Differentiation of Endothelial Cells from Human Induced Pluripotent Stem Cells (hiPSCs)

Endothelial cells (ECs), as important component of blood vessels, are crucial for maintaining vascular tone, and any dysfunction in ECs could lead to many diseases of the vascular system. Due to the limited availability of human samples, most of our research has been limited to animal models. The potential of induced pluripotent stem cells (iPSCs) to differentiate into any cell type including cardiovascular cells, provides us with a great opportunity to study human vascular disease. Here, we describe an efficient, simple, and step-wise method to differentiate iPSCs to endothelial cells (iPSC-ECs) by using small molecules and growth factors.

Figure 1. Differentiation of ECs from hiPSCs.

Materials and Reagents

Preparation of iPSCs

1) Prepare Essential 8 human ESC/iPSC passage medium (E8 medium + 10 μM ROCK inhibitor).
2) Thaw frozen cryovial in a 37°C water bath for 1-2 min. Complete the whole recovery proceed as promptly as possible so as to limit cell death.
3) Prepare 5 mL of iPSC passage medium in a 15 mL conical tube. Use a 1 mL micropipette to transfer the cell solution from the cryovial into the 15 mL conical tube and gently mix.
4) Pellet the cells by centrifugation at 300 g for 3 min.
5) After centrifugation, aspirate medium and resuspend the cell pellet in 1 mL of iPSC passage medium.
6) Have an already prepared 6-well Matrigel-coated plate and dispense 0.5 mL of the cell mixture into each coated well prepared with medium.
7) Place cell plate into the incubator and incubate without disturbance for 24 h.
8) Change medium to Essential 8 culture medium the next day and continue to change medium every day.

Differentiation of Endothelial Cells (ECs)

Human iPSC cells cultured in Essential 8 medium generally will be ready for passaging or differentiation in about 4 days when the cells reach 85-90% confluent.

• Day 0

1) Prepare RPMI + B27-INS medium (RPMI 1640 medium + B27 supplement minus insulin).
2) Aspirate previous medium from iPSC wells and add 2 mL of RPMI + B27-INS medium with GSK-3 inhibitor to each well.
3) Incubate in a 37°C incubator for 48 h.

• Day 2

1) Prepare RPMI + B27-INS medium (RPMI 1640 medium+ B27 supplement minus insulin).
2) Aspirate previous medium from EC wells and add 2 mL of RPMI + B27-INS medium with 2 μM GSK-3 inhibitor to each well. Cells will be induced toward mesodermal stage.
3) Incubate at 37°C in the incubator for 48 h.

• Day 4

1) Prepare EC growth medium supplied with VEGF, bFGF, TGF-beta/Smad inhibitor at the concentration of 50 ng/mL, 20 ng/mL, and 10 μM respectively.
Note: Extra medium can be prepared for future use, but freshly prepared medium is preferred.
2) Aspirate the previous medium from differentiation wells and add 2 mL of EC growth medium supplied with VEGF, bFGF, TGF-beta/Smad inhibitor.
3) Incubate in a 37°C incubator for 48 h.
4) On Days 6, 8, and 10, aspirate previous medium and change to the same EC growth medium supplied with VEGF, bFGF and TGF-beta/Smad inhibitor.

Sorting of ECs

ECs generally are ready for sorting via MACS (magnetic-activated cell sorting) on Day 12 of differentiation.

• Preparation of gelatin plates

1) Produce a 0.2% gelatin coating mixture by adding appropriate ratio of gelatin and DPBS, and mix.
2) Add 1 mL of 0.2% gelatin coating to each well of a 12-well plate and incubate at 37°C in hypoxia-capable incubator.
Note: Preparation of ECs for sorting will provide enough time (≥ 10 min) for gelatin to set and be ready for use.

• Suspension of ECs

1) Aspirate old culture media and wash each well with 1 mL PBS per well.
2) Aspirate PBS and add 1 mL of TrypLE per well then incubate at 37°C in an incubator for 10 min.
3) Use a 1 mL micropipette to pipette suspension up and down to break free cell clusters.
4) Neutralize lysis by adding 1 mL EC growth medium per well.

• Filtering EC cells

1) Pre-wet a 40 μm strainer with 2-3 mL of MACS buffer.
2) Filter cell suspension through a 40 μm strainer into a 50 mL conical tube.
Note: Change to a new strainer when clogging is observed, typically, 1 strainer is used for every 2-4 wells.
3) Centrifuge the collected cells at 300 g for 5 min.
4) Aspirate the medium without disrupting the cell pellet and resuspend the cell pellet in 5 mL of MACS buffer.

• Counting cells and antibody attachment

1) Calculate the total number of cells in suspension with the concentration provided by the cell counter, and then evaluate the amount of MACS buffer and CD144 antibody needed for the sorting solution. Per 10 million cells, add 80 μL of MACS buffer and 20 μL of antibody.
2) Resuspend the cell pellet in sorting solution. Suspension can be transferred into a 15 mL conical tube for convenience.
3) Incubate suspended cells in a dark fridge at 4°C for 15 min.

• Sorting

1) Rinse the cells by diluting the suspension with 1 mL of MACS buffer per 10 million cells.
2) Centrifuge the diluted cell suspension at 300 g for 5 min.
3) Aspirate diluted sorting solution and resuspend cell pellet in 1 mL of MACS buffer.
4) Wet MACS column by passing through 3 mL of MACS buffer.
5) Run 500 μL of resuspended cells per column.
6) Rinse column with 3 mL MACS buffer, repeat 3 times to assure unlabeled cells are flushed out.
Note: Wait for almost all buffer to flow through before adding more.
7) Once rinsed, remove the column from separator and place onto a new 15 mL conical tube.
8) Add 5 mL of MACS buffer and immediately elute labeled cells by forcing buffer through the column with plunger.

(Optional) Additional Sorting of ECs

If cells are still impure after the first sorting, additional sorting can be proceeded. Procedure is the same as Sorting of ECs.

Figure 2. Characterization of endothelial cells induced from hiPSCs.

References

1. Xiaojun Lian, et al. Efficient differentiation of human pluripotent stem cells to endothelial progenitors via small molecule activation of WNT signaling. Stem Cell Reports, 2014, 3(5): 804-816.
2. Liu C. et al. Generation of endothelial cells from human induced pluripotent stem cells. Bio-protocol, 2018, e3086.

For research use only. Not for any other purpose.