Syntactic affixation

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Linguistics and Philosophy, 1984.MICROFICHE COPY AVAILABLE IN ARCHIVES AND HUMANITIES.Bibliography: leaves 260-264.by Nigel Alexander John Fabb.Ph.D

Human pluripotent stem cell derived midbrain PITX3eGFP/w neurons: a versatile tool for pharmacological screening and neurodegenerative modeling

AbstractPITX3 expression is confined to adult midbrain dopaminergic neurons. In this study we describe the generation and basic functional characteristics of midbrain dopaminergic neurons derived from a human pluripotent stem cell line expressing eGFP under the control of the PITX3 promoter. Flow cytometry shows that eGFP is evident in 15% of the neuron population at day 12 of differentiation and this level is maintained until at least day 80. From day 20-80 of differentiation intracellular chloride decreases and throughout this period around ~20% of PITX3eGFP/w neurons exhibit spontaneous Ca2+ transients (from 3.3+/-0.3 to 5.0+/-0.1 min-1, respectively). These neurons also respond to any of ATP, glutamate, acetylcholine or noradrenaline with elevations of intracellular calcium. As neuronal cultures mature more dopamine is released and single PITX3eGFP/w neurons begin to respond to more than one neurotransmitter. MPP+ and tumor necrosis factor(TNF), but not prostaglandin E2, caused death of the ~50% of PITX3eGFP/w neurons (day 80). Tracking eGFP using time lapse confocal microscopy over 24 hours demonstrated significant TNF-mediated neurite retraction over time. These PITX3eGFP/w neurons are amenable to flow cytometry, release dopamine and respond to multiple neurotransmitters with elevations of intracellular calcium, we believe that they represent a versatile system for neuropharmacological and neurotoxicological studies

Extended periods of neural induction and propagation of embryonic stem cell-derived neural progenitors with EGF and FGF2 enhances Lmx1a expression and neurogenic potential

Neural stem (NS) cells are multipotent cells defined by their capacity to proliferate and differentiate into all neuronal and glial phenotypes. NS cells can be obtained from specific regions of the adult brain, or generated from embryonic stem cells (ESCs). NS cells differentiate into neural progenitor (NP) cells and subsequently neural precursors, as transient steps towards terminal differentiation into specific mature neuronal or glial phenotypes. When cultured in EGF and FGF2, ESC-derived NS cells have been reported to be stable and multipotent. Conditions that enable differentiation of NS cells through the committed progenitor and precursor stages to specific neuronal subtypes have not been fully established. In this study we investigated, using Lmx1a reporter ESCs, whether the length of neural induction (NI) dictated the phenotypic potential of cultures of ESC-derived NS cells or NP cells. Following 4, 7 or 10 day periods of NI, ESCs in monolayer culture were harvested and cultured as neurospheres, prior to replating as monolayer cultures for several passages in EGF and FGF2. The NS/NP cultures were then directed towards mature neuronal fates over 16–17 days. 4 and 7-day NS cell cultures could not be differentiated towards dopaminergic, serotonergic or cholinergic fates as determined by the absence of tyrosine hydroxylase, 5-HT or choline acetyltransferase (ChAT) immunolabelling. In contrast NS/NP cultures derived after 10 days of NI were able to generate tyrosine hydroxylase and 5-HT positive neurons (24 ± 6 and 13 ± 1% of the βIII-tubulin positive population, respectively, n = 3). Our data suggest that extended periods of neural induction enhanced the potential of mouse ESC-derived NS/NP cells to generate specific subtypes of neurons. NS/NP cells derived after shorter periods of NI appeared to be lineage-restricted in relation to the neuronal subtypes observed after removal of EGF

DNA-Dependent Protein Kinase Is a Context Dependent Regulator of Lmx1a and Midbrain Specification

<div><p>The identification of small molecules capable of directing pluripotent cell differentiation towards specific lineages is highly desirable to both reduce cost, and increase efficiency. Within neural progenitors, LIM homeobox transcription factor 1 alpha (Lmx1a) is required for proper development of roof plate and cortical hem structures of the forebrain, as well as the development of floor plate and midbrain dopaminergic neurons. In this study we generated homologous recombinant cell lines expressing either luciferase or β-lactamase under the control of the <i>Lmx1a</i> promoter, and used these cell lines to investigate kinase-mediated regulation of Lmx1a activity during neuronal differentiation. A screen of 143 small molecule tyrosine kinase inhibitors yielded 16 compounds that positively or negatively modulated Lmx1a activity. Inhibition of EGF, VEGF and DNA-dependent protein kinase (DNA-PK) signaling significantly upregulated Lmx1a activity whereas MEK inhibition strongly downregulated its activity. Quantitative FACS analysis revealed that the DNA-PK inhibitor significantly increased the number of Lmx1a+ progenitors while subsequent qPCR showed an upregulation of Notch effectors, the basic helix-loop-helix genes, <i>Hes5</i> and <i>Hey1</i>. FACS further revealed that DNA-PK-mediated regulation of Lmx1a+ cells is dependent on the rapamycin-sensitive complex, mTORC1. Interestingly, this DNA-PK inhibitor effect was preserved in a co-culture differentiation protocol. Terminal differentiation assays showed that DNA-PK inhibition shifted development of neurons from forebrain toward midbrain character as assessed by Pitx3/TH immunolabeling and corresponding upregulation of midbrain (<i>En1</i>), but not forebrain (<i>FoxG1</i>) transcripts. These studies show that Lmx1a signaling in mouse embryonic stem cells contributes to a molecular cascade establishing neuronal specification. The data presented here identifies a novel regulatory pathway where signaling from DNA-PK appears to suppress midbrain-specific Lmx1a expression.</p> </div

Modulation of Lmx1a reporter gene expression by combinations of small molecule kinase inhibitors.

<p>Luciferase activity was assayed on day 8 after exposure of differentiating mESCs to combinations of SMAs from day 4-8. (A) Activity of EGF inhibitor plus other SMAs (B) Activity of VEGF inhibitor plus other SMAs, ***p&lt;0.001 and *p&lt;0.05, one-way ANOVA using Bonferroni’s multiple comparison test. Data expressed as mean ± SEM, n=4.</p

Activation of PI3K and PIKK signaling pathways induces Lmx1a reporter gene expression.

<p>Panel A shows the luciferase activity from <i>Lmx1a</i>-<i>luc</i> mESCs on day 8, after incubation with combinations of SMAs from day 4-8. PD169316 and SB220025 are inhibitors of p38 MAPK pathway whereas U-73122 specifically inhibits PLC-γ signaling. Solid colored columns represent incubations with single SMAs whereas striped columns depict combination incubation of SMAs. Western Blot analysis (B and C) revealed that DNA-PKi induces phosphorylation of mTOR, another member of the PIKK superfamily, whilst Rapamycin inhibited its phosphorylation. Membranes were probed for mTOR specifically phosphorylated at Serine 2448 (Ser2448) and β-actin using primary antibodies. Proteins were visualized using donkey anti-rabbit AlexaFluor 680 and donkey anti-mouse IE-800 for mTOR and β-actin, respectively. Data presented as mean ± SEM, ***p&lt;0.001, **p&lt;0.01 and *p&lt;0.05 for one-way ANOVA compared to vehicle using Bonferroni’s post-hoc test, n=3. Abbreviations: PTENi- Phosphatase tensin homologue inhibitor, JNK- Janus Kinase, MEK- Mitogen-activated protein kinase kinase, PDA- Phorbol di-acetate and mTOR- Mammalian target of rapamycin.</p

Small molecule inhibitor screens reveal multiple inhibitor classes effect Lmx1a reporter gene expression.

<p>143 kinase inhibitors were screened yielding 16 small molecule agents (SMAs) for further analysis. Panel A shows Luciferase activity on day 8 of differentiation after incubation of <i>Lmx1a</i>-<i>luc</i> mESCs with SMAs from day 4 to day 8. Panel B shows that incubation with inhibitors that promote Lmx1a activity also increases the generation of Lmx1a+ cells. Color coded bars in A represent different SMAs with known similar kinase inhibitory activity, where black arrows indicate SMAs selected for analysis in panel B See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078759#pone.0078759.s001" target="_blank">Figures <b>S1</b></a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078759#pone.0078759.s002" target="_blank"><b>S2</b></a> for compound libraries used. Data expressed as mean ± SEM of at least four independent experiments. **p&lt;0.01 and *p&lt;0.05 one-way ANOVA with Bonferroni’s post-hoc test, n=4. </p