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Human breast milk (BM) has been nutritionally and immunologically established as a complex and beneficiary fluid to infants. While some components of BM, such as proteins and antibodies have been extensively studied, the cellular profile of BM has yet to be thoroughly characterized. BM contains a heterogeneous population of maternal cells, including leukocytes, epithelial cells, and mammary gland stem cells. These maternal cells can colonize and penetrate the infant gastrointestinal tract; however, the impact of different cell types and their physiological role is not well understood and necessitates further resolution. In this study, we aimed to define and characterize population cell types using single cell RNA sequencing. We sequenced freshly isolated BM cells from 8 new mothers within the first week of birth and again at two weeks of age, covering several gestational periods that range from extremely preterm (<28 weeks), to preterm (28-36 weeks), and term (37-41 weeks) deliveries. Milk cells were pelleted and washed, and the soluble portion of the milk was collected for future tangential analysis. Library preparation was performed according to instruction in the 10X Chromium 3’ single-cell kit. Processing was performed using the 10X Genomics workflow. The Cell Ranger Software Suite was used for demultiplexing, barcoding, and UMI quantification. Samples were pre-processed in aggregate. Cell ranger yielded over 93,000 cells from 11 samples. Processing, clustering and marker identification were handled in R using the Seurat and SingleR packages. Cell types were assigned using CellMarker and Celldex reference datasets. We collected cell type data on over 54,000 cells across 27 clusters. The most abundant cells in BM were epithelial cells, macrophages, and neutrophils. Surprisingly, several neural cell types were found in BM. Future analysis will explore the differences in cell types based on gestational periods and infant health, as well as the cellular composition over time.

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The Cellular Genomics Of Human Breast Milk