A novel and feasible way to cultivate and purify endothelial progenitor cells from bone marrow of children with congenital heart diseases.

BACKGROUND

Endothelial progenitor cells (EPCs) are used in vascular tissue engineering and clinic therapy. Some investigators get EPCs from the peripheral blood for clinic treatment, but the number of EPCs is seldom enough. We have developed the cultivation and purification of EPCs from the bone marrow of children with congenital heart disease, to provide enough seed cells for a small calibre vascular tissue engineering study.

METHODS

The 0.5-ml of bone marrow was separated from the sternum bone, and 5-ml of peripheral blood was collected from children with congenital heart diseases who had undergone open thoracic surgery. CD34+ and CD34+/VEGFR+ cells in the bone marrow and peripheral blood were quantified by flow cytometry. CD34+/VEGFR+ cells were defined as EPCs. Mononuclear cells in the bone marrow were isolated by Ficoll(®) density gradient centrifugation and cultured by the EndoCult Liquid Medium Kit(™). Colony forming endothelial cells was detected. Immunohistochemistry staining for Dil-ac-LDL and FITC-UEA-1 confirmed the endothelial lineage of these cells.

RESULTS

CD34+ and CD34+/VEGFR+ cells in peripheral blood were (0.07 ± 0.05)% and (0.05 ± 0.02)%, respectively. The number of CD34+ and CD34+/VEGFR+ cells in bone marrow were significantly higher than in blood, (4.41 ± 1.47)% and (0.98 ± 0.65)%, respectively (P < 0.0001). Many colony forming units formed in the culture. These cells also expressed high levels of Dil-ac-LDL and FITC-UEA-1.

CONCLUSION

This is a novel and feasible approach that can cultivate and purify EPCs from the bone marrow of children with congenital heart disease, and provide seed cells for small calibre vascular tissue engineering.