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 Laboratory is currently engineering SNV revertant lines, in which the SNV is reverted back to the wildtype allele. Trio sets (heterozygous, homozygous, and revertant) are currently being distributed through the Jackson Laboratory website (https://www.jax.org/jax-mice-and-services/ipsc), along with the parental line. We have also begun to develop gene KO, fluorescently tagged, and functionalized lines and will continue to expand the repository in the next year. Finally, we will identify additional parental lines from diverse ancestral backgrounds into which we will CRISPR-edit a subset of these SNVs in the next year. . We will differentiate these cells into neurons and microglia for large-scale proteomic, transcriptomic, microscopy, and synthetic lethality screening studies on these lines. We have optimized our protocols for cell culture maintenance and differentiation in KOLF2.1J, and these protocols are available on Protocols.IO. In the next period, we will optimize immunostaining, proteomic, and library preparation for CRISPRi protocols. We will assess how these ADRD-related genetic variants impact cell morphology, transcriptomic, proteomic, and genetic interaction networks. We published a manuscript detailing our extensive characterization of the iNDI parental line (Pantazis et al., 2023), and methods for proteomic profiling of the CRISPR-edited lines (Reilly, et al., 2023). We also have an additional preprint on the CRISPR-editing with dCas9 strategy (Skarnes et al.). Pantazis CB, Yang A, Lara E, McDonough JA, Blauwendraat C, Peng L, Oguro H, Kanaujiya J, Zou J, Sebesta D, Pratt G, Cross E, Blockwick J, Buxton P, Kinner-Bibeau L, Medura C, Tompkins C, Hughes S, Santiana M, Faghri F, Nalls MA, Vitale D, Ballard S, Qi YA, Ramos DM, Anderson KM, Stadler J, Narayan P, Papademetriou J, Reilly L, Nelson MP, Aggarwal S, Rosen LU, Kirwan P, Pisupati V, Coon SL, Scholz SW, Priebe T, ttl M, Dong J, Meijer M, Janssen LJM, Lourenco VS, van der Kant R, Crusius D, Paquet D, Raulin AC, Bu G, Held A, Wainger BJ, Gabriele RMC, Casey JM, Wray S, Abu-Bonsrah D, Parish CL, Beccari MS, Cleveland DW, Li E, Rose IVL, Kampmann M, Calatayud Aristoy C, Verstreken P, Heinrich L, Chen MY, Schle B, Dou D, Holzbaur ELF, Zanellati MC, Basundra R, Deshmukh M, Cohen S, Khanna R, Raman M, Nevin ZS, Matia M, Van Lent J, Timmerman V, Conklin BR, Johnson Chase K, Zhang K, Funes S, Bosco DA, Erlebach L, Welzer M, Kronenberg-Versteeg D, Lyu G, Arenas E, Coccia E, Sarrafha L, Ahfeldt T, Marioni JC, Skarnes WC, Cookson MR, Ward ME, Merkle FT. A reference human induced pluripotent stem cell line for large-scale collaborative studies. Cell Stem Cell. 2022 Dec 1;29(12):1685-1702.e22. doi: 10.1016/j.stem.2022.11.004. PMID: 36459969; PMCID: PMC9782786. Reilly, L. et al., A fully automated FAIMS-DIA proteomic pipeline for high-throughput characterization of iPSC-derived neurons. Accepted for publication in Cell Reports Methods. 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 The CARD Advanced Analytics team has supported the development of current best practices for single cell 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. They 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 workbench, code is actively being shared via CARDs GitHub.