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:
PROVIDER: PRJNA644415 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12159538_1.fastq.gz | Fastqsanger.gz | |||
| SRR12159538_2.fastq.gz | Fastqsanger.gz | |||
| SRR12159539_1.fastq.gz | Fastqsanger.gz | |||
| SRR12159539_2.fastq.gz | Fastqsanger.gz | |||
| SRR12159540_1.fastq.gz | Fastqsanger.gz |
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Project description: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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Project description:Milk derived live mammary epithelial cells were characterized by single cell RNA sequencing.
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Project description:Human milk fat globules, by enveloping cell contents during their secretion into milk, are a rich source of mammary cell RNA. Here, we pair this non-invasive mRNA source with RNA sequencing technology to probe the milk fat layer transcriptome during three stages of lactation: colostral, transitional, and mature milk production. We find that transcriptional profiles cluster not by postpartum day, but by milk Na:K ratio, indicating that women sampled during the same postpartum time frame could be at markedly different stages of gene expression. Each stage of lactation is characterized by a dynamic range (105-fold) in transcript abundances not previously observed with microarray technology. We discovered that transcripts for isoferritins and cathepsins are strikingly abundant during colostrum production, highlighting the potential importance of these proteins for neonatal health. Two transcripts, encoding β-casein (CSN2) and a-lactalbumin (LALBA), make up 45% of the total pool of mRNA in mature lactation. Genes significantly expressed across all stages of lactation are associated with making, modifying, transporting, and packaging milk proteins. Stage-specific transcripts are associated with immune defense during the colostral stage, up-regulation of the machinery needed for milk protein synthesis during the transitional stage, and the production of lipids during mature lactation. We observed strong modulation of key genes involved in lactose synthesis and insulin signaling. In particular, PTPRF may serve as a biomarker linking insulin resistance with insufficient milk supply. This study provides the methodology and reference data set to enable future targeted research on the physiological contributors to sub-optimal lactation in humans.
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Project description:Human milk fat globules, by enveloping cell contents during their secretion into milk, are a rich source of mammary cell RNA. Here, we pair this non-invasive mRNA source with RNA sequencing technology to probe the milk fat layer transcriptome during three stages of lactation: colostral, transitional, and mature milk production. We find that transcriptional profiles cluster not by postpartum day, but by milk Na:K ratio, indicating that women sampled during the same postpartum time frame could be at markedly different stages of gene expression. Each stage of lactation is characterized by a dynamic range (105-fold) in transcript abundances not previously observed with microarray technology. We discovered that transcripts for isoferritins and cathepsins are strikingly abundant during colostrum production, highlighting the potential importance of these proteins for neonatal health. Two transcripts, encoding M-NM-2-casein (CSN2) and a-lactalbumin (LALBA), make up 45% of the total pool of mRNA in mature lactation. Genes significantly expressed across all stages of lactation are associated with making, modifying, transporting, and packaging milk proteins. Stage-specific transcripts are associated with immune defense during the colostral stage, up-regulation of the machinery needed for milk protein synthesis during the transitional stage, and the production of lipids during mature lactation. We observed strong modulation of key genes involved in lactose synthesis and insulin signaling. In particular, PTPRF may serve as a biomarker linking insulin resistance with insufficient milk supply. This study provides the methodology and reference data set to enable future targeted research on the physiological contributors to sub-optimal lactation in humans. Milk fat mRNA profiles were generated from Day 2 and mature milk samples obtained from lactating mothers
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Project description:The QSi5 inbred strain of mice was established from an outbred Quackenbush-Swiss strain by full-sib inbreeding and selection on the basis of increased litter size and shortened inter-litter interval in the Department of Veterinary Physiology (later REPROGEN) , University of Sydney (Holt et al., 2004). The strain has an average litter size of more than 13 pups, and females commonly nurse up to 18 pups with greater than 90% survival to weaning. Along with an increased body weight (BW), these traits are clearly indicative of enhanced lactation performance (Knight et al., 1986). Indeed lactation performance, assessed by a weigh-suckle-weigh method, was 3-fold greater in QSi5 mice than the CBA strain (Riley et al., 2006). In this study, we utilize the divergent phenotypes of QSi5 and CBA/CaH mice to identify genes associated with enhanced mammary gland capacity. Experiment Overall Design: Five replicates from each of the two strains CBA and QSi5 were used for gene expression profiling. Mice were force weaned after 9 days of first lactation and the fourth inguinal mammary glands were collected.
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