Cardiomyocyte Stem Cells

Cardiomyocyte Differentiation

Stem cell-derived cardiomyocytes have multiple potential uses in disease modeling and therapy. Signaling pathways important in the control of cardiomyocyte differentiation from pluripotent cells, include BMP, Wnt and TGF-β.

Two distinct methods have been developed for the differentiation of human pluripotent stem cells (hPSCs) into cardiomyocytes: the formation of embryoid bodies (EBs), and the culturing of hPSCs as a monolayer. The EB methodology involves suspending hPSC colonies in media to form spherical aggregates and produces yields of over 70% cardiomyocytes but is complex and time consuming. The monolayer-based method for cardiac differentiation of hPSCs produces a higher yield (>85%) and is easier to use.

The role of Wnt/β-catenin signaling in cardiac induction of stem cells has been studied in both the EB and monolayer techniques. Use of CHIR 99021 (Cat. No. 4423), a small molecule glycogen synthase kinase 3β (GSK-3β) inhibitor, to activate the Wnt/β-catenin pathway, is sufficient to drive hPSCs to differentiate into cardiomyocytes under fully defined, growth factor-free conditions in vitro. In addition, treatment of human PSCs with CHIR 99021 followed by the PORCN inhibitors IWP 2 (Cat. No. 3533) or IWP 4 (Cat. No. 5214) to inhibit Wnt signaling, results in the generation of spontaneously contracting cardiomyocytes that exhibit normal sarcomere organization and a predominantly ventricular-like action potential. These findings suggest that canonical Wnt signaling likely acts as a master regulator of cardiomyocyte specification and that the precise temporal modulation of signaling is important in the determination of cardiac fate.

Cardiomyocytes can be derived from hPSC lines in monolayer culture under cytokine- and serum-free conditions. BIO (Cat. No. 3194) is a GSK-3β inhibitor that promotes cardiomyocyte differentiation from hPSCs. Activation of Wnt signaling using a combination of CHIR 99021 plus BIO during days 0-3 of cardiac differentiation, followed by Wnt signaling inhibition using KY 02111 (Cat. No. 4731) and the tankyrase inhibitor XAV 939 (Cat. No. 3748) from day 3 of differentiation onwards, resulted in the emergence of beating colonies by around day 8-10. This protocol was highly efficient, with up to 98% of resulting cells staining positive for the cardiac marker cardiac troponin T (cTnT), and having well organized sarcomeres and electrophysiological characteristics consistent with cardiomyocytes.

The efficiency of specific cardiomyocyte differentiation protocols shows considerable variability between cell lines. Screening for small molecules that promote cardiac differentiation of stem cells, identified the mTOR (mammalian target of rapamycin) inhibitor Rapamycin (Cat. No. 1292) as a promoter of cardiomyocyte differentiation. When Rapamycin + CHIR 99021 are used in place of BIO + CHIR 99021 in the early stages of cardiomyocyte differentiation, efficiency of cardiomyocytes generation is maintained across different human hESC and hiPSC lines. The addition of Rapamycin inhibits the apoptosis of hESCs in high-density monolayer culture and promotes mesoderm formation suggesting that mTOR is an important regulator of cardiogenesis.

Other compounds that enhance cardiomyocyte differentiation include T3 (triiodothyronine; Cat. No. 6666), which promotes maturation of hiPSC-derived cardiomyocytes, increasing cardiomyocyte size and sarcomere length and improving contractile kinetics. Poly(I:C) (Cat. No. 4287) accelerates cardiomyocyte maturation when used to prime early cardiac progenitors. Epigenetic and transcriptional profiling of primed cardiac progenitor cells reveals increased histone acetylation and activation of epigenetic marks at promoters of cardiac myofilament genes.

Studies have demonstrated how cardiac cells differentiated from iPSCs might be used in regenerative medicine. In one such study, omental (peritoneal) tissue biopsies from patients were separated into cellular and acellular materials. A personalized hydrogel was produced from the extracellular matrix, while the cells were first reprogrammed to iPSCs, then differentiated into endothelial cells and cardiomyocytes. The two different cell types were separately combined with the hydrogel to form two "bioinks", which were used to generate patient-matched, thick, vascularized and perfusable cardiac patches by 3D printing. This approach has potential for engineering personalized tissues for transplantation, eliminating the need for immunosuppression. The technique was also used to generate heart-like structures, with the anticipation that it could eventually lead to the production of human hearts for transplantation.

Cardiomyocyte Transdifferentiation

Cells can be reprogrammed directly from one specialized cell type to another, without first being converted to iPSCs, a process known as transdifferentiation or direct lineage reprogramming. This technique offers an alternative approach for generating cells for cell therapy and research purposes. Transdifferentiation has the advantage that the starting material, i.e. mature somatic cells such as fibroblasts, is readily available. In addition, since the process does not involve the cells entering an induced pluripotent state, the possibility of tumorigenesis is reduced. The production of lineage-specific cells via transdifferentiation therefore has enormous potential in medicine to replace lost or damaged cells, for example following myocardial infarction or cartilage injury, or in neurodegenerative diseases.

Transdifferentiation can be achieved through introduction of exogenous transcription factors via retroviral transduction, but as with reprogramming, this method of converting cells is slow and inefficient. Other methods include activation or silencing of endogenous genes using techniques such as CRISPR/Cas9 or via pharmacological manipulation of the epigenetic environment and signaling pathways using combinations of small molecules. Transdifferentiation has been used to convert fibroblasts into a wide range of different cell types including NSCs (Neural Stem Cells), functional neurons, cardiomyocytes, endothelial cells, hepatocytes, skeletal muscle cells, and pancreatic β cells.

A method to convert human fibroblasts into cardiomyocytes uses a cocktail of nine small molecules (9C). The 9C-treated cells were subsequently cultured in cardiac induction medium and transplanted into mice, where they were converted into cardiomyocyte-like cells. The 9C cocktail comprises: CHIR 99021, Pluripotin, OAC-2, Y-27632 (Cat. No. 1254), BIX 01294 (Cat. No. 3364), A 83-01 (Cat. No. 2939), SU 16f (Cat. No. 3304) and JNJ 10198409 (Cat. No. 6976). This technique is highly efficient, with around a 97% conversion rate from fibroblasts to spontaneously beating chemically induced cardiomyocytes in around 20 days. When transplanted into mice with infarcted hearts, 9C-treated fibroblasts were efficiently converted into cardiomyocyte-like cells (Figure 1).

Figure 1: Transdifferentiation of fibroblasts into cardiomyocytes.

Adapted from Cao et al. (2016) Conversion of human fibroblasts into functional cardiomyocytes by small molecules. Science 352, 1216. PMID: 27127239

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Literature for Cardiomyocyte Stem Cells

Tocris offers the following scientific literature for Cardiomyocyte Stem Cells to showcase our products. We invite you to request* your copy today!

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Stem Cell Research Product Guide

This product guide provides a background to the use of small molecules in stem cell research and lists over 200 products for use in:

• Self-renewal and Maintenance
• Differentiation
• Reprogramming
• Organoid Generation
• GMP and Ancillary Material Grade Products

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Stem Cells Scientific Review

Written by Kirsty E. Clarke, Victoria B. Christie, Andy Whiting and Stefan A. Przyborski, this review provides an overview of the use of small molecules in the control of stem cell growth and differentiation. Key signaling pathways are highlighted, and the regulation of ES cell self-renewal and somatic cell reprogramming is discussed. Compounds available from Tocris are listed.

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Stem Cell Workflow Poster

Stem cells have potential as a source of cells and tissues for research and treatment of disease. This poster summarizes some key protocols demonstrating the use of small molecules across the stem cell workflow, from reprogramming, through self-renewal, storage and differentiation to verification. Advantages of using small molecules are also highlighted.

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Stem Cells Poster

Written by Rebecca Quelch and Stefan Przyborski from Durham University (UK), this poster describes the isolation of pluripotent stem cells, their maintenance in culture, differentiation, and the generation and potential uses of organoids.

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