Human Vascular Progenitor Cells

Chapter 51

Human Vascular Progenitor Cells Ayelet Dar,* Sharon Gerechty and Joseph Itskovitz-Eldor*, ** Sohnis and Forman Stem Cell Center, Faculty of Medicine, Technion e Israel Institute of Technology, Haifa, Israel, yChemical & Biomolecular

*

Engineering, NCI PS-OC Johns Hopkins Engineering in Oncology Center, Johns Hopkins University, Baltimore, MD, USA,

**

Department of

Obstetrics and Gynecology, Rambam Health Care Campus, Haifa, Israel

Chapter Outline Human Vascular Development, Maintenance and Renewal Vasculogenic Embryonic Cells Hemangioblasts/Angiohematopoietic Cells: EndothelialeHematopoietic Common Progenitor Cells and Angioblasts Cardiovascular Progenitor Cells: Cardiace EndothelialeSmooth Muscle Cell Common Progenitors Vascular Progenitors in Adult Tissues Human Pluripotent Stem Cells as a Source of Vascular Progenitors

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Terminology Angiogenesis. Maturation and remodeling of the primitive vascular plexus into a complex network of large and small vessels. Cardiovascular progenitor. A precursor cell that gives rise to cardiomyocytes, endothelial and smooth muscle cells. Embryonic stem cells. Pluripotent cells, derived from the inner cell mass preimplanted blastocytes; capable of unlimited proliferation under specific culture conditions. Embryoid bodies. Aggregates of spontaneously differentiating embryonic stem cells that recapitulate aspects of early embryonic development. Hemangioblast/Hemogenic endothelium. A precursor cell that gives rise to endothelial and hematopoietic cells. Induced pluripotent stem cells. Pluripotent stem cells that are artificially derived from non-pluripotent cells (e.g., adult somatic cells, multipotent precursors) by inducing a forced expression of specific genes. Mesenchymoangioblast. A precursor cell that gives rise to multipotent mesenchymal stem cells and endothelial cells. Mesoderm. The middle embryonic germ layer, between the ectoderm and endoderm, from which connective tissue, muscle, bone, cartilage, and blood vessels develop.

Spontaneous Differentiation Induced Differentiation Induced Differentiation of Hemangioblasts and Vasculogenic Derivatives from hESCs Induced Differentiation of Cardiovascular Progenitor Cells from hPSCs Induced Differentiation of Mesenchymoangioblasts from hPSCs: Common Precursor of Mesenchymal and Endothelial Cells References Further Reading

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Perivascular cells. Compose the outer layer of blood vessels; and refer to pericytes in small blood vessels, and smooth muscle cells in large blood vessels. Vasculogenesis. Generation of new blood vessels in which endothelial cell precursors undergo differentiation, expansion, and coalescence to form a network of primitive tubules.

HUMAN VASCULAR DEVELOPMENT, MAINTENANCE AND RENEWAL During the third week of human embryonic development, blood vessels are formed in conjunction with blood islands within the yolk sac mesoderm. During this process, blood islands develop alongside the endoderm, and segregate into individual hemangioblasts, which are surrounded by flattened endothelial precursor cells. The hemangioblasts mature into the first blood cells, while the endothelial precursors develop into blood vessel endothelium. At the end of the third week, the entire yolk sac, the chorionic villi, and the connecting stalk are vascularized. New vascular formation, termed vasculogenesis, takes place

Handbook of Stem Cells, Two-Volume Set. DOI: http://dx.doi.org/10.1016/B978-0-12-385942-6.00051-2 Copyright Ó 2013 Elsevier Inc. All rights reserved.

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within the embryo on day 18. During this process, the underlying endoderm secretes substances that cause some cells of the splanchnopleuric mesoderm to differentiate into angioblasts. These mesodermal angioblasts then flatten into endothelial cells and coalesce, resulting in small vesicular structures referred to as angiocysts. The latter fuse to form networks of angioblastic cords that later unite, grow, and invade embryonic tissues to create the arterial, venous, and lymphatic channels. The spread of vessel networks occurs by three processes (Carmeliet, 2000; Yancopoulos et al., 2000): 1. Continuous fusion of angiocysts e vasculogenesis. 2. Sprouting of new vessels from existing ones e angiogenesis. 3. Assimilation of new mesodermal cells into walls of existing vessels. Although the yolk sac is the first supplier of blood cells to the embryonic circulation, the role of blood cell production is later regulated by embryonic organs, such as the liver, spleen, thymus, and bone marrow. Therefore, two major propositions have emerged: the bipotential hemangioblast produces the primitive erythroid and endothelial progenitor cells; and the hemogenic endothelium gives rise to hematopoietic stem cells and endothelial progenitors (Nishikawa, 2001; Peault and Tavian, 2003). The capability of these progenitors for hematopoiesis (blood cell repopulation), vasculogenesis, or angiogenesis will be discussed.

Vasculogenic Embryonic Cells Hemangioblasts/Angiohematopoietic Cells: EndothelialeHematopoietic Common Progenitor Cells and Angioblasts In human embryos, homogenic endothelium can be observed and isolated from both the extraembryonic and intraembryonic regions (Peault and Tavian, 2003). Between the 27th and 40th days of human embryonic development, a closely packed population of CD34þ cells adheres to the ventral side of the aortic endothelium. These cells are characterized by the expression of both mature hematopoietic and endothelial cell markers including: CD45, CD34, and CD31; and by the absence of GATA-2, GATA-3, c-kit, and flk-1/KDR. In addition endothelial cells of 3- to 6-week-old human embryos were found to express CD31, CD34, and vascular endothelial cadherin (VE-Cadherin). CD31þCD34þCD45 cells isolated from both yolk sac and aorta were shown to possess the potential to differentiate into myelo-lymphoid cells in culture. Isolated CD34þCD31 cells from 11- to 12-week-old human embryos differentiated into CD34þCD31 and CD34þCD31þ cells. The latter were capable of forming a network of capillary-like structures.

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It was recently discovered that a novel cell surface antigen, BB9 (also expressed by stro-1þ bone marrow stromal cells), can identify the earliest angio-hematopoietic precursors in the human embryo (Zambidis et al., 2008). During human embryonic development, BB9 is expressed in all blood-forming tissues, including the yolk sac (day 19 of gestation), para-aortic splanchnopleura (in CD34CD45 until 26 days of gestation), aortic progenitors (day 27 of gestation), fetal liver, and in bone marrow at later developmental stages. The dynamic expression of BB9 promotes the migration of hemangioblast precursors from the para-aortic splanchnopleura toward the ventral aorta, to further differentiate into BB9þCD34þCD45þ hematopoietic progenitor cells or BB9þCD34þCD45endothelial cells. Transplantation assays that were conducted in an immunodeficient murine experimental model strongly support this notion: only isolated human embryonic BB9þ but not BB9 could reconstitute hematopoiesis and rescue lethally irradiated mice. Of notice, a novel scenario was recently introduced, in which murine hematopoiesis from primitive mesoderm was shown to be devoted of the emergence of hemangioblasts (Kataoka et al., 2011). In the course of primitive mesoderm development hematopoietic or endothelial cells were originated from distinguished precursors that migrate from the extraembryonic to the intraembryonic region wherein angioblasts give rise to endothelial cells and heamogenic endothelial cells give rise to hematopoietic stem cells. These findings demonstrate that the in vitro culture conditions can produce novel cell types, such as hemangioblasts that can not be find in physiological native tissue.

Cardiovascular Progenitor Cells: CardiaceEndothelialeSmooth Muscle Cell Common Progenitors The heart tissue is composed of mesodermal-derived lineages including cardiomyocytes, endothelial cells and smooth muscle cells (SMCs). Clonogenic c-kitþ cardiovascular progenitor cells that were identified in the human myocardium were induced to differentiate in vitro into cardiomyocytes, a-smooth muscle actin (a-SMA)-positive SMCs, and endothelial cells (Bearzi et al., 2007). Alternatively, the presence of cardiovascular-like precursors was also demonstrated for CD133þ cells, isolated from human fetal liver. Preconditioning with vascular endothelial growth factor (VEGF165) and brain-derived nerve growth factor (BDNF) induced myoangiogenic differentiation toward endothelial precursor and striated muscle cells, while hematopoietic growth factors stimulated CD133þ cell commitment toward hematopoietic stem cells. In human fetal hearts, ISL1þ cells were identified as cardiovascular precursors that give rise to cardiomyocytes,

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SMCs, and endothelial cell lineages (Laugwitz et al., 2005).

Vascular Progenitors in Adult Tissues Several studies have attempted to identify adult hemangioblast or angioblasts. With this goal, endothelial progenitor cells were isolated from fat tissue and from lymphoid organs such as bone marrow and spleen (Quirici et al., 2001; Xu, 2007; Ka¨ssmeyer et al., 2009). In addition, CD133þ, CD34þ and CD31þ circulating cells have been isolated from cord blood and peripheral blood of adult human tissues (Salven et al., 2003; Melero-Martin, et al., 2007). Upon initiation of endothelial induced differentiation by various endothelial growth factors (e.g., VEGF, FGF-2, EGF, and ECGF), these populations were capable of endothelial outgrowth with high proliferative capability and endothelial angiogenic functionality in experimental animal models. In the course of endothelial induced differentiation, these cells expressed endothelial specific markers such as VE-cadherin and Eselectin. Furthermore, endothelial precursors were also identified in adult human heart tissue. A subset of multipotent cardiovascular c-kitþ cells, devoid of hematopoietic lineage markers (CD45, CD34, and CD133), differentiated predominantly into cardiac cells; and to a lesser extent into endothelial cells and SMCs (Bearzi, et al., 2007). Less is known about the origin and process of differentiation of SMCs in adulthood. SMCs were only recently found to emerge in vitro and in vivo from CD146þ isolated pericyte-like mesenchymal stem cells (Crisan, et al., 2008). These cells comprise the abluminal endothelial layer of veins or arteries in multiple adult and fetal human organs (e.g., skeletal muscle, pancreas, bone marrow, and placenta) and express SMCs/mesenchymal lineage markers such as CD90, CD105, CD73, and CD44. In addition, CD34þNG2þ progenitor cells from adult vena saphena are proangiogenic perivascular cells with multipotent mesenchymal-like cell features and characteristic vasculogenic properties (Campagnolo, et al., 2010), suggesting that SMCs and pericytes emerge from common precursors in the course of human embryogenesis.

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such as cardiovascular development and signaling pathways that regulate progenitor cell commitment and maturation. In addition, due to virtually unlimited numbers of hPSC derivatives, these cells can be potentially used to improve and to establish new clinical protocols. Both spontaneous and induced differentiation of hPSCs can occur in two-dimensional (2D) and 3D cultures (Levenberg, et al., 2007; Wang, et al., 2007; Yamahara, et al., 2008; Dan, et al., 2012), that is, cell adherence (cellmatrix interactions) and cellecell contact (cellular communication).

Spontaneous Differentiation Human embryoid bodies (hEBs), formed from spontaneous differentiation of hPSCs, comprise multilineage tissues from endodermal, ectodermal, and mesodermal embryonic origins. Several experimental procedures have been developed to derive the endothelial potential of hESCs. Undifferentiated hPSCs form teratomas once injected into severe combined immunodeficient (SCID) mice. Within these teratomas various blood vessels are formed (Figure 51.1). The small-diameter vessels located at the center of the teratomas originate in humans. Therefore, during teratoma formation from hESCs, two parallel vascular processes occur: (1) angiogenesis of host vasculature into the formation of human teratoma and (2) vasculogenesis of spontaneously differentiating hESCs (Gerecht-Nir, et al., 2004). Endothelial cell surface markers, mainly CD34, CD31, and VE-cadherin, have been found to identify subsets of primary endothelium, and have thus been used in attempts

HUMAN PLURIPOTENT STEM CELLS AS A SOURCE OF VASCULAR PROGENITORS Together, human embryonic stem cells (hESCs), which are derived from the inner mass of the blastocyst and artificial human induced pluripotent cells (hIPSCs), which are generated by forced expression of specific genes, comprise an ample source of human pluripotent stem cells (hPSCs) for all primary germ layers (Itskovitz-Eldor, et al., 2000), including vasculogenic derivatives. The identification and induced generation of defined multipotent progenitors in hPSC systems is valuable to various physiological aspects,

FIGURE 51.1 Teratoma vasculature. Human ESCs formed teratomas once injected into severe combined immunodeficient (SCID) mice. Various blood vessels (arrows) can be observed within the formed teratoma, including human and mouse-originating vessels. Bar ¼ 100 mm.

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(A)

(A)

(B)

(B) FIGURE 51.2 Blood vessels in human embryoid bodies (hEBs). Histological sections of 1-month-old hEBs stained with anti-CD34 revealed (A) the formation of relatively small vessels and (B) larger blood vessels. Bar ¼ 100 mm.

to isolate immature and maturing endothelial cells based on the kinetics of their expression by EBs in the course of differentiation. CD34, which is considered to be an early human endothelial marker, is clearly expressed by surrounding endothelial cells that form voids within 1-month-old hEBs (Figure 51.2). Exploration of the early organization of CD34þ cells within 10- to 15-day-old hESC-derived EBs has revealed two types of cell arrangements: the first is a typical three-dimensional (3D) vessel formation, and the second a cryptic arrangement that may not appear to associate with typical 3D-vessel formation (Figure 51.3A). However, elongated and round cells stained for CD34þ represent endothelial and hematopoietic cells, respectively (Figure 51.3B). In another model, CD31, which is highly expressed by immature and mature endothelial cells, appears from day 4 of EB differentiation, and peaks at days 13e15, together with vascular network appearance in hEBs. Sorted out CD34 or CD31 cells are capable of endothelial growth with typical rearrangement in 2D and 3D cultures and vasculogenesis in vivo. Exploring the early organization of CD31þ cells within hIPSC-derived EBs reveals a typical 3D-vascular network formation in different locations within hESC-derived EBs (Figure 51.4).

FIGURE 51.3 CD34D cells in whole-mount human embryoid bodies (hEBs). 10- to 15-day-old EBs stained for CD34 revealed (A) occasionally cryptic, atypical vessel arrangement and (B) positive elongated and round cells.

However, these markers are not exclusive to the endothelial lineage upon the onset of hESC differentiation or EB maturation (e.g., CD34 identifies hematopoietic progenitors as well), raising the need to develop more complex isolation systems based on combinations of several markers and/or levels of expression. Also the in vitro model of differentiating hESCs has been used to study human embryogenesis and developmental vasculogenesis. Concurring with physiological and in vivo data, it has been shown that embryonic body differentiation

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Induced Differentiation Approaches for the promotion of specific vascular differentiation of hESCs are based on single or combined endothelial derivation procedures, and include the following:

FIGURE 51.4 CD31D cells in whole-mount hIPSC-derived human embryoid bodies (hEBs). CD31 labeled (red), 17-day-old-hEBs organized in vascular network. Nuclear staining by DAPI (blue). Bar ¼ 200 mm.

mimics the physiological mesodermal-hemangioblast pathway, whereas development of BB9þACEþ (angiotensin converting enzyme) cells, toward either hematopoietic or endothelial differentiation, is regulated by the renine angiotensin system (Zambidis, et al., 2008). In another hESC model, bone morphogenetic protein-4 (BMP-4) induced the efficient development of KDR/VEGFR2þ progenitor cells; both hematopoietic as well as vascular potential were within 72e96 hours of differentiation. Two distinguished subpopulations emerged from the KDRþ hemangioblasts: one gives rise to primitive erythroid cells, macrophages, and endothelial cells, whereas the other produces endothelial cells and only the primitive erythroid cells (Yang, et al., 2008). Recently, a novel population of CD105þCD3 perivascular multipotent pericytes was identified, which emerges in parallel to hPSC-derived CD105þCD31þVECadherinþ endothelial progenitor cells in spontaneous differentiating EBs (Dar, et al., 2012). Similar to their physiological counterparts, hPSC-derived pericytes express NG2, PDGFR-b, nestin, and CD146, as well as characteristic markers of mesenchymal stem cells, including CD73, CD44, CD29, and CD13, but not the SMC marker, a-SMA. Importantly, hPSC-derived pericytes exhibit vasculogenic properties in vitro and support blood vessel formation and stabilization in vivo, as well as the capacity for mesenchymal stem cell differentiation into osteo-adipo-chondromyogenic lineages. As an entity, hPSC-derived pericytes add to the range of vasculogenic derivatives that can be generated from hPSCs, for use in various combinations, for diverse in vitro models and vascular engineering. Similar to human fetal heart-residing ISL1þ cardiovascular precursors, differentiating hESC-derived, 8-day-old EBs were shown to give rise to equivalent ISL1þ multipotent clonogenic expandable progenitor cells, which further differentiate into ISLCD31þ endothelial cells, ISL SMMHCþ SMCs, and ISLcTNTþ cardiomyocytes in vitro (Bu, et al., 2009).

1. Genetic manipulation, also known as gene targeting e knockin and knockout specific angiogenic receptors and relevant transcription factors. 2. Exogenic factors e administrating the cell cultures with specific and known angiogenic and hematopoietic factors. 3. Matrix-based cultures e culturing the differentiating cells on matrices known to support vascular-cell cultures. 4. Co-culture e culturing the differentiating cells with specific stromal cell lines that promote vascular differentiation.

Induced Differentiation of Hemangioblasts and Vasculogenic Derivatives from hESCs Induction of vascular differentiation from hESCs was initially demonstrated by seeding hESCs on semisolid methylcellulose media supplemented with ascorbic acid and insulin-transferrin-selenium, or alternatively on type IV collagen matrices for 14 days with differentiation medium. Collagen type IV culture conditions induce the expression of all vascular endothelial growth factor (VEGF) isomers, VEGF receptors, growth factor Ang2 and its receptor Tie2, and CD31. Some of the differentiated cells that form muscleevascular arrangements express a-SMA. Induction of lineage-specific differentiation, by administration of human VEGF, platelet-derived growth factor BB (PDGF-BB), or specific hematopoietic cytokines, promotes the maturation of CD34þ hEB-derived progenitor cells toward endothelial cells or SMCs. VEGF treatment induces maturation of CD34þ precursors into endothelial cells, producing von Willebrand factor (vWF) and high lipoprotein metabolism. Upregulation of SMC markers is achieved upon supplementation of human PDGF-BB. In addition, colony formation unit assays reveal the capacity of progenitor populations to form different hematopoietic colonies (Figure 51.5). Formation of tubelike structures and sprouting is observed, subsequent to seeding the differentiated hPSC vasculogenic endothelial cells and perivascular derivatives within 3D collagen and Matrigel gels (Figure 51.6). Alternatively, endothelial subpopulations can be derived by cell sorting from EBs or from blast-forming-cell-derived common progenitors, using multiple marker expression patterns. Another approach to the enhancement of endothelial frequency and differentiation toward mature CD45þ hematopoietic progenitor cells in maturing hPSCs is based

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FIGURE 51.5 Hematopoietic colonies. Enriched progenitor population cultivated in semisolid media formed different types of hematopoietic colonies.

on co-culture of these cells on murine bone marrow and fetal murine hematopoietic organ-derived feeders (Yamashita, et al., 2000; Choi, et al., 2009; Ledran, et al., 2008). Angiohematopoietic cell differentiation of hESCs was increased in the presence of bone-marrow-derived stromal cell line OP9, and to a lesser extent with other murine bone

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marrow cell lines S17 and MS-5, or yolk sac endothelial cell line c166. Of interest, while different hIPSC lines vary somewhat in the efficiency of hematopoietic and endothelial differentiation, the pattern of differentiation tends to be similar in the majority of tested hPSC lines. OP9-derived subpopulations of either CD34þ or VEGFR2þ (KDR/flk) cells are then sorted for further enrichment in culture, or tested in various transplantation assays (e.g., Matrigel plug assay, hind limb ischemia model) for morphology analyses and vasculogenesis evaluations. Two stimuli have been identified as essential for endothelial differentiation: VEGF and mechanical force such as shear stress. Both have been shown to activate the Flk-1-PI3K-Akt signaling pathway (Zeng, et al., 2006). In murine embryo, early periendothelial SMCs were observed in association with embryonic endothelial tubes, originating from a common progenitor. Similarly, a hESC experimental model revealed even more primitive multipotential cardiovascular progenitor cells in a multistep exchange of growth factors throughout the culture period; stimulation of Flk-1þ cells with BMP-4, followed by a combination of BMP-4, bFGF, and activin-A, resulted in development of multipotent progenitor cells that could be directed toward cardiac, endothelial, or smooth muscle cells by adding defined stimulations (Yang, et al., 2008). A more restricted type of progenitor cells, capable of differentiation into endothelial and a-SMAþ smooth muscle cells, are VEGFR2þTRA1VE-Cadherinþ cells, which can be sorted from hESCs that were allowed to differentiate on the murine bone marrow stromal cell line, OP9. Following exposure to VEGF during the culture period, re-sorted VEGFR2þTRA1VE-Cadherin cells were shown to gain the perivascular cell phenotype in the presence of PDGF-BB. Co-transplantation of both cell types augmented neovascularization in a hind limb ischemic mice model (Yamahara, et al., 2008). Endothelial cell generation was enhanced by induction of hESC differentiation in a serum-free culture by means of sequential stimulations with BMP-4, Activin-A, FGF-2, and VEGF-A, as well as inhibition of transforming growth factor b (TGF-b). This gave rise to expandable mature-like endothelial cells, which had a more stable phenotype in long-term culture, compared with CD31þ hESC-derived endothelial progenitor cells (James, et al., 2010). These approaches promote the use of vasculogenic progenitor cells and mature vasculogenic cells at scale and culture conditions relevant for clinical application (Adams, et al., 2007).

Induced Differentiation of Cardiovascular Progenitor Cells from hPSCs A complex multistep induction system of hESC differentiation, based on dynamic exchange of growth factors,

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FIGURE 51.6 Assembly of hESC-derived endothelial cells and pericytes on Matrigel. A mixture of hESC vasculogenic derivatives, CD105þCD31þ endothelial cells (stained for CD31, red) and CD105þCD3 pericytes (CFSE-labeled, green) on Matrigel assemble and sprout to form thick vessel-like structures.

enhances expression of the various markers. For example, a sorted KDRlowc-kitneg subpopulation exhibits cardiovascular potential and gives rise to endothelial cells in the presence of bFGF (Yang, et al., 2008). Further optimization reveals that defined combinations of concentrations of Activin A and BMP-4 are required for efficient specification of hESC-derived KDRþPDGFR-aþ cardiovascular precursors in a NODAL-dependent manner (Kattman, et al., 2011). Of particular note, individual hESC and hIPSC lines require optimization of induction for efficient generation of cardiovascular precursors, due to variations in endogenous signaling between different hPSC lines (Yang, et al., 2008; Kattman, et al., 2011).

Induced Differentiation of Mesenchymoangioblasts from hPSCs: Common Precursor of Mesenchymal and Endothelial Cells The mesoderm is a major source of mesenchymal precursors, giving rise to skeletal, connective tissue, and perivascular cells, including SMCs and pericytes. Early mesodermal precursors were identified in initial stages of hESC differentiation. In the transition from epithelial to mesenchymal tissue, the CD326CD56þ mesodermal subpopulation is generated within 3.5 days of hESCinduced maturation. Subsequent to further stimulation in culture, these precursors are induced to differentiate into CD34þKDRþPDGFR-alow/ hematoendothelial cells, CD34KDRþPDGFR-aþ cardiovascular progenitor cells, or CD34KDRlow/PDGFR-aþCD73þ mesenchymal stem cells (Evseenko, et al., 2010). In another model, the platform of hESCs enabled the identification of a novel

population of mesodermal-derived mesenchymal progenitor cells, expressing Apelin receptor, which can give rise to chondro-osteo-adipogenic lines and bear vasculogenic potential of endothelial and smooth muscle cells (Vodyanik, et al., 2010). Altogether, these findings highlight the unique properties of hESCs as a model for understanding the determinants of germ layer specification, with potential use for tissue engineering. Although substantial progress in generating human developmental vasculogenesis has been achieved via studies of vascular remodeling in human embryos, as well as hPSC-derived vasculogenic derivatives, a number of basic issues still need be addressed: the means of regulation and mechanisms of differentiation, the potential for induction in culture, efficient endothelial-growth scalingup procedures, immunologic studies of hESC and hPSC derivatives for in vitro models, drug screening, and clinical prevention and therapy of vascular disorders based on cell transplantation or tissue engineering strategies.

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FURTHER READING Gerecht-Nir, S., Ziskind, A., Cohen, S., Itskovitz-Eldor, J., 2003. Human embryonic stem cells as an in vitro model for human vascular development and the induction of vascular differentiation. Lab Invest. 83, 1811e1820. Risau, W., Flamme, I., 1995. Vasculogenesis. Annu. Rev. Cell. Dev. Biol. 11, 73e91. Shmelkov, S.V., Meeus, S., Moussazadeh, N., Kermani, P., Rashbaum, W.K., Rabbany, S.Y., et al., 2005. Cytokine preconditioning promotes codifferentiation of human fetal liver CD133þ stem cells into angiomyogenic tissue. Circulation 111, 1175e1183. Kaufman, D.S., Hanson, E.T., Lewis, R.L., Auerbach, R., Thomson, J.A., 2001. Hematopoietic colony-forming cells derived from human embryonic stem cells. Proc. Natl. Acad. Sci. U.S.A. 98, 10716e10721. Larsen, W.J., 1998. Essentials of Human Embryology, 2nd ed. Churchill Livingstone, New York.