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Keywords:

  • angiogenesis;
  • embryoid bodies;
  • embryonic stem cells;
  • vasculogenesis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Differentiation of endothelial cells from ESCs
  5. Sprouting angiogenesis in the EB
  6. EBs as a tool in vascular biology
  7. Comparative studies of sprouting angiogenesis in EBs with in vivo models
  8. Gain- and loss-of function models
  9. Future perspectives, clinical application
  10. Acknowledgments
  11. Disclosure of Conflict of Interests
  12. References

Summary.  Embryonic stem cells have become an established tool in vascular biology to study the details of vasculogenesis as well as angiogenesis. There is also a future potential in using embryonic stem cell-derived endothelial cells for therapeutic purposes. It is important to evaluate this model by comparing features of endothelial cells derived from differentiating stem cells and their responsiveness to external stimuli to those of primary endothelial cells and to in vivo models. Through culture of mouse embryonic stem cell we discovered that differentiating stem cells are highly amenable to analyzing biochemical and cell biologic processes that are independent of flow. Endothelial cell function can be studied in the context of mutations or deletions that are embryonically lethal in vivo. Many, if not all, of the features of sprouting angiogenesis in differentiating stem cells closely mimic the in vivo process.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Differentiation of endothelial cells from ESCs
  5. Sprouting angiogenesis in the EB
  6. EBs as a tool in vascular biology
  7. Comparative studies of sprouting angiogenesis in EBs with in vivo models
  8. Gain- and loss-of function models
  9. Future perspectives, clinical application
  10. Acknowledgments
  11. Disclosure of Conflict of Interests
  12. References

Embryonic stem cells (ESCs) can be established in culture from isolated embryonic day 3–4 blastocysts. In mice, the age of the blastocyst and, therefore, the state of ESC differentiation can be exactly controlled, allowing for a very high degree of reproducibility between different ESCs isolated from the same or from different genetic backgrounds. Cultures of differentiating murine ESCs, denoted embryoid bodies, possess the capacity to establish most, if not all, major cell lineages. This allows the parallel development of several cell types whose interactions can be studied with regard to molecular regulation. Interactions can also be manipulated by loss- and gain-of function strategies. It is furthermore possible to isolate out, or select for particular cell types from the ESC cultures and expand their use for therapeutic purposes. In this contribution, we will highlight features of the EB model that makes it an attractive complement to tissue culture cells and in vivo models.

Differentiation of endothelial cells from ESCs

  1. Top of page
  2. Abstract
  3. Introduction
  4. Differentiation of endothelial cells from ESCs
  5. Sprouting angiogenesis in the EB
  6. EBs as a tool in vascular biology
  7. Comparative studies of sprouting angiogenesis in EBs with in vivo models
  8. Gain- and loss-of function models
  9. Future perspectives, clinical application
  10. Acknowledgments
  11. Disclosure of Conflict of Interests
  12. References

ESCs start to differentiate when removed from feeder cells and leukemia inhibitory factor, which are used during routine culture to maintain the pluripotency of ESCs. Early stages of endothelial cell differentiation can be studied by flow sorting of dispersed, differentiating cultures. The differentiating cells have a strong tendency to aggregate and form embryoid bodies (EBs). Formation of EBs can be controlled through aggregation of ESCs in droplets, hanging on the lid of a tissue culture dish. This is advantageous when comparing the morphology of sprouting vessels formed in different types of cultures as the size and cell number in the aggregates influence the development.

In our standard procedure [1], the hanging drop culture proceeds for a few days to allow EB growth and differentiation, followed by seeding of individual bodies into either a two-dimensional (2D) culture or a 3D collagen gel. The 2D EBs appear as circular cultures with a central accumulation of cells (Fig. 1A) which respond to vascular endothelial growth factor (VEGF) with formation of endothelial cells in a dose-dependent manner. The 3D EB appears as a sphere with a central cavity with angiogenic sprouts emanating from the core (Fig. 1B). The abundance and length of sprouts increase with time of exposure to VEGF. The EB cultures require considerable efforts with meticulous attention to tissue culture details, but allow the study of endothelial cell development in parallel with development of perivascular supporting cells and the formation of a proper vascular basement membrane. Alternatively, ESCs can be differentiated on OP9 feeder cells that secrete VEGF [2] to allow enrichment of endothelial cells. This may be followed by culture on a specific matrix, such as collagen IV, to promote selection for endothelial cells further. This strategy is particularly advantageous when the objective is to obtain a maximal number of endothelial cells, like when needed for therapeutic purposes, rather than to study cellular and molecular interactions within the vascular bed.

image

Figure 1.  Vascularization of EBs. A. Dose-dependent formation of peripheral capillary plexus in 2D EBs treated with VEGFA-165 at 50 and 100 ng mL−1 (scale bar = 500 μm), visualized by immunohistochemical staining for CD31. B. Kinetics of endothelial sprouting induced by VEGF-A165 at 30 ng mL−1 (scale bar = 500 lm) in 3D EBs, visualized by immunofluorescent staining for CD31 at day 10 (d10) and day 15 (d15). C. CD31 and NG2 coimmunostaining of angiogenic sprouts induced by VEGF-A165 at 2.5 nM (95 ng mL−1(scale bar = 100 lm) in 3D EBs).

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De novo formation of endothelial cells during embryogenesis is denoted vasculogenesis. The properties of this process appear to differ dependent on the anatomical location [3]. In the yolk sac, a common precursor for the hematopoietic and endothelial cell lineages, the hemangioblast, has been identified. In clusters of hemangioblasts, denoted blood islands, peripheral cells differentiate to form angioblasts that later give rise to mature endothelial cells. In the center of the blood island, hematopoietic precursors give rise to primitive hematopoiesis. However, the existence of the hemangioblasts has remained a topic of discussion and there is evidence that the extraembryonic vasculature may arise independently from hemangioblasts, by direct differentiation of angioblasts from the mesoderm [4]. In the embryo proper, endothelial cells differentiate directly from angioblasts independently of hematopoiesis. It is not settled at this point if in vitro differentiation of ES-derived endothelial cells is more representative of embryo or yolk sac development.

At day 3 of differentiation of ESCs, angioblasts (alternatively denoted endothelial precursor cells) emerge, signifying the onset of vasculogenesis. The angioblasts markers, T cell acute leukemia 1/stem cell leukemia (TAL/SCL), VEGF receptor (VEGFR-2) and brachyury have been detected on endothelial cells both in murine and human EBs [5,6]. Angioblasts are concentrated in the center of EBs and appear as a sheet, or a primitive plexus, of endothelial cells. The angioblasts undergo sequential maturation to eventually express a set of markers characteristic for mature endothelial cells such as VEGFR-2, CD31, vascular endothelial (VE)-cadherin, Tie-1 and Tie-2. Typically, mature endothelial cells are located in the periphery of the circular 2D EB cultures (see Fig. 1A).

There are early signs of arterial/vein specification of ES-derived angioblasts. Thus, endothelial cells formed from ESCs specifically express either ephrin B2 or EphB4, which are markers for arterial and venous endothelium respectively [7].

Sprouting angiogenesis in the EB

  1. Top of page
  2. Abstract
  3. Introduction
  4. Differentiation of endothelial cells from ESCs
  5. Sprouting angiogenesis in the EB
  6. EBs as a tool in vascular biology
  7. Comparative studies of sprouting angiogenesis in EBs with in vivo models
  8. Gain- and loss-of function models
  9. Future perspectives, clinical application
  10. Acknowledgments
  11. Disclosure of Conflict of Interests
  12. References

The primitive plexus undergoes remodeling from day 6 onwards by sprouting angiogenesis. This process is regulated by growth factors, which may be produced endogenously or added in as exogenous factors [8]. There is a characteristic morphology of the vascular plexus formed in 2D cultures dependent on the growth factor present in the culture: VEGF isoforms VEGF-A121 and VEGF-A165, VEGF-C, fibroblast growth factor-2 (FGF2), platelet derived growth factor (PDGF)-BB, and angiopoietins all promote distinct morphologies of EB vascularization. Typically, VEGF-A165 stimulates the formation of a capillary plexus around the peripheral rim of circular 2D EBs. Endogenous production of growth factors or their presence in serum does not disturb the responsiveness in a clear and reproducible pattern to exogenous growth factors. However, serum is a crucial component and the typical pattern of growth factor responsiveness is dependent on the serum batch. Serum-free cultures are in many ways preferable.

Invasive angiogenesis in 3D collagen gels is preferentially induced by VEGF-A165. Many of the growth factors tested by us this far (VEGFA-121, VEGFC, FGF2 and PDGF-BB) do not induce invasive sprouting. Around day 8, invasive sprouts protrude from the central core of the EB into the surrounding matrix (Fig. 1B). The stalk cells are guided by tip cells with numerous filopodia, a process with striking similarities to vascular development in zebra fish and the retina [9]. Subsequently, the sprouts branch and occasional tip cells fuse with adjacent vessels to form networks. The EC sprouts are surrounded by perivascular cells that share features such as morphology (i.e. close apposition to the endothelial cells) and protein expression pattern [expressing nerve-glia2 (NG2) and/or α-smooth muscle actin (αSMA)] with pericytes seen in vivo, for example, in the retina (Fig. 1C). Furthermore, the sprouts are embedded in a vascular basement membrane whose composition is highly reminiscent of that appearing in vivo [10]. From day 10 of EB differentiation, lumen formation is initiated and, for some sprouts, a mature lumen is evident at day 12. Subsequently, large lumenized vascular networks become established.

EBs as a tool in vascular biology

  1. Top of page
  2. Abstract
  3. Introduction
  4. Differentiation of endothelial cells from ESCs
  5. Sprouting angiogenesis in the EB
  6. EBs as a tool in vascular biology
  7. Comparative studies of sprouting angiogenesis in EBs with in vivo models
  8. Gain- and loss-of function models
  9. Future perspectives, clinical application
  10. Acknowledgments
  11. Disclosure of Conflict of Interests
  12. References

EBs have been a very useful tool to study endothelial cell biology in gene-targeted ESCs, where embryonic development is arrested. This can be exemplified by gene targeting of fgfr1, which leads to early embryonic lethality, prior to establishment of endothelial cells. Analysis of fgfr1−/− EBs showed that vascular development is not arrested; on the contrary, there is increased expansion of the endothelial cell pool in the absence of FGFR-1 [11].

EBs can also be very useful to study the contribution of flow to developmental processes. We analyzed the composition of the vascular basement membrane in wild type EBs and found deposition of alpha4- and alpha5-chain laminins, fibronectin, collagen IV, and HSPGs [10]. We focused on the role of laminins in sprouting angiogenesis. Laminins are composed of heterotrimers which consist of different alpha, beta and gamma chains and assemble to form at least 16 different mammalian isoforms [12]. In EBs established from laminin gamma1-chain-deficient ESCs, no laminin was deposited, as would be expected since laminin gamma1-chain is a component of most laminin isoforms. We examined the capacity of the laminin gamma1-chain deficient EBs to respond to VEGF and found no defect in sprouting capacity. Instead, the main consequence of laminin deposition loss was formation of wider vessel lumen. This is in agreement with the vessel phenotype in laminin alpha4-deficient mice [13]. Laminin alpha4 is specific to the laminin isoform that is found in the vascular basement membrane. The wider vessel diameter in laminin alpha4-deficient mice has been attributed to a weakened vessel wall, which fails to offer resistance to the pressure exerted by the blood flow. However, as there is no flow in EBs, our data instead indicate that loss of laminin-deposition affects lumen width independent of flow and possibly through increased production of fibronectin and enhanced proliferation of endothelial cells.

Comparative studies of sprouting angiogenesis in EBs with in vivo models

  1. Top of page
  2. Abstract
  3. Introduction
  4. Differentiation of endothelial cells from ESCs
  5. Sprouting angiogenesis in the EB
  6. EBs as a tool in vascular biology
  7. Comparative studies of sprouting angiogenesis in EBs with in vivo models
  8. Gain- and loss-of function models
  9. Future perspectives, clinical application
  10. Acknowledgments
  11. Disclosure of Conflict of Interests
  12. References

It is important to validate that molecular mechanisms identified in the EB model can be transferred to other in vivo models of sprouting angiogenesis. As mentioned above, efficient sprouting angiogenesis is exclusively induced by VEGF-A165 in the panel of growth factors examined by us so far. We asked which properties were unique for VEGF-A165 compared to other VEGF isoforms and to other growth factors. Interestingly, VEGF-A165 binds to the transmembrane protein neuropilin1 (NRP1), inducing the formation of a NRP1/VEGFR2 heterocomplex. We tested the effect of a panel of VEGF isoforms, both naturally occurring and recombinant gain- and loss-of function VEGFs and could show that only the NRP1-binding isoforms were able to induce sprouting in EBs [14]. The same panel of growth factors was tested for their ability to induce sprouting angiogenesis in subcutaneous matrigel plugs in mice, as well as in zebra fish embryos. There was a consistent requirement for inclusion of NRP1 in the VEGF-A165/VEGFR-2 signaling complex for sprouting to occur also in these in vivo models. Thus, data on the role of NRP1 in the EB model were validated in two in vivo models, the subcutaneous matrigel plug and the zebrafish embryo.

Gain- and loss-of function models

  1. Top of page
  2. Abstract
  3. Introduction
  4. Differentiation of endothelial cells from ESCs
  5. Sprouting angiogenesis in the EB
  6. EBs as a tool in vascular biology
  7. Comparative studies of sprouting angiogenesis in EBs with in vivo models
  8. Gain- and loss-of function models
  9. Future perspectives, clinical application
  10. Acknowledgments
  11. Disclosure of Conflict of Interests
  12. References

ESCs can be established from mice of all genetic backgrounds with relative ease and from wild type as well as recombinant mice, as long as they are fertile and give rise to E3-4 blastocysts. It is, however, very time-consuming to establish panels of ES cell lines from mouse models. Ideally, one would like to manipulate a given wild type ES cell to create both loss- and gain-of function cell lines. In our experience, lentivirus is the preferable and, perhaps, the only efficient strategy for a stable introduction of genetic material into ESCs. Lentivirus vectors with both general and endothelial cell specific promoters are available [15]. Of note, the commonly employed cytomegalovirus (CMV) promoter gives very inefficient expression in ESCs. Clearly, the endothelial cell-specific promoters are not only preferable but also more challenging. Expression of the transgene has to be screened for in differentiating cells and generally the expression level will be lower. Furthermore, as the endothelial promoters encompass several kilobases, only short inserts can be accommodated or, otherwise, virus packaging is disturbed. For gene silencing, lentiviral vectors expressing certain validated shRNA are available commercially. We have experienced that the shRNA may be silenced during differentiation, and for both gain- and loss-of function strategies, intact expression of the transgene has to be continuously validated.

Future perspectives, clinical application

  1. Top of page
  2. Abstract
  3. Introduction
  4. Differentiation of endothelial cells from ESCs
  5. Sprouting angiogenesis in the EB
  6. EBs as a tool in vascular biology
  7. Comparative studies of sprouting angiogenesis in EBs with in vivo models
  8. Gain- and loss-of function models
  9. Future perspectives, clinical application
  10. Acknowledgments
  11. Disclosure of Conflict of Interests
  12. References

ESCs have an extended self-renewal capacity and can be expanded without limit as long as they are kept strictly undifferentiated. Therefore, ES-cell-derived endothelial cells could be a feasible novel cell source for therapeutic angiogenesis. Both mouse and human ESCs have been used to generate functional endothelial cells that contribute to formation of stable vasculature that connects to the host circulation [16,17]. Clearly, there are striking differences between human and mouse embryonic development, still the hallmarks of endothelial cell development are presented in a similar manner irrespective of species and the properties of ESC-derived endothelial cells appear similar. Therefore, it seems reasonable to use mouse models to explore the potential ESC-derived endothelial cells for therapeutic applications. An important issue concerns the long-term properties of such ESC-derived vessels and if ESCs are a superior source for endothelial cells compared to adult stem cells or progenitors isolated from the patient’s own bone marrow [18,19].

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Differentiation of endothelial cells from ESCs
  5. Sprouting angiogenesis in the EB
  6. EBs as a tool in vascular biology
  7. Comparative studies of sprouting angiogenesis in EBs with in vivo models
  8. Gain- and loss-of function models
  9. Future perspectives, clinical application
  10. Acknowledgments
  11. Disclosure of Conflict of Interests
  12. References
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