Angiopoietin-1 promotes endothelial differentiation from embryonic stem cells and induced pluripotent stem cells by Hyung Joon Joo, Honsoul Kim, Sang-Wook Park, Hyun-Jai Cho, Hyo-Soo Kim, Do-Sun Lim, Hyung-Min Chung, Injune Kim, Yong-Mahn Han, and Gou Young Koh Blood Volume 118(8):2094-2104 August 25, 2011 ©2011 by American Society of Hematology

Ang1 promotes EC differentiation via Tie2 signaling. Ang1 promotes EC differentiation via Tie2 signaling. Flk-1+ MPCs were purified on E4.5 and cultured on OP9 cells. Control buffer (Control), Ang1 (200 ng/mL), sTie2-Fc (sT2, 25μg/mL), RGD peptide (GRGDSP, 25μg/mL), and control peptide (GRADSP, 25μg/mL) were administered alone or together on days 0 and 2, and analyses were performed on day 3. (A) Treatment scheme of Ang1 or inhibitor. (B) Comparison of cell number of each EC colony on day 3 (n = 4). *P < .05 compared with control. (C) Images showing CD144+ EC colonies on day 3. Nuclei were stained with Hoechst. Scale bar represents 200 μm. (D) Dose dependency of Ang1 on the percentage of CD31+/CD144+ ECs from purified Flk-1+ cells on day 3 (n = 3). *P < .05 compared with control. (E,G) Representative FACS analyses of the population of CD31+/CD144+ ECs. Numbers indicate the percentage of CD31+/CD144+ ECs. (F,H) Comparison percentages of CD31+/CD144+ ECs (n = 4). *P < .05 compared with control, GRGDSP, or GRADSP; #P < .01 compared with Ang1. Hyung Joon Joo et al. Blood 2011;118:2094-2104 ©2011 by American Society of Hematology

Ang1 promotes the EC population but reduces the HSC and VSMC populations during Flk-1+ MPC differentiation. Ang1 promotes the EC population but reduces the HSC and VSMC populations during Flk-1+ MPC differentiation. Flk-1+ MPCs were purified on E4.5 and cultured on OP9 cells. Control or Ang1 was administered on days 0, 2, and 4, and analyses were performed on days 1.5, 3, and 6. (A,C,E) Representative FACS analyses of the populations of CD31+/CD144+ ECs, CD45+ HSCs, and α-SMA+ VSMCs. Numbers indicate percentages of ECs, HSCs and VSMCs. (B,D,F) Comparison of percentages of CD31+/CD144+ ECs, CD45+ HSCs, and α-SMA+ VSMCs over time (n = 5). *P < .01 compared with control. Hyung Joon Joo et al. Blood 2011;118:2094-2104 ©2011 by American Society of Hematology

Ang1 increases EC proliferation but suppresses EC apoptosis during Flk-1+ MPC differentiation. Ang1 increases EC proliferation but suppresses EC apoptosis during Flk-1+ MPC differentiation. Control or Ang1 was administered into Flk-1+ MPCs on days 0, 2, and 4 during OP9 coculture. (A) Images showing PHH3+ cells in CD144+ EC colonies on days 1.5, 2, 2.5, 3, and 3.5. Nuclei were stained with Hoechst. Scale bar represents 100 μm. (B) Comparison of number of PHH3+ ECs over time (n = 5). *P < .05 compared with control. (C) Representative FACS analyses of the populations of annexin+ apoptotic Flk-1+ cells and Flk-1− cells on day 3. Numbers indicate percentages of each population. (D) Comparison of percentage of annexin+ apoptotic Flk-1+ cells and Flk-1− cells on day 3 (n = 3). *P < .05 compared with control. Hyung Joon Joo et al. Blood 2011;118:2094-2104 ©2011 by American Society of Hematology

Ang1 drives CD41+ cells to ECs rather than HSCs Ang1 drives CD41+ cells to ECs rather than HSCs. Expression pattern and differentiation potential of CD41+ cells derived from Flk-1+ MPCs were characterized. Ang1 drives CD41+ cells to ECs rather than HSCs. Expression pattern and differentiation potential of CD41+ cells derived from Flk-1+ MPCs were characterized. (A) Representative FACS analyses showing surface expression patterns of CD144, CD31, Tie2, Sca-1, and c-Kit, each compared with CD41 on Flk-1+ MPC–derived cells on day 1.5. (B) Images showing CD144+/CD41+ cells in endothelial colonies on day 1.5. Nuclei were stained with Hoechst. Scale bar represents 20 μm. (C) Experimental scheme for the differentiation potential of CD41+ cells. Sorted CD41+ cells on day 1.5 were replated on the freshly prepared OP9 cells, treated with Control, Ang1, sT2, or Ang1 + sT2 on days 1.5, and 3.5, and analyses were performed on day 6. (D,F) Representative FACS analyses of the populations of CD31+/CD144+ ECs and CD45+ HSCs from the sorted CD41+ cells. Numbers indicate percentages of each population. (E,G) Comparison of percentages of CD31+/CD144+ ECs and CD45+ HSCs from the sorted CD41+ cells (n = 3). *P < .05 compared with control; #P < .05 compared with Ang1. Hyung Joon Joo et al. Blood 2011;118:2094-2104 ©2011 by American Society of Hematology

Ang1 increases EC differentiation from mouse iPSCs and human ESCs Ang1 increases EC differentiation from mouse iPSCs and human ESCs. (A-C) iPSC-derived Flk-1+ MPCs were purified on E4.5 and cultured on OP9 cells with control buffer (Control) or Ang1. Ang1 increases EC differentiation from mouse iPSCs and human ESCs. (A-C) iPSC-derived Flk-1+ MPCs were purified on E4.5 and cultured on OP9 cells with control buffer (Control) or Ang1. Analyses were performed on day 3. (A) Images showing CD144+ EC colonies. Nuclei were stained with Hoechst. Scale bar represents 200 μm. (B) Representative FACS analyses of the population of CD31+/CD144+ ECs. Numbers indicate percentages of ECs from Flk-1+ MPCs during differentiation. (C) Comparison percentages of CD31+/CD144+ ECs from purified Flk-1+ MPCs (n = 3). *P < .05 compared with control. (D-E) hESC-derived CD34+ cells were purified and cultured with Control or Ang1 and analyses were performed 9 days later. (D) Images showing CD144+ EC colonies. Nuclei were stained with Hoechst. Scale bars represent 200 μm. (E) Representative FACS analyses of the population of CD31+/CD105+ ECs. Numbers indicate percentages of ECs from CD34+ cells during differentiation. (F) Comparison percentages of CD31+/CD105+ ECs from purified CD34+ cells (n = 3). *P < .05 compared with control. Hyung Joon Joo et al. Blood 2011;118:2094-2104 ©2011 by American Society of Hematology

Ang1-induced CD31+/CD144+ ECs have a neovasculogenic potential in vitro and in vivo. Ang1-induced CD31+/CD144+ ECs have a neovasculogenic potential in vitro and in vivo. Purified Flk-1+ MPCs differentiated from mouse ESCs were cultured for 3 days on OP9 cells. Ang1 was administered on days 0 and 2. CD31+/CD144+ cells were sorted by FACS on day 3 and used as Ang1-EC-ESCs for further experiments. (A) Phase contrast images showing Ang1-EC-ESCs. Scale bar represents 100 μm. (B) Immunostaining images showing Ang1-EC-ESCs. Nuclei were stained with Hoechst. Scale bar represents 100 μm. (C) Images showing network formations of Ang1-EC-ESCs and HUVECs. Ang1-EC-ESCs and HUVECs were plated on Matrigel-covered plates and incubated in EGM-2. The tube structures were observed after 24 hours. Scale bar represents 200 μm. (D-E) Ang1-mESC-ECs were injected subcutaneously into the dorsal skin of cyclosporine A–treated CD31-deficient mice in Matrigel. The implanted Matrigel was harvested 14 days after injection of FITC-lectin 20 minutes before the mice were killed; The presence of CD31+ vessel-like structures containing FITC-lectin can be identified in regions 1 and 2, indicating that effective perfusion had taken place in the implanted Ang1-EC-ESCs. The bottom 2 panels are the magnified views of indicated regions. Scale bars represent 50 μm. Hyung Joon Joo et al. Blood 2011;118:2094-2104 ©2011 by American Society of Hematology

Schematic diagram of the effect of Ang1 on endothelial and hematopoietic differentiation. Schematic diagram of the effect of Ang1 on endothelial and hematopoietic differentiation. CD41+ cells derived from Flk-1+ MPCs differentiate further into either the endothelial or the hematopoietic lineage. Ang1 potentiates EC differentiation via Ang1/Tie2 signaling, and its mechanisms are: (1) modulating CD41+ cells to differentiate into the endothelial lineage rather than the hematopoietic lineage, and (2) promoting EC proliferation and survival during differentiation. Hyung Joon Joo et al. Blood 2011;118:2094-2104 ©2011 by American Society of Hematology