eprintid: 35410
rev_number: 20
eprint_status: archive
userid: 8438
dir: disk0/00/03/54/10
datestamp: 2024-09-17 10:33:18
lastmod: 2024-09-17 10:33:35
status_changed: 2024-09-17 10:33:18
type: doctoralThesis
metadata_visibility: show
creators_name: Fotopoulou, Foteini
title: Exploring the stochastic nature of ageing: Insights from the hematopoietic system as a model.
subjects: ddc-570
divisions: i-140001
adv_faculty: af-14
abstract: Ageing is traditionally viewed as the deterministic functional decline of organisms. However,
from an evolutionary perspective, aged organisms have surpassed their reproductive phase and
are therefore less subjected to selective pressures, leading to the accumulation of random errors
across various biological tiers of their being. These stochastic errors are crucial in understanding
why ageing manifests differently across individuals.
This thesis generally aimed to explore the stochastic aspects of ageing, using the hematopoietic
system as a model. In the first part, I focused on examining the endogenous mechanisms
facilitating the gradual acquisition of random somatic mutations in hematopoietic stem cells
(HSC) during normal ageing, as these cells are responsible for the lifelong system regeneration.
Somatic mutagenesis is universal and constant over time; however, the mutational rates vary
among different cell types. Here, I used whole genome sequencing (WGS) and mouse models to
inspect single HSC genomes in depth and quantify their mutational burdens, by interrogating
their clonal progeny. Assuming that this acquisition takes place in a linear fashion, as previously
suggested, I estimated that murine HSCs accumulate 44 single nucleotide variants (SNV) per
genome per year, a rate almost three times faster than that observed in human counterparts.
Moreover, the SNV landscapes formed in murine HSCs appeared analogous to those in humans,
shaped by processes reflecting self-renewal activity, metabolic dependencies and cellular state.
However, no further alterations were detected regarding other mutational types.
Apoptosis has been regarded as the guardian of the HSC genome under stress, providing
tumor-suppressive function against cells harboring driver mutations. To understand its impact
during normal ageing, I blocked the intrinsic apoptotic pathway in HSCs using a conditional
Bak -/- Baxδ/δ mouse model. Strikingly, the perturbation did not alter the mutational burdens in
HSCs, challenging the established views about this mechanism. Shifting the focus of analysis on
the states of HSCs, I examined dormant and actively cycling cells, using a label-retention mouse
model that allows the easy separation of these two fractions by flow cytometry. Despite
originating from the same donors, dormant HSCs exhibited significantly fewer mutations than
their active counterparts. Moreover, mutational signature analysis attributed this difference
mainly to the decreased divisional activity of dormant cells; otherwise, their mutational
landscapes appeared similar. This observation highlighted that dormancy, rather than apoptosis,
is the primary mechanism maintaining genomic integrity within the HSC pool during
physiological ageing, while HSC activation allows SNV accumulation over time.
In the second part of my thesis, I aimed to explore the intrinsic origins of the inter-individual
heterogeneity observed in hematological ageing. To address this, I first compiled a
comprehensive murine dataset characterizing extensively the hematological profiles of young, middle-aged and old individuals using flow cytometry, multi-omics and bone histology. With this
dataset in hand, I first explored potential associations among classical indicators of hematological
ageing, such as HSC expansion, myeloid cell production, and HSC functionality, as assessed
through competitive transplantation assays. Interestingly, in young mice, some of the metrics
were interconnected, suggesting a finely regulated system under homeostasis. However, in old
mice, a unique variability of phenotypes emerged, alongside a lack of correlations among the
different metrics.
Transplantations using bone marrow from donors of the three different age groups into
young myeloablated recipients revealed some degree of overlap in hematologic phenotypes
regardless of donor age. This observation pinpointed that the aged phenotype is also regulated
by the niche. Analysis of single-cell RNA-sequencing data from both hematopoietic and niche
cells revealed a remarkable bone marrow remodeling in old mice, including the emergence of a
novel and heterogeneous mesenchymal stromal progenitor population -Adipo-CAR3-, observed
in this study for the first time. This population served as the epicenter of an inflammatory
network involving neutrophils and HSCs. However, the niche phenotype did not correlate with
other hematopoietic metrics in a manner that could explain the overall system behavior,
suggesting that the lack of regulation that characterizes old age may expose each organism to
stochastic events able to determine its phenotype.
In conclusion, this thesis highlighted the often overlooked role of stochasticity as a
fundamental aspect of ageing. The results from the first part challenged conventional beliefs
about how HSCs manage DNA damage and mutagenesis under physiological conditions. Unlike
studies relying on strong stressors to explore genotoxic responses, my findings shed light on
sources of endogenous routine damage that accumulates steadily over time. Furthermore,
through a murine epidemiological study included in the second part of this work, I observed that
as the hematopoietic system ages, it gradually loses its ability to regulate its phenotype.
Ultimately, it is likely that this leads to a transition towards a highly stochastic state, where
random errors can alter its characteristics causing phenotypic variability.
date: 2025
id_scheme: DOI
id_number: 10.11588/heidok.00035410
own_urn: urn:nbn:de:bsz:16-heidok-354105
date_accepted: 2024-07-23
advisor: HASH(0x55de57a389d8)
language: eng
bibsort: FOTOPOULOUEXPLORINGT
full_text_status: restricted
place_of_pub: Heidelberg
citation:   Fotopoulou, Foteini  (2025) Exploring the stochastic nature of ageing: Insights from the hematopoietic system as a model.  [Dissertation]     
document_url: https://archiv.ub.uni-heidelberg.de/volltextserver/35410/1/FotopoulouF_PhDthesis-digital-vol2-13052024.pdf