Protocol to generate endothelial cells, pericytes, and fibroblasts in one differentiation round from human-induced pluripotent stem cells

Summary

Here, we present a protocol for differentiating human-induced pluripotent stem cells into three distinct mesodermal cell types: vascular endothelial cells (ECs), pericytes, and fibroblasts. We describe steps for using monolayer serum-free differentiation and isolating ECs (CD31⁺) and mesenchymal pre-pericytes (CD31⁻) from a single differentiation set. We then differentiate pericytes into fibroblasts using a commercial fibroblast culture medium. The three cell types differentiated in this protocol are useful for vasculogenesis, drug testing, and tissue engineering applications.

For complete details on the use and execution of this protocol, please refer to Orlova et al. (2014).¹

Graphical abstract

Highlights

• •Generate endothelial cells, pericytes, and fibroblasts from the same differentiation
• •Successful cryopreservation and thawing maintaining cell functions
• •Applicable to vascular tissue disease modeling and toxicity/drug testing

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Here, we present a protocol for differentiating human-induced pluripotent stem cells into three distinct mesodermal cell types: vascular endothelial cells (ECs), pericytes, and fibroblasts. We describe steps for using monolayer serum-free differentiation and isolating ECs (CD31+) and mesenchymal pre-pericytes (CD31−) from a single differentiation set. We then differentiate pericytes into fibroblasts using a commercial fibroblast culture medium. The three cell types differentiated in this protocol are useful for vasculogenesis, drug testing, and tissue engineering applications.

Before you begin

iPSCs are available from various commercial sources or institutional core facilities. Be sure to follow the institutional guideline and obtain the institutional review board (IRB) approval. All cells are cultured in standard 5% CO₂ 37°C humidified incubator with room air (∼20% oxygen) and handled in a sterile cell culture biosafety cabinet. All reagents should be prepared under sterile conditions. Undifferentiated iPSCs may be expanded and cryostored before use in differentiation experiments. Regular passages of undifferentiated iPSCs are recommended 1–2 times a week with 1:12 ratio (1 well to 12 wells, or as needed for a given iPSC line). iPSC should be passaged or cryostored before cells reach 90% confluency. Healthy, undifferentiated iPSCs are pivotal to gain high yield and anticipated differentiation results. All differentiation steps and cell culture conditions are summarized in Table 1 including coating substrate, volume of medium to be used, and cell seeding density (Table 1).

Table 1.

Summary of differentiation steps and cell culture conditions

Cell stage	Medium	Coating	Cell culture types	Volume of medium	Seeding density
iPSC	E8	Vitronectin	6 well plates	2 mL	1 × 105 cells/well
APEL D1-7 differentiation	APEL2		6 well plates	2–3 mL
APEL D7 post CD31+	EGM2 + 50 ng/mL VEGF165	Fibronectin (10 μg/mL)	75T	12 mL	0.75 × 106 cells/flask
APEL D7 post CD31−	EGM2	Gelatin (0.1%)	75T	12 mL	1.5 × 106 cells/flask
P0 CD31+ iEC-pre-cryopreserve	Endothelial cell expansion medium	Fibronectin (10 μg/mL)	75T	12 mL	0.5 × 106 cells/flask
P1 CD31− pre-PC	Pericyte differentiation medium	Gelatin (0.1%)	75T	15 mL	0.75–1.5 × 106 cells/flask
P2 CD31− iPC	Pericyte expansion medium	Gelatin (0.1%)	75T	15 mL	0.75–1.5 × 106 cells/flask
P3 CD31− iPC to fibroblasts	FibroLife	No coating	75T	12 mL	1–1.5 × 106 cells/flask
CD31− iFibroblasts	FibroLife	No coating	75T	12 mL	0.5–1 × 106 cells/flask
iEC post thaw	Endothelial cell expansion medium	Fibronectin (10 μg/mL)	75T	12 mL	0.5 × 106 cells/flask
iPC post thaw	Pericyte expansion medium	Gelatin (0.1%)	75T	15 mL	1 × 106 cells/flask
iFibroblast post thaw	FibroLife	No coating	75T	12 mL	0.5 × 106 cells/flask

Institutional permissions

This study is approved by National Eye Institute, NIH (Project number: 11-EI-0245) for generation of iPSC using human samples.

Note: Before you begin to obtain any cellular products or to perform any experiment in your laboratory, all appropriate regulatory standards need to be approved by your institute.

Prepare reagent for induced pluripotent stem cell (iPSC) culture

Timing: 1 day

• 1.
  Prepare Essential 8 (E8) medium.

  • a.
    Thaw E8 supplement 12–15 h at 4°C or 30 min at 17°C–22°C.
  • b.
    Next, add E8 supplement to the E8 basal medium.

    Note: Store complete E8 medium at 4°C maximum 4 weeks.

  • c.
    Make E8 with Rock inhibitor (Y-27632) by adding 25 μL of 10 mM stock solution to 50 mL E8 medium (5 μM final concentration). Store at 4°C maximum 1 week.

    CRITICAL: Do not warm the complete E8 medium at 37°C in regular base. It will decrease basic-FGF half-life. Leave the medium at 17°C–22°C for fifteen minutes before use. If necessary, aliquot to smaller volume (e.g., 50 mL) to avoid warming up the whole bottle.

• 2.Prepare human vitronectin solution as per manufacturer’s recommendation.
  https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2Fvitronectin_truncated_recomb_human_man.pdf.

  • a.
    Thaw one vial of vitronectin stock solution (0.5 mg/mL) at 4°C for 12–15 h or at 17°C–22°C for 10 min.
  • b.
    Aliquot the stock solution to 60 μL per tube and store at -80°C.
  • c.
    For coating 1 of 6-well plate, thaw 1 aliquots and add 1:100 dilution to PBS (e.g., 60 μL into 6 mL of PBS).

    Note: Store the vitronectin solution at 4°C maximum 1 month.

Prepare APEL2 differentiation medium

Timing: 1 day

• 3.
  Prepare APEL2 with supplements for day 1–3 of differentiation.

  • a.
    Thaw APEL2 or APEL2-Li (APEL-Low insulin) medium at 4°C, 12–15 h.²
    See Figure 1 for the comparisons of APEL2 vs. APEL2 Li.
  • b.
    Prepare day 1–3 medium by adding final concentration of 50 ng/mL VEGF165, 30 ng/mL BMP4, 25 ng/mL Activin-A, and 1.5 μM CHIR-99021. For example, to make 100 mL complete medium, include 50 μL of VEGF 165 (100 ng/mL stock solution), 30 μL of BMP4 (100 ng/mL stock solution), 25 μL of Activin-A (100 ng/mL stock solution) and 37.5 μL of CHIR-99021 (4mM stock solution) in 100 mL of APEL2 medium.

    Note: Prepare only the amount needed to be used fresh. This medium should not be stored more than 1 week in 4°C.

• 4.
  Prepare APEL2 with supplements for day 3–7 of differentiation.

  • a.
    Prepare day 3–7 medium by adding final concentration of 50 ng/mL VEGF165 and 10 μM SB-431542. For example, to make 100 mL complete medium, include 50 μL of VEGF165 (100 ng/mL stock solution) and 50 μL of SB431542 (20mM stock solution) in 100 mL of APEL2 medium.

Note: Prepare only the amount needed to be used fresh. This medium should not be stored more than 1 week in 4°C.

Note: APEL2-Li is discontinued from December 2022 by manufacturer’s notice. Figure 1 include the comparison of APEL2 and APEL2-Li. There are no major differences in vascular differentiation.

Figure 1.

Phase contrast images showing compatible use of APEL2 and APEL2-Li medium for the first 7 days of differentiation

Top panel shows undifferentiated iPSC colonies in E8 culture medium to demonstrate the iPSC colony size in corresponding dates. Middle and bottom panel shows vascular differentiation with either APEL2 or APEL2-Li from days 2–7 prior to CD31 sorting. Scale bars are 250 μm.

Prepare reagent for sorting day (Day 7 of differentiation)

Timing: 1–2 days

• 5.
  Prepare MACS buffer.

  • a.
    Prepare MACS buffer by adding final concentration of 0.25% BSA and 1 mM of EDTA. For example, to make 500 mL MACS buffer, include 25 mL of BSA solution (5% stock solution) and 1 mL of EDTA (0.5 M stock solution) to 500 mL of PBS.
  • b.
    Sterilize using 0.22 μm filter system.

Note: Stored MACS buffer at 4°C maximum 8 weeks.

• 6.
  Prepare EGM2 medium.

  • a.
    Thaw supplements at 4°C for 12–15 h, or 17°C–22°C for 30 min.
  • b.
    Add all supplements to the EGM2 Basal medium.

    Note: Store complete EGM2 medium at 4°C maximum 4 weeks.

  • c.
    For CD31⁻ Plating media: Add 5 μM Rock inhibitor to the EGM2 complete medium.
  • d.
    For CD31⁺ Plating media: Add 5 μM Rock inhibitor and 50 ng/mL VEGF to the EGM2 complete medium. For example, add 25 μL of Rock inhibitor and 25 μL of VEGF160 (100 ng/mL stock concentration) in 50 mL of complete EGM2 medium.

• 7.Prepare 1 mg/mL fibronectin solution as per manufacturer recommendation.
  https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2FFibronectin_man.pdf.

  • a.
    Reconstitute 5 mg human fibronectin in 5 mL of cell culture grade H₂O in a sterile manner.
  • b.
    Aliquot 500 μL/tube and store at −20°C maximum 6 months.
• 8.
  Coat flasks with fibronectin for CD31⁺ cells plating.

  • a.
    Thaw one aliquot of 1 mg/mL fibronectin solution at 17°C–22°C.
  • b.
    Add 500 μL 1 mg/mL fibronectin to 49.5 mL of 1× dPBS to make 10 μg/mL fibronectin solution.
  • c.
    Store 10 μg/mL fibronectin solution at 4°C for up to 4 weeks.
  • d.
    Cover all cell culture surface with the fibronectin solution (e.g., add 5 mL of 10 μg/mL fibronectin to a T75-flask) and incubate for minimum of 1 h at 17°C–22°C or until cells are ready to be plated.
  • e.
    If the plates are not used on same day, it can be stored at 4°C and left for 1 h at 17°C–22°C before use.
  • f.
    Remove excess solution before adding medium and cells.
  • g.
    Do not leave the flasks to dry after coating.
• 9.
  Coat flasks with gelatin for CD31⁻ cells plating.

  • a.
    Add 5 mL of 0.1% gelatin solution to each T75 flask to cover entire surface.
  • b.
    Incubate in 37°C incubator minimum 1 h or 12–15 h.
  • c.
    Remove excess solution prior to adding cells or medium.
  • d.
    Do not leave the flasks to dry after coating.

Prepare endothelial cell culture medium

Timing: 1–2 h

• 10.Thaw all frozen components at 4°C for 12–15 h or at 17°C–22°C for 30 min.
• 11.Prepare VascuLife complete medium by adding all components from the kit except FBS and only 10 mL of L-Glutamine.
• 12.Add 50 mL iCell endothelial cell medium supplement to complete VascuLife medium.
• 13.Sterilize using 0.22 μm filter system.
• 14.Store at 4°C maximum 4 weeks.

Prepare pericytes differentiation and expansion medium

Timing: 1–2 days

• 15.Prepare pericyte expansion medium including 500 mL DMEM, 56 mL (10%) heat inactivated FBS, 5.6 mL MEM-NEAA (100×), 5.6 mL L-Glutamine (200 mM stock concentration), 560 μL 2-mercaptoethanol (55 mM stock concentration).
• 16.Sterilize using 0.22 μm filter system.

Note: Store the pericyte expansion medium at 4°C maximum 4 weeks.

• 17.Prepare pericyte differentiation medium by adding 20 ng/mL of rhPDGF-bb ad 10 ng/mL of rhTGF-b3 in the pericyte expansion medium. For example, add 20 μL of rhPDGF-bb and 10 μL of rhTGF-b3 in 100 mL of pericyte expansion medium.

Note: Store the pericyte differentiation medium at 4°C maximum 2 weeks.

Prepare fibroblast expansion medium

Timing: 1–2 days

• 18.Thaw all frozen supplements 12–15 h at 4°C or 30 min at 17°C–22°C.
• 19.Prepare fibroblast expansion medium by adding all components from the FibroLife medium kit to basal medium.
• 20.Sterilize using 0.22 μm filter system.

Note: Store the complete FibroLife medium at 4°C maximum 4 weeks.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
CD31 microbead kit, human (See Table 2 for the dilution)	Miltenyi Biotech	130-091-935
CD31-BV510 (5 μL/reaction)	BD	563454
CD146-BV605 (5 μL/reaction)	BD	743301
CD140b-APC (5 μL/reaction)	BioLegend	323608
CD144-BUV805 (5 μL/reaction)	BD	748542
CD73-Alexa Fluor 350 (5 μL/reaction)	Bio-Techne	FAB5795U
CD31 (1:50)	Agilent Technology	M082301-2
ETV2 (1:100)	Abcam	ab181847
von Willebrand factor (vWF, 1:100)	Agilent Technology	A008229-5
Hoechst33258 (1:1000)	Thermo Fisher Scientific	H3570
Dil-Ac-LDL (5–15 μg/mL)	Thermo Fisher Scientific	L3484
NG2 (0.5 mg/mL)	Abcam	ab83178
aSMA-FITC (1:200)	Sigma-Aldrich	F3777
Collagen-I (1:100)	Thermo Fisher Scientific	PA5-95137
Vimentin (1:25)	Abcam	Ab92547
Connexin-43 (1:70)	Abcam	Ab11370
PDGFR-b (1:20)	Abcam	Ab32570
Chemicals, peptides, and recombinant proteins
Essential 8™ Medium	Thermo Fisher Scientific	A1517001
Versene	Thermo Fisher Scientific	15040066
Rock inhibitor (Y-27632, 10 mM)	Bio-Techne	1254/1
rhVitronectin (0.5 mg/mL)	Thermo Fisher Scientific	A14700
dPBS (1x)	Thermo Fisher Scientific	14190-144
rhBMP4	Bio-Techne	314-BP-050
rhActivin-A	Bio-Techne	338-AC-010
rhVEGF165	Bio-Techne	293-VE-050
rh-basic FGF	Bio-Techne	233-FB
rhCTGF	Bio-Techne	9190-CC
rhPDGF-bb	Bio-Techne	220-BB
rhTGF-b3	Bio-Techne	243-B3
CHIR-99021	Bio-Techne	4423/10
SB-431542	Bio-Techne	1614/10
STEMdiff™ APEL™2 medium	STEMCELL Technologies	05270
5% BSA solution	Miltenyi Biotech	130-091-376
0.5M ETDA	Thermo Fisher Scientific	15575-038
EGM-2 Bullet Kit	Lonza	CC-3162
Human fibronectin, plasma	Thermo Fisher Scientific	33016015
VascuLife Medium	Lifeline Cell Technology	LL-0003
iCell endothelial cells medium supplement	FujiFilm CDI	M1019
FibroLife S2 Medium	Lifeline Cell Technology	LL-0011
EmbryoMax® 0.1% gelatin solution	Millipore Sigma	ES-006-B
DMEM	Thermo Fisher Scientific	14190-144
Heat inactivated FBS	Millipore Sigma	F0926
MEM NEAA (100x)	Thermo Fisher Scientific	11140-050
L-Glutamine, 200 mM	Thermo Fisher Scientific	25030-081
2-Merchaptoethanol, 55 mM	Thermo Fisher Scientific	21985-023
CryoStore CS10	STEMCELL Technologies	07930
Experimental models: Cell lines
iPSC (Study number 11-EI-0245)	In house generated	N/A
Other
6-well plates	Nunc	140675
T-75 flasks	Nunc	156499
20 μm Steriflip cell strainer	Sigma	SCNY00020
0.22 μm filter units-50 mL	Sigma	SCGP00525
0.22 μm filter units-250 mL	Corning	431096
0.22 μm filter units-500 mL	Corning	431118
LS columns	Miltenyi	130-042-401
MidiMACS or QuadroMACS separator	Miltenyi	130-042-302/130-090-976

Table 2.

Suggested conditions for microbead staining and cell separation

Cell number	1 × 107
MACS buffer (μL)	40
Fc Blocker (μL)	30
CD31 microbeads (μL)	30
No. of unsorted cells per LS column	2 × 109
No. of target cells per LS column	1 × 108

Materials and equipment

iPSC culture medium
Reagent	Final concentration	Amount
Essential 8 basal medium	N/A	500 mL
Essential 8 supplement	0.25%	10 mL
Rock inhibitor 10 mM (the day of passage only)	5 μM	6 μL in 12 mL medium

APEL2 differentiation medium
Reagent	Final concentration	Amount
Day 1–3
STEMdiff™ APEL™2 Medium	N/A	100 mL
rhBMP4 (100 ng/mL)	30 ng/mL	30 μL
rhActivin-A (100 ng/mL)	25 ng/mL	25 μL
rhVEGF165 (100 ng/mL)	50 ng/mL	50 μL
CHIR-99021 (4 mM)	1.5 μM	37.5 μL
Day 3–7
STEMdiff™ APEL™2 Medium	N/A	100 mL
rhVEGF165 (100 ng/mL)	50 ng/mL	50 μL
SB-431542 (20 mM)	10 μM	50 μL

MACS buffer (store at 4°C)
Reagent	Final concentration	Amount
dPBS (1x)	N/A	500 mL
BSA solution (5%)	0.25%	25 mL
ETDA (0.5 M)	1 mM	1 mL

EGM2 plating medium (store at 4°C)
Reagent	Final concentration	Amount
CD31+ plating medium
EGM2 complete medium	N/A	100 mL
rhVEGF165 (100 ng/mL)	50 ng/mL	50 μL
Rock inhibitor (10 mM)	5 μM	50 μL
CD31− plating medium
EGM2 complete medium	N/A	100 mL
Rock inhibitor (10 mM)	5 μM	50 μL

Endothelial cell expansion medium (store at 4°C)
Reagent	Final concentration	Amount
VascuLife medium complete (add all components from the kit except FBS and add only 10 mL of L-Glutamine)	N/A	500 mL
iCell Endothelial Cells Medium Supplement	N/A	50 mL
rhVEGF165 (100 ng/mL)	25 ng/mL	25 μL for 100 mL
rh-bFGF (100 ng/mL)	20 ng/mL	20 μL for 100 mL
rhCTGF (100 ng/mL)	10 ng/mL	10 μL for 100 mL

Pericyte differentiation medium (store at 4°C)
Reagent	Final concentration	Amount
DMEM	N/A	500 mL
FBS	N/A	56 mL
MEM NEAA (100x)	1×	5.6 mL
L-Glutamine (200 mM)	2 mM	5.6 mL
2-Merchaptoethanol (55 mM)	55 μM	560 μL
rhPDGF-bb (100 ng/mL)	20 ng/mL	20 μL for 100 mL
rhTGF-b3 (100 ng/mL)	10 ng/mL	10 μL for 100 mL

Pericyte expansion medium (store at 4°C)
Reagent	Final concentration	Amount
DMEM	N/A	500 mL
FBS	N/A	56 mL
MEM NEAA (100×)	1×	5.6 mL
L-Glutamine (200 mM)	2 mM	5.6 mL
2-Merchaptoethanol (55 mM)	55 μM	560 μL

Step-by-step method details

Induced pluripotent stem Cell (iPSC) plating for APEL2 differentiation

Timing: 7 days

This step explains how to begin the differentiation. Undifferentiated iPSC can be plated and next day the differentiation begins. 7 days of differentiation is composed with simple medium changes every 2 days. There are 2 different media: day 1–3 and day 3–7.

• 1.Prepare coating of 6 well plates by adding of 1 mL of vitronectin solution to each well of entire surface and leave at 17°C–22°C for 1 h.

Note: Do not leave in the biosafety laminar flow hood since it can cause drying of the plates. Leave on tabletop if not disturbed. If the plate is not used, store at 4°C refrigerator. Wrap with clean aluminum foil in case needed to avoid contamination.

• 2.Bring E8 medium and versene to 17°C–22°C by leaving on the tabletop for 15 min.

CRITICAL: Do not warm up the E8 medium at 37°C since bFGF half-life in E8 medium is temperature sensitive.

• 3.
  Grow iPSC in iPSC culture media (E8 medium) in a 6-well plate. When iPSC culture reaches to 80% confluency cells can be prepared to initiate differentiation.

  • a.
    Wash cells once in 2 mL PBS/well.
  • b.
    Add 1 mL versene/well and incubate for 5 min at 37°C, 5% CO₂ incubator.
  • c.
    Swirl or gently tap the side of plate to detach cells.

    Note: If the cells don’t lift, do not pipet to detach. Leave another 5 min at 17°C–22°C and swirl or gently tap the side of the plate.

  • d.
    Transfer 80 μL of cells along with versene solution to each well of a new 6-well plate precoated with vitronectin and containing 2 mL of E8 medium including 5 μM of Rock inhibitor.
  • e.
    Prepare 2-3 plates (12–18 wells) for the differentiation set.
  • f.
    Next day, begin the APEL2 (Albumin Poly-vinyl-alcohol Essential Lipids) differentiation process with iPSC cultures that have small colonies and a density of 10%–20% (See Figure 1, Day 2)
• 4.Remove E8 medium from iPSC and add 2.5 mL of day 1–3 differentiation medium for days 1–3. Mark the starting day as day 1.
• 5.Keep the plate in a 37°C incubator until day 3 without changing medium for 48 h.
• 6.On day 3, prepare day 3–7 medium and warm up to 17°C–22°C.
• 7.Remove plate from the 37°C incubator. Swirl it before removing the medium in order to remove most of the floating cells. It is expected to observe significant numbers of floating cells.
• 8.Replace with 2.5 mL of day 3–7 medium at day 3.
• 9.Replace with 3 mL of day 3–7 medium at day 5.

MACS sorting and plating the CD31⁺ and CD31⁻ cells

Timing: 3–4 h

This step explains single cell preparation of differentiating cells, antibody staining for magnetic cell sorting separation, and culture of purified CD31⁻ and CD31⁺ cells.

• 10.
  Cell preparation for MACS

  • a.
    At day 7 of APEL differentiation, wash cells once with 2 mL PBS per well.
  • b.
    Add 1 mL of TrypLE.
  • c.
    Incubate 5 min at 37°C incubator.
  • d.
    Use P1000 pipet to gently detach cells and incubate another 5 min.
  • e.
    Collect cells in 50 mL conical tube that already include 10 mL MACS buffer to stop TrypLE.
  • f.
    Use MACS buffer to wash wells and add to cell suspension.
  • g.
    Make total volume 45 mL with MACS buffer that includes cells from 12–18 wells.

    Note: dilute the cell suspension enough to see through the solution. If the cell solution is too dense, loss of cells at the filtering step will be greater.

  • h.
    Filter cell solution through 20 μm cell strainer.
  • i.
    Centrifuge for 5 min, 100 g.
• 11.
  Antibody staining

  • a.
    Remove the supernatant and tap the tube to dislodge the cell pellet.
  • b.
    Add 5 mL MACS buffer including 5 μM Rock inhibitor final concentration per 6 wells (one 6-well plate) equivalent cells for cell count.

    Note: Expect to yield around 1.5 ± 0.16 × 10⁶ cells (N = 12 experiments) from one well of 6-well plate at this stage.

  • c.
    Centrifuge for 5 min, 100g.
  • d.
    Remove the supernatant and tap the tube to dislodge the cell pellet.
  • e.
    Add MACS buffer including 5 μM Rock inhibitor and FcR blocker (from CD31 microbead kit) and mix well with the cells (See the Table 2 for volume)
  • f.
    Add anti-CD31 microbead to the cell solution and pipet to mix or gently vortex the solution.

    Note: See Table 2 for appropriate volume for staining.

  • g.
    Incubate microbead-cell solution for 15 min at 4°C.
  • h.
    Add 20 mL of MACS buffer for approximately 1 × 10⁷ cells equivalent for washing.
  • i.
    filter through 20 μm cell strainer.

    Note: Magnetic beads tagged microbeads form less than 1 μm in diameter on cell surfaces, even with massive labeling, so they will not interfere with filtration through the 20 μm cell strainer.

  • j.
    Centrifuge for 5 min, 100 g.
• 12.
  Cell separation

  • a.
    During the centrifugation, set the magnetic station, LS separation column, and 15 mL conical tubes below the column.
  • b.
    Add 2 mL of MACS buffer to wash the column.
  • c.
    Once centrifugation is complete, remove the supernatant and tap the tube to dislodge the pellet.
  • d.
    Resuspend cells in 1 mL of MACS buffer including 5 μM Rock inhibitor for less than 2 × 10⁹ cells (Table 2).
  • e.
    Load cells on the top of the LS column.
  • f.
    Collect the CD31⁻ flow through of cells.
  • g.
    Add 3 mL of MACS buffer to the column and collect the 1st wash of cell flow through that will lead to total 6 mL of cell solution (see steps 3b. and 3d.).
  • h.
    Change to new conical tube and wash 2 more times with 3 mL of MACS buffer. Discard these two wash throughs.
  • i.
    Detach the column from the magnetic station and put above a new conical tube labeled CD31⁺ cells.
  • j.
    Add 2 mL of EGM2 medium or MACS buffer including 5 μM Rock inhibitor to the top of LS column.
  • k.
    Immediately flush out the medium to collect CD31⁺ cells.
• 13.
  Cell plating

  • a.
    Centrifuge CD31⁻ cells and resuspend in 1 mL of EGM2 medium per column eluted including 5 μM Rock inhibitor.
    e.g., Resuspend in 4 mL if eluted from 4 LS columns.
  • b.
    Take 10 μL of cell solution and mix with 10 μL of trypan blue to count live cell number.

    Note: Expected to yield 8.3 ± 0.65 × 10⁵ cells (69 ± 1.2% of pre-sort population, N = 5 experiments).

  • c.
    Count CD31⁺ cells with the same method. Additional centrifugation is not necessary if the cells were eluted in CD31⁺ EGM2 plating medium.

    Note: Expected to yield 2.7 ± 0.02 × 10⁵ cells (17.4 ± 1.3% of pre-sort population, N = 7 experiments).

  • d.
    Plate 1 × 10⁴ CD31⁺ cells/cm² on fibronectin coated flasks in CD31⁺ EGM2 plating medium (7.5 × 10⁵ cells/T75 flask).
  • e.
    Plate 2 × 10⁴ CD31⁻ cells/ cm² on gelatin coated flasks in CD31⁻ EGM2 plating medium (maximum plating density 1.5 × 10⁶ cells/T75 flask).

Induced endothelial cell (iEC) expansion, freezing and thawing

Timing: 1–2 weeks

This step explains how to expand iEC prior to cryopreservation, thaw and recover from thawing before used for experiments.

• 14.
  Change the medium

  • a.
    Next day of MACS, change medium of CD31⁺ cells with iEC complete medium including VEGF (25 ng/mL), bFGF (20 ng/mL), and CTGF (10 ng/mL).³
  • b.
    Change medium every 2 or 3 days.
  • c.
    Cells will be 80%–90% confluent about 5–7 days after plating from MACS (Figure 2).
  • d.
    Avoid culturing more than 7 days. Cells will accumulate extracellular matrix (ECM) and will be difficult to detach from the flask.
• 15.
  Expansion of iEC.

  • a.
    When CD31⁺ iEC reach near 80%–90% confluency, cells are washed in 10 mL PBS.
  • b.
    Remove PBS and add 5 mL of TrypLE. Incubate 5 min at 37°C.
  • c.
    Tap the side of flasks to detach cells. Observe under the microscope and incubate another 5 min if some cells are still attached.
  • d.
    Collect all the cells in 5 mL iEC complete medium to stop TrypLE per 75T flasks.
  • e.
    Centrifuge for 5 min, 100 g.
  • f.
    Resuspend in 1 mL iEC medium per 75T flask harvested.
  • g.
    Count the cell number.
  • h.
    Plate to new pre-coated flasks with fibronectin solution with density of 5 × 10⁵ cells/75T-flask or cryopreserve.
• 16.
  Cryopreservation of iEC

  • a.
    When CD31⁺ iEC reach near 80%–90% confluency, cells are washed in 10 mL PBS.
  • b.
    Remove PBS and add 5 mL of TrypLE. Incubate 5 min at 37°C.
  • c.
    Tap the side of flasks to detach cells. Observe under the microscope and incubate another 5 min if some cells are still attached.
  • d.
    Resuspend in 1 mL iEC medium per 75T flask harvested.
  • e.
    Count the cell number.

    Note: Expect to harvest around 2 fold increase from the initial seeding density. E.g., 1.5 ± 0.17 × 10⁶ cells from one 75T (P1, N = 6 experiments) after 7 days of expansion that were seeded with 7.5 × 10⁵ cells/75T flask

  • f.
    Centrifuge for 5 min, 100 g.
  • g.
    Tap to dislodge the pellet and resuspend in CryoStor with the density of 1 million/mL. Label as passage number 0 (P0).
  • h.
    Distribute cell solution into cryovials, recommended at 1 mL/vial.
  • i.
    Freeze in Mr. Frosty with isopropanol or equivalent freezing container at −80°C.
  • j.
    Next day move into liquid nitrogen tank.

    Pause point: Cells are frozen and can be paused at this step.

• 17.
  Thawing iEC

  • a.
    Thaw cells in 37°C water bath for 2 min.
  • b.
    Move cell solution into a conical tube with 10 mL of iEC complete medium added slowly and with frequent agitation.
  • c.
    Centrifuge for 5 min, 100 g.
  • d.
    Resuspend in iEC complete medium containing 5 μM rock inhibitor.
  • e.
    Plate 1 million cells (1 vial) into 2 of T75 flasks precoated with human fibronectin.
  • f.
    Change to iEC complete medium next day and every 2 or 3 days thereafter.
  • g.
    Cells will be 80%–90% confluent in 5–7 days.

Figure 2.

Phase contrast images of MACS isolated CD31⁺ endothelial cells

CD31⁺ cells were plated onto the fibronectin coated culture plate and expanded for 5–7 days considered as passage 0 (P0). Cells were cryopreserved until the experiment. When thawed (P1) and passaged, cells demonstrated typical endothelial cell morphology. It is not recommended to passage cells beyond P3. Scale bars are 250 μm.

Pre-induced pericytes (Pre-iPC) to induced pericyte (iPC) differentiation, cryopreservation, and thawing

Timing: 2–5 weeks

This step explains how to differentiate pre-iPC (P0 in EGM2, P1 in Pericyte differentiation medium) to iPC (P2-P5 in Pericyte expansion medium). Seeding density in each passage is important to follow.

Pre-iPC to iPC differentiation

Timing: 1–2 weeks

• 18.
  Change the medium.

  • a.
    The day after plating CD31⁻ cells from MACS, change EGM2 complete medium and then every 2–3 days.
  • b.
    Cells will be confluent in 4–5 days.
  • c.
    Wash in 10 mL PBS and add 5 mL of TrypLE.
  • d.
    Incubate for 5 min at 37°C.
  • e.
    Tab the side of flasks to detach cells.
  • f.
    Collect cells in 10 mL Pericyte differentiation medium and centrifuge for 5 min, 100 g.
  • g.
    Resuspend cells in Pericyte differentiation medium (see the key resources table) with 5 μM Rock inhibitor.
  • h.
    Count cells and plate 1–2 million cells/T75-flask or 1:1 ratio. Mark as P1.

    Note: Expect to harvest 1–2 million cells from P0. It can be passaged to 1:1 ratio to P1 in Pericyte differentiation medium (e.g., 1 flask to 1 flask). It is recommended to have high number of cells to be carried to the next passage.

  • i.
    Next day, change to 15 mL of Pericyte differentiation medium without Rock inhibitor per 75T flask.
  • j.
    Change medium every 2–3 days.

    Note: Cell morphology will change, and cells will increase in size with high cytoskeleton accumulation (Figure 3).

Figure 3.

Phase contrast images of CD31⁻ MACS isolated and matured iPSC-derived pericyte (iPericytes)

CD31⁻ cells were plated onto the gelatin coated cell culture plate with high cell density. Cells were passaged 3–4 days after into pericyte differentiation medium including DMEM, 10% FBS, PDGF-bb, and TGFβ3. After the first passage (P1), pericytes were grown in Pericyte expansion medium up to passage 4–5. It is not recommended to use cells beyond passage 5. Scale bars are 250 μm.

iPC expansion, cryopreservation, and thawing

Timing: 7 days each passage

• 19.
  Change the medium.

  • a.
    Pre-pericyte (P1) will be confluent in 5–7 days.
  • b.
    Wash in 10 mL PBS and add 5 mL of TrypLE.
  • c.
    Incubate for 5 min at 37°C.
  • d.
    Tab the side of flasks to detach cells.
  • e.
    Collect cells in 10 mL Pericyte expansion medium and centrifuge for 5 min, 100 g.
  • f.
    Resuspend cells in Pericyte expansion medium (see the key resources table) with 5 μM Rock inhibitor.
  • g.
    Count cells and plate 0.75–1 × 10⁶ cells/T75-flask
  • h.
    Next day, change medium to Pericyte expansion medium without Rock inhibitor.
  • i.
    Change medium every 2 or 3 days.
• 20.
  Passage of iPC.

  • a.
    After 7 days of culture from previous passage (P2) or when cells reach 80–90% confluency, wash once in 10 mL PBS and add 5 mL of TrypLE.
  • b.
    Incubate 5 min at 37°C.
  • c.
    Tab the side of flasks to detach cells.
  • d.
    Collect cells in Pericyte expansion medium and centrifuge for 5 min, 100 g.
  • e.
    Resuspend in 1 mL of Pericyte expansion medium per 75T flask harvested.
  • f.
    Count the cell number.
  • g.
    Plate to new pre-coated flasks with gelatin solution with density of 5 × 10⁵ cells/75T-flask or cryopreserve.
  • h.
    Expand up to passage 4–5.

Note: Pericytes demonstrated bigger cell body and low proliferation rate compared to fibroblasts. It was often observed that pericytes showed lower than 2-fold expansion rate after 7 days of expansion.

• 21.
  Cryopreservation of iPC.

  • a.
    When cells are 80%–90% confluent, wash once in 10 mL PBS and add 5 mL of TrypLE.
  • b.
    Incubate 5 min at 37°C.
  • c.
    Tab the side of flasks to detach cells.
  • d.
    Collect cells in Pericyte expansion medium and centrifuge for 5 min, 100 g.
  • e.
    Resuspend in 1 mL of Pericyte expansion medium per 75T flask harvested.
  • f.
    Count the cell number.
  • g.
    Centrifuge for 5 min, 100 g.
  • h.
    Tap to dislodge the pellet and resuspend in CryoStor with the density of 1 × 10⁶ cells/mL.
  • i.
    Distribute cell solution into cryovials, at 1 mL/vial.
  • j.
    Freeze in Mr. Frosty with isopropanol or equivalent freezing container at −80°C.
  • k.
    Next day move the vials into a liquid nitrogen tank.

Pause point: Cells are frozen and can be paused at this step.

• 22.
  Thawing of iPCs.

  • a.
    Coat T75 flasks with 0.1% gelatin solution.
  • b.
    Bring frozen vial(s) to 37°C warm water bath.
  • c.
    Rapidly thaw cells within 2 min.
  • d.
    Move 1 mL of cell solution into conical tubes with 9 mL of Pericyte expansion medium (total 10 mL).
  • e.
    Centrifuge for 5 min, 100 g.
  • f.
    Resuspend in 10 mL of Pericyte expansion medium including 5 μM Rock inhibitor.
  • g.
    Plate 1 vial to 1 of T75 flask (1 × 10⁶ cells/flask).

Note: iPC requires higher seeding density as compared to iEC and iFibroblasts and will demonstrate slower proliferation rate.

iPC to induced-fibroblasts (iFibroblasts) differentiation, cryopreservation, and thawing

Timing: 2–5 weeks

This step explains how to differentiate iPC (P2-P5 in Pericyte expansion medium) to iFibroblast (P2-P8 FibroLife medium).

iPC to iFibroblasts differentiation

Timing: 1–2 weeks

• 23.
  Change medium to differentiate iPC to iFibroblasts.

  • a.
    Between passage 2–5 and when iPC are 70% confluent, change medium to FibroLife instead of Pericyte expansion medium for pre-conditioning.
  • b.
    Two days later wash iPC in 10 mL PBS and add 5 mL of TrypLE.
  • c.
    Incubate 5 min at 37°C.
  • d.
    Tab the side of flasks to detach cells.
  • e.
    Collect cells in FibroLife medium and centrifuge for 5 min, 100 g.
  • f.
    Count the cell number.
  • g.
    Plate iPC into a new T75 flask with density of 1 × 10⁶ cells/ 75T-flask in 12 mL of FibroLife medium including 5 μM Rock inhibitor. Mark as P1.
  • h.
    Change medium the next day to FibroLife without Rock inhibitor and every 2 or 3 days thereafter.
  • i.
    Repeat one more passage with density of 5 × 10⁵ cells/75T flask in 12 mL of FibroLife. medium including 5 μM Rock inhibitor. Mark as P2.

Note: iFibroblasts will demonstrate reduced cell size, elongated cell shape, and higher proliferation rate. It is expected to harvest 3–4 fold expansion.

iFibroblasts expansion, cryopreservation, and thawing

Timing: 7 days each passage

• 24.
  Expansion of iFibroblsts.

  • a.
    When P2 cells are 80%–90% confluent wash once in 10 mL PBS and add 5 mL of TrypLE.
  • b.
    Tab the side of flasks to detach cells.
  • c.
    Collect cells in FibroLife medium and centrifuge for 5 min, 100 g.
  • d.
    Resuspend in 1 mL of FibroLife medium per 75T flask harvested.
  • e.
    Count the cell number.
  • f.
    Plate iPC into a new uncoated T75 flask with density of 5 × 10⁵ cells/ 75T-flask in 12 mL of FibroLife medium including 5 μM Rock inhibitor. Mark as P3.

Note: do not expand beyond passage 7 or 8 in FibroLife and cryopreserve at early passage (e.g., P2-3 in FibroLife).

• 25.
  Cryopreservation of iFibroblasts.

  • a.
    Cryopreserve iFibroblasts after 2–3 passages in Fibroblast fate to ensure this fate is stabilized in cells (Figure 4).
  • b.
    Resuspend iFibroblasts 1 × 10⁶ cells/mL of CryoStore CS10.
  • c.
    Distribute 1 mL each cryovial.
  • d.
    Freeze cryovials in Mr. Frosty with isopropanol or equivalent freezing container at −80°C.
  • e.
    Next day move frozen vials into liquid nitrogen tank.

Pause point: Cells are frozen and can be paused at this step.

• 26.
  Thawing of iFibroblasts.

  • a.
    Bring a frozen vial to 37°C warm water bath.
  • b.
    Rapidly thaw cells within 2 min.
  • c.
    Move cells into conical tubes by slowly adding 9 mL of FibroLife medium with frequent agitation (total volume 10 mL).
  • d.
    Centrifuge for 5 min, 100 g.
  • e.
    Resuspend in 10 mL of FibroLife medium including 5 μM Rock inhibitor to ensure cell viability of cells post thaw.
  • f.
    Plate 1 vial to 2 of T75 flasks.
  • g.
    Next day, replace medium with 12 mL of FibroLife medium without Rock inhibitor.

Figure 4.

Phase contrast images of iPSC-derived fibroblasts (iFibroblast)

Pericytes are differentiated into fibroblasts using FibroLife medium on un-coated 75T-flasks. Cells demonstrated high proliferative rate up to passage 7–8. When cells demonstrate bigger cell size and slow proliferation (P9), it is recommended to discard and use earlier passage. Scale bars are 250 μm.

Expected outcomes

• •Post-sort analysis.

The data is included to demonstrate that there are no major differences between differentiation with APEL2 vs. APEL2-Li for the researchers who used with different medium (Figure 5).

• •iEC.

Figure 5.

Flow cytometry analysis of day 7 APEL differentiated cells

(A and B) (A) Comparisons of APEL2-Li and APEL2 on day 7 differentiating cells, and (B) Post-sort analysis of endothelial (CD31, CD146, CD144) and mesenchymal pericyte (CD146, CD140b, CD73) protein expression. Note: APEL2-Li and APEL2 media behave similar for this differentiation. Either of the two media can be used.

Early passage (P0 to P1) will demonstrate protein expression of CD31, CD146, CD144, vWF, CA4, RGCC.^3,4,5 Functional iEC will uptake acetylated-low density lipoprotein (Ac-LDL) that can be visualized under fluorescent microscopy or flow cytometry when used with fluorochrome tagged e.g., Dil-Ac-LDL (ThermoFisher Scientific, L3484) or Ac-LDL-Alexa488 (ThermoFisher Scientific, L23380) (Figure 6).

• •iPC.

Figure 6.

iPSC derived-endothelial cell (iEC) characterization

(A) iEC express common endothelial cell protein such as CD31, ETV2 and von Willebrand Factor (vWF). Scale bars are 50 μm.

(B) Majority of iECs uptake acetylated low density lipoprotein (Ac-LDL) that are visualized by Dil fluorescent tagged Ac-LDL shown in microscopy and analyzed by flow cytometry. Scale bars are 250 μm.

Established iPC (P2-3 in Pericyte expansion medium) will demonstrate protein expressions of CD140b (PDGFR-beta), CD44, NG2, CD146, and partial expression of alpha-SMA (smooth muscle actin) (Figure 7).^6,7,8,9,10

• •iFibroblast.

Figure 7.

iPSC derived-pericyte (iPC) characterization

(A–C) iPCs demonstrate (A) large high cell body size with highly developed cytoskeletons, clear cell membrane and extensions of cell bodies, express known pericyte markers such as (B) CD140b (a.k.a. PDGFR-b) (C) NG2, collagen type I (Col-I). Portions of iPC include alpha smooth muscle actin (aSMA) expressing cells. Scale bars are (A) 250 μm and (C) 50 μm.

Established iFibroblasts (P4-8 culture in FibroLife medium) will demonstrate protein expression of collagen type-I (Col-I), vimentin, and connexin43¹¹ but lack of expression of neuroglial 2 (NG2), PDGFR-b and aSMA (Figure 8).

Figure 8.

iPSC-derived fibroblasts (iFibroblasts) characterization

iFibroblasts express collagen type-I (Collagen-I), vimentin, connexin43 but not NG2, PDGFRb, and alpha-smooth muscle actin (aSMA). Scale bars are 100 μm for Collagen-I, 250 μm for phase contrast, and 50 μm for all other images.

Limitations

Cell density of iPC is important to maintain steady cell growth and differentiation to fibroblasts. If the plating cell density is too low, iPC growth will be reduced.

With longer cell culture period in each passage (e.g., 8 days instead of 6 days), iEC will produce more ECM and cell detachment using TrypLE or trypsin will be more difficult. It is recommended not to culture more than 7 days in each passage even though the cells do not reach desired cell density.

Troubleshooting

Problem 1

iEC, iPC or iFibroblasts strongly attached in culture plate and cells do not lift with trypLE or trypsin treatment for 5–10 min.

Potential solution 1

Use cell scraper to detach cells instead of pipetting strongly to detach cells.

Problem 2

iFibroblasts do not proliferate and demonstrate large cell bodies.

Potential solution 2

• •Thaw earlier passage or restart differentiation from iPC. Senescent fibroblasts tend not to recover from the senescent stage.
• •Start new fibroblast differentiation with earlier passage number of pericytes (e.g., P2 or P3).

Problem 3

After harvesting iPC, cell pellet was big size but the cell number is low.

Potential solution 3

iPC cell sizes are bigger than iEC and iFibroblasts and you may observe low cell number after cell count even though the cell pellet is big.

Problem 4

After 7–8 days of culture iPC are still not confluent.

Potential solution 4

Although cells are not confluent, passage every 7 days to stimulate cell growth.

Problem 5

CD31⁻ cells are too much. I don’t have enough space and reagent to culture all cells.

Potential solution 5

CD31⁻ cells can be cryopreserved right after MACS purification. Freeze 3–4 million cells/ vial in 1 mL of CryoStore CS10 and resume from the pre-pericyte culture step.

Resource availability

Lead contact

• •Name: Kapil Bharti.
• •Email: kapil.bharti@nih.gov.

Materials availability

• •This study did not generate new unique reagents.
• •APEL2-Li is discontinued by notification of StemCell Technologies. This can be replaced by APEL2 medium (Figure 1).

Data and code availability

This study did not generate datasets or codes.