Human induced pluripotent stem cell line UEFhfiPSC1.4 (36) was derived using lentiviral transduction of Yamanaka transcription factors Oct4, Klf4, Sox2 and c-Myc (18) into fibroblasts isolated from a skin sample taken during cecarean sectioning of a volunteer mother (36). Generation and testing of the UEFhfiPSC1.4 cell line has been described in detail elsewhere and the cells passed all pluripotency tests and differentiated well into any cell type (36, 37). These hiPSCs were cultured in a serum-free stem cell medium supplemented with 20% KnockOut^™ Serum Replacement (GIBCO) and 8 ng/ml basic fibroblast growth factor (FGF-2) (R&D Systems) (38) on a feeder cell layer of mitotically inactivated foreskin fibroblasts (ATCC, CRL-2429) (36, 38), or in Essential 8 hESC cell culture media (Life Technologies) on Matrigel™ basement membrane matrix (Corning, growth factor reduced, phenol red free) supplemented with 50 IU/ml penicillin (Invitrogen) and 50 µg/ml streptomycin (Invitrogen). Medium was changed daily or every other day and cells were mechanically passaged once a week in the presence of 10 µg/ml ROCK inhibitor Y-27632 (Sigma-Aldrich) (39), or approximately 1–2 times a week with EDTA solution (Life Technologies, 0.5 M pH 8.0). To avoid feeder cell contamination before EC differentiation, UEFhfiPSC1.4 cell colonies were moved from a feeder cell layer to a feeder-free culture employing Matrigel® hESC-Qualified Matrix coating (BD Biosciences) and mTeSR^™1 medium (STEMCELL Technologies) supplemented with 50 IU/ml penicillin (Invitrogen) and 50 µg/ml streptomycin (Invitrogen). Culturing of hiPSCs in these conditions was done according to a technical manual of STEMCELL Technologies or Life Technologies (Culturing Pluripotent Stem Cells (PSCs) in Essential 8™ Medium).

Human umbilical vein endothelial cells (HUVEC) were used as positive EC control in flow cytometric analyses and assays for functional EC characterization. Umbilical cords were collected from volunteermothers according to a protocol approved by Ethics Committee of the Kuopio University Hospital (Kuopio, Finland, license number 341/2015).

HUVECs were isolated from umbilical cord samples as previously described (40). HUVECs were cultured in endothelial cell growth medium (Thermo Scientific) on fibronectin-gelatin coating (10 µg/ml, 0.05%; Sigma-Aldrich).

HiPSC colonies were cut with a scalpel from Matrigel-coated dishes under a stereomicroscope and pieces of hiPSC colonies were washed with phosphate buffered saline (PBS). Cells were dissociated with Accutase (Sigma-Aldrich) and approximately 0.7 × 10⁴ cells per cm² were plated on a fibronectin-gelatin (10 µg/ml, 0.05%; Sigma-Aldrich) coated dish. Cells were cultured in serum-free EBM™-2 Endothelial basal medium-2 (Lonza CC-3156) with EGM^™-2 SingleQuots medium supplement with human epidermal growth factor (hEGF), recombinant human long R3 insulin like growth factor-1 (R3-IGF-1), ascorbic acid, hydrocortisone, human FGF-2, heparin and antibiotics GA-1000 (Lonza CC-4176). From this supplement, fetal bovine serum (FBS) and vascular endothelial growth factor (VEGF) were discarded to obtain serum free and VEGF-controlled cell culture conditions. Knockout serum replacement (GIBCO) was used at the final concentration of 20 µl/ml and recombinant human VEGF-A 165 (R&D Systems) was used at the final concentration of 200 ng/ml. Following small molecules and growth factors were used in different combinations and time courses: 10 µM TGFβ inhibitor SB431542 (Tocris Bioscience), 10 µM ROCK inhibitor Y-27632 (Sigma-Aldrich), 20 ng/ml recombinant human BMP-4 (R&D Systems) and 0.25 mM 8-Br-cAMP (SB 431542, Sigma-Aldrich). Cell culture medium was changhed every second day, and differentiating ECs were passaged every 4–6 days using Accutase (Sigma-Aldrich).

In our cell differentiation protocol, we hypothesize that differentiation stages advance from PSC stage through mesodermal commitment (differentiation day 5) towards mature EC stage (differentiation day 15) (22). Different cell culture conditions are described in Table 1. In all groups without the TGFβ inhibitor at day 1 during the differentiation, it was added at day 4 as TGFβ inhibition has been shown to enhance EC vascular identity after mesodermal fate (24). In those groups with BMP-4 at day 1, it was removed at day 4 since it promotes the mesodermal commitment from PSCs (30).

For analyses, cells were detached with Accutase (Sigma-Aldrich) and incubated at +37°C for 5 min. Blocking was done using 2% FBS-PBS at +4°C for 20 min followed by fixing in 1% paraformaldehyde (PFA). Following antibodies (BD Biosciences) were used for staining according to manufacturer’s instructions: mouse anti-human CD31-R-phycoerythrin (PE), mouse anti-human CD309-PE, mouse anti-human CD34-PE, mouse anti-human CD144-fluorescein isothiocyanate (FITC) and mouse anti-endothelial nitric oxide synthase (eNOS)-PE. For intracellular staining of eNOS and anti-Von Willebrand Factor (vWF)-FITC (Abcam), cell permeabilization was done using 0.5% Triton X-100 (Sigma-Aldrich) in 2% FBS-PBS at +4°C for 30 min. Data was captured with a BD FACSCalibur and analyzed with FCS Express 6 Flow Research Edition software (De Novo Software).

The statistical analysis of the data was done using GraphPad Prism software (version 5.04, GraphPad Software Inc., CA, USA). One-way ANOVA tests were done for analyzing the group differences for both expression of endothelial markers between the treatments and as a function of time. If differences were statistically significant with more than 95% confidence, paired differences between the groups were tested with Tukey’s multiple comparison post-tests.

Tube forming capacity of the differentiated ECs at day 14–15 was tested on 10 mg/ml Matrigel Basement Membrane Matrix (BD Biosciences) according to manufacturer’s instructions. Briefly, 300 µl of Matrigel was plated per well of a 24-well culture plate kept on ice. Plates were incubated at +37°C for 50 min and 300 µl of cell suspension with 1.2 × 10⁵ cells in the EC differentiation medium with appropriate supplements were seeded per well. 50 µM L-sulforaphane (Sigma-Aldrich) was used to inhibit specific EC tube formation (41). Pictures of forming tubes were taken by a continuous live cell imaging system, Cell-IQ Analyzer (Chip-Man Technologies) supplied with a 10× objective 12–15 h after cell seeding.

Uptake of acetylated LDL (Ac-LDL) by differentiated ECs at day 14–15 was confirmed by Alexa Fluor^® 488 labeled Ac-LDL (L23380, Molecular Probes) at the final concentration of 1 µg/ml. Fibroblasts (ATCC, CRL-2429) cultured in endothelial cell growth medium (Thermo Scientific) on fibronectin-gelatin coating (10 µg/ml, 0.05%; Sigma-Aldrich) were used as negative controls. Cells were incubated with Ac-LDL for 3 h prior to fixing in 1% PFA - 2% FBS in PBS. Flow cytometric analyses were performed using BD FACSCalibur and FCS Express 6 Flow Research Edition software.

RNA sequencing was performed to compare transcriptional profiles of different EC differentiation groups and hiPSCs. RNA was extracted from cells with RNeasy Mini Kit (Qiagen) and depleted from rRNAs using Ribo-Zero Gold Kit (Illumina). RNA was fragmented using TURBO DNase and RNA fragmentation reagents (Life Technologies) and purified using P-30 columns (Bio-Rad, Hercules, CA, USA). Fragmented RNA was dephosphorylated with polynucleotide kinase (New England Biolabs, Ipswich, MA, USA) followed by heat-inactivation and purification using RNA Clean & Concentrator™−5 kit (Zymo Research Corporation, Irvine, CA, USA). Poly(A)-tailing and reverse transcription were performed as previously described (42). Libraries were amplified using 11 cycles, size-selected (200–350 bp) in 10% TBE gels (Life Technologies) and sequenced using Illumina HiSeq 2000 for 50 cycles according to the manufacturer's instructions.

RNA-Seq results were trimmed to remove 3′ A-stretches originating from the library preparation and poor quality reads were filtered out (minimum 97% of bp over quality cut off 10). Reads were aligned to the hg19 genome using Tophat allowing up to two mismatches and reporting only one alignment for each read. Data analysis was performed using HOMER (43) (http://homer.ucsd.edu/homer/) and the differential gene expression using edgeR (44). Thresholds of FDR < 0.05, reads per kb per million reads >0.5 and fold change >4 were used. Clustering results were generated by Cluster 3.0 (45) by normalizing and centering the gene expression tags to range from −1 to 1. The output from clustering was viewed using Java Treeview (46). The gene ontology (GO) analysis was performed using DAVID Bioinformatics Resources 6.8 (47) and gene lists for selected top functions were exported to generate clustered heatmaps. QIAGEN’s Ingenuity® Pathway Analysis (IPA®, QIAGEN Redwood City, www.qiagen.com/ingenuity) was used to perform Upstream Regulator Analysis with a focus on transcriptional regulators to identify which factors may be causing observed gene expression changes. Gene Expression Dynamics Inspector (GEDI) v2.1 (48) was used to generate self-organizing maps to visualize the expression profiles of different treatments and timepoints. Grid size 26 × 25, 1st phase training iteration 20 and 2nd phase training iteration 80, were used as settings to generate mosaic images. Principal component analysis (PCA) was performed using prcomp function in R environment and 3D plot was prepared using rgl R package.

RNA-Seq data from this study has been submitted to NCBI Gene Expression Omnibus under accession number GSE103945.

In this study, we systematically tested and compared the effects of the most potent signalling factors and small molecules reported to generate ECs from hiPSCs. Tested molecules included ROCK inhibitor indispensable for EC differentiation (25), TGFβ inhibitor that maintains vascular identity after the EC specification and support endothelial cell expansion (24, 49), cAMP analog 8-Br-cAMP that promotes differentiation of ECs (23, 31), and BMP-4 that contributes to mesodermal commitment (30). These molecules were used in seven different combinations (Table 1) added to the serum-free endothelial basal medium. In addition, well-known and essential growth factors for EC differentiation and proliferation, VEGF-A and FGF-2, were always included in EC culture media (50–52).

The optimal time necessary for EC differentiation was determined by culturing hiPSCs in seven different conditions up to 36 days. At the end of the assay, endothelial markers were expressed only in low number of cells (Figure S1). This allowed us to conclude that differentiation of hiPSCs into mature ECs takes approximately 15 days, which is well in accordance with previous literature (53). As seen in Figure 1, a similar trend in cell surface marker expression was seen in all EC treatment groups where the number of cells expressing generally used EC markers, CD31 (PECAM1), CD34, CD309 (vascular endothelial growth factor receptor 2, VEGFR-2, KDR) and CD144 (VE-cadherin, CDH5), was increasing during the first two weeks. Treatments groups did not differ statistically at the end of differentiation (day 15–16) based on all four cell surface markers analyzed although there were statistical differences between single markers. Differentiating ECs also showed similar morphology across different cell culture groups at day 15 resembling the appearance of HUVECs (representative images in Figure 2A–B).

Lack of the ROCK inhibitor in the cell differentiation media resulted in a poor attachment of cells onto fibronectin-gelatin coating. Indeed, ROCK suppression has been shown to be indispensable to EC proliferation and differentiation from PSCs (25), and therefore it was included in all cell culture conditions. Consequently, the treatment group containing only the ROCK inhibitor (R) served as a reference group in the following studies. When the TGFβ inhibitor was included with the ROCK inhibitor from the beginning of the differentiation (RT), EC differentiation was promoted and more than 50% of the cells expressed CD31 and CD144 markers at day 15 (Figure 1). Addition of 8-Br-cAMP (RTC) did not have a major effect on the marker expression in comparison with the RT group. When comparing group RC to the reference group R, especially marker CD309 and CD144 expressions were higher. In contrast, adding BMP-4 at day 1 resulted in the EC marker suppression (RB, RTB and RTCB groups). HUVECs were used as positive controls and almost 100% of the cells were positive for CD31 and CD144. In comparison, the number of HUVECs positive for CD34 and CD309 were approximately 70%.

In addition, expression of intracellular EC markers vWF and eNOS was studied. As shown in Figure 3A, vWF was highly and uniformly expressed in RT, RC, RTB, RTC and RTCB groups and resembled the expression profile seen in HUVECs. eNOS was also expressed in all treatment groups but the number of positive cells was lower than in HUVECs (Figure 3B). The highest percentage of positive cells for eNOS (approximately 96%) was in RC group.

Next, we studied the functional characteristics of ECs originating from different treatment groups. First, tube formation assay was employed to demonstrate the angiogenic activity of ECs. Our analysis demonstrated that a similar tube formation potential on Matrigel was evident in all tested conditions compared to mature ECs (HUVECs) (Figure 2C–D). The tube formation was inhibited using 50 µM sulforaphane (data not shown). Secondly, we studied the capacity of the differentiated ECs to take up Ac-LDL. In all tested EC culture groups, cells were able to internalize Ac-LDL (Figure 4).

The gene expression profiles of differentiating ECs were studied in more detail using RNA-Seq. For transcriptome analysis, cells were treated as described above and RNA was isolated at days 5 and 15 upon differentiation. Single replicates of day 5 were used to visualize the intermediate mesodermal stage, whereas two replicates were used for more throughout analysis of the end point of differentiation at day 15 and for the control hiPSC samples. The two timepoints were clearly separated by principal component analysis (PCA) (Figure S2A), with iPSC cells located next to day 5 samples and day 15 farther apart. Along the PC1 axis, which contributes most to the variance in the dataset (36% vs 17% for PC2), the day 15 samples situated closest to HAEC and HUVEC controls supporting successful reprogramming. Also the two replicates exhibited high reproducibility by PCA (Figure S2B). The expression of EC markers studied by flow cytometry analyses was well in concordance with the sequencing results, suggesting that changes on the transcript levels were translated to a similar profile on the protein level (Figure 5B and Table S1). We also studied the expression of endothelial LDL scavenger receptor SCARF1 responsible for the uptake of Ac-LDL (53–56) and adhesion proteins and enzymes involved in ECM processing related to tube forming ability. At mesodermal phase at day 5, differentiating cells had lower expression of EC-related genes except vWF. Groups RB, RTB and RTCB clustered together at day 15 suggesting a significant connective role of BMP-4 for these groups (Table S1). In these groups, the expression of EC-related genes was the lowest which reflects the results seen visually in flow cytometric analyses even though no statistically significant differences between treatment groups was found (Figure 1). Among them, group RTCB had the lowest expression of EC genes while the expression of MMP1 and PLAU was high in this group. Interestingly, at a differentiation day 15, treatment group RC had more upregulated EC genes (CD34, NOS3, KDR) than the other treatment groups. RC group did not yet express EC surface markers on day 10 as evidenced by FACS (Figure 1) but the expression of EC markers took off by day 11. This suggests that the differentiation process is slower although still successful in this group.

First, we analyzed how the landscape of transcripts was changed in the differentiation groups compared to the hiPSC reference using the Gene Expression Dynamics Inspector (GEDI) analysis (48). Altogether 8535 RefSeq transcripts, corresponding to 4136 different genes, were found differentially regulated in at least one differentiation regimen at day 15 (FDR <0.05, FC > 4, RPKM > 0.5; Table S1). Majority (3690/4136) corresponded to protein-coding accessions (NM_), whereas the remaining 11% represented non-coding RNAs (NR_). GEDI genomic landscape maps demonstrated that the gene expression patterns of hiPSCs and differentiation groups at day 5 were more similar to each other than the day 15 groups (Figure 5A). Additionally, resulting GEDI representations also revealed that a distinctive EC-specific gene expression profile began to emerge in RB, RTB and RTCB groups already at day 5 (Figure 5A). However, by day 15 a more prominent shift in gene expression pattern was seen with shutting down of genes related to regulation of pluripotency and activation of genes related to focal adhesion in all groups (Figure 5A, star/hash, respectively). In line with this, the GO analysis revealed that pathways and signaling related to pluripotency and cellular adhesion, including focal adhesion, cell adhesion molecules, ECM-receptor interactions and tight junctions, were significantly enriched among the differentially regulated genes (Figure 5C). Notably, the pluripotency genes were more highly enriched at day 5 whereas at mature cell level the top GO term was associated with focal adhesion.

In Figure 5D–E, hierarchical clustering of genes related to focal adhesion and pluripotency demonstrated faster gene expression changes for RB, RTB and RTBC groups at day 5 and distinct upregulation of mesodermal genes INHBA, BMP4, MEIS1, HAND1 and TBX3. In contrast, a higher similarity of all differentiation groups was seen especially in pluripotency related gene expression pattern at day 15. However, the RC group demonstrated the highest expression of VEGF-family members, including FLT1, FLT4, KDR and VEGFC in line with the role of cAMP signaling promoting the survival of VEGFR-2 positive cells (31) and TGFβ inhibition in maintaining vascular identity after mesodermal fate (24). In addition, a larger group of adhesion molecules, including integrins and collagens, were activated with the three conditions including BMP-4 treatment at day 15 (Figure 5D). Moreover, TFs, such as mesodermal related MEIS1, HAND1 and TBX3 (57, 58) (http://pathcards.genecards.org/card/mesodermal_commitment_pathway) together with SMAD9, ISL1 and ZFHX3, were highly induced by the BMP-4 groups suggesting distinct differentiation signatures. This suggests that a distinct set of TFs could be induced by different cell culture conditions.

It has been suggested that there are master regulators that control lineage-specific gene expression and such TFs would be highly expressed in differentiated cell types (59–61). Therefore, we studied the expression of differentially regulated TFs between the hiPSC and various EC differentiation conditions. Our analysis identified 186 TFs, which clearly clustered the samples based on the differentiation stage (Figure 6A). For example, pluripotency related TFs POU5F1 (OCT-4), SOX2, JARID2 and GLI1 were highly repressed in all treatment groups except R and RC at day 5. The clustering of R/RC together with hiPSC samples at day 5 also suggests that 8-Br-cAMP does do not promote early cell differentiation. On the other hand, RT/RTC groups already exhibited repression of pluripotency genes, suggesting that early TGFβ inhibition promotes differentiation at day 5. Notably, the most striking difference was seen for the RB, RTB and RTBC groups that clustered away from the other treatment groups at day 5. This was most attributable to the early repression of MYCN, SOX2, SOX21, SOX11, SOX13 and POU3F1 genes, which was evident in the other treatment groups only at day 15. Additionally, these groups also exhibited a high expression level of mesodermal TFs GATA2 and GATA3 at day 5. This likely reflects the ability of exogenous BMP-4 to promote differentiation of pluripotent cells toward mesodermal cells (33).

As a demonstration of the success of differentiation, the major EC-specific TFs, such as ETS1, ETS2, JUNB, ERG, SOX7, SOX17, SOX18, HHEX, ELK3, KLF6, MEF2C and FLI1 (62–68) were highly induced in the majority of differentiation groups at day 15 (Figure 6A). The highest expression of these crucial EC-related TFs was in RC group and the lowest in RTCB group. This suggests that RC, with the highest expression of EC markers (Figure 5B) and EC-specific TFs (Figure 6A) likely represents the most EC-like phenotype. This result was further confirmed by studying the expression of 50 most highly expressed TFs in HUVECs and HAECs, where RC group clustered closest to these mature EC types (Figure S3).

To study how changes in the TF expression could explain the global changes in gene expression patterns, we further searched for other upstream transcriptional regulators using Ingenuity Pathway Analysis (IPA). In line with TF expression, our analysis identified MYCN and SOX2 as the top transcriptional regulators inhibited during differentiation, whereas SMAD3, ETS1 and AP1 signaling was activated (Figure 6B–G). Although not directly linked with the induction of the respective TF, also SMARCA4, GATA6 and KMT2A were identified as possible activated upstream regulators (Figure S4). These TF networks could thus explain gene expression changes during EC differentiation. Interestingly, a similar pattern of activation was also highly associated with a chemical compound tretinoin (Figure S5).