Combination human umbilical cord perivascular and endothelial colony forming cell therapy for ischemic cardiac injury – npj Regenerative Medicine

Ethics approval and umbilical cord collection

The study, including the collection of first-trimester human umbilical cords (8–10 weeks of gestation) from elective pregnancy termination and full-term human umbilical cords, was approved by the University of Toronto Research Ethics Board (REB #28889), and complied with ethical regulations including the Declaration of Helsinki and Tri-Council Policy Statement: Ethical Conduct for Research Involving Humans (TCPS 2 (2022)). Term newborn cords were collected through a third-party source (Lifeline Stem Cell). Written informed consent was obtained from all human participants.

Cell culture and phenotypic characterization

MSC lines from human first-trimester and term umbilical cords were previously established and characterized¹⁹. Cryopreserved HUCPVC (FTM and term HUCPVC) stored in liquid nitrogen were thawed at a constant temperature of 37 °C for 5 min¹⁹. Thawed cell suspensions (1,000,000 cells/ml) were 10x diluted in alpha-modified minimum essential medium eagle (α-MEM) (Gibco), supplemented with 5% human platelet lysate (HPL) (Compass Biomedical) and 1% penicillin/streptomycin (P/S) (Gibco). Cell suspensions were centrifuged at 1600 RPM for 10 min. Cell pellets were resuspended in 10 ml pre-warmed complete culture medium (a-MEM, 5% HPL, 1% P/S) and plated onto culture vessels. Cells were typically seeded at 2.5 × 10³ cells/cm² in 10-cm² culture dishes (Corning) and adjusted to the same ratios for different surface areas. Plated cells were incubated at 37 °C, 5% CO_2, and imaged daily to document cell morphologies and cell densities. HUCPVCs were passaged at 70–80% confluency using TrypleE (Life Technologies) dissociation solution (1 ml/2.5 cm²) for 5 min at 37 °C in 5% CO_2. Dissociated cells were diluted 1:1 with complete culture medium and centrifuged at 1600 RPM for 10 min. Cell pellets were resuspended in 1 ml complete culture medium and HUCPVC were counted using Trypan blue (Life Technologies) staining, in an automated cell counter (Invitrogen). The cells were either plated again for further culture expansion or processed for experimental assays. HUCPVCs at passage 6–7 were utilized for in vitro and in vivo experiments. Experiments were replicated with different cell donors, n values listed in figures.

ECFCs were isolated from bone marrow aspirates from 8-week-old Sprague–Dawley male rats as described previously⁷⁶. All animal procedures were conducted and reported according to ARRIVE guidelines and approved by the Animal Care Committee of the University Health Network (Toronto, Canada). All studies were performed with institutional research ethics board approval (AUP 4276, University of Toronto, Toronto, Canada). Bone marrow aspirates were collected from Sprague–Dawley male rats (8 weeks of age). Animals were euthanized in carbon dioxide chambers set to 20% gas replacement (flow rate = chamber volume × 0.2 per min). Hind limbs were removed by dislocating hip joints and placed in Hank’s balanced salt solution (HBSS) (Thermofisher) supplemented with 1% P/S. Fur and muscle were carefully removed, exposing the femur, tibia and fibula. Only the femur and tibia were utilized for bone marrow extraction. Epiphysis of bones were cut with surgical cutters and bone marrow cavities were flushed with HBSS (1%P/S). Flushed bone marrow cavities were filtered using 70μm filtering mesh (Corning). Diluted bone marrow aspirates were centrifuged at 1600 RPM for 10 min. Cell pellets were reconstituted with EGM (Lonza-same from chapter 2). Cells were seeded onto T75 (Sarstedt) flasks with 15 ml of EGM and incubated at 37 °C in 5% CO₂. Following 48 hours, medium was removed. Flasks were rinsed with PBS (x2) with strong tapping to remove all non-adherent cells. Cells were again cultured with 15 ml of fresh EGM and culture medium was changed every three days. Cell colonies were imaged regularly, where multiple ECFC colonies were observed at day 14 of culture. Depending on the success of bone marrow isolations, ECFC cultures were ready for experiments in 14 ± 2 days. For in vitro experiments, ECFCs were pre-labeled with Cell Tracker Orange™ (CTO) (Life Technologies) prior to cell detachment. 1 ul/ml of CTO was added to ECFC cultures for 30 min at 37 °C in 5% CO_2. Following incubation, culture medium was removed and 8 ml of TrypleE was added. Culture flasks were incubated for 15 min with intermittent tapping to detach ECFC colonies. Detached cell suspensions were diluted (1:1) with EGM and centrifuged for 15 min at 1600 RPM. Cell pellets were reconstituted with EGM and cells were counted using Trypan Blue staining and automated cell counter (Invitrogen). ECFCs were never frozen and used fresh for all experiments.

Both cell types were characterized using flow cytometry prior to experiments (Supplementary Figs. 5–8).

Phenotypic characterization of HUCPVCs by flow cytometry

For flow cytometry (FC) and fluorescence associated-cell sorting (FACS), cell cultures were dissociated and counted as mentioned above. Cell suspensions were incubated with fluorophore-conjugated primary antibodies, according to the provider’s description. For most antibodies, 50,000 cells were incubated with 2.5 μl of antibody for 20 min at 4 °C. FC analysis was performed using either a MACQuant analyzer (Miltenyi Biotec; Create Fertility Centre, Toronto) or digital (LSR II, Canto II, BD; UHN SickKids Flow Cytometry Facility, Toronto) analytical cytometers. FACS was performed using digital cell sorters (MoFlo Astrios, Aria II, UHN SickKids Flow Cytometry Facility, Toronto). Human-specific TRA-1-85⁺ sorted cells were collected in lysis buffer (Qiagen) for qPCR and next-generation sequencing (NGS). Fluorescence signals were gated based on unstained populations and gates were set at 10¹ fluorescence intensity decades. Mean fluorescence intensities were used for analysis. Gating strategies included in figures. Antibodies used for identifying HUCPVC phenotypes included CD90-APC (#130-114-903), CD105-APC (#130-098-778), CD146-FITC (#130-111-323), HLA-G-FITC (130-112-004) at a 1:40 dilution (Miltenyi), and PDGB-R-APC (R&D # FAB1263A) at 1:20 dilution (Supplementary Table 1).

Phenotypic characterization of ECFCs by flow cytometry

For flow cytometry (FC) and fluorescence-associated cell sorting (FACS), cell cultures were dissociated and counted (mentioned above). Cell suspensions were incubated with fluorophore conjugated primary antibodies according to the providers description. For all antibodies, 50,000 cells were incubated with 2.5 μl of antibody for 20 min at 4 °C. FC analysis was performed using the MACQuant analyzer (Miltenyi Biotec; Create Fertility Centre, Toronto). FACS was performed using digital cell sorters (MoFlo Astrios, Aria II, UHN SickKids Flow Cytometry Facility, Toronto). Fluorescence signals were gated based on unstained populations. Gates were set at 10¹ fluorescence intensity decades. Antibodies for ECFC FC were as follows: CD31 (AF3628 R&D-FITC), CD34 (AF6518 R&D-FITC), CD133 (NB120-16518 Novus Biologicals-PE), CD38 (50-0380-80 Thermofisher-APC), KDR (NB200-208 Novus Biologicals-PE), CD146 (560846 BD sciences-FITC), and VE-Cadherin (BS-0878R-A488 BioUSA-FITC) and CD117 (20-1172-u025- Tonbo Science-APC). All antibody titrations were performed and used at a concentration of 1:20 (Supplementary Table 2).

Model of myocardial infarction and cell transplantation

All animal procedures were approved by the Animal Care Committee of the University Health Network, following ARRIVE guidelines (AUP#4276.6, 893.35, 1133.21) (Toronto, Canada). NIH-Foxn1^rnu rats (Charles River) underwent left anterior descending coronary artery (LAD) ligation to induce MI. Eight-week-old, male nude rats (NIH-Foxn1^rnu-Charles River) were anesthetized (2% isoflurane), analgised (0.05 mg/kg buprenorphine), intubated, ventilated (2% isoflurane), and placed on a heated mat. Left-side thoracotomy was performed between the 4^th and 5^th ribs, the pericardium was opened, and the left coronary artery permanently ligated 2–3 mm from its origin with a 7–0 polysuture. White discolouration of the myocardium distal to the ligature confirmed successful infarction. Cyclosporine (5 mg/kg) and buprenorphine (0.05 mg/kg) were administered post-operatively for five days. At 5 days post-MI, cardiac function of all rats was evaluated using echocardiography and those with fractional shortening of 20–40% were randomly separated into 6 further groups (n = 9–12/group): G1: Medium injection; G2: undifferentiated FTM HUPVCs (3 × 10⁶ cells/rat); G3: undifferentiated FTM HUPVCs (1 × 10⁶ cells/rat); G4: FTM HUPVCs (6 × 10⁵ cells/rat) G5: ECFC (2.2 × 10⁶ cells/rat); G6: FTM HUCPVC and ECFC (1:2) (total cells count 3 × 10⁶ cells/rat); and G7: FTM HUCPVC and ECFC (1:4) (total cell count 3 × 10⁶ cells/rat). ECFC single injections included the average number of ECFCs that were injected in the 1:2 and 1:4 treatment groups (2 × 10⁶ and 2.4 × 10⁶ respectively). A total volume of 50 µl of cell suspension or equivalent volume of medium alone was injected into 3 peri-infarct areas, where animals were kept under anesthesia (2% isoflurane). Personnel injecting treatments were blinded to treatment groups. All groups had end-point analyses at 5 days and 4 weeks post cell injections. ECFC for these studies were obtained from bone marrow of 8-week-old Sprague–Dawley (SD) rats. ECFC and FTM HUCPVC were pre-labeled with Qtracker (Q-dot) (Thermofisher) dye prior to myocardial injection for the 5-day study.

Echocardiography

Cardiac function was measured by echocardiography before MI, day of cell injection (Day 0), 5 days, 2- and 4-weeks following cell injection. Rats were anesthetized as described above and echocardiography was undertaken with a GE Vivid 7 ultrasound system (GE Healthcare Canada) with a 10 S transducer (frequency 11.5 MHz, depth 2 cm). Short-axis views were obtained from the parasternal approach. Blinded left ventricle (LV) dimensions (left ventricular end-diastolic internal diameter (LVIDd) and end-systolic internal diameter (LVIDs) were measured in M-mode. Ejection fraction was calculated as (LVIDd³ – LVIDs³) / LVIDd³ × 100. Fractional shortening was calculated as (LVIDd – LVIDs) / LVIDd × 100.

Pressure–volume catheterization

Cardiac function was measured by a pressure-volume catheter as an end point assessment at 4 weeks post cell treatments. Animals were anesthetized (2% isoflurane), left-side thoracotomy was performed between the 4th and 5th ribs, the pericardium was opened, and sternotomy was performed. A 2.0 F micromanometer catheter (SPR8-838 Millar Instruments, Houston TX) was inserted through the myocardium into the left ventricle of anaesthetized animals. Pressure-volume changes were continuously sampled (1000/s) using a pressure-conductance system connected to a PowerLab/4SP analog to digital converter (AD instruments). Resulting pressure-volume loops were used to assess cardiac function (ejection fraction, end-systolic and diastolic volumes, dP/dt and tau) with PVAN 3.3 (Millar Instruments) software. Following this procedure, the aorta was cannulated with a 4 ml vacutainer containing EDTA (367861, BD) and blood collected for subsequent analysis. Hearts were arrested in diastole with 1 ml of 10% KCl and snap frozen in liquid nitrogen or fixed in 2% PFA for biochemical analysis or cryo-sectioning respectively.

Cell retention studies

Five days after cell treatments, hearts were collected, the left ventricle (LV) was excised and snap frozen in liquid nitrogen. For cell retention studies, human DNA content measurement was calibrated against human genomic DNA in rat hearts injected with 600k, 1 million, and 2 million FTM HUCPVC, where left ventricles were isolated within 30 min following injections (Day 0). Cell retention percentages were determined based on relative dose injected at time zero. Alu primers (IDT) were designed and qPCR was used to measure human cDNA in the LV. Ct values less than 28 were excluded.

Histological analysis

Fixed hearts were sectioned beneath the ligature into four 2 mm sections and prepared for cryo-sectioning through 10, 20, and 30% sucrose suspension. Frozen sections were processed by histological staining and immunohistochemistry. Scar size was determined from Masson’s trichrome stains and expressed as a percent of total left ventricle area.

Immunohistochemistry

Sections were washed (2 × 5 min) in tris-buffered saline (TBS) (Thermofisher) containing 0.025% TritonX (Millipore) and blocked in TBS containing 10% normal goat serum (Thermofisher) and 1% BSA for 1 h. Primary antibodies were diluted in TBS (1% BSA) and applied overnight at 4 °C, secondary antibodies were diluted at 1:500 and applied at room temperature for 1 h. Anti-Connexin 43 (Abcam-ab11370), anti-sarcomeric actinin (Abcam-ab32575), anti-PDGFR-β (Abcam-ab62437); Isolectin GS-IB4 (Life technologies-l-21412) and BODIPY Thapsigargin (Life technologies-B-7487); cleaved caspase-3 (Cell Signaling Technologies-9661S). All primary antibodies were used at 1:200 dilution except for IB4 (1:50). (Supplementary Table 3). Respective secondary conjugated antibodies were purchased from Thermofisher and used at a 1:500 dilution (Supplementary Table 4). Samples were coded; therefore, imaging and analysis was performed blinded. Image analysis was conducted using Fiji software (Image J)⁷⁷.

In-situ zymography

DQ Gelatin (Life Technologies) was applied to assess matrix metalloproteinase activity. Sections were renatured with PBS containing 2.5 % triton for 15 min and assay was performed according to the manufacturer’s instructions.

Multiplex luminex elisa assay

Custom Luminex Multiplex Elisa was designed using R&D systems custom tool, targeting 11 human-specific analytes. 200 mg of left ventricle homogenates were further lysed with proteinase inhibitors and 200 µl of PBS (N = 3). Ventricle homogenates were centrifuged at 1600 RPM for 2 min. Supernatants were collected and protein was quantified using Qubit protein assay kit (Thermofisher). Approximately 1.25 mg total protein was processed using manufacturer protocol (R&D). Fluorescent beads (PE) tagged with proteins were processed using flow cytometry (MACQuant analyzer, Miltenyi Biotec) to detect PE signal. Instrument settings were determined by processing control beads untagged with protein. Data analysis was conducted using FlowJo software. Mean fluorescence intensities were normalized to number of beads detected per sample.

Tissue-level cytokine analysis (Rat Proteome Profiler)

In all, 50 mg of left ventricle homogenates were further homogenized in 500 μl PBS and protein inhibitors. Protein was quantified using Qubit protein assay kit (Thermofisher) and ~2.4 mg total protein was processed using Proteome Profiler Rat XL Cytokine Array kit (R&D). Protein arrays were analyzed using the HLImage + + software. Pixel densities were normalized to internal positive controls (100%) and expressed as percentages of positive control.

Matrigel plug assay

Eight-week-old NU-Foxn1^rnu mice were injected subcutaneously with Matrigel combined with either 250 μl FTM HUCPVC-single injection (4 × 10³ cells/ul), ECFC-single injection (4 × 10³ cells/ul), 1:2 or 1:4 combination ratios (4 × 10³ cells/ul total) or cell-free medium injections). Following 14-days, Matrigel plugs were isolated and fixed in formalin for vasculature quantification. For transfection studies, FTM HUCPVCs were transfected with 25 ρM validated small interfering (si) RNAs targeting PDGFR-β, CD146, and control siRNAs using lipofectamine RNAiMAX Transfection Reagent and co-injected with ECFCs.

FTM, TERM HUCPVC, and ECFC were cultured using the methods mentioned above. Treatment groups included FTM:ECFC combinations (1:2, 1:4) using two independent FTM HUCPVC lines, term HUCPVC, HUCPVC, ECFC, and cell-free medium treatments. A total of 1,000,000 cells were prepared for each Matrigel plug injection in a volume of 1000 ml. The ratio of FTM HUCPVC and ECFC were adjusted to a total of 1,000,000. Prior to injections, both cell types were mixed and placed on ice. Prior to injections, 500 µl of cell suspensions were mixed quickly with 500 μl of Matrigel on ice. Matrigel mixed-cell suspensions were injected subcutaneously in FoxN1^nu 8-week-old male mice using a 22.1/4-gauge needle. Injected plugs were held with forceps until the Matrigel plugs polymerized. Each mouse was injected to carry 4 Matrigel plugs, containing a plug from each different cell treatment group (N = 4). Matrigel plugs were retrieved 14-days following injection. Dorsal skin was excised along the vertebral column and carefully opened to visualize Matrigel plugs. Macroscopic images were taken to observe superficial vascularity and blood coagulation within Matrigel plugs. Matrigel plugs were fixed in formalin for 48 hours, embedded in paraffin, and sectioned (5 micron). Sectioned tissue underwent Masson’s Trichrome staining to visualize perfused vasculature and changes in the Matrigel matrix. Blood vessel density was quantified using ImageJ software and stratified based on regions within the Matrigel plugs.

ECFC tube formation assay

Twelve-well plates were pre-coated with 300 μl of Matrigel (phenol red with growth factors) and incubated for 30 min (37 °C, 5% CO₂). Polymerization was attenuated with 200 μl of EBM. HUCPVC and ECFC were pre-stained with Cell Tracker™ Green and Orange respectively for 20 min at 37 °C, 5% CO. Pre-stained cells were washed and resuspended in EGM. HUCPVC and ECFC cell suspensions were mixed at 1:2 (33k:66k) or 1:4 ratio (20k:80k) ratios for a total of 100,000 cells/12-well respectively. Mixed cell suspensions were added to Matrigel-coated wells and topped with 1 ml EGM. For transwell (TW) co-cultures, ECFC were seeded onto Matrigel-coated wells while HUCPVC were seeded on TW inserts (0.4um). TW inserts were placed into ECFC-seeded wells and 500 μl of EGM were placed on top and below TW inserts. Conditioned media was analyzed for human-derived proteins. Cells were isolated and processed for next-generation sequencing using the Ion PGM sequencer as described below.

Imaging and quantification of endothelial networks

For HUCPVC-ECFC co-cultures, bright field (Olympus) microscopy was used to visualize initiation of endothelial structures. At least 4 phase-contrast images were taken to capture ECFC-derived networks. Tubular lengths and thickness were documented for up to 14 days. Quantification of network properties was performed using ImageJ software, utilizing the angiogenesis plugin on day 3 co-cultures. Fluorescence microscopy (EVOS™; LifeTechnologies) was used to visualize physical interactions between HUCPVC and ECFC. Fluorescence images allowed for tracking HUCPVC in relation to ECFC-derived vascular structures.

Human and rat-specific proteome profilers

Conditioned medium from HUCPVC and ECFC co-cultures were collected at different time points throughout assay setup (24 h, 48 h). To determine changes in HUCPVC-secreted proteins following co-culture, the Proteome Profiler™ Human Angiogenesis Array Kit (ARY007, R&D) was used. Conditioned medium was collected, centrifuged and the supernatant was used for arrays. Proteinase inhibitor cocktail (Thermofisher) was added to supernatants and samples were processed according to manufacturer’s protocol. To detect changes in rat endothelial cell secreted proteins following co-culture, the Proteome Profiler Rat XL Cytokine Array Kit (ARY030) was used. Conditioned medium samples were processed as above, and samples were processed according to manufacturer’s protocol. Protein arrays were analyzed using the HLImage + + software. Pixel densities were normalized to internal positive controls (100%) and expressed as a percentage of positive control.

Processing endothelial structures for downstream analysis

Conditioned medium was collected at 24 hours following co-culture in tube formation assays. Conditioned medium was centrifuged at 1600 RPM for 5 min, supernatant collected, and stored at -80 °C for subsequent protein analysis. At assay endpoint, endothelial networks were washed with phosphate-buffered saline (PBS) (Sigma-Aldrich) and disrupted to harvest cells from Matrigel. Briefly, 300 μl of Dispase™ (Stemcell Technologies) were added to each well and incubated at 37 °C, 5% CO for 15 min. Following incubation, quick aspirations and release were conducted to disrupt Matrigel-embedded endothelial networks. Cell suspensions including Dispase and digested Matrigel were collected in 15 ml falcon tubes and this process was repeated (3x). The viability of harvested cells ranged between 55–70% and, approximately 80–100 K cells were harvested. Cell suspensions were centrifuged at 1600 RPM for 6 min. Following the removal of supernatant, cell pellets were resuspended in 3 ml of trypsin and incubated for 10 min (37 °C, 5% CO). EBM was added (1:1) to neutralize trypsin. Cell suspensions were centrifuged at 1600 RPM for 6 min. Supernatants were carefully removed, and cell pellets were either resuspended in PBS supplemented with 3%FBS for FC/FACS or resuspended in lysis buffer (Qiagen) for qPCR/NGS.

Aortic ring assay set-up

The aortic ring assay was established as done previously²⁶. All animal procedures were conducted and reported according to animal research reporting of in vivo experiments (ARRIVE) guidelines and approved by the Animal Care Committee of the University Health Network (Toronto, Canada), (AUP 4276, University of Toronto, Toronto, Canada). Aortic tissues were isolated from Sprague–Dawley female rats of reproductive age (week 9). Animals were euthanized in carbon dioxide chambers set to 20% gas replacement (flow rate = chamber volume × 0.2 per min). Aortas were exposed by an excision through the chest cavity and lung tissue was removed. Aorta’s were identifiable adjacent to the vertebral column. Using surgical tools, the thoracic aorta was excised and cut into ~1 mm sections, yielding approximately 15–20 rings. Matrigel™-phenol red with growth factors (200 μl) (Corning) was coated evenly on 12-well plates (on ice) and then placed in a humidified incubator (37 °C, 5% CO₂) for 30 min. Once the Matrigel was polymerized; a freshly obtained aortic ring was placed at the center of each well. Then 300 μl of Matrigel was carefully applied on top of the aortic ring tissue and incubated for 30 min (37 °C, 5% CO₂). Once polymerized, 1000 μl of pre-warmed endothelial growth medium supplemented with FGF, VEGF, EGF, IGF, hydrocortisone, ascorbic acid, heparin and gentamicin (Endothelial complete medium- EGM, Lonza) were added. EGM was removed 24 hours following incubation and replaced with 1000 μl of endothelial basal medium supplemented with 2% fetal bovine serum (FBS) (Hyclone) and 1% P/S (EBM) for the remainder of the assay and replaced every 2 days. Bright field microscopy was used to monitor endothelial sprouts until endogenous endothelial networks (5–7 days) were ready for co-culture experiments. For direct co-cultures, HUCPVCs were harvested and prepared as single-cell suspensions using TrypLE (Invitrogen) at 37 °C for 5 min. HUCPVC were pre-stained with cell tracker green dye (CTG) (CellTrackerGreen™; Life Technologies) for 30 min, washed (1x) and resuspended in EBM + 2% FBS, 1% P/S. Using an automated cell counter, approximately 10,000 pre-stained HUCPVC were added to the aortic ring assay endothelial networks (Day 0).

Imaging and quantification of endothelial networks

For the aortic ring assay, bright-field (Olympus) microscopy was used to document changes in network growth and structures. Phase-contrast images of four fields (4 quadrants of viewing field) were taken to measure radial network growth and total number of network closed loops up to.

7 days after addition of pre-labeled HUCPVC. Quantification of network growth and network loops was performed using ImageJ software, utilizing the angiogenesis plugin, on day 5 of co-culture. Fluorescence microscopy images (EVOS™; Life Technologies) were taken to evaluate HUCPVC-mediated ECM processing and migratory potential. Fluorescence images could identify preferential homing of HUCPVC to different regions of aortic networks and development of physical interactions between HUCPVC and endothelial cells during co-culture. Investigators were blinded to treatment groups.

Targeted RNA sequencing of human HUCPVC from ECFC co-cultures using next-generation sequencing

RNA was isolated from HUCPVC/endothelial co-cultures (n = 3), HUCPVC alone cultures (n = 3) and ECFC single cultures using the Qiagen RNAeasy kit, according to the manufacturer’s instructions. RNA quantification and quality assessments were performed using the Qubit Fluorometer (ThermoFisher) and Bioanalyzer 2100 (Agilent Genomics) at the Princess Margaret Genomic Centre (Toronto, Canada). RNAseq libraries were prepared with Ion Ampliseq RNA library kit 2.0 (Invitrogen, Carlsbad, California, USA). Briefly, 10 ng of RNA was reverse transcribed using SuperScript IV VILO (Invitrogen Life Technologies, Carlsbad, California, USA) into cDNA and selectively amplified with a targeted RNA sequencing custom panel that included 108 amplicons as outlined in (Supplementary Table 5), according to the manufacturer’s instructions (Invitrogen). Primer sequences were partially digested, and barcode-adaptors (Ion Xpress Barcode Adaptors) were ligated to the amplified cDNA followed by purification using AMPure XP beads (Beckman Coulter) and additional amplification steps. Purified, amplified libraries were equalized to 23 pM using Ion Library TaqMan™ Quantitation Kit (ThermoFisher). Ion 318™ Chip Kit v2 chips (Invitrogen Life Technologies) were used for sequencing. Up to 24 samples were loaded per chip using 23 pM of the pooled libraries. NGS was performed using the Ion PGM sequencer, 400 flows (Invitrogen Life Technologies). Unaligned read files (FASTQ) were uploaded to Partek Flow (Partek) for bioinformatic analysis. Samples were trimmed for quantity, aligned to the rat genome (rn5) using Spliced Transcripts Alignment to a Reference (STAR) (v2.5.2)⁷⁸, and subjected to post-alignment quality control. All unaligned reads were realigned to the human genome (hg19) using STAR (v2.5.2). Aligned files were subjected to post-alignment QC to determine the number of useable reads for each sample, percent alignment, and Phred quality score. Samples with greater than 100,000 total reads, showing greater than 98% alignment, were included in the analysis (and no samples were excluded based on this criteria). Targeted RNASeq analysis was performed by quantifying aligned reads to transcriptome using the Partek/EM platform and Homo sapiens (human) – hg19 canonical Transcripts as the genome build and the custom amplicon panel as the annotation model. Quantification was performed with strict paired-end compatibility and minimal read overlap, with the feature option set at 75% of read length. Transcripts were filtered (excluded if the maximum <5.0) and normalized using trimmed mean of M-values (TMM). Principal component analysis (PCA) was performed to assess the variability of the dataset. Differential gene expression and statistical analysis were performed by comparing each of the untreated HUCPVC to co-cultured HUCPVC. Transcripts were defined as differentially expressed if the fold change (FC) -2 > FC > 2 and False Discovery Rate (FDR) < 0.05.

PDGFR-β silencing

siRNA for PGDFR-B (2 validated constructs s10242, s10241), CD146 (1 validated construct s8572), and scrambled negative control (4390843). All purchased from Thermofisher. Constructs were first diluted by 10 and final concentration used was 25ρmol as suggested by manufacture. FTM HUCPVC (25,000 cells) were plated in each well of a 6-well plate. Cells were cultured in basal α-MEM medium with 2.5% HPL (Compass Biomedical). Following 24 hours, alpha MEM was replaced with Opti-MEM factor-reduced medium (Life Technologies) supplemented with 2.5% HPL. Mixtures of siRNA with lipofectamine (Thermofisher) were administered to cells. Block-it^TM (ThermoFisher) fluorophore-labeled control RNA was used to visualize transfection efficiency. Conditioned medium was collected, and cells of different incubation groups were harvested every 24 h for protein and gene analysis (for 5 days). FTM HUCPVC were processed for flow cytometry and qPCR analysis to confirm successful downregulation of the target protein and its mRNA. To test other methods of silencing PDGFR-β, FTM HUCPVC were first pre-stained with Cell Tracker Green (described previously). Pre-stained FTM HUCPVC were processed similarly to that used for flow cytometry protocols. Approximately 50,000 FTM HUCPVC were incubated with FITC-conjugated, human-specific primary monoclonal mouse IgG antibody for PDGFR-β (2.5ug/100ul) (R&D AB-20-NA) and associated FITC isotype control (BD Biosciences 555748). for 20 min. Following incubation, FTM HUCPVC were washed and approximately 10,000 FTM HUCPVC were co-seeded in the aortic ring assay.

Statistical analysis

Data are expressed as the mean ± standard error of the mean. Inter-group comparisons were performed using analysis of variance. If the analysis of variance F ratio was significant, differences were specified by Tukey post-hoc tests. All statistical analyses were performed using GraphPad Prism 6 (GraphPad). Differences of p < 0.05 were considered statistically significant. Data are displayed as box and whisker plots except proteome array results due to grouped analyses. Description of the box whisker plots: The center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points and outliers. For groups without a whisker suggest either that the lower quartile is equal to the minimum or the higher quartile is equal to the maximum. Next-generation sequencing statistical analysis was performed using Partek Flow (Partek). Transcripts were defined as differentially expressed if the fold change (FC) -2 > FC > 2 and False Discovery Rate (FDR) < 0.05. N values are documented in figure legends for all experiments.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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