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.

Sphere formation has been utilized as a way to isolate multipotent stem/progenitor cells from various tissues. However, very few studies on bone marrow-derived spheres have been published and assessed their multipotentiality. In this study, multipotent marrow cell populations were isolated using a three-step method. First, after elimination of hematopoietic cells, murine marrow-derived adherent cells were cultured in plastic dishes until small cells gradually appeared and multiplied. Cells were then cultured under non-adherent conditions and formed spheres that were immunopositive for a neural precursor marker, nestin. RT-PCR analysis also revealed that the spheres were positive for nestin in addition to PPARgamma, osf2, SOX9, and myoD, which are markers of precursors of adipocytic, osteoblastic, chondrocytic, and skeletal myeloblastic lineages, respectively. Finally, spheres were dissociated into single cells and expanded in adherent cultures. Under appropriate induction conditions, the sphere-derived cells acquired the phenotypic properties in vitro of neurons, skeletal myoblasts, and beating cardiomyocytes, as well as adipocytes, osteoblasts, and chondrocytes. Next, sphere-derived cells were transplanted into murine myocardial infarction models. One month later, they had become engrafted as cardiomyocytes, and cardiac catheterization showed significant functional improvements. Thus, sphere-derived cells represent a new approach to enhance the multi-differentiation potential of murine bone marrow.

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The transplantation of primitive human cells into sublethally irradiated immunedeficient mice is the well-established in vivo system for the investigation of human hematopoietic stem cell function. Although mast cells are the progeny of hematopoietic stem cells, human mast cell development in mice that underwent human hematopoietic stem cell transplantation has not been reported. Here we report on human mast cell development after xenotransplantation of human hematopoietic stem cells into nonobese diabetic severe combined immunodeficient (NOD/SCID)/␥ c null (NOG) mice with severe combined immunodeficiency and interleukin 2 (IL-2) receptor ␥-chain allelic mutation. Supported by the murine environment, human mast cell clusters developed in mouse dermis, but they required more time than other forms of human cell reconstitution. In lung and gastric tract, mucosal-type mast cells containing tryptase but lacking chymase located on gastric mucosa and in alveoli, whereas connective tissue-type mast cells containing both tryptase and chymase located on gastric submucosa and around major airways, as in the human body. Mast cell development was also observed in lymph nodes, spleen, and peritoneal cavity but not in the peripheral blood. Xenotransplantation of human hematopoietic stem cells into NOG mice can be expected to result IntroductionMast cells are recognized as the principal cells which initiate immunoglobulin E (IgE)-dependent immediate hypersensitivity and also as the cells which contribute to innate immunity and tissue remodeling. 1,2 There are 2 phenotypically distinct mast cell subpopulations in rodents: connective tissue-type mast cells (CTMCs) and mucosal-type mast cells (MMCs). These populations differ in location, cell size, staining characteristics, ultrastructure, mediator content, and T-cell dependency. 3 Proliferation of rodent MMCs is dependent on T-cell-derived cytokines, 3,4 whereas that of CTMCs is supported by stem cell factor (SCF). In humans, mast cells are distinguished on the basis of their protease composition, 5,6 MC TC contains tryptase and chymase in its granules and is predominant in skin and intestinal submucosa, like CTMCs in rodents. MC T also contains tryptase, but lacks chymase, and is predominant in the alveolar wall and gastric mucosa, similar to MMCs in rodents. Human mast cells were reported to develop only under the influence of SCF, but T-cell-derived interleukin 3 (IL-3) has little affect on their differentiation. 7 Recently, human intestinal mast cells were reported to respond to IL-3 by enhancing their growth, 8 but SCF is still an indispensable factor for human mast cells. Mast cells are the progenies of hematopoietic stem cells (HSCs). 9,10 In mice, the progenitor cells capable of becoming mast cells leave the bone marrow and enter the circulation but complete their differentiation into mast cells only after arriving in peripheral tissues such as lung, bowel, and skin. 10,11 Unfortunately, the developmental mechanism of human mast cells remains far less clear, possibly bec...

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.

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.

The objectives of this study are to clarify (1) the difference in demographic and clinical variables at initial presentation between acute and chronic idiopathic thrombocytopenic purpura (ITP), and (2) the prognostic factors of patients with chronic ITP. We conducted a retrospective analysis of 247 children with newly diagnosed ITP between April 1991 and March 2006 who visited one of the 12 hospitals belonging to the Kyoto University Pediatric Hematologic Study Group. 180 and 67 cases were classified as the acute type and as the chronic type, respectively. Older age, higher initial platelet count, positive medical history or concomitant medical diagnosis, the absence of preceding infection or vaccination, and the absence of an increase in immunoglobulin were risk factors for the chronicity. The prognostic factors in chronic ITP were evaluated in 53 patients after excluding patients receiving splenectomy or having insufficient follow-up data. The overall time required for 50% resolution in patients with chronic ITP was approximately 5.6 years. Age at presentation of less than 3 years and higher platelet counts at the time of chronic ITP diagnosis were good prognostic factors. On the other hand, gender, initial platelet counts, and preceding infection or vaccination were not associated with the prognosis.

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/gamma(c)null (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 alpha-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.