Enhanced epithelial gap closure and increased angiogenesis in wounds of diabetic mice treated with adult murine bone marrow stromal progenitor cells.

The direct application of bone marrow (BM) can accelerate the healing of chronic wounds. We hypothesized that this effect is due to the presence of stromal progenitor cells (SPCs) found within whole BM preparations. To test this hypothesis, we isolated adult murine SPCs from whole BM and examined their ability to enhance impaired wound healing compared with ficoll separated BM cells in the diabetic (db/db) mouse model. SPCs significantly enhanced reepithelialization, granulation tissue formation, and neovascularization compared with control wounds treated with BM or PBS alone. Higher frequencies of donor SPC cells compared with donor BM cells were observed in treated wounds at 7 days. Transdifferentiation into GFP-positive mature endothelial cells was not observed. These observations suggest that SPCs improve wound healing through indirect mechanisms which lead to enhanced vascularization rather than through direct participation and incorporation into tissue. We conclude that topical application of BM-derived SPCs may represent an effective strategy for the treatment of chronic diabetic wounds.

Murine bone marrow stromal progenitor cells elicit an in vivo cellular and humoral alloimmune response.

Stromal progenitor cells (SPC) exhibit immunosuppressive effects in vitro that have led to speculation regarding their capacity to evade host immune recognition and to treat autoimmune diseases and gravt-versus-host disease. However, there is little in vivo experimental data to support these immunologic claims. To assess immune recognition of SPC in vivo, we evaluated the immune response of animals transplanted with SPC. C57BL/6 (B6) or Balb/c adult, murine, bone marrow-derived SPC (AmSPC) were administered by intraperitoneal injection into B6 recipients. T cell proliferation and alloantibody response was assessed from spleens and peripheral blood harvested from transplanted animals and analyzed by cell proliferation assay and flow cytometry. To assess tolerance induction, transplanted animals also received allogeneic skin grafts. Animals injected with allogeneic AmSPC mounted an accelerated CD4 response to alloantigen compared to syngeneic AmSPC injected and uninjected controls. Allogeneic AmSPC-injected animals also demonstrated high titers (> or =1:1000) of antibody directed against allogeneic AmSPC targets. Animals primed with donor or host-matched AmSPC also failed to induce tolerance, and all animals exhibited rejection of allogeneic skin grafts (n = 7, P < .0001). In contrast to their in vitro behavior, our data demonstrate that AmSPC are recognized by the host immune system in vivo, elicit a cellular and humoral immune response, and fail to induce tolerance. These findings have significant implications for all allogeneic SPC-based therapeutic strategies.

Immunologic consequences of multiple, high-dose administration of allogeneic mesenchymal stem cells to baboons.

Mesenchymal stem cells (MSCs) express low immunogenicity and demonstrate immunomodulatory properties in vitro that may safely allow their transplantation into unrelated immunocompetent recipients without the use of pharmacologic immunosuppression. To test this hypothesis, three groups of baboons (three animals per group) were injected as follows: group 1 animals were injected with vehicle; group 2 animals were injected IV with DiI-labeled MSCs (5 x 106 MSCs/kg body weight) followed 6 weeks later by IM injections of DiO-labeled MSCs (5 x 10(6) MSCs/kg) from the same donor; and group 3 animals were treated similarly as group 2 except that MSCs were derived from two different donors. Muscle biopsies, performed 4 weeks after the second injection of MSCs, showed persistence of DiO-labeled MSCs in 50% of the recipients. Blood was drawn at intervals for evaluation of basic immune parameters (Con A mitogen responsiveness, PBMC phenotyping, immunoglobulin levels), and to determine T-cell and alloantibody responses to donor alloantigens. Host T-cell responses to donor alloantigens were decreased in the majority of recipients without suppressing the overall T-cell response to Con A, or affecting basic parameters of the immune system. All recipient baboons produced alloantibodies that reacted with donor PBMCs. Two of six animals produced alloantibodies that reacted with MSCs. We conclude that multiple administrations of high doses of allogeneic MSCs affected alloreactive immune responses without compromising the overall immune system of recipient baboons. The induction of host T-cell hyporesponsiveness to donor alloantigens may facilitate MSC survival.

T cell responses to allogeneic human mesenchymal stem cells: immunogenicity, tolerance, and suppression.

Human mesenchymal stem cells (MSCs) were evaluated for their ability to activate allogeneic T cells in cell mixing experiments. Phenotypic characterization of MSCs by flow cytometry showed expression of MHC Class I alloantigens, but minimal expression of Class II alloantigens and costimulatory molecules, including CD80 (B7-1), CD86 (B7-2), and CD40. T cells purified from peripheral blood mononuclear cells (PBMCs) did not proliferate to allogeneic MSCs. Lack of response was not due to a deficiency of costimulation, since retroviral transduction of MSCs with either B7-1 or B7-2 costimulatory molecules did not result in lymphoproliferation. Although these results suggested that MSCs were immunologically inert or potentially tolerogenic, T cells cultured with MSCs produced IFN-gamma and displayed secondary kinetics to restimulation with PBMCs, indicating alloantigen priming rather than tolerance induction by the MSCs. To determine whether MSCs suppressed alloreactive T cells, MSCs were added to primary mixed lymphocyte reaction (MLR) cultures. MSCs suppressed cell proliferation when added at the initiation of culture or when added to an ongoing MLR culture. Suppression was dose-dependent, genetically unrestricted, and occurred whether or not MSCs were pretreated with IFN-gamma. MSCs in transwell chambers suppressed primary MLR cultures, indicating that suppression was mediated by soluble molecules. Analysis of cytokines in suppressed MLR cultures demonstrated up-regulation of IFN-gamma and IL-10, and down-regulation of TNF-alpha production relative to control cultures. We conclude that MSCs can initiate activation of alloreactive T cells, but do not elicit T cell proliferative responses due to active suppressive mechanisms.

Mesenchymal stem cells: paradoxes of passaging.

The field of stem cell biology continues to evolve with the ongoing characterization of multiple types of stem cells with their inherent potential for experimental and clinical application. Mesenchymal stem cells (MSC) are one of the most promising stem cell types due to their availability and the relatively simple requirements for in vitro expansion and genetic manipulation. Multiple populations described as "MSCs" have now been isolated from various tissues in humans and other species using a variety of culture techniques. Despite extensive in vitro characterization, relatively little has been demonstrated regarding their in vivo biology and therapeutic potential. Nevertheless, clinical trials utilizing MSCs are currently underway. The aim of this review is to critically analyze the field of MSC biology, particularly with respect to the current paradox between in vitro promise and in vivo efficacy. It is the authors' opinion that until this paradox is better understood, therapeutic applications will remain limited.