Published online before print February 5, 2004, doi: 10.1161/01.ATV.0000120375.51196.73
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© 2004 American Heart Association, Inc.
Vascular Biology |
Embryonic Cell Lines with Endothelial Potential: An In Vitro System for Studying Endothelial Differentiation
From the Department of Obstetrics and Gynaecology (Y.Y., S.-K.L.); Department of Surgery (J.Q., R.M.E.O.); Department of Pathology (M.T.); National University Medical Institute (P.C.), National University of Singapore; and Genome Institute of Singapore (S.-K.L.), Singapore. To better understand endothelial differentiation, an in vitro cellular system was established using embryonic cell lines empirically derived from mouse embryos. When plated on matrigel, the cells readily differentiated to form patent tubular structures that endocytosed acetylated LDL and expressed endothelial specific markers such as CD31, Flk-1, TIE2, and P-selectin.
Correspondence to Dr Sai-Kiang Lim, Genome Institute of Singapore, 60 Biopolis Street, Singapore 138672, Singapore. E-mail limsk{at}gis.a-star.edu.sg
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Abstract |
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Objective— Endothelial differentiation is a fundamental process in angiogenesis and vasculogenesis with implications in development, normal physiology, and pathology. To better understand this process, an in vitro cellular system that recapitulates endothelial differentiation and is amenable to experimental manipulations is required.
Methods and Results— Embryonic cell lines that differentiate exclusively into endothelial cells were derived from early mouse embryos using empirical but reproducible culture techniques without viral or chemical transformation. The cells were not pluripotent and expressed reduced levels of Oct 4 and Rex-1. They were non-tumorigenic with a population doubling time of 
15 hours. When plated on matrigel, they readily differentiated to form patent tubular structures with diameters of 30 to 150 µm. The differentiated cells endocytosed acetylated low-density lipoprotein (LDL) and began to express endothelial-specific markers such as CD34, CD31, Flk-1, TIE2, P-selectin, Sca-1, and thy-1. They also expressed genes essential for differentiation and maintenance of endothelial lineages, eg, Flk-1, vascular endothelial growth factor (VEGF), and angiopoietin-1. When transplanted into animal models, these cells incorporated into host vasculature.
Conclusions— These cell lines can undergo in vitro and in vivo endothelial differentiation that recapitulated known endothelial differentiation pathways. Therefore, they are ideal for establishing an in vitro cellular system to study endothelial differentiation.
Key Words: endothelial • progenitor cells • differentiation • embryonic cell line
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Introduction |
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Tissue vascularization plays pivotal roles in normal physiological processes such as embryonic development and adult tissue repair, as well as pathophysiological processes such as tumor growth. It was previously thought that vasculogenesis or the in situ formation of blood vessels from endothelial progenitor cells (EPCs) or angioblasts was restricted to the embryo, whereas neovascularization in adults occurred from pre-existing mature endothelial cells or angiogenesis.1 However, with the identification of EPCs in several adult tissues such as peripheral blood, bone marrow, and cord blood, it was demonstrated that EPCs or angioblasts also contribute substantially to neovascularization in adult tissues.2 Therefore, most current therapeutic strategies to enhance or inhibit neovascularization have incorporated endothelial progenitor cells (EPC) either as a therapeutic agent to facilitate neovascularization or as a therapeutic target in antiangiogenic therapy of malignant tumors3–5
To better-facilitate the applications of EPC, it is therefore imperative to understand the molecular mechanisms in endothelial differentiation, and detailed molecular dissections of differentiation pathways often require an in vitro cellular system. At present, the cell types commonly used in the study of endothelial cell biology are endothelial cells isolated from blood vessels such umbilical veins and aortas. However, these differentiated endothelial cells cannot be used to study endothelial progenitor cell differentiation. Although putative EPCs have been isolated from embryonic tissues, embryonic stem (ES) cells, adult bone marrow, or peripheral blood using various markers, such as a combination of markers associated with hematopoietic stem cells such as Lin-, CD34+, c-Kit+ and Sca+, flk-1, VEGF and vascular endothelial (VE) cadherin, tie-2, and so on,2,6 the isolation procedures are usually laborious and low-yielding. These cell preparations are often heterogenous and variable. For example, different subsets of bone marrow (BM) cells such as Lin-, c-Kit+; Lin-, CD34-, low c-Kit+, Sca+; CD14-, CD34+; or human G-CSF–mobilized Lin- CD34+lo BM cells have been shown to have angiogenic potential, and when transplanted in experimental models of acute coronary occlusion result in significant recovery of the heart muscle.7–12 However, none of the subsets has been demonstrated to be more superior in inducing angiogenesis. There is some confusion with the identity of the angiogenic BM subfraction, eg, both CD34+ and CD34- subfractions have been used with success in inducing neovascularization.11–13 In isolating EPCs from ES cells, the general method is to induce formation of embryoid bodies and isolating cells from embryoid bodies at different stages of maturation to form blast colonies.14,15 Colonies with endothelial potential are identified by culturing them in the presence of VEGF, c-kit ligand, and conditioned medium from an endothelial cell line, D4T. Alternatively, ES-derived EPCs have been isolated by culturing ES cells on collagen IV-coated plates and then purifying Flk-1+ E-cadherin- cells by fluorescence-activated cell sorter (FACS).16 Therefore, preparation of EPCs from ES cells will require several procedures over a period of several days and impose a constraint on the scale of preparation.
In some aspects, the study of endothelial differentiation is limited by this lack of a suitable cell system with homogenous and expandable cells that can undergo endothelial differentiation in vitro and in vivo. To overcome this obstacle, we describe a simple empirical culture protocol using mouse embryos to derive embryonic cell lines that had the potential to differentiate into vascular tissues with the appropriate endothelial markers when plated on matrigel or when transplanted into suitable animal models. These cell lines are not embryonic stem cells and do not express any endothelial specific markers before differentiation.
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Methods |
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Derivation of RoSH2 Cells
All animal experimentation protocols were approved by National University of Singapore Animal Ethics Research Committee. B6.129S7-GtRosa26 mice were purchased from Jackson Laboratory (Bar Harbor, Me). The 5.5-days post coitum (dpc) delayed blastocysts and 6- to 7.5-dpc embryos were prepared as previously described.17 For 6- to 7.5-dpc embryos, the egg cylinders were dissected out and were placed next to the extra-embryonic tissues during culture. All the embryos were cultured on embryonic fibroblast feeder in ES cell media.17 For the older embryos, the extra-embryonic tissues were removed after the embryos attached and started proliferation. The cultures were fed every 3 or 4 days. After 1 to 2 weeks, colonies of adherent, short, spindle-shaped cells with loosely attached round cells were observed in some of the wells. These colonies were picked when they reached a size visible to the naked eye and were expanded. The colonies were picked by using a pipet tip on Gilson P20 pipet to gently scrape the colony. The detached colony was then transferred into a drop of trypsin and incubated at 37°C for 5 minutes before plating onto a 3-cm feeder plate. The pipetting of large colonies was facilitated by using a large-bore pipet tip or a regular tip that had
2 to 3 mm of the tip cut off. Once the colony had expanded to a confluent 10-cm tissue culture plate, the cells were adapted to growing on gelatinized plates without feeder. Monoclonal cell line was established by plating 2x103 single cells in 100 µL ES media in methylcellulose-based media consisting of 3.9 mL methylcellulose (MethoCult M3134; StemCell Technologies, Vancouver, Canada), 4.2 mL DMEM, 1.5 mL serum, 100 µL diluted monothioglycerol solution (37.8 µL in10 mL PBS) (Sigma, St Louis, Mo), and 100 µL 100xL-glutamine/penicillin/streptomycin stock solution (Life Technologies, Rockville, Md). After 1 week, the colony that had the least number of ring-like cells would be used to establish a line. After the colony expanded, they were designated RoSH1, RoSH2, and so on, in the order of their derivation.
Genomic DNA and Total RNA Analysis by Polymerase Chain Reaction
Genomic DNA and total RNA were prepared using standard protocols and were quantified using, respectively, the RiboGreen RNA Quantification kit and the Pico Green dsDNA Quantification kit (Molecular Probes, Eugene, Ore). Primer set for amplification of the following genes or genomic sequences and the expected amplified fragment size were: Flk-1 5'-ATTGCACACACGGGATTCTG-3' and 5'- CATACAGTACGACACTGACG-3' and 744 bp; VEGF 5'-CCTATGACCACCCACATCCG-3' and 5'-GATGAGGACCA- GAATGAGAGAC-3', 702 bp; TPI 5'-CCCTGGCATGACAAAG-A CTT-3' and 5'-CCTTGCTCCAGTCTTTCACAT-3', 242 bp; Rex-1 5'-ATGTCACTGCTGGTGCTGGA-3' and 5'-TGCTAGCC- AATTCCTCCCAG-3', 409 bp; and Oct 4 5' GGAGCACGAGTGG- AAAGCAAC-3' and 5'-TTCCTCCACCCACTTCTCCAG-3', 327 bp. Primer sets for nestled polymerase chain reaction (PCR) of angiopoietin-1 (ang-1) were: first primer set, 5'-GGAGGAAA AAGAGAAGAAGAG-3' and 5'-ACTTGCTGCTGCAACGGAGAC-3', 770 bp; and second primer set, 5'-GGAGGAAAAAGAGAA-GAAGAG-3' and 5'-TGAAATCAGCACCGTGTAAG-3', 458 bp. PCR and reverse-transcriptase PCR (RT-PCR) was performed as previously described.18 All RT-PCR primers span at least 1 intron. For nestled PCR, the second PCR was performed using the nestled PCR primers and 2 µL of a 10-times diluted amplified reaction sample.
In Vitro Differentiation
To differentiate RoSH2 cells in vitro, cells from confluent RoSH2 cell culture were plated on a matrigel-coated plate at 106 cells per 6-cm tissue culture dish. To coat the plate with matrigel, matrigel (BD Laboratories, Torrey Pines, Calif) was diluted 10 times with ES media before spreading on a tissue culture dish, and excess matrigel was removed. The cells were fed every 3 to 4 days. The formation of tubular structures was highly dependent on batches of matrigel. On average, it took 1 to 2 weeks before distinct tubular structures became obvious. If we were very careful with the culture, then the tubular structures could be maintained for 4 weeks, reaching a diameter of 150 to 200 µm before falling apart, possibly from dying through apoptosis. RoSH cells or RoSH-derived tubular structures were labeled with fluorescent ß-galactosidase (ß-gal)–specific substrate, Imagene Green, according to the manufacturer’s protocol (Molecular Probe, Eugene, Ore), and counterstained with propidium iodide for nuclear staining. Acetylated low-density lipoprotein (LDL) uptake was determined by incubating the tubular structures with diI-labeled acetylated LDL (Molecular Probe) for 24 hours at 37°C, fixed with formalin, and counterstained with SYTOX Green fluorescent stain as per the manufacturer’s protocol.
Immunohistochemistry
Immunofluorescence and immunohistochemistry was performed using standard procedures. Cells and tissues are fixed in 4% paraformaldehyde. Tissues were either embedded in paraffin and sectioned at 4-µm thickness or subjected to whole-mount staining. The primary antibodies used were goat anti-mouse P-selectin, goat anti-mouse CD34, rabbit anti-mouse TIE2, and rabbit anti-mouse Thy-1 (Santa Cruz Biotechnology, Santa Cruz, Calif), rat anti-mouse CD31, rat anti-mouse Ly-6A/E (Sca-1), and rat anti-mouse Flk-1 (BD Pharmingen, San Diego, Calif). For paraffin-embedded tissue sections, the primary antibodies were detected using biotinylated secondary antibodies and streptavidin-conjugated horseradish peroxidase and DAB (Sigma, St Louis, Mo). The sections were counterstained with Mayer hematoxylin. For antibodies that did not react with paraffin-embedded sections, whole-mount in situ immunofluorescence was performed. Briefly, after fixation, the tubular mesh was incubated in sequential order: the first primary antibody, a biotinylated secondary antibody, and then avidin-fluorescein isothiocyanate. The tissues were then counterstained with propidium iodide. The tubular mesh was analyzed by confocal microscopy.
FACS Analysis
Cells were fixed in 2% paraformaldehyde, stained with the antibodies listed, and analyzed on a FACStarplus (Becton Dickinson, San Jose, Calif).
Vascularization of ES Cell-Derived Teratomas
To determine vascularization by RoSH2 in ES cell-derived teratoma, a cellular mix of 1x104 RoSH2 cells and 1x106 CS ES cells (a gift from CS Lin) was injected subcutaneously into Rag1-/- mice. After 3 weeks, the mice were sacrificed and the tumors were removed and cryosectioned at 12 µm for immunohistochemistry staining with rabbit anti-ß-gal antibodies (ICN, Aurora, Ohio).
Incorporation of RoSH2 Cells Into Liver Vasculature During Liver Injury
To demonstrate incorporation of RoSH2 cells into the vasculature during liver injury, mice were anesthetized and 1x106 RoSH2 cells were injected intrasplenically in 20 µL of saline. In sham-transplanted mice, saline was injected in the place of cells. The mice were then injected intraperitoneally with 0.2 µg per gram body weight of hamster anti-fas antibody (BD Pharmingen). After 5 days, the livers were harvested and stained for the presence of ß-gal as previously described.19
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Results |
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Derivation of RoSH Cell Lines
To isolate cells with endothelial potential, we postulated that because vasculogenesis are initiated early in mouse embryogenesis, early embryos would be a good source of these cells. Therefore, 3.5-dpc blastocysts, 5.5-dpc delayed blastocysts, and 6.0- to 7.0-dpc embryos were harvested from pregnant transgenic B6.129S7-GtRosa26 mice. The blastocysts and embryos were cultured as outlined in Figure 1 and in Methods.20 After 2 to 4 weeks, colonies of adherent fibroblastic cells with loosely attached, rapidly dividing round cells and characteristic ring-like cells were observed in some of the cultures and, when present, were present at 1 per culture. Some colonies grew to a size visible to the naked eye and, of these, >95% survived to eventually establish a line. The frequency of these colonies in cultures of delayed blastocysts was
1 in 30, and in that of E6.0 and older embryos was 1 in 10 (data not shown). We did not observe these colonies in cultures of 3.5-dpc blastocysts or embryos older than E8.0. After expanding these colonies as described in Methods, they were plated as single cells in methylcellulose-based media. When the cells had grown to form colonies visible to the naked eye, 5 individual colonies were picked and expanded further in ES media on gelatinized plates. The colony that had the least number of ring-like cells was used to establish RoSH line. The RoSH lines were numerated as RoSH1, RoSH2, and so on, in the order of derivation. Besides having similar cellular morphology, all RoSH cell lines were tested and found to be able to form tubular structures on matrigel.
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To date, we have isolated 13 monoclonal lines, namely RoSH1 to RoSH13, 4 from 5.5-dpc delayed blastocysts, 3 from 6.5-dpc embryos, 3 from 7.0-dpc embryos, and 3 from 7.50-dpc embryos. On gelatinized-coated plates, RoSH cells have a fibroblastic morphology at subconfluency, with 1% to 5% of the cells assuming a von Willebrand factor-reactive ring-like structure (Figure 2); at high confluency, the cells formed a meshwork of cord-like structures (Figure I, available online at http://atvb.ahajournals.org). The cells have a population doubling time of 12 to 15 hours, are not LIF-dependent, and have little morphological resemblance to ES cells. At the mRNA level, they expressed much reduced levels of Oct4 and Rex-121,22 but increased levels of brachyury23 (Figure II, available online at http://atvb.ahajournals.org). Because these cells were derived from B6.129S7-GtRosa26 embryos, they expressed ß-gal in the cytoplasm, as verified by incubating the cells with Imagene Green, a green fluorescent substrate for ß-gal (Figure I).
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Differentiation of RoSH2 Cells
When plated on matrigel, the RoSH cells terminally differentiate to form a mesh of patent tubular structures with extensive branching and sprouting from the main tubular structures and with diameters ranging from an average of 30 to 50 µmol/L to a maximum of 100 to 150 µmol/L (Figure 3A). These tubular structures can be maintained for a few weeks before falling apart, possibly from dying through apoptosis. The cells endocytosed acetylated LDL, a property unique to endothelial cells and macrophages (Figure 3A).24 As typical of vascular structure, flattened monolayer cells lining the lumen were in tight apposition on an amorphous matrix and had polarized plasma membranes, with filamentous structures on the luminal surface and microvesicles underlying the plasma membrane (Figure 3A). The larger structures (100 to 200 µm) also had electron-dense organelles reminiscent of nascent Weibel-Palade bodies, as described in endothelial cells generated from ES cell-derived hemangioblast (Figure 3B).14 They also expressed typical endothelial surface markers such as CD34, PECAM-1 (or CD31), Flk-1, TIE2, Sca-1, Thy-1, and P-selectin (Figure 4). These antigens were, however, not detected on undifferentiated cells by FACS analysis (Figure III, available online at http://atvb.ahajournals.org). Important factors in the regulation of vasculogenesis such as VEGF, VEGF receptor, Flk-1, and angiopoietic factors were also expressed before and after induction of differentiation (Figure IV, available online at http://atvb.ahajournals.org).25–29 However, some of these genes, eg, Flk-1 was not detected on the cell surface before differentiation (Figure 4). We have observed that in the undifferentiated RoSH cells, several endothelial-specific genes such as Flk-1 and c-kit were transcribed but not translated into proteins (unpublished data). To verify that tubular structures formed by other RoSH lines were also endothelial structures, we tested those formed by RoSH1 and RosH3 lines and determined that these were also positive for endothelial-specific markers, eg, Flk-1 and Tie-2 (data not shown). RT-PCR analysis of RNA from RoSH1 and RosH3 lines also indicated that VEGF, VEGF receptor, Flk-1, and angiopoietic factors were also expressed in these lines before and after induction of differentiation (data not shown).
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Transplantation of RoSH2 Cells in Mice to Generate Vascular Tissues
To demonstrate endothelial potential of RoSH cells in vivo, 2 mouse models were used. In the first model, RoSH cells and ESCs were co-transplanted into immunodeficient B6.Rag1-/- mice, with the rationale that the developing ESC-derived teratoma will provide a suitable microenvironment for differentiation of RoSH cells into the entire repertoire of potential lineages. RoSH cells were identified by the presence of ß-gal. At 3 weeks, 4 of 6 mice had highly vascularized hybrid teratomas (Figure 5) whereas mice injected with ES cells alone had similar-size tumors after only 4 to 6 weeks (data not shown). Similar injections of only RoSH cells from RoSH1 to RoSH6 lines into isogenic C57BL6/J or B6.Rag1-/- mice did not cause any tumor formation during a 6-months observation period. Many of the blood-filled vascular spaces were enclosed by a layer of RoSH-derived endothelial cells, as evident by immunohistochemical staining (Figure 5). Based on 20 random high-power (400x) magnification fields per tumor section and 3 tumor sections per tumor (n=4), we estimated that 41.5±19% (SD) of the vascular tissues were derived from RoSH cells. Tumors formed from ES cells alone were smaller, at 50% of the size of those formed from ES and RoSH cells. Not unexpectedly, the smaller tumors were less vascularized. With less vascular spaces, they had a harder, more solid macroscopic and histologic appearance (data not shown). In the second model, the cells were injected intrasplenically into C57BL6/J mice that were subsequently treated with anti-fas antibody to induce liver injury. Five days later, RoSH cells were shown to incorporate into the vasculature of the liver, including the endothelium of hepatic central vein, as demonstrated by X-gal staining (Figure 6).
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-fas antibody to induce apoptosis in hepatocytes, resulting in global liver hepatoxicity. After 5 days, mice were perfused, fixed, and the livers were either stained with X-gal (A) or cryosectioned (B) at 15 µmol/L or 4 µmol/L (Figure V, available online at http://atvb.ahajournals.org) before staining with X-gal. The cells were counterstained with eosin. |
Discussion |
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In this study, we described an empirical protocol using mouse embryos to isolate and establish cell lines that were capable of undergoing endothelial differentiation in vitro and in vivo. Because the cardiovascular system is the first functioning system to be formed during mammalian embryonic development, using delayed blastocysts and early mouse embryos in our isolation protocol essentially increased the probability of isolating cells with endothelial potential.20 These lines were not ES cell lines and expressed much reduced levels of Rex-1 and Oct4, which are genes that are associated with pluripotent stem cells.21,22 It is therefore unlikely that RoSH cells were derived from the inner cell mass as ES cells, but we have yet to confirm the parental embryonic cells from which RoSH cells were derived. We hypothesized that they are likely to be embryonic stem cells with endothelial potential and possibly of mesodermal origin, because they expressed brachyury.23 Consistent with the apparent absence of pluripotency, RoSH cells did not form teratomas when transplanted into immunodeficient Rag-/- mice or isogenic C57BL/6J newborn or adult mice during a 6-month observation period.
The RoSH cells did not display any typical endothelial and hematopoietic markers before differentiation. On differentiation, they displayed endothelial markers such as CD34, PECAM-1 (or CD31), Flk-1, Tie-2, Sca-1, Thy-1, and P-selectin, endocytosed acetylated LDL, and expressed genes essential for differentiation and maintenance of endothelial lineages, eg, Flk-1, VEGF, and angiopoietin-1. In particular, the expression of Flk-1, Tie-2, VEGF, and angiopoietin-1 underscored the recapitulation of some of the known endothelial differentiation pathways. Flk-1, a receptor for VEGF, and VEGF, a critical endothelial growth factor, are essential for vasculogenesis and angiogenesis during embryo development.26–28,30 Tie-2 is a receptor tyrosine kinase for angiopoietin-1 and is expressed almost exclusively in endothelial cells and early hematopoietic cells.29 Although angiopoietin-1 does not directly promote the growth of cultured endothelial cells, it is required for the later stages of vascular remodeling and definitive hematopoiesis.31,32 Its late expression during RoSH cell differentiation was consistent with the branching and sprouting of patent tube-like structures to form a three-dimensional structure that is highly reminiscent of vascular tissue.
Our in vitro data were further verified by in vivo transplantation studies. Using 2 different experimental models, we showed that RoSH2 cells participated in angiogenesis during anti-fas–mediated liver injury and healing and vascularization of ES cell-derived teratomas. It is noteworthy that RoSH2 cells will proliferate and differentiate into endothelial cells only under microenvironment of tissue injury.
In summary, RoSH cell lines can differentiate to form endothelial cells using pathways that recapitulated some of the known endothelial differentiation pathways. They can be easily maintained and manipulated in culture and can be readily induced to form endothelial cells by plating on matrigel. These characteristics fulfilled many of the requirements of an in vitro cell-based model system to understand endothelial differentiation. In particular, they will be important in exploiting recent technological developments in high-volume gene expression and proteomic profiling to understand the roles of different genes and proteins in endothelial differentiation. The rapid proliferation of RoSH cells and their propensity to differentiate almost exclusively into endothelial cells would ensure an abundant supply of RNA and protein from undifferentiated EPCs and endothelial cells for analysis.
To some extent, ES cells also fulfilled many of these requirements. They can differentiate into the endothelial lineage by successive maturation steps recapitulating in vivo events33 and have been used as a model system to study vascular development during embryogenesis.14 However, unlike RoSH cells that differentiate almost exclusively into endothelial cells when plated on matrigel, ES cells often differentiate into complex multi-lineages. Protocols for generating ES cell-derived endothelial cells generally entail several maturation and isolation steps aimed at the isolation of rare endothelial progenitor cells.6 As such, ES cells are generally more useful in studying molecular events in early lineage commitments during embryogenesis rather than in terminal endothelial differentiation. Therefore, we propose that endothelial differentiation of RoSH cell lines represent a more relevant model for in vitro studies on endothelial differentiation.
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Acknowledgments |
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Acknowledgments
We thank Dr Bing Lim (Genome Institute of Singapore/Harvard Medical School) for his critique of the manuscript and for his advice.
Received December 23, 2003; accepted January 16, 2004.
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