Application protocol

Differentiation, isolation, and analysis of cardiomyocytes derived from hPSCs

In this application protocol, we describe the differentiation of human pluripotent stem cells (hPSCs) into cardiomyocytes, the isolation of the resulting cardiomyocytes, and their analysis using flow cytometry or immunofluorescence microscopy.

Materials

The following is a listing of reagents, instruments, and consumables needed for each step of this protocol. These products are for research use only.

For hPSCs differentiation into cardiomyocytes

  • StemMACS™ iPS-Brew XF, human (# 130-104-368)
  • TrypLE™ Select Enzyme (1×), no phenol red, liquid (Life Technologies®)
  • Soybean Trypsin Inhibitor, powder (Life Technologies)
  • StemMACS Thiazovivin (# 130-104-461)
  • RPMI 1640 + L-glutamine (Life Technologies)
  • B-27® Supplement (Life Technologies)
  • B-27 Supplement, minus insulin (Life Technologies)
  • StemMACS CHIR99021 (# 130-103-926)
  • StemMACS IWR-1-edo (# 130-110-491)
  • 12-well plates coated with Matrigel® hESC-Qualified Matrix (Corning®)
  • Dulbecco’s phosphate-buffered saline (DPBS) without Ca²+ and Mg²+ (e.g., Lonza, #BE17-512F)

For cell harvest and preparation

  • Multi Tissue Dissociation Kit 3 (# 130-110-204)
  • MACS® SmartStrainers (70 μm) (# 130-098-462)
  • Cell culture medium with 20 % fetal bovine serum (FBS)
  • Phosphate-buffered saline (PBS), pH 7.4

For cardiomyocyte isolation from hPSCs

  • PSC-Derived Cardiomyocyte Isolation Kit, human (# 130-110-188)
  • PBE buffer: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, 0.5 % bovine serum albumin (BSA), and 2 mM EDTA by diluting MACS BSA Stock Solution (# 130‑091-376) 1:20 with autoMACS® Rinsing Solution (# 130‑091-222). Keep buffer cold (2−8 °C). Degas buffer before use, as air bubbles could block the column.
    ▲ Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD). BSA can be replaced by other proteins such as human serum albumin, human serum, or fetal bovine serum (FBS). Buffers or media containing Ca2+ or Mg2+ are not recommended for use.
  • MACS Columns and MACS Separators: For optimal purity and recovery, the use of LS Columns for depletion of non-cardiomyocytes as well as for the subsequent positive selection of PSC-derived cardiomyocytes is strongly recommended. Positive selection and depletion can also be performed  using the autoMACS Pro Separator.
ColumnMax. number of labeled cellsMax. number of total cellsSeparator
Depletion or positive selection
LS5×10⁶5×10⁶MidiMACS™, QuadroMACS™,
VarioMACS, SuperMACS II
autoMACS1×1071×107autoMACS Pro
▲ Note: Column adapters are required to insert certain columns into the VarioMACS™ or SuperMACS™ II Separators. For details refer to the respective MACS Separator data sheet.

For flow cytometry or immunofluorescence microscopy analysis

For analysis by flow cytometry:

  • MACSQuant® Analyzer 10 (# 130-096-343)
  • Anti-Cardiac Troponin T-FITC, human, mouse, rat; clone REA400 (# 130-106-687)
  • Anti-α-Actinin (Sarcomeric)-VioBlue®, human, mouse, rat; clone REA402 (# 130-106-935)
  • Anti-Myosin Heavy Chain-APC, human, mouse, rat; clone REA399 (# 130-106-215)
  • Anti-MLC2a-APC, human, mouse, rat; clone REA398 (# 130-106-143)
  • Anti-MLC2v-PE, human, mouse, rat; clone REA401 (# 130-106-133)
  • Inside Stain Kit (# 130-090-477)
  • autoMACS Running Buffer – MACS Separation Buffer (# 130-091-221)
  • MACS BSA Stock Solution (# 130-091-376)
  • Dulbecco’s phosphate-buffered saline (DPBS)

For analysis by immunofluorescence microscopy:

  • Anti-α-Actinin (Sarcomeric) pure, human, mouse, rat (# 130‑112-755) or Anti-Cardiac Troponin T pure, human, mouse, rat (# 130-112-756)
  • Phosphate-buffered saline (PBS)
  • autoMACS Running Buffer (# 130-091-221)
  • Secondary antibody, e.g., Anti-IgG (H+L)-Vio® 515, human (# 130‑112‑760)
  • Inside Stain Kit (# 130-090-477) for the fixation and permeabilization of cells

Matching products:

Protocol


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This protocol includes detailed instructions for preparing necessary reagents and materials and then carrying out each step.

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.

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Outline of the differentiation protocol. hPSCs are maintained under xeno-free conditions in StemMACS iPS-Brew XF Medium and differentiated into cardiomyocytes using a monolayer protocol with consecutive activation (CHIR99021) and inhibition (IWR-1) of Wnt signaling.

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.

View details

Cardiomyocytes differentiated from hPSCs and analyzed by flow cytometry and immunofluorescence microscopy. Differentiated cells display a high expression level of cardiomyocyte-specific marker troponin T, as shown by flow cytometry (left). The use of recombinantly engineered REAfinity™ Antibodies against cardiac troponin T, α-actinin, or other cardiomyocyte-specific markers like myosin heavy chain, MLC2a, and MLC2v, allows a detailed analysis of cardiomyocytes and respective subtypes. Immunofluorescence microscopy (right) revealed the typical cardiomyocyte morphology and sarcomeric structure.

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

View details

Strategies for the isolation of hPSC-derived cardiomyocytes based on MACS® technology. Depending on the hPSC differentiation efficiency, different strategies for the magnetic enrichment of hPSC-derived cardiomyocytes may be applicable. For cell populations with low differentiation efficiencies, i.e., at cardiomyocyte ratios of <50 %, we recommend combining strategy A (depletion of non-myocytes) with strategy B (positive selection with cardiomyocyte-specific MicroBeads). At higher differentiation efficiencies (>50 %), strategy A is sufficient to achieve high cardiomyocyte purities.

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Cardiomyocytes isolated from hPSCs. Isolation was performed on day 14 of differentiation using the Multi Tissue Dissociation Kit 3, the PSC-Derived
Cardiomyocyte Isolation Kit, and two LS Columns with a MidiMACS™ Separator. The cells were fluorescently stained with Anti-Cardiac Troponin T-FITC and analyzed by flow cytometry using the MACSQuant® Analyzer. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.

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High purities of cTnT-postive cells and high percentages of MLC2a and MLC2v-positive cells. Cardiomyocytes enriched by depletion of non-cardiomyocytes using the PSC-Derived Cardiomyocyte Isolation Kit, human were characterized by flow cytometry and microscopy. (A) Quantification of cTnT+ and MLC2a/v+ cells in the unseparated population (left bar), enriched cardiomyocytes obtained by positive selection of SIRPα+ cells (second from left) and the corresponding SIRPα fraction (second from right), as well as enriched cardiomyocytes obtained by using the kit for depletion of non-cardiomyocytes (right). Cells were stained with antibodies for cTnT, MLC2a, and MLC2v, and quantified by flow cytometry. (B) Immunofluorescence microscopy of cardiomyocytes enriched by depletion of non-cardiomyocytes using the kit displayed high expression levels of cardiomyocyte-specific markers, N-Cadherin and cTnT.

View details

Functional characterization of cardiomyocyte monolayers derived from BJ-iPSCs after purification using the PCS-Derived Cardiomyocyte Isolation Kit, human. Following purification of hiPSC-cardiomyocytes using the kit, cardiomyocytes were re-plate as confluent monolayers and loaded with an action potential dye (FluoVolt™). In addition to spontaneous activations (top), monolayers were also paced at 1 Hz (center) and 1.5 Hz (bottom). The isolated cardiomyocytes possessed appropriate phenotype and were fully functional.

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.

Flow cytometry

  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.

Immunofluorescence microscopy

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.

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