Isolation and Characterization of Human CD34+ EPCs

Ethical approval was granted by the Institutional Review Board of Mackay Medical College, New Taipei City, Taiwan (reference number: P1000002); informed consent obtained from healthy donors before collection of peripheral blood (80 mL). The peripheral blood mononuclear cells (PBMCs) were fractionated from other blood components by centrifuge on a Ficoll-Paque Plus (Amersham Biosciences, Uppala, Sweden), as per manufacturer's instructions. CD34-positive progenitor cells were obtained from the isolated PBMCs using a CD34 MicroBead kit and a MACS Cell Separation System (Miltenyi Biotec, Bergisch Gladbach, Germany). Maintenance and characterization of CD34-positive EPCs were performed as described previously [45 (link), 46 (link)]. Briefly, human CD34-positive EPCs were maintained and propagated in MV2 complete medium consisting of MV2 basal medium and growth supplement (PromoCell, Heidelberg, Germany), with 20% chemically defined FBS (HyClone, Logan, UT). Cells were seeded onto 1% gelatin-coated plastic ware and cultured in humidified air containing 5% CO₂ at 37°C for further treatment.