Protocol to measure calcium spikes in cardiomyocytes obtained from human pluripotent stem cells using a ready-to-use media

Summary

The derivation of cardiomyocytes from human pluripotent stem cells (hPSCs) is a powerful tool to investigate early cardiogenesis and model diseases in vitro. Here, we present an optimized protocol to obtain contracting hPSCs-derived cardiomyocytes using a ready-to-use kit. We describe steps for hPSC culture and differentiation to cardiomyocytes including the identification of key parameters such as starting cell confluency and temperature. We then detail immunofluorescence, flow cytometry, and the quantification of cardiomyocytes' calcium spikes using live imaging.

For complete details on the use and execution of this protocol, please refer to Astro et al.¹

Graphical abstract

Highlights

• •Protocol to obtain contracting hPSCs-derived cardiomyocytes
• •Key parameters explained for cardiac differentiation such as starting hPSC confluency
• •Eighty percentage of TNNT2-positive cells with a homogeneous beating frequency of 0.5–0.7 Hz
• •Quantification of cardiomyocytes using calcium live imaging

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

The derivation of cardiomyocytes from human pluripotent stem cells (hPSCs) is a powerful tool to investigate early cardiogenesis and model diseases in vitro. Here, we present an optimized protocol to obtain contracting hPSCs-derived cardiomyocytes using a ready-to-use kit. We describe steps for hPSC culture and differentiation to cardiomyocytes including the identification of key parameters such as starting cell confluency and temperature. We then detail immunofluorescence, flow cytometry, and the quantification of cardiomyocytes' calcium spikes using live imaging.

Before you begin

All experiments involving the use of human pluripotent stem cells (hPSCs) must conform to the relevant legal and ethical standards and obtain approval from the institutional ethical committee. The use of hPSCs to perform this study was approved by the Institutional Biosafety and Bioethics Committee of the King Abdullah University of Science and Technology, approval number: 17IBEC14. This protocol allows the derivation of beating cardiomyocytes from hPSCs in about 12–18 days (Figure 1A). Here, we describe in detail how to culture and differentiate hPSCs into cardiomyocytes in 6-well plates and how to characterize them by downstream techniques such as calcium imaging and flow cytometry. Additionally, we explain how to perform the differentiation on coverslips in 24-well plates for subsequent immunofluorescence applications.

Figure 1.

hPSCs differentiation into cardiomyocytes

(A) Differentiation protocol timeline.

(B) Brightfield images of hESCs (H1) and hiPSCs (KAUSTi011-A) derived cardiomyocytes at the indicated time points.

Scale bar, 200 μm.

Coverslips preparation in sodium hydroxide (NaOH)

Timing: 1 day (for steps 1 to 15)

Pre-treated sterile glass coverslips will be required to perform immunofluorescence on hPSC-derived cardiomyocytes (hPSC-CMs). Therefore, it is imperative to wash coverslips with a 1 M NaOH solution to efficiently remove dust and grease-thin film that may interfere with protein coating and cell adhesion.² The following stepwise procedure describes how to clean and sterilize the glass coverslips. Notably, the NaOH pre-treatment is not required if Matrigel or vitronectin-coated 8-well chambered cover glass for cell culture are used.

• 1.Carefully insert around 200 round 13 mm diameter glass coverslips in a 2 L beaker and gently pour 400 mL of 1 M NaOH. The NaOH volume should cover by excess the coverslips.

Note: NaOH solution is a hazardous and corrosive liquid that must be handled using personal protective equipment inside a fume hood.

• 2.Fasten the beaker in an orbital shaker and incubate at 21°C–24°C for 1 h at about 100 rpm. The speed should be sufficient to shake the coverslips without breaking them.
• 3.Recover the NaOH solution into a glass bottle using a strainer and a funnel while keeping the coverslips in the beaker.

Note: The NaOH solution can be stored for up to three years in a closed bottle in the chemical cabinet and reused up to three times for this purpose.

• 4.Wash the coverslips at least three times with 400 mL of tap water.
• 5.Add 400 mL of 70% ethanol and incubate at 21°C–24°C for 10 min at 100 rpm.
• 6.Recover the 70% ethanol while keeping the coverslips in the beaker.
• 7.Wash the coverslips at least three times with 400 mL of MilliQ water.
• 8.With caution, distribute each coverslip on a blotting paper sheet to avoid coverslip overlapping. Use forceps to facilitate this step.
• 9.Let the coverslips dry on the blotting paper at 21°C–24°C.
• 10.Cut several circular pieces of 9 cm diameter blotting paper and place one at the bottom of a 10 cm diameter glass Petri dish.
• 11.Transfer the coverslips using forceps on the round blotting paper placed at the bottom of the glass Petri dish.
• 12.Prepare several layers of round blotting papers in the glass Petri dish and distribute the coverslips in between to avoid the overlap of multiple coverslips.
• 13.Close the glass Petri dish containing the coverslips with the glass lid, wrap it with aluminum foil, and apply autoclave tape.
• 14.Sterilize by autoclaving at 120°C for 15 min and dry it in an oven at 55°C for 12–16 h.
• 15.Store at 21°C–24°C and handle, under sterile conditions, inside the biosafety cabinet.

Coating with extracellular matrix proteins

Timing: 1 h (for step 16)

Timing: 16 h (for step 17)

Timing: 45 min (for step 18)

Human embryonic stem cells (hESCs) are cultured and differentiated into cardiomyocytes on vitronectin-coated dishes, whereas we routinely maintain and differentiate human induced pluripotent stem cells (hiPSCs) on Matrigel-coated plates. Nevertheless, several studies have maintained and differentiated hESCs and hiPSCs into cardiomyocytes using either vitronectin or Matrigel coating (Burridge et al.,³ Liu et al.,⁴ Lin et al.⁵56). Here, we provide the instructions to coat plastic culture plates or glass coverslips with either vitronectin or Matrigel. Due to lower cell attachment on glass coverslips compared to plastic culture plates, we suggest doubling the concentration of coating protein solution for glass coverslips. This will significantly improve cell attachment. The coating must be prepared under sterile conditions and incubated in a humidified incubator at 37°C.

Note: We recommend using the VTN-N or Matrigel coated plates immediately after preparation.

• 16.
  Vitronectin (VTN-N) coating:

  • a.
    Dilution of VTN-N 1:100 for plastic culture plates:

    • i.
      Thaw a 1 mL vial of VTN-N recombinant human protein on ice.
    • ii.
      Dispense VTN-N in 60 μL aliquots into 0.5 mL Eppendorf tubes and store them at −80°C.
    • iii.
      Thaw a 60 μL VTN-N aliquot on ice and dilute it in 6 mL of cold Dulbecco’s phosphate-buffered saline (DPBS).
    • iv.
      Add 1 mL of diluted VTN-N in each well of a 6-well plate and gently shake the plate to ensure that the entire well surface is evenly covered.
    • v.
      Incubate for 45 min in a humidified incubator at 37°C.
    • vi.
      After incubation, discard the VTN-N solution and immediately add 1 mL of E8 Media supplemented with RevitaCell.

      Note: As stated by the manufacturer, discard and do not reuse vitronectin solution. Reuse could result in reduced cell attachment and cell quality.

  • b.
    Dilution of VTN-N 1:50 for glass coverslips:

    • i.
      Inside the biosafety cabinet, using sterile forceps, place a coverslip at the bottom of each well of a 24-well plate.
    • ii.
      Thaw and dilute four 60 μL VTN-N aliquots in 12 mL of cold DPBS.
    • iii.
      Add 500 μL of diluted VTN-N in each well and gently shake the plate to ensure that the coverslips are submerged.
    • iv.
      Incubate for 45 min in a humidified incubator at 37°C.
    • v.
      After incubation, discard the VTN-N solution and immediately add 0.5 mL of E8 Media supplemented with RevitaCell.

• 17.
  Matrigel Aliquoting:

  CRITICAL: Matrigel will gel at a temperature > 10°C. Therefore, it is imperative to work on ice and use pre-chilled materials and solutions to dilute Matrigel.

  • a.
    Place a sterile 1,000 μL tip box at −20°C for 12–16 h and thaw a bottle of hESC-Qualified Matrigel on ice for 12–16 h at 4°C.
  • b.
    Use the pre-chilled tips to aliquot 150 μL of Matrigel into 0.5 mL tubes and store at −20°C.

    Note: The stored aliquots can be used for approximately six months after preparation, but no longer than the expiration date stated in the certificate of analysis for each lot number.

• 18.
  Matrigel coating:

  • a.
    Dilution of Matrigel 1:40 for plastic culture plates:

    • i.
      Thaw and dilute 150 μL of Matrigel into 6 mL of cold DMEM/F12 medium.
    • ii.
      Add 1 mL of diluted Matrigel in each well of a 6-well plate and gently shake the plate to ensure the entire well surface is evenly covered.
    • iii.
      Incubate for 1 h in a humidified incubator at 37°C.
    • iv.
      After incubation, discard the Matrigel solution and immediately add 1 mL of E8 Media supplemented with RevitaCell.
  • b.
    Dilution of Matrigel 1:20 for glass coverslips:

    • i.
      Inside the biosafety cabinet, using sterile forceps, place a coverslip at the bottom of each well of a 24-well plate.
    • ii.
      Thaw and dilute four 150 μL Matrigel aliquots in 12 mL cold DMEM/F12 medium.
    • iii.
      Add 500 μL of diluted Matrigel per well and gently shake the plate to ensure that the coverslips are submerged.
    • iv.
      Incubate for 1 h in a humidified incubator at 37°C.
    • v.
      Discard the Matrigel solution and immediately add 1 mL of E8 Media supplemented with RevitaCell.
    • vi.
      Any unused diluted Matrigel surplus can be stored at 4°C for up to two weeks.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Mouse anti-human Troponin2 (TNNT2) 1:1000	Thermo Fisher Scientific	Cat#A25969
Rabbit anti-human NKX2.5 1:1000	Thermo Fisher Scientific	Cat#A25974
Anti-Cardiac Troponin T antibody [1C11] (FITC) 1:70	Abcam	Cat#ab105439
Mouse IgG1 kappa Isotype Control (P3.6.2.8.1), FITC, eBioscience™ 1:20	Thermo Fisher Scientific	Cat#11-4714-42
Alexa Fluor® 488 donkey anti-mouse 1:250	Thermo Fisher Scientific	Cat#A25972
Alexa Fluor® 594 donkey anti-rabbit 1:250	Thermo Fisher Scientific	Cat#A25970
Chemicals, peptides, and recombinant proteins
Vitronectin (VTN-N) Recombinant Human Protein, Truncated	Thermo Fisher Scientific	Cat#A14700
DPBS, no calcium, no magnesium	Thermo Fisher Scientific	Cat#14190250
hESC-qualified Matrigel	Corning	Cat#CLS354277
DMEM/F12	Thermo Fisher Scientific	Cat#11320-032
Essential 8 Medium	Thermo Fisher Scientific	Cat#A1517001
RevitaCell Supplement	Thermo Fisher Scientific	Cat#A2644501
Accutase	STEMCELL Technologies	Cat#07920
Versene solution	Thermo Fisher Scientific	Cat#15040033
TrypLE	Thermo Fisher Scientific	Cat#12563011
Trypsin-EDTA (0.25%), phenol red	Thermo Fisher Scientific	Cat#25200056
Trypan blue stain (0.4%) for use with the Countess™ Automated Cell Counter	Thermo Fisher Scientific	Cat#T10282
ProLong Gold antifade mounting solution with DAPI	Thermo Fisher Scientific	Cat#P36931
BD Cytofix™ Fixation Buffer	BD Bioscience	Cat#554655
BD Perm/Wash™	BD Bioscience	Cat#554723
Sodium hydroxide solution 1 N	Sigma-Aldrich	Cat#S2567
Bovine serum albumin	Sigma-Aldrich	Cat#A7906
Critical commercial assays
PSC Cardiomyocyte Differentiation Kit	Thermo Fisher Scientific	Cat#A2921201
Human Cardiomyocyte Immunocytochemistry Kit	Thermo Fisher Scientific	Cat#A25973
Fluo-4 NW Calcium Assay Kit	Thermo Fisher Scientific	Cat#F36206
Experimental models: Cell lines
H1 WA01 Human embryonic stem cells (NIHhESC-10-0043)	WiCell	Cat#WAe001-A
KAUSTi011-A iPSC line	KAUST	Cat#KAUSTi011-A
Software and algorithms
ImageJ	Open source	https://imagej.net/ij/download.html
Other
Corning® 35 mm TC-treated culture dish	Corning	Cat#430165
Corning® 100 mm TC-treated culture dish	Corning	Cat#430167
Nunc™ Square BioAssay Dishes	Thermo Fisher Scientific	Cat#240845
Glass Petri dish 100 × 15 mm	VWR	Cat#89000-304
Falcon® 6-well Clear Flat Bottom TC-treated multiwell cell culture plate	Corning	Cat#353046
Costar® 24-well Clear TC-treated multiple well plates	Corning	Cat#3524
8-well chambered coverglass w/ non-removable wells	Thermo Fisher Scientific	Cat#155409PK
Cover slips, round 13 mm, thickness No.1	VWR	Cat#631-0149
Blotting paper, 703	VWR	Cat#28298-20
Color-frosted microscope slides	VWR	Cat#631-1554
Mini Instant Mix 5-Minute General Purpose Epoxy	Loctite	Cat#1975125
Immersion oil Immersol 518 F	Zeiss	Cat#444960-0000-000
Parafilm™ M Laboratory Wrapping Film	Thermo Fisher Scientific	Cat#13-374-12
Fisherbrand™ Lead-Free Autoclave Tape	Thermo Fisher Scientific	Cat#15-901-111
Tweezers Style 5 Super Thin Tips – biological grade	Dumont	Cat#72700-D
Falcon® 5 mL round bottom polystyrene test tube, with cell strainer snap cap	Corning	Cat#352235
100 mL laboratory bottles, narrow neck, with screw cap, DURAN®	VWR	Cat#215-1517
Countess™ Cell Counting Chamber Slides	Thermo Fisher Scientific	Cat#C10283

Materials and equipment

• •
  Complete Essential 8 (E8) Medium (1×):

  • ○
    Thaw a 10 mL E8 supplement vial (50×) at 4°C for 12–16 h.
  • ○
    Mix a bottle of 500 mL E8 basal medium with 10 mL E8 supplement and 5 mL of penicillin-streptomycin (100×).
  • ○
    Prepare 50 mL aliquots of complete E8 and store them at 4°C for up to 2 weeks.
• •
  Complete E8 medium supplemented with RevitaCell (1×):

  • ○
    Thaw a 5 mL RevitaCell Supplement vial (100×) at 4°C.
  • ○
    Prepare 500 μL aliquots and store them at −20°C.
  • ○
    Mix 50 mL of complete E8 Media with 500 μL of RevitaCell and store at 4°C for up to 2 weeks.
• •
  Fluo-4 NW Calcium Assay Kit reagent preparation:

  • ○
    Assay buffer:

    • -
      Thaw a 100 mL assay buffer bottle and prepare 10 mL aliquots.
    • -
      Store at −20°C and protect from light.
  • ○
    Probenecid 250 mM stock solution:

    • -
      Add 1 mL of assay buffer to a probenecid vial and vortex until dissolved.
    • -
      Prepare 100 μL aliquots and store them at −20°C for up to 6 months.
  • ○
    Dye solution:

    • -
      Add a 10 mL aliquot of assay buffer and a 100 μL aliquot of probenecid to a bottle of Fluo-4 dye mix and vortex for 2 min to dissolve completely.
    • -
      Prepare 1 mL aliquots, store them at −20°C, and protect from light.

Step-by-step method details

hPSC culture and dissociation

Timing: 3–4 days; 30 min/day (for steps 1 to 4)

hPSC culture is performed in a biosafety cabinet class 2, and cells are kept in a humidified incubator at 37°C and 5% CO₂. hESCs are cultured at ambient oxygen conditions (21% O₂) on VTN-N and detached with Accutase to obtain single cells, whereas hiPSCs are cultured at 5% O₂ on Matrigel and detached with Versene in cell clumps. hPSCs are cultured for 3–4 days with daily E8 media change and passaged at ratios of 1:6 or 1:8 once the confluency reaches 70% (Figure 1B).

Passaging and maintenance of hESCs:

• 1.
  Passaging: The next procedure describes how to seed a full 6-well plate starting from one 70% confluent well of a 6-well plate.

  • a.
    Warm at least 1 mL of Accutase and 6 mL of complete E8 medium supplemented with RevitaCell at 37°C for 3–5 min.
  • b.
    Remove the old medium and wash with 2 mL of DPBS.
  • c.
    Add 1 mL of Accutase and incubate in a humidified incubator at 37°C for 3 min.
  • d.
    Quickly, observe the cells under an inverted microscope to confirm cell detachment.

    • i.
      Incubate at 37°C for an additional minute if cells have not evenly detached, otherwise proceed to the next step.
  • e.
    Add 1 mL of complete E8 medium supplemented with RevitaCell and gently pipette up and down 2–3 times to obtain a single cell suspension.
  • f.
    Transfer the cell suspension to a 15 mL conical tube and centrifuge at 110 g for 3 min at 21°C–24°C.
  • g.
    Discard the supernatant and resuspend the cell pellet with 6 mL of complete E8 medium supplemented with RevitaCell. Avoid excessive pipetting to reduce cell stress.
  • h.
    Dispense 1 mL of cell resuspension into each well of the vitronectin pre-coated 6-well plate.
  • i.
    Move the plate using a stretched-eight-like motion, followed by a cross-like motion to distribute cells homogeneously in each well.
  • j.
    Place the plate on a 37°C hot plate or flat surface for 5 min to allow cell attachment.
  • k.
    Keep hESCs in a humidified incubator at 37°C, 5% CO₂ and 21% O₂.
• 2.
  Maintenance:

  • a.
    Warm complete E8 medium at 37°C for 3–5 min.
  • b.
    Remove the old medium and add 2 mL of complete E8 medium per well.
  • c.
    Replace the medium daily and passage cells once the confluency reaches 70%.

Passaging and maintenance of hiPSCs:

• 3.
  Passaging: The next procedure describes how to seed a full 6-well plate starting from one 70% confluent well of a 6-well plate.

  • a.
    Warm at least 1 mL of Versene and 6 mL of complete E8 medium supplemented with RevitaCell at 37°C for 3–5 min.

    Note: To passage hiPSCs, EDTA 0.5 mM in DPBS can be used as an alternative to Versene. The use of Accutase may require an adaptation period before routinary usage.

  • b.
    Remove the old medium and wash with 2 mL of DPBS.
  • c.
    Add 1 mL of Versene and incubate at 37°C for 2 min.
  • d.
    Quickly, observe the cells under an inverted microscope to confirm that cells remain attached but slightly separated and colonies have curled edges.

    • i.
      Incubate at 37°C for an additional minute if cells do not show these features, otherwise proceed to the next step.
  • e.
    Remove the Versene with caution to avoid cell detachment and add 2 mL of complete E8 medium supplemented with RevitaCell.
  • f.
    Gently detach the cells from the plate by pipetting 1–2 times using a P1000 pipette and transfer the cell suspension into a 15 mL conical tube. Avoid excessive pipetting to reduce cell stress.
  • g.
    Add 4 mL of complete E8 medium supplemented with RevitaCell to the cell suspension (6 mL total volume).
  • h.
    Mix gently by inverting the tube and dispense 1 mL of cell resuspension into each well of a Matrigel pre-coated 6-well plate. The final volume in each well will be 2 mL.
  • i.
    Move the plate using a stretched-eight-like motion, followed by a cross-like motion to distribute cells homogeneously in each well.
  • j.
    Place the plate on a 37°C hot plate or flat surface for 5 min to allow cell attachment.
  • k.
    Keep hiPSCs in a humidified incubator at 37°C, 5% CO₂ and 5% O₂.
• 4.Maintenance: hiPSCs require the same steps as for hESCs except for the incubation conditions as hiPSCs are maintained in hypoxic conditions (5%O₂).

hPSC differentiation into cardiomyocytes

Timing: 20–40 days; 30 min/day (for steps 5 to 10)

The differentiation procedure is the same for hESCs and hiPSCs, except for the coating substrate. hESCs are differentiated on VTN-N, whereas hiPSCs on Matrigel. hPSCs are passaged at ratios of 1:8, 1:10 or 1:12, and differentiation will be induced once cell confluency reaches around 30%, usually 48 h post-seeding. The medium is changed every two days along the differentiation and cells will start beating by days 12–18 (Figure 1). We observed that 30% confluency is optimal to induce a successful differentiation for multiple hPSC lines. Starting the differentiation at a low confluency allows reaching approximately 100% confluency at day 4 of differentiation. We found that if 100% confluency is reached before or after day 4, the differentiation efficiency will be significantly reduced. This step is not described in the manufacturer protocol and should be implemented to reduce variability in the differentiation efficiency when comparing multiple cell lines. It is crucial to start with high quality karyotypically normal hPSCs, with passage number below 50 and showing a good expression of pluripotency markers. Here, follows a description of how to differentiate hPSCs into cardiac cells in a 6-well plate format.

Note: To characterize hPSC-CMs by immunofluorescence, we recommend seeding hPSCs on pre-coated coverslips in a 24-well plate. A detailed step-by-step protocol to characterize cells differentiated on coverslips is exhaustively described in the subsequent section “hPSC-derived cardiomyocyte immunofluorescence” in steps 11–19.

• 5.Day -2. Passage 70% confluent hPSCs (Figure 1B) as described in the previous section at ratios of 1:8, 1:10 and 1:12.
• 6.Day -1: Remove the old medium containing RevitaCell and add 2 mL of complete E8 medium per well (Figure 1B).
• 7.
  Day 0. Differentiation induction:

  Note: Cell confluency should be around 30% before differentiation starts (Figure 1B).

  • a.
    Carefully, remove E8 medium from the hPSCs.
  • b.
    Add 2 mL of warm Cardiomyocyte Differentiation Medium A per well.

    Note: For details regarding Cardiomyocyte Differentiation Medium A, please refer to the PSC Cardiomyocyte Differentiation Kit manual.

    CRITICAL: We recommend aspirating and adding the differentiation media slowly at every media change to avoid detrimental effects on the differentiation efficiency.

    CRITICAL: At day 0 of differentiation, we advise to set the incubator temperature at 33°C degrees to improve differentiation efficiency and homogeneity of the contracting cells in the dish.

• 8.
  Day 2:

  Note: Cells retain a colony-like morphology, but colonies progressively become opaque with less defined edges. It is expected to observe a consistent number of dead cells in the plate before media change (Figure 1B).

  • a.
    Carefully, remove medium A and add 2 mL of warm Cardiomyocyte Differentiation Medium B per well.

    Note: For details regarding Cardiomyocyte Differentiation Medium B, please refer to the PSC Cardiomyocyte Differentiation Kit manual.

• 9.
  Day 4:

  Note: Increased cell death is observed at this stage, but under the mantle of floating dead cells and debris, the cell monolayer must reach 100% confluency (Figure 1B).

  • a.
    Remove the medium B and add 2 mL of warm Cardiomyocyte Maintenance Medium per well.

    Note: For details regarding Cardiomyocyte Differentiation Medium M, please refer to the PSC Cardiomyocyte Differentiation Kit manual.

    CRITICAL: if cells do not reach 100% confluency at day 4, we expect lower chances of generating beating hPSC-derived cardiomyocytes at the end of the differentiation protocol. Check troubleshooting section, problem 1 for a potential solution.

• 10.Day 6–40: Change the Cardiomyocyte Maintenance Medium every other day. Cells will start contracting by days 12–18 (Figure 1).

Note: If there is no detectable spontaneous beating or heterogenous contraction after 18 days, check the troubleshooting section, problem 2 and 3 for a potential solution.

Note: The longer hPSC-CMs are cultured in Maintenance Medium, the higher the chances for cell detachment. If possible, perform downstream assays before day 24.

hPSC-derived cardiomyocyte immunofluorescence

Timing: 2 days (for steps 11 to 19)

To characterize hPSC-CMs by immunofluorescence, we recommend seeding hPSCs on pre-coated coverslips in a 24-well plate at ratios of 1:6, 1:8, 1:10 to compensate for the lower cell attachment on glass coverslips. This immunofluorescence tests the expression of the transcription factor NKX2.5 as a marker of early cardiac mesoderm and TNNT2/cTNT as a cardiomyocyte marker involved in muscle contraction (Figures 2A and 2B). All reagents to perform the immunofluorescence are included in the Human Cardiomyocyte Immunocytochemistry.

Note: To seed the cells on a 24-well plate (1.9 cm²) starting from hPSCs cultured on a 6-well (9.5 cm²) you need to adjust the dilution ratio accordingly to the different cell area.

• 11.
  Reagent preparation:

  • a.
    Thaw Immunocytochemistry kit reagents and store them at 4°C for up to six months.
  • b.
    Warm the 10× wash buffer at 21°C–24°C and mix well before use.
  • c.
    Prepare 2,100 μL of 1× wash buffer per each well, diluting 210 μL of 10× wash buffer in 1,890 μL of MilliQ water.
• 12.
  Fixation:

  • a.
    Remove the old media from the cells and wash each well with 500 μL of DPBS.
  • b.
    Move with caution the 24-well plate containing the coverslips inside a fume hood, add 300 μL of fixative solution (4% formaldehyde in DPBS) per well and incubate for 15 min at 21°C–24°C.
  • c.
    Remove the fixative solution and add 300 μL of 1× wash buffer per well.

Pause point: Wrap the 24-well plate containing the coverslips with parafilm to prevent drying and store at 4°C for up to a month.

• 13.Remove the wash buffer, add 300 μL of permeabilization solution (1% Saponin in DPBS) in each well and incubate for 15 min at 21°C–24°C.
• 14.Remove the permeabilization solution, add 300 μL of blocking solution and incubate for 30 min at 21°C–24°C.
• 15.
  Primary antibody incubation:

  • a.
    Prepare a humidified chamber by placing a blotting paper soaked in water in a squared plastic dish.
  • b.
    Place a piece of parafilm on top of the soaked paper. The size of the parafilm varies according to the number of coverslips to process.
  • c.
    Use a towel paper to flatten the parafilm on top of the soaked blotting paper ensuring that no wrinkles and bubbles are present. The top side of the parafilm should be dry and clean.
  • d.
    Primary antibody dilution:

    • i.
      Prepare 40 μL of 25× working dilution of each primary antibody (rabbit α-NKX2.5 & mouse α-TNNT2), diluting 1 μL of antibody stock 1000× in 39 μL of blocking solution. Store at 4°C adequately labeled.
    • ii.
      Prepare 25 μL of diluted antibodies (1×) per each coverslip by diluting 1μL of 25× working dilution of each antibody in 23 μL of blocking buffer.
    • iii.
      Dispense a 25 μL drop of the 1× diluted mix containing both antibodies on the parafilm per each coverslip to be stained.
  • e.
    Coverslip handling: proceed with caution using forceps and pay attention to keep the cell side of the coverslips facing up.

    • i.
      Take the coverslip from each well and remove the buffer in excess with a gentle touch of torn blot paper on the coverslip rim. Avoid cell scratching!

      CRITICAL: The coverslips must be handled with extra care to avoid the detachment of the cardiac monolayer. Alternatively, the immunofluorescence can be performed directly inside the 24-well plate without disturbing the coverslip. However, this will require a larger volume of antibodies.

    • ii.
      Place the coverslip on top of the antibody drop with the cell side facing down.
  • f.
    Incubate at 4°C for 12–16 h.
• 16.
  Washing step:

  • a.
    Add 300 μL of 1× wash buffer in each well of a 24-well plate and keep it aside.
  • b.
    Add 100 μL of DPBS at the rim of each coverslip to lift the coverslip from the parafilm.
  • c.
    Take the coverslip from the parafilm and place it in a well containing 1× wash buffer, with the cell-side facing up. Incubate for 3 min at 21°C–24°C.
  • d.
    Remove the wash buffer and perform two additional washes with 300 μL of 1× wash buffer.
• 17.
  Secondary antibody incubation:

  • a.
    Place a new piece of parafilm in the humidified chamber.
  • b.
    Secondary antibody dilution:

    • i.
      Prepare 10 μL of 25× working dilution of each secondary antibody (Alexa Fluor 488 Donkey α-mouse & Alexa Fluor 594 Donkey α-rabbit), diluting 1 μL of antibody stock 250× in 9 μL of blocking solution. Store at 4°C adequately labeled. Protect from light.
    • ii.
      Prepare 25 μL of diluted antibodies (1×) per each coverslip by diluting 1μL of 25× working dilution of each antibody in 23 μL of blocking buffer.
    • iii.
      Dispense a 25 μL drop of the 1× diluted mix containing both antibodies on the parafilm per each sample to stain.
  • c.
    Coverslip handling: proceed as described previously with the primary antibody step (15e).
  • d.
    Incubate for 1 h at 21°C–24°C.
• 18.Washing step: proceed as described previously with the primary antibody step (16).
• 19.
  Mounting coverslip:

  • a.
    Add a drop of ProLong™ Gold Antifade Mounting solution with DAPI on a wipe to remove air bubbles and keep the vial upside down.
  • b.
    Add a drop Mounting solution per sample onto a clean slide. Mount up to 3 samples per slide.
  • c.
    Coverslip handling: proceed as described before at step (15e).

    • i.
      Take the coverslip from the well and remove residual buffer drops with a gentle touch of torn blot paper on the coverslip rim. Avoid cell scratching!
    • ii.
      Place the coverslip on top of the mounting media drop with the cell side facing down. Proceed slowly and gently to avoid the formation of trapped air bubbles.
    • iii.
      Incubate mounted coverslips at 21°C–24°C for 12–16 h at dark.
    • iv.
      Seal the coverslip edges with epoxy glue.
  • d.
    Proceed to acquisition or store the samples at −20°C for long-term storage.

Figure 2.

Characterization of hPSC-CMs

(A and B) Representative immunofluorescence staining of the indicated cardiac markers in (left) hESC- and (right) hiPSC-derived cardiomyocytes. TNNT2 (green), NKX2.5 (red), nuclei (blue). Scale bar, 50 μm.

(C) FACS analysis of TNNT2-positive cells at day 18 of differentiation.

hPSC-derived cardiomyocyte flow cytometry

Timing: 4 h (for step 20 to 24)

We recommend performing flow cytometry analysis on differentiated cardiac cells to quantify the percentage of cells expressing TNNT2, a cardiomyocyte marker involved in muscle contraction⁶ (Figure 2C). The following procedure describes the sample preparation of hPSC-derived cardiomyocytes for flow cytometry analysis using one well of a 6-well plate containing about 2.5 × 10⁵ cells/cm².

Note: Before starting the procedure, harvest undifferentiated hPSCs to be used as an undifferentiated negative control for the antibody staining and process it as described in steps 20g-24.

• 20.
  hPSC-CM harvesting:

  • a.
    Warm 1 mL of trypsin-EDTA 0.25% at 37°C for 3–5 min.
  • b.
    Discard the old media and wash with 2 mL of DBPS.
  • c.
    Add 1 mL of trypsin-EDTA 0.25% and incubate in a humidified incubator at 37°C for 5–15 min.
  • d.
    Check the cells using an inverted microscope every 5 min and only procced to the next step when the cells are detaching from the plate and become round.

    Note: Trypsin-EDTA 0.25% is optimal to obtain a single-cell suspension of hPSC-CMs. We discourage the use of Accutase or TrypLE express, as hPSC-CMs will not detach efficiently and generate cell clumps.

  • e.
    Add 3 mL of DPBS and pipette up and down 2–3 times using a P1000 pipette to obtain a single cell suspension, avoiding excessive pipetting to reduce cell stress and viability.
  • f.
    Transfer the cell suspension into a 15 mL conical tube.
  • g.
    Take 10 μL of cell suspension and mix gently with 10 μL of trypan blue.
  • h.
    Load the mix in a cell counting chamber slide and count cells in the countess automated cell counter.
• 21.
  Fixation and permeabilization:

  • a.
    For each cell line, label four 1.5 mL Eppendorf tubes according to the cell type and antibody as follows:

    • i.
      Undifferentiated: α-mouse TNNT2-FITC.
    • ii.
      Undifferentiated: α-mouse IgG1-FITC.
    • iii.
      Cardiomyocytes: α-mouse TNNT2-FITC.
    • iv.
      Cardiomyocytes: α-mouse IgG1-FITC.
  • b.
    Transfer 1 × 10⁶ cells in each 1.5 mL Eppendorf tube and centrifuge at 200 g for 3 min.
  • c.
    Remove the supernatant and resuspend the cell pellet in 100 μL of BD cytofix fixation buffer. Alternatively, paraformaldehyde 4% in DPBS can be used as a fixative solution.
  • d.
    Incubate for 20 min at 21°C–24°C with gentle agitation in a rocker.
  • e.
    Centrifuge at 500 g for 3 min and discard the supernatant.
  • f.
    Wash twice with 500 μL of DPBS.
  • g.
    BD perm/wash 1× solution preparation:

    • i.
      Mix 350 μL of 10× BD perm/wash solution with 3150 μL of MilliQ water.
  • h.
    Wash twice with 100 μL of 1× BD perm/wash solution.
  • i.
    Resuspend the cell pellet with 100 μL of 1× BD perm/wash solution and incubate for 10 min at 21°C–24°C with gentle agitation in a rocker.
• 22.
  Antibody incubation:

  • a.
    Add 1 μg of the respective antibody to each tube and incubate at 21°C–24°C for 45 min with gentle agitation in a rocker.
  • b.
    Centrifuge at 500 g for 3 min and discard the supernatant.
  • c.
    Wash twice with 500 μL of 1× BD perm/wash solution.
• 23.
  Cell resuspension for flow cytometry analysis:

  • a.
    Bovine serum albumin (BSA) 0.5% in DPBS preparation:

    • i.
      Dissolve 0.1 g of BSA in 20 mL of DPBS by vortexing for 5 min.
  • b.
    Resuspend the cell pellet in 400 μL of BSA 0.5% and transfer the cell suspension into a 5 mL round bottom tube passing through a 35-μm mesh strainer.
• 24.Acquire the data using a flow cytometer equipped with a blue laser (488 nm) and a filter 530/30 nm to record FITC emission.

Calcium live imaging on PSC-derived cardiomyocytes

Timing: 3 h

During cardiomyocyte contraction, a time-dependent transient increase in intracellular calcium levels, known as calcium transients, regulates the excitation-contraction coupling process.⁷ Spontaneous calcium sparks can be observed in cardiomyocytes obtained in vitro from hPSCs. In this section, we describe a simple procedure to record calcium transients of hPSC-CMs using the calcium indicator Fluo-4 acetoxymethyl (AM). This application complements flow cytometry and immunocytochemistry analyses and allows the functional characterization of the derived cardiac cells.

Microscope set up for live imaging

Timing: 10 min

Note: Here, we describe the live imaging acquisition using the EVOS FL Auto 2 Cell Imaging System. Alternatively, any inverted fluorescent microscope equipped with a stage incubator and a camera capable of recording at least 10 frames per second (fps) can be used.

• 25.Fill the water reservoir of the stage incubator up to the maximum level (Figure 3A).
• 26.Turn on the microscope, CO₂ supply, and incubator unit 30 min before use and set the temperature to 37°C and the humidity to 80% in the stage incubator.
• 27.Insert a template plate on the plate holder while the instrument calibrates to reach the correct temperature, CO₂ and humidity values (Figure 3B).
• 28.Launch the EVOS FL Auto 2 software and set the correct incubator parameters in the incubator control panel tab (Figure 3C).
• 29.
  Live cell imaging is performed using an 0.3 NA/10× objective with a long working distance suitable for plastic bottom dishes (WD = 9.2 mm).

  • a.
    Use the option “Live recording”, corresponding to a camera setting of about 20 fps (Figure 3D).
  • b.
    To acquire at a frame rate ≤ 10 fps, use the “as fast as possible” option from the ‘Time Lapse’ tab in the EVOS FL software. Alternatively, set the parameters corresponding to the desired frame rate (Figure 3E).

CRITICAL: The frame rate is an important parameter that can influence the outcome of the calcium live imaging.⁸ To perform this experiment, it is key to use a microscope equipped to track the fast dynamic of calcium reporters. In our hands, the spontaneous beating frequency of hPSC-CMs varies between 0.5-0.7 Hz. Therefore, a camera with a frame rate of 10–20 fps provides a sufficient coverage to describe the shape of the curve of the calcium spikes (Figure 3F). On the contrary, calcium live imaging recorded at 5fps results in the incomplete measurement of the contractility parameters (Figure 3F).

Figure 3.

EVOS FL Auto 2 Microscope set-up for live calcium imaging acquisition

(A and B) (A) Incubator and (B) stage chamber used to control humidity, temperature, and CO₂ levels during live imaging.

(C–E) (C) Incubator control panel tab and (D and E) Time-lapse menu tab settings.

(F) Amplitude of calcium live imaging acquired at three different frame rates (20, 10 and 5 fps). The shape of the amplitude curve loses detail at 10 and 5 fps, where fewer measuring points are describing the shape of a contraction.

Calcium staining

Timing: 1 h

Note: The following procedure is performed on cells seeded on plastic 6-well plates as described in the section “hPSC differentiation into cardiomyocytes” steps 5–10, on hPSCs differentiated into cardiomyocytes at day 14–18 of differentiation. The cell density of the contracting monolayer should be around 2.5 × 10⁵ cells/cm². The indicated volumes are meant for a 6-well or a 35 mm plate format. It is possible to scale down the required volume by differentiating human PSCs into 24-well plates.

• 30.Prepare the Fluo-4 AM NW loading solution as described in the materials and equipment section.
• 31.Carefully remove the culturing media from the beating cardiomyocytes with a P1000 pipette to avoid the detachment of the cell layer and wash once with DPBS.
• 32.Aspirate the DPBS with a P1000 pipette and load 1 mL of Fluo-4 AM NW solution into the well.
• 33.Place the cells in the incubator set at 37°C for 45 min.
• 34.Remove the Fluo-4 AM NW solution from the hPSC-CMs and add 1.5 mL of CMs Maintenance Medium.

Note: The Fluo-4 AM NW (No wash) solution does not require a wash step or a quencher dye. Removing the wash step results in lower variability and higher fluorescence intensity than the standard fluo-3 or fluo-4 assays.

• 35.Before recording, incubate the hPSC-CMs for 10 min inside the stage chamber for equilibration.

Calcium live imaging

Timing: 1 h

• 36.Use the inverted EVOS FL Auto2 microscope equipped with a 10× objective with a pixel size of 0.88 μm to select the first Region of Interest (ROI) in brightfield mode using a low illumination intensity (0.50 msec).

Note: To avoid the biased selection of the ROI and cover the entire plate area, divide the well into five virtual fields and acquire sequential images labelled from one to five (Figure 4A). Apply the same scheme for each condition.

• 37.
  Use the GFP LED light cube to excite the Fluo-4 AM loaded cells at 494 nm and collect the emission fluorescence signal at 516 nm.

  • a.
    Set a low exposure time (about 0.50 msec) to avoid calcium indicator bleaching and reduce cell stress.

Note: If you are unable to visualize the fluorescent signal of the calcium probe, check the troubleshooting section, problem 4 for a potential solution.

CRITICAL: We recommend the use of a microscope equipped with an objective, a sensitive camera, light source, and filter setup that overall provide an optimal and uniform field illumination and a balanced contrast, resulting in a sharp and clear delimitation of the cell boundaries. If you experience loss of signal due to bleaching during the time-lapse, check the troubleshooting section, problem 5 for a potential solution.

• 38.
  Use the fine focus control of the microscope to bring the sample into the sharpest possible focus with a fine adjustment knob.

  • a.
    Identify the plane where the cell edges appear sharp and not blurry (Figure 4B).
  • b.
    Start the live imaging with the 10× objective by clicking “record” (Figure 3D).

Note: Acquisition time is 30 s. During the recording, minimize the vibrations of the microscope table.

• 39.It is advisable to save the time-lapse experiment as a sequence of independent TIFF raw images with a depth of 8-bit.

Note: It is possible to import in ImageJ software TIFF raw images as image sequence and save them with .avi extension (see step 40). We recommend being consistent when saving images and using the same depth (8-bit or 16-bit) for accurate comparison.

Figure 4.

Calcium imaging analysis using ImageJ

(A) Virtual scheme of the fields.

(B) Selection of the optimal focus. The correct focal plane presents sharp and bright cell edges, as shown in the middle image. Scale bar, 275 μm.

(C–I) Kymograph generation using ImageJ to quantify the frequency and amplitude of calcium spikes. (C and D) Draw a straight line of approximately 200 μm using the “line tool” in a random area of the field. (E) In the ROI Manager window, click “Add” to save the coordinates of the straight-line selection. (F) Draw and record the coordinates of multiple lines in the ROI Manager window. Scale bar, 200 μm. (G) Select the line from the ROI Manager menu and use the ImageJ function “Image > Stacks > Reslice to generate the kymograph. (H) Check the options: Rotate 90 degrees and avoid interpolation to visualize the frames over time in the horizontal axes and the distance in the vertical axes. (I) Kymograph showing the spatial position over time of the selected areas.

(J) Interactive plot showing the peaks of MFI over time corresponding to the intensity and duration of the calcium transient in the selected area. The black arrows indicate the summit (F) and the base of one peak (F0), corresponding to the first calcium transient.

Analysis of hPSC-CMs calcium transients

Timing: 40 min

• 40.Launch ImageJ software and open the sequence of TIFF images you want to analyze using the command “FileImportImage sequence”.

Note: It is possible to specify the number of images to import; if not specified, the software will recognize and open all the images with the same extension name.

• 41.To compensate for the non-uniform illumination of the image, we recommend performing a background correction by using the ImageJ software, as described in the step-by-step protocol in Scalzo et al.⁹.
• 42.Adjust the brightness and contrast of the movie to the desired level “ImageAdjustB/C”.

CRITICAL: It is crucial to apply identical image modifications (e.g., brightness and contrast) when comparing different experimental conditions.

• 43.Draw a straight horizontal line of approximately 200 μm using the “line tool” in a random area of the field (Figures 4C and 4D).
• 44.Open the ROI Manager window with the command “AnalyzeToolsROI manager”.
• 45.In the ROI Manager window, click “Add” to save the coordinates of the straight-line selection (Figure 4E).

Note: We recommend recording the coordinates of multiple lines in the ROI Manager window and analyze multiple ROIs to minimize biases due to potential beating differences throughout the plate (Figures 4F and 4G). Specifically, for each sample, we acquire five independent fields of a well of a 6-well plate and analyze five independent ROIs for each field. The arbitrary selection of the ROI in each field could bias the result of this analyses. This is particularly relevant for differentiation experiments characterized by non-homogenous beating throughout the plate. We recommend using the same ROI coordinates for all the samples, to reduce the biased selection of the beating areas.

• 46.
  To obtain a kymograph of the calcium transient, proceed as follows:

  • a.
    Select the line from the ROI Manager menu and use the ImageJ function. “Image Stacks Reslice”
  • b.
    Check the options: "Rotate 90 degrees" and "avoid interpolation" to visualize the frames over time in the horizontal axes and the distance in the vertical axes (Figure 4H).
  • c.
    A new window will open showing the spatial position over time of the selected areas (Figure 4I).
  • d.
    The kymograph can be saved as a TIFF image with the command “FileSave as”

    Note: The line length that identifies the ROI must be identical within all comparisons. The direction of the line will not affect the result of the analyses.

• 47.
  The amplitude and beat rate of each calcium transient can be calculated by measuring the mean fluorescence intensity (MFI) of the area of interest over time from the ROI manager tab, proceeding as follow:

  • a.
    Select the line from the ROI Manager menu and use the ImageJ function.
    “ImageStacksPlot Z-axis Profile”.
  • b.
    An interactive plot will appear showing the peaks of MFI over time corresponding to the intensity and duration of the calcium transient in the selected area (Figure 4J).
  • c.
    To calculate the amplitude of the calcium spike:

    • i.
      Check the maximum and minimum height of the peak by passing the cursor on the summit and at the base of the peak.
    • ii.
      Repeat the same operation for every peak of the plot.
    • iii.
      Calculate the amplitude using the following formula, where “F” is the MFI at the peak summit and “F0” is the MFI at the peak base (Figures 5A–5C):

      $$Amplitude\left(A\right)=\frac{\left(F-F0\right)}{F0}$$
    • iv.
      The Amplitude of the ROI is calculated by averaging the amplitude of all peaks.
  • d.
    To calculate the Frequency (F) in beats per minute (bpm) of the calcium spike:

    • i.
      Count the peaks in each interactive plot.
    • ii.
      Apply the following formula, where P is equivalent to the duration of the movie in seconds:

      $$bpm=\left(\frac{N.Peaks}{P}\right)\ast 60$$

      Note: Approximately 400–500 cells can be analyzed within a 176 μm² (200 μm × 0.88 μm) ROI of an image acquired using a 10× objective with a pixel size of 0.88 μm and a cell density of 2.5 × 10⁵ cells/cm².

Figure 5.

Calcium amplitude calculation using ImageJ

(A) Kymograph showing the ROI displacement over time.

(B) Plot of the peaks corresponding to calcium amplitude. The black arrows indicate the peak summit (F) and the peak base (F0).

(C) Example of a single contraction wave (red rectangle in panel B) showing the amplitude calculation using the formula (F-F0)/F0. The Y-axes represents the duration of the peak (time) in seconds.

Expected outcomes

Here, we describe a protocol to efficiently generate cardiomyocytes in vitro from human iPSCs or hESCs and characterize their differentiation through a functional calcium live imaging assay. Several methodologies for deriving cardiomyocytes from hPSCs have been reported.^3,5,10,11 This protocol is based on an a-ready-to-use media formulation that limits batch effects and operator bias during reagent preparation.

A good quality differentiation experiments should meet the following criteria: i) cells should reach 100% confluency at day 4 of differentiation; ii) the onset of spontaneous contraction should occur around day 12 of differentiation; iii) an homogeneous contracting monolayer of cardiac cells should be visible around day 18; iv) the percentage of cardiac TNNT2-positive cells measured by flow cytometry should be ≥ 80% at day 18; v) the beating rate should be ≥ 0.5 Hz, corresponding to 30 bpm.

We have obtained comparable differentiation results when using Matrigel or vitronectin-coated plates. We typically observe a faint beating in small areas that, in 3–5 more days, evolves in a contraction of the whole dish. The size of the Petri dish that accommodates the cells during the differentiation can also influence the outcome. We recommend using 35 mm Petri dishes or 6-well plates. We also obtained beating cardiomyocytes from 10 cm dishes, but it is more challenging to seed hPSCs homogenously in these plates. The uneven cell distribution results in cardiomyocytes beating only in limited areas.

Cardiomyocytes derived with this protocol can be cultured for up to 40 days. We and others observed increased expression of cardiac markers in cells cultured for extended periods.⁴ The shearing forces exerted on the cells by the continuous beating may lead to the formation of holes in the cell monolayer that, due to contraction forces, will increase in size and eventually detach from the plate. Cells can be detached and reseeded to circumvent this inconvenience, as described by Lin et al.⁵

The differentiation described in this protocol gives rise to cells positive for the cardiomyocyte markers NKX2.5, TBX5, TNNT2, ISL1, and MEF2C. The expression levels of these genes can be monitored by real-time Q-PCR, and protein expression can be evaluated by immunofluorescence and flow cytometry. Here, we used an immunostaining kit for cardiomyocytes, but standard immunofluorescence protocols are a valid alternative.

The study of calcium transients is crucial to investigate the differentiation state of cardiomyocytes and their beating features. Several options exist to monitor cardiomyocytes' calcium signaling-dependent membrane depolarization leading to cell beating. The technique we share is based on the fluorescent calcium sensor Fluo-4 AM NW (no wash), which is a ready-to-use, simple and cheap assay that allows the tracking of fluorescence variation linked to calcium fluctuation.

Importantly, a homogeneous contracting phenotype is a prerequisite to perform downstream analysis such as drug screening and disease modeling of pathological conditions associated with functional and morphological alteration of cardiac cells.¹² For example, we measured the cardiomyocyte response to Epinephrine [10 μM] administered to the cells right after starting the calcium imaging acquisition. The live imaging output is a video recording (Methods video S1) that allows a visual assessment of cardiac contraction and the measurement of several parameters, such as the amplitude and frequency of calcium transients (Figure 6).

Figure 6.

Effects of Epinephrine treatment on PSC-derived cardiomyocytes' calcium signaling

(A) Representative frame of H1 hESCs treated or not with 10 μM Epinephrine. Scale bar, 100 μm.

(B) Plots created with ImageJ showing the sequence of calcium transients in the two conditions. Related to Methods video S1.

Methods video S1. Calcium Imaging of cardiomyocytes derived from H1 hESCs treated with Epinephrine, related to expected outcomes

Time-lapses of cells loaded with Fluo-4-AM and imaged 10 min after 10 μM Epinephrine administration for 30 s at 20 fps. Scale bar, 100 μm.

Limitations

The present protocol provides a relatively easy method to differentiate human PSC into beating cardiomyocytes. Moreover, we offer a quick and straightforward approach to evaluate cardiomyocyte contractility through a calcium live imaging assay. However, there are several technical limitations to consider.

The rate of success of the differentiation can vary with different hPSC batches. Certain cell lines are more efficiently differentiated than others. This may be due mainly to the variation of proliferating rates among hPSCs.

Notably, the quality of hPSC seeding on day -1 (Figure 1) is an important factor influencing the differentiation output. An uneven cell distribution or a non-homogenous size of the cell colony can affect the area occupied by beating cardiomyocytes at the end of the differentiation. To improve the differentiation quality and obtain a whole monolayer of contracting cells, we suggest adjusting the cell density of the hPSCs plated for the differentiation.

An additional limitation is that the method explained here derives immature cardiomyocytes resembling fetal characteristics rather than adult human cardiomyocytes. This limitation is a common bottleneck of the 2D differentiation protocols described so far. Several 3D approaches are now available to improve the in vitro maturation of hPSC-derived cardiomyocytes.¹³

A possible limitation of the calcium live imaging on cardiomyocytes is that the calcium indicators tend to bleach easily. We suggest recording for a short time (30 s–1 min) with high frame rates (>10 fps) rather than acquiring longer movies with low frame rates. Also, short recordings limit phototoxicity and increase cell survival during live imaging.

Troubleshooting

Problem 1

Massive cell death is observed after media B addition (day 4 of differentiation) (step 9). Should I continue the experiment or start a new differentiation modifying the initial hPSC cell density?

Potential solution

• •To observe cell death at day 4 of differentiation should be expected and it is not necessarily a sign of failed differentiation. However, a homogeneous and healthy carpet of cells should always be visible. If, at day 4, the cell confluency is significantly below 100% the experiment is likely to be unsuccessful. In this case, we advise to repeat the experiment starting from the PSCs seeding step and increasing the number of starting cells.

Problem 2

There is no detectable spontaneous beating of cardiomyocytes after 12–18 days of differentiation, or only a few small areas of the plate are contracting (step 10).

Potential solution

• •When the direct differentiation is unsuccessful, the cardiac markers will be lowly or not expressed, and only a few cells will contract in the whole Petri dish. In this case, we suggest adjusting the number of hPSC plated before cardiac induction. It is crucial to start the differentiation with a 20%–30% confluency (Figure 1) because a higher number of cells seeded can negatively affect the differentiation rate. On the contrary, a lower number of seeded cells can result in higher cell death at day 4 and, consequently, a failed differentiation. We recommend starting the differentiation 48 h after seeding for efficient differentiation.
• •Another limiting factor is the detaching reagent used for hPSC detachment. Our protocol is optimized to start with cells seeded as single rather than clumps of cells. You should observe very small cell clusters 24 h after seeding (Day -1 in Figure 1B). We have experienced a reasonable recovery rate using Accutase on hESCs (H1 and H9). Instead, we observed a better recovery of hiPSCs passaged using Versene or TrypleE.
• •To obtain a successful differentiation is critical to use good quality batches of hiPSCs, showing a good expression of pluripotency markers and an excellent colony-shape morphology¹⁴ (Figure 1).
• •We have experienced a homogeneous contraction in the whole plate when hPSCs are differentiated at 5% CO₂ at a lower temperature overall the differentiation timeline (33°C).

Problem 3

The not homogeneous beating of cardiomyocytes after 12–18 days of differentiation (step 10).

Potential solution

• •We suggest detaching and reseeding the cardiomyocytes into new dishes, as described in Lin et al.⁵.
• •If the cells are used for further molecular biology analysis (e.g., ChIP-sequencing), we suggest scratching away with P200 tips or insulin-needles areas populated with no beating cells.

Problem 4

The calcium signal of the cells is too low, and the image is not clearly visible during the acquisition (step 37).

Potential solution

• •The setup of the microscope can be modified to maximize the captured signal. In our hands, an EVOS FL Auto 2 Cell Imaging System, equipped with a high-sensitivity 1.3 MP CMOS monochrome camera with 1328 × 1048 pixels, with a 0.3NA/10× objective (WD 9.2 mm) with an exposure time of 0.50 msec is sufficient to obtain a good signal.
• •We recommend the use of a lens optimized for fluorescent signals, such as the Plan Fluorite objectives.
• •While acquiring at low GFP illumination exposure, such as 0.50 msec, we typically increase the brightness and contrast of the displayed image by using the lower icon of the viewing area (“Image display settings” button, Figure 7). The readers should be aware that the raw images are not modified when tuning the brightness and contrast through the “Image display settings”.

Figure 7.

Graphical user interface of the EVOS FL 2 Auto Software

The red rectangle shows the “Image display settings” window collapsed. This option allows the user to regulate the brightness and contrast of the displayed image during the time-lapse acquisition without modifying the raw images.

Problem 5

High photobleaching of the calcium indicator during the live imaging (step 37).

Potential solution

• •To decrease the photobleaching of the fluorescence calcium die, we suggest lowering the intensity of the illumination during the acquisition and prefer short recordings (<1 min). We usually set the brightness of the GFP LED light cube at 0.50 msec. Increased photobleaching will result in a rapid MFI decrease over time and a biased interpretation of the transient cardiac amplitude.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Antonio Adamo (antonio.adamo@kaust.edu.sa).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This protocol did not generate a code. However, check out our published article to see an example of how to compare cardiomyocyte calcium spikes derived from different genotypes or treatments using our protocol.¹