Establishment of mesenchymal stem cell lines derived from the bone marrow of green fluorescent protein-transgenic mice exhibiting a diversity in intracellular transforming growth factor-β and bone morphogenetic protein signaling

Cytokines and their intercellular signals regulate the multipotency of mesenchymal stem cells (MSCs). The present study established the MSC lines SG-2, -3, and -5 from the bone marrow of green fluorescent protein (GFP)-transgenic mice. These cell lines clearly expressed mouse MSC markers Sca-1 and CD44, and SG-2 and -5 cells retained the potential for osteogenic and adipogenic differentiation in the absence of members of the transforming growth factor (TGF)-β superfamily. By contrast, SG-3 cells only retained adipogenic differentiation potential. Analysis of cytokine and cytokine receptor expression in these SG cell lines showed that bone morphogenetic protein (BMP) receptor 1B was most highly expressed in the SG-3 cells, which underwent osteogenesis in response to BMP, while TGF-β receptor II was most highly expressed in SG-3 and -5 cells. However, it was unexpectedly noted that phosphorylation of Smad 2, a major transcription factor, was induced by TGF-β1 in SG-2 cells but not in SG-3 or -5 cells. Furthermore, TGF-β1 clearly induced the expression of Smad-interacting transcription factor CCAAT/enhancer binding protein-β in SG-2 but not in SG-3 or -5 cells. These results demonstrated the establishment of TGF-β-responsive SG-2 MSCs, BMP-responsive SG-3 MSCs and TGF-β/BMP-unresponsive SG-5 MSCs, each of which was able to be traced by GFP fluorescence after transplantation into in vivo experimental models. In conclusion, the present study suggested that these cell lines may be used to explore how the TGF-β superfamily affects the proliferation and differentiation status of MSCs in vivo.