Generation of two iPS cell lines (HIHDNDi001-A and HIHDNDi001-B) from a Parkinson’s disease patient carrying the heterozygous p.A30P mutation in SNCA

Barbuti, Peter A.; Santos, Bruno F.R.; Dording, Claire M.; Cruciani, Gérald; Massart, François; Hummel, Andreas; Krüger, Rejko

doi:10.1016/j.scr.2020.101951

Cited by 8 publications

(14 citation statements)

References 2 publications

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“…These were coated with high concentration growth factor reduced Matrigel ® (1:100; Corning, New York, NY, USA; 354263) according to the manufacturer instructions. The iPSCs were maintained in E8 media, as previously described [27,28] and Heparin (100 ng/mL; Sigma-Aldrich, St. Louis, MO, USA; H3149). The iPSCs were maintained as colonies and passaged in Dispase ® (5 U/mL; CellSystems GmbH, Troisdorf, Germany; LS02104).…”

Section: Maintenance Of Ipscsmentioning

confidence: 99%

Using High-Content Screening to Generate Single-Cell Gene-Corrected Patient-Derived iPS Clones Reveals Excess Alpha-Synuclein with Familial Parkinson’s Disease Point Mutation A30P

Barbuti

Antony

Santos

et al. 2020

Cells

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The generation of isogenic induced pluripotent stem cell (iPSC) lines using CRISPR-Cas9 technology is a technically challenging, time-consuming process with variable efficiency. Here we use fluorescence-activated cell sorting (FACS) to sort biallelic CRISPR-Cas9 edited single-cell iPSC clones into high-throughput 96-well microtiter plates. We used high-content screening (HCS) technology and generated an in-house developed algorithm to select the correctly edited isogenic clones for continued expansion and validation. In our model we have gene-corrected the iPSCs of a Parkinson’s disease (PD) patient carrying the autosomal dominantly inherited heterozygous c.88G>C mutation in the SNCA gene, which leads to the pathogenic p.A30P form of the alpha-synuclein protein. Undertaking a PCR restriction-digest mediated clonal selection strategy prior to sequencing, we were able to post-sort validate each isogenic clone using a quadruple screening strategy prior to generating footprint-free isogenic iPSC lines, retaining a normal molecular karyotype, pluripotency and three germ-layer differentiation potential. Directed differentiation into midbrain dopaminergic neurons revealed that SNCA expression is reduced in the gene-corrected clones, which was validated by a reduction at the alpha-synuclein protein level. The generation of single-cell isogenic clones facilitates new insights in the role of alpha-synuclein in PD and furthermore is applicable across patient-derived disease models.

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Section: Maintenance Of Ipscsmentioning

confidence: 99%

Using High-Content Screening to Generate Single-Cell Gene-Corrected Patient-Derived iPS Clones Reveals Excess Alpha-Synuclein with Familial Parkinson’s Disease Point Mutation A30P

Barbuti

Antony

Santos

et al. 2020

Cells

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“…A biopsy of dermal fibroblasts was donated with informed consent at 67 years of age. The generation and characterisation of induced pluripotent stem cells (iPSCs) from the dermal fibroblasts has been described 38 and has a unique identifier HIHDNDi001-B (https://hpscreg.eu/cell-line/HIHDNDi001-B). The generation of single-cell gene-corrected patient-derived iPS clones from the A30P patient has also been described 35 .…”

Section: Methodsmentioning

confidence: 99%

“…The maintenance of induced pluripotent stem cells (iPSCs) has been previously described 38 . The generation of ventral midbrain dopaminergic (vmDA) neurons was performed according to the protocol elsewhere described 39 .…”

Section: Methodsmentioning

confidence: 99%

“…The neurons were plated at 200,000 cells/coverslip with the addition of 10 μM Rho-Kinase Inhibitor Y-27632 (10 μM; Abcam, Cambridge, UK; Ab120129). After 48 hours, the neurons were fixed and stained as previously described 38 using primary antibodies for β-3-Tubulin (TUJ1; 1:200; BioLegend, San Diego, CA, USA; 801201) and TH (1:300; Millipore, Burlington, MA, USA; AB152) 35 . For the image acquisition, 10 to 12 Z-stack images per coverslip were acquired using Zeiss spinning disk confocal microscope from four independent directed neuronal differentiations.…”

Section: Methodsmentioning

confidence: 99%

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Gene-corrected Parkinson’s disease neurons show the A30P alpha-synuclein point mutation leads to reduced neuronal branching and function

Barbuti

Santos

Antony

et al. 2020

Preprint

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Parkinson's disease is characterised by the degeneration of A9 dopaminergic neurons and the pathological accumulation of alpha-synuclein. In a patient-derived stem cell model, we have generated dopaminergic neurons from an individual harbouring the p.A30P SNCA mutation and compared those neurons against gene-corrected isogenic control cell lines. We have used confocal microscopy to assess the neuronal network, specifically segmenting dopaminergic neurons and have identified image-based phenotypes showing axonal impairment and reduced neurite branching. We show using multi-electrode array (MEA) technology that the neurons carrying the endogenous p.A30P alpha-synuclein mutation are functionally impaired and identified mitochondrial dysfunction as a pathogenic cellular phenotype. We report that against gene-corrected isogenic control cell lines the neurons carrying the p.A30P SNCA mutation have a deficit and are susceptible to the mitochondrial toxin and environmental pesticide Rotenone. Our data supports the use of isogenic cell lines in identifying image-based pathological phenotypes that can serve as an entry point for future disease modifying compound screenings and drug discovery strategies.

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“…This patient is an affected sibling of the index patient from the 1998 study [4,22]. The generation and characterisation of induced pluripotent stem cells (iPSCs) from the dermal fibroblasts has been described [23] and has a unique identifier HIHDNDi001-B (https://hpscreg.eu/cell-line/HIHDNDi001-B). Ethical approval for the development of and research pertaining to patient-derived cell lines have been given by the National Committee for Ethics in Research, Luxembourg (Comité National d'Ethique dans la Recherche; CNER #201411/05).…”

Section: Cell Line and Ethical Approvalmentioning

confidence: 99%

Using High-Content Screening Technology as a Tool to Generate Single-Cell Patient-Derived Gene-Corrected Isogenic iPS Clones for Parkinson’s Disease Research

Barbuti¹,

Antony²,

Santos³

et al. 2020

Preprint

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The generation of isogenic induced pluripotent stem cell (iPSC) lines using CRISPR-Cas9 technology is a technically challenging, time-consuming process with variable efficiency. Here we use fluorescence-activated cell sorting (FACS) to sort biallelic CRISPR-Cas9 edited single-cell iPS clones into high-throughput 96-well microtiter plates. We used high-content screening (HCS) technology and generated an in-house developed algorithm to select the correctly edited isogenic clones for continued expansion and validation. In our model we have gene-corrected the iPSCs of a Parkinson’s disease (PD) patient carrying the autosomal dominantly inherited heterozygous c.88G>C mutation in the SNCA gene, which leads to the pathogenic p.A30P form of the alpha-synuclein protein. Undertaking a PCR restriction-digest mediated clonal selection strategy prior to sequencing, we were able to post-sort validate each isogenic clone using a quadruple screening strategy. Subsequent transfection with mRNA encoding excision-only transposase allows for the generation of footprint-free isogenic iPSC lines. These monoclonal isogenic iPSC lines retain a normal molecular genotype, express pluripotency markers and have the ability to differentiate into the three germ layers. This combinatory approach of FACS, HCS and post-sorted restriction digestion facilitates the generation of isogenic cell lines for disease modelling to be scaled-up on an automated platform.

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Generation of two iPS cell lines (HIHDNDi001-A and HIHDNDi001-B) from a Parkinson’s disease patient carrying the heterozygous p.A30P mutation in SNCA

Cited by 8 publications

References 2 publications

Using High-Content Screening to Generate Single-Cell Gene-Corrected Patient-Derived iPS Clones Reveals Excess Alpha-Synuclein with Familial Parkinson’s Disease Point Mutation A30P

Using High-Content Screening to Generate Single-Cell Gene-Corrected Patient-Derived iPS Clones Reveals Excess Alpha-Synuclein with Familial Parkinson’s Disease Point Mutation A30P

Gene-corrected Parkinson’s disease neurons show the A30P alpha-synuclein point mutation leads to reduced neuronal branching and function

Using High-Content Screening Technology as a Tool to Generate Single-Cell Patient-Derived Gene-Corrected Isogenic iPS Clones for Parkinson’s Disease Research

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