humans, it is increasingly common (Swett et al., 1940; Munson and Moresco, 2007; Prpar and Dovc, 2013; Harman et al., 2021; Peaker, 2023). These examples illustrate how understanding the genetic and molecular mechanisms that enable evolutionary adaptations may provide useful knowledge for advancements in breast cancer research and for the development of therapies for lactation-related disorders.

Some of the primary obstacles to a deep understanding of mammary gland evolution are the limited access to live specimens, the ethical concerns associated with conducting invasive experiments on animals, and the unsuitability of most species as laboratory animals. As a result, traditional approaches relied heavily on comparative anatomy and histological analysis of preserved tissues, which provided valuable but static information but fell short of capturing the dynamic nature of mammary gland development, lactation and tissue regeneration. Furthermore, important cellular and molecular aspects of mammary gland biology, including the regulation of milk composition and the cellular processes underlying its cyclical regeneration, remain elusive in most species.

Recent advances in organoid technology have enabled the generation of 3D mammary gland tissue from single primary cells isolated from an adult human or mouse (Sokol et al., 2016; Rauner et al., 2021, 2023 preprint; Yuan et al., 2023). The process of organoid development from single cells to mature tissue recapitulates important aspects of mammary tissue development, providing a valuable model for studying mammary gland development and regeneration.

Thus far, complex ductal-lobular 3D organoids of the mammary gland, which capture the branched ductal lobular architecture of the tissue, have not been generated from species other than humans and mice. In this work, we endeavored to grow mammary gland organoids from eight eutherian mammals and one marsupial. We show that individual cells isolated from adult mammary glands of these species proceed to form branched epithelial structures and ductal-lobular networks in a 3D matrix. Notably, this report includes the first branched 3D organoid of a marsupial mammary gland: the gray short-tailed opossum. These organoids can serve as surrogates of animal models that were previously unavailable for studies focused on evolutionary and developmental biology, as well as milk production and breast cancer research.

Furthermore, we identify that the ROCK inhibitor Y-27632 is necessary for the branching of mammary organoids from opossum, cow, goat and rabbit but not from human or rat. Interestingly, human breast organoids exhibited an aberrant branching phenotype when treated with ROCK inhibitor. This finding indicates that ROCK plays a role in regulating branching morphogenesis, that this role may have existed early in mammalian evolution and that it may manifest differently in different mammalian species. This is particularly interesting in the context of the evolutionary transition from the coiled apocrine gland to the branched mammary gland.

Ultimately, the study of mammary gland evolution not only illuminates the biological history of mammals but also unveils the physiological mechanisms that underlie crucial aspects of mammalian life, with the potential to offer significant insights that could be translatable for improving human health.

3D marsupial mammary gland organoids from the gray short-tailed opossum

We aimed to grow organoids of the opossum mammary gland, which could serve as a model to study mammary gland development in a marsupial mammal. The gray short-tailed opossum, Monodelphis domestica, is a pouch-less marsupial that has been used to study mammalian evolution (Frost et al., 2000; Belov et al., 2006; Gentles et al., 2007; Mikkelsen et al., 2007; Urban et al., 2017). The gray short-tailed opossum mammary gland has 13 nipples: 12 arranged in a circle around one (Cockrum, 1962; Rousmaniere et al., 2010; Wessel, 2016). The adult marsupial mammary gland includes an intra-mammary muscle, the ilio marsupialis, which is unique to marsupials and can be seen near the mammary epithelium (Woolley et al., 2002) (Fig. S1A).

To generate organoids, primary mammary epithelial cells were isolated from an adult (17 months old), non-lactating virgin female, and embedded in 3D gels of either Matrigel or a collagen-based extracellular matrix (ECM) hydrogel, supplemented with MEGM, as previously described for the generation of human and mouse mammary organoids (Barcellos-Hoff et al., 1989; Pasic et al., 2011; Sokol et al., 2016).

The organoids that developed were acinar and failed to branch after 13 days in culture (Fig. S1B), indicating that the conditions for the growth of human or mouse mammary organoids did not support the full maturation of the opossum mammary epithelium. To try to induce organoid branching morphogenesis, we added Matrigel to the ECM hydrogels at a range of percentages (0, 25, 50, 75 and 100%), while maintaining the balanced ratio of the other components of the gel (Fig. S1C). Branching was not observed in any of the gel compositions (Fig. S1D). An attempt to increase the laminin concentration 5- or 10-fold did not result in the branching of opossum mammary organoids (Fig. S1E).

We then attempted to induce organoid branching by adding hormones to the media: estrogen and progesterone regulate the expansion of the mammary epithelium; prostaglandin E2 induces aromatase, which is necessary for estrogen biosynthesis (Richards and Brueggemeier, 2003), and its receptors are expressed during pregnancy and lactation in the mouse mammary gland, indicating a role in tissue development (Chang et al., 2004). Adding either hormone to the media did not lead to the branching of opossum mammary organoids (Fig. S1F). Taken together, the failure of our multiple attempts to induce branching and maturation of opossum organoids indicated that it requires significantly different growth conditions compared with human or mouse mammary organoids, and may reflect differences in the developmental biology of this more-primitive mammary gland.

We then attempted to culture opossum mammary epithelial cells in the commercial organoid media Intesticult (06010, StemCell Technologies). The formulation of the media is proprietary but is based on published studies that optimized it for intestinal epithelium organoids (Jung et al., 2011; Sato et al., 2011). Media supplements described in these studies have been successfully used in breast organoid culture medium (Sachs et al., 2018; Dekkers et al., 2021) (Table S1). Remarkably, opossum organoids developed and branched in Intesticult (Fig. 1C). Branching was visible after 7 days, and enlargement of the tips of branches resembling lobules was visible by day 10. However, not all branches had such lobular enlargements even after 3 weeks in culture, and mature lobules (such as those seen in the culture of human breast organoids) were not seen during this time. In situ hybridization for opossum CK14 demonstrated its expression in the basal layer of the organoid, as seen in the eutherian species (Hellmen and Isaksson, 1997; Mikaelian et al., 2006; Santagata et al., 2014) (Fig. 1D).

Previously reported 3D culture models of marsupial mammary organoids highlighted the importance of the ECM composition on mammary epithelial development and function (Wanyonyi et al., 2013a,b, 2017). In their work, Wanyonyi et al. demonstrate that the ECM composition changes with lactation phases and can impact the development of 3D epithelial acini in culture. The organoids that developed in these reports were rudimentary and did not recapitulate a ductal-lobular morphology, possibly owing to the culture method on coated plastic rather than in a floating hydrogel. Combining the best of both systems may prove optimal: embedding epithelial cells in a floating, collagen-based hydrogel into which species-specific ECM components are incorporated. This approach could facilitate an effective investigation of how milk composition is regulated locally in the tammar wallaby and possibly in other mammals.

Generation of 3D mammary gland organoids from eight eutherian mammals

Mammary gland tissue samples were collected from eight eutherian species, representing four mammalian orders: Carnivora (ferret and cat), Artiodactyla (pig, goat and cow), Lagomorpha (rabbit) and Rodentia (rat and hamster) (Fig. S2A). Single primary cells from each species were embedded in ECM hydrogels (Sokol et al., 2016). Organoids were cultured for 11-13 days in identical organoid media (Intesticult).

3D organoids were successfully generated from all eight eutherian mammals (Fig. 2). Varied morphological characteristics were observed between species. Specifically, although branching morphogenesis occurred in organoids from all species analyzed, distinctly shorter ductal elongation was observed for the hamster. Defined alveoli and lobules were visible in organoids of cow, goat, pig and rabbit (artiodactyls and a lagomorph), but not in organoids of cat, ferret, hamster and rat (carnivores and rodents).

Fig. 2.

Generation of mammary gland organoids from diverse eutherian mammals. Bright-field images of hydrogels occupied by mammary tissue organoids from eight eutherian species. Organoid images on the right are enlargements of the areas outlined in the left-hand whole-gel images. Scale bars: 0.5 mm.

Fig. 2.

Generation of mammary gland organoids from diverse eutherian mammals. Bright-field images of hydrogels occupied by mammary tissue organoids from eight eutherian species. Organoid images on the right are enlargements of the areas outlined in the left-hand whole-gel images. Scale bars: 0.5 mm.

We stained the organoids with phalloidin, revealing the distribution of filamentous actin (F-actin) in the epithelial structures that formed (Fig. S2B). F-actin encapsulated the epithelial structures in organoids from cow, rabbit, cat and pig, but not in organoids from ferret, rat, goat and hamster, in which phalloidin stained the organoid mostly internally.

We observed the convergence of adjacent organoids, forming a network, particularly in cow, rat and rabbit organoids. There was a difference in the degree to which the organoids filled the matrix, impacted by organoid number, thickness and spread (Fig. S2C,D). The percentage of matrix occupancy was highest in rat, cow and goat (25.9%, 25.3% and 24.3%, respectively), and lowest in ferret, cat and hamster (5.5%, 4.7% and 4.3%, respectively). Rabbit and pig organoid matrix occupancy were in the mid-range, with 14.6% and 8.6%, respectively.

Organoids have been increasingly developed as models to study normal mammary gland functions (Ciccone et al., 2020; Sumbal et al., 2020; Charifou et al., 2021; Lewis et al., 2022; Yuan et al., 2023). It will be valuable to expand the generation of mammary epithelial organoids to include more species to gain a more comprehensive understanding of mammary gland development across mammals. Of particular interest are species that rarely develop cancer of the mammary gland [e.g. cows, pigs, goats and others (Swett et al., 1940; Munson and Moresco, 2007; Prpar and Dovc, 2013; Harman et al., 2021; Peaker, 2023)], or develop aggressive mammary tumors (e.g. cats; Hughes, 2021). Employing organoids derived from dairy animals can similarly advance our understanding of lactation in agricultural contexts, as had been previously recognized by others (Finot et al., 2021).

ROCK inhibition differentially impacts the branching of mammary organoids from different species

We attempted to identify which factors in Intesticult were necessary for the formation of opossum organoids, by selectively adding, individually or in groups, supplements used in the studies on which the Intesticult formulation is based (Jung et al., 2011; Sato et al., 2011). Fig. S3A lists the supplements included in each of the combinations we tested, and the representative result of each. In all experiments, we used MEGM and the commercial Intesticult media as controls. We identified the commercial supplements B27 and N2 as necessary for the formation of opossum mammary organoids. We also identified that a group of three inhibitors were necessary for growth to occur: Y-27632, an inhibitor of Rho-associated, coiled-coil containing protein kinase (ROCK) (Narumiya et al., 2000); SB202190, a p38 MAPK inhibitor (Davies et al., 2000); and A83-01, an inhibitor of activin receptor-like kinases ALK5, ALK4 and ALK7 (Tojo et al., 2005).

To find which of the inhibitors was necessary for growth, we added them to the media separately or in combinations and quantified the number of organoids that formed in each condition (Fig. S3B). We concluded that A83-01 and SB202190 were each necessary for organoids to form and synergistically increased organoid formation. The ROCK inhibitor Y-27632 was neither necessary nor sufficient for organoid formation, but was necessary for branching (Fig. 3A). Given the role of FGF-2 in mammary gland branching morphogenesis (Zhang et al., 2014; Sumbal and Koledova, 2019), we tested whether FGF-2 would induce branching in opossum mammary organoids. We found that mouse or human recombinant FGF-2 did not induce branching in the absence of Y-27632 and did not enhance branching in its presence (Fig. S3C). Based on these experiments, we formulated a defined media for the culture of opossum organoids (henceforth termed ‘Minimal Media’) that includes the supplements required for growth and branching (Table S2).

Fig. 3.

ROCK inhibition is necessary for the branching of opossum mammary organoids but disrupts the branching of human counterparts. (A) Bright-field images of opossum organoids that developed in the presence of specific chemical inhibitors. Scale bars: 50 μm. (B) Bright-field images of human and opossum mammary gland organoids that developed with or without Y-27632. Scale bars: 50 μm. (C) Quantification of branches per organoid in opossum mammary organoids. Error bars represent s.d. P-value is provided above pairwise comparison; statistical comparison were carried out using an unpaired, two-tailed t-test. (D) A human breast organoid immunostained for CK14 (green) and F-actin (phalloidin, red). Nuclei are stained with DAPI (blue). Scale bars: 100 μm.

Fig. 3.

ROCK inhibition is necessary for the branching of opossum mammary organoids but disrupts the branching of human counterparts. (A) Bright-field images of opossum organoids that developed in the presence of specific chemical inhibitors. Scale bars: 50 μm. (B) Bright-field images of human and opossum mammary gland organoids that developed with or without Y-27632. Scale bars: 50 μm. (C) Quantification of branches per organoid in opossum mammary organoids. Error bars represent s.d. P-value is provided above pairwise comparison; statistical comparison were carried out using an unpaired, two-tailed t-test. (D) A human breast organoid immunostained for CK14 (green) and F-actin (phalloidin, red). Nuclei are stained with DAPI (blue). Scale bars: 100 μm.

Human breast organoids do not require ROCK inhibition for branching (Sokol et al., 2016; Rauner et al., 2021). ROCK inhibitors are routinely added to primary cell cultures in many protocols, to mitigate cell death due to anoikis and to maximize cell viability (Watanabe et al., 2007; Kretzschmar and Clevers, 2016). We cultured human and opossum mammary organoids in Minimal Media, to see whether they exhibit a different response to the presence of ROCK inhibitor. As expected, opossum organoid branching required ROCK inhibitor, whereas human organoids branched in its absence (Fig. 3B,C). Interestingly, in human organoids, ROCK inhibition led to aberrant or hyper-branching.

We stained human breast organoids cultured with or without the ROCK inhibitor for F-actin and the basal marker CK14 and found that ROCK inhibition led to the emergence of CK14-positive cells extending outward from a densely clustered core of epithelial cells in the organoid (Fig. 3D). This starkly contrasted with the presence of a single layer of CK14-positive cells enveloping a branched epithelial structure in the untreated organoids. The hyper-branched phenotype we observed in the human organoid is consistent with the role of ROCK in facilitating branching morphogenesis by maintaining polarization within bi-layered epithelial tissues, as reported by others (Ewald et al., 2008; Walker et al., 2021).

We attempted to culture mammary organoids from the eight eutherian species in Minimal Media or MEGM. These media failed to support the growth of cat, ferret, hamster or pig cells (Fig. S4). Rabbit organoids grew in MEGM; cow, goat and rat organoids grew in the Minimal Media (Table S2). In the four species that could be cultured in a defined media, we tested the effect of ROCK inhibition on branching morphogenesis by adding or removing ROCK inhibitor from the media. Remarkably, we found that, in mammary organoids of cow, goat and rabbit, ROCK inhibition resulted in the development of significantly more branched organoids, whereas rat mammary organoids, like their human counterparts, branched in the absence of ROCK inhibition (Fig. 4A,B). Interestingly, in rat organoids, ROCK inhibition resulted in the development of significantly more organoids (Fig. 4C). An effect of ROCK inhibition on the rate of organoid formation was not observed for the other three species. These findings indicate that the requirement for ROCK inhibition in mammary organoid branching is species dependent and suggests a possible divergence in mammary epithelial branching mechanisms among different mammals. Further investigation may uncover evolutionary or functional explanations behind these observed differences in branching requirements.

Fig. 4.

ROCK inhibition is necessary for the branching of cow, goat and rabbit mammary organoids. (A) Bright-field images of representative mammary organoids from cow, goat and rabbit that developed with or without the ROCK inhibitor Y-27632 treatment. Scale bars: 50 μm. (B) Percentage of branched organoids with or without Y-27632. (C) Total number of organoids per field with or without Y-27632 treatment. Error bars represent s.d. Values above pairwise comparisons indicate P. Statistical testing was carried out using multiple unpaired two-tailed t-tests, with Holm-Sidak corrections.

Fig. 4.

ROCK inhibition is necessary for the branching of cow, goat and rabbit mammary organoids. (A) Bright-field images of representative mammary organoids from cow, goat and rabbit that developed with or without the ROCK inhibitor Y-27632 treatment. Scale bars: 50 μm. (B) Percentage of branched organoids with or without Y-27632. (C) Total number of organoids per field with or without Y-27632 treatment. Error bars represent s.d. Values above pairwise comparisons indicate P. Statistical testing was carried out using multiple unpaired two-tailed t-tests, with Holm-Sidak corrections.

The Rho/ROCK pathway controls actin cytoskeletal organization and plays a role in cell movement and shape in single-cell organisms, such as the amoeba, in the development of sponges, and in complex cellular functions that are important for development, such as cell differentiation and morphogenesis, in vertebrates (Riento and Ridley, 2003; Klopocka and Redowicz, 2004; Wang et al., 2004; Schenkelaars et al., 2016; Narumiya and Thumkeo, 2018). The mechanisms by which ROCK participates in branching are not entirely clear. A role for ROCK proteins in branching morphogenesis was identified in mouse and rat tissues, where ROCK inhibition resulted in reduced or disorganized branching of the mammary gland, submandibular gland and kidneys (Meyer et al., 2006; Ewald et al., 2008; Walker et al., 2021). Our findings indicate that this is not the case in all species; in some species ROCK activity needs to be inhibited for branching to occur.

There are some limitations of this study that should be acknowledged and addressed in future work. Importantly, the composition of the matrix and media can be optimized for each species, as was successfully carried out previously for ECM of human breast organoids (Sokol et al., 2016) and here for the culture medium of opossum organoids. This line of investigation may also reveal new mechanisms of development that are either species specific or are more prominent in some species, which can then serve as animal models for studying these mechanisms. In addition, multiple samples from each species, representing variation in factors such as age and parity, would increase the value of organoids as surrogates of the original tissue.

To conclude, this work describes a key advancement that allows for the investigation of mammary gland development, regeneration, and lactation biology across different species, which can lead to a deeper understanding of the conserved molecular mechanisms involved in mammary gland development in mammals, as well as the identification of unique regenerative or other mechanisms of mammary epithelium in certain species.

Sample collection and processing

Mammary gland tissue from eight eutherian mammals (rabbit, rat, dog, pig, goat, ferret, hamster and cow) was obtained under aseptic conditions, from non-lactating clinically healthy females of each species (age unknown, BioChemed Services). Mammary gland tissue of gray short-tailed opossum was collected from a 17-month-old virgin female, from the colony of the Sears lab at UCLA. Human breast tissue that would otherwise have been discarded as medical waste after reduction mammoplasty surgery was obtained in compliance with all relevant laws, using protocols approved by the institutional review board at Tufts Medical Center. All tissues were anonymized before transfer and could not be traced to specific patients; for this reason, this research was provided exemption status by the Committee on the Use of Humans as Experimental Subjects at Tufts University Health Sciences (IRB# 13521). All patients enrolled in this study signed an informed consent form to agree to participate in this study and for publication of the results.

Tissues were delivered either promptly upon collection or shipped overnight in a saline buffer on ice and immediately processed in the lab. Tissue processing followed the protocol previously described for human breast tissue (Rauner et al., 2021). If present, surrounding adipose tissue was removed and discarded, and the glandular tissue was chopped with scissors into ∼1 mm3 pieces. Tissue was weighed and incubated in an enzyme solution containing 1.5 mg/ml collagenase A (Sigma Aldrich, 11088793001) and 0.3 mg/ml hyaluronidase (Sigma Aldrich Aldrich, H3506) in MEGM (Lonza, CC-3150), at a ratio of 10 ml enzyme solution per 2 g or less of tissue. Tissues were then incubated by rotating for 3 h at 37°C. After incubation, tubes were rested upright for 5 min to allow epithelial tissue fragments to sink to the bottom. Fat that accumulated on the top was removed using a pipette, and the intermediate liquid containing the stromal fraction (SF) was collected separately from the epithelial fragments at the bottom of the tube. The SF and epithelial fragments were washed three times in PBS to remove the enzyme solution, resuspended in MEGM supplemented with 10% DMSO (ThermoFisher Scientific, BP231-100) and cryopreserved in aliquots of 1 ml.

Organoid generation and culture

Cryopreserved samples of enzymatically digested tissue were thawed and further dissociated with trypsin and dispase to obtain a single-cell suspension of mammary epithelial cells, as previously described (Rauner et al., 2021). Briefly, after washing in MEGM to remove DMSO, the samples were resuspended in pre-warmed 0.25% trypsin-EDTA (Thermo Fisher Scientific, 25200-056) and incubated at 37°C for 5 min. A defined trypsin inhibitor (Thermo Fisher Scientific, R007100) was added at 2× trypsin volume. The sample was centrifuged for 4 min at 300 g, and the pellet was resuspended in 5 mg/ml dispase II solution (Sigma Aldrich, 04942078001) supplemented with 0.1 mg/ml DNase I (Sigma Aldrich, 4716728001) to dissolve stringy DNA. After 2 min of incubation in dispase-DNase solution, the suspension was filtered through a 40 μm cell strainer (VWR, 76327-098) to yield a single-cell suspension. Cells were counted with Trypan Blue (Thermo Fisher Scientific, 15250-061) using a cell counter (TC-20 from BioRad), and a cell suspension was prepared at a concentration of 0.5×106 cells/ml. Hydrogels were prepared as previously described (Rauner et al., 2021). Collagen type I (rat tail, Sigma Aldrich, 08-115) was mixed on ice with ice-cold MEGM to a final concentration of 1.7 mg/ml and supplemented with 0.1 N NaOH at a volume that equals 12.5% of the collagen volume. Laminin (Thermo Fisher Scientific, 23017015), fibronectin (Sigma Aldrich, F1056) and hyaluronic acid (Sigma Aldrich, 385908) were added to the collagen mix to yield a final concentration of 25 μg/ml, 25 μg/ml and 12.5 μg/ml, respectively. Cell suspension in MEGM was added to a final concentration of 2.5×104 cells/ml. Hydrogel solution was mixed and deposited as 200 μl gels in four-well chamber slides (Corning, 354104) and allowed to solidify at 37°C for 1 h. After the hydrogels solidified, 1 ml media was added per well, and the gels were mechanically detached from the well surface using a pipette tip. Mammary organoids from eutherian species were cultured in Intesticult human organoid growth media (StemCell Technologies, 06010). Mammary organoids of opossum were cultured in either Intesticult, or opossum organoid media, as described in Table S2. In the relevant experiments, media supplements were added at the concentrations indicated in Table S3. Media was changed three times a week. Primary cultures of opossums are maintained at 32.5°C, as previously described for organotypic cultures derived from developing opossum cortex, because they have a lower body temperature than most placental mammals and other marsupials (Harder et al., 1996; Puzzolo and Mallamaci, 2010; Petrovic et al., 2021).

Immunofluorescence staining

Gels were fixed by 30 min rotation incubation at room temperature in freshly prepared 4% paraformaldehyde (Electron Microscopy Sciences, 15710S) diluted in PBS-T, followed by permeabilization overnight in rotation at room temperature in 0.1% Triton X-100 in PBS-T. DAPI and phalloidin staining was performed by incubating gels for 30 min in 2 μg/ml DAPI (Thermo Fisher Scientific, D1306) and Phalloidin-iFluor 647 (Abcam, ab176759) according to the manufacturer's protocol. Primary antibody for CK14 (Thermo Fisher Scientific, RB-9020-P1) was diluted 1:300, incubated overnight at 4°C, followed by a 2 h incubation with anti-rabbit Alexa-fluor 488-conjugated secondary antibody (Thermo Fisher Scientific, A-11008) diluted 1:500.

Whole mount in situ hybridization

Whole mount in situ hybridization (WISH) for the detection of opossum keratin 14 was performed using HCR RNA-FISH (Molecular Instruments). Fluorescently labeled probes for Monodelphis domestica KRT14 were planned based on the ensemble transcript ENSMODT00000074233, and the hybridization and staining was performed according to the manufacturer's protocol. Fixation of hydrogels for WISH was performed in 4% paraformaldehyde in sterile PBS for 1 h, followed by three washes in sterile PBS-T, dehydration in methanol for 5 min on ice, repeated twice. Hydrogels were stored in methanol at −20°C overnight, and rehydrated with a series of methanol in PBS-T (75%, 50% and 25%), each for 5 min on ice, followed by two washes with PBS-T. Hydrogels were then incubated with proteinase K (10 µg/ml in PBS) for 3 min at room temperature, and washed for 5 min with PBS-T. Fixation in 4% PFA was repeated for 20 min at room temperature, followed by two washes in PBS-T on ice. Subsequently, hydrogels were washed in 1:1 PBS-T:5×SSCT buffer for 5 min on ice. 5×SSCT buffer was prepared by diluting 20×SSC (saline sodium citrate) in DEPC water and adding 0.1% Tween (Sigma Aldrich, P1379). Next, hydrogels were washed in 5×SSCT buffer for 5 min on ice. Hydrogels were then incubated with pre-hybridization and probe solution, washed and incubated with the amplification reagents, as indicated by the kit protocol.

Imaging and analysis

Bright-field images were obtained using a Nikon Eclipse Ti-U inverted microscope equipped with a digital camera and matching software (SPOT Imaging). Full gel images were manually assembled from fields imaged sequentially. DAPI and phalloidin staining images were obtained using a Nikon AXR confocal microscope and NIS Elements software (Nikon Instruments). Gel occupancy was analyzed using ImageJ software (Schneider et al., 2012). WISH images were obtained using a confocal Zeiss LSM 880, with Zen Blue 2.6 software.

Author contributions

Conceptualization: C.K., G.R.; Methodology: C.K., G.R.; Validation: C.K., G.R.; Formal analysis: G.R.; Investigation: H.Y.K., C.K., G.R.; Resources: H.Y.K., I.S., K.E.S., C.K., G.R.; Data curation: G.R.; Writing - original draft: G.R.; Writing - review & editing: H.Y.K., K.E.S., C.K., G.R.; Visualization: G.R.; Supervision: C.K., G.R.; Project administration: H.Y.K., C.K., G.R.; Funding acquisition: C.K., G.R.

Funding

This research was supported by the National Institute of General Medical Sciences (7R01GM124491 to C.K.), the Department of Defense Breast Cancer Research Program (W81XWH2010018 to G.R.) and the Find the Cause – Breast Cancer Foundation (C.K.). Deposited in PMC for release after 12 months.

Data availability

All relevant data can be found within the article and its supplementary information.

Barcellos-Hoff
,
M. H.
,
Aggeler
,
J.
,
Ram
,
T. G.
and
Bissell
,
M. J.
(
1989
).
Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane
.
Development
105
,
223
-
235
.
Belov
,
K.
,
Deakin
,
J. E.
,
Papenfuss
,
A. T.
,
Baker
,
M. L.
,
Melman
,
S. D.
,
Siddle
,
H. V.
,
Gouin
,
N.
,
Goode
,
D.
,
Sargeant
,
T.
,
Robinson
,
M. D.
et al. 
(
2006
).
Reconstructing an ancestral mammalian immune supercomplex from a marsupial major histocompatibility complex
.
PLoS Biol.
4
,
e46
.
Chang
,
S. H.
,
Liu
,
C. H.
,
Conway
,
R.
,
Han
,
D. K.
,
Nithipatikom
,
K.
,
Trifan
,
O. C.
,
Lane
,
T. F.
and
Hla
,
T.
(
2004
).
Role of prostaglandin E2-dependent angiogenic switch in cyclooxygenase 2-induced breast cancer progression
.
Proc. Natl. Acad. Sci. USA
101
,
591
-
596
.
Charifou
,
E.
,
Sumbal
,
J.
,
Koledova
,
Z.
,
Li
,
H.
and
Chiche
,
A.
(
2021
).
A robust mammary organoid system to model lactation and involution-like processes
.
Bio. Protoc.
11
,
e3996
.
Ciccone
,
M. F.
,
Trousdell
,
M. C.
and
Dos Santos
,
C. O.
(
2020
).
Characterization of organoid cultures to study the effects of pregnancy hormones on the epigenome and transcriptional output of mammary epithelial cells
.
J. Mammary Gland Biol. Neoplasia
25
,
351
-
366
.
Cockrum
,
E. L.
(
1962
).
Introduction to Mammalogy
.
New York
:
Ronald Press Co
.
Davies
,
S. P.
,
Reddy
,
H.
,
Caivano
,
M.
and
Cohen
,
P.
(
2000
).
Specificity and mechanism of action of some commonly used protein kinase inhibitors
.
Biochem. J.
351
,
95
-
105
.
Deakin
,
J. E.
,
Graves
,
J. A.
and
Rens
,
W.
(
2012
).
The evolution of marsupial and monotreme chromosomes
.
Cytogenet Genome Res.
137
,
113
-
129
.
Dekkers
,
J. F.
,
van Vliet
,
E. J.
,
Sachs
,
N.
,
Rosenbluth
,
J. M.
,
Kopper
,
O.
,
Rebel
,
H. G.
,
Wehrens
,
E. J.
,
Piani
,
C.
,
Visvader
,
J. E.
,
Verissimo
,
C. S.
et al. 
(
2021
).
Long-term culture, genetic manipulation and xenotransplantation of human normal and breast cancer organoids
.
Nat. Protoc.
16
,
1936
-
1965
.
Ewald
,
A. J.
,
Brenot
,
A.
,
Duong
,
M.
,
Chan
,
B. S.
and
Werb
,
Z.
(
2008
).
Collective epithelial migration and cell rearrangements drive mammary branching morphogenesis
.
Dev. Cell
14
,
570
-
581
.
Finot
,
L.
,
Chanat
,
E.
and
Dessauge
,
F.
(
2021
).
Mammary gland 3D cell culture systems in farm animals
.
Vet. Res.
52
,
78
.
Frost
,
S. B.
,
Milliken
,
G. W.
,
Plautz
,
E. J.
,
Masterton
,
R. B.
and
Nudo
,
R. J.
(
2000
).
Somatosensory and motor representations in cerebral cortex of a primitive mammal (Monodelphis domestica): a window into the early evolution of sensorimotor cortex
.
J. Comp. Neurol.
421
,
29
-
51
.
Gentles
,
A. J.
,
Wakefield
,
M. J.
,
Kohany
,
O.
,
Gu
,
W.
,
Batzer
,
M. A.
,
Pollock
,
D. D.
and
Jurka
,
J.
(
2007
).
Evolutionary dynamics of transposable elements in the short-tailed opossum Monodelphis domestica
.
Genome Res.
17
,
992
-
1004
.
Green
,
B.
,
Newgrain
,
K.
and
Merchant
,
J.
(
1980
).
Changes in milk composition during lactation in the tammar wallaby (Macropus eugenii)
.
Aust. J. Biol. Sci.
33
,
35
-
42
.
Griffiths
,
M.
(
1978
).
The Biology of the Monotremes
.
New York
:
Academic Press
.
Harder
,
J. D.
,
Hsu
,
M. J.
and
Garton
,
D. W.
(
1996
).
Metabolic rates and body temperature of the gray short-tailed opossum (Monodelphis domestica) during gestation and lactation
.
Physiol. Zool.
69
,
317
-
339
.
Harman
,
R. M.
,
Das
,
S. P.
,
Bartlett
,
A. P.
,
Rauner
,
G.
,
Donahue
,
L. R.
and
Van de Walle
,
G. R.
(
2021
).
Beyond tradition and convention: benefits of non-traditional model organisms in cancer research
.
Cancer Metastasis Rev.
40
,
47
-
69
.
Hellmen
,
E.
and
Isaksson
,
A.
(
1997
).
Immunohistochemical investigation into the distribution pattern of myoepithelial cells in the bovine mammary gland
.
J. Dairy Res.
64
,
197
-
205
.
Hughes
,
K.
(
2021
).
Comparative mammary gland postnatal development and tumourigenesis in the sheep, cow, cat and rabbit: Exploring the menagerie
.
Semin. Cell Dev. Biol.
114
,
186
-
195
.
Jung
,
P.
,
Sato
,
T.
,
Merlos-Suárez
,
A.
,
Barriga
,
F. M.
,
Iglesias
,
M.
,
Rossell
,
D.
,
Auer
,
H.
,
Gallardo
,
M.
,
Blasco
,
M. A.
,
Sancho
,
E.
et al. 
(
2011
).
Isolation and in vitro expansion of human colonic stem cells
.
Nat. Med.
17
,
1225
-
1227
.
Klopocka
,
W.
and
Redowicz
,
M. J.
(
2004
).
Rho/Rho-dependent kinase affects locomotion and actin-myosin II activity of Amoeba proteus
.
Protoplasma
224
,
113
-
121
.
Kretzschmar
,
K.
and
Clevers
,
H.
(
2016
).
Organoids: Modeling Development and the Stem Cell Niche in a Dish
.
Dev. Cell
38
,
590
-
600
.
Lewis
,
S. M.
,
Callaway
,
M. K.
and
Dos Santos
,
C. O.
(
2022
).
Clinical applications of 3D normal and breast cancer organoids: A review of concepts and methods
.
Exp. Biol. Med. (Maywood)
247
,
2176
-
2183
.
Meyer
,
T. N.
,
Schwesinger
,
C.
,
Sampogna
,
R. V.
,
Vaughn
,
D. A.
,
Stuart
,
R. O.
,
Steer
,
D. L.
,
Bush
,
K. T.
and
Nigam
,
S. K.
(
2006
).
Rho kinase acts at separate steps in ureteric bud and metanephric mesenchyme morphogenesis during kidney development
.
Differentiation
74
,
638
-
647
.
Mikaelian
,
I.
,
Hovick
,
M.
,
Silva
,
K. A.
,
Burzenski
,
L. M.
,
Shultz
,
L. D.
,
Ackert-Bicknell
,
C. L.
,
Cox
,
G. A.
and
Sundberg
,
J. P.
(
2006
).
Expression of terminal differentiation proteins defines stages of mouse mammary gland development
.
Vet. Pathol.
43
,
36
-
49
.
Mikkelsen
,
T. S.
,
Wakefield
,
M. J.
,
Aken
,
B.
,
Amemiya
,
C. T.
,
Chang
,
J. L.
,
Duke
,
S.
,
Garber
,
M.
,
Gentles
,
A. J.
,
Goodstadt
,
L.
,
Heger
,
A.
et al. 
(
2007
).
Genome of the marsupial Monodelphis domestica reveals innovation in non-coding sequences
.
Nature
447
,
167
-
177
.
Munson
,
L.
and
Moresco
,
A.
(
2007
).
Comparative pathology of mammary gland cancers in domestic and wild animals
.
Breast Dis.
28
,
7
-
21
.
Narumiya
,
S.
and
Thumkeo
,
D.
(
2018
).
Rho signaling research: history, current status and future directions
.
FEBS Lett.
592
,
1763
-
1776
.
Narumiya
,
S.
,
Ishizaki
,
T.
and
Uehata
,
M.
(
2000
).
Use and properties of ROCK-specific inhibitor Y-27632
.
Methods Enzymol.
325
,
273
-
284
.
Nicholas
,
K. R.
(
1988
).
Asynchronous dual lactation in a marsupial, the tammar wallaby (Macropus eugenii)
.
Biochem. Biophys. Res. Commun.
154
,
529
-
536
.
Nilsson
,
M. A.
,
Churakov
,
G.
,
Sommer
,
M.
,
Tran
,
N. V.
,
Zemann
,
A.
,
Brosius
,
J.
and
Schmitz
,
J.
(
2010
).
Tracking marsupial evolution using archaic genomic retroposon insertions
.
PLoS Biol.
8
,
e1000436
.
Oftedal
,
O. T.
(
2002
).
The mammary gland and its origin during synapsid evolution
.
J. Mammary Gland Biol. Neoplasia
7
,
225
-
252
.
Oftedal
,
O. T.
and
Dhouailly
,
D.
(
2013
).
Evo-devo of the mammary gland
.
J. Mammary Gland Biol. Neoplasia
18
,
105
-
120
.
Pasic
,
L.
,
Eisinger-Mathason
,
T. S.
,
Velayudhan
,
B. T.
,
Moskaluk
,
C. A.
,
Brenin
,
D. R.
,
Macara
,
I. G.
and
Lannigan
,
D. A.
(
2011
).
Sustained activation of the HER1-ERK1/2-RSK signaling pathway controls myoepithelial cell fate in human mammary tissue
.
Genes Dev.
25
,
1641
-
1653
.
Peaker
,
M.
(
2023
).
Dairy animals and breast cancer: reflections on a long-term study from the 1970s that was never done
.
J. Dairy Res.
90
,
26
-
27
.
Petrovic
,
A.
,
Ban
,
J.
,
Tomljanovic
,
I.
,
Pongrac
,
M.
,
Ivanicic
,
M.
,
Mikasinovic
,
S.
and
Mladinic
,
M.
(
2021
).
Establishment of long-term primary cortical neuronal cultures from neonatal opossum monodelphis domestica
.
Front. Cell Neurosci.
15
,
661492
.
Prpar
,
S. M.
and
Dovc
,
P.
(
2013
).
Mammary tumors in ruminants
.
Acta Agriculturae Slovenica
102
,
83
-
86
.
Puzzolo
,
E.
and
Mallamaci
,
A.
(
2010
).
Cortico-cerebral histogenesis in the opossum Monodelphis domestica: generation of a hexalaminar neocortex in the absence of a basal proliferative compartment
.
Neural Dev.
5
,
8
.
Rauner
,
G.
,
Jin
,
D. X.
,
Miller
,
D. H.
,
Gierahn
,
T. M.
,
Li
,
C. M.
,
Sokol
,
E. S.
,
Feng
,
Y. X.
,
Mathis
,
R. A.
,
Love
,
J. C.
,
Gupta
,
P. B.
et al. 
(
2021
).
Breast tissue regeneration is driven by cell-matrix interactions coordinating multi-lineage stem cell differentiation through DDR1
.
Nat. Commun.
12
,
7116
.
Rauner
,
G.
,
Traugh
,
N. C.
,
Trepicchio
,
C. J.
,
Parrish
,
M. E.
,
Mushayandebvu
,
K.
and
Kuperwasser
,
C.
(
2023
).
Advancements in human breast organoid culture: modeling complex tissue structures and developmental insights
.
bioRxiv
2023.10.02.560364
.
Richards
,
J. A.
and
Brueggemeier
,
R. W.
(
2003
).
Prostaglandin E2 regulates aromatase activity and expression in human adipose stromal cells via two distinct receptor subtypes
.
J. Clin. Endocrinol. Metab.
88
,
2810
-
2816
.
Riento
,
K.
and
Ridley
,
A. J.
(
2003
).
Rocks: multifunctional kinases in cell behaviour
.
Nat. Rev. Mol. Cell Biol.
4
,
446
-
456
.
Rousmaniere
,
H.
,
Silverman
,
R.
,
White
,
R. A.
,
Sasaki
,
M. M.
,
Wilson
,
S. D.
,
Morrison
,
J. T.
and
Cruz
,
Y. P.
(
2010
).
Husbandry of Monodelphis domestica in the study of mammalian embryogenesis
.
Lab. Anim. (NY)
39
,
219
-
226
.
Sachs
,
N.
,
de Ligt
,
J.
,
Kopper
,
O.
,
Gogola
,
E.
,
Bounova
,
G.
,
Weeber
,
F.
,
Balgobind
,
A. V.
,
Wind
,
K.
,
Gracanin
,
A.
,
Begthel
,
H.
et al. 
(
2018
).
A living biobank of breast cancer organoids captures disease heterogeneity
.
Cell
172
,
373
-
386.e10
.
Santagata
,
S.
,
Thakkar
,
A.
,
Ergonul
,
A.
,
Wang
,
B.
,
Woo
,
T.
,
Hu
,
R.
,
Harrell
,
J. C.
,
McNamara
,
G.
,
Schwede
,
M.
,
Culhane
,
A. C.
et al. 
(
2014
).
Taxonomy of breast cancer based on normal cell phenotype predicts outcome
.
J. Clin. Invest.
124
,
859
-
870
.
Sato
,
T.
,
Stange
,
D. E.
,
Ferrante
,
M.
,
Vries
,
R. G.
,
Van Es
,
J. H.
,
Brink
,
V. d.
,
Van Houdt
,
S.
,
Pronk
,
W. J.
,
Van Gorp
,
A.
,
Siersema
,
J.
(
2011
).
'Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium'
.
Gastroenterology
141
,
1762
-
1772
.
Schenkelaars
,
Q.
,
Quintero
,
O.
,
Hall
,
C.
,
Fierro-Constain
,
L.
,
Renard
,
E.
,
Borchiellini
,
C.
and
Hill
,
A. L.
(
2016
).
ROCK inhibition abolishes the establishment of the aquiferous system in Ephydatia muelleri (Porifera, Demospongiae)
.
Dev. Biol.
412
,
298
-
310
.
Schneider
,
C. A.
,
Rasband
,
W. S.
and
Eliceiri
,
K. W.
(
2012
).
NIH Image to ImageJ: 25 years of image analysis
.
Nat. Methods
9
,
671
-
675
.
Sharp
,
J. A.
,
Lefevre
,
C.
,
Brennan
,
A. J.
and
Nicholas
,
K. R.
(
2007
).
The fur seal-a model lactation phenotype to explore molecular factors involved in the initiation of apoptosis at involution
.
J. Mammary Gland Biol. Neoplasia
12
,
47
-
58
.
Sharp
,
J. A.
,
Wanyonyi
,
S.
,
Modepalli
,
V.
,
Watt
,
A.
,
Kuruppath
,
S.
,
Hinds
,
L. A.
,
Kumar
,
A.
,
Abud
,
H. E.
,
Lefevre
,
C.
and
Nicholas
,
K. R.
(
2017
).
The tammar wallaby: A marsupial model to examine the timed delivery and role of bioactives in milk
.
Gen. Comp. Endocrinol.
244
,
164
-
177
.
Sokol
,
E. S.
,
Miller
,
D. H.
,
Breggia
,
A.
,
Spencer
,
K. C.
,
Arendt
,
L. M.
and
Gupta
,
P. B.
(
2016
).
Growth of human breast tissues from patient cells in 3D hydrogel scaffolds
.
Breast Cancer Res.
18
,
19
.
Sumbal
,
J.
and
Koledova
,
Z.
(
2019
).
FGF signaling in mammary gland fibroblasts regulates multiple fibroblast functions and mammary epithelial morphogenesis
.
Development
146
,
dev185306
.
Sumbal
,
J.
,
Chiche
,
A.
,
Charifou
,
E.
,
Koledova
,
Z.
and
Li
,
H.
(
2020
).
Primary mammary organoid model of lactation and involution
.
Front. Cell Dev. Biol.
8
,
68
.
Swett
,
W. W.
,
Matthews
,
C. A.
and
Graves
,
R. R.
(
1940
).
'Extreme rarity of cancer in the cow's udder: a negative finding of vital interest to the dairy industry and to the consumer'
.
J. Dairy Sci.
23
,
437
-
446
.
Tojo
,
M.
,
Hamashima
,
Y.
,
Hanyu
,
A.
,
Kajimoto
,
T.
,
Saitoh
,
M.
,
Miyazono
,
K.
,
Node
,
M.
and
Imamura
,
T.
(
2005
).
The ALK-5 inhibitor A-83-01 inhibits Smad signaling and epithelial-to-mesenchymal transition by transforming growth factor-beta
.
Cancer Sci.
96
,
791
-
800
.
Urban
,
D. J.
,
Anthwal
,
N.
,
Luo
,
Z.-X.
,
Maier
,
J. A.
,
Sadier
,
A.
,
Tucker
,
A. S.
and
Sears
,
K. E.
(
2017
).
A new developmental mechanism for the separation of the mammalian middle ear ossicles from the jaw
.
Proc. R. Soc. B
284
,
20162416
.
Walker
,
J. L.
,
Wang
,
W.
,
Lin
,
E.
,
Romisher
,
A.
,
Bouchie
,
M. P.
,
Bleaken
,
B.
,
Menko
,
A. S.
and
Kukuruzinska
,
M. A.
(
2021
).
Specification of the patterning of a ductal tree during branching morphogenesis of the submandibular gland
.
Sci. Rep.
11
,
330
.
Wang
,
G.
,
Woods
,
A.
,
Sabari
,
S.
,
Pagnotta
,
L.
,
Stanton
,
L. A.
and
Beier
,
F.
(
2004
).
RhoA/ROCK signaling suppresses hypertrophic chondrocyte differentiation
.
J. Biol. Chem.
279
,
13205
-
13214
.
Wanyonyi
,
S. S.
,
Lefevre
,
C.
,
Sharp
,
J. A.
and
Nicholas
,
K. R.
(
2013a
).
The extracellular matrix regulates MaeuCath1a gene expression
.
Dev. Comp. Immunol.
40
,
289
-
299
.
Wanyonyi
,
S. S.
,
Lefevre
,
C.
,
Sharp
,
J. A.
and
Nicholas
,
K. R.
(
2013b
).
The extracellular matrix locally regulates asynchronous concurrent lactation in tammar wallaby (Macropus eugenii)
.
Matrix Biol.
32
,
342
-
351
.
Wanyonyi
,
S. S.
,
Kumar
,
A.
,
Du Preez
,
R.
,
Lefevre
,
C.
and
Nicholas
,
K. R.
(
2017
).
Transcriptome analysis of mammary epithelial cell gene expression reveals novel roles of the extracellular matrix
.
Biochem. Biophys. Rep.
12
,
120
-
128
.
Watanabe
,
K.
,
Ueno
,
M.
,
Kamiya
,
D.
,
Nishiyama
,
A.
,
Matsumura
,
M.
,
Wataya
,
T.
,
Takahashi
,
J. B.
,
Nishikawa
,
S.
,
Nishikawa
,
S.
,
Muguruma
,
K.
et al. 
(
2007
).
A ROCK inhibitor permits survival of dissociated human embryonic stem cells
.
Nat. Biotechnol.
25
,
681
-
686
.
Wessel
,
G. M.
(
2016
).
The milk line - where mammary gland meets mathematics
.
Mol. Reprod. Dev.
83
,
1
-
1
.
Woolley
,
P. A.
,
Patterson
,
M. F.
,
Stephenson
,
G. M.
and
Stephenson
,
D. G.
(
2002
).
The ilio-marsupialis muscle in the dasyurid marsupial Sminthopsis douglasi: form, function and fibre-type profiles in females with and without suckling young
.
J. Exp. Biol.
205
,
3775
-
3781
.
Yuan
,
L.
,
Xie
,
S.
,
Bai
,
H.
,
Liu
,
X.
,
Cai
,
P.
,
Lu
,
J.
,
Wang
,
C.
,
Lin
,
Z.
,
Li
,
S.
,
Guo
,
Y.
et al. 
(
2023
).
Reconstruction of dynamic mammary mini gland in vitro for normal physiology and oncogenesis
.
Nat. Methods
20
,
2021
-
2033
.
Zhang
,
X.
,
Martinez
,
D.
,
Koledova
,
Z.
,
Qiao
,
G.
,
Streuli
,
C. H.
and
Lu
,
P.
(
2014
).
FGF ligands of the postnatal mammary stroma regulate distinct aspects of epithelial morphogenesis
.
Development
141
,
3352
-
3362
.

Competing interests

G.R. consults for Turtle Tree. C.K. is a co-founder and consultant of Naveris.

Supplementary information