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. Author manuscript; available in PMC: 2023 Jan 1.
Published in final edited form as: Methods Mol Biol. 2022;2549:169–186. doi: 10.1007/7651_2021_377

A High-Efficiency Method for the Production of Endothelial Cells from Human Induced Pluripotent Stem Cells

Maryna Pavlova 1,2, Shennea S McGarvey 1,2, Ganna Bilousova 1,2,3, Igor Kogut 1,2,4
PMCID: PMC8460679  NIHMSID: NIHMS1699894  PMID: 33755906

Abstract

Endothelial cells (ECs) are important components of the circulatory system. These cells can be used for in vitro modeling of cardiovascular diseases and in regenerative medicine to promote vascularization of engineered tissue constructs. However, low proliferative capacity and patient-to-patient variability limit the use of primary ECs in the clinic and disease modeling. ECs differentiated from human induced pluripotent stem cells (iPSCs) can serve as a viable alternative to primary ECs for these applications. This is because human iPSCs can proliferate indefinitely and have the potential to differentiate into a variety of somatic cell lines, providing a renewable source of patient-specific cells. Here, we present an optimized, highly reproducible method for the differentiation of human iPSCs toward vascular ECs. The protocol relies on the activation of the WNT signaling pathway and the use of growth factors and small molecules. The resulting iPSC-derived ECs can be cultured for multiple passages without losing their functionality and are suitable for both in vitro and in vivo studies.

Keywords: Induced pluripotent stem cells, iPSCs, endothelial cells, differentiation, WNT pathway

1. Introduction

Vascular endothelial cells (ECs) line the interior surface of blood vessels in the entire circulatory system and play a key role in many physiological processes and homeostasis. Hence, ECs represent the key cell type for many in vitro disease models (1, 2). These cells have also been utilized in vascular tissue engineering approaches (3, 4) and applied in clinical trials (5). However, the application of primary ECs is limited by their reduced proliferation capacity and the rapid loss of functionality of these cells in culture (6). ECs derived from human induced pluripotent stem cells (iPSCs), potent immature stem cells capable of becoming most cell types in the body (7, 8), can be considered as a viable alternative to primary ECs for clinical and scientific applications (9–11). This is because human iPSCs can proliferate indefinitely, providing an unlimited source of cells for the derivation of ECs. Moreover, evidence suggests that iPSC-derived ECs (iPSC-ECs) can be more potent in promoting vascularization of engineered tissue constructs than widely used human umbilical vein cells (HUVECs) (12), emphasizing the importance of developing efficient protocols for the derivation of ECs from iPSCs.

Multiple protocols for the differentiation of human iPSCs into ECs have been published to date (11–21). The underlying principle of all these protocols is to mimic early developmental stages during iPSC differentiation by activating canonical WNT signaling to induce mesodermal commitment, cardiogenesis, and the formation of vascular cells (13, 17, 22–24). The activation of the canonical WNT signaling pathway in iPSCs promotes the derivation of multi-potential cardiogenic mesodermal intermediates. These intermediates are then stimulated with small molecules and growth factors under selective conditions to induce endothelial specification.

Building on these previously published protocols, we have developed a modified approach for the derivation of human iPSC-ECs as depicted in Fig. 1. In this optimized method, human iPSCs are first treated with CHIR-99021 to activate WNT signaling through GSK-3β inhibition and to induce mesodermal commitment. While many approaches require multiple growth factors to induce mesodermal commitment in iPSCs, our protocol uses only CHIR-99021, which is sufficient to generate high-quality iPSC-ECs as previously described (21). Following CHIR-99021 treatment, the cells are further differentiated toward the EC lineage by applying VEGF-A (Vascular Endothelial Growth Factor A) together with forskolin. The endothelial specification starts at day four. At this point, the mesodermal initiation cocktail is replaced with complete StemPro-34 (SFM) medium supplemented with VEGF-A and forskolin. Forskolin promotes endothelial commitment via cAMP activation. Together with VEGF-A, cAMP ensures that resulting iPSC-ECs maintain their plasticity and can further differentiate toward either arterial or venous lineages (19, 23). While StemPro-34 (SFM) medium (supplemented with VEGF165, the most abundant and potent isoform of VEGF-A) is critical for efficient differentiation of iPSCs toward ECs, prolonged exposure of iPSC-derived intermediates to this medium can lead to the derivation of CD45+ population (15). To avoid this, after magnetic automated cell sorting (MACS) at day seven, the purified CD31+ iPSC-ECs are expanded on collagen-coated plates in low serum endothelial cell growth medium (ECGM). The addition of SB431542 to ECGM at this stage further enhances the derivation of ECs. SB431542 inhibits TGF-β-SMAD2/3 signaling through the blocking of the type I and II receptors. This inhibition promotes VEGF-A-dependent phosphorylation and the upregulation of VEGF receptors, which in turn lead to endothelial cell expansion and the maintenance of long-term vascular identity of iPSC-ECs (25, 26). The inhibition of the TGF-β pathway can also prevent the expansion of undesirable smooth muscles cells (22, 27), which often appear during iPSC differentiation toward ECs and other mesodermal derivatives.

Fig. 1. Schematic representation of the differentiation protocol.

Fig. 1

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Individual steps and the timeline for the differentiation of human iPSCs toward an endothelial cell lineage are depicted

Using the provided protocol, we have successfully differentiated multiple iPSC lines into ECs, including iPSCs derived from individuals with Down Syndrome. We have found that resulting iPSC-ECs exhibit all typical features of ECs, such as the expression of VE-cadherin and PECAM-1, and maintain their functionality in culture for up to fourteen passages. An important advantage of our method is the low-density plating of human iPSCs, which produces a healthy monolayer during differentiation, making the method quantitative and reproducible.

2. Materials

2.1. Human iPSC passaging

  1. Complete mTeSR1 medium: mTeSR1 basal medium supplemented with 5x mTeSR1 supplement according to the manufacturer’s instructions (STEMCELL Technologies).

  2. DPBS (no calcium, no magnesium, Thermo Fisher Scientific).

  3. 0.5 mM EDTA (human iPSC passaging reagent): Add 500 μL of 0.5 M EDTA (pH 8.0) to 500 mL of DPBS (no calcium, no magnesium). Filter-sterilize using a 0.22 μm vacuum filtration system. Store at room temperature (RT).

  4. 1× Pen/Strep/Fungizone (GE Healthcare).

  5. Vitronectin (VTN-N), recombinant human protein, truncated (Thermo Fisher Scientific): upon receipt, thaw the matrix and prepare 60 μL aliquots. Freeze aliquots at −80°C.

  6. 10 cm tissue culture dish.

  7. Cell scraper.

  8. Human iPSC line.

2.2. Plating human iPSCs for differentiation

  1. Complete mTeSR1 medium (see Subheading 2.1, item 1 above).

  2. Y-27632 dihydrochloride (ROCK inhibitor) (STEMCELL Technologies).

  3. DPBS (no calcium, no magnesium, Thermo Fisher Scientific).

  4. 0.5 mM EDTA (human iPSCs passaging reagent) (see Subheading 2.1, item 3 above).

  5. Matrigel human Embryonic Stem Cell (hESC)-Qualified Matrix, LDEV-free (Corning). Matrigel solidifies rapidly at RT. Therefore, it is recommended to aliquot each new batch of the matrix upon arrival following the manufacturer’s instructions. Use prechilled pipette tips, racks and tubes while working with the reagent. Store at −80°C.

  6. Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) medium (Thermo Fisher Scientific).

  7. 10 cm tissue culture dish.

2.3. Mesoderm induction

N2B27/CHIR-99021 medium (day 1–3 mesodermal induction medium): DMEM/F-12 medium mixed with Neurobasal medium at a 1:1 ratio (both from Thermo Fisher Scientific) and supplemented with 0.97% N-2 supplement (Thermo Fisher Scientific), 1.94% B27 minus Vitamin A supplement (Thermo Fisher Scientific), 0.097% 2-Mercaptoethanol (Thermo Fisher Scientific), and 8 μM CHIR-99021 (STEMCELL Technologies).

2.4. Endothelial specification

  1. EndoM1 medium (days 4–5 endothelial specification medium 1): StemPro-34 SFM basal medium containing StemPro-34 Nutrient Supplement (both from Thermo Fisher Scientific), 1× Glutamax (Thermo Fisher Scientific), 1× Pen/Strep/Fungizone (GE Healthcare), 2 μM Forskolin (Sigma), and 200 ng/mL VEGF-A (STEMCELL Technologies).

  2. EndoM2 medium (day 6 endothelial specification medium 2): StemPro-34 SFM basal medium containing StemPro-34 Nutrient Supplement (both from Thermo Fisher Scientific), 1× Glutamax (Thermo Fisher Scientific), 1× Pen/Strep/Fungizone (GE Healthcare), and 200 ng/mL VEGF-A (STEMCELL Technologies).

2.5. Human iPSC-EC harvest and magnetic automated cell sorting (MACS)

  1. Complete StemPro-34 (SFM): StemPro-34 SFM basal medium containing StemPro-34 Nutrient Supplement (both from Thermo Fisher Scientific), 1× Glutamax (Thermo Fisher Scientific), 1× Pen/Strep/Fungizone (GE Healthcare), 50 ng/mL VEGF-A (STEMCELL Technologies).

  2. Accutase (STEMCELL Technologies).

  3. Collagen I stock solution, 3 mg/mL (Advanced BioMatrix).

  4. CD31 MicroBeads (Miltenyi Biotec).

  5. FcR Blocking Reagent (Miltenyi Biotec).

  6. MACS Running Buffer, pH 7.2: To prepare 500 mL, measure 2.5 g of BSA powder (Fisher Scientific), dissolve it in 450 ml of DPBS, and adjust volume to 500 mL. Filter MACS Running Buffer using a 0.22 μm vacuum filtration system and store at 4°C. Before use, warm the buffer to RT and degas using vacuum (see Note 1).

  7. MACS separator.

  8. MACS stand.

  9. MS Miltenyi Columns (Miltenyi Biotec).

  10. 30 μm strainer.

  11. 15 mL conical tubes.

  12. Eppendorf tubes.

2.6. Plating CD31+ iPSC-ECs post-MACS enrichment

  1. Complete ECGM medium: vascular cell basal medium (VCBM) supplemented with Endothelial Cell Growth Kit-VEGF following the manufacturer’s instruction (ATCC).

  2. ECGM/SB medium: complete ECGM from step 1 above supplemented with 10 μM SB SB431542. Add SB431542 just before use.

  3. Medium A: complete StemPro-34 (SFM) (see Subheading 2.5, item 1) mixed with complete ECGM medium from item 1 above at a 3:1 ratio and supplemented with 10 μM SB431542. Add SB431542 just before use.

  4. Medium B: complete StemPro-34 (SFM) (see Subheading 2.5, item 1) mixed with complete ECGM medium from item 1 above at a 1:1 ratio and supplemented with 10 μM SB431542. Add SB431542 just before use.

  5. Medium C: complete StemPro-34 (SFM) (see Subheading 2.5, item 1) mixed with complete ECGM medium from item 1 above at a 1:3 ratio and supplemented with 10 μM SB431542. Add SB431542 just before use.

  6. 6-well plates.

2.7. Expansion of human iPSC-ECs

  1. ECGM/SB medium (see Subheading 2.6, item 2).

  2. Accutase (STEMCELL Technologies).

  3. 10 cm dishes.

2.8. Cryopreservation of human iPSC-ECs

  1. CryoStor CS10 (STEMCELL Technologies).

  2. Cryovials.

  3. Freezing container.

2.9. Equipment

  1. Biological Safety Cabinet.

  2. 37°C water bath or bead bath.

  3. 37°C/5% CO2 humidified normoxic (20% O2) tissue culture incubator.

  4. 37°C/5% CO2/low-O2 (5%) humidified tri-gas tissue culture incubator.

  5. Inverted microscope.

  6. Hemocytometer.

  7. Centrifuge.

  8. Freezing container.

  9. Vacuum aspirator.

  10. MACS sorting apparatus (MACS separator, Miltenyi Columns).

3. Methods

Perform all cell culture procedures and manipulations in a biological safety cabinet using aseptic techniques. Follow institutional biosafety standards for work with human cells.

The provided protocol describes the procedure for the differentiation on a 10 cm tissue culture dish followed by the MACS enrichment of the CD31+ iPSC-EC population. To scale up, recalculate all components according to the size of the tissue culture dish to be used.

In the provided protocol, human iPSCs are cultured on vitronectin-coated tissue culture plates in complete mTeSR1 medium using a tri-gas tissue culture incubator with O2 set to 5% (low-O2). The cells are passaged every 3–5 days using 0.5 mM EDTA. Before proceeding with the differentiation protocol, ensure that human iPSCs are adapted to the above-mentioned culturing conditions (see Note 2).

3.1. Human iPSC passaging (Days −5 to −3)

  1. Prepare one well of a 6-well plate of iPSCs for passaging. Confirm that iPSCs are healthy with no differentiation. iPSC culture should be 70–80% confluent before passaging.

  2. Prewarm 12 mL of complete mTeSR1 to 37°C.

  3. Coat one 10 cm tissue culture dish with vitronectin following the manufacturer’s instruction. One 60 μL aliquot is sufficient to cover the entire 10 cm dish. Seal the dish with parafilm and incubate for 1 h at RT.

  4. Aspirate the vitronectin solution from the coated dish and replace with 9 mL of mTeSR medium. Move the dish to a tri-gas tissue culture incubator with O2 set to 5% (low-O2) for equilibration.

  5. Aspirate the spent cell culture medium from the well with iPSCs to be passaged and add 1 mL of DPBS to rinse. Gently rock the plate to ensure complete coverage of the cells and then aspirate DPBS. Repeat the rinse with DPBS.

  6. Aspirate DPBS and add 1 mL of RT 0.5 mM EDTA. Gently rock the plate to ensure complete coverage of the cells and return the plate to the low O2 incubator. Incubate for 3 min (see Note 3).

  7. Gently remove the plate with iPSCs to be passaged from the incubator and check colony disaggregation under a microscope.

  8. Place the plate in the biosafety cabinet and carefully aspirate 0.5 mM EDTA without disturbing the loosely adherent cells. Add 1 mL of prewarmed complete mTeSR1 and immediately dislodge the colonies with a cell scraper (see Note 4). Pipette with a 5 mL serological pipette 1–5 times to break the iPSC colonies and generate medium-sized iPSC clumps (10–20 cells per clump).

  9. Check cells under a microscope to ensure that all iPSC colonies are lifted and small to medium iPSC clumps are generated.

  10. Collect iPSC clumps into a 15 mL conical tube.

  11. Seed collected human iPSCs without centrifugation into the pre-equilibrated 10 cm dish from step 4 above.

  12. Transfer the dish to a tri-gas tissue culture incubator with O2 set to 5% (low-O2). Once the dish into the incubator, gently but thoroughly disperse the cells in the dish by alternating between a forward/backward then left/right motion. Repeat the motions two more times. Do not swirl the dish to mix.

  13. Change mTeSR1 every other day. In 3–5 days, the culture should be 70–80% confluent, at which point the cells will be ready for differentiation (see Note 5).

3.2. Plating human iPSCs for differentiation (Day 0)

To initiate differentiation in a 10 cm dish, approximately 2.5 × 106 cells are required.

  1. Coat a 10 cm dish with hESC-qualified matrigel following the manufacturer’s instruction (1:30 dilution). Seal plates with parafilm and incubate for 1 h at RT.

  2. Prewarm 20 mL of complete mTeSR1 to 37°C and supplement it with 10 μM Rho-kinase inhibitor Y-27632 (mTeSR1/Rock medium).

  3. Confirm that iPSCs on the 10 cm dish from Subheading 3.1 step 13 are healthy with no differentiation. iPSC colonies should be at approximately 70% confluency before passaging.

  4. Aspirate the spent cell culture medium from the dish with iPSCs to be harvested and add 10 mL of DPBS to rinse. Gently rock the plate to ensure complete coverage of the cells, then aspirate DPBS. Repeat the rinse with DPBS.

  5. Aspirate DPBS and add 4 mL of RT 0.5 mM EDTA. Gently rock the dish to ensure complete coverage of the cells and return the plate to the low O2 incubator. Incubate for 3 min (see Note 3).

  6. Gently remove the dish with iPSCs from the incubator and check colony disaggregation under a microscope.

  7. Place the dish in the biosafety cabinet and carefully aspirate 0.5 mM EDTA without disturbing the loosely adherent cells. Add 4 mL of prewarmed complete mTeSR1 and immediately dislodge the colonies with cell scraper (see Note 4). Pipette with a 5 mL serological pipette 5–7 times to break the iPSC clumps.

  8. Check the cells under a microscope to ensure that all iPSC colonies are lifted (see Note 6).

  9. Collect iPSC clumps into a 15 mL conical tube.

  10. Count cells: mix 20 μL of the human iPSC suspension from step 9 above with 20 μL of Trypan Blue and count cells using a hemocytometer (see Note 6).

  11. After counting, gently but thoroughly resuspend the collected iPSCs one more time. Transfer an aliquot with 1.9 × 106 live cells into a separate 15 mL conical tube. Add mTeSR1/Rock prepared in step 2 above to the cell aliquot to obtain a total volume of 10 mL (see Note 7).

  12. Aspirate the matrigel solution from the 10 cm dish prepared in step 1 above. Do not let the coated surface to dry. Carefully transfer the entire 10 mL of the iPSC suspension in mTeSR1/Rock medium prepared in step 11 above into the matrigel-coated dish.

  13. Place the plated cells into a tissue culture incubator with O2 set to 5% (low-O2). Once the plate is set down, disperse the cells by alternating between a forward/backward then left/right motion. Repeat the motions two more times. Do not swirl the plate to mix.

  14. Incubate the cells for 24 h.

3.3. Mesoderm Induction (Days 1 to 3)

Starting day 1, the cells are always incubated in a normoxic tissue culture incubator (20% O2, 5% CO2, 37°C).

  1. Prewarm 10 mL of N2B27/CHIR-99021 medium to 37°C (see Subheading 2.3 above).

  2. Observe the dish with human iPSCs plated in Subheading 3.2 under a microscope to verify that all cells have attached and look healthy (see Fig. 2a, day 1).

  3. Replace the spent mTeSR1/Rock medium with 10 mL of prewarmed N2B27/CHIR-99021 medium.

  4. Transfer the dish to the regular normoxic tissue culture incubator (20% O2, 5% CO2, 37°C) and incubate for 3 days without medium change.

Fig. 2. The stages of iPSC differentiation during the derivation of endothelial cells.

Fig. 2

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(a) Human iPSCs plated as a monolayer on day 0 of differentiation undergo morphological changes while continue to proliferate. Between day 1 and 4 of differentiation, the density of culture increases, forming multilayers of cells. On day 5, the cells acquire an endothelial cell-like phenotype and can be harvested for magnetic automated cell sorting (MACS) on day 7. (b) Following MACS enrichment, the CD31+ iPSC-ECs can be expanded on collagen-coated plates for multiple passages. Scale bar, 50 μm

3.4. Endothelial Cell Induction (Days 4 to 6)

  1. Prewarm 10 mL of EndoM1 medium to 37°C (see Subheading 2.4, item 1 above).

  2. Replace the spent N2B27/CHIR-99021 medium with 10 mL of EndoM1.

  3. Transfer the dish to the regular normoxic incubator and incubate cells at 5% CO2, 37°C for 2 days, changing medium every day with fresh 10 mL of EndoM1.

  4. On day 6, prewarm 10 mL of EndoM2 medium to 37°C (see Subheading 2.4, item 2 above).

  5. Replace the spent EndoM1 medium from day 5 with 10 mL of EndoM2.

  6. Incubate cells at 20% O2, 5% CO2, 37°C for 24 h.

3.5. Human iPSC-EC harvest and magnetic automated cell sorting (MACS)

On day 7, harvest human iPSC-ECs and perform MACS enrichment of CD31+ iPSC-ECs. At this point, more than 107 iPSC-ECs can be harvested from one 10 cm dish (see Note 8).

  1. Prewarm 5 mL of complete StemPro-34 (SFM) (see Subheading 2.5, item 1) to 37°C in a 15 mL conical tube to collect purified cells from the MACS column.

  2. Prewarm 3 mL of accutase to 37°C.

  3. Coat 6 wells of a 6-well plate with collagen. Due to variabilities in the yield of CD31+ post-MACS enrichment, from one to six wells will be required for plating sorted cells at a density of 1.3 × 104 cells/cm2 (see Note 9).

  4. Resuspend 60 μL of collagen I stock solution in 6 mL of sterile DPBS to a final concentration of 30 μg/mL (1:100 dilution).

  5. Coat each well of a 6-well plate with 1 mL of the collagen solution. Distribute the solution evenly over the surface. Incubate coated plate at 37°C for 1 h.

  6. After incubation, aspirate diluted collagen from the wells. Do not allow the surface of the wells to dry. Add 1 mL of medium A (see Subheading 2.6, item 3 above) into each well.

  7. Transfer the plate into the normoxic tissue culture incubator to equilibrate before plating iPSC-ECs after MACS enrichment. Use the collagen-coated plate within the same day.

  8. Set up the MACS system in a laminar flow hood by placing the MS column in the magnetic field of the MACS separator and by preparing 15 mL conical tubes. Sterilize the MACS separator and conical tubes with 70% ethanol. Clip the MS column into a magnetic separator and attach the separator to the MACS stand at the appropriate height. Take a 15 mL conical tube and put it under the column to collect the flow-through fraction.

  9. Set up a centrifuge at 4°C.

  10. Prechill 50 mL of MACS Running Buffer to 4°C. Keep the remaining 450 mL of degassed MACS Running Buffer at RT.

  11. Aspirate the spent EndoM2 medium from the differentiated iPSC culture and add 10 mL of DPBS to rinse. Gently rock the plate to ensure complete coverage of the cells, then aspirate DPBS. Repeat the rinse with DPBS.

  12. Add 4 mL of prewarmed accutase into iPSC-EC culture. Gently rock the plate to ensure complete coverage of the cells and return the plate to the incubator. Incubate for 2–4 min.

  13. Remove the plate from the incubator and firmly but gently tap the side of the plate to dislodge cells. Check the cells under the microscope. If 90% of the cells are detached and floating, proceed. If many cells are still attached, incubate for another 3 min. Continue to check cells every 3 min until 90% of the cells are detached.

  14. Quickly rinse/collect the detached cells using 10 mL of RT MACS Running Buffer (see Subheading 2.5, item 6 above) to neutralize accutase. Transfer the iPSC suspension into a 15 mL conical tube. Dissociate cell clumps to a single-cell suspension by pipetting 5 times using a 5 mL serological pipet.

  15. Moisten the 30 μm cell strainer with RT MACS Running Buffer. Pass the cell suspension through the strainer to obtain a single cell suspension and remove cell clumps that may clog the MACS column.

  16. Transfer 20 μL of the cell suspension into a 1.5 mL Eppendorf tube and mix it with 20 μL of Trypan Blue. Count cells using a hemocytometer.

  17. Transfer approximately 3 × 105 cells into a separate tube to perform an optional flow cytometry analysis to estimate differentiation efficiency prior MACS enrichment. Keep the cell aliquot at 4°C until ready to process.

  18. Centrifuge approximately 107 cells at 250 × g for 4 min at 4°C. Aspirate supernatant completely. If more than 107 cells are used for MACS enrichment, recalculate the quantity of reagents based on the manufacturer’s instruction.

  19. Loosen cell pellet by gently flicking the tube, resuspend iPSC-ECs with 60 μL of cold MACS Running Buffer per 10⁷ total cells, and transfer the cell suspension to a 1.5 ml Eppendorf tube.

  20. Add 20 μL of cold FcR Blocking Reagent to the cell pellet. Vortex tube briefly and add 20 μL of the cold CD31 MicroBeads per 10⁷ total cells. Shake the beads well before use. The total volume of reaction is 100 μL.

  21. Incubate the bead suspension for 15–20 min in the dark at 4°C. Do not label at RT. Gently shake the suspension every 5 min. Alternatively, apply orbital shaker in a cold room. Do not let the suspension dry.

  22. After incubation, add 1 mL of cold MACS Running Buffer per 10⁷ cells to wash. Centrifuge at 250 × g for 4 min at 4°C. Aspirate supernatant completely.

  23. Resuspend the cell pellet in 0.5 mL of cold MACS Running Buffer. Leave the cells at RT for 2 min to equilibrate.

  24. Wash the MS column by adding 0.5 mL of RT MACS Running buffer into the column and allow the buffer to flow through completely under the gravity. Ensure that the reservoir in the column is empty before proceeding to the next step (see Note 10).

  25. Apply the cell suspension from step 23 above into the column by aiming the micropipette directly on top of the remaining buffer pool of the column. Ensure that the reservoir in the column is empty before proceeding to the next step.

  26. Wash the column 3 times with 0.5 mL of RT MACS Running Buffer. Ensure that the reservoir in the column is empty before each wash.

  27. After the last wash, remove the column from the separator and place it into the 15 ml conical tube containing 5 mL of prewarmed complete StemPro-34 (SFM) from step 1 above.

  28. Pipette 1 mL of RT MACS Running Buffer into the column. Immediately flush out the magnetically labeled cells by firmly pushing the plunger into the column to elute CD31+ human iPSC-ECs into the tube with complete StemPro-34 (SFM).

  29. Take a 20-μL aliquot of eluted CD31+cells for counting. Count cells using a hemocytometer and determine the total number of cells for plating (see Subheading 3.6 below).

  30. Pipette approximately 3 × 105 cells into a separate tube to perform an optional flow cytometry analysis to determine the purity of CD31+ iPSC-ECs. Proceed with plating as described in Subheading 3.6 below. Optional: collect approximately 3 × 105 unlabeled cells that passed through the column for flow cytometry analysis. These cells should be negative for CD31.

3.6. Plating CD31+ iPSC-ECs post-MACS enrichment

  1. Prewarm 10 mL of medium A (see Subheading 2.6, item 3 above) to 37°C.

  2. Centrifuge CD31+ iPSC-ECs obtained in Subheading 3.5, step 28 at 250 × g for 4 min and aspirate supernatant.

  3. Resuspend cells in prewarmed medium A to achieve the final concentration of 1.3 × 105 cells per 1 mL of medium based on the count obtained in Subheading 3.5, step 29 (see Note 9). Set aside.

  4. Remove the collagen-coated plate with pre-equilibrated human iPSC-EC medium A prepared in Subheading 3.5, step 7 above from the incubator. Plate iPSC-ECs at the density of approximately 1.3 × 104 cells/cm2 (1.3 × 105 cells per well of a 6-well dish) by transferring 1 mL of the cell suspension from step 3 into wells with pre-equilibrated medium. If less than 6 wells are used for plating due to a lower cell yield, remove the medium from unused wells.

  5. Place the plate with cells into the normoxic tissue culture incubator and incubate at 37°C for 24 h.

  6. After 24 h, replace medium A with 2 mL/well of fresh prewarmed medium B (see Subheading 2.6, item 4 above) and incubate in the normoxic incubator at 37°C for 24 h.

  7. After 24 h, replace medium B with 2 mL/well of fresh prewarmed medium C (see Subheading 2.6, item 5 above) and incubate in the normoxic incubator at 37°C for 24 h.

  8. After 24 h, replace medium C with 2 mL/well of fresh prewarmed ECGM/SB medium (see Subheading 2.6, item 2 above).

  9. Incubate human iPSC-ECs for another 24–48 h in the normoxic tissue culture incubator at 37°C until the iPSC-ECs reach 70–80% confluency (see Note 11). This is the passage 1 plate (see Fig. 2b).

  10. At this stage, iPSC-ECs can be cryopreserved (see Subheading 3.8 below) or used for functional assays (28).

3.7. Expansion of iPSC-ECs

The enriched iPSC-ECs can be passaged for up to passage 8–14 without losing the endothelial markers expression (see Fig. 3a and 3b) and the ability to create the vasculature network (see Fig. 3c).

Fig. 3. Characterization of human iPSC-ECs.

Fig. 3

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(a) Immunofluorescent staining for CD31 (PECAM-1, green) and CD144 (VE-Cadherin, red) of human iPSC-ECs and human umbilical vein cells (HUVECs). Nuclei are visualized with DAPI (blue). Representative images for 3 independent experiments are shown. Scale bar, 100 μm. (b) Flow cytometry analysis of human iPSC-ECs at passage 1 shows a high percentage of CD31+/CD144+ double-positive cells, confirming the maintenance of the endothelial cell-like phenotype of iPSC-ECs. (c) A tube forming assays validates the functionality of human iPSC-ECs as compared to HUVECs. Scale bar, 50 μm

  1. Ensure that the plate of iPSC-ECs at passage 1 is at 70–80% of confluency (see Subheading 3.6, step 9).

  2. Prewarm 35 mL of ECGM/SB medium (see Subheading 2.6, item 2 above) and 6 mL of accutase to 37°C.

  3. Coat one or two 10 cm dishes with collagen I using the procedure described in Subheading 3.5, steps 4–7. The number of plates depends on the yield of iPSC-ECs post-MACS enrichment (see Note 12). Six mL of the collagen solution is sufficient to coat the entire 10 cm dish. Add 8 ml of prewarmed ECGM/SB medium per each coated 10 cm dish to prevent surface drying. Set aside.

  4. Aspirate the spent ECGM/SB medium from each well with iPSC-ECs and rinse once with 1 mL of DPBS.

  5. Add 1 mL of prewarmed accutase per well and incubate at 5% CO2 37°C for 3–6 min in the normoxic incubator.

  6. Remove the plate from the incubator and firmly but gently tap the side of the plate to dislodge cells. Check the cells under the microscope. If 90% of the cells are detached and floating, proceed. If the majority of cells is still attached, incubate for another minute. Continue to check cells every minute until 90% of the cells are detached.

  7. When all cells are detached, add 2 mL of ECGM/SB per well to neutralize accutase and pipette up and down to make single-cell suspension.

  8. Collect iPSC-ECs from each well into a single 15 ml conical tube.

  9. Centrifuge the suspension at 250 × g for 4 min.

  10. Aspirate supernatant and resuspend the pellet with 1 mL of ECGM/SB medium.

  11. Count cells and plate iPSC-ECs onto collagen dishes with ECGM/SB medium prepared in step 3 above at a density of 1.3 × 104 cells/cm2 (i.e., 0.75 × 106 per each 10 cm dish, see Note 12). Adjust the total volume of ECGM/SB medium in a dish to 10 mL.

  12. Incubate in the normoxic incubator at 37°C for 3–5 days until iPSC-ECs reach 70–80% confluency (see Note 11). This is passage 2.

Human iPSC-ECs can be cryopreserved (see Subheading 3.8 below) or used for functional assays (28). To passage and expand cells, repeat steps 1–12 above adjusting the volume of reagents to the size of a dish used for expansion.

3.8. Cryopreservation of human iPSC-ECs

  1. Label the proper number of cryovials.

  2. Keep the freezing container and labeled cryovials at 4°C.

  3. Collect iPSC-ECs into a 15 mL conical tube as described in Subheading 3.7, steps 4–8 above and count using a hemocytometer. Centrifuge the cell suspension at 250 × g for 4 min.

  4. Using a 1 mL or 5 mL sterile pipet, gently resuspend the pellet in the appropriate volume of CryoStor medium to achieve a concentration of 106 cells per 1 mL (see Note 13).

  5. Transfer 0.5 mL of the iPSC-ECs/CryoStor suspension into each cryovial labeled in step 1 above to freeze 0.5 × 106 cells per vial.

  6. Immediately place the cryovials into a prechilled (2–8°C) freezing container and transfer the container to a −80°C freezer.

Allow the cells to remain at −80°C overnight (16–36 h). Once frozen, transfer the cells on dry ice to an ultra-low temperature storage vessel (liquid nitrogen or −150°C freezer).

4. Notes

  1. Using a standard procedure, degas buffer by applying vacuum. Excessive gas in running buffer will form bubbles in the matrix during separation. This may lead to clogging of the column and decrease in the quality of separation.

  2. For adaptation, the culture should be passaged on vitronectin-coated dishes in complete mTeSR1 for at least 3 passages. Generate medium-sized iPSC clumps during passaging, approximately 10–20 cells per clump.

  3. The incubation time may vary. The majority of human iPSC clone are ready to be collected after 3 min of incubation at 37°C.

  4. Do not scrape the colonies. If the colonies are still attached, extend the incubation time with 0.5 mM EDTA.

  5. Change the medium every day after the culture reaches 50% confluency or if many dead cells are present. Do not allow human iPSCs to overgrow.

  6. At this point, small iPSC agglomerates (containing approximately 2–10 cells) should be visible under a microscope. Individual cells will be visible within these clumps, allowing for counting individual cells. Replate iPSCs as small clumps to ensure the formation of a healthy iPSC monolayer one day after plating.

  7. A cell plating density within a range of 35.7 × 103 – 44.5 × 103/cm2 is desirable for successful differentiation. We determined that the optimal cell plating density for our iPSC lines was 35.7 × 103 iPSCs/cm2, or 1.9 × 106 cells per a 10 cm dish (56 cm2). However, due to inherited variability of iPSC lines, the optimal starting cell density may need to be identified for each line by performing a small-scale differentiation experiment.

  8. The reproducibility of the protocol has been shown by differentiating several human iPSC lines. The differentiation efficiency on day 7 of the protocol varies and can be as high as 25% as determined by flow cytometry. There is a clone-to-clone variability in the total number of cells on day 7 of the protocol. However, the average number is approximately 1.3 × 107 cells. If more than 108 cells are obtained, use the large-scale column (LS) from Miltenyi to enrich for CD31+ cells, adjust the volume of reagents accordingly and follow the manufacturer’s instruction. After MACS enrichment, the majority of derived iPSC-ECs will have the capability to expand between passages 8 and 14 without losing their functionality.

  9. On average, one 10 cm dish of differentiated iPSCs should yield from 0.9 × 105 to 5.85 × 105 CD31+ iPSC-ECs post-MACS enrichment. These cells can be successfully expanded if plated at a density of 1.3–2.6 × 104 cells/cm2 with the density of 1.3 × 104 cells/cm2 being optimal. Due to variability in differentiation capacity of iPSC lines, we recommend to precoat all six wells of a 6-well plate with collagen before performing MACS enrichment. This will allow for enough wells for cell plating if the yield of CD31+ iPSC-ECs is high.

  10. When adding liquid to the column, place the tip of a pipette against the wall of the column close to the opening. Avoid the formation and transfer of air bubbles while pipetting. Note that the top section of the column has a “pool” which keeps magnetic beads in the column wet during repeatable buffer and/or cell dispensation. This liquid does not go away completely.

  11. It usually takes 3–5 days to reach 70–80% confluency after plating cells. The slow growth of cells may be an indicator of cellular senescence.

  12. The number of plates for expansion will depend on the number of wells used for plating MACS purified iPSC-ECs in Subheading 3.6. If 2 – 3 wells were plated, prepare one 10 cm dish for expansion. If 5–6 wells were used, coat two 10 cm dishes. The target cell density for replating should be within the range of 1.3–2.6 × 104 cells/cm2 (or 0.75–1.5 × 106 per a 10 cm dish).

  13. The type of cryoprotectant medium depends on culture conditions and laboratory preferences. We successfully use CryoStor CS10 to cryopreserve iPSC-ECs. Note that Cryostore is light sensitive and can create crystals after repeated bottle reopening.

Acknowledgments

We are grateful for funding support from the National Institutes of Health (R21 AR074642). We also thank the Gates Frontiers Fund and Linda Crnic Institute for Down Syndrome.

Abbreviations:

iPSC

induced pluripotent stem cell

EC

endothelial cell

iPSC-EC

iPSC-derived endothelial cell

HUVEC

human umbilical vein cell

VE-cadherin

vascular endothelial cadherin

PECAM-1

platelet endothelial cell adhesion molecule

VEGF-A

vascular endothelial growth factor A

Flk1

fetal liver kinase 1

cAMP

cyclic adenosine monophosphate

VCBM

vascular cell basal medium

ECGM

endothelial cell growth medium

bFGF

basic fibroblast growth factor

FBS

fetal bovine serum

DPBS

Dulbecco’s phosphate-buffered saline

GSK-3β

glycogen synthase kinase-3β

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