Alpha Omega Alpha Honor Medical Society

Molecular Signaling Pathways in a Spatiotemporal 3D Environment for Endothelial Cell Differentiation of Human Induced Pluripotent Stem Cells

Investigator: Andrew Zhang, University of Minnesota Medical School

Mentor: Jianyi Jay Zhang, M.D., Ph.D.

Introduction:
The field of cardiac regenerative medicine has exploded since the discovery of human induced pluripotent stem cells (hiPSCs). However, the utility of hiPSCs is still limited by the efficiency of lineage-specific differentiation, particularly endothelial cell (EC) differentiation. The Zhang lab recently published a protocol using spatiotemporal 3D fibrin scaffolding rather than monolayer for EC differentiation of hiPSCs. This protocol improved the differentiation efficiency from the field-accepted standard of 20% up to 44%. In this proposal, we studied the molecular pathways underlying this improved differentiation efficiency.

Methods:
Previously established hiPSC lines were utilized. hiPSCs were seeded into a 3D fibrin patch and differentiated using a 2 stage method: the first stage consisted of growth factors Activin-A and BMP4 and the second stage incorporated growth factors VEGF, TGFβ1, and SB- 431542. Differentiation efficiency was evaluated by identifying percentage of cells positive for EC markers CD144 and CD31 using flow cytometry. Various inhibitors along the integrin-linked kinase pathway (Mechanical tension ILK Akt mTOR HIF-1α VEGF MAPK Etv2 angiogenesis) were utilized and differentiation efficiency was analyzed. Additionally, quantitative PCR (qPCR) for VEGFR2 and Etv2 gene expression following seeding to 3D fibrin were evaluated during the course of differentiation.

Results:
VEGFR2 gene expression in hiPSCs was significantly increased by two-fold after 48 hours in the fibrin scaffolding. Etv2 expression increased over 10-fold after 72 hours. αv Integrin antagonist and HIF-1α antagonist did not significantly attenuate the differentiation efficiency, while mTOR antagonist attenuated the differentiation efficiency from 26.8% at baseline to 16.4% with antagonist (p=0.023).

Conclusion:
A spatiotemporal 3D environment plays a significant role in promoting the differentiation of ECs from hiPSCs. We found that the mTOR protein accounted for at least part of the differentiation efficiency through up regulation of Etv2 expression. These molecular insights serve as a foundation for further improvement in endothelial cell differentiation protocols.

Last modified: 2/01/2017

Updated on February 1, 2017.


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