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Identification and Characterization of Endothelial Progenitor Cells from Human Umbilical Cord Blood

This study focuses on the isolation and characterization of endothelial progenitor cells (EPCs) from human umbilical cord blood (UCB) for treating ischemic tissue. We employed a combination of negative immunoselection and flow cytometry to isolate EPCs, analyzing their characteristics, growth rates, and angiogenic potential in vitro and in vivo. The role of aldehyde dehydrogenase (ALDH) activity in distinguishing between two EPC subsets—Alde-High and Alde-Low—is highlighted, suggesting implications for therapeutic angiogenesis and future treatment approaches for ischemic conditions.

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Identification and Characterization of Endothelial Progenitor Cells from Human Umbilical Cord Blood

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

  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.

  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 UCB 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 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. 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. 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 hypoxiaIn Vitro Growth rate

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

  21. 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 hypoxiain Vitro

  23. The Hypoxia inducible pathway

  24. Analyses of gene expression in EPCs under hypoxiaIn 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 hypoxiaIn Vitro

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

  28. 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 2X3 cm EPCs 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.

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