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Making Better Cardiac Pacemaker Cells

How can differentiation into pacemaker cells be boosted?

While human induced pluripotent stem cells (hiPSCs) have been able to deliver the cardiomyocytes necessary for research, efficient differentiation into pacemaker cells remains low at around 10%. This is a serious hurdle to those working to replace pacemaker devices with biological pacemakers to treat human sinus node (hSAN) dysfunctions. Fortunately, researchers from Germany’s Heidelberg University Hospital have come up with multiple protocols for improved differentiation of hiPSCs into cardiac pacemaker cells.

Building beyond the endoderm

The researchers previously established a protocol for pacemaker differentiation from hiPSCs that relied on co-culture mouse visceral endoderm-like (END2) cells. However, the xenogeneic origin of END2 cells raises serious safety concerns regarding the future of these cardiac pacemaker cells in any human clinical setting.

In this new study, Fabrice Darchep’s team developed multiple novel protocols that do not use END2 cells, instead incorporating the activation and inhibition of Wnt signaling pathways. The team compared the transcription profile and expression of key proteins in cardiac pacemaker cells generated by their novel protocols (labeled B-D), plus a commercially developed cardiomyocyte differentiation protocol (labeled E) to that of cardiac pacemaker cells generated by their original protocol (labeled A) to evaluate their respective differentiation efficiencies.

Targeting Wnt for pacemakers

The team’s previous work co-cultured hiPSCs with END2 cells in a fetal bovine serum (FBS)-enriched culture medium. To sidestep the xenogenic and poor scalability issues of END2 cell co-culture, they had to look elsewhere.

Other research had already shown that using the small molecule SB431542 to inhibit NODAL during Wnt signaling deactivation by IWR-1 led to more hiPSC-derived cardiac pacemaker-like cells being produced. Moreover, the Wnt activator CHIR99021, when applied after Wnt inhibition by IWR-1, pushed hiPSC differentiation toward a more atrial- and epicardial-like fate, as well as high expression levels of T-box transcription factor 18 (Tbx18) (a known inducer of cardiac pacemaker tissue). This is where the team turned their attention.

Testing the protocols

The team tested five protocols, A-E (see the paper for full details of these protocols). They evaluated gene expression of pacemaker-specific and myocardium-related transcription factors, as well as ion channels, transporters, connexins, and adrenergic and cholinergic receptors in cardiac pacemaker cells.

They also used a selection of Alomone Labs antibodies to label key proteins in differentiated cells from protocol E. Here they saw pronounced fluorescence signals of the cardiac pacemaker-specific markers HCN1 (Anti-HCN1 Antibody (#APC-056)), HCN4 (Anti-HCN4 Antibody (#APC-052)), NCX1 (Anti-NCX1 (SLC8A1) Antibody (#ANX-011)), Cav1.2, Cx45, Tbx3, Tbx18 and Shox2 (Figure 1A). Conversely, they only saw weak signals of the ventricular-like markers Nav1.5 (Anti-NaV1.5 (SCN5A) (493-511) Antibody (#ASC-005)), Cx43 (Anti-Connexin-43 Antibody (#ACC-201)) and Nkx2.5 (Figure 1B) and moderate signals of the atrial-like markers HCN2 and Cx40 (Figure 1C).

Cardiomyocytes derived from hiPSCs without END2 co-culture

Figure 1. Immunocytochemical analysis of cardiac pacemaker cells differentiated by protocol E. (A): Pacemaker-specific markers. (B): Ventricular-specific markers. (C): Atrial-specific markers. Nuclei were counterstained with DAPI. Scale bars = 10 μm. Image from Darche FF, et al. Int J Mol Sci. 2022;23(13):7318.

Overall, protocol C, which used the NODAL inhibitor SB431542, was more efficient than protocol B, which used the second application of the Wnt activator, CHIR99021, in combination with retinoic acid (RA). Protocol C used culture media and components that induced activation or inhibition of Wnt signaling, supplemented with the NODAL inhibitor SB431542 to enhance hiPSC differentiation toward cardiac pacemaker cells.

The researchers observed significant differences regarding cardiomyocyte differentiation efficiency among protocols B-E. Protocol E upregulated 12 out of 15 cardiac pacemaker-specific genes, while reference protocol A upregulated 11, and protocols B, C, and D upregulated 9, 10, and 8 cardiac pacemaker-specific genes, respectively

Around half of the hiPSC seeded were spontaneously beating following cardiomyocyte differentiation by protocols B–D, which was similar to results from reference protocol A. In contrast, protocol E resulted in at least 80% spontaneously beating areas.

A future without END2 co-culture

The work here has significant implications for future research into biological pacemakers and the novel protocols developed here for the differentiation of hiPSC into cardiac pacemaker cells is an important step toward addressing concerns around END2 cell co-culture.

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