Tomomi Mizokami

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.

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.

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.

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.

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

In vitro expansion of human hematopoietic stem cells has remained cumbersome. This paper demonstrates that umbilical cord blood-derived lineage negative/CD34-positive cells can selectively expand in vitro when cultured on autologous amnion-derived adherent 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.

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.

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.

Management of transvaginal ultrasound-guided absolute ethanol sclerotherapy for ovarian endometriotic cysts.

PurposeEndometriosis and endometriotic ovarian cysts are common gynecologic diseases. Excision of the cyst wall by laparotomy or laparoscopy is the standard treatment for endometriotic ovarian cysts. However, some young patients with cysts would prefer not to have an abdominal incision. We reviewed and assessed the effectiveness of transvaginal ultrasound-guided ethanol sclerotherapy in these patients.MethodsEighteen patients with endometriotic ovarian cysts underwent transvaginal ultrasound-guided aspiration and ethanol sclerotherapy using spinal anesthesia. The contents of the cysts were drained and sent for cytological examination. The cyst cavities were washed with absolute ethanol, and the cysts were then filled with absolute ethanol for 5 min. The patients were followed up with transvaginal ultrasonography.ResultsThe transvaginal ethanol sclerotherapy was completed in all cases. The mean long diameter of the cysts was 50 mm (31–100 mm), and the mean operative duration was 22 min (8–45 min). Malignant cells were not isolated from the aspirated fluid in any case. There were no significant intra- or postoperative complications. Two patients (11.1%) had a recurrence at 3 and 32 months after ethanol sclerotherapy, respectively.ConclusionsTransvaginal ultrasound-guided absolute ethanol sclerotherapy is useful for ovarian endometriotic cysts, particularly in young patients or in patients who would like to become pregnant. However, careful selection based on ultrasonography or magnetic resonance imaging findings and the age of the patients is critical.

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.

Benign endometrial adenofibroma and polyp in patients receiving tamoxifen : findings on transvaginal ultrasonography and magnetic resonance imaging

Tamoxifen, a selective estrogen receptor modulator, is widely used to treat breast cancer, but an association has been reported between tamoxifen and the development of endometrial lesions, including endometrial carcinoma, endometrial polyps, and endometrial hyperplasia. There have also recently been a few reports on the relation between tamoxifen and adenofibroma. We present two case reports, one of a patient with a uterine adenofibroma and one of a patient with an endometrial polyp, both of whom received tamoxifen. Cases 1 and 2 are 75- and 65-year-old postmenopausal women, respectively, undergoing tamoxifen therapy. In both cases, endometrial thickening and many small cysts in the uterine cavity were revealed by transvaginal ultrasonography or magnetic resonance imaging. Postoperative microscopic examination confirmed the mass as an adenofibroma in case 1 and as an endometrial polyp in case 2.