Generation of functional hemangioblasts from human embryonic stem cells

Abstract

Recent evidence suggests the existence of progenitor cells in adult tissues that are capable of differentiating into vascular structures as well as into all hematopoietic cell lineages. Here we describe an efficient and reproducible method for generating large numbers of these bipotential progenitors-known as hemangioblasts-from human embryonic stem (hES) cells using an in vitro differentiation system. Blast cells expressed gene signatures characteristic of hemangioblasts, and could be expanded, cryopreserved and differentiated into multiple hematopoietic lineages as well as into endothelial cells. When we injected these cells into rats with diabetes or into mice with ischemia-reperfusion injury of the retina, they localized to the site of injury in the damaged vasculature and appeared to participate in repair. Injection of the cells also reduced the mortality rate after myocardial infarction and restored blood flow in hind limb ischemia in mouse models. Our data suggest that hES-derived blast cells (hES-BCs) could be important in vascular repair.Although progenitor cells have recently been discovered that can enter the circulation in response to vascular injury and ischemia [1][2][3][4][5] , defining and isolating these cells has proven problematic. Circulating bone marrow-derived cells have also been shown to be important in normal physiologic maintenance and repair of the body's vasculature 6,7 with approximately 1-3% of endothelial cells at any one time being bone marrow-derived. Furthermore, the entire hematopoietic system has been hypothesized to originate from a transient population of hemangioblasts restricted to embryogenesis 8,9 . But recent evidence suggests that hemangioblasts or more mature endothelial progenitors may also exist in adult tissues and umbilical cord blood [2][3][4]10,11 . More direct proof for their existence was provided when the in vitro equivalent of the hemangioblast was isolated using a mouse embryonic stem cell differentiation system 12,13 . Recently a human hemangioblast cell population derived from hES cells was also identified using a procedure that consisted of serum-free differentiation in a mixture of cytokines followed by expansion in serum-containing © 2007 Nature Publishing Group Correspondence should be addressed to R.L. (rlanza@advancedcell.com). Note: Supplementary information is available on the Nature Methods website. COMPETING INTERESTS STATEMENTThe authors declare competing financial interests: details accompany the full-text HTML version of the paper at www.nature.com/ naturemethods.Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions NIH Public Access RESULTS Generation and characterization of blast cells from hES cellsWe developed a simple and efficient method for generating functional blast cells from hES cells with both hematopoietic and endothelial potential in defined media, using a two-step strategy. First, we generated early-stage embryoid bodies (EBs) from hES cells (WA01 (H1)−GFP + ) cultured...

Molecular and developmental biology of the hemangioblast

“…Therefore, the hemogenic endothelial cells have characteristics of definitive hemangioblasts in late somite stage embryos (Jaffredo et al, 2005;Tavian and Peault, 2005). These early observations in chick, mouse, and human embryos play an important role in the evolution of the hemangioblast concept and the ultimate identification of hemangioblasts in mouse and human ESC-derived embryoid bodies (EBs), and mouse and zebrafish embryos as described below (Choi et al, 1998;Lu et al, 2006;Vogeli et al, 2006;Kennedy et al, 2007).…”

“…Most recently, Keller's group has isolated and characterized human BL-CFCs that have almost identical molecular signatures and potential to clonally generate endothelial and hematopoietic lineages as those in mouse BL-CFCs . Independently, Lu and colleagues have also isolated similar human BL-CFCs, and in addition, they have shown that transplanted blast cells could contribute to neo-vascular regeneration in damaged vessels using several animal models (Lu et al, 2006). Because the culture conditions each laboratory used are quite different, the above hemangioblast-like cell populations may not represent the same cells.…”

“…Isolating hemangioblasts and other progenitor cell populations by fluorescence-activated cell sorting (FACS) will help to identify additional players in hemangioblast development with the aid of microarray (Lugus et al, 2007) and proteomics approaches. The blast colony assay in a serum-free system will be useful for isolating growth factors and cytokines to promote hemangioblast development (Adelman et al, 2002;Park et al, 2004;Lu et al, 2006). It is foreseeable that ESCs will continue to play central roles in identifying master genes in hemangioblast development by incorporating the blast colony assay with other cellular, genetic, and genomic approaches.…”

“…This hypothesis is supported by studies using in vitro mouse and human embryonic stem cell (ESC) cultures, and in vivo animal model systems, including zebrafish, chick, and mice. By using mouse and human ESC cultures, blast-colonyforming cells (BL-CFCs) are isolated and characterized clonally to produce both endothelial and hematopoietic cells in the presence of vascular endothelial growth factor-A (VEGF-A) and bone morphogenetic protein-4 (BMP4; Choi et al, 1998;Nishikawa et al, 1998;Lu et al, 2006;Kennedy et al, 2007). Fate mapping in the zebrafish gastrula suggests that hemangioblasts are interspersed with hematopoietic and endothelial progenitors in the ventral-lateral mesoderm (Lee et al, 1994;Vogeli et al, 2006).…”

Early Exposure of Murine Embryonic Stem Cells to Hematopoietic Cytokines Differentially Directs Definitive Erythropoiesis and Cardiomyogenesis in Alginate Hydrogel Three-Dimensional Cultures

“…H ematopoiesis in the early mouse embryo has been recapitulated by in vitro differentiation culture of human and murine embryonic stem cells (mESCs) by which mesodermal cells commit to the hematopoietic lineage before giving rise to other blood cells [1][2][3][4][5][6][7]. During embryogenesis, hematopoietic cells originate from the lateral plate mesoderm in an ordered program of development occurring in conjunction with cells of the cardiovascular system and share a common precursor, the hemangioblast [8][9][10][11].…”