Human embryonic stem cells have the potential to differentiate into various cell types and, thus, may be useful as a source of cells for transplantation or tissue engineering. We describe here the differentiation steps of human embryonic stem cells into endothelial cells forming vascular-like structures. The human embryonic-derived endothelial cells were isolated by using platelet endothelial cell-adhesion molecule-1 (PECAM1) antibodies, their behavior was characterized in vitro and in vivo , and their potential in tissue engineering was examined. We show that the isolated embryonic PECAM1+ cells, grown in culture, display characteristics similar to vessel endothelium. The cells express endothelial cell markers in a pattern similar to human umbilical vein endothelial cells, their junctions are correctly organized, and they have high metabolism of acetylated low-density lipoprotein. In addition, the cells are able to differentiate and form tube-like structures when cultured on matrigel. In vivo , when transplanted into SCID mice, the cells appeared to form microvessels containing mouse blood cells. With further studies, these cells could provide a source of human endothelial cells that could be beneficial for potential applications such as engineering new blood vessels, endothelial cell transplantation into the heart for myocardial regeneration, and induction of angiogenesis for treatment of regional ischemia.

Human embryonic stem (hES) cells hold promise as an unlimited source of cells for transplantation therapies. However, control of their proliferation and differentiation into complex, viable 3D tissues is challenging. Here we examine the use of biodegradable polymer scaffolds for promoting hES cell growth and differentiation and formation of 3D structures. We show that complex structures with features of various committed embryonic tissues can be generated, in vitro , by using early differentiating hES cells and further inducing their differentiation in a supportive 3D environment such as poly(lactic- co -glycolic acid)/poly( l -lactic acid) polymer scaffolds. We found that hES cell differentiation and organization can be influenced by the scaffold and directed by growth factors such as retinoic acid, transforming growth factor β, activin-A, or insulin-like growth factor. These growth factors induced differentiation into 3D structures with characteristics of developing neural tissues, cartilage, or liver, respectively. In addition, formation of a 3D vessel-like network was observed. When transplanted into severe combined immunodeficient mice, the constructs continue to express specific human proteins in defined differentiated structures and appear to recruit and anastamose with the host vasculature. This approach provides a unique culture system for addressing questions in cell and developmental biology, and provides a potential mechanism for creating viable human tissue structures for therapeutic applications.

Transplantation of a tissue-engineered heart muscle represents a novel experimental therapeutic paradigm for myocardial diseases. However, this strategy has been hampered by the lack of sources for human cardiomyocytes and by the scarce vasculature in the ischemic area limiting the engraftment and survival of the transplanted muscle. Beyond the necessity of endothelial capillaries for the delivery of oxygen and nutrients to the grafted muscle tissue, interactions between endothelial and cardiomyocyte cells may also play a key role in promoting cell survival and proliferation. In the present study, we describe the formation of synchronously contracting engineered human cardiac tissue derived from human embryonic stem cells containing endothelial vessel networks. The 3D muscle consisted of cardiomyocytes, endothelial cells (ECs), and embryonic fibroblasts (EmFs). The formed vessels were further stabilized by the presence of mural cells originating from the EmFs. The presence of EmFs decreased EC death and increased EC proliferation. Moreover, the presence of endothelial capillaries augmented cardiomyocyte proliferation and did not hamper cardiomyocyte orientation and alignment. Immunostaining, ultrastructural analysis (using transmission electron microscopy), RT-PCR, pharmacological, and confocal laser calcium imaging studies demonstrated the presence of cardiac-specific molecular, ultrastructural, and functional properties of the generated tissue constructs with synchronous activity mediated by action potential propagation through gap junctions. In summary, this is the first report of the construction of 3D vascularized human cardiac tissue that may have unique applications for studies of cardiac development, function, and tissue replacement therapy.