hiPSC culture and EC differentiation
hiPSCs (passage 25–40) were maintained under feeder-free conditions in defined E8 media (Gibco, A1516901) on tissue culture plates coated with hESC-qualified Matrigel (Corning, 354277) at 37°C in 5% CO2. The culture medium was exchanged daily. Routine passaging of hiPSCs at 1:4, and single-cell dissociation were carried out using Accutase solution (Sigma, A6964). The hiPSCs were cryopreservation with CryStor CS10 (Stemcell, 100–1061). In addition, we performed a mycoplasma contamination test every two weeks to ensure the health of all the cell lines (data not shown).
The EC differentiation protocol was adapted from a published protocol [15]. In brief, hiPSCs were cultured in Essential 8 medium reached ~ 80% confluency, then were dissociated into single cells, and seeded in 6-well plates with 10 µM ROCK inhibitor Y27632 (Selleck, S1049) on D0 at a density of 105 cells/well. On D1, hiPSCs were treated with 8 µM CHIR99021 (Selleck, S2924) in Chemically Defined Medium (CDM) for 3 days. On D4, differentiated cells were treated with 50 ng/ml VEGF (PeproTech, 100 − 20) and 10 mM SB431542 (Selleck, S1067) until cells reached ~ 100% confluent on D6. For cell dissociation, TrypLE Select Enzyme (10×) (Gibco, A1217701) was used for 10 min until most cells became singlets. The cells were then passed through a 40-µm strainer. Cell dissociation was neutralized by adding 2 ml of Endothelial Cell Medium (ECM, ScienCell, 1001), followed by magnetic associated cell sorting (MACS) to purify hiPSC-derived ECs with CD144 (VE-Cadherin) MicroBeads (Miltenyi, 130-097-857) or subjected to flow cytometry analysis. The CDM consists of IMDM(50%); Ham's F12 Nutrient Mix (50%); BSA (0.25%); Lipid concentrate (1X); ITS (0.1%); Ascorbic acid (50 µg/ml); Monothioglycerol (450 µM); and Glutamax (1X).
Generation of human blood VOs
A step-by-step protocol detailing the differentiation of the human VOs can be found in Nature Protocol [16]. Briefly, hiPSCs were dissociated into single cells with Accutase. Subsequently, the cells were resuspended in the E8 medium containing 10 µM Y27632 and seeded at 1,000 cells/well in round bottom ultra-low attachment 96-well plates (Costar, 7007) on D − 1, with a volume of 100 µl per well. The plate was then centrifuged at 100 g for 3 min to assemble hiPSCs into embryoid bodies (EBs) and placed in an incubator at 37°C with 5% CO2. After 24 h (D0), the culture medium was replaced by the N2B27 medium with 12 µM CHIR99021 and 30 ng/ml BMP4 (PeproTech, 120-05). On D3, the N2B27 medium was renewed with 100 ng/ml VEGF and 2 µM forskolin (HY-15371). Starting from D6, the resulting cell aggregates were embedded in Matrigel:Collagen I (1:1) gels and overlaid with ECM containing 15% FBS, 100 ng/mL VEGF and 100ng/ml FGF-2 (PeproTech, 100-18B). This medium was changed every two days. Around D10-12 vascular networks were established, and either directly analyzed or networks from individual cell aggregates were extracted from gels and further cultured in round bottom ultra-low attachment 96-well plates. These vascular networks self-assembled into vascular organoids, and the medium was changed every three days until organoids were ready for analysis.
Establishment of BCL6B overexpression and knockdown hiPSC lines
The human BCL6B coding regions, fused with Flag tags, was amplified through polymerase chain reaction (PCR) using hiPSC cDNA as template. Subsequently, these templates were cloned into adapted FUW-tetO-MCS vector (MiaoLingBio, P48786). Concurently, shRNA constructs were generated using the Tet-pLKO-puro vector (MiaoLingBio, P0171), following previously described methods [17]. For viral production, 10 µg of target vectors (BCL6B-FLAG, BCL6B shRNA, and FUW-M2rtTA (MiaoLingBio, P0521), along with lentiviral envelope and the packaging plasmids psPAX2 (MiaoLingBio, P0261) and pMD2.G (MiaoLingBio, P0262), were transfected into 293T cells using lipofectamine 3000 (Invitrogen, L3000015). After 72 hours, lentivirus supernatants were collected and filtered through a 0.45 µm syringe filter. hiPSCs were coinfected with BCL6B-FLAG and FUW-M2rtTA or infected solely with BCL6B shRNA lentivirus using a spin infection method, as previously described [18]. Two days post-infection, puromycin-resistant clones positive clones were selected using 1 µg/ml puromycin (MCE, HY-B1743A) for 4 days to establish BCL6B overexpression (BCL6B OE) or BCL6B knockdown (BCL6B KD) hiPSC lines, respectively. The successful establishment of hiPSC lines was confirmed by western blotting and then applied for later differentiation.
Immunofluorescence analysis
For hiPSCs-derived ECs, the purified ECs were seeded in a 24-well plate with coverslips and cultured until reaching 100% confluence. The cells were fixed in 4% paraformaldehyde (PFA) for 15 min, permeabilizated with 0.5% Triton X-100 for 10 min, followed by blocking with 10% goat serum for 30 min. Subsequently, the cells were incubated with mouse anti-CD31 (Abcam, ab9498) and rabbit anti-VE Cadherin (ab33168) at 4°C overnight. Secondary antibodies used were Alexa Fluor 488-conjugated goat anti-rabbit (Abcam, ab150077) and Alexa Fluor 647-conjugated goat anti-Mouse (Abcam, ab150115). DAPI (Sigma, D9542) was used to mark cell nuclei. Stained coverslips were mounted with mounting medium and stored at 4°C before imaging. All images were acquired by confocal imaging systems.
For whole-mount staining of organoids, the samples were fixed in 4% PFA for 1 hour at room temperature (RT), followed by three washes with PBS. Subsequently, the organoids were incubated in 0.5% TritonX-100 at RT for 1 hour. After blocking with 5% BSA in 0.1% TritonX-100 at RT for 1 hour, organoids were incubated with primary antibodies at 4°C for 48 hr, washed with PBS, and then incubated with secondary antibodies at 4°C for 48 hr. The stained organoids underwent three washes with PBS before being mounted on glass microscope slides using Mounting Medium (Invitrogen, P36961). To preserve the 3D structure of the organoids before confocal imaging, Polybead Microspheres were placed between the slide and the coverslip.
Flow cytometry analysis
HiPSC-ECs (D6) were dissociated into single-cell suspensions using TrypLE Select Enzyme for 8–10 min. After resuspension in staining buffer (Biolegend, 420201), approximately 0.5 × 106 single cells for each group were incubated with PE anti-human CD144 antibody (Biolegend, 348506) and FITC anti-human CD31 antibody (Biolegend, 303104) for 30 min. The results were analyzed using the BD LSR FortessaX-20 cytometer and FlowJo software.
The vascular organoids (D15) were mechanically disrupted and disaggregated using 3U/mL Dispase (Gibco, 17105041), 2U/mL Liberase (Sigma, 5401020001) and 100U DNAse (Stemcell, 07900) in PBS for 30 min at 37°C while rotating. Subsequently, single cells were stained with the following antibodies for 30 min: PE anti-human CD144 antibody (Biolegend, 348506), FITC anti-human CD31 antibody (Biolegend, 303104) and APC anti-human PDGFRβ antibody (Biolegend, 323608), followed by FACS analysis.
RNA extraction, cDNA synthesis, and qRT-PCR
The total RNA of hiPSCs, hiPSC-ECs and 10 VOs was extracted using Trizol (Invitrogen, 15596026), followed by reverse transcription to generate cDNA using the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, R212-01). qRT-PCR was performed using the Applied Biosystems QuantStudio 5 qPCR system with the ChamQ SYBR qPCR Master Mix (Low ROX Premixed) (Vazyme, Q331-02). Relative mRNA expression was determined by the delta cycle time with human GAPDH serving as the internal control for data normalization. The primer sequences were as follows:
GAPDH: forward, 5′-TCGGAGTCAACGGATTTGGT-3′, reverse, 5′-TTCCCGTTCTCAGCCTTGAC-3′;NANOG: forward, 5′-CAATGGTGTGACGCAGAAGG-3′, reverse, 5′-TGCACCAGGTCTGAGTGTTC-3′;
OCT4: forward, 5′-CTCGAGAAGGATGTGGTCCG-3′, reverse, 5′-TGACGGAGACAGGGGGAAAG-3′;
PECAM1: forward, 5′-AGACGTGCAGTACACGGAAG-3′, reverse, 5′-TTTCCACGGCATCAGGGAC-3′;
VE-Cadherin: forward, 5′-CGCAATAGACAAGGACATAACAC-3′, reverse, 5′-GGTCAAACTGCCCATACTTG-3′;
VWF: forward, 5′-CCCGAAAGGCCAGGTGTA-3′, reverse, 5′-AGCAAGCTTCCGGGGACT-3′;
VEGFR2: forward, 5′-GAGGGGAACTGAAGACAGGC-3′, reverse, 5′-GGCCAAGAGGCTTACCTAGC-3′;
PDGFRβ: forward, 5′-ATCAGCAGCAAGGCGAGC-3′, reverse, 5′-CAGGTCAGAACGAAGGTGCT-3′.
Western blotting
Cell pellets were lysed using RIPA buffer containing 1% PMSF. Protein concentration was measure by the BCA method using a Pierce BCA Protein Assay Kit (23225, Thermo Fisher Scientific). Samples were resolved on 10% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE), followed by transfer to a PVDF membrane at 100v for 120 min. Membranes were blocked with 5% non-fat dry milk in 1xTBST at RT for 1 h and then incubated with primary antibody at 4°C overnight. The following antibodies were used: mouse anti-CD31 (Abcam, ab9498); rabbit anti-VE Cadherin (ab33168); rabbit anti-VWF (ab6994); rabbit anti-BCL6B (Origene, TA369826); rabbit anti-FLAG (Abcam, ab205606); and mouse anti-beta actin (Abcam, ab8226). After appropriate washing with 1xTBST, the membranes were incubated with Goat Anti-Rabbit IgG H&L (HRP) (Abcam, ab6721) or Goat Anti-Mouse IgG H&L (HRP) (Abcam, ab6789) at RT for 1 hr. A chemiluminescent assay was performed using ECL substrates (Abcam, ab133406), and signals were detected with a FUSION Solo S chemiluminescence imaging system (5200 Muti, Tanon)
Tube formation assay
hiPSC-ECs were seeded onto 96-well plates coated with 50 µl matrigel (BD, 356231) at a density of 2×104 cells per well and then incubated for 4 hours. Tubular structures were photographed under a light microscope (Zesis) and analyzed using Image J software (NIH, Bethesda, MD).
Statistical Analysis
Results are presented as mean ± SD from a minimum of three independent experiments. Group comparisons were conducted using the non-paired two-tailed Student's t-test. In cases of non-normal distribution, the two-tailed F test was employed. For comparisons involving more than two groups, ANOVA was applied. A p value of < 0.05 were considered statistically significant.