Showing papers by "Kettering University published in 2009"

Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling

Abstract: Current neural induction protocols for human embryonic stem (hES) cells rely on embryoid body formation, stromal feeder co-culture or selective survival conditions. Each strategy has considerable drawbacks, such as poorly defined culture conditions, protracted differentiation and low yield. Here we report that the synergistic action of two inhibitors of SMAD signaling, Noggin and SB431542, is sufficient to induce rapid and complete neural conversion of >80% of hES cells under adherent culture conditions. Temporal fate analysis reveals the appearance of a transient FGF5(+) epiblast-like stage followed by PAX6(+) neural cells competent to form rosettes. Initial cell density determines the ratio of central nervous system and neural crest progeny. Directed differentiation of human induced pluripotent stem (hiPS) cells into midbrain dopamine and spinal motoneurons confirms the robustness and general applicability of the induction protocol. Noggin/SB431542-based neural induction should facilitate the use of hES and hiPS cells in regenerative medicine and disease modeling and obviate the need for protocols based on stromal feeders or embryoid bodies.

Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs

Abstract: The isolation of human induced pluripotent stem cells (iPSCs) offers a new strategy for modelling human disease. Recent studies have reported the derivation and differentiation of disease-specific human iPSCs. However, a key challenge in the field is the demonstration of disease-related phenotypes and the ability to model pathogenesis and treatment of disease in iPSCs. Familial dysautonomia (FD) is a rare but fatal peripheral neuropathy, caused by a point mutation in the IKBKAP gene involved in transcriptional elongation. The disease is characterized by the depletion of autonomic and sensory neurons. The specificity to the peripheral nervous system and the mechanism of neuron loss in FD are poorly understood owing to the lack of an appropriate model system. Here we report the derivation of patient-specific FD-iPSCs and the directed differentiation into cells of all three germ layers including peripheral neurons. Gene expression analysis in purified FD-iPSC-derived lineages demonstrates tissue-specific mis-splicing of IKBKAP in vitro. Patient-specific neural crest precursors express particularly low levels of normal IKBKAP transcript, suggesting a mechanism for disease specificity. FD pathogenesis is further characterized by transcriptome analysis and cell-based assays revealing marked defects in neurogenic differentiation and migration behaviour. Furthermore, we use FD-iPSCs for validating the potency of candidate drugs in reversing aberrant splicing and ameliorating neuronal differentiation and migration. Our study illustrates the promise of iPSC technology for gaining new insights into human disease pathogenesis and treatment.

Nanodiamond Particles: Properties and Perspectives for Bioapplications

Abstract: Nanodiamonds (NDs) are members of the diverse structural family of nanocarbons that includes many varieties based on synthesis conditions, post-synthesis processes, and modifications. First studied in detail beginning in the 1960s in Russia, NDs have now gained world-wide attention due to their inexpensive large-scale synthesis based on the detonation of carbon-containing explosives, small primary particle size (∼ 4 to 5 nm) with narrow size distribution, facile surface functionalization including bio-conjugation, as well as high biocompatibility. It is anticipated that the attractive properties of NDs will be exploited for the development of therapeutic agents for diagnostic probes, delivery vehicles, gene therapy, anti-viral and anti-bacterial treatments, tissue scaffolds, and novel medical devices such as nanorobots. Additionally, biotechnology applications have shown the prospective use of NDs for bioanalytical purposes, such as protein purification or fluorescent biolabeling. This review critically e...

Aberrant ERG expression cooperates with loss of PTEN to promote cancer progression in the prostate.

Abstract: Pier Paolo Pandolfi and colleagues report that prostate-specific overexpression of ERG in transgenic mice results in no overt phenotype on its own but promotes progression of intraepithelial neoplasia to adenocarcinoma in a PTEN heterozygous background. They also find that human TMPRSS2-ERG–positive tumors are enriched for PTEN loss, suggesting that these two events cooperate in human prostate tumorigenesis. Chromosomal translocations involving the ERG locus are frequent events in human prostate cancer pathogenesis; however, the biological role of aberrant ERG expression is controversial1. Here we show that aberrant expression of ERG is a progression event in prostate tumorigenesis. We find that prostate cancer specimens containing the TMPRSS2-ERG rearrangement are significantly enriched for loss of the tumor suppressor PTEN. In concordance with these findings, transgenic overexpression of ERG in mouse prostate tissue promotes marked acceleration and progression of high-grade prostatic intraepithelial neoplasia (HGPIN) to prostatic adenocarcinoma in a Pten heterozygous background. In vitro overexpression of ERG promotes cell migration, a property necessary for tumorigenesis, without affecting proliferation. ADAMTS1 and CXCR4, two candidate genes strongly associated with cell migration, were upregulated in the presence of ERG overexpression. Thus, ERG has a distinct role in prostate cancer progression and cooperates with PTEN haploinsufficiency to promote progression of HGPIN to invasive adenocarcinoma.

Mouse Kif7/Costal2 is a cilia-associated protein that regulates Sonic hedgehog signaling

Abstract: Mammalian Sonic hedgehog (Shh) signaling is essential for embryonic development and stem cell maintenance and has critical roles in tumorigenesis. Although core components of the Shh pathway are conserved in evolution, important aspects of mammalian Shh signaling are not shared with the Drosophila pathway. Perhaps the most dramatic difference between the Drosophila and mammalian pathways is that Shh signaling in the mouse requires a microtubule-based organelle, the primary cilium. Proteins that are required for the response to Shh are enriched in the cilium, but it is not clear why the cilium provides an appropriate venue for signal transduction. Here, we demonstrate that Kif7, a mammalian homologue of Drosophila Costal2 (Cos2), is a cilia-associated protein that regulates signaling from the membrane protein Smoothened (Smo) to Gli transcription factors. By using a Kif7 mutant allele identified in a reporter-based genetic screen, we show that, similar to Drosophila and zebrafish Cos2, mouse Kif7 acts downstream of Smo and upstream of Gli2 and has both negative and positive roles in Shh signal transduction. Mouse Kif7 activity depends on the presence of cilia and Kif7-eGFP localizes to base of the primary cilium in the absence of Shh. Activation of the Shh pathway promotes trafficking of Kif7-eGFP from the base to the tip of the cilium, and localization to the tip of the cilium is disrupted in a motor domain mutant. We conclude that Kif7 is a core regulator of Shh signaling that may also act as a ciliary motor.

Essential Role for Neutrophils but not Alveolar Macrophages at Early Time Points following Aspergillus fumigatus Infection

Abstract: Alveolar macrophages and neutrophils mediate innate immune defense against the opportunistic fungal pathogen Aspergillus fumigatus and are believed to be essential for host survival following inhalation of fungal spores (conidia). Although alveolar macrophages are postulated to kill inhaled conidia and neutrophils are believed to act against hyphae, the relative contribution of alveolar macrophages and neutrophils to early defense against A. fumigatus remain incompletely defined. To more precisely characterize the contributions of alveolar macrophages and neutrophils in antifungal host defense, we selectively depleted each cell population at different times following pulmonary challenge with conidia. Mice depleted of alveolar macrophages prior to pulmonary A. fumigatus infection recruited neutrophils normally and restricted hyphal tissue invasion. In contrast, neutrophil depletion prior to or within 3 h after infection was associated with high mortality. Neutrophil depletion at later time points, however, was associated with nearly normal survival rates. Our studies suggest that neutrophils, but not alveolar macrophages, provide essential anticonidial defense and that a brief period of influx into the respiratory tree is sufficient to prevent conidial germination and invasive disease.

Abundant primary piRNAs, endo-siRNAs and microRNAs in a Drosophila ovary cell line

Abstract: Piwi proteins, a subclass of Argonaute-family proteins, carry approximately 24-30-nt Piwi-interacting RNAs (piRNAs) that mediate gonadal defense against transposable elements (TEs). We analyzed the Drosophila ovary somatic sheet (OSS) cell line and found that it expresses miRNAs, endogenous small interfering RNAs (endo-siRNAs), and piRNAs in abundance. In contrast to intact gonads, which contain mixtures of germline and somatic cell types that express different Piwi-class proteins, OSS cells are a homogenous somatic cell population that expresses only PIWI and primary piRNAs. Detailed examination of its TE-derived piRNAs and endo-siRNAs revealed aspects of TE defense that do not rely upon ping-pong amplification. In particular, we provide evidence that a subset of piRNA master clusters, including flamenco, are specifically expressed in OSS and ovarian follicle cells. These data indicate that the restriction of certain TEs in somatic gonadal cells is largely mediated by a primary piRNA pathway.

Ascorbic acid increases the yield of dopaminergic neurons derived from basic fibroblast growth factor expanded mesencephalic precursors

Abstract: CNS precursors derived from E12 rat mesencephalon proliferate in the presence of basic fibroblast growth factor and differentiate in vitro into functional dopaminergic neurons, which upon transplantation alleviate behavioral symptoms in a rat model of Parkinson's disease. Here we show that the efficiency of dopaminergic differentiation decreases in the mesencephalic precursors that were proliferated or passaged for extended periods in vitro. Ascorbic acid treatment restored dopaminergic differentiation in these precursors and led to a greater than 10-fold increase in dopamine neuron yield compared with untreated cultures. The effect of ascorbic acid was stereospecific and could not be mimicked by any other antioxidants. The expression of sodium-dependent vitamin C transporter, a recently identified stereospecific ascorbic acid transporter, was maintained in mesencephalic precursors for extended in vitro periods. Pre-treatment of in vitro expanded mesencephalic precursors with ascorbic acid might facilitate the large-scale generation of dopaminergic neurons for clinical transplantation.

The Primary Cilium as a Hedgehog Signal Transduction Machine

Abstract: The Hedgehog (Hh) signal transduction pathway is essential for the development and patterning of numerous organ systems, and has important roles in a variety of human cancers. Genetic screens for mouse embryonic patterning mutants first showed a connection between mammalian Hh signaling and intraflagellar transport (IFT), a process required for construction of the primary cilium, a small cellular projection found on most vertebrate cells. Additional genetic and cell biological studies have provided very strong evidence that mammalian Hh signaling depends on the primary cilium. Here, we review the evidence that defines the integral roles that IFT proteins and cilia play in the regulation of the Hh signal transduction pathway in vertebrates. We discuss the mechanisms that control localization of Hh pathway proteins to the cilium, focusing on the transmembrane protein Smoothened (Smo), which moves into the cilium in response to Hh ligand. The phenotypes caused by loss of cilia-associated proteins are complex, which suggests that cilia and IFT play active roles in mediating Hh signaling rather than serving simply as a compartment in which pathway components are concentrated. Hh signaling in Drosophila does not depend on cilia, but there appear to be ancient links between cilia and components of the Hh pathway that may reveal how this fundamental difference between the Drosophila and mammalian Hh pathways arose in evolution.

Three-Dimensional Modeling and Experimental Study of a High Temperature PBI-Based PEM Fuel Cell

Abstract: This paper investigates the performance of a high temperature proton exchange membrane (PEM) fuel cell. Both experimental work and numerical simulation are conducted. The high temperature proton exchange membrane is based on polybenzimidazole (PBI) doped with phosphoric acid. A single cell with triple serpentine flow channels was operated at steady state at various levels of temperature, pressure, and air stoichiometry. A three-dimensional model was used to simulate the cell performance, and polarization curves were used to validate the experimental values. The theoretical model accurately predicts the experimental results. A sound knowledge of the impact of various variables at various levels of the cell operation is necessary for unraveling the parametric influence and can prove extremely useful for optimizing the cell operation.

A mouse model for Meckel syndrome reveals Mks1 is required for ciliogenesis and Hedgehog signaling.

Abstract: Meckel syndrome (MKS) is a rare autosomal recessive disease causing perinatal lethality associated with a complex syndrome that includes occipital meningoencephalocele, hepatic biliary ductal plate malformation, postaxial polydactyly and polycystic kidneys. The gene mutated in type 1 MKS encodes a protein associated with the base of the cilium in vertebrates and nematodes. However, shRNA knockdown studies in cell culture have reported conflicting results on the role of Mks1 in ciliogenesis. Here we show that loss of function of mouse Mks1 results in an accurate model of human MKS, with structural abnormalities in the neural tube, biliary duct, limb patterning, bone development and the kidney that mirror the human syndrome. In contrast to cell culture studies, loss of Mks1 in vivo does not interfere with apical localization of epithelial basal bodies but rather leads to defective cilia formation in most, but not all, tissues. Analysis of patterning in the neural tube and the limb demonstrates altered Hedgehog (Hh) pathway signaling underlies some MKS defects, although both tissues show an expansion of the domain of response to Shh signaling, unlike the phenotypes seen in other mutants with cilia loss. Other defects in the skull, lung, rib cage and long bones are likely to be the result of the disruption of Hh signaling, and the basis of defects in the liver and kidney require further analysis. Thus the disruption of Hh signaling can explain many, but not all, of the defects caused by loss of Mks1.

Primary Cilia Are Not Required for Normal Canonical Wnt Signaling in the Mouse Embryo

Abstract: Background Sonic hedgehog (Shh) signaling in the mouse requires the microtubule-based organelle, the primary cilium. The primary cilium is assembled and maintained through the process of intraflagellar transport (IFT) and the response to Shh is blocked in mouse mutants that lack proteins required for IFT. Although the phenotypes of mouse IFT mutants do not overlap with phenotypes of known Wnt pathway mutants, recent studies report data suggesting that the primary cilium modulates responses to Wnt signals. Methodology/Principal Findings We therefore carried out a systematic analysis of canonical Wnt signaling in mutant embryos and cells that lack primary cilia because of loss of the anterograde IFT kinesin-II motor (Kif3a) or IFT complex B proteins (Ift172 or Ift88). We also analyzed mutant embryos with abnormal primary cilia due to defects in retrograde IFT (Dync2h1). The mouse IFT mutants express the canonical Wnt target Axin2 and activate a transgenic canonical Wnt reporter, BAT-gal, in the normal spatial pattern and to the same quantitative level as wild type littermates. Similarly, mouse embryonic fibroblasts (MEFs) derived from IFT mutants respond normally to added Wnt3a. The switch from canonical to non-canonical Wnt also appears normal in IFT mutant MEFs, as both wild-type and mutant cells do not activate the canonical Wnt reporter in the presence of both Wnt3a and Wnt5a. Conclusions We conclude that loss of primary cilia or defects in retrograde IFT do not affect the response of the midgestation embryo or embryo-derived fibroblasts to Wnt ligands.

Genome-Wide Association Studies, Field Synopses, and the Development of the Knowledge Base on Genetic Variation and Human Diseases

Abstract: Genome-wide association studies (GWAS) have led to a rapid increase in available data on common genetic variants and phenotypes and numerous discoveries of new loci associated with susceptibility to common complex diseases. Integrating the evidence from GWAS and candidate gene studies depends on concerted efforts in data production, online publication, database development, and continuously updated data synthesis. Here the authors summarize current experience and challenges on these fronts, which were discussed at a 2008 multidisciplinary workshop sponsored by the Human Genome Epidemiology Network. Comprehensive field synopses that integrate many reported gene-disease associations have been systematically developed for several fields, including Alzheimer's disease, schizophrenia, bladder cancer, coronary heart disease, preterm birth, and DNA repair genes in various cancers. The authors summarize insights from these field synopses and discuss remaining unresolved issues -- especially in the light of evidence from GWAS, for which they summarize empirical P-value and effect-size data on 223 discovered associations for binary outcomes (142 with P < 10(-7)). They also present a vision of collaboration that builds reliable cumulative evidence for genetic associations with common complex diseases and a transparent, distributed, authoritative knowledge base on genetic variation and human health. As a next step in the evolution of Human Genome Epidemiology reviews, the authors invite investigators to submit field synopses for possible publication in the American Journal of Epidemiology.

Dicing of viral replication intermediates during silencing of latent Drosophila viruses.

Abstract: Previous studies revealed roles for RNA interference (RNAi) in the immediate cellular response to viral infection in plants, nematodes and flies. However, little is known about how RNAi combats viruses during persistent or latent infections. Our analysis of small RNAs cloned from Drosophila cells latently infected with Flock House Virus (FHV) failed to reveal signatures of bulk degradation of the viral genome. Instead, this + strand virus specifically generated Dicer-2-dependent, 21-nucleotide siRNAs that derived in equal proportion from + and − strands. Curiously, luciferase reporters that are fully complementary to abundant viral siRNAs were poorly repressed. Moreover, although the viral siRNAs that were incorporated into an effector complex associated with Argonaute2, bulk FHV siRNAs in latently infected cells were not loaded into any Argonaute protein. Together, these data suggest that direct dicing of viral replication intermediates plays an important role in maintaining the latent viral state. In addition, the denial of bulk viral siRNAs from effector complexes suggests that criteria beyond the structural competency of RNA duplexes influence the assembly of functional silencing complexes.

Specific recruitment of protein kinase A to the immunoglobulin locus regulates class-switch recombination

Abstract: Immunoglobulin class-switch recombination (CSR) requires activation-induced cytidine deaminase (AID). Deamination of DNA by AID in transcribed switch (S) regions leads to double-stranded breaks in DNA that serve as obligatory CSR intermediates. Here we demonstrate that the catalytic and regulatory subunits of protein kinase A (PKA) were specifically recruited to S regions to promote the localized phosphorylation of AID, which led to binding of replication protein A and subsequent propagation of the CSR cascade. Accordingly, inactivation of PKA resulted in considerable disruption of CSR because of decreased AID phosphorylation and recruitment of replication protein A to S regions. We propose that PKA nucleates the formation of active AID complexes specifically on S regions to generate the high density of DNA lesions required for CSR.

Use of KikGR a photoconvertible green-to-red fluorescent protein for cell labeling and lineage analysis in ES cells and mouse embryos

Abstract: Background The use of genetically-encoded fluorescent proteins has revolutionized the fields of cell and developmental biology and in doing so redefined our understanding of the dynamic morphogenetic processes that shape the embryo. With the advent of more accessible and sophisticated imaging technologies as well as an abundance of fluorescent proteins with different spectral characteristics, the dynamic processes taking place in situ in living cells and tissues can now be probed. Photomodulatable fluorescent proteins are one of the emerging classes of genetically-encoded fluorescent proteins.

BAC transgenesis in human embryonic stem cells as a novel tool to define the human neural lineage.

Abstract: Human embryonic stem cells (hESCs) have enormous potential for applications in basic biology and regenerative medicine. However, harnessing the potential of hESCs toward generating homogeneous populations of specialized cells remains challenging. Here we describe a novel technology for the genetic identification of defined hESC-derived neural cell types using bacterial artificial chromosome (BAC) transgenesis. We generated hESC lines stably expressing Hes5::GFP, Dll1::GFP, and HB9::GFP BACs that yield green fluorescent protein (GFP)(+) neural stem cells, neuroblasts, and motor neurons, respectively. Faithful reporter expression was confirmed by cell fate analysis and appropriate transgene regulation. Prospective isolation of HB9::GFP(+) cells yielded purified human motor neurons with proper marker expression and electrophysiological activity. Global mRNA and microRNA analyses of Hes5::GFP(+) and HB9::GFP(+) populations revealed highly specific expression signatures, suggesting that BAC transgenesis will be a powerful tool for establishing expression libraries that define the human neural lineage and for accessing defined cell types in applications of human disease.

Nap1-mediated actin remodeling is essential for mammalian myoblast fusion.

Abstract: Myoblast fusion is crucial for the formation, growth, maintenance and regeneration of healthy skeletal muscle. Unfortunately, the molecular machinery, cell behaviors, and membrane and cytoskeletal remodeling events that govern fusion and myofiber formation remain poorly understood. Using time-lapse imaging approaches on mouse C2C12 myoblasts, we identify discrete and specific molecular events at myoblast membranes during fusion and myotube formation. These events include rearrangement of cell shape from fibroblast to spindle-like morphologies, changes in lamellipodial and filopodial extensions during different periods of differentiation, and changes in membrane alignment and organization during fusion. We find that actin-cytoskeleton remodeling is crucial for these events: pharmacological inhibition of F-actin polymerization leads to decreased lamellipodial and filopodial extensions and to reduced myoblast fusion. Additionally, shRNA-mediated inhibition of Nap1, a member of the WAVE actin-remodeling complex, results in accumulations of F-actin structures at the plasma membrane that are concomitant with a decrease in myoblast fusion. Our data highlight distinct and essential roles for actin cytoskeleton remodeling during mammalian myoblast fusion, provide a platform for cellular and molecular dissection of the fusion process, and suggest a functional conservation of Nap1-regulated actin-cytoskeleton remodeling during myoblast fusion between mammals and Drosophila.

Thio FCMA Intermediates as Strong Acyl Donors: A General Solution to the Formation of Complex Amide Bonds

Abstract: Novel methodology for the formation of amide bonds under neutral conditions is described. Evidence is presented that the active acyl donors are thio FCMA intermediates, generated from the reactions of thioacids with isonitriles.