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. 2009 May;29(5):718-24.
doi: 10.1161/ATVBAHA.109.184200. Epub 2009 Feb 12.

Signaling hierarchy regulating human endothelial cell development

Melissa A Kelly  1 , Karen K Hirschi
Affiliations

Signaling hierarchy regulating human endothelial cell development

Melissa A Kelly et al. Arterioscler Thromb Vasc Biol. 2009 May.

Abstract

Objective: Our present knowledge of the regulation of mammalian endothelial cell differentiation has been largely derived from studies of mouse embryonic development. However, unique mechanisms and hierarchy of signals that govern human endothelial cell development are unknown and, thus, explored in these studies.

Methods and results: Using human embryonic stem cells as a model system, we were able to reproducibly and robustly generate differentiated endothelial cells via coculture on OP9 marrow stromal cells. We found that, in contrast to studies in the mouse, bFGF and VEGF had no specific effects on the initiation of human vasculogenesis. However, exogenous Ihh promoted endothelial cell differentiation, as evidenced by increased production of cells with cobblestone morphology that coexpress multiple endothelial-specific genes and proteins, form lumens, and exhibit DiI-AcLDL uptake. Inhibition of BMP signaling using Noggin or BMP4, specifically, using neutralizing antibodies suppressed endothelial cell formation; whereas, addition of rhBMP4 to cells treated with the hedgehog inhibitor cyclopamine rescued endothelial cell development.

Conclusions: Our studies revealed that Ihh promoted human endothelial cell differentiation from pluripotent hES cells via BMP signaling, providing novel insights applicable to modulating human endothelial cell formation and vascular regeneration for human clinical therapies.

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Figures

Figure 1
Figure 1. OP9 coculture method generates endothelial cells efficiently
(A) Cells were differentiated and stained with antibodies against VE-cadherin and CD31. (B) Day 14 CD31 positive endothelial cells. DiI-Ac-LDL uptake of CD31 positive cells (C) and lumen (arrowheads) and plexus formation of Day 14 CD31/VE-cadherin positive cells (D) demonstrates functionality.
Figure 2
Figure 2. Vascular differentiation timeline of hES cells
RNA was extracted at the time points indicated, reverse transcribed to cDNA and used for Q-PCR on either genes associated with vascular induction, Ihh, BMP4, Flk1 and VEGF (A), or the hematopoietic marker CD45 (B) or endothelial markers, VE-cadherin and CD31 (C).
Figure 3
Figure 3. Hedgehog signaling is required for vascular differentiation of hES cells
Inhibition of hedgehog signaling reduced the expression of endothelial and hematopoietic markers whereas Ihh treatment enhanced expression. (*, P < 0.05; **, P < 0.01; ***, P < 0.001)
Figure 4
Figure 4. Ihh promotes vascular differentiation of hES cells
Differentiated cells were analyzed via FACS to determine endothelial and hematopoietic cell production. (*, P < 0.05; **, P < 0.01; ***, P < 0.001)
Figure 5
Figure 5. Ihh-mediated vascular differentiation requires BMP signaling
Ihh treated cells were exposed to anti-BMP4 or Noggin. BMP signaling was required for Ihh-mediated upregulation of endothelial markers but not for hematopoietic markers. (*, P < 0.05; **, P < 0.01; ***, P < 0.001)
Figure 6
Figure 6. Ihh promotes mesodermal progenitor differentiation via BMP4
rhBMP4 addition to cyclopamine treated cells rescued endothelial cell formation (A). Molecular regulation of endothelial development from hES cells (B). (*, P < 0.05; **, P < 0.01; ***, P < 0.001)
See this image and copyright information in PMC

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