Isolation and cultivation of endothelial progenitor cells (EPCs)

Circulating bone marrow (BM)–derived endothelial progenitor cells (EPCs) are recruited to the site of tissue regeneration and substantially contribute to neovascularization and re-endothelialization after acute vascular injury. Intravenous infusion of EPCs after ischemia was shown to improve neovascularization and cardiac function in both animal models and pilot clinical studies. Conversely, BM-derived EPCs participate in the pathogenesis of various diseases, such as cancer, retinopathy, and atherosclerosis.  Impaired recruitment of BM-derived endothelial and hematopoietic precursor cells is known to block both tumor angiogenesis and growth.  Injection of BM cells promotes injury-associated retinal angiogenesis. BM-derived progenitors also contribute to the formation of the microvasculature in allograft arteriosclerotic lesions.  Therefore, EPCs have both physiologic and pathologic roles; thus, they have been widely considered for establishing new therapeutic strategies for the treatment of angiogenesis-related diseases.  The number of circulating EPCs may limit the ultimate magnitude of angiogenesis therapies and, therefore, strategies that are based on the administration of ex vivo–expanded populations of EPCs harvested from the patient's circulating blood appear promising.
1. Human umbilical cord blood (HUCB) samples (∼50 mL each) were collected from fresh placentas with attached umbilical cords by gravity flow.

2. EPCs were isolated by density gradient centrifugation over Biocoll (Biochrom, Berlin, Germany) for 30 minutes at 400g and washed 3 times in PBS (Biochrom).

3. One million cells were seeded onto 6-well plates coated with human fibronectin in Primary endothelial basal medium.

4. The medium was supplemented with endothelial growth medium containing fetal bovine serum, human VEGF-A, human fibroblast growth factor-B, human epidermal growth factor, insulin-like growth factor 1 (IGF1), and ascorbic acid in appropriate amounts.

5. After 3 days, non-adherent cells were removed, and fresh culture medium was applied.

6. Cultures were maintained for 7 days.

7. Phenotypic analyses of the cells were performed on days 3, 5, and 7.

8. EPC identification and estimation of culture purity (90%-95%) were determined by staining cells with FITC-labeled Ulex europaeus lectin I and uptake of Dil-conjugated acetylated low-density lipoprotein.

References

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