Chemically defined generation of human cardiomyocytes

Abstract

Existing methodologies for human induced pluripotent stem cell (hiPSC) cardiac differentiation are efficient but require the use of complex, undefined medium constituents that hinder further elucidation of the molecular mechanisms of cardiomyogenesis. Using hiPSCs derived under chemically defined conditions on synthetic matrices, we systematically developed a highly optimized cardiac differentiation strategy, employing a chemically defined medium consisting of just three components: the basal medium RPMI 1640, L-ascorbic acid 2-phosphate, and rice-derived recombinant human albumin. Along with small molecule-based differentiation induction, this protocol produced contractile sheets of up to 95% TNNT2+ cardiomyocytes at a yield of up to 100 cardiomyocytes for every input pluripotent cell, and was effective in 11 hiPSC lines tested. This is the first fully chemically defined platform for cardiac specification of hiPSCs, and allows the elucidation of cardiomyocyte macromolecular and metabolic requirements whilst providing a minimally complex system for the study of maturation and subtype specification.

Accurate nanoelectrode recording of human pluripotent stem cell-derived cardiomyocytes for assaying drugs and modeling disease

“…On the other hand, the ventricular-like cells have much longer APD50 and APD90. These results agree well with our previous study of the same cell type in CDM3 medium 18 and the literature using patch clamp electrophysiology 24 .…”

“…The method of intracellular recording after electroporation is applicable for hESC-CMs (Figure 1e), hiPSC-CMs (Figure 1f), and mouse HL-1 cells (Figure 1g). For the hESC-CMs, we achieved a maximum intracellular action potential amplitude of 25.15 mV (Figure 1h), which is only approximately three times attenuated from the full amplitude recorded by patch clamp 18 and larger than any previous chip-based nanoelectrode recording of mouse HL-1 cardiomyocytes 15,16 . With a peak-to-peak noise level of 30 μV pp , we achieved a signal-to-noise ratio of 838 over a 5 kHz bandwidth.…”

“…Human embryonic stem cells and induced pluripotent stem cells The details of the human pluripotent stem cell generation and cardiac differentiation were presented in our previous work 18 . The hESC line H7 was supplied by WiCell Research Institute 19 .…”

Integrating cardiomyocytes from human pluripotent stem cells in safety pharmacology: has the time come?

“…Protocols are available to direct cell differentiation towards many somatic cell lineages, including CMs (Mummery et al , 2012; Schwach and Passier, 2016). Chemically defined media supplemented with developmentally relevant growth factors [or small molecules that activate similar pathways, (Burridge et al , 2014; Chen et al , 2014; van den Berg et al , 2016)] are now commercially available or sold as complete kits so that deriving CMs from pluripotent stem cells (as hESC‐CM or hiPSC‐CM) is now feasible for most laboratories. Because legal and ethical issues restrict the use of hESC in some countries, hiPSC are preferred by most regulatory authorities seeking to establish reliable cardiotoxicity assays.…”

“…Typical fetal features that they display include automaticity of beating, depolarized diastolic V m , low ion channel expression, delayed excitation‐contraction coupling and low contractile force (Veerman et al , 2015). Defined culture conditions (Burridge et al , 2014; Ribeiro et al , 2015a, 2015b) can be used to improve the maturation of hPSC‐CMs to reveal hidden disease phenotypes (Birket et al , 2015) or to drive differentiation to chamber‐specific cell populations (Devalla et al , 2015), and drug testing might benefit from their standardization. Exogenous stimuli, such as adjusted pacing frequency (Chan et al , 2013), 3D‐microenvironments (Zhao et al , 1999; Nunes et al , 2013; Hirt et al , 2014; Eder et al , 2016) and heterotypic cell co‐culture (Robertson et al , 2013) may all contribute to cell maturation, increasing the expression of ion channels and improving functional properties.…”