Abstract: FR-PO250

Laminar Flow Enhances Endothelial Differentiation from iPS Cells and Stabilizes Endothelial Cell Function

Session Information

  • Stem Cells
    November 03, 2017 | Location: Hall H, Morial Convention Center
    Abstract Time: 10:00 AM - 10:00 AM

Category: Developmental Biology and Inherited Kidney Diseases

  • 402 Stem Cells

Authors

  • Bollin, Robin, Medical School Hannover, Hannover, Germany
  • Kiyan, Yulia, Hannover Medical School, Hannover, Germany
  • Kiyan, Roman, Laser Zentrum Hannover e.V., Hannover, Germany
  • Martin, Ulrich, Medical School Hannover, Hannover, Germany
  • Chichkov, Boris, Laser Zentrum Hannover e.V., Hannover, Germany
  • Haller, Hermann G., Hannover Medical School, Hannover, Germany
Background

Endothelial cells (ECs) have essential roles in organ development and regeneration, and therefore they could be used for regenerative therapies. However, generation of abundant functional endothelium from pluripotent stem cells has been difficult because ECs have limited proliferative potential and display vascular instability. Since stimulation of functional properties may enhance EC differentiation we have tested the hypothesis that laminar flow enhances EC differentiation and analyzed the underlying molecular mechanisms.

Methods

iPSC have been cultured under feeder free conditions for 3 passages, and then seeded in the Geltrex-coated microfluidic chips. Chips contained 4 parallel channels allowing simultaneous analysis of several experimental conditions. Mesoderm differentiation was induced by Bone morphogenetic protein 4 (BMP4) 30 ng ml−1; Activin A 25 ng ml−1; small-molecule inhibitor of glycogen synthase kinase-3β (CHIR) 1.5 μM; Vascular endothelial growth factor (VEGF) 50 ng ml−1 for 4 days. At day 4 of mesoderm differentiation microfluidic chips have been attached to the medium flow and stimulated with VEGF 50 ng ml−1; TGF-β pathway small-molecule inhibitor SB431542 10 μM to induce EC differentiation. Static control chips have been stimulated with the same medium. After 3 days cells were fixed by perfusion with 2% PFA solution, and stained for Sox17, VE-Cadherin, CD31, and heparin sulfate. Confocal microscopy of the cells was performed directly in the microfluidic chips.

Results

Characteristic flow-oriented morphological changes have been observed in the cells incubated under flow but not under static conditions. Control iPSC without flow displayed a low rate of <1% EC differentiation after 3 days of stimulation. In contrast, iPSC cultivated under medium flow conditions showed a more rapid (50.8 ± 4.1% after 3 days) and sustained differentiation to EC. Cells demonstrated expression of EC-markers CD31 and VE-Cadherin, and expressed heparin sulfates on the apical side of the cells indicating terminal differentiation.

Conclusion

Laminar shear stress may directly activate growth factor receptors on stem/progenitor cells, initiating signaling pathways leading toward endothelial cell differentiation. Our results suggest that laminar flow is an important factor in endothelial cell differentiation protocols.