15 results on '"Wichterle, Hynek"'
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2. Ranking reprogramming factors for cell differentiation
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Hammelman, Jennifer, Patel, Tulsi, Closser, Michael, Wichterle, Hynek, and Gifford, David
- Abstract
Transcription factor over-expression is a proven method for reprogramming cells to a desired cell type for regenerative medicine and therapeutic discovery. However, a general method for the identification of reprogramming factors to create an arbitrary cell type is an open problem. Here we examine the success rate of methods and data for differentiation by testing the ability of nine computational methods (CellNet, GarNet, EBseq, AME, DREME, HOMER, KMAC, diffTF and DeepAccess) to discover and rank candidate factors for eight target cell types with known reprogramming solutions. We compare methods that use gene expression, biological networks and chromatin accessibility data, and comprehensively test parameter and preprocessing of input data to optimize performance. We find the best factor identification methods can identify an average of 50–60% of reprogramming factors within the top ten candidates, and methods that use chromatin accessibility perform the best. Among the chromatin accessibility methods, complex methods DeepAccess and diffTF have higher correlation with the ranked significance of transcription factor candidates within reprogramming protocols for differentiation. We provide evidence that AME and diffTF are optimal methods for transcription factor recovery that will allow for systematic prioritization of transcription factor candidates to aid in the design of new reprogramming protocols.
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- 2022
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3. A Stem Cell-Based Screening Platform Identifies Compounds that Desensitize Motor Neurons to Endoplasmic Reticulum Stress
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Thams, Sebastian, Lowry, Emily Rhodes, Larraufie, Marie-Hélène, Spiller, Krista J., Li, Hai, Williams, Damian J., Hoang, Phuong, Jiang, Elise, Williams, Luis A., Sandoe, Jackson, Eggan, Kevin, Lieberam, Ivo, Kanning, Kevin C., Stockwell, Brent R., Henderson, Christopher E., and Wichterle, Hynek
- Abstract
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease selectively targeting motor neurons in the brain and spinal cord. The reasons for differential motor neuron susceptibility remain elusive. We developed a stem cell-based motor neuron assay to study cell-autonomous mechanisms causing motor neuron degeneration, with implications for ALS. A small-molecule screen identified cyclopiazonic acid (CPA) as a stressor to which stem cell-derived motor neurons were more sensitive than interneurons. CPA induced endoplasmic reticulum stress and the unfolded protein response. Furthermore, CPA resulted in an accelerated degeneration of motor neurons expressing human superoxide dismutase 1 (hSOD1) carrying the ALS-causing G93A mutation, compared to motor neurons expressing wild-type hSOD1. A secondary screen identified compounds that alleviated CPA-mediated motor neuron degeneration: three kinase inhibitors and tauroursodeoxycholic acid (TUDCA), a bile acid derivative. The neuroprotective effects of these compounds were validated in human stem cell-derived motor neurons carrying a mutated SOD1 allele (hSOD1A4V). Moreover, we found that the administration of TUDCA in an hSOD1G93Amouse model of ALS reduced muscle denervation. Jointly, these results provide insights into the mechanisms contributing to the preferential susceptibility of ALS motor neurons, and they demonstrate the utility of stem cell-derived motor neurons for the discovery of new neuroprotective compounds.
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- 2019
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4. ALS disrupts spinal motor neuron maturation and aging pathways within gene co-expression networks
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Ho, Ritchie, Sances, Samuel, Gowing, Genevieve, Amoroso, Mackenzie Weygandt, O'Rourke, Jacqueline G, Sahabian, Anais, Wichterle, Hynek, Baloh, Robert H, Sareen, Dhruv, and Svendsen, Clive N
- Abstract
Modeling amyotrophic lateral sclerosis (ALS) with human induced pluripotent stem cells (iPSCs) aims to reenact embryogenesis, maturation and aging of spinal motor neurons (spMNs) in vitro. As the maturity of spMNs grown in vitro compared to spMNs in vivo remains largely unaddressed, it is unclear to what extent this in vitro system captures critical aspects of spMN development and molecular signatures associated with ALS. Here, we compared transcriptomes among iPSC-derived spMNs, fetal spinal tissues and adult spinal tissues. This approach produced a maturation scale revealing that iPSC-derived spMNs were more similar to fetal spinal tissue than to adult spMNs. Additionally, we resolved gene networks and pathways associated with spMN maturation and aging. These networks enriched for pathogenic familial ALS genetic variants and were disrupted in sporadic ALS spMNs. Altogether, our findings suggest that developing strategies to further mature and age iPSC-derived spMNs will provide more effective iPSC models of ALS pathology.
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- 2016
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5. Iterative Role of Notch Signaling in Spinal Motor Neuron Diversification
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Tan, G. Christopher, Mazzoni, Esteban O., and Wichterle, Hynek
- Abstract
The motor neuron progenitor domain in the ventral spinal cord gives rise to multiple subtypes of motor neurons and glial cells. Here, we examine whether progenitors found in this domain are multipotent and which signals contribute to their cell-type-specific differentiation. Using an in vitro neural differentiation model, we demonstrate that motor neuron progenitor differentiation is iteratively controlled by Notch signaling. First, Notch controls the timing of motor neuron genesis by repressing Neurogenin 2 (Ngn2) and maintaining Olig2-positive progenitors in a proliferative state. Second, in an Ngn2-independent manner, Notch contributes to the specification of median versus hypaxial motor column identity and lateral versus medial divisional identity of limb-innervating motor neurons. Thus, motor neuron progenitors are multipotent, and their diversification is controlled by Notch signaling that iteratively increases cellular diversity arising from a single neural progenitor domain.
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- 2016
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6. Protein Prenylation Constitutes an Endogenous Brake on Axonal Growth
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Li, Hai, Kuwajima, Takaaki, Oakley, Derek, Nikulina, Elena, Hou, Jianwei, Yang, Wan Seok, Lowry, Emily Rhodes, Lamas, Nuno Jorge, Amoroso, Mackenzie Weygandt, Croft, Gist F., Hosur, Raghavendra, Wichterle, Hynek, Sebti, Said, Filbin, Marie T., Stockwell, Brent, and Henderson, Christopher E.
- Abstract
Suboptimal axonal regeneration contributes to the consequences of nervous system trauma and neurodegenerative disease, but the intrinsic mechanisms that regulate axon growth remain unclear. We screened 50,400 small molecules for their ability to promote axon outgrowth on inhibitory substrata. The most potent hits were the statins, which stimulated growth of all mouse- and human-patient-derived neurons tested, both in vitro and in vivo, as did combined inhibition of the protein prenylation enzymes farnesyltransferase (PFT) and geranylgeranyl transferase I (PGGT-1). Compensatory sprouting of motor axons may delay clinical onset of amyotrophic lateral sclerosis (ALS). Accordingly, elevated levels of PGGT1B, which would be predicted to reduce sprouting, were found in motor neurons of early- versus late-onset ALS patients postmortem. The mevalonate-prenylation pathway therefore constitutes an endogenous brake on axonal growth, and its inhibition provides a potential therapeutic approach to accelerate neuronal regeneration in humans.
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- 2016
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7. Modeling ALS with motor neurons derived from human induced pluripotent stem cells
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Sances, Samuel, Bruijn, Lucie I, Chandran, Siddharthan, Eggan, Kevin, Ho, Ritchie, Klim, Joseph R, Livesey, Matt R, Lowry, Emily, Macklis, Jeffrey D, Rushton, David, Sadegh, Cameron, Sareen, Dhruv, Wichterle, Hynek, Zhang, Su-Chun, and Svendsen, Clive N
- Abstract
Directing the differentiation of induced pluripotent stem cells into motor neurons has allowed investigators to develop new models of amyotrophic lateral sclerosis (ALS). However, techniques vary between laboratories and the cells do not appear to mature into fully functional adult motor neurons. Here we discuss common developmental principles of both lower and upper motor neuron development that have led to specific derivation techniques. We then suggest how these motor neurons may be matured further either through direct expression or administration of specific factors or coculture approaches with other tissues. Ultimately, through a greater understanding of motor neuron biology, it will be possible to establish more reliable models of ALS. These in turn will have a greater chance of validating new drugs that may be effective for the disease.
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- 2016
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8. Functional Diversity of ESC-Derived Motor Neuron Subtypes Revealed through Intraspinal Transplantation.
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Peljto, Mirza, Dasen, Jeremy S., Mazzoni, Esteban O., Jessell, Thomas M., and Wichterle, Hynek
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CYTOLOGICAL research ,EMBRYONIC stem cells ,CELL culture ,CELL differentiation ,MOTOR neurons - Abstract
Cultured ESCs can form different classes of neurons, but whether these neurons can acquire specialized subtype features typical of neurons in vivo remains unclear. We show here that mouse ESCs can be directed to form highly specific motor neuron subtypes in the absence of added factors, through a differentiation program that relies on endogenous Wnts, FGFs, and Hh-mimicking the normal program of motor neuron subtype differentiation. Molecular markers that characterize motor neuron subtypes anticipate the functional properties of these neurons in vivo: ESC-derived motor neurons grafted isochronically into chick spinal cord settle in appropriate columnar domains and select axonal trajectories with a fidelity that matches that of their in vivo generated counterparts. ESC-derived motor neurons can therefore be programmed in a predictive manner to acquire molecular and functional properties that characterize one of the many dozens of specialized motor neuron subtypes that exist in vivo. [ABSTRACT FROM AUTHOR]
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- 2010
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9. Human Stem Cell-Derived Spinal Cord Astrocytes with Defined Mature or Reactive Phenotypes
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Roybon, Laurent, Lamas, Nuno J., Garcia-Diaz, Alejandro, Yang, Eun Ju, Sattler, Rita, Jackson-Lewis, Vernice, Kim, Yoon A., Kachel, C. Alan, Rothstein, Jeffrey D., Przedborski, Serge, Wichterle, Hynek, and Henderson, Christopher E.
- Abstract
Differentiation of astrocytes from human stem cells has significant potential for analysis of their role in normal brain function and disease, but existing protocols generate only immature astrocytes. Using early neuralization, we generated spinal cord astrocytes from mouse or human embryonic or induced pluripotent stem cells with high efficiency. Remarkably, short exposure to fibroblast growth factor 1 (FGF1) or FGF2 was sufficient to direct these astrocytes selectively toward a mature quiescent phenotype, as judged by both marker expression and functional analysis. In contrast, tumor necrosis factor alpha and interleukin-1β, but not FGFs, induced multiple elements of a reactive inflammatory phenotype but did not affect maturation. These phenotypically defined, scalable populations of spinal cord astrocytes will be important both for studying normal astrocyte function and for modeling human pathological processes in vitro.
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- 2013
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10. Differentiation of Mouse Embryonic Stem Cells to Spinal Motor Neurons
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Wichterle, Hynek and Peljto, Mirza
- Abstract
Controlled differentiation of embryonic stem (ES) cells into clinically relevant cell types is a fundamental goal of stem cell research. This unit describes one of the most efficient protocols for conversion of mouse ES cells into a defined type of nerve cells, the spinal motor neurons. ES cells are separated from feeder mouse embryonic fibroblasts and aggregated to form embryoid bodies (EBs). Two days after the withdrawal of growth factors, EBs reach a stage at which they are responsive to patterning signals and can be effectively induced with retinoic acid (RA) to differentiate into spinal nerve cells. Nascent neural cells become responsive to the ventralizing signal sonic hedgehog (Hh) that controls expression of ventral spinal progenitor markers and initiates the genetic program of motor neuron differentiation. Curr. Protoc. Stem Cell Biol. 5:1H.1.1‐1H.1.9. © 2008 by John Wiley & Sons, Inc.
- Published
- 2008
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11. In utero fate mapping reveals distinct migratory pathways and fates of neurons born in the mammalian basal forebrain
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Wichterle, Hynek, Turnbull, Daniel H., Nery, Susana, Fishell, Gord, and Alvarez-Buylla, Arturo
- Abstract
Recent studies suggest that neurons born in the developing basal forebrain migrate long distances perpendicularly to radial glia and that many of these cells reach the developing neocortex. This form of tangential migration, however, has not been demonstrated in vivo, and the sites of origin, pathways of migration and final destinations of these neurons in the postnatal brain are not fully understood. Using ultrasound-guided transplantation in utero, we have mapped the migratory pathways and fates of cells born in the lateral and medial ganglionic eminences (LGE and MGE) in 13.5-day-old mouse embryos. We demonstrate that LGE and MGE cells migrate along different routes to populate distinct regions in the developing brain. We show that LGE cells migrate ventrally and anteriorly, and give rise to the projecting medium spiny neurons in the striatum, nucleus accumbens and olfactory tubercle, and to granule and periglomerular cells in the olfactory bulb. By contrast, we show that the MGE is a major source of neurons migrating dorsally and invading the developing neocortex. MGE cells migrate into the neocortex via the neocortical subventricular zone and differentiate into the transient subpial granule neurons in the marginal zone and into a stable population of GABA-, parvalbumin- or somatostatin-expressing interneurons throughout the cortical plate.
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- 2001
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12. Sonic hedgehog contributes to oligodendrocyte specification in the mammalian forebrain
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Nery, Susana, Wichterle, Hynek, and Fishell, Gord
- Abstract
This study addresses the role of Sonic hedgehog (Shh) in promoting the generation of oligodendrocytes in the mouse telencephalon. We show that in the forebrain, expression of the early oligodendrocyte markers Olig2, plp/dm20 and PDGFRα corresponds to regions of Shh expression? To directly test if Shh can induce the development of oligodendrocytes within the telencephalon, we use retroviral vectors to ectopically express Shh within the mouse embryonic telencephalon. We find that infections with Shh-expressing retrovirus at embryonic day 9.5, result in ectopic Olig2 and PDGFRα expression by mid-embryogenesis. By postnatal day 21, cells expressing ectopic Shh overwhelmingly adopt an oligodendrocyte identity? To determine if the loss of telencephalic Shh correspondingly results in the loss of oligodendrocyte production, we studied Nkx2.1 mutant mice in which telencephalic expression of Shh is selectively lost. In accordance with Shh playing a role in oligodendrogenesis, within the medial ganglionic eminence ofNkx2.1 mutants, the early expression of PDGFRα is absent and the level of Olig2 expression is diminished in this region. In addition, in these same mutants, expression of both Shh and plp/dm20 is lost in the hypothalamus. Notably, in the prospective amygdala region where Shh expression persists in the Nkx2.1 mutant, the presence of plp/dm20 is unperturbed. Further supporting the idea that Shh is required for the in vivo establishment of early oligodendrocyte populations, expression of PDGFRα can be partially rescued by virally mediated expression of Shh in the Nkx2.1 mutant telencephalon. Interestingly, despite the apparent requirement for Shh for oligodendrocyte specification in vivo, all regions of either wild-type or Nkx2.1 mutant telencephalon are competent to produce oligodendrocytes in vitro. Furthermore, analysis of CNS tissue from Shh null animals definitively shows that, in vitro, Shh is not required for the generation of oligodendrocytes. We propose that oligodendrocyte specification is negatively regulated in vivo and thatShh generates oligodendrocytes by overcoming this inhibition. Furthermore, it appears that a Shh-independent pathway for generating oligodendrocytes exists.
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- 2001
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13. Mutations of Conserved Arginines in the Membrane Domain of Erythroid Band 3 Lead to a Decrease in Membrane-Associated Band 3 and to the Phenotype of Hereditary Spherocytosis
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Jarolim, Petr, Rubin, Hillard L., Brabec, Vaclav, Chrobak, Ladislav, Zolotarev, Alexander S., Alper, Seth L., Brugnara, Carlo, Wichterle, Hynek, and Palek, Jiri
- Abstract
To elucidate the molecular basis of band 3 deficiency in a recently defined subset of patients with autosomal dominant hereditary spherocytosis (HS), we screened band 3 cDNA for single-strand conformation polymorphism (SSCP). In 5 of 17 (29%) unrelated HS subjects with band 3 deficiency, we detected substitutions R760W, R760Q, R808C, and R870W that were all coinherited with the HS phenotype. The involved arginines are highly conserved throughout evolution. To examine whether or not the product of the mutant allele is inserted into the membrane, we studied one HS subject who was doubly heterozygous for the R760Q mutation and the K56E (band 3MEMPHIS) polymorphism that results in altered electrophoretic mobility of the band 3 Memphis proteolytic fragments. We detected only the band 3MEMPHISin the erythrocyte membrane indicating that the protein product of the mutant, R760Q, band 3 allele is absent from the red blood cell membrane. These findings suggest that the R760Q substitution, and probably the other arginine substitutions, produce band 3 deficiency either by precluding incorporation of the mutant protein into the red blood cell membrane or by leading to loss of mutant protein from differentiating erythroid precursors.
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- 1995
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14. Architecture and cell types of the adult subventricular zone: In search of the stem cells
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García-Verdugo, José Manuel, Doetsch, Fiona, Wichterle, Hynek, Lim, Daniel A., and Alvarez-Buylla, Arturo
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Neural stem cells are maintained in the subventricular zone (SVZ) of the adult mammalian brain. Here, we review the cellular organization of this germinal layer and propose lineage relationships of the three main cell types found in this area. The majority of cells in the adult SVZ are migrating neuroblasts (type A cells) that continue to proliferate. These cells form an extensive network of tangentially oriented pathways throughout the lateral wall of the lateral ventricle. Type A cells move long distances through this network at high speeds by means of chain migration. Cells in the SVZ network enter the rostral migratory stream (RMS) and migrate anteriorly into the olfactory bulb, where they differentiate into interneurons. The chains of type A cells are ensheathed by slowly proliferating astrocytes (type B cells), the second most common cell type in this germinal layer. The most actively proliferating cells in the SVZ, type C, form small clusters dispersed throughout the network. These foci of proliferating type C cells are in close proximity to chains of type A cells. We discuss possible lineage relationships among these cells and hypothesize which are the neural stem cells in the adult SVZ. In addition, we suggest that interactions between type A, B, and C cells may regulate proliferation and initial differentiation within this germinal layer. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 234248, 1998
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- 1998
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15. Ligand-dependent dynamics of retinoic acid receptor binding during early neurogenesis
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Mahony, Shaun, Mazzoni, Esteban, McCuine, Scott, Young, Richard, Wichterle, Hynek, and Gifford, David
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Among its many roles in development, retinoic acid determines the anterior-posterior identity of differentiating motor neurons by activating retinoic acid receptor (RAR)-mediated transcription. RAR is thought to bind the genome constitutively, and only induce transcription in the presence of the retinoid ligand. However, little is known about where RAR binds to the genome or how it selects target sites.
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- 2011
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