Yonsei Med J.  2005 Apr;46(2):260-267. 10.3349/ymj.2005.46.2.260.

Isolation of Endothelial Progenitor Cells from Cord Blood and Induction of Differentiation by Ex Vivo Expansion

• Shin JW
• Lee DW
• Kim MJ
• Song KS
• Kim HS
• Kim HO

• KMID: 1734053
• DOI: http://doi.org/10.3349/ymj.2005.46.2.260

Abstract

Endothelial progenitor cells (EPCs) have been reported to possess the capacity to colonize vascular grafts and hold promise for therapeutic neovascularization. However, limited quantities of EPCs have been the major factor impeding effective research on vasculoangiogenesis. In this study, cytokine and culture conditions necessary for the provision of large quantities of endothelial cells (ECs) were investigated. Cord blood was collected from 18 normal full-term deliveries and CD34+ cells were isolated by MACS system (Miltenyi Biotech, Bergish-Gladbach, Germany). To evaluate the effect of cytokines, CD34+ cells were cultured with various cytokine combinations, such as stem cell factor (SCF), flt3-ligand (FL), and thrombopoietin (TPO) with vascular endothelial growth factor (VEGF), interleukin-1beta, fibroblast growth factor-basic (FGF-b) as basic cytokines. The quantities of non-adherent and adherent cells were the greatest with SCF, FL and TPO. The addition of TPO to all other cytokines significantly increased the number of non-adherent and adherent cells (p< 0.05, Wilcoxon rank sum test). After four weeks of culture, adherent cells expressed endothelial specific markers such as KDR, CD31 and CD62E. Typical morphology of ECs was observed during culture, such as cord-like structure and cobblestone appearance, suggesting that the adherent cells were consistent with ECs. In this study, the experimental conditions that optimize the production of ECs for therapeutic neovascularization were described. And it was possibly suggested that TPO plays a major role in differentiation from EPCs to ECs.

Keyword

Endothellial progenitor cells; neovascularization; thrombopoietin

MeSH Terms

Antigens, CD34/analysis
Cell Differentiation/drug effects
Cell Division/drug effects
*Cell Separation
Cells, Cultured
Cytokines/pharmacology
*Endothelial Cells/immunology
Fetal Blood/*cytology
Fetus
Flow Cytometry
Humans
Research Support, Non-U.S. Gov't
*Stem Cells/*immunology
Thrombopoietin/pharmacology

Figure

• 

  Fig. 1 The numbers of non-adherent cells obtained from cultures preformed with or without TPO in the presence of VEGF, IL-1β, FGF-b, SCF and FL in 24-well microplates (n=9). The line represents the mean values. The addition of TPO significantly increased the numbers of non-adherent cells (p < 0.05, Wilcoxon rank sum test).

• 

  Fig. 2 Flow cytometric analysis of endothelial cells obtained from CD34+ cells after 4 weeks of culture. The dark line identifies the cells labeled with isotype control antibody and the faint line identifies the cells labeled with antibodies specific for the surface markers. Cultured cells were positive for KDR, CD31 and CD62E, but negative for CD34, CD117, AC133 and CXCR4 (cut off, 10%).

• 

  Fig. 3 Morphology of cord blood derived adherent cells at days 7(A), 14(B), 21(C), and 28(D) of in vitro culture. A) and B) Cord-like structures resembling the first stages of vasculogenesis; C) and D) Characteristic endothelial cobblestone colonies (Unstained; magnification, ×100).

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