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 µl pipette and organoids were minced with a scalpel. The organoid slurry was transferred to centrifugation tube with 1 ml pre-warmed enzymatic mix of 30 mg/ml Dispase (Gibco, 17105041) and 1 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 25 to 35 min. After the first 5 min, and 60 U/ml DNaseI (RNase-free, NEB, M0303L) 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 3 ml Red Blood Cell Lysis Solution (Promega, Z3141) for 4 min and span down for 5 min at 300g in a pre-cooled centrifuge.  For organoids that had been transplanted in MF1 nu/nu mice, the cell pellet was resuspended in HBSS (-/-) with 2% FBS and nuclei were counterstained with DAPI (Sigma-Aldrich, D9542) for 10 min to exclude dead cells during fluorescence-activated cell sorting (FACS). Samples were spun down for 5 min at 300g in a pre-cooled centrifuge and resuspended in HBSS (-/-) with 2% FBS. This step was repeated for a total of two washes. Cells were strained through a 35µm cell strainer and DAPI-negative singlets were sorted on a BD FACS Aria III using BD FACSDiva 8.0.1 Software.  For organoids that had been transplanted in NSG mice, the cell pellet was resuspended in HBSS (-/-) with 2% FBS, and stained against anti-human CD31 conjugated to APC (BD, 558094) for endothelial cells, and anti-human PDGFR-β conjugated to PE (BD, 558821) for mural cells for 45 min. After 35 min, cells were resuspended and nuclei were counterstained with DAPI for additional 10 min to allow dead cell exclusion during cell sorting. Samples were then spun down for 5 min at 300g in a pre-cooled centrifuge and resuspended in HBSS (-/-) with 2% FBS. This step was repeated for a total of two washes. and cells were strained through a 35µm cell strainer. Debri-free single human CD31- or human PDGFR-β-positive cells were sorted on a BD FACS Aria III using BD FACSDiva 8.0.1 Software using a 100 µm nozzle.  hBVO culture Blood vessel organoids were generated as previously described by Wimmer and colleagues 30, with minor alterations. In brief, cells were washed with DPBS (Gibco, 10010023) and subsequently incubated with TrypLE (Thermo Fisher Scientific, 12605010) for 4 min at room temperature. Cells were collected, span down at 200x g for 5 min and resuspended in Aggregation media consisting of KnockOut DMEM/F12 (Gibco, 12660012), 20% KnockOut Serum Replacement (Gibco, 10828028), 1% GlutaMAX (Gibco, 35050061), 1% NEAA (Gibco, 11140035), 55 µM β-mercaptoethanol (Gibco, 21985023), 100 U/ml penicillin-streptomycin, P/S (Gibco, 15140122), 50 µm Y-27632 (Stem Cell Technologies, 72304). Cells were seeded into Corning Elplasia round-bottom, ultra-low attachment plates at a concentration of 100 to 300 cells per microcavity, depending on the cell line. It has been shown that too small aggregates fail to differentiate or survive, while too large aggregates differentiate inefficiently 30. Therefore, we used a microwell plate-format with a defined, standardized number of cells per microwell. Upon the formation of aggregates of size 50 to 100 µm in diameter (in one to two days), media was exchanged for N2B27 media (day 0). N2B27 media consists of Neurobasal (Gibco, 21103049) : DMEM/F12 (Gibco, 11330031) 1:1, 1% B27 supplement (Gibco, 12587010), 0.5% N2 supplement (Gibco, 17502048), 0.5% GlutaMAX (Gibco, 135050061), 55 µM β-mercaptoethanol (Gibco, 21985023), 100 U/ml P/S (Gibco, 15140122) supplemented with 12 µM CHIR99021 (Tocris Bioscience, 4423) and 30 ng/ml BMP4 (Miltenyi Biotech, 130111165). Three days later (day 3), media was exchanged for N2B27 media supplemented with 2 µm Forskolin (Sigma Aldrich, F3917) and 100 ng/ml VEGF-A (Peprotech, 10020). After two days (day 5), aggregates were embedded in a 12-well plate into Collagen I-Matrigel solution, pH 7.4, consisting of 11.3% v/v 0.1 N NaOH (Sigma Aldrich, S2770), 4.7% v/v 10X DMEM (prepared prior, Sigma Aldrich, D5648-10L), 0.9% v/v HEPES buffer (Gibco, 15630080), 0.7% v/v NaHCO3 (Gibco, 5080094), 0.5% v/v GlutaMAX (Gibco, 25050061), 6.9% v/v Ham's F12 (Gibco, 11765054), 50% v/v PureCol (Advanced BioMatrix,5005), 25% v/v GFR Matrigel (Corning, 356231). From this time on, blood vessel networks and organoids were cultured in blood BVO media - StemPro-34 SFM media, supplemented with StemPro-34 nutrient supplement, 1% GlutaMAX, 100 U/ml penicillin-streptomycin, 15% fetal bovine serum (FBS) (Sigma-Aldrich, F2442-500ML), 100 ng/ml VEGF-A (Peprotech, 10020) and 100 ng/ml FGF2 (Miltenyi Biotech, 1300930841). Media was exchanged fresh every two to three days. Five days after embedding (day 10), gels with blood vessel networks were placed onto a dish lid with sterile lab spoon under a stereomicroscope. Single blood vessel organoids were excised from the matrix manually using sterile 27G needles, and transferred to a 96-well ultra-low attachment plate. BVO media was exchanged fresh every three to four days.  hBVO transplantation  Blood vessel organoids were transplanted under the kidney capsule of 12-15-week-old immunodeficient MF1 nu/nu (one H9 and one NC8 organoid per mouse, 8 mice in total) or NOD/SCID/IL2Rγnull (NSG) mice (3 to 6 H9 or NC8 organoids per mouse, 4 mouse each). All animal experiments were performed under ethical animal license protocols from the Austrian Ministry of Science, Research and Economics (BMWFW).  Generation of iTracer hBVOs and cell scarring Lineage tracing experiments were performed with the iTracer recording system, kindly provided by T. Gerber and R. Petri, following closely the procedure in the original protocol6. The iTracer plasmid used in this study contained a poly-adenylated and barcoded (11 random bases) dTomato reporter driven by the RPBSA promoter. 91 bases away from the RPBSA promoter, in the opposite direction, is a human U6 (hU6) promoter which drived the expression of gRNA targeting region within the 3' portion of a dTomato coding sequence.  In brief, iCRISPR 409-B2 cells were cultivated in mTeSR Plus (Stem Cell Technologies, 05825) supplemented with 100 U/ml P/S on Matrigel-coated T75 tissue culture flasks. Cells were electroporated with 10 µg lineage recorder DNA and 1 µg Sleeping Beauty transposase in 100 µl Human Stem Cell nucleofection buffer (Lonza, VVPH-5022) using the B-016 program of the 4D-Nucleofector (Lonza) following the manufacturer's protocol. Nucleofected cells were plate on Matrigel-coated plates in mTeSR Plus with 100 U/ml P/S and 5 µM Y-27632. Upon reaching around 70% confluency, dTomato-positive cells were sorted on a BD FACS Aria III using BD FACSDiva 8.0.1 Software using a 100 µm nozzle and cultured on Martigel-coated plates in mTeSR Plus supplemented with 100 µg/ml Primocin (InvivoGen, ant-pm-1) and 25 µm Y-27632. After 3 to 4 days, cells were dissociated and used for the generation of blood vessel organoids in one well of a 96-well Elplasia plate, 350 cells per microcavity (27'650 cells in total) as described above.  For scar formation by Cas9, 10 µg/ml doxycycline was added to the blood vessel networks media at day 7. After 24 h media was exchanged with fresh media without doxycycline and organoids were cultured following the protocol described above.   scRNA-seq of iTracer hBVOs and barcode and scar library preparation At day 14 of differentiation, organoids were dissociated, processed for generation of scRNA-seq libraries and sequenced on a NovaSeq 6000, as described above.  Additional enrichment polymerase chain reactions (PCRs) were performed where barcode and scar regions from 60 ng of cDNA remaining from the scRNA-seq preparation were amplified in three separate reactions. First, cDNA was amplified in a PCR broadly targeting the barcode and scar region. Subsequently, two 10 ng portions of the reaction product were used for further separate reactions, one for scar and one for barcode amplification. Finally, Illumina adapter sequences were added. 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 S2.  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.  Multiome ATAC-seq and RNA-seq data generation Single cell Multiome experiments were performed using the 10x Chromium Single Cell Multiome ATAC + Gene Expression (GEX) kit (1000282) following manufacturer's instructions. Briefly, nuclei suspensions were incubated in a transposition mix where DNA in open regions of chromatin was preferentially fragmented and adapter sequences were added to the ends of the DNA fragments. Afterwards, nuclei were mixed with reverse transcription mix, and gel beads and oil were loaded onto a 10x microfluidic chip to be coencapsulated into nanodroplets, forming GEMs. Inside each GEM containing a nucleus, for GEX, first strand cDNA synthesis occurred, where each mRNA was tagged with a UMI and a barcode unique for each nucleus. In the same partition, for ATAC, transposed DNA was uniquely barcoded and P5 adapters were added. Subsequently, the reaction was quenched. The droplets were broken, pooled fractions recovered and purified using Dynabeads MyOne Silane. Barcoded transposed DNA and barcoded full-length cDNA from poly-adenylated mRNA was amplified to fill gaps and generate sufficient amounts for library generation.  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.   Multiome ATAC-seq and RNA-seq data generation The pre-amplified product was used for generation of both GEX and ATAC libraries. Single-cell libraries were generated using fragmentation, end repair, A-tailing and double-sided size selection using SPRIselect. P5 and P7 adaptor sequences were ligated and individual sample indices provided as a Dual Index Plate TT Set A (10x Genomics, 3000431) were used to enable pooling and subsequent demultiplexing of multiple libraries. For ATAC libraries construction, P7 adapter sequences and sample indices from a Sample Index Plate N, Set A (10x Genomics, 3000427) were added to the pre-amplified product. For Gene expression libraries, cDNA was further amplified with the number of PCR cycles depending on the number of nuclei loaded.