Original Investigation Profiling APOL1 Nephropathy Risk Variants in Genome-Edited Kidney Organoids with Single-Cell Transcriptomics Esther Liu,1 Behram Radmanesh,1 Byungha H. Chung,2 Michael D. Donnan,1 Dan Yi,1 Amal Dadi,1 Kelly D. Smith,3 Jonathan Himmelfarb,2 Mingyao Li,4 Benjamin S. Freedman,2,3 and Jennie Lin 1,5 Abstract Background DNA variants in APOL1 associate with kidney disease, but the pathophysiologic mechanisms remain incompletely understood. Model organisms lack the APOL1 gene, limiting the degree to which disease states can be recapitulated. Here we present single-cell RNA sequencing (scRNA-seq) of genome-edited human kidney organoids as a platform for profiling effects of APOL1 risk variants in diverse nephron cell types. Methods We performed footprint-free CRISPR-Cas9 genome editing of human induced pluripotent stem cells (iPSCs) to knock in APOL1 high-risk G1 variants at the native genomic locus. iPSCs were differentiated into kidney organoids, treated with vehicle, IFN-g, or the combination of IFN-g and tunicamycin, and analyzed with scRNA-seq to profile cell-specific changes in differential gene expression patterns, compared with isogenic G0 controls. Results Both G0 and G1 iPSCs differentiated into kidney organoids containing nephron-like structures with glomerular epithelial cells, proximal tubules, distal tubules, and endothelial cells. Organoids expressed detectable APOL1 only after exposure to IFN-g. scRNA-seq revealed cell type–specific differences in G1 organoid response to APOL1 induction. Additional stress of tunicamycin exposure led to increased glomerular epithelial cell dedifferentiation in G1 organoids. Conclusions Single-cell transcriptomic profiling of human genome-edited kidney organoids expressing APOL1 risk variants provides a novel platform for studying the pathophysiology of APOL1-mediated kidney disease. KIDNEY360 1: 203–215, 2020. doi: https://doi.org/10.34067/KID.0000422019 Introduction Human kidney organoids derived from induced Apolipoprotein L1 (APOL1)-mediated kidney disease pluripotent stem cells (iPSCs) can be used to model accounts for a portion of the excess risk of CKD and genetic disease mechanisms in the native genomic ESKD among black patients (1,2). The APOL1 high-risk context and cell-type heterogeneity within the kid- genotype, defined as the presence of two risk alleles ney (5–9). Using CRISPR-Cas9–mediated genome (G1 or G2 coding variants), increases the risk of devel- editing, we engineered iPSCs homozygous for the oping CKD, but not all individuals with the high- G1 risk allele and differentiated these cells into risk genotype develop disease (3,4). Much remains three-dimensional (3D) kidney organoids. To eval- unknown regarding mechanisms and modifiers that uate cell type–specific effects of the APOL1 high- render the disease incompletely penetrant, and com- risk genotype, we also performed single-cell RNA plex interactions underlying these mechanisms are sequencing (scRNA-seq), which we and others have difficult to model outside APOL1’snativegenomic previously leveraged to uncover novel biology of locus. As such, current gaps in knowledge may not be how cell-specific phenotypes contribute to kidney fully addressed by induction of transgenic APOL1 development or disease in organoids and other expression in vivo or in vitro. Additionally, because models (10–14). Here we present the application APOL1 is widely expressed across different cell types, of genome-edited, iPSC-derived kidney organoids studying APOL1 risk variants solely within a specific and single-cell transcriptomics to profile APOL1- type of cell (e.g., podocytes) may not fully capture how mediated effects on kidneyorganoidsrelevantto these variants affect the kidney. disease processes. 1Division of Nephrology and Hypertension, Department of Medicine, Feinberg Cardiovascular and Renal Research Institute, Northwestern University Feinberg School of Medicine, Chicago, Illinois; 2Division of Nephrology, Department of Medicine, Kidney Research Institute, Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington; 3Department of Pathology, University of Washington, Seattle, Washington; 4Department of Biostatistics, Epidemiology, and Informatics, University of Pennsylvania, Philadelphia, Pennsylvania; and 5Section of Nephrology, Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois Correspondence: Dr. Jennie Lin, Northwestern University, 303 E Superior Street, Simpson Querrey Biomedical Research Center 8-405, Chicago, IL 60611. Email: [email protected] www.kidney360.org Vol 1 March, 2020 Copyright © 2020 by the American Society of Nephrology 203 204 KIDNEY360 Materials and Methods 10 mg/ml puromycin was added to iPSC culture to select iPSC Culture for cells expressing the donor plasmid (Supplemental iPSC lines previously derived from fibroblasts from a Figure 1A). Seven days later, iPSC colonies were evaluated non-African ancestry donor (1016SevA; Harvard Stem Cell for insertion of HDR template by PCR and Sanger sequenc- Institute) (15–18) and PBMCs from an African ancestry ing validation. After expansion of the successfully knocked-in donor (Penn134-61-26; WiCell) were maintained in feeder- colonies into separate lines, the piggyBac transposase expression free culture on 10-cm dishes coated with 0.5% Geltrex vector (Transposagen Bio) was introduced by electropo- (Gibco) in Modified Tenneille’s Special Recipe 1 (mTeSR1; ration. Additional screening of genotype was performed STEMCELL Technologies), supplemented with 1% penicil- to validate the puromycin cassette excision. lin/streptomycin (Gibco) and 0.02% Plasmocin (Invivogen). fi iPSCs were con rmed to be mycoplasma-free and below iPSC-Derived Kidney Organoid Differentiation passage 48. They were passaged using 1:3 Accutase (STEM- iPSCs were differentiated into kidney organoids follow- CELL Technologies). ing the previously published protocol by Freedman et al. (5) (Figure 1B). Briefly, iPSCs were dissociated with 1:3 Accu- tase and plated onto 24-well plates precoated with 0.5% CRISPR-Cas9 Genome Editing GelTrex in mTeSR1 supplemented with 10 mM Y-27632 APOL1 G1 risk variants (rs73885319 and rs60910145) ROCK Inhibitor (STEMCELL Technologies). After 24 hours, were introduced into the 1016SevA iPSC line through a another layer of GelTrex at 1.5% was added in mTeSR1 genomic footprint-free approach (Figure 1A, Supplemental media. At the end of day 4, the medium was replaced with fl Figure 1A) (19,20). Brie y, the homology-directed repair Advanced RPMI (Gibco) supplemented with 12 mM CHIR- (HDR) template containing the G1 variants was engineered 99021 and 10 ng/ml noggin (STEMCELL Technologies). using the MV-PGK-Puro-TK vector (Transposagen Bio), Approximately 60 hours later, the medium was changed referred to as the PMV vector, which houses a removable to Advanced RPMI with B27 (Gibco). Organoids were cul- fl puromycin selection cassette anked by two homology tured in this medium until collection at day 25. arms. The puromycin cassette is excisable by a piggyBac transposase, leaving only a “TTAA” sequence behind that Induction of APOL1 Expression and Endoplasmic Reticulum can be seamlessly introduced into a coding sequence by Stress carefully choosing sites where the change would be synon- Day 24 G0 and G1 kidney organoids in identically plated ymous. The G1 variants were engineered by two-step PCR wells of a 24-well plate were treated with IFN-g (25 ng/ml; of G0 genomic DNA (Supplemental Figure 1A, Supplemen- PeproTech) for 24 hours to induce APOL1 expression. This tal Table 1) to create the donor template for homology arm dose approximates previous in vitro IFN-g doses used for A, designed to flank the upstream portion of the puromycin macrophage activation (22,23). Endoplasmic reticulum (ER) selection cassette. Arm B, designed to flank the downstream stress was induced by adding 5 mM tunicamycin (Tocris) for end of the selection cassette, was amplified from G0 24 hours. genomic DNA by traditional PCR. Both arm A and arm B underwent separate TOPO TA cloning reactions (Invitro- gen) for insertion into a stable vector for subsequent sub- scRNA-seq cloning into the PMV vector. Stepwise sequential double We performed scRNA-seq on G0 and G1 day-25 kidney restriction-enzyme digests and homology-arm ligations organoids on the 1016SevA background. The organoids were performed on the PMV vector with the following pairs were treated with vehicle, IFN-g, or both IFN-g and tuni- of restriction enzymes: Not1-High Fidelity (HF) and Bbs1- camycin for 24 hours. Organoids were dissociated from the HF, Nco1-HF, and Bsa1-HF (New England Biolabs). The well with TrypLE Express (Gibco) and processed into single- ends of both homology arms bordering the cassette harbor cell suspension by gentle intermittent pipetting while incu- the TTAA piggyBac transposase cut sequence, thus allowing bating in a ThermoMixer (Eppendorf) for up to 15 minutes. 3 for the transposase to excise the cassette from both ends and Single-cell libraries were prepared using the 10 Genomics 9 leave behind the TTAA sequence in a scarless fashion (Sup- Chromium droplet-based platform and the Single Cell 5 3 plemental Figure 1B). To make this genome-editing event Library Construction Kit (10 Genomics), which was 9 footprint-free, we selected a codon site that would allow the chosen to increase read coverage over the 3 chemistry. TTAA nucleotide sequence to be knocked in without alter- At least three technical replicates (different wells from the ing the APOL1 amino acid