Publications by authors named "Marco Metzger"

76 Publications

Protein Kinase D2 drives chylomicron-mediated lipid transport in the intestine and promotes obesity.

EMBO Mol Med 2021 May 5;13(5):e13548. Epub 2021 May 5.

Rudolf-Virchow-Zentrum, Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany.

Lipids are the most energy-dense components of the diet, and their overconsumption promotes obesity and diabetes. Dietary fat content has been linked to the lipid processing activity by the intestine and its overall capacity to absorb triglycerides (TG). However, the signaling cascades driving intestinal lipid absorption in response to elevated dietary fat are largely unknown. Here, we describe an unexpected role of the protein kinase D2 (PKD2) in lipid homeostasis. We demonstrate that PKD2 activity promotes chylomicron-mediated TG transfer in enterocytes. PKD2 increases chylomicron size to enhance the TG secretion on the basolateral side of the mouse and human enterocytes, which is associated with decreased abundance of APOA4. PKD2 activation in intestine also correlates positively with circulating TG in obese human patients. Importantly, deletion, inactivation, or inhibition of PKD2 ameliorates high-fat diet-induced obesity and diabetes and improves gut microbiota profile in mice. Taken together, our findings suggest that PKD2 represents a key signaling node promoting dietary fat absorption and may serve as an attractive target for the treatment of obesity.
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http://dx.doi.org/10.15252/emmm.202013548DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8103097PMC
May 2021

Triple co-culture and perfusion bioreactor for studying the interaction between and neutrophils: A novel 3D tissue model for bacterial infection and immunity.

J Tissue Eng 2021 Jan-Dec;12:2041731420988802. Epub 2021 Jan 28.

Biocenter, Chair of Microbiology, University of Würzburg, Würzburg, Germany.

Gonorrhea, a sexually transmitted disease caused by the bacteria , is characterized by a large number of neutrophils recruited to the site of infection. Therefore, proper modeling of the interaction with neutrophils is very important for investigating and understanding the mechanisms that gonococci use to evade the immune response. We have used a combination of a unique human 3D tissue model together with a dynamic culture system to study neutrophil transmigration to the site of infection. The triple co-culture model consisted of epithelial cells (T84 human colorectal carcinoma cells), human primary dermal fibroblasts, and human umbilical vein endothelial cells on a biological scaffold (SIS). After the infection of the tissue model with , we introduced primary human neutrophils to the endothelial side of the model using a perfusion-based bioreactor system. By this approach, we were able to demonstrate the activation and transmigration of neutrophils across the 3D tissue model and their recruitment to the site of infection. In summary, the triple co-culture model supplemented by neutrophils represents a promising tool for investigating and other bacterial infections and interactions with the innate immunity cells under conditions closely resembling the native tissue environment.
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http://dx.doi.org/10.1177/2041731420988802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7970704PMC
January 2021

Enteroids Generated from Patients with Severe Inflammation in Crohn's Disease Maintain Alterations of Junctional Proteins.

J Crohns Colitis 2020 Oct;14(10):1473-1487

Department of General, Visceral, Transplant, Vascular and Pediatric Surgery, University Hospital Wuerzburg, Wuerzburg, Germany.

Background: The mechanisms underlying loss of intestinal epithelial barrier [IEB] function in Crohn's disease [CD] are poorly understood. We tested whether human enteroids generated from isolated intestinal crypts of CD patients serve as an appropriate in vitro model to analyse changes of IEB proteins observed in patients' specimens.

Methods: Gut samples from CD patients and healthy individuals who underwent surgery were collected. Enteroids were generated from intestinal crypts and analyses of junctional proteins in comparison to full wall samples were performed.

Results: Histopathology confirmed the presence of CD and the extent of inflammation in intestinal full wall sections. As revealed by immunostaining and Western blot analysis, profound changes in expression patterns of tight junction, adherens junction and desmosomal proteins were observed in full wall specimens when CD was present. Unexpectedly, when enteroids were generated from specimens of CD patients with severe inflammation, alterations of most tight junction proteins and the majority of changes in desmosomal proteins but not E-cadherin were maintained under culture conditions. Importantly, these changes were maintained without any additional stimulation of cytokines. Interestingly, qRT-PCR demonstrated that mRNA levels of junctional proteins were not different when enteroids from CD patients were compared to enteroids from healthy controls.

Conclusions: These data indicate that enteroids generated from patients with severe inflammation in CD maintain some characteristics of intestinal barrier protein changes on a post-transcriptional level. The enteroid in vitro model represents an appropriate tool to gain further cellular and molecular insights into the pathogenesis of barrier dysfunction in CD.
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http://dx.doi.org/10.1093/ecco-jcc/jjaa085DOI Listing
October 2020

Bacterial nanocellulose as novel carrier for intestinal epithelial cells in drug delivery studies.

Mater Sci Eng C Mater Biol Appl 2020 Apr 30;109:110613. Epub 2019 Dec 30.

University Hospital Würzburg, Chair Tissue Engineering and Regenerative Medicine (TERM), Würzburg, Germany; Fraunhofer Institute for Silicate Research (ISC), Translational Center Regenerative Therapies (TLC-RT), Würzburg, Germany. Electronic address:

Synthetic cell carriers (A) represent common scaffold structures for the development of cell-based in vitro models of the human intestine but due to their low porosity or unwanted molecular adhesion effects, synthetic carriers can negatively affect cell function. Alternative scaffolds such as natural extracellular matrices (ECMs) (B) were shown to overcome some of the common drawbacks. However, their fabrication is time-consuming, less well standardized and not entirely conform to the 3R principle (replacement, reduction, refinement). Nowadays, biopolymers such as bacterial nanocellulose (BNC) (C) represent interesting scaffold materials for innovative tissue engineering concepts, as they can be generated in a faster and more standardized process workflow without the need for animal material. In this study, we demonstrate the BNC as suitable carrier for the development of Caco-2-based in vitro models of the human intestine. The BNC-based models exhibit organ-specific properties comprising typical cellular morphologies, characteristic protein expression profiles, representative ultrastructural features and the formation of a tight epithelial barrier. The proof of in vivo-like transport activities further validates the high quality of the BNC-based Caco-2 models. In summary, this illustrates the BNC as alternative bioscaffold of non-animal origin to develop functional organ models in vitro.
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http://dx.doi.org/10.1016/j.msec.2019.110613DOI Listing
April 2020

Matrix decoded - A pancreatic extracellular matrix with organ specific cues guiding human iPSC differentiation.

Biomaterials 2020 06 6;244:119766. Epub 2020 Mar 6.

Chair Tissue Engineering and Regenerative Medicine, University Hospital Würzburg, Würzburg, Germany; Translational Center Regenerative Therapies (TLC-RT), Fraunhofer Institute for Silicate Research ISC, Würzburg, Germany. Electronic address:

The extracellular matrix represents a dynamic microenvironment regulating essential cell functions in vivo. Tissue engineering approaches aim to recreate the native niche in vitro using biological scaffolds generated by organ decellularization. So far, the organ specific origin of such scaffolds was less considered and potential consequences for in vitro cell culture remain largely elusive. Here, we show that organ specific cues of biological scaffolds affect cellular behavior. In detail, we report on the generation of a well-preserved pancreatic bioscaffold and introduce a scoring system allowing standardized inter-study quality assessment. Using multiple analysis tools for in-depth-characterization of the biological scaffold, we reveal unique compositional, physico-structural, and biophysical properties. Finally, we prove the functional relevance of the biological origin by demonstrating a regulatory effect of the matrix on multi-lineage differentiation of human induced pluripotent stem cells emphasizing the significance of matrix specificity for cellular behavior in artificial microenvironments.
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http://dx.doi.org/10.1016/j.biomaterials.2020.119766DOI Listing
June 2020

Insecticide Resistance Status of Aedes aegypti (Diptera: Culicidae) in California by Biochemical Assays.

J Med Entomol 2020 07;57(4):1176-1183

California Department of Public Health, Infectious Diseases Branch/Vector-Borne Disease Section, Marina Bay Parkway, Richmond, CA.

Insecticide resistance in Aedes aegypti mosquitoes poses a major threat to public health worldwide. There are two primary biological mechanisms that can lead to insecticide resistance, target site and metabolic resistance, both of which confer resistance to specific classes of insecticides. Due to the limited number of chemical compounds available for mosquito control, it is important to determine current enzymatic profiles among mosquito populations. This study assessed resistance profiles for three metabolic pathways, α-esterases, β-esterases, and mixed-function oxidases (MFOs), as well as insensitivity of the acetylcholinesterase (iAChE) enzyme in the presence of propoxur, among Ae. aegypti from the Central Valley and southern California. All field-collected Ae. aegypti demonstrated elevated MFOs and iAChE activity, indicating potential development of pyrethroid and organophosphate resistance, respectively. Although regional variations were found among α-esterase and β-esterase activity, levels were generally elevated, further suggesting additional mechanisms for developing organophosphate resistance. Furthermore, mosquito samples from southern California exhibited a higher expression level to all three metabolic enzymes and iAChE activity in comparison to mosquitoes from the central region. These results could help guide future mosquito control efforts, directing the effective use of insecticides while limiting the spread of resistance.
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http://dx.doi.org/10.1093/jme/tjaa031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7334890PMC
July 2020

An Advanced Human Intestinal Coculture Model Reveals Compartmentalized Host and Pathogen Strategies during Infection.

mBio 2020 02 18;11(1). Epub 2020 Feb 18.

Department of Tissue Engineering and Regenerative Medicine, University Hospital Würzburg, Würzburg, Germany

A major obstacle in infection biology is the limited ability to recapitulate human disease trajectories in traditional cell culture and animal models, which impedes the translation of basic research into clinics. Here, we introduce a three-dimensional (3D) intestinal tissue model to study human enteric infections at a level of detail that is not achieved by conventional two-dimensional monocultures. Our model comprises epithelial and endothelial layers, a primary intestinal collagen scaffold, and immune cells. Upon infection, the model mimics human gastroenteritis, in that it restricts the pathogen to the epithelial compartment, an advantage over existing mouse models. Application of dual transcriptome sequencing to the -infected model revealed the communication of epithelial, endothelial, monocytic, and natural killer cells among each other and with the pathogen. Our results suggest that uses its type III secretion systems to manipulate STAT3-dependent inflammatory responses locally in the epithelium without accompanying alterations in the endothelial compartment. Our approach promises to reveal further human-specific infection strategies employed by and other pathogens. Infection research routinely employs cell cultures or mouse models as surrogates of human hosts. Differences between murine and human immunity and the low level of complexity of traditional cell cultures, however, highlight the demand for alternative models that combine the -like properties of the human system with straightforward experimental perturbation. Here, we introduce a 3D tissue model comprising multiple cell types of the human intestinal barrier, a primary site of pathogen attack. During infection with the foodborne pathogen serovar Typhimurium, our model recapitulates human disease aspects, including pathogen restriction to the epithelial compartment, thereby deviating from the systemic infection in mice. Combination of our model with state-of-the-art genetics revealed -mediated local manipulations of human immune responses, likely contributing to the establishment of the pathogen's infection niche. We propose the adoption of similar 3D tissue models to infection biology, to advance our understanding of molecular infection strategies employed by bacterial pathogens in their human host.
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http://dx.doi.org/10.1128/mBio.03348-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7029144PMC
February 2020

A three-dimensional intestinal tissue model reveals factors and small regulatory RNAs important for colonization with Campylobacter jejuni.

PLoS Pathog 2020 02 18;16(2):e1008304. Epub 2020 Feb 18.

Chair of Molecular Infection Biology II, Institute of Molecular Infection Biology, University of Würzburg, Würzburg, Germany.

The Gram-negative Epsilonproteobacterium Campylobacter jejuni is currently the most prevalent bacterial foodborne pathogen. Like for many other human pathogens, infection studies with C. jejuni mainly employ artificial animal or cell culture models that can be limited in their ability to reflect the in-vivo environment within the human host. Here, we report the development and application of a human three-dimensional (3D) infection model based on tissue engineering to study host-pathogen interactions. Our intestinal 3D tissue model is built on a decellularized extracellular matrix scaffold, which is reseeded with human Caco-2 cells. Dynamic culture conditions enable the formation of a polarized mucosal epithelial barrier reminiscent of the 3D microarchitecture of the human small intestine. Infection with C. jejuni demonstrates that the 3D tissue model can reveal isolate-dependent colonization and barrier disruption phenotypes accompanied by perturbed localization of cell-cell junctions. Pathogenesis-related phenotypes of C. jejuni mutant strains in the 3D model deviated from those obtained with 2D-monolayers, but recapitulated phenotypes previously observed in animal models. Moreover, we demonstrate the involvement of a small regulatory RNA pair, CJnc180/190, during infections and observe different phenotypes of CJnc180/190 mutant strains in 2D vs. 3D infection models. Hereby, the CJnc190 sRNA exerts its pathogenic influence, at least in part, via repression of PtmG, which is involved in flagellin modification. Our results suggest that the Caco-2 cell-based 3D tissue model is a valuable and biologically relevant tool between in-vitro and in-vivo infection models to study virulence of C. jejuni and other gastrointestinal pathogens.
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http://dx.doi.org/10.1371/journal.ppat.1008304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7048300PMC
February 2020

Investigation of Cellular Function and DNA Integrity during 2D in vitro Culture of Human Salivary Gland Epithelial Cells.

Cells Tissues Organs 2019;208(1-2):66-75. Epub 2020 Feb 5.

Department of Otorhinolaryngology, Plastic, Aesthetic, and Reconstructive Head and Neck Surgery, University Hospital Würzburg, Würzburg, Germany.

In vitro culture of human salivary gland epithelial cells (SGEC) is still a challenge. A high quantity and quality of cells are needed for the cultivation of 3D matrices. Furthermore, it is known that DNA damage is supposed to be an important factor involved in carcinogenesis. This study investigates cellular function and DNA integrity of human SGEC during 3 passage steps in 2 groups (group 1: n = 10; group 2: n = 9). Cellular function was analyzed by immunofluorescence, transmission electron microscopy (TEM), and quantitative real-time polymerase chain reaction (qPCR). DNA integrity was tested via the comet assay. Immunohistochemistry and qPCR results showed stable α-amylase and pan-cytokeratin levels; TEM revealed functional cells; and no significant DNA damage could be detected in the comet assay during 3 culture steps. The study shows that not only at cellular but also at DNA level human SGEC can be safely quantified over 3 passages for preclinical tissue engineering without loss of differentiation and function.
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http://dx.doi.org/10.1159/000505433DOI Listing
August 2020

Postnatal human enteric neurospheres show a remarkable molecular complexity.

Neurogastroenterol Motil 2019 10 18;31(10):e13674. Epub 2019 Jul 18.

Department of Human Molecular Genetics, Institute of Human Genetics, Heidelberg University Hospital, Heidelberg, Germany.

Background: The enteric nervous system (ENS), a complex network of neurons and glial cells, coordinates major gastrointestinal functions. Impaired development or secondary aberrations cause severe enteric neuropathies. Neural crest-derived stem cells as well as enteric neuronal progenitor cells, which form enteric neurospheres, represent a promising tool to unravel molecular pathomechanisms and to develop novel therapy options. However, so far little is known about the detailed cellular composition and the proportional distribution of enteric neurospheres. Comprehensive knowledge will not only be essential for basic research but also for prospective cell replacement therapies to restore or to improve enteric neuronal dysfunction.

Methods: Human enteric neurospheres were generated from three individuals with varying age. For detailed molecular characterization, nCounter target gene expression analyses focusing on stem, progenitor, neuronal, glial, muscular, and epithelial cell markers were performed. Corresponding archived paraffin-embedded individuals' specimens were analyzed accordingly.

Key Results: Our data revealed a remarkable molecular complexity of enteric neurospheres and archived specimens. Amongst the expression of multipotent stem cell, progenitor cell, neuronal, glial, muscle and epithelial cell markers, moderate levels for the pluripotency marker POU5F1 were observed. Furthermore, besides the interindividual variability, we identified highly distinct intraindividual expression profiles.

Conclusions & Inferences: Our results emphasize the assessment of molecular signatures to be essential for standardized use, optimization of experimental approaches, and elimination of potential risk factors, as the formation of tumors. Our study pipeline may serve as a blueprint implemented into the characterization procedure of enteric neurospheres for various future applications.
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http://dx.doi.org/10.1111/nmo.13674DOI Listing
October 2019

A fast and simple fluorometric method to detect cell death in 3D intestinal organoids.

Biotechniques 2019 07 20;67(1):23-28. Epub 2019 Jun 20.

Department of Biology, University of Konstanz, Konstanz, Germany.

Organoids recapitulate the (patho)physiological processes in certain tissues and organs closer than classical cell lines. Therefore, organoid technology offers great potentials in drug development and testing, and personalized medicine. In particular, organoids can be used to study and predict drug-induced toxicity in certain tissues. However, until today few methods had been reported to analyze cell death in 3D-microtissues in a quantitative manner. Here, we describe a novel fluorometric method for the quantitative measurement of specific organoid cell death. Organoids are stained simultaneously with the cell impermeable nuclear dye propidium iodide and cell permeable Hoechst33342. While Hoechst allows in-well normalization to cell numbers, propidium iodide detects relative proportion of dead cells independent of hydrogel. Measurement and analysis time, as well as usability are drastically improved in comparison to other established methods. Parallel multiplexing of our method with established assays measuring mitochondrial activity further enhances its applicability in personalized medicine and drug discovery.
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http://dx.doi.org/10.2144/btn-2019-0023DOI Listing
July 2019

Neurotrophic factor GDNF regulates intestinal barrier function in inflammatory bowel disease.

J Clin Invest 2019 06 17;129(7):2824-2840. Epub 2019 Jun 17.

Department of General, Visceral, Vascular and Pediatric Surgery, University Hospital Wuerzburg, Wuerzburg, Germany.

Impaired intestinal epithelial barrier (IEB) function with loss of desmosomal junctional protein desmoglein 2 (DSG2) is a hallmark in the pathogenesis of inflammatory bowel disease (IBD). While previous studies have reported that glial cell line-derived neurotrophic factor (GDNF) promotes IEB function, the mechanisms are poorly understood. We hypothesized that GDNF is involved in the loss of DSG2, resulting in impaired IEB function as seen in IBD. In the inflamed intestine of patients with IBD, there was a decrease in GDNF concentrations accompanied by a loss of DSG2, changes of the intermediate filament system, and increased phosphorylation of p38 MAPK and cytokeratins. DSG2-deficient and RET-deficient Caco2 cells revealed that GDNF specifically recruits DSG2 to the cell borders, resulting in increased DSG2-mediated intercellular adhesion via the RET receptor. Challenge of Caco2 cells and enteroids with proinflammatory cytokines as well as dextran sulfate sodium-induced (DSS-induced) colitis in C57Bl/6 mice led to impaired IEB function with reduced DSG2 mediated by p38 MAPK-dependent phosphorylation of cytokeratins. GDNF blocked all inflammation-induced changes in the IEB. GDNF attenuates inflammation-induced impairment of IEB function caused by the loss of DSG2 through p38 MAPK-dependent phosphorylation of cytokeratin. The reduced GDNF in patients with IBD indicates a disease-relevant contribution to the development of IEB dysfunction.
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http://dx.doi.org/10.1172/JCI120261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6597228PMC
June 2019

Induced Pluripotent Stem Cell-Derived Brain Endothelial Cells as a Cellular Model to Study Infection.

Front Microbiol 2019 29;10:1181. Epub 2019 May 29.

Institute of Hygiene and Microbiology, University of Würzburg, Würzburg, Germany.

Meningococcal meningitis is a severe central nervous system infection that occurs when () penetrates brain endothelial cells (BECs) of the meningeal blood-cerebrospinal fluid barrier. As a human-specific pathogen, models are greatly limited and pose a significant challenge. cell models have been developed, however, most lack critical BEC phenotypes limiting their usefulness. Human BECs generated from induced pluripotent stem cells (iPSCs) retain BEC properties and offer the prospect of modeling the human-specific interaction with BECs. Here, we exploit iPSC-BECs as a novel cellular model to study host-pathogen interactions, and provide an overview of host responses to infection. Using iPSC-BECs, we first confirmed that multiple strains and mutants follow similar phenotypes to previously described models. The recruitment of the recently published pilus adhesin receptor CD147 underneath meningococcal microcolonies could be verified in iPSC-BECs. was also observed to significantly increase the expression of pro-inflammatory and neutrophil-specific chemokines , , , , and , and the secretion of IFN-γ and RANTES. For the first time, we directly observe that disrupts the three tight junction proteins ZO-1, Occludin, and Claudin-5, which become frayed and/or discontinuous in BECs upon challenge. In accordance with tight junction loss, a sharp loss in endothelial electrical resistance, and an increase in sodium fluorescein permeability and in bacterial transmigration, was observed. Finally, we established RNA-Seq of sorted, infected iPSC-BECs, providing expression data of -responsive host genes. Altogether, this model provides novel insights into pathogenesis, including an impact of on barrier properties and tight junction complexes, and suggests that the paracellular route may contribute to traversal of BECs.
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http://dx.doi.org/10.3389/fmicb.2019.01181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6548865PMC
May 2019

Identification of Molecular Determinants of Resistance to Pyrethroid Insecticides in Aedes aegypti (Diptera: Culicidae) Populations in California, USA.

J Med Entomol 2019 09;56(5):1353-1358

California Department of Public Health, Infectious Diseases Branch/Vector-Borne Disease Section, Marina Bay Parkway, Richmond, CA.

The first breeding populations of Aedes aegypti (Linnaeus) were identified in California in 2013, and have since been detected in 13 counties. Recent studies suggest two introductions likely occurred, with genetically distinct populations in the central and southern regions of the state. Given the threat of dengue, chikungunya, and Zika virus transmission, it is imperative to understand if these populations harbor genes that could confer resistance to pyrethrin-based insecticides, known as pyrethroids, the most commonly used class of adulticides in the state. In 2017, the California Department of Public Health initiated a pesticide resistance screening program for Ae. aegypti to assess the presence of specific mutations on the sodium channel gene (V1016I and F1534C) associated with knockdown resistance to pyrethroids. Mosquitoes collected between 2015 and 2017 from 11 counties were screened for mutations using real-time polymerase chain reaction assays. Results revealed distinctly different resistance profiles between the central and southern regions. The central population displayed nearly fixed resistant mutations at both loci, whereas the southern population was more variable. The relative proportion of resistant alleles observed in sampled mosquitoes collected in southern California increased each year from 2015 through 2017, indicating potential increases in resistance across this region. The presence of these mutations indicates that these mosquitoes may be predisposed to surviving pyrethroid treatments. Additional biological and biochemical assays will help better elucidate the mechanisms underlying insecticide resistance in California Ae. aegypti and prompt the use of pesticides that are most effective at controlling these mosquitoes.
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http://dx.doi.org/10.1093/jme/tjz076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6736402PMC
September 2019

Human Pluripotent Stem Cell-Derived Multipotent Vascular Progenitors of the Mesothelium Lineage Have Utility in Tissue Engineering and Repair.

Cell Rep 2019 03;26(10):2566-2579.e10

Department of Biochemistry and Molecular Biology and Center for Molecular Medicine, University of Georgia, 325 Riverbend Road, Athens, GA 30605, USA. Electronic address:

In this report we describe a human pluripotent stem cell-derived vascular progenitor (MesoT) cell of the mesothelium lineage. MesoT cells are multipotent and generate smooth muscle cells, endothelial cells, and pericytes and self-assemble into vessel-like networks in vitro. MesoT cells transplanted into mechanically damaged neonatal mouse heart migrate into the injured tissue and contribute to nascent coronary vessels in the repair zone. When seeded onto decellularized vascular scaffolds, MesoT cells differentiate into the major vascular lineages and self-assemble into vasculature capable of supporting peripheral blood flow following transplantation. These findings demonstrate in vivo functionality and the potential utility of MesoT cells in vascular engineering applications.
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http://dx.doi.org/10.1016/j.celrep.2019.02.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6585464PMC
March 2019

Establishment of a Human Blood-Brain Barrier Co-Culture Model Mimicking the Neurovascular Unit Using Induced Pluripotent Stem Cells.

Curr Protoc Stem Cell Biol 2018 11 27;47(1):e62. Epub 2018 Sep 27.

University Hospital Würzburg, Chair Tissue Engineering and Regenerative Medicine.

Human blood-brain barrier (BBB) in vitro models pose a promising tool in drug development and understanding of mechanistic regulations during health and disease. Human-induced pluripotent stem cells (hiPS cells) represent an unlimited cell source to generate functional cells of the neurovascular unit (NVU), independent of variations or limitations during isolation and in vitro cultivation. This unit describes the standardized 2-D differentiation of adherent hiPS cells into BBB endothelial cells and neuronal stem cells (NSCs). Both cell types are combined with primary astrocytes and pericytes to develop complex, physiological BBB in vitro models. The endothelial cells in the apical compartment of the transwell models are separated from the basolateral seeded co-culture mixture by a synthetic membrane, simplifying analyses. The barrier integrity and functionality of the endothelium is improved by the specific mixture of NVU niche cells, determined here by decrease in the paracellular permeability of sodium-fluorescein and transendothelial electrical resistance (TEER) measurement. © 2018 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cpsc.62DOI Listing
November 2018

Obstructed defecation-an enteric neuropathy? An exploratory study of patient samples.

Int J Colorectal Dis 2019 Jan 3;34(1):193-196. Epub 2018 Sep 3.

Department for Tissue Engineering and Regenerative Medicine, University Hospital Würzburg, Würzburg, Germany.

Purpose: Although various strategies exist for chronic constipation therapy, the pathogenesis of chronic constipation is still not completely understood. The aim of this exploratory experimental study is to elucidate alterations of the autonomous enteric nervous system at the molecular level in patients with obstructed defecation, who represent one of the most predominant groups of constipated patients.

Methods: Full-thickness rectal wall samples of patients with obstructed defecation were analyzed and compared with controls. Differential gene expression analyses by RNA-Seq transcriptome profiling were performed and gene expression profiles were assigned to gene ontology pathways by application of different biological libraries.

Results: Analysis of the transcriptome showed that genes associated with the enteric nervous system functions were significantly downregulated in patients with obstructed defecation. These affected functions included developmental processes and synaptic transmission.

Conclusions: Our results therefore indicate that obstructed defecation may represent an enteric neuropathy, comparable to Hirschsprung disease and slow-transit constipation.
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http://dx.doi.org/10.1007/s00384-018-3160-1DOI Listing
January 2019

Altered pancreatic islet morphology and function in SGLT1 knockout mice on a glucose-deficient, fat-enriched diet.

Mol Metab 2018 07 23;13:67-76. Epub 2018 May 23.

Chair Tissue Engineering and Regenerative Medicine, University Hospital Würzburg, 97070, Würzburg, Germany; Translational Center Regenerative Therapies (TLC-RT), Fraunhofer Institute for Silicate Research ISC, 97070, Würzburg, Germany.

Objectives: Glycemic control by medical treatment represents one therapeutic strategy for diabetic patients. The Na+-d-glucose cotransporter 1 (SGLT1) is currently of high interest in this context. SGLT1 is known to mediate glucose absorption and incretin secretion in the small intestine. Recently, inhibition of SGLT1 function was shown to improve postprandial hyperglycemia. In view of the lately demonstrated SGLT1 expression in pancreatic islets, we investigated if loss of SGLT1 affects islet morphology and function.

Methods: Effects associated with the loss of SGLT1 on pancreatic islet (cyto) morphology and function were investigated by analyzing islets of a SGLT1 knockout mouse model, that were fed a glucose-deficient, fat-enriched diet (SGLT1-GDFE) to circumvent the glucose-galactose malabsorption syndrome. To distinguish diet- and Sglt1-dependent effects, wildtype mice on either standard chow (WT-SC) or the glucose-free, fat-enriched diet (WT-GDFE) were used as controls. Feeding a glucose-deficient, fat-enriched diet further required the analysis of intestinal SGLT1 expression and function under diet-conditions.

Results: Consistent with literature, our data provide evidence that small intestinal SGLT1 mRNA expression and function is regulated by nutrition. In contrast, pancreatic SGLT1 mRNA levels were not affected by the applied diet, suggesting different regulatory mechanisms for SGLT1 in diverse tissues. Morphological changes such as increased islet sizes and cell numbers associated with changes in proliferation and apoptosis and alterations of the β- and α-cell population are specifically observed for pancreatic islets of SGLT1-GDFE mice. Glucose stimulation revealed no insulin response in SGLT1-GDFE mice while WT-GDFE mice displayed only a minor increase of blood insulin. Irregular glucagon responses were observed for both, SGLT1-GDFE and WT-GDFE mice. Further, both animal groups showed a sustained release of GLP-1 compared to WT-SC controls.

Conclusion: Loss or impairment of SGLT1 results in abnormal pancreatic islet (cyto)morphology and disturbed islet function regarding the insulin or glucagon release capacity from β- or α-cells, respectively. Consequently, our findings propose a new, additional role for SGLT1 maintaining proper islet structure and function.
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http://dx.doi.org/10.1016/j.molmet.2018.05.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6026318PMC
July 2018

Novel Focus of Sin Nombre Virus in Peromyscus eremicus Mice, Death Valley National Park, California, USA.

Emerg Infect Dis 2018 06;24(6):1112-1115

The deer mouse (Peromyscus maniculatus) is the primary reservoir for Sin Nombre virus (SNV) in the western United States. Rodent surveillance for hantavirus in Death Valley National Park, California, USA, revealed cactus mice (P. eremicus) as a possible focal reservoir for SNV in this location. We identified SNV antibodies in 40% of cactus mice sampled.
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http://dx.doi.org/10.3201/eid2406.180089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6004862PMC
June 2018

Evaluation of a Miniaturized Biologically Vascularized Scaffold in vitro and in vivo.

Sci Rep 2018 03 16;8(1):4719. Epub 2018 Mar 16.

University Hospital of Würzburg, Chair of Tissue Engineering and Regenerative Medicine, 97070, Würzburg, Germany.

In tissue engineering, the generation and functional maintenance of dense voluminous tissues is mainly restricted due to insufficient nutrient supply. Larger three-dimensional constructs, which exceed the nutrient diffusion limit become necrotic and/or apoptotic in long-term culture if not provided with an appropriate vascularization. Here, we established protocols for the generation of a pre-vascularized biological scaffold with intact arterio-venous capillary loops from rat intestine, which is decellularized under preservation of the feeding and draining vascular tree. Vessel integrity was proven by marker expression, media/blood reflow and endothelial LDL uptake. In vitro maintenance persisted up to 7 weeks in a bioreactor system allowing a stepwise reconstruction of fully vascularized human tissues and successful in vivo implantation for up to 4 weeks, although with time-dependent decrease of cell viability. The vascularization of the construct lead to a 1.5× increase in cellular drug release compared to a conventional static culture in vitro. For the first time, we performed proof-of-concept studies demonstrating that 3D tissues can be maintained within a miniaturized vascularized scaffold in vitro and successfully implanted after re-anastomosis to the intrinsic blood circulation in vivo. We hypothesize that this technology could serve as a powerful platform technology in tissue engineering and regenerative medicine.
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http://dx.doi.org/10.1038/s41598-018-22688-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5856827PMC
March 2018

Laser-Capture Microdissection for Layer-Specific Analysis of Enteric Ganglia.

Methods Mol Biol 2018 ;1723:361-369

Department of Tissue Engineering and Regenerative Medicine (TERM), University Hospital Würzburg, Würzburg, Germany.

The enteric nervous system (ENS) is the division of the autonomic nervous system that innervates the gastrointestinal (GI) tract and controls central intestinal functions such as peristalsis and fluid movement. Enteric nerve cell bodies (neurons and glia) are predominantly organized in ganglionated networks that are present along the entire length of the GI tract in multiple tissue layers. Most cell bodies are organized in the myenteric plexus allocated between the longitudinal and the circular muscle layers or in the submucosal plexus between muscle tissue and mucosa. The site-specific characteristics of these enteric nerve cells have traditionally been analyzed via imaging techniques. Laser-capture microdissection (LCM) offers the prospect of site-specifically analyzing the gene expression profiles of these different subpopulations. This protocol addresses critical aspects of handling intestinal tissue for ENS dissection, such as the optimal quick-staining procedure, suitable laser settings, and limits of tissue material required to successfully dissect and analyze tissue layers for gene expression.
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http://dx.doi.org/10.1007/978-1-4939-7558-7_20DOI Listing
January 2019

Development of lacrimal gland spheroids for lacrimal gland tissue regeneration.

J Tissue Eng Regen Med 2018 04 29;12(4):e2001-e2009. Epub 2018 Jan 29.

Laboratory of Experimental Ophthalmology, University Clinic Düsseldorf, Düsseldorf, Germany.

Severe dry eye syndrome resulting from lacrimal gland (LG) dysfunction can cause blindness, yet treatments remain palliative. In vitro reconstruction of LG tissue could provide a curative treatment. We aimed to combine epithelial cells with endothelial cells and mesenchymal stem cells (MSCs) to form a 3D functional unit. Epithelial cells and MSCs were isolated from porcine LG; endothelial cells were isolated from human foreskin. MSCs were characterised (flow cytometry and differentiation potential assays). All 3 cell types were combined on Matrigel and spheroid formation observed. Spheroids were characterised [immunohistochemistry (IHC) and transmission electron microscopy] and function assessed (β-hexosaminidase assay). Spheroids were transferred to decellularised jejunum (SIS-Muc) in dynamic cultures for 1 week before further characterisation. MSCs did not express CD31 but expressed CD44 and CD105 and differentiated towards osteogenic and adipogenic lineages. Spheroids formed on Matrigel within 18 hr, contracting to ~10% of the well area (p < .005). IHC revealed presence of all 3 cells within spheroids. Transmission electron microscopy revealed cell-cell contacts and polarisation at the apical surface. In static cultures, function was increased in spheroids cf. monolayer controls (p < .05) but over 72 hr, spheroid function (p < .05), viability (p < .05), and proliferation decreased, whilst apoptosis increased. On SIS-Muc under dynamic culture, however, spheroids continued to proliferate to repopulate SIS-Muc. IHC revealed LG epithelial cells coexpressing pan-cytokeratin and lysozyme, as well as endothelial cells and MSCs and cells remained capable of responding to carbachol (p < .05). These spheroids could form the basis of a regenerative medicine treatment approach for dry eye syndrome. In vivo studies are required to evaluate this further.
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http://dx.doi.org/10.1002/term.2631DOI Listing
April 2018

Mucus Detachment by Host Metalloprotease Meprin β Requires Shedding of Its Inactive Pro-form, which Is Abrogated by the Pathogenic Protease RgpB.

Cell Rep 2017 Nov;21(8):2090-2103

Institute of Biochemistry, University of Kiel, Kiel, Germany. Electronic address:

The host metalloprotease meprin β is required for mucin 2 (MUC2) cleavage, which drives intestinal mucus detachment and prevents bacterial overgrowth. To gain access to the cleavage site in MUC2, meprin β must be proteolytically shed from epithelial cells. Hence, regulation of meprin β shedding and activation is important for physiological and pathophysiological conditions. Here, we demonstrate that meprin β activation and shedding are mutually exclusive events. Employing ex vivo small intestinal organoid and cell culture experiments, we found that ADAM-mediated shedding is restricted to the inactive pro-form of meprin β and is completely inhibited upon its conversion to the active form at the cell surface. This strict regulation of meprin β activity can be overridden by pathogens, as demonstrated for the bacterial protease Arg-gingipain (RgpB). This secreted cysteine protease potently converts membrane-bound meprin β into its active form, impairing meprin β shedding and its function as a mucus-detaching protease.
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http://dx.doi.org/10.1016/j.celrep.2017.10.087DOI Listing
November 2017

miR-16 and miR-103 impact 5-HT receptor signalling and correlate with symptom profile in irritable bowel syndrome.

Sci Rep 2017 10 31;7(1):14680. Epub 2017 Oct 31.

Institute of Pathology, University of Heidelberg, 69120, Heidelberg, Germany.

Irritable bowel syndrome (IBS) is a gut-brain disorder involving alterations in intestinal sensitivity and motility. Serotonin 5-HT receptors are promising candidates in IBS pathophysiology since they regulate gut motor function and stool consistency, and targeted 5-HTR selective drug intervention has been proven beneficial in subgroups of patients. We identified a single nucleotide polymorphism (SNP) (rs201253747) c.*61 T > C within the 5-HT receptor gene HTR4 to be predominantly present in diarrhoea-IBS patients (IBS-D). It affects a binding site for the miR-16 family and miR-103/miR-107 within the isoforms HTR4b/i and putatively impairs HTR4 expression. Subsequent miRNA-profiling revealed downregulation of miR-16 and miR-103 in the jejunum of IBS-D patients correlating with symptoms. In vitro assays confirmed expression regulation via three 3'UTR binding sites. The novel isoform HTR4b_2 lacking two of the three miRNA binding sites escapes miR-16/103/107 regulation in SNP carriers. We provide the first evidence that HTR4 expression is fine-tuned by miRNAs, and that this regulation is impaired either by the SNP c.*61 T > C or by diminished levels of miR-16 and miR-103 suggesting that HTR4 might be involved in the development of IBS-D.
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http://dx.doi.org/10.1038/s41598-017-13982-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5665867PMC
October 2017

Evaluation of Decellularized Porcine Jejunum as a Matrix for Lacrimal Gland Reconstruction In Vitro for Treatment of Dry Eye Syndrome.

Invest Ophthalmol Vis Sci 2017 10;58(12):5564-5574

Laboratory of Experimental Ophthalmology, University Clinic Düsseldorf, Düsseldorf, Germany.

Purpose: Dry eye syndrome (DES) can cause blindness in severe cases, but mainly palliative treatments exist. A tissue-engineered lacrimal gland (LG) could provide a curative treatment. We aimed to evaluate decellularized porcine jejunum (SIS-Muc) as a scaffold for porcine LG epithelial cells.

Methods: To evaluate SIS-Muc as a potential scaffold, basement membrane proteins in SIS-Muc and native LG were compared (immunohistochemistry [IHC]). Porcine LG epithelial cells cultured on plastic were characterized (immunocytochemistry), and their culture supernatant was compared with porcine tears (proteomics). Epithelial cells were then seeded onto SIS-Muc in either a static (cell crown) or dynamic culture (within a perfusion chamber) and metabolic (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and secretory capacities (β-hexosaminidase assay), protein expression (IHC), and ultrastructure transmission electron microscopy (TEM) compared in each.

Results: Collagen IV and laminin were found in both native LG and SIS-Muc. When cultured on plastic, LG epithelial cells expressed pan-cytokeratin, Rab3D, HexA, and produced mucins, but lysozyme and lactoferrin expression was nearly absent. Some porcine tear proteins (lipocalin-2 and lactoferrin) were found in LG epithelial cell culture supernatants. When LG cells were cultured on SIS-Muc, metabolic and β-hexosaminidase activities were greater in dynamic cultures than static cultures (P < 0.05). In both static and dynamic cultures, cells expressed pan-cytokeratin, Rab3D, lysozyme, and lactoferrin and produced mucins, and TEM revealed cell polarization at the apical surface and cell-cell and cell-scaffold contacts.

Conclusions: SIS-Muc is a suitable scaffold for LG cell expansion and may be useful toward reconstruction of LG tissue to provide a curative treatment for DES. Dynamic culture enhances cell metabolic and functional activities.
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http://dx.doi.org/10.1167/iovs.16-20759DOI Listing
October 2017

Calcium fluoride based multifunctional nanoparticles for multimodal imaging.

Beilstein J Nanotechnol 2017 18;8:1484-1493. Epub 2017 Jul 18.

Fraunhofer Institute for Silicate Research ISC, Neunerplatz 2, 97082 Wuerzburg, Germany.

New multifunctional nanoparticles (NPs) that can be used as contrast agents (CA) in different imaging techniques, such as photoluminescence (PL) microscopy and magnetic resonance imaging (MRI), open new possibilities for medical imaging, e.g., in the fields of diagnostics or tissue characterization in regenerative medicine. The focus of this study is on the synthesis and characterization of CaF:(Tb,Gd) NPs. Fabricated in a wet-chemical procedure, the spherical NPs with a diameter of 5-10 nm show a crystalline structure. Simultaneous doping of the NPs with different lanthanide ions, leading to paramagnetism and fluorescence, makes them suitable for MR and PL imaging. Owing to the Gd ions on the surface, the NPs reduce the MR relaxation time constant as a function of their concentration. Thus, the NPs can be used as a MRI CA with a mean relaxivity of about = 0.471 mL·mg·s. Repeated MRI examinations of four different batches prove the reproducibility of the NP synthesis and determine the long-term stability of the CAs. No cytotoxicity of NP concentrations between 0.5 and 1 mg·mL was observed after exposure to human dermal fibroblasts over 24 h. Overall this study shows, that the CaF:(Tb,Gd) NPs are suitable for medical imaging.
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http://dx.doi.org/10.3762/bjnano.8.148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5530611PMC
July 2017

Development of Human Salivary Gland-Like Tissue In Vitro.

Tissue Eng Part A 2018 02 4;24(3-4):301-309. Epub 2017 Aug 4.

3 Department of Tissue Engineering and Regenerative Medicine (TERM), University Hospital Würzburg , Würzburg, Germany .

The loss of salivary gland function caused by radiation therapy of the head and neck is a serious condition and it affects a patient's quality of life. The current lack of effective therapies demands new options to be explored. This study tested whether human salivary gland epithelial cells (SGECs) could be successfully cultured on a decellularized porcine gut matrix (SIS-muc) in both mono- and coculture with microvascular endothelial cells (mvECs). By performing immunofluorescence imaging, transmission as well as scanning electron microscopy (SEM), quantitative polymerase chain reaction (qPCR), and an amylase enzyme assay, it was investigated as to what extent the three-dimensional (3D)-cultured cells could maintain their molecular differentiation and the production of working α-amylase (α-AMY) compared with two-dimensional (2D) culture. In both 3D mono- and coculture, SGECs were successfully cultured and formed acinar-like structures. Those findings were confirmed by SEM imaging. Immunofluorescence imaging revealed that 3D-cultured cells expressed α-AMY, Claudin-1 (CL-1), and water channel protein aquaporin-5 (AQP-5). Two-dimensional-cultured cells only were positive for α-AMY. Real time (RT)-qPCR analysis showed that α-AMY relative gene expression was higher in both 3D mono- and coculture than in 2D culture. In α-AMY enzyme assay, cocultured SGECs showed about 25 times increased enzyme activity compared with 2D-cultured cells. In conclusion, the SIS-muc combined with endothelial coculture seems a suitable culture setting for the tissue engineering of functional human salivary gland tissue.
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http://dx.doi.org/10.1089/ten.tea.2016.0466DOI Listing
February 2018

Screening Applications to Test Cellular Fitness in Transwell Models After Nanoparticle Treatment.

Methods Mol Biol 2017 ;1601:111-122

Translational Center Würzburg "Regenerative Therapies for Oncology and Musculosceletal Diseases", Branch of Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, Würzburg, 97070, Germany.

Nanoparticles (NPs) in biotechnology hold great promise for revolutionizing medical treatments and therapies. In order to bring NPs into clinical application there is a number of preclinical in vitro and in vivo tests, which have to be applied before. The initial in vitro evaluation includes a detailed physicochemical characterization as well as biocompatibility tests, among others. For determination of biocompatibility at the cellular level, the correct choice of the in vitro assay as well as NP pretreatment is absolutely essential. There are a variety of assay technologies available that use standard plate readers to measure metabolic markers to estimate the number of viable cells in culture. Each cell viability assay has its own set of advantages and disadvantages. Regardless of the assay method chosen, the major factors critical for reproducibility and success include: (1) choosing the right assay after comparing optical NP properties with the read-out method of the assay, (2) verifying colloidal stability of NPs in cell culture media, (3) preparing a sterile and stable NP dispersion in cell culture media used in the assay, (4) using a tightly controlled and consistent cell model allowing appropriate characterization of NPs. This chapter will briefly summarize these different critical points, which can occur during biocompatibility screening applications of NPs.
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http://dx.doi.org/10.1007/978-1-4939-6960-9_10DOI Listing
February 2018

Detection and Establishment of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) Mosquitoes in California, 2011-2015.

J Med Entomol 2017 05;54(3):533-543

Vector-Borne Disease Section, Division of Communicable Disease Control, Center for Infectious Diseases, California Department of Public Health, 1616 Capitol Ave, MS-7307, Sacramento, CA 95814

In 2011, a thriving population of Aedes albopictus (Skuse), the Asian tiger mosquito, was discovered within three cities in Los Angeles County over an estimated 52-km2 urban area. Two years later in 2013, Aedes aegypti (L.), the yellow fever mosquito, was detected within several urban areas of Madera, Fresno, and San Mateo counties. State and local vector control agencies responded with an aggressive effort to eradicate or interrupt the spread of these two invasive mosquitoes; however, known populations continued to expand outward and new infestations were identified at an accelerated pace in central and southern California. By the end of 2015, one or both species had been detected within the jurisdictional boundaries of 85 cities and census-designated places in 12 counties. Herein we report on the discovery and widespread establishment of Ae. aegypti and Ae. albopictus in urban areas of coastal, central, and southern California between 2011 and 2015 and discuss the subsequent rapid changes to the activities and priorities of vector control agencies in response to this unprecedented invasion.
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http://dx.doi.org/10.1093/jme/tjw237DOI Listing
May 2017

Establishment of a Human Blood-Brain Barrier Co-culture Model Mimicking the Neurovascular Unit Using Induced Pluri- and Multipotent Stem Cells.

Stem Cell Reports 2017 04 23;8(4):894-906. Epub 2017 Mar 23.

University Hospital Würzburg, Chair Tissue Engineering and Regenerative Medicine, 97070 Würzburg, Germany; Translational Center Würzburg "Regenerative Therapies for Oncology and Musculoskeletal Diseases", Branch of Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, 97070 Würzburg, Germany. Electronic address:

In vitro models of the human blood-brain barrier (BBB) are highly desirable for drug development. This study aims to analyze a set of ten different BBB culture models based on primary cells, human induced pluripotent stem cells (hiPSCs), and multipotent fetal neural stem cells (fNSCs). We systematically investigated the impact of astrocytes, pericytes, and NSCs on hiPSC-derived BBB endothelial cell function and gene expression. The quadruple culture models, based on these four cell types, achieved BBB characteristics including transendothelial electrical resistance (TEER) up to 2,500 Ω cm and distinct upregulation of typical BBB genes. A complex in vivo-like tight junction (TJ) network was detected by freeze-fracture and transmission electron microscopy. Treatment with claudin-specific TJ modulators caused TEER decrease, confirming the relevant role of claudin subtypes for paracellular tightness. Drug permeability tests with reference substances were performed and confirmed the suitability of the models for drug transport studies.
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http://dx.doi.org/10.1016/j.stemcr.2017.02.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5390136PMC
April 2017
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