Not just a pipe's dream : plumbing the contribution of cell diversity to oesophageal homeostasis

Epithelial barrier tissues like the skin, intestine, and oesophagus form a physical barrier that protects our body from external threats. To accurately fulfil their function, epithelial barriers are subject to constant epithelial cell renewal throughout our lifespan. Recurrent tissue turnover requires the precise control of epithelial cell proliferation and differentiation to maintain homeostasis and health. Adult tissue stem cells residing in epithelial barriers are of pivotal importance for tissue homeostasis and repair. The stem cell niche, composed of a variety of cells, mechanical and chemical elements, provides decisive signalling cues that influence stem cell behaviour and fate. The major part of this thesis investigates behaviour of the mouse oesophageal epithelial progenitor cell and sets out to uncover and characterise the contribution of cellular diversity to oesophageal homeostasis. In paper III, we demonstrated that a subpopulation of oesophageal progenitor cells expressed Tnfrsf19 (Troy) and contributed long-term to oesophageal homeostasis. Using lineage tracing in combination with mathematical modelling we proposed that Troy progenitor cell fate is predominantly symmetrical. In addition, functional TROY knockout in vivo suggested that TROY regulates progenitor proliferation and facilitates differentiation. Thus, TROY might be involved in context dependent cellular decision making processes providing a basis for behavioural progenitor heterogeneity. In paper IV, we characterise regional oesophageal cell composition utilising single cell RNA sequencing. Combining cell-cell communication inference and organoid culture we reveal regionally diverse contributions of fibroblasts and immune cells as well as signalling pathways such as BMP and IGF that differently influence epithelial cell behaviour. In paper II, we developed an organoid co-culture system of oesophageal epithelial cells and fibroblasts that allows for detailed functional investigations of cell-cell communication in vitro. The generation of the stratified squamous epithelium of the skin is governed by intricate and interwoven processes of proliferation, cell cycle exit, differentiation, and stratification. In paper I, we probed the function of ID1 in epidermal development and demonstrated ID1 binding to the transcription factor TCF3. We propose that ID1-CEBPA crosstalk regulates epidermal cell fate decision within a ID1-TCF3-CEBPA axis. The work provided within this thesis demonstrates molecular mechanisms and signalling cues that impinge on epithelial cell behaviour during homeostasis and development

Protocol to establish mouse esophageal epithelial whole mounts for lineage tracing or immunofluorescent analysis

Summary: Herein, we provide a protocol describing the preparation and labeling of cells in mouse esophageal epithelial whole mounts. We include details of tamoxifen administration used to genetically label and trace the fate of basal cells. We demonstrate how EdU incorporation alone or in combination with additional markers can be used to assess in vivo cell cycle progression or stratification dynamics. Finally, we highlight how optimization of cell labeling is required for reproducible analysis.For complete details on the use and execution of this protocol, please refer to Grommisch et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Defining the contribution of Troy-positive progenitor cells to the mouse esophageal epithelium

Progenitor cells adapt their behavior in response to tissue demands. However, the molecular mechanisms controlling esophageal progenitor decisions remain largely unknown. Here, we demonstrate the presence of a Troy (Tnfrsf19)-expressing progenitor subpopulation localized to defined regions along the mouse esophageal axis. Lineage tracing and mathematical modeling demonstrate that Troy-positive progenitor cells are prone to undergoing symmetrical fate choices and contribute to esophageal tissue homeostasis long term. Functionally, TROY inhibits progenitor proliferation and enables commitment to differentiation without affecting fate symmetry. Whereas Troy expression is stable during esophageal homeostasis, progenitor cells downregulate Troy in response to tissue stress, enabling proliferative expansion of basal cells refractory to differentiation and reestablishment of tissue homeostasis. Our results demonstrate functional, spatially restricted progenitor heterogeneity in the esophageal epithelium and identify how dynamic regulation of Troy coordinates tissue generation

ID1 and CEBPA Coordinate Epidermal Progenitor Cell Differentiation

AbstractThe regulatory circuits that coordinate epidermal differentiation during development are still not fully understood. Here we report that the transcriptional regulator ID1 is enriched in basal epidermal progenitor cells and find ID1 expression to be diminished upon differentiation. In utero silencing of Id1 impairs progenitor cell proliferation, leads to precocious delamination of targeted progenitor cells and enables differentiated keratinocytes to retain progenitor markers and characteristics. Transcriptional profiling suggests ID1 acts by mediating adhesion to the basement membrane while inhibiting spinous layer differentiation. Co-immunoprecipitation reveals ID1 binding to transcriptional regulators of the class I bHLH family. We localize bHLH Tcf3, Tcf4 and Tcf12 to epidermal progenitor cells during epidermal stratification and established TCF3 as a downstream effector of ID1-mediated epidermal proliferation. Finally, we identify crosstalk between CEBPA, a known mediator of epidermal differentiation, and Id1 and demonstrate that CEBPA antagonizes BMP-induced activation of Id1. Our work establishes ID1 as a key coordinator of epidermal development, acting to balance progenitor proliferation with differentiation and unveils how functional crosstalk between CEBPA and Id1 orchestrates epidermal lineage progression.</jats:p

Competitive repopulation of an empty microglial niche yields functionally distinct subsets of microglia-like cells

Circulating monocytes can compete for virtually any tissue macrophage niche and become long-lived replacements that are phenotypically indistinguishable from their embryonic counterparts. As the factors regulating this process are incompletely understood, we studied niche competition in the brain by depleting microglia with &gt;95% efficiency using Cx3cr1CreER/+R26DTA/+ mice and monitored long-term repopulation. Here we show that the microglial niche is repopulated within weeks by a combination of local proliferation of CX3CR1+F4/80lowClec12a– microglia and infiltration of CX3CR1+F4/80hiClec12a+ macrophages that arise directly from Ly6Chi monocytes. This colonization is independent of blood brain barrier breakdown, paralleled by vascular activation, and regulated by type I interferon. Ly6Chi monocytes upregulate microglia gene expression and adopt microglia DNA methylation signatures, but retain a distinct gene signature from proliferating microglia, displaying altered surface marker expression, phagocytic capacity and cytokine production. Our results demonstrate that monocytes are imprinted by the CNS microenvironment but remain transcriptionally, epigenetically and functionally distinct

Treatment for Acute Myelogenous Leukemia by Low Dose Irradiation Based Conditioning and Hematopoietic Cell Transplantation from Related and Unrelated Donors.

Abstract Conventional allogeneic hematopoietic cell transplantation (HCT) is effective treatment for AML, but its use has been restricted to younger patients in good medical condition. However, AML is increasingly common with advancing age, and older patients have more adverse prognostic features than younger patients. The vast majority of older patients who reach a CR with chemotherapy relapse within two years from diagnosis and die within three years. The situation is similar in patients with secondary AML or with AML in CR2, more advanced remission status or in partial remission, where median duration of disease-free survival (DFS) is generally &amp;lt;12 months. The use of low-dose irradiation-based preparative regimens has allowed extending allografting to, and exploring graft-versus-tumor effects in, older and medically infirm patients. The present study included 122 patients with AML ineligible for conventional transplants. Their disease stages ranged from CR1 to resistant disease, and their median age was 57.5 (range 17–74) years. The patients were given 2 Gy total body irradiation (TBI) on day 0 with and without preceding fludarabine (30 mg/m2/d from days -4 to -2). After HCT, cyclosporine, 6.25 mg/kg, was given twice daily from day -3, and mycophenolate mofetil, 15 mg/kg, twice daily from day 0. HLA-matched related donors were used in 58, and HLA-matched/partially matched unrelated donors in 64 patients. Among 121 (one died to early after transplant) evaluable patients, 116 (95.8%) had durable engraftment. Cumulative incidences of acute GvHD grade II-IV were 33 % after related and 42% after unrelated HCT. With a median observation time of 17 (range 4–57) months, 58 patients were alive, 53 of whom were in complete remissions. Cumulative non-relapse mortalities (NRM) were 12% and 27%, and the cumulative relapse mortalities were 47% and 34% at 2 years for related and unrelated recipients, respectively. Two-year overall survival (OS) was 41%, and DFS was 35%, with stages of the disease and cytogenetic risks being major determinants for outcomes. Patients transplanted in CR1 had a 2-year OS of 40% after related and 57% after unrelated HCT. We concluded that HCT from related and unrelated donors after low-dose TBI is promising therapy for elderly patients and medically infirm younger patients with AML not eligible for conventional HCT. Due to the lower relapse rates, results in patients with unrelated donors were better than results in patients with related donors. Results have encouraged studies of the role of low-dose irradiation-based conditioning and allogeneic HCT in the primary treatment of patients with AML ≥60 years.</jats:p

BAFF-secreting neutrophils drive plasma cell responses during emergency granulopoiesis

Prolonged infections or adjuvant usage can trigger emergency granulopoiesis (EG), leading to dysregulation in neutrophil blood counts. However, the impact of EG on T and B cell function remains largely unknown. In this study, to address this question, we used a mouse model of neutropenia and studied immune activation after adjuvant administration. The initial neutropenic state fostered an environment of increased dendritic cell activation and T cell-derived IL-17 production. Interestingly, neutropenic lysozyme 2-diphtheria toxin A mice exhibited striking EG and amplified neutrophil recruitment to the lymph nodes (LNs) that was dependent on IL-17-induced prostaglandin activity. The recruited neutrophils secreted a B cell-activating factor that highly accelerated plasma cell generation and antigen-specific antibody production. Reduction of neutrophil functions via granulocyte colony-stimulating factor neutralization significantly diminished plasma cell formation, directly linking EG with the humoral immune response. We conclude that neutrophils are capable of directly regulating T cell-dependent B cell responses in the LN