Maintenance and Differentiation of Stem Cells
in Development and Disease
Projects and Principal Investigators
Section A: Mechanisms of Stem Cell Self-Renewal
Section B: Cell-cell interactions in the stem cell niche
Stem cell plasticity in Hydra
Hydra’s immortality and unlimited regeneration capacity is based on the unique properties of its stem cell system. So far, we unraveled evolutionary conserved, stem-cell based regeneration mechanisms on the level of signaling pathways. In the next funding period, we will focus on the role of germline stem cells. Somatic stem cells can give rise to germline stem cells throughout Hydra’s (unlimited) life span. To analyze this remarkable stem cell plasticity at the mechanistic level, we established cell-type-specific transcriptomes for somatic and germline stem cells. Genes of interest will be analyzed in gain- and loss-of-function studies using read outs such as the switch from somatic to germline stem cells. Our studies will yield important insights into the evolution of germline and adult stem cells and reveal the molecular basis of Hydra’s proposed immortality.
Control of stem cell identity and homeostasis in the ciliary marginal zone of the vertebrate retina
Vertebrate neural stem cells have retained a life-long proliferative potential. Fish and amphibia, in addition, show continuous growth of the central nervous system including the retina. Here, the ciliary marginal zone (CMZ) acts as stem cell niche and hosts multipotent retinal stem cells that ensure differentiation into all retinal cell types while continuously growing. In the CMZ, the transcription factor Rx2 acts as regulatory hub which impacts on fate determination and proliferation. While proliferation control appeared as key determinant for niche homeostasis, our studies uncovered an unexpected link to the immune system likely eliminating stem cells. We will address how niche homeostasis is maintained by the interplay between Rx-mediated proliferation control and stem cell elimination via mononuclear phagocytes.
Interactions of signaling pathways during intestinal stem cell self-renewal and differentiation
During homeostasis, the proliferation and differentiation of intestinal stem cells (ISC) in the Drosophila intestine is tightly controlled. In the past years, several conserved signaling pathways have been implicated in the regulation of ISC proliferation and differentiation decisions, including the Wnt, Notch, Hedgehog, Hippo and Dpp/BMP pathways. Stress responses, infections or inflammation can lead to deregulation of stem cell proliferation, differentiation and apoptosis in the intestinal epithelium, resulting in defects in the homeostatic renewal of the gut epithelium. However, it remains still largely unclear how these different signaling pathways are orchestrated to control stem cell maintenance and differentiation decisions. In the next funding period of the SFB873, (i) we will analyze how different signaling pathways intersect to control maintenance of the epithelium, (ii) study how they impact transcriptional programs in ISCs and progenitor cells and (iii) dissect how pathways interact with the intestinal microbiome during homeostasis.
Mechanisms of Niche – Stem Cell Unit Origin and Maintenance
The stem cell niche is key to maintain stemness in every stem cell population reported in plants and animals. Despite the essential function of stem cell – niche interaction in stem cell biology, little is known about how somatic stem cells and niche get together during organ formation. In the previous funding period, we used neuromasts, sensory organs of the lateral line system in fish, as a minimal model to explore niche-stem cell interaction during organogeneis. We used Cre/LoxP lineage tracing tools and electromicroscopy to identify a system in which neural stem cells induce the formation of their own niche. Our plan for the next funding period is to exploit this system to address niche formation at the molecular level. We will combine our expertise in 4D imaging and lineage analysis with the acquisition of new RNAseq data, to identify molecular determinants critical for niche induction and maintenance during homeostasis and perturbed conditions. Our ultimate goal is to understand how to create a stem cell – niche unit, and to find targets to disrupt the stem cell – niche association.
Studying Lung Stem Cells in Tissue Maintenance, Repair, and Cancer
Despite recent advances in understanding the mesenchymal cell compartment in the lung, cellular and molecular mechanisms of epithelial lung stem and progenitor cell homeostasis and the regulation of their malignant counterparts in lung cancer are still poorly understood. We therefore developed various mouse models that are combined with functional testing and single-cell RNA sequencing to identify novel lung epithelial stem and progenitor cell activity, ascribe this activity to marker defined cell types, and understand regulatory pathways within these cell types. In addition, we determine the potential of these new and known stem and progenitor cells to act as cancer stem cells in KrasG12D-induced lung tumors with and without Stk33 loss.
Functional role of centrosomes and cilia in stem cells
Stem cells divide symmetrically or asymmetrically to renew themselves and to generate daughters with different fates. The correct balance between these two processes is essential for tissue homeostasis and development. Here, we are particularly interested in the function of centrosomes in stem cell division. Centrosomes are microtubule organising centers that are involved in spindle formation and positioning as well as in the formation of signaling centers (namely cilia and flagella). Recent studies, including our own, showed that centrosomes are inherently asymmetric structures that have the potential of working as a scaffold for asymmetric distribution of components, including cell fate determinants. Therefore, the aims of this proposal are to understand how centrosome asymmetry is regulated in stem cells and how this asymmetry is translated into signals to control stem cell division.
Transcriptional complexes and chromatin reorganization in lineage choices and differentiation of embryonic stem cells
Embryonic stem (ES) cells can differentiate into virtually all somatic cell types. The establishment of cell type-specific gene expression patterns requires integrated activity of multiple factors including signaling molecules, transcription factors and epigenetic modifiers. Upon different cues, transcription factors bind cell type-specific enhancers and together with cofactors and epigenetic modifiers play key roles in epigenetic and transcriptional programming instructing the lineage choices and differentiation of embryonic stem cells. The objective of the proposed research program is to characterize the transcriptional and epigenetic events controlling cardiovascular versus hematopoietic lineage choice and cardiovascular differentiation of embryonic stem cells.
Regulatory and phenotypic evolution of the male mammalian germline and underlying stem cells
Our previous gene expression comparisons revealed that the male mammalian germline evolves rapidly at the molecular level. This observation is consistent with the fast evolutionary alterations of sperm cell properties, testis sizes, and sperm production rates and is likely explained by the competition among males to fertilize a female’s egg. Previous work suggests that evolutionary changes of spermatogonial stem cells (SSCs) contribute to the rapid germline evolution. However, our understanding of SSC properties and their developmental emergence is largely confined to rodents. The goal of the first proposed project is therefore to explore in detail the regulatory and phenotypic evolution of SSCs, precursors and resulting spermatogenic cells within a cross-mammalian single-cell gene expression framework. In addition, our lab will lead a highly collaborative project that seeks to explore the evolution of stem cells across all species and systems represented in SFB873.
Towards a mechanistic framework of plant stem cell control
Project B01 will decode the mechanisms underlying the precise spatio-temporal control of gene expression by the stem cell inducing WUSCHEL (WUS) transcription factor in the shoot apical meristem of the reference plant Arabidopsis thaliana. Based on our findings that stem cell function is dependent on WUS protein distribution, selective DNA binding, as well as differential response of individual cells to WUS, we will now elucidate the structural basis for the amazing ability of WUS to bind a diverse set of DNA motifs and to move from cell to cell. Complementing these mechanistic efforts, we will characterize the regulatory landscape encoded in trans by delineating the capacity of shoot meristem cells to respond to stem cell inductive WUS signals by single cell transcript profiling.
Molecular and cellular control of the Drosophila male stem cell niche
Project B02 will elucidate the molecular basis of the interplay between germline stem cells (GSCs) and their support cells under homeostatic and compromised cellular conditions and in collaboration with project A14 will uncover the role of mitotic checkpoint components (MCCs) in monitoring asymmetric stem cell division using the Drosophila testis as a model. The proposed work plan is based on our findings that loss of V-ATPase function in support cells causes non-cell autonomous proliferation defects in the testis germline, that depletion of nucleoporins impacts on stem cell fitness leading to their elimination and that interference with the MCC component Bub3 results in centrosome positioning defects without arresting germline stem cell division indicative for a disengaged checkpoint surveiling centrosome orientation.
Role of WNT signaling in embryonic neural stem cells
The WNT signaling pathway plays a key role in stem cell biology, including neural stem cell prolifera-tion and differentiation. Cyclin Y and Cyclin Y like-1 (CCNY/CCNYL1) are mitotic Cyclins, which regu-late the phosphorylation of the WNT co-receptor LRP6. We have created CCNY/CCNYL1 double knockout (DKO) mice, which display severe embryonic brain defects. Preliminary analysis indicates that CCNY/CCNYL1 are required for progenitor proliferation and differentiation. We plan to systemati-cally characterize DKO embryos and neurosphere cultures at the tissue and cellular level, focusing on cell division and differentiation. We will investigate the role of non-transcriptional WNT/LRP6/GSK3 signaling plays in this context.
A novel level of regulation of normal and leukemic stem cell hierarchies by differential polyadenylation of key transcripts
The hierarchical structure of the hematopoietic system often serves as a paradigm of other stem cell based cellular hierarchies, which form many of the vertebrate tissues and organs. Self-renewing hematopoietic stem cells (HSCs) located atop of the hematopoietic system are crucial for the continuous generation of mature cells and also critical for repair in response to injury cues such as infection, inflammation or chemotherapy. Moreover, multipotent HSCs are the cell-of-origin of leukemias and leukemic stem cells are likely responsible for relapse after initially successful therapy. The stem cell hierarchies are shaped by a complex array of epigenetic mechanisms including differential methylation, chromatin activity, transcription, translation as well as post-transcriptional and post-translational regulation. Here we address the role of differential polyadenylation of transcripts affecting stability and protein expression in normal HSCs and progenitors and explore whether this mechanism is also operational in leukemic stem cells and therapy resistant leukemic clones.
Mechanism of Dynamic Homing and Migration of Human Hematopoietic Stem Cells in Bone Marrow Niche Using Quantitative Tools
Stem cell functions are determined and influenced by their interactions with the niche. In this project, we will quantify the mechanical interactions of healthy hematopoietic stem cells (HSC) and stem cells from leukemia patients with the bone marrow niche. The underlying hypothesis is that leukemia initiating cells could be eliminated by understanding the mechanistic and functional differences in stem cell-niche interactions. Utilizing unique quantitative tools, such as precisely functionalized surrogate surfaces, hydrogel substrates with tunable elasticity, and dynamic phenotyping of active deformation and motion, we will extend our strategies towards three specific aims: (1) biological impacts of extrinsic factors (e.g. pathway specific inhibitors) on adhesion and migration of HSC, (2) influence of 3D niche mechanics on HSC-MSC interactions, and (3) biological impacts of intrinsic factors, such as aging, on adhesion and migration of HSC.
Mathematical Modeling of Stem Cell Renewal and Differentiation
The focus of this project is mathematical modeling of mechanisms and dynamics of cell fate and growth regulation in stem cell based systems in development, tissue regeneration, and cancer. Mathematical modeling is a powerful technique to address key questions in model systems and to provide a mechanistic understanding of the underlying processes. Mathematical models will be built and validated iteratively, based on data provided by collaborating projects. The research will contribute to a better understanding of the underlying mechanisms. In addition, it will also allow comparing design principles of different stem cell systems.
Mechanisms of regulation of stem cells in the adult brain
Adult stem cells impact tissue homeostasis and repair. In the previous funding period we pioneered single cell technologies and used mathematical modeling to gain fundamental knowledge on stem cell activation and differentiation. We first studied the transcriptional and cellular programs governing stem cells in the hippocampus. We initiated a study of the transcriptome and translatome of stem cells in the ventricular-subventricular zone (V-SVZ). In addition, we used the single-cell technologies to find stem cell activity in other locations of the brain and other equally quiescent solid tissue. In the coming period we will extent the molecular analysis of V-SVZ stem cells. This analysis and quantification of stem cells across time will be used to implement a mathematical modeling describing V-SVZ stem cell dynamics. Gained knowledge shall advance our understanding of stem cell biology across the mammalian brain and other solid tissues.
Hematopoietic stem cells (HSC) are widely studied by transplantation into immune- and blood-cell-depleted recipients. To study hematopoiesis in situ, we developed a mouse model enabling inducible genetic labeling of HSC at different time points of embryonic development and in the adult bone marrow (Tie2MeriCreMer). Together with mathematical modeling our HSC fate-mapping experiments provide insights into differentiation rates through stem and progenitor populations, residence times within compartments, estimates on self-renewal rates in compartments, and thus a quantitative framework for studying hematopoiesis in situ. We will apply our fate mapping model to investigate the response of the hematopoietic system to pertubations. We will study HSC, marked by Tie2 expression, to refine the kinetic parameters in terms of stem cell proliferation and differentiation decisions towards a better overall understanding of complex HSC biology in health and disease.
Spatio-temporal Specificity of Cambium Stem Cell Signaling
Radial growth of plant shoots and roots is essential for the formation of wood and of large plant bodies, and thus for the creation of biomass on earth. The process depends on the tissue forming properties of a group of stem cells called the cambium, the activity of which leads to the production of secondary vascular tissue (wood and bast). Here, we want to leverage the unique features of the cambium and establish general principles of stem cell niche organization in plants and beyond. This will be achieved by testing the hypothesis that a spatio-temporal modulation of the effect of the recently discovered plant hormone strigolactone (SL) within the cambium domain is essential for an organized production of tissues during radial plant growth. In close collaboration with project B01, we will in particular investigate the role of the stem cell-specific homeobox transcription factors WOX4 and WUS in promoting SL signaling as one essential feature of plant stem cell systems.
HSCs under acute and chronic inflammatory stress
During inflammatory stress, quiescent hematopoietic stem cells (HSCs) are forced into transient proliferation, presumably to regenerate mature blood cells lost during the stress response. Many open questions remain as to how different inflammatory stimuli impact on the quiescence of HSCs, whether common or distinct mechanisms are involved in the HSC response and what the long-term consequences of stress-induced proliferation are on the function and behaviour of these HSCs. Here, we propose to combine a range of different inflammatory stimuli with loss of function mouse models to interrogate in more detail the mechanisms and short-term and long-term consequences of inflammation-induced activation of quiescent HSCs in vivo. This will allow us to ascertain whether such forms of environmental stress, normally absent in the laboratory setting, have the capacity to impact on HSC function in either a uniform or divergent manner.
Unraveling the mechanisms that regulate vascular properties in neurogenic niches: role of neural stem cells
Blood brain barrier (BBB) properties of blood vessels at the subventricular zone (SVZ) are described to be different to non-neurogenic areas of the brain. These properties seem to be important for allowing diffusion of blood borne substances, important for neural stem cell (NSC) homeostasis. However, how these properties are controlled remains unknown. In this project proposal we hypothesize that NSCs modulate their own microenvironment by controlling vascular properties within their niche. As model system to understand this regulation we will use the mouse SVZ. Using a combination of transgenic mouse lines where NSCs are ablated or increased in number, in vitro co-culture assays and RNA sequencing of NSCs and endothelial cells (ECs), we will determine the cellular and molecular mechanisms leading to the SVZ specific vascular properties. As neurogenesis declines with aging, we are also interested in determining whether specific vascular changes occur in the SVZ upon aging and whether these are due to the reduced number or differential signalling of NSCs.
Central Tasks of the SFB873
The spokesperson's office in charge of all central coordinating activities and organizes seminars, retreats, symposia, prepares and documents the meetings of the steering committee (SC) and is in charge of executing decisions of the SC and the assembly of principal investigators. It is in charge of all financial affairs of th SFB. The coordinator's office handles all internal and external communiciation of the SFB. In essence, the coordinator's office is in charge of all central administrative duties, but its most important task is to establish an atmosphere within the SFB that is conducive to scientific exchange and collaborations between the individual projects for a maximum of scientific synergy and collaborative added value.
Flow Cytometry Core Unit
The mission of the Z02-project is: 1) To provide the consortium with pure and homogenous stem cell or progenitor preparations of outstanding quality as starting material for all subsequent experiments by state-of-the-art cell sorting equipment and qualified personnel as part of the consortium. 2) To develop innovative protocols for the preparation of stem or progentior cells from organisms for which flow cytometry-technology has not yet been standardized (e.g. Hydra, Drosophila, Arabidopsis, Medaka).
Advanced Light Microscopy
The Nikon Imaging Center is a core unit that offers serive in light microscopy within the SFB 873.
Bioinformatic analysis and comparative data mining
This project supports Collaborative Research Centre members with genomic data analysis, including statistical assistance and teaching bioinformatics methods, maintains local
Galaxy instance, and develops requested tools and workflows. In addition, it provides an integrated and comparative analysis of genomic datasets produced in the individual sub-project to derive an understanding of shared and divergent stem cell regulatory mechanisms.
The project scientist is Olga Ermakova