Mouse and human iPSC culture and differentiation
Mouse iPSCs were generated in our laboratory by transfection of pCAG2LMKOSimO vector expressing c-Myc, Klf4, Oct4, and Sox2 into MEFs58,59, and cultured in gelatin-coated flasks (PBS; Life Technologies 10010056 containing 0.02% gelatin from bovine skin; Sigma-Aldrich G1393) in DMEM (ATCC 30-2002) supplemented with 10% fetal bovine serum (FBS) (Embryomax; Millipore ES-009-B), 100 IU/ml penicillin, and 100 μg/ml streptomycin (PenStrep) (Thermo Fisher Scientific 10378-016), 10 ng/ml recombinant human leukemia inhibitory factor (Millipore LIF1010) and 0.1 mM 2-mercaptoethanol (Life Technologies 31350-010) in a humidified incubator supplemented with 5% CO2. Cells were passaged every 2 days at a ratio of 1:6. EC differentiation of iPSCs was induced by seeding the cells on type IV mouse collagen (5 μg/ml; R&D Systems 3410-010-01)-coated dishes in differentiation medium (DM) containing α-MEM (Life Technologies 32571036) supplemented with 10% FBS (Invitrogen 10270106), 0.05 mM 2-mercaptoethanol, 100 units/ml penicillin, and 100 μg/ml streptomycin in the presence of 25 ng/ml VEGF (Thermo Fisher Scientific PMG0111) for the time points indicated. For high-glucose treatment, culture medium was supplemented with D-glucose to reach concentrations indicated in the Figures. L-glucose was used as controls in these experiments.
Diabetic patient-specific iPSCs were generated based on a protocol which we have recently reported30. Mononuclear cells from donor peripheral blood were expanded and reprogrammed into iPS cells by transfection of nonintegrating episomal plasmid vectors pEB-C5 (overexpressing Oct4, Sox2, Klf4, c-Myc, and Lin28), and pEB-Tg vector (overexpressing SV40 large T antigen). The pluripotency and germ layer differentiation capacity of the generated iPS lines were verified. Ethical approval was obtained from the Office for Research Ethics Committees of Northern Ireland (ORECNI) (REC 14/NI/1109).
Verbal and written information about the study was provided to all participants and written informed consent was obtained prior to study procedures from those willing and consenting to take part. Patients with type 1 and type 2 diabetes of more than 15 year standing and age- and sex-matched non-diabetic volunteers, who acted as controls, were recruited to this study. Patients unable to provide informed consent for the study were excluded. All the iPS cells-based in vitro and in vivo studies were confirmed in two independent non-diabetic and two diabetic donors sets based on three biological replicates with n = 3. The QKI-7 expression was also verified in four additional iPS-ECs lines generated from four independent (sex and age-matched) diabetic donors, based on three biological replicates with n = 3. On the day of blood sample collection, a detailed medical history was obtained and blood pressure was measured. A 20 ml sample of peripheral blood was obtained by venepuncture into VACUETTE® K3 EDTA-coated 4-ml tubes (454021, GREINER Bio-One).
Human iPSCs were cultured using mTeSR-1 medium (Stemcell 85850) following the manufacturer’s instructions. For EC differentiation, iPSCs were first cultured for 3 days in a 1:1 mixture of DMEM/F-12 (Thermo Fisher Scientific 31330038) with Neurobasal medium (Thermo Fisher Scientific 21103049) supplemented with N2 (Thermo Fisher Scientific 17502048), B27 (Thermo Fisher Scientific 17504044), 8 µM CHIR99021 (Sigma-Aldrich SML1046), and 25 ng/ml BMP4 (Life Technologies PHC9534). Thereafter, the cells were cultured for 2 days in StemPro 34 medium (Thermo Fisher Scientific 10639011) supplemented with 200 ng/ml hVEGF (Life Technologies PHC9391) and 2 µM Forskolin (Sigma-Aldrich F6886). On day 6, CD144 positive cells were magnetically sorted using MicroBeads Kit (Miltenyi BIotec 130-097-857) and cultured in EGM-2 media (LONZA 00190860). Human coronary artery endothelial cells were purchased from Lonza: HCAEC Coronary Art Endo Cells (CC-2585), DHCAEC Human Coronary Art Endo Cells Diabetic (CC-2921). The cells were cultured in EGM-2 media (LONZA 00190860). All methods regarding the mouse and human iPSC culture and differentiation were performed in accordance with the relevant institutional guidelines and regulations including Declaration of Helsinki and Ethical Guidelines for Medical and Health Research Involving Human Subjects.
Glucose treatment in human fibroblasts
L-Glucose (G5500) or D-glucose (G7021), both from Sigma, were reconstituted in fibroblast media (10% FBS F-12K medium (Kaighn’s modification of Ham’s F-12 medium) by ATCC (30-200). Fibroblasts (HFL1 (ATCC® CCL-153™)) were seeded in 6-well plates in 2 ml media per well at a seeding density of 100,000 or 200,000 cells for a total of 6 and 3 days of glucose treatment, respectively. Cell culture media were substituted on day 2 and on day 4. After the initial glucose treatment on day 0, the treatments were repeated with L- or D-glucose at a final concentration of 30 mM on day 2 and day 4 until harvested for subsequent real-time PCR analysis.
The SiRNA duplex for specific silencing of QKI-7 was synthesized (Sigma-Aldrich). The SiRNA sequence was -GUGAGGAGAUUGGUAUUAGUU-. A total of 1 × 106 iPS-ECs were resuspended in Nucleofector® solution, mixed with 5-µM SiRNA and transferred into an amaxa certified cuvette. A scrambled SiRNA (Thermo Fisher AM4611) was used as control. After 48 h, the cells were harvested for quantification of gene and protein levels of QKI-7.
Constructs for gene manipulation and luciferase assays
QKI-7 ORF was cloned into pReceiver-Lv225 vector to generate QKI-7 overexpression construct (GeneCopoeia). For lentivirus packaging, 1 × 107 293-T cells were transfected with QKI-7 overexpression plasmid, pCMV-dR8.2 (Addgene 8455), pCMV-VSV-G (Addgene 8454), and Endofectin (GeneCopoeia EFM1004). The supernatant containing the lentivirus was harvested twice at 48 and 96 h later, filtered (0.45 µm) and stored at −80 °C. Overexpression constructs for hnRNPM (plasmid #64924), CUG-BP (plasmid #61276) and NLGN1 (plasmid #15260) were obtained from Addgene. TSG6 (NM_007115.3) ORF was cloned into the 3rd generation of lentivirus vector to generate TSG6 overexpression construct (VectorBuilder). 3′ UTR sequence of NLGN1 was obtained by RT-PCR and cloned downstream of luciferase (hLuc) reporter gene in a Firefly/Renilla Duo-Luciferase reporter vector pEZX-MT06 (GeneCopoeia). Plasmid transfections were performed using Endofectin following the manufacturer’s instructions.
Quantitative real-time polymerase chain reaction (RT-PCR)
Total RNA was extracted using the RNeasy Mini Kit (Qiagen 74104) according to the manufacturer’s protocol. Two micrograms of RNA were reverse transcribed into cDNA with random primer by reverse transcriptase (RT) (Thermo Fisher Scientific 4374966). Relative gene expression was determined by quantitative real-time RT-PCR, using 20 ng cDNA (relative to RNA amount) for each sample with the SYBR Green Master Mix (Thermo Fisher Scientific A25742) in a 10 μl reaction. Ct values were measured using a LightCycler 480 sequence detector (Roche). GAPDH served as the endogenous control to normalize the amounts of RNA in each sample. The primers are shown in Supplementary Table 1.
Enzyme-linked immunosorbent assay
The concentration of TSG6 released into the supernatant of 6-day differentiated ECs was detected by TSG6 ELISA (Sigma-Aldrich RAB1092) according to the manufacturer’s procedure.
Cells were harvested and washed with cold PBS, resuspended in lysis buffer (25 mM Tris-Cl pH 7.5, 120 mM NaCl, 1 mM EDTA pH 8.0, 0.5% Triton X-100) supplemented with protease inhibitors (Roche 11697498001) and lysed by ultra-sonication (twice, for 6 s) (Bradson Sonifier150) to obtain whole-cell lysate. The protein concentration was determined using the Bradford Dye Reagent (Bio-Rad 500-0205). Fifty micrograms of whole lysate was applied to SDS-PAGE and transferred to Hybond PVDF membrane (GE Health 15259894), followed by standard western blot procedure.
Flow cytometric analysis
iPSCs derived ECs were dissociated by incubation with 0.05% Trypsin-EDTA (Thermo Fisher Scientific 25300054) for 5 min at 37 °C. A total of 1 × 106 single cells were resuspended in 100 μl FACS buffer (PBS supplemented with 10% FBS) and stained by incubation with 5 μl CD144-APC antibody for 30 min at 4 °C in the dark. The cells were washed twice with PBS before resuspension in 1 ml PBS for FACS experiment on an Attune NxT Flow Cytometer and data were analyzed with Attune software (Thermo Fisher Scientific).
The bound primary antibodies were detected by the use of horseradish peroxidase (HRP)-conjugated secondary antibodies by Bio-Rad (170-6515, 170-6516, 1:3000) and the ECL detection system (GE Health GERPN2232). Primary antibodies include QKI-7 (UC Davis/NIH NeuroMab Facility 73-200, WB 1:1000, ICC 1:100), CD144 (St John’s Laboratory STJ96234, WB 1:1000, ICC 1:200), CD31 (Abcam AB28364, WB 1:1000, ICC 1:20), KDR (R&D Systems MAB3571, WB 1:1000), FLK1 (Thermo Fisher Scientific MA5-15157, WB 1:1000), eNOS (Abcam AB76198, WB 1:1000), NLGN1 (Abcam ab153821, WB 1:1000), β-actin (R&D Systems MAB8928, WB 1:1000), and ZO-1 (Thermo Fisher Scientific 40-2200, ICC 1:200). For immunofluorescence staining, Alexa Fluor secondary antibodies were used (Thermo Fisher Scientific A21202, A11057, A28175, A11004, A11055, A11008, 1:500). For flow cytometry, CD144-APC (Thermo Fisher Scientific 17-1449-42, 1:20) and Mouse IgG1 kappa Isotype Control-APC (Thermo Fisher Scientific 17-4714-82, 1:800) were used.
Luciferase reporter assay
For luciferase reporter assays, 4 × 104/well of iPS-ECs were seeded on collagen-coated wells of a 12-well plate in EGM-2 medium. 0.33 μg/well of reporter plasmids were co-transfected with QKI-7 or control (0.17 μg/well) plasmids using Endofectin Transfection Reagent (GeneCopoeia EFM1004) according to the protocol provided. pGL3-Luc Renilla (0.1 μg/well) was included in all transfection assays as an internal control. Luciferase and Renilla activity assays were detected 48 h after transfection using Dual-Glo Luciferase Assay System (Promega E2920). Relative luciferase units (RLU) was defined as the ratio of luciferase activity to Renilla activity with that of control set as 1.
RNA-binding protein (RBP) immunoprecipitation assays
RBP immunoprecipitation assays were performed on iPS-ECs using the Magna RIP kit from Millipore (17-700) according to the protocol provided. A QKI-7 specific antibody from Millipore, CUG-BP (PA5-85997), hnRNPM (MA1-91607) and mouse or rabbit IgG-purified antibodies were used. The precipitated RNA was subjected to RT-PCR using specific primers for the binding sites of QKI, CUG-BP, and hnRNPM motifs.
Immunofluorescence cell staining
Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS for 10 min and blocked in 5% swine or donkey serum in PBS for 30 min before incubation with primary antibodies, previously mentioned, for 1 h at 37 °C. Alexa Fluor secondary antibodies by Thermo Fisher Scientific were incubated for 45 min at 37 °C. Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich), mounted in Fluoromount-G (Cytomation; DAKO, Glostrup, Denmark), and imaged using a fluorescent (Axioplan 2 imaging; Zeiss) or confocal microscope (SP5, Leica, Germany).
Immunofluorescence tissue staining
Tissues were fixed with cold acetone (4 °C), washed with TBS and blocked in 5% donkey serum in TBS for 2 h at room temperature in a humidified atmosphere before incubating with primary antibodies overnight at 4 °C. The following day, Alexa Fluor secondary antibodies were incubated for 45 min at 37 °C and DAPI solution (62248, Thermo Fischer Scientific) for 5 min at room temperature. Tissues were mounted on VectaShield Antifade Mounting Medium (H-1000) and imaged using a fluorescent (80i Eclipse; Nikon, Japan) or confocal microscope (SP8, Leica, Germany).
Hematoxylin and eosin staining
Tissue sections were rinsed in tap water for 5 min to hydrate and then stained with Harris’ hematoxylin solution (Leica 381560E) for 5 min at room temperature. Tissues were then rinsed in tap water for 3 min. Next, tissues were dipped in 1% acid ethanol (1% HCL + 70% alcohol aq. Leica 3801590E) for 10 s and then soaked into 1% ammonia in H2O for 30 s. Tissues were then placed into filtered eosin solution (Leica 3801590E) for 3 min. Tissues were next dehydrated with 75, 95, and 100% absolute ethanol. Finally, for the clearing step, tissues were rinsed with clearing agent (Leica 3803600E) for 9 min and slides were mount with DPX mountant (Sigma 06522).
Sections were air-dried at RT for 15–20 min. Sections were next rinsed shortly with tap water and incubated for 10 min in cold 1:1 acetone:methanol solution and let them dry for 40 min at RT. Sections were then permeabilized with 0.2% Triton X-100 in TBS for 10 min at RT. And washed with 0.025% TBS-T for another 15 min. Next sections were blocked with BLOXALL® Blocking Solution (30150 ZF0726) for 10 min at RT (Peroxidase blocking) and washed with 0.025% TBS-T for another 15 min at RT. Sections were then blocked for 2 h at room temperature with 5% Donkey serum in 1% BSA in 0.025% TBS-T, and incubated overnight at 4 °C with primary antibody (aSMA Ab5694 and CD31 Ab28364). Next day, sections were washed with 2 × 5 min with TBS and blocked in 0.3% H2O2 in TBS for 15 min at RT. Tissues were then incubated with secondary antibody (goat anti-rabbit, Bio-Rad, Cat. 1706515) in 5% donkey serum, 1% BSA, and in 0.025% TBS-T for 45 min at 37 °C in humidity DAB solution (K4010, lot 015095) and Dab Substrate buffer (K4006 Lot 104224) was then applied to tissue sections for 10 min in the dark, and washed for 15 min with 0.025% TBS-T and for another 5 min rinsed with tap water. Finally, tissues were stained with haematoxylin blue as described in our H&E staining omitting the eosin incubation step.
In vitro tube formation assay
Hundred microliters of Matrigel (Corning 356231) was distributed into wells of a 96-well plate using pre-cooled tips and incubated at 37 °C for 1 h. A total of 1 × 104 iPS-ECs were seeded onto the Matrigel bed of each well and incubated at 37 °C. The formation of tube structures was observed at 6 and 8 h, which was quantified as tube length and meshed area using Image J.
Cell barrier examination
iPS-ECs were seeded on type IV mouse collagen (5 μg/ml; R&D Systems 3410-010-01)-coated E-Plate VIEW 16 (ACEA Biosciences, Inc. 300600890) in EGM-2 medium containing 25 ng/ml VEGF at 40,000 cells/well. Continuous monitor was performed overnight using xCELLigence RTCA DP (ACEA Biosciences, Inc.) in a standard CO2 cell culture incubator until the cell index (CI) reached plateau, which represented the impedance of electron flow caused by adherent ECs. At this stage, the culture medium was replaced by OptiMEM to starve the cells for 2 h. Culture medium was subsequently changed to normal EGM-2 supplemented with 50 ng/ml VEGF and real-time CI monitoring continued for 24 h.
In vivo Matrigel plug assay
Animals used in these studies were housed with free access to standard food and water at a room temperature of 21 ± 2 °C relative humidity of 45 ± 15% and a 12-h-light/dark cycle. All experiments were performed in accordance with the Guidance on the Operation of the Animals (Scientific Procedures) Act, 1986 and approved by the Queen’s University Belfast Animal Welfare and Ethical Review Body. Work was performed under the project license number PPL2821.
In total, 5 × 105 QKI-7-overexpressing iPS-ECs or control cells (iPS-ECs overexpressing an empty vector) were mixed with 150 µl Matrigel and injected subcutaneously into the back or flank of 10-week-old male C57BL/6 mice. Six injections were conducted for each group based on n = 3 biological replicates. Seven days later, the mice were sacrificed and the plugs were harvested and frozen in liquid nitrogen for cryosectioning and immunostaining. Nine plugs for each group were used for quantification.
In total, 2 × 104 iPS-ECs were seeded into wells of a 96-well plate and cultured at 37 °C in 5% CO2 and 95% air humidified atmosphere. After 24 h, 1 × 104 Vybrant-labeled THP-1 cells were added into each well and co-cultured with iPS-ECs for 1 h. Thereafter, the cells were washed three times with PBS to remove the THP-1 cells in suspension. THP-1 adhesion was measured at six different areas under the microscope and the results were expressed as the number of THP-1 cells per mm2.
Streptozocin (STZ)-induced diabetes in mice
About 10 mg/ml STZ solution was prepared with citrate buffer at pH 4.5. Ten-week-old male C57BL/6 mice weighing more than 20 g were fasted for 4 h before intraperitoneal injection of STZ solution at 50 mg/kg (5 µl/g). Injections were performed for 5 consecutive days. One week later the blood glucose level was determined. Blood readings above 15 mmol/l were considered diabetic. All experiments were performed in accordance with the Guidance on the Operation of the Animals (Scientific Procedures) Act, 1986 and approved by the Queen’s University Belfast Animal Welfare and Ethical Review Body. Work was performed under the project license number PPL2821.
Generation of shQKI-7 vector (pLV[miR30-shRNA]-Cd144>EGFP)
shQKI-7 and shNT vectors under the CD144 promoter were designed for use in the in vivo experiments to target QKI-7 in ECs. shRNA sequence can only be driven by polymerase III promoters such as the U6 or H1 promoters. Since the tissue-specific promoter is a Pol II promoter, shRNA transcription was achieved via the miR30-based method, in which shRNA was embedded in a miR30 scaffold to be transcribed as an artificial miRNA expressing cassette. Vector ID VB180427-1084hrb.
Experimental hindlimb ischemia
The mouse hindlimb ischemia model was performed by ligation of femoral artery30,60. For EC transplantation, QKI-7 overexpressing iPS-ECs or control cells were trypsinized and 1 × 106 cells in 100 µl PBS were injected intramuscularly into the adductors of ischemic animals. For in vivo lentivirus infection, 108 transducing units (TU shRNA lentivirus) (non-targeting (NT) or QKI-7 (pLV[miR30-shRNA]-Cd144>EGFP) in a construct containing CD144 promoter, to target ECs) in 100 µl PBS was injected intramuscularly into the adductors. Tissue blood flow of both legs was sequentially assessed by Laser Doppler imaging (moorLDL2-IR) at 7 and 14 days. All experiments were performed in accordance with the Guidance on the Operation of the Animals (Scientific Procedures) Act, 1986 and approved by the Queen’s University Belfast Animal Welfare and Ethical Review Body. Work was performed under the project license number PPL2821.
HRP-DAB immunohistochemical staining of human arterial tissue
Arterial tissue was isolated from patients with critical limb ischemia undergoing a lower-limb amputation. Patients age: 72.7+/−11.3, sex: males 87.4%, degree of atherosclerosis: ***. Arterial plaques were graded according to the Oxford grading system. All human arterial samples were obtained with informed consent and procedures were performed in accordance with institutional guidelines and the Declaration of Helsinki (Ethical reference: 14/NW/1062). The ethics application was approved by the NRES Committee North West – Lancaster and Manchester Metropolitan University Internal Ethics Approval Committee. Tissue was fixed in 4% paraformaldehyde/PBS, processed for wax embedding and cut into 7-µm sections. Immunohistochemical analysis was carried out using the QKI-7 antibody (as above), alongside a mouse non-immune IgG control, and developed using the DAB-peroxidase system (Vector Laboratories) followed by counterstaining with Mayer’s haematoxylin61,62. Sections were imaged using the Panoramic SCAN with Zeiss Plan-apochromat 20×/0.8 objective (3D Histotech/Laser2000, Ringstead, UK).
Biological replicates were performed in all the experiments with n = 3. Data are expressed as the mean ± SEM and were analyzed using GraphPad Prism 5 software with a two-tailed Student’s t test for two groups or ANOVA for more than two groups. A value of *p < 0.05, **p < 0.01, ***p < 0.001 was considered significant.
Further information on research design is available in the Nature Research Reporting Summary linked to this article.