Umbilical cord (UC) is a rich source of rapidly proliferating mesenchymal stem cells (MSCs) that are easily cultured on a large-scale. Clinical applications of UC-MSCs include graft-versus-host disease, and diabetes mellitus types 1 and 2. UC-MSCs should be isolated and proliferated according to good manufacturing practice (GMP) with animal component-free medium, quality assurance, and quality control for their use in clinical applications. This study developed a GMP standard protocol for UC-MSC isolation and culture. UC blood and UC were collected from the same donors. Blood vasculature was removed from UC. UC blood was used as a source of activated platelet rich plasma (aPRP). Small fragments (1-2 mm(2)) of UC membrane and Wharton's jelly were cut and cultured in DMEM/F12 medium containing 1 % antibiotic-antimycotic, aPRP (2.5, 5, 7.5 and 10 %) at 37 °C in 5 % CO2. The MSC properties of UC-MSCs at passage 5 such as osteoblast, chondroblast and adipocyte differentiation, and markers including CD13, CD14, CD29, CD34, CD44, CD45, CD73, CD90, CD105, and HLA-DR were confirmed. UC-MSCs also were analyzed for karyotype, expression of tumorigenesis related genes, cell cycle, doubling time as well as in vivo tumor formation in NOD/SCID mice. Control cells consisted of UC-MSCs cultured in DMEM/F12 plus 1 % antibiotic-antimycotic, and 10 % fetal bovine serum (FBS). All UC-MSC (n = 30) samples were successfully cultured in medium containing 7.5 and 10 % aPRP, 92 % of samples grew in 5.0 % aPRP, 86 % of samples in 2.5 % aPRP, and 72 % grew in 10 % FBS. UC-MSCs in these four groups exhibited similar marker profiles. Moreover, the proliferation rates in medium with PRP, especially 7.5 and 10 %, were significantly quicker compared with 2.5 and 5 % aPRP or 10 % FBS. These cells maintained a normal karyotype for 15 sub-cultures, and differentiated into osteoblasts, chondroblasts, and adipocytes. The analysis of pluripotent cell markers showed UC-MSCs maintained the expression of the oncogenes Nanog and Oct4 after long term culture but failed to transfer tumors in NOD/SCID mice. Replacing FBS with aPRP in the culture medium for UC tissues allowed the successful isolation of UC-MSCs that satisfy the minimum standards for clinical applications.