StemXVivo Cardiomyocyte Differentiation Kit

Catalog #: SC032 Datasheet / COA / SDS
For differentiation of pluripotent stem cells into cardiomyocytes

Discontinued Product

SC032 has been discontinued.
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StemXVivo Cardiomyocyte Differentiation Kit Summary

Kit Summary

For the directed differentiation of human pluripotent stem cells into cardiomyocytes

Key Benefits

  • Optimized components ensure highly enriched cardiomyocyte populations
  • Reproducible differentiation protocols translate into cost and time savings
  • Maximizes workflow efficiency by standardizing cardiomyocyte differentiation
  • Validated for drug toxicity and small molecule screening

 

Why Use Pre-mixed Differentiation Cocktails when Differentiating Human Pluripotent Stem Cells into Cardiomyocytes?

The StemXVivo® Cardiomyocyte Differentiation Kit uses high-quality specialized media and pre-mixed differentiation cocktails to maximize differentiation efficiency and ensure the consistent and reliable generation of scalable amounts of cardiomyocytes. Using optimized reagents and a straightforward protocol, this kit provides a reproducible method for obtaining high-yields of healthy cardiomyocytes while minimizing the cost and time involved in the differentiation process.

Cardiomyocyte differentiation in vitro:

  • Uses premixed differentiation cocktails to optimally drive reproducible differentiation of pluripotent stem cells into cardiomyocytes.
  • Yields a highly enriched and healthy cardiomyocyte population.
  • Produces cardiomyocytes that express Cardiac Troponin T and contract synchronously.
  • Can be part of small molecule and drug toxicity screening workflows.

 

Precautions

This product contains human transferrin. This transferrin was tested at the donor level using an FDA licensed method and found to be non-reactive for anti-HIV-1/2 and Hepatitis B surface antigen. As no testing can offer complete assurance of freedom from infectious agents, these reagents should be handled as if capable of transmitting infection.

Kit Contents

This kit contains the following reagents to drive pluripotent stem cell differentiation into cardiomyocytes and an antibody to verify differentiation status.

  • Stem Cell Qualified RGF BME, Pathclear®
  • Cardiomyocyte Differentiation Base Media Supplement I
  • Cardiomyocyte Differentiation Base Media Supplement II
  • Cardiomyocyte Differentiation Cocktail I
  • Cardiomyocyte Differentiation Cocktail II
  • Cardiomyocyte Differentiation Cocktail III
  • Anti-Human Cardiac Troponin T Antibody

The quantity of each component in this kit is sufficient to differentiate two 24-well plates, or an equivalent surface area, of pluripotent stem cells into cardiomyocytes.

 

Data Examples
Differentiation of Pluripotent Stem Cells into Ectoderm
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Small Molecules Affect the Contraction Rate of Stem Cell-derived Cardiomyocytes.
A rate interval graph shows fluctuations in Fluo-4 fluorescence intensity, indicative of cardiomyocyte contraction, under basal conditions (brown; untreated) and following sequential treatment with Isoproterenol (Catalog # 1747), a beta-adrenergic agonist, and Propranolol (Catalog # 0624), a beta-adrenergic antagonist. B) This table shows the contraction rate (beats per minute) of differentiated cardiomyocytes during each of the treatments in panel A. One beat equals the amount of time between two consecutive Fluo-4 intensity peaks. The concentrations of Isoproterenol and Propranolol used during the experiment are also shown.

Differentiation of Pluripotent Stem Cells into Cardiomyocytes
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Differentiation of Pluripotent Stem Cells into Cardiomyocytes.
BG01V human embryonic stem cells were differentiated into cardiomyocytes using the media supplements included in this kit. Aside from visually observing contracting cells, commitment to the cardiomyocyte cell fate was evaluated by labeling with the Anti-Human Cardiac Troponin T antibody included in the kit. For immunocytochemistry (left panel), the cells were stained using the NorthernLights 557-conjugated Donkey anti-Mouse secondary antibody (R&D Systems, Catalog # NL007; red), and the nuclei were counterstained with DAPI (blue). For flow cytometry (right panel), the cells were stained using Anti-Mouse PE-conjugated secondary antibody (blue histogram; R&D Systems, Catalog # F0102B) and compared to matched isotype control (brown histogram).

ES-Derived Cardiomyocyte Contractions Visualized  Using the Calcium Indicator, Fluo-4.

ES-Derived Cardiomyocyte Contractions Visualized Using the Calcium Indicator, Fluo-4.
Cardiomyocytes were differentiated from BG01V human embryonic stem cells using the StemXVivo® Cardiomyocyte Differentiation Kit and assessed for their ability to contract using the Fluo-4 calcium binding assay.

BG01V human embryonic stem cells are licensed from Viacyte, Inc

Specifications

Storage
Store the unopened product at -70 °C. Use a manual defrost freezer and avoid repeated freeze-thaw cycles. Do not use past expiration date.
Species
Human

Product Datasheets

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Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, human pluripotent stem cells are differentiated into the cardiomyocyte lineage using the following differentiation procedure:

  • Plate cells on coated plates
  • Replace MEF Conditioned Media with Cardiomyocyte Differentiation Base Media I containing RGF BME
  • Replace the media with Day 1 Differentiation Media
  • Replace the media with Day 5 Differentiation Media
  • Replace the media with Cardiomyocyte Differentiation Base Media II
  • Evaluate differentiation status using the included Troponin T antibody
  • Cells are ready for downstream applications
 

 

Reagents Provided

Reagents supplied in the StemXVivo® Cardiomyocyte Differentiation Kit (Catalog # SC032).

  • Stem Cell Qualified RGF BME, Pathclear®
  • Cardiomyocyte Differentiation Base Media Supplement I (50X)
  • Cardiomyocyte Differentiation Base Media Supplement II (50X)
  • Cardiomyocyte Differentiation Cocktail I
  • Cardiomyocyte Differentiation Cocktail II
  • Cardiomyocyte Differentiation Cocktail III
  • Anti-Human Cardiac Troponin T Antibody

 

Other Supplies Required

Reagents

  • RPMI 1640
  • BSA, very low endotoxin
  • D-MEM/F-12 (1X)
  • GlutaMAX (Invitrogen, Catalog # 35050-079 or equivalent)
  • Penicillin-Streptomycin (optional)
  • Phosphate Buffered Saline (PBS)
  • 95% Ethanol
  • 4% Paraformaldehyde
  • 1% BSA in PBS
  • 0.3% Triton X-100, 1% BSA, 10% normal donkey serum in PBS
  • Mounting medium (R&D Systems, Catalog # CTS011)
  • Secondary developing reagent (R&D Systems, Catalog # NL001)
  • Deionized or distilled water

Materials

  • Human pluripotent stem cells
  • 24-well culture plates (or other, as needed)
  • 60 mm culture plates
  • 12 mm coverslips (optional)
  • 15 mL and 50 mL centrifuge tubes
  • 0.2 μm syringe filter
  • 10 mL syringe
  • Pipettes and pipette tips
  • Serological pipettes
  • Glass slides
  • Fine pointed curved forceps
  • FACS tubes
  • Flow Cytometry Fixation/Permeabilization Buffer I (R&D Systems, Catalog # FC007) supplemented with 0.1% Triton X-100
  • Flow Cytometry Permeabilization/Wash Buffer I (R&D Systems, Catalog # FC005)

Equipment

  • 37 °C and 5% CO2 incubator
  • 37 °C water bath
  • Centrifuge
  • Inverted microscope
  • Fluorescence microscope

Precaution: The acute and chronic effects of overexposure to reagents of this kit are unknown. Safe laboratory procedures should be followed and protective clothing should be worn when handling kit reagents.

 

Procedure Overview

This protocol is designed for BG01V human embryonic stem (hES) cells grown in MEF Conditioned Media (Catalog # AR005) and differentiated in 24-well culture dishes on coverslips. If using different cell lines or growth media, the protocol below may need to be modified. If using different culture vessels, additional optimization may be required to determine appropriate volumes of media.

Coat wells with Stem Cell Qualified PathClear® RGF BME (RGF BME).

Incubate at room temperature for 1-2 hours.

SC031B Step 1&2

Plate human pluripotent stem cells onto the coated plates at 8-9 x 104 cells/cm2 in MEF Conditioned Media containing FGF basic.

Culture cells to 80-90% confluency.

SC031B Step 3

Day (-1) of Differentiation

Replace the stem cell culture media with ice cold stem cell culture media containing RGF BME diluted 1:60.

Incubate at 37 °C and 5% CO2 for 18-24 hours.

SC031B Step 4

Day 0 of Differentiation

Replace the media with ice cold Day 0 Differentiation Media containing RGF BME diluted 1:60.

Incubate at 37 °C and 5% CO2 for 24 hours.

SC031B Step 5&6

Day 1 of Differentiation

Replace the media with Day 1 Differentiation Media

Incubate at 37 °C and 5% CO2 for 4 days without media exchange.

SC031B Step 6

Days 5 of Differentiation

Repeat the media with Day 5 Differentiation Media.

Incubate at 37 °C and 5% CO2 for 2 days without media exchange.

SC031B Step 6

Days 7 of Differentiation and Beyond

Repeat the media with Cardiomyocyte Differentiation Base Media II.

Replace the media with Cardiomyocyte Differentiation Base Media II. Incubate at 37 °C and 5% CO2. Replace media every 1-2 days as needed.

SC031B Step 6

FAQs

  1. How many days does it take to generate cardiomyocytes using the StemXVivo® Cardiomyocyte Differentiation Kit (Catalog # SC032B)?

    • Beating cells can be seen as early as Day 10-13 of differentiation. Beating then becomes more widespread throughout the following week.

  2. Have you tried the StemXVivo® Cardiomyocyte Differentiation Kit with other human ES cell lines or human iPS cell lines?

    • This kit has been tested both in-house and by outside groups in multiple iPS cell lines. This kit is expected to work with most cell lines as long as the starting stem cell population is of high quality and has pluripotency. For optimal performance using this kit, it is recommended that pluripotent  stem cells are grown in Mouse Embryonic Fibroblast (MEF) Conditioned Media (Catalog # AR005), however successful differentiation has been observed in cells grown with mTeSR™ medium.  We have had difficulties with cells grown in TeSR™-E8 media.  An anti-human Cardiac Troponin T antibody is provided in the kit to help determine the efficiency of differentiation.

  3. How many days can cardiomyocytes be maintained using StemXVivo® Cardiomyocyte Maintenance Media Supplement (Catalog # AR011)?

    • Stem cell-derived cardiomyocytes can be maintained using StemXVivo® Cardiomyocyte Maintenance Media Supplement (Catalog # AR011) for longer than 200 days. You will notice that the cells will become less adherent to the plate over time, but will continue to beat and express proper cardiac markers.

  4. When using the StemXVivo® Cardiomyocyte Differentiation Kit, when should the switch be made to the StemXVivo® Cardiomyocyte Maintanence Media Supplement (Catalog # AR011)?

    • The switch to maintenance media can be made any time after beating is initiated or upon the completion of the reagents provided in the kit.

  5. Can human Mesenchymal Stem Cells (MSCs) be used with the StemXVivo® Cardiomyocyte Differentiation Kit?

    • This kit is unlikely to work with human MSCs as these cells are at a different point in development and are expected to need different signals.

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