Thursday, 24 January 2019

Stem Cells in Breast Milk - method development

The presence of maternal stem cells in human breast milk became of interest via a number of publications over the last decade.  However, there has been no satisfactory, robust and reliable method for the detection of these cells in the complex matrix of milk, lipid-rich and with considerable cellular debris even in the cell pellet.  Without such a method it is difficult to begin to establish the importance of these cells to the feeding infant's development and whether cell numbers correlate with gestational status.

A new reference method is proposed for the detection of these breast milk-borne stem cells, developed by scientists at the University of Cologne (Uniklinik Köln).  

Having reviewed the previous strategies and discovered their various limitations to reliably enumerate viable putative stem cells (as defined by TRA-1-81 positivity) the authors tested the far-red cell permeant DNA dye DRAQ5™ to resolve intact nucleated cells from the complex matrix (of cellular debris and lipid droplets) and combined this with the spectrally compatible viability dye SYTOX® Blue.  This combination proved successful and was used with antibodies identifying the surface antigens CD11b (-FITC), for monocytes, and TRA-1-81 (-PE), for putative stem cells and, by inference, the double-negative nucleated cell population as other lymphocytes.

There is further scope to use this reagent panel with a marker of epithelial cells that would be expected to present in breast milk. However, the method cannot be used for the detection of stem cell-associated transcription factors e.g. SOX2, Nanog, Oct4 since these require cell permeabilisation.  Nonetheless, phenotypic sorting of the cell populations, as described in this work, could allow molecular interrogation by downstream RNA-seq/transcriptomics to provide significant information.

Reference:
Keller T, Wengenroth L, Smorra D, Probst K, Kurian L, Kribs A, Brachvogel B. Novel DRAQ5™/SYTOX® Blue Based Flow Cytometric Strategy to Identify and Characterize Stem Cells in Human Breast Milk. Cytometry Part B: Clinical Cytometry. 2018 Nov 26.

SYTOX® is a registered trademark of Thermo Fisher Scientific