Isolation, propagation and characterization of mouse testis-derived mesenchymal stromal cells

Abstract

Mesenchymal Stromal/Stem Cells (MSCs) are considered promising tools for regenerative therapy. MSCs are multipotent cells that have been successfully isolated from different species and tissues. Although few studies have reported the isolation of MSCs from the testis, there are no reports on their isolation from the mouse, despite its close genetic similarity to humans. Since the testis is a heterogeneous population, enriching for MSCs will aid in the generation of homogeneous populations from early passages. The aim of our work was to utilize the mouse model for the isolation, propagation and subsequent characterization of testis-derived MSCs (tMSCs). To achieve this aim, we harvested testes from 12 male mice and generated single-cell suspensions using a two-step enzymatic digestion technique. Next, we employed a laminin-based technique to enrich for MSCs. The isolated cells were cultured in vitro and growth kinetics was assessed by calculating the Population Doubling Time (PDT). To characterize the isolated cells, Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was performed on MSC markers (CD44, CD73 and CD29), hematopoietic cell marker (CD45), the pluripotency markers Nanog and Oct4 and the germ cell marker Vasa. Also, the percentages of cells expressing CD44 and CD45 were assessed by immunophenotyping using flow cytometry. Successful cultures of tMSCs were established, and the early passages exhibited high proliferation patterns with average PDT 47.7 hours. RT-PCR data, confirmed by Immunophenotyping, revealed the positive expression of CD44 and the negative expression of CD45 in tMSCs. In addition, tMSCs expressed CD73 and CD29 at the transcript level. Loss of the fibroblast-like morphology in late passages together with the increase in PDT and the decrease in expression levels of CD73 and CD29 suggested that tMSCs started to undergo phenotypic divergence. As for the pluripotency markers, tMSCs were found to express Nanog but not Oct4. Interestingly, a significant variation in the expression patterns of different Nanog variants was observed in the isolated cells. The lack of Vasa expression suggested that tMSCs are, most likely, not of germ cell origin. To our knowledge, this is the first study to report the isolation of Mesenchymal Stromal Cells from the mouse testis. Based on our findings, tMSCs possess characteristics and marker profiles similar to that of MSCs, which makes them a potentially valuable tool in cell therapy.