14 Chapter 1 1.3 Lessons learned from cardiogenesis The important inventions of the microscope, sterile culturing, and defined mediums led to the creation of the first human immortal cell line in 1951.23 These so-famously called HeLa cells were created by a tissue sample taken from a young woman with cervical cancer and quickly became invaluable to medical research. However, until today, due to the limited regenerative capacity of the heart, no successful passaging of adult human primary CMs nor cardiac stem cell isolation has been possible. This made the culturing of adult human CM models a challenge. For many years, signaling pathways that specify cardiac mesoderm and regulate cardiac proliferation have been extensively studied. Hereafter, wingless-related integration site (Wnt) signaling has proven to be essential in heart development 24 and the balance of Wnt regulation appears to play a critical role in cardiogenesis and, later on, shaping the cardiac fields.25,26 In adult organisms, Wnt proteins regulate diverse cellular processes such as gene transcription and cell proliferation, migration, polarity, or division.24 Since the discovery of embryonic stem cells by James Thomson and later, in 2006, the hiPSCs by Shinya Yamanaka have been heralded as major breakthroughs in stem cell research.27,28 The knowledge derived from the developmental studies has been translated into reproducible methods for the efficient generation of cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs). The first attempts to differentiate hiPSCs into CMs about a decade ago resulted in very low numbers of hiPSC-CMs (5-10%), and the re-plating of these cells was a challenge.29 Hereafter, the combination of monolayer cell culture with defined serum-free media, supplemented with growth factors involved in normal human embryological heart development, like Wnt agonists and antagonists, resulted in much higher efficiency (80– 99%).30,31 Altogether, the improvements over the last 70 years have tremendously improved the generation of stem cell-derived cardiomyocytes as a human cell source for in vitro disease modeling. 1.4 Cardiomyocyte generation and expansion The discovery of hiPSCs offers unprecedented opportunities to study early human physiology and pathology at a cellular level.32 Multiple embryonic pathways have been implicated in cardiac differentiation of pluripotent stem cells.33 Understanding the particular role of the Wnt signaling pathway in heart formation has helped to develop pluripotent stem cell differentiation protocols that produce relatively pure cardiomyocyte populations34 Currently, various hiPSC- CM differentiation protocols incorporate Wnt signaling activation via glycogen synthase kinase 3 beta (GSK-3β) inhibition (usually with a small molecule) from days 0 to 3. This is followed by Wnt inhibition via porcupine inhibition at days 3-5 to induce highly pure and dense hiPSC-CM cultures (Figure 1).35
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