Hiroko Hisha

Evidence for migration of donor bone marrow stromal cells into recipient thymus after bone marrow transplantation plus bone grafts: A role of stromal cells in positive selection

Intrathymic T-cell differentiation is characterized by two selection events: positive and negative selection. It has been shown that thymic epithelial cells in the cortex are involved in the positive selection, while macrophages and dendritic cells, derived from hemopoietic stem cells, are involved in the negative selection. Here we investigate whether donor-derived bone marrow stromal cells can migrate into the thymus and participate there in positive selection after bone marrow transplantation plus bone grafts (to recruit bone marrow stromal cells). Allogeneic bone marrow transplantation with or without bone grafts was carried out in the [C57BL/6-->C3H] combination. Fluorescence-activated cell sorter analyses of recipient thymic adherent cells showed that donor-type bone marrow stromal cells exist in the thymus of mice that received bone marrow plus bone grafts but not in the mice that received bone marrow cells alone. Histological examination using confocal microscopy also confirmed the existence of donor-type stromal cells in the thymus of mice that received bone marrow cells plus bones. Both T-cell proliferation and plaque-forming cell assays indicated that the T cells of such mice show donor-type major histocompatibility complex-restriction. These findings strongly suggest that stromal cells can migrate from the bone marrow to the thymus, where they participate in the positive selection of thymocytes.

Major Histocompatibility Complex Restriction Between Hematopoietic Stem Cells and Stromal Cells In Vitro

We have previously found that a significant number of hematopoietic progenitors accumulate in engrafted bones with the same major histocompatibility complex (MHC) as the transplanted bone marrow cells. In the present study, to further clarify the MHC restriction between hematopoietic stem cells (HSC) and microenvironment, we carried out cobblestone colony formation assays by culturing HSCs with MHC‐matched or ‐mismatched stromal cell monolayers. The formation of cobblestone colonies under MHC‐mismatched stromal cells significantly decreased in comparison with MHC‐matched stromal cells. However, the decrease in cobblestone colony formation under MHC‐mismatched stromal cells was not significant when using MHC class I‐deficient HSC or stromal cells. Taken together with the results using B10 congenic strains, it is suggested that the MHC preference is restricted by MHC class Ia molecules. Treatment with monoclonal antibodies (mAbs) against MHC class Ia molecules of stromal cell phenotypes significantly enhanced the cobblestone colony formation, whereas treatment with mAbs against HSC phenotypes significantly inhibited it. The expression of cytokines to promote hematopoiesis was enhanced by the mAbs against stromal cell phenotypes. The enhancement of cytokine expression was also observed when stromal cells and HSCs were MHC‐matched. These results suggest that signaling via the MHC molecules augments stromal cell activity and elicits the MHC restriction.

Characterization of mesenchymal stem cells isolated from mouse fetal bone marrow.

Mesenchymal stem cells (MSCs) are defined as cells that can differentiate into multiple mesenchymal lineage cells. MSCs have some features (surface molecules and cytokine production, etc.) common to so‐called traditional bone marrow (BM) stromal cells, which have the capacity to support hemopoiesis. In the present study, we isolated murine MSCs (mMSCs) from the fetal BM using an anti‐PA6 monoclonal antibody (mAb) that is specific for bone marrow stromal cells. The mMSCs, called FMS/PA6‐P cells, are adherent, fibroblastic, and extensively expanded and have the ability to differentiate not only into osteoblasts and adipocytes but also into vascular endothelial cells. The FMS/PA6‐P cells produce a broad spectrum of cytokines and growth factors closely related to hemopoiesis and show good hemopoiesis‐supporting capacity both in vivo and in vitro, suggesting that they are a component of the hemopoietic stem cell niche in vivo. Interestingly, although the FMS/PA6‐P cells express a high level of the PA6 molecule, which is reactive with anti‐PA6 mAb, they gradually lose their ability to express this molecule during the course of differentiation into osteoblasts and adipocytes, indicating that the PA6 molecule might serve as a novel marker of mMSCs.

A New Method for Bone Marrow Cell Harvesting

To minimize contamination of bone marrow cells (BMCs) with T cells from the peripheral blood, a new “perfusion method” for collecting BMCs is proposed using cynomolgus monkeys. Two BM puncture needles are inserted into a long bone such as the humerus, femur, or tibia. One needle is connected to an extension tube and the end of the tube is inserted into a culture flask to collect the BM fluid. The other needle is connected to a syringe containing 30 ml of phosphate‐buffered saline. The solution is pushed gently from the syringe into the medullary cavity, and the medium containing the BM fluid is collected into the culture flask. There is significantly less contamination with peripheral blood, determined from the frequencies of CD4+ and CD8+ T cells, when using this method (<6%) than when using the conventional method (>20%) consisting of multiple BM aspirations from the iliac crest. Furthermore, the number and progenitor activities of the cells harvested using this “perfusion method” are greater than those harvested using the conventional aspiration method. This perfusion method was carried out 42 times using 15 cynomolgus monkeys, and no complications such as pulmonary infarction or paralysis were observed. These findings suggest that the “perfusion method” is safe and simple and would be of great advantage in obtaining pure BMCs, resulting in a less frequent occurrence of acute graft‐versus‐host‐disease in allogeneic BM transplantation.

Optimal protocol for total body irradiation for allogeneic bone marrow transplantation in mice

We have previously demonstrated, using chimeric resistant MRL/lpr mice, that a fractionated total body irradiation (FTBI) (5 Gy × 2 with a 4 h interval on the day before allogeneic bone marrow transplantation (BMT)) is the best conditioning regimen for the treatment of autoimmune diseases in radiosensitive MRL/lpr mice. In the present study, using various standard strains of mice (not radiosensitive mice), we explore the best protocol for irradiation (doses and intervals) as the conditioning regimen for allogeneic BMT. Recipient mice were exposed to various irradiation regimens: a single total body irradiation (TBI) of 9.5 or 12 Gy and FTBI of (5+5) Gy to (7+7) Gy with a 1 to 24 h interval. The method generally utilized for humans ((2+2) Gy with a 4 h interval for 3 days (total 12 Gy)) was also used. One day after the last irradiation, donor BMCs from BALB/c, C3H, or C57BL/6 (B6) mice were transplanted into C3H or B6 mice. The irradiation protocol of (2+2) Gy for 3 days was found to be insufficient to enable the complete removal of recipient immunocompetent cells, since donor-reactive T cells were observed in the recipient spleens and many recipient-type NK and CD4+ cells were also detected in the recipient hematolymphoid tissues. In all the combinations, the highest survival rate was achieved in the recipients irradiated with (6+6) or (6.5+6.5) Gy with a 4 h interval. In the surviving mice, the hematolymphoid tissues had been fully reconstituted with donor cells.

Experimental autoimmune thyroiditis induced by thyroglobulin-pulsed dendritic cells

Dendritic cells (DCs), which are the most effective professional antigen-presenting cells (APCs), initiate and regulate immune responses. In this report, we examine the role of DCs in the induction of autoimmune thyroiditis. Experimental autoimmune thyroiditis (EAT) induced by immunization with thyroglobulin (Tg) plus adjuvant is considered to be an animal model of autoimmune thyroiditis, and is categorized as a T cell-mediated autoimmune disease. To examine the contribution of DCs to EAT, naive DCs were purified from high responder B10BR mice and pulsed with various concentrations of porcine Tg (pTg). These pTg-pulsed DCs were transferred without adjuvant to syngenic BIOBR mice to induce EAT. Mice that had received pTg-pulsed DCs showed thyroiditis, and the degree of thyroiditis induced was positively correlated to the amounts of pTg used for the incubation (pulsing) of DCs. The severity of thyroiditis was also correlated to the amounts of anti-pTg IgG2a antibodies and IFN-gamma in the recipient sera, but not to IL-4 or IL-10, indicating that Th1 cells are mainly activated by pTg-pulsed DCs and attributable to the pathogenesis of EAT.

Comparison of bone marrow cells harvested from various bones of cynomolgus monkeys at various ages by perfusion or aspiration methods: a preclinical study for human BMT.

Using cynomolgus monkeys, we have previously established a new method for harvesting bone marrow cells (BMCs) with minimal contamination of the BMCs with T cells from the peripheral blood. We originally conducted this new “perfusion method” in the long bones (the humerus, femur, and tibia) of cynomolgus monkeys.

Successful allogeneic bone marrow transplantation (BMT) by injection of bone marrow cells via portal vein: stromal cells as BMT-facilitating cells.

We examined the importance of the coadministration of bone marrow (BM) stromal cells with BM cells via the portal vein. A significant increase in the number of day‐14 colony‐forming unit‐spleen (CFU‐S) was observed in the recipient mice injected with hemopoietic stem cells (HSCs) along with donor BM stromal cells obtained after three to four weeks of culture. Histological examination revealed that hematopoietic colonies composed of both donor hemopoietic cells and stromal cells coexist in the liver of these mice. However, when donor HSCs plus BM stromal cells were administered i.v., neither the stimulatory effects on CFU‐S formation nor the hemopoietic colonies in the recipient liver were observed.

Crucial Role of Donor‐Derived Stromal Cells in Successful Treatment for Intractable Autoimmune Diseases in MRL/lpr Mice by BMT Via Portal Vein

We have recently established a new bone marrow transplantation (BMT) method for the treatment of intractable autoimmune diseases in MRL/lpr mice; the method consists of fractionated irradiation (5.5 Gy × 2), followed by BMT of whole bone marrow cells (BMCs) from allogeneic C57BL/6 mice via the portal vein (abbreviated as 5.5 Gy × 2 + PV). In the present study, we investigate the mechanisms underlying the early engraftment of donor‐derived cells in MRL/lpr mice by this method. In the mice treated with this method, the number of donor‐derived cells possessing the mature lineage (Lin) markers rapidly increased in the BM, spleen, and liver; almost 100% were donor‐derived cells by 14 days after the treatment. The number of donor‐derived hemopoietic progenitor cells (defined as c‐kit+/Lin− cells) increased in the BMCs, hepatic mononuclear cells, and especially spleen cells by 14 days after the treatment. Simultaneously, hemopoietic foci adjoining donor‐derived stromal cells were observed in the liver when injected via the PV, but not via the peripheral vein (i.v.). When adherent cell‐depleted BMCs were injected via the PV, recipients showed a marked reduction in the survival rate. However, when mice were transplanted with adherent cell‐depleted BMCs with cultured stromal cells, all the recipients survived.

Analyses of very early hemopoietic regeneration after bone marrow transplantation: comparison of intravenous and intrabone marrow routes.

In bone marrow transplantation (BMT), bone marrow cells (BMCs) have traditionally been injected intravenously. However, remarkable advantages of BMT via the intra‐bone‐marrow (IBM) route (IBM‐BMT) over the intravenous route (IV‐BMT) have been recently documented by several laboratories. To clarify the mechanisms underlying these advantages, we analyzed the kinetics of hemopoietic regeneration after IBM‐BMT or IV‐BMT in normal strains of mice. At the site of the direct injection of BMCs, significantly higher numbers of donor‐derived cells in total and of c‐kit+ cells were observed at 2 through 6 days after IBM‐BMT. In parallel, significantly higher numbers of colony‐forming units in spleen were obtained from the site of BMC injection. During this early period, higher accumulations of both hemopoietic cells and stromal cells were observed at the site of BMC injection by the IBM‐BMT route. The production of chemotactic factors, which can promote the migration of a BM stromal cell line, was observed in BMCs obtained from irradiated mice as early as 4 hours after irradiation, and the production lasted for at least 4 days. In contrast, sera collected from the irradiated mice showed no chemotactic activity, indicating that donor BM stromal cells that entered systemic circulation cannot home effectively into recipient bone cavity. These results strongly suggest that the concomitant regeneration of microenvironmental and hemopoietic compartments in the marrow (direct interaction between them at the site of injection) contributes to the advantages of IBM‐BMT over IV‐BMT.