[Generation and Evaluation of Human Umbilical Cord Derived Mesenchymal Stem Cells with Antioxidant Capacity]

To prepare mesenchymal stem cells with antioxidant capacity (AO-MSC) from human umbilical cords and evaluate its cell biological properties.

In control group, mesenchymal stem cells (MSC) were isolated by digesting human umbilical cord Wharton's Jelly tissues with 0.2% collagenase II, and the released cells were collected and cultured in an animal serum-free culture medium. In AO-MSC group, incompletely collagenase II-digested tissue debris were allowed to adhere to flusk flat bottoms and the AO-MSC was harvested by adherent culture. The conventional digestion and culture method was used as control. MSC colony forming ability was evaluated by fibroblast colony forming assay (CFU-F). MSC proliferative capacity was evaluated by CCK-8 assay. The MSC surface markers were detected by using flow cytometry and immunofluorescence staining. The adipogenic and osteogenic capacity of MSC was evaluated by multi-differentiation in vitro, and the mRNA expression of genes that control adipogenic and osteogenic differentiation was detected by real-time fluorescence quantitative PCR (RT-qPCR); Moreover, the mRNA expression of antioxidant substances such as SOD-1, GSH, GAT, and NQO1 in MSC was also evaluated by RT-qPCR.

The AO-MSC isolated by this strategy reached a confluence of 80%-90% at around 18 days and grew in a swirling pattern. Flow cytometry and immunofluorescence staining assays showed that CD73, CD29, CD105, CD90 were highly expressed and CD31, CD45, HLA-DR were scarcely expressed in AO-MSC. AO-MSC exhibited stronger self-renewal and differentiation ability compared to MSC. However, the in vitro adipogenic-osteogenic capacity of MSC in the control group was stronger than that of AO-MSC. RT-qPCR assay showed that AO-MSC expressed higher mRNA levels of antioxidant substances compared to MSC.

Human AO-MSC is successfully prepared from human umbilical cord without animal serum.