Isolation of trophoblast Hoescht side populations from human first trimester villi (#222)
OBJECTIVES: We currently understand very little about the cell fate decisions in early placental development. Whether placental villi contain trophoblast stem cells has not yet been established. When stained with Hoescht and analysed by flow cytometry, many stem cells form a ‘side-population’ as a result of their ability to rapidly efflux the stain. We used this trait to examine whether trophoblast stem cells reside in first trimester villi and to isolate these cells to use as a model of human trophoblast differentiation. METHODS: Villi from first trimester placentae were dissected from the membranes, digested overnight in trypsin, and harvested by repeated washing with PBS. Cells were stained with Hoescht-33342 and Propidium Iodide. Live Hoescht side-populations were isolated using a Fluorescence Activated Cell Sorter. Expression of cytokeratin, vimentin, HLA-G and FGFR4 were determined by immunohistochemistry. RESULTS: 94.7% (±2.3%SE, n=3) of live cells obtained from villi were cytokeratin positive. 99.5% of live cells obtained from villi were HLA-G negative (n=4), indicating that these cells were primarily cytotrophoblasts. 1.04% (±0.3SE, range 0.1-3.9%, n=13) of these cells formed a Hoescht side-population. There was no effect of gestational age on the proportion of cells that formed a side-population (p=0.38, n=13). 98.4% (±0.2% SE, n=3) of side-population cells were cytokeratin positive and vimentin negative, confirming they were trophoblasts. Hoescht side-population cells express FGFR4. CONCLUSION: We have isolated a trophoblast population from first trimester villi that form a Hoescht side-population characteristic of stem cells. Side-population cells expressed FGFR4, which is characteristic of adult stem cells, but this receptor is ubiquitously expressed in placenta, highlighting that trophoblast stem cells may have a phenotype that lies between embryonic and adult stem cell populations.Future characterisation trophoblast side-population cells will determine whether they express key stem cell markers and can differentiate into syncytiotrophoblast and extravillous trophoblasts.