tissue cannot grow beyond the size of a pinhead without a blood supply. Formation of new blood vessels is essential for embryonic development and adult tissue repair. With their capacity for self-renewal and differentiation into various cell types, embryonic stem cells have opened up exciting vistas for both developmental biology and regenerative medicine. The goals of my research are to elucidate the mechanisms of blood vessel formation and to create vascularized grafts for tissue repair using embryonic stem cells.
Formation of blood vessels takes place via one of two processes: vasculogenesis, in which progenitor cells differentiate into endothelial cells that construct a nascent vascular network; and angiogenesis, the formation of new blood vessels from existing vessels via sprouting and remodeling. Vasculogenesis mainly occurs during embryonic development and gives rise to the heart and the first primitive vasculature inside the embryo and in its surrounding membranes, as the yolk sac circulation. In adults, ischemic insults can also recruit a small number of endothelial progenitors from the bone marrow, which participate in the re-vascularization of ischemic tissues, such as an infarcted heart.
In the embryo, blood vessels provide growing organs with the necessary oxygen and nutrients to develop. The early blood vessels assemble from mesoderm-derived angioblasts, ancestors of vascular endothelial cells. Fluorescence imaging of blood vessel formation in the early embryo with labeled antibodies that recognize endothelial cells and their progenitors has elucidated the following essential steps in the vasculogenetic process: (1) the birth of angioblasts; (2) angioblasts aggregating to organize into blood islands, structures consisting of angioblasts surrounding blood cells; (3) angioblasts elongating and migrating to form isolated cord-like endothelial segments; (4) endothelial segments coalescing into a continuous network; and (5) lumen formation transforming solid endothelial cords into vascular channels.
Embryonic stem (ES) cell differentiation has been used as a tractable model for vascular development and as a source of endothelial precursors for potential therapeutic vascular repair. When cultured in suspension without leukemia inhibiting factor, mouse ES cell aggregates develop into embryoid bodies, spherical structures that recapitulate the steps of early embryonic development, including endoderm formation, basement membrane assembly, epiblast (primitive ectoderm) differentiation and cavitation (Figure 1A). Our studies have shown that further culturing of the cystic embryoid bodies in suspension can induce the differentiation of mesoderm, from which PECAM-1 positive angioblasts are derived (Figure 1B). These endothelial precursors proliferate and migrate to form interconnecting solid endothelial cords. Subsequently the endothelial cell generates numerous vesicles or sacs inside the cell that fuse at the center of the endothelial cord to create a vascular lumen. Alternatively, vascular development can be studied in embryoid bodies cultured in suspension for 7 days and then grown on a thin layer of collagen gel. Angioblast aggregates appear in the epiblast region after another 6 days of culturing. Next, these endothelial precursors are switched to a migratory phenotype and guided by environmental cues to form a dense vasculature (Figure 1c). The beauty of the embryoid body system lies in that it provides a proper microenvironment, involving three-dimensional interactions between endothelial cells and adjacent supporting cells and extracellular matrix, that are known to be vital in the regulation of vasculogenetic processes.
My research is focused on elucidating the extracellular cues and the intracellular signaling events that regulate vasculogenesis during embryonic development and ES cell differentiation. Using differentiating ES cells assembled into embryoid bodies, we demonstrated that vascular endothelial growth factor, which is produced by angioblasts and adjacent cells, markedly enhanced vascular network formation when added to the culture medium. We further found that the basement membrane, a specialized extracellular matrix that underlies the endothelium of mature vessels, also formed on the surface of aggregated angioblasts in the early stage of vasculogenesis. This basement membrane contains laminin and perlecan but not type IV collagen. To test the hypothesis that the basement membrane assembly might potentiate the signaling induced by vascular endothelial growth factor, we isolated angioblasts from embryoid bodies and induced aggregation of these cells in hanging drops. Forced assembly of a basement membrane on the surface of the angioblast aggregates with exogenous laminin significantly augmented vascular endothelial growth factor-induced activation of Cdc42 and Rac1, small GTPases involved in the regulation of many cellular processes including migration and survival. Furthermore, targeted deletion of the Cdc42 gene in ES cells blocked angioblast migration and vascular network formation, despite the fact that endothelial lineage commitment was unaffected. Finally, ablation of Rac1 induced massive apoptosis of newly formed angioblasts. These findings have brought to focus the importance of the interplay between the extracellular matrix, vascular endothelial growth factor and small GTPases in regulating the migration and survival of endothelial precursors during vasculogenesis. However, many questions remain. How do angioblasts interpret the extracellular matrix cues and relay them to Cdc42 and Rac1? What are the downstream effectors of Cdc42 that control angioblast migration? How does active Rac1 regulate the apoptosis machinery to protect angioblasts from programmed cell death? We are currently addressing these questions using a combination of genetic and molecular cell biology approaches.
Another focus of my research is to translate our basic research findings to clinical tools for better human health. We are especially interested in engineering complex tissue structures containing vasculature that can fuse with in vivo circulation. We have differentiated mouse ES cells to cardiomyocytes that organized into a cyst with synchronized contraction in embryoid body cultures. With proper growth factor induction, vascular channels can be induced in the vicinity of these contractile structures. The long-term goal of this study is to create a cardiac patch with blood vessels from human ES cells to repair damaged heart tissue.
Shaohua Li received his Doctor of Medicine degree from Southeast University Medical School in China. In 1994, he was awarded a National Research Council research associateship and studied vascular dysfunctions in sepsis at NIH and the Naval Medical Research Institute in Maryland. He was recruited to UMDNJ in 2000 and worked with Dr. Peter Yurchenco to examine basement membrane assembly during ES cell differentiation. He joined the Department of Surgery at UMDNJ-Robert Wood Johnson Medical School in January 2006.