Kenji Nagao

Generation of Induced Pluripotent Stem Cells by Efficient Reprogramming of Adult Bone Marrow Cells

Reprogramming of somatic cells provides potential for the generation of specific cell types, which could be a key step in the study and treatment of human diseases. In vitro reprogramming of somatic cells into a pluripotent embryonic stem (ES) cell-like state has been reported by retroviral transduction of murine fibroblasts using four embryonic transcription factors or through cell fusion of somatic and pluripotent stem cells. Here we show that mouse adult bone marrow mononuclear cells (BM MNCs) are competent as donor cells and can be reprogrammed into pluripotent ES cell-like cells. We isolated BM MNCs and mouse embryonic fibroblasts (MEFs) from Oct4-GFP transgenic mice, fused them with ES cells, or infected them with retroviruses expressing Oct4, Sox2, Klf4, and c-Myc. Fused BM MNCs formed more ES-like colonies than did MEFs. Infected BM MNCs gave rise to induced pluripotent stem (iPS) cells, although transduction efficiencies were not high. It was more efficient to pick up iPS colonies as compared with MEFs. BM-derived iPS (BM iPS) cells expressed ES cell markers, formed teratomas, and contributed to chimera mice with germ line development. Clonal analysis revealed that BM iPS clones had diversity, although some clones were found to be genetically identical with different phenotypes. Our findings imply that BM MNCs have potential advantages to generate iPS cells for the clinical application.

Craniofacial malformation in R-spondin2 knockout mice.

In vertebrates, craniofacial formation is accomplished by synergistic interaction of many small elements which are generated independently from distinct germ layers. Because of its complexity, the imbalance of one signaling cascade such as Wnt/beta-catenin pathway easily leads to craniofacial malformation, which is the most frequent birth defect in humans. To investigate the developmental role of a newly identified activator of Wnt/beta-catenin signaling, Rspo2, we generated and characterized Rspo2(-/-) mice. We found CLP with mild facial skeletal defects in Rspo2(-/-) mice. Additionally, Rspo2(-/-) mice also exhibited distal limb loss and lung hypoplasia, and died immediately after birth with respiratory failure. We showed the apparent reduction of Wnt/beta-catenin signaling activity at the branchial arch and the apical ectodermal ridge in Rspo2(-/-) mice. These findings indicate that Rspo2 regulates midfacial, limb, and lung morphogenesis during development through the Wnt/beta-catenin signaling.

Direct Generation of Induced Pluripotent Stem Cells from Human Nonmobilized Blood

The use of induced pluripotent stem cells (iPSCs) is an exciting frontier in the study and treatment of human diseases through the generation of specific cell types. Here we show the derivation of iPSCs from human nonmobilized peripheral blood (PB) and bone marrow (BM) mononuclear cells (MNCs) by retroviral transduction of OCT3/4, SOX2, KLF4, and c-MYC. The PB- and BM-derived iPSCs were quite similar to human embryonic stem cells with regard to morphology, expression of surface antigens and pluripotency-associated transcription factors, global gene expression profiles, and differentiation potential in vitro and in vivo. Infected PB and BM MNCs gave rise to iPSCs in the presence of several cytokines, although transduction efficiencies were not high. We found that 5 × 10(5) PB MNCs, which corresponds to less than 1 mL of PB, was enough for the generation of several iPSC colonies. Generation of iPSCs from MNCs of nonmobilized PB, with its relative efficiency and ease of harvesting, could enable the therapeutic use of patient-specific pluripotent stem cells.

Involvement of a Novel Q-SNARE, D12, in Quality Control of the Endomembrane System

The cellular endomembrane system requires the proper kinetic balance of synthesis and degradation of its individual components, which is maintained in part by a specific membrane fusion apparatus. In this study, we describe the molecular properties of D12, which was identified from a mouse expression library. This C-terminal anchored membrane protein has sequence similarity to both a yeast soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE), Use1p/Slt1p, and a recently identified human syntaxin 18-binding protein, p31. D12 formed a tight complex with syntaxin 18 as well as Sec22b and bound to α-SNAP, indicating that D12 is a SNARE protein. Although the majority of D12 is located in the endoplasmic reticulum and endoplasmic reticulum-Golgi intermediate compartments at steady state, overexpression or knockdown of D12 had no obvious effects on membrane trafficking in the early secretory pathway. However, suppression of D12 expression caused rapid appearance of lipofuscin granules, accompanied by apoptotic cell death without the apparent activation of the unfolded protein response. The typical cause of lipofuscin formation is the impaired degradation of mitochondria by lysosomal degradative enzymes, and, consistent with this, we found that proper post-Golgi maturation of cathepsin D was impaired in D12-deficient cells. This unexpected observation was supported by evidence that D12 associates with VAMP7, a SNARE in the endosomal-lysosomal pathway. Hence, we suggest that D12 participates in the degradative function of lysosomes.

Enhanced degradation of MDM2 by a nuclear envelope component, mouse germ cell-less

A mouse homologue of Drosophila germ cell less, mouse germ cell less-1 (mgcl-1), encodes a nuclear envelope component essential for nuclear integrity. To analyze the molecular function of mGCL-1, we carried out two hybrid screening and found that mGCL-1 bound to the gene product of tumor susceptibility gene 101 (tsg101). Effects of mGCL-1 on the expression of MDM2-p53 axis were examined, since TSG101 has been shown to elevate the amount of MDM2 by inhibiting the ubiquitination. mGCL-1 significantly reduced the amount of MDM2 probably by changing the sub-cellular localization of the MDM2 and facilitating the ubiquitination of MDM2. In addition, the amount of p53 was increased and transactivation by p53 was enhanced by mGCL-1. Thus, mGCL-1 turned out to be a factor modulating MDM2-p53 axis by enhanced degradation of MDM2.

Expression profile analysis of aorta-gonad-mesonephros region-derived stromal cells reveals genes that regulate hematopoiesis

The aorta-gonad-mesonephros (AGM) region is involved in the generation and maintenance of the first definitive hematopoietic stem cells (HSCs). A mouse AGM-derived cell line, AGM-S3, was shown to support the development of HSCs. To elucidate the molecular mechanisms regulating early hematopoiesis, we obtained subclones from AGM-S3, one of which was hematopoiesis supportive (S3-A9) and the other one of which was non-supportive (S3-A7), and we analyzed their gene expression profiles by gene chip analysis. In the present study, we found that Glypican-1 (GPC1) was highly expressed in the supportive subclone AGM-S3-A9. Over-expression of GPC1 in non-supportive cells led to the proliferation of progenitor cells in human cord blood when cocultured with the transfected-stromal cells. Thus, GPC1 may have an important role in the establishment of a microenvironment that supports early events in hematopoiesis.