Biology subjects

Schuele, B.

Publications and source records attributed to Schuele, B..

4 recordsLinked to original sources

bioRxiv2026

Optical genome mapping identifies source-associated structural variant differences across early-passage human iPSCs

BackgroundInduced pluripotent stem cells (iPSCs) are an important model for studying human diseases in vitro. However, previous studies have shown that iPSC reprogramming and extended cell culture can introduce genomic structural variants (SVs). Technologies like karyotyping, CNV microarrays, and whole-genome sequencing have limitations in resolution, sensitivity, or the ability to detect large and complex structural variants compared to optical genome mapping (OGM). OGM is a genome-wide structural variant detection method that analyzes fluorescently labeled ultra-high-molecular-weight DNA molecules to identify copy-number and balanced rearrangements. At sufficient coverage, OGM can detect SVs at approximately [≥]2 kbp and identify mosaic events supported by molecule-level evidence, offering higher resolution than conventional karyotyping or SNP-array-based QC. Here, we compared iPSC clones derived from peripheral blood mononuclear cells (PBMCs) and fibroblasts (FBCs) to determine whether starting somatic cell source is associated with differences in structural variant burden and SV-type profiles after nuclear reprogramming into iPSCs. ResultsWe analyzed 73 low-passage iPSC clones generated from 25 parental lines using OGM. Compared with PBMC-iPSCs, FBC-iPSCs showed higher SV burden with the enrichment of duplications [≥]100 kbp, more frequent overlap with protein-coding genes, fragile sites, and recurrent chromosomal hotspot regions. In contrast, PBMC-iPSCs showed fewer SVs overall, and a higher proportion of clones without detectable clone-specific SVs. ConclusionsOGM provides a high-resolution approach for post-reprogramming genomic quality control by detecting clone-specific structural variants at approximately [≥]2 kbp, including events below the resolution of conventional cytogenetic and SNP-array-based assays. In these early passage iPSCs, SVs overlapped protein-coding genes, fragile sites, and recurrent culture-associated chromosomal regions, underscoring the need for clone-level genomic assessment before downstream applications. FBC-derived iPSCs showed a higher SV burden, including more frequent and larger duplications, whereas PBMC-derived iPSCs more often lacked detectable clone-specific SVs. These findings suggest that PBMC-iPSCs and FBC-iPSCs can differ in post-reprogramming SV profiles and support the use of OGM as a QC strategy during iPSC generation and selection.

genomics↗

bioRxiv2023

Nuclease-dead S. aureus Cas9 downregulates alpha-synuclein and reduces mtDNA damage and oxidative stress levels in patient-derived stem cell model of Parkinson's disease

Parkinsons disease (PD) is one of the most common neurodegenerative diseases, but no disease modifying therapies have been successful in clinical translation presenting a major unmet medical need. A promising target is alpha-synuclein or its aggregated form, which accumulates in the brain of PD patients as Lewy bodies. While it is not entirely clear which alpha-synuclein protein species is disease relevant, mere overexpression of alpha-synuclein in hereditary forms leads to neurodegeneration. To specifically address gene regulation of alpha-synuclein, we developed a CRISPR interference (CRISPRi) system based on the nuclease dead S. aureus Cas9 (SadCas9) fused with the transcriptional repressor domain Krueppel-associated box to controllably repress alpha-synuclein expression at the transcriptional level. We screened single guide (sg)RNAs across the SNCA promoter and identified several sgRNAs that mediate downregulation of alpha-synuclein at varying levels. CRISPRi downregulation of alpha-synuclein in iPSC-derived neuronal cultures from a patient with an SNCA genomic triplication showed functional recovery by reduction of oxidative stress and mitochondrial DNA damage. Our results are proof-of-concept in vitro for precision medicine by targeting the SNCA gene promoter. The SNCA CRISPRi approach presents a new model to understand safe levels of alpha-synuclein downregulation and a novel therapeutic strategy for PD and related alpha-synucleinopathies.

neuroscience↗

bioRxiv2022

Multiplex imaging of human induced pluripotent stem cell-derived neurons with CO-Detection by indEXing (CODEX) technology

BackgroundHuman induced pluripotent stem cell (iPSC) models have been hailed as a breakthrough for understanding disease and developing new therapeutics. The major advantage of iPSC-derived neurons is that they carry the genetic background of the donor, and as such could be more predictive for clinical translation. However, the development of these cell models is time-consuming and expensive and it is thus critical to maximize biomarker readout from every model that is developed. One option is to use a highly multiplexed biomarker imaging assay, like CO-Detection by indEXing (CODEX), which allows detection of 50+ targets in situ at single-cell resolution. New MethodThis paper describes the development of CODEX in neuronal cell cultures derived from human iPSCs. ResultsWe differentiated human iPSCs into mixed neuronal and glial cultures on glass coverslips. We then developed and optimized a panel of 21 antibodies to phenotype iPSC-derived neuronal subtypes of cortical, dopaminergic, and striatal neurons, as well as astrocytes, and pre-and postsynaptic proteins. Comparison with existing methodsCompared to standard immunocytochemistry, CODEX oligoconjugated fluorophores circumvent antibody host interactions and allow for highly customized multiplexing. ConclusionWe show that CODEX can be applied to iPSC neuronal cultures and developed fixation and staining protocols for the neurons to sustain the multiple wash-stain cycles of the technology. Furthermore, we demonstrate both cellular and subcellular resolution imaging of multiplexed biomarkers in the same samples. CODEX is a powerful technique that complements other single-cell omics technologies for in-depth phenotype analysis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/479039v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@68d49dorg.highwire.dtl.DTLVardef@5ea424org.highwire.dtl.DTLVardef@16a763forg.highwire.dtl.DTLVardef@7950fc_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOC_FLOATNO CODEX(R) Multiplex Imaging in human iPSC neurons [A-D] Schematic drawings of the tools and steps used for Co-Detection by indEXing (CODEX) imaging. [A] Target-specific antibodies are conjugated to unique DNA oligonucleotide barcodes. Fluorescent reporter (excitation wavelength at 488 nm, 550 nm, or 647 nm/Cy5) carrying the complementary DNA (to the barcode) enables barcode-specific binding of the reporter to the antibody and detection by fluorescence microscopy. [B] Neuronal cell cultures are prepared for the CODEX staining and imaging by several fixation steps with different PFA concentrations followed by incubation with 100% acetone. Residual acetone is removed by drying the sample. After rehydration with PBS, autofluorescence is quenched by exposure to broad-spectrum LED light. Following a pre-staining fixation step, the sample is incubated with a mix of all conjugated primary antibodies. Excessive, unbound antibodies are removed by a washing step, leaving only the bound antibodies followed by a final post-staining fixation. [C] The CODEX Instrument Manager performs the multicycle run and controls the microscope software for automated addition of reporters, imaging, and washing of the samples (pre-stained with primary antibodies) to remove reporters from each cycle. After imaging, bound reporters are removed without damaging the tissue using a solvent, and the next set of reporters (conjugated to different barcodes) are added. [D] CODEX(R) Processor processes raw files and performs stitching, deconvolution, background subtraction, and cell segmentation. The processed images can be viewed and analyzed with the CODEX(R) Multiplex Analysis Viewer (MAV) plugin using Fiji software. C_FIG

neuroscience↗

bioRxiv2019

LRRK2 modifies α-syn pathology and spread in mouse models and human neurons

Progressive aggregation of the protein alpha-synuclein (-syn) and loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) are key histopathological hallmarks of Parkinsons disease (PD). Accruing evidence suggests that -syn pathology can propagate through neuronal circuits in the brain, contributing to the progressive nature of the disease. Thus, it is therapeutically pertinent to identify modifiers of -syn transmission and aggregation as potential targets to slow down disease progression. A growing number of genetic mutations and risk factors have been identified in studies of both familial and sporadic forms of PD. However, how these genes affect -syn aggregation and pathological spread, and whether they can be targeted for therapeutic interventions, remains unclear. We performed a targeted genetic screen of risk genes associated with PD and parkinsonism for modifiers of -syn aggregation, using an -syn preformed-fibril (PFF) induction assay. We found that decreased expression of Lrrk2 and Gba modulated -syn aggregation in mouse primary neurons. Conversely, -syn aggregation increased in primary neurons from transgenic mice expressing the PD-linked LRRK2 G2019S mutation. In vivo, we investigated -syn spreading in a LRRK2 G2019S transgenic mouse model and observed acceleration of -syn aggregation and degeneration of dopaminergic neurons in the SNpc. Furthermore, LRRK2 G2019S transgenic mice displayed more pronounced degeneration-associated neuroinflammation and exacerbated behavioral deficits. To validate our findings in a human context, we established a human -syn transmission model using induced pluripotent stem cell (iPS)-derived neurons (iNs). Recombinant human -syn PFFs triggered aggregation of endogenous -syn in a time-dependent manner in human iNs. In isogenic lines of human iNs, the G2019S mutation enhanced -syn aggregation, whereas loss of LRRK2 decreased aggregation. Collectively, these findings establish a strong interaction between the PD risk gene LRRK2 and -syn transmission across mouse and human models. Since clinical trials of LRRK2 inhibitors in PD are currently underway, our findings raise the possibility that these may be effective in PD broadly, not just in the cases caused by LRRK2 mutations.

neuroscience↗