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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.

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.

The support of hematopoiesis declines with age and it was previously shown that aging hematopoietic cells show cytoskeletal deregulation. Also, efficient autophagy is required to maintain cellular functions with age. Here, we describe a new mouse model with reduced support of hematopoiesis and age-related cytoskeletal changes in which the Wnt5a gene is deleted in Osterix (SP7)+ niche cells (O5AD/D mice). Considering that in these mice Wnt5a is deleted in niche cells, we here investigate niche cells from O5AD/D and control mice treated with 5-fluorouracil (5FU). We found that multipotent stromal cells (MSC) from the bone marrow of 5FU-treated O5AD/D mice show reduced CFU-F frequencies with preferential adipogenic differentiation. In these cells, the F-actin network is disorganized and associated with an elevated activity of the actin assembly-stimulating CDC42. Moreover, we found more and perinuclear accumulated ATG7+ and LC3+ autophagosomes in O5AD/D MSC. Also, LAMP1 expression was elevated and lysosomal diameters increased. But, we found no colocalization of LC3 and LAMP1 in O5AD/D MSC and concomitant autophagic flux, suggesting a defect in autophagy. Since in deregulated MSC from 5FU-treated O5AD/D mice CDC42 activity was increased and protein location was different, we additionally treated these mice with the CDC42 inhibitor CASIN. This treatment restored the CDC42 localization in O5AD/D MSCs and their F-actin stress fibers. Furthermore, LC3 and LAMP1 colocalization, mitochondrial and lysosomal diameters, and autophagic flux were restored. Most importantly, CASIN treatment restored the presence of the hematopoietic system and rescued the ability of O5AD/D mice to survive serial 5FU treatments.

We have previously found that a Sfrp1 knock-out environment fails to support the regeneration of hematopoetic stem cells (HSC) with serial to repopulate secondary recipients Renstrom et al., [1] . In order to dissect cell specific requirements of the Sfrp1 gene we established the Sfrp1flox/flox mouse strain and deleted Sfrp1 gene in Osterix+ (Sp7) osteolineage cells. In these Osx-Cre, Sfrp1 (OS1) mice, the number of MSCs is reduced, but these show an increased proportion of colony forming units (CFU-F). Furthermore, stromal cells grown ex-vivo show increased senescence-associated beta-galactosidase staining. In addition, the CFU-F-derived stromal cells differentiated spontaneously into adipocytes. These findings indicate altered functionality of stromal cells from OS1 mice. In the hematopoietic compartment of these mice, we found a decrease in myeloid progenitors in the bone marrow (BM) with concomitant increase in CD11b+ GR1hi granulocytes in peripheral blood. Although the number of primitive CD34- CD48- CD150+ HSCs (LT-HSCs) in the BM was unchanged, LT-HSCs from OS1 mice failed to repopulate in wild type recipients. In single cell cultures, we found that LT-HSCs from OS1 mice show decreased proliferation with concomitant increased differentiation into mature myeloid cells, which was associated with increased DNA damage as shown with comet tail assays and staining for gammaH2.AX. On a molecular level, we found that LT-HSCs from OS1 mice show increased CTNNB1 protein levels. CTNNB1 regulates cell differentiation and proliferation of stem cells by binding to Ep300 or CBP respectively Miyabayashi et al., [2] . Interestingly, CBP protein level was decreased in LT-HSCs from OS1 mice while Ep300 was increased, suggesting overactivation of the CTNNB1/Ep300 axis. Indeed, blocking CTNNB1/Ep300 binding with specific PP2A inhibitor IQ-1, we not only rescued the aberrant behavior of OS1 LT-HSC in vitro, but we also restored the repopulating activity of these LT-HSCs in vivo. Our results suggest that deletion of stromal SFRP1 diminishes the repopulating activity of LT-HSCs by increasing differentiation through PP2A-mediated dephosphorylation of the phospho-Ep300-binding site with CTNNB1.

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.

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.