Bone marrow contains a population of rare progenitor cells capable of differentiating into osteoblasts, chondrocytes, adipocytes, myoblasts, and hematopoiesis-supporting stromal cells. These cells, referred to as mesenchymal progenitor cells (MPCs), can be purified and cultureexpanded from animals and humans. Using bone-marrowconditioned medium combined with basic fibroblast growth factor, we cultured a relatively homogeneous population of MPCs from murine bone marrow, which uniformly expressed stem cell antigen-1, CD29, CD44, c-kit, and CD105, while being negative for expression of CD45, CD31, and CD34. In vitro differentiation assays showed the tripotential differentiation capacities of these cells toward adipogenic, osteogenic, and chondrogenic lineages. Most importantly, immunophenotypic analyses demonstrated that MPCs did not express major histocompatibility complex class II molecules or the T-cell costimulatory molecules CD80 and CD86, consistent with further investigation showing that MPCs failed to elicit a proliferative response from allogeneic lymphocytes. Moreover, when allogeneic or third-party MPCs were added to T cells stimulated by allogeneic lymphocytes or the potent T-cell mitogen concanavalin-A, a significant reduction in T-cell proliferation was observed. In conclusion, our data demonstrate that we successfully isolated and cultureexpanded a relatively homogeneous population of MPCs from adult murine bone marrow. Additionally, these primary cells could suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. This immunoregulatory feature of MPCs strongly implies that they may have potential applications in allograft transplantation.

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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

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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.