[Frontiers in Bioscience 17, 2247-2258, June 1, 2012]

Cobalt chloride enhances angiogenic potential of CD133+ cells

Tao Zan1, Zijing Du1, Hua Li1, QingFeng Li1, Bin Gu1

1Department of Plastic and Reconstructive Surgery, Shanghai Ninth People's Hospital, Shanghai Medical College of Jiao Tong University, Shanghai, 200011, China

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods 3.1. Human CD133+ cell isolation
3.2. Cell culture
3.3. Acetylated LDL uptake and UEA-1 lectin binding site labeling
3.4. Tubule formation
3.5. Flow cytometric analysis of cell surface markers
3.6. Migration assay
3.7. Cell wound healing
3.8. Cell proliferation Assay
3.9. Measurement of VEGF, FGF, and HIF-1a protein by ELISA
3.10. Real time PCR
3.11. Western blot analysis
3.12. Statistical analysis
4. Results
4.1. Characteristics of isolated CD133+ positive cells from umbilical cord blood
4.2. Cell migration
4.3. Proliferation
4.4. Tubule formation ability
4.5. Secretion of growth factors by CD133+
4.6. Hypoxia induced the protein expression
4.7. HIF-1a and downstream gene expression
5. Discussion
6. Conclusion
7. Acknowledgements
8. References

1. ABSTRACT

Umbilical cord blood-derived CD133+ cells differentiate into endothelial cells and induce new blood vessel growth. Hypoxia-inducible factor-1 (HIF-1), a regulator of hypoxia or hypoxia-mimetic agent, activates the SDF-1/CXCR4 signaling pathway and plays an important role in angiogenesis in vivo. In this study, we determined whether CD133+ cells enhance angiogenic potential through hypoxia or hypoxia mimetic agent (CoCl2) in vitro. The CD133+ cells were cultured respectively, in normoxia (20% O2), or hypoxia (10% O2, 3% O2), or with various concentrations of CoCl2 (50 microM/L, 100 microM /L, 200 microM /L). We showed here that both hypoxia and CoCl2 induced hypoxia cause a significant increase of CD133+ cell migration, proliferation, and tubule-like structure formation. Under conditions of hypoxia or CoCl2 induced hypoxia, the HIF-1a , SDF-1, and VEGF protein and gene expression level are elevated as compared to those under normoxia conditions. These data suggest that a hypoxia mimetic agent can be used to enhance the angiogenic potential of CD133+ cells.