Authors: Source: Blood, July 2007.

1. Identification of functional endothelial progenitor cells suitable for the treatment of ischemic tissue using human umbilical cord blood Authors: Source: Blood, July 2007.

2. Outlines 1. Background : a. Endothelial progenitor cell ( EPC ) b. Aldehyde dehydrogenase activity ( ALDH ) 2. Experimental design & Results a. Isolation of EPC b. Characterization of EPC c. Function assays In vivo & In vitro 3. Conclusion

3. Endothelial progenitor cells ( EPC ) ◆ originally identified from human peripheral blood ( PB ) ◆ also isolated from bone marrow , fetal liver, and umbilical cord blood.

4. Endothelial progenitor cells ( EPC ) Limb ischemia Myocardial infarction ◆ Physiologic functions: ◆ Therapeutic angiogenesis :

5. The definition of an EPC

6. The definition of an EPC ◆ Hur et al. ( Arteriosclerosis Thrombosis , and Vascular Biology.2004 ) ◆Ingram et al.( Blood,2004) ● divided subpopulations according to clonogenic and proliferative potential. ●Highly & Low proliferative endothelial potential-colony-forming cells ( HPP-ECFCs & LPP-ECFCs ) ◆ Yoder et al ( Blood,2007 ) ● Progeny of CD45+CD14+ cells are not EPCs but hematopoietic-derived myeloid progenitor cells.

7. Aldehyde dehydrogenase ( ALDH ) ◆Functions: ● Oxidized intercellular aldehyde and involved in ethanol, vitamin A , and cyclo- phosphamide metabolism. ● High levels in hematopoietic progenitor and stem cells ( HPC & HSC ). ●The higher ALDH activity HSC expressed, the better progenitor function and repopulation activity worked. ◆Detection: ● Fluorescent aldehyde substrate (Dansyl aminoacetaldehyde, Aldefluor ) by flow cytometry.

8. Aim: To develop an appropriate procedure for isolating EPCs from UCB to improve therapeutic efficacy and eliminate the expansion of nonessential cells.

9. Isolation of EPCs Step 1 Isolation of UCB-derived EPCs by negative immunoselection Red blood cell surface marker: glycophorin A

10. Isolation of UCB-derived EPCs by negative immunoselection UCB Hematopoietic cell surface markers: CD3, CD14, CD19, CD38, CD66b. Red blood cell marker: glycophorin A

11. Characterization of EPCs by uptake of Dil-Ac-LDL Cell morphology Cobblestone-like clusters Bright field Dark field PE-conjugated Dil-Ac-LDL marker: a. Dil-acetylated low-density lipoprotein b. Uptake of Dil-Ac-LDL by endothelial cells & macrophages as scavengers.

12. Characterization of EPCs by flow cytometry sorting Step 2 CD45- / Ac-LDL+ CD31+ / Ac-LDL+ CD45: Hematopoietic stem cell surface marker Ac-LDL+/CD31+/CD45- cells EC-like morphology

13. Analysis of endothelial tube formation of EPCs in Matrigel Matrigel : A. Solubilized basement membrane matrix . B. Rich in extracellular matrix proteins. C. Endothelial cells formed capillary tube in matrigel. Ac-LDL+/CD31+/CD45- cells Capillary tube-like structure on Matrigel

14. Conclusion Characterization of isolated EPCs • Endothelial cell morphology • Ac-LDL+/CD31+/CD45- cells • Capillary tube formation in matrigel

15. Separation of EPCs according to the ALDH activity Aldefluor : ALDH substrate Alde-High EPC Alde-Low EPC

16. Characterization of Alde-High & Alde-Low EPCs Endothelial cell–specific cell surface markers

17. Characterization of Alde-High & Alde-Low EPCs Hematopoietic stem cell surface markers

18. Conclusion • EPCs can divide two groups according to ALDH activity. • Alde-High & Alde-Low EPCs : • EC-specific markers • No hematopoietic stem cells

19. Growth rate of Alde-High & Alde-Low EPCs under hypoxiaIn Vitro Growth rate

20. Capillary formation of Alde-High & Alde-Low EPCs under hypoxiaIn Vitro Capillary networks formation in Matrigel

21. The assay of migration activity of EPCs by transwell culture in Vitro Transwell culture system EPCs SDF-1 SDF-1 : Homing factor

22. The assay of migration activity of EPCs under hypoxiain Vitro

23. The Hypoxia inducible pathway

24. Analyses of gene expression in EPCs under hypoxiaIn Vitro VEGF: Vascular endothelial growth factor KDR : VEGF receptor 2 Flt-1: VEGF receptor 1 CXCR4: SDF-1 receptor Glut-1: Glucose transporter-1

25. The Hypoxia inducible pathway

26. Protein expression in HIF-1α & 2α under hypoxiaIn Vitro

27. Hypoxia-inducible gene & protein expression Less More Conclusion Under hypoxia Alde-High EPCs Alde-Low EPCs V.S. Lower Faster Growth rate Tube numbers formation More Less Migration cell numbers Less More

28. The functional assay for neovascularization of EPCs in vivo A murine stem cell virus (MSCV)–internal ribosomal entry site–enhanced GFP Flap ischemia mice model 2X3 cm EPCs Tail vein 7 days Ischemia recovery

29. The effect of EPCs in neovascularization in vivo

30. Tracking the Alde-Low EPCs location in the ischemia tissue Neovascularization Newly formed vessels TRITC-Lectin: glycoprotein binding protein

31. Tracking the Alde-Low EPCs location in the ischemia tissue Re-endothelialization Dorsal ischemia skin

32. Conclusion • A novel method for isolating EPCs from UCB by a combination of negative immunoselection and cell culture techniques. • ALDH activity may serve as an excellent marker for isolating EPCs from UCB for clinical cell therapy. • Alde-Low EPCs possess a greater ability to proliferate and migrate compared to those with Alde-High EPCs . • Introduction of Alde-Low EPCs may be a potential strategy for inducing rapid neovascularization and regeneration of ischemic tissues.