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.
Table 1: Overview of the procedures covered in this application protocol.
|PSC differentiation into cardiomyocytes||StemMACS CardioDiff Kit XF (# 130-125-289)||1||8 days|
|Long-term culturing||StemMACS Cardiac Cultivation Medium XF (# 130-125-287)||2.1||User-dependent, up to more than 30 days|
|Cell harvest||Multi Tissue Dissociation Kit 3 (# 130-110-204)||2.2||30 minutes|
|Cell seeding||StemMACS Cardiac Cultivation Medium XF (# 130-125-287)||2.3||15 minutes|
|Cell freezing and thawing||StemMACS Cryo-Brew (# 130-109-558)||2.4||30 minutes|
|(Optional) Isolation of cardiomyocytes||PSC-Derived Cardiomyocyte Isolation Kit, human (# 130-110-188)||3||2 hours|
|Flow cytometry or microscopy analysis of cardiomyocytes||REAfinity™ Antibodies panel||4||1 hour|
Table 2: Timeline of the differentiation protocol.
|Day –1||Coat 12-well plates|
|Day 0||Prepare media|
Seed cells in Mesoderm Induction Medium
|Day 1||Wash and replace medium with Cardiac Cultivation Medium|
|Day 2||Wash and replace medium with Cardiac Induction Medium|
|Day 3||Wash and replace medium with Cardiac Cultivation Medium|
|Day 4 – Day 8||Replace medium with Cardiac Cultivation Medium daily|
With this 8-days protocol, cardiomyocytes can be generated from hPSCs with high efficiency. Depending on the cellular line, first contracting cardiomyocytes can be observed already after 6 days of culture.
After directed differentiation using StemMACS CardioDiff Kit XF (# 130-125-289), PSC-derived cardiomyocytes can be maintained in culture in StemMACS Cardiac Cultivation Medium XF for more than 30 days.
▲Note: To achieve an efficient differentiation, it is important to start with high quality PSCs. Monitor regularly the pluripotency status of the stem cell cultures by observing their morphology and staining for pluripotency markers. The PSCs culture should show a confluency of 75–85% before starting.
▲Note: Perform all steps under sterile conditions.
▲Note: The starting cell number is strongly line-dependent. Therefore, when using a stem cell clone for the first time it is recommended to perform a titration experiment in order to determine the best starting PSC concentration for differentiation. Suggested cell numbers are 125,000 cells/cm², 250,000 cells/cm², 300,000 cells/cm², and 400,000 cells/cm².
Table 3: Example of plating scheme for titration experiment.
|1.25 × 105 cells/cm2||1.25 × 105 cells/cm2||2.5 × 105 cells/cm2||2.5 × 105 cells/cm2|
|3.0 × 105 cells/cm2||3.0 × 105 cells/cm2||4.0 × 105 cells/cm2||4.0 × 105 cells/cm2|
|Negative control||Negative control|
1. Prepare complete media as indicated in table 4.
Table 4: Preparation of complete media.
|Complete media||Component 1||Component 2||Total volume|
|Mesoderm Induction Medium||StemMACS CardioDiff Basal Medium XF||+||StemMACS CardioDiff Mesoderm Induction Supplement XF||100 mL|
|95 mL||5 mL|
|Cardiac Cultivation Medium||StemMACS CardioDiff Basal Medium XF||+||StemMACS CardioDiff Cardiac Cultivation Supplement XF||800 mL|
|784 mL||16 mL|
|Cardiac Induction Medium||StemMACS CardioDiff Basal Medium XF||+||StemMACS CardioDiff Cardiac Induction Supplement XF||100 mL|
|95 mL||5 mL|
▲Note: Volumes given below are for hPSCs originally cultivated in a 6-well plate and transferred to a 12-well plate for induction. If using other culture ware adjust the volumes accordingly.
▲Note: (Optional) Antibiotic solutions might be added to prevent contamination, e.g. 100 U/mL penicillin and 100 μg/ mL streptomycin.
2. Aspirate cell culture medium and wash each well of the 6-well plate with 2 mL of D-PBS without Ca²+ and Mg²+.
3. Add 1 mL/well TrypLE™ Select Enzyme. Gently rock the plate to ensure even distribution of the solution.
4. Incubate for 5 minutes in the dark at 37 °C.
5. Stop enzymatic reaction by adding 1 mL/well of Soybean Trypsin Inhibitor (0.5 mg/mL).
6. Using a 5 mL serological pipette, dissociate to a single-cell suspension by carefully pipetting up and down.
7. Determine the cell number and viability. Viability should be >95%.
8. Transfer the desired cell number into a new tube. Centrifuge at 300×g for 5 minutes. Aspirate supernatant completely.
9. Resuspend the cells in a sufficient amount of Mesoderm Induction Medium. Per well, 2 mL of Mesoderm Induction Medium are needed.
10. Transfer cells into a freshly-coated 12-well plate with 2 mL/well. Place the plate into the incubator (37 °C, 5% CO2).
▲Note: Depending on the PSC line, first contractions can be observed already on day 6 of differentiation.
After directed differentiation using StemMACS CardioDiff Kit XF, PSC-derived cardiomyocytes can be maintained in culture in StemMACS Cardiac Cultivation Medium XF (# 130-125-287) for more than 30 days. – Media change needs to be performed every second day.
Prepare complete media
After day 10 cells can be harvested for:
- Re-plating for long-term culturing.
▲Note: Cells should be transferred into a freshly coated culture ware just if necessary, e.g. when cells show signs of detachment from the plate.
- Cell isolation
- Flow cytometry
Cell harvest with Multi Tissue Dissociation Kit 3
To achieve best performance, dissociate the monolayer cultures into a single-cell suspension using the Multi Tissue Dissociation Kit 3 (# 130-110-204). The Multi Tissue Dissociation Kit 3 has been developed for the gentle, rapid, and effective generation of single-cell suspensions and results in high yield of viable cells.
▲Note: Volumes below are given for 12-well plates.
Cell harvest with TrypLE™ Select Enzyme
After differentiation with StemMACS Cardio Diff Kit it is possible to further enrich the cardiomyocyte culture by isolating cardiomyocytes with the PSC-Derived Cardiomyocyte Isolation Kit, human (# 130-110-188). Moreover, prolonged culturing can increase the purity of the cardiomyocyte population.
It is recommended to perform the isolation step in case:
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
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.
Besides analysis by flow cytometry, cardiomyocytes are often examined by microscopy using immunofluorescence staining. Recombinantly engineered REAfinity™ Antibodies enable unambiguous analysis of cardiomyocytes.
The following is a list of relevant resources that might be of interest:
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Cell freezing and thawing
|Column||Max. number of labeled cells||Max. number of total cells||Separator|
|Depletion or positive selection|
▲ Note: Column adapters are required to insert certain columns into the SuperMACS™ II Separators. For details refer to the respective MACS Separator data sheet.
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