Production Of Soluble Factors Research Articles

Comparative study of the production of soluble factors in human placenta-derived mesenchymal stromal/stem cells grown in adherent conditions or as aggregates in a catheter-like device

Different approaches have been studied in both preclinical and clinical settings to develop cell-based therapies and/or engineered cell-based therapies to better integrate grafts with the host. In these techniques, much attention is addressed to the use of adult stem cells such as mesenchymal stem cells (MSCs), but identifying and obtaining sufficient numbers of therapeutic cells, and the right route of administration, is often a challenge.In this study, we tested the feasibility of encapsulating human amnion-derived MSCs (hAMSCs) in a semipermeable and biocompatible fiber as a new approach for regenerative medicine. Our data showed that hAMSCs aggregated in the device constitutes an effective system for enhancing, or at least for maintaining, the paracrine activity of these cells in order to better promote tissue regeneration in an immune isolated state. In our new experimental approach, the hAMSCs retained their therapeutic potential, as shown by both the production of specific immunomodulatory/angiogenic factors and immunomodulatory and angiogenic ability observed in vitro.Unlike cell infusion methods, the use of encapsulated-cells leads to minimally invasive approaches, avoiding a direct interaction with the host. Therefore, the potentiality of an allograft or xenograft without the need for immunosuppression, and the lack of tumorigenesis is very intriguing.

Micro-vibrations at 30 Hz on bone cells cultivated in vitro produce soluble factors for osteoclast inhibition and osteoblast activity

ObjetiveIt has been claimed that micro-pulse vibration can accelerate the rate of tooth movement during orthodontic treatment; however, the underlying cellular mechanism has yet to be elucidated. The purpose of this study was to understand the mechanisms underlying tooth movement acceleration by measuring alterations in a panel of intercellular signalling molecules and markers of osteoblast/osteoclast function following micro-pulse vibration for 20 min at 30 Hz. DesignPrimary BALB/c mouse calvarial osteoblasts were cultivatedin vitro and subjected to micro-pulse vibration (0.25 N; 30 Hz) with the AcceleDent® Aura appliance for 20 min and assayed for IL-4, IL-13, IL-17, OPG, soluble RANKL and TGF-β protein by ELISA; for PCNA in osteoblasts and caspase 3/7 in osteoclasts by immunohistochemistry; for IL-4, IL-13, and Il-17 in osteoclasts by ELISA; and for cathepsin K by flow cytometry. ResultsAfter micro-pulse vibration, the murine osteoblast culture supernatant showed increased IL-4, IL-13, IL-17, OPG and TGF-β levels and decreased RANKL levels; PCNA in osteoblasts and caspase 3/7 in osteoclasts were also upregulated. The osteoclast culture supernatant had increased levels of IL-4, IL-13 and IL-17, and cathepsin K was upregulated in the treatment group compared with the control group. ConclusionsMicro-pulse vibration promotes the production of soluble factors that inhibit osteoclasts, promote apoptosis and activate osteoblasts in vitro, which could increase bone mineral density. Further studies should be conducted in order to understand the biological mechanism of how micro-vibration might influence tooth movement during orthodontic treatment.

High-intensity ultrasound: A novel technology for the development of probiotic and prebiotic dairy products.

High-intensity ultrasound (HIUS) can be used as a mild-preservation technology in dairy products, due to its ability to inactivate pathogenic microorganisms and enzymes. In addition, it can result in physical and chemical alterations in the products and has impact on the probiotic viability and metabolic activity. This review provides an overview of the effects of HIUS on dairy products manufactured with probiotics and prebiotics. Furthermore, it presents perspectives of HIUS application on paraprobiotics and postbiotics products. HIUS has been proven to be a potential technology and its application to fermented dairy products can result in shorter processing time, increased probiotic viability, and products with low lactose content, higher oligosaccharides concentration, less undesirable taste (lower propionic and acetic acids content) and reduced ingredients (no need of prebiotic addition or β-galactosidase inclusion). In cheeses, HIUS can reduce the ripening time and accelerate proteolysis, resulting in products with better sensory, textural and nutritional (bioactive peptides) characteristics. Furthermore, it can change the prebiotic structure, facilitating the access for the probiotics. The impact of the HIUS is highly dependent on the process parameters (frequency, power, processing time, pulse mode and duration), type of probiotic culture and food composition. Therefore, HIUS process parameters must be precisely quantified and controlled. The HIUS can also be applied to the inactivation of probiotic cultures and development of paraprobiotic products or to the improvement in the production of soluble factors (postbiotics) with health effects. Further researches should be conducted to evaluate the efficiency of this methodology in the cases of paraprobiotic and postbiotic products.

E-105 Mesenchymal stromal cells: Mediators of inflammation in cancer

Cancer-associated fibroblasts (CAF) have been identified in the tumor microenvironment (TME) of many cancers. CAF represent a heterogeneous population of cells that can have pro-tumorigenic and anti-tumorigenic functions. We have isolated from primary human neuroblastoma tumors, a population of CAF that share phenotypic and functional characteristics of bone marrow-derived mesenchymal stromal cells (MSC). These cells are CD73+, CD90+, CD105+, CD34−, CD45−, CD14−, CD19−, CD31−, and VEGFR2−, and retain the ability to differentiate into osteoblasts, chondrocytes and adipocytes, a feature of MSC. However, we show that these cells also express fibroblast activation protein alpha (FAP/DPPIV), fibroblast specific protein 1 (FSP1/S1004A) and alpha smooth muscle actin (SMA), markers of cancer-associated fibroblasts. When in the presence of tumor cells, these MSC/CAF stimulate the production of multiple pro-tumorigenic cytokines and chemokines including interleukin (IL)-6, IL-8, CCL2/monocyte chemoattractant protein 1, CXCL-12/stromal-derived factor 1 (SDF1), CXCL1, vascular endothelial growth factor (VEGF-A), plasminogen activator inhibitor 1 (PAI-1) and transforming growth factor (TGF) β. The production of these cytokines/chemokines by MSC/CAF is stimulated by the production of soluble factors by tumor cells including galectin-3 binding protein and extracellular vesicles. In addition to stimulating angiogenesis, the recruitment and M2 polarization of macrophages, and to suppress immune cells, MSC/CAF have a direct effect on tumor cells by stimulating tumor cell growth, survival and resistance to chemotherapy in an ERK1/2 and STAT3-dependent manner. We then show that targeting ERK1/2 and STAT3 or targeting MSC in combination with chemotherapy or immunotherapy in mice orthotopically implanted with human neuroblastoma tumors, inhibits tumor growth and extends survival. In contrast, targeting a single cytokine like IL-6 has no significant effect. These data thus identify a new type of CAF sharing characteristics of bone marrow-derived MSCs that contribute to a pro-tumorigenic inflammatory reaction in the TME and provide preclinical evidence that targeting these cells or the pathways they activated in tumor cells is a valuable therapeutic approach.

Phorbol esters induce PLVAP expression via VEGF and additional secreted molecules in MEK1-dependent and p38, JNK and PI3K/Akt-independent manner.

Endothelial diaphragms are subcellular structures critical for mammalian survival with poorly understood biogenesis. Plasmalemma vesicle associated protein (PLVAP) is the only known diaphragm component and is necessary for diaphragm formation. Very little is known about PLVAP regulation. Phorbol esters (PMA) are known to induce de novo PLVAP expression and diaphragm formation. We show that this induction relies on the de novo production of soluble factors that will act in an autocrine manner to induce PLVAP transcription and protein expression. We identified vascular endothelial growth factor‐A (VEGF‐A) signalling through VEGFR2 as a necessary but not sufficient downstream event as VEGF‐A inhibition with antibodies and siRNA or pharmacological inhibition of VEGFR2 only partially inhibit PLVAP upregulation. In terms of downstream pathways, inhibition of MEK1/Erk1/2 MAP kinase blocked PLVAP upregulation, whereas inhibition of p38 and JNK MAP kinases or PI3K and Akt had no effect on PMA‐induced PLVAP expression. In conclusion, we show that VEGF‐A along with other secreted proteins act synergistically to up‐regulate PLVAP in MEK1/Erk1/2 dependent manner, bringing us one step further into understanding the genesis of the essential structures that are endothelial diaphragms.

Phenotyping of circulating extracellular vesicles (EVs) in obesity identifies large EVs as functional conveyors of Macrophage Migration Inhibitory Factor

ObjectiveObesity-associated metabolic dysfunctions are linked to dysregulated production of adipokines. Accumulating evidence suggests a role for fat-derived extracellular vesicles (EVs) in obesity-metabolic disturbances. Since EVs convey numerous proteins we aimed to evaluate their contribution in adipokine secretion. MethodsPlasma collected from metabolic syndrome patients were used to isolate EV subtypes, namely microvesicles (MVs) and exosomes (EXOs). Numerous soluble factor concentrations were measured successively on total, MV- and EXO-depleted plasma by multiplexed immunoassays. ResultsCirculating MVs and EXOs were significantly increased with BMI, supporting a role of EVs as metabolic relays in obesity. Obesity was associated with dysregulated soluble factor production. Sequential depletion of plasma MVs and EXOs did not modify plasma levels of these molecules, with the exception of Macrophage Migration Inhibitory Factor (MIF). Half of plasma MIF circulated within MVs, and this MV secretory pathway was conserved over different MIF-producing cells. Although MV-associated MIF triggered rapid ERK1/2 activation in macrophages, these functional MV-MIF effects specifically relied on MIF tautomerase activity. ConclusionOur results emphasize the importance of reconsidering MIF-metabolic actions with regard to its MV-associated form and opening new EV-based strategies for therapeutic MIF approaches.

USP18 (UBP43) Abrogates p21-Mediated Inhibition of HIV-1.

The host intrinsic innate immune system drives antiviral defenses and viral restriction, which includes the production of soluble factors, such as type I and III interferon (IFN), and activation of restriction factors, including SAMHD1, a deoxynucleoside triphosphohydrolase. Interferon-stimulated gene 15 (ISG15)-specific ubiquitin-like protease 43 (USP18) abrogates IFN signaling pathways. The cyclin-dependent kinase inhibitor p21 (CIP1/WAF1), which is involved in the differentiation and maturation of monocytes, inhibits human immunodeficiency virus type 1 (HIV-1) in macrophages and dendritic cells. p21 inhibition of HIV-1 replication is thought to occur at the reverse transcription step, likely by suppressing cellular deoxynucleoside triphosphate (dNTP) biosynthesis and increasing the amount of antivirally active form of SAMHD1. SAMHD1 strongly inhibits HIV-1 replication in myeloid and resting CD4+ T cells. Here, we studied how USP18 influences HIV-1 replication in human myeloid THP-1 cells. We found that USP18 has the novel ability to inhibit the antiviral function of p21 in differentiated THP-1 cells. USP18 enhanced reverse transcription of HIV-1 by downregulating p21 expression and upregulating intracellular dNTP levels. p21 downregulation by USP18 was associated with the active form of SAMHD1, phosphorylated at T592. USP18 formed a complex with the E3 ubiquitin ligase recognition factor SKP2 (S-phase kinase associated protein 2) and SAMHD1. CRISPR-Cas9 knockout of USP18 increased p21 protein expression and blocked HIV-1 replication. Overall, we propose USP18 as a regulator of p21 antiviral function in differentiated myeloid THP-1 cells.IMPORTANCE Macrophages and dendritic cells are usually the first point of contact with pathogens, including lentiviruses. Host restriction factors, including SAMHD1, mediate the innate immune response against these viruses. However, HIV-1 has evolved to circumvent the innate immune response and establishes disseminated infection. The cyclin-dependent kinase inhibitor p21, which is involved in differentiation and maturation of monocytes, blocks HIV-1 replication at the reverse transcription step. p21 is thought to suppress key enzymes involved in dNTP biosynthesis and activates SAMHD1 antiviral function. We report here that the human USP18 protein is a novel factor potentially contributing to HIV replication by blocking the antiviral function of p21 in differentiated human myeloid cells. USP18 downregulates p21 protein expression, which correlates with upregulated intracellular dNTP levels and the antiviral inactive form of SAMHD1. Depletion of USP18 stabilizes p21 protein expression, which correlates with dephosphorylated SAMHD1 and a block to HIV-1 replication.

Adaptive Regulation of Osteopontin Production by Dendritic Cells Through the Bidirectional Interaction With Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSCs) exert immunosuppressive effects on immune cells including dendritic cells (DCs). However, many details of the bidirectional interaction of MSCs with DCs are still unsolved and information on key molecules by which DCs can modulate MSC functions is limited. Here, we report that osteopontin (OPN), a cytokine involved in homeostatic and pathophysiologic responses, is constitutively expressed by DCs and regulated in the DC/MSC cocultures depending on the activation state of MSCs. Resting MSCs promoted OPN production, whereas the production of OPN was suppressed when MSCs were activated by proinflammatory cytokines (i.e., TNF-α, IL-6, and IL-1β). OPN induction required cell-to-cell contact, mediated at least in part, by β1 integrin (CD29). Conversely, activated MSCs inhibited the release of OPN via the production of soluble factors with a major role played by Prostaglandin E2 (PGE2). Accordingly, pretreatment with indomethacin significantly abrogated the MSC-mediated suppression of OPN while the direct addition of exogenous PGE2 inhibited OPN production by DCs. Furthermore, DC-conditioned medium promoted osteogenic differentiation of MSCs with a concomitant inhibition of adipogenesis. These effects were paralleled by the repression of the adipogenic markers PPARγ, adiponectin, and FABP4, and induction of the osteogenic markers alkaline phosphatase, RUNX2, and of the bone-anabolic chemokine CCL5. Notably, blocking OPN activity with RGD peptides or with an antibody against CD29, one of the OPN receptors, prevented the effects of DC-conditioned medium on MSC differentiation and CCL5 induction. Because MSCs have a key role in maintenance of bone marrow (BM) hematopoietic stem cell niche through reciprocal regulation with immune cells, we investigated the possible MSC/DC interaction in human BM by immunohistochemistry. Although DCs (CD1c+) are a small percentage of BM cells, we demonstrated colocalization of CD271+ MSCs with CD1c+ DCs in normal and myelodysplastic BM. OPN reactivity was observed in occasional CD1c+ cells in the proximity of CD271+ MSCs. Altogether, these results candidate OPN as a signal modulated by MSCs according to their activation status and involved in DC regulation of MSC differentiation.

Chondrocytes Contribute to Alphaviral Disease Pathogenesis as a Source of Virus Replication and Soluble Factor Production.

Arthritogenic alphavirus infections often result in debilitating musculoskeletal disorders that affect the joints, muscle, and bone. In order to evaluate the infection profile of primary human skeletal muscle and chondrocyte cells to Ross River virus (RRV) in vitro, cells were infected at a multiplicity of infection (MOI) of 1 over a period of two days. Viral titers were determined by plaque assay and cytokine expression by Bio-Plex® assays using the supernatants harvested. Gene expression studies were conducted using total RNA isolated from cells. Firstly, we show that RRV RNA is detected in chondrocytes from infected mice in vivo. Both human primary skeletal muscle and chondrocyte cells are able to support productive RRV infection in vitro. We also report the production of soluble host factors including the upregulation of heparanase (HPSE) and inflammatory host factors such as interleukin-6 (IL-6), monocyte chemoattractant protein 1 (MCP-1), RANTES (regulated on activation, normal T cell expressed and secreted), interferon gamma (IFN-γ), and tumor necrosis factor alpha (TNF-α), which are also present during clinical disease in humans. Our study is the first to demonstrate that human chondrocyte cells are permissive to RRV infection, support the production of infectious virus, and produce soluble factors including HPSE, which may contribute to joint degradation and the pathogenesis of disease.

Regulatory Mechanism of The Induction of Regulatory T Cells through Growth Factors Released by Human Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) undergo self-renewal and differentiation into multiple lineages. They exert anti-inflammatory and immunomodulatory effects via either cell-cell contact or via the production of soluble factors. Various anti-inflammatory and immunomodulatory mechanisms have been identified using experimental murine MSCs. The most representative mechanism is the induction of FOXP3+ regulatory T cells (Treg cells) via the production of transforming growth factor (TGF)-β, which has also been reported in human MSCs. Recent studies have demonstrated that insulin-like growth factor (IGF) is also involved in the induction of FOXP3+ Treg cells. We previously demonstrated that the induction of FOXP3+ Treg cells by IGF is suppressed by the expression of IGF-binding protein-4 (IGFBP-4), which is an inhibitor of IGF. Because human MSCs produce both IGFs and IGFBP4, they may be considered to maintain immunological homeostasis by positive regulation of Treg cells through the production of growth factors as well as by a negative regulatory mechanism. Although human MSCs have already been applied in regenerative therapy and autoimmune disease treatment, control of their positive and negative regulatory mechanisms is expected to lead to more efficient clinical applications.

Mesenchymal stem cell-derived factors: Immuno-modulatory effects and therapeutic potential.

Stem cell-based therapy is considered to be a new hope in transplantation medicine. Among stem cells, mesenchymal stem cells (MSCs) are, due to their differentiation and immuno-modulatory characteristics, the most commonly used as therapeutic agents in the treatment of immune-mediated diseases. MSCs migrate to the site of inflammation and modulate immune response. The capacity of MSC to alter phenotype and function of immune cells are largely due to the production of soluble factors which expression varies depending on the pathologic condition to which MSCs are exposed. Under inflammatory conditions, MSCs-derived factors suppress both innate and adaptive immunity by attenuating maturation and capacity for antigen presentation of dendritic cells, by inducing polarization of macrophages towards alternative phenotype, by inhibiting activation and proliferation of T and B lymphocytes and by reducing cytotoxicity of NK and NKT cells. In this review, we emphasized current findings regarding immuno-modulatory effects of MSC-derived factors and emphasize their potential in the therapy of immune-mediated diseases. © 2017 BioFactors, 43(5):633-644, 2017.

Cytokine Production in Mixed Cultures of Mesenchymal Stromal Cells from Wharton's Jelly and Peripheral Blood Lymphocytes.

We compared the production of 19 humoral factors in mixed cultures of mesenchymal stromal cells from Wharton's jelly and allogenic peripheral blood lymphocytes. For evaluation of the specificity of immunosuppressive activity of mesenchymal stromal cells, comparative analysis of the production of these humoral factors in mixed cultures of lymphocytes and epithelial BxPC-3 cells was conducted. The production of soluble factors in both mono- and mixed cultures significantly correlated (p<0.05). The maximum production was found for proinflammatory chemokine IP-10 and IFN-γ and anti-inflammatory cytokine IL-10. The major difference of mesenchymal stromal cells from epithelial BxPC-3 cells was 7-fold higher production of IL-10, which can explain the immunosuppressive effect of mesenchymal stromal cells.

Pseudomonas aeruginosa outcompetes other bacteria in the manifestation and maintenance of a biofilm in polyvinylchloride tubing as used in dental devices.

In a PVC tube as a model system for dental devices, Pseudomonas aeruginosa outcompetes Staphylococcus aureus and Klebsiella pneumoniae for the biofilm formation. P. aeruginosa has advantage over the other strains due to higher tolerance for low-nutrient situations or direct killing by the production of soluble factors like pyocyanin.

Proinflammatory T cells and IL-17 stimulate osteoblast differentiation

The local immune response is important to consider when the aim is to improve bone regeneration. Recently T lymphocytes and their associated cytokines have been identified as regulators in fracture callus formation, but it is not known whether T cells affect bone progenitor cells directly. The goal of this in vitro study was to investigate the role of different T cell subsets and their secreted factors on the osteogenic differentiation of human mesenchymal stem cells (MSCs). Significant increases in the alkaline phosphatase activity and the subsequent matrix mineralization by MSCs were found after their exposure to activated T cells or activated T cell-derived conditioned medium. Blocking IFN-γ in the conditioned medium abolished its pro-osteogenic effect, while blocking TGF-β further enhanced osteogenesis. The relative contribution of an anti- or proinflammatory T cell phenotype in MSC osteogenic differentiation was studied next. Enrichment of the fraction of anti-inflammatory regulatory T cells had no beneficial osteogenic effect. In contrast, soluble factors derived from enriched T helper 17 cells upregulated the expression of osteogenic markers by MSCs. IL-17A, and IL-17F, their main proinflammatory cytokines, similarly exhibited strong osteogenic effects when exposed directly to MSCs. IL-17A in particular showed a synergistic action together with bone morphogenetic protein 2. These results indicate that individual T cell subsets, following their activation, affect osteoblast maturation in a different manner through the production of soluble factors. From all T cells, the proinflammatory T cells, including the T helper 17 cells, are most stimulatory for osteogenesis.

Pre-eclampsia Protectors and Promoters

A new study identifies genes linked to the risk for preeclampsia in a population from sub-Saharan Africa. These genes encode for human leukocyte antigen (HLA) ligands on trophoblasts and receptors that recognize these ligands, which are present on maternal Natural Killer (NK) cells [1]. Trophoblast invasion of the uterine tissue leads to the remodeling of uterine arteries into large vessels, thereby tapping the mother’s blood supply. This is a carefully equilibrated event. Too aggressive of an invasion can result in outsized babies, exposure to excess nutrients and oxygen in utero, and placental overgrowth conditions that can lead to hemorrhage. Poor invasion can result in such conditions as fetal growth restriction and pre-eclampsia. Maternal NK cells modulate the activity of trophoblasts, determining the extent of invasion in humans. This modulation occurs via HLA-C ligands on trophoblast cells, which recognize a key family of receptors on NK cells: killer-cell immunoglobulin-like receptors (KIRs). In previous studies, Ashley Moffett and colleagues found that genetic risk for pre-eclampsia, and some related conditions such as fetal growth restriction, is linked to various genetic variants at the KIR and HLA-C loci. But their studies, conducted primarily in European populations, limit their analysis. In the new study, the researchers turned to women of sub-Saharan African ancestry, who have elevated rates of pre-eclampsia and also a high diversity of KIR genotypes, and found some parallels and some differences with European women. Moffett and colleagues studied pregnant women at a hospital in Kampala, Uganda: 251 with pre-eclampsia and 483 without the condition. As with Europeans, they found elevated risk for pre-eclampsia in women homozygous for one of two major haplotypes, the KIR A haplotype. This risk was even higher if the fetus also had particular HLA-C alleles, which encode C2 epitopes (all HLA-C alleles encode either a C1 or C2 epitope). The researchers also identified three KIR genes that seemed to protect against pre-eclampsia in the women in Kampala—alleles that are rare in Europeans. A KIR gene known to be protective in Europeans was found at only low frequency in the women from Kampala. The study highlights the need for global genetic studies to assess pre-eclampsia risk—curiously, distinct human populations seem to have evolved to have distinct mixes of favorable KIR-HLA-C combinations. Future research should flesh out the biological mechanisms. One study, for instance, showed that favorable KIR-HLA-C combinations result in increased production of soluble factors that promote trophoblast invasion.

Mesenchymal Stromal Cells Affect Disease Outcomes via Macrophage Polarization.

Mesenchymal stromal cells (MSCs) are multipotent and self-renewable cells that reside in almost all postnatal tissues. In recent years, many studies have reported the effect of MSCs on the innate and adaptive immune systems. MSCs regulate the proliferation, activation, and effector function of T lymphocytes, professional antigen presenting cells (dendritic cells, macrophages, and B lymphocytes), and NK cells via direct cell-to-cell contact or production of soluble factors including indoleamine 2,3-dioxygenase, prostaglandin E2, tumor necrosis factor-α stimulated gene/protein 6, nitric oxide, and IL-10. MSCs are also able to reprogram macrophages from a proinflammatory M1 phenotype toward an anti-inflammatory M2 phenotype capable of regulating immune response. Because of their capacity for differentiation and immunomodulation, MSCs have been used in many preclinical and clinical studies as possible new therapeutic agents for the treatment of autoimmune, degenerative, and inflammatory diseases. In this review, we discuss the central role of MSCs in macrophage polarization and outcomes of diseases such as wound healing, brain/spinal cord injuries, and diseases of heart, lung, and kidney in animal models.

Bone Marrow Mesenchymal Stem and Progenitor Cells Are Altered in Sipa1 Deficient Mice before Initiation of Myeloproliferative Neoplasm

Hematopoiesis is tightly regulated by bone marrow (BM) stromal cells through the production of soluble factors, matrix proteins, and intracellular signals initiated by cell-cell adhesive interactions. Despite the involvement of mesenchymal stem and progenitor cells in the maintenance of normal hematopoiesis and the development of hematological disorders have been reported (Mendez-Ferrer et al., 2010, Nature)(Raaijmakers et al., 2010, Nature), little is known how the different BM stromal cell fractions contribute to the processes.Signal-induced proliferation-associated gene 1 (Sipa1), a principal Rap1 GTPase activating protein, is expressed in hematopoietic cells (Ishida et al., 2003, Cancer Cell). Deletion of Sipa1 results in age-dependent development of myeloproliferative neoplasm (MPN) in mice, resembling human chronic myelogenous leukemia (Ishida et al., 2003, Cancer Cell). However, it is not known whether the deletion of Sipa1 affects BM cellular niche components, which could lead to the development of the MPN. Knowledge of this is important for better understanding the role of BM stromal cells in the initiation and progression of MPN.In the present study, we first analyzed Sipa1 expression by multicolor fluorescent activated cell sorting (FACS) using hematopoietic cell marker CD45 and TER119, mesenchymal stem cell (MSC) associated marker CD51 and SCA1, and endothelial cell marker CD31. We found that Sipa1 gene was also expressed in different BM stromal cell subsets including MSCs (CD45-TER119-CD31-CD44-CD51+SCA1+), more differentiated mesenchymal progenitor cells (CD45-TER119-CD31-CD44-CD51+SCA1-), and endothelial cells (CD45-TER119-CD31+). Among all the cell types, Sipa1 expression seemed to be higher in MSCs and endothelial cells than other cell types. We next studied whether the gene deficiency led to the alteration of BM stromal cell composition and function by phenotypically, functionally, and molecularly analyzing BM stromal cell subsets in young (<3 months) Sipa1-/- mice where hematopoiesis remained normal. Phenotypic multicolor FACS analysis showed significant increase in percentages and numbers of BM stromal cell (CD45-TER119-) subsets including endothelial cells, MSCs, and the mesenchymal progenitor cells in Sipa1-/- mice compared to age and sex-matched Sipa1+/+ mice (P<0.05). The alteration in the frequency of BM MSCs in the Sipa1-/- mice was further confirmed by colony-forming unit-fibroblast assay (CFU-F) on unfractionated BM mononuclear cells (P=0.0302) and FACS-sorted MSCs (P=0.0066). It is important to note that most (75% on average) of CFU-Fs generated from FACS-sorted Sipa1-/- stromal cells contained simultaneously differentiated adipocytes whereas around 57% of CFU-Fs from Sipa1+/+ stromal cells did (P=0.0184). In keeping with this, in vitro differentiation assays revealed greater adipocyte differentiation potential of Sipa1-/- MSCs. Similarly, an increase of adipocytes associated gene FABP4 expression was determined by Q-PCR. Our preliminary Q-PCR data indicated differential expression of Kit Ligand and Angipoietinlike 1 in the Sipa1-/- MSCs and the CD51+SCA1- cells compared to that in the Sipa1+/+ cells. Global gene expression analysis by RNA sequencing and more multilineage differentiation assays on FACS-sorted different stromal cell subsets of Sipa1-/- and Sipa1+/+ BM are currently ongoing.Taken together, our data suggested the BM stromal cell composition and function in the Sipa1-/- mice have already been altered before the initiation of MPN. Transplantations of normal hematopoietic stem cells into Sipa1 deficient BM have been initiated for examining if the Sipa1-/- BM niches play an instructive role in the initiation of MPN. DisclosuresNo relevant conflicts of interest to declare.

Brain endothelial cells increase the proliferation of Plasmodium falciparum through production of soluble factors

We here describe the novel finding that brain endothelial cells in vitro can stimulate the growth of Plasmodium falciparum through the production of low molecular weight growth factors. By using a conditioned medium approach, we show that the brain endothelial cells continued to release these factors over time. If this mirrors the in vivo situation, these growth factors potentially would provide an advantage, in terms of enhanced growth, for sequestered parasitised red blood cells in the brain microvasculature. We observed this phenomenon with brain endothelial cells from several sources as well as a second P. falciparum strain. The characteristics of the growth factors included: <3kDa molecular weight, heat stable, and in part chloroform soluble. Future efforts should be directed at identifying these growth factors, since blocking their production or actions might be of benefit for reducing parasite load and, hence, malaria pathology.

Anaplastic large cell lymphoma-propagating cells are detectable by side population analysis and possess an expression profile reflective of a primitive origin.

Cancer stem cells or tumour-propagating cells (TPCs) have been identified for a number of cancers, but data pertaining to their existence in lymphoma so far remain elusive. We show for the first time that a small subset of cells purified from human anaplastic lymphoma kinase (ALK)-positive and -negative, anaplastic large cell lymphoma cell lines and primary patient tumours using the side population (SP) technique have serial tumour-propagating capacity both in vitro and in vivo; they give rise to both themselves and the bulk tumour population as well as supporting growth of the latter through the production of soluble factors. In vivo serial dilution assays utilising a variety of model systems inclusive of human cell lines, primary human tumours and nucleophosmin (NPM)-ALK-induced murine tumours demonstrate the TPC frequency to vary from as many as 1/54 to 1/1336 tumour cells. In addition, the SP cells express higher levels of pluripotency-associated transcription factors and are enriched for a gene expression profile consistent with early thymic progenitors. Finally, our data show that the SP cells express higher levels of the NPM-ALK oncogene and are sensitive to an ALK inhibitor.

Tumor microenvironment and therapeutic resistance process

Over the last decade, it has become clear that cancer is not just a disease of the genes, and that the tumor microenvironment (TME) plays an important role in cancer progression. Interactions between tumor cells and the TME, made of the extracellular matrix (ECM) and of non-transformed cells (designated here as stromal cells), promote cancer cell survival and drug resistance. Many of the mechanisms involved are known and are either contact-dependent or contact-independent. Contact between tumor cells and the ECM or stromal cells as well as the production of soluble factors and microvesicles all contribute. The bone marrow plays a special role in environment-mediated drug resistance as it is not only a sanctuary protecting tumor cells from cytotoxic drugs, but also a source of many stromal cells that colonize primary tumors and contribute to the pre-metastatic niche. As our understanding of the mechanisms by which the tumor microenvironment promotes therapeutic resistance progresses, clinical trials testing agents that disrupt the interaction between tumor cells and the stroma have been initiated. This new avenue of therapy is promising.