Vitamin A-Retinoic Acid Signaling Regulates Hematopoietic Stem Cell Dormancy.

Dormant hematopoietic stem cells (dHSCs) are atop the hematopoietic hierarchy. The molecular identity of dHSCs and the mechanisms regulating their maintenance or exit from dormancy remain uncertain. Here, we use single-cell RNA sequencing (RNA-seq) analysis to show that the transition from dormancy toward cell-cycle entry is a continuous developmental path associated with upregulation of biosynthetic processes rather than a stepwise progression. In addition, low Myc levels and high expression of a retinoic acid program are characteristic for dHSCs. To follow the behavior of dHSCs in situ, a Gprc5c-controlled reporter mouse was established. Treatment with all-trans retinoic acid antagonizes stress-induced activation of dHSCs by restricting protein translation and levels of reactive oxygen species (ROS) and Myc. Mice maintained on a vitamin A-free diet lose HSCs and show a disrupted re-entry into dormancy after exposure to inflammatory stress stimuli. Our results highlight the impact of dietary vitamin A on the regulation of cell-cycle-mediated stem cell plasticity. VIDEO ABSTRACT.

Ubiquitination of hnRNPA1 by TRAF6 links chronic innate immune signaling with myelodysplasia.

Toll-like receptor (TLR) activation contributes to premalignant hematologic conditions, such as myelodysplastic syndromes (MDS). TRAF6, a TLR effector with ubiquitin (Ub) ligase activity, is overexpressed in MDS hematopoietic stem/progenitor cells (HSPCs). We found that TRAF6 overexpression in mouse HSPC results in impaired hematopoiesis and bone marrow failure. Using a global Ub screen, we identified hnRNPA1, an RNA-binding protein and auxiliary splicing factor, as a substrate of TRAF6. TRAF6 ubiquitination of hnRNPA1 regulated alternative splicing of Arhgap1, which resulted in activation of the GTP-binding Rho family protein Cdc42 and accounted for hematopoietic defects in TRAF6-expressing HSPCs. These results implicate Ub signaling in coordinating RNA processing by TLR pathways during an immune response and in premalignant hematologic diseases, such as MDS.

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.

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.

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.

Loss of epigenetic polarity is a hallmark of hematopoietic stem cell aging.

Changes of polarity in somatic stem cells upon aging or disease lead to a functional deterioration of stem cells and consequently loss of tissue homeostasis, likely due to changes in the mode (symmetry versus asymmetry) of stem cell divisions. Changes in polarity of epigenetic markers (or 'epi-polarity') in stem cells, which are linked to alterations in chromatin architecture, might explain how a decline in the frequency of epipolar stem cells can have a long-lasting impact on the function of especially aging stem cells. The drift in epipolarity might represent a novel therapeutic target to improve stem cell function upon aging or disease. Here we review basic biological principles of epigenetic polarity, with a special focus on epipolarity and aging of hematopoietic stem cells.

Yap1-Scribble polarization is required for hematopoietic stem cell division and fate.

Yap1 and its paralogue Taz largely control epithelial tissue growth. We have identified that hematopoietic stem cell (HSC) fitness response to stress depends on Yap1 and Taz. Deletion of Yap1 and Taz induces a loss of HSC quiescence, symmetric self-renewal ability, and renders HSC more vulnerable to serial myeloablative 5-fluorouracil treatment. This effect depends on the predominant cytosolic polarization of Yap1 through a PDZ domain-mediated interaction with the scaffold Scribble. Scribble and Yap1 coordinate to control cytoplasmic Cdc42 activity and HSC fate determination in vivo. Deletion of Scribble disrupts Yap1 copolarization with Cdc42 and decreases Cdc42 activity, resulting in increased self-renewing HSC with competitive reconstitution advantages. These data suggest that Scribble/Yap1 copolarization is indispensable for Cdc42-dependent activity on HSC asymmetric division and fate. The combined loss of Scribble, Yap1, and Taz results in transcriptional upregulation of Rac-specific guanine nucleotide exchange factors, Rac activation, and HSC fitness restoration. Scribble links Cdc42 and the cytosolic functions of the Hippo signaling cascade in HSC fate determination.

An aged bone marrow niche restrains rejuvenated hematopoietic stem cells.

Aging-associated leukemia and aging-associated immune remodeling are in part caused by aging of hematopoietic stem cells (HSCs). An increase in the activity of the small RhoGTPase cell division control protein 42 (Cdc42) within HSCs causes aging of HSCs. Old HSCs, treated ex vivo with a specific inhibitor of Cdc42 activity termed CASIN, stay rejuvenated upon transplantation into young recipients. We determined in this study the influence of an aged niche on the function of ex vivo rejuvenated old HSCs, as the relative contribution of HSCs intrinsic mechanisms vs extrinsic mechanisms (niche) for aging of HSCs still remain unknown. Our results show that an aged niche restrains the function of ex vivo rejuvenated HSCs, which is at least in part linked to a low level of the cytokine osteopontin found in aged niches. The data imply that sustainable rejuvenation of the function of aged HSCs in vivo will need to address the influence of an aged niche on rejuvenated HSCs.

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.

TLR4-dependent shaping of the wound site by MSCs accelerates wound healing.

We here address the question whether the unique capacity of mesenchymal stem cells to re-establish tissue homeostasis depends on their potential to sense pathogen-associated molecular pattern and, in consequence, mount an adaptive response in the interest of tissue repair. After injection of MSCs primed with the bacterial wall component LPS into murine wounds, an unexpected acceleration of healing occurs, clearly exceeding that of non-primed MSCs. This correlates with a fundamental reprogramming of the transcriptome in LPS-treated MSCs as deduced from RNAseq analysis and its validation. A network of genes mediating the adaptive response through the Toll-like receptor 4 (TLR4) pathway responsible for neutrophil and macrophage recruitment and their activation profoundly contributes to enhanced wound healing. In fact, injection of LPS-primed MSCs silenced for TLR4 fails to accelerate wound healing. These unprecedented findings hold substantial promise to refine current MSC-based therapies for difficult-to-treat wounds.

Osteopontin attenuates aging-associated phenotypes of hematopoietic stem cells.

Upon aging, hematopoietic stem cells (HSCs) undergo changes in function and structure, including skewing to myeloid lineages, lower reconstitution potential and loss of protein polarity. While stem cell intrinsic mechanisms are known to contribute to HSC aging, little is known on whether age-related changes in the bone marrow niche regulate HSC aging. Upon aging, the expression of osteopontin (OPN) in the murine bone marrow stroma is reduced. Exposure of young HSCs to an OPN knockout niche results in a decrease in engraftment, an increase in long-term HSC frequency and loss of stem cell polarity. Exposure of aged HSCs to thrombin-cleaved OPN attenuates aging of old HSCs, resulting in increased engraftment, decreased HSC frequency, increased stem cell polarity and a restored balance of lymphoid and myeloid cells in peripheral blood. Thus, our data suggest a critical role for reduced stroma-derived OPN for HSC aging and identify thrombin-cleaved OPN as a novel niche informed therapeutic approach for ameliorating HSC phenotypes associated with aging.

Aging alters the epigenetic asymmetry of HSC division.

Hematopoietic stem cells (HSCs) balance self-renewal and differentiation to maintain homeostasis. With aging, the frequency of polar HSCs decreases. Cell polarity in HSCs is controlled by the activity of the small RhoGTPase cell division control protein 42 (Cdc42). Here we demonstrate-using a comprehensive set of paired daughter cell analyses that include single-cell 3D confocal imaging, single-cell transplants, single-cell RNA-seq, and single-cell transposase-accessible chromatin sequencing (ATAC-seq)-that the outcome of HSC divisions is strongly linked to the polarity status before mitosis, which is in turn determined by the level of the activity Cdc42 in stem cells. Aged apolar HSCs undergo preferentially self-renewing symmetric divisions, resulting in daughter stem cells with reduced regenerative capacity and lymphoid potential, while young polar HSCs undergo preferentially asymmetric divisions. Mathematical modeling in combination with experimental data implies a mechanistic role of the asymmetric sorting of Cdc42 in determining the potential of daughter cells via epigenetic mechanisms. Therefore, molecules that control HSC polarity might serve as modulators of the mode of stem cell division regulating the potential of daughter cells.

Exit from dormancy provokes DNA-damage-induced attrition in haematopoietic stem cells.

Haematopoietic stem cells (HSCs) are responsible for the lifelong production of blood cells. The accumulation of DNA damage in HSCs is a hallmark of ageing and is probably a major contributing factor in age-related tissue degeneration and malignant transformation. A number of accelerated ageing syndromes are associated with defective DNA repair and genomic instability, including the most common inherited bone marrow failure syndrome, Fanconi anaemia. However, the physiological source of DNA damage in HSCs from both normal and diseased individuals remains unclear. Here we show in mice that DNA damage is a direct consequence of inducing HSCs to exit their homeostatic quiescent state in response to conditions that model physiological stress, such as infection or chronic blood loss. Repeated activation of HSCs out of their dormant state provoked the attrition of normal HSCs and, in the case of mice with a non-functional Fanconi anaemia DNA repair pathway, led to a complete collapse of the haematopoietic system, which phenocopied the highly penetrant bone marrow failure seen in Fanconi anaemia patients. Our findings establish a novel link between physiological stress and DNA damage in normal HSCs and provide a mechanistic explanation for the universal accumulation of DNA damage in HSCs during ageing and the accelerated failure of the haematopoietic system in Fanconi anaemia patients.

Aged murine hematopoietic stem cells drive aging-associated immune remodeling.

Aging-associated remodeling of the immune system impairs its functional integrity and contributes to increased morbidity and mortality in the elderly. Aging of hematopoietic stem cells (HSCs), from which all cells of the adaptive immune system ultimately originate, might play a crucial role in the remodeling of the aged immune system. We recently reported that aging of HSCs is, in part, driven by elevated activity of the small RhoGTPase Cdc42 and that aged HSCs can be rejuvenated in vitro by inhibition of the elevated Cdc42 activity in aged HSCs with the pharmacological compound CASIN. To study the quality of immune systems stemming selectively from young or aged HSCs, we established a HSC transplantation model in T- and B-cell-deficient young RAG1-/- hosts. We report that both phenotypic and functional changes in the immune system on aging are primarily a consequence of changes in the function of HSCs on aging and, to a large extent, independent of the thymus, as young and aged HSCs reconstituted distinct T- and B-cell subsets in RAG1-/- hosts that mirrored young and aged immune systems. Importantly, aged HSCs treated with CASIN reestablished an immune system similar to that of young animals, and thus capable of mounting a strong immune response to vaccination. Our studies further imply that epigenetic signatures already imprinted in aged HSCs determine the transcriptional profile and function of HSC-derived T and B cells.

HPRT and Purine Salvaging Are Critical for Hematopoietic Stem Cell Function.

Adult hematopoietic stem cells (HSCs) maintain tissue homeostasis and regenerative capacity of the hematopoietic system through self-renewal and differentiation. Metabolism is recognized as an important regulatory entity controlling stem cells. As purine nucleotides are essential for metabolic functions, we analyzed the role of hypoxanthine guanine phosphoribosyl transferase (HPRT)-associated purine salvaging in HSCs. Here, we demonstrate that hematopoietic stem and progenitor cells (HSPCs) show a strong dependence on HPRT-associated purine salvaging. HSPCs with lower HPRT activity had a severely reduced competitive repopulation ability upon transplantation. Strikingly, HPRT deficiency resulted in altered cell-cycle progression, proliferation kinetics and mitochondrial membrane potential primarily in the HSC compartment, whereas more committed progenitors were less affected. Our data thus imply a unique and important role of HPRT and the purine salvage pathway for HSC function. Stem Cells 2019;37:1606-1614.

Hematopoietic Stem Cell Dynamics Are Regulated by Progenitor Demand: Lessons from a Quantitative Modeling Approach.

The prevailing view on murine hematopoiesis and on hematopoietic stem cells (HSCs) in particular derives from experiments that are related to regeneration after irradiation and HSC transplantation. However, over the past years, different experimental techniques have been developed to investigate hematopoiesis under homeostatic conditions, thereby providing access to proliferation and differentiation rates of hematopoietic stem and progenitor cells in the unperturbed situation. Moreover, it has become clear that hematopoiesis undergoes distinct changes during aging with large effects on HSC abundance, lineage contribution, asymmetry of division, and self-renewal potential. However, it is currently not fully resolved how stem and progenitor cells interact to respond to varying demands and how this balance is altered by an aging-induced shift in HSC polarity. Aiming toward a conceptual understanding, we introduce a novel in silico model to investigate the dynamics of HSC response to varying demand. By introducing an internal feedback within a heterogeneous HSC population, the model is suited to consistently describe both hematopoietic homeostasis and regeneration, including the limited regulation of HSCs in the homeostatic situation. The model further explains the age-dependent increase in phenotypic HSCs as a consequence of the cells' inability to preserve divisional asymmetry. Our model suggests a dynamically regulated population of intrinsically asymmetrically dividing HSCs as suitable control mechanism that adheres with many qualitative and quantitative findings on hematopoietic recovery after stress and aging. The modeling approach thereby illustrates how a mathematical formalism can support both the conceptual and the quantitative understanding of regulatory principles in HSC biology.

Latexin regulation by HMGB2 is required for hematopoietic stem cell maintenance.

Hematopoietic stem cells provide life-long production of blood cells and undergo self-renewal division in order to sustain the stem cell pool. Homeostatic maintenance of hematopoietic stem cell pool and blood cell production is vital for the organism to survive. We previously reported that latexin is a negative regulator of hematopoietic stem cells in mice. Its natural variation in the expression is inversely correlated with hematopoietic stem cell number. However, the molecular mechanisms regulating latexin transcription remain largely unknown, and the genetic factors contributing to its natural variation are not clearly defined. Here we discovered a chromatin protein, high-mobility group protein B2, as a novel transcriptional suppressor of latexin by using DNA pull-down and mass spectrometry. High-mobility group protein B2 knockdown increases latexin expression at transcript and protein levels, and decreases hematopoietic stem cell number and regeneration capacity in vivo Concomitant blockage of latexin activation significantly reverses these phenotypic changes, suggesting that latexin is one of the downstream targets and functional mediators of high-mobility group protein B2. We further identified a functional single nucleotide polymorphism, rs31528793, in the latexin promoter that binds to high-mobility group protein B2 and affects the promoter activity. G allelic variation in rs31528793 associates with the higher latexin expression and lower hematopoietic stem cell number, whereas C allele indicates the lower latexin expression and higher stem cell number. This study reveals for the first time that latexin transcription is regulated by both transacting (high-mobility group protein B2) and cis-acting (single nucleotide polymorphism rs31528793) factors. It uncovers the functional role of naturally occurring genetic variants, in combination with epigenetic regulator, in determining differential gene expression and phenotypic diversity in the hematopoietic stem cell population.

A canonical to non-canonical Wnt signalling switch in haematopoietic stem-cell ageing.

Many organs with a high cell turnover (for example, skin, intestine and blood) are composed of short-lived cells that require continuous replenishment by somatic stem cells. Ageing results in the inability of these tissues to maintain homeostasis and it is believed that somatic stem-cell ageing is one underlying cause of tissue attrition with age or age-related diseases. Ageing of haematopoietic stem cells (HSCs) is associated with impaired haematopoiesis in the elderly. Despite a large amount of data describing the decline of HSC function on ageing, the molecular mechanisms of this process remain largely unknown, which precludes rational approaches to attenuate stem-cell ageing. Here we report an unexpected shift from canonical to non-canonical Wnt signalling in mice due to elevated expression of Wnt5a in aged HSCs, which causes stem-cell ageing. Wnt5a treatment of young HSCs induces ageing-associated stem-cell apolarity, reduction of regenerative capacity and an ageing-like myeloid-lymphoid differentiation skewing via activation of the small Rho GTPase Cdc42. Conversely, Wnt5a haploinsufficiency attenuates HSC ageing, whereas stem-cell-intrinsic reduction of Wnt5a expression results in functionally rejuvenated aged HSCs. Our data demonstrate a critical role for stem-cell-intrinsic non-canonical Wnt5a signalling in HSC ageing.