Cell Types and Metabolites in Human Milk Change Over Lactation

Written by: Alla Katsnelson, Ph.D | Issue # 114 | 2023

Human milk is a highly nourishing food for infants and babies—not only does it fulfill their nutritional needs, but it also provides extensive health benefits. Although human milk is known to contain a diverse population of cells and metabolites (1), scientists are just beginning to characterize these components in the hope of better understanding their health-promoting properties as well as the relationship between maternal and infant wellness. 

Advanced molecular techniques allow researchers to identify milk’s cellular components in unprecedented detail. A few years ago, researchers first used an approach called single cell RNA sequencing (scRNAseq) to investigate gene expression in individual cells from milk of two post-partum women, identifying a surprising diversity of cell types (2). Since then, a handful of studies have also investigated the cellular fraction of human milk using this technique. “All of the studies have nicely validated each other,” said Britt Goods, an assistant professor of engineering at Dartmouth College in Hanover, New Hampshire. 

In one such study published last year, Goods and her colleagues used scRNAseq to track how the cellular components of human milk changed at different stages of lactation (3). The researchers analyzed milk samples from 15 women taken over approximately 21 months at multiple timepoints during lactation. The samples included the early milk that comes in during the first 5 days postpartum, transitional milk that is present 6-14 days after delivery during a period of rapid change in breast tissue, mature milk that comes in after about 2 weeks, and late-stage milk from mothers who are weaning their babies to solid foods. 

The researchers identified two different populations of epithelial cells called lactocytes whose proportions shift over the duration of breastfeeding, suggesting structural changes occurring in the breast during that time (3). One of these lactocyte populations, called LC1, increased as a function of time postpartum while the other, called LC2, decreased. Gene expression profiles from the cells in each group suggest that cells in the LC1 cluster provide structural support for the breast during later stages of lactation—for example, playing a role in the formation of tight junctions between cells—whereas LC2 cells produce milk components. Goods cautions, however, that she and her colleagues have not yet validated this hypothesis and that more milk samples would be needed to draw firm conclusions. Day care attendance and the use of hormonal birth control were also associated with the predominance of LC1 levels.

The researchers also identified a significant presence of immune cells called macrophages, which play a role in responding to tissue injury. “We think immune cells are taking on these anti-inflammatory phenotypes to support the breast as it’s responsible for making this tremendous amount of milk,” Goods said. There’s no evidence currently that these cells transfer to the infant, but it is possible that they do, and that they secrete molecules there that support gut health, she added.

Overall, the researchers found a highly diverse population of epithelial cells in human milk, and found that this diversity increased in later stages of lactation. “This could suggest some specialization of these cells happening over time, where these cells might be making or secreting substances or dividing the labor a bit differently in earlier compared to later time points,” Goods said. She and her colleagues are now following up on some of these findings, particularly functional differences in the LC1 and LC2 populations. “The big question now is what does this all mean from a biology standpoint,” Goods said.

Meanwhile, other studies are exploring the cellular content of human milk by analyzing its metabolome, that is, by characterizing the sugars, lipids, amino acids, and other molecules it contains. One study, published in 2022, analyzed 101 milk samples collected from 59 mothers over 25 months. The researchers observed a dynamic pattern over the course of lactation, with some metabolites present at higher levels earlier in lactation and others at higher levels later on [4]. “Overall, we find that the metabolome of [human milk] continuously varies throughout lactation and some changes are distinctive for milk excreted after 1 to 2 months of lactation,” the authors write. 

A more recent study, published in 2023, took a slightly different approach (5). Here, the researchers analyzed 15 human milk samples collected at different postnatal times in the first year of lactation. However, unlike in the previous study, where researchers extracted metabolites from the entire milk sample, in this study they first isolated the cells and then extracted the metabolites only from the milk’s cell fraction. 

The team noted that cell counts in the samples decreased along lactation (5), an observation that confirmed previous work (6). Because the overall number of cells was low, it was difficult to conduct a full metabolic analysis, said Isabel Ten-Doménech, a postdoctoral researcher studying neonatal health at the Health Research Institute Hospital La Fe in Valencia, Spain, and the first author of that work. Overall, however, the team found a decrease in the number of metabolites detected over time. Out of the 53 total metabolites identified, the study found two that became more prevalent during the sampling year [5]. “We hypothesize that this is because these two metabolites are related with cell death,” said Ten-Doménech. She also noted that the study’s small sample size made its conclusions preliminary. 

Just as nursing infants continually change, the cells and metabolites of their mother’s milk also change. Better understanding of these changes in milk will yield tools for promoting healthy lactation and improving infant nutrition.

References

1. Witkowska-Zimny M, Kaminska-El-Hassan E. Cells of human breast milk. Cell Mol Biol Lett. 2017;22(1):1-11.
2. Martin Carli JF, Trahan GD, Jones KL, Hirsch, N, Rolloff, KP, Dunn, EZ, Friedman, JE. Barbour, LA. Hernandez, TL, MacLean, PS. Monks, J. Single cell RNA sequencing of human milk-derived cells reveals sub-populations of mammary epithelial cells with molecular signatures of progenitor and mature states: a novel, non-invasive framework for investigating human lactation physiology. J Mammary Gland Biol Neoplasia. 2020;25(4):367-387.
3. Nyquist SK, Gao P, Haining TKJ, Retchin MR, Golan Y, Drake RS, Kolb K, Mead BE, Ahituv N, Martinez ME, Shalek AK, Berger B, Goods BA. Cellular and transcriptional diversity over the course of human lactation. Proc Natl Acad Sci U S A. 2022; 119(15):e2121720119.
4. Poulsen KO, Meng F, Lanfranchi E, Young JF, Stanton C, Ryan CA, Kelly AL, Sundekilde UK. Dynamic changes in the human milk metabolome over 25 weeks of lactation. Front Nutr. 2022;9:917659.
5. Ten-Doménech I, Cascant-Vilaplana MM, Navarro-Esteve V, Felderer B, Moreno-Giménez A, Rienda I, Gormaz M, Moreno-Torres M, Pérez-Guaita D, Quintás G, Kuligowski J. Metabolomic diversity of human milk cells over the course of lactation-a preliminary study. Nutrients. 2023 Feb 22;15:1100.
6. Hassiotou F. Hepworth AR, Metzger P, Tat Lai C, Trengove N, Hartmann PE, Filgueira L. Maternal and Infant Infections Stimulate a Rapid Leukocyte Response in Breastmilk. Clin Transl Immunol. 2013 Apr 12; 2(4), e3.

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