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Kaykas, A.

Publications and source records attributed to Kaykas, A..

7 recordsLinked to original sources

Deep Learning Analysis on Images of iPSC-derived Motor Neurons Carrying fALS-genetics Reveals Disease-Relevant Phenotypes

Amyotrophic lateral sclerosis (ALS) is a devastating condition with very limited treatment options. It is a heterogeneous disease with complex genetics and unclear etiology, making the discovery of disease-modifying interventions very challenging. To discover novel mechanisms underlying ALS, we leverage a unique platform that combines isogenic, induced pluripotent stem cell (iPSC)-derived models of disease-causing mutations with rich phenotyping via high-content imaging and deep learning models. We introduced eight mutations that cause familial ALS (fALS) into multiple donor iPSC lines, and differentiated them into motor neurons to create multiple isogenic pairs of healthy (wild-type) and sick (mutant) motor neurons. We collected extensive high-content imaging data and used machine learning (ML) to process the images, segment the cells, and learn phenotypes. Self-supervised ML was used to create a concise embedding that captured significant, ALS-relevant biological information in these images. We demonstrate that ML models trained on core cell morphology alone can accurately predict TDP-43 mislocalization, a known phenotypic feature related to ALS. In addition, we were able to impute RNA expression from these image embeddings, in a way that elucidates molecular differences between mutants and wild-type cells. Finally, predictors leveraging these embeddings are able to distinguish between mutant and wild-type both within and across donors, defining cellular, ML-derived disease models for diverse fALS mutations. These disease models are the foundation for a novel screening approach to discover disease-modifying targets for familial ALS.

neuroscience↗

A Pooled Cell Painting CRISPR Screening Platform Enables de novo Inference of Gene Function by Self-supervised Deep Learning

Pooled CRISPR screening has emerged as a powerful method of mapping gene functions thanks to its scalability, affordability, and robustness against well or plate-specific confounders present in array-based screening 1-6. Most pooled CRISPR screens assay for low dimensional phenotypes (e.g. fitness, fluorescent markers). Higher-dimensional assays such as perturb-seq are available but costly and only applicable to transcriptomics readouts 7-11. Recently, pooled optical screening, which combines pooled CRISPR screening and microscopy-based assays, has been demonstrated in the studies of the NFkB pathway, essential human genes, cytoskeletal organization and antiviral response 12-15. While the pooled optical screening methodology is scalable and information-rich, the applications thus far employ hypothesis-specific assays. Here, we enable hypothesis-free reverse genetic screening for generic morphological phenotypes by re-engineering the Cell Painting 16 technique to provide compatibility with pooled optical screening. We validated this technique using well-defined morphological genesets (124 genes), compared classical image analysis and self-supervised learning methods using a mechanism-of-action (MoA) library (300 genes), and performed discovery screening with a druggable genome library (1640 genes) 17. Across these three experiments we show that the combination of rich morphological data and deep learning allows gene networks to emerge without the need for target-specific biomarkers, leading to better discovery of gene functions.

genomics↗

CellSIUS provides sensitive and specific detection of rare cell populations from complex single cell RNA-seq data

Comprehensive benchmarking of computational methods for single-cell RNA sequencing (scRNA-seq) analysis is scarce. Using a modular workflow and a large dataset with known cell composition, we benchmarked feature selection and clustering methodologies for scRNA-seq data. Results highlighted a methodology gap for rare cell population identification for which we developed CellSIUS (Cell Subtype Identification from Upregulated gene Sets). CellSIUS outperformed existing approaches, enabled the identification of rare cell populations and, in contrast to other methods, simultaneously revealed transcriptomic signatures indicative of the rare cells function. We exemplified the use of our workflow and CellSIUS for the characterization of a human pluripotent cell 3D spheroid differentiation protocol recapitulating deep-layer corticogenesis in vitro. Results revealed lineage bifurcation between Cajal-Retzius cells and layer V/VI neurons as well as rare cell populations that differ by migratory, metabolic, or cell cycle status, including a choroid plexus neuroepithelial subgroup, revealing previously unrecognized complexity in human stem cell-derived cellular populations.

bioinformatics↗

Genome-wide screens in accelerated human stem cell-derived neural progenitor cells identify Zika virus host factors and drivers of proliferation

Neural progenitor cells (NPCs) are essential to brain development and their dysfunction is linked to several disorders, including autism, Zika Virus Congenital Syndrome, and cancer. Understanding of these conditions has been improved by advancements with stem cell-derived NPC models. However, current differentiation methods require many days or weeks to generate NPCs and show variability in efficacy among cell lines. Here, we describe human Stem cell-derived NGN2-accelerated Progenitor cells (SNaPs), which are produced in only 48 hours. SNaPs express canonical forebrain NPC protein markers, are proliferative, multipotent, and like other human NPCs, are susceptible to Zika-mediated death. We further demonstrate SNaPs are valuable for large-scale investigations of genetic and environmental influencers of neurodevelopment by deploying them for genome-wide CRISPR-Cas9 screens. Our studies expand knowledge of NPCs by identifying known and novel Zika host factors, as well as new regulators of NPC proliferation validated by re-identification of the autism spectrum gene PTEN.

neuroscience↗

Genome-Scale CRISPR Screening Identifies Novel Human Pluripotent Gene Networks

Human pluripotent stem cells (hPSCs) generate a wide variety of disease-relevant cells that can be used to improve the translation of preclinical research. Despite the potential of hPSCs, their use for genetic screening has been limited because of technical challenges. We developed a renewable Cas9/sgRNA-hPSC library where loss-of-function mutations can be induced at will. Our inducible-mutant hPSC library can be used for an unlimited number of genome-wide screens. We screened for novel genes involved in 3 of the fundamental properties of hPSCs: Their ability to self-renew/survive, their capacity to differentiate into somatic cells, and their inability to survive as single-cell clones. We identified a plethora of novel genes with unidentified roles in hPSCs. These results are available as a resource for the community to increase the understanding of both human development and genetics. In the future, our stem cell library approach will be a powerful tool to identify disease-modifying genes.\n\nVISUAL ABSTRACT\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC=\"FIGDIR/small/323436_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (48K):\norg.highwire.dtl.DTLVardef@5101e4org.highwire.dtl.DTLVardef@5321f2org.highwire.dtl.DTLVardef@1d9c94corg.highwire.dtl.DTLVardef@12ebd53_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

P53 toxicity is a hurdle to CRISPR/CAS9 screening and engineering in human pluripotent stem cells

CRISPR/Cas9 has revolutionized our ability to engineer genomes and to conduct genome-wide screens in human cells. While some cell types are easily modified with Cas9, human pluripotent stem cells (hPSCs) poorly tolerate Cas9 and are difficult to engineer. Using a stable Cas9 cell line or transient delivery of ribonucleoproteins (RNPs) we achieved an average insertion or deletion efficiency greater than 80%. This high efficiency made it apparent that double strand breaks (DSBs) induced by Cas9 are toxic and kill most treated hPSCs. Cas9 toxicity creates an obstacle to the high-throughput use CRISPR/Cas9 for genome-engineering and screening in hPSCs. We demonstrated the toxic response is tp53-dependent and the toxic effect of tp53 severely reduces the efficiency of precise genome-engineering in hPSCs. Our results highlight that CRISPR-based therapies derived from hPSCs should proceed with caution. Following engineering, it is critical to monitor for tp53 function, especially in hPSCs which spontaneously acquire tp53 mutations.

molecular biology↗

ADAM17 Is The Main Sheddase For The Generation Of Human Triggering Receptor Expressed In Myeloid Cells (hTREM2) Ectodomain And Cleaves TREM2 After Histidine 157

Triggering receptor expressed in myeloid cells (TREM2) is a member of the immunoglobulin superfamily and is expressed in macrophages, dendritic cells, microglia, and osteoclasts. TREM2 plays a role in phagocytosis, regulates release of cytokine, contributes to microglia maintenance, and its ectodomain is shed from the cell surface. Using both pharmacological and genetic approaches we report here that the main protease contributing to the release of TREM2 ectodomain is ADAM17, (a disintegrin and metalloproteinase domain containing protein, also called TACE, TNF converting enzyme) while ADAM10 plays a minor role. Using mutational analysis, we demonstrate that the main cleavage site of the sheddases is located within the stalk region of TREM2 proximal to the plasma membrane. Complementary biochemical experiments reveal that cleavage occurs between histidine 157 and serine 158. Shedding is not altered for the R47H-mutated TREM2 protein that confers an increased risk for the development of Alzheimers disease. O-glycosylation is detected within the stalk region, but distant to the cleavage site. These findings reveal a link between shedding of TREM2 and its regulation during inflammatory conditions or chronic neurodegenerative disease like AD in which activity or expression of sheddases might be altered.

cell biology↗