Standardized Reporter Systems for Purification and Imaging of Human Pluripotent Stem Cell-derived Motor Neurons and Other Cholinergic Cells

Garcia-Diaz, Alejandro; Efe, Gizem; Kabra, Khushbu; Patel, Amar; Lowry, Emily; Shneider, Neil A.; Corneo, Barbara; Wichterle, Hynek

doi:10.1016/j.neuroscience.2020.06.028

Cited by 12 publications

(13 citation statements)

References 25 publications

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“…Aging represents an important determinant of disease onset in vivo that remains challenging to assess in living cells. Aged cells display defects in metabolism, nuclear structure, and nuclear Recombinases such as Cre activate reporter expression by excising premature stop signals or flipping a gene into an active orientation [113,114]. Event recording may also be implemented using CRISPR-Cas9 [115,116].…”

Section: A Vision For Circuit-based Screening In Neurodegenerative Diseasesmentioning

confidence: 99%

Synthetic gene circuits as tools for drug discovery

Beitz

Oakes

Galloway

2022

Trends in Biotechnology

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Within mammalian systems, there exists enormous opportunity to use synthetic gene circuits to enhance phenotype-based drug discovery, to map the molecular origins of disease, and to validate therapeutics in complex cellular systems. While drug discovery has relied on marker staining and high-content imaging in cell-based assays, synthetic gene circuits expand the potential for precision and speed. Here we present a vision of how circuits can improve the speed and accuracy of drug discovery by enhancing the efficiency of hit triage, capturing disease-relevant dynamics in cell-based assays, and simplifying validation and readouts from organoids and microphysiological systems (MPS). By tracking events and cellular states across multiple length and time scales, circuits will transform how we decipher the causal link between molecular events and phenotypes to improve the selectivity and sensitivity of cell-based assays. Enhancing phenotypic drug discovery with synthetic biologyDevelopment of new drugs requires identification and optimization of functional molecules that often proceeds sequentially through high-throughput discovery screening, hit validation, hit expansion and optimization, and preclinical disease modeling (see Glossary) (Figure 1, Key figure)[1]. Target-based drug discovery (TDD) programs aim to identify drug candidates that modulate a defined biological target that is hypothesized to play a causal role in initiating or sustaining a disease. TDD programs offer precision by directly screening for drug candidate efficacy using purified molecular targets [2]. By contrast, phenotypic drug discovery (PDD) programs aim to identify drug candidates that modulate a physiologically-relevant biological system or cellular signaling pathway and screen for drug efficacy using cell-based assays [2]. Cell-based assays are target-agnostic and provide the opportunity to identify therapeutics that resolve diseaseassociated phenotypic deficits even when the disease etiology remains obscure and targets are undefined. Thus, PDD has recently received renewed interest for its potential to identify first-in-class drugs that act via a novel mechanism of action (MoA) as well as identify drugs for disease with complex or unknown etiology [1][2][3][4]. In order to identify drug candidates that translate to effective therapeutics in the clinic, PDD programs require cell-based assays that faithfully recapitulate and report on disease-relevant processes. To efficiently and effectively screen in phenotypic models, assay development for initial discovery screens must balance throughput with the ability to capture these disease processes [1]. As PDD probes a larger biological space than TDD to identify potential therapeutics, the number of hits from an initial discovery screen in a PDD program may be very large [2]. Following initial discovery screens, hit validation and triage are required to produce a small number of high-confidence candidates to proceed to the resource-intensive stages of hit expansion, compound optimization, and hig...

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Section: A Vision For Circuit-based Screening In Neurodegenerative Diseasesmentioning

confidence: 99%

Synthetic gene circuits as tools for drug discovery

Beitz

Oakes

Galloway

2022

Trends in Biotechnology

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“…This has led to enormous challenges using these cultures, especially in MN survival studies, since ongoing neurogenesis occurs, leading to important confounding effects (Lamas et al, 2014). Using laborious and expensive methodologies this problem can, however, be efficiently circumvented using cell sorting strategies [for example, employing fluorescence activated cell sorting (FACS)] and adequate culturing conditions (Lamas et al, 2014;Garcia-Diaz et al, 2020).…”

Section: Generation Of Human Motor Neurons From Pluripotent Stem Cellsmentioning

confidence: 99%

Harnessing the Potential of Human Pluripotent Stem Cell-Derived Motor Neurons for Drug Discovery in Amyotrophic Lateral Sclerosis: From the Clinic to the Laboratory and Back to the Patient

Lamas

Roybon

2021

Front. Drug. Discov.

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Amyotrophic Lateral Sclerosis (ALS) is a motor neurodegenerative disorder whose cellular hallmarks are the progressive death of motor neurons (MNs) located in the anterior horn of the spinal cord, brainstem and motor cortex, and the formation of intracellular protein aggregates. Over the course of the disease, progressive paralysis takes place, leading to patient death within 3–5 years after the diagnosis. Despite decades of intensive research, only a few therapeutic options exist, with a limited benefit on the disease progression. Preclinical animal models have been very useful to decipher some aspects of the mechanisms underlying ALS. However, discoveries made using transgenic animal models have failed to translate into clinically meaningful therapeutic strategies. Thus, there is an urgent need to find solutions to discover drugs that could impact on the course of the disease, with the ultimate goal to extend the life of patients and improve their quality of life. Induced pluripotent stem cells (iPSCs), similarly to embryonic stem cells (ESCs), have the capacity to differentiate into all three embryonic germ layers, which offers the unprecedented opportunity to access patient-specific central nervous system cells in an inexhaustible manner. Human MNs generated from ALS patient iPSCs are an exciting tool for disease modelling and drug discovery projects, since they display ALS-specific phenotypes. Here, we attempted to review almost 2 decades of research in the field, first highlighting the steps required to efficiently generate MNs from human ESCs and iPSCs. Then, we address relevant ALS studies which employed human ESCs and iPSC-derived MNs that led to the identification of compounds currently being tested in clinical trials for ALS. Finally, we discuss the potential and caveats of using patient iPSC-derived MNs as a platform for drug screening, and anticipate ongoing and future challenges in ALS drug discovery.

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“…However, an accurate motor neuron reporter system is critical for live-cell imaging to compare the function and viability between motor neurons and other types of neurons in live cells. To target the spinal motor neurons specifically, the HB9 gene expressed specifically in motor neurons at early stages has been used to construct reporter lines [110]. Another gene named vesicular acetylcholine transferase (VACHT) expressed in all cholinergic neurons has also been selected for generating transgenic lines.…”

Section: The Use Of Gene Editing and Stem Cell Models For Studying Smamentioning

confidence: 99%

“…Another gene named vesicular acetylcholine transferase (VACHT) expressed in all cholinergic neurons has also been selected for generating transgenic lines. These reporter lines are created either by knocking in a fluorescent reporter gene (TdTomato) or Cre/CreERT2 recombinase into the reporter genomic loci [110]. The HB9-tdTomato reporter line is compatible with Fluorescent Activated Cell Sorting (FACS) and capable of live imaging with high magnification, while the fluorescence is weak under low-intensity illumination.…”

Section: The Use Of Gene Editing and Stem Cell Models For Studying Smamentioning

confidence: 99%

“…The tamoxifen-inducible HB9-CreERT2 reporter cell line has brighter fluorescence although only 30-40% HB9 + neurons are effectively labeled, suggesting that this inducible system needed further improvement. Interestingly, the fluorescence intensity of the VACHT-Cre/tdTomato reporter cell line was twice in magnitude compared to the HB9-tdTomato line, which makes it a better choice for live cell imaging [110]. As VACHT is expressed in all cholinergic neurons, combination with other motor neuron markers will be needed to confirm the spinal motor neuron identity.…”

Section: The Use Of Gene Editing and Stem Cell Models For Studying Smamentioning

confidence: 99%

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Stem Cell Models and Gene Targeting for Human Motor Neuron Diseases

Karpe

Chen

2021

Pharmaceuticals

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Motor neurons are large projection neurons classified into upper and lower motor neurons responsible for controlling the movement of muscles. Degeneration of motor neurons results in progressive muscle weakness, which underlies several debilitating neurological disorders including amyotrophic lateral sclerosis (ALS), hereditary spastic paraplegias (HSP), and spinal muscular atrophy (SMA). With the development of induced pluripotent stem cell (iPSC) technology, human iPSCs can be derived from patients and further differentiated into motor neurons. Motor neuron disease models can also be generated by genetically modifying human pluripotent stem cells. The efficiency of gene targeting in human cells had been very low, but is greatly improved with recent gene editing technologies such as zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and CRISPR-Cas9. The combination of human stem cell-based models and gene editing tools provides unique paradigms to dissect pathogenic mechanisms and to explore therapeutics for these devastating diseases. Owing to the critical role of several genes in the etiology of motor neuron diseases, targeted gene therapies have been developed, including antisense oligonucleotides, viral-based gene delivery, and in situ gene editing. This review summarizes recent advancements in these areas and discusses future challenges toward the development of transformative medicines for motor neuron diseases.

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Standardized Reporter Systems for Purification and Imaging of Human Pluripotent Stem Cell-derived Motor Neurons and Other Cholinergic Cells

Cited by 12 publications

References 25 publications

Synthetic gene circuits as tools for drug discovery

Synthetic gene circuits as tools for drug discovery

Harnessing the Potential of Human Pluripotent Stem Cell-Derived Motor Neurons for Drug Discovery in Amyotrophic Lateral Sclerosis: From the Clinic to the Laboratory and Back to the Patient

Stem Cell Models and Gene Targeting for Human Motor Neuron Diseases

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