Application of CRISPR/Cas9 editing and digital droplet PCR in human iPSCs to generate novel knock-in reporter lines to visualize dopaminergic neurons

Überbacher, Christa; Obergasteiger, Julia; Volta, Mattia; Venezia, Serena; Müller, Stefan; Pesce, Isabella; Pizzi, Sara; Lamonaca, Giulia; Picard, Anne; Cattelan, Giada; Malpeli, Giorgio; Zoli, Michèle; Beccano-Kelly, Dayne; Flynn, Rowan; Wade‐Martins, Richard; Pramstaller, Peter P.; Hicks, Andrew A.; Cowley, Sally A.; Corti, Corrado

doi:10.1016/j.scr.2019.101656

Cited by 15 publications

(9 citation statements)

References 23 publications

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“…To overcome this issue, we adapted an established strategy ( Xia et al, 2017 ; Calatayud et al, 2019 ; Hong and Daadi, 2019 ; Uberbacher et al, 2019 ) and engineered an mCherry-based TH reporter iPSC line, which enables visual identification of cells expressing TH, the rate-limiting enzyme in dopamine synthesis. The subsequent differentiation into DN cultures was performed according to two well-established procedures to evaluate if and how protocol-specific conditions affect the electrophysiological characteristics of DNs.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, a series of elegant studies demonstrated how some of these limitations can be circumvented. These studies described the generation of TH reporter iPSC lines engineered to express fluorescent proteins such as mOrange ( Calatayud et al, 2019 ), GFP ( Hong and Daadi, 2019 ; Uberbacher et al, 2019 ) or RFP ( Xia et al, 2017 ) which were introduced into the endogenous TH locus, right before TH’s stop codon using a CRISPR/Cas9-based editing strategy. Immunochemistry analysis and transcriptional assays confirmed the neuronal identity of TH-expressing (TH + ) cells, however, the electrophysiological properties of TH + and TH – subpopulations were not evaluated in these studies.…”

Section: Introductionmentioning

confidence: 99%

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Electrophysiological Properties of Induced Pluripotent Stem Cell-Derived Midbrain Dopaminergic Neurons Correlate With Expression of Tyrosine Hydroxylase

Raković

Voß

Vulinović

et al. 2022

Front. Cell. Neurosci.

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Induced pluripotent stem cell (iPSC)-based generation of tyrosine hydroxylase-positive (TH+) dopaminergic neurons (DNs) is a powerful method for creating patient-specific in vitro models to elucidate mechanisms underlying Parkinson’s disease (PD) at the cellular and molecular level and to perform drug screening. However, currently available differentiation paradigms result in highly heterogeneous cell populations, often yielding a disappointing fraction (<50%) of the PD-relevant TH+ DNs. To facilitate the targeted analysis of this cell population and to characterize their electrophysiological properties, we employed CRISPR/Cas9 technology and generated an mCherry-based human TH reporter iPSC line. Subsequently, reporter iPSCs were subjected to dopaminergic differentiation using either a “floor plate protocol” generating DNs directly from iPSCs or an alternative method involving iPSC-derived neuronal precursors (NPC-derived DNs). To identify the strategy with the highest conversion efficiency to mature neurons, both cultures were examined for a period of 8 weeks after triggering neuronal differentiation by means of immunochemistry and single-cell electrophysiology. We confirmed that mCherry expression correlated with the expression of endogenous TH and that genetic editing did neither affect the differentiation process nor the endogenous TH expression in iPSC- and NPC-derived DNs. Although both cultures yielded identical proportions of TH+ cells (≈30%), whole-cell patch-clamp experiments revealed that iPSC-derived DNs gave rise to larger currents mediated by voltage-gated sodium and potassium channels, showed a higher degree of synaptic activity, and fired trains of mature spontaneous action potentials more frequently compared to NPC-derived DNs already after 2 weeks in differentiation. Moreover, spontaneous action potential firing was more frequently detected in TH+ neurons compared to the TH– cells, providing direct evidence that these two neuronal subpopulations exhibit different intrinsic electrophysiological properties. In summary, the data reveal substantial differences in the electrophysiological properties of iPSC-derived TH+ and TH– neuronal cell populations and that the “floor plate protocol” is particularly efficient in generating electrophysiologically mature TH+ DNs, which are the most vulnerable neuronal subtype in PD.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Electrophysiological Properties of Induced Pluripotent Stem Cell-Derived Midbrain Dopaminergic Neurons Correlate With Expression of Tyrosine Hydroxylase

Raković

Voß

Vulinović

et al. 2022

Front. Cell. Neurosci.

View full text Add to dashboard Cite

show abstract

“…Despite its merits, the use of ddPCR still holds some disadvantages, such as higher operation cost, reagent costs, and availability, when compared to conventional methods [44,95]. Despite these limitations, the high accuracy and resolution of ddPCR has led it to be widely used in food sciences [31,45,[47][48][49]51,52,67,68,91,116,117] and disease and evolution studies [23,46,53,66,69,73,85,90,[92][93][94]99,101,[118][119][120][121][122][123]. These studies are continually developing the technology and creating an extensive collection and recording of the optimization process for different genes.…”

Section: Discussionmentioning

confidence: 99%

Competitiveness of Quantitative Polymerase Chain Reaction (qPCR) and Droplet Digital Polymerase Chain Reaction (ddPCR) Technologies, with a Particular Focus on Detection of Antibiotic Resistance Genes (ARGs)

Park

Rana

Sung

et al. 2021

Applied Microbiology

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With fast-growing polymerase chain reaction (PCR) technologies and various application methods, the technique has benefited science and medical fields. While having strengths and limitations on each technology, there are not many studies comparing the efficiency and specificity of PCR technologies. The objective of this review is to summarize a large amount of scattered information on PCR technologies focused on the two majorly used technologies: qPCR (quantitative polymerase chain reaction) and ddPCR (droplet-digital polymerase chain reaction). Here we analyze and compare the two methods for (1) efficiency, (2) range of detection and limitations under different disciplines and gene targets, (3) optimization, and (4) status on antibiotic resistance genes (ARGs) analysis. It has been identified that the range of detection and quantification limit varies depending on the PCR method and the type of sample. Careful optimization of target gene analysis is essential for building robust analysis for both qPCR and ddPCR. In our era where mutation of genes may lead to a pandemic of viral infectious disease or antibiotic resistance-induced health threats, this study hopes to set guidelines for meticulous detection, quantification, and analysis to help future prevention and protection of global health, the economy, and ecosystems.

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“…The use of reporter gene that is selectively expressed in post-mitotic DA neurons is a convenient solution to detect these cells in heterogenous cultures. Thus, reporter iPS cell lines expressing GFP [ 87 , 88 ] or the brighter reporter protein mOrange [ 89 ] under the control of TH promoter were developed using the CRISPR Cas9 genome editing system. These genetic modifications facilitated the identification of DA neurons in mixed neuron cultures and xenotransplants, and even enabled their isolation in a viable state by FACS [ 89 ].…”

Section: Directed Differentiation Of Es and Ips Cells Into The Midmentioning

confidence: 99%

Current State-of-the-Art and Unresolved Problems in Using Human Induced Pluripotent Stem Cell-Derived Dopamine Neurons for Parkinson’s Disease Drug Development

Antonov

Новосадова

2021

IJMS

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Human induced pluripotent stem (iPS) cells have the potential to give rise to a new era in Parkinson’s disease (PD) research. As a unique source of midbrain dopaminergic (DA) neurons, iPS cells provide unparalleled capabilities for investigating the pathogenesis of PD, the development of novel anti-parkinsonian drugs, and personalized therapy design. Significant progress in developmental biology of midbrain DA neurons laid the foundation for their efficient derivation from iPS cells. The introduction of 3D culture methods to mimic the brain microenvironment further expanded the vast opportunities of iPS cell-based research of the neurodegenerative diseases. However, while the benefits for basic and applied studies provided by iPS cells receive widespread coverage in the current literature, the drawbacks of this model in its current state, and in particular, the aspects of differentiation protocols requiring further refinement are commonly overlooked. This review summarizes the recent data on general and subtype-specific features of midbrain DA neurons and their development. Here, we review the current protocols for derivation of DA neurons from human iPS cells and outline their general weak spots. The associated gaps in the contemporary knowledge are considered and the possible directions for future research that may assist in improving the differentiation conditions and increase the efficiency of using iPS cell-derived neurons for PD drug development are discussed.

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Application of CRISPR/Cas9 editing and digital droplet PCR in human iPSCs to generate novel knock-in reporter lines to visualize dopaminergic neurons

Cited by 15 publications

References 23 publications

Electrophysiological Properties of Induced Pluripotent Stem Cell-Derived Midbrain Dopaminergic Neurons Correlate With Expression of Tyrosine Hydroxylase

Electrophysiological Properties of Induced Pluripotent Stem Cell-Derived Midbrain Dopaminergic Neurons Correlate With Expression of Tyrosine Hydroxylase

Competitiveness of Quantitative Polymerase Chain Reaction (qPCR) and Droplet Digital Polymerase Chain Reaction (ddPCR) Technologies, with a Particular Focus on Detection of Antibiotic Resistance Genes (ARGs)

Current State-of-the-Art and Unresolved Problems in Using Human Induced Pluripotent Stem Cell-Derived Dopamine Neurons for Parkinson’s Disease Drug Development

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