organism: Homo sapiens
description: Protocols: For the experiment we differentiated the hIPSC line A1AT-RR, derived as previously described in the following publications: Yusa, K., et al., 2011. Targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells. Nature 478, 391-394 and Rashid, S.T., et al., 2010. Modeling inherited metabolic disorders of the liver using human induced pluripotent stem cells. J Clin Invest. 2010 Sep;120(9):3127-36. hPSCs were maintained on plates coated with 10 μg/ml Vitronectin and cultured in E6 media supplemented with 2 ng/ml TGFβ and 25 ng/ml FGF2. Cells were maintained at 37 °C and 5% CO2, and passaged every 5-6 days by dissociation with 0.5 mM EDTA. For coating plates, 10 μg/ml Vitronectin in PBS was applied for at least 1 hour at room temperature. Differentiation was performed at 5% O2. Briefly, a serum-free differentiation (SFD) medium was used, consisting of 75% Iscove's modified Dulbecco's medium and 25% Ham's F12 medium supplemented with 1% N2, 0.5% B27, 0.05% BSA, 1 mM ascorbic acid (2-phospho-L-ascorbic acid trisodium salt), 4.5x10-4 monothioglycerol, 2 mM L-glutamine, 150 μg/ml transferrin and 10 ng/ml penicillin/streptomycin. Undifferentiated cells were dissociated using 0.5 mM EDTA and small aggregates were resuspended in SFD medium supplemented with 10 ng/ml BMP4 and cultured as EBs on non-treated plates. After 24 hours, an equivalent volume of SFD was added directly on top to not perturb the small EBs, supplemented with final concentrations of 10 ng/ml BMP4 and 5 ng/ml FGF2. At day 2, developing EBs were collected, washed, resuspended in SFD supplemented with 10 ng/ml BMP4, 5 ng/ml FGF2 and 3 μM CHIR99021, and seeded back on the plate. After 24 hours, EBs were again collected and washed, and resuspended in StemPro-34 SFM (ThermoFisher Scientific) supplemented with 1 mM ascorbic acid, 4.5x10-4 monothioglycerol, 2 mM L-glutamine, 150 μg/ml transferrin, 10 ng/ml penicillin/streptomycin (from now on referred to as complete SP34) containing 5 ng/ml FGF2, 15 ng/ml VEGF, 10 ng/ml IL-6 and 5 ng/ml IL-11, and cultured for 48 hours. At day 5, EBs were again collected, washed and resuspended in complete SP34 with 5 ng/ml FGF2, 15 ng/ml VEGF, 10 ng/ml IL-6, 5 ng/ml IL-11, 50 ng/ml SCF, 5 ng/ml IGF-1, 2 U/ml EPO and cultured until day 8. At this stage, EBs were collected, dissociated to single cells and prepared for sorting by flow cytometry of the CD34+/CD43- fraction. This population was resuspended at a density of 10^6 cells/ml in complete SP34 with 10 ng/ml BMP4, 5 ng/ml FGF2, 5 ng/ml VEGF, 10 ng/ml IL-6, 5 ng/ml IL-11, 100 ng/ml SCF, 25 ng/ml IGF-1, 30 ng/ml TPO, 30 ng/ml IL-3, 10 ng/ml Flt3-L, 20 ng/ml SHH, 10 ng/ml angiotensin II and 100 μM losartan potassium. Cells were transferred to a non-treated round-bottom 96 well plate, 200 μl/well, centrifuged 3 minutes at 300 g, and incubated overnight to allow the cells to re-aggregate. On the following day, marking EHT D1, the small aggregates were plated on Matrigel. For this, they were gently transferred to thin-layer Matrigel-coated wells, with a density of 2x10^5 cells/well in a 24 well plate, and cultured for additional 2-4 days using the same media, replaced every 2 days. For coating plates, Matrigel was diluted into cold medium with a concentration of 35 μg/cm^2 of surface to be coated, and applied overnight at 37 °C. For treatments with cell cycle inhibitors, 1 μM PD0332991 (Tocris), 4 μM Roscovitine (Sigma-Aldrich), 10 μM RO3306 (Sigma-Aldrich) or 0.1% DMSO were added at EHT D3 for 48 hours. Cells were differentiated until the emergence of haematopoietic clusters at endothelial-to-haematopoietic transition D3, when the first samples were collected and processed for scRNAseq (CTRL1). Alternatively, they were further cultured for 48 hours applying one of the following treatments: - 0.1% DMSO (CTRL2) - 1 μM PD0332991 - 10 μM RO3306 - 4 μM Roscovitine - 2 μM GSK126 At the end of the treatment, cells were collected and processed for scRNAseq. Conversion was performed following the 10X protocol (https://www.10xgenomics.com/) Chromium 10X v2 platform. Constructed by core facility at Wellcome Sanger Institute.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2020-05-17T17:04:59Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-08-02T15:58:56Z
attributes.tag: External Id
attributes.value: SAMEA5843836
attributes.tag: INSDC center name
attributes.value: Wellcome Sanger Institute
attributes.tag: INSDC first public
attributes.value: 2020-05-17T17:04:59Z
attributes.tag: INSDC last update
attributes.value: 2019-08-02T15:58:56Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-8205:4823STDY7231844
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: A1AT-RR
attributes.tag: cell type
attributes.value: haematopoietic stem cell
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: Alpha-1-antitrypsin deficiency
attributes.tag: genotype
attributes.value: corrected Glu342Lys mutation
attributes.tag: organism part
attributes.value: skin
attributes.tag: progenitor cell type
attributes.value: induced pluripotent stem cell
attributes.tag: sample name
attributes.value: E-MTAB-8205:4823STDY7231844
attributes.tag: scientific_name
attributes.value: Homo sapiens
attributes.tag: sex
attributes.value: male
attributes.tag: single cell quality
attributes.value: OK