Improved Sendai viral system for reprogramming to naive pluripotency.

Naive human induced pluripotent stem cells (iPSCs) can be generated by reprogramming somatic cells with Sendai virus (SeV) vectors. However, only dermal fibroblasts have been successfully reprogrammed this way, and the process requires culture on feeder cells. Moreover, SeV vectors are highly persistent and inhibit subsequent differentiation of iPSCs.

Simple derivation of skeletal muscle from human pluripotent stem cells using temperature-sensitive Sendai virus vector.

Human pluripotent stem cells have the potential to differentiate into various cell types including skeletal muscles (SkM), and they are applied to regenerative medicine or in vitro modelling for intractable diseases. A simple differentiation method is required for SkM cells to accelerate neuromuscular disease studies. Here, we established a simple method to convert human pluripotent stem cells into SkM cells by using temperature-sensitive Sendai virus (SeV) vector encoding myoblast determination protein 1 (SeV-Myod1), a myogenic master transcription factor.

Generation of Motor Neurons from Human ESCs/iPSCs Using Sendai Virus Vectors.

Human motor neurons are important materials for the research of the pathogenesis and drug discovery of motor neuron diseases. Various methods to generate motor neurons (MNs) from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) by the addition of signaling molecules have been reported. However, they require multiple steps and complicated processes.

iPSC screening for drug repurposing identifies anti-RNA virus agents modulating host cell susceptibility.

Human pathogenic RNA viruses are threats to public health because they are prone to escaping the human immune system through mutations of genomic RNA, thereby causing local outbreaks and global pandemics of emerging or re-emerging viral diseases. While specific therapeutics and vaccines are being developed, a broad-spectrum therapeutic agent for RNA viruses would be beneficial for targeting newly emerging and mutated RNA viruses. In this study, we conducted a screen of repurposed drugs using Sendai virus (an RNA virus of the family Paramyxoviridae), with human-induced pluripotent stem cells (iPSCs) to explore existing drugs that may present anti-RNA viral activity.

Isolation and propagation of enteric neural crest progenitor cells from mouse embryonic stem cells and embryos.

Neural crest is a source of diverse cell types, including the peripheral nervous system. The transcription factor Sox10 is expressed throughout early neural crest. We exploited Sox10 reporter and selection markers created by homologous recombination to investigate the generation, maintenance and expansion of neural crest progenitors.

Promotion of reprogramming to ground state pluripotency by signal inhibition.

Induced pluripotent stem (iPS) cells are generated from somatic cells by genetic manipulation. Reprogramming entails multiple transgene integrations and occurs apparently stochastically in rare cells over many days. Tissue stem cells may be subject to less-stringent epigenetic restrictions than other cells and might therefore be more amenable to deprogramming.

Generation of osteoblasts and chondrocytes from embryonic stem cells.

The efficient generation of mesenchymal cells such as adipocytes, osteoblasts, and chondrocytes from embryonic stem cells is achieved by following sequential steps: embryoid body formation, retinoic acid (RA) treatment, and exposure to specific reagents for differentiation. RA treatment of embryoid bodies is critical for subsequent mesengenesis. Adipogenesis, osteogenesis, and chondrogenesis occur by culturing outgrowths for 2-3 wk with insulin/triiodothyronine, bone morphogenetic protein/dexamethasone-beta/glycerophosphate/ascorbic acid, and transforming growth factor-beta3/parathyroid hormone/1% fetal bovine serum, respectively.

Osteogenic and chondrogenic differentiation of embryonic stem cells in response to specific growth factors.

Reliable in vitro conversion of pluripotent embryonic stem (ES) cells into bone and cartilage-forming cells would expand opportunities for experimental investigations of skeletogenesis and could also provide new cellular sources for pharmaceutical screening and for cell therapy applications. Here, we evaluate the generation of mesenchymal cell lineages from mouse ES cells following treatment of embryoid bodies with retinoic acid, previously reported to induce development of adipocyte precursors. We find that retinoic acid reduces mesodermal differentiation but enhances expression of markers of neural crest, an alternative origin of mesenchymal elements.

Overexpression of cadherins suppresses pulmonary metastasis of osteosarcoma in vivo.

Osteosarcoma by nature shows aggressive pulmonary metastasis; however, the underlying molecular mechanisms remain unclear. We previously showed that N-cadherin and cadherin-11 (OB-cadherin), which are highly expressed in normal osteoblasts, are anomalously expressed in human osteosarcoma (Kashima et al., Am J Pathol 1999;155:1549-55).

Visualization of whole-mount skeletal expression patterns of LacZ reporters using a tissue clearing protocol.

Gene targeting or trapping constructs that utilize the lacZ gene encoding beta-galactosidase activity to trap promoter expression have become an increasingly important way to disrupt gene function and monitor gene expression. A number of genes targeted in this way have revealed both expected and unexpected developmental abnormalities of the skeleton. The use of X-gal staining to monitor gene expression in developing skeletal structures is hampered in these mutants because, during the critical latter stages of mouse embryonic development, visualization is hindered by the opacity of overlying soft tissue.