Jun Hayakawa

Generation of a Chimeric Mouse Reconstituted with Green Fluorescent Protein-Positive Bone Marrow Cells: A Useful Model for Studying the Behavior of Bone Marrow Cells in Regeneration In Vivo

Studies have indicated that bone marrow contains both hematopoietic stem cells and mesenchymal stem cells that can differentiate into a variety of mesenchymal tissues, such as bone, cartilage, muscle, and adipose tissue. Therefore, bone marrow cells are thought to be very useful for cell and gene therapy for various diseases. However, the multipotentiality of these cells remains unclear. To address this issue, we established a chimeric model mouse stably reconstituted with green fluorescent protein (GFP)-marked bone marrow cells. We injected bone marrow cells from GFP-transgenic C57BL/6 mice into the tail veins of recipient wild-type C57BL/6 mice that had been irradiated with a lethal dose of 10 Gy from a cesium source. Microscopic examination and fluorescence-assisted cell sorter (FACS) analysis showed that bone marrow cells, including mesenchymal cells, were almost completely reconstituted with GFP+ cells 5 weeks after transplantation. FACS analysis with lineage-specific antibodies confirmed that the GFP+ cells could differentiate into all types of blood cells. To confirm the usefulness of this mouse model, we studied the role of circulating bone marrow—derived cells in healing of damaged intestine. We performed amputation and anastomosis of the jejunum 10 cm from the pyloric region of the stomach. On the third day after operation, a large number of GFP+ cells were infiltrated in the area of anastomosis, and these cells were positive for CD45 and F4/80 antigens. In 7 days, several cells became negative for CD45 and F4/80 and positive for a smooth muscle actin antigen, which is specific for smooth muscle. This finding suggested that bone marrow-derived cells had differentiated into smooth muscle. Because reconstituted bone marrow cells, as opposed to injected bone marrow cells, behave naturally, this model is ideal for studying the multipotentiality of bone marrow cells in vivo.

Cardiomyocyte regeneration from circulating bone marrow cells in mice.

We investigated the role of circulating bone marrow cells (BMC) in cardiomyocyte regeneration. BMC, isolated from transgenic mice expressing enhanced green fluorescent protein (GFP), were transplanted into lethally irradiated C57BL6 mice. Five weeks after bone marrow transplantation (BMT), flow cytometric analysis for GFP-positive cells confirmed reconstitution of transplanted bone marrow. Bone marrow transplant mice subsequently underwent left coronary artery ligation (myocardial infarction) or sham-operation, and were killed at 1 mo or 3 mo after operation. Infarct size was similar in bone marrow transplant mice at 1 mo (47.1 ± 5.9%) and at 3 mo (45.3 ± 7.8%), and echocardiography at 2 and 8 wk revealed decreasing left ventricular function. In infarcted heart, GFP-positive cells that expressed desmin and troponin T-C were identified by confocal microscopy. GFP and troponin T-C double-positive cells were predominantly in the peri-infarcted region (1 mo, 365 ± 45 cells/50 sections; 3 mo: 458 ± 100 cells/50 sections; p < 0.05 versus noninfarct, infarct, and sham-operated regions). Furthermore, BMC mobilization and differentiation into cardiomyocytes was found to be complete within 1 mo after myocardial infarction. These results demonstrate that circulating BMC undergo mobilization and differentiation in cardiac cells after myocardial infarction. Future studies are required to determine the molecular signaling mechanisms responsible for this phenomenon.

5% Dimethyl sulfoxide (DMSO) and pentastarch improves cryopreservation of cord blood cells over 10% DMSO

BACKGROUND: Cell number and viability are important in cord blood (CB) transplantation. While 10% dimethyl sulfoxide (DMSO) is the standard medium, adding a starch to freezing medium is increasingly utilized as a cytoprotectant for the thawing process. Similar to hetastarch, pentastarch has the advantages of faster renal clearance and less effect on the coagulation system.

Role of Bone Marrow Cells in the Healing Process of Mouse Experimental Glomerulonephritis

Recent studies have shown bone marrow (BM) cells to differentiate into a variety of cell types and to thereby participate in the reconstitution of damaged organs. In the present study, we examined the extent to which BM-derived cells are incorporated into glomeruli during recovery from experimentally induced nephritis. To investigate the localization of BM cells in glomeruli, chimeric mice were prepared by transplanting BM cells from green fluorescent protein (GFP) transgenic mice into wild-type mice. Five weeks later, glomerulonephritis was induced by intravenous injection of Habu snake venom. Groups of mice were then killed every few days for 42 d, and harvested kidney samples were subjected to immunohistochemical and immunoelectron microscopic analyses with the aim of detecting the presence of GFP(+) cells within glomeruli. Chimeric animals injected with Habu venom developed proliferative glomerulonephritis within 1–3 d. The lesion gradually subsided and the glomerular structure returned to normal within 42 d. Consistent with the disease course, large numbers of GFP(+) cells were present within glomeruli on d 1–3, but most had disappeared by d 7. Nevertheless, some GFP(+) cells did remain within glomeruli showing mesangial proliferative changes, and were found to express thrombomodulin (TM), a specific endothelial cell marker. These GFP-TM–double-positive cells accounted for a mean of 1.31–2.24% of the total glomerular nuclei from d 7 through d 42, levels that remained stable for at least 12 mo. It thus appears that BM cells can give rise to endothelial cells that participate in the remodeling of glomeruli.

Transient In Vivo β-Globin Production After Lentiviral Gene Transfer to Hematopoietic Stem Cells in the Nonhuman Primate

Inherited disorders of globin synthesis remain desirable targets for hematopoietic stem cell (HSC)-based therapies. Gene transfer using retroviral vectors offers an alternative to allogeneic HSC transplantation by the permanent integration of potentially therapeutic genes into primary autologous HSCs. Although proof of principle has been demonstrated in humans, this approach has been met by formidable obstacles, and large-animal models have become increasingly important for the preclinical development of gene addition strategies. Here we report lentiviral gene transfer of the human beta-globin gene under the control of the globin promoter and large fragments of the globin locus control region (LCR) in the nonhuman primate. Using an HIV-1, vesicular stomatitis virus glycoprotein G (VSV-G)-pseudotyped vector, modified to overcome a species-specific restriction to HIV-1, gene transfer to colony-forming units (CFU) derived from mobilized peripheral blood (PB) rhesus CD34+ cells was 84.4 +/- 2.33%. Erythroid cells derived from transduced rhesus CD34+ cells expressed human beta-globin at high levels as assessed by flow cytometry with a human beta-globin-specific antibody. Two rhesus macaques (RQ3586 and RQ3583) were transplanted with mobilized PB CD34+ cells transduced with our modified HIV vector at a multiplicity of infection of 80. High gene transfer rates to CFUs were achieved in vitro (RQ3586, 87.5%; RQ3583, 83.3%), with efficient human beta-globin expression among erythroid progeny generated in vitro. Early posttransplantation, gene transfer rates of 5% or higher were detectable and confirmed by genomic Southern blotting, with equivalent-level human beta-globin expression detected by flow cytometry. Long-term gene marking levels among mononuclear cells and granulocytes assessed by quantitative polymerase chain reaction gradually decreased to about 0.001% at 2 years, likely due to additional HIV-1 restrictive elements in the rhesus macaque. No evidence of clonal hematopoiesis has occurred in our animals in up to 2 years. Current efforts are aimed at developing a lentiviral vector capable of efficiently transducing both human and rhesus HSCs to allow preclinical modeling of globin gene transfer.

Zanamivir is an Effective Treatment for Influenza in Children Undergoing Therapy for Acute Lymphoblastic Leukemia

We diagnosed influenza infection in 2 children receiving maintenance treatment for acute lymphoblastic leukemia. Both patients received zanamivir within 1 d of the onset of fever and their symptoms of influenza were rapidly alleviated. We conclude that inhaled zanamivir seems to be an effective treatment for influenza infection in immunocompromised patients.

Apoptotic Cell Death and Regeneration in the Newborn Retina After Irradiation Prior to Bone Marrow Transplantation

Purpose: We studied the contribution made by circulating bone marrow (BM)-derived cells to the newborn and mature retinas of BM-transplanted mice. Methods: Newborn and adult C57BL/6J mice were administered a lethal dose of total-body irradiation, after which pathologic changes to the retinas were periodically assessed. In addition, mice received BM cells from 8-week-old green fluorescent protein (GFP) transgenic mice, and the subsequent differentiation of GFP+ cells was studied. Results: Within 5 hr after irradiation of newborn mice, retinal cells began to die due to apoptosis. By contrast, irradiation of adult mice elicited no histologic changes in the retina. BM cells generally did not differentiate in adult mice, but numerous GFP+ BM cells were integrated into the retinal tissue of newborn mice, where they expressed various cell type–specific markers. Finally, examination of whole retina mounts showed that GFP+ cells also contributed to retinal vascularization. Conclusions: Our findings underscore the importance of careful evaluation of the biological effects of irradiation in models making use of BM transplantation.

Serum KL-6 and surfactant protein D in children with 2009 pandemic H1N1 influenza infection

Background:  A global pandemic influenza A (H1N1) outbreak occurred in 2009. Rapid progress of respiratory distress is one of the characteristic features of pandemic influenza A (H1N1) infection. The physiologic mechanism causing hypoxia in pandemic influenza A (H1N1) infection, however, has not been elucidated.

Recipient non-hematopoietic bone marrow cells in the intestinal graft after fetal small intestinal transplantation.

We examined whether non-hematopoietic BM cells can migrate into the intestinal graft after fetal small intestinal transplantation (FSITx). Fetal small intestine from donor C57BL/6 mice was transplanted into the rectus abdominis of recipient C57BL/6 mice with only green fluorescent protein (GFP) BM cells (syngeneic FSITx). Intestinal grafts were harvested on days 5, 10, and 30 after FSITx and stained immunohistochemically using anti-CD45 antibody (a marker for hematopoietic BM cells). Although there were no GFP-positive cells identified in the epithelium of the graft intestinal villi, there were a few cells positive for both GFP and CD45 in the lamina propria on day 5 after FSITx, and many present on days 10 and 30. In some grafts there were only cells that were GFP positive/CD45 negative (i.e., non-hematopoietic BM cells) found in the lamina propria on days 10 and 30. These data indicate that non-hematopoietic BM cells as well as hematopoietic BM cells can migrate from the recipient’s bone marrow, suggesting that recipient mesenchymal stem cells may be strongly implicated in graft regeneration and development after FSITx.

Unusual findings in single-photon emission computed tomography in a 1-year-old boy with acute necrotizing encephalopathy.

Acute necrotizing encephalopathy (ANE) is characterized by multifocal symmetrical lesions in the thalami and other re gions, including the brainstem tegmentum, cerebral periventricular white matter and cerebellar medulla. 1 The pathogenesis of this disease in children is unknown, but approximately 20% of babies and young children who develop infl uenzaassociated encephalopathy exhibit clinical symptoms that closely resemble those of ANE, 2 and the outcomes are generally very poor. Indeed, the mortality rate among ANE patients is approximately 30%. 3