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) that include skeletal progenitors from mice undergoing HSCT. Such MSPCs showed reduced CFU-F frequency, increased DNA damage and enhanced occurrence of cellular senescence, while there was a reduced bone volume in animals that underwent HSCT. This reduced MSPC function correlated with elevated activation of the small RhoGTPAse 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.

Remodeling of the bone marrow microenvironment in chronic inflammation and in aging reduces hematopoietic stem cell (HSC) function. To assess the mechanisms of this functional decline of HSC and find strategies to counteract it, we established a model in which the Sfrp1 gene was deleted in Osterix+ osteolineage cells (OS1Δ/Δ mice). HSC from these mice showed severely diminished repopulating activity with associated DNA damage, enriched expression of the reactive oxygen species pathway and reduced single-cell proliferation. Interestingly, not only was the protein level of Catenin beta-1 (bcatenin) elevated, but so was its association with the phosphorylated co-activator p300 in the nucleus. Since these two proteins play a key role in promotion of differentiation and senescence, we inhibited in vivo phosphorylation of p300 through PP2A-PR72/130 by administration of IQ-1 in OS1Δ/Δ mice. This treatment not only reduced the b-catenin/phosphop300 association, but also decreased nuclear p300. More importantly, in vivo IQ-1 treatment fully restored HSC repopulating activity of the OS1Δ/Δ mice. Our findings show that the osteoprogenitor Sfrp1 is essential for maintaining HSC function. Furthermore, pharmacological downregulation of the nuclear b-catenin/phospho-p300 association is a new strategy to restore poor HSC function.

Bone marrow transplantation (BMT) is a widely used treatment for a variety of hematological diseases. However, prior to BMT, recipients are conditioned with total body irradiation (TBI) and/or chemotherapy to eliminate their hematopoietic system, which can also damage the niche, including mesenchymal stem and progenitor cells (MSPCs). The niche is an important microenvironment for the maintenance and regulation of stem cell function, including hematopoietic stem cells. The aim of our study was to investigate the effects of BMT on MSPCs and their subsequent recovery. We found that 10 months after BMT, MSPCs exhibited long-term damage, including decreased frequency (CFU-F), accumulation of DNA damage and an increase in senescent cells. In addition, we observed over-activation of CDC42 and damage to the actin cytoskeleton in BMT MSPCs, resulting in abnormal mitochondrial control and cytoskeletal reorganisation. These abnormalities lead to impaired differentiation and regenerative capacity of MSPCs, resulting in a delay in tissue recovery and an increased risk of long-term complications. To investigate this, we tested the effect of CDC42 inhibition in BMT mice during the first week after transplantation. Attenuation of CDC42 overactivation prevented cytoskeletal changes, improved bone volume and reduced osteoporotic changes, resulting in improved osteogenesis after BMT. This finding suggests that inhibition of CDC42 may be a potential strategy to improve long-term tissue recovery after transplantation in aging transplant recipients. In summary, our study shows that MSPCs from the conditioned niche are damaged, leading to long-term defects in tissue regenerative capacity. Our results shed light on the mechanisms underlying age-related factors that impede BMT and highlight potential strategies to improve long-term tissue recovery after transplantation. Bone marrow transplantation (BMT) is a widely used treatment for a variety of hematological diseases. However, prior to BMT, recipients are conditioned with total body irradiation (TBI) and/or chemotherapy to eliminate their hematopoietic system, which can also damage the niche, including mesenchymal stem and progenitor cells (MSPCs). The niche is an important microenvironment for the maintenance and regulation of stem cell function, including hematopoietic stem cells. The aim of our study was to investigate the effects of BMT on MSPCs and their subsequent recovery. We found that 10 months after BMT, MSPCs exhibited long-term damage, including decreased frequency (CFU-F), accumulation of DNA damage and an increase in senescent cells. In addition, we observed over-activation of CDC42 and damage to the actin cytoskeleton in BMT MSPCs, resulting in abnormal mitochondrial control and cytoskeletal reorganisation. These abnormalities lead to impaired differentiation and regenerative capacity of MSPCs, resulting in a delay in tissue recovery and an increased risk of long-term complications. To investigate this, we tested the effect of CDC42 inhibition in BMT mice during the first week after transplantation. Attenuation of CDC42 overactivation prevented cytoskeletal changes, improved bone volume and reduced osteoporotic changes, resulting in improved osteogenesis after BMT. This finding suggests that inhibition of CDC42 may be a potential strategy to improve long-term tissue recovery after transplantation in aging transplant recipients. In summary, our study shows that MSPCs from the conditioned niche are damaged, leading to long-term defects in tissue regenerative capacity. Our results shed light on the mechanisms underlying age-related factors that impede BMT and highlight potential strategies to improve long-term tissue recovery after transplantation.

GATA2 zinc-finger (ZF) mutations are associated with distinct entities of myeloid malignancies. The specific distribution of these mutations points toward different mechanisms of leukemogenesis depending on the ZF domain affected. In this study, we compared recurring somatic mutations in ZF1 and ZF2. All tested ZF mutants disrupted DNA binding in vitro. In transcription assays, co-expression of FOG1 counteracted GATA2-dependent transcriptional activation, while a variable response to FOG1-mediated repression was observed for individual GATA2 mutants. In primary murine bone marrow cells, GATA2 wild-type (WT) expression inhibited colony formation, while this effect was reduced for both mutants A318T (ZF1) and L359V (ZF2) with a shift toward granulopoiesis. In primary human CD34+ bone marrow cells and in the myeloid cell line K562, ectopic expression of GATA2 L359V, but not A318T or G320D, caused a block of erythroid differentiation accompanied by downregulation of GATA1, STAT5B, and PLCG1. Our findings may explain the role of GATA2 L359V during the progression of chronic myeloid leukemia and the collaboration of GATA2 ZF1 alterations with CEBPA double mutations in erythroleukemia.

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.

We have previously found that Sfrp1 expression is required for the maintenance of hematopoietic stem cell (HSC) function. To assess the mechanisms of HSC functional decline and find strategies to counteract these, we established a model in which the Sfrp1 gene was conditionally deleted. Since Sfrp1 is mainly expressed in osteoblastic cells, we deleted Sfrp1 expression specifically in osteoprogenitors of the BM niche (OS1∆/∆ mice). HSCs from these mice showed severely diminished repopulating activity with associated DNA damage, enriched expression of the `ROS pathway´, and reduced single cell proliferation. On the protein level, Catenin beta-1 (beta-catenin) was elevated. In a complex with the phosphorylated transcription factor p300, catenin beta-1 promotes differentiation and senescence. A detailed analysis of p300 and pSer89-p300, showed elevated levels of both p300 and pSerS89-p300, particularly in the nucleus. Importantly, we noted nuclear accumulation of the catenin beta-1/pSer89-p300 complex. To disrupt this transcriptional complex and restore HSC function, we modulated in vivo phosphorylation of p300 through PP2A/PR72-130 with a short treatment with the PR72-130 inhibitor IQ-1 in OS1∆/∆ mice. The IQ-1 treatment reduced both nuclear p300 and the catenin-beta-1/p300 complex. More importantly, IQ-1 fully restored HSC repopulating activity of treated mice, both in primary and secondary recipients. Our findings show that Sfrp1 expression in osteoprogenitors is essential for maintaining HSC function by reducing formation of the nuclear catenin beta-1/pSer89-p300 complex. Furthermore, pharmacological downregulation of phosphorylated p300 is a new strategy to restore poor HSC function. We have previously found that Sfrp1 expression is required for the maintenance of hematopoietic stem cell (HSC) function. To assess the mechanisms of HSC functional decline and find strategies to counteract these, we established a model in which the Sfrp1 gene was conditionally deleted. Since Sfrp1 is mainly expressed in osteoblastic cells, we deleted Sfrp1 expression specifically in osteoprogenitors of the BM niche (OS1∆/∆ mice). HSCs from these mice showed severely diminished repopulating activity with associated DNA damage, enriched expression of the `ROS pathway´, and reduced single cell proliferation. On the protein level, Catenin beta-1 (beta-catenin) was elevated. In a complex with the phosphorylated transcription factor p300, catenin beta-1 promotes differentiation and senescence. A detailed analysis of p300 and pSer89-p300, showed elevated levels of both p300 and pSerS89-p300, particularly in the nucleus. Importantly, we noted nuclear accumulation of the catenin beta-1/pSer89-p300 complex. To disrupt this transcriptional complex and restore HSC function, we modulated in vivo phosphorylation of p300 through PP2A/PR72-130 with a short treatment with the PR72-130 inhibitor IQ-1 in OS1∆/∆ mice. The IQ-1 treatment reduced both nuclear p300 and the catenin-beta-1/p300 complex. More importantly, IQ-1 fully restored HSC repopulating activity of treated mice, both in primary and secondary recipients. Our findings show that Sfrp1 expression in osteoprogenitors is essential for maintaining HSC function by reducing formation of the nuclear catenin beta-1/pSer89-p300 complex. Furthermore, pharmacological downregulation of phosphorylated p300 is a new strategy to restore poor HSC function.

The bone marrow (BM) microenvironment, also called the BM niche, is essential for the maintenance of fully functional blood cell formation (hematopoiesis) throughout life. Under physiologic conditions the niche protects hematopoietic stem cells (HSCs) from sustained or overstimulation. Acute or chronic stress deregulates hematopoiesis and some of these alterations occur indirectly via the niche. Effects on niche cells include skewing of its cellular composition, specific localization and molecular signals that differentially regulate the function of HSCs and their progeny. Importantly, while acute insults display only transient effects, repeated or chronic insults lead to sustained alterations of the niche, resulting in HSC deregulation. We here describe how changes in BM niche composition (ecosystem) and structure (remodeling) modulate activation of HSCs in situ . Current knowledge has revealed that upon chronic stimulation, BM remodeling is more extensive and otherwise quiescent HSCs may be lost due to diminished cellular maintenance processes, such as autophagy, ER stress response, and DNA repair. Features of aging in the BM ecology may be the consequence of intermittent stress responses, ultimately resulting in the degeneration of the supportive stem cell microenvironment. Both chronic stress and aging impair the functionality of HSCs and increase the overall susceptibility to development of diseases, including malignant transformation. To understand functional degeneration, an important prerequisite is to define distinguishing features of unperturbed niche homeostasis in different settings. A unique setting in this respect is xenotransplantation, in which human cells depend on niche factors produced by other species, some of which we will review. These insights should help to assess deviations from the steady state to actively protect and improve recovery of the niche ecosystem in situ to optimally sustain healthy hematopoiesis in experimental and clinical settings.

Hematopoietic stem cell self-renewal, proliferation, and differentiation are independently regulated by intrinsic as well as extrinsic mechanisms. We previously demonstrated that murine proliferation of hematopoietic stem cells is supported in serum-free medium supplemented with two growth factors, stem cell factor and interleukin 11. The survival of hematopoietic stem cells is additionally improved by supplementing this medium with two more growth factors, neural growth factor and collagen 1 (four growth factors) or serum-free medium conditioned by the hematopoietic stem cell-supportive stromal UG26-1B6 cells1. Here, we describe a robust and versatile alternative source of conditioned medium from mouse embryonic fibroblasts. We found that this conditioned medium supports survival and phenotypical identity of hematopoietic stem cells, as well as cell cycle entry in single cell cultures of CD34- CD48- CD150+ Lineage- SCA1+ KIT+ cells supplemented with two growth factors. Strikingly, in comparison with cultures in serum-free medium with four growth factors, conditioned medium from mouse embryonic fibroblasts increases the numbers of proliferating clones and the number of Lineage- SCA1+ KIT+ cells, both with two and four growth factors. In addition, conditioned medium from mouse embryonic fibroblasts supports self-renewal in culture of cells with short- and long-term hematopoiesis-repopulating ability in vivo. These findings identify conditioned medium from mouse embryonic fibroblasts as a robust alternative serumfree source of factors to maintain self-renewal of in vivo-repopulating hematopoetic stem cells in culture.

Successful tissue regeneration depends on the regenerative potential of the graft and its integration in recipients tissue. In bone marrow transplantation (BMT), age-related factors negatively impact on BMT. To dissect the separate contributions of recipient conditioning and aging for the success of BMT, we compared in vitro behavior and transcriptomes of multipotent stromal cells (MSCs) from young (Y: 3 months old), middle aged mice (A: 13 months old) with mice 10 months after BMT (BMT; total age of 13 months). Our experiments show that although HSC numbers are similar in A, and BMT mice, repopulating activity is significantly reduced in BMT HSC. This is accompanied by a reduction of (CD45/Ter119) - CD31 - CD166 - SCA1 + MSCs in the BM with a strongly reduced CFU-F frequency, indicating a functional compromise of BMT MSC. On the cellular level, BMT MSC show a reduced number of mitochondria with increased ROS production compared to A mice. In addition, BMT MSC show disorganized F-actin stress fibers compared to both Y and A MSC. To understand the mechanisms underlying the compromised function of BMT MSC, we analyzed the transcriptome of uncultured primary MSCs from the BM of Y, A, and BMT mice. These analyses show that in comparison to Y and A MSC, BMT MSC downregulate genes involved in intracellular nutrient transport and mitochondrial clearance, but upregulate metabolic processes, lysosomal genes and calcium-dependent non-canonical Wnt signaling. In experiments to assess similar changes in human BMT MSC, we found that MSC from patients undergoing BMT (2 and 4 weeks after BMT) show a severe dysregulation of F-Actin organization, compared to MSC from young and aged healthy donors (HD). Our results show deregulation of HSCs and MSCs in murine or human recipients undergoing BMT. Our results further suggest underlying defects in mitochondrial clearance and metabolic activation with deregulated F-actin organization. Our findings help to dissect and understand mechanisms of age-related factors hampering BMT and could help in devising strategies to improve long-term restoration of tissues after transplantation in aging graft recipients. Successful tissue regeneration depends on the regenerative potential of the graft and its integration in recipients tissue. In bone marrow transplantation (BMT), age-related factors negatively impact on BMT. To dissect the separate contributions of recipient conditioning and aging for the success of BMT, we compared in vitro behavior and transcriptomes of multipotent stromal cells (MSCs) from young (Y: 3 months old), middle aged mice (A: 13 months old) with mice 10 months after BMT (BMT; total age of 13 months). Our experiments show that although HSC numbers are similar in A, and BMT mice, repopulating activity is significantly reduced in BMT HSC. This is accompanied by a reduction of (CD45/Ter119) - CD31 - CD166 - SCA1 + MSCs in the BM with a strongly reduced CFU-F frequency, indicating a functional compromise of BMT MSC. On the cellular level, BMT MSC show a reduced number of mitochondria with increased ROS production compared to A mice. In addition, BMT MSC show disorganized F-actin stress fibers compared to both Y and A MSC. To understand the mechanisms underlying the compromised function of BMT MSC, we analyzed the transcriptome of uncultured primary MSCs from the BM of Y, A, and BMT mice. These analyses show that in comparison to Y and A MSC, BMT MSC downregulate genes involved in intracellular nutrient transport and mitochondrial clearance, but upregulate metabolic processes, lysosomal genes and calcium-dependent non-canonical Wnt signaling. In experiments to assess similar changes in human BMT MSC, we found that MSC from patients undergoing BMT (2 and 4 weeks after BMT) show a severe dysregulation of F-Actin organization, compared to MSC from young and aged healthy donors (HD). Our results show deregulation of HSCs and MSCs in murine or human recipients undergoing BMT. Our results further suggest underlying defects in mitochondrial clearance and metabolic activation with deregulated F-actin organization. Our findings help to dissect and understand mechanisms of age-related factors hampering BMT and could help in devising strategies to improve long-term restoration of tissues after transplantation in aging graft recipients.