scholarly journals Mouse embryonic stem cells can differentiate via multiple paths to the same state

2017 ◽  
Author(s):  
James A. Briggs ◽  
Victor C. Li ◽  
Seungkyu Lee ◽  
Clifford J. Woolf ◽  
Allon M. Klein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Motor Neuron ◽  
Motor Neurons ◽  
Embryonic Stem ◽  
State Transitions ◽  
Standard Protocol ◽  
Transitional State ◽  
Single Cell Rna Sequencing ◽  
Multiple Paths ◽  
Alternative Routes

AbstractIn embryonic development, cells must differentiate through stereotypical sequences of intermediate states to generate mature states of a particular fate. By contrast, direct programming can generate similar fates through alternative routes, by directly expressing terminal transcription factors. Yet the cell state transitions defining these new routes are unclear. We applied single-cell RNA sequencing to compare two mouse motor neuron differentiation protocols: a standard protocol approximating the embryonic lineage, and a direct programming method. Both undergo similar early neural commitment. Then, rather than transitioning through spinal intermediates like the standard protocol, the direct programming path diverges into a novel transitional state. This state has specific and abnormal gene expression. It opens a ‘loop’ or ‘worm hole’ in gene expression that converges separately onto the final motor neuron state of the standard path. Despite their different developmental histories, motor neurons from both protocols structurally, functionally, and transcriptionally resemble motor neurons from embryos.

scholarly journals Mouse embryonic stem cells can differentiate via multiple paths to the same state

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
James Alexander Briggs ◽  
Victor C Li ◽  
Seungkyu Lee ◽  
Clifford J Woolf ◽  
Allon Klein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Motor Neuron ◽  
Motor Neurons ◽  
Embryonic Stem ◽  
The Novel ◽  
Transitional State ◽  
Single Cell Rna Sequencing ◽  
Intermediate States ◽  
Multiple Paths ◽  
Alternative Routes

In embryonic development, cells differentiate through stereotypical sequences of intermediate states to generate particular mature fates. By contrast, driving differentiation by ectopically expressing terminal transcription factors (direct programming) can generate similar fates by alternative routes. How differentiation in direct programming relates to embryonic differentiation is unclear. We applied single-cell RNA sequencing to compare two motor neuron differentiation protocols: a standard protocol approximating the embryonic lineage, and a direct programming method. Both initially undergo similar early neural commitment. Later, the direct programming path diverges into a novel transitional state rather than following the expected embryonic spinal intermediates. The novel state in direct programming has specific and uncharacteristic gene expression. It forms a loop in gene expression space that converges separately onto the same final motor neuron state as the standard path. Despite their different developmental histories, motor neurons from both protocols structurally, functionally, and transcriptionally resemble motor neurons isolated from embryos.

scholarly journals Modeling Poliovirus Infection Using Human Engineered Neural Tissue Enriched With Motor Neuron Derived From Embryonic Stem Cells

2021 ◽  
Vol 8 ◽  
Author(s):  
Érika Cosset ◽  
Youssef Hibaoui ◽  
Sten Ilmjärv ◽  
Pierre-Yves Dietrich ◽  
Caroline Tapparel ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Motor Neuron ◽  
Motor Neurons ◽  
Embryonic Stem ◽  
Neural Tissue ◽  
Enveloped Virus ◽  
Standardized Protocol ◽  
Molecular Events

Poliomyelitis is caused by poliovirus (PV), a positive strand non-enveloped virus. Since its discovery in the 1950s, several cell culture and molecular methods have been developed to detect and characterize the various strains of PV. Here, we provide an accurate and standardized protocol to differentiate human embryonic stem cells (hESCs) toward engineered neural tissue enriched with motor neurons (MN ENTs). These MN ENTs expressed markers of motor neuron CHAT and Hb-9 as revealed by immunofluorescence staining and quantitative RT-PCR. Interestingly, our results suggest that motor neurons are responsible for the permissiveness of poliovirus within the MN ENTs. Moreover, our study revealed the molecular events occurring upon PV-3 infection in the MN ENTs and highlighted the modulation of a set of genes involved in EGR-EP300 complex. Collectively, we report the development of a reliable in vitro model to investigate the pathophysiology of PV infection, allowing to both design and assess novel therapeutic approaches against PV infection.

scholarly journals Modeling motor neuron resilience in ALS using stem cells

2018 ◽  
Author(s):  
Ilary Allodi ◽  
Jik Nijssen ◽  
Julio Aguila Benitez ◽  
Christoph Schweingruber ◽  
Andrea Fuchs ◽  
...  
Keyword(s):  
Stem Cells ◽  
Motor Neuron ◽  
Motor Neurons ◽  
Embryonic Stem ◽  
Spinal Motor Neurons ◽  
Neuronal Progenitors ◽  
Oculomotor Neurons ◽  
Bona Fide

SUMMARYOculomotor neurons, which regulate eye movement, are resilient to degeneration in the lethal motor neuron disease amyotrophic lateral sclerosis (ALS). It would be highly advantageous if motor neuron resilience could be modeled in vitro. Towards this goal, we generated a high proportion of oculomotor neurons from mouse embryonic stem cells through temporal overexpression of Phox2a in neuronal progenitors. We demonstrate, using electrophysiology, immunocytochemistry and RNA sequencing, that in vitro generated neurons are bona fide oculomotor neurons based on their cellular properties and similarity to their in vivo counterpart in rodent and man. We also show that in vitro generated oculomotor neurons display a robust activation of survival-promoting Akt signaling and are more resilient to the ALS-like toxicity of kainic acid than spinal motor neurons. Thus, we can generate bona fide oculomotor neurons in vitro which display a resilience similar to that seen in vivo.

scholarly journals Human Motor Neuron Progenitor Transplantation Leads to Endogenous Neuronal Sparing in 3 Models of Motor Neuron Loss

2011 ◽  
Vol 2011 ◽  
pp. 1-11 ◽  
Author(s):  
Tanya J. Wyatt ◽  
Sharyn L. Rossi ◽  
Monica M. Siegenthaler ◽  
Jennifer Frame ◽  
Rockelle Robles ◽  
...  
Keyword(s):  
Animal Models ◽  
Motor Neuron ◽  
Motor Neurons ◽  
Embryonic Stem ◽  
Active Growth ◽  
Neuron Loss ◽  
Cord Injury ◽  
Human Motor ◽  
Motor Neuron Loss

Motor neuron loss is characteristic of many neurodegenerative disorders and results in rapid loss of muscle control, paralysis, and eventual death in severe cases. In order to investigate the neurotrophic effects of a motor neuron lineage graft, we transplanted human embryonic stem cell-derived motor neuron progenitors (hMNPs) and examined their histopathological effect in three animal models of motor neuron loss. Specifically, we transplanted hMNPs into rodent models of SMA (Δ7SMN), ALS (SOD1 G93A), and spinal cord injury (SCI). The transplanted cells survived and differentiated in all models. In addition, we have also found that hMNPs secrete physiologically active growth factorsin vivo, including NGF and NT-3, which significantly enhanced the number of spared endogenous neurons in all three animal models. The ability to maintain dying motor neurons by delivering motor neuron-specific neurotrophic support represents a powerful treatment strategy for diseases characterized by motor neuron loss.

scholarly journals Altered Gene Expression, Mitochondrial Damage and Oxidative Stress: Converging Routes in Motor Neuron Degeneration

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Luisa Rossi ◽  
Cristiana Valle ◽  
Maria Teresa Carrì
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Motor Neuron ◽  
Motor Neurons ◽  
Mitochondrial Damage ◽  
Energy Deficit ◽  
Control Of Gene Expression ◽  
Altered Gene Expression ◽  
Altered Gene ◽  
And Oxidative Stress

Motor neuron diseases (MNDs) are a rather heterogeneous group of diseases, with either sporadic or genetic origin or both, all characterized by the progressive degeneration of motor neurons. At the cellular level, MNDs share features such as protein misfolding and aggregation, mitochondrial damage and energy deficit, and excitotoxicity and calcium mishandling. This is particularly well demonstrated in ALS, where both sporadic and familial forms share the same symptoms and pathological phenotype, with a prominent role for mitochondrial damage and resulting oxidative stress. Based on recent data, however, altered control of gene expression seems to be a most relevant, and previously overlooked, player in MNDs. Here we discuss which may be the links that make pathways apparently as different as altered gene expression, mitochondrial damage, and oxidative stress converge to generate a similar motoneuron-toxic phenotype.

scholarly journals MicroRNAs organize intrinsic variation into stem cell states

2020 ◽  
Vol 117 (12) ◽  
pp. 6942-6950 ◽  
Author(s):  
Meenakshi Chakraborty ◽  
Sofia Hu ◽  
Erica Visness ◽  
Marco Del Giudice ◽  
Andrea De Martino ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Regulatory Network ◽  
Regulatory Network ◽  
Target Genes ◽  
Embryonic Stem ◽  
State Transitions ◽  
Environmental Signals ◽  
Morphogen Gradients ◽  
Developmental Gene Expression ◽  
Gene Regulatory

Pluripotent embryonic stem cells (ESCs) contain the potential to form a diverse array of cells with distinct gene expression states, namely the cells of the adult vertebrate. Classically, diversity has been attributed to cells sensing their position with respect to external morphogen gradients. However, an alternative is that diversity arises in part from cooption of fluctuations in the gene regulatory network. Here we find ESCs exhibit intrinsic heterogeneity in the absence of external gradients by forming interconverting cell states. States vary in developmental gene expression programs and display distinct activity of microRNAs (miRNAs). Notably, miRNAs act on neighborhoods of pluripotency genes to increase variation of target genes and cell states. Loss of miRNAs that vary across states reduces target variation and delays state transitions, suggesting variable miRNAs organize and propagate variation to promote state transitions. Together these findings provide insight into how a gene regulatory network can coopt variation intrinsic to cell systems to form robust gene expression states. Interactions between intrinsic heterogeneity and environmental signals may help achieve developmental outcomes.

scholarly journals A microfluidic approach to rescue ALS motor neuron degeneration using rapamycin

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Phaneendra Chennampally ◽  
Ambreen Sayed-Zahid ◽  
Prabakaran Soundararajan ◽  
Jocelyn Sharp ◽  
Gregory A. Cox ◽  
...  
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Mouse Model ◽  
Motor Neuron ◽  
Motor Neurons ◽  
Embryonic Stem ◽  
Immunosuppressive Drug ◽  
Motor Neuron Degeneration ◽  
Neuron Degeneration ◽  
Lateral Sclerosis

AbstractTAR DNA-binding protein-43 (TDP-43) is known to accumulate in ubiquitinated inclusions of amyotrophic lateral sclerosis affected motor neurons, resulting in motor neuron degeneration, loss of motor functions, and eventually death. Rapamycin, an mTOR inhibitor and a commonly used immunosuppressive drug, has been shown to increase the survivability of Amyotrophic Lateral Sclerosis (ALS) affected motor neurons. Here we present a transgenic, TDP-43-A315T, mouse model expressing an ALS phenotype and demonstrate the presence of ubiquitinated cytoplasmic TDP-43 aggregates with > 80% cell death by 28 days post differentiation in vitro. Embryonic stem cells from this mouse model were used to study the onset, progression, and therapeutic remediation of TDP-43 aggregates using a novel microfluidic rapamycin concentration gradient generator. Results using a microfluidic device show that ALS affected motor neuron survival can be increased by 40.44% in a rapamycin dosage range between 0.4-1.0 µM.

scholarly journals Absolute Scaling of Single-Cell Transcriptomes Reveals Pervasive Hypertranscription in Adult Stem and Progenitor Cells

2021 ◽  
Author(s):  
Yun-Kyo Kim ◽  
Miguel Ramalho-Santos
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Ribosome Biogenesis ◽  
Progenitor Cell ◽  
Dynamic Range ◽  
Tissue Injury ◽  
Embryonic Stem ◽  
State Transitions ◽  
Single Cell Rna Sequencing ◽  
Transcriptional Output

Hypertranscription facilitates biosynthetically demanding cellular state transitions through global upregulation of the nascent transcriptome. Despite its potential widespread relevance, documented examples of hypertranscription remain few and limited predominantly to early development. This limitation is in large part due to the fact that modern sequencing approaches, including single-cell RNA sequencing (scRNA-seq), generally assume similar levels of transcriptional output per cell. Here, we use molecule counting and spike-in normalization to develop absolute scaling of single-cell RNA sequencing data. Absolute scaling enables an estimation of total transcript abundances per cell, which we validate in embryonic stem cell (ESC) and germline data and apply to adult mouse organs at steady-state or during regeneration. The results reveal a remarkable dynamic range in transcriptional output among adult cell types. We find that many different multipotent stem and progenitor cell populations are in a state of hypertranscription, including in the hematopoietic system, intestine and skin. Hypertranscription marks cells with multilineage potential in adult organs, is redeployed in conditions of tissue injury, and can precede by 1-2 days bursts of proliferation during regeneration. In addition to the association between hypertranscription and the stem/progenitor cell state, we dissect the relationship between transcriptional output and cell cycle, ploidy and secretory behavior. Our analyses reveal a common set of molecular pathways associated with hypertranscription across adult organs, including chromatin remodeling, DNA repair, ribosome biogenesis and translation. Our findings introduce an approach towards maximizing single-cell RNA-seq profiling. By applying this methodology across a diverse collection of cell states and contexts, we put forth hypertranscription as a general and dynamic cellular program that is pervasively employed during development, organ maintenance and regeneration. 

scholarly journals Temporal expression profiles of lncRNA and mRNA in human embryonic stem cell-derived motor neurons during differentiation

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e10075
Author(s):  
Xue-Jiao Sun ◽  
Ming-Xing Li ◽  
Chen-Zi Gong ◽  
Jing Chen ◽  
Mohammad Nasb ◽  
...  
Keyword(s):  
Motor Neuron ◽  
Motor Neurons ◽  
Time Course ◽  
Target Genes ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Differentially Expressed ◽  
Neuron Development ◽  
Qrt Pcr

Background Human embryonic stem cells (hESC) have been an invaluable research tool to study motor neuron development and disorders. However, transcriptional regulation of multiple temporal stages from ESCs to spinal motor neurons (MNs) has not yet been fully elucidated. Thus, the goals of this study were to profile the time-course expression patterns of lncRNAs during MN differentiation of ESCs and to clarify the potential mechanisms of the lncRNAs that are related to MN differentiation. Methods We utilized our previous protocol which can harvest motor neuron in more than 90% purity from hESCs. Then, differentially expressed lncRNAs (DElncRNAs) and mRNAs (DEmRNAs) during MN differentiation were identified through RNA sequencing. Bioinformatic analyses were performed to assess potential biological functions of genes. We also performed qRT-PCR to validate the DElncRNAs and DEmRNAs. Results A total of 441 lncRNAs and 1,068 mRNAs at day 6, 443 and 1,175 at day 12, and 338 lncRNAs and 68 mRNAs at day 18 were differentially expressed compared with day 0. Bioinformatic analyses identified that several key regulatory genes including POU5F1, TDGF1, SOX17, LEFTY2 and ZSCAN10, which involved in the regulation of embryonic development. We also predicted 283 target genes of DElncRNAs, in which 6 mRNAs were differentially expressed. Significant fold changes in lncRNAs (NCAM1-AS) and mRNAs (HOXA3) were confirmed by qRT-PCR. Then, through predicted overlapped miRNA verification, we constructed a lncRNA NCAM1-AS-miRNA-HOXA3 network.

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