Prevention of premature ovarian failure and osteoporosis induced by irradiation using allogeneic ovarian/bone marrow transplantation.

BACKGROUND: Two side effects of irradiation are premature ovarian failure (POF) and osteoporosis, both of which are concerns not only clinically, for patients, but also experimentally, for animals. We examine whether bone marrow transplantation (BMT) can correct the POF induced by radiation and also address whether allogeneic ovarian transplantation (OT) can modulate the adverse effects of radiotherapy. METHODS: Eight-week-old female C57BL/6 mice were lethally irradiated with 6 Gy x2, and then injected with allogeneic bone marrow cells into their bone marrow cavity using our previously described intrabone marrow (IBM)-BMT technique. Allogeneic ovaries were simultaneously transplanted under the renal capsules of the mice. RESULTS: Three months after the transplantation, we noted that hematopoietic and lymphoid cells had been successfully reconstituted. The ovaries transplanted under the renal capsules demonstrated signs of development with a large number of differentiating follicles at different stages of development. Importantly, the total bone mineral density of the tibia in the "IBM-BMT+OT" (BMT/OT) group remained normal. However, the reproductive function of the recipient mice was not restored, despite the presence of many immature oocytes in the host ovaries in the BMT/OT group. In the BMT group, no oocytes were found in the host ovaries. CONCLUSIONS: These findings suggest that IBM-BMT with ovarian allografts can be advantageous for young women with POF and osteopenia or osteoporosis that is due to chemotherapy and radiotherapy for malignant diseases.

Mouse mesenchymal stem cells can support human hematopoiesis both in vitro and in vivo: the crucial role of neural cell adhesion molecule.

BACKGROUND: We previously established a mesenchymal stem cell line (FMS/PA6-P) from the bone marrow adherent cells of fetal mice. The cell line expresses a higher level of neural cell adhesion molecule and shows greater hematopoiesis-supporting capacity in mice than other murine stromal cell lines. DESIGN AND METHODS: Since there is 94% homology between human and murine neural cell adhesion molecule, we examined whether FMS/PA6-P cells support human hematopoiesis and whether neural cell adhesion molecules expressed on FMS/PA6-P cells contribute greatly to the human hematopoiesis-supporting ability of the cell line. RESULTS: When lineage-negative cord blood mononuclear cells were co-cultured on the FMS/PA6-P cells, a significantly greater hematopoietic stem cell-enriched population (CD34(+)CD38(-) cells) was obtained than in the culture without the FMS/PA6-P cells. Moreover, when lineage-negative cord blood mononuclear cells were cultured on FMS/PA6-P cells and transplanted into SCID mice, a significantly larger proportion of human CD45(+) cells and CD34(+)CD38(-) cells were detected in the bone marrow of SCID mice than in the bone marrow of SCID mice that had received lineage-negative cord blood mononuclear cells cultured without FMS/PA6-P cells. Furthermore, we found that direct cell-to-cell contact between the lineage-negative cord blood mononuclear cells and the FMS/PA6-P cells was essential for the maximum expansion of the mononuclear cells. The addition of anti-mouse neural cell adhesion molecule antibody to the culture significantly inhibited their contact and the proliferation of lineage-negative cord blood mononuclear cells. CONCLUSIONS: These findings suggest that neural cell adhesion molecules expressed on FMS/PA6-P cells play a crucial role in the human hematopoiesis-supporting ability of the cell line.

Transformation potential of bone marrow stromal cells into undifferentiated high-grade pleomorphic sarcoma.

PURPOSE: Bone marrow adherent cells contain conventional bone marrow stromal cells and mesenchymal stem cells and these cells constitute the hematopoietic microenvironment. Mesenchymal stem cells have the capacity to give rise to multiple mesenchymal lineage cells and even ectodermal lineage cells. In the present study, we investigated what types of tumor cells are inducible from BM adherent cells by chemical carcinogens. METHODS: Bone marrow cells from neonatal C3H/HeN mice were collected within 24 h after birth and then cultured. Four days later, bone marrow adherent cells were obtained and the cells were treated with 3-methylcholanthrene. RESULTS: By this treatment, some transformed clones consisting of large spindle cells were obtained. The transformed cells were highly positive for CD44 and were positive for Sca-1, CD49d and CD106, whereas the cells were negative for hematolymphoid markers. The cell clones had the ability to support hematopoiesis in vitro. These results indicate that the transformed cell lines have the characteristics of BM stromal cells/mesenchymal stem cells. Moreover, during culture of the transformed cells, spontaneous bone nodule formation was observed. When the transformed cells were inoculated into immunodeficient mice subcutaneously, the neoplasms grew in the subcutaneous tissue of the mice. Microscopically and ultrastructurally, the neoplasms showed the typical morphology of undifferentiated high-grade pleomorphic sarcoma (UHGPS). Bone-related genes have been found to be expressed in both transformed cells and UHGPSs. CONCLUSION: The present study suggests that UHGPSs are derived from BM stromal cells, probably mesenchymal stem cells.

Preferential expansion of human umbilical cord blood-derived CD34-positive cells on major histocompatibility complex-matched amnion-derived mesenchymal stem cells.

BACKGROUND: We previously found in a murine hematopoietic system that hematopoietic stem cells show high differentiation and proliferation capacity on bone marrow-derived mesenchymal stem cells/stromal cells (microenvironment) with "self" major histocompatibility complex (MHC). DESIGN AND METHODS: We examined whether amnion-derived adherent cells have the characteristics of mesenchymal stem cells, and whether these adherent cells can support the proliferation of umbilical cord blood-derived lineage-negative and CD34-positive cells (Lin(-)CD34(+) cells) obtained from the same fetus to a greater extent than those derived from other fetuses. RESULTS: Culture-expanded amnion-derived adherent cells expressed mesenchymal stem cell markers and HLA-ABC molecules and could differentiate into osteoblasts, adipocytes and chondrocyte-like cells, indicating that the cells have the characteristics of mesenchymal stem cells. The Lin(-)CD34(+) cells purified from the frozen umbilical cord blood were strongly positive for HLA-ABC, and contained a large number of hematopoietic stem cells. When the Lin(-)CD34(+) cells were cultured on the autologous (MHC-matched) or MHC-mismatched amnion-derived adherent cells in short-term assays (hematopoietic stem cell-proliferation) and long-term culture-initiating cell assays, greater expansion of the Lin(-)CD34(+) cells was observed in the MHC-matched combination than in MHC-mismatched combinations. The concentration of granulocyte-macrophage colony-stimulating factor in the culture supernatants of the long-term culture-initiating cell assays was significantly higher in the MHC-matched combination than in MHC-mismatched combinations. CONCLUSIONS: IT is likely that a MHC restriction exists between hematopoietic stem cells and mesenchymal stem cells/stromal cells in the human hematopoietic system and that granulocute-macropage colony-stimulating factor contributes to some extent to the preferential hematopoiesis-supporting ability of the MHC-matched amnion-derived adherent cells.

Facilitation of hematopoietic recovery by bone grafts with intra-bone marrow-bone marrow transplantation.

We have previously shown that T cells can acquire donor-type major histocompatibility complex (MHC) restriction and can interact with both donor-type antigen-presenting cells (APCs) and B cells, when adult donor bones are co-grafted with intravenous (IV) injection of bone marrow cells (BMCs) in order to supply donor bone marrow (BM) stromal cells. We have also found that the direct injection of donor BMCs into recipient BM (intra-bone marrow-bone marrow transplantation: IBM-BMT) produces more rapid reconstitution (including T-cell functions) and higher survival rates than IV injection (IV-BMT) even in chimerism-resistant combinations. In the present study, we show that the co-administration of bones from suckling (2-3 days old) donor mice is also effective in the IBM-BMT system. Even when a relatively low number of BMCs were injected into adult (more than 15 weeks old) mice, complete reconstitution was achieved in the mice that had received IBM-BMT+bone grafts, but not in the mice that had received IBM-BMT alone. Most BM and splenic adherent cells obtained from the recipients that had received IBM-BMT+bone grafts were reconstituted by donor-type cells. Both T-cell proliferation and plaque-forming cell assays indicated that the T cells of such mice showed donor-type MHC restriction. Moreover, the analyses of thymic sections using confocal microscopy revealed that donor BM stromal cells had migrated into the thymus. Thus, the co-administration of donor bones has great advantages for allogeneic BMT in adult mice.

Contribution of neural cell adhesion molecule (NCAM) to hemopoietic system in monkeys.

Neural cell adhesion molecules (CD56) are important adhesion molecules that are mainly expressed on neural cells and natural killer cells. Although freshly isolated cynomolgus monkey bone marrow cells (BMCs) contained only a few CD56-positive cells, almost all the BM adherent cells (obtained after a 2- to 3-week culture of the BMCs) were stained positively with anti-CD56 monoclonal antibody (mAb). The BM adherent cells showed uniformly fibroblastic morphology and were negative for hematolymphoid markers (CD4, CD8, CD11b, CD14, CD34, and CD45). Adipogenesis and osteogenesis were observed under inductive culture conditions. The BM adherent cells had the ability to support hemopoiesis of hemopoietic stem cells (HSCs) in vitro, and the proliferation of HSCs was significantly inhibited by the addition of anti-CD56 mAb to the coculture system. CD56 molecules were also expressed on HSCs because about 20% of an HSC-enriched population (lineage-negative and blast-gated cells) was positive for CD56. In addition, the immunostaining of monkey BM sections revealed that many stromal cells were CD56-positive, and some CD56-positive stromal cells came into direct contact with CD56-positive hemopoietic cells. These results indicate that the CD56 molecule is expressed on both HSCs and BM stromal cells (containing MSCs) in monkeys, and therefore it can be speculated that CD56 also contributes to the human hematopoietic system.

Successful acceptance of adult liver allografts by intra-bone marrow-bone marrow transplantation.

Previously, we have shown that liver allografts obtained from the fetus or young mice are accepted when bone marrow cells (BMCs) from adult mice of the same strain are co-grafted. However, for practical clinical use, it is more convenient to obtain both BMCs and liver from the same adult donors. C57BL/6 mice were irradiated with a single high-dose irradiation or two low-dose irradiations and injected with donor BALB/c (8 weeks old) BMCs intravenously (IV-BMT) or directly into the recipient BM cavity (IBM-BMT). Liver tissues taken from the same donor were, on the same day, engrafted under the kidney capsules. Higher survival rates and more complete reconstitution of donor cells were achieved in the IBM-BMT group than in the IV-BMT group, and this was the case in both irradiation protocols. The acceptance of donor liver tissue was seen in all mice in which hematolymphoid cells were replaced by donor-type cells. The liver grafts of the reconstituted mice showed normal morphology and stained positively with anti-albumin antibody and Periodic Acid Schiff (PAs) staining, indicating that the grafted livers were accepted, had grown, and were functioning. These results demonstrate that the acceptance of allogeneic liver can be achieved by cografting donor BMCs via the IBM route.

Analysis of tolerance induction using triple chimeric mice: major histocompatibility complex-disparate thymus, hemopoietic cells, and microenvironment.

BACKGROUND: Although bone marrow transplantation (BMT) has become a valuable strategy for the treatment of various intractable diseases in recent years, success rates remain low in elderly patients because of low thymic function. We have previously shown that fetal thymus transplantation (TT) with BMT is effective for elderly recipients in mice. METHODS: We performed fully major histocompatibility complex (MHC)-mismatched fetal TT from B6 (H-2) mice plus allogeneic BMT from C3H/HeN (H-2) mice by intra-bone marrow-BMT (IBM-BMT) using congenitally athymic nude (nu/nu) BALB/c (H-2), or BALB/c adult-thymectomized recipients to obtain triple chimeras. We next carried out the IBM-BMT+TT using senescence-accelerated mouse P1 strain (SAMP1) to examine whether this method would be applicable to aging mice. RESULTS: Triple chimeric mice survived for a long period with sufficient T-cell functions comparable to the mice treated with BMT plus MHC-matched TT, whereas those without TT survived for a short period with insufficient T-cell reconstitution. Almost all the hematolymphoid cells were derived from donor bone marrow cells. Interestingly, they showed tolerance to all three types of MHC determinants with donor-derived thymic dendritic cells in TT. Triple chimeric SAMP1 also survived for long periods with T-cell functions restored in contrast to non-TT SAMP1 recipients. CONCLUSION: These findings suggest that third party combined TT with allogeneic IBM-BMT may be more advantageous for elderly recipients with low thymic function, than IBM-BMT alone (without TT).

Long-term donor-specific tolerance in rat cardiac allografts by intrabone marrow injection of donor bone marrow cells.

BACKGROUND: Donor-specific central tolerance in cardiac allograft can be induced by hematopoietic chimerism via conventional intravenous bone marrow transplantation (IV-BMT). However, there are problems with IV-BMT, such as the risk of graft failure and of the toxicity from conditioning regimens. METHODS: A new method for heart transplantation is presented. This method consists of administration of fludarabine phosphate (50 mg/kg) and fractionated low-dose irradiation (3.5 Gyx2 or 4.0 Gyx2), followed by intrabone marrow injection of whole bone marrow cells (IBM-BMT) plus heterotopic heart transplantation. RESULTS: Cardiac allografts with IBM-BMT were accepted and survived long-term (>10 months) showing neither acute rejection nor chronic rejection including cardiac allograft vasculopathy by such conditioning regimens. In contrast, cardiac allografts with conventional IV-BMT were rejected within 1 month after the treatment with irradiation of 3.5 Gyx2 or within 3 months after the treatment with irradiation of 4.0 Gyx2. Macrochimerism (>70%) was favorably established and stably maintained by IBM-BMT but not IV-BMT. Low levels of transient mixed chimerism (<7%) were induced by IV-BMT with fludarabine plus 4.0 Gyx2, but the chimerism was lost within 1 month after the treatment. CONCLUSIONS: These findings indicate that IBM-BMT is a feasible strategy for the induction of persistent donor-specific tolerance, enables the use of reduced radiation doses as conditioning regimens, and obviates the need for immunosuppressants.

Prevention of osteoporosis and hypogonadism by allogeneic ovarian transplantation in conjunction with intra-bone marrow-bone marrow transplantation.

BACKGROUND: We investigated the effects of ovarian allograft in conjunction with intra-bone marrow-bone marrow transplantation (IBM-BMT) on estrogen deficiency in mice. METHODS: Female C57BL/6 mice underwent ovariectomy (OvX). After 3 months, the mice were irradiated at 9.5 Gy, and the bone marrow cells (BMCs) of female BALB/c mice (8 weeks old) were then injected into the bone cavity of the B6 mice. Simultaneously, allogeneic ovaries from BALB/c mice were transplanted under the renal capsules of the B6 mice. RESULTS: Three months after the transplantation, the hematolymphoid cells were found to be completely reconstituted with donor-derived cells. The transplanted ovary tissues under the renal capsules were accepted without using immunosuppressants; there were a large number of growing follicles at different stages of development. Atrophic endometrium and its glands were also recovered by ovarian transplantation (OT). The transplanted allogeneic ovaries secreted estrogen at normal levels. Furthermore, bone loss was prevented to a certain extent. CONCLUSIONS: These findings suggest that IBM-BMT+OT will become a valuable strategy for young women with malignant tumors to prevent premature senescence, including hypogonadism and osteoporosis, after radiochemotherapy.

Extensive studies on perfusion method plus intra-bone marrow-bone marrow transplantation using cynomolgus monkeys.

The collection of bone marrow cells (BMCs) using a perfusion method has been advantageous not only because of the low contamination of BMCs with T cells from the peripheral blood but also the enrichment of stromal cells, which support hemopoiesis. Before the application of this new method to humans, its safety needed to be confirmed using cynomolgus monkeys. We therefore performed the perfusion method on more than 100 cynomolgus monkeys using the long bones (such as the humerus and femur) and also the iliac bones (for human application); in the more than 150 trials to date, there have been no accidental deaths. Furthermore, the technical safety of a new method for the intra-bone marrow (IBM) injection of BMCs (termed IBM-bone marrow transplantation) has also been confirmed using 30 monkeys. Disclosure of potential conflicts of interest is found at the end of this article.

The characteristics of hematopoietic stem cells from autoimmune-prone mice and the role of neural cell adhesion molecules in abnormal proliferation of these cells in MRL/lpr mice.

BACKGROUND AND OBJECTIVES: Using various animal models for autoimmune diseases, we have previously shown that such diseases are stem cell disorders.1 In order to understand how autoimmune diseases develop, we investigated the distinct qualitative differences between hematopoietic stem cells (HSC) from normal and autoimmune-prone mice. DESIGN AND METHODS: We studied the major histocompatibility complex (MHC) restriction between HSC and stromal cells in vitro and in vivo. We also examined the ability of HSC to adhere to a stromal cell line and, using flow cytometry, analyzed the expression of various adhesion molecules in HSC before and after the onset of autoimmune disease. In addition, the effect of antibodies to anti-adhesion molecules on the proliferation of HSC was investigated. RESULTS: The abnormal HSC of MRL/lpr mice showed no MHC restriction (or preference) with stromal cells either in vitro or in vivo, although there was MHC restriction between normal HSC and stromal cells, as we previously reported.2,3 The abnormal HSC of MRL/lpr mice exhibited enhanced adhesion to stromal cells in vitro and expressed a higher amount of adhesion molecules such as neural cell adhesion molecule (NCAM). Interestingly, the proliferation of HSC in MRL/lpr mice was significantly suppressed by anti-NCAM monoclonaal antibodies. INTERPRETATION AND CONCLUSIONS: Abnormal HSC of MRL/lpr mice are more resilient than normal HSC. Furthermore, among various adhesion molecules, only NCAM shows increased expression on HSC of MRL/lpr mice after the onset of autoimmune diseases, and these molecules contribute to the enhanced proliferation capacity of abnormal HSC in MRL/lpr mice. The present findings suggest that there are intrinsic qualitative differences between HSC from normal and autoimmune-prone MRL/lpr mice.

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. Disclosure of potential conflicts of interest is found at the end of this article.

Lin(-)CD34(-) bone marrow cells from adult mice can differentiate into neural-like cells.

Numerous studies have shown that some populations of bone marrow cells (BMCs) have the capacity to differentiate into neural cells, which is useful for repairing brain lesions. In this paper, we analyze neural differentiation features of lineage-negative/CD34-negative (Lin(-)CD34(-)) cells in the bone marrow of adult mice. The population of Lin(-)CD34(-) in BMCs was isolated by magnetic bead sorting and fluorescence-activated cell sorter (FACS) using specific lineage (CD4, CD8a, CD11b, CD45R, Gr-1 and TER-119) antibodies and CD34 antibody. First, we cultured Lin(-)CD34(-) BMCs in the presence of RNIF: vitamin A derivative retinoic acid (RA) and neural-inducing factors (platelet-derived growth factor BB (PDGF-BB), epidermal growth factor (EGF) and fibroblast growth factor-basic (FGF-b)). Analyses of RT-PCR and immunocytochemistry indicated that RNIF-treated Lin(-)CD34(-) BMCs expressed neural phenotypes as well as neurogenic transcription factors. When we implanted the Lin(-)CD34(-) BMCs isolated from enhanced green fluorescent protein (eGFP) transgenic mice into the subventricular zone (SVZ) of postnatal mice, eGFP-positive cells survived 3 weeks after the injection in the various brain regions, some of which expressed the neural phenotypes. Our data suggest that certain subsets in the CD34(-) populations of adult bone marrow could have the capacity to differentiate into neural cells in a suitable environment.

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.

Neural cell adhesion molecule contributes to hemopoiesis-supporting capacity of stromal cell lines.

To clarify mechanisms underlying cell-to-cell interactions between hemopoietic stem cells (HSCs) and stromal cells, we established a stromal cell line (FMS/PA6-P) from day-16 fetal bone marrow (BM) adherent cells using an anti-PA6 monoclonal antibody (mAb) specific for BM stromal cells. Importantly, this FMS/PA6-P cell line, showing homogenous fibroblastic morphology, is absent from hematolymphoid and endothelial lineage markers and maintains a high level of expression of PA6 molecule, recognized by the anti-PA6 mAb, for approximately 20 passages. Further, the cell line expressing a high level of PA6 molecule has a better hemopoiesis-supporting capacity in vitro than other stromal cell lines such as PA6 and MS-5. In fact, the PA6 molecule is closely related to the hemopoiesis-supporting capacity of the stromal cells because the proliferation of HSCs was suppressed to a great extent by the anti-PA6 mAb. Affinity chromatography and mass peptide fingerprinting revealed that the protein reacting with the anti-PA6 mAb is neural cell adhesion molecule (NCAM). The frequencies of long-term cobblestone area-forming cells and long-term culture-initiating cells were significantly suppressed by repression of NCAM in the FMS/PA6-P cells using NCAM small interfering RNA. Our findings clearly indicate that NCAM functions on the maintenance of HSCs.

Induction of tolerance in quadruple chimeric mice.

Human cord blood (CB) contains hematopoietic stem cells and progenitors. Because the major limitation to a widespread use of CB for transplantation lies in its limited volume, it is necessary to combine the CB from several donors. In this study, we show that lethally irradiated mice can be reconstituted with the injection of a mixture of T cell-depleted bone marrow cells (BMCs; total, 3 x 10(6)) obtained from three fully allogeneic mouse strains in two different mouse combinations. A higher survival rate was obtained in the triple injection group than in mice injected with BMCs (1 x10(6)) obtained from a single mouse strain. In the mixed chimeric mice, three kinds of donor-type and recipient-type cells were detected in all the hematopoietic organs 1 month after bone marrow transplantation (BMT). Mixed-lymphocyte reaction showed that the tolerance to both recipient-type and donor-type major histocompatibility complex determinants was induced in the chimeric mice. In the peripheral blood (PB) of these mice, only one type of cells from the three different donor strains became dominant in most chimeric mice and reached a stable level about 4 months after BMT. Polymerase chain reaction analyses, however, revealed that the skins from all the donors were accepted even when no cells with their phenotypes could be detected in the PB. These results suggest that both hemato-lymphoid reconstitution and stable tolerance to not only the recipient strain but also all the donor strains can be achieved in chimeric mice, indicating the possibility of mixed CB transplantation in humans.