scholarly journals Hindbrain V2a neurons impose rhythmic activity on motor neurons in anin vitroreticulospinal circuit

2019 ◽  
Author(s):  
Adele V Bubnys ◽  
Hagar Kandel ◽  
Lee-Ming Kow ◽  
Donald W Pfaff ◽  
Inna Tabansky
Keyword(s):  
Spinal Cord ◽  
Motor Neurons ◽  
Complex Mixture ◽  
Cell Types ◽  
Neuron Activity ◽  
Neuronal Cultures ◽  
Cell Type ◽  
Medullary Reticular Formation ◽  
Spinal Motor

AbstractThe reticulospinal system is an evolutionarily conserved pathway among vertebrates that relays locomotor control signals from the hindbrain to the spinal cord. Recent studies have identified specific hindbrain cell types that participate in this circuit, including Chx10+neurons of the medullary reticular formation, which project to the spinal cord and are active during periods of locomotion. To create a system in which reticulospinal neurons communicate with spinal motor effectors, we have constructed anin vitromodel using two purified excitatory neuronal subtypes: HB9+spinal motor neurons and Chx10+hindbrain neurons. Cultured separately, these neurons exhibit cell type-specific patterns of activity; the Chx10+cultures developed regular, synchronized bursts of activity that recruited neurons across the entire culture, whereas motor neuron activity consisted of an irregular pattern. A combination of the two subtypes produced cultures in which Chx10+neurons recruited the motor neurons into synchronized network bursts, which were dependent on AMPA receptors. In addition to demonstrating that the activity ofin vitronetworks can depend on the developmental identity of their constituent neurons, we provide a new model with genetically specified nerve cell types to study the activity of a reticulospinal circuit.Significance statementModels of the brain that use cultured neurons are usually comprised of a complex mixture of different kinds of cells, making it hard to determine how each cell type contributes to the overall pattern of activity. We made a simplified culture containing two cell types known to form a reticulospinal circuitin vivo. While in isolated cultures, each cell type had a distinct pattern of activity, in coculture the activity of one cell type came to dominate, indicating that the patterns observed in complex neuronal cultures arise in part from the distinctive properties of the constituent neurons.

scholarly journals Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1–9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical Neurons

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Thu T. Duong ◽  
James Lim ◽  
Vidyullatha Vasireddy ◽  
Tyler Papp ◽  
Hung Nguyen ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Transgene Expression ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Cell Types ◽  
Egfp Expression ◽  
Cell Type ◽  
Egfp Gene ◽  
Rat Cortical Neurons

Recombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ acrossin vitroand ex vivo cellular transduction strategies. This study compared 11 rAAV serotypes, carrying one reporter transgene cassette containing a cytomegalovirus immediate-early enhancer (eCMV) and chicken beta actin (CBA) promoter driving the expression of an enhanced green-fluorescent protein (eGFP) gene, which was transduced into four different cell types: human iPSC, iPSC-derived RPE, iPSC-derived cortical, and dissociated embryonic day 18 rat cortical neurons. Each cell type was exposed to three multiplicity of infections (MOI: 1E4, 1E5, and 1E6 vg/cell). After 24, 48, 72, and 96 h posttransduction, GFP-expressing cells were examined and compared across dosage, time, and cell type. Retinal pigmented epithelium showed highest AAV-eGFP expression and iPSC cortical the lowest. At an MOI of 1E6 vg/cell, all serotypes show measurable levels of AAV-eGFP expression; moreover, AAV7m8 and AAV6 perform best across MOI and cell type. We conclude that serotype tropism is not only capsid dependent but also cell type plays a significant role in transgene expression dynamics.

Achyranthes bidentata polypeptide k enhances the survival, growth and axonal regeneration of spinal cord motor neurons in vitro

Neuroreport ◽  
2021 ◽  
Vol 32 (6) ◽  
pp. 518-524
Author(s):  
Ergai Cai ◽  
Qiong Cheng ◽  
Shu Yu ◽  
Fei Ding
Keyword(s):  
Spinal Cord ◽  
Motor Neurons ◽  
Axonal Regeneration ◽  
Achyranthes Bidentata

A novel epithelial cell from neonatal rat lung: isolation and differentiated phenotype

1990 ◽  
Vol 259 (6) ◽  
pp. L415-L425 ◽  
Author(s):  
P. E. Roberts ◽  
D. M. Phillips ◽  
J. P. Mather
Keyword(s):  
Epithelial Cell ◽  
Molecular Mechanisms ◽  
Basal Medium ◽  
Fold Increase ◽  
Cell Types ◽  
Cell Number ◽  
Neonatal Rat ◽  
Rat Lung ◽  
Cell Type

A novel epithelial cell from normal neonatal rat lung has been isolated, established, and maintained for multiple passages in the absence of serum, without undergoing crisis or senescence. By careful manipulation of the nutrition/hormonal microenvironment, we have been able to select, from a heterogeneous population, a single epithelial cell type that can maintain highly differentiated features in vitro. This cell type has characteristics of bronchiolar epithelial cells. A clonal line, RL-65, has been selected and observed for greater than 2 yr in continuous culture. It has been characterized by ultrastructural, morphological, and biochemical criteria. The basal medium for this cell line is Ham's F12/Dulbecco's modified Eagle's (DME) medium plus insulin (1 micrograms/ml), human transferrin (10 micrograms/ml), ethanolamine (10(-4) M), phosphoethanolamine (10(-4) M), selenium (2.5 x 10(-8) M), hydrocortisone (2.5 x 10(-7) M), and forskolin (5 microM). The addition of 150 micrograms/ml of bovine pituitary extract to the defined basal medium stimulates a greater than 10-fold increase in cell number and a 50- to 100-fold increase in thymidine incorporation. The addition of retinoic acid results in further enhancement of cell growth and complete inhibition of keratinization. We have demonstrated a strategy that may be applicable to isolating other cell types from the lung and maintaining their differentiated characteristics for long-term culture in vitro. Such a culture system promises to be a useful model in which to study cellular events associated with differentiation and proliferation in the lung and to better understand the molecular mechanisms involved in these events.

scholarly journals Establishment of a Human Induced Pluripotent Stem Cell-Derived Neuromuscular Co-Culture Under Optogenetic Control

2020 ◽  
Author(s):  
Elliot W. Swartz ◽  
Greg Shintani ◽  
Jijun Wan ◽  
Joseph S. Maffei ◽  
Sarah H. Wang ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Calcium Flux ◽  
Electrode Arrays ◽  
Spinal Motor Neurons ◽  
Culture Model ◽  
Skeletal Myotubes ◽  
Induced Pluripotent

SummaryThe failure of the neuromuscular junction (NMJ) is a key component of degenerative neuromuscular disease, yet how NMJs degenerate in disease is unclear. Human induced pluripotent stem cells (hiPSCs) offer the ability to model disease via differentiation toward affected cell types, however, the re-creation of an in vitro neuromuscular system has proven challenging. Here we present a scalable, all-hiPSC-derived co-culture system composed of independently derived spinal motor neurons (MNs) and skeletal myotubes (sKM). In a model of C9orf72-associated disease, co-cultures form functional NMJs that can be manipulated through optical stimulation, eliciting muscle contraction and measurable calcium flux in innervated sKM. Furthermore, co-cultures grown on multi-electrode arrays (MEAs) permit the pharmacological interrogation of neuromuscular physiology. Utilization of this co-culture model as a tunable, patient-derived system may offer significant insights into NMJ formation, maturation, repair, or pathogenic mechanisms that underlie NMJ dysfunction in disease.

scholarly journals Understanding axial progenitor biology in vivo and in vitro

Development ◽  
2021 ◽  
Vol 148 (4) ◽  
pp. dev180612
Author(s):  
Filip J. Wymeersch ◽  
Valerie Wilson ◽  
Anestis Tsakiridis
Keyword(s):  
Spinal Cord ◽  
Vertebral Column ◽  
Cell Types ◽  
Disease Modelling ◽  
Recent Developments ◽  
The Common ◽  
Key Features ◽  
Trunk Musculature

ABSTRACTThe generation of the components that make up the embryonic body axis, such as the spinal cord and vertebral column, takes place in an anterior-to-posterior (head-to-tail) direction. This process is driven by the coordinated production of various cell types from a pool of posteriorly-located axial progenitors. Here, we review the key features of this process and the biology of axial progenitors, including neuromesodermal progenitors, the common precursors of the spinal cord and trunk musculature. We discuss recent developments in the in vitro production of axial progenitors and their potential implications in disease modelling and regenerative medicine.

scholarly journals Single cell transcriptome profiling of the human developing spinal cord reveals a conserved genetic programme with human specific features

2021 ◽  
Author(s):  
Teresa Rayon ◽  
Rory J. Maizels ◽  
Christopher Barrington ◽  
James Briscoe
Keyword(s):  
Gene Expression ◽  
Spinal Cord ◽  
Single Cell ◽  
Motor Neurons ◽  
Neural Tube ◽  
Transcriptome Profiling ◽  
Cell Types ◽  
Human Embryos ◽  
Neuronal Populations ◽  
Cell Transcriptome

AbstractThe spinal cord receives input from peripheral sensory neurons and controls motor output by regulating muscle innervating motor neurons. These functions are carried out by neural circuits comprising molecularly and physiologically distinct neuronal subtypes that are generated in a characteristic spatial-temporal arrangement from progenitors in the embryonic neural tube. The systematic mapping of gene expression in mouse embryos has provided insight into the diversity and complexity of cells in the neural tube. For human embryos, however, less information has been available. To address this, we used single cell mRNA sequencing to profile cervical and thoracic regions in four human embryos of Carnegie Stages (CS) CS12, CS14, CS17 and CS19 from Gestational Weeks (W) 4-7. In total we recovered the transcriptomes of 71,219 cells. Analysis of progenitor and neuronal populations from the neural tube, as well as cells of the peripheral nervous system, in dorsal root ganglia adjacent to the neural tube, identified dozens of distinct cell types and facilitated the reconstruction of the differentiation pathways of specific neuronal subtypes. Comparison with existing mouse datasets revealed the overall similarity of mouse and human neural tube development while highlighting specific features that differed between species. These data provide a catalogue of gene expression and cell type identity in the developing neural tube that will support future studies of sensory and motor control systems and can be explored at https://shiny.crick.ac.uk/scviewer/neuraltube/.

scholarly journals CellMap: Characterizing the types and composition of iPSC-derived cells from RNA-seq data

2021 ◽  
Author(s):  
Zhengyu Ouyang ◽  
Nathanael Bourgeois ◽  
Eugenia Lyashenko ◽  
Paige Cundiff ◽  
Patrick F Cullen ◽  
...  
Keyword(s):  
Single Cell ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Model Systems ◽  
Rna Seq ◽  
Cell Type ◽  
Fine Grained ◽  
Single Nucleus ◽  
Induced Pluripotent

Induced pluripotent stem cell (iPSC) derived cell types are increasingly employed as in vitro model systems for drug discovery. For these studies to be meaningful, it is important to understand the reproducibility of the iPSC-derived cultures and their similarity to equivalent endogenous cell types. Single-cell and single-nucleus RNA sequencing (RNA-seq) are useful to gain such understanding, but they are expensive and time consuming, while bulk RNA-seq data can be generated quicker and at lower cost. In silico cell type decomposition is an efficient, inexpensive, and convenient alternative that can leverage bulk RNA-seq to derive more fine-grained information about these cultures. We developed CellMap, a computational tool that derives cell type profiles from publicly available single-cell and single-nucleus datasets to infer cell types in bulk RNA-seq data from iPSC-derived cell lines.

scholarly journals Revisiting the role of IRF3 in inflammation and immunity by conditional and specifically targeted gene ablation in mice

2018 ◽  
Vol 115 (20) ◽  
pp. 5253-5258 ◽  
Author(s):  
Hideyuki Yanai ◽  
Shiho Chiba ◽  
Sho Hangai ◽  
Kohei Kometani ◽  
Asuka Inoue ◽  
...  
Keyword(s):  
Immune Cell ◽  
Cell Types ◽  
Type I ◽  
Type I Ifn ◽  
Cell Type ◽  
Gene Ablation ◽  
Cell Type Specific

IFN regulatory factor 3 (IRF3) is a transcription regulator of cellular responses in many cell types that is known to be essential for innate immunity. To confirm IRF3’s broad role in immunity and to more fully discern its role in various cellular subsets, we engineered Irf3-floxed mice to allow for the cell type-specific ablation of Irf3. Analysis of these mice confirmed the general requirement of IRF3 for the evocation of type I IFN responses in vitro and in vivo. Furthermore, immune cell ontogeny and frequencies of immune cell types were unaffected when Irf3 was selectively inactivated in either T cells or B cells in the mice. Interestingly, in a model of lipopolysaccharide-induced septic shock, selective Irf3 deficiency in myeloid cells led to reduced levels of type I IFN in the sera and increased survival of these mice, indicating the myeloid-specific, pathogenic role of the Toll-like receptor 4–IRF3 type I IFN axis in this model of sepsis. Thus, Irf3-floxed mice can serve as useful tool for further exploring the cell type-specific functions of this transcription factor.

Chagas Disease Chemotherapy: What Do We Know So Far?

2021 ◽  
Vol 27 ◽  
Author(s):  
Aline Araujo Zuma ◽  
Wanderley de Souza
Keyword(s):  
Host Cell ◽  
Chagas Disease ◽  
Chronic Phase ◽  
Cell Types ◽  
Neglected Tropical Disease ◽  
Cell Type ◽  
Cure Rate ◽  
New Compounds

: Chagas disease is a Neglected Tropical Disease (NTD), and although endemic in Latin America, affects around 6-7 million people infected worldwide. The treatment of Chagas disease is based on benznidazole and nifurtimox, which are the only available drugs. However, they are not effective during the chronic phase and cause several side effects. Furthermore, BZ promotes cure in 80% of the patients in the acute phase, but the cure rate drops to 20% in adults in the chronic phase of the disease. In this review, we present several studies published in the last six years, which describes the antiparasitic potential of distinct drugs, from the synthesis of new compounds aiming to target the parasite, as well as the repositioning and the combination of drugs. We highlight several compounds for having shown results that are equivalent or superior to BZ, which means that they should be further studied, either in vitro or in vivo. Furthermore, we stand out the differences in the effects of BZ on the same strain of T. cruzi, which might be related to methodological differences such as parasite and cell ratios, host cell type and the time of adding the drug. In addition, we discuss the wide variety of strains and also the cell types used as a host cell, which makes it difficult to compare the trypanocidal effect of the compounds.

Engrailed-1 and netrin-1 regulate axon pathfinding by association interneurons that project to motor neurons

Development ◽  
1999 ◽  
Vol 126 (19) ◽  
pp. 4201-4212 ◽  
Author(s):  
H. Saueressig ◽  
J. Burrill ◽  
M. Goulding
Keyword(s):  
Spinal Cord ◽  
Motor Neurons ◽  
Molecular Mechanisms ◽  
Axon Growth ◽  
Cell Types ◽  
Second Phase ◽  
Axon Pathfinding ◽  
Cell Type Specific ◽  
Embryonic Spinal Cord ◽  
Ventrolateral Funiculus

During early development, multiple classes of interneurons are generated in the spinal cord including association interneurons that synapse with motor neurons and regulate their activity. Very little is known about the molecular mechanisms that generate these interneuron cell types, nor is it known how axons from association interneurons are guided toward somatic motor neurons. By targeting the axonal reporter gene τ-lacZ to the En1 locus, we show the cell-type-specific transcription factor Engrailed-1 (EN1) defines a population of association neurons that project locally to somatic motor neurons. These EN1 interneurons are born early and their axons pioneer an ipsilateral longitudinal projection in the ventral spinal cord. The EN1 interneurons extend axons in a stereotypic manner, first ventrally, then rostrally for one to two segments where their axons terminate close to motor neurons. We show that the growth of EN1 axons along a ventrolateral pathway toward motor neurons is dependent on netrin-1 signaling. In addition, we demonstrate that En1 regulates pathfinding and fasciculation during the second phase of EN1 axon growth in the ventrolateral funiculus (VLF); however, En1 is not required for the early specification of ventral interneuron cell types in the embryonic spinal cord.

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