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   Integrated, automated maintenance, expansion and differentiation of 2D and 3D patient-derived cellular models for high throughput drug screeningBoussaad, Ibrahim  ; Cruciani, Gérald ; Bolognin, Silvia et alin Scientific Reports (2021) Detailed reference viewed: 357 (5 UL) 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.; ; Dording, Claire et alin Stem cell research (2020), 48 Dermal fibroblasts from a patient carrying a heterozygous c.88G > C mutation in the SNCA gene that encodes alpha-synuclein were reprogrammed to pluripotency by retroviruses. This pathogenic mutation ... [more ▼] Dermal fibroblasts from a patient carrying a heterozygous c.88G > C mutation in the SNCA gene that encodes alpha-synuclein were reprogrammed to pluripotency by retroviruses. This pathogenic mutation generates the p.A30P form of the alpha-synuclein protein leading to autosomal dominantly inherited Parkinson's disease (PD). Two clonal iPS cell lines were generated (A30P-3 and A30P-4) and characterised by validating the silencing of viral transgenes, the expression of endogenous pluripotency genes, directed differentiation into three germ layers in-vitro and a stable molecular genotype. These iPSC lines will serve as a valuable resource in determining the role of the p.A30P SNCA mutation in PD pathogenesis. [less ▲] Detailed reference viewed: 121 (2 UL)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, Peter ; Antony, Paul ; Rodrigues Santos, Bruno et alin Cells (2020), 9(9), 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 ... [more ▼] 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. [less ▲] Detailed reference viewed: 190 (5 UL) |
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