Cellular immune response to intrastriatally implanted allogeneic bone marrow stromal cells in a rat model of Parkinson's disease.

BACKGROUND: Marrow stromal cells (MSC), the non-hematopoietic precursor cells in bone marrow, are being investigated for therapeutic potential in CNS disorders. Although in vitro studies have suggested that MSC may be immunologically inert, their immunogenicity following transplantation into allogeneic recipients is unclear. The primary objective of this study was to investigate the cellular immune response to MSC injected into the striatum of allogeneic recipients (6-hydroxydopamine [6-OHDA]-hemilesioned rats, an animal model of Parkinson's disease [PD]), and the secondary objective was to determine the ability of these cells to prevent nigrostriatal dopamine depletion and associated motor deficits in these animals. METHODS: 5-Bromo-2-deoxyuridine (BrdU) - labeled MSC from two allogeneic sources (Wistar and ACI rats) were implanted into the striatum of adult Wistar rats at the same time as 6-OHDA was administered into the substantia nigra. Behavioral tests were administered one to two weeks before and 16-20 days after 6-OHDA lesioning and MSC transplantation. Immunocytochemical staining for T helper and T cytotoxic lymphocytes, microglia/macrophages, and major histocompatibility class I and II antigens was performed on post-transplantation days 22-24. MSC were detected with an anti-BrdU antibody. RESULTS: Tissue injury due to the transplantation procedure produced a localized cellular immune response. Unexpectedly, both sources of allogeneic MSC generated robust cellular immune responses in the host striatum; the extent of this response was similar in the two allograft systems. Despite these immune responses, BrdU+ cells (presumptive MSC) remained in the striatum of all animals that received MSC. The numbers of remaining MSC tended to be increased (p = 0.055) in rats receiving Wistar MSC versus those receiving ACI MSC. MSC administration did not prevent behavioral deficits or dopamine depletion in the 6-OHDA-lesioned animals. CONCLUSION: MSC, when implanted into the striatum of allogeneic animals, provoke a marked immune response which is not sufficient to clear these cells by 22-24 days post-transplantation. In the experimental paradigm in this study, MSC did not prevent nigrostriatal dopamine depletion and its associated behavioral deficits. Additional studies are indicated to clarify the effects of this immune response on MSC survival and function before initiating trials with these cells in patients with PD or other neurodegenerative disorders.

Bone marrow stromal cells protect oligodendrocytes from oxygen-glucose deprivation injury.

Oligodendrocyte (OLG) damage leads to demyelination, which is frequently observed in ischemic cerebrovascular diseases. In this study, we investigated the effect of bone marrow stromal cells (BMSCs) on OLGs subjected to oxygen-glucose deprivation (OGD). N20.1 cells (mouse OLG cell line) were transferred into an anaerobic chamber for 3 hr in glucose-free and serum-free medium. After OGD incubation, OLG cultures were divided into the following groups: 1) OGD alone, 2) OLG cocultured with BMSCs, 3) treatment with the phosphoinostide 3-kinase (PI3k) inhibitor LY294002, 4) LY294002-treated OLGs with BMSC cocultured, and 5) anti-p75 antibody-treated OLGs. After an additional 3 hr of reoxygenation incubation, OLG viability and apoptosis were measured. The mRNA expression in the BMSCs and OLGs was analyzed using quantitative real-time PCR (RT-PCR). Serine/threonine-specific protein kinase (Akt), phosphorylated Akt (p-Akt), p75, and caspase 3 protein expressions in OLGs were measured by Western blot. Our results suggest that BMSCs produce growth factors, activate the Akt pathway, and increase the survival of OLGs. BMSCs also reduce p75 and caspase 3 expressions in the OGD-OLGs, which leads to decreased OLG apoptosis. BMSCs participate in OLG protection that may occur with promoting growth factors/PI3K/Akt and inhibiting the p75/caspase pathways. Our study provides insight into white matter damage and the therapeutic benefits of BMSC-based remyelinating therapy after stroke and demyelinating diseases.

Axonal sprouting into the denervated spinal cord and synaptic and postsynaptic protein expression in the spinal cord after transplantation of bone marrow stromal cell in stroke rats.

We investigated whether compensatory reinnervation in the corticospinal tract (CST) and the corticorubral tract (CRT) is enhanced by the administration of bone marrow stromal cells (BMSCs) after experimental stroke. Adult male Wistar rats were subjected to permanent right middle cerebral artery occlusion (MCAo). Phosphate-buffered saline (PBS, control, n=7) or 3x10(6) BMSCs in PBS (n=8) were injected into a tail vein at 1 day postischemia. The CST of the left sensorimotor cortices was labeled with DiI 2 days prior to MCAo. Functional recovery was measured. Rats were sacrificed at 28 days after MCAo. The brain and spinal cord were removed and processed for vibratome sections for laser-scanning confocal analysis and paraffin sections for immunohistochemistry. Normal rats (n=4) exhibited a predominantly unilateral pattern of innervation of CST and CRT axons. After stroke, bilateral innervation occurred through axonal sprouting of the uninjured CRT and CST. Administration of BMSCs significantly increased the axonal restructuring on the de-afferented red nucleus and the denervated spinal motoneurons (p<0.05). BMSC treatment also significantly increased synaptic proteins in the denervated motoneurons. These results were highly correlated with improved functional outcome after stroke (r>0.81, p<0.01). We conclude that the transplantation of BMSCs enhances axonal sprouting and rewiring into the denervated spinal cord which may facilitate functional recovery after focal cerebral ischemia.

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.

Studies of the route of administration and role of conditioning with radiation on unrelated allogeneic mismatched mesenchymal stem cell engraftment in a nonhuman primate model.

OBJECTIVE: The aim of this study was to examine the effects of the route of administration [intrabone marrow (IBM) vs intravenous (IV)] and the role of conditioning with irradiation in optimizing mesenchymal stem cell (MSC) transplantation. MATERIALS AND METHODS: To determine if irradiation resulted in depletion of colony-forming unit fibroblasts (CFU-F), which might favor the engraftment of donor MSC, the number of CFU-Fs was assayed from animals receiving either hemibody irradiation (HBI) or total body irradiation (TBI). RESULTS: TBI resulted in a marked reduction of CFU-F numbers that spontaneously resolved, whereas animals receiving HBI did not experience depletion of CFU-F. Animals receiving MSC grafts by the IV route had higher numbers of marrow CFU-F. MSC were transduced using retroviral vectors encoding the neomycin resistance gene (Neo(R)) and a second gene encoding either the human soluble tumor necrosis factor receptor (hsTNFRII) or beta-galactosidase (beta-Gal). MSCs were administered by either the IV or IBM route to animals receiving HBI. The Neo(R) transgene was detectable in hematopoietic tissues of all animals and nonhematopoietic tissues in a single animal. Evidence of transgene expression was documented by detection of beta-Gal(+) cells in BM smears and transiently elevated serum levels of hsTNFRII. CONCLUSION: These studies indicate that 1) MSC possess the ability to engraft and persist in an unrelated mismatched allogeneic hosts; 2) 250-cGy HBI did not favor engraftment of MSC; 3) the IBM route was not more effective than the IV route in delivering MSC grafts; and 4) transplanted MSC preferentially localized to the marrow rather than nonhematopoietic tissues.

Immunogenicity of allogeneic adipose-derived stem cells in a rat spinal fusion model.

Adipose-derived stem cells (ASCs) express a nonimmunogenic profile as shown by in vitro studies that demonstrate a lack of T cell proliferation to allogeneic ASCs as well as ASC-mediated suppression of mixed lymphocyte reactions. To determine whether these observations would translate in vivo, immune monitoring studies were carried out in conjunction with a rat spinal fusion study. ASCs derived from Fischer or ACI strain rats were loaded onto scaffolds and implanted in Fischer recipients that had undergone the following treatments: (1) No treatment; (2) Scaffold only; (3) Syngeneic ASCs+Scaffold; or (4) Allogeneic ASCs+Scaffold. Half of each group was sacrificed at 4 weeks postimplantation, and the remaining animals were sacrificed at 8 weeks. As determined in a separate study, allogeneic and syngeneic ASCs were equally efficacious in accelerating spinal fusion compared to No treatment and Scaffold only control groups. To determine whether donor ASCs induced an immune response in recipient rats, lymph nodes were harvested for T cell proliferation studies and serum was collected to assess antibody responses. Although T cell priming was not detected to donor alloantigens in recipients at either time point, significant antibody responses were detected to ACI ASCs in animals implanted with syngeneic or allogeneic ASCs. Antibodies were of the IgG isotype, noncytotoxic in the presence of complement, and reactive to fetal bovine serum. These results support the use of allogeneic ASCs for spinal fusion.

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/mm(3)) 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.

Bone marrow stromal cell therapy reduces proNGF and p75 expression in mice with experimental autoimmune encephalomyelitis.

Demyelination is prominent in experimental autoimmune encephalomyelitis (EAE). The receptor p75 and its high affinity ligand proNGF are required for oligodendrocyte death after injury. We hypothesize that bone marrow stromal cells (BMSCs) provide therapeutic benefit in EAE mice by reducing proNGF/p75 expression. PBS or BMSCs (2 x 10(circumflex)6) were administered intravenously on the day of EAE onset. Neurological function and demyelination areas were measured. Immunohistochemical staining was used to measure apoptotic oligodendrocytes, expression of proNGF and p75, and the relationship between proNGF and p75 in neural cells. proNGF was used to treat oligodendrocytes in culture with or without BMSCs. EAE mice exhibited neurological function deficit and demyelination, and expression of proNGF and p75 was increased. BMSC treatment improved functional recovery, reduced demyelination area and apoptotic oligodendrocytes, decreased expression of proNGF and p75 compared with PBS treatment. proNGF(+) cells colocalized with neural cell markers, while p75 colocalized with an oligodendrocytic marker, and proNGF colocalized with p75. proNGF induced apoptosis of oligodendrocytes in vitro, and p75 antibody blocked this apoptotic activity. BMSCs reduced p75 expression and apoptotic activity in oligodendrocytes with proNGF treatment. BMSC treatment benefits on EAE mice may be fostered by decreasing the cellular expression of proNGF and p75, thereby reducing oligodendrocyte death.

Bone marrow stromal cells increase oligodendrogenesis after stroke.

Oligodendrocytes are sensitive to ischemic damage. The Sonic hedgehog (Shh) pathway is critical in oligodendrogenesis; Gli1 is the principal effector of Shh signaling. We investigated oligodendrogenesis and Shh/Gli1 pathway activation after bone marrow stromal cell (BMSC) treatment of stroke in rats. Rats were subjected to the middle cerebral artery occlusion (MCAo). BMSCs have been shown to promote functional recovery post stroke. A therapeutic dose of BMSC (3 x 10(6) cells) treatment was initiated 1 day after MCAo. Immunohistochemistry was carried out to measure the oligodendrocyte progenitor cells, oligodendrocytes, myelin, and expressions of Shh and Gli1 at 14 days after MCAo. Gene expression of Shh and Gli1 was tested at 2 days after MCAo. An in vitro study was used to investigate the effects of BMSC on a premature oligodendrocyte cell line (N20.1 cells). BMSC treatment significantly increased O4(+) oligodendrocytes, MBP(+) area, and bromodeoxyuridine (BrdU)(+), NG2(+), BrdU(+)-NG2(+) cells, and mRNA and protein expressions of Shh and Gli1 in the ipsilateral brain of the MCAo rats than that in phosphate buffered saline (PBS)-treated rats. BMSCs promoted N20.1 cell proliferation and Gli1 mRNA expression, and these effects were abolished by the Shh pathway inhibitor cyclopamine. These data indicate that the BMSC treatment stimulates oligodendrogenesis by activation of the Shh/Gli1 pathway post stroke.

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.

Mesenchymal stem cells home to injured tissues when co-infused with hematopoietic cells to treat a radiation-induced multi-organ failure syndrome.

BACKGROUND: Recent studies have suggested that ex vivo expansion of autologous hematopoietic cells could be a therapy of choice for the treatment of bone marrow failure. We investigated the potential of a combined infusion of autologous ex vivo expanded hematopoietic cells with mesenchymal (MSCs) for the treatment of multi-organ failure syndrome following irradiation in a non-human primate model. METHODS: Hematopoietic cells and MSCs were expanded from bone marrow aspirates. MSCs were transduced with the gene encoding for the green fluorescent protein (e-GFP), in order to track them following infusion. Twelve animals were studied. Nine animals received total-body irradiation at 8 Gy from a neutron/gamma source thus resulting in heterogeneous exposure; three animals were sham-irradiated. The animals were treated with expanded hematopoietic stem cells and MSCs, expanded hematopoietic stem cells alone, or MSCs alone. Unmanipulated bone marrow cell transplants were used as controls. RESULTS: Depending on the neutron/gamma ratio, an acute radiation sickness of varying severity but of similar nature resulted. GFP-labeled cells were found in the injured muscle, skin, bone marrow and gut of the treated animals via PCR up to 82 days post-infusion. CONCLUSIONS: This is the first evidence of expanded MSCs homing in numerous tissues following a severe multi-organ injury in primates. Localization of the transduced MSCs correlated to the severity and geometry of irradiation. A repair process was observed in various tissues. The plasticity potential of the MSCs and their contribution to the repair process in vivo remains to be studied.