A defined synthetic substrate for serum-free culture of human stem cell derived cardiomyocytes with improved functional maturity identified using combinatorial materials microarrays

Abstract

Cardiomyocytes from human stem cells have applications in regenerative medicine and can provide models for heart disease and toxicity screening. Soluble components of the culture system such as growth factors within serum and insoluble components such as the substrate on which cells adhere to are important variables controlling the biological activity of cells. Using a combinatorial materials approach we develop a synthetic, chemically defined cellular niche for the support of functional cardiomyocytes derived from human embryonic stem cells (hESC-CMs) in a serum-free fully defined culture system. Almost 700 polymers were synthesized and evaluated for their utility as growth substrates. From this group, 20 polymers were identified that supported cardiomyocyte adhesion and spreading. The most promising 3 polymers were scaled up for extended culture of hESC-CMs for 15 days and were characterized using patch clamp electrophysiology and myofibril analysis to find that functional and structural phenotype was maintained on these synthetic substrates without the need for coating with extracellular matrix protein. In addition, we found that hESC-CMs cultured on a co-polymer of isobornyl methacrylate and tert-butylamino-ethyl methacrylate exhibited significantly longer sarcomeres relative to gelatin control. The potential utility of increased structural integrity was demonstrated in an in vitro toxicity assay that found an increase in detection sensitivity of myofibril disruption by the anti-cancer drug doxorubicin at a concentration of 0.05 µM in cardiomyocytes cultured on the co-polymer compared to 0.5 µM on gelatin. The chemical moieties identified in this large-scale screen provide chemically defined conditions for the culture and manipulation of hESC-CMs, as well as a framework for the rational design of superior biomaterials.

Cardiomyocytes from human pluripotent stem cells: From laboratory curiosity to industrial biomedical platform

“…This was concurrent with increases in expression of MLC2v and integrin α7, as well as a modest level of isoform switch from foetal ssTnI to the postnatal cTnI [121]. Patel and co-workers [122] screened almost 700 polymers for their utility as growth substrates for hPSC-CMs. These were refined down to identify chemically-defined methacrylate co-polymers (isobornyl and tert-butylamino-ethyl) on which hPSC-CMs exhibited a 6-fold faster upstroke velocity and significantly longer sarcomeres relative to gelatin controls.…”

“…These imaging approaches have been extended to hPSC-CMs (Table 4), allowing identification of polymers that facilitate maturation [122]. Further progress in using imaging to define hPSC-CM structure was made by Pasqualini and co-workers [160].…”

Intensive care for human hearts in pluripotent stem cell models

“…Two further initiatives with hPSC-CMs for safety pharmacology are the Japan iPS Cardiac Safety Assessment (JiCSA) 46,47 and the CRACK IT InPulse Challenge [48][49][50] . Key lessons from JiCSA, which likewise is focused on arrhythmic risk, have included fidelity of the relationship between MEA-measured field potential duration and interspike interval in hPSC-CMs to the QT-RR relation deduced from clinical electrocardiograms in the Framingham Heart Study 46 .…”

“…The InPulse academic-industry consortium, by contrast, has emphasized developing a robust in vitro platform to monitor cardiac contractility, with cells that are phenotypically mature [48][49][50] . (See the Challenges and Prospects section for a detailed discussion of this concern.)…”

Stem Cell Therapy of Myocardial Infarction: A Promising Opportunity in Bioengineering

“…Patel et al. reported a synthetic substrate composed of a co‐polymer of isobornyl methacrylate and tert‐ butylaminoethyl methacrylate that supported hESC‐derived CM growth for more than 15 days, in addition to showing significantly lengthened sarcomeres compared to the control grown on gelatin . Chun et al.…”