Fraction Of Bone Marrow Cells Research Articles

RESULTS OF INTRAMYOCARDIAL ADMINISTRATION OF A MONONUCLEAR FRACTION OF AUTOLOGOUS BONE MARROW CELLS IN CHD PATIENTS WITH CONCOMITANT CARDIAINSUFFICIENCY

Aim . Evaluation of long-term results of drug therapy and intramyocardial administration of a mononuclear fraction of bone marrow cells in CHD patients with chronic cardiac insuf fi ciency. Materials and methods . 109 patients were randomized into two groups by using an envelope method. Intramyocardial administration of a mononuclear fraction of autologous bone marrow cells and cardiac insuf fi ciency therapy were performed for the 1st group (n = 55), while the 2nd group (n = 54) received drug therapy only. All patients underwent clinical examination at admission and at 6 and 12 months after the onset of the study. Results. In the 1st group the angina functional class was reliably lowered (from 3.3 ± 0.2 at the onset of the study down to 2.5 ± 0.1 after 12 months). The distance covered during a 6-minute walk test increased from the initial 185 ± 39 meters up to 359 ± 69 me- ters by the end of the 12th month. The angina class decreased from 3.1 ± 0.4 at the onset of the study down to 1.6 ± 0.4 by the end of the 12th month. Minnesota Life Quality Index reduced from 65.3 ± 21 points down to 22.4 ± 6 points in the fi rst group, while in the control one it decreased down to 59.9 ± 16 points. On the contrary, cardiac insuf fi ciency in patients of the second group tended to continually progress: from NYHA FC 3.5 ± 0.1 at the beginning of the study up to 3.9 ± 0.1 in the course of 12-month observation. The angina class remained the same (3.5 ± 0.5 at the beginning and 3.5 ± 0.4 after 12 months respectively). Conclusion. Intramyocardial implantation of a mononuclear fraction of autologous bone marrow cells is a safe method that contributes to the improvement of the left ventricular function, clinical data and prognosis.

Guest editorial: leukemia stem cell

Leukemia is a clonal, malignant hematological disorder. Recent studies have shown that at least in acute myelogenous leukemia (AML), the clonogenic and leukemogenic activities reside in a minor, primitive fraction of the leukemia cell population. In 1996, Dick et al. showed that the CD34CD38, but not other fractions of bone marrow cells from AML patients can reconstitute human AML in immunodeficient mice, demonstrating direct evidence for the presence of leukemia stem cells (LSCs). After this finding, the concept of the LSC becomes critical to understand the pathogenesis of leukemia. In this issue, four excellent review articles [1–4] focus on developmental mechanisms of LSCs to discuss how to eradicate them to obtain absolute cure of leukemia. Cancer should result from the accumulation of multiple genetic abnormalities. In hematopoiesis, the cells accumulating such mutations need to survive for a long time in the human body, and therefore, the main target for mutations should be hematopoietic stem cells (HSCs) that are capable of self-renewal. HSCs harboring multiple mutations can produce a large number of proliferating progenitor populations with identical mutations, which further increases the chance to get another critical hit to transform into LSCs. This outline of leukemia development reasonably fits the concept of blastic crisis from chronic myelogenous leukemia (CML). In CML, a HSC acquires BCR-ABL, resulting in clonal expansion of BCR-ABLexpressing hematopoietic progenitors and mature cells. These HSCs accumulate additional mutations, and their expanding myeloid or lymphoid progenitors finally transform into LSCs of myeloid or lymphoid blastic clones, respectively. In this context, BCR-ABL is an early mutation, and HSCs in CML patients could be regarded as ‘‘preLSCs’’. Imatinib mesylate, an inhibitor for kinase activity of BCR-ABL, induces hematological and cytogenetic remissions in nearly every patient in chronic phase of CML, preventing the transformation into blastic crisis, but the cessation of this drug results in relapse of CML in the majority of patients. Therefore, it is important to understand the molecular mechanisms to maintain BCR-ABLexpressing residual HSCs (pre-LSCs) in CML. Ito [1] summarizes key players mediating maintenance of such CML stem cells. Recent technological advances in genomics have revealed recurrent driver mutations in AML, demonstrating existence of the similar evolutionary processes for LSC development, in which genetic diversity caused by multiple mutations and clonal selection of a population with growth advantage should be critical. In this selection process, it is now clear that pre-LSCs possessing multiple mutations but still retaining ability to differentiate into all mature blood cells progressively become dominant. Chan and Majeti [2] especially focus on mutations related to epigenetic regulation, one of important groups of driver mutations, and summarize the current understanding of their roles in leukemogenesis. Targeting the LSC should be the ultimate therapeutic strategy to cure leukemias. Xenogeneic transplantation of human cells into immunodeficient mice has enabled to identify cell fractions enriched for LSCs. Ishikawa [3] summarizes the history of xenotransplantation models, and describes his NSG mouse model that is a powerful tool to discover functional properties of LSCs including their cell cycle status and location in the bone marrow. Using the K. Akashi (&) Department of Medicine and Biosystemic Sciences, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-Ku, Fukuoka 812-8582, Japan e-mail: akashi@med.kyushu-u.ac.jp

Neural Crest Cells Isolated from the Bone Marrow of Transgenic Mice Express JCV T-Antigen

JC virus (JCV), a common human polyomavirus, is the etiological agent of the demyelinating disease, progressive multifocal leukoencephalopathy (PML). In addition to its role in PML, studies have demonstrated the transforming ability of the JCV early protein, T-antigen, and its association with some human cancers. JCV infection occurs in childhood and latent virus is thought to be maintained within the bone marrow, which harbors cells of hematopoietic and non-hematopoietic lineages. Here we show that non-hematopoietic mesenchymal stem cells (MSCs) isolated from the bone marrow of JCV T-antigen transgenic mice give rise to JCV T-antigen positive cells when cultured under neural conditions. JCV T-antigen positive cells exhibited neural crest characteristics and demonstrated p75, SOX-10 and nestin positivity. When cultured in conditions typical for mesenchymal cells, a population of T-antigen negative cells, which did not express neural crest markers arose from the MSCs. JCV T-antigen positive cells could be cultured long-term while maintaining their neural crest characteristics. When these cells were induced to differentiate into neural crest derivatives, JCV T-antigen was downregulated in cells differentiating into bone and maintained in glial cells expressing GFAP and S100. We conclude that JCV T-antigen can be stably expressed within a fraction of bone marrow cells differentiating along the neural crest/glial lineage when cultured in vitro. These findings identify a cell population within the bone marrow permissible for JCV early gene expression suggesting the possibility that these cells could support persistent viral infection and thus provide clues toward understanding the role of the bone marrow in JCV latency and reactivation. Further, our data provides an excellent experimental model system for studying the cell-type specificity of JCV T-antigen expression, the role of bone marrow-derived stem cells in the pathogenesis of JCV-related diseases and the opportunities for the use of this model in development of therapeutic strategies.

Prospective Isolation of the Earliest Common Myeloid Progenitor in Adult Hematopoiesis

Abstract 768In murine hematopoiesis, hematopoietic stem cells (HSCs) and multi-potent progenitors (MPPs) have been identified within the LSK (Lin− Sca-1+ c-kit+) fraction of bone marrow cells (Morrison SJ and Weissman IL Immunity 1994). We have proposed that the first commitment step at the myeloid versus lymphoid bifurcation occurred outside the LSK fraction, where myeloid or lymphoid lineage-committed progenitors such as common myeloid progenitors (CMPs) and common lymphoid progenitors (CLPs) are prospectively isolated (Akashi et al Nature 2000). However, recent studies revealed that the initial commitment step might occur within LSK fraction. Adolfsson et al showed that a fraction of LSK cells expressing Flt3 at a high level have lost megakaryocyte/erythroid (MegE) potential and were largely primed for granulocyte/monocyte (GM) and lymphoid cells. This population was named lymphoid-primed multipotent progenitors (LMPPs) (Adolfsson et al Cell 2005). In our hands, by utilizing mice harboring a fluorescent reporter for GATA-1 transcription factor, we demonstrated that the initial upregulation of GATA-1 transcription factor was observed within MPP population, which was defined as CD34+ LSK fraction, and GATA-1+ MPPs were capable of generating only myelo-erythroid cells but lacked lymphoid potential (Arinobu et al Cell Stem Cell 2007). This result strongly suggested that the initial myelo-erythroid commitment occurs at the MPP stage.To isolate the earliest myelo-erythroid LSK progenitors in normal mice without utilizing the GATA-1 reporter system, we conducted expression profiling of GATA-1+ MPP population by cDNA microarray analyses and following validation by FACS analyses. We found that a cell-surface antigen CD41 was specifically expressed at a high level in GATA-1+ MPP cells, indicating that CD41hi MPP cells might be the corresponding population to GATA-1+ MPP cells.CD41hi MPPs gave rise exclusively to GM and MegE colonies in vitro, but no lymphoid colonies were observed even under lymphoid-inducible conditions such as co-culture with OP9 or OP9-DL1 stromal cell layer. In vivo, CD41hi MPPs showed differentiation potential into GM cells, erythroid cells and platelets, lacking B or T lymphoid cell-producing potential in congenic transplantation assay. While the GM cell-reconstitution potential of LMPPs and CMPs peaked around day10 and day4 after transplantation, respectively, CD41hi MPPs had their peak around 3 weeks and relatively long-lasting reconstitution potential, presumably reflecting their immaturity. To further evaluate differentiation potential more directly and quantitatively, CD41hi MPPs with LMPPs or CMPs were injected together into one individual recipient mouse. In this head-to-head competitive assay system, CD41hi MPPs generated a plenty of mature GM cells, whose numbers were nearly ten times of those produced from the original CMPs or LMPPs in a reconstitution setting. Thus CD41hi MPPs possess potent restricted-lineage differentiation potential into GM and MegE lineages.In addition, the gene expression analysis by single-cell quantitative real-time PCR and cDNA microarray revealed that the CD41hi MPP population expresses both GM- and MegE-affiliated genes, but not lymphoid genes, reflecting their lineage restriction. More importantly, each single CD41hi MPP cell expresses either one or both of GATA-1 and PU.1 at a low level, suggesting that the promiscuous expression of these transcription factors might play a critical role in myelo-erythroid lineage commitment (Miyamoto et al Developmental Cell 2002).Furthermore, to evaluate the functional importance of CD41hi MPP population, we created the systemic infectious model mice, mimicking the severe peritonitis. CD41hi MPP pool as well as GMP expands dramatically in response to an increased demand for GM cells, while LMPP does not; thus, CD41hi MPPs compose physiologically important pathway in myelo-erythropoiesis.Accordingly, CD41hi MPP population might represent the earliest branch point for myelo-erythroid development, which resides upstream of the conventional CMP. Based on these data, we propose to redefine the true CMP as CD41hi MPP population. This CD41hi earliest myelo-erythroid progenitor should be useful to investigate the molecular mechanisms for hematopoietic lineage fate decision. Disclosures:No relevant conflicts of interest to declare.

Fractionation of mouse bone-marrow cells limits functional efficacy in non-reperfused mouse model of acute myocardial infarction

Background and objectives. Clinical trials of bone-marrow (BM)-derived cells for therapy after acute myocardial infarct (MI) have been controversial. The most commonly used cells for these trials have been mononuclear cells (MNC), obtained by fractionation of BM cells (BMCs) via different protocols. In this study, we performed a head-to-head comparison of: 1) whole BMC; 2) fractionated BM (fBM) using the commonly used Ficoll protocol; 3) the extract derived from the fBM (fBM extract) versus 4) saline (HBSS) control for treatment of acute MI. Methods. In total, 155 male C57BL/6J (10–12-week old) mice were included. Echocardiography was performed at baseline and 2 days after permanent ligation of the left anterior descending artery to induce MI. Echocardiography and histology were employed to measure outcome at 28 days post-MI. Results. Whole BMC therapy improved left ventricular ejection fraction (LVEF) post-MI, but fBM or fBM extract was not beneficial compared to control (change of LVEF of 4.9% ±4.6% (P = 0.02), –0.4% ±5.8% (P = 0.86), –2.0% ±6.2% (P = 0.97) versus −1.4% ±5.3%, respectively). The histological infarct size or numbers of arterioles or capillaries at infarct or border zone did not differ between the groups. Conclusions. Clinical studies should be performed to test whether whole BMC therapy translates into better outcome also after human MI.

Abstract 17476: Transplantation of an Antigenically-Defined Subpopulation of Bone Marrow Mesenchymal Stem Cells Results in Superior Cardiac Repair After a Reperfused Myocardial Infarction

Although bone marrow mesenchymal stem cells (MSCs) have been used quite extensively for cardiac repair, the outcomes have been highly variable. We investigated whether antigenically-defined subpopulations of MSCs would promote superior repair after myocardial infarction (MI). Unfractionated MSCs were obtained from the adherent fraction of bone marrow cells from C57BL/6 mice using expansion culture, and subpopulations were obtained using two-step FACS isolation. Our in vitro studies identified a Sca-1+/CD45-/c-kit-/Thy1+/CD105- MSC population (CD105- MSCs) that exhibited greater mRNA expression of angiogenic molecules (VEGF, HGF), SDF-1, and reduced expression of deleterious molecules, including TNF-a (Figure). The CD105- MSCs also exhibited greater propensity toward cardiac differentiation. The efficacy of these cells for cardiac repair in vivo was tested in a mouse model of reperfused MI. Wild-type (C57BL/6) mice underwent a 30-min coronary occlusion followed by reperfusion, and 48 h later received intramyocardial injection of vehicle (Vehicle, n=8), unfractionated MSCs (MSCs, n=8), or CD105- MSCs (CD105- MSCs, n=6). Echocardiography was performed 4 d prior to coronary occlusion/reperfusion (BSL1) and at 48 h (BSL2) and 35 d after cell injection. Mice were sacrificed at 35 d. LV ejection fraction (EF) was significantly and similarly reduced in all groups at 48 h after MI (Figure). At 35 d after MI, mice injected with CD105- MSCs exhibited greater EF (Figure) and smaller LV end-diastolic volume compared with vehicle-treated and unfractionated MSC-treated mice. We conclude that compared with unfractionated MSCs, intramyocardial transplantation of CD105- MSCs after a reperfused MI results in improved cardiac repair. The use of this MSC subpopulation may potentially improve the outcomes of cell therapy in humans.

Stem/progenitor cells in closed suction drainage fluid after hip arthroplasty

Background and purpose Drainage after surgery is commonly used, and the contents are generally discarded as clinical waste. We analyzed closed suction drainage fluid from hip arthroplasty patients to determine whether any multipotent stem cells were present that could be used as a source of cells for tissue regeneration.Methods Drainage fluid was obtained from 14 patients after hip arthroplasty on the day of surgery, the next day, and 2 days after surgery. Peripheral blood and bone marrow from the iliac crest were also obtained from the same patients during surgery. These samples were examined using regular flow cytometric profiling, and we performed quantitative immunoassays of stromal-derived factor-1 (SDF1) levels in the plasma. Mononuclear cells (MNCs) from these samples were also isolated and cultured. Fibroblastic adherent cells from MNC fractions were cultured in an osteogenic and a chondrogenic differentiation medium and were then evaluated for multipotentency.Results and interpretation Fibroblastic adherent cells were isolated from the mononuclear cell fraction of bone marrow and drainage fluid on the day of surgery, but they were not present in either the mononuclear cell fraction of the peripheral blood or the drainage fluid on the next day and 2 days after surgery. The cells from the drainage fluid on the day of surgery could differentiate in vitro into osteogenic and chondrogenic cells. SDF1 was elevated on the day of surgery, while CXCR4 was elevated on that day and the next day. This suggests that locally-induced SDF1 contributes to the mobilization of circulating CXCR4-positive cells. These results show that the drainage fluid collected on the day of surgery contains stem/progenitor cells that could be used for autologous cell-based therapy.

Decreased collagen types I and IV, laminin, CK-19 and α-SMA expression after bone marrow cell transplantation in rats with liver fibrosis

Bone marrow cells have frequently been tested in animal models of liver fibrosis to assess their role in hepatic regeneration. The mononuclear fraction of bone marrow cells is of particular interest, as many studies show that these cells may be beneficial to treat hepatic fibrosis. In this study, we used the bile duct ligation model to induce hepatic fibrosis in an irreversible manner, and rats were treated with bone marrow mononuclear (BMMN) cells after fibrosis was established. Analysis of collagen types I and IV, laminin and α-SMA showed a decreased expression of these proteins in fibrotic livers after 7 days of BMMN cell injection. Moreover, cytokeratin-19 analysis showed a reduction in bile ducts in the BMMN cell-treated group. These results were accompanied by ameliorated levels of hepatic enzymes GPT (Glutamic-pyruvic transaminase), GOT (glutamic-oxaloacetic transaminase) and alkaline phosphatase (AP). Therefore, we showed that BMMN cells decrease hepatic fibrosis by significantly reducing myofibroblast numbers and through reduction of the collagen and laminin-rich extracellular matrix of fibrotic septa and hepatic sinusoids.

Adipose Tissue–derived Mesenchymal Stem Cells Expressing Prodrug-converting Enzyme Inhibit Human Prostate Tumor Growth

The ability of human adipose tissue-derived mesenchymal stem cells (AT-MSCs), engineered to express the suicide gene cytosine deaminase::uracil phosphoribosyltransferase (CD::UPRT), to convert the relatively nontoxic 5-fluorocytosine (5-FC) into the highly toxic antitumor 5-fluorouracil (5-FU) together with their ability to track and engraft into tumors and micrometastases makes these cells an attractive tool to activate prodrugs directly within the tumor mass. In this study, we tested the feasibility and efficacy of these therapeutic cells to function as cellular vehicles of prodrug-activating enzymes in prostate cancer (PC) therapy. In in vitro migration experiments we have shown that therapeutic AT-MSCs migrated to all the prostate cell lines tested. In a pilot preclinical study, we observed that coinjections of human bone metastatic PC cells along with the transduced AT-MSCs into nude mice treated with 5-FC induced a complete tumor regression in a dose dependent manner or did not even allow the establishment of the tumor. More importantly, we also demonstrated that the therapeutic cells were effective in significantly inhibiting PC tumor growth after intravenous administration that is a key requisite for any clinical application of gene-directed enzyme prodrug therapies.

The role for interferon regulatory factor-2 on mouse hematopoietic stem cells in an inflammation state

Megakaryopoiesis is associated with inflammation, and certain inflammatory cytokines stimulate hematopoietic progenitors to differentiate into megakaryocytes. Interferon-γ ( IFN-γ) is an inflammatory cytokine that stimulates megakaryocyte development, and interferon regulatory factors (IRFs), IRF-1 and IRF-2 are typical transcription factors that are involved in the IFN-γ response. We investigate the role of IRFs in megakaryopoiesis. Mouse bone marrow hematopoietic stem cells (HSCs) were prepared and stimulated with IFN-γ. IFN-γ treatment induced IRF-2 expression as well as that of CD41 although induction levels of both were much lower than those of IRF-1. When IRF expression levels were studied in mouse bone marrow cell fractions, IRF-2 expression was relatively high in HSCs. An in vitro clonogenic assay showed that IRF-2 overexpressed cells increased the number of megakaryocytic colonies, but IRF-1 overexpressed cells did not, suggesting that IRF-2 is involved in megakaryopoiesis. Mechanistic analysis showed that IRF-2 transfection up-regulated CD41 promoter activity in hematopoietic cell lines through its binding to an ISRE-like site in the CD41 promoter. These findings suggest that IRF-2 plays an important role in megakaryocytic cell commitment or differentiation from HSCs by regulating CD41 expression under inflammation conditions.

P-ERK1/2 Is a Predictive Factor of Response to ESA Treatment in Myelodysplastic Syndromes.

Abstract 294Endogenous serum erythropoietin (sEPO) less than 500UI/L and a transfusion requirement lower than 2 units per month are the best predictive factors for response to treatment by erythropoiesis-stimulating agents (ESA) in low/int-1 myelodysplastic syndromes (MDS). However, the highest response rate hardly reaches 60% suggesting that other factors may influence the response. To investigate the biological signature of response to ESA, we enrolled 100 low/int-1 MDS patients in a prospective study of erythropoiesis at diagnosis before they were treated with ESA. According to the IWG 2006 criteria, 43 patients were non-responders. These patients had significantly higher serum EPO level, higher number of transfusion per month, and lower number of bone marrow-deriving BFU-E and CFU-E than responders. Analysis of CD34+-deriving erythroid progenitors by in vitro liquid culture, demonstrated that all MDS patients (n=54) had an increased apoptosis and a delayed expression of erythroid marker, glycophorin A (GPA). A collapse of EPO-induced DNA synthesis was observed in non-responders, while EPO-dependent erythroid cell differentiation and survival to Fas-induced apoptosis was equivalent in the two groups. Thus, non-responders exhibited an early and isolated default in EPO-induced cell proliferation, suggesting a defect in EPO-R signaling. Immunofluorescence to p-ERK1/2 before and after EPO-R stimulation in immature erythroblasts was negative in 6/8 non-responders, and positive in all 11 responders. Immunohistochemistry to p-ERK1/2 on bone marrow biopsies in 5 non-responders was negative and positive in immature cells in 4 responders. By flow cytometry, p-ERK1/2 expression in the CD71+/GPA− bone marrow cell fraction corresponding to immature erythroblasts (n=30) was significantly lower in non-responders (n=16) than in responders (n=14; Wilcoxon-test: p<0.0001). Receiver operator curve (ROC) analysis of the flow cytometry test demonstrated a good predictive value for the response to ESA with a 0.96 area under the curve (AUC) [95%CI: 0.89 – 1.00]. ROC were also constructed for BFU-E number, serum EPO level, and number of transfusion per month and the AUC were computed. p-ERK1/2 was equivalent to BFU-E and superior to serum EPO level or number of transfusion in predicting the response to ESA. Although requiring validation in a larger cohort, these results suggest that p-ERK1/2 is a ready tool available for the prediction of response to ESA in MDS patients. Disclosures:No relevant conflicts of interest to declare.

Interactions Between Megakaryocytes and Tumour Cells at the Bone Marrow Vascular Stem Cell Niche Promote Tumour Growth and Metastasis.

Abstract 470 Introduction:Specialized microenvironments, or niches, of the bone marrow (BM) maintain and regulate physiological haematopoietic stem/progenitor cells and have been implicated as areas of preferential engraftment and ‘sanctuary sites' for leukaemic cells and metastasizing cells of solid tumours. The cellular and molecular factors that comprise the BM vascular niche have not been well described. Megakaryocytes (MKs) reside in close association with the BM sinusoidal endothelium. The aim of this study was to investigate whether MKs play a role in the homing and engraftment of malignant cells in the BM. Methods:C57Bl/6 wild type and thrombopoietin (TPO)-/- mice (which have <10% of the normal number of MKs and platelets) received flank or tail vein injections of the syngeneic B16-F10 melanoma or EL4 lymphoma cell lines fluorescently labelled with mCherry or GFP. The MK-vascular niche was examined using citrulline, MECA32, and TSP1 immunostaining. MKs were cultured from the Lin-Sca1+cKit+-enriched fraction of BM cells from flushed femurs and tibias, using 50ng/ml TPO + 20ng/ml SCF. Costar transwell co-culture plates and Neuroprobe chemotaxis chambers were used; cellular proliferation was assessed using the CellTitre MTS assay. RNA was extracted from flushed BM cells and in vitro cell cultures using Qiagen RNeasy columns/Trizol and gene expression was analyzed by RT-qPCR. Results:In wild type mice, the majority of BM sinusoids were surrounded by one or more large MKs forming the MK-vascular niche, with MKs tightly abutting the vascular endothelium. In TPO-/- mice, MKs were largely absent from the BM, blood vessels appeared more tortuous and the mean vessel diameter in the BM was significantly larger than in wild type mice (P<0.01). Expression of the angiogenic regulatory proteins platelet factor 4 (PF4) and thrombospondin 1 (TSP1) was markedly lower in BM from TPO-/- mice than wild type; expression of VEGF and TGFb was also reduced. Together these findings suggest that MKs support the integrity of the vascular niche and that homeostasis of the niche may be disrupted in the absence of MKs. Wild type mice injected with either B16-F10 melanoma or EL4 lymphoma had increased numbers of MKs and a larger mean vessel diameter. Although there was no increase in platelet count, the mean platelet volume was significantly increased by day 18 (p=0.002), suggesting increased thrombopoiesis. Furthermore, there was a linear decrease in PF4 in response to tumour, reaching a 3-fold reduction by late-stage tumour growth. This finding was consistent with the increase in MK-vascular niches as PF4 normally acts as an autocrine inhibitor of megakaryopoiesis and inhibits endothelial cell proliferation and migration. Consistent with a modulatory effect of MKs and/or the MK-vascular niche on tumour phenotype, tumour growth in TPO-/- mice was markedly retarded and there was reduced metastasis to the BM and lung. To investigate the mechanism of these effects, MK-conditioned medium (MCM) was added to in vitro cultures of B16 cells. MCM significantly enhanced the proliferation rate of B16 melanoma cells (P<0.001). Further, MCM was highly chemotactic for B16 cells (P<0.001). This effect was found to be mediated by pertussis toxin-sensitive Gi-protein receptors and reduced but not entirely abrogated in the absence of TSP1 (using MCM generated from TSP1-/- mice). To investigate the interactions between tumour cells and MKs, MKs were cocultured with B16 cells. Coculture increased MK expression of proangiogenic factors VEGF and TGFb while cocultured B16 cells displayed increased expression of alpha integrins, a4, a5 and a6. Moreover, coculturing B16 cells with MKs prior to tail vein injection enhanced tumour cell engraftment in the lung. A pilot study of BM trephine biopsies from 8 patients with carcinoma (breast, lung, prostate, bladder and kidney) supported these preclinical findings. MKs in 3/3 patients with BM metastasis and 3/5 patients without BM metastasis showed a variable excess of MKs, some in loose clusters, with abnormal morphology and localization. Conclusions:These findings suggest that MKs contribute to the integrity and functionality of the BM vascular niche in homeostasis and in malignancy, and that cellular/molecular cross-talk between MKs and tumour cells at the vascular niche may promote metastasis. Targeting these interactions may be a useful as adjunctive therapy to prevent dissemination of cancer to the BM. Disclosures:No relevant conflicts of interest to declare.

Mass Spectrometry

Mass Spectrometry

Serpina1 (α1-AT) is synthesized in the osteoblastic stem cell niche

Previously, we identified Serpina1 as a potent inhibitor of hematopoietic stem and progenitor cell (HSC/HPC) mobilization. Serpina1 protein is found in the bone marrow (BM) extracellular fluid and concentrations are decreased during granulocyte colony-stimulating factor-induced HSC/HPC mobilization in mice. In addition, administration of exogenous Serpina1 protein inhibits HSC/HPC mobilization. BM cells responsible for production and secretion of Serpina1 remain unknown. Here, we examined the expression of Serpina1 in order to identify cell populations of the BM that synthesize Serpina1. Osteoblast (OB) and hematopoietic BM cell fractions were isolated from femurs, tibias, and humeri obtained from untreated mice. Subsequently, each BM fraction was examined for the production of Serpina1 messenger RNA and protein by quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry. Quantitative real-time polymerase chain reaction analysis showed that Serpina1 messenger RNA is produced at high levels by OB compared to hematopoietic BM cells. Furthermore, Western blot analysis indicated that Serpina1 protein was secreted by OB. In contrast, no Serpina1 protein could be detected in the supernatant obtained from overnight cultured hematopoietic BM cells. Finally, in BM sections obtained from the femurs of untreated mice, Serpina1 protein was detected in OB cells lining the bone. Serpina1 protein in the BM extracellular fluid is predominantly produced by OB. This indicates that Serpina1 may play a regulatory role in the maintenance of HSC in the OB stem cell niche.

NK cells in the CD19 − B220 + bone marrow fraction are increased in senescence and reduce E2A and surrogate light chain proteins in B cell precursors

E2A encoded proteins, key transcriptional regulators in B lineage specification and commitment, have been shown to decrease in B cell precursors in old age. E2A regulates genes encoding the surrogate light chain proteins λ5 and VpreB. In old age, B cell precursors express less surrogate light chain and this results in compromised pre-B cell receptor function and diminished expansion of new pre-B cells in senescence. Herein, we show that aged bone marrow has increased Hardy Fraction A (CD19 − B220 +) cells, including NK cells, that can inhibit both E47 (E2A) protein and surrogate light chain protein expression in B cell precursors. In vitro, NK-associated inhibition of E47 protein is contact-independent and partially reversed by neutralization of TNFα. In vivo, depletion of NK cells in aged mice by treatment with anti-asialo GM1 antibody led to restoration of surrogate light chain protein levels to that typical of young B cell precursors. These studies suggest that NK cells, within the CD19 − B220 + bone marrow cell fraction, may contribute to a bone marrow microenvironment that has the potential to negatively regulate E47 (E2A) as well as surrogate light chain levels in B cell precursors in old age.

Isolation of Human Bone Marrow Mesenchymal Stem Cells Using Different Membrane Markers: Comparison of Colony/Cloning Efficiency, Differentiation Potential, and Molecular Profile

Bone marrow (BM) mesenchymal stem cells (MSCs) represent an interesting field of research for their in vitro properties and the in vivo therapeutic applications. In the present study, we compared the clonogenic and differentiation capacity of MSCs present in three BM-derived populations-namely, the CD105(+)/CD45(-) cells, the glycophorin A (GlycoA)(-)/CD45(-) cells, and the BM mononuclear cells (BMMCs)-by growing/expanding clones from single colony-forming unit fibroblasts (CFU-F). We also quantified the Oct-4 and Nanog mRNA in the CD105(+)/CD45(-) and GlycoA(-)/CD45(-) cells to define the fraction containing more immature MSCs. We found that basic-fibroblast growth factor (bFGF) favors the long-term survival and growth of the more immature MSCs but has no significant effect on MSC clonogenic potential. CFU-F number and clone recovery were higher in CD105(+)/CD45(-) compared to GlycoA(-)/CD45(-) (p < 0.0001 and p = 0.0364, respectively) cells or BMMCs (p < 0.0001 and p = 0.0007, respectively). The relative mRNA expression of Oct-4 and Nanog was significantly increased in CD105(+)/CD45(-) compared to GlycoA(-)/CD45(-) cells (p < 0.0001 and p < 0.0001, respectively). No significant difference was found in the immunophenotypic characteristics and differentiation potential of clones derived from all three cellular sources. These data suggest that the CD105(+)/CD45(-) BM cell fraction is enriched in immature MSCs and, accordingly, represents an appropriate source for MSC culture initiation.

Frequent in Vivo redundancy of C/EBPβ and C/EBPε during Myeloid Development

CCAAT/enhancer binding protein (C/EBP) transcription factors are involved in a variety of cellular responses including proliferation and differentiation. Although C/EBPβ and C/EBPε are believed to be most important for macrophage and granulocyte activity, respectively, experiments by others and ourselves suggest a possible overlap in their function in myelopoiesis. In order to explore further this potential redundancy, we assessed the in vivo and in vitro function of both transcription factors by generating a double knockout (KO) germline murine model (C/EBPβ/ε−/−/−/−) and compared their hematopoiesis to those of single deficient (C/EBPβ−/−, C/EBPε−/−) and wild-type (WT) mice. Gene expression analysis of bone marrow cells showed expression of C/EBPβ in C/EBPε−/− and WT mice, and vice versa. The weight of the double-KO mice was significantly less as measured at 4 weeks of age (11.5 ± 0.9 g) compared to WT (13.4 ± 0.6 g), C/EBPβ−/− (14.5 ± 1.4 g), and C/EBPε−/− mice (15.4 ± 2.3 g) (p < 0.05). The double-KO mice were prone to infections of the eyes, lungs, liver, and peritoneum. In contrast, C/EBPβ−/−, C/EBPε−/− and WT mice demonstrated no signs of infection. Microscopic imaging of peripheral blood showed metamyelocytes and myelocytes in the double-KO mice. FACS analysis found that the fraction of bone marrow cells which were Lin(−) (no expression of B220, CD3, Gr1, Ter119, and Mac1) were modestly elevated in double-KO and C/EBPβ−/− mice (8.42 % and 8.1 %, respectively) compared to C/EBPε−/− (4.24 %) and WT (3.93 %) mice. A subanalysis highlighted an elevated level of B220(−)/Gr1(−) bone marrow cells in the double-KO mice (54 %) compared to the levels in the C/EBPβ−/− (31 %), C/EBPε−/− (33 %) and WT (21.5 %) mice. Moreover, the proportion of hematopoietic stem cells in the bone marrow were significantly increased in the hematopoietic stem cell compartment [Sca1(+)/c-Kit(+)] in the double-KO mice (20.8 %) compared to the C/EBPβ−/− (6.9 %), C/EBPε−/− (5.9 %) and WT (6.9 %) mice. When given a cytotoxic stress (5-FU) to kill cycling hematopoietic progenitor cells, the mean neutrophil count at their nadir (day 4) was 0.14 × 109 cells/L in the double-KO mice compared to 0.71 × 109 cells/L in the WT mice (p < 0.001); both reached normal values again on day 10. Taken together, these results indicated a relatively higher percentage of immature hematopoietic cells in the double-KO mice compared to the WT mice. Nevertheless, clonogenic assays in methylcellulose using bone marrow cells of the double-KO showed a significant decreased number of myeloid colonies. For example, in the presence of G-CSF, GM-CSF, and SCF, a mean of 83 ± 10 hematopoietic colonies formed in the double-KO mice compared to 135 ± 6 in C/EBPβ−/−, 159 ± 12 in C/EBPε−/− and 165 ± 2 in WT mice (p < 0.001, double-KO vs. WT). Similar clonogenic results occurred when bone marrow cells were stimulated with either G-CSF, GM-CSF or SCF/G-CSF alone. Although our in vitro experiments suggested that double-KO mice had a decreased clonogenic response to G-CSF, their bone marrow cells had normal levels of phosphorylated STAT3 protein when stimulated with G-CSF. Hence, the G-CSFR and its secondary signaling pathway seemed to be intact. In further experiments, downstream targets of the C/EBP transcription factors were examined. Bone marrow macrophages activated with LPS and IFNγ from both double-KO and C/EBPβ−/− mice had decreased gene expression of IL6, IL12p35, TNFα, and G-CSF compared to the levels detected in macrophages of C/EBPε−/− and WT. Interestingly, expression levels of cathelicidin antimicrobial peptide (CAMP) were similarly robust in the macrophages from C/EBPβ−/−, C/EBPε−/−, and WT mice. In sharp contrast, CAMP expression was undetectable in the activated macrophages of the double-KO mice. In conclusion, the phenotype of the double-KO mice was often distinct from the C/EBPβ−/− and C/EBPε−/− mice suggesting a redundancy of activity of both transcription factors in myeloid hematopoiesis.

T-Cell Receptor Complementarity Determining Region Analysis of Peripheral Blood and Bone Marrow T-Lymphocyte Subsets and Quantitative Evaluation of T-Regulatory Cells in Patients with Chronic Idiopathic Neutropenia

Clonal/oligoclonal expansions of cytotoxic T-lymphocytes (CTLs) and quantitative/qualitative abnormalities of T-regulatory cells (Tregs) have been implicated in the pathophysiology of acquired bone marrow (BM) failure syndromes such as aplastic anemia, myelodysplastic syndromes and large granular lymphocyte proliferative disease. Chronic Idiopathic Neutropenia (CIN) is an acquired disorder of granulopoiesis characterised by increased apoptosis of BM CD34+/CD33+ granulocytic progenitor cells. We have previously provided evidence for oligoclonal T-cell expansions with possible pathogenetic significance in CIN. The aim of the current study is (a) to define and compare the magnitude of CD4+ and CD8+ T-cell responses and (b) to evaluate the number of Tregs, in the peripheral blood (PB) and BM of CIN patients. Eighty five CIN patients and 85 healthy controls, age- and sex-matched to the patients, were studied after informed consent. All patients had PB neutrophil counts below 1800/μL (mean 1410 ± 330 neutrophils/μL, range 100–1799 neutrophils/μL), displayed negative anti-neutrophil antibody activity and were satisfying the previously reported diagnostic criteria for the disease.The T-cell receptor (TCR)-Vβ repertoire was analysed by flow cytometry (Vβ spectratyping) in the PB and BM CD3+, CD4+ and CD8+ cells. The size distribution of the TCR-Vβ complementarity determining region 3 (CDR3) was analysed in immunomagnetically sorted PB and BM CD4+ and CD8+ cells by PCR according to the -2 protocol using a fluorescence-based DNA sequencer (CDR3 spectratyping). Vβ family expansions were defined as above of 2 standard deviations from the mean values of the 85 healthy controls. CDR3 oligoclonal/monoclonal patterns were defined upon comparison with the normal Gaussian-type size distribution. The Treg cell frequency was defined as the proportion of FOXP3+ cells within the CD4+/CD25high PB and BM cell fraction by flow cytometry. We found that 69.41% and 82.61% of CIN patients displayed one or more predominant TCR-Vβ family expansions within the CD3+ cell fraction of PB and BM, respectively. None of the controls displayed clonal/oligoclonal expansions in either PB or BM (P<0.001 and P<0.001, respectively). Further analysis showed that 58.62% and 74.19% of CIN patients displayed a skewed TCR-Vβ repertoire in PB CD4+ and CD8+ cells, respectively. A higher proportion of patients i.e. 93.75% and 75.0% displayed a skewed TCR-Vβ repertoire in BM CD4+ and CD8+ cells, respectively. The total sum of predominant TCR-Vβ clones was statistically significant increased in patients' BM compared to PB in all subpopulations studied, namely the CD3+, CD4+ and CD8+ cells (P<0.05, P<0.05, P<0.05, respectively). Interestingly, CDR3 spectratyping identified a skewed (oligoclonal) pattern in PB and/or BM CD8+ cells in 100% of patients. PCR analysis of PB and BM CD4+ cells revealed a normal Gaussian-like CDR3 size distribution in all but three patients. None of the controls displayed a skewed CDR3 profile in either CD4+ or CD8+ cells. The percentage of FOXP3+ cells within the PB CD4+CD25high cell fraction was significantly decreased in CIN patients (57.77%±15.77%) compared to controls (72.95±12.23%; P=0.0004). A parallel analysis of Treg cell proportion in patients' PB and BM showed statistically significant increased percentage of FOX3+ cells within the CD4+/CD25high T-cells of BM (68.51%±11.88%) compared to PB (52.20%±12.77%) (P=0.0005). No statistically significant difference was found in the percentage of FOX3+ cells within the CD4+/CD25high T-cells of BM compared to PB in healthy controls. These data suggest that oligoclonal CTL expansions are uniformly identified in CIN patients. The polarized cell subsets are more frequent in BM than the PB suggesting the possible existence of “target” within the BM. Whether, however, the CD34+/CD33+ BM cells represent the target or the innocent bystanders of this immune process, remains elusive. The low frequency of Tregs in the PB and the accumulation of these cells in patients' BM represents probably a compensatory control mechanism aiming to suppress the local immune reactions. All these data substantiate further the concept that CIN belongs to the spectrum of T-cell mediated BM failure syndromes.

CD97 is differentially expressed on murine hematopoietic stem-and progenitor-cells

CD97 is a member of the epidermal growth factor-seven transmembrane (EGF-TM7) family of adhesion receptors and is broadly expressed on hematopoietic cells. The aim of this study was to investigate the expression of CD97 on hematopoietic stem- and progenitor cells (HSC/HPC). CD97 expression on hematopoietic stem- and progenitor cells was studied in BALB/c, C57BL/6 and DBA/1 mice using flow cytometry. Functional hematopoietic stem- and progenitor cell characteristics were investigated in vitro and in vivo by progenitor cell assays, cobblestone area forming cell assays and bone marrow cell transplantation. Analysis of CD97 expression on murine bone marrow cells showed three major populations i.e. CD97(HI), CD97(INT) and CD97(NEG) cells. Functional studies revealed that radioprotective capacity and cobblestone area forming cell day 28-35 activity resides in the CD97(INT) bone marrow cell fraction while CFU-GM colony-forming capacity mainly resides in the CD97(NEG) population in all strains. In C57BL/6 and DBA/1 mice CD97(NEG) and CD97(HI) bone marrow cells show hematopoietic stem cell characteristics as well. Further functional analysis of BALB/c CD97(INT) bone marrow cells revealed that c-Kit(HI)CD97(INT) bone marrow cells exhibit HSC activity and are 1.5-fold enriched for cobblestone area forming cell-day 35 activity compared to c-Kit(HI) bone marrow cells. Moreover, phenotypical analysis showed that BALB/c and C57BL/6 HSC are CD97(INT), while DBA/1 HSC are CD97(HI). CD97 is differentially expressed on hematopoietic stem cells and hematopoietic progenitor cells. Committed progenitor cell activity is largely comprised in the CD97(NEG) fraction, while the CD97(INT) population contains hematopoietic stem cell activity. In BALB/c mice, CD97 expression can be applied to almost completely separate colony-forming cells and cells exhibiting radioprotective capacity. In addition we propose that the CD97(INT)c-Kit(HI) phenotype allows simple and rapid purification of murine hematopoietic stem cells.

Development of Interactive Immunoregulatory Gr‐1+ Subsets Induced by Mycobacterial Products: Role of IL‐23 in Mobilization from Bone Marrow.

Similar to the regulation of vasodilation, the balance between immunosuppressive nitric oxide (NO) and its antagonist superoxide (O2−) regulates expansion of activated T cells in mice. Reduction of NO levels by O2− is essential for T cell expansion and development of autoimmunity. Mice primed with heat‐killed Mycobacterium develop granulocytic Ly‐6G+ and monocytic Ly‐6G− Gr‐1+ subsets in the spleen, which, upon stimulation by activated T cells, produce O2− or NO, respectively, which regulate T‐cell expansion. Here, we report on the development of this ‘Greg system’ in bone marrow (BM). Unlike spleen, naïve BM expressed high levels of Gr‐1+ cells and weak immunoregulatory activity. Upon priming, BM cells expressed strong NO and O2− activity in the Gr‐1 enriched fraction, similar to the spleen, but in addition developed NO activity in the Gr‐1 depleted fraction as well, unlike the spleen. As expected, Ly‐6G+ splenocytes developed poorly in primed IL‐23−/− mice and produced no O2− Unexpectedly, splenocytes expressed reduced NO‐mediated suppression by Gr‐1+ cells as well, indicating a general role for IL‐23 in Greg development. Surprisingly, strong immunoregulatory NO and O2− activities both developed in BM of IL‐23−/− mice. While O2− production was restricted to the Gr‐1+ BM cell fraction, suppressive NO production was strongest in the Gr‐1‐depleted fraction. We conclude that IL‐23 functions at different levels during development of Greg subsets, i.e. mobilization of O2− producing counter‐suppressive granulocytic Greg from BM, and optimal development of suppressive Gr‐1+ monocytic cells within the BM prior to mobilization.