title: Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes Under Defined Conditions.
abstract: Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) can differentiate to cardiomyocytes in vitro, offering unique opportunities to investigate cardiac development and disease as well as providing a platform to perform drug and toxicity tests. Initial cardiac differentiation methods were based on either inductive co-culture or aggregation as embryoid bodies, often in the presence of fetal calf serum. More recently, monolayer differentiation protocols have evolved as feasible alternatives and are often performed in completely defined culture medium and substrates. Thus, our ability to efficiently and reproducibly generate cardiomyocytes from multiple different hESC and hiPSC lines has improved significantly.We have developed a directed differentiation monolayer protocol that can be used to generate cultures comprising ~50% cardiomyocytes, in which both the culture of the undifferentiated human pluripotent stem cells (hPSCs) and the differentiation procedure itself are defined and serum-free. The differentiation method is also effective for hPSCs maintained in other culture systems. In this chapter, we outline the differentiation protocol and describe methods to assess cardiac differentiation efficiency as well as to identify and quantify the yield of cardiomyocytes.
authors.affiliation: Department of Anatomy & Embryology, Leiden University Medical Center, Einthovenweg 20, Leiden, 2333 ZC, The Netherlands.
authors.forename: Cathelijne W
authors.initials: CW
authors.lastname: van den Berg
authors.affiliation: Murdoch Childrens Research Institute, The Royal Children's Hospital, 50 Flemington Road, Parkville, VIC, 3052, Australia.
authors.forename: David A
authors.initials: DA
authors.lastname: Elliott
authors.affiliation: Pluriomics BV, Biopartner Building 3, Galileiweg 8, Leiden, 2333 BD, The Netherlands.
authors.forename: Stefan R
authors.initials: SR
authors.lastname: Braam
authors.affiliation: Department of Anatomy & Embryology, Leiden University Medical Center, Einthovenweg 20, Leiden, 2333 ZC, The Netherlands.
authors.forename: Christine L
authors.initials: CL
authors.lastname: Mummery
authors.affiliation: Department of Anatomy & Embryology, Leiden University Medical Center, Einthovenweg 20, Leiden, 2333 ZC, The Netherlands. r.p.davis@lumc.nl.
authors.forename: Richard P
authors.initials: RP
authors.lastname: Davis
mesh_terms: D028341:Activins / Q000494:pharmacology; D015415:Biomarkers / Q000378:metabolism; D055415:Bone Morphogenetic Protein 4 / Q000494:pharmacology; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D065150:Cellular Reprogramming; D003094:Collagen / Q000737:chemistry; D004338:Drug Combinations; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015870:Gene Expression; D006575:Heterocyclic Compounds, 3-Ring / Q000494:pharmacology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology; D007797:Laminin / Q000737:chemistry; D057054:Molecular Imaging; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D061251:Primary Cell Culture; D011509:Proteoglycans / Q000737:chemistry; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D020107:Troponin T / Q000235:genetics / Q000378:metabolism
chemical_list: C525809:BMP4 protein, human; D015415:Biomarkers; D055415:Bone Morphogenetic Protein 4; C473711:Chir 99021; D004338:Drug Combinations; D004791:Enzyme Inhibitors; D006575:Heterocyclic Compounds, 3-Ring; D036341:Intercellular Signaling Peptides and Proteins; D007797:Laminin; D011509:Proteoglycans; D011725:Pyridines; D011743:Pyrimidines; C545568:TNNT2 protein, human; D020107:Troponin T; C544261:XAV939; C422405:activin A; D028341:Activins; C059485:matrigel; D003094:Collagen
keywords: Cardiac differentiation; Cardiomyocyte characterization; Human embryonic stem cells; Human induced pluripotent stem cells