Atrial fibrillation is the most commonly observed arrhythmia in clinics, afflicting six million patients in Europe and accounting for 30% of strokes. The widespread presence of atrial fibrillation is continually growing as a result of an aging population, and it is anticipated to double during the next fifty years.
In accordance with the requirement for developing safer and more powerful anti-arrhythmic therapeutics, significant attention has been paid to understanding the cellular mechanisms of the disease and translating this understanding into cutting-edge treatments.
Conventional approaches to drug discovery in the atrial fibrillation sector are premised on preclinical animal and non-cardiac cell models. Unfortunately, these are often insufficient for providing a reliable prediction of treatment efficacy and safety in patients, as shown by the absence of new drugs licensed in the recent past.
Although human atrial cardiomyocyte primary cells represent a more encouraging option, the viability and yield of live cells from human hearts is low, and research can only be undertaken on a restricted amount of material. An additional limiting factor for the utility of primary cells are the problems associated with maintaining the cells in culture for long periods. The whitepaper will examine the advantages of utilizing iPSC-derived atrial cardiomyocytes to overcome some of these problems.
iPSC-derived Atrial Cardiomyocytes: A Better Model For Atrial Fibrillation Research
Human induced pluripotent stem cell (iPSC)-derived atrial cardiomyocytes represent a perfect platform for modeling atrial fibrillation for two important reasons. Firstly, they are human atrial cells, and are thus more relevant than animal models and alternative human cell types.
Secondly, they offer a continuous and abundant source of cells which with to work (overcoming the restrictions imposed by utilizing primary cells). In spite of their advantages, the production of human iPSC cells brings its own challenges. To permit their utilization as a physiologically relevant tool for supporting translational atrial fibrillation research, manufacturing processes must be able to deliver a regular and scalable supply.
To overcome this problem, Axol’s team has developed a line of iPSC-derived atrial cardiomyocytes that can be utilized for supporting drug discovery in this sector. The cells were derived from iPSCs using footprint-free episomal reprogramming methodologies, via the integration of specific differentiation factors at critical time points in order to drive atrial fate.
Immunocytochemical analysis of the consequent atrial cardiomyocytes demonstrates strong expression of cardiac and atrial-specific proteins. Figure 1 indicates that the cells are richly labeled by antibodies directed against Troponin T and myosin light chain (MLC), showing that they are cardiac myocytes expressing contractile proteins linked to muscle sarcomeres. Their atrial differentiation is demonstrated by enrichment for atrial MLC (over ventricular MLC), and strong labeling for atrial natriuretic peptide (ANP), which is specifically secreted by atrial myocytes upon cell stretching.
Figure 1: Visualization of cardiac and atrial-specific protein markers in hiPSC-atrial cardiomyocytes by immunocytochemistry.
Genotypic Characterization of iPSC-derived Atrial Cardiomyocytes
For atrial cardiomyocytes to be effective as translationally predictive and reliable models in disease research, reproducibility and consistency are critical. The atrial cardiomyocyte genotype of Axol’s Human iPSC-derived Atrial Cardiomyocytes was characterized utilizing gene chip microarray analysis of cardiac and atrial-specific markers (Figure 2).
Figure 2: Gene chip microarray expression for A) genes that signify pluripotency compared to iPS cells B) cardiac genes (red), atrial-specific genes (dark blue), cardiac ion channel genes (light blue), and ventricular-specific genes (yellow), compared to signals detected in iPSC-derived ventricular cardiomyocytes.
Important iPSC markers KLF4, MYC, NANOG, POU5F1 and SOX2, were each down-regulated in human iPSC-derived atrial cardiomyocytes, indicating that these cardiomyocytes are no longer pluripotent and are approaching terminal differentiation (Figure 2A). The data proves that important cardiac genes and contractile proteins (both displayed in red) are up-regulated in the iPSC-derived atrial cardiomyocytes, in comparison to iPSC-derived ventricular cardiomyocytes (Figure 2B).
A number of atrial-specific genes (displayed in dark blue) are also up-regulated in the iPSC-derived atrial cardiomyocytes, in comparison to iPSC-derived ventricular cardiomyocytes from the same donor. Significantly, the expression of atrial-specific ion channels KCNA5, KCNJ3 and KCNJ5 is also selectively augmented in the iPSC-derived atrial cells in comparison to iPSC-derived ventricular cardiomyocytes, indicating that they embody the right biophysics and pharmacology for atrial fibrillation drug discovery and disease modeling. Comparatively, ventricular-specific genes (displayed in yellow) are down-regulated in the iPSC-derived atrial cardiomyocytes, consistent with the production of differentiated iPSC-derived atrial cardiomyocytes.
iPSC-derived Atrial Cardiomyocytes: Biophysical And Pharmacological Characterization
Ion channels and electrical signaling are critical features of healthy atrial cardiomyocyte function and atrial fibrillation disease processes. To establish consistency, Axol’s researchers also performed biophysical and pharmacological validation of the iPSC-derived atrial cardiomyocytes, utilizing manual patch clamp electrophysiology recordings.
The biophysical properties of the iPSC-derived atrial cardiomyocytes were established utilizing current clamp recordings of action potential (AP) parameters (Figure 3). At room temperature, the cells spontaneously beat and fired APs with a frequency of 0.3 Hz, enabled by a negative resting potential of –73 mV and a powerful upstroke velocity, suggestive of good Nav1.5 channel availability, and providing strongly overshooting AP amplitudes.
Figure 3: Biophysical characteristics of spontaneous iPSC-derived atrial cardiac APs. Data generated in collaboration with Metrion Biosciences.
The functional expression of relevant cardiac ion channels was confirmed utilizing selective pharmacological ligands. The presence of the core panel of Cav1.2, Nav1.5 and hERG channels, which are proven to be expressed in the iPSC-derived atrial cardiomyocytes by gene microarray research (highlighted in Figure 2), was confirmed by the effects of the selective inhibitors E-4031, lidocaine and nifedipine (Figure 4).
These reagents generated the predicted alterations in AP upstroke and amplitude, reinforcing the functionality of these ion channels. Moreover, following application to spontaneously beating iPSC-derived atrial cardiomyocytes, moderate concentrations of the hERG blocker dofetilide induced arrhythmia, an observation that accords with native human cardiomyocytes.
Figure 4: Core cardiac ion channel pharmacology of iPSC-derived atrial cardiomyocytes. Data generated in collaboration with Metrion Biosciences.
The atrial phenotype of the iPSC-derived cardiomyocytes was given additional confirmation via observation of the characteristic effects of modulators of atrial-specific ionic currents, which are proven to be up-regulated in the gene microarray data. For instance, activation of the acetylcholine-activated inward-rectifying potassium current mimicked the negative chronotropic effect of vagal tone to decelerate spontaneous activity, whilst inhibition of the ultra-rapid delayed rectifier potassium current prolonged APD20. In this way, the functional ion channel characteristics of the human iPSC-derived atrial cardiomyocytes are strongly reflective of those observed in native human tissue, thus confirming the expression of ion channel targets of relevance to atrial fibrillation drug discovery.
Approaching a More Robust Model of Atrial Fibrillation
Batch-to-batch consistency is critical in cell reagents and assay applications being utilized for drug discovery screening and disease modeling. The biophysical and pharmacological profiles of Axol’s human iPSC-derived atrial cardiomyocytes were shown to be particularly consistent between pilot batches and commercial scale-up materials (to understand more, refer to the new white paper). Consequently, they are likely to represent an extremely reliable source of cells for utilization in both.
In light of the significant potential of human iPSC-derived cells as translationally useful tools for enhancing our knowledge of atrial fibrillation, such first-rate characterization results prove that Axol’s iPSC-derived atrial cardiomyocytes represent an especially reproducible and consistent platform that can underpin a broad array of applications across drug discovery and preclinical studies.
Download the whitepaper, written in partnership with Metrion Biosciences, to find out more about how Axol’s validated iPSC-derived atrial cardiomyocytes can assist with any research program.
About AXOL Biosciences
Axol specializes in human cell culture.
Axol produces high quality human cell products and critical reagents such as media and growth supplements. We have a passion for great science, delivering epic support and innovating future products to help our customers advance faster in their research.
Our expertise includes reprogramming cells to iPSCs and then differentiating to various cell types. We supply differentiated cells derived from healthy donors and patients of specific disease backgrounds. As a service, we also take cells provided by customers (primary or iPSC) and then do the reprogramming (when necessary) and differentiation. Clearly, by offloading the burden of generating cells, your time is freed up to focus on the research. Axol holds the necessary licenses that are required to do iPSC work.
The package wouldn't be complete without optimized media, coating solutions and other reagents. Our in-house R&D team works hard to improve on existing media and reagents as well as innovate new products for human cell culture. We also supply a growing range of human primary cells; making Axol your first port of call for your human cell culture needs.
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