Troubleshooting Guide for STEMdiff™ Cardiomyocyte Kits
Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) offer an attractive model for studying cardiac diseases, drug toxicity, and regeneration. This Tech Tip provides potential solutions to common problems encountered when generating cardiomyocytes from human embryonic stem (ES) and induced pluripotent stem (iPSC) cells using STEMdiff™ Ventricular Cardiomyocyte Differentiation Kit (Catalog #05010) or STEMdiff Atrial Cardiomyocyte Differentiation Kit (Catalog #100-0215). For details of the workflow, refer to the Product Information Sheets found under the respective product pages.
Figure 1. Cardiomyocyte Differentiation Protocol
Two days before the differentiation protocol (Day -2), hPSC colonies are harvested and seeded as single cells at 350,000 - 800,000 cells/well in a 12-well format in TeSR™ medium. After one day (Day -1), the medium is replaced with fresh TeSR™ medium. The following day (Day 0), the TeSR™ medium is replaced with Medium A (STEMdiff™ Cardiomyocyte Differentiation Basal Medium containing Supplement A) to begin inducing the cells toward a cardiomyocyte fate. On Day 2, a full medium change is performed with fresh Medium B (STEMdiff™ Cardiomyocyte Differentiation Basal Medium containing Supplement B). On Days 4 and 6, full medium changes are performed with fresh Medium C (STEMdiff™ Cardiomyocyte Differentiation Basal Medium containing Supplement C). On Day 8, medium is switched to STEMdiff™ Cardiomyocyte Maintenance Medium with full medium changes on Days 10, 12, and 14, to promote further differentiation into cardiomyocyte cells. Small beating areas of cardiomyocytes can be seen as early as Day 8, progressing to a full lawn of beating cardiomyocytes that can be harvested as early as Day 15.
- Do not continue incubation. It is CRITICAL that cells reach > 95% confluency within 48 hours before starting the differentiation protocol. Repeat and seed a range of densities to account for differences in hPSC lines and variations in their rates of proliferation during maintenance culture.
- Do not assume optimal densities for one hPSC line will work across all cell lines.
- Supplement the plating media with 10 μM Y-27632 (Catalog #72302) on Day -2 when hPSCs were plated down as single cells.
- Assess pluripotency by checking the following parameters:
- Observe morphology and markers of undifferentiated state (OCT3/4 and TRA-1-60) to ensure hPSCs are of high quality. If OCT3/4 and TRA-1-60 are low (< 90%), restart with high-quality hPSCs.
- Assess the trilineage differentiation potential of starting hPSCs (e.g. using the STEMdiff™ Trilineage Differentiation Kit Catalog #05230).
- Detect the presence of karyotypic abnormalities (e.g. using the hPSC Genetic Analysis Kit Catalog #07550).
- Start with high-quality hPSCs (with < 10% differentiated areas) from an earlier passage number. Remove areas of differentiation before starting.
- Passage cells when they are no more than 70 - 80% confluent to reduce spontaneous differentiation of cultures.
- Use the Gentle Cell Dissociation Reagent (Catalog #100-0485) and incubate at 37°C and 5% CO2 for 8 - 10 minutes. The use of other reagents, such as Accutase or TrypLE, are not optimal and may require further optimization by the user.
- Ensure cultureware was coated with Corning® Matrigel® hESC-Qualified Matrix (Corning Catalog #354277) at the correct dilution factor. Details can be found in the Technical Manual for ձ™1, mTeSR™ Plus, ձ™-, or ձ™-8™.
- If Matrigel® is on backorder, Geltrex can be used. Other matrices (e.g. Vitronectin) have been tested and found not to perform well. If hPSCs were maintained on Vitronectin beforehand, transition them onto Matrigel® and maintain them for a couple of passages before initiating differentiation.
- Ensure matrigel is added to STEMdiff™ Ventricular or Atrial Cardiomyocyte Differentiation Medium A at 1 in 100 dilution on Day 0 of the differentiation workflow.
- As recommended in hPSC cell quality section.
- Be gentle when removing or adding media. Use a pipettor at all times (e.g. Corning® Lambda™ Plus Pipettor, Catalog #38058); DO NOT aspirate.
- If cells are detaching, refer to the recommendations regarding cell detachment above
- Do not skip media feeds; follow the feeding schedule outlined in the PIS of the STEMdiff™ Ventricular Cardiomyocyte Differentiation Kit or STEMdiff™ Atrial Cardiomyocyte Differentiation Kit depending on which protocol you are following.
- Continue incubation and changing media using STEMdiff™ Cardiomyocyte Maintenance Medium as per the PIS. Larger areas of beating cardiomyocytes should be visible over time, around Day 15.
- Repeat by seeding a range of densities to ensure > 95% confluency is reached within 48 hours. Refer to the recommendations above.
- Check hPSCs quality as recommended above; restart the experiment.
- Dissociate with correct reagents as recommended above.
- Instead of assessing beating by the naked eye, use a microelectrode array (MEA) assay device to measure the beat rate. Refer to the suggested protocol here.
- Perform immunocytochemistry or flow staining for common cardiomyocyte markers, such as cardiac troponin T (cTNT).
- Feed cultures as per PIS. At the end of 48 hours, before media change, nutrients may be depleted and pH may be acidic, causing CMs to slow their beating. Feed cells as normal, put them back in the incubator, and observe again after a couple of hours or on the next day.
- Don’t panic if the beating disappears right after adding fresh media. After a couple of hours in the incubator, cells should beat again.
- Assess for expression of common cardiac markers, such as cTNT.
With the help of the troubleshooting guide, you should be able to get beating cultures as shown below:
Figure 2. Representative Morphology and Beating of hPSC-Derived Ventricular Cardiomyocytes
Ventricular cardiomyocytes were generated from hPSCs using STEMdiff™ Ventricular Cardiomyocyte Differentiation Kit (Catalog #05010). (A) 48 hours after plating as single cells, hPSCs are at > 95% confluency and are ready for Day 0 of differentiation according to the . (B) By Day 15 of differentiation, monolayer cultures show hPSC-derived ventricular cardiomyocytes that exhibit coordinated beating behavior.
Additional Resources
Request Pricing
Thank you for your interest in this product. Please provide us with your contact information and your local representative will contact you with a customized quote. Where appropriate, they can also assist you with a(n):
Estimated delivery time for your area
Product sample or exclusive offer
In-lab demonstration
