Periodic Reporting for period 4 - STEMpop (Mechanisms of stem cell population dynamics and reprogramming)
Reporting period: 2022-11-01 to 2023-10-31
The overarching aim of this project was to understand how mammalian tissues are formed, maintained, and regenerated through the collective action of tissue-resident stem cells. As a self-renewing organ maintained by a distinct stem cell population, the hair follicle represent outstanding clinically relevant paradigms to address these questions.
Ste, cells reside in spatially distinct microenvironments termed niches that consist of neighboring cells, extracellular matrix and signals derived from these compartments. Niches integrate signals that control the balanced response of stem cellss to the needs of organisms, prevent stem cell depletion while at the same time restrict excessive stem cell expansion into the surrounding tissue. Although the critical importance of niches in stem regulation has been established, the complexity of mammalian stem cell niches has prevented identification of the precise nature of the niche-derived signals and hindered mechanistic studies of adult stem regulation.
The project used a range of methods from mouse genetics to stem cell organoids, live imaging and next generation sequencing to understand how skin stem cell fate is regulated on the single cell level, and how these single cell fates are coordinated on the population level by the stem cell niche to ensure appropriate responses of the stem cell pool to the changing need of the tissue. Answering these questions will aid us to understanding of how issues are maintained during the lifetime of the organism and lay important foundations for future research in the field of stem cell therapies, tissue regeneration and repair.
The main aims of the project were to:
1. Understand how the local tissue microenvironment, in particular its geometrical shapes and mechanical properties, control stem cell behavior
2. Identify the transcriptional networks and epigenetic barriers that control stem cell fate and plasticity
3. Identify drugs that could be used to boost stem cell function as means to enhance tissue repair or prevent age-dependent loss of regenerative potential
Ste, cells reside in spatially distinct microenvironments termed niches that consist of neighboring cells, extracellular matrix and signals derived from these compartments. Niches integrate signals that control the balanced response of stem cellss to the needs of organisms, prevent stem cell depletion while at the same time restrict excessive stem cell expansion into the surrounding tissue. Although the critical importance of niches in stem regulation has been established, the complexity of mammalian stem cell niches has prevented identification of the precise nature of the niche-derived signals and hindered mechanistic studies of adult stem regulation.
The project used a range of methods from mouse genetics to stem cell organoids, live imaging and next generation sequencing to understand how skin stem cell fate is regulated on the single cell level, and how these single cell fates are coordinated on the population level by the stem cell niche to ensure appropriate responses of the stem cell pool to the changing need of the tissue. Answering these questions will aid us to understanding of how issues are maintained during the lifetime of the organism and lay important foundations for future research in the field of stem cell therapies, tissue regeneration and repair.
The main aims of the project were to:
1. Understand how the local tissue microenvironment, in particular its geometrical shapes and mechanical properties, control stem cell behavior
2. Identify the transcriptional networks and epigenetic barriers that control stem cell fate and plasticity
3. Identify drugs that could be used to boost stem cell function as means to enhance tissue repair or prevent age-dependent loss of regenerative potential
Adult somatic stem cells fuel tissue renewal, repair, and remodeling to maintain organ structure and function by tuning their proliferation and differentiation rates to match the changing needs of their resident tissues. Given their potency, even incremental alterations in stem cell behavior could lead to substantial changes in tissue size and architecture. Yet, these types of effects are strikingly rare, strongly implying that stem cells are under tight homeostatic regulation that allows the system to react rapidly to disturbances and to efficiently restore proper functions. The main results of this project were identification of such mechanisms.
We had previously established an organoid system that, for the first time, allows expansion and long-term maintenance of multipotent hair follicle SCs (HFSCs). Strikingly, the system promotes de novo generation of HFSCs from non-HFSCs, and vice versa, in a dynamic self-organizing process (Chacón-Martínez et al., EMBO J 2017). Using this system, we discovered that ability of progenitors to return to the HFSC state requires capability to suppress a metabolic switch from glycolysis to oxidative phosphorylation and glutamine metabolism that occurs during early HFSC differentiation. Mechanistically, HFSC fate reversibility and modulation of mitochondrial metabolism are regulated by the mTORC2-Akt signaling axis that is active in the HFSC niche (Kim et al., 2020 Cell Metabolism). Recently, we have upscaled the organoid cultures to allow drug screening to identify compounds that promote stem cell fate (Biggs et al., in preparation).
To understand the role of tissue architecture, we first focused on studying how the hair follicle stem cell compartment forms. Here, we identified a key role for coordinated mechanical forces stemming from contractile, proliferative, and proteolytic activities across the epithelial and mesenchymal compartments in generating the initial hair follicle invagination, the placode (Villeneuve, et al., BiorXiv 2021; in press Nature Cell Biology)
In parallel we focused on understanding how mechanical forces control global patterns of gene expression to mediate lineage decisions, but also more generally explores the role of mechanical force in regulating nuclear and genome architecture. We discovered covered how mechanical deformation induces calcium-dependent nuclear softening driven by loss of H3K9me3-marked heterochromatin proximal to the nuclear lamina in epidermal stem cells. The resulting changes in chromatin mobility, viscoelasticity, and architecture are required to insulate genetic material from mechanical force. Failure to mount this nuclear mechanoresponse results in DNA damage (Nava et al., 2020 Cell). Importantly, persisting mechanical signals to the nucleus are also relevant for ageing-related decline of stem cell function, since ageing-induced changes in extracellular matrix deposition and crosslinking lead to increased tissue stiffening. The resulting mechanical stress on stem cells results in transcriptional repression, compromising the ability of stem cells to become rapidly activated in response to regenerative signals, thus attenuating tissue renewal (Koester et al., 2021 Nat Cell Biol). Collectively our results thus reveal how mechanical signals integrate nuclear architecture, chromatin organization and transcriptional regulation to control lineage commitment and facilitate generation of specific tissue patterns.
We had previously established an organoid system that, for the first time, allows expansion and long-term maintenance of multipotent hair follicle SCs (HFSCs). Strikingly, the system promotes de novo generation of HFSCs from non-HFSCs, and vice versa, in a dynamic self-organizing process (Chacón-Martínez et al., EMBO J 2017). Using this system, we discovered that ability of progenitors to return to the HFSC state requires capability to suppress a metabolic switch from glycolysis to oxidative phosphorylation and glutamine metabolism that occurs during early HFSC differentiation. Mechanistically, HFSC fate reversibility and modulation of mitochondrial metabolism are regulated by the mTORC2-Akt signaling axis that is active in the HFSC niche (Kim et al., 2020 Cell Metabolism). Recently, we have upscaled the organoid cultures to allow drug screening to identify compounds that promote stem cell fate (Biggs et al., in preparation).
To understand the role of tissue architecture, we first focused on studying how the hair follicle stem cell compartment forms. Here, we identified a key role for coordinated mechanical forces stemming from contractile, proliferative, and proteolytic activities across the epithelial and mesenchymal compartments in generating the initial hair follicle invagination, the placode (Villeneuve, et al., BiorXiv 2021; in press Nature Cell Biology)
In parallel we focused on understanding how mechanical forces control global patterns of gene expression to mediate lineage decisions, but also more generally explores the role of mechanical force in regulating nuclear and genome architecture. We discovered covered how mechanical deformation induces calcium-dependent nuclear softening driven by loss of H3K9me3-marked heterochromatin proximal to the nuclear lamina in epidermal stem cells. The resulting changes in chromatin mobility, viscoelasticity, and architecture are required to insulate genetic material from mechanical force. Failure to mount this nuclear mechanoresponse results in DNA damage (Nava et al., 2020 Cell). Importantly, persisting mechanical signals to the nucleus are also relevant for ageing-related decline of stem cell function, since ageing-induced changes in extracellular matrix deposition and crosslinking lead to increased tissue stiffening. The resulting mechanical stress on stem cells results in transcriptional repression, compromising the ability of stem cells to become rapidly activated in response to regenerative signals, thus attenuating tissue renewal (Koester et al., 2021 Nat Cell Biol). Collectively our results thus reveal how mechanical signals integrate nuclear architecture, chromatin organization and transcriptional regulation to control lineage commitment and facilitate generation of specific tissue patterns.
The project addresses a fundamental problem in the field of developmental and regenerative biology: what determines stemness and how can this phenotype be dynamically adapted to the changing need of an organ/organism. The combined use of innovative and controlled in vitro systems such as organoids and mechanical manipulation bioreactors has allowed us to uncover new mechanisms of fate regulation both by mechanical forces as well by metabolic signals within the stem cell niche. The remained of this project will focus on the research identifying druggable targets of hair follicle stem cell fate using compund screens and subsequent mechanistic analyses. The results from these studies will lay important foundations for future research in the field of SC therapies, tissue regeneration and repair.