2025-03-01
ENA first public
ERC000011
ENA-CHECKLIST
D-BSSE, ETH Zurich
INSDC center name
public
INSDC status
ERS23286527
SRA accession
E-MTAB-14839:Signaling_G_10ng_VEGF_DAPT
Submitter Id
ArrayExpress
broker name
H9
cell_line
blood vessel
cell_type
not collected
collection date
human
common name
Protocols: Different protocols were used for tissue dissociation, depending on the time point of differentiation. For the generation single cell RNA-seq time course of H9 and NC8 organoid differentiation, a similar number of cells or organoids were pooled together prior to dissociation. For dissociation of pluripotent stem cell culture, cells were washed once with DPBS and incubated with Accutase (Gibco, A1110501) for 3 to 5 min at 37°C. The reaction was stopped with cold KnockOut DMEM/F12 (Gibco, 12660012) with 10% FBS and span down for 5 min at 300g at 4°C. The cell pellet (on ice) was resuspended in HBSS (without Ca2+, without Mg2+, -/-) with 2% FBS, filtered through a 30 µm cell strainer and centrifuged again for 5 min at 300g at 4°C. The pellet was resuspended in HBSS (-/-) with 2% FBS, counted using Trypan Blue assay on an automated cell counter Countess (Thermo Fisher) and diluted to the appropriate concentration for 10,000 cells to be obtained per lane of the 10x microfluidic chip. For mesoderm aggregates (age 3 to 5 days), 20-30 aggregates were washed twice with HBSS (without Ca2+, without Mg2+) and incubated in a mixture of one part Accutase and three parts EDTA (Invitrogen, cat. no. AM9261) on a Mini Rocker Shaker at 37°C at 30 rpm for 30 to 40 min, with gentle trituration with a P1000 pipette every 5 min. When most cells were single, the reaction was stopped with cold KnockOut DMEM/F12 (Gibco, 12660012) with 10% FBS and span down for 5 min at 300g in a pre-cooled centrifuge. The cell pellet (on ice) was resuspended in HBSS (without Ca2+, without Mg2+, -/-) with 2% FBS, filtered through a 30 µm cell strainer and centrifuged again for 5 min at 300g at 4°C. The pellet was resuspended in HBSS (-/-) with 2% FBS, counted, diluted to the appropriate concentration if needed and counted again. Cells were loaded on the 10x microfluidic chip aiming for 8,000 cells. For vascular networks (age 6 to 10 days), one well of a 12-well plate was used. Excess gel was excised with a scalpel, networks were transfer to a 15 ml centrifuge tube, and incubated with cold Cell Recovery Solution (Corning, 35423) for 5 min at 4°C. The networks were triturated gently with a P1000 pipette with a wide-bore tip, centrifuged for 3 min at 300g at 4°C and resuspended in 1 ml pre-warmed enzymatic mix of 5 mg/ml Dispase (Gibco, 17105041) and 0.5 mg/ml Liberase TH Research Grade (Roche, 05401151001) in DPBS (Gibco, 10010023), sterile filtered through a 0.22 µm gaze. The reaction was incubated on a Mini Rocker Shaker at 37°C at 30 rpm for 20 to 30 min. After the first 5 min, and 60 U/ml DNaseI were added and the organoids were triturated gently with a P1000 pipette with a wide-bore tip. Organoids were triturated every 5-7 min and regularly checked under the microscope until a single-cell suspension was achieved. The reaction was stopped with cold KnockOut DMEM/F12 (Gibco, 12660012) with 10% FBS and spun down for 5 min at 300g in a pre-cooled centrifuge. The cell pellet (on ice) was resuspended in HBSS (-/-) with 2% FBS, filtered through a 30 µm cell strainer and centrifuged again for 5 min at 300g at 4°C. The pellet was washed once more with HBSS (-/-) with 2% FBS, then resuspended in HBSS (-/-) with 2% FBS, counted and the concentration was adjusted if necessary. Cells were loaded on the 10x microfluidic chip aiming for 10,000 cells. For single-cell dissociation of organoids aged 14 days or more, 15 to 20 blood vessel organoids were pooled (20-30 organoids if FACS was to be performed). Organoids were washed once with HBSS (-/-) and subsequently placed onto the lid of a cell culture dish. Excess liquid was aspirated carefully with a 200 µl pipette and organoids were minced with a scalpel. The organoid slurry was transferred to an enzymatic mix of 5 mg/ml Dispase (Gibco, 17105041) and 0.5 mg/ml Liberase TH Research Grade (Roche, 05401151001) in DPBS (Gibco, 10010023). The reaction was incubated on a Mini Rocker Shaker at 37°C at 30 rpm for 20 to 30 min. After the first 5 min, and 60 U/ml DNaseI added and the organoids were triturated gently with a P1000 pipette with a wide-bore tip. Organoids were triturated every 5-7 min and regularly checked under the microscope until a single-cell suspension was achieved. The reaction was stopped with cold KnockOut DMEM/F12 (Gibco, 12660012) with 10% FBS and spun down for 5 min at 300g in a pre-cooled centrifuge. The cell pellet (on ice) was resuspended in HBSS (-/-) with 2% FBS, filtered through a 30 µm or a 40 µm cell strainer and centrifuged again for 5 min at 300g at 4°C. The pellet was washed once more in HBSS (-/-) with 2% FBS, then resuspended in HBSS (-/-) with 2% FBS, counted and the concentration was adjusted if necessary. Cells were loaded on the 10x microfluidic chip aiming for 10,000 cells.  Generation of mosaic hBVOs with genetic perturbations For generation of mosaic perturbed vascular organoids, 409-B2 iCRISPR cells were grown on Matrigel-coated plates in mTeSR Plus (Stem Cell Technologies, 100-027) and dissociated into single cells using TrypLE (Thermo Fisher Scientific, 12605010) when at 60% - 80% confluency. 2.1 Mln cells in mTeSR Plus supplemented with 100 U/ml P/S and 5 µm Y-27632 were plated per T75 tissue culture flask pre-coated with Matrigel. After 24 h, cells were transduced at a low multiplicity of infection (MOI), so that no more than 3% of the cells were GFP-positive in order to reduce the possibility of cells receiving more than one lentivirus (and therefore, one gRNA). The virus was added to the cell culture media consisting of mTeSR Plus, 100 U/ml P/S and 5 µm Y-27632. 24 h later media was exchanged to mTeSR Plus and 100 U/ml P/S. After 2 to 4 days, GFP-positive cells were sorted on a BD FACS Aria III using BD FACSDiva 8.0.1 Software using a 100 µm nozzle and subsequently cultured on Martigel-coated plates in mTeSR Plus supplemented with 100 µg/ml Primocin and 25 µm Y-27632. 24 h later media was exchanged to mTeSR Plus supplemented with 100 µg/ml Primocin, 5 µm Y-27632 and 2 µg/ml doxycycline (Sigma-Aldrich, D9891). For the next 4 to 6 days, until reaching 70% confluency, cells were cultured in mTeSR Plus supplemented with 100 µg/ml Primocin and 2 µg/ml doxycycline. Blood vessel organoids were generated either from GFP-positive cells only (samples in batches 1 and 2) or from 20% GFP-positive cells and 80% non-transduced cells (samples in batch 2).  scRNA-seq of mosaic hBVOs with genetic perturbations scRNA-seq experiments were performed with organoids at day 7, 14, or 21 following the procedure described in the dissociation into single cells subsection and the scRNAseq GEX libraries generation section of this study. Two batches of organoids with multiple samples per batch were run. Organoids from batch 1 were dissociated and cells were directly loaded onto a 10x Genomics chip for subsequent scRNA-seq. Organoids from batch 2 were dissociated, GFP-positive cells were sorted on a BD FACS Aria III using BD FACSDiva 8.0.1 Software using a 100 µm nozzle and and only GFP-positive cells were loaded onto a 10x Genomics chip.   Enrichment of gRNA-containing transcripts from scRNA-seq gRNA was amplified from 60 ng cDNA remaining from the scRNA-seq preparation in three separate PCRs, similar to others 55,190. First, the gRNA region was amplified in a PCR broadly targeting the outer part of the human U6 promoter. Second, the inner part of the U6 promoter was amplified and a Truseq Illumina i5 adapter was added. A Truseq Illumina i7 adapter was added in the last reaction. After every PCR, samples were purified with SPRIselect beads (Beckman Coulter, B23319). The libraries were sequenced on a NovaSeq 6000. Primers for the PCRs can be found in Table S5.   Generation of single-cell transcriptome of MECOM-KO and WT organoids MECOM-KO and WT vascular networks at day 7 and vascular organoids at day 21 from two independent batches were dissociated using an enzymatic mix of Liberase and Dispase as described above. Final cell suspension in HBSS (-/-) with 2% FBS was counted, diluted to the appropriate concentration if needed and counted again.  scRNA-seq GEMs and libraries were generated using Chromium Single Cell 3’ v3.1 kit, targeting 9,000 cells, and sequenced on a NovaSeq 6000 following the manufacturer’s recommendations.  Signaling pathways modulation of blood vessel organoids Generation of hBVOs with pharmacological perturbations For studying the importance of Notch and VEGF signaling pathways in blood vessel development, we performed a screen with 5 different conditions (see Table S7 for details), varying the concentration of VEGF and testing the effect of DAPT, a γ-secretase inhibitor (Sigma-Aldrich, D5942) on blood vessel organoid development.   scRNA-seq of pharmacologically perturbed hBVOs For scRNA-seq experiments, 10-day-old blood vessel organoid networks were dissociated into single cells as described in the tissue dissociation for scRNA-seq section of this article and loaded on the 10x microfluidic chip aiming for 10,000 cells.  Organotypic hBVOs  Generation of organotypic hBVOs  For the generation of organotypic hBVOs, hBVOs generated from the ESC line H9 under 11 different conditions and the original protocol as control (see Table S8 for details) were generated. The small molecules RepSox (Stem Cell Technologies, 73794), Wnt3a (R&D Systems, 5036-WN-010), Wnt7a (PeproTech, 120-31), retinoic acid (Sigma-Aldrich, R2625-50MG), CHIR99021 (Tocris Bioscience, 4423) were added to the original blood vessel organoid media in different combinations between days 5 and 10 of differentiation. As some small molecules are dissolved in DMSO, all conditions were adjusted to have the same final DMSO concentration.   scRNA-seq of organotypic hBVOs For scRNA-seq experiments, 10-day-old blood vessel organoid networks were dissociated into single cells as described in the tissue dissociation for scRNA-seq section of this article and all samples were stained using conjugated hashtag oligos, pooled, sequenced and demultiplexed with regard to their hashtag, based on the original protocol for cell hashing192. In brief, after filtering through a 40 µm Flowmi mesh, cells from each condition were suspended in Staining Buffer at a concentration of 7,777 cells/µl (final volume of 45 µl) to which 5 µl TruStain Fc Receptor Blocking Solution (Biolegend, 422301) was added. The cell suspension was incubated for 10 min at 4°C, followed by the addition of 2 µl (1 µg) TotalSeq anti-human Hashtag antibody (hashtag oligo, HTO) (individual for each sample; Biolegend, catalog numbers 394601, 394603, 394605, 394607, 394609, 394611, 394613, 394615,  394617, 394619, 394623) and incubation for 30 min at 4°C, with manual shaking of the tubes every 10 min. Cell suspensions were washed with 1 ml Staining buffer (0.5% BSA in DPBS) followed by centrifugation at 300 x g for 5 min. Washing steps were repeated for a total of three times. Finally, cells were counted, resuspended to 1,000 cells/µl and 10 µl of each sample were added to a final mix. Two lanes on the 10x microfluidic chip (Chromium Single Cell 3’ v3.1 kit) were loaded with 25 µl of the final mix, targeting a total of 25,000 per lane, and sequenced on a NovaSeq 6000 following the manufacturer’s recommendations.  cDNA libraries were prepared following 10X Genomics standard protocol, described in more detail above. For preparation of the additional hashtag libraries,  the 80 µl supernatant from the cDNA cleanup was further size-selected with 70 µl SPRIselect reagent (2.0X) and eluted in a final volume of 40 µl Buffer EB. A PCR reaction was set up with 5 µl of the eluted hashtag fraction, 2.5 µl i5 dual index, 1.5 µl i7 dual index, 50 µl Kapa Hifi Hotstart Ready Mix (Kapa Biosystems, KK2602) and 40 µl nuclease-free water with the following program: 2 min at 98°C, 19 cycles of (20 sec at 98°C, 30 sec at 64°C, 20 sec at 72°C), 5 min at 72°C in a BioRad C1000 Thermal Cycler. The final PCR product was size-selected with 1.2X SPRIselect reagent, cleaned up and eluted in 40 µl water.   Generation of hBVOs with TF overexpression Generation of rtTA and GOI lentivirus For lentivirus generation, HEK293T cells were cultured in DMEM (Gibco, 41965039) supplemented with 1% GlutaMAX (Gibco, 25050061), 10% FBS and 100 U/ml P/S (Gibco, 15140122). One day before transfection, cells were split to a 10 cm dish in 10 ml media, so as to reach confluency of 80% on the next day. On day 2, media was exchanged to DMEM with 1% GlutaMAX, 10% FBS and 10 U/ml P/S. TFs were commercially available: ETS1 (Nexus, cesbBroadEn 10809), NR3C1 (Horizon Discovery, OHS1770-202322291), LEF1 (Nexus, OHS5894-202500619), FOXQ1 (AddGene, 101628). Per reaction, 6 µg of the construct with rtTA or GOI were mixed with 1.5 µg gagpol, VSVG, Rev and Tat each and vortexed. Then, 36 µl TransIT-293 Transfection Reagent reagent (Mirus, MIR 2705) was added, mixed well and incubated at room temperature for 17 min. This mix was added dropwise to cells with gentle shaking of the plate. On the next morning, media was exchanged with 6.5 ml DMEM with 1% GlutaMAX, 30% FBS and 10 U/ml P/S per dish. On the next day, in the afternoon, media was collected and spun down at 2,000 rpm for 5 min. The clear supernatant was transferred to a sterile conical tube, 1 vol of Lenti-X Concentrator (Takara Bio, 631232) was mixed with 3 vol of supernatant (i.e. 2 ml Concentrator with 6 ml supernatant), mixed by gentle inversion and incubated for 24 h at 4℃. Samples were centrifuged at 1,500 rpm for 45 min at 4℃. Supernatant was removed without disturbing the white pellet at the bottom. The pellet was resuspended to 1/500th of the original volume using PBS and prepared virus was stored long-term in single-use aliquots at -80℃.  Generation of rtTA ESC line For the generation of hBVOs with induced TF expression, first the ESC line H9 was transduced with lentivirus packaged rtTA plasmid. Briefly, 300,000 cells were seeded onto a Matrigel-coated 6-well plate well with 1 ml mTesR Plus (Stem Cell Technologies, 100-0276) containing CloneR2 (1:10 dilution) (Stem Cell Technologies, 100-0691), 100 U/ml P/S and 5 µl rtTA virus. After 48 h, media was exchanged to mTeSR Plus with 100 U/ml P/S. When confluent, cells were split and seeded sparsely as single cells on Matrigel-coated plates in mTeSR Plus with 100 U/ml with 5 µM Y-27632 (Stem Cell Technologies, 72304). After 24 h, media was exchanged to mTeSR Plus supplemented with 5 mg/ml G418 (Sigma Aldrich, G8168-10ML) for selection. 3 days later, when control non-transfected cells were dead, colonies from single cells were picked and expanded. A single clone was used for further experiments.  Generation of rtTA-GOI ESC line ESCs with inducible GOI (GFP-TF or GFP only as control) were generated from the H9 rtTA clone using the approach used for generation of the rtTA cell line, but using lentivirus with packaged GOI under the TRE and minimal promoter. As this plasmid contains a Neomycin resistance cassette, selection for positive clones was performed using 0.1 mg/ml Hygromycin (InvivoGen, ant-hg-1) for 96 h in the presence of 5 mg/ml G418. Selected single clones were tested by inducing expression with 2 µg/ml Doxycycline and detection of GFP signal with epifluorescent Revolve Echo microscope.  Generation of rtTA-GOI hBVOs hBVOs were generated following the original hBVO protocol. Between day 5 and day 10, media was further supplemented with 4 µg/ml Doxycycline. On day 10, organoids were dissociated, GFP-positive cells were sorted on a BD FACS Aria III using BD FACSDiva 8.0.1 Software using a 100 µm nozzle and and only GFP-positive cells were loaded onto a 10x Genomics chip.   Modeling diabetic vasculopathy in hBVOs in vitro Modeling diabetes in hBVOs For mimicking inflammation and diabetes (here represented as hyperglycemic condition in addition to inflammatory condition), hBVOs were cultured with the original protocol mentioned above until day 8 . On day 8, vascular networks were cultured in the original protocol media (as mentioned above; 17 mM glucose) with the following supplementation: nondiabetic control - 58 mM D-Mannitol (VWR chemicals, 25314.264) as osmotic control; inflammatory media - 58 mM D-Mannitol, 1 ng/ml interleukin-6 (IL-6; PeroTech, 200-06) and 1 ng/ml tumor necrosis factor alpha (TNF-alpha; Gibco, PHC30111); diabetic media - 75 mM glucose final concentration (D-Glucose, AMRESCO, 0188-1KG), 1 ng/ml IL-6 and 1 ng/ml TNF-alpha. Organoids were cultured with the respective media until day 22.   Whole-mount immunohistochemistry of 22-day-old organoids was performed as described above, with any modifications to the procedure mentioned here. Stained BVOs were cleared with RapiClear® CS mounting solution (SUNJin Lab, RCCS002) overnight and subsequently mounted into 1 mm iSpacers (SUNJin Lab, IS003) in DAKO fluorescent mounting medium (Sigma Aldrich, F4680). Organoids were analyzed on an Olympus spinning disk confocal microscope (Olympus IX83 inverted microscope equipped with a dual-camera Yokogawa W1 spinning disk).   scRNA-seq of diabetic hBVOs For scRNA-seq experiments, 22-day-old hBVOs were dissociated following the procedure above. The pellet was resuspended in HBSS (-/-) with 2% FBS, counted, diluted to the appropriate concentration if needed and counted again. Cells were loaded on the 10x microfluidic chip aiming for 7,000 cells (Chromium Single Cell 3’ v3.1 kit). A total of two batches were sequenced for a total of two control samples, three inflammatory samples and three diabetic samples.   scRNA-seq data generation Single-cell experiments were conducted using the 10x Chromium Single Cell 3’ v3 (1000075) or v3.1 (1000121) kit following the manufacturer’s instructions. In brief, cells mixed with reverse transcription mix, gel beads and oil were loaded onto a 10x microfluidic chip to be co-encapsulated into nanodroplets into so-called Gel Beads-in-emulsion (GEMs). Inside each GEM containing a cell, first strand cDNA synthesis occurred, where each mRNA was tagged with a unique molecular identifier (UMI) and a barcode unique for each cell. The droplets were broken, pooled fractions recovered and purified using Dynabeads MyOne Silane (Thermo Fisher, 37002D). cDNA was amplified with a number of PCR cycles depending on the loaded number of cells. scRNA-seq data generation  Single-cell libraries were generated using fragmentation, end repair, A-tailing and double-sided size selection using SPRIselect Reagent (Beckman Coulter, B23318). P5 and P7 adapters were ligated and individual sample indices provided as a Single Index Plate T Set A (10x Genomics, 2000240) were used to enable pooling and subsequent demultiplexing of multiple libraries.
description
day 10
developmental stage
Healthy
disease
Control
genotype
not collected
geographic location (country and/or sea)
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
BVO
organism part
Homo sapiens
scientific_name
Signaling_G_10ng_VEGF_DAPT
title