| 研究実績の概要 |
The goal of this study was to find optimal conditions for formation of placenta-like structures in differentiated trophoblast cells derived from human ES cells grown on a solid matrix. We tested whether providing a shaped scaffold (micro-bumps of polydimethylsiloxane, PDMS) encouraged the outgrowth of trophoblast cells from human ES cells, but found human cells would not attach to the matrix.
In the final year of this study, we found that plasma treatment of the PDMS mold greatly enhanced the ability of human cells to adhere and grow. Using plasma-treated molds we succeeded in attaching human ES cells and differentiating them into trophoblast-like precursors. Further, we developed conditions for culturing trophoblast cells on these molds that could recapitulate placental structures. We found that growing cells underneath a PDMS mold floating on the media surface encouraged outgrowth of villus-like structures and shedding of syncitial knot-like clusters of multinucleated cells. The villus structures were positive for trophoblast markers PL1 and cytokeratin 7. Differentation to a syncitiotrophoblast state was further supported by the cell proliferation marker KI67, which was present in the underlying cells but not found in the upper layer, as is found in syncitiotrophoblast in vivo. The clusters of cells found at the bottom of the dish were multinucleated and of the same apparent size as actual syncitial knots. We are currently cytologically characterizing these clusters to determine whether they are actual differentiated syncitial knots.
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