No abstract

It has been demonstrated that bone marrow (BM)-derived pluripotent stem cells can be incorporated into muscle, bone, nerve, lung, stomach, intestine, and skin. Fibrin-based biodegradable microbeads (FMB) were developed for culturing, in suspension, a high density of cells, mostly of mesenchymal origin. In the current study, FMB were used to isolate and expand mesenchymal progenitor cells from BM of mice and rats. Cells from BM isolated on FMB (FMB-BM cells) were visualized by fluorescent confocal microscopy and quantified by a modified MTS colorimetric assay. Downloading the BM cells from FMB onto plastic induced their differentiation into islets of cells with osteogenic phenotype that secreted mineralized extracellular matrix. This was augmented by inducers of osteogenesis, such as ascorbic acid, beta-glycerophosphate, and dexamethasone, or osteoblast-growth peptides (OGP). Implanting FMB-BM cells under the kidney capsule in mouse tested the osteogenic potential of these cells in vivo. Thirty days after implantation, bone structures with typical BM elements were seen in 8/53 kidneys in 6-Gy-irradiated mice and in 1/10 kidneys in nonirradiated recipients; bone formation was verified by soft x-ray imaging and elemental analysis that showed elevated Ca and Fe in the implant region. FMB-BM cells - downloaded onto plastic flasks, cultured for 2 weeks, mechanically harvested and then implanted - induced 100% bone formation in both irradiated (6/6) and nonirradiated (3/3) mice. Histology revealed well-organized bone structures under the kidney capsule, including osteoblasts and typical elements of BM. Our findings demonstrate that FMB are capable of isolating and expanding progenitor cells from BM for osteogenesis and possibly for regenerating other mesenchymal tissues.

Protective autoimmunity was only recently recognized as a mechanism for attenuating the progression of neurodegeneration. Using a rat model of optic nerve crush or contusive spinal cord injury, and a mouse model of neurodegenerative conditions caused by injection of a toxic dose of intraocular glutamate, we show that a single low dose of whole-body or lymphoid-organ gamma-irradiation significantly improved the spontaneous recovery. Animals with severe immune deficiency or deprived of mature T cells were unable to benefit from this treatment, suggesting that the irradiation-induced neuroprotection is immune mediated. This suggestion received further support from the findings that irradiation was accompanied by an increased incidence of activated T cells in the lymphoid organs and peripheral blood and an increase in mRNA encoding for the pro-inflammatory cytokines interleukin-12 and interferon-gamma, and that after irradiation, passive transfer of a subpopulation of suppressive T cells (naturally occurring regulatory CD4(+)CD25(+) T cells) wiped out the irradiation-induced protection. These results suggest that homeostasis-driven proliferation of T cells, induced by a single low-dose irradiation, leads to boosting of T cell-mediated neuroprotection and can be utilized clinically to fight off neurodegeneration and the threat of other diseases in which defense against toxic self-compounds is needed.

Highly specialized hard tissues, such as cartilage, bone, and stromal microenvironment supporting hematopoiesis, originate from a common type of mesenchymal progenitor cell (MPC). We hypothesized that MPCs present in bone marrow cell suspension and demineralized bone matrix (DBM) that possess natural conductive and inductive features might constitute a unit containing all the essential elements for purposive bone and cartilage induction. Using a rodent preclinical model, we found that implantation of a composite comprising DBM and MPCs into A) a damaged area of a joint; B) an ablated bone marrow cavity, and C) a calvarial defect resulted in the generation of A) a new osteochondral complex comprising articular cartilage and subchondral bone; B) trabecular bone and stromal microenvironment supporting hematopoiesis, and C) flat bone, respectively. The new tissue formation followed differentiation pathways controlled by site-specific physiological conditions, thus developing tissues that precisely met local demands.

T cell depletion prevents graft-versus-host disease (GVHD) but also removes T cell-mediated support of hematopoietic cell engraftment. A chimeric molecule composed of IL-2 and caspase-3 (IL2-cas) has been evaluated as a therapeutic modality for GVHD and selective ex vivo depletion of host-reactive T cells. IL2-cas does not affect hematopoietic cell engraftment and significantly reduces the clinical and histological severity of GVHD. Early administration of IL2-cas reduced the lethal outcome of haploidentical transplants, and survivor mice displayed markedly elevated levels of X-linked forkhead/winged helix (FoxP3(+); 50%) and CD25(+)FoxP3(+) T cells (35%) in the lymph nodes. The chimeric molecule induces in vitro apoptosis in both CD4(+)CD25(-) and CD4(+)CD25(+) subsets of lymphocytes from alloimmunized mice, and stimulates proliferation of cells with highest levels of CD25 expression. Adoptive transfer of IL2-cas-pretreated viable splenocytes into sublethally irradiated haploidentical recipients resulted in 60% survival after a lethal challenge with lipopolysaccharide, which is associated with elevated fractions of CD25(high)FoxP3(+) T cells in the lymph nodes of survivors. These data demonstrate that ex vivo purging of host-presensitized lymphocytes is effectively achieved with IL2-cas, and that IL-2-targeted apoptotic therapy reduces GVHD severity in vivo. Both approaches promote survival in lethal models of haploidentical GVHD. The mechanism of protection includes direct killing of GVHD effectors, prevention of transition to effector/memory T cells, and induction of regulatory T cell proliferation, which becomes the dominant subset under conditions of homeostatic expansion.