Yoshiro Nakajima

Development of Defective and Persistent Sendai Virus Vector A UNIQUE GENE DELIVERY/EXPRESSION SYSTEM IDEAL FOR CELL REPROGRAMMING

The ectopic expression of transcription factors can reprogram differentiated tissue cells into induced pluripotent stem cells. However, this is a slow and inefficient process, depending on the simultaneous delivery of multiple genes encoding essential reprogramming factors and on their sustained expression in target cells. Moreover, once cell reprogramming is accomplished, these exogenous reprogramming factors should be replaced with their endogenous counterparts for establishing autoregulated pluripotency. Complete and designed removal of the exogenous genes from the reprogrammed cells would be an ideal option for satisfying this latter requisite as well as for minimizing the risk of malignant cell transformation. However, no single gene delivery/expression system has ever been equipped with these contradictory characteristics. Here we report the development of a novel replication-defective and persistent Sendai virus (SeVdp) vector based on a noncytopathic variant virus, which fulfills all of these requirements for cell reprogramming. The SeVdp vector could accommodate up to four exogenous genes, deliver them efficiently into various mammalian cells (including primary tissue cells and human hematopoietic stem cells) and express them stably in the cytoplasm at a prefixed balance. Furthermore, interfering with viral transcription/replication using siRNA could erase the genomic RNA of SeVdp vector from the target cells quickly and thoroughly. A SeVdp vector installed with Oct4/Sox2/Klf4/c-Myc could reprogram mouse primary fibroblasts quite efficiently; ∼1% of the cells were reprogrammed to Nanog-positive induced pluripotent stem cells without chromosomal gene integration. Thus, this SeVdp vector has potential as a tool for advanced cell reprogramming and for stem cell research.

TIF1β regulates the pluripotency of embryonic stem cells in a phosphorylation-dependent manner

Transcription networks composed of various transcriptional factors specifically expressed in undifferentiated embryonic stem (ES) cells have been implicated in the regulation of pluripotency in ES cells. However, the molecular mechanisms responsible for self-renewal, maintenance of pluripotency, and lineage specification during differentiation of ES cells are still unclear. The results of this study demonstrate that a phosphorylation-dependent chromatin relaxation factor, transcriptional intermediary factor–1β (TIF1β), is a unique regulator of the pluripotency of ES cells and regulates Oct3/4–dependent transcription in a phosphorylation-dependent manner. TIF1β is specifically phosphorylated in pluripotent mouse ES cells at the C-terminal serine 824, which has been previously shown to induce chromatin relaxation. Phosphorylated TIF1β is partially colocalized at the activated chromatin markers, and forms a complex with the pluripotency-specific transcription factor Oct3/4 and subunits of the switching defective/sucrose nonfermenting, ATP-dependent chromatin remodeling complex, Smarcad1, Brg-1, and BAF155, all of which are components of an ES-specific chromatin remodeling complex, esBAF. Phosphorylated TIF1β specifically induces ES cell–specific genes and enables prolonged main-tenance of an undifferentiated state in mouse ES cells. Moreover, TIF1β regulates the reprogramming process of somatic cells in a phosphorylation-dependent manner. Our results suggest that TIF1β provides a phosphorylation-dependent, bidirectional platform for specific transcriptional factors and chromatin remodeling enzymes that regulate the cell differentiation process and the pluripotency of stem cells.

Prohibitin 2 Regulates the Proliferation and Lineage-Specific Differentiation of Mouse Embryonic Stem Cells in Mitochondria

Background The pluripotent state of embryonic stem (ES) cells is controlled by a network of specific transcription factors. Recent studies also suggested the significant contribution of mitochondria on the regulation of pluripotent stem cells. However, the molecules involved in these regulations are still unknown. Methodology/Principal Findings In this study, we found that prohibitin 2 (PHB2), a pleiotrophic factor mainly localized in mitochondria, is a crucial regulatory factor for the homeostasis and differentiation of ES cells. PHB2 was highly expressed in undifferentiated mouse ES cells, and the expression was decreased during the differentiation of ES cells. Knockdown of PHB2 induced significant apoptosis in pluripotent ES cells, whereas enhanced expression of PHB2 contributed to the proliferation of ES cells. However, enhanced expression of PHB2 strongly inhibited ES cell differentiation into neuronal and endodermal cells. Interestingly, only PHB2 with intact mitochondrial targeting signal showed these specific effects on ES cells. Moreover, overexpression of PHB2 enhanced the processing of a dynamin-like GTPase (OPA1) that regulates mitochondrial fusion and cristae remodeling, which could induce partial dysfunction of mitochondria. Conclusions/Significance Our results suggest that PHB2 is a crucial mitochondrial regulator for homeostasis and lineage-specific differentiation of ES cells.

Isolation and expression of the retinoid X receptor from last instar nymphs and adult females of the soft tick Ornithodoros moubata (Acari: Argasidae).

Retinoid X receptors (RXR) exist broadly from invertebrates to vertebrates, and play essential roles in physiological processes of these organisms. In arthropods, RXRs form a complex with the ecdysteroid receptor (EcR) and ecdysteroids to mediate the regulation of ecdysis and reproduction. Compared to EcR, RXR and its homologue ultraspiracle (USP) are much less well understood. Therefore, we identified RXR of the soft tick Ornithodoros moubata (OmRXR) and used real-time PCR to examine the expression of OmRXR. This is the first report of RXR from a soft tick. OmRXR showed higher homology to hard tick, crustacean and vertebrate RXRs than insect RXRs and USPs. OmRXR expression was observed during molting in the last instar nymphs coinciding with EcR expression and increases in ecdysteroid titers. Tick vitellogenesis normally occurs soon after engorgement and OmRXR expression coinciding with EcR expression and ecdysteroid titers in engorged females occurred before vitellogenin (Vg) synthesis and egg maturation. The ecdysteroid/EcR/RXR complex appears to be important in the regulation of molting and vitellogenesis of soft ticks.

Novel cell surface genes expressed in the stomach primordium during gastrointestinal morphogenesis of mouse embryos.

The mechanisms of gastrointestinal morphogenesis in mammals are not well understood. This is partly due to the lack of appropriate markers that are expressed with spatiotemporal specificity in the gastrointestinal tract during development. Using mouse embryos, we surveyed markers of the prospective stomach region during gastrointestinal morphogenesis. The initiation of organ bud formation occurs at E10.5 in mice. These primordia for the digestive organs protrude from a tube-like structured endoderm and have their own distinct morphogenesis. We identified 3 cell surface genes -Adra2a, Fzd5, and Trpv6 - that are expressed in the developing stomach region during gastrointestinal morphogenesis using a microarray-based screening. These novel genes will be useful in expanding our understanding of the mechanisms of gastrointestinal development.

Mechanobiology During Vertebrate Organ Development

Neural crest cells are one of many migrating cell types found in vertebrate tissues. Neural crest specification occurs between the neural plate and epidermal region during vertebrate embryogenesis, and is regulated at the gene level by appropriate concentrations of cell signaling proteins such as bone morphogenetic protein (BMP), fibroblast growth factor (FGF), and Wnt (reviewed by Meulemans and Bronner-Fraser 2004). Many genes participate in this regional network to specify the neural crest. After neural tube closure, neural crest cells start migrating to the ventral half of the embryo, and the resultant epithelial-to-mesenchymal transformation dissociates the neural crest cells from the epidermis. The dissociated neural crest cells then start to move ventrally.