Joerg Galle

A Comprehensive Model of the Spatio-Temporal Stem Cell and Tissue Organisation in the Intestinal Crypt

We introduce a novel dynamic model of stem cell and tissue organisation in murine intestinal crypts. Integrating the molecular, cellular and tissue level of description, this model links a broad spectrum of experimental observations encompassing spatially confined cell proliferation, directed cell migration, multiple cell lineage decisions and clonal competition. Using computational simulations we demonstrate that the model is capable of quantitatively describing and predicting the dynamic behaviour of the intestinal tissue during steady state as well as after cell damage and following selective gain or loss of gene function manipulations affecting Wnt- and Notch-signalling. Our simulation results suggest that reversibility and flexibility of cellular decisions are key elements of robust tissue organisation of the intestine. We predict that the tissue should be able to fully recover after complete elimination of cellular subpopulations including subpopulations deemed to be functional stem cells. This challenges current views of tissue stem cell organisation.

Noise-Driven Stem Cell and Progenitor Population Dynamics

Background The balance between maintenance of the stem cell state and terminal differentiation is influenced by the cellular environment. The switching between these states has long been understood as a transition between attractor states of a molecular network. Herein, stochastic fluctuations are either suppressed or can trigger the transition, but they do not actually determine the attractor states. Methodology/Principal Findings We present a novel mathematical concept in which stem cell and progenitor population dynamics are described as a probabilistic process that arises from cell proliferation and small fluctuations in the state of differentiation. These state fluctuations reflect random transitions between different activation patterns of the underlying regulatory network. Importantly, the associated noise amplitudes are state-dependent and set by the environment. Their variability determines the attractor states, and thus actually governs population dynamics. This model quantitatively reproduces the observed dynamics of differentiation and dedifferentiation in promyelocytic precursor cells. Conclusions/Significance Consequently, state-specific noise modulation by external signals can be instrumental in controlling stem cell and progenitor population dynamics. We propose follow-up experiments for quantifying the imprinting influence of the environment on cellular noise regulation.

Low oxygen expansion improves subsequent chondrogenesis of ovine bone-marrow-derived mesenchymal stem cells in collagen type I hydrogel.

Background/Objective: A crucial factor when investigating cartilage tissue engineering using mesenchymal stem cells (MSCs) is their application in large-animal models and preclinical trials. However, in vitro studies using cells of these model organisms must proceed. Considering that oxygen tension is an important parameter for stem cell culture, we investigated the effect of low oxygen tension during the expansion of ovine MSCs on colony-forming unit-fibroblast (CFU-F) formation, senescence and subsequent chondrogenesis in pellet culture and a collagen I hydrogel which is in clinical use for matrix-associated autologous chondrocyte transplantation (MACT). Materials and Methods: Ovine MSCs were isolated from bone marrow aspirates and cultured at 5 and 20% O2 in monolayer. CFU-F formation was detected by Giemsa staining. Senescence was analyzed by detection of senescence-associated β-galactosidase and flow cytometry. Chondrogenic differentiation was carried out in pellet and collagen I hydrogel culture and assessed by gene expression, immunohistochemistry and measurement of sulfated glycosaminoglycans (sGAG). Results: MSCs expanded at 5% O2 revealed a 2-fold higher CFU-F potential and diminished senescence compared to those expanded at 20% O2. Most notably, our results show enhanced chondrogenic differentiation in both pellet culture and the MACT-approved collagen I hydrogel. Conclusion: The findings demonstrate that physiologically low oxygen tension during monolayer expansion of ovine MSCs is advantageous in order to improve cartilage tissue engineering in a sheep model. The ovine system is shown to represent an appropriate basis for large-animal studies and preclinical trials on MSC-based cartilage repair.

From single cells to tissue architecture-a bottom-up approach to modelling the spatio-temporal organisation of complex multi-cellular systems.

Collective phenomena in multi-cellular assemblies can be approached on different levels of complexity. Here, we discuss a number of mathematical models which consider the dynamics of each individual cell, so-called agent-based or individual-based models (IBMs). As a special feature, these models allow to account for intracellular decision processes which are triggered by biomechanical cell–cell or cell–matrix interactions. We discuss their impact on the growth and homeostasis of multi-cellular systems as simulated by lattice-free models. Our results demonstrate that cell polarisation subsequent to cell–cell contact formation can be a source of stability in epithelial monolayers. Stroma contact-dependent regulation of tumour cell proliferation and migration is shown to result in invasion dynamics in accordance with the migrating cancer stem cell hypothesis. However, we demonstrate that different regulation mechanisms can equally well comply with present experimental results. Thus, we suggest a panel of experimental studies for the in-depth validation of the model assumptions.

Individual cell-based models of the spatial-temporal organization of multicellular systems--achievements and limitations.

Computational approaches of multicellular assemblies have reached a stage where they may contribute to unveil the processes that underlie the organization of tissues and multicellular aggregates. In this article, we briefly review and present some new results on a number of 3D lattice free individual cell‐based mathematical models of epithelial cell populations. The models we consider here are parameterized by bio‐physical and cell‐biological quantities on the level of an individual cell. Eventually, they aim at predicting the dynamics of the biological processes on the tissue level. We focus on a number of systems, the growth of cell populations in vitro, and the spatial‐temporal organization of regenerative tissues. For selected examples we compare different model approaches and show that the qualitative results are robust with respect to many model details. Hence, for the qualitative features and largely for the quantitative features many model details do not matter as long as characteristic biological features and mechanisms are correctly represented. For a quantitative prediction, the control of the bio‐physical and cell‐biological parameters on the molecular scale has to be known. At this point, slide‐based cytometry may contribute. It permits to track the fate of cells and other tissue subunits in time and validated the organization processes predicted by the mathematical models.

Impact of oxygen environment on mesenchymal stem cell expansion and chondrogenic differentiation

Introduction:  In vitro expansion and differentiation of mesenchymal stem cells (MSC) rely on specific environmental conditions, and investigations have demonstrated that one crucial factor is oxygen environment.

On the biomechanics of stem cell niche formation in the gut – modelling growing organoids

In vitro culture of intestinal tissue has been attempted for decades. Only recently did Sato et al. [Sato, T., Vries, R. G., Snippert, H. J., van de Wetering, M., Barker, N., Stange, D. E., van Es, J. H., Abo, A., Kujala, P., Peters, P. J., et al. (2009) Nature459, 262–265] succeed in establishing long‐term intestinal culture, demonstrating that cells expressing the Lgr5 gene can give rise to organoids with crypt‐like domains similar to those found in vivo. In these cultures, Paneth cells provide essential signals supporting stem cell function. We have recently developed an individual cell‐based computational model of the intestinal tissue [Buske, P., Galle, J., Barker, N., Aust, G., Clevers, H. & Loeffler, M. (2011) PLoS Comput Biol7, e1001045]. The model is capable of quantitatively reproducing a comprehensive set of experimental data on intestinal cell organization. Here, we present a significant extension of this model that allows simulation of intestinal organoid formation in silico. For this purpose, we introduce a flexible basal membrane that assigns a bending modulus to the organoid surface. This membrane may be re‐organized by cells attached to it depending on their differentiation status. Accordingly, the morphology of the epithelium is self‐organized. We hypothesize that local tissue curvature is a key regulatory factor in stem cell organization in the intestinal tissue by controlling Paneth cell specification. In simulation studies, our model closely resembles the spatio‐temporal organization of intestinal organoids. According to our results, proliferation‐induced shape fluctuations are sufficient to induce crypt‐like domains, and spontaneous tissue curvature induced by Paneth cells can control cell number ratios. Thus, stem cell expansion in an organoid depends sensitively on its biomechanics. We suggest a number of experiments that will enable new insights into mechano‐transduction in the intestine, and suggest model extensions in the field of gland formation.

Lgr5(+) gastric stem cells divide symmetrically to effect epithelial homeostasis in the pylorus

The pyloric epithelium continuously self-renews throughout life, driven by limited reservoirs of resident Lgr5+ adult stem cells. Here, we characterize the population dynamics of these stem cells during epithelial homeostasis. Using a clonal fate-mapping strategy, we demonstrate that multiple Lgr5+ cells routinely contribute to epithelial renewal in the pyloric gland and, similar to what was previously observed in the intestine, a balanced homeostasis of the glandular epithelium and stem cell pools is predominantly achieved via neutral competition between symmetrically dividing Lgr5+ stem cells. Additionally, we document a lateral expansion of stem cell clones via gland fission under nondamage conditions. These findings represent a major advance in our basic understanding of tissue homeostasis in the stomach and form the foundation for identifying altered stem cell behavior during gastric disease.

Mesenchymal Stem Cells in Cartilage Repair: State of the Art and Methods to monitor Cell Growth, Differentiation and Cartilage Regeneration

Degenerative joint diseases caused by rheumatism, joint dysplasia or traumata are particularly widespread in countries with high life expectation. Although there is no absolutely convincing cure available so far, hyaline cartilage and bone defects resulting from joint destruction can be treated today by appropriate transplantations. Recently, procedures were developed based on autologous chondrocytes from intact joint areas. The chondrocytes are expanded in cell culture and subsequently transplanted into the defect areas of the affected joints. However, these autologous chondrocytes are characterized by low expansion capacity and the synthesis of extracellular matrix of poor functionality and quality. An alternative approach is the use of adult mesenchymal stem cells (MSCs). These cells effectively expand in 2D culture and have the potential to differentiate into various cell types, including chondrocytes. Furthermore, they have the ability to synthesize extracellular matrix with properties mimicking closely the healthy hyaline joint cartilage. Beside a more general survey of the architecture of hyaline cartilage, its composition and the pathological processes of joint diseases, we will describe here which advances were achieved recently regarding the development of closed, aseptic bioreactors for the production of autologous grafts for cartilage regeneration based on MSCs. Additionally, a novel mathematical model will be presented that supports the understanding of the growth and differentiation of MSCs. It will be particularly emphasized that such models are helpful to explain the well-known fact that MSCs exhibit improved growth properties under reduced oxygen pressure and limited supply with nutrients. Finally, it will be comprehensively shown how different analytical methods can be used to characterize MSCs on different levels. Besides discussing methods for non-invasive monitoring and tracking of the cells and the determination of their elastic properties, mass spectrometric methods to evaluate the lipid compositions of cells will be highlighted.

Individual fates of mesenchymal stem cells in vitro

BackgroundIn vitro cultivated stem cell populations are in general heterogeneous with respect to their expression of differentiation markers. In hematopoietic progenitor populations, this heterogeneity has been shown to regenerate within days from isolated subpopulations defined by high or low marker expression. This kind of plasticity has been suggested to be a fundamental feature of mesenchymal stem cells (MSCs) as well. Here, we study MSC plasticity on the level of individual cells applying a multi-scale computer model that is based on the concept of noise-driven stem cell differentiation.ResultsBy simulation studies, we provide detailed insight into the kinetics of MSC organisation. Monitoring the fates of individual cells in high and low oxygen culture, we calculated the average transition times of individual cells into stem cell and differentiated states. We predict that at low oxygen the heterogeneity of a MSC population with respect to differentiation regenerates from any selected subpopulation in about two days. At high oxygen, regeneration becomes substantially slowed down. Simulation results on the composition of the functional stem cell pool of MSC populations suggest that most of the cells that constitute this pool originate from more differentiated cells.ConclusionsIndividual cell-based models are well-suited to provide quantitative predictions on essential features of the spatio-temporal organisation of MSC in vitro. Our predictions on MSC plasticity and its dependence on the environment motivate a number of in vitro experiments for validation. They may contribute to a better understanding of MSC organisation in vitro, including features of clonal expansion, environmental adaptation and stem cell ageing.