In contrast to the considerable amount of data that documents the biological properties of mesenchymal progenitor cells from human and other species, there is still paucity of information about mouse counterparts, as their purification and culture expansion procedures remain rudimentary. In the present study, murine mesenchymal progenitor cell (muMPC) culture was developed by explant culture of collagenase-digested bone fragments after removal of the released cells. During cultivation, fibroblastoid cells sprouted and migrated from the fragments, followed by adherent monolayer development. The cells exhibited homogenous surface antigen profile and presented in vitro multipotential differentiation along osteocyte, chondrocyte, and adipocyte lineages, as evaluated by matched cell or matrix staining and reverse transcription polymerase chain reaction techniques. Also, the surface antigenic epitope changed and potential of proliferation and multidifferentiation decreased with successive subculturing. Functional investigations demonstrated that these cells supported in vitro hematopoiesis and suppressed lymphocyte cell proliferation triggered by ConA or allogeneic splenocytes. Furthermore, muMPCs prolonged the mean survival time of skin grafts across the major histocompatibility barrier (H2 b 3 H2 d ), suggestive of the immunosuppressive effects in vivo. The findings demonstrate that muMPCs obtained with this simple protocol are similar in property to their marrow counterparts, and thus, the protocol described here could be used for further investigations in mouse physiological and pathological models. STEM CELLS 2006;24:992-1000

Mesenchymal stem cells (MSCs) are characterized by their hematopoiesis-supporting and immunosuppressive capacity, while osteoclasts are main cell components in the endosteal hematopoietic stem cell niche and pivotal players in osteoimmunology. To clarify the association of these 2 kinds of cells, mouse CD11b(+) monocytes were cultured onto MSC layers in the presence or absence of macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL). The results showed that MSCs independently supported osteoclast development and this effect was enhanced by M-CSF and RANKL. Interestingly, tumor necrosis factor-alpha (TNF-alpha)-stimulated MSCs turned to inhibit osteoclast formation and protect tusk slices from osteoclastic resorption. Real-time PCR and ELISA assays demonstrated that osteoprotegerin expression at both mRNA and protein levels in TNF-alpha-stimulated MSCs was up-regulated, at least partially by activating the mitogen-activated protein kinase pathway. Furthermore, TNF-alpha-stimulated MSCs maintained their immunophenotypic, multipotential differentiation and immunosuppressive characteristics. Moreover, MSCs treated with synovial fluid from rheumatoid arthritis patients modulated osteoclast generation in close relation with the TNF-alpha levels. This study suggests that MSCs exhibit dual modulatory function on osteoclasts and the result might shed light on understanding the involvement of MSCs in the inflammatory diseases.

Previous data have demonstrated that mesenchymal stem cells (MSCs) can exert immunomodulatory activity in vitro, in which of the process nearly all kinds of immune cell subsets are involved. However, there is still a paucity of information about whether and why MSCs inhibit the ongoing immune responses in vivo. Working in a murine splenocyte transfusion model across the major histocompatibility barrier (C57BL/6 → BALB/c, H2 b → H2 d ), we have found that MSC coinfusion prolongs the mean survival time (MST) of the recipient mice in a dose-dependent manner and reduces graft-versus-host-associated histopathology in comparison to the allosplenocyte transfusion controls. In vivo eGFP tracing with polymerase chain reaction analysis revealed that grafted MSCs could migrate and settle into the lungs, spleen, liver, intestine, and skin shortly after administration. Further investigations into the functional characteristics of intrasplenic lymphocytes showed that their proliferation and cytotoxic activity against P815 cells (H2 d ) were significantly restrained by MSC cotransfer. FACS analysis demonstrated that MSC infusion not only increased the proportion of CD4 + subset but also decreased that of CD8 + cells at the belated observation points, resulting in the increase of the ratio of CD4 + /CD8 + cells. Also, in contrast to the slight increase of the proportion of CD4 + CD25 + T regulatory cells (Tregs) in MSC cotransfer mice, the ratio of Tregs/CD8 + cells was dramatically elevated. Furthermore, RT-PCR analysis on the cytokine array of IL-2, IL-4, IL-12, TNF-α, and TGF-β in recipient splenocytes implied the Th1 to Th2 polarization. Therefore, it is deducible that alteration in the proportions of different T-lymphocyte subsets may be one of the main mechanisms by which grafted MSCs suppress the ongoing immune responses in vivo. The study here might provide some new clues for the design of therapeutic approaches for MSC transplantation.

The online version of this article has a Supplementary Appendix. BackgroundThe hemangioblast is a bi-potential precursor cell with the capacity to differentiate into hematopoietic and vascular cells. In mouse E7.0-7.5 embryos, the hemangioblast can be identified by a clonal blast colony-forming cell (BL-CFC) assay or single cell OP9 co-culture. However, the ontogeny of the hemangioblast in mid-gestation embryos is poorly defined. Design and MethodsThe BL-CFC assay and the OP9 system were combined to illustrate the hemangioblast with lymphomyeloid and vascular potential in the mouse aorta-gonad-mesonephros region. The colony-forming assay, reverse transcriptase polymerase chain reaction analysis, immunostaining and flow cytometry were used to identify the hematopoietic potential, and Matrigel-or OP9-based methods were employed to evaluate endothelial progenitor activity. ResultsFunctionally, the aorta-gonad-mesonephros-derived BL-CFC produced erythroid/myeloid progenitors, CD19 + B lymphocytes, and CD3 + TCRβ + T lymphocytes. Meanwhile, the BL-CFCderived adherent cells generated CD31 + tube-like structures on OP9 stromal cells, validating the endothelial progenitor potential. The aorta-gonad-mesonephros-derived hemangioblast was greatly enriched in CD31 + , endomucin + and CD105 + subpopulations, which collectively pinpoints the endothelial layer as the main location. Interestingly, the BL-CFC was not detected in yolk sac, placenta, fetal liver or embryonic circulation. Screening of candidate cytokines revealed that interleukin-3 was remarkable in expanding the BL-CFC in a dose-dependent manner through the JAK2/STAT5 and MAPK/ERK pathways. Neutralizing interleukin-3 in the aorta-gonad-mesonephros region resulted in reduced numbers of BL-CFC, indicating the physiological requirement for this cytokine. Both hematopoietic and endothelial differentiation potential were significantly increased in interleukin-3-treated BL-CFC, suggesting a persistent positive influence. Intriguingly, interleukin-3 markedly amplified primitive erythroid and macrophage precursors in E7.5 embryos. Quantitative polymerase chain reaction analysis demonstrated declined Flk-1 and elevated Scl and von Willebrand factor transcription upon interleukin-3 stimulation, indicating accelerated hemangiopoiesis. ConclusionsThe hemangioblast with lymphomyeloid potential is one of the precursors of definitive hematopoiesis in the mouse aorta-gonad-mesonephros region. Interleukin-3 has a regulatory role with regards to both the number and capacity of the dual-potential hemangioblast.Key words: interleukin-3, hemangioblast, AGM, definitive hematopoiesis, hematopoietic stem cells. Lan Y, Yao H-Y, Li Z, Wang X-Y, Li X-S, Zhang J-Y, Zhang Y, Liu B, and Mao N. Interleukin-3 promotes hemangioblast development in mouse aorta-gonad-mesonephros region. Haematologica 2010;95:875-883. doi:10.3324/haematol.2009 This is an open-access paper. Citation: He W-Y, Interleukin-3 promotes hemangioblast development in mouse aorta-gonad-mesonephros region

As a novel power steering technology, Differential Drive Assisted Steering (DDAS) technology for the independent-wheel-drive electric vehicle has gradually appealed to researcher's attention. However, the previous experimental results show that its assistance quality cannot be fully accepted due to its caused sensitive steering wheel torque fluctuation in actual work environment. According to the working principle of the DDAS system, it is founded that the road roughness, the front wheel alignment parameters and sensor noise are the main factors that influence the quality of assisted steering and driver's road feel. Hence the three factors are added as interference into the ideal vehicle model. The simulation results and its comparison with the previous real vehicle tests confirm this causality between these factors considered and the steering wheel torque fluctuation of the DDAS system. Then a robust H ∞ loop-shaping controller is designed to solve the issue caused by these inner interferences and outer noises. Simulations results validate the proposed controller and show better steering wheel torque performance than the traditional anti-windup PID controller published in the literature earlier.The proposed robust controller is further verified via real vehicle tests and the results are similar to the simulation results which can effectively suppress the steering wheel torque ripple, improve the anti-interference ability of DDAS system and greatly improve the driver's road feel.