title: Fate and State Transitions during Human Blood Vessel Organoid Development: Time Course
design: Fate and State Transitions during Human Blood Vessel Organoid Development: Time Course
library_construction_protocol: Cell and tissue dissociation for single cell RNA-seq and single-cell multiome (scMultiome) experiments 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. For 10x Multiome experiments, single nuclei were extracted from dissociated mesoderm aggregates at day 3 and day 4 and vascular networks at day 7. After obtaining a single-cell solution, as described above, cells were resuspended in Lysis Buffer containing 10 mM Tris-HCl, pH 7.4 (Sigma-Aldrich, T2194), 10 mM Sodium Chloride (Sigma-Aldrich, 59222C), 3 mM Magnesium Chloride (Sigma-Aldrich, M1028), 0.01% Tween-20 (Bio-Rad, 1662404), 0.01% NP40 Substitute (Sigma-Aldrich, 74385), 0.005% Digitonin (Thermo Fisher Scientific, BN2006), 1% BSA (Miltenyi Biotec, 130-091-376), 1 mM DTT (Sigma-Aldrich, 646563), RNase inhibitor (Takara, 2313A) and nuclease-free water (Ambion, AM9937). The suspension was incubated for 10 min on ice. The reaction was stopped with Wash Buffer (10 mM Tris-HCl, pH 7.4, 10 mM Sodium Chloride, 3 mM Magnesium Chloride, 1% BSA, 0.1% Tween-20, 1 mM DTT, 1 U/µl RNase inhibitor, and nuclease-free water) and span down for 5 min at 500g at 4°C. The pellet was resuspended in Wash buffer, filtered through a 40 µm Flowmi cell strainer and again span down for 5 min at 500g at 4°C. The pellet was resuspended in 1X Diluted Nuclei Buffer consisting of 1X Nuclei Buffer (stock of 20X, 10x Genomics, 2000207), 1 mM DTT, 1 U/µl RNase inhibitor and nuclease-free water and nuclei concentration was determined. Nuclei were span down for 5 min at 500g at 4°C and resuspended in the respective volume of 1X Diluted Nuclei Buffer according to the 10x Multiome user guide for targeting 10,000 nuclei.  scRNA-seq of transplanted hBVOs For single cell experiments, 8 weeks post-organoid transplantation, mice were sacrificed and organoids were excised from the mouse tissue. 10 to 15 organoids were pooled for each dissociation reaction.  Organoids were collected in a 15-ml centrifugation tube, washed three times with DPBS and transferred to the lid of a 10 cm cell culture dish. Excess liquid was aspirated carefully with a 200 µ