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Abstract
Over the past decade, the cellular content of human milk has been a focus in lactation research due to the benefit a potential non-invasive stem cell compartment could provide either to the infant or for therapeutic applications. Despite an increase in the number of studies in this field, fundamental knowledge in regard to milk cell identification and characterisation is still lacking. In this project, we investigated the nature, morphology and content of membrane enclosed structures (MESs) and explored different methods to enrich human milk cells (HMCs) whilst reducing milk fat globule (MFG) content. Using both flow cytometry and immunofluorescence imaging, we confirmed previous reports and showed that nucleated HMCs make up a minority of milk-isolated MESs and are indistinguishable from MFGs without the use of a nuclear stain. HMC heterogeneity was demonstrated by differential uptake of nuclear stains Hoechst 33258 and DRAQ5™ using a novel technique of imaging milk MESs (by embedding them in agar), that enabled examination of both extracellular and intracellular markers. We found that MESs often contain multiple lipid droplets of various sizes and for the first time report that late post-partum human milk contains secretory luminal binucleated cells found across a number of participants. After investigation of different techniques, we found that viably freezing milk cells is an easy and effective method to substantially reduce MFG content of samples. Alternatively, milk MESs can be filtered using a MACS® filter and return a highly viable, though reduced population of milk cells. Using the techniques and findings we’ve developed in this study; future research may focus on further characterising HMCs and the functional secretory mammary epithelium during lactation.
Highlights
Breastfeeding provides rich and adaptive milk constituents to the developing infant promoting brain development and cognitive functioning as well as reducing the lifetime risk for the infant acquiring diseases such as diabetes and obesity [1,2,3]
Using both flow cytometry and fluorescence microscopy, we found that whilst a majority of membrane enclosed structures (MESs) isolated from human milk absorb lipid stain Nile red, only a small subset are nucleated and incorporate DNA stains DRAQ5TM and/or Hoechst 33258 (Fig. 1, Fig. 2)
Flow cytometry revealed that Hoechst 33258 stained significantly less MESs than DRAQ5TM (1.7% of total MESs compared to 7.3% of total MESs, P = 0.007) where only 0.9% of MESs stained positive for both nuclear stains (Fig. 1A, C)
Summary
Breastfeeding provides rich and adaptive milk constituents to the developing infant promoting brain development and cognitive functioning as well as reducing the lifetime risk for the infant acquiring diseases such as diabetes and obesity [1,2,3]. Studies suggest that a proportion of this cell fraction might survive the passage of the infant’s gastrointestinal tract and may play an important role in providing benefits to the breastfeeding new born [5, 10,11,12,13]. Based on these findings, human clinical trials have been initiated to investigate the potential beneficial effects of administrating human milk through the intranasal cavity of tube-fed pre-term infants [14]
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