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 : 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

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 )
◆ Physiologic functions:
◆ Therapeutic angiogenesis :
Limb ischemia
Myocardial infarction

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.
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

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
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
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. Characterization of isolated EPCs
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. Hematopoietic stem cell surface markers
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 hypoxia In Vitro

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

21. 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

22. The assay of migration activity of EPCs under hypoxia in Vitro

23. The Hypoxia inducible pathway

24. 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

25. The Hypoxia inducible pathway

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

27. 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

28. 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

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.