Kevin R. Mcintosh

Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo

OBJECTIVE Mesenchymal stem cells (MSCs), multipotential cells that reside within the bone marrow, can be induced to differentiate into various components of the marrow microenvironment, such as bone, adipose, and stromal tissues. The bone marrow microenvironment is vital to the development, differentiation, and regulation of the lymphohematopoietic system. We hypothesized that the activities of MSCs in the bone marrow microenvironment might also include immunomodulatory effects on lymphocytes. METHODS Baboon MSCs were tested in vitro for their ability to elicit a proliferative response from allogeneic lymphocytes, to inhibit an ongoing allogeneic response, and to inhibit a proliferative response to potent T-cell mitogens. In vivo effects were tested by intravenous administration of donor MSCs to MHC-mismatched recipient baboons prior to placement of autologous, donor, and third-party skin grafts. RESULTS MSCs failed to elicit a proliferative response from allogeneic lymphocytes. MSCs added into a mixed lymphocyte reaction, either on day 0 or on day 3, or to mitogen-stimulated lymphocytes, led to a greater than 50% reduction in proliferative activity. This effect could be maximized by escalating the dose of MSCs and could be reduced with the addition of exogenous IL-2. In vivo administration of MSCs led to prolonged skin graft survival when compared to control animals: 11.3 +/- 0.3 vs 7 +/- 0. CONCLUSIONS Baboon MSCs have been observed to alter lymphocyte reactivity to allogeneic target cells and tissues. These immunoregulatory features may prove useful in future applications of tissue regeneration and stem cell engineering.

Characterization and Functionality of Cell Surface Molecules on Human Mesenchymal Stem Cells

We have characterized adhesion molecules on the surface of multipotential human mesenchymal stem cells (hMSCs) and identified molecules whose ligands are present on mature hematopoietic cells. Flow cytometric analysis of hMSCs identified the expression of integrins: alpha1, alpha2, alpha3, alpha5, alpha6, alphav, beta1, beta3, and beta4, in addition to ICAM-1, ICAM-2, VCAM-1, CD72, and LFA-3. Exposure of hMSCs to IL-1alpha, TNFalpha or IFNgamma up-modulated ICAM-1 surface expression, whereas only IFNgamma increased both HLA-class I and -class II molecules on the cell surface. Whole cell-binding assays between the hMSCs and hematopoietic cell lines showed that T lymphocytic lines bound hMSCs with higher affinity than lines of either B lymphocytes or those of myeloid lineage. Experiments using autologous T lymphocytes isolated from peripheral blood mononuclear cells showed that hMSCs exhibited increased affinity for activated T-lymphocytes compared to resting T cells by quantitative whole cell binding and rosetting assays. Flow cytometric analysis of rosetted cells demonstrated that both CD4+ and CD8+ cells bound to hMSCs. To determine the functional significance of these findings, we tested the ability of hMSCs to present antigen to T lymphocytes. hMSCs pulsed with tetanus toxoid stimulated proliferation and cytokine production (IL-4, IL-10, and IFNgamma) in a tetanus-toxoid-specific T cell line. Maximal cytokine production correlated with maximal antigen-dependent proliferation. These data demonstrate physiological outcome as a consequence of interactions between hMSCs and human hematopoietic lineage cells, suggesting a role for hMSCs in vivo to influence both hematopoietic and immune function(s).

The Immunogenicity of Human Adipose‐Derived Cells: Temporal Changes In Vitro

Regenerative medical techniques will require an abundant source of human adult stem cells that can be readily available at the point of care. The ability to use unmatched allogeneic stem cells will help achieve this goal. Since adipose tissue represents an untapped reservoir of human cells, we have compared the immunogenic properties of freshly isolated, collagenase‐digested human adipose tissue‐derived stromal vascular fraction cells (SVFs) relative to passaged, plastic‐adherent adipose‐derived stem cells (ASCs). Parallel studies have shown that adherence to plastic and subsequent expansion of human adipose‐derived cells selects for a relatively homogeneous cell population based on immunophenotype. Consistent with these findings, the presence of hematopoietic‐associated markers (CD11a, CD14, CD45, CD86, and histocompatible locus antigen‐DR [HLA‐DR]) detected on the heterogeneous SVF cell population decreased upon subsequent passage of the ASCs. In mixed lymphocyte reactions (MLRs), SVFs, and early passage ASCs stimulated proliferation by allogeneic responder T cells. In contrast, the ASCs beyond passage P1 failed to elicit a response from T cells. Indeed, late passage ASCs actually suppressed the MLR response. Although these results support the feasibility of allogeneic human ASC transplantation, confirmatory in vivo animal studies will be required.

Human mesenchymal stem cells: progenitor cells for cartilage, bone, fat and stroma.

Bone marrow provides the rich milieu necessary to maintain myeloid and lymphoid progenitor cells throughout the life of an organism. At least two stem cell populations have been identified in marrow, the hematopoietic stem cell (HSC) and the mesenchymal stem cell (MSC). The HSC has been characterized in many ways, but much remains to be learned about its intrinsic potential and interactions with other cells of the marrow environment. We have studied the human stem cell population for mesenchymal tissues that resides in adult bone marrow. These MSCs potentially have the ability to differentiate to all mesenchymal cell types, including osteocytic, chondrocytic, adipocytic, myocytic, tenocytic, and also dermal and stromal lineages (1, 2). We have sought to understand the potential role(s) that MSCs play in healthy individuals and their response to trauma, disease or aging.

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 × 106 MSCs/kg body weight) followed 6 weeks later by IM injections of DiO-labeled MSCs (5 × 106 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.

Mesenchymal stem cells combined with biphasic calcium phosphate ceramics promote bone regeneration

The reconstruction and repair of large bone defects, resulting from trauma, cancer or metabolic disorders, is a major clinical challenge in orthopaedics. Clinically available biological and synthetic grafts have clear limitations that necessitate the development of new graft materials and/or strategies. Human mesenchymal stem cells (MSCs), obtained from the adult bone marrow, are multipotent cells capable of differentiating into various mesenchymal tissues. Of particular interest is the ability of these cells to differentiate into osteoblasts, or bone-forming cells. At Osiris, we have extensively characterized MSCs and have demonstrated MSCs can induce bone repair when implanted in vivo in combination with a biphasic calcium phosphate, specifically hydroxyapatite/tricalcium phosphate. This article reviews previous and current studies utilizing mesenchymal stem cells and biphasic calcium phosphates in bone repair.

Allogeneic bone marrow stromal cells promote glial-axonal remodeling without immunologic sensitization after stroke in rats.

We evaluated the effects of allogeneic bone marrow stromal cell treatment of stroke on functional outcome, glial-axonal architecture, and immune reaction. Female Wistar rats were subjected to 2 h of middle cerebral artery occlusion. Rats were injected intravenously with PBS, male allogeneic ACI--or syngeneic Wistar--bone marrow stromal cells at 24 h after ischemia and sacrificed at 28 days. Significant functional recovery was found in both cell-treated groups compared to stroke rats that did not receive BMSCs, but no difference was detected between allogeneic and syngeneic cell-treated rats. No evidence of T cell priming or humoral antibody production to marrow stromal cells was found in recipient rats after treatment with allogeneic cells. Similar numbers of Y-chromosome+ cells were detected in the female rat brains in both groups. Significantly increased thickness of individual axons and myelin, and areas of the corpus callosum and the numbers of white matter bundles in the striatum were detected in the ischemic boundary zone of cell-treated rats compared to stroked rats. The areas of the contralateral corpus callosum significantly increased after cell treatment compared to normal rats. Processes of astrocytes remodeled from hypertrophic star-like to tadpole-like shape and oriented parallel to the ischemic regions after cell treatment. Axonal projections emanating from individual parenchymal neurons exhibited an overall orientation parallel to elongated radial processes of reactive astrocytes of the cell-treated rats. Allogeneic and syngeneic bone marrow stromal cell treatment after stroke in rats improved neurological recovery and enhanced reactive oligodendrocyte and astrocyte related axonal remodeling with no indication of immunologic sensitization in adult rat brain.

Acceleration of spinal fusion using syngeneic and allogeneic adult adipose derived stem cells in a rat model

Posterolateral spinal fusion is the standard treatment for lumbar compression fractures. Adult adipose tissue‐derived stem cells (ASCs) promote osteogenesis in vivo and in vitro. The hypothesis tested in this study was that syngeneic and allogeneic ASCs on a biomaterial scaffold composed of tricalcium phosphate and collagen I will accelerate spinal fusion in a rat model. ASCs from male Fischer or ACI rats were loaded onto scaffolds (53,571 cells/mm3) and cultured in stromal media for 48 h. Male Fisher rats were assigned to 4 cohorts (n = 14/cohort) after bilateral decortication of the L4 and L5 transverse processes: (1) No treatment; (2) scaffold only; (3) scaffold + syngeneic ASCs; or (4) scaffold + allogeneic ASCs. Half of each cohort was harvested 4 or 8 weeks after surgery. Spinal fusion was evaluated with radiographs, microcomputed tomography, and light microscopy. Callus did not form in spines without scaffolds. There were no significant differences in callus formation among scaffold cohorts 4 weeks after surgery. Callus formation was more mature in both ASC cohorts versus scaffold alone 8 weeks after surgery based on microstructure as well as radiographic and microcomputed tomographic evidence of active bone formation. Inflammatory cell infiltrate was significantly lower in both ASC cohorts (syngeneic = 18.3 ± 0.85%; allogeneic = 23.5 ± 2.33%) versus scaffold alone (46.8 ± 11.8%) 4 weeks after surgery. Results of this study support syngeneic and allogeneic ASC acceleration of posterior lumbar spinal fusion in a rat model.

Evolution and future prospects of adipose-derived immunomodulatory cell therapeutics.

Over the past two decades, tissue engineering and regenerative medicine have evolved from what many considered a theoretical science to what is now a clinical reality. Tissue engineering combines biomaterial scaffolds, growth factors and stem or progenitor cells to repair damaged tissues. Adipose tissue, an abundant and easily accessed tissue, is a potential source of stromal/stem cells for regenerative therapeutic applications. Like bone marrow-derived mesenchymal stem cells, adipose-derived stromal/stem cells display both immunomodulatory and immunosuppressive properties. The adipose cells exert these actions, in part, through their secretion of paracrine growth factors. This review highlights recent developments in the isolation, characterization and preclinical application of adipose-derived cells and the challenges facing their translation into clinical practice.

Tendon Regeneration Using Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) have the potential to differentiate into the tenocyte lineage and regenerate diseased or injured tendons. Because there is a limited base of research on the tendon lineage, illustrative examples from the bone and cartilage lineage are included to define more clearly the clinical potential of this cell-based approach to medicine. The concluding sections of this chapter consider the emerging data that support the use of allogeneic/universal MSCs as a cost-effective and practical approach for clinical delivery of MSCs.