title: Sample 20
organism: Homo sapiens
description: Protocols: Cells from kidney organoids were collected at day 0, 7, 12, 19 and 25 of the differentiation protocol. CRISPR/Cas9 gene editing was used to mutate HNF1B in MAN13 hESCs (PMID: 28583200). We first constructed a pair of plasmids expressing the nickase (D10A) version of Cas9 (Cas9 n ) and a gRNA each. The two gRNAs targeted the Cas9 n to nick at position 231 of the coding strand of HNF1B and at position 171 of the complementary strand, resulting in a deletion of 58 bases (plus/minus any indels) near the beginning of the coding sequence of the gene (Fig. 1A). Each gRNA-coding insert was designed as a pair of oligonucleotides that, upon annealing, produced overhangs that allowed cloning into the pX461 plasmid vector, (Addgene, #48140), under the control of a U6 promoter. The vector also expresses Cas9 n , and a GFP tag for cell sorting. The primers used to create the inserts are listed in Table S2. Each pair of oligonucleotides was annealed and phosphorylated, using T4 Polynucleotide Kinase (NEB, #M0201S), then ligated into BbsI-digested pX461, to produce pX461-gRNA(HNF1B/231+) and pX461-gRNA(HNF1B/177-). 4x10 5 MAN13 hESCs were nucleofected with both pX461-gRNA(HNF1B/231+) and pX461-gRNA(HNF1B/177-), using the AmaxaTM P3 Primary Cell 4D-NucleofectorTM X Kit L (Lonza, #V4XP-3024), in 100μl nucleofection buffer according to the manufacturer's instructions, on a Lonza 4D-Nucleofector (program DN100). 1ml of TeSR1 medium, containing 10 μM ROCK inhibitor, was then added to the cell mix and the cells were plated on a well of a 12-well plate coated with Vitronectin. The medium was replaced 16h later with 1ml fresh TeSR1 medium. Two days post-nucleofection, the cells were collected by TrypLE treatment (Life Technlogies, #12605-028) for 2min at 37 o C and sorted for GFP fluorescence on a BD FACSAria Fusion flow cytometer. Two aliquots of the sorted, GFP+ cells of 5,000 and 10,000 cells were then plated in one Vitronectin-coated well each in a 6-well plate. The rest of the GFP+ cells (approximately 35,000) were plated on a separate well and – when confluent – they were stored in liquid N 2 , as backup. After 8-15 days, separate cell colonies had emerged on the sparsely seeded plates. Each was manually passaged, using a flame-pulled glass Pasteur pipette, into a separate well of a 24-well plate. Each clonal line thus produced was expanded further by EDTA passaging when confluent, and genomic DNA (gDNA) was extracted for genotyping using the Wizard® Genomic DNA Purification Kit (Promega, #A1120). A 781bp fragment around the expected mutation site was amplified by PCR, using Herculase II polymerase (Agilent, #600675) and was then sequenced to verify the presence/absence of the deletion. Of 21 clones isolated and sequenced, 3 were heterozygous mutants and one (IBM13-19) was studied further. This clone carried a frameshift in exon 1 (Fig. 1B) such that mutant mRNA would contain a premature stop codon 51 nucleotides downstream. A wild-type clone (IBM13-08) constituted an isogenic non-mutant control line. Samples were taken at time points in the differentiation of stem cells to organoids. The first differentiation steps occur in 2D over 7 days when dissociated cells are plated onto the air/medium interface of semipermeable membranes andthen maintained in 3D organoid culture for a further 18 days. Differentiation of cells into kidney in 2D and 3D (organoids) was performed as described previously (PMID: 29429961; PMID: 26444236), substituting APELTM with APELTM2 medium, supplemented with 1% (v/v) PFHM-II Protein-Free Hybridoma Medium (Thermo Fisher Scientific, # 11370882), hereafter referred to as APELTM2. For 2D differentiation, stem cells were plated on Vitronectin-coated plates, at a density of 18,000 cells cm -2 in culture medium containing 10μM Y-27632. The following day the medium was replaced with STEMdiffTM APELTM2 (StemCell Technologies, #05270), containing 8 μM CHIR-99021 (Tocris, #4423) for three days, followed by APELTM2 supplemented with 200 ng ml −1 FGF9 (Peprotech, #100-23) and 1 μg ml −1 heparin (Sigma, #3149) for a further 10 days. Subsequently, the cells were cultured in basal APELTM2 medium which was changed daily. For 3D organoid cultures was performed according as described (PMID: 26444236). Cells were differentiated for the first 7 days as in the 2D protocol above, except that APEL/CHIR-99021 medium was used for the first four days. On day 7, cells were dissociated with TrypLE. Aliquots of 2x10 5 cells were centrifuged at 400g for 2 min and the pellets were subsequently cultured on a medium-air interface by transfer onto a MilliCell cell culture insert (0.4μm pore size; Millipore, #PICM03050) inserted into the wells of 6-well plates. Upon transfer, the cells were exposed to APELTM2 medium containing 5μM CHIR-99021 for 1h, before reverting back to APELTM2 with FGF9/Heparin for a further 5 days. Finally, the 3D cultures were maintained in basal APELTM2 medium for up to another 13 days. Total RNA was extracted using the miRVana miRNA isolation kit (Thermo Fisher, AM1560) without using the optional miRNA-enriching protocol Truseq stranded mRNA Assay. Paired end.