Periodic Reporting for period 1 - REVERT (REVERT Regeneration as a Vulnerable State for Microbe-Driven Injury and Tumorigenesis)
Reporting period: 2023-01-01 to 2025-06-30
Tissues with high turnover are hierarchically organized and rely on long-lived stem cells that are protected by a variety of mechanisms. In the gastrointestinal tract, highly active stem cells are located in the base of crypts, where differentiated cells shield them from environmental threats. It has recently emerged that mucosal injuries initiate regenerative repair programs that promote a disruption of cellular hierarchies and reversal of differentiated cells back to the proliferative stem cell state. While this remarkable plasticity enables rapid injury repair, I propose that the recruitment of differentiated cells to the stem cell pool represents a critical event for the accumulation of genetic and epigenetic alterations because differentiated cells are more exposed to the environment and less equipped to repair DNA damage. Particularly in the colon with its dense and potentially harmful microbiota, injury-driven de-differentiation may be linked to loss of cell functions that control the microbiota and direct exposure of “de novo stem cells” to bacteria and their genotoxic virulence factors. REVERT will investigate the long-term consequences of such transient interactions on molecular, cellular, and tissue levels and explore the impact of the regenerative state on mucosal microbial ecology and function.
REVERT will combine stem cell biology approaches such as, lineage tracing, organoids, and assembloids with microbiology techniques such as gnotobiotic infection models, and integrate complex systems biology technologies to build up a picture of dynamic tissue responses to injuries and the ability of microbes to interfere with them.
REVERT has the potential to establish fundamental new knowledge of principles that govern mucosal integrity and reveal its vulnerabilities in the context of injury. It has the potential to drastically expand our understanding of processes that drive chronic tissue dysfunction and carcinogenesis.
REVERT will combine stem cell biology approaches such as, lineage tracing, organoids, and assembloids with microbiology techniques such as gnotobiotic infection models, and integrate complex systems biology technologies to build up a picture of dynamic tissue responses to injuries and the ability of microbes to interfere with them.
REVERT has the potential to establish fundamental new knowledge of principles that govern mucosal integrity and reveal its vulnerabilities in the context of injury. It has the potential to drastically expand our understanding of processes that drive chronic tissue dysfunction and carcinogenesis.
We have been able to dissect the different steps of epithelial regeneration in detail and identify specific signaling pathways that drive each step. Specifically, the process of regeneration upon injury starts with the loss of the stem cell compartment, followed by a phase with high proliferation and epithelial reprogramming into a fetal-like state and finally results in termination of regeneration.
We have explored the first step, namely the loss of stem cells and found that it is linked to interferon-gamma signaling. Mice that lack interferon gamma receptors fail to show the loss of stem cells in the colon normally observed upon injury induced by the toxin DSS. Our in-depth analysis has revealed that the loss of stem cells is part of a complex crypt remodeling process that also involves various other cell types: Upon injury, recruited T cells release interferon, which causes rapid death and loss of surface colonocytes. These are then replaced by the stem cell compartment, where all cells transition into “de-novo colonocytes”. These de-novo colonocytes differ from homeostatic colonocytes and the resulting alterations in their signaling activity lead to the de-suppression of regenerative pathways in the surrounding stroma, culminating in the secretion of novel factors that further stimulate tissue regeneration (Heuberger, Liu et al, Nature Communications in second revision).
We have also explored the process whereby regeneration is terminated again. Here we found a critical role of p53 signaling: Upon entry into the fetal-like regenerative state, the epithelium activates p53. Although p53 is not required for epithelial regeneration itself, it is required for re-entry into the homeostatic state. When we induced loss of p53, the epithelium became locked into the regenerative state upon injury, unable to restore its normal architecture and function. Mechanistically, we found that p53 triggers the reversal from regenerative to homeostatic crypt state by downregulating the high levels of Wnt and Yap signaling that drive the regenerative state, and by reversing the alterations in cellular metabolism that fuel the high cell turnover during regeneration. (Hartl et al., Science Advances 2024)
We have explored the first step, namely the loss of stem cells and found that it is linked to interferon-gamma signaling. Mice that lack interferon gamma receptors fail to show the loss of stem cells in the colon normally observed upon injury induced by the toxin DSS. Our in-depth analysis has revealed that the loss of stem cells is part of a complex crypt remodeling process that also involves various other cell types: Upon injury, recruited T cells release interferon, which causes rapid death and loss of surface colonocytes. These are then replaced by the stem cell compartment, where all cells transition into “de-novo colonocytes”. These de-novo colonocytes differ from homeostatic colonocytes and the resulting alterations in their signaling activity lead to the de-suppression of regenerative pathways in the surrounding stroma, culminating in the secretion of novel factors that further stimulate tissue regeneration (Heuberger, Liu et al, Nature Communications in second revision).
We have also explored the process whereby regeneration is terminated again. Here we found a critical role of p53 signaling: Upon entry into the fetal-like regenerative state, the epithelium activates p53. Although p53 is not required for epithelial regeneration itself, it is required for re-entry into the homeostatic state. When we induced loss of p53, the epithelium became locked into the regenerative state upon injury, unable to restore its normal architecture and function. Mechanistically, we found that p53 triggers the reversal from regenerative to homeostatic crypt state by downregulating the high levels of Wnt and Yap signaling that drive the regenerative state, and by reversing the alterations in cellular metabolism that fuel the high cell turnover during regeneration. (Hartl et al., Science Advances 2024)
To better mimic the crosstalk between epithelial and stromal cells in the native tissue, we have recently established an in vitro colonic assembloid model derived from adult murine primary epithelial and stromal cells1. These assembloids encompass most stromal cell subpopulations, including different fibroblast subpopulations (trophocytes and telocytes) and myofibroblasts as well as small numbers of endothelial cells, neurons, and glial cells. Crucially, we found that combining epithelial and stromal cells in this way enables self-organization of both compartments to take place. The model allowed us to determine that epithelium and stroma directly shape each other through reciprocal signaling loops involving predominantly BMP agonists and antagonists. The resulting signaling gradients establish a spatial organization of the stroma along the axis of the emerging epithelial crypts, consisting of different subpopulations with different functions. These different stromal subpopulations are in turn able to drive the establishment of a fully mature crypt structure: Once self-organization has taken place, the system is able to maintain itself, with stromal cells producing appropriate growth factors for neighboring epithelial cells. Crucially, the resulting independence from external growth factors supplied via the culture medium allows these localized signals to control cell fate and establish a native crypt structure with stem cells at the base and fully mature cells at the surface. This is in stark contrast to existing colonic organoid technology, where cell fate is controlled by medium supplementation, effectively preventing mature and proliferating cell types from being maintained simultaneously.
Several other gastrointestinal co-culture systems have recently been developed, based on adult primary cells, cell lines, or pluripotent stem cells cultured in different systems, e.g. conventional static co-culture in basement membrane matrix, air-liquid interface, organ-on-a-chip, and pluripotent stem cell-derived organoids. While these approaches allow important insights into cell communication, they fail to fully recapitulate the self-organization of the adult gastrointestinal mucosa with a mature crypt structure and compartmentalized multicomponent stromal niche. Our assembloid system also offers important advantages to facilitate broad application within the scientific community: It is low-tech and comparatively low-cost, requiring only basic equipment without the need for engineered scaffolds, microfluidic devices, or bioreactors.
The data were published in Lin et al., Nature Communications 2023.
Several other gastrointestinal co-culture systems have recently been developed, based on adult primary cells, cell lines, or pluripotent stem cells cultured in different systems, e.g. conventional static co-culture in basement membrane matrix, air-liquid interface, organ-on-a-chip, and pluripotent stem cell-derived organoids. While these approaches allow important insights into cell communication, they fail to fully recapitulate the self-organization of the adult gastrointestinal mucosa with a mature crypt structure and compartmentalized multicomponent stromal niche. Our assembloid system also offers important advantages to facilitate broad application within the scientific community: It is low-tech and comparatively low-cost, requiring only basic equipment without the need for engineered scaffolds, microfluidic devices, or bioreactors.
The data were published in Lin et al., Nature Communications 2023.