Immunophenotypic characterization, multi-lineage differentiation and aging of zebrafish heart and liver tissue-derived mesenchymal stem cells as a novel approach in stem cell-based therapy

Abstract

Mesenchymal stem cells (MSCs) are a good model for preclinical and clinical investigations, and alternative sources of MSCs are subject to intensive experiments. In this study, mesenchymal stem cells (MSCs) were isolated from heart and liver tissue of Zebrafish (Danio rerio). The flow-cytometry as well as RT-PCR were used to analyze the expression of a panel of cell surface markers CD44, CD90, CD31 and CD34. In the following, alizarin red, oil red-O and toluidine blue staining were carried out to evaluate the multi-lineage differentiation of zebrafish heart and liver tissue-derived MSCs. Subsequently, the gene and protein expression of Oct4, Sox2 and Nanog as pluri-potent markers were analyzed by RT-PCR and western blotting, respectively. In addition, MTT assay was used for cell proliferation potential and population doubling time (PDT) assessment. Also, the aging of cells was investigated by β-galactosidase activity assay. The results showed that, like other MSCs, zebrafish heart and liver tissue-derived MSCs were positive for mesenchymal, negative for hematopoietic markers and expressed pluripotent markers Oct4, Sox2 and Nanog. Moreover, these cells were differentiated to osteocyte, adipocyte, and chondrocyte lineages following directed differentiation. It was found that PDT of zebrafish heart and liver tissue-derived MSCs were 50.67 and 46.61 h, respectively. These cells had significantly more rapid growth on day 4. Our results show that zebrafish heart and liver tissue-derived MSCs exhibited typical MSC characteristics including fibroblast morphology, multi-lineage differentiation capacity, pluripotency potential and expression of a typical set of classic MSC surface markers.