Directed induction of anterior and posterior primitive streak by Wnt from embryonic stem cells cultured in a chemically defined serum-free medium.

Formation of the primitive streak (PS) is the initial specification step that generates all the mesodermal and endodermal tissue lineages during early differentiation. Thus, a therapeutically compatible and efficient method for differentiation of the PS is crucial for regenerative medicine. In this study, we developed chemically defined serum-free culture conditions for the differentiation of embryonic stem (ES) cells into the PS-like cells. Cultures supplemented with Wnt showed induction of expression of a PS marker, the brachyury gene, followed by induction of the anterior PS markers goosecoid and foxa2, a posterior PS marker, evx1, and the endoderm marker sox17. Similar differentiation of PS by Wnt was also observed in human ES cells. Moreover, we revealed that the activation of the Wnt canonical pathway is essential for PS differentiation in mouse ES cells. These results demonstrated that Wnt is an essential and sufficient factor for the induction of the PS-like cells in vitro. These conditions of induction could constitute the initial step in generating therapeutically useful cells of the definitive endoderm lineage, such as hepatocytes and pancreatic endocrine cells, under chemically defined conditions.

Development of a practical sandwich assay to detect human pluripotent stem cells using cell culture media.

Human pluripotent stem cells are considered to be ideal cell sources for regenerative medicine, but their clinical and industrial application is hindered by their tumorigenic potential. Previously we have identified a pluripotent stem cell-specific lectin rBC2LCN recognizing podocalyxin as a cell surface ligand. More recently, podocalyxin was found to be a soluble ligand of rBC2LCN that is secreted specifically from human pluripotent stem cells into cell culture media. Taking advantage of this phenomenon, we have previously developed a sandwich assay targeting the soluble podocalyxin using rBC2LCN as a capturing probe and another lectin rABA as an overlay probe to detect human pluripotent stem cells residing in cell therapy products derived from human pluripotent stem cells. A drawback to this, however, was that cell culture media containing fetal bovine serum was found to cause a substantial background signal to the sandwich assay. To reduce the background and increase the sensitivity, we screened different overlay probes to detect the soluble podocalyxin. Among them, an anti-keratan sulfate monoclonal antibody called R-10G showed the highest sensitivity and provided a low background signal to fetal bovine serum. The established sandwich assay using rBC2LCN and R-10G was proved to be powerful, which allowed the high-sensitive detection of human induced pluripotent stem cells residing among clinical-grade cardiomyocytes and neural stem cells, both derived from human induced pluripotent stem cells. The developed method has a possibility to be a standard technology to detect human induced pluripotent stem cells resided in various types of cell therapy products.

Measurements of weak interactions between truncated substrates and a hammerhead ribozyme by competitive kinetic analyses: implications for the design of new and efficient ribozymes with high sequence specificity.

Exploitation of ribozymes in a practical setting requires high catalytic activity and strong specificity. The hammerhead ribozyme R32 has considerable potential in this regard since it has very high catalytic activity. In this study, we have examined how R32 recognizes and cleaves a specific substrate, focusing on the mechanism behind the specificity. Comparing rates of cleavage of a substrate in a mixture that included the correct substrate and various substrates with point mutations, we found that R32 cleaved the correct substrate specifically and at a high rate. To clarify the source of this strong specificity, we quantified the weak interactions between R32 and various truncated substrates, using truncated substrates as competitive inhibitors since they were not readily cleaved during kinetic measurements of cleavage of the correct substrate, S11. We found that the strong specificity of the cleavage reaction was due to a closed form of R32 with a hairpin structure. The self-complementary structure within R32 enabled the ribozyme to discriminate between the correct substrate and a mismatched substrate. Since this hairpin motif did not increase the Km (it did not inhibit the binding interaction) or decrease the kcat (it did not decrease the cleavage rate), this kind of hairpin structure might be useful for the design of new ribozymes with strong specificity and high activity.

Cleavage of an inaccessible site by the maxizyme with two independent binding arms: an alternative approach to the recruitment of RNA helicases.

To overcome obstacles to target site selection, we recently created a novel hybrid ribozyme that could access any chosen site by the recruitment of intracellular RNA helicases [Warashina et al. (2001) Proc. Natl. Acad. Sci. USA 98, 5572-5577; Kawasaki et al. (2002) Nat. Biotech. 20, 376-380]. We also demonstrated previously that pol III-driven maxizymes with two substrate-binding arms that were directed against two different sites within a target mRNA formed very active heterodimers in vivo [Kuwabara, et al. (2000) Trends Biotechnol. 18, 462-468; Tanabe et al. (2001) Nature 406, 473-474]. Despite the complicated dimerization process, all the maxizymes that we tested in cultured cells had greater catalytic activity than the parental ribozymes. To investigate the action of maxizymes in cells, we designed a specific maxizyme with two substrate-binding arms that was directed against endogenously expressed LTR-luciferase chimeric mRNA, where LTR refers to the long terminal repeat of HIV-1. One substrate-binding arm of the maxizyme was designed to bind to a site within HIV-1 TAR RNA that is known to form a stable stem structure that normally prevents binding of a ribozyme. The other substrate-binding arm was directed against a relatively accessible site within the luciferase gene. As expected, the conventional ribozyme failed to cleave the TAR region in vivo because of the latter's stable secondary structure. However, to our surprise, the maxizyme cleaved the TAR region within the stem with high efficiency in vivo. The enhanced cleavage in vivo by the maxizyme might have resulted from an entropically favorable, intramolecular, second binding process that occurred during the breathing of the stem structure of the target mRNA. Importantly, our data suggest that this maxizyme technology might be used as an alternative approach to the recruitment of RNA helicases in cleaving sites previously found to be inaccessible.

Helicase-attached novel hybrid ribozymes.

Ribozymes have potential as therapeutic agents and in functional studies of genes of interest. The activities of ribozymes in vivo depend on the accessibility of ribozymes to a cleavage site in the target RNA. At present, the selection of a target site for ribozymes is often based on a computer-aided structural analysis of the target RNA or trial-and-error experiments in which vast numbers of ribozymes are tested systematically. To overcome this problem, we have engineered intracellularly produced ribozymes with unwinding activity in vivo. We found that attachment to ribozymes (hybrid ribozymes) of an RNA motif with the ability to interact with intracellular RNA helicases, which create hybrid ribozymes, enhances ribozyme activity significantly in vivo. Thus, hybrid ribozymes can catalyze cleavage at the specified target site within an RNA in vivo almost independently of the secondary or tertiary structure of the target RNA around the cleavage site.

A medium hyperglycosylated podocalyxin enables noninvasive and quantitative detection of tumorigenic human pluripotent stem cells.

While human pluripotent stem cells are attractive sources for cell-replacement therapies, a major concern remains regarding their tumorigenic potential. Thus, safety assessment of human pluripotent stem cell-based products in terms of tumorigenicity is critical. Previously we have identified a pluripotent stem cell-specific lectin probe rBC2LCN recognizing hyperglycosylated podocalyxin as a cell surface ligand. Here we demonstrate that hyperglycosylated podocalyxin is secreted from human pluripotent stem cells into cell culture supernatants. We establish a sandwich assay system, named the GlycoStem test, targeting the soluble hyperglycosylated podocalyxin using rBC2LCN. The GlycoStem test is sufficiently sensitive and quantitative to detect residual human pluripotent stem cells. This work provides a proof of concept for the noninvasive and quantitative detection of tumorigenic human pluripotent stem cells using cell culture supernatants. The developed method should increase the safety of human pluripotent stem cell-based cell therapies.

Insulin biosynthesis in neuronal progenitors derived from adult hippocampus and the olfactory bulb.

In the present study, we demonstrated that insulin is produced not only in the mammalian pancreas but also in adult neuronal cells derived from the hippocampus and olfactory bulb (OB). Paracrine Wnt3 plays an essential role in promoting the active expression of insulin in both hippocampal and OB-derived neural stem cells. Our analysis indicated that the balance between Wnt3, which triggers the expression of insulin via NeuroD1, and IGFBP-4, which inhibits the original Wnt3 action, is regulated depending on diabetic (DB) status. We also show that adult neural progenitors derived from DB animals retain the ability to give rise to insulin-producing cells and that grafting neuronal progenitors into the pancreas of DB animals reduces glucose levels. This study provides an example of a simple and direct use of adult stem cells from one organ to another, without introducing additional inductive genes.

Reduction of N-glycolylneuraminic acid in human induced pluripotent stem cells generated or cultured under feeder- and serum-free defined conditions.

BACKGROUND: The successful establishment of human induced pluripotent stem cells (hiPSCs) has increased the possible applications of stem cell research in biology and medicine. In particular, hiPSCs are a promising source of cells for regenerative medicine and pharmacology. However, one of the major obstacles to such uses for hiPSCs is the risk of contamination from undefined pathogens in conventional culture conditions that use serum replacement and mouse embryonic fibroblasts as feeder cells. METHODOLOGY/PRINCIPAL FINDINGS: Here we report a simple method for generating or culturing hiPSCs under feeder- and serum-free defined culture conditions that we developed previously for human embryonic stem cells. The defined culture condition comprises a basal medium with a minimal number of defined components including five highly purified proteins and fibronectin as a substrate. First, hiPSCs, which were generated using Yamanaka's four factors and conventional undefined culture conditions, adapted to the defined culture conditions. These adapted cells retained the property of self renewal as evaluated morphologically, the expression of self-renewal marker proteins, standard growth rates, and pluripotency as evaluated by differentiation into derivatives of all three primary germ layers in vitro and in vivo (teratoma formation in immunodeficient mice). Moreover, levels of nonhuman N-glycolylneuraminic acid (Neu5Gc), which is a xenoantigenic indicator of pathogen contamination in human iPS cell cultures, were markedly decreased in hiPSCs cultured under the defined conditions. Second, we successfully generated hiPSCs using adult dermal fibroblast under the defined culture conditions from the reprogramming step. For a long therm culture, the generated cells also had the property of self renewal and pluripotency, they carried a normal karyotype, and they were Neu5Gc negative. CONCLUSION/SIGNIFICANCE: This study suggested that generation or adaption culturing under defined culture conditions can eliminate the risk posed by undefined pathogens. This success in generating hiPSCs using adult fibroblast would be beneficial for clinical application.

Induction of neural crest cells from mouse embryonic stem cells in a serum-free monolayer culture.

The neural crest (NC) is a group of cells located in the neural folds at the boundary between the neural and epidermal ectoderm. NC cells differentiate into a vast range of cells,including neural cells, smooth muscle cells, bone and cartilage cells of the maxillofacial region, and odontoblasts. The molecular mechanisms underlying NC induction during early development remain poorly understood. We previously established a defined serum-free culture condition for mouse embryonic stem (mES) cells without feeders. Here, using this defined condition, we have developed a protocol to promote mES cell differentiation into NC cells in an adherent monolayer culture. We found that adding bone morphogenetic protein (BMP)-4 together with fibroblast growth factor (FGF)-2 shifts mES cell differentiation into the NC lineage. Furthermore, we have established a cell line designated as P0-6 that is derived from the blastocysts of P0-Cre/Floxed-EGFP mice expressing EGFP in an NC-lineage-specific manner. P0-6 cells cultured using this protocol expressed EGFP. This protocol could be used to help clarify the mechanisms by which cells differentiate into the NC lineage and to assist the development of applications for clinical therapy.

Evaluation of BACE1 Silencing in Cellular Models.

Beta-secretase (BACE1) is the major enzyme participating in generation of toxic amyloid-beta (Abeta) peptides, identified in amyloid plaques of Alzheimer's disease (AD) brains. Its downregulation results in decreasing secretion of Abeta. Thus, BACE1 silencing by RNAi represents possible strategy for antiamyloid therapy in the treatment of AD. In this study, a series of newly designed sequences of synthetic and vector-encoded siRNAs (pSilencer, pcPURhU6, and lentivirus) were tested against overexpressed and endogenous BACE1 in several cell lines and in adult neural progenitor cells, derived from rat hippocampus. SiRNAs active in human, mouse, and rat cell models were shown to diminish the level of BACE1. In HCN A94 cells, two BACE1-specific siRNAs did not alter the expression of genes of BACE2 and several selected genes involved in neurogenesis (Synapsin I, betaIII-Tubulin, Calbidin, NeuroD1, GluR2, CREB, MeCP2, PKR), however, remarkable lowering of SCG10 mRNA, coding protein of stathmin family, important in the development of nervous system, was observed.

Wnt-mediated activation of NeuroD1 and retro-elements during adult neurogenesis.

In adult hippocampus, new neurons are continuously generated from neural stem cells (NSCs), but the molecular mechanisms regulating adult neurogenesis remain elusive. We found that Wnt signaling, together with the removal of Sox2, triggered the expression of NeuroD1 in mice. This transcriptional regulatory mechanism was dependent on a DNA element containing overlapping Sox2 and T-cell factor/lymphoid enhancer factor (TCF/LEF)-binding sites (Sox/LEF) in the promoter. Notably, Sox/LEF sites were also found in long interspersed nuclear element 1 (LINE-1) elements, consistent with their critical roles in the transition of NSCs to proliferating neuronal progenitors. Our results describe a previously unknown Wnt-mediated regulatory mechanism that simultaneously coordinates activation of NeuroD1 and LINE-1, which is important for adult neurogenesis and survival of neuronal progenitors. Moreover, the discovery that LINE-1 retro-elements embedded in the mammalian genome can function as bi-directional promoters suggests that Sox/LEF regulatory sites may represent a general mechanism, at least in part, for relaying environmental signals to other nearby loci to promote adult hippocampal neurogenesis.

RNA interference in silencing of genes of Alzheimer's disease in cellular and rat brain models.

Accumulation of insoluble aggregates of beta-amyloid peptide, a cleavage product of amyloid precursor protein, is thought to be a central step in the pathogenesis of Alzheimer's disease. The major enzymes required for the generation of toxic amyloid-beta peptide are beta-(BACE1) and gamma-secretases. Here, we present the rational design and the application of synthetic and lentivirus vector-encoded siRNAs for specific and efficient knockdown of overexpressed and endogenous BACE1, both in dividing and neural stem cells and in a rat brain. We also tested an approach to anti-amyloid therapy by the use of the allele-specific siRNAs to silence the mutant presenilin 1 (L392V PS-1), the main component of gamma-secretase, responsible for development of Familial Alzheimer's disease. Reducing the level of beta-amyloid accumulation in the brain could be beneficial for metabolic studies as well as potential therapeutic approach for prevention and treatment of Alzheimer's disease.

The NRSE smRNA specifies the fate of adult hippocampal neural stem cells.

Recently we found that the nuclear localized small modulatory double-stranded (ds) RNA (smRNA) coding NRSE sequences triggered activation of transcription of NRSE genes in adult hippocampal neural stem cells. NRSE smRNA, which are non-coding dsRNAs about 20 bp in length, reside in the nucleus and play a critical role in mediating neuronal differentiation. These smRNAs carry the sequence of NRSE/RE1, which is recognized by the NRSF/REST transcription factor. The NRSE sequences are embedded widely in the genomic region, typically in promoters of neuron-specific genes. The mechanism of action appears to be mediated through a specific interaction between dsRNA and DNA/protein interaction, rather than through siRNA or miRNA. The discovery of smRNAs extends the important contribution of non-coding RNAs as key regulators of cell fate choice for adult neurogenesis.

Chemistry-based RNA technologies: demonstration of usefulness of libraries of ribozymes and short hairpin RNAs (shRNAs).

Mechanism of action of hammerhead ribozymes has been investigated and their intracellular activities have been improved. Based on the improved ribozymes and more recently discovered natural RNAi, we have created libraries of both ribozymes and short hairpin RNAs (shRNAs). The introduction of a library of active ribozymes or shRNAs into cells, and the subsequent screening for phenotypic changes, allows the rapid identification of gene function.

Analysis of the conserved P9-G10.1 metal-binding motif in hammerhead ribozymes with an extra nucleotide inserted between A9 and G10.1 residues.

Hammerhead ribozymes (Rz) have catalytically important tandem G:A pairs in the core region, and we recently demonstrated that the P9-G10.1 motif (a sheared-type G:A pair with a guanine residue on the 3' side of the adenine residue) with several flanking base pairs is sufficient for capture of divalent cations, such as Mg(2+) and Cd(2+) ions that are important to maintain full activities (Tanaka et al. J. Am. Chem. Soc. 2002, 124, 4595-4601; Tanaka et al. J. Am. Chem. Soc. 2004, 126, 744-752). We also found that mutant hammerhead ribozymes that have an additional G residue inserted between A9 and G10.1 residues (the metal-binding P9-G10.1 motif) have significant catalytic activities. In this study, we demonstrate that the hammerhead ribozymes are capable of maintaining the catalytically competent structure even when the tandem, sheared-type G:A pairs were perturbed by an insertion of an additional nucleotide, whereas the chirality of the phosphorothioate at the P9 position significantly influenced the enzymatic activity for both the natural and G-inserted ribozymes.

A novel strategy by the action of ricin that connects phenotype and genotype without loss of the diversity of libraries.

We present a novel strategy for connection of phenotype and genotype in vitro that can be used for the selection of functional proteins even at room temperature. The strategy involves generation of a stable complex between a ribosome, an mRNA, and its translated protein, without removal of the termination codon, as a result of the action of the ricin A chain during translation. We demonstrate the potential selection capacity of this novel strategy by isolating such complexes that contain newly synthesized streptavidin and glutathione-S-transferase (GST) using appropriate ligands. The technique requires no transfection, no chemical synthesis, no ligation, and no removal of the termination codon. Thus our novel "Ribosome-Inactivation Display System (RIDS)" should provide, without loss of the pool population, a reliable, simple, and robust selection system for in vitro evolution of the properties of proteins in a predictable direction by a combination of randomization and appropriate selection strategies.

Novel method for selection of tRNA-driven ribozymes with enhanced stability in mammalian cells.

Intracellular stability is a critical determinant of the activity of a ribozyme in vivo. In previous studies, we succeeded in constructing an effective system for the expression of ribozymes using the promoter of a human gene for tRNA(Val). The resultant tRNA(Val)-driven ribozymes (tRNA-ribozymes) had a half-life of approximately 100 minutes. In the present study, we established a novel system for the selection of tRNA-ribozymes that were more stable than a previously generated optimally designed tRNA-ribozyme, and we confirmed that the newly selected tRNA-ribozymes worked well. Selective pressure was applied by treating cells that expressed tRNA-ribozymes with actinomycin D, and the system yielded tRNA-ribozymes with enhanced stability. The sequences isolated after selection exhibited some similarities. Furthermore, some selected tRNA-ribozymes had almost the same activity as or higher activity than that of the optimally designed tRNA-ribozyme despite the fact that the selective pressure was not aimed at enhancing the cleavage activity. Our approach might be very useful for selection not only of ribozymes with enhanced stability but also of other functional nucleic acids in vivo.

Evaluating sandwich immunoassays in microarray format in terms of the ambient analyte regime.

BACKGROUND: Conceptionally, antibody microarrays are simply multiplexed sandwich immunoassays in a miniaturized format. However, from the amounts of capture antibodies used, it is not apparent whether such assays are ambient analyte (Ekins. Clin Chem 1998;44:2015-30) or mass-sensing devices (Silzel et al. Clin Chem 1998;44:2036-43). We evaluated multiplexed microarray sandwich assays for 24 mouse serum proteins in these terms within the boundaries of our experimental setup and based on theoretical considerations of the law of mass action. METHODS: Capture antibodies for 24 mouse serum proteins were printed on planar microarray substrates. After incubation with mixtures of purified antigens for 1 or 18 h, mixtures of biotinylated detection antibodies were used. High assay sensitivity was achieved by use of resonance-light-scattering particles for signal generation. Titration curves were generated for assay volumes of 20, 40, and 80 microL, and detection limits were calculated and compared. The assays were modeled theoretically based on the amounts of capture antibodies and the assay volumes used. RESULTS: As predicted, experimental variations of the assay volume by up to fourfold did not appreciably affect detection. Even for the most sensitive assay, < 2% of the analyte molecules present in the sample were captured and generated signal at the detection limit. However, increasing the sample incubation time from 1 to 18 h on average lowered the detection limit threefold. CONCLUSIONS: In our experimental setup, all 24 sandwich microarray assays fulfill the criteria of the "ambient analyte" regime because depletion of analyte molecules from the assay volume is insignificant.