【Objective】To optimize the isolation and expansion protocol of human umbilical cord mesenchymal stem cells (hUC-MSCs). 【Methods】 Human UC-MSCs were isolated from 20 umbilical cords by combined enzyme digestion (n=12) or by sequential enzyme digestion (n=8). The derivation efficiency

primary cell culture period as well as cell yield were compared. The hUC-MSCs proliferation capacity was compared between complete low glucose-Dulbecco’s modified eagle medium (LG-DMEM) condition and LD-Mesen medium (a medium mixed with MesenPro RSTM (Invitrogen) and LG-DMEM) condition. The phenotypic characteristics of hUC-MSCs were determined by FACS and multi-lineage differentiation capacity was confirmed by induced adipogenesis and osteogenesis.【Results】 The hUC-MSCs derivation efficiency using combined enzymatic digestion was 100% (12/12)

significantly higher than that of sequential enzymatic digestion [12.5% (1/8)

P=1.03×10-4]. With combined enzymatic digestion

(1.30±0.14)×106 cells can be obtained during a mean primary cell culture period of (14.17±1.14) d. Moreover

2×105 hUC-MSCs were cultured for 12 days

more cells can be obtained using LD-Mesen culture medium than using LG-DMEM culture medium [(14.86±0.08)×106 vs (5.08±0.08)×106

P=1.38×10-8]. hUC-MSCs express CD73

CD105

CD90

CD29

and CD44 surface markers

but do not express CD31

CD34

CD45 and HLA-DR. They can be also differentiated into osteoblasts and adipocytes in vitro. 【Conclusion】 hUC-MSCs can be efficiently derived by combined enzymatic digestion from Wharton’s Jelly and expanded by LD-Mesen expansion system

which is significantly superior to the conventional protocol.