Scaffold-assisted tissue engineering of early human placenta derived from embryonic stem cells

2015 Fiscal Year Research-status Report

• Project/Area Number: 15K14525
• Research Institution: Kyoto University
• Principal Investigator: Carlton Peter 京都大学, 物質－細胞統合システム拠点, 特定准教授 (20571813)
• Project Period (FY): 2015-04-01 – 2017-03-31
• Keywords: trophoblast cells / tissue engineering / placenta

Outline of Annual Research Achievements

We constructed a PDMS scaffold for cell growth based on a 3D-printed resin mold. To match the observed size of extraembryonic mesoderm villi, we set the width of the individual scaffold protuberances to between 100 and 500 microns, and the depth to between 300 and 500 microns. Due to resolution limitations of the 3D printer, the actual PDMS protuberance height did not exceed 150 microns at the small diameters attempted. Coating these scaffolds with gelatin or matrigel allowed the attachment and growth of mouse trophoblast stem cells (mTSC). mTSC showed evidence of stratified organization on our scaffolds. Human embryonic stem cells (hESC) were unable to attach to these scaffolds, but instead settled in the troughs between protuberances, where they developed into embryoid bodies.

Current Status of Research Progress

3: Progress in research has been slightly delayed.

Reason

The attachment and differentiation of mTSC to our PDMS scaffolds could not be replicated with human cells, despite changing both coating and culturing conditions. The fulfilment of this proposal's specific aims depends on being able to use human cells; therefore, our progress has been delayed until an alternate method could be found.

We became aware of a paper reporting success at culturing human induced pluripotent stem cells on a mesh screen, which led directly to trophoblast-like differentiation and the production of cysts [Okeyo, K. O., et al., Cell Adhesion Minimization by a Novel Mesh Culture Method Mechanically Directs Trophoblast Differentiation and Self-Assembly Organization of Human Pluripotent Stem Cells. Tissue Eng. Part C Methods 21, 1105-1115 (2015)], which was a specific aim of our proposed research. These results have led us to revise our scaffold design and consider mesh-like scaffolds.

Strategy for Future Research Activity

Since hESC have failed to attach to and grow on our PDMS scaffold, we will switch to using a mesh-type scaffold that has been previously demonstrated to allow the culture of hESC cells and their spontaneous differentiation to the trophoblast lineage. We will also use our trophoblast differentiation protocol in conjunction with culturing on a mesh substrate, to determine whether ES->TS differentiation can be accelerated.

When growth of human TS cells on a mesh substrate can be achieved, we will perform long-term live imaging of the resulting changes in differentiation and cellular arrangement. Over the course of several days of imaging we will observe the hallmarks of in vivo placental villus development, such as differentiated organization of cytotrophoblast and syncitiotrophoblast layers, cell fusion in the syncitiotrophoblasts, and shedding of cysts.

If switching to a mesh substrate still does not allow human trophoblast-like cells to be cultured in our hands, we will return to mouse trophoblast stem cells on our original PDMS mold substrate, and investigate how cell-substrate interactions influence the organization of cytotrophoblast and syncitiotrophoblast cells.