Identification and isolation of neural progenitor cells from the human enteric nervous system (ENS) is currently hampered by the lack of reliable, specific markers. Here, we define the Wnt-receptor frizzled-4 as a marker for the isolation of enteric neural progenitor cells derived from paediatric gut samples. We show that the Wnt-receptor frizzled-4 is expressed in the human colon and in Tunica muscularis-derived enterospheres. To obtain a purified culture, we carried out fluorescence-activated cell sorting (FACS) using PE-conjugated frizzled-4 antibodies. Frizzled-4positive cells gave rise to neurosphere-like bodies and ultimately differentiated into neurons as revealed by BrdU-proliferation assays and immunocytochemistry, whereas in frizzled-4negative cultures we did not detect any neuronal and glial cells. By using a patch-clamp approach, we also demonstrated the expression of functional sodium and potassium channels in frizzled-4positive cell cultures after differentiation in vitro.

BACKGROUND & AIMS:Neural stem and progenitor cells from the enteric nervous system (ENS) might serve as a source of cells for treatment of neurogastrointestinal disorders. Before we can use these cells, we must increase our understanding of the signaling mechanisms that regulate proliferation and differentiation. We systematically evaluated the effects of canonical Wnt signaling on proliferation and differentiation of cultured ENS progenitor cells from neonatal mice and humans.METHODS:We isolated ENS progenitors from tunica muscularis of the small intestine of newborn (postnatal day 0) wild-type C57BL/6 mice as well as from Wnt1-Cre2 reporter mice. We also obtained intestinal tissue samples from infants (2 and 7 months old) undergoing surgery for imperforate anus or focal intestinal perforation and isolated ENS cells. ENS cells were cultured under proliferation conditions leading to formation of 3-dimensional spheres, which we activated with Wnt3a and SB216763 in order to activate the β-catenin-dependent canonical Wnt pathway. We used immunoblot and quantitative polymerase chain reaction to evaluate the molecular response to Wnt stimuli and immunohistochemistry, proliferation, and cell death assays to identify new neurons.RESULTS:In proliferating enterospheres derived from ENS progenitor cells, we verified the expression of Wnt receptors frizzled 1-10 and the co-receptors low-density lipoprotein receptor-related proteins 5 and 6. Pharmacologic stimulation with Wnt agonists led to intracellular accumulation of Wnt-dependent β-catenin and up-regulated expression of known Wnt target genes axin2, lef1, and lgr5. Activation of the canonical Wnt pathway promoted growth of ENS cell spheres during cell expansion and increased the number of newborn neurons derived from mouse and human progenitor cells.CONCLUSIONS:In studies of human and mouse ENS progenitors, we found activation of the Wnt signaling pathway to promote neurogenesis of the ENS in vitro. The neurogenic effect of Wnt agonists on ENS progenitors supports their use in generation of cell pools for autologous cell replacement therapies.

Over the last 20 years, there has been increasing focus on the development of novel stem cell based therapies for the treatment of disorders and diseases affecting the enteric nervous system (ENS) of the gastrointestinal tract (so-called enteric neuropathies). Here, the idea is that ENS progenitor/stem cells could be transplanted into the gut wall to replace the damaged or absent neurons and glia of the ENS. This White Paper sets out experts' views on the commonly used methods and approaches to identify, isolate, purify, expand and optimize ENS stem cells, transplant them into the bowel, and assess transplant success, including restoration of gut function. We also highlight obstacles that must be overcome in order to progress from successful preclinical studies in animal models to ENS stem cell therapies in the clinic.

Novel techniques, like CLARITY and PACT, render large tissue specimens transparent and thereby suitable for microscopic analysis. We used these techniques to evaluate their potential in the intestine as an exemplary organ with a complex tissue composition. Immunohistochemistry, light sheet- and confocal scanning-microscopy enabled us to follow complex three-dimensional structures, like nerve fibers, vessels and epithelial barriers throughout the entire organ. Moreover, in a systematic electron microscopic study, we analyzed the morphology and preservation of tissue on ultrastructural level during the clearing process. We also connect tissue clearing with classical histology and demonstrate that cleared tissues can be stained with Hematoxylin-Eosin and Heidenhain’s Azan stain, suggesting potential use in histopathology. These experiments showed that a neutral pH during the clearing process results in much better preservation of tissue ultrastructure and standard stainability. Volume changes of specimens were monitored and quantified during the course of the protocol. Additionally, we employed the technique to visualize the enteric nervous system and the epithelial barrier in post mortem human gut preparations. Our data show the high potential of tissue clearing throughout different tissue types supporting its usefulness in research and diagnosis and contribute to the technical discussion of ultrastructural tissue-retention.

The complex functions of the gastrointestinal tract rely on the coordinated interplay of several cell and tissue types involving epithelium, connective tissue, smooth muscles as well as cells of the immune and nervous system. It is therefore obvious, that these functions can hardly be investigated sufficiently using cell lines or two-dimensional cell cultures.Here, we describe an easy to produce three-dimensional organotypical explants culture from fetal and neonatal murine colon. This model is suitable for in vitro testing of intestinal function or the evaluation of developmental or pathological processes.

Postnatal neural progenitor cells of the enteric nervous system are a potential source for future cell replacement therapies of developmental dysplasia like Hirschsprung's disease. However, little is known about the molecular mechanisms driving the homeostasis and differentiation of this cell pool. In this work, we conducted Affymetrix GeneChip experiments to identify differences in gene regulation between proliferation and early differentiation of enteric neural progenitors from neonatal mice. We detected a total of 1333 regulated genes that were linked to different groups of cellular mechanisms involved in cell cycle, apoptosis, neural proliferation, and differentiation. As expected, we found an augmented inhibition in the gene expression of cell cycle progression as well as an enhanced mRNA expression of neuronal and glial differentiation markers. We further found a marked inactivation of the canonical Wnt pathway after the induction of cellular differentiation. Taken together, these data demonstrate the various molecular mechanisms taking place during the proliferation and early differentiation of enteric neural progenitor cells.

The Wnt signalling pathway plays a crucial role in the development of the nervous system. This signalling cascade is initiated upon binding of the secreted Wnt ligand to a member of the family of frizzled receptors. In the present study, we analysed the presence of frizzled-4 in the enteric nervous system of human infants. Frizzled-4 could be identified by immunohistochemistry in a subpopulation of enteric neuronal and glial cells in the small and large intestine. Detection of frizzled-4 in the tunica muscularis by RT-PCR confirmed this receptor's expression on the mRNA level. Interestingly, we observed distinct cell populations that co-expressed frizzled-4 with the intermediate filament protein nestin and the neurotrophin receptor p75NTR, which have been reported to be expressed in neural progenitor cells. Flow cytometry analysis revealed that 60% of p75NTR positive cells of the tunica muscularis were positive for frizzled-4. Additionally, in pathological samples of Hirschsprung's disease, the expression of this Wnt receptor correlated with the number of myenteric ganglion cells and decreased from normoganglionic to aganglionic areas of large intestine. The expression pattern of frizzled-4 indicates that this Wnt receptor could be involved in postnatal development and/or function of the enteric nervous system.

Ein neuer Bioreaktortyp, der Nebelkammer-Reaktor, wird vorgestellt. Im Unterschied zu den bisherig verwendeten submersen Kultur- bzw. Bioreaktorverfahren werden im Nebelkammer-Reaktor Zellverbande mittels eines Aerosols aus ultraschallzerstaubtem Nahrmedium umhullt. Die kontinuierlich erzeugten Aerosoltropfchen setzen sich auf den Zellen ab und ermoglichen so eine besonders effektive und scherstressarme Versorgung der Zellen mit Gasen und Nahrstoffen sowie eine gro ss ere Unabhangigkeit von Form und Gro ss e der eingesetzten Konstrukte.The mist chamber reactor is described as a new bioreactor type in which cell structures, in contrast to conventional submerged culture systems, are surrounded by an aerosol of a culture medium derived by ultrasonic vaporization. The continuously produced aerosol droplets sediment on the cells, which leads to an effective supply with gases and nutrients with low shear stress as well as greater independence of shape and size of the constructs used.

ABSTRACT In this article we describe the development, the characterization and the evaluation of a novel bioreactor type for the cultivation of different pro‐ and eukaryotic cell‐systems: the mist‐chamber bioreactor. This innovative bioreactor meets the demand of cultivation systems for shear stress sensitive cells with high requirements for gas supply. Within the mist‐chamber bioreactor the cells are cultivated inside an aerosol of vaporized medium generated by ultrasonic vaporization. In contrast to many established bioreactor systems the mist‐chamber bioreactor offers an environment with an excellent gas supply without any impeller or gas bubble induced shear stress. A mist‐chamber bioreactor prototype has been manufactured and characterized during this work. In the technical and chemical characterization we evaluated the vaporization process, resulting in a vaporization performance of 32 mL/h at working conditions. On this basis we calculated a biomass of 1.4 g (S. cerevisiae, q s = 3.45 × 10–3 mol/g/h ) and 3.4 g (Aspergillus niger, q s = 1.33 × 10–3 mol/g/h ) where the growth rate becomes limited by transport processes. Additionally, we determined a homogenous cultivation area to a height of 3 cm giving a total volume of 0.45 L for the cultivation. Medium components were examined according to their stability during vaporization with the result that all components are stable for at least 5 days. After the technical characterization we demonstrated the feasibility to cultivate S. cerevisiae and F. velupites in the mist‐chamber bioreactor. The results demonstrated that the mist‐chamber bioreactor is able to transport a sufficient amount of nutrients consistently to the cell samples and offers an excellent oxygen supply without any shear stress inducing aeration. Furthermore we successfully cultivated F. velupites in a solid state cultivation in a long term experiment. The data indicate that the new bioreactor concept can contribute to improve various fermentations and cell culture processes depending on the cultured cell types. Biotechnol. Bioeng. 2015;112: 1155–1164. © 2014 Wiley Periodicals, Inc.

Biotechnology and BioengineeringVolume 112, Issue 6 p. fmi-fmv ContentsFree Access Biotechnology and Bioengineering: Volume 112, Number 6, June 2015 First published: 27 April 2015 https://doi.org/10.1002/bit.25403AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Volume112, Issue6June 2015Pages fmi-fmv RelatedInformation

The demand for scaffolds comprised of natural materials such as collagen has increased in recent years. However, many scaffolds rely on chemical or physical modifications in order to comply with the necessary requirements for biomedical engineering. We evaluated the in vivo biocompatibility and biodegradation of a novel, thin, mechanically stable, and chemically non-crosslinked collagen cell carrier (CCC). CCC was implanted subcutaneously into 25 adult Lewis rats and biopsies were taken on days 7, 14, 21, 42, and 84 after surgery. For histological analysis, paraffin sections of implanted skin were immunolabeled for CD68 and stained by hematoxylin-eosin and Masson-Goldner's trichrome method. Macroscopic analysis of skin surface during wound healing process showed a normal physiological reaction. Biodegradation of CCC was completed 42 days after subcutaneous implantation. Histological evaluation revealed no evidence of encapsulation, scar formation, or long-term vascularization and inflammation. The collagen type I based biomaterial demonstrated a high in vivo biocompatibility, low irritability, complete resorption, and replacement by autologous tissue. The in vivo biocompatibility and degradation behavior encourage for further evaluation of CCC in surgical applications and regenerative medicine.

The authors have provided new data to correct errors presented in this Article. Nature has peer-reviewed all evidence provided by the authors to the editors.

Neural stem or progenitor cells have been proposed to restore gastrointestinal function in patients suffering from congenital or acquired defects of the enteric nervous system. Various, mainly embryonic cell sources have been identified for this purpose. However, immunological and ethical issues make a postnatal cell based therapy desirable. We therefore evaluated and quantified the potential of progenitor cells of the postnatal murine enteric nervous system to give rise to neurons and glial cells in vitro. Electrophysiological analysis and BrdU uptake studies provided direct evidence that generated neurons derive from expanded cells in vitro. Transplantation of isolated and expanded postnatal progenitor cells into the distal colon of adult mice demonstrated cell survival for 12 weeks (end of study). Implanted cells migrated within the gut wall and differentiated into neurons and glial cells, both of which were shown to derive from proliferated cells by BrdU uptake. This study indicates that progenitor cells isolated from the postnatal enteric nervous system might have the potential to serve as a source for a cell based therapy for neurogastrointestinal motility disorders. However, further studies are necessary to provide evidence that the generated cells are capable to positively influence the motility of the diseased gastrointestinal tract.

INTRODUCTION:Thyroid hormones play important roles in the development of neural cells in the central nervous system. Even minor changes to normal thyroid hormone levels affect dendritic and axonal outgrowth, sprouting and myelination and might even lead to irreversible damages such as cretinism. Despite our knowledge of the influence on the mammalian CNS, the role of thyroid hormones in the development of the enteric nervous system (ENS) still needs to be elucidated. In this study we have analyzed for the first time the influence of 3,5,3'-triiodothyronine (T3) on ENS progenitor cells using cell biological assays and a microarray technique. RESULTS:In our in vitro model, T3 inhibited cell proliferation and stimulated neurite outgrowth of differentiating ENS progenitor cells. Microarray analysis revealed a group of 338 genes that were regulated by T3 in differentiating enterospheres. 67 of these genes are involved in function and development of the nervous system. 14 of them belong to genes that are involved in axonal guidance or neurite outgrowth. Interestingly, T3 regulated the expression of netrin G1 and endothelin 3, two guidance molecules that are involved in human enteric dysganglionoses. CONCLUSION:The results of our study give first insights how T3 may affect the enteric nervous system. T3 is involved in proliferation and differentiation processes in enterospheres. Microarray analysis revealed several interesting gene candidates that might be involved in the observed effects on enterosphere differentiation. Future studies need to be conducted to better understand the gene to gene interactions.

Introduction Split-thickness skin grafting is often associated with poor skin quality. In this context, a new collagen cell carrier (CCC), as an underlayment in split skin graft (SSG) transplantation for covering tissue defects in rats, has been evaluated as an improving agent. Methods Twenty-eight full-thickness wounds were covered with SSGs, applying the CCC as an underlayment in 14 rats and using SSG transplantation alone in control group. Postgraft skin areas were assessed using an instrument that measures mechanical properties of skin. Three parameters were considered for skin elasticity analysis: total skin deviation (R0), gross elasticity (R2) and viscoelasticity (R8). Measurements were performed every 10 similar to days for 80 similar to days after grafting. Biopsies were taken subsequently for histologic evaluation. Results The results demonstrated significantly superior elasticity values in CCC-supplemented SSGs in terms of gross elasticity and viscoelasticity (R2/R8) starting from day 60 after grafting to the end of the measuring period. There was no histologic evidence of inflammation, adverse host tissue reaction, or scar tissue formation. Conclusion Split skin grafting is associated with poor dermal quality, but CCC may offer unique opportunities in complex wound management in terms of skin graft quality improvement.

Chemie Ingenieur TechnikVolume 84, Issue 8 p. 1329-1329 PosterFree Access Nebelkammerbioreaktor – Evaluation eines neuen Reaktorsystems B. Tscheschke, Corresponding Author B. Tscheschke Tscheschke@iftc.uni-hannover.de Gottfried Wilhelm Leibniz Universität Hannover, Institut für Technische Chemie, Callinstraße 5, D-30167 Hannover, GermanyGottfried Wilhelm Leibniz Universität Hannover, Institut für Technische Chemie, Callinstraße 5, D-30167 Hannover, GermanySearch for more papers by this authorJ. Dreimann, J. Dreimann Gottfried Wilhelm Leibniz Universität Hannover, Institut für Technische Chemie, Callinstraße 5, D-30167 Hannover, GermanySearch for more papers by this authorJ. von der Ruhr, J. von der Ruhr Zentrum für Regenerationsbiologie und Regenerative Medizin (ZRM), Anatomisches Institut, Österbergstraße 3, D-72074 Tübingen, GermanySearch for more papers by this authorDr. T. Schmidt, Dr. T. Schmidt Zentrum für Regenerationsbiologie und Regenerative Medizin (ZRM), Anatomisches Institut, Österbergstraße 3, D-72074 Tübingen, GermanySearch for more papers by this authorDr. L. Just, Dr. L. Just Zentrum für Regenerationsbiologie und Regenerative Medizin (ZRM), Anatomisches Institut, Österbergstraße 3, D-72074 Tübingen, GermanySearch for more papers by this authorDr. F. Stahl, Dr. F. Stahl Gottfried Wilhelm Leibniz Universität Hannover, Institut für Technische Chemie, Callinstraße 5, D-30167 Hannover, GermanySearch for more papers by this authorProf. Dr. T. Scheper, Prof. Dr. T. Scheper Gottfried Wilhelm Leibniz Universität Hannover, Institut für Technische Chemie, Callinstraße 5, D-30167 Hannover, GermanySearch for more papers by this author B. Tscheschke, Corresponding Author B. Tscheschke Tscheschke@iftc.uni-hannover.de Gottfried Wilhelm Leibniz Universität Hannover, Institut für Technische Chemie, Callinstraße 5, D-30167 Hannover, GermanyGottfried Wilhelm Leibniz Universität Hannover, Institut für Technische Chemie, Callinstraße 5, D-30167 Hannover, GermanySearch for more papers by this authorJ. Dreimann, J. Dreimann Gottfried Wilhelm Leibniz Universität Hannover, Institut für Technische Chemie, Callinstraße 5, D-30167 Hannover, GermanySearch for more papers by this authorJ. von der Ruhr, J. von der Ruhr Zentrum für Regenerationsbiologie und Regenerative Medizin (ZRM), Anatomisches Institut, Österbergstraße 3, D-72074 Tübingen, GermanySearch for more papers by this authorDr. T. Schmidt, Dr. T. Schmidt Zentrum für Regenerationsbiologie und Regenerative Medizin (ZRM), Anatomisches Institut, Österbergstraße 3, D-72074 Tübingen, GermanySearch for more papers by this authorDr. L. Just, Dr. L. Just Zentrum für Regenerationsbiologie und Regenerative Medizin (ZRM), Anatomisches Institut, Österbergstraße 3, D-72074 Tübingen, GermanySearch for more papers by this authorDr. F. Stahl, Dr. F. Stahl Gottfried Wilhelm Leibniz Universität Hannover, Institut für Technische Chemie, Callinstraße 5, D-30167 Hannover, GermanySearch for more papers by this authorProf. Dr. T. Scheper, Prof. Dr. T. Scheper Gottfried Wilhelm Leibniz Universität Hannover, Institut für Technische Chemie, Callinstraße 5, D-30167 Hannover, GermanySearch for more papers by this author First published: 25 July 2012 https://doi.org/10.1002/cite.201250081AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume84, Issue8Special Issue: ProcessNet-Jahrestagung 2012 und 30. Jahrestagung der BiotechnologenAugust, 2012Pages 1329-1329 RelatedInformation