Leukemia inhibitory factor (LIF) is a pleiotropic cytokine that belongs to the interleukin-6 family and is expressed by multiple tissue types. This study analyzed the effect of LIF on osteoblast differentiation using primary murine bone marrow stromal cells (BMSCs). Colony-forming unit-osteoblast formation by BMSCs was significantly suppressed by LIF treatment. To clarify the mechanism underlying the LIF suppressive effect on osteoblast differentiation, we analyzed the downstream signaling pathway of LIF. LIF/signal transducer and activator of transcription 3 (STAT3) signaling induces the expression of suppressor of cytokine signaling 3 (SOCS3). SOCS3 knockdown experiments have previously demonstrated that short-hairpin SOCS3-BMSCs reversed the LIF suppressive effect. Our results demonstrated that LIF suppresses osteoblast differentiation through the LIF/STAT3/SOCS3 signaling pathway.
We recently developed a simple strategy for the enrichment of mesenchymal stem cells (MSCs) with the capacity for osteoblast, chondrocyte, and adipocyte differentiation. On transplantation, the progenitor-enriched fraction can regenerate bone with multiple lineages of donor origin. Although comprising multiple precursor cell types, the population is enriched >100-fold in osteoprogenitors, hence the name "highly purified osteoprogenitors" (HipOPs). To establish a new modified method of purifying pure MSCs, it is useful to know the expression patterns of surface markers on heterogeneous MSCs and committed cells such as osteoblasts, adipocytes, and chondrocytes. However, calcium deposition by osteoblasts is a critical obstacle in visualizing the expression patterns of surface markers. We now report a new method of separating differentiated osteoblastic HipOPs (OB-HipOPs) from calcium deposits using the Percoll density gradient centrifugation technique. After centrifuge separation, calcium deposits were observed at the bottom of the centrifuge tube, and living OB-HipOPs were harvested from the 10-70% fractions. However, there were no living cells in the 70-80% fraction. We concluded that living OB-HipOPs are separated by one 10-70% Percoll gradient. Furthermore, we analyzed the expression patterns of putative MSC markers on differentiated HipOPs. FACS analysis revealed that Sca-1, CD44, CD73, CD105, and CD106 were decreased in OB-HipOPs. In adipogenic- and chondrogenic-HipOPs, Sca-1, CD73, CD105, and CD106 were decreased. This new technique is a helpful tool to identify MSC surface markers and to clarify in more detail the differentiation stages of osteoblasts.
We recently succeeded in purifying a novel multipotential progenitor or stem cell population from bone marrow stromal cells (BMSCs). This population exhibited a very high frequency of colony forming units-osteoblast (CFU-O; 100 times higher than in BMSCs) and high expression levels of osteoblast differentiation markers. Furthermore, large masses of mineralized tissue were observed in in vivo transplants with this new population, designated highly purified osteoprogenitors (HipOPs). We now report the detailed presence and localization of HipOPs and recipient cells in transplants, and demonstrate that there is a strong relationship between the mineralized tissue volume formed and the transplanted number of HipOPs.
Studies describing the effects of leukemia inhibitory factor (LIF) on adipocyte differentiation in murine cells have shown varying results. For example, LIF has been reported to have a suppressive effect on adipocyte differentiation in the 3T3-L1 cell line, whereas it promoted adipocyte differentiation in the Ob1771 and 3T3-F442A cell lines. Thus, it is possible that the effects of LIF on adipogenesis vary with the developmental stage of the cells or tissues, but the details remain unclear. To further elucidate the role of LIF in adipogenesis, we investigated the effects of LIF on murine bone marrow stromal cells at the early and late stages of adipogenesis. LIF decreased the number of lipid foci and suppressed the expression levels of adipocyte differentiation markers at day 5; however, it enhanced these same traits at day 15. A previous report showed that the expression levels of Wnt signaling molecules are different at the early and late differentiation stages; therefore, we investigated the relationship between LIF and Wnt signaling. LIF affected the mRNA expression levels of different Wnt signaling molecules but inhibited the expression level of β-catenin protein at both days 5 and 15. Our data suggest that LIF has reciprocal roles during the early and late stages of adipocyte differentiation, regulating the Wnt signaling pathway.
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