Effect of human endoderm, and of products secreted by ischemic myocardium, on the growth and cardiac differentiation of pluripotent stem cells

Loss of cardiac tissue due to myocardial infarction is an irreversible process that leads to heart failure and death. Functional defects caused by the human heart's inability to regenerate have been circumvented in the clinics only via organ transplant. The development of technology to isolate and propagate human embryonic stem cells (ESCs) has provided a potential therapeutic approach toward the regeneration of cardiac tissue. Furthermore, recent advances resulting in the ability to induce pluripotent cells from patient tissues have raised possibilities for the creation of pluripotent cells and their derivatives that are patient-specific. However, to safely use these cells in a clinical setting, several questions must be addressed. This doctoral thesis addresses two of these issues. First, which growth factors are capable of inducing pluripotent cells to differentiate into mature cardiac cells? Second; how might the environment of the injured myocardium affect the behavior of transplanted stem cells?;Regarding the first question, several laboratories have reported methods to derive cardiac cells from pluripotent ESCs. These methods generally rely on growth factors that have been implicated in heart development in the embryo. However, these methods are only ∼50% efficient, a level considered unsatisfactory for clinical replacement therapy. In order to refine these protocols, additional cues informed from embryonic heart development should be useful. In the embryo of lower animals, endoderm residing adjacent to the developing cardiac mesoderm is known to secrete factors that induce cardiac differentiation; however, only a handful of these factors have been identified. Clearly, a demonstration that human endoderm derived from pluripotent stem cells can induce cardiomyogenesis in pluripotent cells would provide a starting point to identify cardiomyogenic factors which may increase the efficiency of cardiomyogenic differentiation. Here we report that human definitive endoderm (hDE), which is conveniently and efficiently generated from human ESCs, secretes factors capable of inducing cardiac myogenesis in pluripotent ESCs. These factors include cerberus, which has been implicated in heart development in lower animals. The proteomic characterization of factors secreted by hDE should inform new approaches toward the refinement of human cardiomyogenic protocols.;As background to the second question, the past decade has observed results from several laboratories indicating that transplantation of pluripotent as well as partly differentiated ESCs into infarcted rodent hearts modestly improves cardiac function. However, whether improved function reflects the effects of paracrine secretions and/or engraftment by transplanted stem cells has not been resolved. Clarification of these issues demands an understanding of how the environmental milieu of the ischemic/infarcted myocardium affects transplanted ESCs. The structural and biochemical complexity presented by the ischemic/infarcted heart precludes the ability to conveniently and directly monitor ESC behavior post-transplantation in vivo. To circumvent these problems, we have utilized an in vitro model enabling direct observation of ESC behavior in the presence of myocardial interstitial fluid (MIF), which is isolated from in vivo canine myocardium following ischemia. Using MIF secreted by chronic ischemic myocardium over a three week period to supplement ESC culture medium, we have observed that ischemic myocardium secretes factors which limit expansion, as well as cardiac myocyte differentiation, in ESCs, a situation that may favor myocardial re-vascularization over re-muscularization. This in vitro approach to simulate the injured myocardial environment is useful for determining how ischemic factors, as well as factors resultant from other myocardial injuries or diseases, affect other types of transplanted cells.