Biotechnologische Prozesse und Verfahren

Efficient and cost effective method to generate cardiomyocytes

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Background

Human pluripotent stem cells – embryonic stem cells or induced pluripotent stem cells – can be differentiated into cells with a cardiomyocyte phenotype. These cardiomyocytes present a novel source of cells for (patient-specific) toxicity testing, drug discovery, disease modelling, as well as regenerative medicine. Several of these applications however require a routine supply of large quantities of cells with a defined phenotype, and protocols have been developed to improve this process.
Current methods can achieve high efficiency, but often require additional enrichment procedures, or expensive media additives/growth factors, or suffer from lack of reproducibility.

Technology

Our scientists have developed a cost-effective, efficient, robust, and scalable protocol to produce cells with a cardiomyocyte phenotype from human pluripotent stem cells. Briefly, the protocol involves the controlled overexpression of a T-box transcription factor (EOMES, eomesodermin) from a transgenic cassette stably integrated into a work horse cell line (embyonic stem cells or induced pluripotent stem cells), followed by small molecule-based inhibition of the WNT-pathway.

Notably, the resulting programmed cardiomyocytes (pCMs) compared very well with conventionally CMs generated by conventional procedures. Hence, they displayed robust staining for cardiac markers at the protein level and showed a pronounced striated sarcomeric pattern in most cells after just 3 weeks. Further, pCMs also showed key physiological responses to chronotropic drugs and ion channel blockers, such as beat rate acceleration in response to isoprenaline, an adrenergic agonist, and slowdown after addition of propranolol which is a beta blocker.

Taken together, this method provides a facilitated means for generating physiological cardiomyocyte-like cells from human pluripotent stem cells in a controlled and cost-effective manner.

EOMES programs human ESCs (hESCs) into functional CMs at high efficiency

  1. Illustration of growth factor-mediated and optimized EOMES induction-based cardiac differentiation protocols. Bottom right: Immunoblot validating doxycycline-dependent EOMES expression in a transgenic EOMES KO/E.TET-ON hESC line.
  2. Typical yields of hESC-CMs (left, flow cytometry) and NKX2.5 (early cardiomyocyte marker) expression (right, immunoblot) obtained with the two protocols (day 10).
  3. Immunostainings 21 days after the initiation of EOMES induction. Weak perinuclear ANP staining is typical in overall MLC2v-positive hESC-CMs. Scale bars: 25 (top) and 50 μm (bottom).
  4. Acceleration and slowdown of spontaneous beat rates in pCMs following exposure to 10 μM isoprenaline and 10 μM propranolol, respectively, on multielectrode arrays.

We are now looking for either a licensing partner, or a collaboration partner to further develop this project.

Publication

DOI: 10.1038/s41467-017-02812-6

Patent Information


The PCT application was filed August 2018.

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Efficient and cost effective method to generate cardiomyocytes

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