Jens Przybilla

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.

Transcriptional regulation by histone modifications: towards a theory of chromatin re-organization during stem cell differentiation

Chromatin-related mechanisms, as e.g. histone modifications, are known to be involved in regulatory switches within the transcriptome. Only recently, mathematical models of these mechanisms have been established. So far they have not been applied to genome-wide data. We here introduce a mathematical model of transcriptional regulation by histone modifications and apply it to data of trimethylation of histone 3 at lysine 4 (H3K4me3) and 27 (H3K27me3) in mouse pluripotent and lineage-committed cells. The model describes binding of protein complexes to chromatin which are capable of reading and writing histone marks. Molecular interactions of the complexes with DNA and modified histones create a regulatory switch of transcriptional activity. The regulatory states of the switch depend on the activity of histone (de-) methylases, the strength of complex-DNA-binding and the number of nucleosomes capable of cooperatively contributing to complex-binding. Our model explains experimentally measured length distributions of modified chromatin regions. It suggests (i) that high CpG-density facilitates recruitment of the modifying complexes in embryonic stem cells and (ii) that re-organization of extended chromatin regions during lineage specification into neuronal progenitor cells requires targeted de-modification. Our approach represents a basic step towards multi-scale models of transcriptional control during development and lineage specification.

Understanding epigenetic changes in aging stem cells – a computational model approach

During aging, a decline in stem cell function is observed in many tissues. This decline is accompanied by complex changes of the chromatin structure among them changes in histone modifications and DNA methylation which both affect transcription of a tissue‐specific subset of genes. A mechanistic understanding of these age‐associated processes, their interrelations and environmental dependence is currently lacking. Here, we discuss related questions on the molecular, cellular, and population level. We combine an individual cell‐based model of stem cell populations with a model of epigenetic regulation of transcription. The novel model enables to simulate age‐related changes of trimethylation of lysine 4 at histone H3 and of DNA methylation. These changes entail expression changes of genes that induce age‐related phenotypes (ARPs) of cells. We compare age‐related changes of regulatory states in quiescent stem cells occupying a niche with those observed in proliferating cells. Moreover, we analyze the impact of the activity of the involved epigenetic modifiers on these changes. We find that epigenetic aging strongly affects stem cell heterogeneity and that homing at stem cell niches retards epigenetic aging. Our model provides a mechanistic explanation how increased stem cell proliferation can lead to progeroid phenotypes. Adapting our model to properties observed for aged hematopoietic stem cell (HSC) clones, we predict that the hematopoietic ARP activates young HSCs and thereby retards aging of the entire HSC population. In addition, our model suggests that the experimentally observed high interindividual variance in HSC numbers originates in a variance of histone methyltransferase activity.

Modeling the dynamic epigenome: from histone modifications towards self-organizing chromatin

Epigenetic mechanisms play an important role in regulating and stabilizing functional states of living cells. However, in spite of an increasing amount of experimental data, models of transcriptional regulation by epigenetic processes, in particular by histone modifications, are rather rare. In this article, we focus on epigenetic modes of transcriptional regulation based on histone modifications and their potential dynamical interplay with DNA methylation and higher-order chromatin structure. The main purpose of this article is to review recent formal modeling approaches to the dynamics and propagation of histone modifications and to relate them to available experimental data. We evaluate their assumptions with respect to recruitment of relevant modifiers, establishment and processing of modifications, and compare the emerging stability properties and memory effects. Theoretical predictions that await experimental validation are highlighted and potential extensions of these models towards multiscale models of self-organizing chromatin are discussed.

Is adult stem cell aging driven by conflicting modes of chromatin remodeling

Epigenetic control of gene expression by chromatin remodeling is critical for adult stem cell function. A decline in stem cell function is observed during aging, which is accompanied by changes in the chromatin structure that are currently unexplained. Here, we hypothesize that these epigenetic changes originate from the limited cellular capability to inherit epigenetic information. We suggest that spontaneous loss of histone modification, due to fluctuations over short time scales, gives rise to long‐term changes in DNA methylation and, accordingly, in gene expression. These changes are assumed to impair stem cell function and, thus, to contribute to aging. We discuss cell replication as a major source of fluctuations in histone modification patterns. Gene silencing by our proposed mechanism can be interpreted as a manifestation of the conflict between the stem cell plasticity required for tissue regeneration and the permanent silencing of potentially deleterious genomic sequences.

Genomic and transcriptomic heterogeneity of colorectal tumors arising in Lynch Syndrome

Colorectal cancer (CRC) arising in Lynch syndrome (LS) comprises tumours with constitutional mutations in DNA mismatch repair genes. There is still a lack of whole‐genome and transcriptome studies of LS‐CRC to address questions about similarities and differences in mutation and gene expression characteristics between LS‐CRC and sporadic CRC, about the molecular heterogeneity of LS‐CRC, and about specific mechanisms of LS‐CRC genesis linked to dysfunctional mismatch repair in LS colonic mucosa and the possible role of immune editing. Here, we provide a first molecular characterization of LS tumours and of matched tumour‐distant reference colonic mucosa based on whole‐genome DNA‐sequencing and RNA‐sequencing analyses. Our data support two subgroups of LS‐CRCs, G1 and G2, whereby G1 tumours show a higher number of somatic mutations, a higher amount of microsatellite slippage, and a different mutation spectrum. The gene expression phenotypes support this difference. Reference mucosa of G1 shows a strong immune response associated with the expression of HLA and immune checkpoint genes and the invasion of CD4+ T cells. Such an immune response is not observed in LS tumours, G2 reference and normal (non‐Lynch) mucosa, and sporadic CRC. We hypothesize that G1 tumours are edited for escape from a highly immunogenic microenvironment via loss of HLA presentation and T‐cell exhaustion. In contrast, G2 tumours seem to develop in a less immunogenic microenvironment where tumour‐promoting inflammation parallels tumourigenesis. Larger studies on non‐neoplastic mucosa tissue of mutation carriers are required to better understand the early phases of emerging tumours. Copyright

Histone modifications control DNA methylation profiles during ageing and tumour expansion

The stem cell epigenome reflects a delicate balance of chromatin (de-)modification processes. During cancer development this balance becomes disturbed, resulting in characteristic changes. Among them are changes of the DNA methylation profile. It has been demonstrated that these changes share common features with changes observed during stem cell ageing. Recently, we proposed that chromatin remodelling during stem cell ageing originates in the limited cellular capability to inherit histone modification states, and thus is linked to cell proliferation. This suggests that increased proliferation activity and associated loss of histone modification may represent a generic cause of the changes observed in cancer DNA methylation profiles. Moreover, additional changes of these profiles due to mutations of chromatin modifiers are expected to act on this background. We study the implications of these assumptions, introducing a computational model which describes transcriptional regulation by cis-regulatory networks, trimethylation of histone 3 at lysine 4 and 9 and DNA methylation. By simulation of the model we analyse how mutations that change the proliferation activity and histone de-modification rates impact DNA methylation profiles. We demonstrate that our hypotheses are consistent with experimental findings, and thus may provide a mechanistic explanation of DNA methylation changes during ageing and tumour expansion.

Bistable Epigenetic States Explain Age‐Dependent Decline in Mesenchymal Stem Cell Heterogeneity

The molecular mechanisms by which heterogeneity, a major characteristic of stem cells, is achieved are yet unclear. We here study the expression of the membrane stem cell antigen‐1 (Sca‐1) in mouse bone marrow mesenchymal stem cell (MSC) clones. We show that subpopulations with varying Sca‐1 expression profiles regenerate the Sca‐1 profile of the mother population within a few days. However, after extensive replication in vitro, the expression profiles shift to lower values and the regeneration time increases. Study of the promoter of Ly6a unravels that the expression level of Sca‐1 is related to the promoter occupancy by the activating histone mark H3K4me3. We demonstrate that these findings can be consistently explained by a computational model that considers positive feedback between promoter H3K4me3 modification and gene transcription. This feedback implicates bistable epigenetic states which the cells occupy with an age‐dependent frequency due to persistent histone (de‐)modification. Our results provide evidence that MSC heterogeneity, and presumably that of other stem cells, is associated with bistable epigenetic states and suggest that MSCs are subject to permanent state fluctuations. Stem Cells 2017;35:694–704

Stem cell competition in the gut: insights from multi-scale computational modelling

Three-dimensional (3D) computational tissue models can provide a comprehensive description of tissue dynamics at the molecular, cellular and tissue level. Moreover, they can support the development of hypotheses about cellular interactions and about synergies between major signalling pathways. We exemplify these capabilities by simulation of a 3D single-cell-based model of mouse small intestinal crypts. We analyse the impact of lineage specification, distribution and cellular lifespan on clonal competition and study effects of Notch- and Wnt activation on fixation of mutations within the tissue. Based on these results, we predict that experimentally observed synergistic effects between autonomous Notch- and Wnt signalling in triggering intestinal tumourigenesis originate in the suppression of Wnt-dependent secretory lineage specification by Notch, giving rise to an increased fixation probability of Wnt-activating mutations. Our study demonstrates that 3D computational tissue models can support a mechanistic understanding of long-term tissue dynamics under homeostasis and during transformation.

Different in vivo and in vitro transformation of intestinal stem cells in mismatch repair deficiency

Mutations in mismatch repair (MMR) genes result in microsatellite instability (MSI) and early onset of colorectal cancer. To get mechanistic insights into the time scale, sequence and frequency of intestinal stem cell (ISC) transformation, we quantified MSI and growth characteristics of organoids of Msh2-deficient and control mice from birth until tumor formation and related them to tissue gene expression. Although in Msh2-deficient organoids MSI continuously increased from birth, growth characteristics remained stable at first. Months before tumor onset, normal Msh2-deficient tissue contained tumor precursor cells forming organoids with higher MSI, cystic growth and growth rates resembling temporarily those of tumor organoids. Consistently, Msh2-deficient tissue exhibited a tumor-like gene signature. Normal Msh2-deficient organoids showed increased inheritable transient cyst-like growth, which became independent of R-spondin. ISC transformation proceeded faster in vitro than in vivo independent of the underlying genotype but more under MMR deficiency. Transient cyst-like growth but not MSI was suppressed by aspirin. In summary, as highlighted by organoids, molecular alterations continuously proceeded long before tumor onset in MMR-deficient intestine, thus increasing its susceptibility for ISC transformation.