Identification of functional endothelial progenitor Identification of functional endothelial progenitor cells suitable for the treatment of ischemic tissue using cells suitable for the treatment of ischemic tissue using

human umbilical cord bloodhuman umbilical cord bloodAuthors: Authors:

Source: Source: Blood, July 2007. Blood, July 2007.

1. Background :1. Background :

a. Endothelial progenitor cell ( EPC )a. Endothelial progenitor cell ( EPC )

b. Aldehyde dehydrogenase activity ( ALDH )b. Aldehyde dehydrogenase activity ( ALDH )

2. Experimental design & Results2. Experimental design & Results

a. Isolation of EPCa. Isolation of EPC

b. Characterization of EPCb. Characterization of EPC

c. Function assays c. Function assays In vivoIn vivo & & In vitroIn vitro

3. Conclusion3. Conclusion

OutlinesOutlines

Endothelial progenitor cells ( EPC )Endothelial progenitor cells ( EPC )

◆◆ originally identified from human peripheral blood ( PB )originally identified from human peripheral blood ( PB )

◆◆ also isolated from bone marrow , fetal liver, and umbilical cord blood.also isolated from bone marrow , fetal liver, and umbilical cord blood.

Limb ischemia Myocardial infarction◆◆ Therapeutic angiogenesis :Therapeutic angiogenesis :

Endothelial progenitor cells ( EPC )Endothelial progenitor cells ( EPC )

◆◆ Physiologic functions:Physiologic functions:

The definition of an EPCThe definition of an EPC

◆◆ Hur et al. ( Arteriosclerosis Thrombosis , and Vascular Biology.2004 ) Hur et al. ( Arteriosclerosis Thrombosis , and Vascular Biology.2004 )

◆◆ Ingram et al.( Blood,2004)Ingram et al.( Blood,2004) ●● divided subpopulations according to clonogenic and proliferative potential.divided subpopulations according to clonogenic and proliferative potential. ●● Highly & Low proliferative endothelial potential-colony-forming cells ( HPP-ECFCs & LPP-Highly & Low proliferative endothelial potential-colony-forming cells ( HPP-ECFCs & LPP-ECFCs )ECFCs )

◆◆ Yoder et al ( Blood,2007 )Yoder et al ( Blood,2007 ) ●● Progeny of CD45Progeny of CD45++CD14CD14+ + cells are not EPCs but hematopoietic-derived myeloid progenitor cells. cells are not EPCs but hematopoietic-derived myeloid progenitor cells.

　　 SourceSourceExponential Exponential

growthgrowthSurface markerSurface marker

Early Early EPCEPC

Adult peripheral Adult peripheral blood blood

mononuclear mononuclear cellscells

2 to 3 weeks2 to 3 weeks CD45,CD14CD45,CD14

Late Late EPCEPC

4 to 8 weeks4 to 8 weeksCD31,CD34,VEGFR2 , and VE-CD31,CD34,VEGFR2 , and VE-

cadherincadherin

The definition of an EPCThe definition of an EPC

Aldehyde dehydrogenase ( ALDH ) 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:Aim: To develop an appropriate procedure for isolating To develop an appropriate procedure for isolating EPCs from UCB to improve therapeutic efficacy and EPCs from UCB to improve therapeutic efficacy and

eliminate the expansion of nonessential cells.eliminate the expansion of nonessential cells.

Isolation of UCB-derived EPCs by negative immunoselectionIsolation of UCB-derived EPCs by negative immunoselection

Isolation of EPCsIsolation of EPCs

Red blood cell surface marker: glycophorin A

Step 1Step 1

Isolation of UCB-derived EPCs by negative immunoselectionIsolation of UCB-derived EPCs by negative immunoselection

Hematopoietic cell surface markers:Hematopoietic cell surface markers:CD3, CD14, CD19, CD38, CD66bCD3, CD14, CD19, CD38, CD66b..

Red blood cell marker:Red blood cell marker:glycophorin Aglycophorin A

UCB

PE-conjugated Dil-Ac-LDL marker:PE-conjugated Dil-Ac-LDL marker: a. Dil-acetylated low-density lipoproteina. Dil-acetylated low-density lipoprotein b. Uptake of Dil-Ac-LDL by endothelial cells & macrophages b. Uptake of Dil-Ac-LDL by endothelial cells & macrophages as scavengers.as scavengers.

Characterization of EPCs by uptake of Dil-Ac-LDLCharacterization of EPCs by uptake of Dil-Ac-LDL

Bright fieldBright field Dark fieldDark field

Cell morphology Cell morphology Cobblestone-like clustersCobblestone-like clusters

Characterization of EPCs by flow cytometry sortingCharacterization of EPCs by flow cytometry sorting

CD45CD45- - / Ac-LDL/ Ac-LDL++ CD31CD31+ + / Ac-LDL/ Ac-LDL++

Ac-LDLAc-LDL++/CD31/CD31++/CD45/CD45-- cells cells

EC-like morphologyEC-like morphology

CD45: CD45: Hematopoietic stem Hematopoietic stem cell surface markercell surface marker

Step 2Step 2

Capillary tube-like structure on MatrigelCapillary tube-like structure on Matrigel

Matrigel :Matrigel : A. Solubilized basement membrane matrix . A. Solubilized basement membrane matrix . B. Rich in extracellular matrix proteins. B. Rich in extracellular matrix proteins. C. Endothelial cells formed capillary tube in matrigel.C. Endothelial cells formed capillary tube in matrigel.

Analysis of endothelial tube formation of EPCs in MatrigelAnalysis of endothelial tube formation of EPCs in Matrigel

Ac-LDLAc-LDL++/CD31/CD31++/CD45/CD45-- cells cells

ConclusionConclusion

Endothelial cell morphologyEndothelial cell morphology

Ac-LDLAc-LDL++/CD31/CD31++/CD45/CD45- - cellscells

Capillary tube formation in matrigelCapillary tube formation in matrigel

Characterization of isolated EPCsCharacterization of isolated EPCs

Separation of EPCs according to the ALDH activitySeparation of EPCs according to the ALDH activityAldefluor : Aldefluor : ALDH substrateALDH substrate

Alde-High EPCAlde-High EPC

Alde-Low EPCAlde-Low EPC

Characterization of Alde-High & Alde-Low EPCsCharacterization of Alde-High & Alde-Low EPCs

Endothelial cell–specific cell surface markersEndothelial cell–specific cell surface markers

Hematopoietic stem cell surface markersHematopoietic stem cell surface markers

Characterization of Alde-High & Alde-Low EPCsCharacterization of Alde-High & Alde-Low EPCs

ConclusionConclusion

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

Growth rateGrowth rate

Capillary networks formation in MatrigelCapillary networks formation in Matrigel

Capillary formation of Alde-High & Alde-Low EPCs under Capillary formation of Alde-High & Alde-Low EPCs under hypoxiahypoxia In VitroIn Vitro

The assay of migration activity of EPCs by transwell culture The assay of migration activity of EPCs by transwell culture in Vitro in Vitro

Transwell culture systemTranswell culture system

SDF-1

SDF-1 :SDF-1 : Homing factorHoming factor

EPCsEPCs

The assay of migration activity of EPCs under The assay of migration activity of EPCs under hypoxiahypoxia in Vitroin Vitro

The Hypoxia inducible pathwayThe Hypoxia inducible pathway

Analyses of gene expression in EPCs under Analyses of gene expression in EPCs under hypoxiahypoxia In VitroIn Vitro

VEGF: Vascular endothelial growth factorVEGF: Vascular endothelial growth factor

KDR : VEGF receptor 2KDR : VEGF receptor 2

Flt-1: VEGF receptor 1Flt-1: VEGF receptor 1

CXCR4: SDF-1 receptorCXCR4: SDF-1 receptor

Glut-1: Glucose transporter-1 Glut-1: Glucose transporter-1

The Hypoxia inducible pathwayThe Hypoxia inducible pathway

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

ConclusionConclusion

Alde-High EPCsAlde-High EPCs Alde-Low EPCsAlde-Low EPCs

Growth rateGrowth rate

Tube numbers formationTube numbers formation

Under Under hypoxiahypoxia

FasterFasterLowerLower

LessLessMoreMore

Hypoxia-inducible geneHypoxia-inducible gene &&

protein expressionprotein expressionMoreMoreLessLess

Migration cell numbersMigration cell numbers

V.S.V.S.

MoreMoreLessLess

The functional assay for neovascularization of EPCs The functional assay for neovascularization of EPCs in vivoin vivo

2X3 cm2X3 cm

Flap ischemia mice modelFlap ischemia mice model

Tail veinTail vein

A murine stem cell virus A murine stem cell virus (MSCV)–internal ribosomal (MSCV)–internal ribosomal entry site–enhanced entry site–enhanced GFP GFP

EPCsEPCs

7 days7 daysIschemia Ischemia recovery recovery

The effect of EPCs in neovascularization The effect of EPCs in neovascularization in vivoin vivo

Newly formed vesselsNewly formed vessels

Tracking the Alde-Low EPCs location in the ischemia tissueTracking the Alde-Low EPCs location in the ischemia tissue

TRITC-Lectin: TRITC-Lectin: glycoprotein binding glycoprotein binding proteinprotein

NeovascularizationNeovascularization

Dorsal ischemia skinDorsal ischemia skinRe-endothelializationRe-endothelialization

Tracking the Alde-Low EPCs location in the ischemia tissueTracking the Alde-Low EPCs location in the ischemia tissue

ConclusionConclusion

A novel method for isolating EPCs from UCB by a combination of negative A novel method for isolating EPCs from UCB by a combination of negative

immunoselection and cell culture techniques.immunoselection and cell culture techniques.

ALDH activity may serve as an excellent marker for isolating EPCs from ALDH activity may serve as an excellent marker for isolating EPCs from

UCB for clinical cell therapy.UCB for clinical cell therapy.

Alde-Low EPCs possess a greater ability to proliferate and migrate compared Alde-Low EPCs possess a greater ability to proliferate and migrate compared

to those with Alde-High EPCs .to those with Alde-High EPCs .

Introduction of Alde-Low EPCs may be a potential strategy for inducing rapid Introduction of Alde-Low EPCs may be a potential strategy for inducing rapid

neovascularization and regeneration of ischemic tissues.neovascularization and regeneration of ischemic tissues.