Novel DRAQ5™/SYTOX® Blue Based Flow Cytometric Strategy to Identify and Characterize Stem Cells in Human Breast Milk

Abstract

Background: Human breast milk could be an important stem cell source for the development of newborn and preterm infants, but quantitative data on the stem cell content in breast milk at various gestational stages are needed to determine the clinical value of breast milk as a source of stem cells. Breast milk also contains milk fat globules, lipid droplets of different sizes, debris and dead cells and these components hamper flow cytometry analysis of human breast milk samples.Methods: Here, we originally used standard protocols for flow cytometry to characterize cell populations in human breast milk but failed to discriminate between cells and noncellular components. We then applied a centrifugation protocol to separate cream and skim milk from the cell-containing pellet and used a novel staining protocol with DRAQ5™ and SYTOX ® blue dye as well as antibodies to characterize cells within the pellet fraction.Results: Flow cytometry analysis identified viable DRAQ5™ + /SYTOX ® Blue − cells and determined the content of CD11b + monocytes and TRA-1-81 + putative stem cells in human breast milk samples.Conclusions: Hence, we developed a novel and reliable flow cytometry based-approach to quantify subpopulation of cells in human breast milk with a high content of milk fat globules, lipid droplets, and particles. This approach will improve the identification and quantification of breast milk cells and allow standardizing the flow cytometry-based evaluation of the stem cell content.

Morphological Analysis of Human Milk Membrane Enclosed Structures Reveals Diverse Cells and Cell-like Milk Fat Globules

“…Whilst past publications may have considered all MESs in the pelleted fraction of milk as cells, our data corroborates recent findings suggesting that merely 9% of all pelleted MESs are HMCs [17]. Indeed, using both flow cytometry and fluorescence microscopy, we found that whilst a majority of MESs isolated from human milk absorb lipid stain Nile red, only a small subset are nucleated and incorporate DNA stains DRAQ5™ and/or Hoechst 33258 ( Fig.…”

“…However, recovered live HMCs ranged from 5 to 27.6% of total expected HMCs, thereby lower than the previously reported number of live cells per sample (8 to 81% of HMCs) [17]. Further analysis should thereby assess whether any unaccounted live cells are retained inside of the column.…”

“…Christina H. Scheel and Alecia-Jane Twigger shared senior authorship Together with earlier publications [15,16], a recent study by Keller et al [17] suggests that only a minority of pelleted structures obtained by centrifugation of human milk are in fact nucleated. On average only 9% (range 0.7-63%) of recorded events were found to stain positive for nuclear dye DRAQ5™ and of these 8 to 81% represent viable cells that excluded dead cell stain Sytox™ Blue [17]. Remarkably, it was found that a majority of structures isolated from human milk are nonnucleated particles that absorb neutral lipid stain Nile red [17] suggesting they likely represent milk fat globules (MFGs).…”

“…Due to the high concentration of MFGs in milk, accurate characterization and quantification of HMCs may be impaired. MFGs are directly secreted by HMCs (specifically secretory epithelial cells within the alveoli of the lactating mammary gland) and often contain HMC-derived cytoplasm, membrane proteins of different cellular origin, mRNA and organelles [6,15,17,21,27,[33][34][35], making it difficult to molecularly distinguish between the MESs. Despite these limitations, it still remains necessary to purify and quantify viable single cells from human milk, to effectively conduct single cell analysis or quantify colony forming ability of HMCs.…”

Stem cells in human breast milk

“…Keller et al have developed an improved protocol that use a recently developed staining agents DRAQ5™ far red and SYTOX ® blue for flow cytometry analysis of breast cells in human milk. This novel and reliable approach allowed to identify and quantify subpopulation of CD11b + monocytes as well as putative stem cells positive for cell surface markers TRA-1-81 and SSEA-4 [ 52 ]. Interestingly, for TRA-1-81 + putative stem cells the high variations were detected in samples without significant differences between the gestational age groups.…”