Mammals have two orthologs, Mll and Trx2, for the Drososphila protein Trithorax (TRX), which is the founding member of the trithorax group (TrxG) of epigenetic regulators. TrxG proteins are characterized by an evolutionary conserved SET domain. A major function of all SET domain- containing proteins is to modulate gene activity, but the underlying mechanisms are poorly understood. Apparently TRX, Mll and Trx2 are histone H3 lysine 4 specific methyltransferases. So far all evidence points to roles in expression of specific target genes. However, target genes and function of the epigenetic regulator Trx2 were still unknown. Homozygous trx2 mutant embryos arrest in development because of severe and widespread defects {Glaser, 2005 #296}. Thus mouse embryonic stem (ES) cells carrying a null mutation of trx2 were used as an alternative model system to address the implication of Trx2 in differentiation. This study showed that Trx2 is redundant for ES cell self-renewal. Homozygous trx2 knockout ES cells did not exhibit cell cycle defects. However, loss of Trx2 resulted in reduced proliferation and increased apoptosis rates in trx2-/- ES cells. Due to the fact that differentiation requires an appropriate rate of population growth, trx2-/- cells were affected adversely upon in vitro differentiation. Neurogeneic differentiation of trx2 mutant cells generated fewer mature neurons than wild type cells. Moreover a temporal delay in the developmental progression to differentiation became apparent. Cardiac differentiation of trx2-/- cells confirmed the developmental defect and temporal delay. Notably differentiation of trx2-/- cells was merely delayed or impaired but it was not absent, implying that Trx2 is not required for gene expression programs specific for neurons or cardiac myocytes. We propose that differentiation of trx2-/- ES cells is impaired because apoptosis is disturbing differentiation. Apart from analyzing the phenotype of trx2 mutant cells, this work was focused on the identification of Trx2 target genes. Oligonucleotide expression arrays were used to identify genes whose expression levels were affected by the absence of Trx2. In general, loss of Trx2 function resulted in more genes with decreased than increased expression levels. This is consistent with the hypothesis that Trx2 functions as a transcriptional activator. Comparison of gene expression profiles for constitutive and conditional trx2 mutant cells enabled a distinction between direct and indirect target genes for Trx2. As a result Magoh2 was identified as the key candidate target gene for Trx2. Interaction between Trx2 and Magoh2 suggested a potential regulatory role for Trx2 in alternative splicing. Furthermore this work provided evidence that Trx2 could be involved in the maintenance of CpG island promoter gene expression, thus providing a potent regulatory mechanism for ubiquitously expressed genes.

The development of new therapeutical approaches, devised to foster the regeneration of neuronal circuits after injury and/or in neurodegenerative diseases, is of great importance. The impairment of dopaminergic projections is especially severe, because these projections are involved in crucial brain functions such as motor control, reward and cognition. In the work presented here, organotypic brain slice co-cultures of (a) the mesostriatal and (b) the mesocortical dopaminergic projection systems consisting of tissue sections of the ventral tegmental area/substantia nigra (VTA/SN), in combination with the target regions of (a) the striatum (STR) or (b) the prefrontal cortex (PFC), respectively, were used to evaluate different approaches to stimulate neurite outgrowth: (i) inhibition of cAMP/cGMP turnover with 3’,5’ cyclic nucleotide phosphodiesterase inhibitors (PDE-Is), (ii) blockade of calcium currents with nimodipine, and (iii) the co-cultivation with bone marrow-derived mesenchymal stromal/stem cells (BM-MSCs). The neurite growth-promoting properties of the tested substances and cell populations were analyzed by neurite density quantification in the border region between the two brain slices, using biocytin tracing or tyrosine hydroxylase labeling and automated image processing procedures. In addition, toxicological tests and gene expression analyses were conducted. (i) PDE-Is were applied to VTA/SN+STR rat co-cultures. The quantification of neurite density after both biocytin tracing and tyrosine hydroxylase labeling revealed a growth promoting effect of the PDE2A-Is BAY60-7550 and ND7001. The application of the PDE10-I MP-10 did not alter neurite density in comparison to the vehicle control. (ii) The effects of nimodipine were evaluated in VTA/SN+PFC rat co-cultures. A neurite growth-promoting effect of 0.1 µM and 1 µM nimodipine was demonstrated in a projection system of the CNS. In contrast, the application of 10 µM nimodipine did not alter neurite density, compared to the vehicle control, but induced the activation of the apoptosis marker caspase 3. The expression levels of the investigated genes, including Ca2+ binding proteins (Pvalb, S100b), immediate early genes (Arc, Egr1, Egr2, Egr4, Fos and JunB), glial fibrillary acidic protein, and myelin components (Mal, Mog, Plp1) were not significantly changed (with the exception of Egr4) by the treatment with 0.1 µM and 1 µM nimodipine. (iii) Bulk BM-MSCs that were classically isolated by plastic adhesion were compared to the subpopulation Sca-1+Lin-CD45--derived MSCs (SL45-MSCs). The neurite growth-promoting properties of both MSC populations were quantified in VTA/SN+PFC mouse co-cultures. For this purpose, the MSCs were seeded on glass slides that were placed underneath the co-cultures. A significantly enhanced neurite density within the co-cultures was induced by both bulk BM-MSCs and SL45-MSCs. SL45-MSCs increased neurite density to a higher degree. The characterization of both MSC populations revealed that the frequency of fibroblast colony forming units (CFU-f ) is 105-fold higher in SL45-MSCs. SL45-MSCs were morphologically more homogeneous and expressed higher levels of nestin, BDNF and FGF2 compared to bulk BM-MSCs. Thus, this work emphasizes the vast potential for molecular targeting with respect to the development of therapeutic strategies in the enhancement of neurite regrowth.

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Tumors exhibit complex organization and contain a variety of cell populations. The realization that the regenerative properties of a tumor may be largely confined to a cell subpopulation (cancer stem cell) is driving a new era of anti-cancer research. Cancer stem cells from Glioblastoma Multiforme tumors express markers that are also expressed in non-cancerous neural stem cells, including nestin and Sox2. We previously showed that the transcription factor Hes3 is a marker of neural stem cells, and that its expression is inhibited by JAK activity. Here we show that Hes3 is also expressed in cultures from glioblastoma multiforme which express neural stem cell markers, can differentiate into neurons and glia, and can recapitulate the tumor of origin when transplanted into immunocompromised mice. Similar to observations in neural stem cells, JAK inhibits Hes3 expression. Hes3 RNA interference reduces the number of cultured glioblastoma cells suggesting a novel therapeutic strategy.

Both platelet-rich plasma (PRP) and vascular endothelial growth factor (VEGF) can promote regeneration. The aim of this study was to compare the effects of these two elements on bone formation and vascularization in combination with bone marrow stromal cells (BMSC) in a critical-size bone defect in rabbits. The critical-size defects of the radius were filled with: (1) a calcium-deficient hydroxyapatite (CDHA) scaffold + phVEGF165-transfected BMSC (VEGF group), (2) CDHA and PRP, or (3) CDHA, autogenous BMSC, and PRP. As controls served: (4) the CDHA scaffold alone and (5) the CDHA scaffold and autogenous BMSC. The volume of new bone was measured by means of micro-CT scans, and vascularization was assessed in histology after 16 weeks. Bone formation was higher in the PRP + CDHA, BMSC + CDHA, and PRP + BMSC + CDHA groups than in the VEGF group (p < 0.05). VEGF transfection significantly promoted vascularization of the scaffolds in contrast to BMSC and PRP (p < 0.05), but was similar to the result of the CDHA + PRP + BMSC group. The results show that VEGF-transfected BMSC as well as the combination of PRP and BMSC improve vascularization, but bone healing was better with the combination of BMSC and PRP than with VEGF-transfected BMSC. Expression of VEGF in BMSC as a single growth factor does not seem to be as effective for bone formation as expanded BMSC alone or PRP which contains a mixture of growth factors. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Zur Therapie von Knochendefekten, die nach Traumata, nach Infektion oder Knochennekrosen ohne das Einbringen von Knochenersatz nicht zu heilen sind, wird versucht, Knochenersatz oder Knochenersatzgewebe mit Hilfe des Tissue Engineerings zu züchten. Da eine vollständige Vaskularisation für das Einheilen großer Ersatzgewebe unabdingbar ist, wird durch verschiedene Strategien versucht eine Blutgefäßversorgung solcher Knochenersatzgewebe zu erzielen. Das Ziel dieser Arbeit ist die Generierung axial vaskularisierten Knochenersatzgewebes mit einer β-Trikalziumphosphat-Hydroxylapatitkeramik, mesenchymalen Stammzellen und BMP-2 (bone morphogenetic protein 2) im Großtiermodell des Schafes. Um das Potential zur Knochenbildung bei Verwendung einer β-Trikalziumphosphat-Hydroxylapatitkeramik mit und ohne Zugabe des Wachstumsfaktors bone morphogenetic protein 2 (BMP-2) von ovinen mesenchymalen Stammzellen sowohl als direkt reimplantierte oder vorher expandierte Zellquelle zu untersuchen, wurden im ersten Teil dieser Studie die mesenchymalen Stammzellen des Schafes isoliert und durch Durchflusszytometrie auf Proteinebene und PCR-Untersuchung auf Genebene charakterisiert. Die für die Induktion der Knochenbildung nötige Konzentration von BMP-2 in Kombination mit der Knochenersatzmatrix und Fibrinkleber im Schaf wurde durch subkutane Implantation evaluiert. Nach zwölf Wochen Implantationsdauer wurden die Konstrukte mit 2,5 μg/ml, 12,5 μg/ml und 60 μg/ml BMP-2 im Fibrinkleber durch histologische Auswertung untersucht. Es konnte festgestellt werden, dass für die Ausbildung von lamellärem Knochengewebe 60μg/ml BMP-2 nötig waren. Als nächster Schritt wurden zur Untersuchung des Verhaltens der mesenchymalen Stammzellen für unterschiedliche Implantationsdauern sowohl direkt reimplantierte mesenchymale Stammzellen als auch expandierte Stammzellen im subkutanen Modell des Schafes untersucht. Das Verhalten der verschieden prozessierten Stammzellen war bezüglich ihres Proliferations- und Apoptoseverhaltens, das immunhistologisch untersucht wurde, ähnlich. Das Potential zur ektopen Knochenbildung der direkt reimplantierten mesenchymalen Stammzellen alleine oder in Kombination mit 60 μg/ml BMP-2 und der expandierten mesenchymalen Stammzellen jeweils mit β-Trikalziumphosphat-Hydroxylapatitkeramik und Fibrinkleber wurde nach zwölfwöchiger subkutaner Implantation untersucht. Durch PCR-Untersuchungen konnte gezeigt werden, dass in allen drei Gruppen Knochengene im Vergleich zur Kontrollgruppe mit β-Trikalziumphosphat-Hydroxylapatitkeramik und Fibrinkleber hochreguliert waren. Die quantitative histologische Auswertung ergab, dass die innerhalb der Konstrukte gebildete Knochenfläche im Vergleich zu den anderen Gruppen in den Konstrukten mit expandierten mesenchymalen Stammzellen signifikant größer war. Zur Generierung axial vaskularisierten Knochengewebes wurde das AV-Loop-Modell der Ratte auf das Schaf übertragen und das Potential von direkt reimplantierten mesenchymalen Stammzellen mit β-Trikalziumphosphat-Hydroxylapatitkeramik allein und mit der Zugabe von 60 μg/ml BMP-2 untersucht. Neben der Analyse der Vaskularisierung dieser Konstrukte durch immunhistologische und bildgebende Methoden wurde die Knochenbildung anhand histologischer Präparate quantifiziert. Die AV-Loop-Konstrukte beider Gruppen waren nach zwölfwöchiger Implantationsdauer vollständig vaskularisiert. Die für die Gruppe mit BMP-2 als Wachstumsfaktor durchgeführten intravitalen MRT-Untersuchungen zeigten, dass der Zuwachs an Gefäßen vor allem zwischen der vierten und achten Woche stattfand. Die quantitative Auswertung der neugebildeten Knochenfläche wurde semiautomatisch an histologischen Präparaten durchgeführt. In der Gruppe mit β-Trikalziumphosphat-Hydroxylapatitkeramik, direkt reimplantierten mesenchymalen Stammzellen und 60 μg/ml BMP-2 war die Knochenfläche signifikant größer als in der Gruppe ohne Wachstumsfaktor. Mit dieser Studie konnte erstmals im Großtiermodell gezeigt werden, dass mesenchymale Stammzellen nach direkter Reimplantation im subkutanen Modell und im AV-Loop-Modell zur Induktion der Knochenbildung fähig sind. Durch die Generierung axial vaskularisierten Knochenersatzgewebes im Großtiermodell des Schafes könnte es gelingen, die Größenlimitierung der durch Tissue Engineering gezüchteten Ersatzgeweben zu überwinden.

Over the last decade a huge amount of experimental data on biological systems has been generated by modern high-throughput methods. Aided by bioinformatics, the '-omics' (genomics, transcriptomics, proteomics, metabolomics and interactomics) have listed, quantif ed and analyzed molecular components and interactions on all levels of cellular regulation. However, a comprehensive framework, that does not only list, but links all those components, is still largely missing. The biology-based but highly interdisciplinary field of systems biology aims at such a holistic understanding of complex biological systems covering the length scales from molecules to whole organisms. Spanning the length scales, it has to integrate the data from very different fields and to bring together scientists from those fields. For linking experiments and theory, hypothesis-driven research is an indispensable concept, formulating a cycle of experiment, modeling, model predictions for new experiments and, fi nally, their experimental validation as the start of the new iteration. On the hierarchy of length scales certain unique entities can be identi fied. At the nanometer scale such functional entities are molecules and at the micrometer level these are the cells. Cells can be studied in vitro as independent individuals isolated from an organism, but their interplay and communication in vivo is crucial for tissue function. Control over such regulation mechanisms is therefore a main goal of medical research. The requirements for understanding cellular interplay also illustrate the interdisciplinarity of systems biology, because chemical, physical and biological knowledge is needed simultaneously. Following the notion of cells as the basic units of life, the focus of this thesis are mathematical multi-scale models of multi-cellular systems employing the concept of individual (or agent) based modeling (IBM). This concept accounts for the entity cell and their individuality in function and space. Motivated by experimental observations, cells are represented as elastic and adhesive spheres. Their interaction is given by a model for elastic homogeneous spheres, which has been established for analysis of the elastic response of cells, plus an adhesion term. Cell movement is modeled by an equation of motion for each cell which is based on the balance of interaction, friction and active forces on the respective cell. As a fi rst step the model was carefully examined with regard to the model assumptions, namely, spherical shape, homogeneous isotropic elastic body and apriori undirected movement. The model examination included simulations of cell sorting and compression of multicellular spheroids. Cell sorting could not be achieved with only short range adhesion. However, it sorting completed with long range interactions for small cell numbers, but failed for larger aggregates. Compression dynamics of multi-cellular spheroids was apparently reproduced qualitatively by the model. But in a more detailed survey neither the time scales nor the rounding after compression could be reproduced. Based on these results, the applications consistent with the assumed simpli cations are discussed. One already established application is colony growth in two-dimensional cell cultures. In order to model cell growth and division, a two-phase model of the cell cycle was established. In a growth phase the cell doubles its volume by stochastic increments, and in a mitotic phase it divides into two daughter cells of equal volume. Additionally, control of the cell cycle by contact inhibition is included in the model. After examination of its applicability, the presented model is used for simulations of in vitro growth of mesenchymal stem cells (MSC) and subsequent cartilage formation in multi-cellular spheroids. A main factor for both processes is the oxygen concentration. Experimental results have shown, that i) MSC grow much better in vitro at low than at high oxygen concentrations and ii) the MSC progeny harvested from low oxygen culture produce higher amounts of the cartilage components aggrecan and collagen II in multicellular spheroids than the ones from high oxygen culture. In order to model these processes, IBM was extended by a stochastic model for cellular differentiation. In this model cellular differentiation is captured phenomenologically by two additional individual properties, the degree of differentiation and the lineage or cell type, which are subject to fl uctuations, that are state and environment dependent. After fitting the model parameters to the experimental results on MSC growth in monoclonal expansion cultures at low and high oxygen concentrations, the resulting simulated cell populations were used for initialization of the simulations of cartilage formation in multi-cellular spheroids. The model nicely reproduced the experimental results on growth dynamics and the observed number of functional cells in the spheroids and suggests the following explanation for the difference between the two expansion cultures: due to the stronger pre-differentiation found after expansion in high oxygen, the plasticity of these cells is smaller and less cell adopt the chondrogenic phenotype and start to produce cartilage. Moreover, the model predicts an optimal oxygen concentration for cartilage formation independent of expansion culture and a de-differentiating effect of low oxygen culture within 24h. Because all simulations comply with the concept of hypothesis-driven research and follow closely the experimental protocols, they can easily be tested and are currently used for optimization of a bioreactor for cartilage production. Cell populations are composed of individual cells and regulation of population properties is performed by individual cell, but knowledge about individual cell fates is largely missing due to the problem of single cell tracking. The IBM modeling approach used for modeling MSC growth and differentiation generically includes information of each individual cell and is therefore perfectly suited for tackling this question. Based on the validated parameter set, the model was used to generate predictions on plasticity of single cells and related population dynamics. Single cell plasticity was quantifi ed by calculating transition times into stem cell and differentiated cell states at high and low oxygen concentrations. At low oxygen the results predict a frequent exchange between all subpopulations, while at high oxygen a quasi-deterministic differentiation is found. After quantifying the plasticity of single cells at low and high oxygen concentration, the plasticity of a cell population is addressed in a simulation closely following a regeneration experiment of populations of hematopoietic progenitor cells. In the simulation the regeneration of the distribution of differentiation states in the population is monitored after selection of subpopulations of stem cells and differentiated cells. Simulated regeneration occurs on the time scales estimated from the single cell transition times except the unexpectedly fast regeneration from differentiated cells in the high oxygen environment, which favors differentiation. The latter case emphasizes the importance of single outlier cells in such system, which in this case repopulate less differentiated states with their progeny. In general, cell proliferation and regeneration behavior are in uenced by biomechanical and geometrical properties of the environment e.g. matrix stiffness or cell density. Because in the model cells are represented as physical objects, a variation of friction is linked to cell motility. The cultures of less motile cells become denser at the same size and the effects of contact inhibition of growth more pronounced. This variation of friction coe fficients allows the comparison of cultures with varying degrees of contact inhibition regarding their differentiation structure and the results suggest, that stalled proliferation is su fficient to explain the well-known differentiation effects in confl uent colonies. In addition, the composition of the simulated stem cell pool was analyzed regarding differentiation. In contrast to the established pedigree models, where stem cell can only be produced by asymmetric division, this model predicts that most of the cells in stem cell states descend from progenitor cells of intermediate differentiation states. A more detailed analysis of single cell derived clones revealed properties that could not be described by the model so far. First, a differentiation gradient was observed in larger colonies, that was the opposite of the one predicted by the model. Second, the proliferative activity turned out to depend not only on oxygen, but also to be a property of individual clones persisting over many generations. Because the relation slow growth/pre-differentiation also holds for single cell derived clones, the general model of differentiation is extended by another heritable individual property. Motivated by the decline of proliferation and differentiation in culture and the high metabolic and epigenetic activity during cell division, each division event is assumed to de-stabilize stem cell states. Consequently, in the model the cells age in terms of cell divisions determines the fl uctuations in stem cell states and the environment the mean fl uctuation strength. Including this novel concept, that links aging to growth and differentiation dynamics, into the model reproduces the experimental results regarding differentiation gradient and persistent clonal heterogeneity. The spatial differentiation pattern can largely be explained by the spatio-temporal growth pattern of the mono-clonal cell assembly: cells close to the border of the cell assembly have undergone more cell divisions than those in the interior and therefore their stem cell states are less stable. Heterogeneity of single-cell derived clones depends on the age of the first cell in the clone. When the stem cell fluctuations equal the mean fl uctuations strength, the proliferative activity passes a maximum at a certain age due to the destabilization of stem cell states. Thereafter the proliferative activity decreases, because more time is spent in non-proliferative differentiated states. Considering the number of divisions the cells have already undergone in vivo and after the initial expansion in vitro, it can be assumed that all cells have already passed this maximum. Interestingly, the model also predicts an optimal age for directed differentiation, when cells stably differentiate, but have not lost the required plasticity. According to the model, this clonal heterogeneity may be caused purely in vitro, but hypothetical simulation of in vivo aging yielded results consistent with experiments on MSC from rats of varying age. Finally, the detailed molecular regulation mechanisms in a multi-scale tissue model of liver zonation was studied, in which the key molecular components were explicitly modeled. Hence, this model resolved the intracellular regulation in higher resolution than the above considered differentiation models which had summarized the intracellular control and differentiation mechanisms by a few phenomenological, dynamical variables. The metabolic zonation of the liver is essential for many of the complex liver functions. One of the vitally important enzymes, glutamine synthetase, (GS) is only synthesized in a strictly defi ned pattern. Experimental evidence has shown that a particular pathway, the canonical wnt pathway, controls expression of the gene for GS. A model for transport, receptor dynamics and intracellular regulation mechanism has been set up for modeling the spatio-temporal formation of this pattern. It includes membrane-bound transport of the morphogen and an enzyme kinetics approach to fibeta-catenin-regulation in the interior of the cell. As an IBM this model reproduces the results of co-culture experiments in which two-dimensional arrangements of liver cells and an epithelial liver cell line give rise to different patterns of GS synthesis. The two main predictions of the model are: First, GS-synthesis requires a certain local cell number of wnt releasing cells. And second, a simple inversion of geometry explains the difference between the specifi c GS pattern found in the liver and in the co-culture experiments. Summarizing the results presented in this thesis, it can be concluded that properties such as the occurrence of memory effects and single cells pursuing fates far off the population average could be essential for biological function. Considering the role of single cells in many tissues, the use of individual based methods, that are able to take such effects into account, can be expected to be a very valuable tool for the problems of systems biology.