Contractile Defect Caused by Mutation in MYBPC3 Revealed under Conditions Optimized for Human PSC-Cardiomyocyte Function.

Maximizing baseline function of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is essential for their effective application in models of cardiac toxicity and disease. Here, we aimed to identify factors that would promote an adequate level of function to permit robust single-cell contractility measurements in a human induced pluripotent stem cell (hiPSC) model of hypertrophic cardiomyopathy (HCM). A simple screen revealed the collaborative effects of thyroid hormone, IGF-1 and the glucocorticoid analog dexamethasone on the electrophysiology, bioenergetics, and contractile force generation of hPSC-CMs. In this optimized condition, hiPSC-CMs with mutations in MYBPC3, a gene encoding myosin-binding protein C, which, when mutated, causes HCM, showed significantly lower contractile force generation than controls. This was recapitulated by direct knockdown of MYBPC3 in control hPSC-CMs, supporting a mechanism of haploinsufficiency. Modeling this disease in vitro using human cells is an important step toward identifying therapeutic interventions for HCM.

Expansion and patterning of cardiovascular progenitors derived from human pluripotent stem cells.

The inability of multipotent cardiovascular progenitor cells (CPCs) to undergo multiple divisions in culture has precluded stable expansion of precursors of cardiomyocytes and vascular cells. This contrasts with neural progenitors, which can be expanded robustly and are a renewable source of their derivatives. Here we use human pluripotent stem cells bearing a cardiac lineage reporter to show that regulated MYC expression enables robust expansion of CPCs with insulin-like growth factor-1 (IGF-1) and a hedgehog pathway agonist. The CPCs can be patterned with morphogens, recreating features of heart field assignment, and controllably differentiated to relatively pure populations of pacemaker-like or ventricular-like cardiomyocytes. The cells are clonogenic and can be expanded for >40 population doublings while retaining the ability to differentiate into cardiomyocytes and vascular cells. Access to CPCs will allow precise recreation of elements of heart development in vitro and facilitate investigation of the molecular basis of cardiac fate determination. This technology is applicable for cardiac disease modeling, toxicology studies and tissue engineering.

Cardiomyocytes from human and mouse embryonic stem cells.

Human and mouse embryonic stem (ES) cells have the potential to differentiate to cardiomyocytes in culture. They are therefore of interest for studying early human and mouse heart development, as well as properties of cardiomyocytes from both species, including their responses to cardiac drugs, and, at some point in the future, may represent a source of transplantable cells for cardiac muscle repair. The differentiation protocols that are effective depend in part on the species from which the ES cell lines were derived, and in part on the individual cell lines and the methods used for their propagation prior to differentiation. Here, several methods for generating and characterizing cardiomyocytes from mouse and human ES cells are described, as well as methods for dissociation of cardiomyocytes into single-cell suspensions which are useful both for characterizing cells by antibody staining and electrophysiological measurements, as well as preparing cells for transplantation into (animal) hearts.

Differentiation of human embryonic stem cells to cardiomyocytes by coculture with endoderm in serum-free medium.

Many of the applications envisaged for human embryonic stem cells (hESC) undergoing cardiomyogenesis require that the differentiation procedure is robust and high yield. For many hESC lines currently available this is a challenge; beating areas are often obtained but subsequent analysis shows only few (<1%) cardiomyocytes actually present. Here the authors provide a protocol based on serum-free coculture with a mouse endoderm-like cell line (END2), which yields cultures containing on average 25% cardiomyocytes for two widely available hESC lines, hES2 and hES3. The authors also provide a variant on the protocol based on growth of hESC aggregates/embryoid bodies in END2-conditioned medium and a method for dissociating beating aggregates without compromising cardiomyocyte viability so that they can be used for transplantation into animals or further (electrophysiological) analysis.

Human embryonic stem cells: genetic manipulation on the way to cardiac cell therapies.

Almost 7 years after their first derivation from human embryos, a pressing urgency to deliver the promises of therapies based on human embryonic stem cells (hESC) has arisen. Protocols have been developed to support long-term growth of undifferentiated cells and partially direct differentiation to specific cell lineages. The stage has almost been set for the next step: transplantation in animal models of human disease. Here, we review the state-of-the-art with respect to the transplantation of embryonic stem cell-derived heart cells in animals. One problem affecting progress in this area and functional analysis in vivo in general, is the availability of genetically marked hESC. There are only a few cell lines that express reporter genes ubiquitously, and none is associated with particular lineages; a major hurdle has been the resistance of hESC to established infection and chemical transfection methodologies to introduce ectopic genes. The methods that have been successful are reviewed. We also describe the processes for generating a new, genetically-modified hESC line that constitutively expresses GFP as well as some of its characteristics, including its ability to form cardiomyocytes with electrophysiological properties of ventricular-like cells.