Reagents and antibodies
The following antibodies were used at the specified concentrations: mouse anti-human CD34-APC 1:10 (130–046–703, Miltenyi), mouse anti-human CD133/2 (293C3)-PE 1:10 (130–090–853, Miltenyi), mouse anti-human CD144-PE 1:10 (VE-cadherin; 560410, BD biosciences), mouse anti-human CD144-APC 1:10 (VE-cadherin; 348507, Biolegend), CD105-PE 1:10 (endoglin; ab60902, Abcam), mouse anti-human CD105-PE 1:10 (endoglin; 560839, BD biosciences), CD31-FITC 1:10 (555445, BD biosciences), CD117-PeCy7 1:10 (c-Kit; 339195, BD biosciences), VEGFR2-PE 1:10 (KDR; 560494, BD biosciences), mouse anti-human CD45-FITC 1:10 (130–080–202, Miltenyi), anti-human CD235a-PE 1:10 (340947, BD biosciences), mouse APC isotype control 1:10 (555751, BD biosciences), mouse FITC isotype control 1:10 (555748, BD biosciences), PeCy7 isotype control 1:10 (557872, BD biosciences), PE isotype control 1:10 (555749, BD biosciences), VE-Cadherin 1:500 (555661, BD biosciences), Endoglin 1:500 (M3527, DAKO), Anti-von Willebrand Factor 1:200 (vWF; 7356, Millipore), calponin 1:500 (Dako, M3556), α-SMA 1:500 (AB56994, Abcam), α-SMA 1:1000 (A5228, Sigma), PECAM-1 (M-20) 1:100 (CD31; sc1506, Santa Cruz Biotechnology) anti-Human Nuclei 1:100 (MAB1281, Millipore), DAPI [5 mg ml−1] 1:2000 (D1306, Invitrogen), Hoechst 33342 [5mg ml−1] 1:2000 (B2261, Sigma), Alexa fluor 488 goat anti-mouse (A11001, Invitrogen), Alexa fluor 488 Donkey anti-goat (A11055, Invitrogen), Alexa fluor 568 Donkey anti-mouse (A10037, Invitrogen), Alexa-fluor 568 Donkey anti-rabbit (A10042, Invitrogen).
Human ES cells, H1 (WA1, WiCell), HuES 9 (http://www.mcb.harvard.edu/melton/hues/
) and Human iPS cells CBiPS27
(KIPS 4F#2, CBiPS 2F#4) (passage 25–45) were cultured in chemically defined hES/hiPS growth media (mTeSR29
on growth factor reduced matrigel (35623, BD biosciences) coated plates). Briefly, 70–80% confluent hES/iPS cells were treated with dispase (Invitrogen) for 7 minutes at 37°C, colonies were dispersed to small clusters and lifted carefully using a 5 ml glass pipette, at a ratio of ~1:4. Neonatal human fibroblasts (HFF-1, BJ; ATCC) and adult human dermal fibroblasts (HDF-693) were cultured in DMEM containing 10% FBS, 2 mM GlutaMAX (Invitrogen), 50 U ml−1
penicillin and 50 mg ml−1
streptomycin (Invitrogen). Human Umbilical Vein Endothelial Cells (HUVEC) were purchased from Promocell and cultured in EBM medium supplemented with EGM-2 singlequots (cc-3162, Lonza), 2% FBS, hEGF 10 μg ml−1
, Heparin 100 μg ml−1
(Sigma). iPS/ES-derived endothelial cells were cultivated in EBM-2 medium supplemented with EGM-2 singleQuot kit (cc-3162, Lonza). iPS/ES-derived smooth muscle cells were cultured in SmBM medium supplemented with SmGM-2 singleQuot kit (cc-3182, Lonza). All the cells were grown in collagen I coated plates (BD biosciences). All cell lines were maintained in an incubator (37°C, 5% CO2) with media changes every day (hES/iPS) or every second day (HUVEC/Fibroblasts).
Directed differentiation of hES/hiPS cells in chemically defined conditions
Human ES/iPS cells cultured as described above were freshly split on matrigel-coated plates, making sure the sub-colonies were of small size (~300–500 cells/colony). After 24 hours of recovery in mTeSR, the cells were gently washed using DMEM:F12 (Invitrogen) and allowed to grow in chemically defined differentiation media (Mesodermal Induction Media or MIM), which consists of DMEM:F12, 15 mg ml−1 stem cell grade BSA (MP biomedicals), 17.5 μg ml−1 Human Insulin (SAFC bioscience), 275 μg ml−1 Human holo-transferrin (Sigma Aldrich), 20 ng ml−1 bFGF (Stemgent), 50 ng ml−1 Human VEGF-165 aa (Humanzyme), 25 ng ml−1 human BMP4 (Stemgent), 450 μM monothioglycerol (Sigma Aldrich), 2.25 mM each L-Glutamine and Non-essential amino acids (Invitrogen), systematically designed by titration of growth factors and culture conditions. Media was changed every second day with addition of half the volume of media every other day.
Single Cell Differentiation Assays
Upon MIM differentiation for 8 days, CD34+ Angioblast-like cells (Angioblast-like) were sorted and plated in collagen I coated 48-well plates at a density of one cell/well in either EBM-2 (endothelial differentiation) or SmBM (Smooth Muscle differentiation) supplemented as described above. After 7 days in the respective differentiation conditions cells were washed once with PBS and fixed with 4% Paraformaldehyde (PFA) in 1X PBS. Following fixation, cells were blocked and permeabilized for 1 hour at 37° C with 5% BSA/5% appropriate serum/1X PBS in the presence of 0.1% Triton X100. Subsequently, cells were incubated overnight at 4° C with an anti-endoglin antibody in case of cells in EBM-2/EGM-2 or with an anti-calponin antibody in the case of cells in SmBM/SmGM-2. Cells were then washed thrice with 1X PBS, incubated for 1 hour at 37° C with the respective secondary antibodies and 20 minutes with DAPI for nuclear staining. Following incubation, cells were washed thrice with 1X PBS before microscopy analysis and scoring.
Conversion of human fibroblasts into angioblast-like CD34+ progenitor cells
For retroviral infection, 75,000/well human fibroblast cells (HFF-1, BJ, HFF-693) were plated on matrigel-coated 6-well plates. The next day, cells were infected with an equal ratio of either a combination of 4 pMX-derived retroviruses encoding Oct4, SOX2, KLF4 and c-Myc (4F) or 5 pMX-derived retroviruses encoding Oct4, SOX2, KLF4, c-Myc and miRs302–367 (4F/miRs). Scramble miRNA control (PMIRH000PA-1, SBI) was used whenever appropriate. The plates were infected by spinfection of the cells at 1850 rpm for 1 hour at room temperature in the presence of polybrene (4 μg ml−1) and put back in the incubator without media change. 24 hours later, the media was switched to WiCell media composed of DMEM/F12 (Invitrogen), 20% Knockout serum replacement, 10 ng ml−1 bFGF, 1 mM GlutaMax, 0.1 mM non-essential amino acids and 55 μM β-mercaptoethanol; with media changes every day. After 6 days, cells were split at a ratio of 1:3 on to matrigel coated 6-well plates supplemented with WiCell media for another 2 days. The cells were then washed once with DMEM/F12 and induced for differentiation for 8 days in the presence of MIM. Media was changed every second day with addition of half the volume of media every other day.
For episomal transfection, 2.106
cells were transfected with 1.5μg each of pCXLE-episomal vectors encoding for Oct4
(#27077, #27078 and #27080, addgene) with and without addition of pcDNA3.1 encoding for miRs302–367 (6F or 6F/miRs). Fibroblasts were transfected by nucleofection (Amaxa NHDF nucleofector kit, # VPD-1001) according to manufacturer’s instructions, and plated back on to matrigel-coated wells. After 6 days resting in DMEM/F12 supplemented with 10% FBS, 0.1 mM non-essential amino acids and 2 mM GlutaMAX, the media was switched to WiCell media with media changes every day. After 6 days, cells were split at a ratio of 1:3 on to matrigel coated 6-well plates with WiCell media for another 2 days. The cells were then washed once with DMEM:F12 and induced for differentiation for 8 days in the presence of MIM. Media was changed every second day with addition of half the volume of media every other day.
RNA isolation and real time-PCR analysis
Total cellular RNA was isolated using Trizol Reagent (Invitrogen) according to the manufacturer’s recommendations. 2μg of DNAse1 (Invitrogen) treated total RNA was used for cDNA synthesis using the SuperScript II Reverse Transcriptase kit for RT-PCR (Invitrogen). Real-time PCR was performed using the SYBR-Green PCR Master mix (Applied Biosystems). The levels of expression of respective genes were normalized to corresponding GAPDH values and are shown as fold change relative to the value of the control sample. All the samples were done in triplicate. The list of the primers used for real time-PCR experiments are listed in Supplementary Table 3
Flow cytometry analysis
Human ES/iPS cells undergoing directed differentiation, lineage converted CD34+ cells or their respectively derived endothelial cells were harvested using TripLE (Invitrogen), washed once with PBS and further incubated with the corresponding antibodies in the presence of FACS blocking buffer (1xPBS/10%FCS) for 1 hour on ice in the absence of light. After incubation, cells were washed thrice with 1 ml of FACS blocking buffer and resuspended in a total volume of 200 μl prior to analysis. A minimum of 10,000 cells in the living population were analyzed by using a BD LSRII flow cytometry machine equipped with 5 different lasers and the BD FACSDiva software. Percentages are presented after subtracting isotype background and referring to the total living population analyzed. Results are representative of at least three independent experiments with a minimum of two technical replicates per experiment.
After 8 days of differentiation CD34+ cells were stained as described above and sorted by using a BDAria II FACS sorter (BD Biosystems). Alternatively, CD34+ cells were enriched using anti-CD34 conjugated magnetic beads (Miltenyi) according to the manufacturer’s instructions with slight modifications. Briefly, up to 109 cells were incubated with constant mixing at 4°C with 100 μl of the corresponding magnetic beads in the presence of 100 μl of Fc-blocking solution in a total volume of 500 μl FACS blocking buffer. After 1 hour, cells were sorted by two consecutive rounds of column separation in order to increase purity by applying MACS separation magnets. Shortly, cells were passed through the first MS separation column allowing binding of labeled cells. Non-labeled cells were washed thoroughly with 3 ml FACS blocking buffer prior to elution of the labeled fraction. Eluted labeled cells were then subjected to a second purification step as described above.
Differentiation of CD34+ cells to endothelial cells
PSC- and lineage converted-CD34+ cells, isolated by MACS or by FACS sorting after 8 days of differentiation in MIM, were plated in collagen I coated plates (50,000 cells well−1 of a 12 well plate) and cultured in EBM-2/EGM-2 (Lonza) with media changes every day. After 5–8 days in culture, upon reaching 90% confluence, cells were split 1:4, using TripLE (Invitrogen). The cells were cultured for at least 8 passages.
Differentiation of CD34+ cells to smooth muscle cells
PSC- and lineage converted-CD34+ cells, isolated by MACS or by FACS sorting after 8 days of differentiation in MIM, were plated in collagen I coated plates (50,000 cells well−1 of a 12-well plate) and cultured in SmBM/SmGM-2 (Lonza) with media changes every day. After 5–8 days in culture, upon reaching 90% confluence, cells were split 1:4, using TripLE (Invitrogen). The cells were cultured for at least 8 passages.
DNA Methylation Analysis
Illumina 450K Infinium Methylation Arrays were normalized and pre-processed in Genome Studio. Probes with missing values were removed. A filter for average Beta value difference between groups (PSCs, Fibroblasts, primary human Arterial Smooth Muscle Cells (PriSMC), primary human Umbilical Vein Endothelial Cells (PriEC), PSC → CD34+ Progenitor Cells, PSC → Endothelial Cells (iECs), converted Endothelial Cells (cECs), PSC → Smooth Muscle Cells (iSMCs), converted Smooth Muscle Cells (cSMCs)) of ≥0.3 was applied. The resulting probes were used for ANOVA analysis using R scripts with a p-value filter of <0.0001)(at this point, 30,000 probes remained). Probes with beta value difference of at least 0.3 ((max - min) >= 0.3) were used. ANOVA test (p<0.05; Var 0.58) was applied to obtain statistically significant differentially methylated probes among the 5 groups in both SMC and EC sample group. The resulting probes (see Supplementary Data
) were used for hierarchical clustering using Cluster 3.0 with complete linkage. Venn Diagram Plotter (http://omics.pnl.gov/software/VennDiagramPlotter.php
) was used to generate “area-proportional Venn Diagrams”.
Gene Expression microarray analysis
The following groups were analyzed: PSCs, Fibroblasts, primary human Arterial Smooth Muscle Cells (PriSMC), primary human Umbilical Vein Endothelial Cells (PriEC), PSC → Endothelial Cells (iECs), converted Endothelial Cells (cECs), PSC → Smooth Muscle Cells (iSMCs) and converted Smooth Muscle Cells (cSMCs). Briefly, total RNA was extracted from collected sample pellets (Ambion mirVana; Applied Biosystems) according to the manufacturer’s protocol. RNA quantity (Qubit™
RNA BR Assay Kits; Invitrogen) and quality (RNA6000 Nano Kit; Agilent) was determined to be optimal for each sample prior to further processing. 200ng RNA per sample was amplified using the Illumina® Total Prep™
RNA Amplification Kit according to manufacturer’s protocol and quantified as above. 750ng RNA/sample was hybridized to Illumina HT-12v3 Expression BeadChips, scanned with an Illumina iScan Bead Array Scanner and quality controlled in GenomeStudio and the lumi bioconductor package. All RNA processing and microarray hybridizations were performed in-house according to manufacturer’s protocols. Differential expression was defined as a minimum 2x fold-change and multiple-testing corrected p<0.05 by ANOVA. The resulting probes (see Supplementary Data
) were used for hierarchical clustering using Cluster 3.0 with complete linkage. Probes with minimum gene expression differences between groups of 2× fold-change were obtained (see Supplementary Data
). Venn Diagram Plotter (http://omics.pnl.gov/software/VennDiagramPlotter.php
) was used to generate “area-proportional Venn Diagrams”.
Determination of copy number by quantitative PCR
Briefly, total DNA was extracted using the Qiagen DNeasy Blood & Tissue kit (QIAGEN). The purity and quantity of DNA was measured using a NanoDrop 8000 spectrophotometer (Thermo Scientific), and then used as templates for absolute quantitation by qPCR assay22
. The primers used are listed above and their amplification efficiencies, as well as specificity, were checked by performing standard curve and melting curve analyses.
Immunocytochemistry and fluorescence Microscopy
Briefly, cells were washed thrice with PBS and fixed using 4% PFA in 1X PBS. After fixation, cells were blocked and permeabilized for 1 hour at 37° C with 5% BSA/5% appropriate serum/1X PBS in the presence of 0.1% Triton X100. Subsequently, cells were incubated with the indicated primary antibody either for 1 hour at room temperature or overnight at 4° C. The cells were then washed thrice with 1X PBS and incubated for 1 hour at 37° C with the respective secondary antibodies and 20 minutes with DAPI or Hoechst 33342. Cells were washed thrice with 1X PBS before analysis. Sections were analyzed by using an Olympus 1X51 upright microscope equipped with epifluorescence and TRITC, FITC, and DAPI filters. Confocal image acquisition was performed using a Zeiss LSM 780 laser scanning microscope (Carl Zeiss Jena, Germany) with 20×, 40× or 63× immersion objectives.
Acetylated-LDL uptake assay and vascular tube-like structure formation assay
In short, 80% confluent endothelial cells derived from human ES/iPS cells were incubated with 10μg ml−1 Dil-Ac-LDL (L23380, Molecular Probes) for 3 hours in DMEM:F12. The cells were washed 3 times with PBS, dissociated using TripLE and analyzed by flow cytometry. Briefly, to assess the formation of capillary structures, a suspension of 4.105 cells ml−1 endothelial cells in the presence EBM-2/EGM-2 was prepared. Subsequently, 100 μl well−1 were dispensed on flat bottom 96 well plates coated with Matrigel (BD biosciences). Tube formation was observed after 24 hours of incubation and a minimum of three replicates per experiment analyzed.
Hematopoietic colony-forming assays
Hematopoietic clonogenic assays were performed in 35-mm low adherent plastic dishes (Stem Cell Technologies, Vancouver, BC, Canada) using 1.1 ml dish−1 of methylcellulose semisolid medium (MethoCult H4434 classic, Stem Cell Technologies) according to the manufacturer’s instructions. Briefly, enriched CD34+ cells were sorted and immediately plated at various densities: 1.5 × 103 ml−1, 3 × 103 ml−1 and 6 × 103 ml−1. All assays were performed in duplicate. After 21 days of incubation plates were analyzed for the presence of both, Colony-forming units (CFU) and Burst-forming units (BFU).
All murine experiments were conducted with approval of The Salk Institute Institutional Animal Care and Use Committee (IACUC). NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice (or NOD-Scid IL2rγ null abbreviated as NSG; age, 7 weeks; weight, 20 g) were purchased from Charles River Laboratories, housed in air-flow racks on a restricted access area and maintained on a 12 hour light/dark cycle at a constant temperature (22 ±1°C).
Matrigel plug assay
Anaesthesia was induced using a mixture of Xylazine (Rompun® 2%, Bayer) at 10 mg kg−1 and Ketamine (Imalgene1000, Merial) at 100 mg kg−1 in NaCl at 0.9% i.p injected at a dose of 10 ml kg−1. The animals’ backs were shaved, swabbed with Hexomedine®. Prior injection, HUVECs, HUES9-, KiPS-, BJ 6F- and BJ 6F/miRs- derived endothelial cells were harvested using TripLE (invitrogen). A total of 1.106 cells were resuspended in 500 μl of cold matrigel (Matrigel basement membrane matrix from BD Biosciences adjusted to 9.8 mg ml−1 PBS) supplemented with 150 ng of bFGF. Cell and no cell containing matrigel solutions were then injected subcutaneously in the back of mice, carefully positioning the needle between the epidermis and the muscle layer. Seventeen days later, mice were sacrificed and the matrigel plugs were removed by a wide excision of the back skin, including the connective tissues (skin and all muscle layers).
For immunohistochemistry (IHC), in situ hybridization (ISH) or immunofluorescence (IF) analysis, cell-containing implants with associated connective tissues were fixed with Accustain® (SIGMA) for 24 hours, dehydrated through an ethanol series and then processed for paraffin embedding before being sliced with a microtome. Slices from paraffin-embedded samples were stained with appropriate antibodies or probes. Alternatively, plugs were harvested and fixed with a 4% paraformaldehyde solution overnight at 4°, washed thrice in PBS and then incubated in a glucose solution (30%) for another 48 hours before being sliced (45μm) with a cryostat (Leica). Both methodologies were equally successful to identify neovasculature derived from human cells.
For IHC, slides were stained with an anti human-CD31 monoclonal antibody and then incubated with biotin-labeled secondary antibody followed by incubation with streptavidin-HRP (Ventana Roche).
For ISH, slides were hybridized according to the manufacturer’s protocol with an Alu probe (780–2845, Ventana Roche) and then labeled with the ISH iView Blue Detection kit (760–092, Ventana Roche).
For IHC and ISH, images were then captured with a camera mounted on a light microscope (Nikon E-800).
For immunofluorescence assays, slides were stained with either rhodamine-labeled Ulex Europaeus Agglutinin I (UEA I, a marker for human endothelial cells from Vector Laboratories) or PECAM-1 (M-20) (CD31; sc1506, Santa Cruz Biotechnology) and anti-Human Nuclei 1:100 (MAB1281, Millipore) counterstained with DAPI. Images were captured with confocal microscopes (Zeiss, LSM 510 or LSM780).
Calcium live cell imaging
Subconfluent cells were washed with DMEM:F12 and incubated for 45 minutes with 1 μM Fluo-4/AM (Molecular Probes) in 0.5% BSA, DMEM:F12 in an incubator at 37 °C, 95% CO2. After washing to remove unloaded dye, cells responses to 100 μM carbachol or vehicle (water) were imaged in HEPES-buffered, phenol red-free DMEM:F12 in a wide field fluorescent microscope (Olympus BX61WI) equipped for fast fluorescent imaging. Image capture was performed with Metamorph and an EM-CCD camera (Hamamatsu). Image analysis was carried out with Metamorph and Fiji software. To determine functional SMC contraction after stimulations, the cell surface area was determined before and after carbachol exposure.
Statistical analyses of all endpoints were performed by using standard unpaired Student t test (one-tailed, 95% confidence intervals) using the SPSS/PC + statistics 11.0 software (SPSS Inc.). All data are presented as mean ± standard deviation (s.d.) or standard error of the mean (s.e.m.) where indicated and represent a minimum of two independent experiments with at least two technical duplicates.