TITLE: Simulation of cardiac arrhythmias in human induced pluripotent stem cell-derived cardiomyocytes
AUTHORS: Thea Bommer, Maria Knierim, Julia Unsöld, Dominic Riedl, Laura Stengel, Michael Paulus, Thomas Körtl, Norman Liaw, Lars S. Maier, Katrin Streckfuss-Bömeke, Samuel Sossalla, Steffen Pabel

[DESCRIPTION]
The effects and mechanisms of cardiac arrhythmias are still incompletely understood and an important subject of cardiovascular research. A major difficulty for investigating arrhythmias is the lack of appropriate human models. Here, we present a protocol for a translational simulation of different types of arrhythmias using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) and electric cell culture pacing. The protocol comprises the handling of ventricular and atrial hiPSC-CM before and during in vitro arrhythmia simulation and possible arrhythmia simulation protocols mimicking clinical arrhythmias like atrial fibrillation. Isolated or confluent hiPSC-CM can be used for the simulation. In vitro arrhythmia simulation did not impair cell viability of hiPSC-CM and could reproduce arrhythmia associated phenotypes of patients. The use of hiPSC-CM enables patient-specific studies of arrhythmias, genetic interventions, or drug-screening. Thus, the in vitro arrhythmia simulation protocol may offer a versatile tool for translational studies on the mechanisms and treatment options of cardiac arrhythmias.

[STEPS]
SECTION: Differentiation of ventricular and atrial hiPSC-CM
1. Differentiation of hiPSC-CM was performed as previously described [1,2]. Briefly, hiPSCs were cultured feeder-free and adherent on cell culture dishes in the presence of chemically defined medium E8. 

SECTION: Differentiation of ventricular and atrial hiPSC-CM
2. Cardiac differentiation of hiPSCs was performed by sequential targeting of the WNT pathway. Undifferentiated hiPSCs were cultured as a monolayer on Geltrex-coated 12-well dishes to a confluence of 85%-95%. 

SECTION: Differentiation of ventricular and atrial hiPSC-CM
4. After 48h, medium was changed to fresh media supplemented with 5 µmol/L of the inhibitor of Wnt production-2 for two days. For atrial subtype specification, 1 μmol/L retinoic acid was added between day 3 and day 6 [3].

SECTION: Differentiation of ventricular and atrial hiPSC-CM
3. Medium was changed to cardio differentiation medium composed of RPMI medium supplemented with L-Ascorbic Acid 2-Phosphate and albumin including the GSK3 inhibitor CHIR99021 (d0). 

SECTION: Differentiation of ventricular and atrial hiPSC-CM
5. Cardiac contraction is usually observed on day 7. From day 8 (atrial) or day 10 (ventricular), the cells were cultured in cardio culture medium, which was changed every 2-3 days. 

SECTION: Differentiation of ventricular and atrial hiPSC-CM
6. HiPSC-CM were purified after 20-30 (atrial) or 20-40 (ventricular) days of differentiation by metabolic selection for 4-5 days using lactate (4 mmol/L, Sigma Aldrich) as carbon source. In contrast to undifferentiated iPSCs the differentiated cardiomyocytes have the ability to effectively uptake and use lactate as a carbon source [4]. Thereby, substitution of glucose by lactate allows differentiated cardiomyocytes to grow whereas undifferentiated iPSCs die. 

SECTION: Differentiation of ventricular and atrial hiPSC-CM
7. Following differentiation, purity of hiPSC-CM was determined by flow analysis (∼90% cardiac troponin T +), cardiac immuno-fluorescence, morphology and qPCR for cardiac sub-type marker as previously described [1, 2, 5, 6].

SECTION: Preparation of the 6 well culture plates and dishes
8. 
 

Prepare 0.1 % gelatin solution in distilled water.

SECTION: Preparation of the 6 well culture plates and dishes
9. Autoclave at 120 °C and 1 bar for 20 min .

SECTION: Preparation of the 6 well culture plates and dishes
10. Coat plates with 1 mL gelatin solution per well or dish.

SECTION: Preparation of the 6 well culture plates and dishes
11. Incubate coated plates either 30 min at 4 °C or for 30 min at Room temperature

SECTION: Preparation of the 6 well culture plates and dishes
12. Extract the fluid supernatant.

SECTION: Preparation of the 6 well culture plates and dishes
13. Dry plates for a minimum 30 min until no wetness is left in the wells or dishes.

 

SECTION: Digestion and plating of the hiPSC-CM
14. 
 

For plating the cells, we perform a digestion protocol using trypsin as described before [2]. Briefly:

SECTION: Digestion and plating of the hiPSC-CM
15. Add 1 mL-2 mL Trypsin to confluent hiPSC-CM cultures.

SECTION: Digestion and plating of the hiPSC-CM
16. Incubate for 3 min-10 min at 37 °C .

SECTION: Digestion and plating of the hiPSC-CM
17. Stop reaction by adding 2 mL of the digestion medium when cells dissolve. 

SECTION: Digestion and plating of the hiPSC-CM
18. Transfer suspension into a tube that already contains 2 mL digestion medium.

SECTION: Digestion and plating of the hiPSC-CM
19. Centrifuge at  200 x g, 5 min .

SECTION: Digestion and plating of the hiPSC-CM
20. Extract supernatant and resuspend the pellet in digestion medium.

SECTION: Digestion and plating of the hiPSC-CM
21. Count cells (i.e. via Neubauer chambers) and distribute them according to the targeted cell numbers in coated plates.

SECTION: Digestion and plating of the hiPSC-CM
22. Plating is performed based on the planned experiments:

SECTION: Digestion and plating of the hiPSC-CM
22.1. For assays requiring larger cell numbers (100 000 – 500 000 cells, i.e. Western Blots, Proteomics or gene sequencing) plate hiPSC-CM in the wells of the 6 well plate. 

SECTION: Digestion and plating of the hiPSC-CM
22.2. For investigation of single cells (i.e. patch clamp, fluorescence microscopy), plate a cell number of 20 000 – 40 000 hiPSC-CM in 35 mm glass bottom dishes, which are placed within the wells of the 6 well plate.

SECTION: Digestion and plating of the hiPSC-CM
23. Fill every well with 2 mL and every dish with 1.5 mL digestion medium.

SECTION: Digestion and plating of the hiPSC-CM
24. Resuspend cells very gentle to avoid damaging the cells and add needed cell number to the prepared medium.

 

SECTION: Digestion and plating of the hiPSC-CM
25. After 2 days: replace the digestion medium by culture medium

 

SECTION: Digestion and plating of the hiPSC-CM
26. Change medium every two days.

 

SECTION: Electrode preparation
27. Electrode preparation and cleaning of the electrodes is described in the manufacturer’s guidelines to ensure full technical support. We prepared the electrodes as follows:

SECTION: Electrode preparation
27.1. Autoclave electrodes at 120 °C for 20 minat 1 bar.

SECTION: Electrode preparation
27.2. After cleaning, the electrodes can be placed on 6 wells with or without inserted glass bottom dishes.

 

SECTION: Electrode preparation
28. 
 

The single glass bottom dishes fit into the 6 well but still leave enough space for the electrodes. Therefore, direct measurements of single cells without another digestion/plating can be conducted after arrhythmia simulation since the dishes can be directly transferred to the microscope.

SECTION: Setting the electrical stimulus pulse
29. After plating and resting, electrical stimulation of the cells was started:

SECTION: Setting the electrical stimulus pulse
31. Connect each plate via ribbon cable to one channel of the pacer.

 

SECTION: Setting the electrical stimulus pulse
30. Place clean electrodes into the wells in the sterile bench.

SECTION: Setting the electrical stimulus pulse
32. Stimulate every plate individually according to the planned experiments by programming the single channels of the pacer.

SECTION: Setting the electrical stimulus pulse
33. Adjust the stimulation pulse for starting the pacing.

 

SECTION: Setting the electrical stimulus pulse
34. Control the contraction of the cells under the microscope:

SECTION: Setting the electrical stimulus pulse
34.1. Right at the start of the stimulation.

SECTION: Setting the electrical stimulus pulse
34.2. For the first two hours: frequently repeated, at least every30 min .

SECTION: Setting the electrical stimulus pulse
34.3. At the first day: control/readjust the stimulation for around every 60 min  for the following 360 min .

SECTION: Setting the electrical stimulus pulse
34.4. The following days: check cells for vitality (typical shape? detachment?) and visible contraction of the cardiomyocytes minimum two times a day.

SECTION: Setting the electrical stimulus pulse
34.5. Protocol pacing settings and cell contraction (i.e. percentage of beating cardiomyocyte in a prespecified area).

 

SECTION: Medium changes
35. 
 

Disinfect all material that is used thoroughly with ethanol.

SECTION: Medium changes
36. Warm up fresh culture medium at 37 °C.

SECTION: Medium changes
37. Remove the electrodes together with the top of the 6 well plate under the sterile working bench.

SECTION: Medium changes
39. Add 2 mL fresh medium per well and 1.5 mL per dish. 

 

SECTION: Medium changes
38. Remove old medium.

SECTION: Monitoring of apoptosis during culture pacing
40. To evaluate if the cells get stressed or damaged during culture pacing we perform assays for detecting apoptosis. One assay we used is the APC Annexin V Apoptosis Detection Kit (BioLegend). It was used as previously described [5,7]. In short:

SECTION: Monitoring of apoptosis during culture pacing
43. Analyse apoptosis by flow cytometry with dual excitation at 488 nm and 635 nm.

SECTION: Monitoring of apoptosis during culture pacing
42. Expose cells to Annexin V Binding Buffer. 

SECTION: Monitoring of apoptosis during culture pacing
41. Dissociate cells from the culture plates.

SECTION: Monitoring of apoptosis during culture pacing
45. Calculate apoptosis rate as the percentage of events positive for APC and negative for propidium iodide.

 

SECTION: Monitoring of apoptosis during culture pacing
44. Gate events using a positive control with staurosporine (1 mmol/l).