ToxSci Advance Access published January 26, 2015 TOXICOLOGICAL SCIENCES, 2014, 1–13 doi: 10.1093/toxsci/kfu264 Advance Access Publication Date: December 15, 2014

mESC-based in vitro Differentiation Models to Study Vascular Response and Functionality Following Genotoxic Insults

*Institute of Toxicology, Heinrich-Heine-University Du¨sseldorf, †Institute of Neuro- and Sensory Physiology, Heinrich-Heine-University Du¨sseldorf, Moorenstrasse 5 and ‡Institute of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University Du¨sseldorf, Universita¨tsstrasse 1, 40225 Du¨sseldorf, Germany 1

These authors contributed equally to this study. To whom correspondence should be addressed at Institute of Toxicology, Heinrich-Heine-University Du¨sseldorf, Moorenstrasse 5, 40225 Du¨sseldorf, Germany. Fax: þ49-211-8113013. E-mail: [email protected].

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ABSTRACT Because of high exposure to systemic noxae, vascular endothelial cells (EC) have to ensure distinct damage defense and regenerative mechanisms to guarantee vascular health. For meaningful toxicological drug assessments employing embryonic stem cell (ESC)-based in vitro models, functional competence of differentiated progeny and detailed knowledge regarding damage defense mechanisms are essential. Here, mouse ESCs (mESC) were differentiated into functionally competent vascular cells (EC and smooth muscle cells [SMC]). mESC, EC, and SMC were comparatively analyzed regarding DNA repair and DNA damage response (DDR). Differentiation was accompanied by both congruent and unique alterations in repair and DDR characteristics. EC and SMC shared the downregulation of genes involved cell cycle regulation and repair of DNA double-strand breaks (DSBs) and mismatches, whereas genes associated with nucleotide excision repair (NER), apoptosis, and autophagy were upregulated when compared with mESC. Expression of genes involved in base excision repair (BER) was particularly low in SMC. IR-induced formation of DSBs, as detected by nuclear cH2AX foci formation, was most efficient in SMC, the repair of DSBs was fastest in EC. Together with substantial differences in IR-induced phosphorylation of p53, Chk1, and Kap1, the data demonstrate complex alterations in DDR capacity going along with the loss of pluripotency and gain of EC- and SMC-specific functions. Notably, IR exposure of early vascular progenitors did not impair differentiation into functionally competent EC and SMC. Summarizing, mESC-based vascular differentiation models are informative to study the impact of environmental stressors on differentiation and function of vascular cells. Key words: mouse embryonic stem cells; differentiation; vascular cells; DNA damage response; DNA repair; ionizing radiation

Mechanisms of DNA repair and DNA damage response (DDR) are key factors promoting the preservation of genomic integrity and survival following genotoxic stress (Christmann et al., 2003; Harper and Elledge, 2007; Roos and Kaina, 2013; Zhou and Elledge, 2000). Correspondingly, stem cells harbor complex and robust mechanisms of DNA repair and DDR to avoid genomic instability (Fan et al., 2011; Momcilovic et al., 2010; Rocha et al.,

2013; Stambrook and Tichy, 2010). In general, the repair capacity of stem cells decreases with ongoing differentiation. For instance, the expression of DNA repair factors involved in base excision repair (BER) declines during differentiation of hematopoietic progenitor cells (Bauer et al., 2011; Briegert et al., 2007; Briegert and Kaina, 2007) as well as during terminal differentiation of muscle cells (Narciso et al., 2007). Comparative analysis

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Tatiana Hennicke*,1, Katja Nieweg†,1, Nicole Brockmann‡, Matthias U. Kassack‡, Kurt Gottmann†, and Gerhard Fritz*,2

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factors determining the susceptibility of stem cells and their differentiated progeny toward damaging agents are characterized. We hypothesize that the DNA damage defense capacity of mESC and their differentiated vascular progeny varies and, moreover, that genotoxic stress impairs the differentiation potency of vascular progenitors. To survey these hypotheses, we comparatively investigated the DNA repair and DDR capacities of mouse ESCs (mESC) and thereof derived vascular cell types, that is endothelial-like (EC) and smooth muscle-like (SMC) cells. To strengthen the significance of the analyses, we carefully made sure that the differentiated EC and SMC express prototypical cell-type-specific features and functions before investigating DNA repair and DDR under basal conditions and following treatment with ionizing radiation (IR), respectively. The rationale why we used IR for the initial characterization of our in vitro vascular differentiation models is that IR represents a prototypical and both environmentally and clinically relevant genotoxic noxae. Additionally, we examined whether irradiation of mesenchymal progenitors impacts their differentiation into EC and SMC. The data obtained show that differentiation of mESC into EC and SMC is accompanied by complex cell-typespecific alterations in DNA repair and DDR capacities and is not blocked by exposure of progenitor cells to a single low dose of IR.

MATERIALS AND METHODS Materials. Mouse ESC (LF2) isolated from mouse strain 129J (Nichols et al., 1990) were obtained by A. Smith (Oxford, UK). For direct vascular differentiation they were cultured with morphogens and small molecules as described (Chiang and Wong, 2011). VEGF, Bmp4, FGF2, Activin A originate from PeproTech (Hamburg, Germany), Forskolin from Sigma Aldrich (Munich, Germany), Gski from Calbiochem (Darmstadt, Germany), and Alki from Sellek Chemicals LLC (Munich, Germany). Antibodies originate from the following companies: Actin, PARP, ERK2, Hmox1, Smc1 (pS957), p53 (Santa Cruz, California), cH2AX (pS139) (Millipore, Billerica, Massachusetts), Msh2, Rad51 (Abcam, Cambridge, UK), Brca1, p53 (pS15), Chk1 (pS345), Chk1, GAPDH (Cell Signaling, Beverly, Massachusetts), Kap1 (pS824) (Bethyl Laboratories, Inc, Montgomery), Flk-1 (BD Bioscience, Heidelberg, Germany), VE-Cadherin (eBioscience, Frankfurt, Germany), alpha-smooth muscle actin (a-SMA), platelet endothelial adhesion molecule-1 (Pecam-1) (Abcam, Cambridge, UK). Immortalized mouse ECs (H5V) originate from A. Vecchi (Milan, Italy) (Garlanda et al., 1994). mESC culture and vascular differentiation. Mouse ESCs (LF2) were cultured in feeder-free conditions using Knock-out Dulbecco’s Modified Eagle Medium (KO-DMEM) supplemented with 10% fetal calf serum, penicillin/streptomycin (1%), serum replacement (5%), L-glutamin (1%), b-mercaptoethanol (5  105 M) and leukemia inhibitory factor (LIF) (1.000 U/ml). For differentiation a modified protocol according to Chiang and Wong was used (Chiang and Wong, 2011). Briefly, cells were split (0.15  105 cells/6-well) in modified N2B27 medium (50% DMEM/F12, 50% neurobasal medium, 0.5% glutamax, 1% B27 supplement, 0.5% neuropan2 (104 M), b-mercaptoethanol (104 M), 3 mg/ml heparin, and 1% penicillin/streptomycin) supplemented with varying combinations of growth factors (see Fig. 1A) on fibronectincoated plates. On Day 2, Activin A (4 ng/ml), Fgf2 (12.5 ng/ml), Bmp4 (5 ng/ml), and Gski (6 mM) were added (¼ N2B27-Mi). Fgf2 (12.5 ng/ml), VEGF (20 ng/ml), Forskolin (10 mM), and Alki (2 mM) were added at Day 4 (¼ N2B27-VM). At Day 6, differentiation into EC was complete. To stimulate differentiation into SMC, cells

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of mouse embryonic stem cells (mESCs) and isogenic mouse embryonic fibroblasts (MEFs) show that, apart from a decrease in DNA repair activity, reduction of apoptotic capacity also parallels the loss of pluripotency (Tichy and Stambrook, 2008; Tichy et al., 2010, 2011, 2012). Thus, mESCs are hypersensitive to DNA alkylating drugs when compared with MEFs (Roos et al., 2007). Under in vivo situation, cells are exposed to a plethora of DNA damaging noxae originating from endogenous or exogenous sources. Exposure to harmful agents together with an insufficient repair of damage or induction of cell death can result in substantial cytotoxicity in terminally differentiated cells. Additionally, impaired organ- and tissue-specific functions caused by environmental noxae can also rest on stem cell depletion or stalled differentiation of progenitor cells, eventually leading to an insufficient regenerative capacity of tissues. It is well known that constrictions in DNA repair capacity contribute to both aging (Lopez-Otin et al., 2013) and tumor formation (Hanahan and Weinberg, 2011). Hence, it is conceivable that the capability of stem and progenitor cells to adequately respond to genotoxic insult plays a pivotal role for the final outcome of genotoxin exposure. Factors determining the sensitivity/resistance of stem cells and multipotent progenitors to damaging agents are likely quite complex and both cell type and agent specific. For instance, the radioresistance of mesenchymal stromal cells is determined by multiple DDR mechanisms (Sugrue et al., 2013). Thus, detailed information regarding the molecular determinants of the susceptibility of stem cells, lineage-specific progenitor cells, and their differentiated progeny toward DNA damaging noxae is required for toxicological assessments. Due to their barrier function, vascular endothelial cells (ECs) are exposed to the highest concentrations of systemically present noxae. Apart from regulating nutrient exchange, ECs are also crucial for the regulation of angiogenesis and blood pressure as well as inflammatory processes and clotting. Because of a permanent exposure to noxious substances, it is conceivable that the vascular endothelium has to ensure effective repair and/or regenerative processes to preserve vascular health. Erroneous repair of damage and/or incomplete regeneration eventually ends up in pathophysiological alterations, which manifest as endothelial dysfunction. Such malfunction of the endothelium, which typically corresponds with increased oxidative stress and subsequent decrease in nitric oxide (NO) bioavailability (Munzel et al., 2008; Schulz et al., 2011), is the main reason for atherosclerosis, a major risk factor for heart attack and stroke. Previous reports demonstrated that endothelial dysfunction is related to oxidative DNA damage (Forstermann, 2008; Mahmoudi et al., 2006; Pernice et al., 2006). Correspondingly, oxidative stress resulting from cigarette smoking aggravates endothelial dysfunction (Heitzer et al., 1996). Notably, polymorphisms of DNA repair factors are related to the risk of large artery atherosclerotic stroke when smoking cigarettes (Shyu et al., 2012). Based on these reports, it appears feasible that an insufficient response to DNA damage impacts endothelial functions and compromises the regenerative capacity of the endothelium, thereby eventually favoring diseases that are related to a loss of vascular functionality. The ability to predict potentially deleterious health effects resulting from acute or chronic exposures to chemical noxae is highly challenging due to the complexity of biology. The more it is evident that toxicological drug assessments employing ESC-based in vitro model systems, which support the global intended 3Rconcept of reduction, refinement, and replacement of animal experiments, are particular meaningful if (i) the functional competence of the differentiated cell types is assured and (ii) the

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cell morphology observed 6 days after the onset of differentiation (removal of LIF; Day 0). Differentiated cells show the typical cobblestone-like morphology of EC. Magnification: 10. Scale bar: 50 mm. C, mRNA expression of the stem cell factors Klf4, Nanog, and Oct4 in endothelial-like cells (EC) at Day 6 after the onset of the differentiation process. Relative mRNA expression in non-differentiated mESC was set to 1.0. Data shown are from 2 independent experiments each performed in triplicate. D, Quantitative RT-PCR analysis of the mRNA expression of prototypical endothelial marker genes at Days 6–9 after the onset of differentiation (see Fig. 1A). Endothelial markers used are Flk-1, Pecam-1, and VE-cadherin. Smooth-muscle markers analyzed are calponin and alpha smooth muscle actin (a-SMA). mRNA expression was related to that of mESC, which was set to 1.0. Data shown are the mean 6 SD of 3 independent experiments, each performed in triplicate. E, Quantitative RTPCR analysis of the mRNA expression of the venous maker Ephb4 and the arterial marker Efnb2. Relative mRNA levels in non-differentiated mESC cells were set to 1.0. Data shown are the mean 6 SD from 3 independent experiments. F, Immunocytochemical analysis of the protein expression of the endothelial marker proteins Flk-1, VE-Cadherin, and Pecam-1 in differentiated ECs (Day 6) when compared with mESC. The nucleus is stained by DAPI. Shown is the result of one representative experiment out of more than or equal to 3 independent experiments.

were further cultured for additional 3 days in N2B27-VM before they were splitted (1:3) onto gelatin-coated plates in endothelial growth medium 2 (Lonza, Cologne, Germany) at Day 9. RNA isolation and quantitative polymerase chain reaction. Total RNA was purified using the RNeasy Mini Kit (Qiagen, Hilden, Germany). The reverse transcriptase (RT) reaction was performed by use of the OmniScript Kit (Qiagen) or the high capacity cDNA reverse transcription Kit (Applied Biosystems, Darmstadt, Germany) using 1000–2000 ng of RNA. Quantitative RT-polymerase chain reaction (PCR) runs were done as follows: (1) 95 C, 2 min; 2. 95 C, 30 s—55–60 C, 30 s—72 C, 40 s; 3. 72 C, 10 min. qRT-PCR analyses were performed in duplicates using SensiMix Mastermix (BioLine) or PowerSYBRGreen PCR Mastermix (Applied Biosystems, Darmstadt, Germany) and a MyIQ Thermal Cycler (BioRad, Munich, Germany). To analyze

the mRNA expression of a subset of susceptibility relevant genes, a semi-customized 96-well format PCR-array was employed (Fritz et al., 2011). Melting curves were analyzed to ensure the specificity of the amplification product. Primers used for mRNA expression analyses are listed in Supplementary Tables 1–3. mRNA expression levels were normalized to that of the housekeeping genes GAPDH and b-actin. Relative gene expression in the corresponding controls (ie, mESC or untreated cells) was set to 1.0. Changes in gene expression of less than or equal to 0.5 and more than or equal to 2-fold are considered as biologically relevant. Semi-quantitative RT-PCR. Isolation of total RNA and subsequent cDNA synthesis were performed as described above. The RTPCR was performed by using 10 ml of the 2 QuantiTect-SYBR Green-Master-Mix (Qiagen GmbH, Hilden, Germany), 1.5 ml of

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FIG. 1. Expression of prototypical endothelial markers in differentiated EC. A, Schematical illustration of the protocol used for endothelial differentiation. B, Changes in

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diluted cDNA, 0.8 ml of forward and reverse primer (125 mM) in a final volume of 20 ml using the Mastercycler personal, (Eppendorf AG, Hamburg, Germany) (95 C for 120 s, followed by 40 cycles at 94 C for 20 s, 57 C for 30 s, and 72 C for 60 s). PCR products were separated on a 2.0% agarose gel by electrophoresis and visualized by GelRed staining using the INTAS Gel iX Imager (INTAS Science Imaging, Go¨ttingen, Germany). GAPDH was used as housekeeping gene.

Analysis of the DDR. To detect DNA damage, the formation of nuclear foci consisting of S139-phosphorylated histone H2AX (cH2AX) was analyzed microscopically. cH2AX is a well accepted and highly sensitive surrogate marker of the DDR stimulated by DNA double-strand breaks (DSBs) (Olive, 2004; Rogakou et al., 1998). Cells were seeded onto cover slips and treated with IR (1 Gy) (Cs137 source) 24 h later. Up to 6 h after irradiation, they were fixed with 4% paraformaldehyde (15 min, RT), followed by incubation with ice-cold methanol (20 C, 20 min). After blocking for 1 h (5% BSA in PBS/0.3% Triton X-100), incubation with cH2AX antibody (1:500) was performed (overnight, 4 C). Incubation with the secondary antibody (1:500, Alexa Fluor 488; Invitrogen) was done for 1 h at room temperature. After washing with PBS and staining of nuclear DNA by diamidino-2phenylindole dihydrochloride (DAPI), the number of cH2AX foci per nucleus was determined by microscopical analysis (Olympus Bx43). Data presented are the mean 6 SD from 3 independent experiments with 50 cells being analyzed in each experiment (n ¼ 3; N ¼ 150). ATM/ATR-catalyzed phosphorylation of p53, Chk1, Kap1, and Smc1 was analyzed by Western blot analysis following irradiation of cells with 10 Gy. When compared with the cH2AX foci analysis (see above), the investigation of DDR mechanisms by western blot analysis is a more insensitive method that requires higher doses of IR. Immunofluorescence analyses. Adherent cells were fixed with 4% paraformaldehyde (15 min, RT). After washing with PBS, cells were blocked with PBS/3% BSA for 1 h, followed by incubation with primary antibodies (1:100) directed against fetal liver kinase-1 (Flk-1), VE-Cadherin, a-SMA or platelet EC adhesion molecule-1 (Pecam-1) at 4 C overnight. After washing with PBS, the fluorescence-labeled secondary antibody (Alexa Fluor 488 or 532 goat polyclonal to mouse, rabbit or rat) was added (1:1000) (2 h, RT). After washing with PBS and staining of nuclear DNA by DAPI, microscopic slides were analyzed by fluorescence microscopy (Olympus BX43). Analysis of Ca21 release and contractility. The presence of functional muscarinic acetylcholine receptors on mESC-derived SMC was determined by measuring their capacity to provoke a rise of [Ca2þ] in response to carbachol, a potent agonist for muscarinic and nicotinic receptors. Calcium fluorescence

Analysis of tube formation by matrigel assay. Frozen matrigel matrix (BD Matrigel Basement Membrane Matrix, Growth factor reduced) was thawed on ice at 4 C overnight. 200 ml of chilled matrigel/well was applied to a 24-well plate and incubated for 1 h at 37 C. After 6 days of endothelial differentiation, cells were trypsinized and plated onto the matrigel-coated 24-well plates (2  104 cells per well) and were further incubated for 16 h. Tube formation was assayed by microscopical analysis. Immortalized mouse ECs (H5V cells) were included for control. Analysis of low-density lipoprotein-uptake and lectin binding. Direct fluorescent staining was used to detect the dual binding of fluorescein-isothiocyanate (FITC)-conjugated Ulex europaeus agglutinin lectin (UEA-1; Sigma-Aldrich, St Louis, Missouri) and 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine-labeled ac etylated LDL (DiI-acLDL; Invitrogen, Grand Island, New York). 10 mg/ml of acetylated low-density DiI complex was added to the culture medium. 4 h later, the medium was removed, cells were washed with PBS and then fixed with 4% cold paraformaldehyde (15 min, RT), followed by incubation with UEA-1 (10 mg/ ml, 1 h). Cells were counterstained with the nuclear dye DAPI and examined for the uptake of DiI-acLDL and binding of UEA-1 by fluorescence microscopy (Olympus Bx43). Statistical analysis. The data were analyzed by the 1-way analysis of variance with Bonferroni post hoc test and Student’s t test. P  0.05 were considered as statistically significant.

RESULTS Characterization of EC Differentiated in vitro from mESC The protocol used for differentiation of EC from mESC is shown in Figure 1A. Cells with a cobblestone-like morphology (Fig. 1B), which is typical for EC (Martin-Ramirez et al., 2012; Nourse et al., 2010) were observed 6 days after withdrawal of LIF and supplementation of the growth medium with a mixture of morphogens and small molecules (Fig. 1A). Gain of EC-like morphology was accompanied by a large decrease in the mRNA expression of various stem cell-specific transcription factors such as octamer-binding transcription factor 4 (Oct4), Kruppel-like factor 4 (Klf4), and the homeobox transcription factor Nanog as expected (Fig. 1C) (Jauch and Kolatkar, 2013). To further confirm successful differentiation of mESC into EC, mRNA and protein expression of a subset of prototypical endothelial factors (Glaser et al., 2011) was analyzed. EC revealed a stable upregulation of the mRNA expression of the endothelial markers VE-cadherin, Flk-1, and Pecam-1, as analyzed at Days 6–9 after the start

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Western blot analysis. Total cell extracts were prepared by lysing of an equal number of cells in Roti-Load buffer (Carl Roth GmbH, Karlsruhe, Germany). After heating (95 C, 5 min), 20–30 mg of protein was separated by SDS-PAGE (12% gel) and transferred onto nitrocellulose membrane. After blocking (5% non-fat milk in TBS/0.1% Tween 20; 1 h at RT), incubation with the primary antibodies (1:200-1000) was performed over night at 4 C. After washing with TBS/0.1% Tween 20, incubation with peroxidase-conjugated secondary antibody (1:2000, Rockland Immunochemicals Inc, Gilbertsville) was performed (2 h, RT). For visualization, the Fusion FX7 imaging system (Peqlab, Erlangen, Germany) was used.

measurements were performed using Oregon Green 488 BAPTA1/AM as described (Kassack et al., 2002). Cells were harvested with 0.05% trypsin and rinsed with culture medium containing 10% FBS. Cells were resuspended in fresh medium and kept under 5% CO2 at 37 C for 15 min. After washing with KrebsHEPES buffer (KHB: 118.6 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 4.2 mM NaHCO3, 11.7 mM D-glucose, 10 mM HEPES (free acid), 1.3 mM CaCl2, and 1.2 mM MgSO4, pH 7.4), cells were loaded with 1.5 mM Oregon Green 488 BAPTA-1/AM (Molecular Probes) under shaking (45 min, RT) in the same buffer containing 0.03% Pluronic F-127 (Sigma Aldrich). Afterwards, cells were rinsed with KHB and plated into 96-well plates at a density of 75.000 cells per well. Plates were kept at 37 C and treated with different types of purinergic receptor agonists. Changes in fluorescence were measured using a NOVOstar plate reader with a built-in pipettor system (BMG LabTechnologies, Offenburg, Germany).

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LDL and a largely increased lectin binding activity when compared with mESC (Fig. 2B), supporting the note that functionally competent EC have been differentiated from mESC. In addition, mESC-derived ECs were able to form vessel-like structures (tube formation) (Fig. 2C), which is another prototypical function of ECs (Vishnubalaji et al., 2012). Tube formation of ECs was similar effective as observed for immortalized mouse H5V ECs (Fig. 2C) and primary human umbilical vein ECs (HUVECs) (data not shown). Characterization of SMC Differentiated in vitro from mESC In order to compare the damage defense capacity of mESCderived EC with that of another (isogenic) type of vascular cell, we considered SMC as highly suitable. SMC were differentiated from EC according to the protocol shown in Figure 3A (Emmanuel et al., 2013). A reduced expression of the stem cellspecific transcription factors Klf4, Nanog, and Oct4 was confirmed on the mRNA level (Supplementary Fig. 2) as anticipated. SMC revealed an increased mRNA expression of the smoothmuscle markers calponin, a-SMA, smoothelin, and TagIn (Fig. 3B) (Lachaud et al., 2013). Expression of a-SMA protein was confirmed by immunocytochemistry (Fig. 3C). In addition, SMC responded to treatment with the muscarinergic agonist carbachol with an increase in the intracellular Ca2þ concentration (Fig. 3D). This finding demonstrates that the differentiated SMC harbor smooth-muscle-specific functions (Lachaud et al., 2013).

FIG. 2. EC that are differentiated from mESC are functionally competent. A, EC (d6) were left untreated or were treated with pro-inflammatory cytokines (IL1b/TNFa) (10 ng/ml). Up to 2 h after treatment, the mRNA expression of E-selectin, Icam-1, iNos, and Nos3 was analyzed by qRT-PCR. For control, cells were exposed to ionizing radiation (IR) (5 Gy) and mRNA expression was analyzed 30 min later. Relative mRNA expression in untreated EC was set to 1.0. Data shown are the mean 6 SD of 3 independent experiments. B, LDL-uptake and lectin-binding (UEA-1, FITC-conjugated) in mESC and thereof derived EC (d6) was analyzed as described in Materials and Methods. Shown are representative data from more than or equal to 3 independent experiments. C, The angiogenic potential (tube formation) of differentiated EC (d6) cells was analyzed in vitro as described in Materials and Methods. Immortalized mouse EC (H5V cells) (Garlanda et al., 1994) and human EC (HUVEC) (data not shown) were used as positive controls. Shown are the morphological changes observed 16 h after plating of EC and H5V on matrigel. The result of one representative experiment (out of more than or equal to 3 independent experiments) is shown.

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of differentiation (Fig. 1D). Protein expression of these endothelial surface markers was confirmed by immunocytochemistry (Fig. 1F). Moreover, EC revealed a largely increased mRNA expression of E-selectin, Vcam-1, and of endothelial nitric oxide synthase 3 (NOS3) (Supplementary Fig. 1), which are additional EC markers. The differentiated EC revealed a favored mRNA expression of venous Ephb4 over arterial Efnb2 (Fig. 1E) (Lindskog et al., 2014). Notably, the mRNA expression of the smooth muscle cell (SMC)-specific markers a-SMA and calponin was not upregulated in EC (Fig. 1D), further supporting the note that specific differentiation of mESC into EC was achieved. To scrutinize that the differentiated EC are functionally competent, their response to both inflammatory and genotoxic stress was analyzed. Treatment of EC with the inflammatory cytokines TNFa/IL1b caused a substantial upregulation of the mRNA expression of the endothelial adhesion molecules Eselectin and Icam-1 as well as of endothelial Nos3 and the inducible NO-synthase (iNOS) (Fig. 2A), as anticipated (Nourse et al., 2010). The mRNA expression of cell adhesion molecules was also simulated by IR (Fig. 2A), which is in line with data reported for human EC in vitro (Nuebel et al., 2004) and in mouse pulmonary vascular endothelium in vivo (Hallahan et al., 1997). Increased uptake of LDL was used for the identification and isolation of functionally competent ECs (Hirschi et al., 2008; Voyta et al., 1984). Accordingly, EC revealed a much higher uptake of

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mRNA expression of the smooth muscle-specific markers calponin (Calp), a-SMA, smoothelin (Smo), and transgelin (Tagln) was analyzed by qRT-PCR at Days 10 (d10) and 15 (d15). Data shown are the mean 6 SD of 3 independent experiments. C, Protein expression of a-SMA was analyzed in SMC at Day 15 of the differentiation process by immunocytochemistry. The result of 1 representative experiment (out of more than or equal to 3 independent experiments) is shown. D, SMC (Day 15 of differentiation) were treated with the indicated concentrations of the vasoactive agonists carbachol (Carba, 1 mM or 0.316 mM), adenosin-triphosphate (ATP, 10 mM), or endothelin (Endot, 50 nM). Increase in intracellular Ca2þ concentration was determined as described in Materials and Methods. Data shown are mean values from a representative experiment performed in triplicate.

Additionally, we analyzed whether changes in the expression of purinergic receptors, which are functionally relevant for mesenchymal stem cells (Ulrich et al., 2012; Zippel et al., 2012) as well as EC and SMC (Burnstock, 2012; Burnstock and Ralevic, 2014), do occur in the course of differentiation. When compared with mESC, differentiated EC and SMC unanimously showed an increased mRNA expression of the purinergic receptor subtypes P2X2, P2X7, and P2Y1 (see Supplementary Fig. 3). Measuring intracellular Ca2þ concentrations following stimulation of purinergic receptors with ATP, ADP, and UTP revealed lower EC50 concentrations in EC and SMC when compared with mESC (Supplementary Fig. 3).

mRNA Expression of Susceptibility-Related Factors in mESC, EC, and SMC To examine whether mESC and the functionally competent EC and SMC differ from each other in the mRNA expression pattern of susceptibility factors that are related to DNA repair, cell cycle regulation, and death, a semi-customized 96-well-based PCR array was used (Fritz et al., 2011). Quantitative RT-PCR-based mRNA expression analyses revealed that a subset of genes was concordantly down- or upregulated in both EC and SMC when compared with mESC. A total of 15 genes were upregulated in both vascular cell types, including genes coding for factors involved in DNA repair by nucleotide excision repair (NER) (Ercc1, Xpa), cell death (c-IAP, Bcl2, Fas-L), and autophagy (Atg3, Atg7) (Fig. 4 and Supplementary Fig. 4). By contrast, the expression of 10 genes, including genes encoding proteins involved in DNA repair by NER (Ddb2), mismatch repair (MMR) (Msh2), homologous recombination (HR) (Rad51), and translesion

synthesis (Pole) as well as cell cycle regulation (CyclinA1, CyclinE1, Wee1) was downregulated in EC and SMC (Fig. 4 and Supplementary Fig. 4). On the other hand, EC and SMC also differed from each other regarding the expression of multiple susceptibility-related genes (Fig. 4B). For instance, the expression of DNA repair factors Lig4 and Xrcc4 was upregulated in EC but not in SMC. In addition, the mRNA expression of various DNA repair (Brca1, Brca2, Fancc, Fen1, Lig1, Mre11a, Wrn, Xpc, Xrcc1, Xrcc3) and cell cycle regulatory factors (Cdc25A, Cdkn1b, CyclinB1, Chk1, Chk2, Pcna) was specifically downregulated in SMC but not in EC (Supplementary Fig. 4).

IR-Induced DDR of mESC and Differentiated EC and SMC To investigate whether the cellular responsiveness to induced DNA damage has changed upon differentiation, mESC, EC, and SMC cells were treated with IR and the DDR was analyzed up to 6 h later. As a prototypical surrogate marker of the DDR, we monitored the level of S139 phosphorylated histone H2AX (cH2AX) (Olive, 2004; Rogakou et al., 1998; Stiff et al., 2004). S139 phosphorylation of H2AX is catalyzed by the PI3-like protein kinases ATM/ATR, which are the key regulators of the DDR (Harper and Elledge, 2007). The formation of nuclear cH2AX foci is indicative of DNA DSBs (Kinner et al., 2008; Olive, 2004; Rogakou et al., 1998; Stiff et al., 2004). The basal number of cH2AX foci was highest in mESC (Figs. 5A and 5B) and increased by about 4-fold within 30–60 min after irradiation (Figs. 5B and 5C). When compared with mESC, both EC and SMC revealed a significantly stronger increase in the number of nuclear cH2AX foci following irradiation (Fig. 5C). The strongest effect was detected 30–60 min after irradiation of SMC (Fig. 5C). As

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FIG. 3. Differentiation of functional competent smooth muscle cells (SMC) from mESC. A, Schematical illustration of the protocol used for differentiation of SMCs. B,

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changes in basal mRNA levels between mESC, EC, and SMC. Only alteration in relative gene expression of more than 2.0 (¼ upregulation) and less than 0.5 (¼ downregulation) were considered as biologically relevant. Relative mRNA expression in non-differentiated mESC was set to 1.0. Quantitative data are based on 3 independent experiments. A, Number of genes that are upregulated (left panel) or downregulated (right panel) in either EC, SMC, or both cell types when compared with mESC. B, Scatter plot illustrating the differences in basal gene expression observed in EC when compared with mESC, SMC when compared with mESC, and EC when compared with SMC. C and D, Detailed presentation of genes that are commonly upregulated (C) or downregulated (D) in EC and SMC when compared with mESC. Genes that show a specific up- or downregulation in EC or SMC are depicted in Supplementary Figure 4.

concluded from the time-dependent decrease in cH2AX foci, which reflects the repair of DSBs, EC and SMC possess a higher DSB repair capacity when compared with mESC (Fig. 5D). The number of residual foci determined 6 h after irradiation was lowest in EC (Fig. 5D). In addition, the protein expression of selected DNA repair and DDR factors was analyzed under basal conditions and after irradiation by Western blot analyses. These analyses showed that the basal expression of Rad51 protein is lower in EC and SMC when compared with mESC (Fig. 6). Following IR exposure, increase in Msh2 protein expression was observed in mESC only but not in EC or SMC (Fig. 6). Basal Msh2 expression was reduced in differentiated vascular cells (Fig. 6). Protein expression of the antioxidative factor heme oxygenase-1 (Hmox1) was highest in mESC and lowest in SMC (Fig. 6). Following IR exposure, Hmox1 protein expression was clearly increased 6 h after irradiation in EC but not in mESC or SMC (Fig. 6). Furthermore, we analyzed the level of phosphorylated p53, checkpoint kinase-1 (Chk1), KRAB domain-associated protein-1 (Kap1), and structural maintenance of chromosome protein-1 (Smc1). Both mESC and EC

responded to IR with an increase in the level of S15 phosphorylated p53, which was not observed in SMC (Fig. 6). Activation of checkpoint kinase-1 by irradiation was only detectable in mESC (Fig. 6). Basal level of p-Chk1, however, was highest in EC. mESC revealed a much stronger ATM-catalyzed S473 phosphorylation of Kap1, which controls DNA repair in heterochromatin (White et al., 2006, 2012), than EC. Again, SMC failed to show elevated protein levels of p-Kap1 in response to irradiation. Regarding the phosphorylation status of Smc proteins, highest levels of pSmc1 were found in mESC, lowest levels in SMC. Taken together, mESC, EC, and SMC revealed large differences in basal and radiation inducible expression of factors related to DNA repair, DDR, and antioxidative defense. Impact of IR on Vascular Differentiation and Function Vascular progenitor cells were irradiated with a single low dose of IR (1 Gy) at Day 4 of the differentiation process (Fig. 7A). Importantly, at this time, the mESC progeny does not yet show mRNA or protein expression of the prototypical endothelial markers Flk-1, Pecam-1, or VE-cadherin (Figs. 7B and 7C). Two

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FIG. 4. Differentiation-dependent alterations in the mRNA expression of susceptibility-related genes. The mRNA expression of a selected subset of genes affecting the sensitivity of cells to genotoxins was analyzed by quantitative RT-PCR using a semi-customized 96-well array as described in Materials and Methods. Shown are

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nuclei being scored per experiment (n ¼ 3, N ¼ 150). A, Representative microscopic images illustrating the formation and disappearance of nuclear cH2AX foci following irradiation of mESC, EC, and SMC. Shown are single cells with cH2AX foci. B, Number of cH2AX foci/nucleus formed without (Control) and after irradiation. The histogram shows the mean 6 SD from 3 independent experiments with 50 nuclei being evaluated per experiment. C, Fold changes in the number of nuclear cH2AX foci formed after irradiation. The relative number of cH2AX foci in the corresponding non-irradiated controls (Con) was set to 1.0. The dashed line indicates the cell linespecific reduction of the maximum number of cH2AX foci by 50%. *P < 0.05 (when compared with mESC). D, Kinetik analysis of cH2AX foci following irradiation of mESC, EC, and SMC. The maximum number of foci observed was set to 100%. The decrease in the number of cH2AX foci observed 4–6 h after irradiation is indicative of DSB repair. *P < 0.05 (mESC when compared with EC and SMC); #P < 0.05 (EC when compared with SMC).

days after irradiation, the expression of endothelial marker proteins and endothelial functionality was analyzed. Irradiation of EC progenitors did not block the expression of the endothelial marker proteins in differentiated EC (Figs. 7B and 7C). Identical results were obtained for SMC (Figs. 7D and 7E). To substantiate these result, we further investigated whether the EC and SMC that were derived from either non-irradiated or irradiated progenitor cells are functional. Regarding EC, LDL uptake and tube formation were not prohibited by irradiation of progenitors (Figs. 8A and 8B). Moreover, Ca2þ-release of SMC following stimulation with carbachol or ATP was also not prevented following irradiation (Fig. 8C). Hence, low dose radiation treatment does not block the differentiation of progenitor cells into functionally competent EC and SMC.

DISCUSSION mESC and thereof derived differentiated progeny are a powerful in vitro tool for toxicological drug assessments in accordance with the 3R principle and, furthermore, for molecular analysis

of differentiation-associated alterations of genetic stability/ instability factors. Genetic stability is a prerequisite for proper lineage-specific differentiation, effective regenerative processes, and the maintenance of cell-type-specific functionality. Most studies available so far have comparatively analyzed mESC and MEFs isolated from isogenic mouse strains. In our study, we generated EC and SMC, both of which are key components of the vascular system, by direct differentiation from mESC. Notably, the EC generated express endothelial-specific marker proteins and respond to exogenous stress (ie, inflammatory cytokines and IR) as reported for primary EC. Furthermore, they show prototypical endothelial functions such as LDLuptake, lectin binding, and tube formation. Likewise we were able to generate SMC from mESC, which, apart from expressing smooth muscle-specific marker proteins, responded to treatment with the muscarinergic agonist carbachol with an substantial Ca2þ release, demonstrating their functional competence (Lachaud et al., 2013). As purinergic signaling plays important roles in promoting migration and proliferation of both vascular smooth muscle and EC via P1 and P2Y receptors

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FIG. 5. Induction and repair of DNA double-strand breaks (DSBs) in mESC and differentiated EC and SMC. 10 minutes (100 ) up to 6 h after irradiation of mESC, EC, and SMC with 1 Gy, the number of nuclear cH2AX foci was analyzed by immunocytochemistry. Data shown are the mean 6 SD from 3 independent experiments with 50

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mESC, EC, and SMC were irradiated with 10 Gy and cells were harvested up to 6 h after irradiation. Protein expression of a selected subset of DNA repair factors (DNA repair), detoxifying factor Hmox-1 (oxidative stress), and the phosphorylation status of proteins involved in the DDR were analyzed by Western blot analysis using the indicated antibodies. In addition, total protein levels of p53 and Chk1 were analyzed. Protein expression of ERK2, b-actin, and GAPDH were determined as protein loading controls.

during angiogenesis and vessel remodeling (Burnstock and Ralevic, 2014), differentiation associated changes in their mRNA expression were analyzed as well. The regulation of the vascular tone by ATP is dual (Burnstock and Kennedy, 1986). For instance, ATP acts on P2X (ion channel) receptors of SMC to stimulate contraction, whereas ATP released from EC acts on endothelial P2Y (G-protein-coupled) receptors to release NO (Burnstock, 1990, 2008). Comparative analysis of the mRNA expression of various purinoceptor subtypes showed complex, both qualitative and quantitative differences between mESC, EC, and SMC. These data further support the bottom line that direct differentiation of EC and SMC from mESC is accompanied by cell-type-specific gain of functions, which enables the differentiated cells an adequate response to purinergic stimuli. Thus, EC and SMC that we have differentiated from mESC harbor key features and functions of corresponding primary endothelial and smooth-muscle cells and, hence, are a particular valid in vitro model for toxicological studies addressing the question of differentiation-dependent alterations in damage defense mechanisms. Quantitative mRNA expression analyses of susceptibility factors that are related to DNA repair, cell cycle regulation, and death revealed that a subset of genes was concordantly downor upregulated in both EC and SMC when compared with mESC. The overlap in the genes that revealed a reduced expression in differentiated EC and SMC is hypothesized to reflect the loss of pluripotency/loss of stem cell character that is linked with the differentiation of mESC. Reduced expression of factors involved in the repair of DSBs, oxidative base damage, and mismatches that we observed in EC and SMC has also been described for mESC when compared with MEFs (Tichy et al., 2010, 2011). On the level of the protein, we observed that the expression of Rad51, a key player in DSB repair through HR, is lower in EC and SMC than in mESC. Reduced protein expression of Rad51 was also reported for MEFs when compared with mESC (Tichy et al., 2010, 2012), supporting the hypothesis that the ability of stem

cells to efficiently repair DSBs by error-free HR generally decreases with differentiation (Stambrook and Tichy, 2010; Tichy et al., 2010). On the other hand, we observed an increased expression of Xrcc4 and Ligase IV specifically in EC, pointing to an EC-specific gain of DSB repair by non-homologous end-joining. The basal expression of the MMR factor Msh2 was reduced in differentiated EC and SMC, which again is in line with other reports showing higher MMR capacity in mESC when compared with MEFs (Roos et al., 2007; Tichy et al., 2011). Apart from factors of DSB repair and MMR, BER seems to be majorly altered during differentiation. For instance, a decrease in the expression of BER factors Ligase I, Ligase III, and Xrcc1 was found in terminally differentiated mouse muscle cells when compared with proliferating cells (Narciso et al., 2007). Increased BER activity and levels of Ligase III, Xrcc1, and Ape were also described in mESC when compared with MEFs (Tichy et al., 2011). In the human system, reduced expression of Fen1 and Ligase III was reported in hESC when compared with fibroblasts (Maynard et al., 2008). On the other hand, differentiation of human monocytes into dendritic cells and macrophages is paralleled by a large increase in the BER factors Xrcc1, Ligase III, and PARP (Bauer et al., 2011; Briegert and Kaina, 2007). It appears that constriction of BER capacity is not imperative for differentiation. In line with this hypothesis, we found a specific downregulation of Ligase I, Fen1, and Xrcc1 in SMC only, but not in EC. This finding supports the note that confinements in BER capacity is not mandatory for differentiation in general, but rather is a lineagespecific phenomenon. Spontaneous differentiation of human stem cells is accompanied by increased oxidative stress, which is thought to result from downregulation of antioxidative functions, notably Sod2 and Gpx2 (Saretzki et al., 2008). Accordingly, we found a decrease in the mRNA expression of antioxidative factors Gpx1, Sod1, and Gstm1 as well as of protein expression of Hmox-1 following differentiation of mESC into EC and SMC. A compensatory gain of specific repair capacity for (mutagenic) oxidative DNA lesions, which might be considered as

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FIG. 6. Basal and IR-induced protein expression of DNA repair factors and activation of the DNA damage response (DDR) in EC and SMC when compared with mESC.

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or were irradiated with 1 Gy (þIR). Expression of EC- and SMC-specific markers was analyzed on the mRNA and protein level at Day 6 (EC) and Day 15 (SMC). A, Schematical illustration of the experimental protocol. B, qRT-PCR-based analysis of the mRNA expression of endothelial marker genes Flk-1, Pecam-1, and VE-Cadherin. Relative mRNA expression of mESC was set to 1.0. Data shown are mean values from a representative experiment performed in triplicate. C, Protein expression of endothelial markers Flk1, Pecam-1, and VE-cadherin was analyzed by immunocytochemistry. Shown are representative images. D, qRT-PCR-based analysis of the mRNA expression of the smooth muscle marker genes calponin (Calp), a-SMA, smoothelin (Smo), and transgelin (Tagln). Relative mRNA expression of mESC was set to 1.0. Data shown are mean values 6 SD from a single experiment performed in triplicate. E, Protein expression of the SMC marker a-SMA was analyzed by immunohistochemistry.

preferential to counteract the accumulation of mutations in differentiated cells under situation of elevated oxidative stress, was not found. Regarding differentiation-related alterations in NER factors, only few data are available so far. For instance, higher expression of Xpa has been reported in fibroblasts when compared with hESC, whereas differences in Xpc levels were not observed (Maynard et al., 2008). In our study, we also noticed a differentiation-related increase in the mRNA expression of the NER factors Xpa and Ercc1 in EC and SMC. On the other hand, we found that the levels of Xpc and Ddb2 mRNA were downregulated in SMC when compared with ESC. The data indicate that NER is subject to complex alterations during vascular differentiation. Analyzing the formation of DSBs, as reflected by nuclear cH2AX foci, we found the highest basal level of cH2AX foci in mESC. As replicative stress is higher in mESC than in differentiated EC and SMC, we assume that the DSBs in mESC result from replication-associated DNA strand breaks. Recently, it was reported that the topoisomerase II-binding protein 1 (TopBP1), which is a key activator of the DDR kinase ATR (Cimprich and Cortez, 2008; Kumagai et al., 2006; Mordes et al., 2008), protects from replicative DNA damage, thereby maintaining genomic integrity of early neuronal progenitors during murine neurogenesis (Lee et al., 2012). Whether TopBP1 is relevant for maintaining the genomic stability of endothelial and smooth-muscle

progenitors is unknown. Inactivation of p53 rescued the disrupted neurogenesis in TopBP1-depleted tissue (Lee et al., 2012). We observed a downregulation of various cyclins, Pcna, and checkpoint kinases in EC and SMC, which is indicative of a decrease in replicative stress after differentiation is completed. The expression of Topo II isoforms and members of the p53 transcription factor family (p53, p63, p73) varied with vascular differentiation. Although Topo IIb was upregulated in EC, expression of Topo IIa was specifically downregulated in SMC. p53, p63, and p73 expression was downregulated following differentiation of mESC into SMC and EC. Following irradiation, EC and SMC showed a stronger increase in the number of cH2AX foci than mESC. The repair of DSBs, as reflected by the time-dependent disappearance of cH2AX foci, was most pronounced in EC. These findings indicate that the differentiated cell types repair IR-induced DSBs more efficiently than mESC. SMC did not respond to irradiation with an increase in p-p53 or p-Kap1 levels, whereas EC and mESC showed this response. The IR-induced rise in p-p53, p-Chk1, and p-Kap1 levels was most distinctive in mESC, pointing to a particular efficient activation of cell cycle checkpoints in stem cells. Overall, the data show that differentiation of mESC into vascular cell types is accompanied by complex, both qualitative and quantitative, alterations in DDR capacities. By contrast, differentiation of immortalized human neural stem cells (ihNSC)

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FIG. 7. IR does not block phenotypical differentiation of EC and SMC from progenitor cells. On Day 4 after withdrawal of LIF (EC (d4)) cells were either left untreated (IR)

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left untreated (IR) or were irradiated with 1 Gy (þIR). Expression of EC and SMC-specific markers was analyzed on the mRNA and protein level at Day 6 (EC) and Day 15 (SMC). The function of the differentiated EC (EC d6) or SMC (day 15) derived from non-irradiated (IR) or irradiated (þIR) progenitors was analyzed as described in Materials and Methods. For experimental protocol, see Figure 7A. A, LDL-uptake in endothelial progenitor cells (EC d4) and thereof derived EC (d6). The red color indicates the presence of LDL. Nuclei were counterstained with DAPI. B, Angiogenic potential of EC (EC d6) differentiated from non-irradiated (IR) or irradiated (þIR) progenitors. Phase contrast images are shown on the left part of the figure. The right panel reveals tube formation after calcein staining of the cells. Pictures were taken 16 h after seeding of the cells on matrigel as described in Materials and Methods. C, Calcium release of SMC that were differentiated from either non-irradiated or irradiated precursor cells following treatment with the vasoactive agonists carbachol (Carba, 0.316 mM) or adenosin-triphosphate (ATP, 10 mM). After incubation period of 30 s, intracellular Ca2þ concentration was determined as described in Materials and Methods. Data shown are mean values from a representative experiment performed in triplicate.

did not result in major alterations in IR-induced phosphorylation of ATM and its substrates Nbs1, Smc1, Chk2, and p53 or the kinetics of cH2AX foci formation (Carlessi et al., 2009). In this human system, neuronal differentiation was accompanied by upregulation of ATM and p53 and downregulation of ATR and Chk1 (Carlessi et al., 2009). Apparently, differentiation-associated variations in DDR facilities are highly species and cell-type specific. Stem cell depletion or inhibition of differentiation is considered as a major mechanism of impaired regenerative capacity of tissues (Lopez-Otin et al., 2013). Therefore, we further asked the question whether irradiation during the early phase of the differentiation process influences the development of vascular progenitor cells into terminally differentiated and functionally competent EC and SMC. IR (1 Gy) did not impact the appearance of functionally competent EC and SMC. Lack of radiation effects were also observed regarding the osteogenic differentiation potential of human MSC (Nicolay et al., 2013). Therefore, threshold radiation doses that block differentiation, for instance by triggering cell death or senescence of progenitor cells, need to be determined in forthcoming studies. Taken together, the data provide evidence that differentiation of mESC into functionally competent EC and SMC is accompanied by both overlapping and cell-type-specific complex alterations (ie, increase and decrease) in DNA repair and DDR capabilities. It is rational to assume that these changes reflect (1) a modified DNA damage defense capacity resulting from the loss of pluripotency and (2) the establishment of cell-type-specific repair/damage defense mechanisms that go along with lineage-specific differentiation. Surprisingly, exposure of progenitors to sublethal genotoxic stress, as provided by low radiation dose, does not impede differentiation into

functionally competent progeny. The results of our study provide novel insight into the complex changes in DNA repair and DDR-related networks that come along with the differentiation of pluripotent mESC into different vascular cell types. Moreover, the findings highlight the potential of mESC-based in vitro differentiation systems for (1) meaningful toxicological drug assessment and (2) thorough characterization of the impact of DNA repair and DDR mechanisms for angiogenesis and vascular functionality under situation of chronic environmental stress.

SUPPLEMENTARY DATA Supplementary data are available online at http://toxsci. oxfordjournals.org/.

ACKNOWLEDGMENTS We thank A. Smith (Department of Biochemistry, University of Oxford, UK) for providing us with the mESC cell line LF2 and A. Vecchi (Institute Mario Negri, Milan, Italy) for the H5V mouse EC line. The authors declare no conflict of interest.

FUNDING Strategic Research Fund of the Heinrich-Heine-University Du¨sseldorf (Germany) (GRK-1921 initiative).

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FIG. 8. IR does not prevent differentiation of functionally competent EC and SMC from progenitor cells. On Day 4 after withdrawal of LIF (EC (d4)) cells were either

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mESC-based in vitro differentiation models to study vascular response and functionality following genotoxic insults.

Because of high exposure to systemic noxae, vascular endothelial cells (EC) have to ensure distinct damage defense and regenerative mechanisms to guar...
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