Biology subjects

Coon, S. L.

Publications and source records attributed to Coon, S. L..

4 recordsLinked to original sources

bioRxiv2021

A reference induced pluripotent stem cell line for large-scale collaborative studies

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate iPSC lines and deeply characterised their genetic properties using whole genome sequencing, their genomic stability upon CRISPR/Cas9-based gene editing, and their phenotypic properties including differentiation to commonly-used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and hundreds of its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field. SummaryThe authors of this collaborative study deeply characterized human induced pluripotent stem cell (iPSC) lines to rationally select a clonally-derived cell line that performs well across multiple modalities. KOLF2.1J was identified as a candidate reference cell line based on single-cell analysis of its gene expression in the pluripotent state, whole genome sequencing, genomic stability after highly efficient CRISPR-mediated gene editing, integrity of the p53 pathway, and the efficiency with which it differentiated into multiple target cell populations. Since it is deeply characterized and can be readily acquired, KOLF2.1J is an attractive reference cell line for groups working with iPSCs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/472643v6_ufig1.gif" ALT="Figure 1"> View larger version (84K): org.highwire.dtl.DTLVardef@cef106org.highwire.dtl.DTLVardef@31d5d8org.highwire.dtl.DTLVardef@1ce6d8corg.highwire.dtl.DTLVardef@17a19d2_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2021

Transcriptomic heterogeneity of Sox2-expressing pituitary cells

The mammalian pituitary gland is a complex organ consisting of hormone-producing cells (HPC), nonhormonal folliculostellate cells (FSC) and pituicytes, vascular pericytes and endothelial cells, and putative Sox2-expressing stem cells. Here, we used scRNAseq analysis of adult female rat pituitary cells to study the heterogeneity of pituitary cells with a focus on evaluating the transcriptomic profile of the Sox2-expressing population. Samples containing whole pituitary and separated anterior and posterior lobe cells allowed the identification of all expected pituitary resident cell types and lobe-specific subpopulations of vascular cells. Sox2 was expressed uniformly in all FSC, pituicytes, and a fraction of HPC. FSC comprised two subclusters; FSC1 contained more cells but expressed less genetic diversity compared to FSC2. The latter contained proliferative cells, expressed genes consistent with stem cell niche formation, including tight junctions, and shared genes with HPC. The FSC2 transcriptome profile was also consistent with the activity of pathways regulating cell proliferation and stem cell pluripotency, including the Hippo and Wnt pathways. The expression of other stem cell marker genes was common for FSC and pituicytes (Sox9, Cd9, Hes1, Vim, S100b) or cell type-specific (FSC: Prop1, Prrx1, Pitx1, Pitx2, Lhx3; pituicytes: Fgf10, Tbx3, Lhx2, Nkx2-1, Rax). FSC and pituicytes also expressed other astroglial marker genes, some common and other distinct, consistent with their identities as astroglial cells of the pituitary. These data suggest functional heterogeneity of FSC, with a larger fraction representing classical FSC, and a smaller fraction containing active stem-like cells and HPC-committed progenitors.

neuroscience↗

bioRxiv2021

A fully automated FAIMS-DIA proteomic pipeline for high-throughput characterization of iPSC-derived neurons

Fully automated proteomic pipelines have the potential to achieve deep coverage of cellular proteomes with high throughput and scalability. However, it is important to evaluate performance, including both reproducibility and ability to provide meaningful levels of biological insight. Here, we present an approach combining high field asymmetric waveform ion mobility spectrometer (FAIMS) interface and data independent acquisition (DIA) proteomics approach developed as part of the induced pluripotent stem cell (iPSC) Neurodegenerative Disease Initiative (iNDI), a large-scale effort to understand how inherited diseases may manifest in neuronal cells. Our FAIMS-DIA approach identified more than 8000 proteins per mass spectrometry (MS) acquisition as well as superior total identification, reproducibility, and accuracy compared to other existing DIA methods. Next, we applied this approach to perform a longitudinal proteomic profiling of the differentiation of iPSC-derived neurons from the KOLF2.1J parental line used in iNDI. This analysis demonstrated a steady increase in expression of mature cortical neuron markers over the course of neuron differentiation. We validated the performance of our proteomics pipeline by comparing it to single cell RNA-Seq datasets obtained in parallel, confirming expression of key markers and cell type annotations. An interactive webapp of this temporal data is available for aligned-UMAP visualization and data browsing (https://share.streamlit.io/anant-droid/singlecellumap). In summary, we report an extensively optimized and validated proteomic pipeline that will be suitable for large-scale studies such as iNDI.

neuroscience↗

bioRxiv2021

Detection of precisely edited CRISPR/Cas9 alleles through cointroduced restriction-fragment length polymorphisms

CRISPR/Cas9 is a powerful tool for producing genomic insertions and deletions (indels) to interrogate gene function. Modified CRISPR/Cas9 protocols can produce targeted genetic changes that are more precise than indels, but founder recovery is less efficient. Focusing on producing missense mutations in zebrafish using single-stranded oligo deoxynucleotide (ssODN) donor templates, we pioneered a strategy of adding synonymous changes to create novel restriction-enzyme (RE) sites, allowing detection of rare precise edits in a modified fluorescent-PCR fragment assay. We have named this process TIARS (test for incorporation of added recognition sites). Aided by TIARS, we induced two distinct amino-acid substitutions (T979I and P1387S) in the atp7a gene among somatic tissues of CRISPR-Cas9-treated F0 zebrafish. One of these F0s transmitted the allele to atp7aT979I/+ F1 progeny, and trans-heterozygosity of this allele against a null atp7a allele causes hypopigmentation, consistent with more severe pigment deficits in zebrafish or humans carrying only null mutations in atp7a/ATP7A. Design of ssODNs with novel RE recognition sites is labor-intensive, so we developed an in silico tool, TIARS Designer, and performed bioinformatic validation indicating that TIARS should be generalizable to other genes and experimental systems that employ donor template DNA.

genetics↗