Isolation and characterization of CD133 + CD34 + VEGFR-2 + CD45 − fetal endothelial cells from human term placenta

Microvascular Research 84 (2012) 65–73

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Isolation and characterization of CD133 + CD34 + VEGFR-2 + CD45 − fetal endothelial cells from human term placenta☆ Elisabeth Sölder a, 1, Barbara C. Böckle b,⁎, 1, Van Anh Nguyen b, Christina Fürhapter b, Petra Obexer d, Martin Erdel c, Hella Stössel b, Nikolaus Romani b, Norbert T. Sepp b a

Department of Obstetrics and Gynaecology, Innsbruck Medical University, Anichstrasse 35, A-6020 Innsbruck, Austria Department of Dermatology and Venereology, Innsbruck Medical University, Anichstrasse 35, A-6020 Innsbruck, Austria Department of Medical Biology and Genetics, Innsbruck Medical University, Anichstrasse 35, A-6020 Innsbruck, Austria d Department of Pediatrics IV, Innsbruck Medical University, Anichstrasse 35, A-6020 Innsbruck, Austria b c

a r t i c l e

i n f o

Article history: Accepted 15 March 2012 Available online 23 March 2012

a b s t r a c t The phenotypes and functions of endothelial cells (EC), a heterogeneous cell population, vary along the vascular tree and even in the same organ between different vessels. The placenta is an organ with abundant vessels. To enhance further knowledge concerning placenta derived EC, we develop a new method for isolation, purification and culture of these EC. Moreover, in order to investigate the peculiarity of placenta derived EC we compare their phenotypic and functional characteristics with human dermal lymphatic endothelial cells (HDLEC) and human umbilical vein endothelial cells (HUVEC). Freshly isolated placenta derived EC displayed an elongated shape with pale cytoplasm and showed the typical cobblestone pattern of EC but also a swirling pattern when confluent. FISH-analyses of the isolated EC from placentae of male fetus revealed an XY genotype strongly indicating their fetal origin. Characterisation of placenta derived fetal EC (fEC) underlined their blood vessel phenotype by the expression of vWF, Ulex europaeus lectin-1, HLA-class I molecules, CD31, CD34, CD36, CD51/61, CD54, CD62E, CD105, CD106, CD133, CD141, CD143, CD144, CD146, VEGFR-1, VEGFR-2, EN-4, PAL-E, BMA120, Tie-1, Tie-2 and α-Tubulin. In contrast to previous reports the expression of lymphatic markers, like VEGFR-3, LYVE-1, Prox-1 and Podoplanin was consistently negative. Haematopoietic surface markers like CD45 and CD14 were also always negative. Various functional tests (Dil-Ac-LDL uptake, Matrigel assay and TNF-α induced upregulation of CD62E and CD54) substantiated the endothelial nature of propagated fEC. At the ultrastructural level, fEC harboured numerous microvilli, micropinocytic vesicles at their basis, were rich in intermediate filaments and possessed typical Weibel - Palade bodies. In conclusion, the placenta is a plentiful source of fetal, microvascular, blood EC with an expression profile (CD34+, CD133+, VEGFR-2+, CD45-) suggestive of an endothelial progenitor phenotype. © 2012 Elsevier Inc. All rights reserved.

Introduction Endothelial cells (EC), the inner lining of blood and lymphatic vessels, have many functions and play a central role in the maintenance of blood fluidity (control of coagulation and thrombolysis), vascular tone, permeability, inflammation, tissue repair, angiogenesis and vasculogenesis. Remarkably, although EC share certain common functions, they also display heterogeneity in structure and function within the same species and occasionally in the same organ as depending on the vascular bed of origin and the vessel size (Aird, 2007; Yano et al., 2007). Placental vasculature represents

☆ The manuscript has not been published previously and is not under consideration for publication elsewhere. ⁎ Corresponding author. Fax: + 43 512 504 22967. E-mail address: [email protected] (B.C. Böckle). 1 These authors contributed equally to this article. 0026-2862/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.mvr.2012.03.005

a distinct territory and therefore culturing placenta derived fetal endothelial cells (fEC) provides a system for studying the role of these cells in the complex activities of the placenta, an organ, which has recently come into the focus as a potential source of progenitor cells and therefore a reservoir of cells for regenerative medicine (Evangelista et al., 2008). Besides that, recent studies revealed conflicting observations concerning the expression of lymphatic-associated markers by placenta derived fEC (Bockle et al., 2008; Gu et al., 2006; Wang et al., 2011). These include VEGFR-3, the tyrosine kinase receptor for vascular endothelial growth factor VEGF-C and VEGF-D; (Kaipainen et al., 1995) podoplanin, a glomerular podocytes membrane mucoprotein (Weninger et al., 1999) and a hyaluronan receptor LYVE-1 (Banerji et al., 1999). Because of its high degree of vascularisation the human placental tissue is a plentiful and readily available source of fEC (Challier et al., 1995; Drake and Loke, 1991; Jinga et al., 2000; Kacemi et al., 1997;

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Lang et al., 2003, 2008; Leach et al., 1994; Schutz and Friedl, 1996). This prompted us to develop an improved method for isolation, purification and culture of fEC from human term placenta in order to characterise in detail these fEC. Hence, we compared in the present study the phenotypic and functional properties of placenta derived fEC with human dermal lymphatic endothelial cells (HDLEC) and human umbilical vein endothelial cells (HUVEC). Materials and methods Isolation and culture of human fEC from human placenta Full term placentae (n = 12; clinically uncomplicated pregnancies undergoing an optional caesarean section at term) were collected under sterile conditions after obtaining written informed consent from patients according to the guidelines of the local ethical committee of Innsbruck Medical University. Samples were generally processed within 24 h of collection. After removal of decidua, fetal membranes and the chorionic plate, finely minced specimens from fresh placental cotyledons were repeatedly washed in Hank's buffered salt solution (HBSS) (Invitrogen-Gibco, Paisley, Scotland) to remove residual blood. Specimens were then incubated overnight in 1.2 U/ml dispase II (Roche Applied Science, Basel, Switzerland) per milliliter Puck's solution (Sigma Chemicals, St. Louis, MO) at 4 °C. Afterwards, specimens were gently pressed through syringes as described for the isolation of HDLEC (Nguyen et al., 2009). Released EC were plated in tissue culture flasks (Greiner Bio One, Frickenhausen, Germany) that had been pre-coated with 0.1% gelatin (Sigma) and

cultured in EC basal medium (EBM; Clonetics Corp., Walkersville, MD, USA) supplemented with 20% normal heat inactivated human serum (Biologica, Atlanta, GA), 5 ng/ml epidermal growth factor (EGF; Clonetics Corp.), 1 μg/ml hydrocortisone acetate, 50 μM dibutyryl cyclic adenosine monophosphate (Sigma), 2-mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 250 μg/ml amphotericin B (all purchased from Irvine Scientific, Santa Ana, CA, USA) without addition of further angiogenetic growth factors at 37 °C with 5% CO2 in a humidified atmosphere. At day one after plating, the non-adherent cells were removed and fresh EBM medium was applied. To maintain optimal culture conditions, media were changed every third day. After one day small colonies of EC were detected. Cells had an elongated shape and pale cytoplasm. 2 weeks of primary culture resulted in a monolayer of cells. ECs from primary cultures were purified using immunomagnetic beads recognizing CD31, according to the manufacturer's protocol (Dynabeads, Invitrogen-Dynal). Cells were passaged and were grown in media. For morphological, immunophenotypic and functional analyses, fEC at passage 3 (on average at 8 weeks of culture) were used. Isolation and culture of HDLEC and HUVEC HDLEC were isolated as described previously (Nguyen et al., 2009). HUVEC, a gift from Dr. G. Wick, were isolated, cultured and assessed by immunofluorescence as described by Sgonc et al. (2000). The resulting cultures were consistently pure without contaminating fibroblasts as assessed by morphological and immunologic criteria. HDLEC and HUVEC were cultured through passage 3.

Table 1 Monoclonal and polyclonal antibodies. Antibody specifity PECAM-1 Thrombospondin receptor Leukocyte common antigen Vitronectin receptor ICAM-1 E-Selectin P-Selectin Macrophages Endoglin VCAM-1 Prominin-1 Angiotensin-converting enzyme VE cadherin Melanoma cell adhesion molecule VEGFR-1 (Flt-1) VEGFR-2 (KDR/Flk-1) VEGFR-3 (Flt-4) PAL-E EN4 HLA class I HLA class II Thrombomodulin Podoplanin LYVE-1 Prox-1 von Willebrand Factor (vWF, FVIII) Glut-1 Tie-1 Tie-2 BMA120 Ulex europaeus lectin-1 Vimentin Smooth muscle actin Fibroblast

CD14 CD31 CD34 CD36 CD45 CD51/61 CD54 CD62E CD62P CD68 CD105 CD106 CD133 CD143 CD144 CD146

Clone

Isotype dilution

rmC5-3 WM-59 581 SMO 4B2/HB-196 23C6 HA58 1.2 B6 1E.3 KP1 266 1.G11B1 AC133 mca2055 55-7H1 S-Endo 1 Polyclonal Monoclonal Monoclonal

IgG1 IgG1 IgG1 IgM IgG2a IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 rabbit IgG1 goat IgG IgG2a IgG2a IgG1 IgG2a IgG1 IgG1 IgG1 rabbit serum rabbit serum rabbit serum IgG1 rabbit serum rabbit rabbit IgG1 rabbit serum IgG2a IgG2a IgG1

PV-1 G46-2.6 G46-6 55-7H1 gp36 D2-40 Polyclonal Polyclonal Polyclonal F8/86 Polyclonal Polyclonal Polyclonal Monoclonal Polyclonal Vim3B4 1A4 5B5

Source 1:5 1:5 1:5 1:20 1:10 1:20 1:25 1:25 1:25 1:25 1:20 1:25 1:25 1:10 1:25 1:10 1:200 1:100 1:50 1:10 1:10 1:5 1:5 1:25 1:100 1:10 1:100 1:400 1:100 1:50 1:50 1:200 1:200 1:10 1:100 1:10 1:50 1:200

BD Pharmingen, San Diego, CA BD Pharmingen, San Diego, CA BD Pharmingen, San Diego, CA Ancell Corp., Bayport, MN ATCC BD Pharmingen, San Diego, CA Dako, Glostrup, Denmark Dako, Glostrup, Denmark Dako, Glostrup, Denmark Dako, Glostrup, Denmark BD Pharmingen, San Diego, CA Ancell, Bayport, MN Miltenyi Biotec, Bergisch Gladbach, Germany Serotec, Oxford, UK BD Pharmingen, San Diego, CA Alexis Biochemicals, Vienna Austria, Santa Cruz Biotechnology, Inc., CA Santa Cruz Biotechnology, Inc., CA R & D Systems, Inc., Tustin, CA Harlan Seralab Longborough, England Harlan Seralab Longborough, England BD Pharmingen, San Diego, CA BD Pharmingen, San Diego, CA Dako, Glostrup, Denmark RDI, Research Diagnostics, Inc, Flanders Convance RDI, Research Diagnostics, Inc, Flanders DCS Hamburg, Germany RDI, Research Diagnostics, Inc, Flanders Dako, Glostrup, Denmark Abcam United, Cambridge, UK Santa Cruz Biotechnology, Inc., CA Santa Cruz Biotechnology, Inc., CA Behring, Vienna Austria Abcam United, Cambridge, UK Dako, Glostrup, Denmark Dako, Glostrup, Denmark Abcam United, Cambridge, UK

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Characterization of fEC

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visualised on a conventional fluorescence microscope. HDLEC served as control.

Flow cytometric analysis of surface molecules on fEC The panel of antibodies listed in Table 1 was used to determine the levels of expression of surface molecules on human fEC. EDTAdetached fECs were harvested by centrifugation, washed in PBS with 0.5% BSA (Boehringer Ingelheim, Germany), counted and aliquoted at 100.000 cells/tube for antibody staining. Unconjugated or FITC-/PE-conjugated antibodies at a concentration of 1 μg/ml were incubated on ice with the cells. After 30 minutes incubation, cells were washed in PBS with 0.5% BSA. For indirect fluorescent staining, cells were further incubated with PE-labeled F(ab)2 fragments of sheep anti-mouse IgG for 30 min, washed as above and resuspended in PBS with 0.5% BSA. Negative controls were stained with isotypematched IgGs. Surface marker expression was examined in a FACS Calibur flow cytometer (Becton Dickinson, Mountain View, CA) measuring a total of 5.000 to 10.000 events per aliquot. Cytokine stimulation of fEC Confluent cultures of fEC were stimulated with TNF-α (PeproTech, London, UK) at a concentration of 500 U/ml for 4 h. Thereafter, surface molecule expression of CD54, CD62E and CD31 was examined by flow cytometry (see above). Immunofluorescent staining of fECs on cytospin preparations After transferring cells to glass slides by cytospin centrifugation, they were air-dried for at least 24 h, fixed with acetone and subsequently incubated with human IgG (Beriglobin P™, Aventis Behring; Beringwerke Marburg, Germany; final concentration 0.6 mg/ml) for blocking Fc-receptors. Afterwards, we applied the diluted unconjugated primary antibodies (Table 1) and proceeded as described. [5] For negative controls, the primary antibodies were replaced by isotype-matched immunoglobulins of irrelevant specificity and at equal concentrations. Finally, cells were mounted in Vectashield (Vector Laboratories, Burlingame, CA) and viewed on a conventional fluorescence microscope (OLYMPUS BX 60, Japan). Staining cytospins with various antibodies was additionally controlled by applying the same antibodies on placenta tissue specimens or human skin specimens. Dil-Ac-LDL uptake Acetylated low density lipoproteins labelled with 1,1-dioctadecyl3,3,3′,3′tetramethylin-docarbocyanine perchlorate (DiI-Ac-LDL) (Invitrogen/Molecular Probes Inc., Eugene, OR, USA) was diluted to 2 μg/ml in the culture medium, added to the cells grown in Labtec™ chambers and incubated for 4 h at 37 °C. After washing in PBS, the cells were rinsed in distilled water, mounted in Vectashield and fluorescence was

Matrigel-assay (in vitro capillary tube formation) Matrigel basement membrane matrix (Becton Dickinson, San Jose, CA, USA) was thawed at 4 °C overnight and was mixed with cooled pipettes to homogeneity. Matrigel was added to 48-well plates in a volume of 150 μl and allowed to solidify at 37 °C for 30 min. 100 000 cells were added to each well and incubated at 37 °C with humidified 95% air/5% CO2 for 24 h. Thereafter, the medium was removed and capillary tube formation of vessel-like tube in Matrigel was observed under an inverted microscope.

Western blot analysis Identical numbers of HUVECs, HDMEC and fEC were lysed on ice in CelLytic™-M Mammalian Cell Lysis/Extraction Reagent (Sigma Chemicals) containing a protease inhibitor cocktail (Sigma Chemicals) and centrifuged at 14000 rpm. Protein concentration was determined with “Protein Reagent” (Bio-Rad Laboratories, Hercules, CA). The supernatant was then mixed with 4 x SSB containing 20% ß-mercaptoethanol and boiled. Samples were separated with SDS-PAGE on 7.5-10% polyacrylamide gels, and transferred to nitrocellulose membranes (Whatman-Schleicher & Schuell, Dassel, Germany) by a NOVEX blotter apparatus. The membranes were blocked with PBS blocking buffer containing 0.1% Tween20 and 5% nonfat dry milk, incubated with primary antibodies specific for Tie-1, Tie-2, VEGFR-1, VEGFR-2, Podoplanin, VEGFR-3 (Table 1) and α-Tubulin (Calbiochem/Oncogene Research, Cambridge, MA, USA), washed and incubated with antimouse or anti-rabbit horseradish-peroxidase-conjugated secondary antibodies (GE Healthcare, Piscataway, NJ, USA). The blots were developed by enhanced chemoluminescence (GE Healthcare, USA) according to the manufacturer's instructions and analyzed with the chemoluminescence system of ultraviolet laboratory products (UVP). To demonstrate equal protein loading, the same blot was reprobed with anti-α-Tubulin antibody. Transmission electron microscopy fEC were grown on gelatine-coated coverslips and fixed in a mixture of 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1-M cacodylate buffer (pH 7.4) for 15 min. at room temperature. After washing in cacodylate buffer (15 min), the samples were post-fixed in OsO4 for 15 min, contrasted en bloc with 0.5% veronal-buffered uranyl acetate and dehydrated in ascending concentrations of ethanols followed by embedding in Epon 812 resin. Ultrathin sections were stained with lead citrate and viewed in the transmission electron microscope at 80 kV (Philips EM400, FEI Company, Eindhoven, The Netherlands).

Fig. 1. In vitro growth pattern of cultured placenta-derived fEC examined by phase contrast light microscopy; (A) 1 day after isolation from human placenta: besides other placental cells small fEC colonies with cells demonstrating pale cytoplasm were observed; (B) cobblestone and swirling pattern of confluent monolayers of fEC after 4 weeks of culturing; Original magnification: × 20.

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Fluorescence In Situ Hybridization Analysis (FISH) for human X/Y chromosomes Cells grown in flasks were detached and suspended by trypsin treatment. After hypotonic treatment, fixation was effected and slides were made using standard protocols (Clouston, 2008). For FISH analysis placentae of male fetus were used in order to distinguish between maternal EC and fEC. FISH detection of gender was performed with the use of the directly labelled dual colour chromosome X/Y satellite cocktail probe (Qbiogene, France), following the manufacturer's instructions. At least 200 interphase nuclei were observed on a Zeiss Axioplan 2 microscope (Oberkochen, Germany) using appropriate single and dual band pass filters set to detect the amount of normal male and female cells. Results Isolation and culture of fEC from human placenta 24 hours after isolation, in addition to other placental cells, small colonies of large fEC with typical pale cytoplasm were observed by phase contrast microscopy (Fig. 1A). After culturing for 2 weeks, confluent cultures of fEC were obtained. In the next step, fEC were purified with immunomagnetic beads recognizing CD31. Purified fECs were again plated, grown in media and passaged. Morphological analysis using phase contrast light microscopy revealed a cell population following 2–3 weeks of culture. They showed an elongated, spindle and sometimes also a polygonal shape and confluent fEC cultures displayed a swirling cell morphology varying with the typical cobblestone pattern (Fig. 1B) known from HDLEC and HUVEC (Nguyen et al., 2009). Characterization of fEC In order to determine the phenotype of fEC, we performed flow cytometry and immunofluorescence analysis using a selected set of markers and compared it to that obtained in HDLEC and HUVEC. Flow cytometric analysis of surface antigens Purified, cultured fEC expressed common endothelial cell markers like CD31, CD34, CD36, CD105, CD133, CD144 and PAL-E (Fig. 2). In addition, fEC were positive for CD51/61, CD54, CD62E, CD106, CD141, CD143, BMA120 and EN4 (data not shown). The EC phenotype of fEC was further associated with the expression of HLA class I antigens, but not with HLA class II antigens (Fig. 2). Moreover, fEC expressed VEGFR-2, but showed consistent lack of VEGFR-1 and VEGFR-3 expression (Fig. 2). As expected fEC cultures did not contain monocytes or macrophages as attested by the absence of CD14, CD68 (data not shown) and HLA class II molecules. These CD34 + VEGFR-2+ CD133+ fECs were constantly negative for common leukocyte antigen CD45 (data not shown). Several cell surface endothelial markers were similarly expressed on HUVEC and fEC. Among them were CD31, CD34, CD105, CD144, PAL-E, VEGFR-2 and HLA class I molecules. Both, HDLEC and fEC were positive for microvascular marker CD36, whereas macrovascular HUVECs were expectably negative. Interestingly, CD133 was still present on the surface of fEC and HDLEC. However, HUVEC lacked expression of CD133 (Fig. 2). Immunofluorescent characterization of fEC on cytospin preparations and LDL-uptake The phenotype of fEC was further characterised using immunofluorescence (Fig. 3). fEC were positive for common endothelial markers like: Ulex europaeus-lectin-1 displaying a characteristically homogenous staining pattern (Fig. 3A), vWF exhibiting a perinuclear

Fig. 2. Histograms of flow cytometry analyses demonstrating the expression of surface molecules of placenta-derived fEC compared with HDLEC and HUVEC. The grey histograms show the region of fluorescent intensity of the specific antibody and bold empty histograms represent staining of respective isotype-matched control immunoglobulins.

granular staining pattern counterstained with CD31 (Fig. 3B) and even melanoma cell adhesion molecule CD146 counterstained with vWF (Fig. 3E). Moreover, analyses on cytospins revealed that CD31 positive fECs were additionally positive for vimentin (Fig. 3D). Therefore, placenta derived fECs cultures did not contain trophoblasts as attested by the expression of vimentin. The lymphatic endothelial cell-specific markers LYVE-1 (Fig. 3C), Prox-1 and Podoplanin were always negative. Furthermore, cytospins containing cultured fEC continuously lacked leukocyte common antigen CD45 (Fig. 3F) and contamination with smooth muscle cells and fibroblasts was

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Fig. 3. Immunfluorescence stainings of cytospin preparations of placenta-derived fEC. Cells were positively stained for Ulex-europaeus lectin-1 (A), double labelling of fECs with granular staining pattern of vWF (green) and homogenous staining pattern of CD31 (red) (B), fEC were negative for the lymphatic cell-specific marker LYVE-1 (C); (D) doublelabelling for CD31 (green) and vimentin (red): CD31 positive fEC co-express vimentin (merge — orange); (E) fEC were positively stained for vWF (red) with a granular immunofluorescence and bright homogenous staining with CD146 (green) and no staining was observed for CD45 (F); original magnifications: ×40 (A–C, F), ×20 (D), ×100 (E).

excluded using anti-α-smooth muscle actin and fibroblast antibodies, respectively. Acetylated low density lipoproteins labelled with 1.1-dioctadecyl-3.3.3′.3′tetramethylin-docarbocyanine perchlorate (DiI-Ac-LDL) were incorporated into all cells which designates another EC function (Fig. 4.3). Dil-Ac-LDL uptake was also observed in HDLEC and HUVEC.

markers like Tie-1 (140kD), Tie-2 (126kD), VEGFR-1 and VEGFR-2 (195/ 235kD). fEC and HUVEC lacked expression of podoplanin (95kD) and VEGFR-3 (64kD). These lymphatic markers were observed in HDLEC which served as a positive control for both lymphatic markers. Additionally, these results suggest that fEC and HUVEC show no phenotypical features of lymphatic endothelial cells (Fig. 5).

Cytokine stimulation of fEC

Ultrastructural findings

TNF-α stimulation (500 U/l for 4 hours) resulted in an obvious upregulation of CD62E and CD54, but not of CD31 (Fig. 4.1), a characteristic feature of EC (Sepp et al., 1994). Treatment with HDLEC and HUVEC with TNF-α resulted in a similar dose and time response.

Cells of variable sizes were found; some cells were extraordinarily large. They were often polarized in that a dense network of villi protruded from one pole of the cell. Sometimes the main cell body and the villous part were separated by a "neck". At the basis of these villi micropinocytic vesicles could be detected. Sometimes they were abundant thus resembling dermal endothelial cells in situ. However, they were only found in substantial numbers in these particular cells; they did not occur, lined up one after the other, along extended stretches of membrane such as in EC in situ. Cells possessed large nuclei that were deeply indented one- or several fold, again similar to EC in situ. Most of the cells contained many autophagic vacuoles filled with small membrane vesicles (reminiscent of multivesicular bodies) and often large membrane whorls (myelinoid figures). Occasionally medium to small lipid droplets occurred, presumably a consequence of the long culture period. The cells were

Matrigel assay The Matrigel assay displayed another functional test of typical EC behaviour. Within 24 hours of incubation fEC formed a network of capillaries on a Matrigel-coated surface (Fig. 4.2). Western blot analysis Western blot studies of fEC and HUVEC, the latter as control cells, revealed in both cell types positive bands for characteristic endothelial

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Fig. 4. Functional characterisation of fEC; Fig. 4.1: Upregulation of surface molecules on placenta-derived fEC in response to TNF-α. Stimulation with TNF-α at 500 U/ml for 4 h (c,d) resulted in an upregulation of CD62E (compare a versus c), whereas CD31 surface expression on fEC remained unchanged (compare b versus d). Fig. 4.2.: Matrigel-assay: within 24 hours fEC formed netlike capillary structures on matrigel. Original magnification: × 20 Fig. 4.3.: Dil-ac-LDL uptake: placenta-derived fEC internalized fluorochrome-conjugated Dilac-LDL. Original magnification: × 100.

rich in ribosomes, both as free ribosomes and as ribosomes attached to the endoplasmic reticulum. Intermediate filaments were easily detected. Sometimes they were arranged in the parallel, "spaghetti-like" fashion as known from EC in situ. Importantly, unequivocal Weibel-Palade bodies were readily found in roughly half of the inspected cell profiles (Fig. 6).

FISH-analysis FISH analysis using X and Y chromosome probes was performed in order to confirm the fetal origin of cultured EC. Genetic analysis of the purified EC derived from placentae from male fetus revealed that more than 97% of the cells after passage 3 displayed an XY genotype, strongly indicating that propagated EC originate from fetal but not maternal tissue (data not shown).

Discussion Here, we present a systematic investigation of the phenotypic, morphologic and functional characteristics of placental fEC compared with microvascular HDLEC and macrovascular HUVEC. In contrast to previous techniques isolating EC from human placenta (Challier et al., 1995; Drake and Loke, 1991; Jinga et al., 2000; Kacemi et al., 1997; Leach et al., 1994; Schutz and Friedl, 1996), we incubated fine specimens of cotyledons with dispase overnight and squeezed the cells out of the vessels - a technique known from isolating HDLEC from foreskins (Nguyen et al., 2009). Using placentae from mothers with male fetus followed by FISH analysis gave evidence that the isolated fECs were exclusively of fetal origin. The performance of this simple isolation technique was evaluated by observing the cell morphology and especially by exposing fEC to several functional tests. Placenta derived fEC take up DiI-Ac-LDL and

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Fig. 5. Western blot analysis of fEC, HDLEC and HUVEC; column 1: fEC, column 2: HDLEC, column 3: HUVEC; lane a: Positive bands recognizing podoplanin were seen in HDLEC, but not in fEC and HUVEC. Lane b: VEGFR-1 was increasingly expressed by HUVEC in contrast to fEC and was not detected in HDLEC. Lane c, lane d and lane g: Endothelial markers like Tie-1, Tie-2 and !-Tubulin were expressed in all types of endothelial cells. Lane e: VEGF-3 expression was restricted to HDLEC. This marker was completely absent in HUVEC and fEC. Lane f: VEGFR-2 is more pronounced expressed in HDLEC and fEC than in HUVEC. Lane g: α Tubulin was used as loading control.

showed positive Ulex europaeus-1 agglutinin binding. However, the specifity of these tests has been discredited (Graziano et al., 2001; Suzuki et al., 1990). Therefore, further tests like Matrigel assay and TNF-α induced upregulation of CD62E and CD54 (Wiese et al., 2009) confirmed EC identity of our propagated fEC. A contamination with trophoblasts, fibroblast cells, macrophages/ monocytes or smooth muscle cells was excluded because staining with specific antibodies revealed negative results. According to previous results (Lang et al., 2003) fEC show morphologically a spindle cell shape and confluent monolayers display a characteristic swirling cell morphology as well as a polygonal cell shape resulting in classical cobblestone morphology. These results are in accordance with the findings of Schutz et al. (Schutz and Friedl, 1996) and reflects another feature in order to distinguish fEC from other EC like HDLEC or HUVEC that show only the typical cobblestone pattern. By transmission electron microscopy, we identified Weibel-Palade bodies within fEC which is another typical feature of EC. Remarkably, fEC exhibited numerous microvilli at the cell surface and at the basis of these villi micropinocytic vesicles could be detected. This corroborates the important metabolic activity of placenta derived fEC. Concerning the phenotypic characteristics fEC shared phenotypic similarities as well as differences with the two other compared EC types, indicating the phenotypic heterogeneity of these placenta specific fEC. In fact, fEC expressed a surface molecule profile investigated

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by FACS analysis of blood EC-specific antigens, including CD34, CD105 and PAL-E (Keuschnigg et al., 2009). Accordingly, lymphatic cell specific marker VEGFR-3 was not detected in fEC in contrast to HDLEC. In addition, western blot analysis supported blood vascular phenotype of our fEC by the expression of Tie-1, Tie-2, VEGFR-1 and VEGFR-2 and by the lack of lymphatic markers Podoplanin and VEGFR-3. Wang et al. (2011) found VEGFR-3 protein expression in tissue homogenate samples of human term placenta as well as VEGFR-3 expression in both villous core fetal vessel EC and trophoblasts. In contrast to Wang et al. immunofluorescence stainings of our fEC cytospins as well as placental tissue specimens (unpublished own results) revealed always negative results for VEGFR-3. Immunfluorescence stainings of cytospin preparations further sustained the blood endothelial phenotype of fECs. According to our previous results on placental tissue specimens (Bockle et al., 2008) LYVE-1, Prox-1 and Podoplanin was also not observed on cultured fEC after adequate blocking of placental Fc-receptors (Honig et al., 2005). This observation is in sharp contrast to the report by Gu et al. (2006), who demonstrated expression of LYVE-1 and Prox-1 by fEC of placentae and syncytiotrophoblast. Such observed discrepancies may arise from the variability of antibodies and methods, which were used. One explanation for the different results may be the peculiarity of human placenta. Numerous Fc-receptors were expressed by fEC and by the syncytiotrophoblast. Therefore, inadequate or absent blocking may lead to unspecific binding of antibodies to placental Fc-receptors (Honig et al., 2005). Swerlick et al. used CD36 for the first time in order to discriminate macrovascular from microvascular ECs. Accordingly, HUVECs do not express CD36 in vitro and in vivo. The consistent surface expression of CD36 on fECs, like on HDLEC, suggests a microvascular origin (Swerlick et al., 1992). Together with the lack of expression of VEGFR-1 on the isolated cells by flow cytometer and the results of Western blot analysis which demonstrated low VEGFR-1 expression on fEC in contrast to the huge expression of VEGFR-1 in HUVEC, we emphasize in accordance to previous results (Lang et al., 2003) that the isolated fEC are different to macrovascular HUVEC. Investigations with flow cytometer revealed that fECs expressed CD34, VEGFR-2 and CD133. Interestingly, the latter molecule was also expressed by HDLEC but not by HUVEC. In 1997, Asahara et al. (1997) proposed the new concept of neovasculogenesis and endothelial progenitor cells (EPCs). Since then, lots of EPC-related articles have been published and the definition, identification, characterisation and the role of EPC remain controversial (Timmermans et al., 2009). Because CD34 + VEGFR-2+ phenotype may represent mature EC, Peichev et al. suggested the addition of CD133 to differentiate EPC from mature EC (Peichev et al., 2000). Through the process of differentiation into mature EC, EPC down-regulate the expression of CD34 and CD133 and continuous culturing increases the expression of mature EC markers (Nguyen et al., 2009). Interestingly, during cultivation of our placental derived fEC CD133 and CD34 expression was not lost and simultaneously fEC expressed mature EC markers. In fact, CD34 + VEGFR-2+ CD133+ were recently shown to be haematopoietic progenitors instead of circulating EPC (Case et al., 2007; Timmermans et al., 2009). A haematopoietic origin of the isolated CD133 + fEC was excluded due to the lack of expression of CD45. However, mature EC also lack CD45 (Khakoo and Finkel, 2005). Moreover, CD133+ fEC showed positive vimentin staining. This is important since CD133 may be also expressed by trophoblasts that are vimentin negative (Potgens et al., 2002). According to more recent reports two distinct subpopulations of EPC were discriminated (Sipos et al., 2010): the hematopoietic subpopulation (circulating angiogenetic cells - CAC) and the endothelial colony forming cells (ECFC). The latter share many features with our placental derived CD34 + VEGFR-2 + CD133 + CD45- fEC because they have more endothelial-like phenotypic characteristics as well as their functional behaviour (form capillary-like tubes, incorporate

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Fig. 6. Ultrastructure of placenta-derived cultured fEC. Low power views (A–C) show the polarized appearance of the cells. Abundant microvilli are confined to one area of the cell surface. Panel (D) depicts the often encountered organelles that presumably represent autophagic vacuoles. Areas filled with densely packed intermediate filaments (E) and an abundance of pinocytotic vesicles (F) are typical for EC. Several examples of Weibel–Palade bodies, the defining organelles for EC, are marked with asterisks in panels (E) and (G–I). Magnifications and scale bars: 4.500 ×/2 μm in A–C; 17.000 ×/1 μm in D,F; 32.000 ×/100 nm in E; 51.000 ×/100 nm in G,I; 55.000 ×/100 nm in H.

DiI-Ac-LDL and stain with UAE-1, vWF) (Sipos et al., 2010). Notably, previous reports observed expression of development-associated genes by fEC and demonstrate an adipogenic and osteogenic differentiation potential of fEC (Lang et al., 2008). In accordance with these findings we speculate that placenta derived fEC may display tissue resident endothelial progenitors. To summarize, our data suggest that fEC isolated from human placentae are microvascular (CD36+), fetal (FISH-analyses) cells with distinct features of progenitor cells (CD133+, CD34+, VEGFR-2+) committed to differentiate into EC (all EC-markers are positive as well as their functional behaviour). Human placenta, a readily available organ with a high degree of vascularisation, may therefore be an ideal and abundant source for ECFC for the purpose of cell culture. They may serve as a tool to readily study progenitor function with regard to their cell development, their pivotal role in pregnancy diseases like eclampsia, growth retardation and their relation to other progenitor cells of different sources. Moreover, these fECs may be used as a substrat for diagnostic procedures, e.g. detection of antiendothelial cell antibodies. Further investigations of their biological behaviour might be important for several diseases, especially preeclampsia and fetal distress syndrome.

Acknowledgments This work was supported by the Tyrolean Provincial Hospital Company (Tilak Ges.m.b.H.) and the Children's Cancer Research Institute (CCRI). The authors confirm that there are no conflicts of interest.

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