Identification of a familial cleidocranial dysplasia with a novel RUNX2 mutation and establishment of patient-derived induced pluripotent stem cells.

Cleidocranial dysplasia (CCD) is an autosomal dominant hereditary disease associated with the gene RUNX2. Disease-specific induced pluripotent stem cells (iPSCs) have emerged as a useful resource to further study human hereditary diseases such as CCD. In this study, we identified a novel CCD-specific RUNX2 mutation and established iPSCs with this mutation. Biopsies were obtained from familial CCD patients and mutation analyses were performed through Sanger sequencing and next generation sequencing. CCD-specific human iPSCs (CCD-hiPSCs) were established and maintained under completely defined serum, feeder, and integration-free condition using a non-integrating replication-defective Sendai virus vector. We identified the novel mutation RUNX2_c.371C>G and successfully established CCD-hiPSCs. The CCD-hiPSCs inherited the same mutation, possessed pluripotency, and showed the ability to differentiate the three germ layers. We concluded that RUNX2_c.371C>G was likely pathogenic because our results, derived from next generation sequencing, are supported by actual clinical evidence, familial tracing, and genetic data. Thus, we concluded that hiPSCs with a novel CCD-specific RUNX2 mutation are viable as a resource for future studies on CCD.

Formation of taste-active pyroglutamyl peptide ethyl esters in sake by rice koji peptidases.

Formation of taste-active pyroglutamyl (pGlu) peptide ethyl esters in sake was investigated: 2 enzymes (A and B) responsible for the esterification were purified from a rice koji extract. MADLI-TOF/TOF analysis after deglycosylation identified enzyme (A) as peptidase S28 (GenBank accession number OOO13707.1) and enzyme (B) as serine-type carboxypeptidase (accession number AO090010000534). Both enzymes hydrolyzed pGlu peptides and formed ethyl esters under sake mash conditions: acidic pH (3-4) and in ethanol (5%-20% v/v) aqueous solutions. Enzyme (A) formed pGlu penta-peptide ethyl esters from pGlu undeca-peptides by a prolyl endo-type reaction. Enzyme (B) formed (pGlu) deca-peptide and its ethyl esters from pGlu undeca-peptides in an exo-type reaction. We are the first to report the enzymatic ethyl esterification reaction in the formation of pGlu peptides by rice koji peptidases.

Yip1A, a novel host factor for the activation of the IRE1 pathway of the unfolded protein response during Brucella infection.

Brucella species replicate within host cells in the form of endoplasmic reticulum (ER)-derived vacuoles. The mechanisms by which the bacteria are sequestered into such vacuoles and obtain a continuous membrane supply for their replication remain to be elucidated. In the present study, we provided several lines of evidence that demonstrate the mechanism by which B. abortus acquires the ER-derived membrane. First, during Brucella infection, the IRE1 pathway, but not the PERK and ATF6 pathways, of the unfolded protein response (UPR) was activated in a time-dependent manner, and the COPII vesicle components Sar1, Sec23, and Sec24D were upregulated. Second, a marked accretion of ER-derived vacuoles was observed around replicating bacteria using fluorescent microscopy and electron microscopy. Third, we identified a novel host factor, Yip1A, for the activation of the IRE1 pathway in response to both tunicamycin treatment and infection with B. abortus. We found that Yip1A is responsible for the phosphorylation of IRE1 through high-order assembly of Ire1 molecules at ER exit sites (ERES) under the UPR conditions. In Yip1A-knockdown cells, B. abortus failed to generate the ER-derived vacuoles, and remained in endosomal/lysosomal compartments. These results indicate that the activation of the IRE1 pathway and the subsequent formation of ER-derived vacuoles are critical for B. abortus to establish a safe replication niche, and that Yip1A is indispensable for these processes. Furthermore, we showed that the autophagy-related proteins Atg9 and WIPI1, but not DFCP1, were required for the biogenesis of the ER-derived membrane compartments. 　On the basis of our findings, we propose a model for intracellular Brucella replication that exploits the host UPR and ER-derived vacuole formation machineries, both of which depend on Yip1A-mediated IRE1 activation.

JNK1/2-dependent phosphorylation of angulin-1/LSR is required for the exclusive localization of angulin-1/LSR and tricellulin at tricellular contacts in EpH4 epithelial sheet.

Tricellular tight junctions (tTJs) are specialized structural variants of tight junctions within tricellular contacts of an epithelial sheet and comprise several transmembrane proteins including lipolysis-stimulated lipoprotein receptor (angulin-1/LSR) and tricellulin. To elucidate the mechanism of its formation, we carried out stepwise screening of kinase inhibitors followed by RNAi screening to identify kinases that regulate intracellular localization of angulin-1/LSR to the tTJs using a fluorescence image-based screen. We found that the activity of JNK1 and JNK2, but not JNK3, was required for the exclusive localization of angulin-1/LSR at the tTJs. Based on a bioinformatics approach, we estimated the potential phosphorylation site of angulin-1/LSR by JNK1 to be serine 288 and experimentally confirmed that JNK1 directly phosphorylates angulin-1/LSR at this site. We found that JNK2 was also involved in the phosphorylation of angulin-1/LSR. Furthermore, GFP-tagged angulin-1/LSR(S288A), in which serine 288 was substituted by alanine, was observed to be dispersed to bicellular junctions, indicating that phosphorylation of Ser288 is crucial for the exclusive localization of angulin-1/LSR and tricellulin at tTJs. Our fluorescence image-based screening for kinases inhibitor or siRNAs combined with the phosphorylation site prediction could become a versatile and useful tool to elucidate the mechanisms underlying the maintenance of tTJs regulated by kinase networks.

Unique bone histology of modern giant salamanders: a study on humeri and femora of Andrias spp.

The osteohistology of Andrias spp. is a pivotal analogue for large fossil non-amniotes (e.g., Temnospondyli), and the endangered status of this taxon underlines the importance of gathering information on its growth. We here present the first osteohistological study by petrographic thin sections of an ontogenetic series of humeri and femora of eight individuals of varying sizes (28.5-104 cm) and ages (2.5-32 years) of Andrias japonicus from the Hiroshima City Asa Zoological Park, Japan. In addition, two individuals of A. cf. davidianus of unknown age but of different size (62 cm and 94 cm) were studied. All samples of Andrias spp. show a primary avascular periosteal cortex made of parallel-fibred tissue around the ossification center in the petrographic thin sections. Mainly in small individuals, the fibers forming this tissue are very coarse and loosely organized. With increasing size and age, the coarse tissue is irregularly intermixed and later replaced with finer and better organized fibers. This histologic change is accompanied by a change from diffuse annuli in the inner cortex to distinct lines of arrested growth (LAGs) in the outer cortex. We interpret these changes in tissue and the appearance of distinct growth marks as indicating the onset of active reproduction. The lack of primary vascularization around the ossification center in our Andrias spp. sample is striking and contradicts other observations. Vascularity may be prone to plasticity and further studies are necessary. We hypothesize that the large osteocyte lacunae and the dense networks of canaliculi observed in our sample may have nourished the tissue instead of primary vascular canals. We measured the size of osteocyte lacunae of Andrias spp. in comparison to other Lissamphibia, and found them to be significantly larger throughout ontogeny. The periosteal cortex contains a high amount of thick Sharpey's fibers all around the midshaft cross sections. The two samples of Andrias cf. davidianus show tissue and growth mark distribution similar to that observed in A. japonicus. However, the large individual of A. cf. davidianus differed in its extremely osteosclerotic condition and the retention of a small layer of calcified cartilage in the endosteal region of the femur. It remains unclear whether these differences are related to plasticity, taxonomy, sex, exogenous factors, or attributable to a regenerated but fully regrown leg. Although the present study is based on zoo-kept and not wild, animals, it yields important insights into osteohistological plasticity and growth patterns in giant salamanders.

Peptide mimetic NC114 induces growth arrest by preventing PKCδ activation and FOXM1 nuclear translocation in colorectal cancer cells.

The peptide mimetic, NC114, is a promising anticancer compound that specifically kills colorectal cancer cells without affecting normal colon epithelial cells. In our previous study, we observed that NC114 inhibited the Wnt/ß-catenin pathway, with significant downregulation of both Ser 675-phosphorylated ß-catenin and its target genes, cyclin D1 and survivin. However, the molecular mechanism responsible for its cytotoxic effect has not yet been fully characterized. In the present study, we demonstrated that NC114 prevented cell cycle progression from S to G2/M phase by downregulating cell cycle-related gene expression, and also induced growth arrest in SW480 and HCT-116 colorectal cancer cells. A novel covariation network analysis combined with transcriptome analysis revealed a series of signaling cascades affected by NC114 treatment, and identified protein kinase C-δ (PKCδ) and forkhead box protein M1 (FOXM1) as important regulatory factors for NC114-induced growth arrest. NC114 treatment inhibits the activation of PKCδ and its kinase activity, which suppresses MEK/ERK signaling. Attenuated MEK/ERK signaling then results in a reduction in FOXM1 phosphorylation and subsequent nuclear translocation of FOXM1 and ß-catenin. Consequently, formation of a T-cell factor-4 (TCF4)/ß-catenin transcription complex in the nucleus is inhibited and transcription of its target genes, such as cell cycle-related genes, is downregulated. The efficacy of NC114 on tumor growth was confirmed in a xenograft model. Collectively, elucidation of the mechanism by which NC114 induces growth arrest in colorectal cancer cells should provide a novel therapeutic strategy for colorectal cancer treatment.

Individual identification of endangered amphibians using deep learning and smartphone images: case study of the Japanese giant salamander (Andrias japonicus).

Information obtained via individual identification is invaluable for ecology and conservation. Physical tags, such as PIT tags and GPS, have been used for individual identification; however, these methods could impact on animal behavior and survival rates, and the tags may become lost. Although non-invasive methods that do not affect the target species (such as manual photoidentification) are available, these techniques utilize stripes and spots that are unique to the individual, which requires training, and applying them to large datasets is challenging. Many studies that have applied deep learning for identification have focused on species-level identification, but few have addressed individual-level identification. In this study, we developed an image-based identification method based on deep learning that uses the head spot pattern of the Japanese giant salamander (Andrias japonicus), an endemic and endangered species in Japan. We trained and evaluated a dataset collected over two days from 11 individuals in captivity, which included 7075 images taken by a smartphone camera. Individuals were photographed three times a day at approximately 11:00 (morning), 15:00 (afternoon), and 18:00 (evening). As a result, individual identification by our method, which used the EfficientNetV2 achieved 99.86% accuracy, kappa coefficient of 0.99, and an F1 score of 0.99. Performance was lower for the evening  model than for the morning and afternoon models, which were trained and evaluated using photographs taken at the corresponding time of the day. The proposed method does not require direct contact with the target species, and the effect on the animals is minimal; moreover, individual-level information can be obtained under natural conditions. In the future, smartphone images can be applied to citizen science surveys and individual-level big data collection, which is difficult using current methods.

PKM2 controls the translation of TFE3 to maintain the integrity of the Golgi apparatus for the survival of HeLa and ME-180 cervical cancer cells.

The M2 isoform of pyruvate kinase (PKM2) is abundantly expressed in various cancer cells and associated with tumorigenesis, tumour proliferation and tumour progression. However, the role of PKM2 in these oncological processes is not fully understood. In the present study, we depleted PKM2 expression using RNA interference (RNAi), which induced apoptotic cell death and was accompanied by the downregulation of GM130, giantin, and p115 in HeLa and ME-180 cervical cancer cells. The decreased expression of these proteins caused structural and functional disturbances in the Golgi apparatus, which manifested as the dispersion of the Golgi apparatus and delayed anterograde trafficking from the ER to the Golgi. The transcription factor, TFE3, which functions in the Golgi stress response, was responsible for the expression of GM130, giantin, and p115 that maintained the integrity of the organelle under normal growth conditions. In PKM2-knockdown cells, the translation of TFE3 was markedly reduced. Knockdown of TFE3 by RNAi resulted in the downregulation of GM130, giantin, and p115, dispersion of the Golgi apparatus, and apoptotic cell death, similar to those observed following PKM2 knockdown. Conversely, the exogenous expression of TFE3 in PKM2 knockdown cells partially mitigated the aforementioned effects. We also demonstrated that PKM2 bound to the 5' UTR on TFE3 mRNA and promoted translation. This study is the first to identify a new function for PKM2, which activates the basal Golgi stress response to maintain the integrity of the Golgi apparatus through the translation of TFE3 and promote cancer cell survival.

Identification of hybrids between the Japanese giant salamander (Andrias japonicus) and Chinese giant salamander (Andrias cf. davidianus) using deep learning and smartphone images.

Human-mediated hybridization between native and non-native species is causing biodiversity loss worldwide. Hybridization has contributed to the extinction of many species through direct and indirect processes such as loss of reproductive opportunity and genetic introgression. Therefore, it is essential to manage hybrids to conserve biodiversity. However, specialized knowledge is required to identify the target species based on visual characteristics when two species have similar features. Although image recognition technology can be a powerful tool for identifying hybrids, studies have yet to utilize deep learning approaches. Hence, this study aimed to identify hybrids between the native Japanese giant salamander (Andrias japonicus) and the non-native Chinese giant salamander (Andrias cf. davidianus) using EfficientNetV2 and smartphone images. We used smartphone images of 11 individuals of native A. japonicus (five training and six test images) and 20 individuals of hybrids between A. japonicus and A. cf. davidianus (five training and 15 test images). In our experimental environment, an AI model constructed with EfficientNetV2 exhibited 100% accuracy in identifying hybrids. In addition, gradient-weighted class activation mapping revealed that the AI model was able to classify A. japonicus and hybrids between A. japonicus and A. cf. davidianus on the basis of the dorsal head spot patterning. Our approach thus enables the identification of hybrids against A. japonicus, which was previously considered difficult by non-experts. Furthermore, since this study achieved reliable identification using smartphone images, it is expected to be applied to a wide range of citizen science projects.

BMP4-SMAD1/5/9-RUNX2 pathway activation inhibits neurogenesis and oligodendrogenesis in Alzheimer's patients' iPSCs in senescence-related conditions.

In addition to increasing ß-amyloid plaque deposition and tau tangle formation, inhibition of neurogenesis has recently been observed in Alzheimer's disease (AD). This study generated a cellular model that recapitulated neurogenesis defects observed in patients with AD, using induced pluripotent stem cell lines derived from sporadic and familial AD (AD iPSCs). AD iPSCs exhibited impaired neuron and oligodendrocyte generation when expression of several senescence markers was induced. Compound screening using these cellular models identified three drugs able to restore neurogenesis, and extensive morphological quantification revealed cell-line- and drug-type-dependent neuronal generation. We also found involvement of elevated Sma- and Mad-related protein 1/5/9 (SMAD1/5/9) phosphorylation and greater Runt-related transcription factor 2 (RUNX2) expression in neurogenesis defects in AD. Moreover, BMP4 was elevated in AD iPSC medium during neural differentiation and cerebrospinal fluid of patients with AD, suggesting a BMP4-SMAD1/5/9-RUNX2 signaling pathway contribution to neurogenesis defects in AD under senescence-related conditions.

SPR sensing of bisphenol A using molecularly imprinted nanoparticles immobilized on slab optical waveguide with consecutive parallel Au and Ag deposition bands coexistent with bisphenol A-immobilized Au nanoparticles.

A slab-type optical waveguide (s_OWG)-based microfluidic SPR measurement system for bisphenol A was developed. This s_OWG possesses consecutive parallel gold and silver deposition bands in the line of plasmon flow, allowing two individual SPR signals to be independently obtained as a result of the difference in resonant reflection spectra of these metals. As a molecular recognition element, molecularly imprinted polymer nanoparticles (MIP-Np) were employed and immobilized on the surface of each of the gold and silver deposition bands. The resonant reflection spectra were measured on the MIP-Np-immobilized consecutive parallel gold and silver deposition bands coexistent with BPA-AuNp. The Ag-based SPR spectra showed a red shift (0.7 nm) when free BPA (0.1 mM) was passed over the BPA-AuNp/immobilized MIP-Np complexes formed on the s_OWG, unlike the case for the Au deposition band, while a large excess of BPA induced a blue shift due to the competitive desorption of BPA-AuNp from the immobilized MIP-Np on the s_OWG. By using the proposed detection system, binding events of other small molecules could be monitored in conjunction with the use of MIP-Np and labeled-AuNp.

Evaluation of Oral Mucosal Lesions Using the IllumiScan® Fluorescence Visualisation Device: Distinguishing Squamous Cell Carcinoma.

We evaluated whether fluorescence intensity (FI) and its coefficient of variation (CV) can be used to diagnose squamous cell carcinoma (SCC) through IllumiScan®, an oral mucosa fluorescence visualisation (FV) device. Overall, 190 patients with oral mucosal lesions (OMLs; SCC, 59; non-SCC OMLs, 131) and 49 patients with normal oral mucosa (NOM) were enrolled between January 2019 and March 2021. The FI of the images was analysed using image analysis software. After establishing regions of interest for SCC, non-SCC, and NOM, the average FI, standard deviation (SD), and CV were compared. There was a significant difference in the average FI for all pairs of comparisons. The SD was not significantly different between the SCC and NOM groups (p = 0.07). The CV differed significantly for NOM (p < 0.001) and non-SCC groups (p < 0.001) relative to the SCC group but was not different between NOM and non-SCC groups (p = 0.15). Univariate analysis of SCC and non-SCC groups showed significant differences for all factors, except age. However, multivariate analysis showed a significant intergroup difference only in the CV (p = 0.038). Therefore, analysing the CV in FV images of OML may be useful for the diagnosis of oral cancer.

Development of Low-Molecular-Weight Compounds Targeting the Cancer-Associated KLF5 Transcription Factor.

Krüppel-like factor 5 (KLF5) is a potential target for anticancer drugs. However, as an intrinsically disordered protein (IDP) whose tertiary structure cannot be solved, innovative strategies are needed. We focused on its hydrophobic α-helix structure, defined as an induced helical motif (IHM), which is a possible interface for protein-protein interaction. Using mathematical analyses predicting the α-helix's structure and hydrophobicity, a 4-amino-acid site (V-A-I-F) was identified as an IHM. Low-molecular-weight compounds that mimic the main chain conformation of the α-helix with the four side chains of V-A-I-F were synthesized using bicyclic pyrazinooxadiazine-4,7-dione. These compounds selectively suppressed the proliferation and survival of cancer cells but not noncancer cells and decreased the protein but not mRNA levels of KLF5 in addition to reducing proteins of Wnt signaling. The compounds further suppressed transplanted colorectal cancer cells in vivo without side effects. Our approach appears promising for developing drugs against key IDPs.

Statistical studies of photonic heterostructure nanocavities with an average Q factor of three million.

We have measured the quality (Q) factors and resonant wavelengths for 80 photonic crystal nanocavities with the same heterostructure. In this statistical evaluation, the Q factors varied according to a normal distribution centered at 3 million and ranging between 2.3 million and 3.9 million. The resonant wavelengths also fluctuated but with a standard deviation of only 0.33 nm. Such a high average Q factor and highly controlled resonant wavelength will be important for the development of advanced applications of photonic crystal nanocavities. Comparing the experimental values with calculated values suggests that factors other than structural variations of air holes, which decrease the Q factor, are indeed present in the fabricated nanocavities.

Novel prosurvival function of Yip1A in human cervical cancer cells: constitutive activation of the IRE1 and PERK pathways of the unfolded protein response.

Cancer cells are under chronic endoplasmic reticulum (ER) stress due to hypoxia, low levels of nutrients, and a high metabolic demand for proliferation. To survive, they constitutively activate the unfolded protein response (UPR). The inositol-requiring protein 1 (IRE1) and protein kinase RNA-like ER kinase (PERK) signaling branches of the UPR have been shown to have cytoprotective roles in cancer cells. UPR-induced autophagy is another prosurvival strategy of cancer cells, possibly to remove misfolded proteins and supply nutrients. However, the mechanisms by which cancer cells exploit the UPR and autophagy machinery to promote survival and the molecules that are essential for these processes remain to be elucidated. Recently, a multipass membrane protein, Yip1A, was shown to function in the activation of IRE1 and in UPR-induced autophagy. In the present study, we explored the possible role of Yip1A in activation of the UPR by cancer cells for their survival, and found that depletion of Yip1A by RNA interference (RNAi) induced apoptotic cell death in HeLa and CaSki cervical cancer cells. Intriguingly, Yip1A was found to activate the IRE1 and PERK pathways of the UPR constitutively in HeLa and CaSki cells. Yip1A mediated the phosphorylation of IRE1 and also engaged in the transcription of PERK. The activation of these signaling pathways upregulated the expression of anti-apoptotic proteins and autophagy-related proteins. These events might enhance resistance to apoptosis and promote cytoprotective autophagy in HeLa and CaSki cells. The present study is the first to uncover a key prosurvival modulator, Yip1A, which coordinates IRE1 signaling with PERK signaling to support the survival of HeLa and CaSki cervical cancer cells.

Locomotion pattern and trunk musculoskeletal architecture among Urodela.

We comparatively examined the trunk musculature and prezygapophyseal angle of mid-trunk vertebra in eight urodele species with different locomotive modes (aquatic Siren intermedia, Amphiuma tridactylum, Necturus maculosus and Andrias japonicus; semi-aquatic Cynops pyrrhogaster, Cynops ensicauda; and terrestrial Hynobius nigrescens, Hynobius lichenatus and Ambystoma tigrinum). We found that the more terrestrial species were characterized by larger dorsal and abdominal muscle weight ratios compared with those of the more aquatic species, whereas muscle ratios of the lateral hypaxial musculature were larger in the more aquatic species. The lateral hypaxial muscles were thicker in the more aquatic species, whereas the M. rectus abdominis was more differentiated in the more terrestrial species. Our results suggest that larger lateral hypaxial muscles function for lateral bending during underwater locomotion in aquatic species. Larger dorsalis and abdominal muscles facilitate resistance against sagittal extension of the trunk, stabilization and support of the ventral contour line against gravity in terrestrial species. The more aquatic species possessed a more horizontal prezygapophyseal angle for more flexible lateral locomotion. In contrast, the more terrestrial species have an increasingly vertical prezygapophyseal angle to provide stronger column support against gravity. Thus, we conclude trunk structure in urodeles differs clearly according to their locomotive modes.

Generation of human induced pluripotent stem (Ips) cells in serum- and feeder-free defined culture and TGF-Β1 regulation of pluripotency.

Human Embryonic Stem cells (hESCs) and human induced Pluripotent Stem cells (hiPSCs) are commonly maintained on inactivated mouse embryonic fibroblast as feeder cells in medium supplemented with FBS or proprietary replacements. Use of culture medium containing undefined or unknown components has limited the development of applications for pluripotent cells because of the relative lack of knowledge regarding cell responses to differentiating growth factors. In addition, there is no consensus as to the optimal formulation, or the nature of the cytokine requirements of the cells to promote their self-renewal and inhibit their differentiation. In this study, we successfully generated hiPSCs from human dental pulp cells (DPCs) using Yamanaka's factors (Oct3/4, Sox2, Klf4, and c-Myc) with retroviral vectors in serum- and feeder-free defined culture conditions. These hiPSCs retained the property of self-renewal as evaluated by the expression of self-renewal marker genes and proteins, morphology, cell growth rates, and pluripotency evaluated by differentiation into derivatives of all three primary germ layers in vitro and in vivo. In this study, we found that TGF-ß1 increased the expression levels of pluripotency markers in a dose-dependent manner. However, increasing doses of TGF-ß1 suppressed the growth rate of hiPSCs cultured under the defined conditions. Furthermore, over short time periods the hiPSCs cultured in hESF9 or hESF9T exhibited similar morphology, but hiPSCs maintained in hESF9 could not survive beyond 30 passages. This result clearly confirmed that hiPSCs cultured in hESF9 medium absolutely required TGF-ß1 to maintain pluripotency. This simple serum-free adherent monoculture system will allow us to elucidate the cell responses to growth factors under defined conditions and can eliminate the risk might be brought by undefined pathogens.

Long-term serial cultivation of mouse induced pluripotent stem cells in serum-free and feeder-free defined medium.

Mouse embryonic stem (mES) cells and mouse induced pluripotent stem (miPS) cells are commonly maintained on inactivated mouse embryonic fibroblast feeder cells in medium supplemented with fetal bovine serum or proprietary replacements. An undefined medium containing unknown quantities of reagents has limited the development of applications for pluripotent cells because of the relative lack of knowledge regarding cell responses to differentiating growth factors. Therefore we developed a serum-free medium, designated ESF7, in which mES cells can be maintained in an undifferentiated state without feeder cells. The medium was tested for culturing miPS cells. The miPS cells have been maintained in ESF7 medium for more than 3 years with an undifferentiated phenotype manifested by the expression of pluripotency marker genes and alkaline phosphatase, and these cells exhibited largely normal karyotypes. Furthermore, we found that fibroblast growth factor-2 (FGF-2) with heparin induced miPS cell differentiation into neuronal cells, both in an adherent monolayer and in embryoid body suspension culture. Moreover, we found that FGF-2 with bone morphogenetic protein 2 induced miPS cell differentiation into cardiomyocytes in embryoid body suspension culture. Furthermore, we transplanted subcutaneously miPS cells maintained in ESF7 into the dorsal flanks of SCID mice; all of the transplants produced tumors with tissues derived from all three embryonic germ layers. As this simple serum-free adherent monoculture system supports the long-term propagation of pluripotent iPS cells in vitro, it will allow us to elucidate cell responses to growth factors under defined conditions, and it should provide useful information for differentiation protocols for human iPS cells.