1. Differentiation, Survival, and Function of Embryonic Stem Cell–Derived Endothelial Cells for Ischemic Heart Disease by Zongjin Li, Jenny C. Wu, Ahmad Y. Sheikh, Daniel Kraft, Feng Cao, Xiaoyan Xie, Manishkumar Patel, Sanjiv S. Gambhir, Robert C. Robbins, John P. Cooke, and Joseph C. Wu Circulation Volume 116(11 suppl):I-46-I-54 September 11, 2007 Copyright © American Heart Association, Inc. All rights reserved.

2. Figure 1. ES cell differentiation into endothelial-like cells. a, Schema of the endothelial cell lineage-specific promoter with VE-cadherin promoter driving eGFP and SV40 promoter driving neomycin resistant gene for antibiotic selection of stable clones. b, After 4 days on collagen IV plates, Flk-1+–positive cells can be identified by FACS. The outgrowth from these Flk-1+ cells exhibit predominantly 2 morphologies: cobblestone-like endothelial colony and striated smooth muscle-like cells as shown on brightfield microscopy (100× magnification). c, After another 4 days of culturing on differentiation medium, ≈36% of cells express both VE-cadherin surface marker and intracellular eGFP. Zongjin Li et al. Circulation. 2007;116:I-46-I-54 Copyright © American Heart Association, Inc. All rights reserved.

3. Figure 2. In vitro characterization of murine ESC-ECs. a, FACS analysis shows increased expression of common endothelial cell markers (VE-cadherin, Flk-1, CD34) in differentiated ESC- ECs compared with undifferentiated ES cells. Zongjin Li et al. Circulation. 2007;116:I-46-I-54 Copyright © American Heart Association, Inc. All rights reserved.

4. Figure 3. Transduction of ES cells with double fusion (DF) reporter gene. a, Schema of the DF reporter gene with ubiquitin promoter driving Fluc and mRFP joined by a 14-amino acid linker (LENSHASAGYQAST). b, FACS analysis showing lentiviral transduction efficiency (≈21%) after 48 hours. Zongjin Li et al. Circulation. 2007;116:I-46-I-54 Copyright © American Heart Association, Inc. All rights reserved.

5. Figure 4. Molecular imaging of ESC-EC fate after transplantation. a, A representative animal injected with 5×105 ESC-ECs shows significant bioluminescence activity at day 2, which decreases progressively over the following 8 weeks. Zongjin Li et al. Circulation. 2007;116:I-46-I-54 Copyright © American Heart Association, Inc. All rights reserved.

6. Figure 5. Engraftment of ESC-ECs can improve neovascularization. a, i-iii, Representative histology of a heart transplanted with ESC-ECs and harvested at week 2. Zongjin Li et al. Circulation. 2007;116:I-46-I-54 Copyright © American Heart Association, Inc. All rights reserved.

7. Figure 6. Teratoma formation after transplantation with undifferentiated ES cells. a, A representative SV129 mouse injected with 5×105 undifferentiated ES cells shows the lowest bioluminescence signals at day 7 which increase dramatically between week 2 and week 4. Zongjin Li et al. Circulation. 2007;116:I-46-I-54 Copyright © American Heart Association, Inc. All rights reserved.