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A major limitation for many
tissue engineering strategies
is poor nutrient supply due to
a lack of a functional
vasculature. To overcome
these limitations, there is a
need for improved
understanding of the biological
signals that induce blood
vessel formation and
strategies to promote
revascularization of
engineered tissues. Here, we
describe a strategy for
tailoring poly(ethylene glycol)
(PEG) hydrogels to promote
and optimize vascular network
formation using human
endothelial cells (EC) cultured
in 3D in vitro environments.
Synthetic extracellular matrix for investigating 3D
vascular network formation Michael P. Schwartz1, Jue Zhang3, Zhonggang Hou3, Victor Ruotti3, David G. Belair1, Angela W. Xie1,
Matthew R. Zanotelli1, Eric H. Nguyen1, James A. Thomson3-5, and William L. Murphy1, 2 Depts. of 1. Biomedical Engineering, 2. Orthopedics and Rehabilitation, and 3. Cell and Regenerative Biology, University of Wisconsin-Madison; 4. Morgridge Institute for
Research, Madison, WI; 5. Department of Molecular, Cellular, and Developmental Biology, University of California-Santa Barbara
Acknowledgements • National Science Foundation (CAREER award #0745563)
• National Institutes of Health (R01HL093282)
• National Institutes of Health (1UH2TR000506-01)
Sequestering/delivery of biomolecules (Soluble cues)
Synthetic ECM (Insoluble cues)
Multicellular organization (Cellular cues)
• Impellitteri et al., Biomat. 2012, 33 3475-3484 • Toepke et al., Adv. Healthcare Mater. 2012, 1, 457–460 • Toepke et al., Macromolec. Mater. Engineering, 2013, doi: 10.1002/mame.201200119
light
I2959
Strategies for investigating and manipulating the 3D microenvironment
B.D. Fairbanks, M.P. Schwartz, et al. 2009, Adv. Mater. 21, 5005.
Cysteine
Poly(ethylene glycol)-
Norbornene
CRGDS Adhesion
“Thiol-ene” coupling
MMP degradable
CGPQG*IWGQC
Synthetic Extracellular Matrix
HUVECs in synthetic ECM
3D Monoculture
(synthetic ECM) 2D Culture
CD31 CD31
Human pluripotent stem cell-derived endothelial cells. • 5 day differentiation procedure.
• 50-80% efficiency multiple pluripotent stem cell lines (ESCs and iPSCs).
• CD31+/CD34+ and CD31+/CD34- populations generated.
• CD31 and VE-cadherin (CD144) expressing
• Uptake LDL
• Form networks in Matrigel.
Conclusions •Synthetic ECM based on PEG-NB chemistry is suitable for promoting vascular network formation
•Compatible with several endothelial cell types.
• Pluripotent stem cell-derived endothelial cells (both ESC and iPSC) and HUVECs.
• Synthetic ECM provides control over biochemical and biophysical matrix properties.
• Tune to optimize, manipulate cell function.
Human induced pluripotent stem cell-derived endothelial cells (iPSC-derived ECs, iCell® ECs, Cellular Dynamics)
are well-suited for investigating vascular network formation and sprouting.
Sprouting in synthetic ECM (3D): iPSC-derived ECs
iPSC-derived ECs
HUVECs and iPSC-derived ECs on Matrigel (2D)
iCell® iPSC-derived ECs
(Cellular Dynamics)
Human umbilical cord
endothelial cells (HUVECs)
Live Dead
Day 1 > 90% Viability (to at least day 5)
Calcein DAPI
CD31 DAPI
Day 4 Day 4
iPSC-derived ECs encapsulated in synthetic ECM (3D)
Day 2 to Day 3 Vascular network formation: iPSC-derived ECs encapsulated in synthetic ECM (3D)
RNA sequencing
• Statistically upregulated genes vs.
human pluripotent stem cells.
• iPSC-derived ECs characterized by
similar or higher expression of EC
and blood vessel development
genes compared to HUVECs.
CD31/SM22a
1 day 2 days
3 days 10 days
• ESC-derived ECs form vascular networks in synthetic ECM.
• Vascular network formation improved with pericyte coculture
(pericytes incorporate into networks).
• Vascular network density is dependent on RGD
concentration (adhesion), crosslinking density (stiffness).
• Vascular networks are stable for > 2 weeks.
4 mM CRGDS 2 mM CRGDS 0 mM CRGDS
Adhesion-dependence
60% Crosslinking 35% Crosslinking 45% Crosslinking
“Stiffness”-dependence
J. Zhang, M.P. Schwartz, Z. Zhu, V. Ruotti, W.L. Murphy, and J.A. Thomson, Submitted for Publication
CD31/ SM22a
Cocultures:
ESC-derived ECs
Pericytes