An efficient piling up of pluripotent stem cell-derived cardiac tissue-like sheets that robustly promotes cell engraftment and ameliorates cardiac dysfunction after myocardial infarction

Abstract

Purpose: Sufficient engraftment of transplanted cardiovascular cell populations as a regenerated heart tissue is desirable for successful cardiac cell therapy. We recently reported that the transplantation of three-layered cardiac tissue sheet bioengineered with mouse embryonic stem cell (ESC)-derived defined cardiac cell populations (Yamashita, Nature 2000) ameliorated cardiac dysfunction, mainly through neovascularization rather than direct contribution of regenerated myocardium (Masumoto, Stem Cells 2012). This result indicates that additional technology is still needed to realize regeneration of neo-myocardium. Gelatin hy-drogel is a biodegradable biomaterial which possesses excellent capacity and feasibility which supplements oxygen and nutrients and improves cell survival (Tabata, Acta Biomater 2011). Here we attempted to extend this biomaterial-based technology to our sheets to efficiently generate a stuck stem cell-derived cardiac sheets in vitro and in vivo. Methods and results: Five mouse ESC-derived cardiac sheets were stacked with gelatin hydrogel microspheres in vitro. The stacked cell sheets with gelatin hydrogel showed more sustained self-pulsation compared to those without it. The thickness of viable stacked sheets and live cell area among the sheets were significantly greater compared to those without gelatin hydrogel (thickness; 725.4±6.1 vs. 204.7±9.5μm, n=5, p < 0.0001, live cell area; 0.4746±0.041 vs. 0.03446±0.0053mm2 n=3, p=0.0090). Transplantation of the cardiac tissue-like structure incorporated with gelatin hydrogel for sub-acute nude rat myocardial infarction model showed highly efficient engraftment of the cardiac cell populations compared to those without gelatin hydrogel (average engrafted area; 1.874 vs. 0.4090 mm2 n=2). Systolic function was significantly improved (FAC; 59.0±5.6 vs. 34.7±2.8%, n=3, p=0.0220) and non-contracting length was significantly attenuated (AL; 0.4±0.7 vs. 14.9±5.1%, n=3, p < 0.01) after transplantation of stacked cardiac sheets with gelatin hydrogel compared to those without gelatin hydrogel. Masson's trichrome staining showed more limited infarct wall thinning, indicating more prominent potential for attenuation of LV remodeling. Conclusions: Incorporation of gelatin hydrogel is amenable to efficiently generate pluripotent stem cell-derived multi-layered cardiac cell sheet as a viable tissue-like structure in vitro and partly realized neo-myocardium after transplantation to ischemic heart with functional benefits. Here we have developed a promising bioengineered technology for pluripotent stem cell-based cardiac regeneration.