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.
Human embryonic stem cells Human induced pluripotent stem cells Cardiac differentiation Cardiomyocyte characterization
This is a preview of subscription content, log in to check access.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
We thank Dorien Ward-van Oostwaard for providing cells. Financial support was from the Netherlands Institute of Regenerative Medicine (NIRM, FES0908; C.W.v.d.B., S.R.B., C.L.M.), the Netherlands Proteomics Consortium (050-040-250; R.P.D., C.L.M.), the Australian National Health and Medical Research Council (NHMRC; D.A.E.), and the European Research Council (StemCardioVasc; C.L.M.).
Davis RP, van den Berg CW, Casini S et al (2011) Pluripotent stem cell models of cardiac disease and their implication for drug discovery and development. Trends Mol Med 17(9):475–484CrossRefPubMedGoogle Scholar
Moretti A, Laugwitz KL, Dorn T et al (2013) Pluripotent stem cell models of human heart disease. Cold Spring Harb Perspect Med 3(11):1–11CrossRefGoogle Scholar
Sterneckert JL, Reinhardt P, Scholer HR (2014) Investigating human disease using stem cell models. Nat Rev Genet 15(9):625–639CrossRefPubMedGoogle Scholar
Davis RP, Casini S, van den Berg CW et al (2012) Cardiomyocytes derived from pluripotent stem cells recapitulate electrophysiological characteristics of an overlap syndrome of cardiac sodium channel disease. Circulation 125(25):3079–3091CrossRefPubMedGoogle Scholar
Bellin M, Casini S, Davis RP et al (2013) Isogenic human pluripotent stem cell pairs reveal the role of a KCNH2 mutation in long-QT syndrome. EMBO J 32(24):3161–3175PubMedCentralCrossRefPubMedGoogle Scholar
Mummery C, Ward-van Oostwaard D, Doevendans P et al (2003) Differentiation of human embryonic stem cells to cardiomyocytes: role of coculture with visceral endoderm-like cells. Circulation 107(21):2733–2740CrossRefPubMedGoogle Scholar
Mummery CL, Zhang J, Ng ES et al (2012) Differentiation of human embryonic stem cells and induced pluripotent stem cells to cardiomyocytes: a methods overview. Circ Res 111(3):344–358PubMedCentralCrossRefPubMedGoogle Scholar
Elliott DA, Braam SR, Koutsis K et al (2011) NKX2-5(eGFP/w) hESCs for isolation of human cardiac progenitors and cardiomyocytes. Nat Methods 8(12):1037–1040CrossRefPubMedGoogle Scholar
Ng ES, Davis R, Stanley EG et al (2008) A protocol describing the use of a recombinant protein-based, animal product-free medium (APEL) for human embryonic stem cell differentiation as spin embryoid bodies. Nat Protoc 3(5):768–776CrossRefPubMedGoogle Scholar
Ungrin MD, Joshi C, Nica A et al (2008) Reproducible, ultra high-throughput formation of multicellular organization from single cell suspension-derived human embryonic stem cell aggregates. PLoS One 3(2):e1565PubMedCentralCrossRefPubMedGoogle Scholar
Bauwens CL, Song H, Thavandiran N et al (2011) Geometric control of cardiomyogenic induction in human pluripotent stem cells. Tissue Eng Part A 17(15–16):1901–1909CrossRefPubMedGoogle Scholar
Lian X, Hsiao C, Wilson G et al (2012) Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling. Proc Natl Acad Sci U S A 109(27):E1848–E1857PubMedCentralCrossRefPubMedGoogle Scholar
Paige SL, Osugi T, Afanasiev OK et al (2010) Endogenous Wnt/beta-catenin signaling is required for cardiac differentiation in human embryonic stem cells. PLoS One 5(6):e11134PubMedCentralCrossRefPubMedGoogle Scholar
Dambrot C, Buermans HP, Varga E et al (2014) Strategies for rapidly mapping proviral integration sites and assessing cardiogenic potential of nascent human induced pluripotent stem cell clones. Exp Cell Res 327(2):297–306CrossRefPubMedGoogle Scholar
Den Hartogh SC, Schreurs C, Monshouwer-Kloots JJ et al (2015) Dual reporter MESP1(mCherry/w)-NKX2-5(eGFP/w) hESCs enable studying early human cardiac differentiation. Stem Cells 33(1):56–67Google Scholar
Costa M, Sourris K, Hatzistavrou T et al (2008) Expansion of human embryonic stem cells in vitro. Curr Protoc Stem Cell Biol 5(C):1C.1.1–1C.1.7Google Scholar
Skelton RJ, Costa M, Anderson DJ et al (2014) SIRPA, VCAM1 and CD34 identify discrete lineages during early human cardiovascular development. Stem Cell Res 13(1):172–179CrossRefPubMedGoogle Scholar
Watanabe K, Ueno M, Kamiya D et al (2007) A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol 25(6):681–686CrossRefPubMedGoogle Scholar
Braam SR, Nauw R, Ward-van Oostwaard D et al (2010) Inhibition of ROCK improves survival of human embryonic stem cell-derived cardiomyocytes after dissociation. Ann N Y Acad Sci 1188:52–57CrossRefPubMedGoogle Scholar
Kattman SJ, Witty AD, Gagliardi M et al (2011) Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell Stem Cell 8(2):228–240CrossRefPubMedGoogle Scholar
Anderson D, Self T, Mellor IR et al (2007) Transgenic enrichment of cardiomyocytes from human embryonic stem cells. Mol Ther 15(11):2027–2036CrossRefPubMedGoogle Scholar
Huber I, Itzhaki I, Caspi O et al (2007) Identification and selection of cardiomyocytes during human embryonic stem cell differentiation. FASEB J 21(10):2551–2563CrossRefPubMedGoogle Scholar
Bu L, Jiang X, Martin-Puig S et al (2009) Human ISL1 heart progenitors generate diverse multipotent cardiovascular cell lineages. Nature 460(7251):113–117CrossRefPubMedGoogle Scholar