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Neuroblastoma (NB) is the second most common solid cancer in childhood, accounting for 15% of cancer-related deaths in children. In high-risk NB patients, the majority suffers from metastasis. Despite intensive multimodal treatment, long-term survival remains

Osteolineage cell-derived extracellular vesicles (EVs) play a regulatory role in hematopoiesis and have been shown to promote the ex vivo expansion of human hematopoietic stem and progenitor cells (HSPCs). Here, we demonstrate that EVs from different human osteolineage sources do not have the same HSPC expansion promoting potential. Comparison of stimulatory and non-stimulatory osteolineage EVs by next-generation sequencing and mass spectrometry analyses revealed distinct microRNA and protein signatures identifying EV-derived candidate regulators of ex vivo HSPC expansion. Accordingly, the treatment of umbilical cord blood-derived CD34+ HSPCs with stimulatory EVs-altered HSPC transcriptome, including genes with known roles in cell proliferation. An integrative bioinformatics approach, which connects the HSPC gene expression data with the candidate cargo in stimulatory EVs, delineated the potentially targeted biological functions and pathways during hematopoietic cell expansion and development. In conclusion, our study gives novel insights into the complex biological role of EVs in osteolineage cell-HSPC crosstalk and promotes the utility of EVs and their cargo as therapeutic agents in regenerative medicine.

Aim Preclinical work indicates that low-density lipoprotein cholesterol (LDL-C) not only drives atherosclerosis by directing the innate immune response at plaque level but also augments proinflammatory monocyte production in the bone marrow (BM) compartment. In this study, we aim to unravel the impact of LDL-C on monocyte production in the BM compartment in human subjects. Methods and results A multivariable linear regression analysis in 12 304 individuals of the EPIC-Norfolk prospective population study showed that LDL-C is associated with monocyte percentage (β = 0.131 [95% CI: 0.036–0.225]; P = 0.007), at the expense of granulocytes (β = −0.876 [95% CI: −1.046 to −0.705]; P < 0.001). Next, we investigated whether altered haematopoiesis could explain this monocytic skewing by characterizing CD34+ BM haematopoietic stem and progenitor cells (HSPCs) of patients with familial hypercholesterolaemia (FH) and healthy normocholesterolaemic controls. The HSPC transcriptomic profile of untreated FH patients showed increased gene expression in pathways involved in HSPC migration and, in agreement with our epidemiological findings, myelomonocytic skewing. Twelve weeks of cholesterol-lowering treatment reverted the myelomonocytic skewing, but transcriptomic enrichment of monocyte-associated inflammatory and migratory pathways persisted in HSPCs post-treatment. Lastly, we link hypercholesterolaemia to perturbed lipid homeostasis in HSPCs, characterized by lipid droplet formation and transcriptomic changes compatible with increased intracellular cholesterol availability. Conclusions Collectively, these data highlight that LDL-C impacts haematopoiesis, promoting both the number and the proinflammatory activation of circulating monocytes. Furthermore, this study reveals a potential contributory role of HSPC transcriptomic reprogramming to residual inflammatory risk in FH patients despite cholesterol-lowering therapy., Graphical Abstract LDL-C impacts hematopoiesis, promoting both the number and the proinflammatory activation of circulating monocytes.

Recently, we and others have illustrated that extracellular vesicles (EVs) have the potential to support hematopoietic stem and progenitor cell (HSPC) expansion; however, the mechanism and processes responsible for the intercellular communication by EVs are still unknown. In the current study, we investigate whether primary human bone marrow derived mesenchymal stromal cells (BMSC) EVs isolated from two different origins, fetal (fEV) and adult (aEV) tissue, can increase the relative low number of HSPCs found in umbilical cord blood (UCB) and which EV-derived components are responsible for ex vivo HSPC expansion. Interestingly, aEVs and to a lesser extent fEVs, showed supportive ex vivo expansion capacity of UCB-HSPCs. Taking advantage of the two BMSC sources with different supportive effects, we analyzed the EV cargo and investigated how gene expression is modulated in HSPCs after incubation with aEVs and fEVs. Proteomics analyses of the protein cargo composition of the supportive aEV vs. the less-supportive fEV identified 90% of the Top100 exosome proteins present in the ExoCarta database. Gene Ontology (GO) analyses illustrated that the proteins overrepresented in aEVs were annotated to oxidation-reduction process, mitochondrial ATP synthesis coupled proton transport, or protein folding. In contrast, the proteins overrepresented in fEVs were annotated to extracellular matrix organization positive regulation of cell migration or transforming growth factor beta receptor (TGFBR) signaling pathway. Small RNA sequencing identified different molecular signatures between aEVs and fEVs. Interestingly, the microRNA cluster miR-99b/let-7e/miR-125a, previously identified to increase the number of HSPCs by targeting multiple pro-apoptotic genes, was highly and significantly enriched in aEVs. Although we identified significant differences in the supportive effects of aEVs and fEVs, RNAseq analyses of the 24 h treated HSPCs indicated that a limited set of genes was differentially regulated when compared to cells that were treated with cytokines only. Together, our study provides novel insights into the complex biological role of EVs and illustrates that aEVs and fEVs differentially support ex vivo expansion capacity of UCB-HSPCs. Together opening new means for the application of EVs in the discovery of therapeutics for more efficient ex vivo HSPC expansion.

Ribonucleoproteins (RNPs) are frequently applied for therapeutic gene editing as well as fundamental research, because the method is fast, viral free, and does not rely on clonal selection.We evaluated various parameters to genetically engineer human hematopoietic stem progenitor cells (HSPCs) using spCas9-RNPs and achieve gene editing efficiencies up to 80%. We find that single guide RNA (sgRNA) design is critical to achieve high gene editing efficiencies. However, finding effective sgRNAs for HSPCs can be challenging, while the contribution of numerous in silico models is unclear.Here we established a time- and cost-efficient in vitro transcribed sgRNA screening model in K562 cells to identify sgRNAs that are effective in HSPCs using RNP delivery. We show that this simple screening method outperforms all in silico prediction models. Our data demonstrates that most in silico sgRNA prediction models are ineffective and we make recommendations to potentially improve their accuracy.We report that gene editing is equally efficient in distinct CD34+ HSPC subpopulations. Furthermore, no effects on cell proliferation, differentiation or in vitro hematopoietic lineage commitment were observed. Finally, no upregulation of p21 expression was found, suggesting unperturbed HSPC homeostasis.Key pointsIn vitro transcribed single sgRNAs (IVTsgRNA) screening in K562 outperforms in silico modelingHematopoietic stem and progenitor cells are equally targeted by Ribonucleoproteins (RNPs)Hematopoietic stem and progenitor cells show no induction of p21 expression or effects on differentiation, proliferation and lineage commitmentGraphical abstract

Bone marrow endothelium plays an important role in the homing of hematopoietic stem and progenitor cells upon transplantation, but surprisingly little is known on how the bone marrow endothelial cells regulate local permeability and hematopoietic stem and progenitor cells transmigration. We show that temporal loss of vascular endothelial-cadherin function promotes vascular permeability in BM, even upon low-dose irradiation. Loss of vascular endothelial-cadherin function also enhances homing of transplanted hematopoietic stem and progenitor cells to the bone marrow of irradiated mice although engraftment is not increased. Intriguingly, stabilizing junctional vascular endothelial-cadherin in vivo reduced bone marrow permeability, but did not prevent hematopoietic stem and progenitor cells migration into the bone marrow, suggesting that hematopoietic stem and progenitor cells use the transcellular migration route to enter the bone marrow. Indeed, using an in vitro migration assay, we show that human hematopoietic stem and progenitor cells predominantly cross bone marrow endothelium in a transcellular manner in homeostasis by inducing podosome-like structures. Taken together, vascular endothelial-cadherin is crucial for BM vascular homeostasis but dispensable for the homing of hematopoietic stem and progenitor cells. These findings are important in the development of potential therapeutic targets to improve hematopoietic stem and progenitor cell homing strategies.

Culture expanded mesenchymal stromal cells (MSCs) are being extensively studied for therapeutic applications, including treatment of graft-versus-host disease, osteogenesis imperfecta and for enhancing engraftment of hematopoietic stem cells after transplantation. Thus far, clinical trials have shown that the therapeutic efficiency of MSCs is variable, which may in part be due to inefficient cell migration. Here we demonstrate that human MSCs display remarkable low migratory behaviour compared to other mesodermal-derived primary human cell types. We reveal that specifically in MSCs the nucleus is irregularly shaped and nuclear lamina are prone to wrinkling. In addition, we show that expression of Lamin A/C is relatively high in MSCs. We further demonstrate that in vitro MSC migration through confined pores is limited by their nuclei, a property that might correlate to the therapeutic inefficiency of administered MSC in vivo. Silencing expression of Lamin A/C in MSCs improves nuclear envelope morphology, promotes the protrusive activity of MSCs through confined pores and enhances their retention in the lung after intravenous administration in vivo. Our findings suggest that the intrinsic nuclear lamina properties of MSCs underlie their limited capacity to migrate, and that strategies that target the nuclear lamina might alter MSC-based cellular therapies.

Item does not contain fulltext Mesenchymal stromal cells (MSCs) are applied as novel therapeutics for their regenerative and immune-suppressive capacities. Clinical applications, however, require extensive expansion of MSCs. Fetal bone marrow-derived MSCs (FBMSCs) proliferate faster than adult bone marrow-derived MSC (ABMSCs). To optimize expansion and function of MSC in general, we explored the differences between ABMSC and FBMSC. Gene expression profiling implicated differential expression of genes encoding proteins in the Wnt signaling pathway, including excreted inhibitors of Wnt signaling, particularly by ABMSC. Both MSC types had a similar basal level of canonical Wnt signaling. Abrogation of autocrine Wnt production by inhibitor of Wnt production-2 (IWP2) reduced canonical Wnt signaling and cell proliferation of FBMSCs, but hardly affected ABMSC. Addition of exogenous Wnt3a, however, induced expression of the target genes lymphocyte enhancer-binding factor (LEF) and T-cell factor (TCF) faster and at lower Wnt3a levels in ABMSC compared to FBMSC. Medium replacement experiments indicated that ABMSC produce an inhibitor of Wnt signaling that is effective on ABMSC itself but not on FBMSC, whereas FBMSC excrete (Wnt) factors that stimulate proliferation of ABMSC. In contrast, FBMSC were not able to support hematopoiesis, whereas ABMSC displayed hematopoietic support sensitive to IWP2, the inhibitor of Wnt factor excretion. In conclusion, ABMSC and FBMSC differ in their Wnt signature. While FBMSC produced factors, including Wnt signals, that enhanced MSC proliferation, ABMSC produced Wnt factors in a setting that enhanced hematopoietic support. Thus, further unraveling the molecular basis of this phenomenon may lead to improvement of clinical expansion protocols of ABMSCs.

Bone marrow (BM) endothelium plays an important role in homing of hematopoietic stem and progenitor cells (HSPCs) upon transplantation, but surprisingly little is known on how endothelial cells regulate local permeability and HSPC transmigration. We show that temporal loss of VE-cadherin function promotes vascular permeability in BM, even upon low dose irradiation and strongly enhanced homing of transplanted HSPCs to BM of irradiated mice. Intriguingly, stabilizing junctional VE-cadherinin vivoreduced BM permeability, but did not prevent HSPC migration into the BM, suggesting that HSPCs enter the bone marrow by transcellularly crossing the endothelium. Indeed, HSPCs induce podosomes to cross human BM endothelial monolayers in a transcellular manner. By contrast, HSPC rather use the paracellular route when VE-cadherin function is inhibited. Taken together, VE-cadherin is crucial for BM vascular homeostasis and HSPC homing, and may therefore serve as a potential therapeutic target to improve HSPC homing strategies.

Bone marrow (BM) mesenchymal stromal cells (MSCs) provide microenvironmental support to hematopoietic stem and progenitor cells (HSPCs). Culture-expanded MSCs are interesting candidates for cellular therapies due to their immunosuppressive and regenerative potential which can be further enhanced by pretreatment with interferon-gamma (IFN-γ). However, it remains unknown whether IFN-γ can also influence hematopoietic support by BM-MSCs. In this study, we elucidate the impact of IFN-γ on the hematopoietic support of BM-MSCs. We found that IFN-γ increases expression of interleukin (IL)-6 and stem cell factor by human BM-MSCs. IFN-γ-treated BM-MSCs drive HSPCs toward myeloid commitment in vitro, but impair subsequent differentiation of HSPC. Moreover, IFN-γ-ARE-Del mice with increased IFN-γ production specifically lose their BM-MSCs, which correlates with a loss of hematopoietic stem cells' quiescence. Although IFN-γ treatment enhances the immunomodulatory function of MSCs in a clinical setting, we conclude that IFN-γ negatively affects maintenance of BM-MSCs and their hematopoietic support in vitro and in vivo.

The interactions of hematopoietic stem and progenitor cells (HSPCs) with extracellular matrix (ECM) components and cells from the bone marrow (BM) microenvironment control their homeostasis. Regenerative BM conditions can induce expression of the ECM protein transforming growth factor beta-induced gene H3 (TGFBI or BIGH3) in murine HSPCs. In this study, we examined how increased or reduced TGFBI expression in human HSPCs and BM mesenchymal stromal cells (MSCs) affects HSPC maintenance, differentiation, and migration. HSPCs that overexpressed TGFBI showed accelerated megakaryopoiesis, whereas granulocyte differentiation and proliferation of granulocyte, erythrocyte, and monocyte cultures were reduced. In addition, both upregulation and downregulation of TGFBI expression impaired HSPC colony-forming capacity of HSPCs. Interestingly, the colony-forming capacity of HSPCs with reduced TGFBI levels was increased after long-term co-culture with MSCs, as measured by long-term culture-colony forming cell (LTC-CFC) formation. Moreover, TGFBI downregulation in HSPCs resulted in increased cobblestone area-forming cell (CAFC) frequency, a measure for hematopoietic stem cell (HSC) capacity. Concordantly, TGFBI upregulation in HSPCs resulted in a decrease of CAFC and LTC-CFC frequency. These results indicate that reduced TGFBI levels in HSPCs enhanced HSC maintenance, but only in the presence of MSCs. In addition, reduced levels of TGFBI in MSCs affected MSC/HSPC interaction, as observed by an increased migration of HSPCs under the stromal layer. In conclusion, tight regulation of TGFBI expression in the BM niche is essential for balanced HSPC proliferation and differentiation.

Maintenance of haematopoietic stem cells and differentiation of committed progenitors occurs in highly specialized niches. The interactions of haematopoietic stem and progenitor cells (HSPCs) with cells, growth factors and extracellular matrix (ECM) components of the bone marrow (BM) microenvironment control homeostasis of HSPCs. We only start to understand the complexity of the haematopoietic niche(s) that comprises endosteal, arterial, sinusoidal, mesenchymal and neuronal components. These distinct niches produce a broad range of soluble factors and adhesion molecules that modulate HSPC fate during normal hematopoiesis and BM regeneration. Adhesive interactions between HSPCs and the microenvironment will influence their localization and differentiation potential. In this review we highlight the current understanding of the functional role of ECM- and adhesion (regulating) molecules in the haematopoietic niche during homeostatic and regenerative hematopoiesis. This knowledge may lead to the improvement of current cellular therapies and more efficient development of future cellular products.

To investigate whether the migratory ability of peripheral blood-derived CD34+ cells of patients undergoing autologous peripheral blood stem cell transplantation is related to the homing efficiency of these cells, the migration in vitro of these cells was determined and correlated with in vivo hematopoietic recovery. Large inter-individual differences of the in vitro migratory ability of the CD34+ cells were observed, ranging from 1.1% to 16.4% for spontaneous migration and 6.2% to 40.8% for SDF-1–induced (100 ng/mL) migration. Significantly faster hematologic recovery was observed in those patients who received transplanted CD34+cells that showed high spontaneous and SDF-1–induced migration in vitro (P

Hepatocyte growth factor (HGF), also known as scatter factor (SF), is produced by mesenchymal cells, including bone marrow (BM) stromal cells, and has mitogenic and motogenic effects on a variety of cell types. Recently, a role has been assigned to HGF/SF and its receptor, c-MET, in both normal and malignant hemopoiesis. We investigated the function of HGF/SF on hemopoietic mononuclear cells (MNC) from patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) with circulating blasts. In contrast to results with normal MNC, HGF/SF alone stimulated the proliferation and colony formation of MNC from these patients. MNC from some (4/13) of the AML patients also produced HGF/SF (0.1-0.2 ng/ml/day), while we could not detect HGF/SF in cultures from normal MNC. Furthermore, it appeared that HGF/SF induced migration of leukemic cells in Boyden using KG1a cells as a model for leukemic blasts. The membranes dividing the two compartments of the Boyden chambers were coated with fibronectin. HGF/SF significantly promoted migration in 3/5 samples of MDS patients and in 5/7 samples of AML patients. Supernatant of human BM stromal cells, which is chemoattractive for normal human hemopoietic progenitor cells, also promoted migration of MNC from 4/5 MDS patients and 6/7 AML patients. Since HGF/SF is one of the growth factors produced by BM stromal cells, a neutralizing antibody directed against HGF/SF was added to the BM stroma supernatant, which reduced migration significantly in 2/3 MDS and in 3/6 AML responders to BM stroma supernatant. In conclusion, HGF/SF promotes proliferation and migration of hemopoietic cells from AML and MDS patients in vitro and may therefore contribute to the malignant potential of these cells.

Cell adhesion and migration are important determinants of homing and development of hematopoietic stem and progenitor cells (HSPCs) in bone marrow (BM) niches. The extracellular matrix protein transforming growth factor-β (TGF-β) inducible gene H3 (BIGH3) is involved in adhesion and migration, although the effect of BIGH3 is highly cell type-dependent. BIGH3 is abundantly expressed by mesenchymal stromal cells, while its expression in HSPCs is relatively low unless induced by certain BM stressors. Here, we set out to determine how BIGH3 modulates HSPC adhesion and migration. We show that primary HSPCs adhere to BIGH3-coated substrates, which is, in part, integrin-dependent. Overexpression of BIGH3 in HSPCs and HL60 cells reduced the adhesion to the substrate fibronectin in adhesion assays, which was even more profound in electrical cell-substrate impedance sensing (ECIS) assays. Accordingly, the CXCL12 induced migration over fibronectin-coated surface was reduced in BIGH3-expressing HSPCs. The integrin expression profile of HSPCs was not altered upon BIGH3 expression. Although expression of BIGH3 did not alter actin polymerization in response to CXCL12, it inhibited the PMA-induced activation of the small GTPase RAC1 as well as the phosphorylation and activation of extracellular-regulated kinases (ERKs). Reduced activation of ERK and RAC1 may be responsible for the inhibition of cell adhesion and migration by BIGH3 in HSPCs. Induced BIGH3 expression upon BM stress may contribute to the regulation of BM homeostasis.

One of the most remarkable characteristics of stem cells is their ability to perpetuate themselves through self-renewal while concomitantly generating differentiated cells. In the hematopoietic system, stem cells balance these mechanisms to maintain steady-state hematopoiesis for the lifetime of the organism, and to effectively regenerate the system following injury. Defects in the proper control of self-renewal and differentiation can be potentially devastating and contribute to the development of malignancies. In this review, we trace the emerging role of Wnt signaling as a critical regulator of distinct aspects of self-renewal and differentiation, its contribution to the maintenance of homeostasis and regeneration, and how the pathway can be hijacked to promote leukemia development. A better understanding of these processes could pave the way to enhancing recovery after injury and to developing better therapeutic approaches for hematologic malignancies.

Aims Experimental work posits that acute ischaemic events trigger haematopoietic activity, driving monocytosis, and atherogenesis. Considering the chronic low-grade inflammatory state in atherosclerosis, we hypothesized that haematopoietic hyperactivity is a persistent feature in cardiovascular disease (CVD). Therefore, we aimed to assess the activity of haematopoietic organs and haematopoietic stem and progenitor cells (HSPCs) in humans. Methods and results First, we performed 18F-fluorodeoxyglucose positron emission tomographic (18F-FDG PET) imaging in 26 patients with stable atherosclerotic CVD (ischaemic event >12 months ago), and 25 matched controls. In splenic tissue, 18F-FDG uptake was 2.68 ± 0.65 in CVD patients vs. 1.75 ± 0.54 in controls (1.6-fold higher; P < 0.001), and in bone marrow 3.20 ± 0.76 vs. 2.72 ± 0.46 (1.2-fold higher; P = 0.003), closely related to LDL cholesterol levels (LDLc, r = 0.72). Subsequently, we determined progenitor potential of HSPCs harvested from 18 patients with known atherosclerotic CVD and 30 matched controls; both groups were selected from a cohort of cancer patients undergoing autologous stem cell transplantation. In CVD patients, the normalized progenitor potential, expressed as the number of colony-forming units-granulocyte/monocyte (CFU-GM) colonies/CD34+ cell, was 1.6-fold higher compared with matched controls ( P < 0.001). Finally, we assessed the effects of native and oxidized lipoproteins on HSPCs harvested from healthy donors in vitro . Haematopoietic stem and progenitor cells displayed a 1.5-fold increased CFU-GM capacity in co-culture with oxidized LDL in vitro ( P = 0.002), which was inhibited by blocking oxidized phospholipids via E06 ( P = 0.001). Conclusion Collectively, these findings strengthen the case for a chronically affected haematopoietic system, potentially driving the low-grade inflammatory state in patients with atherosclerosis.

Hematopoietic stem cells (HSCs) respond to injury by rapidly proliferating and regenerating the hematopoietic system. Little is known about the intracellular programs that are activated within HSCs during this regenerative process and how this response may be influenced by alterations in signals from the injured microenvironment. Here we have examined the regenerating microenvironment and find that following injury it has an enhanced ability to support HSCs. During this regenerative phase, both hematopoietic and stromal cell elements within the bone marrow microenvironment show increased expression of Wnt10b, which can function to enhance growth of hematopoietic precursors. In addition, regenerating HSCs show increased activation of Wnt signaling, suggesting that microenvironmental changes in Wnt expression after injury may be integrated with the responses of the hematopoietic progenitors. Cumulatively, our data reveal that growth signals in the hematopoietic system are re-activated during injury, and provide novel insight into the influence of the microenvironment during regeneration. Disclosure of potential conflicts of interest is found at the end of this article.

The hemopoietic microenvironment consists of a diverse repertoire of cells capable of providing signals that influence hemopoietic stem cell function. Although the role of osteoblasts and vascular endothelial cells has recently been characterized, the function of the most abundant cell type in the bone marrow, the adipocyte, is less defined. Given the emergence of a growing number of adipokines, it is possible that these factors may also play a role in regulating hematopoiesis. Here, we investigated the role of adiponectin, a secreted molecule derived from adipocytes, in hemopoietic stem cell (HSC) function. We show that adiponectin is expressed by components of the HSC niche and its’ receptors AdipoR1 and AdipoR2 are expressed by HSCs. At a functional level, adiponectin influences HSCs by increasing their proliferation, while retaining the cells in a functionally immature state as determined by in vitro and in vivo assays. We also demonstrate that adiponectin signaling is required for optimal HSC proliferation both in vitro and in long term hemopoietic reconstitution in vivo. Finally we show that adiponectin stimulation activates p38 MAPK, and that inhibition of this pathway abrogates adiponectin’s proliferative effect on HSCs. These studies collectively identify adiponectin as a novel regulator of HSC function and suggest that it acts through a p38 dependent pathway.

All the cells present in the blood are derived from the hematopoietic stem cell (HSC). Because mature blood cells have a limited life span, HSCs must perpetuate themselves through self-renewal to maintain a functional hematopoietic compartment for the lifetime of an organism. This review focuses on studies that identify the Wnt signaling pathway as a mediator of HSC self-renewal and maintenance and analyzes its potential influence in context of the HSC niche. Recent findings The Wnt signaling pathway has emerged as a potential regulator of self-renewal for HSCs. Recent reports have demonstrated that Wnt signaling can directly promote HSC self-renewal and ability to reconstitute the hematopoietic system of lethally irradiated mice. The recent findings that osteoblasts are an important regulatory component of the HSC microenvironment, and that elements of the Wnt signaling pathway can influence osteoblast frequency, raise the possibility that Wnt signaling may influence HSC function indirectly through the niche as well. Summary In this review, the authors evaluate the experimental evidence for a direct role of Wnt signaling HSCs as well as an indirect role through its influence on the HSC niche. Defining the mechanism of action of Wnt signaling in HSC maintenance in context of the surrounding microenvironment and determining how this signal may integrate with other niche derived signals represents the next challenge in HSC biology.

Abstract 3414 Mesenchymal stromal cells (MSC) are of promising therapeutic use to suppress immunogenic responses following transplantation, and to support expansion of hematopoietic stem- and progenitors cells (HSPC) from small transplants derived for instance from cord blood. Culture-expanded MSC produce a wide variety and quantity of Wnt-proteins and the crucial role of Wnt-signaling in the hematopoietic stem cell niche is well established. However, studies addressing Wnt-signaling in MSC have (i) only focused on culture-expanded MSC and (ii) did not discriminate between phenotypically distinct subpopulations which are present in bulk cultures of expanded MSC. Recently we identified three new subpopulations of MSC in human bone marrow (BM) based on expression of CD271 and CD146: CD271brightCD146−, CD271brightCD146+, CD271−CD146+. These fractions co-express the “classical” MSC markers CD90 and CD105 and lack expression of CD45 and CD34 (Maijenburg et al, Blood 2010, 116, 2590). We and others demonstrated that the adult BM-derived CD271brightCD146− and CD271brightCD146+ cells contain all colony forming units-fibroblasts (Maijenburg et al, Blood 2010, 116, 2590; Tormin et al, Blood 2010, 116, 2594). To investigate how these primary subsets functionally compare to conventional, culture-expanded MSC, we investigated their Wnt-signature and hematopoietic support capacity. To this end, we sorted CD271brightCD146− and CD271brightCD146+ cells from human adult BM (n=3) and compared their Wnt-signatures obtained by Wnt-PCR array to the profiles from cultured MSC from the same donors. Fifteen genes were consistently differentially expressed in the two sorted uncultured subsets compared to their conventionally cultured counterparts. Expression of CCND1, WISP1 and WNT5B was strongly increased, and WNT5A was only detected in the conventionally cultured MSC. In contrast, WNT3A was exclusively expressed by sorted primary CD271brightCD146− and CD271brightCD146+ cells, that also expressed higher levels of JUN, LEF1 and WIF1. The differences in Wnt (target)-gene expression between CD271brightCD146− and CD271brightCD146+ cells were more subtle. The Wnt-receptors LRP6 and FZD7 were significantly higher expressed in CD271brightCD146+ cells, and a trend towards increased expression in the same subset was observed for CTNNB1, WNT11 and MYC. When the sorted subsets were cultured for 14 days (one passage), the differences in Wnt-related gene expression between the subsets was lost and the expanded sorted cells acquired an almost similar Wnt-signature as the MSC cultured from BM mononuclear cells from the same donors. The cultured subsets lost the expression of Wnt3a and gained the expression of Wnt5a, similar to the unsorted MSC cultured from the same donors in parallel. Despite the loss of a distinct Wnt-signature, co-culture experiments combining the sorted MSC subsets with human HSPC revealed that CD271brightCD146+ cells have a significantly increased capacity to support HSPC in short-term co-cultures (2 weeks) compared to CD271brightCD146− cells (p In conclusion, we demonstrate for the first time that primary sorted uncultured MSC subsets have a distinct Wnt-signature compared to cultured unsorted MSC and display differences in hematopoietic support. As it was recently shown that CD271brightCD146− and CD271brightCD146+ MSC localize to separate niches in vivo (Tormin et al, Blood 2011), our data indicate that the two MSC subsets are not necessarily distinct cell types and that the different Wnt-signature may be a reflection of these distinct microenvironments. Cell culturing for only one passage dramatically changed the Wnt-signature of the sorted MSC subsets, indicating that Wnt-signaling in in vitro expanded MSC does not resemble the Wnt-signature in their tissue resident counterparts in vivo. Disclosures: No relevant conflicts of interest to declare.

Abstract 2590 Mesenchymal stromal cells (MSC) in bone marrow (BM) consist of a heterogeneous population of cells. MSC orchestrate the BM microenvironment, and therefore have a pivotal role in hematopoietic support. Since MSC produce a large quantity and variety of Wnt proteins, it is likely that Wnts are involved in their hematopoietic support function Recent studies show enrichment for BM-derived MSC by sorting for CD271+ or CD146+ cells. However, it is unclear whether these markers are co-expressed with classical markers MSC markers CD73, CD105 and CD90. In addition mainly adult samples were studied, while it is known that MSC frequency declines with age. Most in-vitro studies with MSC as well as all therapeutic applications have been performed with a heterogeneous population of cultured cells. In this study we first characterized the different MSC subpopulations in BM from 81 donors (adult n=55 mean age 62 (19-82), pediatric n=22 mean age 4 (0-16) and fetal n=6, second trimester) by 6-color FACS analysis. Next we analyzed the subsets with respect to their distribution in different age groups, colony forming ability, and Wnt (target)-gene expression compared to cultured MSC. Three putative MSC subsets were identified in CD45−/dimCD34− cells from adult BM, based on expression of CD271 and/or CD146, CD105 and CD90. These populations were CD271brightCD146−CD105+CD90+, CD271brightCD146+CD105+CD90+ and CD271−CD146+CD105+CD90+, hereafter referred to CD271 single positive (sp) (0.011±0.001% from the mononuclear cell fraction (MNC)), double positive (0.003±0.001% from MNC) and CD146 sp (0.007±0.001%). The largest population of putative MSC in adult BM was CD271 sp (n=55, 55.9±24.3%). In contrast to adult BM, in pediatric BM the dominant population was double positive (n=22, 78.2±20.9%, 0.0115±0.003% MNC count), while the CD271 sp (0.0005±0.001% from MNC) and CD146 sp (0.0005±0.0001% from MNC) populations contained less than 25% of the CD271 and/or CD146+ cells. Remarkably, in fetal BM CD146 sp cells accounted for 35.5±8.8% (0.107±0.004% from MNC) of all presumptive MSC. Double positive cells represented 60.8±8.2% (0.1739±0.025% from MNC). CD271 sp cells, dominant in adults, were nearly absent (0.0115% ±0.004% from MNC)in fetal BM. The proportion of CD271 sp cells in BM was positively correlated with donor age (n=83, R2=0.41, p Disclosures: No relevant conflicts of interest to declare.

Abstract 2591 Adult hematopoietic stem cells (HSC) reside in dedicated niches in the bone marrow (BM). Within this specialized microenvironment, the various interactions of HSC with adhesion molecules on neighbouring cells and extracellular matrix (ECM) components are critical for the maintenance of the HSC population and the concomitant development of the distinct blood cell lineages. Comparative gene-expression profiling of purified HSC identified ECM proteins that are differentially expressed in homeostatic and regenerative conditions. The ECM protein βig-h3 was one of the proteins upregulated in regenerative conditions. Therefore, we characterized the role of βig-h3 in the regulation of HSC self-renewal and differentiation. A comparison between human CD34+ hematopoietic stem/progenitor cells (HSPC) isolated from BM, mobilized peripheral blood (MPB) and umbilical cord blood (UCB), revealed the highest βig-h3 expression in BM-HSPC (3.9-fold increased compared to MPB, 1.7-fold increased compared to CB), which may implie a role for βig-h3 in retaining HSC in the BM. To examine the functional relevance of βig-h3 in HSC, we first increased βig-h3 expression by transducing human HSPC with a lentiviral βig-h3-SIN-GFP expression vector or a control SIN-GFP vector. Over-expression of βig-h3 (80-fold) in HSPC decreased colony-forming-unit-granulocyte-monocyte (CFU-GM) formation from 130 (SEM=47) to 73 (SEM=19, n=3) CFU-GM per 500 plated CD34+ cells, while megakaryopoiesis was accelerated and the number of mature megakaryocytic cells increased from 16% (SEM=6%) to 30% (SEM=7%, n=4) at day 14 of culture. Ectopic expression of βig-h3 did not affect differentiation along the erythroid or granulopoietic lineage. The development of megakaryocytes at the cost of pluripotent CFU-GM suggests that βig-h3 drives differentiation. In addition, βig-h3 expression in HSPC was reduced by two different short-hairpin-RNAs (shRNA) expressed from lentiviral vectors, which resulted in decreased proliferation (from 19.6- to 5.8-fold per input cell at day 13) and increased apoptosis (from 13.5% to 25.3% at day 13) in liquid HSPC cultures, as analyzed by Annexin V staining. Similarly, knock-down of βig-h3 in various cell lines also resulted in a decreased proliferation and increased apoptosis. Knock-down of βig-h3 in primary HSPC dramatically reduced CFU-GM from 73 (SEM=8.7) to 31 (SEM=14.4, n=6) CFU-GM per 500 CD34+ cells plated, and reduced colony-forming-unit-erythrocyte (CFU-E) formation from 30 (SEM=6.5) to 9 (SEM=1.6, n=4) CFU-E per 500 CD34+ cells plated. This can be explained by increased apoptosis of βig-h3 knock-down cells. Notably, co-culture of βig-h3 knock-down HSPC with stromal feeder cells, known to express high levels of βig-h3, showed no difference compared to control HSPC in cobblestone area formation within two weeks, indicating that stromal cells can counteract apoptosis in βig-h3 knock-down cells. Remarkably, long-term-culture CFU-GM (LTC-CFU) formation of HSPC that were co-cultured with stromal cells during two weeks, was even significantly increased (1.9-fold, n=2) in βig-h3 knock-down cells, indicating that decreased endogenous levels of βig-h3 stimulates the maintenance or expansion of HSPC on stroma. In conclusion, ectopic expression of βig-h3 decreased CFU-GM in HSPC and accelerated differentiation towards megakaryocytes, suggesting that βig-h3 might drive lineage commitment of HSC. Conversely, knock-down of βig-h3 in HSPC stimulated LTC-CFU formation, indicating that decreased βig-h3 levels in HSPC maintain their undifferentiated state. In absence of stroma, however, knock-down of βig-h3 induces apoptosis, indicating βig-h3 as an essential survival factor, which expression levels regulate differentiation and maintenance of HSC. Disclosures: No relevant conflicts of interest to declare.

Abstract 3640 Poster Board III-576 Adult hematopoietic stem cells (HSC) reside in the bone marrow (BM) in so-called niches. Within this specialized microenvironment, the interactions of HSC with adhesion molecules on neighbouring cells and extracellular matrix (ECM) components are thought to be critical for the maintenance of the HSC population. Comparative gene-expression profiling of purified HSC in homeostatic and regenerative conditions allowed the identification of a set of differentially expressed ECM proteins. One of these proteins was the novel ECM protein βg-h3, which plays a role in cell-ECM interactions, by binding to type I, II and IV collagens and cellular integrins. We postulated that βig-h3 could have a role in HSC biology by being both a homeostatic and regenerative regulator of HSC self-renewal and differentiation. First we analyzed the mRNA expression in human CD34+ hematopoietic stem/progenitor cells (HSPC) isolated from BM, mobilized peripheral blood (MPB) and umbilical cord blood (UCB). The expression of βig-h3 was found to be significantly higher in BM-CD34+ cells as compared to MPB-CD34+ cells, suggesting a role for this ECM protein in retaining HSC in the BM. To determine expression of βig-h3 on the various subsets within the heterogeneous CD34+ population, the expression was compared between sorted sub-populations of BM-CD34+ cells: megakaryocyte-erythrocyte-progenitors (MEP: CD38+/CD110+/CD45RA−), common myeloid progenitors (CMP: CD38+/CD110−/CD45RA−), granulocyte-monocyte-progenitors (GMP: CD38+/CD110−/CD45RA+) and more immature CD34+/CD38− HSC. The purity of the sub-populations was analyzed by colony forming assays. These data indicate that at least the mRNA expression of βig-h3 was highest in GMPs. Analysis of different human cell types revealed that the highest βig-h3 mRNA expression is measured in monocytes, dendritic cells and mesenchymal stromal cells (MSC), while its expression in megakaryocytes and HUVEC is comparable to that in HSPC. In addition, cell surface expression of the βig-h3 protein was determined by flowcytometry. βig-h3 was found to be expressed on the cell surface of only a subpopulation of BM derived CD34+ cells (0.5%), monocytes (5%), MSCs (11%) and megakaryocytes (30%). Intracellular flowcytometry staining revealed that βig-h3 is expressed inside CD34+ cells derived from all sources. Since there is evidence in several other cell types that βig-h3 plays a role in enhancing cell adhesion and migration, adhesion experiments using CD34+ cells were performed. These experiments show a significant (p The functional relevance of the target proteins in HSC differentiation and self-renewal was studied by lentiviral mediated overexpression. We used a βig-h3-SIN-GFP vector or a control SIN-GFP vector to transduce CD34+ cells isolated from MPB or UCB and cultured them towards a megakaryocytic lineage using TPO, SCF, Flt3 and IL6. Overexpression of βig-h3 in MPB and UCB-CD34+ cells resulted in an acceleration of the megakaryopoiesis and in an increased percentage of mature megakaryocytic cells (i.e. CD41+) two weeks after transduction. In conclusion, βig-h3 is an adhesive protein for HSPCs and GMP's express significantly more βig-h3 as compared to other CD34+ subsets. Moreover, ectopic expression of βig-h3 in CD34+ cells accelerates differentiation towards megakaryocytes. These data suggest that upregulation of βig-h3 in HSCs may be a vital element driving lineage commitment of HSCs in homeostatic or regenerative conditions. Disclosures: No relevant conflicts of interest to declare.

Current methods for the cryopreservation of peripheral blood stem cell transplants (PBSCTs) have been developed empirically. Although the use of cryopreserved PBSCTs is successful and usually leads to rapid hematopoietic recovery, the freeze-thawing process is known to induce a significant amount of cell death. Furthermore, the infusion of DMSO, which is used to protect the cells against damage induced by freezing, can cause morbidity. Freezing methods may be improved by using a fundamental cryobiological approach, addressing the putative causes of cell injury during freezing and thawing. Different cell types may have different optimal cooling rates. Cooling rates above this optimum may cause ‘fast cooling damage’, e.g. by lethal intracellular ice formation, while cooling rates below the optimum may lead to ‘slow cooling damage’, These cryoinjuries relate to the osmotic changes during freezing and thawing, and the resulting fluxes of water and cryoprotectant across the cell membrane. Mathematic modeling of these osmotic events can be used to predict the optimal cooling rates for specific cell types. Woelders and Chaveiro* have developed a model that calculates the ‘compromise’ cooling rate for every subzero temperature, resulting in non-linear freezing curves. In the present study, this model was applied to predict ‘optimal’ freezing curves for PBSCTs with 10% and 5% DMSO, respectively, using values for the membrane permeability coefficients and related parameters as published earlier for cord blood HPCs. These predicted curves were tested empirically and compared to the presently used standard linear freezing curve. CD34+ selected and unselected PBSCs were cryopreserved using the standard or the new freezing curves. Post-thaw quality was evaluated by cell viability, CFU-GM formation and megakaryocyte outgrowth. With 10% DMSO, the use of the predicted optimal freezing curve compared to the currently used freezing curve resulted in increased post-thaw viability of CD34+ cells (mean±SEM; 78.4%±6.6% versus 72.0%±6.1% for unselected CD34+ cells and 92.0%±0.6% versus 83.9%±2.5% [p

In addition to hematopoietic cells, the receptor for thrombopoietin (Tpo), c-Mpl, is expressed on human umbilical vein endothelial cells (HUVEC). In this study, we demonstrated that Mpl mRNA and protein can also be detected in immortalized human bone marrow endothelial cells (HBMEC). Furthermore, it was investigated whether Tpo affects endothelial monolayer integrity by either cell spreading or cell-cell contacts. By continuously monitoring the electrical resistance of freshly plated HUVEC or HBMEC with ECIS (electric cell-substrate impedance sensing), we observed a significant increase in the electrical resistance when endothelial cells were treated with 100 ng/ml of Tpo just prior to seeding, as shown in the figure below. This increase in electrical resistance was not due to increased cell adhesion or proliferation of the endothelial cells. However, a significant increase in initial spreading could be detected. Besides its effect on spreading, Tpo increased the electrical resistance of a confluent endothelial monolayer when compared to untreated monolayers. This indicates that Tpo promotes the formation of endothelial cell-cell contacts. Indeed, a decrease in immunostaining levels of phosphotyrosine could be observed in monolayers treated with Tpo. Also the junctional protein VE-cadherin showed diminished co-localization with phosphotyrosine staining when cells were treated with Tpo. The small GTPase Rap1 plays an important role in the formation of endothelial cell-cell contacts. In a pull-down experiment, using RalGDS as bait for active Rap1, we could demonstrate that Tpo transiently activates Rap1 in HUVEC. Thus, these results may put Tpo forward as a regulator of endothelial cell-cell contacts, possibly after vascular damage and subsequent release of Tpo by activated platelets. To our knowledge, Tpo is the first cytokine exerting this effect on endothelial cells. Download : Download high-res image (72KB) Download : Download full-size image Figure

Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare disorder that presents with severe thrombocytopenia and absence of megakaryocytes in the bone marrow. The disease may develop into bone marrow aplasia. In vitro, CD34-positive hematopoietic progenitor cells from CAMT patients did not show any megakaryocyte formation in a Tpo-driven expansion culture. We and others found genetic defects in the gene encoding the Tpo receptor, c-mpl (Van den Oudenrijn et al., Br J Haematol.2002, 117: 390–398 and Ballmaier et al., Ann N Y Acad Sci.2003, 996: 17–25). In our patients, we found four mutations that predicted amino-acid substitutions, of which three in the extracellular domain; Arg102Pro, Pro136His and Arg257Cys, and one in the intracellular signaling domain (Pro635Leu), which may result in either defective Tpo-binding and/or signaling. To investigate this, we transfected full-length Mpl (wt and mutants) into the erythroleukemic cell line K562 and truncated Mpl (encompassing the extracellular domain; wt and mutants) into Baby Hamster Kidney (BHK) cells. In the K562 cells, the mRNA level (RQ-PCR) of the Pro136His mutant was severely decreased compared to the wt transfectant, while the mRNA level of the other mutants was comparable to that of wt. On Western blot, wt Mpl migrated as two, presumably differently glycosylated, bands of 75 kD and 72 kD. The mutants showed an altered migration pattern, which might result from differences in glycosylation. With the Pro635Leu mutant lower signals were obtained when equal amounts of total protein were loaded. Since the Mpl mRNA level was comparable to that of wt, this suggests a higher level of protein degradation. Upon transfection of the Arg102Pro and the Arg257Cys mutants in BHK cells, we observed that these mutants did not gain endo-H resistency, which suggests an aberrant processing of these mutant Mpls through the Golgi apparatus and retention in the ER. However, in cell fractionation experiments with surface-biotinylated K562 cells, biotinylated wt Mpl and mutant Mpl (except Pro136His) could be detected. Apparently, in K562 cells, the amino-acid substitutions do not impair membrane expression completely. To examine whether the mutant receptors were still able to signal after Tpo incubation, K562 cells were serum-starved and subsequently stimulated with 50 ng/ml rhTpo for 5 to 30 minutes. All mutants, including Pro136His, showed increased ERK phosphorylation after 5 minutes. To summarize, the Pro136His mutant is hardly expressed in the K562 expression model, presumably because of instability of the mRNA, but is still able to induce signaling. In contrast to the results obtained in the BHK model, the Arg102Pro and Arg257Cys mutants, showed cell-surface expression in the K562 cell line. The obtained cell-surface expression in the K562 model may have been significantly increased compared to the in vivo situation on hematopoietic stem cells, because of artificially induced efficient expression. Finally, with a super-physiological concentration of rhTpo, we obtained evidence that all Mpl mutants were able to signal upon Tpo binding. Whether impaired signaling by the Mpl mutants in the presence of physiological levels of Tpo may contribute to the development of CAMT, will be investigated.

In an effort to identify the molecular changes during hematopoietic stem cell (HSC) mobilization we have analyzed genome wide changes in gene expression in HSCs in response to the chemotherapeutic agent cyclophosphamide (Cy) and the growth factor granulocyte colony stimulating factor (G). Cy/G treatment leads to an initial loss of proliferating precursors, followed by a rapid expansion of stem and progenitor cells and their migration to the peripheral blood. While this approach has been capitalized on for harvesting hematopoietic stem cells in clinical therapy, the molecular regulation of this process remains less well understood. To analyze changes in gene expression that occur in vivo as HSCs proliferate and prepare to migrate out of the bone marrow, we enriched for HSC fractions by isolating cells that express c-kit (K), Sca-1(S) and low levels of Thy1.1(T) but do not express lineage (L) markers (KTLS) from untreated mice and mice treated with Cy, G or Cy/G. The gene expression profile of these cells was examined by microarray analysis, relative to HSCs isolated from untreated mice. Interestingly, among the genes upregulated by Cy/G, a large majority (>60%) were a consequence of synergistic activity between Cy and G, while the rest of the upregulated genes could be attributed to activation by Cy or G alone. To test whether this screen allowed identification of novel candidates that regulate HSC function we analyzed the role of transforming growth factor beta induced gene (Big-h3), a gene highly upregulated after Cy/G treatment. Big-h3 is an extracellular matrix protein that mediates the adhesion and migration of various cell types, however its role in HSCs is unknown. Our experiments indicate that overexpression of Big-h3 in HSCs leads to enhanced adhesion to fibronectin as well as upregulation of specific integrins. These experiments suggest that changes in adhesion are an integral component of HSC mobilization, and that Big-h3 is one regulator of such processes. Further functional analysis of other candidates will likely lead to identification of other novel regulators of HSC development and mobilization.