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The trophoblast cell lineage is essential for the survival of the mammalian embryo in utero. This lineage is specified before implantation into the uterus and is restricted to form the fetal portion of the placenta. A culture of mouse blastocysts or early postimplantation trophoblasts in the presence of fibroblast growth factor 4 (FGF4) permitted the isolation of permanent trophoblast stem cell lines. These cell lines differentiated to other trophoblast subtypes in vitro in the absence of FGF4 and exclusively contributed to the trophoblast lineage in vivo in chimeras.

Agonist-induced activation of peroxisome proliferator-activated receptor gamma (PPAR gamma) is known to cause adipocyte differentiation and insulin sensitivity. The biological role of PPAR gamma was investigated by gene targeting. Homozygous PPAR gamma-deficient embryos died at 10.5-11.5 dpc due to placental dysfunction. Quite unexpectedly, heterozygous PPAR gamma-deficient mice were protected from the development of insulin resistance due to adipocyte hypertrophy under a high-fat diet. These phenotypes were abrogated by PPAR gamma agonist treatment. Heterozygous PPAR gamma-deficient mice showed overexpression and hypersecretion of leptin despite the smaller size of adipocytes and decreased fat mass, which may explain these phenotypes at least in part. This study reveals a hitherto unpredicted role for PPAR gamma in high-fat diet-induced obesity due to adipocyte hypertrophy and insulin resistance, which requires both alleles of PPAR gamma.

Acute myelogenous leukemia (AML) is the most common adult leukemia, characterized by the clonal expansion of immature myeloblasts initiating from rare leukemic stem (LS) cells. To understand the functional properties of human LS cells, we developed a primary human AML xenotransplantation model using newborn nonobese diabetic/severe combined immunodeficient/interleukin (NOD/SCID/IL)2r gamma(null) mice carrying a complete null mutation of the cytokine gamma c upon the SCID background. Using this model, we demonstrated that LS cells exclusively recapitulate AML and retain self-renewal capacity in vivo. They home to and engraft within the osteoblast-rich area of the bone marrow, where AML cells are protected from chemotherapy-induced apoptosis. Quiescence of human LS cells may be a mechanism underlying resistance to cell cycle-dependent cytotoxic therapy. Global transcriptional profiling identified LS cell-specific transcripts that are stable through serial transplantation. These results indicate the potential utility of this AML xenograft model in the development of novel therapeutic strategies targeted at LS cells.

DNA methylation is an important means of epigenetic gene regulation and must be carefully controlled as a prerequisite for normal early embryogenesis. Although global demethylation occurs soon after fertilization, it is not evenly distributed throughout the genome. Genomic imprinting and epigenetic asymmetry between parental genomes, that is, delayed demethylation of the maternal genome after fertilization, are clear examples of the functional importance of DNA methylation. Here, we show that PGC7/Stella, a maternal factor essential for early development, protects the DNA methylation state of several imprinted loci and epigenetic asymmetry. After determining that PGC7/Stella binds to Ran binding protein 5 (RanBP5; a nuclear transport shuttle protein), mutant versions of the two proteins were used to examine exactly when and where PGC7/Stella functions within the cell. It is likely that PGC7/Stella protects the maternal genome from demethylation only after localizing to the nucleus, where it maintains the methylation of several imprinted genes. These results demonstrate that PGC7/Stella is indispensable for the maintenance of methylation involved in epigenetic reprogramming after fertilization.

Whereas humanized mouse models have contributed significantly to human immunology research, human T cells developing in mouse thymic environment fail to demonstrate HLA-restricted function. To achieve HLA-restricted human immune response, we created an immune-compromised non-obese diabetic/SCID/IL2rg null strain (NSG) with homozygous expression of HLA class I heavy chain and light chain (NSG-HLA-A2/HHD). Transplantation of purified Lin−CD34+ CD38− human hematopoietic stem cells into NSG-HLA-A2/HHD newborns resulted in the development of human CD4+ and CD8+ TCRαβ+ T cells and CD4−CD8− and CD8+ TCRγδ+ cells in recipient bone marrow and spleen. Human cytotoxic T lymphocytes (CTLs) become functionally mature, as evidenced by the production of granzyme corresponding to phenotypic transition from naïve to effector memory CTLs. In these recipients, human Th17 cells developed along with Th1 and Th2 cells. Epstein-Barr virus (EBV) infection in the humanized NSG-HLA-A2/HHD recipients resulted in the formation of lymphoproliferative lesions consisting mainly of human B cells with scattered human T cells. Human CTLs developing in the recipients recognized EBV-derived peptides in an HLArestricted manner and exerted HLA-restricted cytotoxicity against EBV-infected human B cells. The HLA-expressing humanized mouse with functional HLA-restricted T cells and consistent representation of rare T-cell subsets overcomes a major constraint in human immunology, and serves as a useful model for investigation of human immune responses against pathogens and for the development of therapeutic strategies against human diseases.human immunology | T-cell development | HLA restriction | Epstein-Barr virus

Human acute myeloid leukemia (AML) originates from rare leukemia stem cells (LSCs). Because these chemotherapy-resistant LSCs are thought to underlie disease relapse, effective therapeutic strategies specifically targeting these cells may be beneficial. Here, we report identification of a primary human LSC gene signature and functional characterization of human LSC-specific molecules in vivo in a mouse xenotransplantation model. In 32 of 61 (53%) patients with AML, either CD32 or CD25 or both were highly expressed in LSCs. CD32-or CD25-positive LSCs could initiate AML and were cell cycle-quiescent and chemotherapy-resistant in vivo. Normal human hematopoietic stem cells depleted of CD32-and CD25-positive cells maintained long-term multilineage hematopoietic reconstitution capacity in vivo, indicating the potential safety of treatments targeting these molecules. In addition to CD32 and CD25, quiescent LSCs within the bone marrow niche also expressed the transcription factor WT1 and the kinase HCK. These molecules are also promising targets for LSC-specific therapy.

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