Mitsutaka Shiota

Development of primitive and definitive hematopoiesis from nonhuman primate embryonic stem cells in vitro

Although information about the development of primitive and definitive hematopoiesis has been elucidated in murine embryos and embryonic stem (ES) cells, there have been few in vitro studies of these processes in primates. In this study, we investigated hematopoietic differentiation from cynomolgus monkey ES cells grown on OP9, a stromal cell line deficient in macrophage colony-stimulating factor. Primitive erythrocytes (EryP) and definitive erythrocytes (EryD) developed sequentially from ES cells in the culture system; this was confirmed by immunostaining and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of embryonic, fetal and adult globin genes. EryP were detected on day 8 without exogenous erythropoietin (EPO), whereas EryD appeared on day 16 and had an indispensable requirement for exogenous EPO. RT-PCR analysis of the cultures revealed a sequential expression of genes associated with primitive and definitive hematopoietic development that was equivalent to that seen during primate ontogeny in vivo. Vascular endothelial growth factor (VEGF) increased, in a dose-dependent manner, not only the number of floating hematopoietic cells, but also the number of adherent hematopoietic cell clusters containing CD34-positive immature progenitors. In colony assays, exogenous VEGF also had a dose-dependent stimulatory effect on the generation of primitive erythroid colonies. More efficient primitive and definitive erythropoiesis was induced by re-plating sorted CD34-positive cells. Thus, this system reproduces early hematopoietic development in vitro and can serve as a model for analyzing the mechanisms of hematopoietic development in primates.

Rapid detoxification of cereulide in Bacillus cereus food poisoning.

Bacillus cereus is recognized as a major pathogenic bacterium that causes food poisoning and produces gastrointestinal diseases of 2 types: emetic and diarrheal. The emetic type, which is often linked to pasta and rice, arises from a preformed toxin, cereulide, in food. Rapid and accurate diagnostic methods for this emetic toxin are important but are limited. Here we describe 3 patients with B cereus food poisoning in which cereulide was detected and measured sequentially. Three family members began to vomit frequently 30 minutes after consuming reheated fried rice. After 6 hours, a 1-year-old brother died of acute encephalopathy. A 2-year-old sister who presented with unconsciousness recovered rapidly after plasma exchange and subsequent hemodialysis. Their mother recovered soon by fluid therapy. From leftover fried rice and the childrens stomach contents, B cereus was isolated. Serum cereulide was detected in both children; it decreased to an undetected level in the sister. These cases highlight the importance of measuring the value of cereulide, which would reflect the severity of B cereus emetic food poisoning. The cases also suggest the possible role of blood-purification therapy in severe cases.

Direct visualization of transplanted hematopoietic cellreconstitution in intact mouse organs indicates the presence of a niche

OBJECTIVE The temporal and spatial behavior of transplanted hematopoietic stem cells (HSCs) within bones remains to be clarified. Our goal is to examine in vivo reconstitution processes and candidate niches in all bones in the mouse body using a new visualization method. MATERIALS AND METHODS Using bone marrow cells from green fluorescent protein (GFP) transgenic mice, the reconstitution processes of transplanted hematopoietic cells (HCs) under myeloablative or nonmyeloablative conditions were observed sequentially from outside the bones with a fluorescent stereomicroscope. RESULTS In case of myeloablative transplantation, GFP(+) spots were first detected at the epiphysis of femurs, and in some ribs and vertebrae among all intact bones. Thereafter, engrafted cells proliferated and spread into other bones. In case of nonmyeloablative transplantation with lin(-)Sca-1(+)c-kit(+) cells into W/Wv neonates, characterized by vacant niches because of stem cell defects, GFP(+) cells localized at the epiphysis of femurs and in some vertebrae and ribs, but not in all bones even 4 months after transplantation. CONCLUSION Our findings show that transplanted HSCs or their immature progenies engraft preferentially at the epiphysis of the femurs or short and flat bones such as ribs and vertebrae. The transplanted cells remain quiescent for at least 4 months under nonmyloablative conditions, which implies the presence of stem cells in a niche. Our approach for the first time graphically demonstrates the kinetics of HCs in vivo and should facilitate analysis of HSC behavior in a three-dimensional mode.

Human cord blood CD34+ cells develop into hepatocytes in the livers of NOD/SCID/γcnull mice through cell fusion

Several studies have shown that hepatocytes can be generated from hematopoietic stem cells, but this event is believed to be rare and to require hepatic damage. To investigate this phenomenon in human cells, we used a NOD/SCID/γcnull (NOG) mouse model that can achieve a tremendously high level of chimerism when transplanted with human hematopoietic cells. Even without hepatotoxic treatment other than irradiation, human albumin and α‐1‐antitrypsin‐positive cells were invariably detected in the livers of NOG mice after i.v. transplantation of human cord blood CD34+ cells. Human albumin was detected in the murine sera, indicating functional maturation of the human hepatocytes. Flow cytometric analysis of recipient liver cells in single‐cell suspension demonstrated that human albumin‐positive cells were also positive for both murine and human MHC and were negative for human CD45. PCR analysis of recipient livers revealed the expression of a wide variety of human hepatocyte‐ or cholangiocyte‐specific mRNAs. These results show that human CD34+ cells fuse with hepatocytes of NOG mice without liver injury, lose their hematopoietic phenotype, and begin hepatocyte‐specific gene transcription. These phenomena were not observed when CD34− cells were transplanted. Thus, our model revealed a previously unidentified pathway of human hematopoietic stem/progenitor cell differentiation.—Fujino, H., Hiramatsu, H., Tsuchiya, A., Niwa, A., Noma, H., Shiota, M., Umeda, K., Yoshimoto, M., Ito, M., Heike, T., Nakahata, T. Human cord blood CD34+ cells develop into hepatocytes in the livers of NOD/SCID/γcnull mice through cell fusion. FASEB J. 21, 3499–3510 (2007)

Identification and characterization of hemoangiogenic progenitors during cynomolgus monkey embryonic stem cell differentiation

We identified intermediate‐stage progenitor cells that have the potential to differentiate into hematopoietic and endothelial lineages from nonhuman primate embryonic stem (ES) cells. Sequential fluorescence‐activated cell sorting and immunostaining analyses showed that when ES cells were cultured in an OP9 coculture system, both lineages developed after the emergence of two hemoangiogenic progenitor‐bearing cell fractions, namely, vascular endothelial growth factor receptor (VEGFR)‐2high CD34− and VEGFR‐2high CD34+ cells. Exogenous vascular endothelial growth factor increased the proportion of VEGFR‐2high cells, particularly that of VEGFR‐2high CD34+ cells, in a dose‐dependent manner. Although either population of VEGFR‐2high cells could differentiate into primitive and definitive hematopoietic cells (HCs), as well as endothelial cells (ECs), the VEGFR‐2high CD34+ cells had greater hemoangiogenic potential. Both lineages developed from VEGFR‐2high CD34−or VEGFR‐2high CD34+ precursor at the single‐cell level, which strongly supports the existence of hemangioblasts in these cell fractions. Thus, this culture system allows differentiation into the HC and EC lineages to be defined by surface markers. These observations should facilitate further studies both on early developmental processes and on regeneration therapies in human.

Successful T-cell-replete peripheral blood stem cell transplantation from HLA-haploidentical microchimeric mother to daughter with refractory acute lymphoblastic leukemia using reduced-intensity conditioning

Summary:A 16-year-old girl with refractory acute lymphoblastic leukemia underwent reduced-intensity hematopoietic stem cell transplantation from her two-locus-mismatched haploidentical mother, who was microchimeric for the patients hematopoietic cells. The conditioning regimen comprised melphalan, fludarabine, and low-dose total body irradiation. Non-T-cell-depleted peripheral blood stem cells were infused with graft-versus-host disease (GVHD) prophylaxis consisting of tacrolimus, prednisolone, and short-course methotrexate. Complete donor-type engraftment without evidence of residual leukemia was confirmed on day 22. Severe GVHD was not observed despite rapid cessation of immunosuppression. The patient remains well in continuous remission 15 months after transplant. This successful experience suggests that maternal hematopoietic stem cell transplants for children, in the presence of microchimerism, may be associated with hyporesponsiveness to the inherited paternal HLA antigens (IPA); preventing severe GVHD.

Two Different Roles of Purified CD45+c‐Kit+Sca‐1+Lin− Cells After Transplantation in Muscles

Recent studies have indicated that bone marrow cells can regenerate damaged muscles and that they can adopt phenotypes of other cells by cell fusion. Our direct visualization system gave evidence of massive muscle regeneration by green fluorescent protein (GFP)–labeled CD45+c‐Kit+Sca‐1+Lin− cells (KSL cells), and we investigated the role of KSL cells in muscle regeneration after transplantation with or without lethal irradiation. In the early phase, GFP signals were clearly observed in all the muscles of only irradiated mice. Transverse cryostat sections showed GFP+ myosin+ muscle fibers, along with numerous GFP+ hematopoietic cells in damaged muscle. These phenomena were temporary, and GFP signals had dramatically reduced 30 days after transplantation. After 6 months, GFP+ fibers could hardly be detected, but GFP+ c‐Met+ mononuclear cells were located beneath the basal lamina where satellite cells usually exist in both conditioned mice. Immunostaining of isolated single fibers revealed GFP+ PAX7+, GFP+ MyoD+, and GFP+ Myf5+ satellite‐like cells on the fibers. Single‐fiber cultures from these mice showed proliferation of GFP+ fibers. These results indicate two different roles of KSL cells: one leading to regeneration of damaged muscles in the early phase and the other to conversion into satellite cells in the late phase.

Somatic Mosaicism for a NRAS Mutation Associates with Disparate Clinical Features in RAS-associated Leukoproliferative Disease: a Report of Two Cases

RAS-associated leukoproliferative disease (RALD) is a newly classified disease; thus its clinical features and management are not fully understood. The cases of two patients with characteristic features of RALD are described herein. Patient 1 was a 5-month-old female with clinical features typical of autoimmune lymphoproliferative syndrome (ALPS) and markedly elevated TCRαβ+CD4−CD8− T cell numbers. Genetic analyses failed to detect an ALPS-related gene mutation; however, whole exome sequencing and other genetic analyses revealed somatic mosaicism for the G13D NRAS mutation. These data were indivative of NRAS-associated RALD with highly elevated αβ-double-negative T cells. Patient 2 was a 12-month-old girl with recurrent fever who clearly met the diagnostic criteria for juvenile myelomonocytic leukemia (JMML). Genetic analyses revealed somatic mosaicism, again for the G13D NRAS mutation, suggesting RALD associated with somatic NRAS mosaicism. Notably, unlike most JMML cases, Patient 2 did not require steroids or hematopoietic stem cell transplantation. Genetic analysis of RAS should be performed in patients fulfilling the diagnostic criteria for ALPS in the absence of ALPS-related gene mutations if the patients have elevated αβ-double-negative-T cells and in JMML patients if autoimmunity is detected. These clinical and experimental data increase our understanding of RALD, ALPS, and JMML.

Early diagnosis improves the quality of life in MYH9 disorder.

To the Editor: MYH9 disorder is a hereditary macrothrombocytopenia, characterized by a triad of giant platelets, thrombocytopenia, and Döhle-body-like cytoplasmic inclusion bodies in granulocytes, possibly with renal failure, deafness, and/or cataracts. The causative MYH9 gene encodes the non-muscle myosin heavy chain-IIA (NMMHC-IIA) [1]. MYH9 disorder is typically of autosomal dominant inheritance, but 35% show de novo mutations. It is often misdiagnosed as idiopathic thrombocytopenic purpura (ITP), and the patient might receive unnecessary therapies including corticosteroids and splenectomy [2,3]. A 1-year-old male with an unremarkable medical history was admitted for surgery of an open fracture of right great toe. On

Severe Hemolytic Jaundice in a Neonate with a Novel COL4A1 Mutation

We report our experience with a preterm infant with severe hemolytic jaundice who required exchange transfusion just after birth. The patient was negative for alloimmune hemolysis as a result of maternal-fetal blood type incompatibility, and tests for inherited defects in erythrocyte metabolism, membrane function, and hemoglobin synthesis were normal. We also performed a bone marrow examination, but could not identify the cause of hemolysis. The patient had several other complications, including porencephaly, epilepsy, elevated serum levels of creatine kinase, and persistent microscopic hematuria. Later, we detected a genetic mutation in COL4A1, which was recently found to be associated with hemolytic anemia. We therefore believe that all of the patients clinical features, including hemolytic anemia, were due to the mutation in COL4A1. Genetic testing for COL4A1 mutations is recommended in neonates who exhibit hemolytic disease of unknown etiology, especially when other complications compatible with COL4A1-related disorders are present.