PRPF8 CLIP seq for iPSC, kidney organoid, retinal organoid, and retinal epithelium derived from c.6926A>C RPE with Retinitis Pigmentosa patients and CRISPR/Cas9 corrected
PRPF8 CLIP seq for iPSC, kidney organoid, retinal organoid, and retinal epithelium derived from c.6926A>C RPE with Retinitis Pigmentosa patients and CRISPR/Cas9 corrected
Human Subjects Informed consent for research studies, according to the protocols approved by Yorkshire and the Humber Research Ethics Committee (REC ref: 15/YH/0365), was obtained for all study participants. Information on the patient and control participants is provided in Table S1.  Derivation and characterisation of RP13-iPSCs We ascertained three related RP type 13 patients with a PRPF8 c.6926A>C (p.H2309P) heterozygous missense mutation characterised by night blindness in the first decade of life, and one patient with a de novo PRPF8 c.6926A>C heterozygous mutation displaying night blindness at the age of 8 years (Table S1) (Towns et al., 2010). Dermal skin fibroblasts were reprogrammed to iPSCs using a non-integrative RNA-based Sendai virus system (Figure S1A), as described in Buskin et al. 2018. Several clones from each RP13 participant were tested for expression of pluripotent markers (Figure S1B), clearance of transgenes (Figure S1C) and genome stability (Figure S1D). Apart from clone RP13-1A1, which displayed a decreased copy number in the Chr 12p region, all the other clones displayed the expected copy number in the loci that are most susceptible to changes in pluripotent stem cells. Based on these data, one iPSC clone from each participant was selected for further studies and referred to as RP13-1A, 1B, 1C, and 2 as shown in Table S1. All iPSCs were maintained in Matrigel-coated plates with mTeSR™ Plus media and passaged using Versene (EDTA 0.02%) in 1:3 -1: 6 ratio. All cultures were maintained at 37°C, in a humidified environment, with 5% CO2. Cells were cryopreserved with freezing media containing 90% foetal bovine serum (FBS), 10% dimethyl sulfoxide and 10 µM Y26732.  Sendai Virus Clearance assay Total RNA was extracted from 5 × 106 iPSCs using TRIzol® reagent following the manufacturer's instructions. Reverse transcription was carried out using 1 µg of total RNA and the SuperScript cDNA Synthesis Kit following the manufacturer's instructions. Polymerase chain reaction (PCR) was carried out using the cDNA from the reverse transcription reaction with GoTaq DNA polymerase. The PCR products were analysed using 2% agarose gel electrophoresis. All primer details are shown in Table S13.   Detection of pluripotency markers by flow cytometry  iPSCs were dissociated by incubation with Accutase for 5 minutes at 37℃. A total of 1x106 single cells were resuspended in PBS supplemented with 5% fetal calf serum (FCS) and stained with anti-TRA-1-60 antibody (directly conjugated to FITC, 1:50) and anti-NANOG antibody (directly conjugated to AlexFluor647 conjugate, 1:50) for two hours at 2-8°C followed by PBS washing and subjected to flow cytometry analysis using FACSCanto flow cytometer (BD Biosciences). At least 10,000 cells were acquired, and the data was analysed using the FACSDiva software.  Pluripotest analysis  100 ng total RNA extracted from each RP13 and isogenic control iPSCs was used to prepare the GeneChip® for the PluriTest™ analysis to generate a Pluripotency and Novelty Score. The Pluripotency Score is based on many samples (pluripotent, somatic, and tissues) in the stem cell model matrix which consists of an extensive reference set of >450 cell/tissue types, including 223 hESC lines, 41 iPSCs, somatic cells, and tissues. Samples with positive Pluripotency Scores are more similar to pluripotent samples in the model matrix than to all other classes of samples. The Novelty Score is based on well-characterised iPSCs in the stem cell model matrix.  Genome stability assays Genome stability was assessed using the hPSC Genetic Analysis kit or the Karyostat assay (Thermofisher). hPSC Genetic Analysis Kit: Genomic DNA from the patient iPSCs was harvested using genomic DNA extraction kit. Genomic DNA was mixed with 2x qPCR master mix and ROX reference dye for qPCR analysis. Nine pairs of primers were applied for the examination of Chromosome regions 1q, 4p, 8q, 10p, 12p, 12q, 18q, 20q and Xp. qPCR ∆∆Ct values were normalised to normal control iPSCs. A copy number < 1.8 or > 2.2 with a p-value <0.05 may indicate the presence of a cytogenetic abnormality. Karyostat Assay: To verify the genome stability of derived iPSCs, 250ng total genomic DNA extracted from each cell line was used in GeneChip® for KaryoStat™ assays for assessment of potential copy number variants or single nucleotide polymorphisms across the genome. The KaryoStat™ assay allows digital visualisation of chromosome aberrations with a resolution similar to G-band karyotyping. The size of structural aberration that can be detected is >2Mb for chromosomal gains and >1Mb for chromosomal losses. The KaryoStat™ array is optimised for balanced whole-genome coverage with a low-resolution DNA copy number analysis. The assay covers all 36,000 RefSeq genes, including 14,000 OMIM® targets, allowing the detection of aneuploidies, submicroscopic aberrations, and mosaic events.  Correction of PRPF8 mutation in RP13-iPSCs using CRISPR/Cas9 To fully understand the impact of PRPF8 mutation in RPE and retinal cells, CRISPR/Cas9 genome editing was used to correct the PRPF8 c.6926A>C genetic mutation in all patient iPSCs using protocols described in Buskin et al. 2018. For in situ gene correction, a guide RNA was designed to include the PRPF8 c. 6926A>C mutation in the 3' region as well as an asymmetric ssODN template with wild-type PRPF8 sequence with the PAM sequence silenced to prevent repeated cleavage by Cas9 (Figure S2A). 100 iPSC clones from each patient were selected and tested by restriction site analysis using ApaL1, which cleaves the wild-type sequence “GTGCAC”. Approximately 20% of the clones showed complete digestion by ApaL1 (Figure S2B): these were further analysed by Sanger sequencing, which demonstrated the correction of the heterozygous mutation “A/C” to homozygous wild type “A” (Figure S2C). To exclude possible off-targeting effects, we tested five candidate genomic regions containing sequences partially matched to the gRNA (Table S10). All the candidate regions showed DNA sequences that match the wild type without insertion or deletion, indicating that no off-targeting occurred during the CRISPR/Cas9 genome editing procedure (Figure S2D). KaryoStat™ analysis, which assesses copy number variants and single nucleotide polymorphisms across the genome (Figure S2E) revealed the lack of genomic instabilities in the corrected clones. The CRISPR/Cas9 RP13-iPSC clones displayed high pluripotency scores in the PluriTest™ analysis (Figure S2F), confirming their pluripotent phenotype. From herein, unedited iPSCs that harbour the pathogenic c.6926A>C mutation in PRPF8 are collectively referred to as “RP13”. Similarly, the edited isogenic controls (c.6926C>A) are referred to as “RP13-Cas9”.  Differentiation of iPSCs to RPE Cells Differentiation was performed as previously described (Regents et al., 2019). In brief, RP13 and RP13-Cas9 iPSCs were grown on 6 well plates coated with growth factor-reduced Matrigel™, as recommended by the manufacturer's. Cells were incubated at 37⁰C, 5% CO2 and fed every second day with 2 ml mTeSR™ Plus per well. Once iPSCs achieved 80-95% confluency, mTeSR™ Plus media was replaced with 3 ml of differentiation medium (DMEM/F-12, 50 μM β-mercaptoethanol, 1X MEM NEAA, 1X penicillin streptomycin, 20% knockout serum replacement and 10 mM nicotinamide). Differentiation media was partially changed every second day. On days 8 and day 14 media composition was adjusted, by omitting nicotinamide and adding 100 ng/ml Activin A or 3 µM CHIR99021, respectively (Figure 1A). RPE patches formed during this time and were manually excised using a scalpel and transferred to a Falcon tube containing differentiation media. Next, media was aspirated, and patches were washed quickly with PBS. Then patches were dissociated by incubation at 37°C with TryplE Select (10X) for approximately 20 minutes followed by passage through a 40 µm cell strainer. TryplE Select was neutralised with RPE media and cells were centrifuged for 5 minutes at 300 x g. Media was aspirated and replaced with fresh RPE maintenance media (DMEM/F-12, 50 µM β-mercaptoethanol, 1X MEM NEAA, 1X penicillin streptomycin, and 4% knockout serum replacement). iPSC-RPE were counted using a haemocytometer and plated at density of 1.5x105/cm2 on 6 well plates coated with growth factor-reduced Matrigel™ in 3 ml RPE maintenance media. Media was partially replaced every 2-3 days until cells were pigmented and had a typical “cobblestone” epithelial cell morphology. Cells were then dissociated using TryplE Select (10X) as described, and plated onto 24-well and 12-well PET 0.4 µm pore Transwell ThinCerts™.  Immunofluorescent Staining of iPSC-RPE cells iPSC-RPE cells grown on 24-well ThinCerts™ were washed with PBS and incubated with 4% paraformaldehyde (PFA) for 20 minutes at room temperature. PFA was then removed, cells were rinsed twice with PBS and stored in PBS at 4°C until required. The transwell culture surface was isolated and placed on Superfrost+ microscope slide. Pigmentation of RPE cells was removed using Melanin Bleach Kit, as recommended by the manufacturer. RPE cells were blocked in PBS supplemented with 10% normal donkey serum and 0.3% Triton-X100, for 1 hour at room temperature. For ezrin immunostaining, RPE cells were not bleached, and Triton X-100 was substituted for a 5-minute incubation at -20°C with methanol, prior to blocking. The RPE cells were incubated with primary antibodies overnight at 4°C. Following three washing with PBS, RPE cells were incubated with secondary antibodies diluted in antibody dilution (PBS and 1% normal donkey serum) with Hoechst for 1 hour at room temperature. Then, iPSC-RPE cells were washed 3X for 5 minutes with PBS before mounting with Vectashield and sealed with a #1 coverslip. RPE cells were imaged using an AxioImager upright microscope with Apotome structured illumination fluorescence using 20x, 40x and 63x oil objectives (Zeiss, Germany). Three to five images per patient were processed using ZEN 2.5 Lite software (Zeiss). For cilia studies, RPE cells were imaged using an 100x objective on an A1R confocal microscope (Nikon). Images were analysed using Fiji (ImageLab) to assess co-localisation of ciliary proteins, and Huygens software (Scientific Volume Imaging) to determine cilia length. Data were analyzed and processed in Prism 7 (GraphPad).  Measurement of trans-epithelial resistance (TER) Trans-epithelial resistance (TER) was measured in Transwells (containing RPE cells by inserting the tip of the short electrode in the inner side of the Transwell insert, and the tip of the longer electrode in the outer side of the Transwell insert. Also, the resistance of a blank TW containing PBS was measured. Three measurements were obtained from each Transwell insert. After that, the readings from the blank Transwell was subtracted from the readings obtained from Transwells containing RPE cells. Based on the formula Unit area resistance = Resistance (Ω) x Effective Membrane Area (cm2), the results were multiplied by 0.3, which is the membrane area of a Transwell, and final values were given in ohms (Ω). TER measurements were performed one day after feeding the RPE cells to avoid variability in the TER readings.  Phagocytosis assay Bovine rod photoreceptor outer segments (POSs) were centrifuged at 2600 × g for 4 minutes, and the pellet was resuspended with 0.4 mg/ml FITC for 1 hour at room temperature followed by agitation in the dark. Following centrifugation at 2600 × g for 4 minutes and washing of the POSs three times with PBS, POSs were resuspended in AdRPMI 1640 media supplemented with B-27 Supplement and FBS. The staining of POSs was confirmed under a Bioscience Axiovert microscope. RPE cells were treated with unlabelled and FITC-labelled POSs (20 POSs per cell) and incubated for 4 hours at 37°C. For control, RPE cells were treated with FITC-labelled POSs and were incubated at 4°C for 4 hours. Both control and RP13-RPE cells were washed with PBS and were detached from the wells using 200μl of TryplE Select (10X) for 15 minutes. Then, control and RP13-RPE cells were resuspended in flow buffer (PBS with 2% FBS) and were transferred into Eppendorf tubes followed by centrifugation at 300 × g. To distinguish cells from debris and unbound POSs, RPE cells were incubated with 5 mM DRAQ5 for 5 minutes. To quench fluorescence from unbound POSs, cells were incubated with 0.2% Trypan Blue solution for 10 minutes. RPE cells were washed 3 times with PBS, and cell pellets were resuspended in flow buffer. Samples were run on a LSRII flow cytometer, and 10,000 events were collected per sample. Results were analysed using FacsDiva software.   RPE cytokine secretion studies Medium from basal and apical chambers of transwell inserts were collected from RPE cells of healthy controls and patients. The levels of PEDF and VEGF secretion were measured by using human PEDF-ELISA Kit (Cusabio, CSB-E08818h) and human VEGF-ELISA Kit (Life Technologies) according to the manufacturer's instructions.  Retinal organoids differentiation from iPSCs 7000 iPSCs were seeded into each well of Lipidure-coated 96-well U-bottomed plate and cultured for 48 hours to form Embryonic Bodies before start of differentiation. From day 0 to day 18, the differentiation medium was added into the culture containing 41% IMDM, 41% HAM's F12, 15% KOSR, 1% GlutaMAX, 1% Chemically defined, lipid concentrate, 1% Pen/Strep, 450 μM (20 μl of stock) 1-Thioglycerol. 1.5nM BMP4 was added from day 6 to day 9 (Figure S4A). From day 18, the culture medium was changed to 10% FBS in DMEM/F12 supplemented with 1x N2, 0.1mM taurine and 0.5 μM retinoic acid until day 150.   Immunofluorescent Staining of iPSC-Retinal Organoids  Ten 10 µm thickness sections of retinal organoids were collected on glass cover slips and were separated using an ImmEdge pen. Sections were air-dried for 20 minutes at room temperature and washed with PBS to remove OCT 91 from the tissue. Retinal organoids sections were incubated with a blocking solution (5% normal goat or donkey serum, 0.3% Triton-X in PBS) for 1 hour at room temperature, followed by overnight incubation with primary polyclonal non-conjugated antibodies shown in the Key Resources Table at 4°C. Primary antibodies were diluted in antibody diluent solution containing PBS, 1% bovine serum albumin (BSA) and 0.3% Triton-X. Following overnight incubation, retinal sections were washed three times with PBS for 15 minutes each and incubated with secondary antibodies conjugated to fluorophores and diluted in antibody diluent for 1 hour at room temperature. Retinal organoids sections were then washed three times with PBS for 10 minutes each to remove unspecific binding. To detect nuclear staining, sections were covered by Hoechst nuclear stain diluted in Vectashield at 1:2000, followed by mounting of the slides by a coverslip.  Kidney organoid differentiation from iPSCs Kidney cell and organoid culture was based on established protocols (Takasato et al., 2015). In summary, RP13 and RP13-Cas9 iPSCs were cultured in mTeSR™ Plus as described above. To start differentiation into kidney cells, iPSCs were dissociated into single cells with TryplE. 400,000 cells were plated in Matrigel-coated 6-well plate with 2 ml mTeSR™ Plus containing 10 μM ROCK inhibitor (Y-27632) and incubated overnight (day -1). The next day, cells were at 50% confluency and media was changed to APEL2 containing 5% protein-free hybridoma medium (PFHM-II) (complete medium; henceforth called APEL2+) and 8 μM CHIR99021 (day 0). The same media change was done on day 2. On day 4 of differentiation, media was changed to APEL2+ containing 200 ng/ml FGF9 and 1 μg/ml heparin. Media was changed every other day. On day 7 of differentiation, kidney cells were trypsinised, resuspended into a single cell suspension and counted. 500,000 cells were transferred to an Eppendorf tube and spun down at 800 x g for 3 minutes at room temperature three times, with gentle tube agitation after each spin. 3D kidney organoid cultures were grown on an air-liquid interphase, on cell culture inserts called Thincerts (Greiner Bio-One Ltd). Kidney cell pellets were transferred into the inner part of the Thincert, and kidney organoids were allowed to mature. APEL2+ containing 5 μM CHIR99021 was added to the outer part of the Thincert and incubated for 1 hour at 37°C, following media change to APEL2+ containing 1x antibiotic/antimycotic (complete media; henceforth called APEL2++), 200 ng/ml FGF9 and 1 μg/ml heparin. Media containing growth factors was changed every other day throughout the differentiation. Growth factors were withdrawn on day 12 and culture with APEL2++ media was carried on until day 25.  Cyst induction A 24-well plate was coated with Lipidure by placing 500 μl of 0.5% w/v Lipidure solution in each well and allowing complete evaporation at room temperature in sterile conditions. On day 22, kidney organoids were moved from Thincerts into a non-adherent plate with APEL2++ media containing 30 μM forskolin, 12.5 μM blebbistatin or DMSO. Organoids were grown in suspension with drugs for 72 hours.  Kidney organoids processing On day 25 organoids were washed with PBS and snap-frozen in liquid nitrogen for biochemical and proteomic assays. Organoids for RNA sequencing were suspended in 1 ml Trizol and snap-frozen. Organoids for immunofluorescent staining were fixed with 4% PFA for a minimum of 4 hours, followed by three PBS washes. They were permeabilized and blocked using 2% BSA in PBS containing 0.3% Triton X-100 overnight at 4°C. After three PBS washes, organoids were incubated in primary antibodies diluted in 1% BSA overnight at 4°C. Following three PBS washes, organoids were incubated in secondary antibodies diluted in 1% BSA overnight at 4°C. On the final day, organoids were washed three times with PBS and imaged with a confocal A1R microscope (Nikon) at x4 magnification. Organoids for OCT embedding were fixed with 4% PFA, washed with PBS and soaked in 30% (w/v) sterile sucrose overnight at 4°C on a roller. Following a PBS wash, organoids were placed in moulds, submerged in OCT and frozen. Frozen OCT blocks were cryosectioned at 10 μm thickness on a Leica CM1950 cryostat. Sectioned organoids were stored at -80°C until use. Further processing was as described below. Images were processed in Fiji (ImageJ). Image processing included the use of the following plugins: “Colocalisation Threshold”, “CiliaQ” and “Mitochondria Analyser”.  7000 iPSCs were seeded into each well of Lipidure-coated 96-well U-bottomed plate and cultured for 48 hours to form Embryonic Bodies before start of differentiation. From day 0 to day 18, the differentiation medium was added into the culture containing 41% IMDM, 41% HAM's F12, 15% KOSR, 1% GlutaMAX, 1% Chemically defined, lipid concentrate, 1% Pen/Strep, 450 μM (20 μl of stock) 1-Thioglycerol. 1.5nM BMP4 was added from day 6 to day 9 (Figure S4A). From day 18, the culture medium was changed to 10% FBS in DMEM/F12 supplemented with 1x N2, 0.1mM taurine and 0.5 μM retinoic acid until day 150.  Non CRISPR/CAS9 correction, of PRPF8 H2309P mutation. then proceed to different growth protocols for development of different organoids. Trizol suspensions of iPSCs, RPEs, kidney and retinal organoids were homogenized using 26-gauge syringe needles followed by standard Trizol RNA extraction. Extracted RNA was further purified using DNAse TURBO, and RNA purity and concentration was evaluated on TapeStation (Agilent Technologies).  RNA libraries were prepared using TruSeq Stranded mRNA libraries