Several studies have documented hematopoietic development in hES cell cultures. Differentiation was achieved by serum induction of cells either through coculture with mouse bone marrow stromal cells (Kaufman et al. 2001) or the generation of EBs (Chadwick et al. 2003; Cerdan et al. 2004). While differentiation was serum-induced, hematopoietic development was augmented in the EBs by the addition to the cultures of BMP4, VEGF, and a mixture of hematopoietic cytokines (Cerdan et al. 2004). Kinetic analysis revealed the development of definitive erythroid and myeloid progenitors following 2 wk of differentiation, a pattern that suggests that hematopoietic induction, under the conditions used, is slower than observed in mouse ES cell differentiation cultures. Distinct primitive and definitive erythroid populations have not yet been identified in the human cultures, although changes in patterns of hemoglobin expression within the ES-cell-derived erythroid lineages have been documented (Cerdan et al. 2004). These changes suggest that at least some aspects of globin switching are taking place, reflecting the changes found during normal fetal development (Stamatoyannopoulos and Grosveld 2001). The hematopoietic potential of hES cells has been recently extended to include the lymphoid lineages, with the observation that cells expressing B-cell markers develop from CD34+ cells following extensive culture on stromal cells (Vodyanik et al. 2005). Analysis of the earliest stages of hematopoietic development in the human system identified a CD45– Flk-1+ VE-cad+ CD31+ population at day 10 of differentiation that generated CD45+ hematopoietic cells following further culture (L. Wang et al. 2004). These findings suggest that human hematopoietic development within the EBs parallels that of the mouse in that the earliest hematopoietic progenitors express endothelial markers prior to their maturation to CD45+ cells. Together, the findings from this limited number of studies indicate that it is possible to generate hematopoietic cells from hES cells in culture and that the sequence of developmental events may reflect the onset of hematopoiesis in the early embryo.
The early appearance of the BL-CFC in ES cell differentiation cultures not only defines the onset of hematopoietic commitment, but also represents the earliest stages of vascular development. This pattern of vascular commitment was anticipated, as endothelial cells can be detected early in the yolk sac blood islands of the embryo (Haar and Ackerman 1971). As with the hematopoietic lineages, vascular development appears to be quite efficient in serum-stimulated differentiation cultures (Wang et al. 1992; Bautch et al. 1996; Vittet et al. 1996; Kabrun et al. 1997; Hirashima et al. 1999). Kinetic analysis of endothelial development in intact EBs revealed a sequential up-regulation of the following markers associated with the development and maturation of the lineage; flk-1, CD31, tie2, tie1, and VE-cad (Vittet et al. 1996). This pattern is similar to that observed in the early embryo, suggesting that development of the endothelial lineage in vitro recapitulates its development in vivo. A similar pattern of differentiation was observed when Flk-1+ progenitors were isolated from the differentiation cultures and recultured on type IV collagen-coated dishes or on OP9 stromal cells (Hirashima et al. 1999). Further analysis of this ES-cell-derived Flk-1 population revealed that it also displayed the capacity to generate cells of the VSM lineage. Clonal analysis demonstrated that both the endothelial and VSM lineages develop from a common progenitor, a cell that can be considered to be a vascular progenitor (Yamashita et al. 2000). A comparable progenitor was also identified in the yolk sac of the embryo. These findings are important as they define a new branch point within the vascular system and, in doing so, further highlight the power of the ES cell system in studying early lineage commitment.
Studies on the regulation of vascular commitment have revealed that factors essential for the development and maturation of the endothelial lineage in the early embryo, including VEGF (Carmeliet et al. 1996; Ferrara et al. 1996) and the receptors Flt1 (Fong et al. 1995) and Flk-1 (Shalaby et al. 1995), are also required for the establishment of the lineage in ES cell differentiation cultures (Bautch et al. 2000; Kearney et al. 2002; Zippo et al. 2004). The regulation of endothelial and VSM development in ES cell cultures is also controlled, in part, by the transcription factor Scl/tal-1. In the absence of Scl/tal-1, ES cells favor the VSM pathway, while when expressed, they differentiate to both the endothelial and VSM lineages (Ema et al. 2003; D'Souza et al. 2005).
The functional capacity of the ES-cell-derived vascular progenitors has been evaluated both in culture and in animal models following transplantation. ES-cell-derived vascular cells are able to organize into vessel-like structures in EBs (Doetschman et al. 1985), in explant cultures (Bautch et al. 1996), or when cultured on collagen I (Yamashita et al. 2000). When transplanted into tumor-bearing mice, ES-cell-derived progenitors incorporated into the newly formed vessel in the tumors, indicating that they can function as vascular cells and participate in neovascularization in vivo (Marchetti et al. 2002; Yurugi-Kobayashi et al. 2003). These observations are significant as they demonstrate that the ES cell differentiation system can be used as a model for investigating the mechanisms regulating angiogenesis in tumor formation.
Endothelial differentiation has also been demonstrated in hES cell differentiation cultures (Levenberg et al. 2002). The cells that develop in these cultures express markers associated with the endothelial lineage, form tube-like structures in matrigel in vitro, and generate capillary structures when embedded in sponges and transplanted into SCID mice. Taken together, the findings from studies on vascular development of ES cells demonstrate that the lineages develop efficiently and that the cells that are generated display properties of normal endothelial and VSM cells.