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Mammary Gland Organoids Reveal Species-specific Traits

    Stem cells building up organoids in solution

    Written by: Jyoti Madhusoodanan, Ph.D. | Issue # 119 | 2024

    • Stem cells from diverse mammals can be grown in labs to form branched mammary gland organoids with lobes and ducts.
    • Culture conditions differ and reveal key growth factors necessary for some species but not others. 
    • Mammary gland organoids could help understand cancer susceptibility and factors that influence milk composition. 

    Mammary glands have evolved to produce a vast array of milks to support offspring. In ancient mammals such as monotremes, mammary glands are simply hairy patches. Some species of wallabies and kangaroos can produce distinct kinds of milk from different teats to support offspring of different ages. In humans and mice, these same glands take on elaborate structures with several ducts. These diverse paths to milk production have been difficult to study in laboratories, in part because of a lack of model systems. 

    In a new study [1], Gat Rauner, a developmental biology researcher at Tufts University in Boston, and her colleagues successfully cultivated 3-D organoids from mammary cells of several different species: one kind of opossum, cows, goats, hamsters, rats, rabbits, cats, ferrets, and pigs. 

    Rauner had wanted to study the mammary glands of diverse species ever since she learned as a graduate student that cows and other species were curiously resistant to cancer. “The mechanisms are really unknown and under-studied,” she said.  

    Such research has been challenging because of a lack of feasible laboratory models. Many species with different susceptibility or resistance to cancer are either wild or large animals and thus difficult to maintain in laboratories for studies. Mammary glands pose an additional hurdle because they undergo dramatic, temporary changes during lactation, which can be difficult to study in a laboratory if animals only reproduce infrequently or don’t do so in captivity. 

    If they could be grown, organoids would help circumvent the problem of needing an entire animal in the lab. But earlier attempts at producing mammary gland organoids from species other than humans and mice resulted in blobs of cells that lacked the characteristic features and functions of mammary tissue. “A lot of organoid models didn’t really have the morphology that is so characteristic of the mammary gland,” Rauner said.

    During her postdoctoral research, Rauner began collecting stem cells from mammary glands of various species. Nudging these cells to form glands requires a precise combination of growth conditions. Organoids from other species didn’t grow under the same culture conditions that work for stem cells from humans and mice. After many failed attempts, Rauner and her team discovered that tweaking the cell culture medium and the rigidity of the extracellular matrix, a gel-like support structure necessary for cells growth, was the key to success. 

    Using Intesticult, a growth medium designed to culture intestinal epithelial cells, worked best for all the species tested in this study, resulting in organoids with the branched, lobular- ductal structures seen in mature animals. When Rauner and her colleagues examined the ingredients of Intesticult individually, they found that components that inhibited the ROCK protein were important for stem cells to form branched structures. When grown in minimal media, opossum cells required the presence of the ROCK inhibitor to form branched organoids. This same culture condition caused human and rat stem cells to produce abnormal, hyper-branched organoids. Cow, goat, and rabbit organoids, on the other hand, required ROCK inhibition for successful growth. “By parsing out the specific needs of each species to activate the proper developmental program, we can learn what signals are necessary for each and compare that between species,” Rauner said. “In this sense, it’s the experimental process that teaches us about the biology.” 

    In the future, such models could also help study components of milk produced by different species at various stages of lactation, Rauner said. “We don’t actually have a good grasp of how milk composition is regulated,” she explained. Small sugary molecules known as oligosaccharides in milk are crucial modulators of an infant’s development and immune health. In humans, these molecules, known as human milk oligosaccharides (HMOs), mediate gut and brain development, predict allergy risk, and confer protection against infections. However, oligosaccharides are formed by modifying carbohydrates and proteins after synthesis, so their characteristics can’t be deduced by studying genetic or protein sequences. “You have to be able to collect them from milk and then look at them,” Rauner said. 

    Being able to secrete milk from organoids, collect it, and examine how different growth conditions impact oligosaccharides and other components could help researchers learn how milk composition is regulated – even in the absence of a live, lactating animal. “Having these organoids really opens up the possibility to study these questions without actually having access to the animals themselves,” Rauner said.

    References

    Kim HY, Sinha I, Sears KE, Kuperwasser C, Rauner G. Expanding the evo-devo toolkit: generation of 3D mammary tissue from diverse mammals. Development. 2024 Jan 15;151(2).