With this protocol, cardiomyocytes can be generated from hPSCs with efficacies of up to 80%, giving rise to contracting cells in 9–12 days.

▲ Note: Testing of various cell numbers and CHIR99021 concentrations is recommended to optimize differentiation for each individual hPSC line. The diagram below provides a graphical overview of the procedure.

Day –3: Harvest cells

1. Use hPSCs at 90 % confluence. Remove culture medium and wash cells once with DPBS w/o Ca²⁺ and Mg²⁺.
2. Add 1 mL TrypLE™ Select Enzyme to each well containing hPSCs (6-well plate).
3. Incubate for 5 minutes at 37 °C.
4. Stop enzymatic reaction by adding 1 mL/well of Soybean Trypsin Inhibitor.
5. Dissociate cell layer to obtain a single-cell suspension by pipetting up and down using a 5 mL serological pipette.
   

Day –3: Seed cells

1. Determine cell number using the MACSQuant® Analyzer 10.
2. Centrifuge cell suspension at 125×g for 5 minutes to collect cells. Aspirate supernatant completely.
3. Adjust cell densities (1.5–7×10⁵ cells/mL) in StemMACS™ iPS-Brew XF + 2 μM StemMACS Thiazovivin.
4. Seed cells into a Matrigel® Matrix–coated 12-well plate with a final volume of 1 mL per well.

▲Note: The effectiveness of differentiation depends on the number of seeded cells. Therefore, it is crucial to determine the cell number that leads to optimal differentiation. Different cell numbers might be required for different hPSC lines.

Day –2/–1: Medium exchange

Replace medium in each well with 1 mL StemMACS iPS-Brew XF.

Day 0: Adjustment of CHIR99021 concentration

1. Prepare medium RPMI 1640 + L-glutamine + B-27® Supplement, minus insulin with different CHIR99021 concentrations (8–12 μM).
2. Replace medium in each well with 1 mL of the prepared CHIR99021-containing medium.
   ▲Note: Cells should be confluent at this point.
3. Incubate with CHIR99021 for exactly 24 hours.

Day 1: Medium exchange

Replace medium in each well with 2 mL of RPMI 1640 + L-glutamine + B-27 Supplement, minus insulin.

Day 3: Addition of IWR-1

1. Remove 1 mL/well of the old cell culture medium and transfer it into a tube.
2. Add 1 mL/well fresh RPMI 1640 + L-glutamine + B-27 Supplement, minus insulin into the tube.
3. Add 5 μM IWR-1 into the tube.
4. Replace medium in each cell culture well with 2 mL of the conditioned medium created in steps 1–3.

Day 5: Medium exchange

Replace medium in each well with 2 mL of RPMI 1640 + L-glutamine + B-27 Supplement, minus insulin.

Day 7: Medium exchange

Replace medium in each well with 2 mL of RPMI 1640 + L-glutamine + B-27 Supplement w/insulin, and continue to do so every 2–3 days.

The development of highly efficient cardiac-directed differentiation methods makes it possible to generate large numbers of cardiomyocytes.^1,4 However, due to varying differentiation efficiencies, cardiomyocyte populations must be further enriched for downstream applications.

To achieve highest possible purity and recovery during cell separation, first dissociate the monolayer cultures into a single-cell suspension using the Multi Tissue Dissociation Kit 3.

▲ Note:  Perform all steps under sterile conditions.
▲ Note:  The dissociation protocol is optimized for use with 12-well or 6-well plates.

1. Remove cell culture supernatant from the cultured cells.
2. Wash each well containing cells 3 times with the appropriate amount of PBS:
   12-well plate: 1 mL 
   6-well plate: 2 mL
3. Prepare enzyme mix by adding Enzyme T to Buffer X of the Multi Tissue Dissociation Kit 3 in a ratio of  1:10,  for  example,  add 50 μL of Enzyme T to 450 μL of Buffer X.
4. Add to each well containing cells the appropriate amount of enzyme mix:
   12-well plate: 400 μL 
   6-well plate: 1 mL
5. Incubate cells for 10 minutes at 37 °C.
6. Add to each well containing cells the appropriate amount of cell culture medium with 20 % FBS:
   12-well plate: 600 μL
   6-well plate: 1 mL
7. Gently detach cells from the dish by pipetting 3 times up and down using a 1 mL pipette.
   ▲Note: For late stage differentiation it may be necessary to pipette more often.
8. Apply the cells to a MACS® SmartStrainer (70 μm) placed on a 50 mL tube.
9. Wash each well with the appropriate amount of cell culture medium with 20 % FBS and also apply to the MACS SmartStrainer (70 μm):
   12-well plate: 1 mL
   6-well plate: 2 mL
10. Wash MACS SmartStrainer (70 μm) with the appropriate amount of cell culture medium with 20 % FBS:
    12-well plate: 1 mL
    6-well plate: 2 mL
11. Determine the cell number and proceed to "Isolation of cardiomyoctes from hPSCs."
    

Separate the cardiomyocytes using the PSC-Derived Cardiomyocyte Isolation Kit, human. Follow the protocol of the kit data sheet.

The isolation is performed either as a single-step or two-step protocol, depending on the initial differentiation efficiency (see isolation strategy below). As a general guideline, we recommend performing the second (positive selection) step if the culture contains <50 % of cardiomyocytes.

The standard protocol enables enrichment of cardiomyocytes from up to 5×10⁶ total cells, and upscaling is possible for up to 1×10⁷ total cells.

Download kit data sheet

PSC-Derived Cardiomyocyte Isolation Kit, human

In recent years, various protocols to differentiate PSCs into cardiomyocytes have been published. However, the resulting populations are not homogeneous, but rather composed of a variety of cardiomyocyte subtypes or subpopulations, as well as non-cardiomyocyte cell types.

Different cardiomyocyte subpopulations can be identified and analyzed using techniques such as patch clamp, immunocytochemistry or flow cytometry. Compared to other techniques, flow cytometry is a fast and efficient method to analyze and quantify different cell types within a cell population, without difficult techniques or genetic engineering of the starting material.

1. Harvest cultured purified cardiomyocytes. See "Harvest and preparation of cells."
2. Count isolated cardiomyocytes by flow cytometry using the MACSQuant® Analyzer 10.
3. Transfer 5×10⁵ cells per staining into a 1.5 mL microtube and centrifuged at 300×g for 5 minutes.
4. Remove the supernatant and resuspend the pellet in InsideFix (a component of the Inside Stain Kit) 1:1 diluted in DPBS.
5. Incubate cells for 10 minutes at room temperature.
6. Wash the cells with 1 mL autoMACS® Running Buffer – MACS® Separation Buffer and centrifuge at 300×g for 5 minutes.
7. Remove the supernatant.
8. Resuspend cells in 80 μL Inside Perm (a component of the Inside Stain Kit). Add 10 μL each of the following antibodies:
   Anti-Cardiac Troponin T-FITC and Anti-α-Actinin (Sarcomeric)-VioBlue®
   Anti-Cardiac Troponin T-FITC and Anti-Myosin Heavy Chain-APC
   Anti-Cardiac Troponin T-TnT-FITC and Anti-MLC2a-APC
   Anti-Cardiac Troponin T-TnT-FITC and Anti-MLC2v-PE
   Anti-MLC2a-APC and Anti-MLC2v-PE
9. Incubate cells for 10 minutes at room temperature and in the dark.
10. Wash cells with 1 mL Inside Perm and centrifuge at 300×g for 5 minutes.
11. Remove the supernatant and resuspend the cell pellet in 250 μL autoMACS Running Buffer – MACS Separation Buffer.
12. Analyze the stained cells on the MACSQuant Analyzer 10.
    

Besides analysis by flow cytometry, cardiomyocytes are often examined by microscopy using immunofluorescence staining. Recombinantly engineered REAfinity™ Antibodies enable unambiguous analysis of cardiomyocytes.

1. Wash cells cultures twice with PBS.
2. Dilute Inside Fix (from the Inside Stain Kit) 1:1 with PBS.
3. Fix cells with Inside Fix for 10 minutes in the dark at room temperature.
4. Wash cells twice with PBS.
   ▲Note: Fixed cells can be stored in azide-containing buffer at 2–8 °C for up to 1 week.
5. Add the Anti-α-Actinin (Sarcomeric) pure or Anti-Cardiac Troponin T pure antibody 1:100 to Inside Perm, e.g., 1 μL antibodies to 99 μL Inside Perm.
6.  Add the antibody solution from step 5 to cells and incubate for 10 minutes in the dark at room temperature.
7. Wash cells twice with PBS.
8. Add an appropriate secondary antibody, e.g., Anti-IgG (H+L)-Vio® 515, 1:100 to Inside Perm. Add the solution to the cells.
   ▲Note: If using a cell nucleus marker, e.g., DAPI, add it at this point as well.
9. Incubate for 10 minutes in the dark at room temperature.
10. Wash cells twice with PBS.
11. Cells are now ready for immunofluorescence microscopy.
    ▲Note: Samples can be stored at 2–8 °C in the dark for up to one week.

1. Lian, X. et al. (2012) Proc. Natl. Acad. Sci. U.S.A. 109: E1848–E1857.
2. Lian, X. et al. (2013) Nat. Protoc. 8: 162–175.
3. Burridge, P.W. et al. (2014) Nat. Methods 11: 855–860.
4. Zhang, J. et al. (2012) Circ. Res. 111: 1125–1136.

The following is a list of relevant resources that might be of interest:

• Cardiovascular research solutions – Conveniently groundbreaking
• PSC-derived Cardiomyocyte Isolation Kit, human – Gentle and fast purification of hPSC-derived cardiomyocytes
• REAfinity™ Recombinant Antibodies – Flow cytometry is in their genes
• REAfinity Recombinant Antibodies – Frequently Asked Questions
• Application: Efficient generation of cardiomyocytes from human pluripotent stem cells
• Application: Cardiomyocyte purification from pluripotent stem cells
• Application: Characterization by flow cytometry of PSC-derived cardiomyocyte subtypes
• Immunofluorescence staining with Anti-α-Actinin (Sarcomeric) or Anti-Cardiac Troponin T antibodies for microscopy (includes protocols for cultivated cells, acetone-fixed and formalin-fixed tissue sections).
  
• Noack, K., et al. (2016) Highly efficient immunomagnetic purification of cardiomyocytes derived from human pluripotent stem cells. ISSCR. San Francisco, USA.

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