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Cellular disease models are useful tools for Alzheimer's disease (AD) research. Pluripotent stem cells, including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), are promising materials for creating cellular models of such diseases. In the present study, we established cellular models of AD in hESCs that overexpressed the mutant Presenilin 1 (PS1) gene with the use of a site-specific gene integration system. The overexpression of PS1 did not affect the undifferentiated status or the neural differentiation ability of the hESCs. We found increases in the ratios of amyloid-β 42 (Aβ42)/Aβ40 and Aβ43/Aβ40. Furthermore, synaptic dysfunction was observed in a cellular model of AD that overexpressed mutant PS1. These results suggest that the AD phenotypes, in particular, the electrophysiological abnormality of the synapses in our AD models might be useful for AD research and drug discovery.

Summary Utilizing human pluripotent stem cells (hPSCs) in cell-based therapy and drug discovery requires large-scale cell production. However, scaling up conventional adherent cultures presents challenges of maintaining a uniform high quality at low cost. In this regard, suspension cultures are a viable alternative, because they are scalable and do not require adhesion surfaces. 3D culture systems such as bioreactors can be exploited for large-scale production. However, the limitations of current suspension culture methods include spontaneous fusion between cell aggregates and suboptimal passaging methods by dissociation and reaggregation. 3D culture systems that dynamically stir carrier beads or cell aggregates should be refined to reduce shearing forces that damage hPSCs. Here, we report a simple 3D sphere culture system that incorporates mechanical passaging and functional polymers. This setup resolves major problems associated with suspension culture methods and dynamic stirring systems and may be optimal for applications involving large-scale hPSC production., Graphical Abstract, Highlights • Suspended sphere culture of hPSCs with expansion rates similar to adherent culture • Uniform hPSC spheres made by mechanical passaging and adding methylcellulose • 3D hPSC sphere culture suspended with gellan gum polymer without dynamic agitation • Proof of principle 3D hPSC culture using 200 ml culture bag for large-scale trial, Application of human pluripotent stem cells (hPSCs) in cell-based therapy and drug discovery requires large-scale cell production. Here, Nakatsuji and colleagues developed a simple 3D sphere culture system by utilizing functional polymers. It can enable robust expansion of uniform hPSC spheres without dynamic agitation. This system would be an optimal method for producing large-scale and uniform hPSCs for various applications.

SummaryA small-molecule fluorescent probe specific for human pluripotent stem cells would serve as a useful tool for basic cell biology research and stem cell therapy. Screening of fluorescent chemical libraries with human induced pluripotent stem cells (iPSCs) and subsequent evaluation of hit molecules identified a fluorescent compound (Kyoto probe 1 [KP-1]) that selectively labels human pluripotent stem cells. Our analyses indicated that the selectivity results primarily from a distinct expression pattern of ABC transporters in human pluripotent stem cells and from the transporter selectivity of KP-1. Expression of ABCB1 (MDR1) and ABCG2 (BCRP), both of which cause the efflux of KP-1, is repressed in human pluripotent stem cells. Although KP-1, like other pluripotent markers, is not absolutely specific for pluripotent stem cells, the identified chemical probe may be used in conjunction with other reagents.

We are recently developing novel technologies for large-scale production of high-quality human pluripotent stem cells (hPSCs) by development of defined and robust adhesion molecules for hPSCs as well as other culture technologies. We are also working on control of stem cells with chemical compounds and micro-fabricated materials for growth and differentiation of ES/iPS cells in collaboration with chemical biology groups and micro-engineering groups. So far, we have identified novel small molecules which can induce efficient and robust cardiomyocyte differentiation from many human ES and iPS cell lines in completely defined and xeno-free conditions. Finally, we have created and investigated various model cells from human ES and iPS cell lines. They include neurodegenerative disease model cells, such as Alzheimer, ALS and Huntington disease models, which are produced by genetic modification of stem cell lines and differentiation into relevant cells in each disease.

Alzheimer’s disease (AD) is the most common form of dementia. Cellular AD models derived from human pluripotent stem cells are promising tools in AD research. We recently developed human embryonic stem cell-derived AD models which overexpress mutant Presenilin1 genes, and which exhibit AD phenotypes, including synaptic dysfunction. In this study, we found that our AD models showed reduced levels of RAB3A and SV2B proteins in the pre-synapses, which is a possible cause of electrophysiological abnormalities. Through the screening of chemical compounds using our AD models, we have identified Aβ peptide inhibitors which decrease the concentration of Aβ in culture supernatant. Among these, BMS-708163 and Nilotinib were found to improve the expression levels of RAB3A and SV2B proteins and to recover the electrophysiological function in our AD models. These results suggest that the AD models we developed are promising materials for the discovery of AD drugs that target the expression of pre-synaptic proteins and synaptic function.

The generation of amyotrophic lateral sclerosis (ALS) disease models is an important subject for investigating disease mechanisms and pharmaceutical applications. In transgenic mice, expression of a mutant form of superoxide dismutase 1 (SOD1) can lead to the development of ALS that closely mimics the familial type of ALS (FALS). Although SOD1 mutant mice show phenotypes similar to FALS, dissimilar drug responses and size differences limit their usefulness to study the disease mechanism(s) and identify potential therapeutic compounds. Development of an in vitro model system for ALS is expected to help in obtaining novel insights into disease mechanisms and discovery of therapeutics. We report the establishment of an in vitro FALS model from human embryonic stem cells overexpressing either a wild-type (WT) or a mutant SOD1 (G93A) gene and the evaluation of the phenotypes and survival of the spinal motor neurons (sMNs), which are the neurons affected in ALS patients. The in vitro FALS model that we developed mimics the in vivo human ALS disease in terms of the following: (a) selective degeneration of sMNs expressing the G93A SOD1 but not those expressing the WT gene; (b) susceptibility of G93A SOD1-derived sMNs to form ubiquitinated inclusions; (c) astrocyte-derived factor(s) in the selective degeneration of G93A SOD1 sMNs; and (d) cell-autonomous, as well as non-cell-autonomous, dependent sMN degeneration. Thus, this model is expected to help unravel the disease mechanisms involved in the development of FALS and also lead to potential drug discoveries based on the prevention of neurodegeneration.

Low efficiencies of gene targeting via homologous recombination (HR) have limited basic research and applications using human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Here, we show highly and equally efficient gene knockout and knock-in at both transcriptionally active (HPRT1, KU80, LIG1, LIG3) and inactive (HB9) loci in these cells using high-capacity helper-dependent adenoviral vectors (HDAdVs). Without the necessity of introducing artificial DNA double-strand breaks, 7–81% of drug-resistant colonies were gene-targeted by accurate HR, which were not accompanied with additional ectopic integrations. Even at the motor neuron-specific HB9 locus, the enhanced green fluorescent protein (EGFP) gene was accurately knocked in in 23–57% of drug-resistant colonies. In these clones, induced differentiation into the HB9-positive motor neuron correlated with EGFP expression. Furthermore, HDAdV infection had no detectable adverse effects on the undifferentiated state and pluripotency of hESCs and hiPSCs. These results suggest that HDAdV is one of the best methods for efficient and accurate gene targeting in hESCs and hiPSCs and might be especially useful for therapeutic applications.

Familial amyotrophic lateral sclerosis (fALS) accounts for 10% of ALS cases, and about 25% of fALS cases are due to mutations in superoxide dismutase 1 (SOD1). Mutant SOD1-mediated ALS is caused by a gain of toxic function of the mutant protein, and the SOD1 level in nonneuronal neighbors, including astrocytes, determines the progression of ALS (non-cell-autonomous toxicity). Therefore, the authors hypothesized that small molecules that reduce SOD1 protein levels in astrocytes might slow the progression of mutant SOD1-mediated ALS. They developed and optimized a cell-based, high-throughput assay to identify low molecular weight compounds that decrease SOD1 expression transcriptionally in human astrocyte-derived cells. Screening of a chemical library of 9600 compounds with the assay identified two hit compounds that selectively and partially downregulate SOD1 expression in a dose-dependent manner, without any detectable cellular toxicity. Western blot analysis showed that one hit compound significantly decreased the level of endogenous SOD1 protein in H4 cells, with no reduction in expression of β-actin. The assay developed here provides a powerful strategy for discovering novel lead molecules for treating familial SOD1-mediated ALS.

Biologists rely on morphology, function and specific markers to define the differentiation status of cells. Transcript profiling has expanded the repertoire of these markers by providing the snapshot of cellular status that reflects the activity of all genes. However, such data have been used only to assess relative similarities and differences of these cells. Here we show that principal component analysis of global gene expression profiles map cells in multidimensional transcript profile space and the positions of differentiating cells progress in a stepwise manner along trajectories starting from undifferentiated embryonic stem (ES) cells located in the apex. We present three 'cell lineage trajectories', which represent the differentiation of ES cells into the first three lineages in mammalian development: primitive endoderm, trophoblast and primitive ectoderm/neural ectoderm. The positions of the cells along these trajectories seem to reflect the developmental potency of cells and can be used as a scale for the potential of cells. Indeed, we show that embryonic germ cells and induced pluripotent cells are mapped near the origin of the trajectories, whereas mouse embryo fibroblast and fibroblast cell lines are mapped near the far end of the trajectories. We suggest that this method can be used as the non-operational semi-quantitative definition of cell differentiation status and developmental potency. Furthermore, the global expression profiles of cell lineages provide a framework for the future study of in vitro and in vivo cell differentiation.

Whether SWI/SNF chromatin remodeling complexes play roles in embryonic stem (ES) cells remains unknown. Here we show that SWI/SNF complexes are present in mouse ES cells, and their composition is dynamically regulated upon induction of ES cell differentiation. For example, the SWI/SNF purified from undifferentiated ES cells contains a high level of BAF155 and a low level of BAF170 (both of which are homologs of yeast SWI3 protein), whereas that from differentiated cells contains nearly equal amounts of both. Moreover, the levels of BAF250A and BAF250B decrease during the differentiation of ES cells, whereas that of BRM increases. The altered expression of SWI/SNF components hinted that these complexes could play roles in ES cell maintenance or differentiation. We therefore generated ES cells with biallelic inactivation of BAF250B and found that these cells display a reduced proliferation rate and an abnormal cell cycle. Importantly, these cells are deficient in the self-renewal capacity of undifferentiated ES cells and exhibit certain phenotypes of differentiated cells, including reduced expression of several pluripotency-related genes and increased expression of some differentiation-related genes. These data suggest that the BAF250B-associated SWI/SNF is essential for mouse ES cells to maintain their normal proliferation and pluripotency. The work presented here underscores the importance of SWI/SNF chromatin remodeling complexes in pluripotent stem cells. Disclosure of potential conflicts of interest is found at the end of this article.

Background: Use of induced pluripotent stem cells (hiPSC) is promising to establish cardiac regenerative medicine in clinical arena; however, safety is concerned by regulatory scientific view, including use of xeno-biological materials for production of the graft. We have explored synthetic small molecules, KY, instead of standard xeno-materials to induce cardiomyogenic differentiation, such as BMP4, Activin A or insulin. We herein studied therapeutic efficacy of hiPSC-derived cardiomyocytes (CMs) generated by xeno-free condition on chronic myocardial infarction (MI) in rat. Methods: Lentivirus vector carrying Oct3/4, Sox2 and Klf4 was transfected in human dermal fibroblasts to establish hiPSC cell-line. KY was added to the hiPSCs, which displayed 80±10%-positivity in cardiac-troponin T. Scaffold-free cardiac graft was produced by hiPSC-CMs that were cultured in thermoresponsive dishes, and transplanted over the cardiac surface of athymic nude rats that were subjected to left coronary artery ligation 2 weeks prior to the treatment, or sham operation was performed. Results: Echocardiographically, ejection fraction recovered after the treatment in the hiPSC-CM group (48±4% at baseline, 55±4% at 1 week, 55±4% at 2 weeks, and 56±3% at 4 weeks), whereas the sham group showed gradual reduction in ejection fraction (46±5% at baseline, 42±5% at 1 week, 38±4% at 2 weeks, and 34±6% at 4 weeks). Immunohistochemistry showed that cardiac-troponin T and human nuclear antigen double positive-hiPSC-CMs were present in the lateral cardiac surface in the hiPSC-CM hearts. In the hiPSC-CM group, collagen fibers significantly decreased in the infarct-border and remote areas (p Conclusion: Transplantation of scaffold-free human iPSC-CMs grafts produced by xeno-free synthetic small molecules was effective on rat chronic MI heart, warranting further studies to optimize production of iPSC-based artificial cardiac graft for cardiac regeneration therapy.

A catalog of mouse genes expressed in early embryos, embryonic and adult stem cells was assembled, including 250000 ESTs, representing approximately 39000 unique transcripts. The cDNA libraries, enriched in full-length clones, were condensed into the NIA 15 and 7.4K clone sets, freely distributed to the research community, providing a standard platform for expression studies using microarrays. They are essential tools for studying mammalian development and stem cell biology, and to provide hints about the differential nature of embryonic and adult stem cells.

Transplantation of nuclei (NT) from engineered mouse ES cells is a potentially powerful and rapid route to create knockout mice, obviating the need for matings to obtain germ-line chimeras. However, such an application is currently impossible, because NT often results in abnormalities in embryo and placenta. Although the epigenetic instability of several imprinted genes in ES cells and ES-derived NT mice has been demonstrated, it is not clear yet what causes the abnormalities. To gain perspective on the extent and types of changes, we have done gene expression profiling for mouse placentas produced by NT of ES cells and compared them with the expression profiles of placentas produced by NT of one-cell embryos. Based on microarray studies with the NIA 15K mouse cDNA collection, we report five principal aberrant events: (1) inappropriate expression of imprinted genes; (2) altered expression of regulatory genes involved in global gene expression, such as DNA methyltransferase and histone acetyltransferase; (3) increased expression of oncogenes and growth promoting genes; (4) overexpression of genes involved in placental growth, such as Plac1; and (5) identification of many novel genes overexpressed in ES-derived NT mouse placentas, including Pitrm1, a new member of the metalloprotease family. The results indicate that placentomegaly in ES-derived NT mice is associated with large-scale dysregulation of normal gene expression patterns. The study also suggests the presence of two regulatory pathways that may lead to histologically discernable placentomegaly. The discovery of groups of genes with altered expression may provide potential targets for intervention to mimic natural regulation more faithfully in NT mice.

A set of 7407 cDNA clones (NIA mouse 7.4K) was assembled from >20 cDNA libraries constructed mainly from early mouse embryos, including several stem cell libraries. The clone set was assembled from embryonic and newborn organ libraries consisting of ∼120,000 cDNA clones, which were initially re-arrayed into a set of ∼11,000 unique cDNA clones. A set of tubes was constructed from the racks in this set to prevent contamination and potential mishandling errors in all further re-arrays. Sequences from this set (11K) were analyzed further for quality and clone identity, and high-quality clones with verified identity were re-arrayed into the final set (7.4K). The set is freely available, and a corresponding database was built to provide comprehensive annotation for those clones with known identity or homology, and has been made available through an extensive Web site that includes many link-outs to external databases and analysis servers.[The sequence data from this study have been submitted to GenBank under accession nos. BQ550036–BQ563104.]

Two cDNA fragments induced in developing zygotes ofDictyostelium discoideum were isolated by mRNA differential display. the relevant genes were also found to be expressed during asexual development, suggesting that sexual and asexual development share common molecular mechanisms inD. discoideum.

The sexual cycle of the cellular slime mold, Dictyostelium discoideum, offers a suitable experimental system to analyze sexual cell interactions. We have been analyzing molecular mechanisms involved in sexual cell fusion using complementary heterothallic strains in D. discoideum and have identified several cell surface proteins involved in the process. One of them, gp138 is present in strains of both mating types and considered to be responsible for membrane fusion itself. Two genes with high mutual homology, GP138A and GP138B, have been identified so far as encoding this protein. Expression of antisense RNA for GP138B has been shown to suppress sexual cell fusion, confirming the critical importance of these genes in sexual cell fusion. However, neither the functional relationship of the two gp138 genes nor the possibility of the existence of more genes that encode gp138 has been determined yet. In the present study, GP138A and GP138B were disrupted by homologous recombination in an effort to clarify these points. Analysis of the double knock-out mutants suggested the presence of a third gene for gp138.

The sexual cycle of the cellular slime mold Dictyostelium discoideum offers a suitable system to analyze the mechanism of cell recognition during mating. Sexual cell fusion in D. discoideum typically occurs between complementary heterothallic strains. In addition, several bisexual strains are known which undergo sexual cell fusion with heterothallic strains of either mating type, but cannot do so by themselves. In the present study, trypsin digestion of cell surface molecules was found to induce self-fusion in a bisexual strain WS2162, suggesting the presence on the cell surface of a self-recognition molecule whose homophilic interaction interferes with the cell fusion mechanism.

Sexually competent cells of a cellular slime mold Dictyostelium discoideum strain NC4, were mixed with fusion-competent cells of either HM1, or WS2162, and mixed cell suspensions were incubated with rotation in the dark. Cells 30min after mixing NC4 and SW2162 contained giant cells and smaller cells. Mitochondria of giant cells were charastenstically distributed to the cell periphery. This trend was more evident in giant cells as nucleus number increased in a cell. At 24hr after mixing, blue fluorescences from cell wall, and dark brown fluorescence in the center of and around squashed structures were eminent, while both fluorescences were not detected in 30min cell. Western blot analysis using rabbit polyclonal antibody against bacterial maltose-binding protein detected a major 40 kD and a minor 43 kD peptides in total protein from cells at 24 and 66 hr after mixing NC4 and HN1 cells, while these bands were not detected in two types of gametes before mixing and cells 30min after mixing.

Cre and FLP recombinases are the most widely used site-specific recombinases in genome engineering. Both are members of the tyrosine class of recombinases and catalyze the reversible, site-specific recombination between two identical sequences of 34 bp length in the absence of accessory factors. The substrate sequences for Cre and Flp recombinases are called loxP and FRT sites respectively. Cre recombinase was discovered in the E. coli bacteriophage P1 where it plays a crucial role in the life cycle of P1 while Flp recombinase was originally derived from the 2 μ circle of Saccharomyces cerevisiae and catalyzes recombination between inverted repeats within the 2 μ plasmid. This chapter shall provide a brief historical perspective on the discovery and early development of the Cre/loxP and FRT/FLP systems citing key studies that paved the way for the application of these site-specific recombination technologies to the engineering of mammalian genomes. Also included are discussions on the mechanisms of Cre/loxP and FRT/FLP systems application of these site-specific recombinase technologies to the introduction of transgenes in human pluripotent stem cells. Certain studies using mouse pluripotent stem cells will also be discussed in order to highlight the possibility of adopting the same strategy in their human counterparts. Lastly, future prospects for these two site-specific recombinases will be presented.

Research using human embryonic stem cell (hESC) lines has expanded dramatically because of two attractive capacity; self-renewal and differentiation into almost all cell types. For therapeutic purposes, many researchers are trying to establish methods for maintaining pluripotency in defined xeno-free conditions and scalable culture systems. Banking of hESC lines is important for the wide spread of personalized cell therapy and transplantation. We introduced the ongoing clinical trials using hESC-derived cells in patients with subacute spinal cord injury and Stargardt's macular dystrophy. We also discussed opportunities and an example for the use of hESC in drug discovery. Finally, we introduced transgenic hESC as a disease model.

Human embryonic stem cells (hESCs) are promising materials for both basic and applied research fields such as human developmental biology and drug discovery because of their capacity for long-term self-renewal and differentiation into virtually all types of cells and tissues (Thomson et al., 1998; Hoffman & Carpenter, 2005). For example, with regard to their applications for drug development research, these cells can be used test whether drugs under development are efficacious against specific diseases by using hESCs-derived disease models which show disease phenotypes (Barbaric et al., 2010; Laustriat et al., 2010). Genetic manipulation is a useful strategy for generating cellular disease models. Random integration of exogenous genes into the genome of hESCs is the most straightforward and easiest method. Random integration, however, often results in inactivation or silencing of integrated genes in hESCs (Ellis, 2005; Liew et al., 2007; Xia et al., 2007) and also might alter the cellular phenotypes due to insertional mutagenesis, which disrupts gene functions in undefined gene regions (Hacein-Bey-Abina et al., 2003; Nienhuis et al., 2006). Furthermore, the reliability of data may be compromised by differences in the transgene integration sites when comparing multiple transgenic cell lines. Introducing transgenes by gene targeting is one way to avoid undesired gene silencing and insertional mutagenesis, but the targeting efficiency in hESCs is notoriously low (Zwaka and Thomson, 2003; Urbach et al, 2004; Irion et al, 2007; Di Domenico et al, 2008; Ruby et al, 2009). Therefore, a lot of time and effort may be required to obtain gene-targeted hESC clones. To address these issues, several strategies have been explored. Researchers have identified usable native sites for gene integration in the human genome. Bacteriophage phiC31 integrase, or adeno-associated virus type 2 (AAV2), can mediate plasmid integration into pseudo-attP sites or the adeno-associated virus integration site 1 locus in the genome, respectively (Thyagarajan et al., 2008; Smith et al., 2008). However, the human genome possesses 23 different pseudo-attP sites, so the integration site cannot be specifically controlled, and the AAV2-mediated targeting efficiency is still low (4.16%). In contrast, foreign sequences such as bacterial loxP and yeast FRT are useful for gene integrations into the genome. Both loxP and lox2272 sequences can

Sexual cell fusion occurs between NC4 and HM1, heterothallic strains of Dictyostelium discoideum. Several cell surface proteins relevant to the process have been identified. One of them, gp138, exists in fusion-competent cells of both NC4 and HM1, and is considered to be more concerned with membrane fusion than gamete recognition. In this study, we raised monoclonal antibodies against gp138 and examined gp138 distribution among strains and species of cellular slime moulds to confirm its importance in sexual cell fusion. All heterothallic and bisexual D. discoideum strains examined were found to possess gp138, while asexual and homothallic strains lacked it. The anti-gp138 monoclonal antibody detected several distinct proteins in homothallic strains and one in an asexual strain. Some of the former proteins appeared together with the increase in binucleated cells. Cells of Dictyostelium mucoroides and Polysphondylium pallidum did not possess proteins reactive to the monoclonal antibody. These results indicate that gp138 is common among, but restricted to, cross-matable strains of D. discoideum. Our results also support previously published molecular phylogenetic studies which suggest that homothallic and asexual strains of D. discoideum are remote from other strains of D. discoideum but are less distantly related to them than other species are.

Sexual cell fusion occurs between NC4 and HM1, the heterothallic strains in Dictyostelium discoideum. Cells of these strains are fusion incompetent when cultured on agar plates in the light and become fusion competent upon cultivation in a liquid medium in darkness. Two cell-surface components, gp70 and gp138, have been identified and characterized as being relevant to sexual cell fusion. Both are glycoproteins, and the former is detected only in fusion-competent HM1 cells, while the latter is detected both in fusion-competent HM1 and fusion-competent NC4 cells. We therefore suspect gp70 to be responsible for cell recognition and gp138, for membrane fusion. Therefore, NC4 cells are expected to possess specific surface molecule(s) that can be recognized by HM1 cells. In the present study, we raised monoclonal antibodies (mAbs) against membrane fractions of NC4 cells and selected fusion-inhibiting mAbs to identify novel molecules related to sexual cell fusion in D. discoideum. Out of the five mAbs we obtained three, DEI, GG6, and HH9, were characterized. DEI recognized antigens that specifically existed in fusion-competent NC4 cells but not in fusion-incompetent NC4 or HM1 cells. GG6 recognized cell-surface proteins with approximate molecular weights of 125 and 32 kDa in both fusion-competent NC4 and fusion-competent HMI cells. In addition GG6 also recognised other proteins commonly present in fusion-incompetent cells. The 125 kDa protein appeared to be the same as gp138. The epitope recognized by HH9 was sodium dodecyl sulfate (SDS)-sensitive. In an immunoblot of nondenatured polyacrylamide-gel electrophoresis (PAGE), HH9 detected two protein bands that were common to fusion-competent NC4 and fusion-competent HMI cells, but were distinct from gp138. These results suggest that other protein species in addition to gp138 are involved in sexual cell fusion between NC4 and HMI cells, with one having the properties of putative recognition molecules.

During the preimplantation phase of pregnancy the endometrial stroma differentiates into decidua, a process that implies numerous morphological changes and is an example of physiological transdifferentiation. Here we show that UIII rat endometrial stromal cells cultured in the presence of calf serum acquired morphological features of decidual cells and expressed decidual markers. To identify genes involved in decidualization we compared gene expression patterns of control and decidualized UIII cells using cDNA microarray. We found 322 annotated genes exhibiting significant differences in expression (>3-fold, fold discovery rate (FDR) >0.005), of which 312 have not been previously related to decidualization. Analysis of overrepresented functions revealed that protein synthesis, gene expression, and chromatin architecture and remodeling are the most relevant modified functions during decidualization. Relevant genes are also found in the functional terms differentiation, cell proliferation, signal transduction, and matrix/structural proteins. Several of these new genes involved in decidualization (Csdc2, Trim27, Eef1a1, Bmp1, Wt1, Aes, Gna12, and Men1) are shown to be also regulated in uterine decidua during normal pregnancy. Thus, the UIII cell culture model will allow future mechanistic studies to define the transcriptional network regulating reprogramming of stromal cells into decidual cells.

BACKGROUND: There are no cures or efficacious treatments for severe motor neuron diseases. It is extremely difficult to obtain naïve spinal motor neurons (sMNs) from human tissues for research due to both technical and ethical reasons. Human embryonic stem cells (hESCs) are alternative sources. Several methods for MN differentiation have been reported. However, efficient production of naïve sMNs and culture cost were not taken into consideration in most of the methods. METHODS/PRINCIPAL FINDINGS: We aimed to establish protocols for efficient production and enrichment of sMNs derived from pluripotent stem cells. Nestin+ neural stem cell (NSC) clusters were induced by Noggin or a small molecule inhibitor of BMP signaling. After dissociation of NSC clusters, neurospheres were formed in a floating culture containing FGF2. The number of NSCs in neurospheres could be expanded more than 30-fold via several passages. More than 33% of HB9+ sMN progenitor cells were observed after differentiation of dissociated neurospheres by all-trans retinoic acid (ATRA) and a Shh agonist for another week on monolayer culture. HB9+ sMN progenitor cells were enriched by gradient centrifugation up to 80% purity. These HB9+ cells differentiated into electrophysiologically functional cells and formed synapses with myotubes during a few weeks after ATRA/SAG treatment. CONCLUSIONS AND SIGNIFICANCE: The series of procedures we established here, namely neural induction, NSC expansion, sMN differentiation and sMN purification, can provide large quantities of naïve sMNs derived from human and monkey pluripotent stem cells. Using small molecule reagents, reduction of culture cost could be achieved.

Sexual cell fusion in the cellular slime mold Dictyostelium discoideum occurs between cells of opposite (heterothallic system) or same (homothallic system) mating types. It also requires certain environmental conditions such as darkness and abundance of water, and thus offers an interesting model system for analyzing mechanisms of cell recognition and of cellular response to environmental factors. We have been studying the mechanism of sexual cell fusion, using two heterothallic strains, NC4 and HM1 of D. discoideum. Two cell-surface glycoproteins, gp70 and gp138, have been identified as relevant molecules in the cell fusion of these strains. The former is specific to mat a cells (HM1) and the latter, common to both mat a and mat A (NC4). Involvement of cell-surface carbohydrates has also been suggested. However, the fuctions of the above fusion-related molecules are still elusive. In the present study, we isolated fusion-deficient mutants from a mutagenized mat A strain of D. discoideum to set up combined genetic and biochemical analyses. Among the three nonconditional mutants obtained, two were normal in the fruiting-body formation, asexual development, but one was aggregateless (agg−). Further analysis of these mutants would provide detailed information on the mechanism of sexual cell fusion.

Background Target genes of a transcription factor (TF) Pou5f1 (Oct3/4 or Oct4), which is essential for pluripotency maintenance and self-renewal of embryonic stem (ES) cells, have previously been identified based on their response to Pou5f1 manipulation and occurrence of Chromatin-immunoprecipitation (ChIP)-binding sites in promoters. However, many responding genes with binding sites may not be direct targets because response may be mediated by other genes and ChIP-binding site may not be functional in terms of transcription regulation. Results To reduce the number of false positives, we propose to separate responding genes into groups according to direction, magnitude, and time of response, and to apply the false discovery rate (FDR) criterion to each group individually. Using this novel algorithm with stringent statistical criteria (FDR < 0.2) to a compendium of published and new microarray data (3, 6, 12, and 24 hr after Pou5f1 suppression) and published ChIP data, we identified 420 tentative target genes (TTGs) for Pou5f1. The majority of TTGs (372) were down-regulated after Pou5f1 suppression, indicating that the Pou5f1 functions as an activator of gene expression when it binds to promoters. Interestingly, many activated genes are potent suppressors of transcription, which include polycomb genes, zinc finger TFs, chromatin remodeling factors, and suppressors of signaling. Similar analysis showed that Sox2 and Nanog also function mostly as transcription activators in cooperation with Pou5f1. Conclusion We have identified the most reliable sets of direct target genes for key pluripotency genes – Pou5f1, Sox2, and Nanog, and found that they predominantly function as activators of downstream gene expression. Thus, most genes related to cell differentiation are suppressed indirectly.

Cross-species comparison of gene expression is a powerful approach for discovering genes that have been conserved throughout evolution. Conserved genes are presumably very important in the mechanisms related to the unique molecular functions in oocytes. The objective of this study was to identify genes expressed in the oocyte and conserved across three diverse vertebrate species. We report the global gene expression profiles of Bos taurus and Xenopus laevis oocytes on an NIA mouse development microarray that consists of 60-mer oligonucleotide probes representing more than 20,000 mouse transcripts derived from stem cell, oocyte, and early embryo cDNA libraries. Analysis based on intensity values revealed that 9853 and 10,046 genes are expressed in bovine and Xenopus oocytes respectively. Furthermore, previously published microarray data on preimplantation development in the mouse were used for a comparative analysis of global oocyte gene expression profiles. Interestingly, a substantial proportion of the genes expressed in mouse oocytes is conserved between the three species (74%, 7275 genes). Moreover, functional annotation of these conserved oocyte-expressed genes confirmed that certain functions are conserved among the three species. RNA metabolism and cell cycle were among the over-represented Gene Ontology terms in the biological process category. Finally, a pattern-matching analysis identified 208 conserved maternally expressed genes. Results from these cross-species hybridizations allowed numerous genes expressed in oocytes and conserved between Mus musculus, B. taurus, and X. laevis to be identified. This comparative analysis of oocyte transcript profiles revealed a high degree of conservation among species.

We previously reported that Zscan4 showed heterogeneous expression patterns in mouse embryonic stem (ES) cells. To identify genes that show similar expression patterns, we carried out high-throughput in situ hybridization assays on ES cell cultures for 244 genes. Most of the genes are involved in transcriptional regulation, and were selected using microarray-based comparisons of gene expression profiles in ES and embryonal carcinoma (EC) cells versus differentiated cell types. Pou5f1 (Oct4, Oct3/4) and Krt8 (EndoA) were used as controls. Hybridization signals were detected on ES cell colonies for 147 genes (60%). The majority (136 genes) of them showed relatively homogeneous expression in ES cell colonies. However, we found that two genes unequivocally showed Zscan4-like spotted expression pattern (spot-in-colony pattern; Whsc2 and Rhox9). We also found that nine genes showed relatively heterogeneous expression pattern (mosaic-in-colony pattern: Zfp42/Rex1, Rest, Atf4, Pa2g4, E2f2, Nanog, Dppa3/Pgc7/Stella, Esrrb, and Fscn1). Among these genes, Zfp42/Rex1 showed unequivocally heterogeneous expression in individual ES cells prepared by the CytoSpin. These results show the presence of different types or states of cells within ES cell cultures otherwise thought to be undifferentiated and homogeneous, suggesting a previously unappreciated complexity in ES cell cultures.

Large-scale systematic gene expression analyses of early embryos and stem cells provide useful information to identify genes expressed differentially or uniquely in these cells. We review the current status of various approaches applied to preimplantation embryos and stem cells: expressed sequence tag, serial analysis of gene expression, differential display, massively parallel signature sequencing, DNA microarray (DNA chip) analysis, and chromatin-immunoprecipitation microarrays. We also discuss the biological questions that can only be addressed by the analysis of global gene expression patterns, such as so-called stemness and developmental potency. As the emphasis now shifts from expression profiling to functional studies, we review the genome-scale functional studies of genes: expression cloning, gene trapping, RNA interference, and gene disruptions. Finally, we discuss the future clinical application of such methodologies.

To understand global features of gene expression changes during in vitro neural differentiation, we carried out the microarray analysis of embryonic stem cells (ESCs), embryonal carcinoma cells, and adult neural stem/progenitor (NS) cells. Expression profiling of ESCs during differentiation in monolayer culture revealed three distinct phases: undifferentiated ESCs, primitive ectoderm-like cells, and neural progenitor cells. Principal component (PC) analysis revealed that these cells were aligned on PC1 over the course of 6 days. This PC1 represents approximately 4,000 genes, the expression of which increased with neural commitment/differentiation. Furthermore, NS cells derived from adult brain and their differentiated cells were positioned along this PC axis further away from undifferentiated ESCs than embryonic stem–derived neural progenitors. We suggest that this PC1 defines a path to neural fate, providing a scale for the degree of commitment/differentiation.

This chapter discusses some technical aspects of genomic approaches such as complementary DNA (cDNA) analyses and expression profiling. It presents two examples of such applications: expression profiling and comparison among various types of stem cells; and expression profiling between cloned mice and normal mice. The fundamental information required for genomics approaches are the sequences and structures of the genes under study. This information is provided by Expressed Sequence Tag projects, in which cDNA libraries are first made from a variety of tissues and randomly selected individual cDNA clones are sequenced from their 5'- or 3'-ends. Two forms of microarrays are currently available: cDNA microarrays and oligonucleotide microarrays. Various bioinformatics and statistical analysis tools have been developed and utilized for the analysis of expression profiles of a large number of genes. In particular, hierarchical clustering, self-organizing map and k-means clustering analyses have been widely used.

Applications of microarray technologies to mouse embryology/genetics have been limited, due to the nonavailability of microarrays containing large numbers of embryonic genes and the gap between microgram quantities of RNA required by typical microarray methods and the miniscule amounts of tissue available to researchers. To overcome these problems, we have developed a microarray platform containing in situ-synthesized 60-mer oligonucleotide probes representing approximately 22,000 unique mouse transcripts, assembled primarily from sequences of stem cell and embryo cDNA libraries. We have optimized RNA labeling protocols and experimental designs to use as little as 2 ng total RNA reliably and reproducibly. At least 98% of the probes contained in the microarray correspond to clones in our publicly available collections, making cDNAs readily available for further experimentation on genes of interest. These characteristics, combined with the ability to profile very small samples, make this system a resource for stem cell and embryogenomics research.[Supplemental material is available online atwww.genome.org and at the NIA Mouse cDNA Project Web site,http://lgsun.grc.nia.nih.gov/cDNA/cDNA.html.]

Rats fed a Zn-deficient diet develop a characteristic cyclic variation in feed intake (Mills et al., Am J Clin Nutr 22: 1240-1249 (1969). A preliminary analysis (Tamaki et al., Br J Nutr 73: 711-722 (1995)) of the cyclic variations was followed with a personal computer. Cosinor analysis revealed that the cyclic period of the feed intake of male rats was 3.5 +/- 0.05 d. The mesor, amplitude and acrophase value were 10.0 +/- 0.3 g/d, 4.4 +/- 0.2 g/d and 3.5 +/- 0.3 radian, respectively. The cycle of body-weight change of the Zn-deficient rats was well synchronized with that of feed intake. The parameters of the feed intake cycle had a high correlation to the corresponding parameters of body-weight change (mesor: r = 0.846; amplitude: r = 0.771; period: r = 0.925; acrophase: r = 0.452). With the supplementation of Zn (0.95-3.80 mg/kg of the Zn-deficient diet), cyclic variations in feed intake and body-weight change were also found. The mesor, amplitude and period of feed intake cycle were is good correlation with Zn intake (r = 0.856, p < 0.001, r = 0.804, p < 0.001 and r = 0.613, p < 0.01, respectively). The cycle of feed intake of the rats fed a Zn-free diet was simulated to be: mesor 9.7 +/- 0.1 g/d, amplitude 6.5 +/- 0.1 g/d and period 3.4 +/- 0.02 d. The concentration of Zn intake given the half-maximal value of the amplitude was assumed to be 56 +/- 1 microgram/d.

Sexual development of Dictyostelium discoideum is a unique and useful system for the study of sexual phenomena. We have been studying molecular mechanisms of sexual cell fusion in D. discoideum and have identified several relevant cell-surface proteins. One of the proteins, gp138, was identified as a target molecule for fusion-blocking antibodies, and two genes for gp138, GP138A and GP138B, were cloned. The participation of gp138 in the sexual cell fusion was confirmed by antisense RNA mutagenesis, but it is unclear which of the genes encodes gp138. Moreover, the presence of a third gene for gp138 was indicated by gene disruption. In the present study, we generated strains of D. discoideum overexpressing either GP138A or GP138B to investigate the products of these genes. The transformants overexpressing GP138A and GP138B overproduced glycoproteins with molecular masses of 135 and 130 kDa, respectively. Although their molecular masses were different from that of gp138, the results of peptide mapping and amino acid sequencing showed that they are related to proteins, suggesting that the proteins encoded by GP138A and GP138B are isoforms of gp138 protein.

A glycoprotein, gp138, is implicated in the sexual cell fusion of Dictyostelium discoideum. We previously cloned and sequenced the two genes encoding the gp138 protein, GP138A and GP138B (Fang et al. (1993) Dev. Biol. 156, 201–208). Here, we have constructed a vector producing antisense RNA for the gp138 genes and have transformed the vector into Dictyostelium cells. The transformed cells showed a reduction in the amounts of gp138 mRNA and protein and their sexual cell fusion activity was considerably repressed.