Tatyana B. Prigozhina

Fibrin Microbeads for Isolating and Growing Bone Marrow–Derived Progenitor Cells Capable of Forming Bone Tissue

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.

Low‐dose γ‐irradiation promotes survival of injured neurons in the central nervous system via homeostasis‐driven proliferation of T cells

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 γ‐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‐γ, 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.

Transplantation of allogeneic or xenogeneic bone marrow within the donor stromal microenvironment

Successful engraftment of hematopoietic stem cells requires a supportive hematopoietic stromal microenvironment (HSM). Defects in the HSM associated with aplastic anemia, myelofibrosis, or caused by intensive ionizing radiation and chemotherapy generally result in failure of bone marrow (BM) engraftment. Transplantation of donor BM within donor HSM may therefore provide optimal conditions for allogeneic BM transplantation. We have transplanted donor hematopoietic cells together with their own HSM to improve acceptance of allogeneic or xenogeneic BM. The non-myeloablative treatment used induced tolerance to murine allografts and provided conditions for the life-long acceptance of allogeneic HSM. Allogeneic BM transplanted within its own HSM under the kidney capsule caused less graft-versus-host disease than BM transplanted i.v. Tolerance in mice to xenogeneic (rat) HSM was less complete. Ectopic ossicles were small and contained fewer hematopoietic cells. However, simultaneous transplantation of rat BM and HSM to preconditioned mice improved engraftment of rat BM compared with transplantation of BM alone. Donor hematopoietic cells survived longer on their own HSM than on HSM of recipients.

Reconstruction of Cartilage, Bone, and Hematopoietic Microenvironment with Demineralized Bone Matrix and Bone Marrow Cells

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.

Nonmyeloablative conditioning to induce bilateral tolerance after allogeneic bone marrow transplantation in mice

We recently described a new nonmyeloablative method to induce stable and specific transplantation tolerance to allogeneic tissues in adult mice. It included total lymphoid irradiation (TLI) of recipients with six fractions of 200 cGy each, inoculation with donor bone marrow (BM) cells and cyclophosphamide (Cy) for selective elimination or inactivation of residual donor-reactive cells of the host, and infusion with T-cell depleted donor BM cells after Cy. Here, we investigated the possibility to induce stable bilateral graft-vs-host and host-vs-graft transplantation tolerance using non-T-cell depleted allogeneic BM. Our results show that the dose of BM required for the induction of transplantation tolerance was inversely correlated with the intensity of the conditioning. Transfer of a low dose (3 x 10(6)) of total donor BM cells to recipients preconditioned with a less intensive regimen (two or three TLI fractions instead of six) diminished graft-vs-host disease (GVHD)-related mortality of recipients to 40% and converted 89% of the survivors into GVHD-free mixed hematopoietic chimeras that maintained donor skin allografts >180 days. A tenfold increase in the number of donor BM cells (3 x 10(7) instead of 3 x 10(6)) reduced the rate of GVHD-related mortality of recipients to 20% and resulted in bilateral transplantation tolerance in 100% of nonirradiated survivors.

Permanent and specific transplantation tolerance induced by a nonmyeloablative treatment to a wide variety of allogeneic tissues: I. Induction of tolerance by a short course of total lymphoid irradiation and selective elimination of the donor-specific host lymphocytes.

The long-term success of organ transplantation is limited by complications resulting from consistent nonspecific immunosuppression. Induction of stable, donor-specific tolerance remains the main goal of transplantation immunology. In this article, a new, nonmyeloablative method is described for induction of transplantation tolerance to fully mismatched bone marrow cells (BMC), bone marrow stromal precursors, heart muscle, and skin allografts. The method is based on pretransplant conditioning with no postgraft immunosuppression, and consists of a short course (six daily fractions of 200 cGy) of total lymphoid irradiation (sTLI), followed by selective elimination of donor-specific alloreactive cells of the host escaping low-dose sTLI. Donor-specific alloreactive cells were activated by intravenous inoculation with a high dose of donor BMC (3 x 10(7) cells) 1 day after sTLI, and eliminated by a single intraperitoneal dose (200 mg/kg) of cyclophosphamide given 1 day after cell transfer. Infusion of a low number of T cell-depleted BMC (3 x 10(6) cells) after tolerogenic preconditioning converted recipients to stable mixed chimeras free of graft-versus-host disease. The same treatment provided long-lasting acceptance of heterotopically transplanted allografts of the heart muscle and of the stromal precursors to the hematopoietic microenvironment. This treatment also led to acceptance and life-long survival of full-thickness donor skin allografts. However, skin allografts survived only in mice that received donor T cell-depleted BMC after cyclophosphamide and had 20-50% donor cells in the blood. Our results suggest that after sTLI, additional selective clonal deletion of residual host cells induces a state of long-lasting specific tolerance to a wide variety of donor-derived tissues.

Nonmyeloablative allogeneic bone marrow transplantation as immunotherapy for hematologic malignancies and metastatic solid tumors in preclinical models

OBJECTIVE We previously demonstrated that a combination of mild total lymphoid irradiation (TLI) with selective depletion of the hosts donor-reactive cells allows for stable and graft-vs-host disease (GVHD)-free engraftment of allogeneic bone marrow (BM). In this study, we investigated the efficacy of this nonmyeloablative strategy for BM transplantation (BMT) as immunotherapy for minimal residual disease. MATERIALS AND METHODS BALB/c mice inoculated with leukemia (BCL1) or breast carcinoma (4T1) cells were conditioned for BMT with TLI (200 cGy) followed by priming with donor (C57BL/6) BM cells on day 1, and by injection with 200 mg/kg cyclophosphamide on day 2. After conditioning (day 3), recipients were transplanted with BM cells from the same donor. Treated animals were monitored for 230 days for survival, development of leukemia/solid tumor, and GVHD. RESULTS BMT converted the mice to complete chimeras and prevented development of leukemia in 90% of recipients and locally growing breast carcinoma in 40% of the mice. Immunization of donors of the second BM with 4T1 cells prevented development of breast carcinoma in 80% of 4T1 inoculated mice. Fewer animals treated for malignancy by nonmyeloablative BMT died of GVHD than those treated by myeloablative BMT. However, late GVHD-related mortality in mice treated for leukemia was higher than after nonmyeloablative BMT to naive recipients (p < 0.00001). Infusion of host-type anti-donor immune lymphocytes 8 days after BMT improved the survival of recipients treated for leukemia without affecting engraftment and the graft-vs-leukemia potential of donor BM. CONCLUSIONS Effective eradication of malignant cells can be achieved following allogeneic BMT after nonmyeloablative conditioning.

IL-2-targeted therapy ameliorates the severity of graft-versus-host disease: ex vivo selective depletion of host-reactive T cells and in vivo therapy.

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.

CD40 ligand-specific antibodies synergize with cyclophosphamide to promote long-term transplantation tolerance across MHC barriers but inhibit graft-vs-leukemia effects of transplanted cells

OBJECTIVES We previously demonstrated that allogeneic bone marrow transplantation (BMT) after low-dose total lymphoid irradiation (200 cGy) and depletion of donor-reactive cells with cyclophosphamide (Cy) converted recipients to graft-vs-host disease (GVHD)-free chimeras tolerant to donor skin grafts. BMT also generated strong graft-vs-leukemia (GVL) response. However, clinical application of the protocol was hampered by the requirement for a relatively high dose of Cy (200 mg/kg). In this study we have tried to minimize the Cy dose by a concomitant blockade of CD40-CD40L interaction. MATERIALS AND METHODS Mildly irradiated BALB/c mice were primed with C57BL/6 BM cells (BM(1)) and skin graft on day 0, injected with Cy (200 mg/kg or less) on day 1, and transplanted with a second C57BL/6 BM cell inoculum (BM(2)) on day 2. CD40L-specific antibody (MR1) was given with BM(1), BM(2), and 2 days later. Treated animals were monitored for survival, chimerism, and skin allograft rejection. The GVL potential of transplanted cells was examined in mice inoculated with BCL1 leukemia cells before irradiation. RESULTS Blocking CD40-CD40L interaction with MR1 mAb allowed the reduction of a tolerance-generating Cy dose by 50%. Unfortunately, adding MR1 to the protocol reduced the GVL potential of the transplanted cells. Neither low-dose Cy nor antibodies alone could downregulate donor or recipient immune response. CONCLUSIONS CD40L-specific antibodies synergize with Cy to induce bilateral transplantation tolerance. Therefore, their use may be beneficial for safer allogeneic BMT for nonmalignant indications. However, due to MR1-associated reduction of GVL effects, MR1 should be considered with caution as conditioning for BMT for leukemia-bearing recipients.

MSC for the improvement of hematopoietic engraftment.

A recent issue of Bone Marrow Transplantation included the results of a clinical trial from Minnesota of 15 pediatric patients who were transplanted with umbilical cord blood in combination with pre-expanded MSCs in an attempt to accelerate hematopoietic recovery.1 According to our present understanding, MSCs, osteoblasts and other stromal cells, including sinusoidal and endothelial, are responsible for supporting hematopoiesis and controlling HSC number. Therefore a co-transplantation of these cells, together with HSC, can be more effective in the reconstitution of hematopoiesis. This approach is especially attractive for cases with a predicted poor engraftment due to both an insufficient number of transplanted HSCs or impaired stroma.