Bone Marrow Marrow Mononuclear Cells Research Articles

The expressions of VEGF and VEGFR signaling pathway in the bone marrow mononuclear cells with chronic mountain sickness

Objective: This study was aimed at investigating the levels and relationships of vascular endothelial growth factor (VEGF) and its receptor(VEGFR) in the bone marrow mononuclear cells (MNC) of chronic mountain sickness (CMS). Methods: A total of 34 patients with CMS and 30 controls residing at altitudes of 3 000-4 500 m were recruited for this study. The levels of VEGF, VEGFR1 and VEGFR2 in bone marrow MNC were detected by flow cytometry technique and RT-qPCR. Results: The percentage of VEGFR2 positive cells in the bone marrow MNC of CMS were higher than that of the controls[20.7% (8.1%, 67.6%) vs 8.1% (2.2%, 14.9%), P<0.05], but that of VEGFR1-positive and VEGF-positive were similar in CMS and controls. The mRNA levels of VEGFR2 were higher in the bone marrow MNC of CMS than in the controls[1.7(1.0, 5.1) vs 1.0(0.4, 2.7), P<0.05], while VEGF and VEGFR1 mRNA levels were similar between the two groups. The percentage of VEGFR2 positive cells in CMS were significantly correlated with hemoglobin (r=0.453, P=0.007) and the percentage of VEGF-positive cells (r=0.373, P=0.030). Conclusions: Bone marrow MNC of CMS may show enhanced activity of the VEGF-VEGFR2 pathway, and it appears to be involved in the pathogenesis of CMS.

Abstract T MP22: Ly6g/c Monocytes Within The Spleen May Be An Important Target For Bone Marrow Mononuclear Cell Therapy In Stroke

Background: We and other investigators have demonstrated that autologous bone marrow mononuclear cells (MNCs) improve stroke recovery in animal models. We assessed whether the spleen, a major homing organ after MNC administration, is a key target organ associated with the benefits of MNCs. Methods: C57 mice underwent stroke and were treated with autologous bone marrow MNCs or saline IV at 24 hours after stroke. At various time points, spleens were harvested and flow cytometry of the splenocytes was performed (n=4, per group at each time point). In a separate experiment, C57 mice underwent splenectomy two weeks prior to stroke, and then were treated with autologous MNCs IV or saline at 24 hours after stroke (n=10 each group), compared with mice who had intact spleens and were treated with autologous MNCs or saline at 24 hrs after stroke (n=8 to 12 each group). Behavior testes were evaluated up to the 28 days after stroke using corner and ladder rung tests. Results: Compared to the saline group, MNC treatment did not change the proportion of B cells, CD4 T helper cells, CD8 T cytotoxic cells, Treg cells or NK cells among splenocytes at any time points tested. However, there was an increased proportion of monocytes in MNC treated animals after stroke compared with saline treated animals ( p= 0.01 at 1 hour, 0.07 at 3hrs, 0.04 at 6 hrs and 0.09 at 24hrs). The Ly6G/C positive cell population among the monocytes were elevated in the MNC group ( p =0.01 at 1 hour, 0.09 at 3hrs, 0.08 at 6 hrs and 0.04 at 24hrs). Neural deficits and lesion size were lower in mice without spleens compared to mice with intact spleens. In mice without spleens, MNCs compared with saline had no effect on functional recovery. Conclusion: Treatment with autologous MNCs may alter specific subpopulations of monocytes in the spleen. The spleen might be a key target organ associated with recovery after treatment with MNCs for stroke.

Clinical Application of Vascular Regenerative Therapy for Peripheral Artery Disease

Prognosis of peripheral artery disease (PAD), especially critical limb ischemia, is very poor despite the development of endovascular therapy and bypass surgery. Many patients result in leg amputation and, therefore, vascular regenerative therapy is expected in this field. Gene therapy using vascular endothelial growth factor is the first step of vascular regenerative therapy, but did not confirm effectiveness in a large-scale randomized comparative study. Based on animal experiments, bone marrow mononuclear cells (MNCs), peripheral blood MNCs were used as the cell source for regenerative therapy. Those cells were confirmed to be effective to decrease rest pain and ulcer size, but its effect was not fully satisfied. Mesenchymal stem cells (MSCs) are expected as an effective cell source for vascular regeneration and clinical studies are ongoing, because the cells are able to differentiate into various cell types and produce a significant amount of vascular growth factors. Of vascular regeneration therapy, peripheral MNCs and bone marrow MNCs were recognized as advanced medical technology but do not attain to the standard therapy. However, clinical use of MSCs have already started, and induced pluripotent stem cells are surely promising tool for vascular regeneration therapy although further basic studies are required for clinical application.

Abstract 2791: Bone Marrow Derived Mononuclear Cells Are Neuroprotective By Modulating Microglia In A Rodent Model Of Ischemic Stroke

Background: Bone marrow mononuclear cells (MNCs) are an investigational, autologous cell-based therapy for acute ischemic stroke. These cells are easily obtained by bone marrow harvest and exert neuroprotective and immunomodulatory effects in the injured rodent brain after stroke. We have investigated a new mechanism in which MNCs exert neuroprotective effects by modulating microglia. Methods: Microglia and neurons were isolated and cultured from E20 pregnant rats. Microglia were activated by oxygen-glucose deprivation (OGD) and then were cultured with neurons (n=4). At the time microglia were placed into culture with neurons, bone marrow MNCs or media vehicle was injected directly into the co-cultures or placed in transwells with the neuron-microglia cultures. The number of NeuN positive cells was quantified at 24 hrs afterwards. In another experiment, adult rats were subjected to middle cerebral artery occlusion for 90 minutes, and then treated with autologous bone marrow MNCs or saline IV at 24 hours after stroke (N=5). Three days later, brain samples were analyzed by immunohistochemistry to investigate the microglial response. Results: Adding activated microglia to neuronal cultures resulted in 36.2%±1.8% neuronal survival while adding non-activated microglia led to 85.1±15.6% neuronal survival ( p =0.001). Treatment with MNCs improved survival of neurons cultured with activated microglia to 64.0%±19.9% ( p =0.037) compared with neuronal-microglia cultures treated with media vehicle alone (36% neuronal survival). However, MNCs in transwell did not alter neuronal death as there was 31.9%±11.7% neuronal survival in co-cultures with activated microglia. All data were normalized to controls. In the in vivo study, there were significantly more OX-42 positive microglial cells in the peri-infarct area from animals treated with MNCs compared with saline treatment (43±3.9 vs.29±3.1 per high power field, p &lt;0.05). However, the percentage of iNOS positive cells among OX-42 positive cells was less in the same regions from animals treated with MNCs compared with saline treatment (9.1%±1.1% vs. 61.5%±7.8%, p &lt;0.05). There were also significantly more cells co-stained with Iba-1 and SDF-1α (13.8±1.5 vs. 7.4±0.8, p &lt;0.05) in the peri-infarct region of animals treated with MNCs compared with saline controls. Conclusion: MNCs administered 24h after stroke increase the number of microglia in the peri-infarct area. MNCs decrease the number of pro-inflammatory microglia and increase the number of SDF-1a expressing microglia. Furthermore, MNCs reduce microglial-mediated neurotoxicity in vitro . Thus, the results of these studies suggest that MNCs may exert cytoprotective effects in animal models of stroke by modulating microglia. MNCs may protect neurons not only by directly reducing microglial toxicity but also by upregulating neuroprotective microglia in the post-ischemic brain.

Synergistic Action of the Human Inhibitory KIR Antibody IPH2102, and the Human CD38 Antibody Daratumumab to Enhance the Lysis of Primary Multiple Myeloma (MM) Cells in the Bone Marrow Mononuclear Cells (MNCs) From Myeloma Patients

Abstract 1865Despite significant improvements in the treatment of multiple myeloma (MM), this progressive malignancy of antibody-producing clonal plasma cells is still considered incurable. New innovative treatments need to be developed to improve long term outcomes. Recent successes of CD20 antibodies in the clinical lymphoma management indicate that targeted immunotherapy can represent a powerful therapeutical strategy for hematological malignancies. Towards developing a similar strategy for MM, we have recently generated a novel human monoclonal antibody, daratumumab (DARA), which targets the CD38 molecule expressed at high levels on MM cells. We have demonstrated that DARA mediates the lysis of CD38+ MM cells via direct apoptosis, complement mediated lysis and antibody-dependent cell mediated cytotoxicity (ADCC). Natural killer (NK) cells appeared important effector cells mediating the ADCC effect. Since NK cell activity against tumor cells is regulated by the balance of signals generated by inhibitory or activating receptors of NK cells (KIRs), we now explored whether blocking the inhibitory KIRs would improve the NK cell mediated DARA dependent lysis of MM cells. Thus, we evaluated the potential benefits of combining DARA with a novel human anti KIR monoclonal antibody, IPH2102, which blocks the inhibitory KIR2DL1/2/3 receptors (HLA-C specific KIRs), and has been shown to augment NK cell function against MM cells.We recently developed FACS-based ex vivo MM cell lysis assays, in which DARA-dependent NK cell-mediated lysis of MM cells can be directly measured in bone marrow MNCs, thus without separating the malignant cells from autologous NK cells and other accessory cells. Using these, we investigated whether the addition of IPH2102 would augment the DARA dependent lysis of MM cells.As expected, DARA induced lysis of MM cells in bone marrow MNCs isolated from MM patients (n=10). Mean lysis at 10 μg/ml DARA was 27.6% (range 11.3–48.1%). IPH2102 showed little or no lysis of MM cells (at 0.3, 1, 3 and 10 μg/ml) in this setting. The combination of 10 μg/ml IPH2102 with 3 and 10 μg/ml DARA significantly enhanced cytotoxicity against primary MM tumor cells compared to DARA alone (p=0.013 and p=0.028 respectively). Mean lysis of MM tumor cells at 10 μg/ml DARA and 10 μg/ml IPH2102 was 38%.These data confirm our previous findings that NK-cell mediated killing is an important mechanism of action of DARA. We demonstrate a clear synergy between DARA and IPH2102 to achieve effective lysis of MM cells directly in the bone marrow MNC of MM patients, indicating that complementary effects may be achieved by combining IPH2102 and DARA in clinical MM management. Disclosures:Weers:Genmab: Employment. Andre:Innate Pharma: Employment. Lokhorst:Genmab: Research Funding. Parren:Genmab: Employment. Morel:Innate Pharma: Employment. Mutis:Genmab: Research Funding.

Ruxolitinib, a Janus Kinase (JAK)-1 and -2 Inhibitor Modulates Microrna (miR) Levels in Patients with Myelofibrosis (MF): miR Levels Might Predict MF Treatment Outcome

Abstract 1744Myeloproliferative neoplasms (MPN) are a class of stem cell–derived hematologic malignancies, characterized by an expansion of one or more myeloid lineages with resulting bone marrow (BM) hypercellularity. A gain-of-function mutation in JAK2, detected in most patients with MPN, induces constitutive activation of JAK2, stimulation of downstream signaling pathways, and activation of several cytokine and growth factor genes. Recent phase I/II clinical trial revealed that ruxolitinib (INCB018424), an oral inhibitor of JAK1 and JAK2, induced marked and durable clinical benefits in patients with primary or secondary MF (Verstovsek S et al. N Engl J Med 12:1117, 2010). Although clinically effective, ruxolitinib induced a modest (13%) suppression of the JAK2V617F allele burden after 12 cycles of therapy and the response did not correlate with the presence of JAK2V617 mutation. Therefore other, yet unknown, mechanisms might be operative in attaining these clinical benefits.Alterations in gene and protein levels affect miRs levels, and miRs have been shown to regulate protein levels, thereby playing a role in cancer pathogenesis (Calin G, et al. PNAS 99:15524, 2002; Merritt MW et al. N Engl J Med 25:2641, 2008; Munker R, et al, Clin Sci 121:141, 2011). Therefore, in MF patients we investigated changes in the levels of RNase III enzymes Dicer and Drosha, which are the two main regulators of miR biogenesis. Deregulation of their expression has been indicative of possible miR alterations in various cancers. We also investigated expression of several miRs (h-miR-16, 21, 29a, 29b1, 29b2, 29c, 155, 181a, and 451). Bone marrow mononuclear cells (MNC) from six best responders in the ruxolitinib study, and MNC from six worst responders were collected (at baseline, one year and two years on therapy), plus six normal control bone marrow MNC, and used for this study. Using qRT-PCR we found that the levels of miR-16, -21, -29 (a, b1, b2, c), -155, -188a and -451 were significantly higher in MF than in normal controls. MiR-155 levels were significantly higher at baseline in best responders than worst responders (P = 0.024). However, after two years of treatment they significantly declined (P = 0.055). The expression of miR-188a was also significantly higher in the worst responders at baseline, as compared to the best responders, but stayed elevated during two years of therapy (P = 0.0021). The levels of RNase III enzymes Dicer and Drosha were significantly lower at baseline in MF MNC as compare to normal MNC. However, after two years of treatment, Dicer level significantly increased in the best responders but not in the worst responders (P = 0.0001). In conclusion, ruxolitinib treatment modulates miR levels and associated enzymes; miR profiling might be useful in predicting response to ruxolitinib. Disclosures:Verstovsek:Incyte Corporation: Research Funding.

Mobilization efficiency of granulocyte colony-stimulating factor and stem cell factor to bone marrow mononuclear cells and mechanisms

The mobilization efficiency of granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) to bone marrow mononuclear cells (MNCs) in mice was observed, and the changes of CXCL12/CXCR4 signal were detected in order to find out the mobilization mechanism of stem cells. Kunming mice were randomly divided into two groups. The mice in treatment group were subjected to subcutaneous injection of G-CSF at a dose of 100 microg/kg and SCF at a dose of 25 microg/kg every day for 5 days, and those in control group were given isodose physiological saline. The MNCs were separated, counted and cultured, and the colony-forming unit-fibroblast (CFU-F) was evaluated. CD34+CXCR4+ MNCs were sorted by flow cytometry. The expression of CXCL12 protein in bone marrow extracellular fluid was detected by ELISA, and that of CXCL12 mRNA in bone marrow was measured by RT-PCR. The results showed that the counts of MNCs in peripheral blood and bone marrow were increased after administration of G-CSF/SCF (P<0.01). The factors had a dramatic effect on the expansion capability of CFU-F (P<0.05). Flow cytometric of bone marrow MNCs surface markers revealed that CD34+CXCR4+ cells accounted for 44.6%+/-8.7% of the total CD34+ MNCs. Moreover, G-CSF/SCF treatment induced a decrease in bone marrow CXCL12 mRNA that closely mirrored the fall in CXCL12 protein. In this study, it is evidenced that G-CSF/SCF can effectively induce MNCs mobilization by disrupting the balance of CXCL12/CXCR4 signaling pathway in the bone marrow and down-regulating the interaction of CXCL12/CXCR4.

Autologous Bone Marrow Mononuclear Cells Enhance Recovery after Acute Ischemic Stroke in Young and Middle-Aged Rats

We investigated intra-arterially administered autologous bone marrow mononuclear cells (MNCs) in rats with acute ischemic stroke. Long Evans rats (2 to 3 months or 12 months old) underwent tandem reversible common carotid artery (CCA)/middle cerebral artery (MCA) occlusion (CCAo/MCAo) for 3 h and then 24 h later underwent tibial bone marrow harvest. Ten million or 4 million cells were re-injected by an intra-carotid infusion. Control animals underwent marrow needle insertion and then saline injection into the carotid artery. Animals were assessed on a battery of neurological tests. MNCs in the ischemic brain were tracked using Q-dot nanocrystal labeling. Infarct volume and cytokines in the ischemia-affected brain were analyzed. Cell-treated animals in the younger and older groups showed improvement from 7 to 30 days after stroke compared with vehicle-treated animals. MNCs significantly reduced infarct volume compared with saline. There was a significant reduction in tumor necrosis factor-alpha, interleukin-1alpha (IL-1alpha), IL-beta, IL-6, and a significant increase in IL-10 in injured brains harvested from the cell-treated groups compared with saline controls. Labeled MNCs were found in the peri-infarcted area at 1 h and exponentially decreased over the ensuing week after injection. Autologous bone marrow MNCs can be safely harvested from rodents after stroke, migrate to the peri-infarct area, enhance recovery, and modulate the post-ischemic inflammatory response.

Human rib bone marrow mononuclear cells spontaneously synthesize and secrete IgEin vitro

We have examined spontaneous secretion of IgE by human rib bone marrow mononuclear cells (MNC). Bone marrow MNC from nine out of 12 rib specimens synthesized and secreted substantial amounts of IgE during 14 days of in vitro culture. The 14-day supernatants from these bone marrow MNC contained a mean of 2589 pg/ml of IgE (n = 12) with a maximum production of 15,408 pg/ml of IgE compared with small amounts of IgE (80-200 pg/ml) produced by similarly cultured normal and inflammatory bowel disease intestinal lamina propria MNC. Using two rib specimens, time-course studies revealed spontaneous secretion of IgE to be minimal during the first 2 days of culture (152 pg/ml), followed by a steady increase between days 4 (517 pg/ml) and 14 (3588 pg/ml). The addition of pokeweed mitogen resulted in 72% suppression of spontaneous IgE production by bone marrow MNC. The bone marrow MNC isolated from the ribs consisted of 22% Leu12+ (B) cells of which 3.2% were surface IgE positive. Staining for cytoplasmic immunoglobulin revealed 1% of the bone marrow MNC to be cytoplasmic IgE+. The presence of IgE-bearing and IgE-secreting MNC in human bone marrow is consistent with the observation that allergen-specific IgE-mediated hypersensitivity is adoptively transferred by human bone marrow transplantation and demonstrates the usefulness of human bone marrow MNC for examination of IgE secretory and regulatory events.

Gfi-1 Is Overexpressed on Chronic Myeloid Leukemic Cells.

Gfi-1 oncogene was first discovered as an integration site for Moloney murine leukemia virus(MoMulv) in virally infected cells that were selected for interleukin-2 (IL-2) independence; In synergy with pim-1 and L-Myc genes, Gfi-1 was involved in the pathogenesis of lymphoid malignancies. Recent studies also indicated that Gfi-1 regulates self-renewal and engraftment of hematopoietic stem cells and the development of neutrophils. Normally, Gfi-1 zinc finger protein is expressed almost exclusively on thymocytes or stem cells. Chronic myeloid leukemia (CML) is a typical hematopoietic stem cell disease, we ask whether Gfi-1 is expressed on CML cells and the possible role in the leukemogenesis. By RT-PCR and flow cytometry, we analysed the expression of Gfi-1 in the transcription and the protein levels respectively on bone marrow mononuclear cells(MNC) from 53 CML patients. Control samples were taken from the ribs of 5 patients undergoing thoracic surgery for lung cancer. Results Gfi-1 mRNA was detected in K562 cell line and all the MNCs from CML patients but not or very weak in control MNCs; Flow cytometry study show that both the positive percentage (87.2±7.5% Vs 12.4±6.6%, p<0.01) and the relative fluorescence intensity (represented by mean fluorescence intensity, 58.94 ±22.09 Vs 18.67±7.88, p<0.01) were significantly increased in CML MNCs. Considering the complexity of the composition of bone marrow MNCs, we also sorted CD34+ cells from CML or control samples and examined by Rt-PCR and flow cytometry; The Gfi-1 was found to be overexpressed in CD34+ cells of CML; On confocal microscopy, Gfi-1 was mainly sublocalized in nucleus. Conclusion Gfi-1 was overexpressed in MNCs or CD34+ cells from CML patients; Gfi-1 may be implicated in the leukemogenesis of CML and potentially be targeted in the future for treatment of CML.

Local implantation of autologous mononuclear cells from bone marrow and peripheral blood for treatment of ischaemic digits in patients with connective tissue diseases

CD34-positive bone marrow mononuclear cells (MNCs) have been successfully used for regeneration of small arteries in Buerger's disease. The objective of this study is to examine the angiogenetic potential of autologous MNCs from bone marrow and peripheral blood implanted into the ischaemic digits from patients with connective tissue diseases. Three patients with systemic sclerosis, two with mixed connective tissue disease, and one with CREST syndrome were enrolled who had painful ischaemic digits with necrosis refractory to several vasodilators including intravenous prostaglandins. MNCs obtained from 7 ml/kg bone marrow blood and 400 ml peripheral blood were implanted into 20 different sites in palms and/or soles. The study was performed open-labelled. Pain in the numeric rating scale improved remarkably up to 1 month after implantation of bone marrow or peripheral MNCs to the same extent, although no significant differences were found in transcutaneous oxygen pressure and thermogram before and after the implantation. Bone marrow MNCs increased blood flow of the hand determined by intra-arterial digital subtraction angiography, while peripheral MNCs did not. Implantation of autologous MNCs from peripheral and bone marrow into the ischaemic digits was so effective in pain-relief and more clinical trials would be warranted to see whether this could be a new treatment modality for angiogenesis in connective tissue diseases as in Buerger's disease.

Rescue of Hematopoietic Stem Cells Following High-Dose Radiation Injury Using Ex Vivo Culture on Endothelial Monolayers

Military personnel are at significant risk for potentially lethal myeloablative injury secondary to nuclear accident, nuclear attack, or chemical weapons attack. In an attempt to develop culture conditions in which hematopoietic stem cells might be rescued from the effects of radiation, we irradiated (1,000 cGy split dose) 6-week-old C57BL/6 (Ly 5.1) and syngeneic C57BL/6 (Ly 5.2) mice and tested whether bone marrow mononuclear cells (MNCs) harvested postirradiation could be rescued via coculture in porcine microvascular endothelial cell (PMVEC) monolayers. We found that a subpopulation of bone marrow MNCs exposed to 1,000 cGy could be maintained and expanded over 10 days in a PMVEC culture (3.8-fold expansion), whereas liquid suspension culture did not maintain a significant number of hematopoietic cells postirradiation. Colony-forming assays demonstrated that murine MNCs exposed to 1,000 cGy did not give rise to granulocyte/macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-e), or CFU-Mix in 14-day cultures, whereas irradiated MNCs that were subsequently cultured in PMVEC-generated CFU-GM, BFU-e, and CFU-Mix at cloning efficiencies of 1.3%, 0.2%, and 0.2%, respectively. In survival studies, we found that 78% of mice that were irradiated and then transplanted with irradiated/PMVEC-expanded MNCs were alive at day 50, compared with 18% of irradiated control mice (p < 0.05). We also observed that mice transplanted with irradiated/PMVEC-expanded MNCs showed complete hematologic recovery. At 8 weeks post-transplant, we found evidence of Ly 5.1 donor cells in both the bone marrow and the spleen of the transplanted animals, but the levels of engraftment were low (range, 0-5.1%; mean, 1.9%). These results demonstrate that a subpopulation of bone marrow stem cells are capable of surviving the effects of high-dose radiation if these cells are placed in coculture with endothelial cell monolayers.

Non-T, non-B acute lymphocytic leukemia cells enhance the mitogen induced proliferation of normal lymphocytes

Peripheral blood and bone marrow mononuclear cells (MNC) from patients with untreated non-T, non-B acute lymphocytic leukemia (ALL) were assessed for capacity to respond to the mitogens phytohemagglutinin (PHA), pokeweed (PWM) and staphylococcal enterotoxin A (SEA). At low mitogen concentrations considerable reactivity was observed to PHA and SEA, but not to PWM by patient MNC including specimens comprised of at least 90% leukemic cells. The responding cells were identified in cell separation experiments and by surface marker analysis as residual normal T-cells. The non-T, non-B leukemic cells enhanced the mitogen induced proliferation of responding lymphocytes as determined by co-culture experiments in which 5 × 10 3 or 10 4 MNC from normal donors were cultured with feeder layers of 3 × 10 5 mitomycin C treated MNC from autologous or unrelated normal donors or from ALL patients. In contrast to MNC feeder layers from normal donors, feeder layers of non-T, non-B leukemic cells enhanced normal lymphocyte reactivity to PHA and SEA but not to PWM as determined by 3H-thymidine uptake and cell survival. The potentiating effect of non-T, non-B leukemic cells was not mediated entirely by an allogeneic interaction as shown by the demonstration that frozen-thawed non-T, non-B leukemic cells enhanced the mitogen response of autologous MNC obtained during remission. Mitomycin treated feeder layers of leukemia cells from patients with acute myelogenous leukemia as well as bone marrow MNC from a normal donor or from lymphoma patients without bone marrow involvement failed to enhance PHA and SEA reactivity of normal lymphocytes. In addition, leukemia cells from patients with T-cell ALL did not potentiate normal lymphocyte reactivity in contrast to non-T, non-B ALL cells, providing a functional distinction between these two subtypes of ALL.