Homo sapiens
Protocols: Untransplanted and transplanted organoids generated using the hiPSC-MAFB line were dissociated to single cells at d7+13 and d7+20 of differentiation. At d7+13, 94 untransplanted and 75 bisected transplanted organoids (150 chicken embryos) were dissociated, and at d7+20, 49 untransplanted and 24 bisected transplanted organoids (48 chicken embryos) were used. Organoids were incubated in a collagenase I buffer consisting of 600U ml-1 collagenase Type I (Worthington) and 0,75U ml-1 DNAse (Sigma Aldrich) in HBSS with calcium and magnesium (Thermo Fisher Scientific) for 40 minutes in a water bath at 37°C with repeated pipetting. Cell suspensions were centrifuged for 7 minutes at 300G and resuspended in a TrypLE buffer consisting of 5U ml-1 DNAse I (Sigma Aldrich) and 4µg ml-1 heparin (Sigma Aldrich) in 80% TrypLE select 10x (Thermo Fisher Scientific) in DPBS (Thermo Fisher Scientific). They were incubated for another 5 minutes at 37°C before dissociation was stopped through addition of cold HBSS+/+ with 10% FCS and further diluted with HBSS+/+. Cells were centrifuged for 5 minutes at 400G and resuspended in wash buffer (PBS + 0,1% BSA). After dissociation, all cells from organoids from the same condition were pooled and diluted in wash buffer. Viable ECs were identified and sorted on a FACSAria II or III cell sorter using a staining for human CD31 combined with a viability dye: rganoid cells were incubated with CD31-PE (1:100, BD Biosciences 555446) and viability dye eFluor 780 (1:5000, ThermoFisher Scientific 65-0865-14) in FACS buffer (PBS + 0,1% BSA + 2mM EDTA) for 30 minutes at 4⁰C. Cooked cells (20 minutes at 60⁰C) and cells from dissociated organoids derived from the LUMC0072iCTRL01 line were used for compensation. Viable endothelial cells (d7+13 untransplanted: 23060, transplanted: 6843. D7+20 untransplanted: 6697, transplanted: 36444) were sorted using a FACS Aria II 3L or FACS Aria III 4L (BD Biosciences) and collected in wash buffer. Cells were centrifuged for 6 minutes at 400G and resuspended in 10X buffer (PBS + 0,04% BSA). Cell number and viability were assessed using a Bio-Rad TC20 cell counter after staining with Trypan Blue. Due to the low number of sorted cells from transplanted d7+13 and untransplanted d7+20 organoids, these were not counted. The single cell suspensions were converted to barcoded scRNA-seq libraries with a targeted cell recovery of 6000 cells / condition. Nucleic acid extraction was performed in single cells using the Chromium Single Cell 3' v3 Library, Gel Bead & Multiplex Kit and Chip Kit (10x Genomics) according to manufacturer's instructions. The single cell suspensions obtained from dissociated kidney organoids were converted to barcoded scRNA-seq libraries using the Chromium Single Cell 3' Library, Gel Bead & Multiplex Kit and Chip Kit (10x Genomics). Samples were processed with 10x Genomics' V3 barcoding chemistry. Cells from each sample were treated with the same master mix and in the same reaction vessel, by processing every single sample in a single well of a PCR plate.
disease
normal
organism
Homo sapiens
organism
Homo sapiens
collection date
not collected
scientific_name
Homo sapiens
common name
human
organism part
kidney organoid
developmental stage
fetal stage
genotype
wild type genotype
isolate
not applicable
growth condition
in vitro cultured
cell_line
hiPSC reporter MAFB:mTagBFP2
progenitor cell type
induced pluripotent stem cell
cell_type
kidney cell
geographic location (country and/or sea)
not collected
ENA-LAST-UPDATE
2023-12-03