It has been suggested that marrow stromal cells (MSCs) may be immunoprivileged and can engraft in allogeneic recipients with intact immune systems. We determined if the implantation of murine MSCs engineered to release erythropoietin (Epo) would be feasible in major histocompatibility complex (MHC)-mismatched allogeneic mice without immunosuppression, and we monitored hematocrit (Hct) as a reporter of MSC graft survival. MSCs from C57Bl/6 mice were engineered to release murine Epo (Epo ؉ MSCs) and implanted subcutaneously in either syngeneic C57Bl/6 mice or MHC-mismatched Balb/c mice. In syngeneic recipients, the Hct rapidly rose from baseline level and remained higher than .88 (88%) for more than 200 days. However, in MHCmismatched recipient Balb/c mice, the Hct rose transiently and rapidly declined to baseline values. Repeat implantations in these same mice were associated with an acquired refractoriness in the Hct response consistent with alloimmunization to donor Epo ؉ MSCs. Allogeneic MSC implants had an increased proportion of host-derived lymphoid CD8 ؉ , natural killer T (NKT), and NK infiltrating cells compared with syngeneic controls, and splenocytes isolated from Balb/c mice that had received implants also displayed a significant interferon-gamma (IFN␥) response to C57Bl/6 MSCs in vitro. These IntroductionBone marrow stromal cells (MSCs), 1 also sometimes referred to as mesenchymal stem cells, are promising for regenerative medicine due to their innate ability to differentiate into various cell types. [2][3][4][5] Thus, autologous MSCs and their genetically engineered progeny have potential use in several preclinical regenerative medicine scenarios, such as cardiovascular regeneration, 6-14 brain and spinal cord regeneration, 15,16 as well as bone and cartilage repair. [17][18][19][20][21][22][23] Furthermore, genetically engineered MSCs may operate as cellular vehicles for the delivery of therapeutic proteins in hereditary and acquired metabolic, endocrine, and malignant diseases. 4,[24][25][26][27][28][29][30] In vitro studies on human, baboon, and murine MSCs have revealed that MSCs are immunosuppressive. [31][32][33][34] Depending upon the experimental circumstances, suppression of mixed lymphocyte reaction (MLR) in vitro between major histocompatibility complex (MHC)-mismatched stimulator and responder cells by MSCs appears to arise from both contact-dependent 35 and soluble factors including, but not limited to, [36][37][38] hepatocyte growth factor (HGF) and transforming growth factor 1 (TGF-1). 32 However, alternative experimental settings suggest that MSCs may also behave as nonprofessional antigen-presenting cells (APCs). 39,40 In support of their in vivo immunosuppressive features are the observations that allogeneic MSCs may prolong skin allograft survival in immunocompetent baboons, 31 prevent the rejection of allogeneic B16 mouse melanoma cells in immunocompetent C3H mice, 34 and attenuate graft-versus-host disease in mice and humans. 38,41 The sum of these observations supports the...
The ease of isolation and ex vivo culture of marrow-derived stromal cells (MSCs) from adult bone marrow renders them a very promising source of adult stem cells for gene transfer and cell therapy. However, little is known about the signaling pathways that control their in vivo mobilization and subsequent biodistribution. Platelet-derived sphingosine-1-phosphate (S1P), a bioactive lipid that acts via G-protein-coupled-receptors, exerts strong chemoattraction upon MSCs through yetuncharacterized signaling pathways. We show that the S1P-induced migration and morphological changes of MSCs in vitro require the activities of extracellular signal-regulated kinase (ERK), Rho kinase (ROCK), and matrix metalloproteinase (MMP) signaling molecules. Specifically, S1P-induced remodeling of the MSC cytoskeleton led to the rapid (<1 minute) formation of actin stress fibers via activation of the RhoA/ ROCK pathway and required the catalytic activity of MMPs. S1P-induced activation of the mitogen-activated protein kinase kinase-1 (MEK1)/ERK pathway also contributed to the induction of the actin stress fibers and to the redistribution of paxillin at the focal adhesions through tyrosine phosphorylation of focal adhesion kinase in an MMP-dependent manner. Moreover, MMP-and ROCK-dependent molecular events are implicated in the regulation of the S1P-induced activation of ERK. Our results demonstrate that MSC mobilization in response to S1P requires cooperation between MMP-mediated signaling events and the RhoA/ROCK and MEK1/ERK intracellular pathways. Therefore, the characterization of the cellular factors and the intracellular signaling pathways underlying MSC mobilization is crucial to achieve high efficacy in therapeutic use.
Marrow stromal cells (MSCs) can be easily gene-modified and clonally expanded making them ideal candidates for transgenic cell therapy. However, recent reports suggest that MSCs possess immunosuppressive effects, which may limit their clinical applications. We investigated whether interleukin (IL)-2 gene-modified MSCs can be used to mount an effective immune response against the poorly immunogenic B16 melanoma model. We first show that primary MSCs mixed with B16 cells and injected subcutaneously in syngeneic recipients do not affect tumor growth. On the other hand, IL-2-producing MSCs mixed with B16 cells significantly delayed tumor growth in an IL-2 dose-dependent manner. Furthermore, we observed that matrix-embedded IL-2-producing MSCs injected in the vicinity of preestablished B16 tumors led to absence of tumor growth in 90% of treated mice (p < 0.001). We demonstrated that tumor-bearing mice treated with IL-2-producing MSCs developed CD8-mediated tumor-specific immunity and significantly delayed tumor growth of a B16 cell challenge (p < 0.05). In addition, treatment of cd8-/-, cd4-/- and beige mice revealed that CD8+ and natural killer (NK) cells, but not CD4+ cells, were required to achieve antitumor effect. In conclusion, MSCs can be exploited to deliver IL-2 and generate effective immune responses against melanoma in mice with normal immune systems.
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