Organotypical tissue cultures from adult murine colon as an in vitro model of intestinal mucosa

Together with animal experiments, organotypical cell cultures are important models for analyzing cellular interactions of the mucosal epithelium and pathogenic mechanisms in the gastrointestinal tract. Here, we introduce a three-dimensional culture model from the adult mouse colon for cell biological investigations in an in vivo-like environment. These explant cultures were cultured for up to 2 weeks and maintained typical characteristics of the intestinal mucosa, including a high-prismatic epithelium with specific epithelial cell-to-cell connections, a basal lamina and various connective tissue cell types, as analyzed with immunohistological and electron microscopic methods. The function of the epithelium was tested by treating the cultures with dexamethasone, which resulted in a strong upregulation of the serum- and glucocorticoid-inducible kinase 1 similar to that found in vivo. The culture system was investigated in infection experiments with the fungal pathogen Candida albicans. Wildtype but not Δcph1/Δefg1-knockout Candida adhered to, penetrated and infiltrated the epithelial barrier. The results demonstrate the potential usefulness of this intestinal in vitro model for studying epithelial cell-cell interactions, cellular signaling and microbiological infections in a three-dimensional cell arrangement

Comparative Microarray Analysis of Proliferating and Differentiating Murine ENS Progenitor Cells

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

Expression of the Wnt Receptor Frizzled-4 in the Human Enteric Nervous System of Infants

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

Wnt Receptor Frizzled-4 as a Marker for Isolation of Enteric Neural Progenitors in Human Children

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

Isolation, expansion and transplantation of postnatal murine progenitor cells of the enteric nervous system.

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

Bone Marrow-Derived Human Mesenchymal Stem Cells Express Cardiomyogenic Proteins But Do Not Exhibit Functional Cardiomyogenic Differentiation Potential

Despite their paracrine activites, cardiomyogenic differentiation of bone marrow (BM)-derived mesenchymal stem cells (MSCs) is thought to contribute to cardiac regeneration. To systematically evaluate the role of differentiation in MSC-mediated cardiac regeneration, the cardiomyogenic differentiation potential of human MSCs (hMSCs) and murine MSCs (mMSCs) was investigated in vitro and in vivo by inducing cardiomyogenic and noncardiomyogenic differentiation. Untreated hMSCs showed upregulation of cardiac tropopin I, cardiac actin, and myosin light chain mRNA and protein, and treatment of hMSCs with various cardiomyogenic differentiation media led to an enhanced expression of cardiomyogenic genes and proteins; however, no functional cardiomyogenic differentiation of hMSCs was observed. Moreover, co-culturing of hMSCs with cardiomyocytes derived from murine pluripotent cells (mcP19) or with murine fetal cardiomyocytes (mfCMCs) did not result in functional cardiomyogenic differentiation of hMSCs. Despite direct contact to beating mfCMCs, hMSCs could be effectively differentiated into cells of only the adipogenic and osteogenic lineage. After intramyocardial transplantation into a mouse model of myocardial infarction, Sca-1(+) mMSCs migrated to the infarcted area and survived at least 14 days but showed inconsistent evidence of functional cardiomyogenic differentiation. Neither in vitro treatment nor intramyocardial transplantation of MSCs reliably generated MSC-derived cardiomyocytes, indicating that functional cardiomyogenic differentiation of BM-derived MSCs is a rare event and, therefore, may not be the main contributor to cardiac regeneration