Efficient Differentiation of Human Pluripotent Stem Cells Into Contracting Cardiomyocytes

J. Owens, Y. Tan, & J. Aho

ABSTRACT

Functionally mature cardiomyocytes produced from a renewable cell source, such as human pluripotent stem cells, are in high demand because of their potential contributions to developmental research, disease modeling, high throughput toxicity drug screening, and clinical therapies. Here we introduce our StemXVivo® Cardiomyocyte Differentiation Kit which will guide human pluripotent stem cells through the necessary cell fate decisions that recapitulate embryonic development to produce functional cardiomyocytes. Our differentiation protocol has been verified on multiple pluripotent cell lines, including human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells. The differentiated cardiomyocytes are validated by immunocytochemistry, flow cytometry, and visualization of calcium fluctuations during cellular contraction. We also show that ES-differentiated cardiomyoctyes can be used as a tool for investigation, including functional assays for small molecule screening, cardiotoxicity, and cardiac hypertrophy.

StemXVivo® Cardiomyocyte Differentiation Kit

  • Three-stage directed differentiation followed by an extended maturation and maintenance phase
  • Contracting cells are typically observed as early as day 11–13; widespread and synchronous contractions are observed by day 24
  • Verified to differentiate multiple human ES and iPS cell lines (in-house and beta-tested in independent academic laboratories)
  • Protocol designed to recapitulate embryonic development of cardiomyocytes
DIFFERENTIATION
Progression of marker expression during kit-differentiated cardiomyocyte development
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Figure 1: Progression of marker expression during kit-differentiated cardiomyocyte development. BG01V human embryonic stem cells were differentiated with the StemXVivo Cardiomyocyte Differentiation Kit (Catalog # SC032) and assessed at select time points for stage-specific marker expression. The pluripotency marker Oct-4 (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, Brachyury (Goat Anti-Human/Mouse Brachyury Polyclonal Antibody; Catalog # AF2085), 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 less prevalent in cells during early (Day 0) and intermediate (Day1) differentiation and become more highly expressed during the later stages of differentiation (Day 7, Day 25–30). Brachyury and NKX2.5 primary antibodies were visualized with the NorthernLights™ (NL)557-conjugated Donkey Anti-Goat IgG Secondary Antibody (Catalog # NL001). Oct-4 and Troponin T were visualized with the NL557-conjugated Donkey Anti-Mouse IgG Secondary Antibody (Catalog # NL007) .
VERIFICATION
Cardiac-specific marker expression in terminally differentiated cardiomyocytes
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Figure 2: Cardiac-specific marker expression in terminally differentiated cardiomyocytes. BG01V human embryonic stem cells were differentiated into cardiomyocytes using the StemXVivo Cardiomyocyte Differentiation Kit (Catalog # SC032). A-B) Cells were harvested from culture, fixed, permeabilized, and incubated with antibodies for (A) Troponin I (Mouse Anti-Human Cardiac Troponin I Monoclonal Antibody; Biospacific; Catalog # A34440), (B) Troponin T (Mouse Anti-Human Cardiac Troponin T Monocolonal Antibody; Catalog # MAB1874), or with isotype control primary antibodies Mouse IgG1 (Catalog # MAB002) and Mouse IgG2A (Catalog # MAB003), respectively. The cells were stained with a PE-conjugated Goat Anti-Mouse IgG secondary antibody (Catalog # F0102B), and expression was quantified by flow cytometry. C) Differentiated cardiomyocytes were fixed and processed for immunocytochemical analysis of atrial-specific (MLC2a) and ventricle-specific (MLC2v) marker expression.
Cardiomyocyte contraction visualized using the calcium indicator, Fluo-4
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Figure 3: Cardiomyocyte contraction visualized using the calcium indicator, Fluo-4. BG01V human embryonic stem cells were differentiated into cardiomyocytes with the StemXVivo Cardiomyocyte Differentiation Kit (Catalog # SC032). Cells were then loaded with the calcium indicator, Fluo-4, which fluoresces upon calcium binding. A) Representative still images of resting and contracting cardiomyocytes, taken from a time-lapse video capturing calcium influx during contraction. B) Rate-interval graph demonstrating cardiomyoctye contraction as measured by recording changes in Fluo-4 fluorescence intensity. The contraction interval was determined as the time between low Fluo-4 fluorescence (relaxed cells) and high Fluo-4 fluorescence (contracted cells).
INVESTIGATION
Small molecules affect the rate of cardiomyocyte contraction
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Figure 4: Small molecules affect the rate of cardiomyocyte contraction. Cardiomyoctyes were differentiated from the BG01V human embryonic stem cells using the StemXVivo Cardiomyocyte Differentiation Kit (Catalog # SC032) and assessed for their ability to contract using the Fluo-4 calcium binding assay. A) Rateinterval graph show fluctuations in Fluo-4 fluorescence intensity, indicative of cardiomyocyte contraction, under basal conditions (brown; untreated) and following sequential treatment with Isoproterenol (blue; Catalog # 1747), a beta-adrenergic agonist, and Propranolol (tan; Catalog # 0624), a beta-adrenergic antagonist. Small molecules affect the rate of cardiomyocyte contraction) Table showing drug concentrations and contraction rate of cardiomyocytes during each treatment. The contraction rate of differentiated cardiomyocytes was determined as the number of contractions (contraction-interval plus relaxation interval) per minute.
Reduced viability of differentiated cardiomyocytes exposed to cardiotoxic small molecules
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Figure 5: Reduced viability of differentiated cardiomyocytes exposed to cardiotoxic small molecules. The viability of differentiated cardiomyocytes was assessed using a Resazurin metabolism assay. A) Untreated cells metabolize Resazurin (Catalog # AR002) to produce resorufin, a fluorescent molecule that can be measured using a fluorometric plate reader. Resorufin fluorescence accumulated in untreated cardiomyocytes. Cells treated with the cardiotoxic small molecules Staurosporine (blue; Catalog # 1285), a non-selective protein kinase inhibitor, or Doxazosin (tan; Catalog # 2964), an alpha-1 antagonist, did not metabolize Resazurin, as lack of fluorescence accumulation, indicating a loss of viability upon treatment. B) Cell morphology of untreated, Staurosporine-treated, and Doxazosin-treated cardiomyocytes was assessed by brightfield microscopy.
Differentiated cardiomyocytes respond physiologically to Endothelin-1 induced hypertrophy
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Figure 6: Differentiated cardiomyocytes respond physiologically to Endothelin-1 induced hypertrophy. Cardiomyoctyes differentiated from BG01V human embryonic stem cells were treated with Endothelin-1 (Et-1; Catalog # 1160) for 28 hours to induce cardiac hypertrophy. Levels of Pro-Atrial Natriuretic peptide (ANP) secretion, which is known to be elevated during cardiac hypertrophy, were assessed using ELISA (Coming Soon from R&D Systems). As expected, detection of Pro-ANP increased proportionally with the concentration of Endothelin-1 treatment.

CONCLUSIONS

  • The StemXVivo® Cardiomyocyte Differentiation Kit efficiently directs human pluripotent stem cells into functional cardiomyocytes
  • Pluripotent stem cells, terminally-differentiated cardiomyocytes, and differentiation intermediates, express stage-appropriate markers throughout the course of the differentiation protocol
  • Differentiated cells exhibit function and morphology characteristic of cardiomyocytes
  • Differentiated cardiomyocytes can be utilized as a tool for high throughput drug screening

For research use only. Not for use in diagnostic procedures.

BG01V human embryonic stem cells are licensed from ViaCyte, Inc.

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