Date Published: March 7, 2018
Publisher: Public Library of Science
Author(s): Julia Dahlmann, George Awad, Carsten Dolny, Sönke Weinert, Karin Richter, Klaus-Dieter Fischer, Thomas Munsch, Volkmar Leßmann, Marianne Volleth, Martin Zenker, Yaoyao Chen, Claudia Merkl, Angelika Schnieke, Hassina Baraki, Ingo Kutschka, George Kensah, Austin John Cooney.
The possibility to generate cardiomyocytes from pluripotent stem cells in vitro has enormous significance for basic research, disease modeling, drug development and heart repair. The concept of heart muscle reconstruction has been studied and optimized in the rat model using rat primary cardiovascular cells or xenogeneic pluripotent stem cell derived-cardiomyocytes for years. However, the lack of rat pluripotent stem cells (rPSCs) and their cardiovascular derivatives prevented the establishment of an authentic clinically relevant syngeneic or allogeneic rat heart regeneration model. In this study, we comparatively explored the potential of recently available rat embryonic stem cells (rESCs) and induced pluripotent stem cells (riPSCs) as a source for cardiomyocytes (CMs). We developed feeder cell-free culture conditions facilitating the expansion of undifferentiated rPSCs and initiated cardiac differentiation by embryoid body (EB)-formation in agarose microwell arrays, which substituted the robust but labor-intensive hanging drop (HD) method. Ascorbic acid was identified as an efficient enhancer of cardiac differentiation in both rPSC types by significantly increasing the number of beating EBs (3.6 ± 1.6-fold for rESCs and 17.6 ± 3.2-fold for riPSCs). These optimizations resulted in a differentiation efficiency of up to 20% cTnTpos rPSC-derived CMs. CMs showed spontaneous contractions, expressed cardiac markers and had typical morphological features. Electrophysiology of riPSC-CMs revealed different cardiac subtypes and physiological responses to cardio-active drugs. In conclusion, we describe rPSCs as a robust source of CMs, which is a prerequisite for detailed preclinical studies of myocardial reconstruction in a physiologically and immunologically relevant small animal model.
Laboratory rats are a fundamental tool in the investigation of heart physiology, heart failure and myocardial injury with profound advantages over mice. Open-chest cardiac surgery and invasive hemodynamic assessment are easier to pursue in this larger rodent model [1,2]. Rat models have been used extensively for proof-of-principle cell and tissue based regenerative studies of the heart [3–6] and continue to be highly attractive, especially since the induced pluripotent stem cell (iPSC) technology has driven the focus even more towards the strategy of stem cell-mediated heart repair [7–11]. A significant limitation is the fact that published studies invariably represent xenogeneic models, e.g. using either human or murine iPSC-derived cardiomyocytes for transplantation. In these settings, significance is impaired by species differences in immunology, genetics and cardiac physiology. It is only recently that germline competent rat embryonic stem cells (rESCs) [12,13], and induced pluripotent stem cells (riPSCs) [14,15] are available. In combination with novel possibilities of generating transgenic rat strains via novel genome editing strategies , this will increase opportunities for the refinement of treatment strategies of various degenerative diseases. However, until now, data on the cardiac differentiation potential of rat pluripotent stem cells are limited, with only one study describing the generation of functional cardiomyocytes from rESCs . The potential of riPSCs to generate cardiomyocytes (CMs) remains elusive. One possible reason for this lack of knowledge is that conditions for culturing and genetic modification of undifferentiated rPSCs had to be optimized [18,19]. Additionally, formation of stable embryoid bodies (EBs) of undifferentiated rPSCs to induce differentiation was a general problem reported by several groups [12,19,20]. In contrast to murine and human PSCs, where defined protocols meanwhile result in high cardiac differentiation efficiencies with e.g. 40–60% CMs for mESCs [21,22] and >80% for hESCs and hiPSCs [23,24], the reported efficiency for rESCs is relatively low. Here, an average of 6.6% cardiac Troponin T (cTnT)pos CMs has been observed in the differentiated population . This is hardly enough to provide sufficient numbers of PSC-derived CMs for allogeneic or syngeneic transplantation studies. We assume that, depending on the delivery method, at least 2-10×106 PSC-CMs per animal will be required to achieve relevant restoration of infarcted myocardium [7,25].
Although germline-competent riPSCs are available, there are still no detailed reports on their cardiac differentiation potential. In this study, we assessed a simplified expansion technique and a scalable cardiac differentiation approach in a side-by-side comparison between rESCs and riPSCs. The major findings are: (I) rPSCs can be expanded in Geltrex-2iLIF conditions without losing pluripotency, (II) ascorbic acid 2-phosphate significantly enhances the development of functional CMs in both rPSC types, (III) stable cardiac differentiation is feasible employing agarose microwells to induce EB formation with subsequent dynamic suspension culture, (IV) differentiating rESCs and riPSCs show comparable cardiac gene expression profiles (V) riPSC-CMs display morphological features, physiological properties and pharmacological responses comparable to what is reported for primary rat fetal CMs and rESC-CMs . Therefore, the presented approach may be useful for future myocardial reconstruction studies in vivo.