S3 includes data from chemical- and immune-mediated chronic liver injury models. Arrows indicate YFP+ cells. Write a Review. S3 C). Nevertheless, they play a central role in maintaining tissue homeostasis (Hu et al., 2014; Rafii et al., 2016; Augustin and Koh, 2017). (NO AWARD), Seventh Framework Programme S1, F and G), indicating that both cell populations possess a similar proliferation capacity. For gene expression analysis, microarrays were performed by the German Cancer Research Center Genomics Core Facility. Similar findings have recently been reported during large vessel regeneration in an aortic injury model (McDonald et al., 2018). Author contributions: M. Singhal, X. Liu, H.G. 3 C and Fig. Welcome back. CAG-GFP (chicken β-actin promoter and cytomegalovirus enhancer regulate expression of enhanced GFP) mice were purchased from the Jackson Laboratory. Your job seeking activity is only visible to you. S2 D). This causes an early cytopathic effect on hepatocytes and a secondary adaptive immune response against infected hepatocytes during later stages of the experiment, as illustrated by enhanced plasma ALT/AST values (Fig. Approval for experiments involving animals was granted by the local Animal Welfare Committee (Regierungspraesidium Karlsruhe, Germany, reference numbers A‐38/11, G‐221/12, G‐296/14, and G‐217/18). Save job. D. Inverso and M. Singhal performed Imaris-based image analysis. Karlsruhe was an old small ship, but in those days, every unit capable of evacuating people to the west was important. Further, flow cytometry analysis revealed no significant changes in the frequencies of YFP+ liver ECs between mice treated with either oil or CCl4 (Fig. Consequently, transplantation of healthy bone marrow into a preirradiated host resulted in BMDMC recruitment and incorporation as bona fide liver ECs to restore the injured endothelium. Fc receptors were blocked by incubating cells in 5% FCS with purified mouse IgG (500 mg/ml; Jackson ImmunoResearch). Augustin); the European Research Council Advanced Grant “AngioMature” (787181 to H.G. All animal experiments were approved by the local animal ethics committee (Regierungspräsidium Karlsruhe; Ministerium für Landwirtschaft, Umwelt und ländliche Räume, Kiel, Germany). 4 A), i.e., before proliferation of liver ECs. These compelling preclinical observations have stimulated clinical stem cell therapeutic approaches for patients with end-stage liver disease. Experiments involving animals were performed in accordance with the guidelines and regulations set forth by the German Animal Welfare Act and the Regierungspräsidium Karlsruhe (Germany). Augustin from Regierungspräsidium Karlsruhe, Germany). S1 E). Arrowheads indicate GFP+ hematopoietic cells in the shielded liver. To quantitatively evaluate whether BMDMCs incorporate into the liver vasculature following irradiation, GFP+ bone marrow cells were transplanted into lethally irradiated recipients. These data recapitulate the observations from the REALISTIC trial, as neither direct infusion of bone marrow cells in NSG mice nor G-CSF–mediated mobilization of progenitor cells in WT mice showed any BMDMC contribution to the regenerating liver vasculature. Regierungspräsidium Karlsruhe Karlsruhe, Baden-Württemberg, Germany 3 weeks ago Be among the first 25 applicants. 3 D). To this end, we applied a fate mapping strategy with VECad-CreERT2xRosa26-YFPfl/fl mice, in which transient tamoxifen administration resulted in permanent YFP labeling of the adult vasculature (Fig. 4 B). Quantitative PCR analysis revealed higher expression of Bax, an apoptotic activator, in livers of irradiated but not partially hepatectomized mice compared with nonirradiated and partially hepatectomized mice (Fig. (D) Representative images of liver sections of control or irradiated mice costained with cleaved caspase-3 (CC3) and CD31 (EC-specific surface marker). Scale bars, 25 µm. Bone marrow chimeric mice (Fig. All animal procedures were approved by the Regierungspräsidium Karlsruhe (AZ 35-9185.82/A-2/06, AZ53-9185.81/G-32/03). Additional labeling of proliferating cells with EdU revealed YFP+ resident ECs to be proliferation efficient, as they constituted up to 95% of the EdU+ liver EC population (Fig. 3 A; Kamran et al., 2013). Finally, the percentage of GFP+ or YFP+ cells in the liver EC population was measured using either Beckman Coulter Cytoflex or BD Canto-II. Augustin); the European Union FP7 project “Early warning signals of aging in human stem cells and age-related disorders” (306240 to H.G. The animal studies were approved by the responsible authority for animal experiments (Regierungspraesidium Karlsruhe). (A’ and A’’) Zoomed-in images illustrating GFP+ cells incorporated into the liver vasculature. "to" : ["more", "e-mail", "sms", "facebook","twitter","googleplus","linkedin"], The care and experimental use of animals were in accordance with institutional guidelines and in compliance with the Association for Research in Vision and Ophthalmology statement. These data demonstrate that bone marrow–derived ECs are functionally indistinguishable from the resident liver ECs. 5 A). Likewise, resident ECs were previously reported to mediate adult neovascularization following cardiac injury (He et al., 2017) and during tumor progression (Peters et al., 2005; Purhonen et al., 2008). The study was approved by the Committee of Animal Care of the Regierungspräsidium Karlsruhe, Germany. Blood samples were taken from the Vena facialis after 4 hours of fasting during the day cycle. — die Eintragung in das Berufsregister ihres Sitzes oder Wohnsitzes. Add photos. After a 2-h rest, the mice were injected with bone marrow cells or 5,000 LSK cells isolated from CAG-GFP transgenic mice via the tail vein. Fig. 3 E) were subjected to PHx to induce liver regeneration. Welfare Office and Regierungspraesidium Karlsruhe). Augustin, and J. Hu analyzed and interpreted data. GSE116377. Further, we did not detect a CD133+ fraction of the liver EC, which was previously described as sinusoidal progenitor cells (Wang et al., 2012), in either sham-operated or PHx mice. However, neither LSK cells in the bone marrow nor mononuclear cells in the peripheral blood were YFP labeled (Fig. (C) The ratio of CD133+ ECs in the livers of sham-operated and PHx mice was analyzed by FACS (mean ± SD, n = 5 mice). PHx was performed according to the methods described by Mitchell and Willenbring (2008) to induce liver regeneration. The circulatory systems of WT and CAG-GFP mice were surgically conjoined, and both mice were subjected to PHx to induce liver regeneration. (C) The percentage of GFP+ ECs in livers of mice irradiated in the presence or absence of a liver shield was analyzed by FACS (mean ± SD, n = 6 mice). (NO AWARD), European Molecular Biology Organization Afterwards, mice were subjected to two-thirds PHx. Angaben, ob ein Insolvenzverfahren oder ein vergleichbares gesetzlich geregeltes Verfahren eröffnet oder die Eröffnung beantragt worden ist oder der Antrag mangels Masse abgelehnt wurde oder ein Insolvenzplan rechtskräftig bestätigt wurde. Blood samples were drawn from the tail veins and kept in anticoagulant EDTA-K2–coated tubes. Following a recovery period of 2 wk after CCl4 treatment, Sirius red staining was performed on the liver sections of CCl4-treated mice, which showed a strong deposition of collagen, an indication of liver fibrosis (Fig. Arrows indicate Ki67+ ECs. 76131 Karlsruhe. Bone marrow–derived cells have previously been reported to supposedly constitute ∼25% of total liver ECs following PHx (Wang et al., 2012; DeLeve, 2013). In contrast to most studies that use multiple cocaine-cue conditioning sessions, … S. Bartels contributed to the irradiation-primed bone marrow transplantation model. The adult liver is the only organ that can completely regenerate after injury or partial resection. All stainings were performed in 5% FCS on ice for 30 min with optimal dilutions of commercially prepared antibodies. In brief, magnetic beads (Dynabeads; Dynal) were coated with anti-CD31 antibodies (Mec13.3; BD Biosciences). Future studies involving stringent and mechanistic preclinical experimental approaches will need to address these questions to possibly establish a scientific rationale for bone marrow cell–involved stem cell therapies to interfere with liver dysfunction. / B. Eng. Cell isolation and culture. Additionally, our data cannot exclude the impact of the immune system on the success of BMDMC contribution toward liver parenchyma. PanRosaYFP mice were used as donors. 8-wk-old WT mice were lethally irradiated with a total dose of 9 Gy (split dose, 2 × 4.5 Gy). Plates were further incubated for 4 hours at 37 C and the formazan crystals formed were dissolved by adding 110μl/well acidified 2-propanol (0.04 N HCl). Photos and Videos. S3 B). After 8 d, the mice were euthanized, and blood and livers were collected for FACS analysis or immunostaining. In brief, inclusion criteria were study enrollment within 24 h after symptom onset. Our data clearly suggest that stem cell infusion/mobilization therapies do not physically contribute to the regeneration of liver vasculature in mice with healthy remaining vasculature. After the last treatment, mice were allowed to recover for 2 wk, and, thereafter, mice were euthanized, and livers were collected for FACS analysis or immunostaining. Experimental animals were generated by breeding of hemizygous βENaC-TG mice with C3B6 F1 WT mice (Charles River … (D) Experimental outline for transplantation of LSK cells into Rag2−/−γc−/−KitW/Wv mice. W. Li and L. Hui contributed to the mouse model of irradiation-induced liver injury. S1 C, upper panel). RM, GK, KK, and RP: data acquisition. A radioprotective shield reduces the recruitment of BMDMCs to the liver vasculature. However, a subsequent randomized controlled trial involving 58 patients with decompensated alcoholic liver disease resulted in no additional benefit from autologous BMDMC infusion combined with standard medical therapy as compared with standard medical therapy alone (Spahr et al., 2013). Germany. All experiments were carried out according to the German guidelines on the care and use of animals in research and with the European Community Council Directive 2010/63/EU and were approved by the local animal care committee (Regierungspräsidium, Karlsruhe, Germany; animal experimentation license T‐16/19). While several conditional systems were successful in invertebrates, in mice the Cre/loxP recombination system and the tet-controlled transcription activation system are predominant. Therefore, NF-κB is a good candidate to convert psychosocial stress into cellular activation. Fig. Log In. After sedation with azaperon (5 mg/kg IM; Elanco, … (A) Experimental outline of PHx-induced liver regeneration in NSG mice. In both WT (Para-WT) and CAG-GFP (Para-GFP) mice, approximately half of the mononuclear fraction of blood, which contains endothelial progenitor cells (Aoki et al., 2004), was GFP+, indicating that the circulatory systems of the two mice had successfully fused (Fig. For transfer experiments, lymphocyte-deficient We have in the present study employed multiple irradiation-based myeloablative and nonmyeloablative mouse models that allowed us to unambiguously evaluate the contribution of different cellular sources to the regenerating liver vasculature following two-thirds PHx. Cell. The animal studies were approved by the responsible authority for animal experiments (Regierungspraesidium Karlsruhe). C57BL/6 Rosa26-YFPfl/fl mice were crossed with C57BL/6 VECad-CreERT2 mice to specifically label ECs upon tamoxifen application. 3 F), indicating that YFP+ BMDMCs did not integrate into the regenerating liver vasculature following PHx. BLOOD PARAMETERS. M. Singhal and X. Liu performed most of the experiments. Bisected kidney basins were used as enrichment. H.G. The potential therapeutic manipulation of visceral organs has been complicated by the carbohydrate surface layer—here, called the mesopolysaccharide (MPS)—that … Yet, a growing number of studies suggest that a small population of bone marrow–derived mononuclear cells (BMDMCs), which express a variety of endothelial surface markers and have thus been designated as endothelial progenitor cells, could promote neovascularization in adults (Asahara et al., 1997; Shi et al., 1998; Peichev et al., 2000; Wang et al., 2012). After hearing B, the Regierungspräsidium Karlsruhe (Regional Council, Karlsruhe, Germany) determined, by decision of 25 November 2014, adopted on the basis of Paragraph 6(5) of the FreizügG/EU in conjunction with Article 28(3)(a) of Directive 2004/38, that B had lost his right of entry to and residence in Germany. Thus, under nonvascular-damaging conditions, the vascular regeneration proceeds exclusively by the expansion of preexisting tissue-resident ECs. Whole thoracic irradiation was administrated to 8–10 weeks old female C57BL/6 mice as described previously. Taken together, the data reconcile many of the discrepancies in the literature and highlight that the cellular source of regenerating endothelium depends on the fitness of the residual vasculature. To prevent infection and to relieve pain, the skin wounds were treated with hydrogel containing lincomycin hydrochloride (5 mg/g) and lidocaine (4 mg/g) for 3 d. 1 mo later, blood was collected from the tail veins of all the operated mouse pairs for FACS analysis to confirm the blood chimerism. A microarray analysis comparing the bone marrow–recruited ECs and the corresponding resident liver ECs revealed that BMDMCs incorporated as bona fide liver ECs after irradiation (Fig. Augustin and J. Hu contributed equally to this work. Little is known about the mechanisms converting psychosocial stress into cellular dysfunction. Das … All experiments comply with the regulations for animal usage set out by The … FORSTBW MANAGEMENT Headquarter Ministry of Rural Affairs and Consumer Protection Kernerplatz 10 . We are shaping the future We are shaping the future. Mahak Singhal, Xiaoting Liu, Donato Inverso, Kai Jiang, Jianing Dai, Hao He, Susanne Bartels, Weiping Li, Ashik Ahmed Abdul Pari, Nicolas Gengenbacher, Eva Besemfelder, Lijian Hui, Hellmut G. Augustin, Junhao Hu; Endothelial cell fitness dictates the source of regenerating liver vasculature. Zoomed-in images are shown on the right. It includes microarray-based gene expression analyses to compare the resident and the bone marrow–derived liver ECs. The microarray data with the description are deposited under GEO accession no. Stem cells: Stem cell therapy for liver cirrhosis unREALISTIC? Little is known about the mechanisms converting psychosocial stress into cellular dysfunction. number: G-252/17). — Sicherung Spritzbeton bis d= 30cm = 2 500 m2. We conclude that both preexisting liver ECs and BMDMCs can act as a potential source of new vessels depending on the vascular fitness. Most of the GFP+ cells in the livers of Para-WT-PHx mice were CD45+ and maintained their hematopoietic identity (Fig. Thereafter, RNA was isolated with the Arcturus PicoPure RNA Isolation Kit (Life Technologies), and RNA quality and quantity were analyzed on an Agilent Bioanalyzer. Donor chimerism of blood cells was determined 1 mo after transplantation. The regenerated liver lobes were stained with an EdU Flow Cytometry Kit (Baseclick GmbH) and analyzed by FACS. Then, the left lateral lobe and the median lobe were ligated with 4–0 silk sutures and resected. Sections were blocked and permeabilized with PBS containing 10% normal donkey serum and 0.3% Triton for 2 h at room temperature. The regenerated livers of Rag2−/−γc−/−KitW/Wv mice showed a similar vascular microstructure as WT controls 10 d after PHx, suggesting that liver regeneration was not impaired in Rag2−/−γc−/−KitW/Wv mice (Fig. Official name: Regierungspräsidium Karlsruhe, Referat 47.2 – Baureferat Mitte Postal address: Schlossplatz 4-6 Town: Karlsruhe NUTS code: DE122 Karlsruhe Postal code: 76131 Country: Germany E-mail: abteilung4@rpk.bwl.de Telephone: +49 721926-0 Fax: +49 72193340240 Internet address(es): Main address: www.rp-karlsruhe.de. A longitudinal skin incision was made to expose the elbow and knee joint. Animals were housed in the same room with a 12-h/12-h light/dark cycle in Makrolon Type II (360 cm 2 ) cages with standard bedding, either alone or in groups, and had ad libitum access to standard chow and water. In certain experiments, recombinant murine TNFα (R&D systems, Minneapolis, USA) was injected intrascrotally at a dose of 500 ng per mouse 3 hours before intravital microscopy. Scale bars, 100 µm. M. Singhal, H.G. In brief, 2.5 μg of nuclear extracts isolated from cell pellets with Nuclear Extract Kit (Active Motif, Inc., Carlsbad, USA) were incubated with a synthetic oligonucleotide containing the c-MYC consensus sequence. Dass sich das Unternehmen bei der Berufsgenossenschaft angemeldet hat. S2, B and C). 3 H). Publications Lines of Disconnection: The Penguin London Tube Anniversary Series ... this article analyses how Edwidge Danticat’s Claire of the Sea Light and Junot Díaz’s The Brief Wondrous Life of Oscar Wao re-write the stereotype of the paradisiacal and picturesque islands. These mice lack T and B cells (Rag2−/−) and natural killer cells (γc−/−, common gamma chain of the Il2r) and have an impaired HSC self-renewal capacity (KitW/Wv). S3 A). Augustin from Regierungspräsidium Karlsruhe, Germany). 4–5-wk-old animals were administered four doses of tamoxifen (100 mg/kg) twice a week. Embryonic tissue was used for genotyping. Male mice (8–10 wk of age) were used in this study unless otherwise indicated. Preparation of primary cortical neurons Cortical neurons from newborn C57BL/6NCrl mice of both sexes were prepared and maintained as described previously [15]. (B) The ratio of GFP+ ECs in the livers of Para-WT (sham operated and PHx) as well as Para-GFP (sham operated and PHx) mice was analyzed by FACS (mean ± SD, n = 3–4 mice). The study was conducted in accordance with national guidelines for the use of experimental animals and the protocols were approved by the governmental committees (Regierungspraesidium Karlsruhe, Germany). We detected that you disabled the JavaScript feature in your browser. 50-µm sections were prepared with a vibratome (Leica VT1000S). We hypothesized that if BMDMCs contribute to the regenerating vasculature, then there should be GFP+ ECs incorporated into the regenerated liver in Para-WT mice after PHx. Yet, we did not observe any significant expansion or recruitment of CD133+ liver ECs when comparing PHx to sham-operated mice following G-CSF administration (Fig. All procedures were performed according to the Guide of the Care and Use of Laboratory Animals published by the National Academy of Sciences and were approved by the local authorities (Regierungspräsidium Karlsruhe AZ: 35-9185.81/G—19/12, 35-9185.81/G—229/12). Initial case studies and proof-of-concept trials with the administration of autologous stem cell grafts showed improvement in liver function and accelerated hepatic regeneration (Huebert and Rakela, 2014; Moore et al., 2014). the regional licensing body (“Regierungspräsidium Karlsruhe”) and the specific experiment protocol was approved by the ethics committee attached to Regierungspräsidium Karlsruhe. S3 E). To generate bone marrow chimeras without damaging the liver vasculature, a radio-protective shield was applied over the upper abdomen while irradiating the animals (Halder et al., 1998; Fig. Exclusion criteria were recent clinical infection (fever, cough, antibiotic treatment, laboratory markers) within 14 d before admission, a chronic immunological disorder, preexisting immunosuppression, cancer, and pregnancy. Primers (Bax: 5′-AGACAGGGGCCTTTTTGCTAC-3′, forward, and 5′-AATTCGCCGGAGACACTCG-3′, reverse; Icam1: 5′-CTGGATCTCAGGCCGCAAG-3′, forward, and 5′-TGTCGAGCTTTGGGATGGTAG-3′, reverse) were ordered from Sangon Biotech. Next, VECad-CreERT2xRosa26-YFPfl/fl mice with labeled ECs were injected with 1011 viral particles of empty replication-deficient adenovirus. To induce maximal liver regeneration in the parabiotic mice, both Para-WT and Para-GFP mice were simultaneously subjected to PHx. The YFP+ EC ratio of both the resected and the regenerated liver lobes of an individual mouse was analyzed to precisely evaluate the contribution of YFP+ BMDMCs to vascular regeneration. Location & Hours. 3 Nr. Timed matings and analyses of embryos and placental morphology Placental and embryonic tissues were obtained from timed matings of p45NF-E2+/– females and p45NF-E2–/– males on a C57Bl/6 background. Results from initial uncontrolled clinical trials indicated increased serum albumin levels and an overall improvement in several clinical parameters such as the Child-Pugh-Turcotte score or the model for end-stage liver disease score (Huebert and Rakela, 2014). 3 G). — Auf Verlangen ist die Urkalkulation mit den Grundlagen der Preisermittlung für die Hauptunternehmer und aller Nachunternehmer sowie Ermittlung der Kalkulationssumme (Formblatt 222) zu übergeben.