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 :
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 )
Source
Early
EPC
Late
EPC
Adult peripheral
blood
mononuclear
cells
Exponential
growth
Surface marker
2 to 3 weeks
CD45,CD14
4 to 8 weeks
CD31,CD34,VEGFR2 , and VEcadherin
◆ 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.
Aldehyde dehydrogenase ( ALDH )
◆ Functions:
● Oxidized intercellular aldehyde and involved in ethanol, vitamin A , and cyclophosphamide 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
UCB
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
Bright field
Cobblestone-like clusters
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
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
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
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
Growth rate
Capillary formation of Alde-High & Alde-Low EPCs under hypoxia In Vitro
Capillary networks formation in Matrigel
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
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
Less
More
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
Tail vein
7 days
Ischemia
recovery
2X3 cm
EPCs
The effect of EPCs in neovascularization in vivo
Tracking the Alde-Low EPCs location in the ischemia tissue
Neovascularization
TRITC-Lectin:
glycoprotein binding
protein
Newly formed vessels
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.