Abstract
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.
Highlights
Laboratory rats are a fundamental tool in the investigation of heart physiology, heart failure and myocardial injury with profound advantages over mice
Undifferentiated cells were maintained on gamma-irradiated murine embryonic fibroblasts in serum-free N2B27-2iLIF medium (MEF-2iLIF) as previously described [13]. Expansion under these conditions resulted in floating or loosely attached colonies, which were positive for pluripotency markers alkaline phosphatase (AP), stage specific embryonic antigen 1 (SSEA1) and octamer-binding factor 4 (Oct4) (Fig 1A and 1B)
Both cell types had a diploid karyotype in 83% of rat embryonic stem cells (rESCs) (Passage 14) and 73% of riPSC (Passage 27) metaphases, respectively (Fig 1C)
Summary
Laboratory rats are a fundamental tool in the investigation of heart physiology, heart failure and myocardial injury with profound advantages over mice. 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. 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 [17]. An average of 6.6% cardiac Troponin T (cTnT)pos CMs has been observed in the differentiated population [17] 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 2-10x106 PSC-CMs per animal will be required to achieve relevant restoration of infarcted myocardium [7,25]
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