Showing papers by "Kettering University published in 2007"

Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells

Abstract: Vertebrate neural crest development depends on pluripotent, migratory precursor cells. Although avian and murine neural crest stem (NCS) cells have been identified, the isolation of human NCS cells has remained elusive. Here we report the derivation of NCS cells from human embryonic stem cells at the neural rosette stage. We show that NCS cells plated at clonal density give rise to multiple neural crest lineages. The human NCS cells can be propagated in vitro and directed toward peripheral nervous system lineages (peripheral neurons, Schwann cells) and mesenchymal lineages (smooth muscle, adipogenic, osteogenic and chondrogenic cells). Transplantation of human NCS cells into the developing chick embryo and adult mouse hosts demonstrates survival, migration and differentiation compatible with neural crest identity. The availability of unlimited numbers of human NCS cells offers new opportunities for studies of neural crest development and for efforts to model and treat neural crest-related disorders.

Directed differentiation and transplantation of human embryonic stem cell-derived motoneurons.

Abstract: Motoneurons represent a specialized class of neurons essential for the control of body movement. Motoneuron loss is the cause of a wide range of neurological disorders including amyotrophic lateral sclerosis and spinal muscular atrophy. Embryonic stem cells are a promising cell source for the study and potential treatment of motoneuron diseases. Here, we present a novel in vitro protocol of the directed differentiation of human embryonic stem cells (hESCs) into engraftable motoneurons. Neural induction of hESCs was induced on MS5 stromal feeders, resulting in the formation of neural rosettes. In response to sonic hedgehog and retinoic acid, neural rosettes were efficiently directed into spinal motoneurons with appropriate in vitro morphological, physiological, and biochemical properties. Global gene expression analysis was used as an unbiased measure to confirm motoneuron identity and type. Transplantation of motoneuron progeny into the developing chick embryo resulted in robust engraftment, maintenance of motoneuron phenotype, and long-distance axonal projections into peripheral host tissues. Transplantation into the adult rat spinal cord yielded neural grafts comprising a large number of human motoneurons with outgrowth of choline acetyltransferase positive fibers. These data provide evidence for in vivo survival of hESC-derived motoneurons, a key requirement in the development of hESC-based cell therapy in motoneuron disease. Disclosure of potential conflicts of interest is found at the end of this article.

MyD88-mediated signals induce the bactericidal lectin RegIIIγ and protect mice against intestinal Listeria monocytogenes infection

Abstract: Listeria monocytogenes is a food-borne bacterial pathogen that causes systemic infection by traversing the intestinal mucosa. Although MyD88-mediated signals are essential for defense against systemic L. monocytogenes infection, the role of Toll-like receptor and MyD88 signaling in intestinal immunity against this pathogen has not been defined. We show that clearance of L. monocytogenes from the lumen of the distal small intestine is impaired in MyD88−/− mice. The distal ileum of wild-type (wt) mice expresses high levels of RegIIIγ, which is a bactericidal lectin that is secreted into the bowel lumen, whereas RegIIIγ expression in MyD88−/− mice is nearly undetectable. In vivo depletion of RegIIIγ from the small intestine of wt mice diminishes killing of luminal L. monocytogenes , whereas reconstitution of MyD88-deficient mice with recombinant RegIIIγ enhances intestinal bacterial clearance. Experiments with bone marrow chimeric mice reveal that MyD88-mediated signals in nonhematopoietic cells induce RegIIIγ expression in the small intestine, thereby enhancing bacterial killing. Our findings support a model of MyD88-mediated epithelial conditioning that protects the intestinal mucosa against bacterial invasion by inducing RegIIIγ.

Morphology, molecular codes, and circuitry produce the three-dimensional complexity of the cerebellum.

Abstract: The most noticeable morphological feature of the cerebellum is its folded appearance, whereby fissures separate its anterior-posterior extent into lobules. Each lobule is molecularly coded along the medial-lateral axis by parasagittal stripes of gene expression in one cell type, the Purkinje cells (PCs). Additionally, within each lobule distinct combinations of afferents terminate and supply the cerebellum with synchronized sensory and motor information. Strikingly, afferent terminal fields are organized into parasagittal domains, and this pattern bears a close relationship to PC molecular coding. Thus, cerebellum three-dimensional complexity obeys a basic coordinate system that can be broken down into morphology and molecular coding. In this review, we summarize the sequential stages of cerebellum development that produce its laminar structure, foliation, and molecular organization. We also introduce genes that regulate morphology and molecular coding, and discuss the establishment of topographical circuits within the context of the two coordinate systems. Finally, we discuss how abnormal cerebellar organization may result in neurological disorders like autism.

Derivation of engraftable skeletal myoblasts from human embryonic stem cells

Abstract: Human embryonic stem cells (hESCs) are a promising source for cell therapy in degenerative diseases. A key step in establishing the medical potential of hESCs is the development of techniques for the conversion of hESCs into tissue-restricted precursors suitable for transplantation. We recently described the derivation of multipotent mesenchymal precursors from hESCs. Nevertheless, our previous study was limited by the requirement for mouse feeders and the lack of in vivo data. Here we report a stroma-free induction system for deriving mesenchymal precursors. Selective culture conditions and fluorescence-activated cell sorting (FACS)-mediated purification yielded multipotent mesenchymal precursors and skeletal myoblasts. Skeletal muscle cells undergo in vitro maturation resulting in myotube formation and spontaneous twitching. We found that hESC-derived skeletal myoblasts were viable after transplantation into the tibialis anterior muscle of SCID/Beige mice, as assessed by bioluminescence imaging. Lack of teratoma formation and evidence of long-term myoblast engraftment suggests considerable potential for future therapeutic applications.

Inhibition of ischemic cardiomyocyte apoptosis through targeted ablation of Bnip3 restrains postinfarction remodeling in mice.

Abstract: Following myocardial infarction, nonischemic myocyte death results in infarct expansion, myocardial loss, and ventricular dysfunction. Here, we demonstrate that a specific proapoptotic gene, Bnip3, minimizes ventricular remodeling in the mouse, despite having no effect on early or late infarct size. We evaluated the effects of ablating Bnip3 on cardiomyocyte death, infarct size, and ventricular remodeling after surgical ischemia/reperfusion (IR) injury in mice. Immediately following IR, no significant differences were observed between Bnip3(-/-) and WT mice. However, at 2 days after IR, apoptosis was diminished in Bnip3(-/-) periinfarct and remote myocardium, and at 3 weeks after IR, Bnip3(-/-) mice exhibited preserved LV systolic performance, diminished LV dilation, and decreased ventricular sphericalization. These results suggest myocardial salvage by inhibition of apoptosis. Forced cardiac expression of Bnip3 increased cardiomyocyte apoptosis in unstressed mice, causing progressive LV dilation and diminished systolic function. Conditional Bnip3 overexpression prior to coronary ligation increased apoptosis and infarct size. These studies identify postischemic apoptosis by myocardial Bnip3 as a major determinant of ventricular remodeling in the infarcted heart, suggesting that Bnip3 may be an attractive therapeutic target.

Bacterial DNA repair by non-homologous end joining.

Abstract: The capacity to rectify DNA double-strand breaks (DSBs) is crucial for the survival of all species. DSBs can be repaired either by homologous recombination (HR) or non-homologous end joining (NHEJ). The long-standing notion that bacteria rely solely on HR for DSB repair has been overturned by evidence that mycobacteria and other genera have an NHEJ system that depends on a dedicated DNA ligase, LigD, and the DNA-end-binding protein Ku. Recent studies have illuminated the role of NHEJ in protecting the bacterial chromosome against DSBs and other clastogenic stresses. There is also emerging evidence of functional crosstalk between bacterial NHEJ proteins and components of other DNA-repair pathways. Although still a young field, bacterial NHEJ promises to teach us a great deal about the nexus of DNA repair and bacterial pathogenesis.

Cerebellum morphogenesis: the foliation pattern is orchestrated by multi-cellular anchoring centers

Abstract: Background The cerebellum has a striking morphology consisting of folia separated by fissures of different lengths. Since folia in mammals likely serve as a broad platform on which the anterior-posterior organization of the sensory-motor circuits of the cerebellum are built, it is important to understand how such complex morphology arises.

Long-term impact of radiation on the stem cell and oligodendrocyte precursors in the brain.

Abstract: Background The cellular basis of long term radiation damage in the brain is not fully understood. Methods and Findings We administered a dose of 25Gy to adult rat brains while shielding the olfactory bulbs. Quantitative analyses were serially performed on different brain regions over 15 months. Our data reveal an immediate and permanent suppression of SVZ proliferation and neurogenesis. The olfactory bulb demonstrates a transient but remarkable SVZ-independent ability for compensation and maintenance of the calretinin interneuron population. The oligodendrocyte compartment exhibits a complex pattern of limited proliferation of NG2 progenitors but steady loss of the oligodendroglial antigen O4. As of nine months post radiation, diffuse demyelination starts in all irradiated brains. Counts of capillary segments and length demonstrate significant loss one day post radiation but swift and persistent recovery of the vasculature up to 15 months post XRT. MRI imaging confirms loss of volume of the corpus callosum and early signs of demyelination at 12 months. Ultrastructural analysis demonstrates progressive degradation of myelin sheaths with axonal preservation. Areas of focal necrosis appear beyond 15 months and are preceded by widespread demyelination. Human white matter specimens obtained post-radiation confirm early loss of oligodendrocyte progenitors and delayed onset of myelin sheath fragmentation with preserved capillaries. Conclusions This study demonstrates that long term radiation injury is associated with irreversible damage to the neural stem cell compartment in the rodent SVZ and loss of oligodendrocyte precursor cells in both rodent and human brain. Delayed onset demyelination precedes focal necrosis and is likely due to the loss of oligodendrocyte precursors and the inability of the stem cell compartment to compensate for this loss.

SCAR/WAVE and Arp2/3 are crucial for cytoskeletal remodeling at the site of myoblast fusion

Abstract: Myoblast fusion is crucial for formation and repair of skeletal muscle. Here we show that active remodeling of the actin cytoskeleton is essential for fusion in Drosophila. Using live imaging, we have identified a dynamic F-actin accumulation (actin focus) at the site of fusion. Dissolution of the actin focus directly precedes a fusion event. Whereas several known fusion components regulate these actin foci, others target additional behaviors required for fusion. Mutations in kette/Nap1, an actin polymerization regulator, lead to enlarged foci that do not dissolve, consistent with the observed block in fusion. Kette is required to positively regulate SCAR/WAVE, which in turn activates the Arp2/3 complex. Mutants in SCAR and Arp2/3 have a fusion block and foci phenotype, suggesting that Kette-SCAR-Arp2/3 participate in an actin polymerization event required for focus dissolution. Our data identify a new paradigm for understanding the mechanisms underlying fusion in myoblasts and other tissues.

The Drosophila homolog of the Exo84 exocyst subunit promotes apical epithelial identity.

Abstract: The polarized architecture of epithelial tissues involves a dynamic balance between apical and basolateral membrane domains. Here we show that epithelial polarity in the Drosophila embryo requires the exocyst complex subunit homolog Exo84. Exo84 activity is essential for the apical localization of the Crumbs transmembrane protein, a key determinant of epithelial apical identity. Adherens junction proteins become mislocalized at the cell surface in Exo84 mutants in a pattern characteristic of defects in apical, but not basolateral, components. Loss of Crumbs from the cell surface precedes the disruption of Bazooka and Armadillo localization in Exo84 mutants. Moreover, Exo84 mutants display defects in apical cuticle secretion that are similar to crumbs mutants and are suppressed by a reduction in the basolateral proteins Dlg and Lgl. In Exo84 mutants at advanced stages of epithelial degeneration, apical and adherens junction proteins accumulate in an expanded recycling endosome compartment. These results suggest that epithelial polarity in the Drosophila embryo is actively maintained by exocyst-dependent apical localization of the Crumbs transmembrane protein.

The carboxy terminus of NBS1 is required for induction of apoptosis by the MRE11 complex

Abstract: The MRE11 complex (MRE11, RAD50 and NBS1) and the ataxia-telangiectasia mutated (ATM) kinase function in the same DNA damage response pathway to effect cell cycle checkpoint activation and apoptosis. The functional interaction between the MRE11 complex and ATM has been proposed to require a conserved C-terminal domain of NBS1 for recruitment of ATM to sites of DNA damage. Human Nijmegen breakage syndrome (NBS) cells and those derived from multiple mouse models of NBS express a hypomorphic NBS1 allele that exhibits impaired ATM activity despite having an intact C-terminal domain. This indicates that the NBS1 C terminus is not sufficient for ATM function. We derived Nbs1(DeltaC/DeltaC) mice in which the C-terminal ATM interaction domain is deleted. Nbs1(DeltaC/DeltaC) cells exhibit intra-S-phase checkpoint defects, but are otherwise indistinguishable from wild-type cells with respect to other checkpoint functions, ionizing radiation sensitivity and chromosome stability. However, multiple tissues of Nbs1(DeltaC/DeltaC) mice showed a severe apoptotic defect, comparable to that of ATM- or CHK2-deficient animals. Analysis of p53 transcriptional targets and ATM substrates showed that, in contrast to the phenotype of Chk2(-/-) mice, NBS1(DeltaC) does not impair the induction of proapoptotic genes. Instead, the defects observed in Nbs1(DeltaC/DeltaC) result from impaired phosphorylation of ATM targets including SMC1 and the proapoptotic factor, BID.

Systematic review of discography as a diagnostic test for spinal pain: an update

Abstract: Background The intervertebral disc has been implicated as an etiology of chronic spine pain based on clinical, basic science, and epidemiological research. There is currently no way to determine with absolute certainty whether or not the disc is a spinal pain generator. At our current level of understanding, discography is thought of as the best tool to evaluate disc-related pain. Study design A systematic review. Objective To systematically assess the diagnostic accuracy of discography with respect to chronic spinal pain. Methods A systematic review of the literature was performed to assess the diagnostic accuracy of discography with respect to chronic spinal pain. Study inclusion/exclusion criteria were based on the modern practice of discography. Selected studies were then subjected to two rating instruments for diagnostic accuracy studies (AHRQ and QUADAS). Specific data were then culled from these studies and tabulated. Evidence was then classified into five levels: conclusive, strong, moderate, limited, or indeterminate. Results Evidence is strong for the diagnostic accuracy of discography as an imaging tool. Evidence is also strong for the ability of discography to evoke pain. There is strong evidence supporting the role of discography in identifying that subset of patients with lumbar discogenic pain. There is moderate evidence supporting the role of discography in identifying a subset of patients with cervical discogenic pain. There is limited evidence supporting the role of discography in identifying a subset of patients with thoracic discogenic pain. Conclusion Discography is a useful imaging and pain evaluation tool in identifying a subset of patients with chronic spinal pain secondary to intervertebral disc disorders.

Fully synthetic carbohydrate HIV antigens designed on the logic of the 2G12 antibody.

Abstract: Di- and trivalent glycopeptide mimics of the HIV 2G12 epitope have been synthesized and evaluated for their comparative 2G12 binding characteristics. The epitope mimics consist of a cyclic peptide scaffold (unrelated to gp120 peptide sequences) attached via aspartate linkages to two or three copies of the high-mannose glycan, Man9GlcNAc2. The synthesis has been achieved via high-yielding double and triple Lansbury aspartylations of Man9GlcNAc2-NH2 with peptides containing, respectively, two and three aspartate residues. Conjugation of such constructs with an immunogenic carrier protein, OMPC, has been accomplished through the peptide's cysteine sulfhydryl function, and Biacore assays have shown that binding affinity for 2G12 increases with increasing valency.

Mutations in the BMP pathway in mice support the existence of two molecular classes of holoprosencephaly.

Abstract: Holoprosencephaly (HPE) is a devastating forebrain abnormality with a range of morphological defects characterized by loss of midline tissue. In the telencephalon, the embryonic precursor of the cerebral hemispheres, specialized cell types form a midline that separates the hemispheres. In the present study, deletion of the BMP receptor genes, Bmpr1b and Bmpr1a, in the mouse telencephalon results in a loss of all dorsal midline cell types without affecting the specification of cortical and ventral precursors. In the holoprosencephalic Shh-/- mutant, by contrast, ventral patterning is disrupted, whereas the dorsal midline initially forms. This suggests that two separate developmental mechanisms can underlie the ontogeny of HPE. The Bmpr1a;Bmpr1b mutant provides a model for a subclass of HPE in humans: midline inter-hemispheric HPE.

The FERM protein Epb4.1l5 is required for organization of the neural plate and for the epithelial-mesenchymal transition at the primitive streak of the mouse embryo.

Abstract: During early mouse development, a single-layered epithelium is transformed into the three germ layers that are the basis of the embryonic body plan. Here we describe an ENU-induced mutation, limulus ( lulu ), which disrupts gastrulation and the organization of all three embryonic germ layers. Positional cloning and analysis of additional alleles show that lulu is a null allele of the FERM-domain gene erythrocyte protein band 4.1-like 5 ( Epb4.1l5 ). During gastrulation, some cells in lulu mutants are trapped in the primitive streak at an intermediate stage of the epithelial-mesenchymal transition; as a result, the embryos have very little paraxial mesoderm. Epithelial layers of the later lulu embryo are also disrupted: definitive endoderm is specified but does not form a gut tube, and the neural plate is broad and forms ectopic folds rather than closing to make the neural tube. In contrast to zebrafish and Drosophila , in which orthologs of Epb4.1l5 control the apical localization and activity of Crumbs proteins, mouse Crumbs proteins are localized normally to the apical surface of the lulu mutant epiblast and neural plate. However, the defects in both the lulu primitive streak and neural plate are associated with disruption of the normal organization of the actin cytoskeleton. We propose that mouse Lulu (Epb4.1l5) helps anchor the actin-myosin contractile machinery to the membrane to allow the dynamic rearrangements of epithelia that mediate embryonic morphogenesis.

The current provision of specialist health services to people with learning disabilities in England and Wales

Abstract: Considerable change has taken place in the provision of specialist health services to people with learning disabilities with the move away from institutions and an emphasis on care in the community. There is diversity between services in different parts of the country, but no existing source of information by which NHS Trusts can benchmark their own services against the national norm. This project was undertaken in order to ascertain the current provision of specialist health services in England and Wales. A questionnaire was returned by 83.9% of 161 NHS Trusts, who quantified their service in terms of in-patient and community resources. Results were analysed and presented where possible as cumulative frequencies, to facilitate benchmarking.

Validation of time-distance helioseismology by use of realistic simulations of solar convection

Abstract: Recent progress in realistic simulations of solar convection have given us an unprecedented opportunity to evaluate the robustness of solar interior structures and dynamics obtained by methods of local helioseismology. We present results of testing the time-distance method using realistic simulations. By computing acoustic wave propagation time and distance relations for different depths of the simulated data, we confirm that acoustic waves propagate into the interior and then turn back to the photosphere. This demonstrates that in numerical simulations properties of acoustic waves (p-modes) are similar to the solar conditions, and that these properties can be analyzed by the time-distance technique. For surface gravity waves (f-modes), we calculate perturbations of their travel times caused by localized downdrafts and demonstrate that the spatial pattern of these perturbations (representing so-called sensitivity kernels) is similar to the patterns obtained from the real Sun, displaying characteristic hyperbolic structures. We then test time-distance measurements and inversions by calculating acoustic travel times from a sequence of vertical velocities at the photosphere of the simulated data and inferring mean three-dimensional flow fields by performing inversion based on the ray approximation. The inverted horizontal flow fields agree very well with the simulated data in subsurface areas up to 3 Mm deep, but differ in deeper areas. Due to the cross talk effects between the horizontal divergence and downward flows, the inverted vertical velocities are significantly different from the mean convection velocities of the simulation data set. These initial tests provide important validation of time-distance helioseismology measurements of supergranular-scale convection, illustrate limitations of this technique, and provide guidance for future improvements.