No abstract

To study the biodistribution of MSCs, we labeled adult murine C57BL/6 MSCs with firefly luciferase and DsRed2 fluorescent protein using nonviral Sleeping Beauty transposons and coinfused labeled MSCs with bone marrow into irradiated allogeneic recipients. Using in vivo whole-body imaging, luciferase signals were shown to be increased between weeks 3 and 12. Unexpectedly, some mice with the highest luciferase signals died and all surviving mice developed foci of sarcoma in their lungs. Two mice also developed sarcomas in their extremities. Common cytogenetic abnormalities were identified in tumor cells isolated from different animals. Original MSC cultures not labeled with transposons, as well as independently isolated cultured MSCs, were found to be cytogenetically abnormal. Moreover, primary MSCs derived from the bone marrow of both BALB/c and C57BL/6 mice showed cytogenetic aberrations after several passages in vitro, showing that transformation was not a strain-specific nor rare event. Clonal evolution was observed in vivo, suggesting that the critical transformation event(s) occurred before infusion. Mapping of the transposition insertion sites did not identify an obvious transposonrelated genetic abnormality, and p53 was not overexpressed. Infusion of MSC-derived sarcoma cells resulted in malignant lesions in secondary recipients. This new sarcoma cell line, S1, is unique in having a cytogenetic profile similar to human sarcoma and contains bioluminescent and fluorescent genes, making it useful for investigations of cellular biodistribution and tumor response to therapy in vivo. More importantly, our study indicates that sarcoma can evolve from MSC cultures. STEM CELLS 2007;25:371-379

The gene for acute myeloid leukemia-1 (AML-1) is one of the most frequently translocated genes in human cancer. It is targeted by t(8;21) and t(3;21) in AML and by t(12;21) in acute lymphocytic leukemia (39). AML-1 is also indirectly targeted by inv(16), which disrupts core binding factor beta, an AML-1-interacting protein. AML-1 binds the enhancer core motif (TGT/cGGT) and regulates a variety of viral and cellular genes in concert with other factors (31). t(8;21) is one of the most frequent translocations found in AML, comprising 10 to 15% of cases with discernible translocations (39). The t(8;21) fusion protein AML-1/ETO acts as a repressor of transcription in transient-transfection assays (10,31,32,43). When expressed during development, the t(8;21) fusion protein yielded the same phenotype as AML-1 deficiency (37, 45).Although eight-twenty-one (ETO; also known as MTG8 [myeloid tumor gene 8] [8,34]) was identified at the breakpoint of t(8;21), little is known about the normal function of the protein. ETO is the human homologue of the Drosophila Nervy protein (9), and it shares four homologous domains with the Nervy protein. These include a region with extensive homology to a Drosophila coactivator, transcription-activating factor 110 (TAF110), a predicted hydrophobic heptad repeat (HHR), a small domain with no other homology, termed the Nervy domain (27), and the MYND (myeloid-Nervy-DEAF-1 [12]) domain. The MYND domain is present in numerous human, murine, Caenorhabditis elegans, and Drosophila proteins and contains two putative zinc finger (ZF) motifs (9,12,31). ETO is expressed in hematopoietic cells and in the brain, but another closely related family member is ubiquitously expressed (19). A third closely related factor, MTG16, is fused to AML-1 by t(16;21) (20).AML-1 is a site-specific DNA binding protein that can both activate and repress transcription (2, 28, 36). The t(8;21) fusion protein AML-1/ETO contains the N-terminal 177 amino acids of AML-1, including the DNA binding domain, fused to nearly all of ETO (7,8,34). The fusion protein inhibits AML-1-dependent transactivation (10, 32). AML-1/ETO also repressed both basal transcription and Ets-1-dependent activation of the multidrug resistance 1 promoter (27). Similarly, AML-1/ETO inhibited both AML-1 and C/EBP␣-dependent transactivation of the neutrophil protein 3 (NP-3) promoter (44). AML-1/ETO-mediated repression is dependent on both the DNA binding domain of AML-1 and ETO sequences (24). AML-1/ETO acts at substoichiometric levels and thus does not compete with AML-1 for DNA binding sites within promoters, nor does it act to "squelch" transcription (24). Thus, we hypothesized that ETO recruits a corepressor or normally functions as a corepressor to inhibit transcription (30,31,33).Several corepressor proteins have been recently described that associate with histone deacetylases (HDACs) to repress transcription (3,5,13,17,38,40,42). The nuclear hormone corepressor N-CoR was identified through interactions with the thyroid hormone receptor and associa...

Under most conditions, resorbed bone is nearly precisely replaced in location and amount by new bone. Thus, it has long been recognized that bone loss through osteoclast-mediated bone resorption and bone replacement through osteoblast-mediated bone formation are tightly coupled processes. Abundant data conclusively demonstrate that osteoblasts direct osteoclast differentiation. Key questions remain, however, as to how osteoblasts are recruited to the resorption site and how the amount of bone produced is so precisely controlled. We hypothesized that osteoclasts play a crucial role in the promotion of bone formation. We found that osteoclast conditioned medium stimulates human mesenchymal stem (hMS) cell migration and differentiation toward the osteoblast lineage as measured by mineralized nodule formation in vitro. We identified candidate osteoclast-derived coupling factors using the Affymetrix microarray. We observed significant induction of sphingosine kinase 1 (SPHK1), which catalyzes the phosphorylation of sphingosine to form sphingosine 1-phosphate (S1P), in mature multinucleated osteoclasts as compared with preosteoclasts. S1P induces osteoblast precursor recruitment and promotes mature cell survival. Wnt10b and BMP6 also were significantly increased in mature osteoclasts, whereas sclerostin levels decreased during differentiation. Stimulation of hMS cell nodule formation by osteoclast conditioned media was attenuated by the Wnt antagonist Dkk1, a BMP6-neutralizing antibody, and by a S1P antagonist. BMP6 antibodies and the S1P antagonist, but not Dkk1, reduced osteoclast conditioned media-induced hMS chemokinesis. In summary, our findings indicate that osteoclasts may recruit osteoprogenitors to the site of bone remodeling through SIP and BMP6 and stimulate bone formation through increased activation of Wnt/BMP pathways.one is a dynamic tissue that continuously remodels and can undergo regeneration throughout life. This continuous remodeling occurs through a dynamic process of breakdown by osteoclasts and rebuilding by osteoblasts. Bone mass in adults is maintained by a local balance between osteoclastic bone resorption and osteoblastic activities that are mediated via various factors such as hormones, growth factors, cytokines, and matrix proteins. Under most conditions, resorbed bone is nearly precisely replaced in location and amount by new bone. Thus, it has long been recognized that bone loss through osteoclast-mediated bone resorption and bone replacement through osteoblastmediated bone formation are tightly coupled. We now have clear evidence of how osteoblasts direct osteoclast differentiation through RANK and RANKL as well as other pathways (1). Questions remain as to how osteoblasts are recruited to the site after the resorption phase and how the amount of bone produced is controlled. This has led to consideration of how osteoclasts and/or their activity could promote bone formation.Mouse models and humans in whom osteoclastogenesis is perturbed have provided important insights into the role of b...