Hisanori Fujino

Selective infection of CD4+ effector memory T lymphocytes leads to preferential depletion of memory T lymphocytes in R5 HIV-1-infected humanized NOD/SCID/IL-2Rγnull mice

To investigate the events leading to the depletion of CD4(+) T lymphocytes during long-term infection of human immunodeficiency virus type 1 (HIV-1), we infected human CD34(+) cells-transplanted NOD/SCID/IL-2Rgamma(null) mice with CXCR4-tropic and CCR5-tropic HIV-1. CXCR4-tropic HIV-1-infected mice were quickly depleted of CD4(+) thymocytes and both CD45RA(+) naïve and CD45RA(-) memory CD4(+) T lymphocytes, while CCR5-tropic HIV-1-infected mice were preferentially depleted of CD45RA(-) memory CD4(+) T lymphocytes. Staining of HIV-1 p24 antigen revealed that CCR5-tropic HIV-1 preferentially infected effector memory T lymphocytes (T(EM)) rather than central memory T lymphocytes. In addition, the majority of p24(+) cells in CCR5-tropic HIV-1-infected mice were activated and in cycling phase. Taken together, our findings indicate that productive infection mainly takes place in the activated T(EM) in cycling phase and further suggest that the predominant infection in T(EM) would lead to the depletion of memory CD4(+) T lymphocytes in CCR5-tropic HIV-1-infected mice.

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)

Neutrophil differentiation from human-induced pluripotent stem cells

Induced pluripotent stem (iPS) cells are of potential value not only for regenerative medicine, but also for disease investigation. The present study describes the development of a neutrophil differentiation system from human iPS cells (hiPSCs) and the analysis of neutrophil function and differentiation. The culture system used consisted of the transfer of hiPSCs onto OP9 cells and their culture with vascular endothelial growth factor (VEGF). After 10 days, TRA 1‐85+CD34+VEGF receptor‐2 (VEGFR‐2)high cells were sorted and co‐cultured with OP9 cells in the presence of hematopoietic cytokines for 30 days. Floating cells were collected and subjected to morphological and functional analysis. These hiPSC‐derived neutrophils were similar to peripheral blood mature neutrophils in morphology, contained functional neutrophil specific granules, and were equipped with the basic functions such as phagocytosis, superoxide production, and chemotaxis. In the process of differentiation, myeloid cells appeared sequentially from immature myeloblasts to mature segmented neutrophils. Expression patterns of surface antigen, transcription factors, and granule proteins during differentiation were also similar to those of granulopoiesis in normal bone marrow. In conclusion, differentiation of mature neutrophils from hiPSCs was successfully induced in a similar process to normal granulopoiesis using an OP9 co‐culture system. This system may be applied to elucidate the pathogenesis of various hematological diseases that affect neutrophils. J. Cell. Physiol. 226: 1283–1291, 2011.

Neurodegenerative central nervous system disease as late sequelae of Langerhans cell histiocytosis. Report from the Japan LCH Study Group

Langerhans’ cell histiocytosis can affect the central nervous system, where it frequently manifests as diabetes insipidus. Cerebellar ataxia and other neurological defects can represent late sequelae of this disorder. Clinical features, brain magnetic resonance imaging findings and EDSS scores of 11 patients with neurodegenerative central nervous system Langerhans cell histiocytosis were analyzed in Japan. All patients initially had multi-system-type Langerhans cell histiocytosis; 8 at 1–2 years of age and 3 at a later age. Neurodegenerative central nervous system largermans cell histiocytosis disease developed after a median time interval of 3.9 years from initial diagnosis. With a median follow-up of 4.5 years, 6 patients showed progression of disease with an EDSS score >3. This study demonstrates the importance of early detection of neurodegenerative central nervous system Langerhans cell histiocytosis by brain magnetic resonance imaging, particularly in the follow-up of patients who developed multi-system-type Langerhans cell histiocytosis in early infancy.

Long‐Term Culture of Postnatal Mouse Hepatic Stem/Progenitor Cells and Their Relative Developmental Hierarchy

Few studies on the long‐term culture of postnatal mouse hepatic stem/progenitor cells have been reported. We successfully adapted a serum‐free culture system that we employed previously to expand fetal mouse hepatic stem/progenitor cells and maintained them in culture over long periods. The expanded postnatal cells contained immature α‐fetoprotein‐positive cells along with hepatocytic and cholangiocytic lineage‐committed cells. These cells expressed CD49f but not CD45, CD34, Thy‐1, c‐kit, CD31, or flk‐1, and oncostatin M induced their differentiation. This heterogeneous population contained side population (SP) cells, which express the ATP‐binding cassette transporter ABCG2, and sca‐1+ cells. As mice aged, the frequency of SP and sca‐1+ cells decreased along with the ability of cultured cells to expand. Approximately 20%–40% of the SP cells expressed sca‐1, but only a few sca‐1+ cells were also SP cells. Analysis of colonies derived from single SP or sca‐1+ cells revealed that, although both cells had dual differentiation potential and self‐renewal ability, SP cells formed colonies more efficiently and gave rise to SP and sca‐1+ cells, whereas sca‐1+ cells generated only sca‐1+ progeny. Thus, SP cells are more characteristic of stem cells than are sca‐1+ cells. In regenerating livers, ABCG2+ cells and sca‐1+ cells were detected around or in the portal area (the putative hepatic stem cell niche). The expanded cells share many features of fetal hepatic stem/progenitor cells or oval cells and may be useful in determining the mechanisms whereby hepatic stem cells self‐renew and differentiate.

Identification of Hepatic Niche Harboring Human Acute Lymphoblastic Leukemic Cells via the SDF-1/CXCR4 Axis

In acute lymphoblastic leukemia (ALL) patients, the bone marrow niche is widely known to be an important element of treatment response and relapse. Furthermore, a characteristic liver pathology observed in ALL patients implies that the hepatic microenvironment provides an extramedullary niche for leukemic cells. However, it remains unclear whether the liver actually provides a specific niche. The mechanism underlying this pathology is also poorly understood. Here, to answer these questions, we reconstituted the histopathology of leukemic liver by using patients-derived primary ALL cells into NOD/SCID/Yc null mice. The liver pathology in this model was similar to that observed in the patients. By using this model, we clearly demonstrated that bile duct epithelial cells form a hepatic niche that supports infiltration and proliferation of ALL cells in the liver. Furthermore, we showed that functions of the niche are maintained by the SDF-1/CXCR4 axis, proposing a novel therapeutic approach targeting the extramedullary niche by inhibition of the SDF-1/CXCR4 axis. In conclusion, we demonstrated that the liver dissemination of leukemia is not due to nonselective infiltration, but rather systematic invasion and proliferation of leukemic cells in hepatic niche. Although the contribution of SDF-1/CXCR4 axis is reported in some cancer cells or leukemic niches such as bone marrow, we demonstrated that this axis works even in the extramedullary niche of leukemic cells. Our findings form the basis for therapeutic approaches that target the extramedullary niche by inhibiting the SDF-1/CXCR4 axis.

Direct Development of Functionally Mature Tryptase/Chymase Double‐Positive Connective Tissue‐Type Mast Cells from Primate Embryonic Stem Cells

Conditions that influence the selective development or recruitment of connective tissue‐type and mucosal‐type mast cells (MCs) are not well understood. Here, we report that cynomolgus monkey embryonic stem (ES) cells cocultured with the murine aorta‐gonad‐mesonephros‐derived stromal cell line AGM‐S1 differentiated into cobblestone (CS)‐like cells by day 10–15. When replated onto fresh AGM‐S1 with the addition of stem cell factor, interleukin‐6, and Flt3 ligand, these CS‐like cells displayed robust growth and generated almost 100% tryptase/chymase double‐positive MCs within 3 weeks. At all time points, the percentage of tryptase‐positive cells did not exceed that of chymase‐positive cells. These ES‐derived MCs were CD45+/Kit+/CD31+/CD203c+/HLA‐DR− and coexpressed a high‐affinity IgE receptor on their surface, which was upregulated after IgE exposure. Electron microscopy showed that they contained many electron dense granules. Moreover, ES‐derived MCs responded to stimulation by via IgE and substance P by releasing histamine. These results indicate that ES‐derived MCs have the phenotype of functionally mature connective tissue‐type MCs. The rapid maturation of ES‐derived MCs suggests a unique embryonic pathway in primates for early development of connective tissue‐type MCs, which may be independent from the developmental pathway of mucosal‐type MCs.

Second transplantation from HLA 2-loci-mismatched mother for graft failure due to hemophagocytic syndrome after cord blood transplantation

A 7-year-old girl with acute myelogenous leukemia with multilineage dysplasia received unrelated cord blood transplantation but developed hemophagocytic syndrome (HPS) after sepsis with methicillin-resistant coagulase-negative staphylococci before engraftment. Bone marrow aspiration on day 20 revealed a markedly increased number of activated macrophages showing hemophagocytosis. The presence of donor-type chimera in the bone marrow was confirmed at that time. We therefore quickly started immunosuppressive and antibacterial treatment. Although her condition gradually improved, the patient suf-fered graft failure due to HPS. She received peripheral blood stem cell transplantation from her HLA 2-loci-mismatched mother on day 54 and continued in complete remission 12 months after the second transplantation. The results in this case sug-gested that because of fetomaternal microchimerism it may be useful to select an HLA-haploidentical mother as a backup donor for stem cell transplantation.

Rapid Improvement of Paraplegia Caused by Epidural Involvements of Burkitt’s Lymphoma With Chemotherapy

Study Design. Case report. Objective. The authors present a case of atypical Burkitt’s lymphoma with multiple epidural involvements. Summary of Background Data. Spinal cord compression in children is an emergency that requires urgent attention to minimize neurologic dysfunction. Although it is not life-threatening in most patients, cord compression can cause severe neurologic morbidity. Materials and Methods. Because the patient showed rapid neurologic deterioration, we started chemotherapy and high-dose steroids without laminectomy or radiotherapy immediately after a tumor biopsy from the left mandible. Result. The combined therapies were very effective and his neurologic symptoms improved immediately. The epidural involved masses disappeared in imaging studies after the first course of chemotherapy including methylprednisolone (20 mg/kg per day for 3 consecutive days and gradually tapered off over 2 weeks), vincristine (1.5 mg/m2 per day), cyclophosphamide (2 g/m2 per day for 2 days) and pirarubicin (40 mg/m2 per day). After completing seven courses of chemotherapy, the patient is now fully ambulant. Conclusion. Considering the severe late effects of laminectomy and radiotherapy, chemotherapy should be considered as a first choice of treatment for spinal cord compression caused by malignant lymphoma.

Clonal selection in xenografted TAM recapitulates the evolutionary process of myeloid leukemia in Down syndrome

Transient abnormal myelopoiesis (TAM) is a clonal preleukemic disorder that progresses to myeloid leukemia of Down syndrome (ML-DS) through the accumulation of genetic alterations. To investigate the mechanism of leukemogenesis in this disorder, a xenograft model of TAM was established using NOD/Shi-scid, interleukin (IL)-2Rγ(null) mice. Serial engraftment after transplantation of cells from a TAM patient who developed ML-DS a year later demonstrated their self-renewal capacity. A GATA1 mutation and no copy number alterations (CNAs) were detected in the primary patient sample by conventional genomic sequencing and CNA profiling. However, in serial transplantations, engrafted TAM-derived cells showed the emergence of divergent subclones with another GATA1 mutation and various CNAs, including a 16q deletion and 1q gain, which are clinically associated with ML-DS. Detailed genomic analysis identified minor subclones with a 16q deletion or this distinct GATA1 mutation in the primary patient sample. These results suggest that genetically heterogeneous subclones with varying leukemia-initiating potential already exist in the neonatal TAM phase, and ML-DS may develop from a pool of such minor clones through clonal selection. Our xenograft model of TAM may provide unique insight into the evolutionary process of leukemia.