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

Outlines 1. Background : 2. Experimental design & Results 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

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

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

The definition of an EPC

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. Source Exponential growth Surface marker Early EPC Adult peripheral blood mononuclear cells 2 to 3 weeks CD45,CD14 Late EPC 4 to 8 weeks CD31,CD34,VEGFR2 , and VE-cadherin

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.

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

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

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

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.

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

Analysis of endothelial tube formation of EPCs in 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

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

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

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

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

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

Growth rate of Alde-High & Alde-Low EPCs under hypoxia In Vitro

Capillary formation of Alde-High & Alde-Low EPCs under hypoxia In Vitro Capillary networks formation in Matrigel

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

The assay of migration activity of EPCs under hypoxia in Vitro

The Hypoxia inducible pathway

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

The Hypoxia inducible pathway

Protein expression in HIF-1α & 2α under hypoxia In Vitro

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

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

The effect of EPCs in neovascularization in vivo

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

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

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.