Enrichment, Propagation, and Characterization of Mouse Testis-Derived Mesenchymal Stromal Cells

The therapeutic potential of multipotent stromal cells (MSCs) largely depends on the isolation and expansion methods used. In this study, we propose a laminin-based technique to select and enrich for MSCs isolated from the mouse testis. Primary cell cultures were prepared from juvenile mouse testes and the capacity to generate colony forming units together with population doubling time (PDT) during expansion were determined. The identity of MSCs was assayed using reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry for the active expression of cell surface markers, such as CD44, CD73, and CD29; absence of the CD45 hematopoietic cell marker; and in vitro differentiation of the cells into osteoblasts and adipocytes. Testis-derived MSCs (tMSCs) displayed self-renewal properties and in the early passages, exhibited high proliferation patterns with an average PDT of 44.1 hours. The lack of Vasa expression implied that the tMSCs were not of germ cell origin. The RT-PCR data, which were confirmed by immunophenotyping, revealed high expression of CD44 and the absence of CD45 expression in tMSCs. The strong Alizarin Red stain in tMSCs that were stimulated into making bone cells was indicative of the presence of calcium-producing cells (osteoblasts). Likewise, the adipogenic potential of tMSCs was demonstrated based on Oil Red O staining of lipid vacuoles in differentiated cells. Loss of fibroblast-like morphology in late passage cells along with the increase in PDT and the decrease in the mRNA levels of CD73 and CD29 suggested that the tMSCs developmental program is reformed at this stage.