New advances of NG2-expressing cell subset in marrow mesenchymal stem cells as novel therapeutic tools for liver fibrosis/cirrhosis.

BACKGROUND

Bone marrow-derived mesenchymal stem cell (MSC)-based therapy has become a major focus for treating liver fibrosis/cirrhosis. However, although these cell therapies promote the treatment of this disease, the heterogeneity of MSCs, which causes insufficient efficacy during clinical trials, has not been addressed. In this study, we describe a novel Percoll-Plate-Wait procedure (PPWP) for the isolation of an active cell subset from MSC cultures that was characterized by the expression of neuroglial antigen 2 (NG2/MSCs).

METHODS

By using the key method of PPWP and other classical biological techniques we compared NG2/MSCs with parental MSCs in biological and functional characteristics within a well-defined diethylnitrosamine (DEN)-induced liver fibrosis/cirrhosis injury male C57BL/6 mouse model also in a culture system. Of note, the pathological alterations in the model is quite similar to humans'.

RESULTS

The NG2/MSCs revealed more advantages compared to parentalMSCs. They exhibited greater proliferation potential than parental MSCs, as indicated by Ki-67 immunofluorescence (IF) staining. Moreover, higher expression of SSEA-3 (a marker specific for embryonic stem cells) was detected in NG2/MSCs than in parental MSCs, which suggested that the "stemness" of NG2/MSCs was greater than that of parental MSCs. In vivo studies revealed that an injection of NG2/MSCs into mice with ongoing DEN-induced liver fibrotic/cirrhotic injury enhanced repair and functional recovery to a greater extent than in mice treated with parental MSCs. These effects were associated with the ability of NG2/MSCs to differentiate into bile duct cells (BDCs). In particular, we discovered for the first time that NG2/MSCs exhibit unique characteristics that differ from those of parental MSCs in terms of producing liver sinusoidal endothelial cells (LSECs) to reconstruct injured blood vessels and sinusoidal structures in the diseased livers, which are important for initiating hepatocyte regeneration. This unique potential may also suggest that NG2/MSCs could be an novel off-liver progenitor of LSECs. Ex vivo studies revealed that the NG2/MSCs exhibited a similar trend to that of their in vivo in terms of functional differentiation responding to the DEN-diseased injured liver cues. Additionally, the obvious core role of NG2/MSCs in supporting the functions of MSCs in bile duct repair and BDC-mediated hepatocyte regeneration might also be a novel finding.

CONCLUSIONS

Overall, the PPWP-isolated NG2/MSCs could be a novel effective cell subset with increased purity to serve as a new therapeutic tool for enhancing treatment efficacy of MSCs and special seed cell source (BDCs, LSECs) also for bioliver engineering.