StemXVivo Cardiomyocyte Differentiation Kit

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SC032B
Product Details
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StemXVivo Cardiomyocyte Differentiation Kit Summary

Kit Summary

Reagents for the directed differentiation of human pluripotent stem cells into the cardiomyocyte lineage.

Key Benefits

  • Optimized reagents generate more robust and uniform beating of differentiated cardiomyocytes.
  • Provides consistent differentiation across pluripotent stem cell lines
  • Reproducible differentiation protocols translate into cost and time savings
  • Maximizes workflow efficiency by standardizing cardiomyocyte differentiation
  • Applicable 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. This kit has been tested on iPSC cell lines derived from both blood and fibroblasts.

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

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.

 

 

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 IIA
  • Cardiomyocyte Differentiation Cocktail IIB
  • 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.

Stability and Storage

Store unopened kit at < -70 °C in a manual defrost freezer. Do not use past kit expiration date.

Limitations

  • FOR LABORATORY RESEARCH USE ONLY. NOT FOR DIAGNOSTIC USE.
  • Do not mix or substitute reagents with those from other lots or sources.
  • The safety and efficacy of this product in diagnostic or other clinical uses has not been established.
  • The quality and differentiation potential of human pluripotent stem cells at the onset of the differentiation protocol are of paramount importance to the efficiency of differentiation.
 

 

Data Examples

Synchronized Contractile Beating of iPSC-Derived Cardiomyocytes.
Cardiomyocytes were differentiated from JOY1 human induced pluripotent stem cells using the StemXVivo® Cardiomyocyte Differentiation Kit. Waves of beating cardiomyocytes were visualized using brightfield microscopy.

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

iPSC-Derived Cardiomyocyte Contractions Visualized Using the Calcium Indicator, Fluo-4.
Cardiomyocytes were differentiated from JOY1 human induced pluripotent stem cells using the reagents and protocol provided in this kit. They were assessed for their ability to contract using the Fluo-4 calcium binding assay.

Differentiation of Pluripotent Stem Cells into Cardiomyocytes.
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Differentiation of Pluripotent Stem Cells into Cardiomyocytes.
JOY6 (left panel) and iBJ6 (right panel) human induced pluripotent 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 this kit. For visualization, the cells were stained using NorthernLights 557-conjugated Donkey anti-Mouse Secondary Antibody (R&D Systems, Catalog # NL007; red), and the nuclei were counterstained with DAPI (blue).

Improved Efficiency of iPSC Differentiation Across Pluripotent Stem Cell Lines.
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Improved Efficiency of iPSC Differentiation Across Pluripotent Stem Cell Lines.
Multiple established human iPS cell lines and one embryonic stem cell (ESC) line were differentiated into beating cardiomyocytes using either the new and enhanced version of the StemXVivo® Cardiomyocyte Differentiation Kit (New Kit) or the previous version of the kit (SC032). After 21 days of differentiation beating quality was scored based on percentage of cells beating and beating intensity. Differentiation with the new kit results in more robust and uniform differentiation based on beat quality. The new kit also increases the likelihood of differentiation success across ES and iPS cell lines.

Endothelin-1 Induces Expression of Atrial Natriuretic Peptide in Cardiomyoctyes Derived from iPSCs Using the New Kit.
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Endothelin-1 Induces Expression of Atrial Natriuretic Peptide in Cardiomyoctyes Derived from iPSCs Using the New Kit.
JOY6 human iPSCs were differentiated into beating cardiomyocytes using the new and enhanced version of the StemXVivo® Cardiomyocyte Differentiation Kit. After 21 days of differentiation cardiomyocytes were treated with 5 nM of Endothelin-1 (Catalog # 1160) for 25.5 hours to induce cardiac hypertrophy. The level of secreted Pro-Atrial Natriuretic peptide (ANP), which is known to be elevated during cardiac hypertrophy, was assessed using (A) Western Blot and (B) ELISA (Human NTProANP Quantikine® ELISA; Catalog # DANP00). As expected, detection of Pro-ANP was increased in Endothelin-1 treated cells.

Kit-Derived Cardiomyocytes Express Atrial and Ventricular Markers.
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Kit-Derived Cardiomyocytes Express Atrial and Ventricular Markers.
JOY6 human iPSCs were differentiated into cardiomyocytes using the new and enhanced version of the StemXVivo® Cardiomyocyte Differentiation Kit. The iPSC-derived cardiomyocytes were fixed and processed for immunocytochemical analysis of atrialspecific (MLC2a) and ventricle-specific (MYH7; Rabbit Anti-Human MYH7 Monoclonal Antibody; Catalog # MAB90961) marker expression.

Kit-Derived Cardiomyocytes Express Stage-specific Markers During Differentiation.
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Kit-Derived Cardiomyocytes Express Stage-specific Markers During Differentiation.
JOY6 human iPSCs were differentiated with the new StemXVivo® Cardiomyocyte Differentiation Kit and assessed at select time points for stage-specific marker expression. The pluripotency marker Oct-4A (Mouse Anti-Human Oct-4A Monoclonal Antibody; Catalog # MAB17591) is highly expressed during early differentiation (Day 0) and is subsequently downregulated. Expression of the mesoderm marker, Snail (Goat-Anti-Human Snail Polyclonal Antibody; Catalog # AF3639), is expressed intermediately during differentiation (Day 1). The cardiomyocyte markers NKX2.5 (Goat Anti-Human NKX2.5 Polyclonal Antibody; Catalog # AF2444) and Troponin T (Mouse Anti-Human Cardiac Troponin T Monocolonal Antibody; Catalog # MAB1874) are not present in cells during early (Day 0) and intermediate (Day1) differentiation and become more highly expressed during the later stages of differentiation (Day 7, Day 30). Snail and NKX2.5 primary antibodies were visualized with the NorthernLights (NL)557-conjugated Donkey Anti-Goat IgG Secondary Antibody (Catalog # NL001). Oct-4A and Troponin T were visualized with the NL557-conjugated Donkey Anti-Mouse IgG Secondary Antibody (Catalog # NL007).

Embryonic stem (ES) cells are pluripotent stem cells derived from the inner cell mass of pre-implantation embryos. Induced pluripotent stem (iPS) cells can be generated by somatic cell reprogramming following the exogenous expression of specific transcription factors (Oct-3/4, KLF4, SOX2, and c-Myc). These cell types are capable of unlimited, undifferentiated proliferation in vitro and still maintain the capacity to differentiate into a wide variety of somatic cells. In this capacity, pluripotent stem cells have widespread clinical potential for the treatments of heart disease, diabetes, spinal cord injury, and a variety of neurodegenerative disorders.

R&D Systems offers a wide range of products to support pluripotent stem cell culture and differentiation. Mouse embryonic fibroblasts may be used to maintain and expand pluripotent stem cells in an undifferentiated state. We also offer defined culture media, which are specifically optimized for use with human or rodent pluripotent stem cells. In addition, R&D Systems offers a variety of products to assess differentiation status and identify specific stem cell types of interest, including panels of marker antibodies, primer pairs, multi-color flow cytometry kits, and specialized verification kits.

Specifications

Shipping Conditions
The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
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

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
  • Cardiomyocyte Differentiation Base Media Supplement II
  • Cardiomyocyte Differentiation Cocktail I
  • Cardiomyocyte Differentiation Cocktail IIA
  • Cardiomyocyte Differentiation Cocktail IIB
  • 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 has been tested on human pluripotent stem cells cultured in either MEF Conditioned Media (R&D Systems, Catalog # AR005) or equivalent. The quality and differentiation potential of human pluripotent stem cells at the onset of the differentiation protocol are of paramount importance to the efficiency of differentiation. Human pluripotent stem cells must be > 95% positive for OCT-3/4.

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

Incubate at room temperature for 1-2 hours.

SC032B Step 1

Plate human pluripotent stem cells onto the coated plates at 3-4 x 104 cells/cm2 in Pluripotent Stem Cell Maintenance Media.

Culture cells to 80-90% confluency.

SC032B Step 2

Day (-1) of Differentiation

Replace the stem cell culture media with ice cold Pluripotent Stem Cell Maintenance Media containing RGF BME diluted 1:60.

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

SC032B Step 3

Day 0 of Differentiation

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

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

SC032B Step 4

Day 1 of Differentiation

Replace the media with Day 1 Cardiomyocyte Differentiation Media

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

SC032B Step 5

Day 5 of Differentiation

Replace the media with Day 5 Cardiomyocyte Differentiation Media.

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

SC032B Step 6

Day 7 of Differentiation and Beyond

Replace the media with Cardiomyocyte Differentiation Base Media I.

Incubate at 37 °C and 5% CO2. Replace media every 1-2 days as needed.

SC032B Step 7

Day 12 of Differentiation and Beyond

Replace the media with Cardiomyocyte Differentiation Base Media II.

Incubate at 37 °C and 5% CO2. Replace media every 1-2 days as needed.

SC032B Step 8

FAQs

No product specific FAQs exist for this product, however you may

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Cell Culture Media and Supplements

Mycoplasma Detection

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