Bone Marrow Cell Populations Research Articles

Abstract 046: Obesity-induced Oxidative Stress in Hematopoietic Stem and Progenitor Cells Allows a Sustained Myelopoiesis and Persistent Inflammation in Mouse Peripheral Artery Disease

Peripheral artery disease (PAD) is linked with obesity-induced complications. Obesity induced by a high-fat diet (HFD) modulates myelopoiesis of hematopoietic stem and progenitor cells (HSPCs) and innate immune responses in a long-term. Here we show that, in a mouse hindlimb ischemia, normal mice transplanted HSPCs from HFD mice exhibit persistent inflammation and impaired healing using three-dimensional magnetic resonance images, compared to mice transplanted HSPCs from mice fed a normal diet (ND). Bone marrow cell population analysis revealed HFD induces expansion of myeloid progenitor cells in steady state and prolonged activity of more primitive HSPCs following hindlimb ischemia. Ex vivo culture of HSPCs revealed that HFD increases monocyte generation by damage-associated toll-like receptor stimulation, suggesting that HFD promotes myelopoiesis in a HSPC-intrinsic manner. This notion was confirmed by adoptive transfer of HSPCs from HFD mice in a mouse after hindlimb ischemia that contribute to monocyte generation in vivo. Mechanistically, HFD-induced oxidative stress is associated with increased histone-3 lysine-4 trimethylation (H3K4me3), an activating histone methylation, in HSPCs potentially through KDM5 histone demethylase, as a reduction of oxidative stress by cyclosporine A or MitoTEMPO decreased H3K4me3 and increased KDM5 activity in HSPCs. In myeloid progeny of HSPCs from HFD mice, we found enhanced gene expressions of inflammatory Tnfa and Il6, and increased H3K4me3-marks in KDM5 targets, compared to those from ND mice. Our results suggest that HFD-induced oxidative stress in HSPCs modulates their myelopoiesis and phenotype of their progeny via epigenetic reprogramming that, in turn, persistent inflammatory monocyte/macrophage response in an acute ischemic event of PAD.

Mesenchymal stromal cell lineages in synovium and their contribution to osteoarthritis and cartilage repair

In recent years, landmark studies using genetic lineage tracing models in mice have identified and functionally characterised skeletal stem and progenitor cell populations in bone marrow, providing important insights in the regulation of bone homeostasis and repair, as well as the control of haematopoiesis. By comparison, the stem and progenitor cells that are resident in the synovial joint and maintain and repair joint tissues such as articular cartilage remained poorly defined, but recent work from our lab and others is starting to shed light on this. Lineage tracing of progenitor cells in cartilage has provided important new insights into the process of articular cartilage formation and maintenance. Our work has focused on characterising stem and progenitor cells in the synovial membrane, and how these cells respond to cartilage injury. Tracing of cells from the embryonic joint interzone, using Gdf5 as driver, showed that Gdf5-lineage cells persist in adult knee synovium up to at least one year of age as a mesenchymal stromal cell population with progenitor activity, which is largely distinct from the skeletal stem/progenitor cell populations identified in bone marrow. After purification, Gdf5-lineage cells display a differentiation potency in vitro that is skewed towards formation of cartilage and a synovial lining-like layer, while showing poor osteogenic ability. Cartilage injury in vivo triggers Gdf5-lineage cells in synovium to undergo proliferation leading to synovial lining hyperplasia, a process that is dependent on the activity of the transcriptional co-factor Yap in these cells. Gdf5-lineage cells also contribute to repair of traumatic joint surface defects, though they do not seem to be the sole cell population able to populate the repair tissue. We further find that in pathology, using the destabilisation of the medial meniscus model of osteoarthritis, Gdf5-lineage cells contribute to chondro-osteophyte formation and subchondral bone remodeling. In conclusion, recent findings are starting to unravel distinct mesenchymal stromal cell subsets in the adult synovium, identifying the key players in joint pathophysiology and promising therapeutic targets for cartilage repair and treatment of osteoarthritis.

An Automated Image Analysis Method to Quantify Veterinary Bone Marrow Cellularity on H&E Sections.

Bone marrow toxicity is a common finding when assessing safety of drug candidate molecules. Standard hematoxylin and eosin (H&E) marrow tissue sections are typically manually evaluated to provide a semiquantitative assessment of overall cellularity. Here, we developed an automated image analysis method that allows quantitative assessment of changes in bone marrow cell population in sternal bone. In order to test whether the method was repeatable and sensitive, we compared the automated method with manual subjective histopathology scoring of total cellularity in rat sternal bone marrow samples across 17 independently run studies. The automated method was consistent with manual scoring methodology for detecting altered bone marrow cellularity and, in multiple cases, identified changes at lower doses. The image analysis method allows rapid and more quantitative assessment of bone marrow toxicity compared to manual examination of H&E slides, making it an excellent tool to aid detection of bone marrow cell depletion in preclinical toxicologic studies.

Role of the angiotensin-converting enzyme in the G-CSF-induced mobilization of progenitor cells.

In addition to being a peptidase, the angiotensin-converting enzyme (ACE) can be phosphorylated and involved in signal transduction. We evaluated the role of ACE in granulocyte-colony-stimulating factor (G-CSF)-induced hematopoietic progenitor cell (HPC) mobilization and detected a significant increase in mice-lacking ACE. Transplantation experiments revealed that the loss of ACE in the HPC microenvironment rather than in the HPCs increased mobilization. Indeed, although ACE was expressed by a small population of bone-marrow cells, it was more strongly expressed by endosteal bone. Interestingly, there was a physical association of ACE with the G-CSF receptor (CD114), and G-CSF elicited ACE phosphorylation on Ser1270 in vivo and in vitro. A transgenic mouse expressing a non-phosphorylatable ACE (ACES/A) mutant demonstrated increased G-CSF-induced HPC mobilization and decreased G-CSF-induced phosphorylation of STAT3 and STAT5. These results indicate that ACE expression/phosphorylation in the bone-marrow niche interface negatively regulates G-CSF-induced signaling and HPC mobilization.

Dural Cells Release Factors Which Promote Cancer Cell Malignancy and Induce Immunosuppressive Markers in Bone Marrow Myeloid Cells.

Thirty per cent of cancer patients develop spine metastases with a substantial number leading to spinal cord compression and neurological deficits. Many demonstrate a propensity toward metastasis to the posterior third of the vertebral body. The dura, the outer layer of the meninges, lies in intimate contact with the posterior border of the vertebral body and has been shown to influence adjacent bone. The effects of the dura on bone marrow and cancer cells have not been examined. Understanding the biology of spinal metastasis will provide insights into mechanisms of cancer growth and allow for new treatment strategies. To examine the extent to which dura influences bone marrow/tumor cell metastatic characteristics. Dura conditioned media (DCM) from primary dura was examined for the ability to stimulate tumor cell proliferation/invasion and to alter bone marrow cell populations. RNA sequencing of dural fibroblasts was performed to examine expression of cytokines and growth factors. DCM induced a significant increase in invasion and proliferation of multiple tumor cell lines, and of patient-derived primary spinal metastatic cells. DCM also increased the proliferation of bone marrow myeloid cells, inducing expression of immunosuppressive markers. RNA sequencing of dural fibroblasts demonstrated abundant expression of cytokines and growth factors involved in cancer/immune pathways. Factors released by primary dural cells induce proliferation of tumor cells and alter bone marrow to create a fertile environment for tumor growth. The dura therefore may play an important role in the increased incidence of metastases to adjacent bone.

Abstract B063: Targeting the pathogenic mesenchymal stem cells as a new strategy for preventing immune dysfunction in cancer therapy

Abstract The immune system is exhausted by persistent and chronic inflammation due to internal and external stimuli, and ultimately loses the functions contributing to the pathogenesis of cancer. T-cell dysfunction is fatal to immunotherapy designed to elicit and intensify immune responses against cancer, and it is a crucial issue beyond immunosuppression that is recoverable by breaking immune checkpoint pathways. Although increase of exhausted T cells has been demonstrated in inflammatory cancer and aging, the reasons for the immune dysfunction and the methods for overcoming it remain undefined. In this study, we attempted to elucidate the underlying mechanisms involved in the immune exhaustion. We compared cell population and immune responses of spleen cells and bone marrow cells between young (1- to 4-month-old) and aged (18- to 24-month-old) C57BL/6 mice implanted with/without murine tumor cells such as melanoma B16-F10 and lung cancer 3LL. We found that CD45-CD166+ activated mesenchymal stromal/stem cells (MSCs) increased in the spleen and bone marrow of mice with age, correlatively with increase of CD3+PD1+Tim3+ exhausted T cells having no cytotoxic activities, and were further expanded by implantation with tumor cells. The MSCs sorted from aged mice generated the CD3+PD1+Tim3+ exhausted T cells as well as immunosuppressive Tregs and MDSCs when cocultured in vitro, and transfer with these MSCs promoted in vivo tumor growth via systemic expansion of exhausted T cells in young mice, although none or less of such properties were seen using the young MSCs. Interestingly, the aged MSCs frequently differentiated into hypertrophic adipocytes with a large lipid droplet containing a huge amount of inflammatory cytokines, implying acceleration of the immune exhaustion after adipogenic differentiation in aged mice. These suggest the key role of the increasing MSCs in deterioration of T-cell responses with age. We identified the specific molecules significantly upregulated in the aged MSCs by utilizing cDNA microarray analysis. Blocking the gene expressions using the specific siRNAs corrected unbalanced immunity through decline of immune suppression and exhaustion in the aged mice, and generated potent tumor-specific CTLs resulting in tumor regression, although immune checkpoint inhibitors are ineffective in the same settings. These results suggest that increase of the pathogenic MSCs might be a risk factor of resistance to immune checkpoint inhibitors, and targeting these MSCs may be a promising strategy for creating a suitable immune status for eliminating cancer. Citation Format: Chie Kudo-Saito, Yamato Ogiwara, Kazunori Aoki. Targeting the pathogenic mesenchymal stem cells as a new strategy for preventing immune dysfunction in cancer therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B063.

Immunotoxic and hepatotoxic effects of perfluoro-n-decanoic acid (PFDA) on female Harlan Sprague–Dawley rats and B6C3F1/N mice when administered by oral gavage for 28 days

Poly- and perfluoroalkyl substances (PFAS) are chemically and thermally stable, hydrophobic, lipophobic compounds used in stain repellants and water and oil surfactants, and associated with immunosuppression and peroxisome proliferator activity. Perfluoro-n-decanoic acid (PFDA, (CF3(CF2)8COOH), a fluorinated straight chain fatty acid compound, is reported to induce thymic atrophy and reversible bone marrow hypocellularity in rodent models. The objective of this study was to assess potential immunotoxicity of PFDA, due to its structural similarity to other immunosuppressive PFASs. Female Harlan Sprague–Dawley rats were exposed to 0–2.0 mg PFDA/kg by oral gavage daily for 28 d. Female B6C3F1/N mice were exposed once/week to 0–5.0 mg PFDA/kg by gavage for 4 weeks. Animals were evaluated for effects on immune cell populations in spleen and bone marrow, and innate, humoral-, and cell-mediated immunity. Mice were also evaluated for resistance to Influenza virus. Treatment-related hepatocyte necrosis and hepatomegaly were observed in rats treated with 0.5 mg PFDA/kg/d. In mice, hepatomegaly (26–89%) was observed following exposure to ≥0.625 mg PFDA/kg/week, while splenic atrophy (20%) was observed at 5.0 mg PFDA/kg/week. At 5.0 mg PFDA/kg/week, total spleen cells, and Ig + and NK + cells were decreased (17.6–27%). At ≥ 1.25 mg PFDA/kg/week the numbers of splenic CD3+, CD4+, CD8+, and Mac3+ cells were decreased (10.5–39%). No changes were observed in leukocyte subpopulations in PFDA-exposed rats. Phagocytosis by fixed-tissue macrophages was decreased in liver (specific activity, 24–39%) at ≥0.25 mg PFDA/kg/d in rats. PFDA-induced effects on humoral- and cell-mediated immunity, host resistance, and bone marrow progenitor cells were limited. These data suggest that exposure to PFDA may induce adverse effects in rat liver in a manner consistent with the PFAS class, and may also alter the balance of immune cell populations in lymphoid tissues in mice.

Abstract 201: High Fat Diet Selectively Decreases the Bone Marrow Lin-/c-kit+ Cell Population in Aging Mice Through Increased ROS Production

Bone marrow-derived c-kit-positive (c-kit + ) cells are a potential source for cell-based therapy and tissue repair and regeneration. The ability of tissue repair and regeneration is significantly decreased in the aging population for various reasons. Western diet usually has increased level of fat. The present study was designed to evaluate the effect of long-term high fat diet on bone marrow c-Kit positive cells and the role of ROS in aging mice. Aging male wild-type (WT) C57BL/6 mice (40 weeks) were fed with high fat diet (HFD) for 3 months with regular diet as the control. To evaluate the role of reactive oxygen species (ROS) in mediating the effect of HFD on bone marrow cell population, a separate group of mice were treated with N-acetylcysteine (NAC) to reduce ROS production. Bone marrow (BM) and blood cells were harvested and prepared in the mice after 3 months of HFD treatment for flowcytometry analysis for Sca-1+, or c-Kit+, or CD133+ cells. The lineage negative (Lin-) and c-Kit positive (Lin-/c-Kit+) cell population was significantly decreased in the BM, not in blood, in the mice with HFD, while no significant change was observed in Sca-1+ or CD133+ cell populations in the BM or blood. The BM Lin-/c-Kit+ cells also exhibited increased intracellular ROS level, increased level of apoptosis, and deceased level of proliferation in HFD-treated mice. NAC treatment significantly decreased intracellular ROS level and normalized the BM Lin-/c-Kit+ cell population in HFD mice. In conclusion, the present study demonstrated that long-term HFD selectively decreased BM Lin-/c-Kit+ cell population in the aging mice through increased ROS production.

Study of Correlation of Structural Features in the Porous TiNi-based Materials Obtained by Sintering with the Integration Process of Bone Marrow Cell Populations

The structural features of porous-permeable TiNi-based materials obtained by sintering in depending on the temperature have been studied. It is shown that a material obtained at the sintering temperature of T2=1250 °C and time t=40 min has an optimal degree of sintering and relates to finely porous materials with the porosity of 55 %. A positive dynamics of development of bone marrow cells in the TiNi-based sintered material was proved. It is noted that the finely porous macrostructure with developed rough surface of pore walls provides favorable conditions for development of cell populations. When the 21 day of cultivation a dense formed tissue on basis of bone marrow cells observes in the material pores.

Persistent and inducible neogenesis repopulates progenitor renin lineage cells in the kidney

Renin lineage cells (RLCs) serve as a progenitor cell reservoir during nephrogenesis and after renal injury. The maintenance mechanisms of the RLC pool are still poorly understood. Since RLCs were also identified as a progenitor cell population in bone marrow we first considered that these may be their source in the kidney. However, transplantation experiments in adult mice demonstrated that bone marrow-derived cells do not give rise to RLCs in the kidney indicating their non-hematopoietic origin. Therefore we tested whether RLCs develop in the kidney through neogenesis (de novo differentiation) from cells that have never expressed renin before. We used a murine model to track neogenesis of RLCs by flow cytometry, histochemistry, and intravital kidney imaging. During nephrogenesis RLCs first appear at e14, form a distinct population at e16, and expand to reach a steady state level of 8-10% of all kidney cells in adulthood. De novo differentiated RLCs persist as a clearly detectable population through embryogenesis until at least eight months after birth. Pharmacologic stimulation of renin production with enalapril or glomerular injury induced the rate of RLC neogenesis in the adult mouse kidney by 14% or more than three-fold, respectively. Thus, the renal RLC niche is constantly filled by local de novo differentiation. This process could be stimulated consequently representing a new potential target to beneficially influence repair and regeneration after kidney injury.

Effects of a tissue-selective estrogen complex on B lymphopoiesis and B cell function

Background/objective17β-estradiol (E2) has major effects on the immune system. It induces thymic atrophy, inhibits both T and B lymphopoiesis and stimulates antibody production treatment with E2 has protective effects on the skeleton but is associated with negative side effects in reproductive organs. A tissue-selective estrogen complex (TSEC) comprise of estrogens combined with a selective estrogen receptor modulator (SERM). TSEC therapy displays the bone-protective effects of estrogen, while the negative side effects on reproductive organs are blocked by the SERM. In a recent publication we showed that treatment with the TSEC E2+bazedoxifene (bza) potently inhibits experimental arthritis and associated osteoporosis. In order to elucidate immunological mechanisms involved in those effects, the aim of this study was to investigate how E2+bza treatment affects the healthy immune system. MethodsOvariectomized C57BL/6N mice were treated with vehicle, E2, bza or E2+bza. Weights of uterus and thymus were determined and fluorescence-activated cell sorting was used to analyze B cell populations in bone marrow and spleen. Immunoglobulin production from B cells in bone marrow and spleen were determined using ELISPOT. ResultsAddition of bza to E2-treatment totally antagonized the E2-mediated proliferative effect on uterus. On the contrary, addition of bza to E2-treatment did not block the E2-induced thymic atrophy or inhibition of B lymphopoiesis, and did not block the E2-induced increase in immunoglobulin secretion from bone marrow B cells. ConclusionsAddition of bza to E2-treatment blocks E2-induced uteroproliferation but does not alter E2-mediated effects on thymus, B lymphopoiesis or B cell function.

The skeletal impact of the chemotherapeutic agent etoposide.

Chemotherapeutics target rapidly dividing tumor cells yet also impact hematopoietic and immune cells in an off target manner. A wide array of therapies have negative side effects on the skeleton rendering patients osteopenic and prone to fracture. This study focused on the pro-apoptotic chemotherapeutic agent etoposide and its short- and long-term treatment effects in the bone marrow and skeleton. Six- to 16-week-old mice were treated with etoposide (20-25mg/kg) or vehicle control in short-term (daily for 5-9days) or long-term (3×/week for 17days or 6weeks) regimens. Bone marrow cell populations and their phagocytic/efferocytic functions were analyzed by flow cytometry. Blood cell populations were assessed by CBC analysis. Bone volume and area compartments and osteoclast numbers were measured by microCT, histomorphometry, and TRAP staining. Biomarkers of bone formation (P1NP) and resorption (TRAcP5b) were assayed from serum. Gene expression in bone marrow was assessed using qPCR. Flow cytometric analysis of the bone marrow revealed short-term etoposide reduced overall cell numbers and B220+ cells, with increased marrow apoptotic (AnnexinV+PI-) cells, mesenchymal stem-like cells, and CD68+, CD45+, and CD11b+ monocyte/myeloid cells (as a percent of the total marrow). After 6weeks, the CD68+, Gr1+, CD11b+, and CD45+ cell populations were still relatively increased in etoposide-treated bone marrow. Skeletal phenotyping revealed etoposide decreased bone volume, trabecular thickness, and cortical bone volume. Gene expression in the marrow for the leptin receptor and CXCL12 were reduced with short-term etoposide, and an increased ratio of RANKL/OPG mRNA was observed. In whole bone, Runx2 and osteocalcin gene expressions were reduced, and in serum, P1NP was significantly reduced with etoposide. Treatment with the antiresorptive agent zoledronic acid prior to etoposide increased bone volume and improved the etoposide-induced decrease in skeletal parameters. These data suggest that etoposide induces apoptosis in the bone marrow and significantly reduces parameters of bone formation with rapid reduction in bone volume. Pre-treatment with an antiresorptive agent results in a preservation of bone mass. Preventive approaches to preserving the skeleton should be considered in human clinical studies.

PEGylated liposomes associate with Wnt3A protein and expand putative stem cells in human bone marrow populations.

To fabricate PEGylated liposomes which preserve the activity of hydrophobic Wnt3A protein, and to demonstrate their efficacy in promoting expansion of osteoprogenitors from human bone marrow. PEGylated liposomes composed of several synthetic lipids were tested for their ability to preserve Wnt3A activity in reporter and differentiation assays. Single-molecule microspectroscopy was used to test for direct association of protein with liposomes. Labeled Wnt3A protein directly associated with all tested liposome preparations. However, Wnt3A activity was preserved or enhanced in PEGylated 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes but not in PEGylated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes. PEGylated Wnt3A liposomes associated with skeletal stem cell populations in human bone marrow and promoted osteogenesis. Active Wnt protein-containing PEGylated liposomes may have utility for systemic administration for bone repair.

Tyrosine kinase inhibitor therapy-induced changes in humoral immunity in patients with chronic myeloid leukemia.

Tyrosine kinase inhibitors (TKIs) have well-characterized immunomodulatory effects on T and NK cells, but the effects on the humoral immunity are less well known. In this project, we studied TKI-induced changes in B cell-mediated immunity. We collected peripheral blood (PB) and bone marrow (BM) samples from chronic myeloid leukemia (CML) patients before and during first-line imatinib (n = 20), dasatinib (n = 16), nilotinib (n = 8), and bosutinib (n = 12) treatment. Plasma immunoglobulin levels were measured, and different B cell populations in PB and BM were analyzed with flow cytometry. Imatinib treatment decreased plasma IgA and IgG levels, while dasatinib reduced IgM levels. At diagnosis, the proportion of patients with IgA, IgG, and IgM levels below the lower limit of normal (LLN) was 0, 11, and 6% of all CML patients, respectively, whereas at 12 months timepoint the proportions were 6% (p = 0.13), 31% (p = 0.042) and 28% (p = 0.0078). Lower initial Ig levels predisposed to the development of hypogammaglobulinemia during TKI therapy. Decreased Ig levels in imatinib-treated patients were associated with higher percentages of immature BM B cells. The patients, who had low Ig levels during the TKI therapy, had significantly more frequent minor infections during the follow-up compared with the patients with normal Ig values (33% vs. 3%, p = 0.0016). No severe infections were reported, except recurrent upper respiratory tract infections in one imatinib-treated patient, who developed severe hypogammaglobulinemia. TKI treatment decreases plasma Ig levels, which should be measured in patients with recurrent infections.

The study of bone marrow cells in rats according to their age and caloric intake

The bone marrow is a central organ of haemopoiesis and a source of pluripotent hemopoietic stem cells. The age-related changes in bone marrow induce the disorder of coordination between parenchyma and stroma of the organ that negatively influences proliferative potential and differentiation of the cells. That is why it is urgent to study the age-related changes of bone marrow and influence of caloric intake limitation on it.Aim. The aim of presented work was the study of proliferative potential, index of viability and percentage ratio of the bone marrow cells in rats depending on their age and caloric intake.Methods. The following methods were used in the study: selection and cultivation of the bone marrow stem cells, assessment of proliferative potential and viability of cells, analysis of percentage ratio of cellular types.Result. It was established, that the number of cells in bone marrows increases with age. The limitation of caloric intake favors the decrease of cells number in bone marrow despite the rats’ age. The received data demonstrated that the limitation of caloric intake increases the proliferative potential of the bone marrow only in old animals. It was established, that the viability of bone marrow cells was high during all period of cultivation. The morphological population of bone marrow cells in rats of different age was heterogenic, not depending on the conditions of their feeding. It was demonstrated, that the limitation of feeding caused the increase of the number of non-differentiated blasts in bone marrow of old animals that can be connected with the activation of the processes of self-activation of stem cells pool and non-differentiated predecessors of immune system cells. The morphological analysis of the bone marrow cells culture of all animal groups despite the age and nutrition regime demonstrated that the cultivation favors the selective decrease of cells heterogeneity. At that the cells of erythroid and megakaryocytic sprouts were absent in cellular cultures.Conclusions. As a result, the limitation of caloric intake of old rats favors the restoration or activation of proliferative potential of immunocompetent cell of bone marrow in vitro system

The evolving view of the hematopoietic stem cell niche.

Hematopoietic stem cells (HSCs) reside in specialized microenvironments known as niches. The niche is essential to support HSC function and to maintain a correct balance between self-renewal and differentiation. Recent advances in defining different mesenchymal and endothelial bone marrow cell populations, as well as hematopoietic stem and progenitor cells, greatly enhanced our understanding of these niches and of the molecular mechanisms by which they regulate HSC function. In addition to the role in maintaining HSC homeostasis, the niche has also been implicated in the pathogenesis of blood disorders including hematological malignancies. Characterizing the extrinsic regulators and the cellular context in which the niches interact with HSCs will be crucial to define new strategies to enhance blood regeneration. Furthermore, a better understanding of the role of the niche in leukemia development will open new possibilities for the treatment of these disorders by using therapies aiming to target the leukemic niche specifically. To update on recent findings on this topic, the International Society for Experimental Hematology (ISEH) organized a webinar, presented by Prof. Sean J. Morrison and Dr. Simón Méndez-Ferrer and moderated by Dr. Cristina Lo Celso, entitled "The evolving view of the hematopoietic stem cell niche," which we summarize here.

IKAROS expression in distinct bone marrow cell populations as a candidate biomarker for outcome with lenalidomide-dexamethasone therapy in multiple myeloma.

Immunomodulatory drugs (IMiDs) are a cornerstone in the treatment of multiple myeloma (MM), but specific markers to predict outcome are still missing. Recent work pointed to a prognostic role for IMiD target genes (e.g. CRBN). Moreover, indirect activity of IMiDs on immune cells correlated with outcome, raising the possibility that cell populations in the bone marrow (BM) microenvironment could serve as biomarkers. We therefore analysed gene expression levels of six IMiD target genes in whole BM samples of 44 myeloma patients treated with lenalidomide-dexamethasone. Expression of CRBN (R = 0.30, P = .05), IKZF1 (R = 0.31, P = .04), IRF4 (R = 0.38, P = .01), MCT-1 (R = 0.30, P = .05), and CD147 (R = 0.38, P = .01), but not IKZF3 (R = -0.15, P = .34), was significantly associated with response. Interestingly, IKZF1 expression was elevated in BM environmental cells and thus selected for further investigation by multicolor flow cytometry. High IKAROS protein levels in total BM mononuclear cells (median OS 83.4 vs. 32.2 months, P = .02), CD19+ B cells (median OS 71.1 vs. 32.2 months, P = .05), CD3+ CD8+ T cells (median OS 83.4 vs 19.0 months, P = .008) as well as monocytes (median OS 53.9 vs 18.0 months, P = .009) were associated with superior overall survival (OS). In contrast, IKAROS protein expression in MM cells was not predictive for OS. Our data therefore corroborate the central role of immune cells for the clinical activity of IMiDs and built the groundwork for prospective analysis of IKAROS protein levels in distinct cell populations as a potential biomarker for IMiD based therapies.

Influence of Plasma Cell Niche Factors on the Recruitment and Maintenance of IRF4hi Plasma Cells and Plasmablasts in Vaccinated, Simian Immunodeficiency Virus-Infected Rhesus Macaques with Low and High Viremia.

As key antibody producers, plasma cells and plasmablasts are critical components of vaccine-induced immunity to human immunodeficiency virus type 1 (HIV-1) in humans and SIV in the macaque model; however, few have attempted to examine the role of these cells in viral suppression postinfection. Our results suggest that plasmablast trafficking to and retention in the bone marrow play a previously unappreciated role in viral control and contrast the potential contribution of mucosal plasma cells to mediate protection at sites of infection with that of bone marrow plasmablasts and plasma cells to control viremia during chronic infection. Manipulation of niche factors influencing the distribution and maintenance of these critical antibody-secreting cells may serve as potential therapeutic targets to enhance antiviral responses postvaccination and postinfection.

Donor chimerism and minimal residual disease monitoring in leukemia patients after allo-HSCT

In present research the comparative analysis of donor chimerism (DC) using different tests was performed to improve the diagnostic tool in patients with malignant hematological disorders after allo-HSCT. The RBC antigen typing, identification of ABO blood type and quantitative analysis of InDel-, STR-, Y-polymorphisms were carried out for detection of DC. In addition, the expression of well-known oncogenes and CD-markers for monitoring MRD was evaluated to predict relapse and clinical outcome. According to our research, the analysis of InDel polymorphism using AlleleSEQR-PCR is more sensitive test for estimation of DC as compared with other assays. Moreover, the sensitivity of AlleleSEQR-PCR may be increased after isolation of the CD34 cell population in bone marrow. Nevertheless, observation of high levels in DC (³95%) in some leukemia patients (ALL, Ph+, bcr-abl/p190+) during first 6 months after HSCT cannot exclude the possibility of relapse. Thus, the combined monitoring of both DC (InDel) and MRD (oncogenes, WT1 and CD-markers) is a more advisable and useful test in managing hematologic malignancies and predicting relapse risk after allo-HSCT.

Shipping mouse bone marrow: Keep it in the bone.

Sharing reagents is of self-evident value in life science research, however, primary cell populations often do not cryopreserve well or can require extensive preparation by collaborators, making shipping difficult. Here we report an evaluation of different conditions for the storage shipment of mouse bone marrow (BM) cells that would best preserve the number, viability, and frequency of different hematopoietic lineages, as well as functionality of progenitor populations. Bones were either crushed to release BM cells or stored intact in one of three media: Phosphate buffered saline (PBS) supplemented with 2% fetal bovine serum (FBS), Plasmalyte, or RPMI at 4°C. Cell numbers, viability, phenotype, and functionality were assessed 16 hours and 40 hours later and compared to freshly prepared samples. Whereas BM cells stored in suspension for 16 hours and BM cells kept in bone for 40 hours suffered major losses in cell number, hematopoietic lineages that were kept in the bone for 16 hours had only minor differences compared to fresh cells. With no significant differences among the different media used, intact long bones stored in media, Plasmalyte, or PBS 2% FBS for up to 16 hours provided a reasonable means of preserving bone marrow cell populations.