eprintid: 34654 rev_number: 15 eprint_status: archive userid: 8055 dir: disk0/00/03/46/54 datestamp: 2024-04-16 07:49:19 lastmod: 2024-05-07 11:00:15 status_changed: 2024-04-16 07:49:19 type: doctoralThesis metadata_visibility: show creators_name: itani, Abdul Rahman title: Stress Signaling Heterogeneity in Normal and Malignant Hematopoiesis subjects: ddc-500 subjects: ddc-570 divisions: i-140001 adv_faculty: af-14 cterms_swd: Chronisch-myeloische Leukämie cterms_swd: Interferon cterms_swd: Hämatopoese cterms_swd: Exosom abstract: Hematopoietic stem cells (HSCs), positioned at the apex of the hematopoietic lineage, have long been considered a homogeneous population characterized by multipotency and self-renewal. Recent breakthroughs, however, have unveiled profound functional and molecular heterogeneity within this seemingly uniform group. Research from our group has identified a stable and heritable diversity in the intrinsic expression levels of interferon- stimulated genes (ISGs) in HSCs, termed intrinsic IFN signaling heterogeneity. Yet, it remains unclear whether this heterogeneity is established and sustained at the HSC level, and its potential role in the development and progression of clonal hematopoietic malignancies remains unexplored. In my thesis, I explore the intrinsic IFN signaling signature in tissue-resident macrophages (TRMs) (aim 1). Utilizing flow cytometry and gene expression analysis, I demonstrated the presence of IFN signaling heterogeneity in TRMs. Importantly, as TRMs originate embryonically and precede HSC development, this finding suggests that IFN signaling heterogeneity in the hematopoietic system is established before the emergence of HSCs. To assess the impact of intrinsic IFN signaling heterogeneity in disease (aim 2), I developed a humanized chronic myeloid leukemia (CML) mouse model on the background of a unique ISRE-eGFP reporter mouse. This reporter mouse features the interferon-stimulated response element (ISRE) upstream of eGFP, which enables the identification of HSCs expressing low or high levels of ISGs. In this context, the CML-ISG reporter mouse model facilitated the study of clonal expansion of CML leukemia stem cells (LSCs) relative to the intrinsic IFN signaling status of the parent HSC clone in vivo. Total BM transplantation experiments revealed a significant expansion of eGFPlow leukemic clones in the peripheral blood, together with a quiescent eGFPlow LSC pool in the BM suggesting a more aggressive leukemia clone. Survival analysis revealed that 100% eGFPlow leukemic chimeras succumbed to leukemia significantly faster that eGFPhigh chimeras confirming my findings. Additionally, I established a reliable method for isolating exosome-enriched small extracellular vesicles (sEVs) through serial centrifugation followed by size exclusion chromatography (SEC) (aim 3) in order to explore the role of sEV signaling in the BM (aim 4). My findings indicated a shift in sEV profile and content upon inflammatory stress, with specific sorting of inflammatory response proteins into vesicles exhibiting an inhibitory effect on hematopoietic stem and progenitor cells (HSPCs) proliferation in vitro. Subsequent in vivo experiments I performed using different mouse models demonstrated a dynamic release of sEVs in response to acute inflammatory stress, suggesting the hematopoietic compartment as a major source of sEVs during such conditions. In summary, my research has provided new insight on the origins of intrinsic IFN signaling heterogeneity and its consequential influence on disease progression in CML. Furthermore, it advanced our understanding of the intricate role played by sEV signaling in modulating the acute inflammatory response within the BM. date: 2024 id_scheme: DOI id_number: 10.11588/heidok.00034654 ppn_swb: 1888010894 own_urn: urn:nbn:de:bsz:16-heidok-346545 date_accepted: 2024-02-27 advisor: HASH(0x55fc35f795d0) language: eng bibsort: ITANIABDULSTRESSSIGN2024 full_text_status: public citation: itani, Abdul Rahman (2024) Stress Signaling Heterogeneity in Normal and Malignant Hematopoiesis. [Dissertation] document_url: https://archiv.ub.uni-heidelberg.de/volltextserver/34654/1/thesis%202024-4-02.pdf