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.

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.

Rejuvenation of the reconstitution potential and reversal of myeloid bias of aged HSCs upon pH treatment.

Aged hematopoietic stem cells (HSCs) show reduced reconstitution potential, limiting their use in transplantation settings in the clinic. We demonstrate here that exposure of aged HSCs ex vivo to a pH of 6.9 instead of the commonly used pH of 7.4 results in enhanced HSCs potential that is consistent with rejuvenation, including attenuation of the myeloid bias of aged HSC and restoration of a youthful frequency of epigenetic polarity. Rejuvenation of aged HSCs by pH 6.9 is, at least in part, due to alterations in the polyamine/methionine pathway within pH 6.9 HSCs, and consequently, attenuation of the production of spermidine also attenuated aging of HSCs. Exposure of aged HSCs to pH 6.9, or pharmacological targeting of the polyamine pathway, might thus extend the use of HSCs from aged donors for therapeutic applications.

Targeting CDC42 reduces skeletal degeneration after hematopoietic stem cell transplantation.

ABSTRACT: Osteopenia and osteoporosis are common long-term complications of the cytotoxic conditioning regimen for hematopoietic stem cell transplantation (HSCT). We examined mesenchymal stem and progenitor cells (MSPCs), which include skeletal progenitors, from mice undergoing HSCT. Such MSPCs showed reduced fibroblastic colony-forming units frequency, increased DNA damage, and enhanced occurrence of cellular senescence, whereas there was a reduced bone volume in animals that underwent HSCT. This reduced MSPC function correlated with elevated activation of the small Rho guanosine triphosphate hydrolase CDC42, disorganized F-actin distribution, mitochondrial abnormalities, and impaired mitophagy in MSPCs. Changes and defects similar to those in mice were also observed in MSPCs from humans undergoing HSCT. A pharmacological treatment that attenuated the elevated activation of CDC42 restored F-actin fiber alignment, mitochondrial function, and mitophagy in MSPCs in vitro. Finally, targeting CDC42 activity in vivo in animals undergoing transplants improved MSPC quality to increase both bone volume and trabecular bone thickness. Our study shows that attenuation of CDC42 activity is sufficient to attenuate reduced function of MSPCs in a BM transplant setting.

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).

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.

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.

KRasG12D expression in the bone marrow vascular niche affects hematopoiesis with inflammatory signals.

The bone marrow (BM) niche is an important milieu where hematopoietic stem and progenitor cells (HSPCs) are maintained. Previous studies have indicated that genetic mutations in various components of the niche can affect hematopoiesis and promote hematologic abnormalities, but the impact of abnormal BM endothelial cells (BMECs), a crucial niche component, on hematopoiesis remains incompletely understood. To dissect how genetic alterations in BMECs could affect hematopoiesis, we have employed a novel inducible Tie2-CreERT2 mouse model, with a tdTomato fluorescent reporter, to introduce an oncogenic KRasG12D mutation specifically in the adult endothelial cells. Tie2-CreERT2;KRasG12D mice had significantly more leukocytes and myeloid cells in the blood with mostly normal BM HSPC populations and developed splenomegaly. Genotyping polymerase chain reaction revealed KRasG12D activation in BMECs but not hematopoietic cells, confirming that the phenotype is due to the aberrant BMECs. Competitive transplant assays revealed that BM cells from the KRasG12D mice contained significantly fewer functional hematopoietic stem cells, and immunofluorescence imaging showed that the hematopoietic stem cells in the mutant mice were localized farther away from BM vasculature and closer to the endosteal area. RNA sequencing analyses found an inflammatory gene network, especially tumor necrosis factor α, as a possible contributor. Together, our results implicate an abnormal endothelial niche in compromising normal hematopoiesis.

Haematopoietic stem cells in perisinusoidal niches are protected from ageing.

With ageing, intrinsic haematopoietic stem cell (HSC) activity decreases, resulting in impaired tissue homeostasis, reduced engraftment following transplantation and increased susceptibility to diseases. However, whether ageing also affects the HSC niche, and thereby impairs its capacity to support HSC function, is still widely debated. Here, by using in-vivo long-term label-retention assays we demonstrate that aged label-retaining HSCs, which are, in old mice, the most quiescent HSC subpopulation with the highest regenerative capacity and cellular polarity, reside predominantly in perisinusoidal niches. Furthermore, we demonstrate that sinusoidal niches are uniquely preserved in shape, morphology and number on ageing. Finally, we show that myeloablative chemotherapy can selectively disrupt aged sinusoidal niches in the long term, which is linked to the lack of recovery of endothelial Jag2 at sinusoids. Overall, our data characterize the functional alterations of the aged HSC niche and unveil that perisinusoidal niches are uniquely preserved and thereby protect HSCs from ageing.