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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

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Abstract

Cells in human milk are an untapped source, as potential “liquid breast biopsies”, of material for investigating lactation physiology in a non-invasive manner. We used single cell RNA sequencing (scRNA-seq) to identify milk-derived mammary epithelial cells (MECs) and their transcriptional signatures in women with diet-controlled gestational diabetes (GDM) with normal lactation. Methodology is described for coordinating milk collections with single cell capture and library preparation via cryopreservation, in addition to scRNA-seq data processing and analyses of MEC transcriptional signatures. We comprehensively characterized 3740 cells from milk samples from two mothers at two weeks postpartum. Most cells (>90%) were luminal MECs (luMECs) expressing lactalbumin alpha and casein beta and positive for keratin 8 and keratin 18. Few cells were keratin 14+ basal MECs and a small immune cell population was present (<10%). Analysis of differential gene expression among clusters identified six potentially distinct luMEC subpopulation signatures, suggesting the potential for subtle functional differences among luMECs, and included one cluster that was positive for both progenitor markers and mature milk transcripts. No expression of pluripotency markers POU class 5 homeobox 1 (POU5F1, encoding OCT4) SRY-box transcription factor 2 (SOX2) or nanog homeobox (NANOG), was observed. These observations were supported by flow cytometric analysis of MECs from mature milk samples from three women with diet-controlled GDM (2–8 mo postpartum), indicating a negligible basal/stem cell population (epithelial cell adhesion molecule (EPCAM)−/integrin subunit alpha 6 (CD49f)+, 0.07%) and a small progenitor population (EPCAM+/CD49f+, 1.1%). We provide a computational framework for others and future studies, as well as report the first milk-derived cells to be analyzed by scRNA-seq. We discuss the clinical potential and current limitations of using milk-derived cells as material for characterizing human mammary physiology.

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Data Availability

To review GEO accession GSE153889, go to https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE153889.

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Acknowledgements

This work was supported by grants from the National Institutes of Health, including the National Institute of Diabetes and Digestive and Kidney Diseases (P30 DK048520 – PSM, JFMC and MCR, T32 DK007658-28 – JFMC, 5R01DK101659 – TLH and LAB, K01 DK109079 – MCR, & R03 DK122189-01 – MCR), the Eunice Kennedy Shriver National Institute of Child Health and Human Development (5T32HD007186-40 – JFMC & 5R01HD093729-02 – JLM and JM), the National Institute of Aging (U54 AG062319 – PSM) and the National Center for Advancing Translational Sciences (UL1 TR002535 – MCR). The authors wish to thank the anonymous reviewers who clarified and strengthened the analysis described here. We also thank the study participants for donating their precious milk samples and David Orlicky for numerous insightful discussions related to this study.

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Authors and Affiliations

  1. Department of Pediatrics, Section of Nutrition, University of Colorado Anschutz Medical Campus, Aurora, CO, USA

    Jayne F. Martin Carli

  2. Department of Pediatrics, Section of Hematology, Oncology, and Bone Marrow Transplant, University of Colorado Anschutz Medical Campus, Aurora, CO, USA

    G. Devon Trahan & Kenneth L. Jones

  3. Department of Cell Biology, Oklahoma University Health Sciences Center, Oklahoma City, OK, USA

    Kenneth L. Jones

  4. Department of Medicine, Division of Endocrinology, Metabolism and Diabetes, University of Colorado Anschutz Medical Campus, Aurora, CO, USA

    Nicole Hirsch, Kristy P. Rolloff, Emily Z. Dunn, Linda A. Barbour, Teri L. Hernandez, Paul S. MacLean & Michael C. Rudolph

  5. Department of Pediatrics, Section of Neonatology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA

    Jacob E. Friedman

  6. Department of Physiology, Oklahoma University Health Sciences Center, Oklahoma City, OK, USA

    Jacob E. Friedman & Michael C. Rudolph

  7. Department of Obstetrics & Gynecology, Division of Maternal-Fetal Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA

    Linda A. Barbour

  8. College of Nursing, University of Colorado Anschutz Medical Campus, Aurora, CO, USA

    Teri L. Hernandez

  9. Department of Obstetrics & Gynecology, Division of Reproductive Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO, USA

    Jenifer Monks & James L. McManaman

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  1. Jayne F. Martin Carli
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Contributions

JFMC and MCR conceived of the project and JFMC, MCR, JLM and JM designed the experiments with input from LAB, TLH, JEF and PSM. NH, KPR and EZD collected the samples. JFMC performed the experiments and JFMC, GDT and MCR analyzed the data. JFMC, GDT, MCR, JLM, JM and KLJ prepared the manuscript, which all other authors critically reviewed and approved.

Corresponding author

Correspondence to Jayne F. Martin Carli.

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The authors declare that there are no conflicts of interest.

Ethics Approval

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Milk-derived cells were collected from deidentified samples obtained for Clinical Trial #NCT02244814. This trial was approved by the Colorado Multiple Institution Review Board (COMIRB #14–1538).

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All participants gave their informed consent for milk collection and samples were deidentified prior to experimentation for this study.

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All authors declare their consent to the publication of this work.

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All code has been submitted in the supplemental methods file (“Martin_Carli_MEC_scRNA-seq_Markdown.html”).

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Martin Carli, J.F., Trahan, G.D., Jones, K.L. et al. 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 25, 367–387 (2020). https://doi.org/10.1007/s10911-020-09466-z

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