Human pluripotent stem cell culture
Human induced pluripotent stem cell line NP0040-8 (kindly provided by Dr. Tomo Saric, University of Cologne, Medical Faculty, Institute for Neurophysiology) was cultured on Matrigel Matrix (hESC-qualified, Corning, # 734–1440) pre-coated six-well plates at 10 μg / cm2 growth area in E8 medium consisting of DMEM/F12 (1:1) + Glutamax (Thermo Fisher Scientific, # 31,331–028) supplemented with 64 μg/ml L-ascorbic acid phosphate magnesium n-hydrate (Wako Chemicals Europe, # 013–12,061), 20 μg/ml insulin (Lilly Deutschland GmbH, “Humalog 100I.E.”), 5 μg/ml transferrin (Sigma-Aldrich, # T3705), 14 ng/mL sodium selenite (Sigma-Aldrich, # S5261), 100 ng/mL heparin sodium salt (Sigma-Aldrich, # H3149), 100 ng/mL basic fibroblast growth factor 2 (Peprotech, # 100-18B) and 2 ng/mL transforming growth factor β (Peprotech, # 100–21) in a humidified incubator at 37 °C with 5% CO2.
Every 3–4 days hiPSCs were sub-cultured when cell culture reached 70 – 80% confluency. The medium was removed, and the culture dish was washed by 1 mL DPBS (-/-), 1 mL ReLeSR™ (Stemcell Technologies, # 05,872) was added per well of six-well plates and incubated for 1 min at RT, the ReLeSR™ was removed, and the plate was incubated for another 2 min in a humidified incubator at 37 °C with 5% CO2. The plate was knocked against the bench horizontally to produce a uniform hiPSCs cluster suspension. 50–200 μL hiPSCs cluster suspension was transferred into fresh wells of Matrigel-coated six-well plates, 2 mL E8 medium per well was added supplemented with 5 μM of Rho Kinase (ROCK) inhibitor (Y27632, Adooq, # A11001-5), and the plate was incubated in a humidified incubator 37 °C with 5% CO2. Every 24–48 h a complete medium change was performed into E8 medium without ROCK.
Generation of hiPSC-ECs in 2D monolayer culture
For differentiation of hiPSCs into ECs, a hiPSCs single cells suspension was prepared. Once cell culture reaches 70—80% culture confluency, the cell culture was washed by 1 mL DBPS (-/-), 1 mL ReLeSR™ per well of six-well plates was added, the plate was incubated in a humidified incubator at 37 °C with 5% CO2 for 7–10 min. After that, 2 mL E8 medium supplemented with 5 μM ROCK was added per well of a six-well plate. In order to form a cell suspension, the well content was pipetted up and down up to 10 times. Next, the cell suspension was filtered through a 40 μm cell strainer (Greiner Bio-One, # 542,040), centrifuged at 120 g, re-suspended into 1 mL E8 medium supplemented with 5 μM ROCK, and the cell number was counted by an automatic cell counter (NaNoEnTek, Korea). 0.021 × 106 hiPSCs were added per cm2 growth area in 2 mL E8 medium supplemented with 5 μM ROCK per well of a six-well plate. This day was counted as day -2 and starting day of human endothelial cell differentiation, and hiPSCs were cultured in E8 medium from day − 2 for 48 h.
Next, on day 0, mesodermal induction was performed by a complete medium change into E6 medium supplemented with 6 μM CHIR99021 (LC Laboratories, # C-6556) for 24 h. The E6 medium composition was similar to E8 medium, but without supplementation of bFGF and TGFβ growth factors. On day 1, a complete medium change was performed into E6 without CHIR99021 supplementation.
Afterward, on day 2, endothelial induction was performed by a complete medium change into E6 supplemented with 300 ng / mL human vascular endothelial growth factor A165 (VEGF) (Peprotech, # 100–20), 200 ng / mL bFGF, 1 mM 8-Bromoadenosine 3′,5′-cyclic monophosphate sodium salt monohydrate (8Bro) (Sigma-Aldrich, # 1,002,183,637), and 50 μM Melatonin (Mel; Sigma-Aldrich, #M5250) for 48 h. From day 4 onward, the culture medium was changed every 48 h into E6 medium supplemented with 10 ng / mL VEGF and 10 ng / mL bFGF, and 10 μM hydrocortisone (Sigma-Aldrich, # H4001).
Later, on day 6, the culture dish was washed by 1 mL DPBS (-/-), and 1 mL 0.05% Trypsin–EDTA (Thermo Fisher Scientific, # 25,200–056) per well of a six-well plate, and the plate was then incubated in humidified incubator 37 °C with 5% CO2 for 10 min to dissociate EC cells. The ECs suspension was prepared by pipetting up to 10 times with a 1000 µL pipette. The ECs suspension was filtered through a 40 µm cell strainer, and ECs were counted by a fully automated counter with aid of trypan blue dye.
Generation of hiPSC-ECs in 3D bioreactor culture
Bioreactor (DASGIP, Eppendorf) assembling and preparing has been described previously . The 3D culture system was continuously agitated with 60 revolutions per minute (r.p.m.), 3 standard liter gas overlay per hour, 5% CO2 and 37 °C temperature from the beginning to the end of hiPSC-ECs differentiation. To differentiate hiPSC-ECs in a stirred bioreactor, 30 × 106 NP0040 hiPSCs were inoculated in 100 mL E8 medium supplemented with 5 μM ROCK for 2 days. On day 0, culture medium was completely changed into 100 mL E6 medium supplemented with 6 μM CHIR99021 for 24 h. After 24 h, the culture medium was changed into E6 medium without CHIR99021 for another 24 h.
On day 2, the culture medium was refreshed with 100 mL E6 medium supplemented with 300 ng / mL human VEGF, 200 ng / mL bFGF, 1 mM 8Bro and 50 μM Mel for 48 h. From day 4 onward, the culture medium was changed into E6 medium supplemented with 10 ng / mL VEGF and 10 ng / mL bFGF for 48 h. On day 6, embryoid bodies of hiPSC-ECs (EB-ECs) were collected into 25 mL E6 medium for preparing EB-ECs suspension. 5 mL of EB-ECs suspension was used for dissociation of EB-ECs with 1 mL 0.05% Trypsin–EDTA per 2 mL EB-ECs suspension. Trypsin activity was blocked with 25% FBS in DMEM / F12; hiPSC-ECs suspension was filtered by a 40 µm strainer and counted.
Characterization of hiPSC-ECs by flow cytometry
hiPSC-EC was dissociated into single cells, 0.25 × 106 EC single cells were re-suspended into 0.5 mL of 0.5% BSA and 2 mM EDTA in DPBS (-/-) in a 1.5 mL Eppendorf tube and centrifuged at 300 g for 1 min at 4 °C, and supernatant was removed. The pellet was re-suspended in 50 μL of 0.5% BSA and 2 mM EDTA in DPBS (-/-) containing 1:50 of Anti-SSEA4-PE, human (Miltenyi Biotec, # 130–098-369) for characterizing hiPSC. For mesodermal cell characterization, single cells were fixed with 4% Paraformaldehyde (PFA; Polysciences, # 1884) in DPBS (-/-) and permeabilized with 0.5% t-Octylphenoxypolyethoxyethanol (Triton X-100; Sigma-Aldrich, # 9002–93-1) in DPBS (-/-). Cells were incubated with anti-brachyury (D2Z3J) rabbit mAb (Cell Signaling Technology, # 81,694). Secondary detection was done by 1:1000 dilution of Alexa 555 conjugated goat anti-rabbit IgG (Life technology, # A21430) for 20 min. 1:50 dilutions of α-human-CD31-APC (Miltenyi Biotec, # 130–110-670), α-human-VE-Cadherin-PE (Miltenyi Biotec, # 130–100-716), CD34-FITC, human (Miltenyi Biotec, # 130–113-178) and α-human-CD184-PE-Vio770 (Miltenyi Biotec, # 130–103-798) were used for characterization of ECs. After the staining process, 0.5 mL of 0.5% BSA and 2 mM EDTA in DPBS (-/-) were added and centrifuged and supernatant was removed. The pellet was re-suspended in 250 μL of 0.5% BSA and 2 mM EDTA in DPBS (-/-), and samples were stored at 4 °C in the dark until analysis. Prior analysis flow cytometer compensation was performed to prevent spillover of fluorochromes. Data were acquired by flow cytometry (LSR Fortessa Analyzer, BD Biosciences). FCSexpress 6 (De Novo Software, Glendale, CA) was used for data analyzation and graphical presentation.
Characterization of hiPSC-ECs by immunofluorescent staining
0.2 × 106 ECs were plated on 10 μg / cm2 Matrigel-coated round glass cover slips (Carl Roth, # D-76185) in 48-well plates (Greiner-Bio-One, # 677,180), maintained in E6 medium supplemented with 10 ng / mL VEGF, 10 ng / mL bFGF, 10 ng / mL EGF, 10 μM hydrocortisone (Sigma-Aldrich, # H4001), 50 μM Mel and incubated at 37 °C with 5% CO2 for 24 h.
The medium was removed, and ECs were fixed by 4% PFA in DPBS (-/-) for 10 min at RT. Fixed ECs were permeabilized by 0.5% Triton X-100 in DPBS (-/-) for 10 min at RT. The well with cover slip attached ECs was washed by 3% bovine serum albumin (BSA) (Sigma-Aldrich, # A2153) in DBPS (-/-), and ECs were blocked with 3% BSA in DPBS for 60 min at RT.
The blocking solution was removed, and 150 μL of 3% BSA was added, containing 1:150 dilution ratio of α-CD31-APC (Miltenyi Biotec, # 130–110-670), α-VE-Cadherin-AF488 (Santa Cruz, # sc9989) and α-vWF (Dako, # A008229-2) antibodies and 1:100 dilution ratio of Rabbit α-LDL receptor primary antibody (Abcam LDL uptake assay kit (cell-based), # ab133127), as well as 1:10 α-eNOS (BD bioscience, #610,297) against nitric oxide synthase overnight at 4 °C.
Following primary antibody binding, the well was washed twice with 3% BSA in DBPS (-/-) and 150 μL of 3% BSA was added containing 1:1000 Alexa Flour 555 goat α-rabbit (Life Technologies, # A21430), 1:100 DyLight 488-conjugated goat α-rabbit IgG secondary antibody (Abcam LDL uptake assay kit (cell-based), 1:100 Alexa Flour 555 goat α-mouse IgG1 (Life Technologies, # A21422) and Hoechst 33,342 for 60 min at RT in the dark. Three washing steps were performed with 3% BSA in DPBS. The cover slips with stained cells were taken out from plate and mounted upside down on glass slides with aid of 5 µL SlowFade™ Diamond Antifade Mountant (Life Technologies, # S36972). Finally, slides with attached cover slips were examined by confocal fluorescent microscopy (SP8, Leica) for obtaining images.
Endothelial vascular tube formation
0.5 × 106 HUVEC or hiPSC-ECs were re-suspended in 1 mL E6 medium supplemented with 10 ng / mL VEGF, 10 ng / mL bFGF, 10 ng / mL EGF, 10 μM hydrocortisone, 50 μM Mel and 15% FBS. 100 μL of cell suspensions was added into 96-well plates (Greiner Bio-One, # 655,180) pre-coated with 30 µL Matrigel per well, and 100 μL of E6 medium was added additionally. The plate was incubated for 24 h in humidified incubator at 37 °C with 5% CO2, and images were captured by light microscopy after 5 h and 24 h.
Determination low-density lipoprotein uptake of hiPSC-ECs
0.25 × 106 either HUVEC or hiPSC-ECs were seeded in 48-well plates on Matrigel-coated cover slips (Carl Roth, #219,827,535) in 0.5 mL E6 medium supplemented with 10 ng / mL VEGF, 10 ng / mL bFGF, 10 ng / mL EGF, 10 μM hydrocortisone and 50 μM Mel. After 24 h, 1 μL / 100 μL medium of LDL-DyLight 550 (Abcam LDL uptake assay kit, # ab133127) was added into the well that contained attached hiPSC-ECs or HUVEC separately, and the plate was incubated at 37 °C with 5% CO2 for 5 h.
Next, the culture medium was discarded, and the wells were washed with DPBS (-/-) and fixed by 4% PFA for 10 min. After fixation, cell permeabilization was performed by 0.5% Triton-X in DPBS (-/-) for 10 min as well as blocking that was performed by 3% BSA for 1 h. 1:100 rabbit α-LDL receptor primary antibody (Abcam LDL uptake assay kit) was then added and incubated overnight at 4–8 °C in the dark. Finally, 1:100 DyLight 488-conjugated goat α-rabbit IgG secondary antibody (Abcam LDL uptake assay kit) and 1.2 µg / mL Hoechst dye were added and incubated at RT for 30 min in the dark. Afterward, coverslips were embedded with 5 μL of SlowFade™ Diamond Antifade mounting medium on the glass slides and cover slips were evaluated by SP8 confocal microscopy.
Determination of nitric oxide synthase expression
To determine endothelial cell nitric oxide synthase production, on day 6 of hiPSC-ECs differentiation, 0.25 × 106 hiPSC-ECs parallel to HUVEC were seeded on 10 μg / cm2 Matrigel-coated coverslip in 48-well plates containing 0.5 mL E6 medium supplemented with 10 ng / mL VEGF, 10 ng / mL bFGF, 10 ng / mL EGF, 10 μM hydrocortisone, and 50 μM Mel. After 24 h, hiPSC-ECs were fixed by 4% PFA in DPBS for 10 min at RT, permeabilized by 0.5% Triton X-100 in DPBS (-/-) for 10 min at RT. Later, hiPSC-ECs were blocked with 3% BSA in DPBS (-/-) for 60 min at RT. After that, 1:10 α-eNOS (BD Bioscience, #610,297) in 3% BSA was added and incubated overnight at RT. Next, hiPSC-ECs were washed with DPBS (-/-) twice and stained by 1:100 Alexa Flour 555 goat α-mouse IgG1 and 1.2 µg / mL Hoechst dye in 3% BSA in DPBS (-/-) for 60 min at RT in the dark. At the end, hiPSC-ECs on cover slips were washed with DPBS three times, embedded and evaluated by confocal microscopy.
Transcriptomic whole genome analysis
Total RNA from ECs (human induced pluripotent stem cell-derived endothelial cells, hiPSC-ECs; human coronary artery endothelial cells, HCAEC; human cardiac microvascular endothelial cells, HCMEC; human umbilical vein endothelial Cells, HUVEC; human saphenous vein endothelial cells, HSaVEC; human dermal microvascular endothelial cells, HDMEC; human pulmonary microvasculature endothelial cell, HPMEC) was isolated using the PureLink RNA Mini Kit (Life Technologies, # 12183018A). Samples of 106 primary ECs each in RNAlater were obtained from PromoCell.
Briefly, 1 × 106 ECs were added to RNase-free 1.5 tubes, washed with PBS (-/-) and centrifuged at 300 g for 2 min at 4 °C. The pellet was re-suspended in 1 ml TRIzol (Life Technologies, # 15,596,026). By repeated pipetting and mixing on a vortex mixer, the ECs were lysed for 30–60 s. The lysate was incubated at RT for 5 min. To separate the RNA from phenol, 0.2 ml chloroform (Sigma, # C-2432) was added per tube, mixed gently by hand and centrifuged at 12,000 g for 15 min at 4 °C. 350 μL of the colorless supernatants was transferred into new 1.5 ml RNase-free tubes, and 350 µL of absolute ethanol (Carl Roth, # 9065.3) was added per tube and vortexed well. 700 μL of the mixed sample was passed through a centrifuge cartridge with collection tubes (included in the kit) and centrifuged at 12,000 g for 1 min at RT. The centrifuge cartridge was washed with 500 µL wash buffer II (Life Technologies, #12183018A) and centrifuged at 12,000 g for 15 s at RT. 22 μL RNase-free water was used to elute RNA into 1.5 RNase-free tubes, and RNA aliquots were stored at − 80 °C.
For transcriptome analysis, 5.5 µg of fragmented biotin-labeled double-stranded cDNA from endothelial cells was hybridized on Clariom™ S arrays (Clariom™ S Arrays, Human Applied Biosystems by Thermo Fisher Scientific). After staining, the arrays were scanned with Affymetrix Gene-Chip Scanner-3000-7G, while quality control analysis was performed with Affymetrix GCOS software. Transcriptome analysis was performed at the Transcriptomics Core Facility of the Center for Molecular Medicine Cologne (CMMC).
The gene-level views of the human transcriptome with Clarion S Assays for the endothelial samples from different human cell types of our experiments were obtained. After the RMA normalization, only significantly expressed probesets were chosen using FDR F test. k-means cluster analysis was performed after transcript-wise normalization of signal values to a mean of 0 and an SD of 1 using Euclidean distance measurement, with the Cluster 3.0 tool from the Eisen laboratory . The number of clusters was decided based on manual clustering for different values of k, varying k from 1 to 15 clusters. K = 12 was found optimal, while higher numbers of clusters other clusters did not result in a better separation of samples. The raw data are available at the NCBI GEO database (Home—GEO—NCBI (nih.gov)) under accession number GSE200399.
Enzyme-Linked Immunosorbent Assay (ELISA) of eNOS
To validate eNOS expression of hiPSC-ECs, both NP0040 and NP0040-R (a transgenic subclone of NP0040 expressing mCherry) were differentiated into ECs with or without Mel supplementation from day 2 to day 4. Next, hiPSC-ECs were collected inside 1.5 mL reaction tubes on day 4 and day 6, respectively.
After that, cells were lysed with mammalian cell lysis kit (Sigma-Aldrich, # MCL1-1KT). eNOS was then detected in the lysate by ELISA (Human eNOS DeoSet ELISA; R&D systems, # DY950-05). An ELISA reader (Tecan, # 1,502,004,958) was used for quantification according to the manufacturer’s instructions.
Sprouting EB-ECs assay
To observe capillary sprouting from EB-ECs, 0.3 × 106 cells either 789-O renal tumor cell line (ATCC® CRL-1932™) or NP0040-ECs per 1 mL E6 medium supplemented with 10 ng/mL VEGF, 10 ng/mL bFGF, 10 ng/mL EGF, 10 μM hydrocortisone, 50 μM Mel and 15% FBS were plated per well of a six-well plate separately. The plate was agitated at 40 r.p.m. and incubated for 24 h in a humidified incubator at 37 °C with 5% CO2. After 24 h, equal numbers of 789-O spheroids and hiPSC-ECs spheroids were pooled in 100 μL E6 medium supplemented with 10 ng/mL VEGF, 10 ng/mL bFGF, 10 ng/mL EGF, 10 μM hydrocortisone, 50 μM Mel and 15% FBS. The pooled spheroids were cultured on top of 50 μL Matrigel per well of a 96-well plate that was pre incubated at 37 °C with 5% CO2 for 1 h, and images were captured by light microscope after 1 min, 24 h, and 48 h.
Three-dimensional (3D) culture of hiPSC-ECs and 789-O renal tumor spheroids (EBs)
NP0040 and 789-O cell lines were genetically engineered to express mCherry (NP0040-R) and green fluorescence proteins (789-O-GFP), respectively. NP0040-R was differentiated into ECs inside six-well plates by applying the 2D monolayer method. At day six of differentiation, NP0040-R-ECs were dissociated into single cells with 0.05% trypsin–EDTA. In parallel, 789-O-GFP cells were also dissociated with 0.05% trypsin–EDTA, filtered through a 40 µm cell strainer and counted.
NP0040-R-ECs and 789-O-GFP were re-suspended in 15% DMEM to achieve 20 µL cell suspension containing 1000 and 500 single cells, respectively. Drops of 20 µL cell suspension per drop were cultured over night as hanging drops on the lid of square tissue culture dishes to generate cell spheroids. Next, NP0040-R-ECs and 789-O-GFP spheroids were collected in a 15 mL falcon tube and spun down at 120 g for 1 min. Both types of spheroids were re-suspended in 20 µL ice-cold 15% DMEM. 60 µL of 4 °C cool Matrigel was then mixed with 20 µL EBs suspension using pre-chilled 100 µL pipette tips. Matrigel-spheroid suspension was poured into wells of a 96-well that were pre-coated 1 h before with 30 µL Matrigel per well, thereby forming a gel layer inside the wells. 45 min post-casting EBs-Matrigel suspension, 200 µL of 15% DMEM was added per well of the 96-well plate. The plate was incubated at 37 °C with 5% CO2. Every 24 h the plate was examined on a Carl Zeiss fluorescence microscope and/or SP8 Leica microscope.
De Novo software version FCS Express 6 was used for flow cytometry analysis. GraphPad Prism software version 5 was used for graph drawing and statistical analysis. To find statistical difference between groups, one-way analysis of variance (ANOVA) was used, and Bonferroni's test also was used as a post hoc test. Data were represented as mean ± standard deviation (mean ± SD) when biological independent replications were three to six (n = 3–6) and significant difference value was less than 0.05 (P < 0.05).
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. The transcriptomics data are available as a supplementary file.