Center for Alzheimer's and Related Dementias (CARD): iPSC Neurodegenerative Diseases Initiative

National Institute On Aging

Investigators

Linked publications & trials

Abstract

We have selected 134 single nucleotide variants (SNVs) associated with ADRDs across 73 genes and have developed mutant lines from our parental cell line that are heterozygous and homozygous for each SNV, through partnerships with external organizations. The development of these heterozygous and homozygous mutant lines has been completed, and Jackson Laboratories is currently engineering SNV revertant lines, in which the SNV is reverted back to the wildtype allele. We will begin to distribute trio sets (heterozygous, homozygous, and revertant) of these lines, along with the parental line through a distribution center Jackson Laboratories and additionally funded by the Chan Zuckerberg Initiative. In the next period, we will expand our collection of available trio sets, engineer HaloTag and gene knockout lines, and identify additional parental lines from diverse ancestral backgrounds into which we will CRISPR-edit a subset of these SNVs. We will differentiate these cells into neurons and microglia for large-scale proteomic, transcriptomic, microscopy, and synthetic lethality screening studies on these lines. We are currently optimizing our protocols for cell culture maintenance and differentiation, immunostaining, proteomic profiling, and library preparations for CRISPRi screens. We will assess how these ADRD-related genetic variants impact cell morphology, transcriptomic, proteomic, and genetic interaction networks. We have three pre-prints that detail our parental line characterization (Pantazis et al.), CRISPR-editing with dCas9 strategy (Skarnes et al.), and methods for proteomic profiling of the CRISPR-edited lines (Reilly et al.). 1. Pantazis, C.B., et al., A reference induced pluripotent stem cell line for large-scale collaborative studies. https://doi.org/10.1101/2021.12.15.472643. 2. Skarnes, W.C., et al., (2021). Controlling homology-directed repair outcomes in human stem cells with dCas9. https://doi.org/10.1101/2021.12.16.472942 3. Reilly, L. et al., A fully automated FAIMS-DIA proteomic pipeline for high-throughput characterization of iPSC-derived neurons. https://doi.org/10.1101/2021.11.24.469921 As part of the CARD Advanced Analytics teams efforts to standardize and harmonize workflows across projects for increased user friendliness and interoperability, we have supported the development of current best practices for single cell ATAC-seq, RNA-seq and proteomics. The work in this space synthesizes code from collaborators and key opinion leaders in these areas to build easy to use and reproducible analysis pipelines to empower CARD staff. We are also building novel semi-supervised learning tools to support the cell imaging and cell painting components of iNDI. In addition to passing prototype iNDI data to online repositories like the Alzheimers disease data workbench (https://fair.addi.ad-datainitiative.org/#/data/datasets/a_reference_induced_pluripotent_stem_cell_line_for_large_scale_collaborative_studies), code is actively being shared via CARDs GitHub (https://github.com/NIH-CARD/scOmicPipeline and https://github.com/nuan604/proteomics-data-analysis-for-iNDI)