Isolation of stem cells from human umbilical cord tissue by enzymatic digestion

Objective To investigate the possibility of mesenchymal stem cells derived from human umbilical cord tissue by enzymatic digestion.Methods The umbilical cord tissue was taken from volunteers,and treated with collagenase Ⅱ and trypsin.Then,the isolated cells were cultured and undergone osteogenic,adipogenic and cardiomyogenic differentiation.The morphology of the cells was observed.At the end of culture,differentiation was examined with Kossa or Oil red-O solution stainingand immunocytochemistry.Phenotypes of isolated cells were identified by flow cytometry.Results When the isolated ceils were initially plated,they appeared round shape.After 72 hours' plating,the cells were adherent and appeared elongated and spindle shape.By flow cytometry,the cells were identified positive for CD13,CD29,CD44,CD90,CD105,CD166,and MHC-Ⅰ,but negative for CD31,CD34,CD45,CD106 and MHC-Ⅱ.After cultivation in osteogenic and adipogenic differentiation medium.thecells were positive to Kossa staining and had ability to accumulate different amounts of lipid vacuoles.Additionally,immunocytochemistry showed that differentiated cells were strongly positive for cardiacsarcomeric α-actin and Troponin T after 4 weeks'5-azacytidine treatment. Conclusions Human umbilical cord tissue as the source of mesenchymal stem cells can be successfully isolated by enzymatic digestion. Key words: Umbilical cord; Stem cells; Cell separation; Collagenase