[Skin aging-cellular senescence : What is the future?] / Hautalterung ­ zelluläre Seneszenz : Wohin geht die Reise?

BACKGROUND: Cellular senescence is the main cause of skin and organ aging and is associated with a wide range of aging-related diseases. OBJECTIVES: To understand which senolytics, senomorphics, and cell-based therapies have been developed to alleviate and even rejuvenate skin aging and reduce cellular senescence. METHODS: Basic literature for the mode of action of senolytics and senomorphics and their clinical perspectives in daily routine are discussed. RESULTS: Various causes lead to mitochondrial dysfunction and the activation of pro-aging signaling pathways, which eventually lead to cellular senescence with degradation of structural proteins of the dermal connective tissue and severe suppression of regenerative stem cell niches of the skin. CONCLUSIONS: Depletion of senescent cells suppress skin aging and enforce rejuvenation of skin and other organs and their function. The removal of senescent cells by cells of the native immune system is severely disturbed during aging. Selected senolytics and senomorphics are approved and are already on the market.

Induction of mitochondrial recycling reverts age-associated decline of the hematopoietic and immune systems.

Aging compromises hematopoietic and immune system functions, making older adults especially susceptible to hematopoietic failure, infections and tumor development, and thus representing an important medical target for a broad range of diseases. During aging, hematopoietic stem cells (HSCs) lose their blood reconstitution capability and commit preferentially toward the myeloid lineage (myeloid bias)1,2. These processes are accompanied by an aberrant accumulation of mitochondria in HSCs3. The administration of the mitochondrial modulator urolithin A corrects mitochondrial function in HSCs and completely restores the blood reconstitution capability of 'old' HSCs. Moreover, urolithin A-supplemented food restores lymphoid compartments, boosts HSC function and improves the immune response against viral infection in old mice. Altogether our results demonstrate that boosting mitochondrial recycling reverts the aging phenotype in the hematopoietic and immune systems.

pH regulates hematopoietic stem cell potential via polyamines.

Upon ex vivo culture, hematopoietic stem cells (HSCs) quickly lose potential and differentiate into progenitors. The identification of culture conditions that maintain the potential of HSCs ex vivo is therefore of high clinical interest. Here, we demonstrate that the potential of murine and human HSCs is maintained when cultivated for 2 days ex vivo at a pH of 6.9, in contrast to cultivation at the commonly used pH of 7.4. When cultivated at a pH of 6.9, HSCs remain smaller, less metabolically active, less proliferative and show enhanced reconstitution ability upon transplantation compared to HSC cultivated at pH 7.4. HSCs kept at pH 6.9 show an attenuated polyamine pathway. Pharmacological inhibition of the polyamine pathway in HSCs cultivated at pH 7.4 with DFMO mimics phenotypes and potential of HSCs cultivated at pH 6.9. Ex vivo exposure to a pH of 6.9 is therefore a positive regulator of HSC function by reducing polyamines. These findings might improve HSC short-term cultivation protocols for transplantation and gene therapy interventions.

A critical role of RUNX1 in governing megakaryocyte-primed hematopoietic stem cell differentiation.

As a transcription factor in the RUNT domain core-binding factor family, RUNX1 is crucial in multiple stages of hematopoiesis, and its mutation can cause familial platelet disorder with a predisposition to acute myeloid leukemia. Previous work has established that RUNX1 is involved in the maturation of megakaryocytes (MKs) and the production of platelets. Recent studies have shown that there exists a subpopulation of hematopoietic stem cells (HSCs) with relatively high expression of von Willebrand factor and CD41 at the apex of the HSC hierarchy, termed MK-HSCs, which can give rise to MKs without going through the traditional differentiation trajectory from HSC via MPP (multipotent progenitors) and MEP (megakaryocyte-erythroid progenitor). Here, by using Runx1F/FMx1-Cre mouse model, we discovered that the MK-HSC to MK direct differentiation can occur within 1 cell division, and RUNX1 is an important regulator in the process. Runx1 knockout results in a drastic decrease in platelet counts and a severe defect in the differentiation from MK-HSCs to MKs. Single cell RNA sequencing (RNAseq) analysis shows that MK-HSCs have a distinct gene expression signature compared with non-MK-HSCs, and Runx1 deletion alters the platelet and MK-related gene expression in MK-HSCs. Furthermore, bulk RNAseq and Cut&Run analyses show that RUNX1 binds to multiple essential MK or platelet developmental genes, such as Spi1, Selp, and Itga2b and regulates their expressions in MK-HSCs. Thus, by modulating the expression of MK-related genes, RUNX1 governs the direct differentiation from MK-HSCs to MKs and platelets.

Transplanting rejuvenated blood stem cells extends lifespan of aged immunocompromised mice.

One goal of regenerative medicine is to rejuvenate tissues and extend lifespan by restoring the function of endogenous aged stem cells. However, evidence that somatic stem cells can be targeted in vivo to extend lifespan is still lacking. Here, we demonstrate that after a short systemic treatment with a specific inhibitor of the small RhoGTPase Cdc42 (CASIN), transplanting aged hematopoietic stem cells (HSCs) from treated mice is sufficient to extend the healthspan and lifespan of aged immunocompromised mice without additional treatment. In detail, we show that systemic CASIN treatment improves strength and endurance of aged mice by increasing the myogenic regenerative potential of aged skeletal muscle stem cells. Further, we show that CASIN modifies niche localization and H4K16ac polarity of HSCs in vivo. Single-cell profiling reveals changes in HSC transcriptome, which underlie enhanced lymphoid and regenerative capacity in serial transplantation assays. Overall, we provide proof-of-concept evidence that a short systemic treatment to decrease Cdc42 activity improves the regenerative capacity of different endogenous aged stem cells in vivo, and that rejuvenated HSCs exert a broad systemic effect sufficient to extend murine health- and lifespan.

Epigenetic Clocks for Mice Based on Age-Associated Regions That are Conserved Between Mouse Strains and Human.

Aging of mice can be tracked by DNA methylation changes at specific sites in the genome. In this study, we used the recently released Infinium Mouse Methylation BeadChip to compare such epigenetic modifications in C57BL/6 (B6) and DBA/2J (DBA) mice. We observed marked differences in age-associated DNA methylation in these commonly used inbred mouse strains, indicating that epigenetic clocks for one strain cannot be simply applied to other strains without further verification. In B6 mice age-associated hypomethylation prevailed with focused hypermethylation at CpG islands, whereas in DBA mice CpG islands revealed rather hypomethylation upon aging. Interestingly, the CpGs with highest age-correlation were still overlapping in B6 and DBA mice and included the genes Hsf4, Prima1, Aspa, and Wnt3a. Notably, Hsf4 and Prima1 were also top candidates in previous studies based on whole genome deep sequencing approaches. Furthermore, Hsf4, Aspa, and Wnt3a revealed highly significant age-associated DNA methylation in the homologous regions in human. Subsequently, we used pyrosequencing of the four relevant regions to establish a targeted epigenetic clock that provided very high correlation with chronological age in independent cohorts of B6 (R 2 = 0.98) and DBA (R 2 = 0.91). Taken together, the methylome differs extensively between B6 and DBA mice, while prominent age-associated changes are conserved among these strains and even in humans. Our new targeted epigenetic clock with 4 CpGs provides a versatile tool for other researchers analyzing aging in mice.

Fast and high-fidelity in situ 3D imaging protocol for stem cells and niche components for mouse organs and tissues.

Quantitative 3D imaging of organ-wide cellular and subcellular components is central for revealing and understanding complex interactions between stem cells and their microenvironment. Here, we present a gentle but fast whole-mount immunofluorescence staining protocol for 3D confocal microscopy (iFAST3D) that preserves the 3D structure of the entire tissue and that of subcellular structures with high fidelity. The iFAST3D protocol enables reproducible and high-resolution 3D imaging of stem cells and various niche components for many mouse organs and tissues. For complete details on the use and execution of this protocol, please refer to Saçma et al. (2019).

A Limited Role for AMD3100 Induced Stem Cell Mobilization for Modulation of Thoracic Trauma Outcome.

ABSTRACT: Thoracic trauma is a major cause of mortality due to the associated inflammatory acute respiratory distress syndrome and morbidity due to impaired tissue regeneration. Trauma-induced lung inflammation is characterized by the early recruitment of cells with pro- or anti-inflammatory activity to the lung. Therapeutic interventions reducing the level of tissue inflammation may result in decreased tissue damage and improved healing and recovery. Stem cells might be able to improve trauma outcome via immunomodulation or by enhancing tissue regeneration.Here, we describe the migratory dynamics of murine mesenchymal, hematopoietic and endothelial stem and progenitor cells (SPCs) as well as mature inflammatory cells (monocytes, neutrophils, lymphocytes) to peripheral blood (PB) and lung tissue between 0.2 and 48 h post-blunt chest trauma (TXT). We demonstrate that the kinetics of immune cell and SPC distribution upon trauma are both cell-type and tissue-dependent. We identified a transient, early increase in the number of inflammatory cells in PB and lung at 2 h post-TXT and a second wave of infiltrating SPCs in lungs by 48 h after TXT induction, suggesting a role for SPCs in tissue remodeling after the initial inflammatory phase. Cxcl12/Cxcr4 blockade by AMD3100 within the first 6 h after TXT, while inducing a strong and coordinated mobilization of SPCs and leukocytes to PB and lung tissue, did not significantly affect TXT associated inflammation or tissue damage as determined by inflammatory cytokine levels, plasma markers for organ function, lung cell proliferation and survival, and myofibroblast/fibroblast ratio in the lung. Further understanding the dynamics of the distribution of endogenous SPCs and inflammatory cells will therefore be indispensable for stem cell-based or immunomodulation therapies in trauma.

Reduced adhesion of aged intestinal stem cells contributes to an accelerated clonal drift.

Upon aging, the function of the intestinal epithelium declines with a concomitant increase in aging-related diseases. ISCs play an important role in this process. It is known that ISC clonal dynamics follow a neutral drift model. However, it is not clear whether the drift model is still valid in aged ISCs. Tracking of clonal dynamics by clonal tracing revealed that aged crypts drift into monoclonality substantially faster than young ones. However, ISC tracing experiments, in vivo and ex vivo, implied a similar clonal expansion ability of both young and aged ISCs. Single-cell RNA sequencing for 1,920 high Lgr5 ISCs from young and aged mice revealed increased heterogeneity among subgroups of aged ISCs. Genes associated with cell adhesion were down-regulated in aged ISCs. ISCs of aged mice indeed show weaker adhesion to the matrix. Simulations applying a single cell-based model of the small intestinal crypt demonstrated an accelerated clonal drift at reduced adhesion strength, implying a central role for reduced adhesion for affecting clonal dynamics upon aging.

Aging of intestinal stem cells.

The intestine is one of the organs that relies on stem cell function for maintaining tissue homeostasis. Recent findings on intestinal aging show that intestinal architecture, such as villus length, crypt size, and cell composition changes in the aged crypts. The correspondent decline in the regenerative capacity of the intestine is mainly due to a decline in intestinal stem cell function upon aging, as the underlying mechanisms of aging intestinal stem cells are beginning to unravel. This review summarizes our current knowledge on stem cell-intrinsic mechanisms of aging of intestinal stem cells and their connection to extrinsic factors, such as niche cells and microbiota and will introduce recent approaches to attenuate or even revert the aging of intestinal stem cells.

Autophagy in mesenchymal progenitors protects mice against bone marrow failure after severe intermittent stress.

The cellular mechanisms required to ensure homeostasis of the hematopoietic niche and the ability of this niche to support hematopoiesis upon stress remain elusive. We here identify Wnt5a in Osterix+ mesenchymal progenitor and stem cells (MSPCs) as a critical factor for niche-dependent hematopoiesis. Mice lacking Wnt5a in MSPCs suffer from stress-related bone marrow (BM) failure and increased mortality. Niche cells devoid of Wnt5a show defective actin stress fiber orientation due to an elevated activity of the small GTPase CDC42. This results in incorrect positioning of autophagosomes and lysosomes, thus reducing autophagy and increasing oxidative stress. In MSPCs from patients from BM failure states which share features of peripheral cytopenia and hypocellular BM, we find similar defects in actin stress fiber orientation, reduced and incorrect colocalization of autophagosomes and lysosomes, and CDC42 activation. Strikingly, a short pharmacological intervention to attenuate elevated CDC42 activation in vivo in mice prevents defective actin-anchored autophagy in MSPCs, salvages hematopoiesis and protects against lethal cytopenia upon stress. In summary, our study identifies Wnt5a as a restriction factor for niche homeostasis by affecting CDC42-regulated actin stress-fiber orientation and autophagy upon stress. Our data further imply a critical role for autophagy in MSPCs for adequate support of hematopoiesis by the niche upon stress and in human diseases characterized by peripheral cytopenias and hypocellular BM.

Aging of human hematopoietic stem cells is linked to changes in Cdc42 activity.

In this study, we characterize age-related phenotypes of human hematopoietic stem cells (HSC). We report increased frequencies of HSC, hematopoietic progenitor cells and lineage negative cells in the elderly but a decreased frequency of multi-lymphoid progenitors. Aged human HSC further exhibited a delay in initiating division ex vivo though without changes in their division kinetics. The activity of the small RhoGTPase Cdc42 was elevated in aged human hematopoietic cells and we identified a positive correlation between Cdc42 activity and the frequency of HSC upon aging. The frequency of human HSC polar for polarity proteins was, similar to the mouse, decreased upon aging, while inhibition of Cdc42 activity via the specific pharmacological inhibitor of Cdc42 activity, CASIN, resulted in re-polarization of aged human HSC with respect to Cdc42. Elevated activity of Cdc42 in aged HSC thus contributed to age-related changes in HSC. Xenotransplant, using NBSGW mice as recipients, showed elevated chimerism in recipients of aged compared to young HSC. Aged HSC treated with CASIN ex vivo displayed an engraftment profile similar to recipients of young HSC. Taken together, our work reveals strong evidence for a role of elevated Cdc42 activity in driving aging of human HSC, and similar to mice, this presents a likely possibility for attenuation of aging in human HSC.

Septins in Stem Cells.

Septins were first described in yeast. Due to extensive research in non-yeast cells, Septins are now recognized across all species as important players in the regulation of the cytoskeleton, in the establishment of polarity, for migration, vesicular trafficking and scaffolding. Stem cells are primarily quiescent cells, and this actively maintained quiescent state is critical for proper stem cell function. Equally important though, stem cells undergo symmetric or asymmetric division, which is likely linked to the level of symmetry found in the mother stem cell. Due to the ability to organize barriers and be able to break symmetry in cells, Septins are thought to have a significant impact on organizing quiescence as well as the mode (symmetric vs asymmetric) of stem cell division to affect self-renewal versus differentiation. Mechanisms of regulating mammalian quiescence and symmetry breaking by Septins are though still somewhat elusive. Within this overview article, we summarize current knowledge on the role of Septins in stem cells ranging from yeast to mice especially with respect to quiescence and asymmetric division, with a special focus on hematopoietic stem cells.

Reconstructing Boolean network ensembles from single-cell data for unraveling dynamics in the aging of human hematopoietic stem cells.

Regulatory dependencies in molecular networks are the basis of dynamic behaviors affecting the phenotypical landscape. With the advance of high throughput technologies, the detail of omics data has arrived at the single-cell level. Nevertheless, new strategies are required to reconstruct regulatory networks based on populations of single-cell data. Here, we present a new approach to generate populations of gene regulatory networks from single-cell RNA-sequencing (scRNA-seq) data. Our approach exploits the heterogeneity of single-cell populations to generate pseudo-timepoints. This allows for the first time to uncouple network reconstruction from a direct dependency on time series measurements. The generated time series are then fed to a combined reconstruction algorithm. The latter allows a fast and efficient reconstruction of ensembles of gene regulatory networks. Since this approach does not require knowledge on time-related trajectories, it allows us to model heterogeneous processes such as aging. Applying the approach to the aging-associated NF-κB signaling pathway-based scRNA-seq data of human hematopoietic stem cells (HSCs), we were able to reconstruct eight ensembles, and evaluate their dynamic behavior. Moreover, we propose a strategy to evaluate the resulting attractor patterns. Interaction graph-based features and dynamic investigations of our model ensembles provide a new perspective on the heterogeneity and mechanisms related to human HSCs aging.

Cdc42-Borg4-Septin7 axis regulates HSC polarity and function.

Aging of hematopoietic stem cells (HSCs) is caused by the elevated activity of the small RhoGTPase Cdc42 and an apolar distribution of proteins. Mechanisms by which Cdc42 activity controls polarity of HSCs are not known. Binder of RhoGTPases proteins (Borgs) are known effector proteins of Cdc42 that are able to regulate the cytoskeletal Septin network. Here, we show that Cdc42 interacts with Borg4, which in turn interacts with Septin7 to regulate the polar distribution of Cdc42, Borg4, and Septin7 within HSCs. Genetic deletion of either Borg4 or Septin7 results in a reduced frequency of HSCs polar for Cdc42 or Borg4 or Septin7, a reduced engraftment potential and decreased lymphoid-primed multipotent progenitor (LMPP) frequency in the bone marrow. Taken together, our data identify a Cdc42-Borg4-Septin7 axis essential for the maintenance of polarity within HSCs and for HSC function and provide a rationale for further investigating the role of Borgs and Septins in the regulation of compartmentalization within stem cells.

Persistent JunB activation in fibroblasts disrupts stem cell niche interactions enforcing skin aging.

Fibroblasts residing in the connective tissues constitute the stem cell niche, particularly in organs such as skin. Although the effect of fibroblasts on stem cell niches and organ aging is an emerging concept, the underlying mechanisms are largely unresolved. We report a mechanism of redox-dependent activation of transcription factor JunB, which, through concomitant upregulation of p16INK4A and repression of insulin growth factor-1 (IGF-1), initiates the installment of fibroblast senescence. Fibroblast senescence profoundly disrupts the metabolic and structural niche, and its essential interactions with different stem cells thus enforces depletion of stem cells pools and skin tissue decline. In fact, silencing of JunB in a fibroblast-niche-specific manner-by reinstatement of IGF-1 and p16 levels-restores skin stem cell pools and overall skin tissue integrity. Here, we report a role of JunB in the control of connective tissue niche and identified targets to combat skin aging and associated pathologies.

KDM6A, a histone demethylase, regulates stress hematopoiesis and early B-cell differentiation.

Histone methylases and demethylases regulate gene expression programs in hematopoiesis. The molecular function of the demethylase KDM6A in normal hematopoiesis and, in particular, for the hematopoietic stem and progenitor cell (HSPC) compartment remains only partially understood. Female but not male Kdm6a-/- HSPCs were functionally impaired in adoptive transfer experiments as well as upon proliferative stress induced by 5-fluorouracil. Loss of Kdm6a affected primarily early B cells and erythroid and myeloid progenitor cells with respect to both number and function. Global gene expression analyses revealed a shared altered gene signature in Kdm6a-/- pro-B and pre-B cells that is also present in HSPCs, supporting that altered B-cell differentiation in Kdm6a-/- animals is already initiated in HSPCs. Interestingly, loss of KDM6A did not affect the global level of methylation of H3K27, its presumed target, in hematopoietic cells. Our data indicate a critical role for KDM6A in the regulation of hematopoietic differentiation and differentiation-specific gene expression programs, with a prominent role in early B-cell differentiation that is likely independent of H3K27 methylation status.

Suppression of elevated Cdc42 activity promotes the regenerative potential of aged intestinal stem cells.

Homeostasis in the intestinal epithelium is maintained by Lgr5-positive intestinal stem cells (ISCs) located at the base of the crypt. The function of ISCs is reduced upon aging which leads to a decline of regeneration of the intestinal epithelium. We report that aged intestinal crypts present with an elevated activity of the small RhoGTPase Cdc42. Elevation of Cdc42 activity in young animals by genetic means causes premature ISC aging, whereas pharmacological suppression of elevated Cdc42 activity restores organoid formation potential in vitro. Consistent with a critical role of elevated Cdc42 activity in aged ISCs for a reduced regenerative capacity of aged ISCs, suppression of Cdc42 activity in vivo improves crypt regeneration in aged mice. Thus, pharmacological reduction of Cdc42 activity can improve the regeneration of aged intestinal epithelium.

Inflammation rapidly recruits mammalian GMP and MDP from bone marrow into regional lymphatics.

Innate immune cellular effectors are actively consumed during systemic inflammation, but the systemic traffic and the mechanisms that support their replenishment remain unknown. Here, we demonstrate that acute systemic inflammation induces the emergent activation of a previously unrecognized system of rapid migration of granulocyte-macrophage progenitors and committed macrophage-dendritic progenitors, but not other progenitors or stem cells, from bone marrow (BM) to regional lymphatic capillaries. The progenitor traffic to the systemic lymphatic circulation is mediated by Ccl19/Ccr7 and is NF-κB independent, Traf6/IκB-kinase/SNAP23 activation dependent, and is responsible for the secretion of pre-stored Ccl19 by a subpopulation of CD205+/CD172a+ conventional dendritic cells type 2 and upregulation of BM myeloid progenitor Ccr7 signaling. Mature myeloid Traf6 signaling is anti-inflammatory and necessary for lymph node myeloid cell development. This report unveils the existence and the mechanistic basis of a very early direct traffic of myeloid progenitors from BM to lymphatics during inflammation.

When the body becomes infected with disease-causing pathogens, such as bacteria, the immune system activates various mechanisms which help to fight off the infection. One of the immune system's first lines of defense is to launch an inflammatory response that helps remove the pathogen and recruit other immune cells. However, this response can become overactivated, leading to severe inflammatory conditions that damage healthy cells and tissues. A second group of cells counteract this over inflammation and are different to the ones involved in the early inflammatory response. Both types of cells ­ inflammatory and anti-inflammatory ­ develop from committed progenitors, which, unlike stem cells, are already destined to become a certain type of cell. These committed progenitors reside in the bone marrow and then rapidly travel to secondary lymphoid organs, such as the lymph nodes, where they mature into functioning immune cells. During this journey, committed progenitors pass from the bone marrow to the lymphatic vessels that connect up the different secondary lymphoid organs, and then spread to all tissues in the body. Yet, it is not fully understood what exact route these cells take and what guides them towards these lymphatic tissues during inflammation. To investigate this, Serrano-Lopez, Hegde et al. used a combination of techniques to examine the migration of progenitor cells in mice that had been treated with lethal doses of a bacterial product that triggers inflammation. This revealed that as early as one to three hours after the onset of infection, progenitor cells were already starting to travel from the bone marrow towards lymphatic vessels. Serrano-Lopez, Hegde et al. found that a chemical released by an "alarm" immune cell already residing in secondary lymphoid organs attracted these progenitor cells towards the lymphatic tissue. Further experiments showed that the progenitor cells travelling to secondary lymphoid organs were already activated by bacterial products. They then follow the chemical released by alarm immune cells ready to respond to the immune challenge and suppress inflammation. These committed progenitors were also found in the inflamed lymph nodes of patients. These findings suggest this rapid circulation of progenitors is a mechanism of defense that contributes to the fight against severe inflammation. Altering how these cells migrate from the bone marrow to secondary lymphoid organs could provide a more effective treatment for inflammatory conditions and severe infections. However, these approaches would need to be tested further in the laboratory and in clinical trials.