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Profiling of mature-stage human breast milk cells identifies six unique lactocyte subpopulations.

ABSTRACT: Breast milk is chock-full of nutrients, immunological factors, and cells that aid infant development. Maternal cells are the least studied breast milk component, and their unique properties are difficult to identify using traditional techniques. Here, we characterized the cells in mature-stage breast milk from healthy donors at the protein, gene, and transcriptome levels. Holistic analysis of flow cytometry, quantitative polymerase chain reaction, and single-cell RNA sequencing data identified the predominant cell population as epithelial with smaller populations of macrophages and T cells. Two percent of epithelial cells expressed four stem cell markers: SOX2, TRA-1-60, NANOG, and SSEA4. Furthermore, milk contained six distinct epithelial lactocyte subpopulations, including three previously unidentified subpopulations programmed toward mucosal defense and intestinal development. Pseudotime analysis delineated the differentiation pathways of epithelial progenitors. Together, these data define healthy human maternal breast milk cells and provide a basis for their application in maternal and infant medicine.

SUBMITTER: Gleeson JP

PROVIDER: S-EPMC9242445 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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Profiling of mature-stage human breast milk cells identifies six unique lactocyte subpopulations.

Gleeson John P JP Chaudhary Namit N Fein Katherine C KC Doerfler Rose R Hredzak-Showalter Patricia P Whitehead Kathryn A KA

Science advances 20220629 26

Breast milk is chock-full of nutrients, immunological factors, and cells that aid infant development. Maternal cells are the least studied breast milk component, and their unique properties are difficult to identify using traditional techniques. Here, we characterized the cells in mature-stage breast milk from healthy donors at the protein, gene, and transcriptome levels. Holistic analysis of flow cytometry, quantitative polymerase chain reaction, and single-cell RNA sequencing data identified t ...[more]

PMID: 35767604

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Project description:BackgroundIn this work we aimed at sequencing and assembling the goat milk transcriptome corresponding at colostrum and 120 days of lactation. To reconstruct transcripts we used both the genome as reference, and a de novo assembly approach. Additionally, we aimed at identifying the differentially expressed genes (DEGs) between the two lactation stages and at analyzing the expression of genes involved in oligosaccharides metabolism.ResultsA total of 44,635 different transcripts, organized in 33,757 tentative genes, were obtained using the goat genome as reference. A significant sequence similarity match was found for 40,353 transcripts (90%) against the NCBI NT and for 35,701 (80%) against the NR databases. 68% and 69% of the de novo assembled transcripts, in colostrum and 120 days of lactation samples respectively, have a significant match with the merged transcriptome obtained using Cufflinks/Cuffmerge. CSN2, PAEP, CSN1S2, CSN3, LALBA, TPT1, FTH1, M-SAA3, SPP1, GLYCAM1, EEF1A1, CTSD, FASN, RPS29, CSN1S1, KRT19 and CHEK1 were found between the top fifteen highly expressed genes. 418 loci were differentially expressed between lactation stages, among which 207 and 122 were significantly up- and down-regulated in colostrum, respectively. Functional annotation and pathway enrichment analysis showed that in goat colostrum somatic cells predominate biological processes involved in glycolysis, carbohydrate metabolism, defense response, cytokine activity, regulation of cell proliferation and cell death, vasculature development, while in mature milk, biological process associated with positive regulation of lymphocyte activation and anatomical structure morphogenesis are enriched. The analysis of 144 different oligosaccharide metabolism-related genes showed that most of these (64%) were more expressed in colostrum than in mature milk, with eight expressed at very high levels (SLCA3, GMSD, NME2, SLC2A1, B4GALT1, B3GNT2, NANS, HEXB).ConclusionsTo our knowledge, this is the first study comparing goat transcriptome of two lactation stages: colostrum and 120 days. Our findings suggest putative differences of expression between stages and can be envisioned as a base for further research in the topic. Moreover because a higher expression of genes involved in immune defense response, carbohydrate metabolism and related to oligosaccharide metabolism was identified in colostrum we here corroborate the potential of goat milk as a natural source of lactose-derived oligosaccharides and for the development of functional foods.

Project description:Besides nutritional components, breast milk contains diverse microbes, which may be involved in colonization of the infant gut. Expressed milk is often stored for few days in the refrigerator. The aim of this study was to determine the abundance, prevalence and diversity of facultative and strict anaerobic bacteria using culture-dependent and -independent methods, and to determine changes in milk microbial and chemical composition during storage. Samples of mature breast milk from 21 women were collected 3-6 months post-partum and were analyzed fresh or after anaerobic storage for 6 days at 4°C. The cultivable bacterial population was analyzed using the most probable number (MPN) method or plate counts and different media. The abundance of major bacterial groups was determined using quantitative PCR and 16S rRNA gene sequencing. Lactose, lactate, short chain fatty acids (SCFA) and human milk oligosaccharides (HMO) were analyzed using chromatography techniques. Highest mean viable cell counts were obtained in yeast casitone fatty acids (YCFA) broth supplied with mucin (log 4.2 ± 1.8 cells/ml) and lactose (log 3.9 ± 1.4 cells/ml), or Columbia broth (log 3.0 ± 0.7 cells/ml). Mean total bacterial counts estimated by qPCR was 5.3 ± 0.6 log cells/ml, with Firmicutes being the most abundant phylum. The most prevalent bacterial groups were Streptococcus spp. (15/19 samples), Enterobacteriaceae (13/19) and Lactobacillus/Lactococcus/Pediococcus group (12/19). While the average total number of bacterial cells did not change significantly during storage, the prevalence of strict anaerobic Bacteroidetes increased threefold, from 3/19 to 9/19, while in 7 samples Clostridium clusters IV or XIVa became detectable after storage. Major HMO were not degraded. Lactate was present in 18/21 samples after storage (2.3-18.0 mM). Butyrate was detected in 15/21 and 18/21 samples before and after storage, respectively, at concentrations ranging from 2.5 to 5.7 mM. We demonstrate enhanced prevalence and/or abundance of viable strict anaerobes from the Bacteroidetes and Clostridiales after 6-day anaerobic storage of human milk. Our data indicate that anaerobic cold storage did not markedly change total viable bacterial load, while HMO profiles were stable. Anaerobic cold storage of human milk for up to 6 days may be suitable for preserving milk quality for potential microbial transfer to the infant gut.

Project description:BackgroundThe interaction between the human breast milk microbiota and human milk oligosaccharides (HMOs) plays a crucial role in the healthy growth and development of infants. We aimed to clarify the link between the breast milk microbiota and HMOs at two stages of lactation.MethodsThe microbiota and HMOs of 20 colostrum samples (C group, 1-5 days postpartum) and 20 mature milk samples (S group, 42 days postpartum) collected from postpartum mothers were analyzed using 16S rRNA gene high-throughput sequencing and high-performance liquid chromatography-tandem mass spectrometry.ResultThe total average HMO content was significantly higher in the C group than in the S group (6.76 ± 1.40 g/L vs. 10.27 ± 2.00 g/L, p < 0.05). Among the HMOs, the average values of 2'-fucosyllactose (2'-FL, 1.64 ± 1.54 g/L vs. 3.03 ± 1.79 g/L), 3'-sialyllactose (3'-SL, 0.10 ± 0.02 g/L vs. 0.21 ± 0.06), 6'-SL (0.22 ± 0.09 g/L vs. 0.33 ± 0.11 g/L), and lacto-N-triaose 2 (LNT2, 0.03 ± 0.01 g/L vs. 0.16 ± 0.08 g/L) were significantly lower in the S group than in the C group (p < 0.05), while that of 3'-FL was significantly higher in the S group than in the C group (1.35 ± 1.00 g/L vs. 0.41 ± 0.43 g/L, p < 0.05). The diversity and structure of the microbiota in the S and C groups were also significantly different (p < 0.05). Comparative analysis of the microbial communities revealed that Proteobacteria and Firmicutes were the most abundant phyla, in both groups, with the keystone species (Serratia, Streptococcus and Staphylococcus) of breast milk closely interacting with HMOs, including 3'-SL, 6'-SL, and LNT2. In PICRUSt2 functional prediction analysis, the S group exhibited significant reduction in the expression of genes involved in several infectious disease pathways.DiscussionOur findings support the recognition of human milk as a synbiotic comprising beneficial bacteria and prebiotic HMOs.

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Profiling of mature-stage human breast milk cells identifies six unique lactocyte subpopulations.

Publications

Profiling of mature-stage human breast milk cells identifies six unique lactocyte subpopulations.

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