Anton Simeonov Ph.D. National Institute of Health

This optimized cardiac differentiation strategy uses a chemically defined medium consisting of just three components.

Production of large numbers (hundreds of millions to billions) of cells destined for use in regenerative medicine places huge demands on existing protocols and reagents, which remain extremely expensive, difficult to scale up, and prone to mostly unexplained variability (though some of the variability in production outcome has been traced to the reliance on animal/human-derived media components that are extremely difficult to standardize). To this end, Burridge and colleagues researched methods to simplify the culture medium used in production of cardiomyocytes derived from induced pluripotent stem cells (iPSCs). Starting with an evaluation of several combinations of media components and focusing on the RPMI medium with B27 complex without insulin, the team methodically deconvoluted the B27 complex by subtracting each of the 21 components, one at a time, and evaluating the resulting media. The team additionally evaluated the role of all pathways associated with in vivo cardiac differentiation and also assessed the role of matrix on the cell culturing and differentiation. Of the 21 components in B27, only albumin was required, and the simple CDM3 medium consisting of three components (recombinant albumin, ascorbic acid, and the basal medium RPMI 1640) was shown to work just as well in cardiac differentiation as the complete RPMI+ B27− ins medium. The authors demonstrated reproducibility and scalability of the new protocol (first figure) by validating it on 11 human iPSC lines and dozens of different stages of passaging. While the new protocol supported scaled-up production with high efficiency and reproducibility, detailed electrophysiological study revealed that cells differentiated in this manner possessed a largely immature predominantly ventricular phenotype (second figure). Although further evaluation of the protocol/media is needed to understand the level of maturity reached by the so-derived cardiac cells, the provision and initial characterization of this simple medium should spur multiple scale-up applications.

Chemically defined cardiac differentiation of hiPSCs. Schematic of optimized chemically defined cardiac differentiation protocol. EDTA, EDTA for clump cell passaging, using a 1 to 12 ratio; TZV, thiazovivin; VTN, vitronectin.

Electrophysiological characterization of cardiomyocytes. (a) Representative action potential recordings using whole-cell patch of three cardiomyocyte subtypes produced from day 15–20 cells (n=21 cells) and day 30–35 cells (n=13 cells). (b) Proportions of cardiomyocyte subtypes at day 15–20 (n=21 cells) and day 30–35 (n=13 cells). (c) Patch clamp recordings of differentiation day (d) 15–20 and day 30–35 cells, demonstrating maximum diastolic potential, peak voltage, action potential amplitude (APA), action potential duration (APD) at different levels of repolarization (90% and 50%) and dV/dtmax (maximal rate of depolarization). (d) Action potential traces of ventricular-like and atrial-like subtypes of cells differentiated in CDM3 at day 30 of differentiation and action potential morphology used to classify cardiomyocyte subtype, measured using an MEA-based nanopillar. n values are as above. (e) Percentages of ventricular-like and atrial-like cells measured by nanopillar; n=20 cells. (f) Depolarization time, APD, APD at different levels of repolarization (50% and 90%). Error bars, SEM.

*Abstract from Nat Methods 2014, Vol. 11: 855–860

Existing methods for human induced pluripotent stem cell (hiPSC) cardiac differentiation are efficient but require 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 an optimized cardiac differentiation strategy, using a chemically defined medium consisting of just three components: the basal medium RPMI1640, L-ascorbic acid 2-phosphate, and rice-derived recombinant human albumin. Along with small molecule–based induction of differentiation, 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 hiPSClines tested. This chemically defined platform for cardiac specification of hiPSCs will allow the elucidation of cardiomyocyte macromolecular and metabolic requirements and will provide a minimal system for the study of maturation and subtype specification.

Anton Simeonov, Ph.D., works at the NIH.

ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a "Literature Search and Review" column that identifies published papers of note and discusses their importance. GEN presents here one article that was analyzed in the "Literature Search and Review" column, a paper published in Analytical Chemistry titled "Nanodiscs and electrospray ionization mass spectrometry: a tool for screening glycolipids against proteins." Authors of the paper are Leney AC, Fan X, Kitova EN, and Klassen JS.

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