Identification of functional Identification of functional endothelial progenitor cells suitable endothelial progenitor cells suitable for the treatment of ischemic tissue for the treatment of ischemic tissue using human umbilical cord blood using human umbilical cord blood Authors: Authors: Source: Source: Blood, July 2007. Blood, July 2007.
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 ) - PowerPoint PPT Presentation
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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.
◆◆ 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-
● 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
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
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
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.