A Universal System for Highly Efficient Cardiac Differentiation of Human Induced Pluripotent Stem Cells That Eliminates Interline Variability

Abstract

BackgroundThe production of cardiomyocytes from human induced pluripotent stem cells (hiPSC) holds great promise for patient-specific cardiotoxicity drug testing, disease modeling, and cardiac regeneration. However, existing protocols for the differentiation of hiPSC to the cardiac lineage are inefficient and highly variable. We describe a highly efficient system for differentiation of human embryonic stem cells (hESC) and hiPSC to the cardiac lineage. This system eliminated the variability in cardiac differentiation capacity of a variety of human pluripotent stem cells (hPSC), including hiPSC generated from CD34+ cord blood using non-viral, non-integrating methods.Methodology/Principal FindingsWe systematically and rigorously optimized >45 experimental variables to develop a universal cardiac differentiation system that produced contracting human embryoid bodies (hEB) with an improved efficiency of 94.7±2.4% in an accelerated nine days from four hESC and seven hiPSC lines tested, including hiPSC derived from neonatal CD34+ cord blood and adult fibroblasts using non-integrating episomal plasmids. This cost-effective differentiation method employed forced aggregation hEB formation in a chemically defined medium, along with staged exposure to physiological (5%) oxygen, and optimized concentrations of mesodermal morphogens BMP4 and FGF2, polyvinyl alcohol, serum, and insulin. The contracting hEB derived using these methods were composed of high percentages (64–89%) of cardiac troponin I+ cells that displayed ultrastructural properties of functional cardiomyocytes and uniform electrophysiological profiles responsive to cardioactive drugs.Conclusion/SignificanceThis efficient and cost-effective universal system for cardiac differentiation of hiPSC allows a potentially unlimited production of functional cardiomyocytes suitable for application to hPSC-based drug development, cardiac disease modeling, and the future generation of clinically-safe nonviral human cardiac cells for regenerative medicine.

Highly efficient delivery of functional cargoes by the synergistic effect of GAG binding motifs and cell-penetrating peptides

“…As there are stepwise phenotypic changes observed during pluripotency induction (28,29) and in directed-differentiation protocols (30), it seems likely that individual transcription factors play distinct, stage-specific roles. The unprecedented potential for temporal control over individual factor function afforded by GET technology should enable these variables to be tested to improve efficiency and kinetics of cell fate control.…”

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

“…However, the success of these approaches will, nevertheless, be determined by progress in improving the maturation status of hPSC‐CMs, that is, the more hPSC‐CMs resemble adult CMs, the less they will tolerate invasive techniques. For this reason, less invasive alternatives are being already being developed in parallel, with the interest in voltage‐sensitive dyes (Burridge et al , 2011) and optogenetics (Park et al , 2014; Chang Liao et al , 2015; Song et al , 2015) significantly rising (Dempsey et al , 2016). At present, their widespread implementation in drug screening is limited by the slow kinetics and the relatively low signal/noise ratio of voltage sensors, with data still requiring proper validation by low‐throughput patch clamp analyses.…”

“…High line‐to‐line variability (although only modest clonal variability in cells from one line) is still a widespread problem that requires joint efforts to solve, although recent, robust, methodologies have contributed to mitigating the issues (Burridge et al , 2011; Hartjes et al , 2014; Denning et al , 2016). The reproducibility of the results in terms of the absolute values of parameters measured may indeed be due to limited standardization of experimental conditions: few studies carry out head‐to‐head comparisons of cells from different hiPSC lines derived under identical conditions.…”

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

“…This model system provides unique opportunities to study the molecular pathways that control cardiac lineage commitment and cell fate specification. In recent years, the ability to efficiently generate cardiomyogenic cells from hPSCs has greatly improved [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] . However, among protocols there is cell line variation with respect to the efficiency in generating cardiomyogenic cells and timing at which the cells express chamber-specific markers (e.g., ventricle and atria).…”

“…In contrast to previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or Bone morphogenetic protein 4(BMP4) and robustly generates cultures highly positive for TNNI3, TNNT2, IRX4, MLC2v, and MLC2a by day 10 cells across all hESC and hiPSC lines tested to date. The strategy is technically simple to implement, especially compared to three-dimensional cultures, mass culture, or embryoid body based strategies [4][5][6][7][8][9] , and was recently defined in a study that describes a small molecule with selective toxicity to hPSCs (Boheler et al) Flow cytometry is a powerful analytical tool for assessing the quality of cells in culture and determining subpopulation homogeneity, and with proper experimental design, can provide quantitative measurements. As with all antibody-based strategies, accurate interpretation of experimental results requires that elements of the assay design including antibody concentration and fixation and permeabilization conditions (when targeting intracellular antigens) are carefully tested for each antibody as sub-optimal conditions significantly affect efficiency of antibody binding, and therefore, interpretation of results.…”