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Institute
- Lehrstuhl für Tissue Engineering und Regenerative Medizin (129) (remove)
Sonstige beteiligte Institutionen
Growth and Differentiation Factor 5 (GDF5) is a secreted growth factor that belongs to the Bone Morphogenetic Protein (BMP) family and plays a pivotal role during limb development. GDF5 is a susceptibility gene for osteoarthritis (OA) and mutations in GDF5 are associated with a wide variety of skeletal malformations ranging from complex syndromes such as acromesomelic chondrodysplasias to isolated forms of brachydactylies or multiple synostoses syndrome 2 (SYNS2). Here, we report on a family with an autosomal dominant inherited combination of SYNS2 and additional brachydactyly type A1 (BDA1) caused by a single point mutation in GDF5 (p.W414R). Functional studies, including chondrogenesis assays with primary mesenchymal cells, luciferase reporter gene assays and Surface Plasmon Resonance analysis, of the GDF5 W-414R variant in comparison to other GDF5 mutations associated with isolated BDA1 (p.R399C) or SYNS2 (p.E491K) revealed a dual pathomechanism characterized by a gain-and loss-of-function at the same time. On the one hand insensitivity to the main GDF5 antagonist NOGGIN (NOG) leads to a GDF5 gain of function and subsequent SYNS2 phenotype. Whereas on the other hand, a reduced signaling activity, specifically via the BMP receptor type IA (BMPR1A), is likely responsible for the BDA1 phenotype. These results demonstrate that one mutation in the overlapping interface of antagonist and receptor binding site in GDF5 can lead to a GDF5 variant with pathophysiological relevance for both, BDA1 and SYNS2 development. Consequently, our study assembles another part of the molecular puzzle of how loss and gain of function mutations in GDF5 affect bone development in hands and feet resulting in specific types of brachydactyly and SYNS2. These novel insights into the biology of GDF5 might also provide further clues on the pathophysiology of OA.
Background
Bone morphogenetic protein (BMP)-2 and growth and differentiation factor (GDF)-5 are two related transforming growth factor (TGF)-β family members with important functions in embryonic development and tissue homeostasis. BMP-2 is best known for its osteoinductive properties whereas GDF-5—as evident from its alternative name, cartilage derived morphogenetic protein 1—plays an important role in the formation of cartilage. In spite of these differences both factors signal by binding to the same subset of BMP receptors, raising the question how these different functionalities are generated. The largest difference in receptor binding is observed in the interaction with the type I receptor BMPR-IA. GDF-5, in contrast to BMP-2, shows preferential binding to the isoform BMPR-IB, which is abrogated by a single amino acid (A57R) substitution. The resulting variant, GDF-5 R57A, represents a “BMP-2 mimic” with respect to BMP receptor binding. In this study we thus wanted to analyze whether the two growth factors can induce distinct signals via an identically composed receptor.
Results
Unexpectedly and dependent on the cellular context, GDF-5 R57A showed clear differences in its activity compared to BMP-2. In ATDC-5 cells, both ligands induced alkaline phosphatase (ALP) expression with similar potency. But in C2C12 cells, the BMP-2 mimic GDF-5 R57A (and also wild-type GDF-5) clearly antagonized BMP-2-mediated ALP expression, despite signaling in both cell lines occurring solely via BMPR-IA. The BMP-2- antagonizing properties of GDF-5 and GDF-5 R57A could also be observed in vivo when implanting BMP-2 and either one of the two GDF-5 ligands simultaneously at heterotopic sites.
Conclusions
Although comparison of the crystal structures of the GDF-5 R57A:BMPR-IAEC- and BMP-2:BMPR-IAEC complex revealed small ligand-specific differences, these cannot account for the different signaling characteristics because the complexes seem identical in both differently reacting cell lines. We thus predict an additional component, most likely a not yet identified GDF-5-specific co-receptor, which alters the output of the signaling complexes. Hence the presence or absence of this component then switches GDF-5′s signaling capabilities to act either similar to BMP-2 or as a BMP-2 antagonist. These findings might shed new light on the role of GDF-5, e.g., in cartilage maintenance and/or limb development in that it might act as an inhibitor of signaling events initiated by other BMPs.
In situ guided tissue regeneration, also addressed as in situ tissue engineering or endogenous regeneration, has a great potential for population-wide “minimal invasive” applications. During the last two decades, tissue engineering has been developed with remarkable in vitro and preclinical success but still the number of applications in clinical routine is extremely small. Moreover, the vision of population-wide applications of ex vivo tissue engineered constructs based on cells, growth and differentiation factors and scaffolds, must probably be deemed unrealistic for economic and regulation-related issues. Hence, the progress made in this respect will be mostly applicable to a fraction of post-traumatic or post-surgery situations such as big tissue defects due to tumor manifestation. Minimally invasive procedures would probably qualify for a broader application and ideally would only require off the shelf standardized products without cells. Such products should mimic the microenvironment of regenerating tissues and make use of the endogenous tissue regeneration capacities. Functionally, the chemotaxis of regenerative cells, their amplification as a transient amplifying pool and their concerted differentiation and remodeling should be addressed. This is especially important because the main target populations for such applications are the elderly and diseased. The quality of regenerative cells is impaired in such organisms and high levels of inhibitors also interfere with regeneration and healing. In metabolic bone diseases like osteoporosis, it is already known that antagonists for inhibitors such as activin and sclerostin enhance bone formation. Implementing such strategies into applications for in situ guided tissue regeneration should greatly enhance the efficacy of tailored procedures in the future.
Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy
(2012)
Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 °C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 °C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.
Age-related gene expression analysis in enteric ganglia of human colon after laser microdissection
(2014)
The enteric nervous system (ENS) poses the intrinsic innervation of the gastrointestinal tract and plays a critical role for all stages of postnatal life. There is increasing scientific and clinical interest in acquired or age-related gastrointestinal dysfunctions that can be manifested in diseases such as gut constipation or fecal incontinence. In this study, we sought to analyze age-dependent changes in the gene expression profile of the human ENS, particularly in the myenteric plexus. Therefore, we used the laser microdissection technique which has been proven as a feasible tool to analyze distinct cell populations within heterogeneously composed tissues. Full biopsy gut samples were prepared from children (4–12 months), middle aged (48–58 years) and aged donors (70–95 years). Cryosections were histologically stained with H&E, the ganglia of the myenteric plexus identified and RNA isolated using laser microdissection technique. Quantitative PCR was performed for selected neural genes, neurotransmitters and receptors. Data were confirmed on protein level using NADPH-diaphorase staining and immunohistochemistry. As result, we demonstrate age-associated alterations in site-specific gene expression pattern of the ENS. Thus, in the adult and aged distal parts of the colon a marked decrease in relative gene expression of neural key genes like NGFR, RET, NOS1 and a concurrent increase of CHAT were observed. Further, we detected notable regional differences of RET, CHAT, TH, and S100B comparing gene expression in aged proximal and distal colon. Interestingly, markers indicating cellular senescence or oxidative stress (SNCA, CASP3, CAT, SOD2, and TERT) were largely unchanged within the ENS. For the first time, our study also describes the age-dependent expression pattern of all major sodium channels within the ENS. Our results are in line with previous studies showing spatio-temporal differences within the mammalian ENS.
Age-related macular degeneration (AMD) is the leading cause of blindness in the Western world. With an ageing population, it is anticipated that the number of AMD cases will increase dramatically, making a solution to this debilitating disease an urgent requirement for the socioeconomic future of the European Union and worldwide. The present paper reviews the limitations of the current therapies as well as the socioeconomic impact of the AMD. There is currently no cure available for AMD, and even palliative treatments are rare. Treatment options show several side effects, are of high cost, and only treat the consequence, not the cause of the pathology. For that reason, many options involving cell therapy mainly based on retinal and iris pigment epithelium cells as well as stem cells are being tested. Moreover, tissue engineering strategies to design and manufacture scaffolds to mimic Bruch's membrane are very diverse and under investigation. Both alternative therapies are aimed to prevent and/or cure AMD and are reviewed herein.
Integrated approaches using different in vitro methods in combination with bioinformatics can (i) increase the success rate and speed of drug development; (ii) improve the accuracy of toxicological risk assessment; and (iii) increase our understanding of disease. Three-dimensional (3D) cell culture models are important building blocks of this strategy which has emerged during the last years. The majority of these models are organotypic, i.e., they aim to reproduce major functions of an organ or organ system. This implies in many cases that more than one cell type forms the 3D structure, and often matrix elements play an important role. This review summarizes the state of the art concerning commonalities of the different models. For instance, the theory of mass transport/metabolite exchange in 3D systems and the special analytical requirements for test endpoints in organotypic cultures are discussed in detail. In the next part, 3D model systems for selected organs liver, lung, skin, brain are presented and characterized in dedicated chapters. Also, 3D approaches to the modeling of tumors are presented and discussed. All chapters give a historical background, illustrate the large variety of approaches, and highlight up- and downsides as well as specific requirements. Moreover, they refer to the application in disease modeling, drug discovery and safety assessment. Finally, consensus recommendations indicate a roadmap for the successful implementation of 3D models in routine screening. It is expected that the use of such models will accelerate progress by reducing error rates and wrong predictions from compound testing.
Recent advances in the in vitro characterization of human adult enteric neural progenitor cells have opened new possibilities for cell-based therapies in gastrointestinal motility disorders. However, whether these cells are able to integrate within an in vivo gut environment is still unclear. In this study, we transplanted neural progenitor-containing neurosphere-like bodies (NLBs) in a mouse model of hypoganglionosis and analyzed cellular integration of NLB-derived cell types and functional improvement. NLBs were propagated from postnatal and adult human gut tissues. Cells were characterized by immunohistochemistry, quantitative PCR and subtelomere fluorescence in situ hybridization (FISH). For in vivo evaluation, the plexus of murine colon was damaged by the application of cationic surfactant benzalkonium chloride which was followed by the transplantation of NLBs in a fibrin matrix. After 4 weeks, grafted human cells were visualized by combined in situ hybridization (Alu) and immunohistochemistry (PGP9.5, GFAP, SMA). In addition, we determined nitric oxide synthase (NOS)-positive neurons and measured hypertrophic effects in the ENS and musculature. Contractility of treated guts was assessed in organ bath after electrical field stimulation. NLBs could be reproducibly generated without any signs of chromosomal alterations using subtelomere FISH. NLB-derived cells integrated within the host tissue and showed expected differentiated phenotypes i.e. enteric neurons, glia and smooth muscle-like cells following in vivo transplantation. Our data suggest biological effects of the transplanted NLB cells on tissue contractility, although robust statistical results could not be obtained due to the small sample size. Further, it is unclear, which of the NLB cell types including neural progenitors have direct restoring effects or, alternatively may act via 'bystander' mechanisms in vivo. Our findings provide further evidence that NLB transplantation can be considered as feasible tool to improve ENS function in a variety of gastrointestinal disorders.
The mono-6-deoxy-6-azides of 2,6-di-O-methyl-beta-cyclodextrin (DIMEB) and randomly methylated-beta-cyclodextrin (RAMEB) were conjugated to propargylated hydroxyethyl starch (HES) by Cu+-catalysed [2 + 3] cycloaddition. The resulting water soluble polymers showed lower critical solution temperatures (LCST) at 52.5 degrees C (DIMEB-HES) and 84.5 degrees C (RAMEB-HES), respectively. LCST phase separations could be completely avoided by the introduction of a small amount of carboxylate groups at the HES backbone. The methylated CDs conjugated to the HES backbone exhibited significantly lower cytotoxicities than the corresponding monomeric CD derivatives. Since the binding potentials of these CD conjugates were very high, they are promising candidates for new oral dosage forms of anaesthetic actives.