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