Differentiation of human embryonic stem cells and induced pluripotent stem cells to cardiomyocytes: a methods overview

Abstract

Since human embryonic stem cells were first differentiated to beating cardiomyocytes a decade ago, interest in their potential applications has increased exponentially. This has been further enhanced over recent years by the discovery of methods to induce pluripotency in somatic cells, including those derived from patients with hereditary cardiac diseases. Human pluripotent stem cells have been among the most challenging cell types to grow stably in culture, but advances in reagent development now mean that most laboratories can expand both embryonic and induced pluripotent stem cells robustly using commercially available products. However, differentiation protocols have lagged behind and in many cases only produce the cell types required with low efficiency. Cardiomyocyte differentiation techniques were also initially inefficient and not readily transferable across cell lines, but there are now a number of more robust protocols available. Here, we review the basic biology underlying the differentiation of pluripotent cells to cardiac lineages and describe current state-of-the-art protocols, as well as ongoing refinements. This should provide a useful entry for laboratories new to this area to start their research. Ultimately, efficient and reliable differentiation methodologies are essential to generate desired cardiac lineages to realize the full promise of human pluripotent stem cells for biomedical research, drug development, and clinical applications.

Figure 1. Model of Differentiation of Human PSC via Sequential Progenitors to Cardiomyocytes

Based on available data, this simplified model shows the cardiomyocyte lineage hierarchy progressing through sequential progenitors identified by key transcription factors as well as known cell-surface markers. Some of the best characterized signaling pathways responsible for the sequential transitions in cell fate are shown.

Figure 2. Current Methods for Cardiac Differentiation of Human PSCs

The three major approaches for differentiation of human PSCs to CMs are summarized: embryoid body (EB), monolayer culture, and inductive co-culture. Methods for forming EBs range from a simple enzymatic partial dissociation of hPSC colonies to various methods to more precisely control EB cell number and EB size using microwells with forced aggregation (centrifugation), microwells to first expand hPSC colonies to a defined size, and micropatterned hPSC colonies of defined sizes. Alternatively, propagating hPSCs as monolayers on Matrigel with defined media can be used for cardiogenesis. For both EBs and monolayer approaches, stage-specific application of key growth factors (GFs) in defined media are required for optimal cardiogenesis, although some protocols use fetal bovine serum (FBS) or small molecules to induce cardiogenesis (see text for details of specific protocols). Co-culture of mechanically passaged hPSCs with visceral endodermal-like END2 cells takes advantage of cell signaling from END2 cells to promote cardiogenesis.

Figure 3. Basic Characterization of Human PSC-derived Cardiomyocytes

A) Flow cytometry provides a quantitative method to evaluate the relative yield and purity of CMs resulting from differentiation protocols by measuring the number of cells expressing cardiac-specific proteins such as cardiac troponin T (cTnT). B) RT-PCR can be used as a first assessment for changes in gene expression typical of cardiogenesis by assaying for the expression of cardiac transcription factors, myofilament proteins, and Ca²⁺-cycling proteins. C) Immunofluorescence microscopy using antibodies specific for myofilament proteins determine if cells exhibit organized sarcomeres typical of CMs. D) Functional assessment of CMs can be provided by cellular electrophysiology measurements to determine if cardiac action potentials typical of different types of CMs are present. E) Extracellular field potential measurements by multielectrode arrays provide another method to detect spontaneous electrical activity in the CMs preparations. F) Detection of Ca²⁺ transients typical of CMs provides another assessment of the functional integrity of the differentiating CMs. Cells loaded with the Ca²⁺ indicator Fluo-3 were imaged using laser scanning confocal microscopy in the line scan mode with Ca²⁺ transients displayed and time versus normalized Ca²⁺ transient intensity (F/F0) shown below. (Panels B and D modified from Zhang et al., 2009)