Abstract 4020: The Novel TrkB Agonist, 7,8-Dihydroxyflavone Enhances Stem Cell Mobilization After Stroke.

Stroke ◽  
2012 ◽  
Vol 43 (suppl_1) ◽  
Author(s):  
J Mocco ◽  
Aqeela Afzal ◽  
Nagheme Thomas ◽  
Zuha Warraich ◽  
Jeffery Kleim
Keyword(s):  
Bone Marrow ◽  
Mononuclear Cells ◽  
Critical Role ◽  
Stroke Recovery ◽  
The Novel ◽  
Hematopoietic Stem ◽  
Post Surgery ◽  
Cell Mobilization ◽  
Neurotropic Factor ◽  
Oxidative Effects

Background: Increasing levels of circulating Hematopoietic Stem Cells (HSC)/Hematopoietic Progenitor Cells (HPC), bone marrow derived mononuclear cells that promote repair in areas of injury, have been demonstrated to correlate with improved neurological function following stroke, suggesting a potentially critical role for HSC/HPC’s in limiting stroke injury and/or facilitating stroke recovery. Flavonoids, found in plants and fruit, exert anti-oxidative effects. Recent studies have demonstrated that 7,8 Dihydroxyflavone (DHF) is a potent TrkB agonist mimicking Brain Derived Neurotropic Factor, thus making it a powerful potential tool for treating neurological disorders. Stromal Derived Growth Factor 1-Alpha (SDF1-A) along with its receptor CXCR4 is a potent chemo attractant released by areas of injury. SDF1-A has been shown to mobilize HSC/HPC from the bone marrow to the blood and lead to ‘homing’ of the cells to an area of injury. We investigated the effect of DHF on HSC/HPC function following cerebral ischemia. Methods: Ischemic damage was induced in adult male Long Evans hooded rats (350-400g) with a peri-MCA injection of the vasoconstriction peptide ET-1. The rats were sacrificed at 24 hours post surgery and their bone marrow and blood HSC/HPC enriched using nanoparticles tagged with LIN negative and CD90 markers. Results: Stroked animals showed an increase in bone marrow production of HSC/HPC versus control animals (31.9±7 versus 2±0.5, p<0.05). The mobilization of the HSC/HPC from the bone marrow to the blood was also significantly higher in the stroked animals versus control animals (43±19 versus 3.6±0.3, p<0.05). Following stroke, DHF pre-treated HSC/HPC’s demonstrated significantly improved migration along an SDF-1 gradient compared to controls (129±1.0 versus 108±1.15, p<0.05), despite the fact that DHF alone provided no independent migratory stimulus. Conclusions: The results suggest that DHF may be a viable compound to facilitate HSC/HPC migration post-stroke.

scholarly journals The Role of the Bone Marrow Microenvironment in the Response to Infection

2020 ◽  
Vol 11 ◽  
Author(s):  
Courtney B. Johnson ◽  
Jizhou Zhang ◽  
Daniel Lucas
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Immune Cells ◽  
Critical Role ◽  
Primary Source ◽  
Bone Marrow Microenvironment ◽  
Future Research ◽  
Multipotent Stem Cells ◽  
Hematopoietic Stem

Hematopoiesis in the bone marrow (BM) is the primary source of immune cells. Hematopoiesis is regulated by a diverse cellular microenvironment that supports stepwise differentiation of multipotent stem cells and progenitors into mature blood cells. Blood cell production is not static and the bone marrow has evolved to sense and respond to infection by rapidly generating immune cells that are quickly released into the circulation to replenish those that are consumed in the periphery. Unfortunately, infection also has deleterious effects injuring hematopoietic stem cells (HSC), inefficient hematopoiesis, and remodeling and destruction of the microenvironment. Despite its central role in immunity, the role of the microenvironment in the response to infection has not been systematically investigated. Here we summarize the key experimental evidence demonstrating a critical role of the bone marrow microenvironment in orchestrating the bone marrow response to infection and discuss areas of future research.

scholarly journals Bone Marrow Homeostasis Is Impaired via JAK/STAT and Glucocorticoid Signaling in Cancer Cachexia Model

Cancers ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 1059
Author(s):  
Jinyeong Yu ◽  
Sanghyuk Choi ◽  
Aran Park ◽  
Jungbeom Do ◽  
Donghyun Nam ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Animal Model ◽  
Bone Loss ◽  
Cancer Cachexia ◽  
Mononuclear Cells ◽  
Bone Homeostasis ◽  
Bone Marrow Mscs ◽  
Hematopoietic Stem ◽  
Glucocorticoid Signaling

Cancer cachexia is a multifactorial systemic inflammation disease caused by complex interactions between the tumor and host tissues via soluble factors. However, whether cancer cachexia affects the bone marrow, in particular the hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs), remains unclear. Here, we investigated the bone marrow and bone in a cancer cachexia animal model generated by transplanting Lewis lung carcinoma cells. The number of bone marrow mononuclear cells (BM-MNCs) started to significantly decrease in the cancer cachectic animal model prior to the discernable loss of muscle and fat. This decrease in BM-MNCs was associated with myeloid skewing in the circulation and the expansion of hematopoietic progenitors in the bone marrow. Bone loss occurred in the cancer cachexia animal model and accompanied the decrease in the bone marrow MSCs that play important roles in both supporting HSCs and maintaining bone homeostasis. Glucocorticoid signaling mediated the decrease in bone marrow MSCs in the cancer cachectic environment. The cancer cachexia environment also skewed the differentiation of the bone marrow MSCs toward adipogenic fate via JAK/STAT as well as glucocorticoid signaling. Our results suggest that the bone loss induced in cancer cachexia is associated with the depletion and the impaired differentiation capacity of the bone marrow MSCs.

scholarly journals Enrichment of Rabbit Primitive Hematopoietic Cells via MACS Depletion of CD45+ Bone Marrow Cells

2021 ◽  
Vol 7 (1) ◽  
pp. 11
Author(s):  
Jaromír Vašíček ◽  
Andrej Baláži ◽  
Miroslav Bauer ◽  
Andrea Svoradová ◽  
Mária Tirpáková ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mononuclear Cells ◽  
Bone Marrow Cells ◽  
Quantitative Polymerase Chain Reaction ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Rabbit Bone ◽  
Hematopoietic Disorders ◽  
Novel Strategy

Hematopoietic stem and progenitor cells (HSC/HPCs) of human or few animal species have been studied for over 30 years. However, there is no information about rabbit HSC/HPCs, although they might be a valuable animal model for studying human hematopoietic disorders or could serve as genetic resource for the preservation of animal biodiversity. CD34 marker is commonly used to isolate HSC/HPCs. Due to unavailability of specific anti-rabbit CD34 antibodies, a novel strategy for the isolation and enrichment of rabbit HSC/HPCs was used in this study. Briefly, rabbit bone marrow mononuclear cells (BMMCs) were sorted immunomagnetically in order to remove all mature (CD45+) cells. The cells were depleted with overall purity about 60–70% and then cultured in a special medium designed for the expansion of CD34+ cells. Quantitative Polymerase Chain Reaction (qPCR) analysis confirmed the enrichment of primitive hematopoietic cells, as the expression of CD34 and CD49f increased (p < 0.05) and CD45 decreased (p < 0.001) at the end of culture in comparison to fresh BMMCs. However, cell culture still exhibited the presence of CD45+ cells, as identified by flow cytometry. After gating on CD45− cells the MHCI+MHCII−CD38+CD49f+CD90−CD117− phenotype was observed. In conclusion, rabbit HSC/HPCs might be isolated and enriched by the presented method. However, further optimization is still required.

scholarly journals T Cell Subsets and Associated Cytokines in Chronic Graft-Versus-Host-Disease Using G-CSF Primed Bone Marrow in Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4580-4580
Author(s):  
Monica M Rivera Franco ◽  
Eucario Leon Rodriguez ◽  
Diana Gomez Martin ◽  
Javier Merayo Chalico ◽  
Jorge Alcocer Varela
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Th17 Cells ◽  
Mononuclear Cells ◽  
Chronic Gvhd ◽  
The Other ◽  
Hematopoietic Stem ◽  
Graft Versus Host ◽  
Ifn Γ

Abstract Background Graft versus host disease (GVHD) is the major complication of allogeneic hematopoietic stem cell transplantation. It is characterized by an imbalance between the effector and regulatory arms of the immune system which results in the over production of inflammatory cytokines. Regulatory T (T regs) cells and T helper 17 (Th17) cells are two recently described lymphocyte subsets with opposing actions. Both can develop from naïve CD4+ T cell precursors under the influence of TGFβ1. Th17 lymphocytes, are key effector cells in rodent models of human diseases including GVHD. The other subset, T regs, is essential for dominant immunologic tolerance. At our institution, patients transplanted using G-CSF primed bone marrow (G-BM), have a lower incidence of acute and chronic GVHD when compared to those transplanted with peripheral blood and not primed bone marrow. Some microenvironment characteristics of this hematopoietic stem cells (HSC) source remain unknown, as well as the difference between Tregs, Th17 and cytokine levels in patients who develop GVHD and those who do not. Objective To analyze the characteristics of thirty-eight G-BM donor samples, identifying lymphocytes subsets and associated cytokines, and comparing patients who developed chronic GVHD (cGVHD) and those who did not. Materials and Methods A prospective analysis was performed in 38 G-BM samples from donors from 1999 to 2016. Mononuclear cells were defrosted, counted, and viability was evaluated. A 24 hour resting with RPMI, and posterior activation with PMA (50 ng/ml) for 48 hours was performed. Cells were harvested and cytokines were evaluated by flow cytometry (CBA assay). From each sample, one million mononuclear cells were permeabilized, fixed, and stained with CD4-FITC, IL17A-PE, IFN-γ APC, and IL-4 PECy7, for their posterior phenotipication by flow cytometry. The samples were obtained in a BD LSR Fortessa cytometry, and analyzed with the Flow-Jo software. Patients (recipients) information was analyzed using SPSS v.21. Results GVHD incidence was reported as following: Three (8%) patients developed acute GVHD (2 grade II, and 1 grade IV), 11 patients (29%) developed chronic GVHD (9% extensive, and 91% limited), and 24 patients did not present either. Mononuclear cells from G-BM from donors of patients who developed cGVHD showed a pro inflammatory response, characterized by an increased concentration of IL-17A (15.5 vs 0.71 pg/mL, p=0.013), TNF-α (80.27 vs 0.13 pg/mL, p=0.001), and IL-6 (4953.6 vs 11.75 pg/mL, p=0.025), after a mitogenic stimulation, compared to cells from donors of patients who did not developed GVHD. On the other hand, a decreased IL-10 production (2.62 vs 52.81 pg/mL, p=0.001) was documented in mononuclear cells from donors of patients who developed chronic GVHD, compared to donor cells of patients who did not. No significant difference in the production of IL-2, IL-4, and IFN-γ was observed. There was no difference in Th1 and Th2 between both groups, but mononuclear cells from donors of patients who developed chronic GVHD had a higher percentage of Th17 (1.02% vs 0.46%, p<0.001), and less Tregs (0.88% vs 1.95%, p<0.001), compared to those who did not developed GVHD. Conclusions Patients who develop cGVHD (29%) are characterized by a pro inflammatory response with an increased production of IL-17A, IL-6, and IFN-γ, and also a major percentage of Th17 cells. Also, a decreased suppressive response was documented with reduced IL-10 and Tregs levels. The low incidence of cGVHD show that G-CSF primed bone marrow is an excellent source for allogeneic HSC transplantations, and would be useful to compare these results with other HSC sources. Disclosures No relevant conflicts of interest to declare.

Abstract 13: Plasminogen Is Required for Hematopoietic Stem Cell-Mediated Cardiac Repair After Myocardial Infarction

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Yanqing Gong ◽  
Jane Hoover-Plow ◽  
Ying Li
Keyword(s):  
Myocardial Infarction ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Tissue Repair ◽  
Critical Role ◽  
Cardiac Repair ◽  
Internal Diameter ◽  
Hematopoietic Stem

Ischemic heart disease, including myocardial infarction (MI), is the primary cause of death throughout the US. Granulocyte colony-stimulating factor (G-CSF) is used to mobilize hematopoietic progenitor and stem cells (HPSC) to improve cardiac recovery after MI. However, poor-mobilization to G-CSF is observed in 25% of patients and 10-20% of healthy donors. Therefore, a better understanding of the underlying mechanisms regulating G-CSF-induced cardiac repair may offer novel approaches for strengthening stem cell-mediated therapeutics. Our previous studies have identified an essential role of Plg in HPSC mobilization from bone marrow (BM) in response to G-CSF. Here, we investigate the role of Plg in G-CSF-stimulated cardiac repair after MI. Our data show that G-CSF significantly improves cardiac tissue repair including increasing neovascularization in the infarct area, and improving ejection fraction and LV internal diameter by echocardiogram in wild-type mice. No improvement in tissue repair and heart function by G-CSF is observed in Plg -/- mice, indicating that Plg is required for G-CSF-regulated cardiac repair after MI. To investigate whether Plg regulates HPSC recruitment to ischemia area, bone marrow transplantion (BMT) with EGFP-expressing BM cells was performed to visualize BM-derived stem cells in infarcted tissue. Our data show that G-CSF dramatically increases recruitment of GFP+ cells (by 16 fold) in WT mice but not in Plg -/- mice, suggesting that Plg is essential for HPSC recruitment from BM to the lesion sites after MI. In further studies, we investigated the role of Plg in the regulation of SDF-1/CXCR-4 axis, a major regulator for HPSC recruitment. Our results show that G-CSF significantly increases CXCR-4 expression in infarcted area in WT mice. While G-CSF-induced CXCR-4 expression is markedly decreased (80%) in Plg -/- mice, suggesting Plg may regulate CXCR-4 expression during HSPC recruitment to injured heart. Interestingly, Plg does not affect SDF-1 expression in response to G-CSF treatment. Taken together, our findings have identified a critical role of Plg in HSPC recruitment to the lesion site and subsequent tissue repair after MI. Thus, targeting Plg may offer a new therapeutic strategy to improve G-CSF-mediated cardiac repair after MI.

scholarly journals SETDB1 mediated histone H3 lysine 9 methylation suppresses MLL-fusion target expression and leukemic transformation

Haematologica ◽  
2019 ◽  
Vol 105 (9) ◽  
pp. 2273-2285 ◽  
Author(s):  
James Ropa ◽  
Nirmalya Saha ◽  
Hsiangyu Hu ◽  
Luke F. Peterson ◽  
Moshe Talpaz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Target Genes ◽  
Critical Role ◽  
High Expression ◽  
Leukemia Cells ◽  
Biological Impact ◽  
Hematopoietic Stem ◽  
Acute Myeloid

Epigenetic regulators play a critical role in normal and malignant hematopoiesis. Deregulation, including epigenetic deregulation, of the HOXA gene cluster drives transformation of about 50% of acute myeloid leukemia. We recently showed that the Histone 3 Lysine 9 methyltransferase SETDB1 negatively regulates the expression of the pro-leukemic genes Hoxa9 and its cofactor Meis1 through deposition of promoter H3K9 trimethylation in MLL-AF9 leukemia cells. Here, we investigated the biological impact of altered SETDB1 expression and changes in H3K9 methylation on acute myeloid leukemia. We demonstrate that SETDB1 expression is correlated to disease status and overall survival in acute myeloid leukemia patients. We recapitulated these findings in mice, where high expression of SETDB1 delayed MLL-AF9 mediated disease progression by promoting differentiation of leukemia cells. We also explored the biological impact of treating normal and malignant hematopoietic cells with an H3K9 methyltransferase inhibitor, UNC0638. While myeloid leukemia cells demonstrate cytotoxicity to UNC0638 treatment, normal bone marrow cells exhibit an expansion of cKit+ hematopoietic stem and progenitor cells. Consistent with these data, we show that bone marrow treated with UNC0638 is more amenable to transformation by MLL-AF9. Next generation sequencing of leukemia cells shows that high expression of SETDB1 induces repressive changes to the promoter epigenome and downregulation of genes linked with acute myeloid leukemia, including Dock1 and the MLL-AF9 target genes Hoxa9, Six1, and others. These data reveal novel targets of SETDB1 in leukemia that point to a role for SETDB1 in negatively regulating pro-leukemic target genes and suppressing acute myeloid leukemia.

Shp-2 Heterozygous Hematopoietic Stem Cells Have Deficient Repopulating Activity Due to a Self-Renewal Defect.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1690-1690
Author(s):  
Rebecca J. Chan ◽  
Yanjun Li ◽  
Chris Shelley ◽  
Mervin C. Yoder
Keyword(s):  
Bone Marrow ◽  
Mononuclear Cells ◽  
Tyrosine Phosphatase ◽  
Embryonic Stem ◽  
Low Density ◽  
Loss Of Function ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Split Dose ◽  
Significant Difference

Abstract The protein tyrosine phosphatase, Shp-2, has been shown to be necessary for normal hematopoiesis based on embryonic stem (ES) cell-based assays; however, due to the early lethality of the homozygous Shp-2 mutant mice (Shp-2−/−) the role of Shp-2 in adult hematopoietic stem cell (HSC) function has never been examined. The Shp-2 heterozygous mice (Shp-2+/−) bear a mutant allele of the Shp-2 gene resulting in the production of a mutant protein lacking amino acids 46–110, which confers a loss of function. To test the hypothesis that Shp-2 is required for normal HSC activity, we compared the competitive repopulating ability of Shp-2+/− bone marrow-derived cells with WT cells. Total adult bone marrow low density mononuclear cells were isolated from Shp-2+/− and WT littermate controls (test cells, C57Bl/6 background, CD45.2+), mixed with a common pool of competitor (comp) cells (BoyJ background, CD45.1+), and administered to lethally irradiated (1100 cGy split dose) Gpi/BoyJ recipients. Based on peripheral blood chimerism, the repopulating ability of the Shp-2+/− cells was significantly lower than that of the WT cells (Figure 1, *p<0.0001 Shp-2+/− v. WT at ratio 1:2; **p=0.001 Shp-2+/− v. WT at ratio 1:1). We next converted the chimerism to repopulating units using the formula [competitor number x 105] X [% 45.2]/100 − [% 45.2] to quantitatively asses the repopulating defect in Shp-2+/− HSCs. We observed that the repopulating units of the Shp-2+/− cells was approximately 3-fold lower than that of the WT cells at both cell doses administered (Figure 2, *p=0.003 Shp-2+/− v. WT at ratio 1:2; **p=0.03 comparing Shp-2+/− v. WT at ratio 1:1). Multi-lineage analysis using two color fluorescence cytometry revealed a significantly lower contribution of Shp-2+/− cells to all lineages tested (B220, GR1, Mac, and CD4/8) compared to WT cells. As Shp-2 has been shown to participate in cell migration, we sought to rule out a homing deficiency of the Shp-2+/− HSCs. We performed short term homing assays and observed no difference in spleen-homed or bone marrow-homed Shp-2+/− and WT lin- cells twenty hours following transplantation. To evaluate self-renewal potential, we conducted serial transplantation experiments. Total bone marrow low density mononuclear cells were isolated from primary or seconary recipient mice with equal chimerism and transplanted into lethally irradiated (1100 cGy split dose) Gpi/BoyJ recipients. While no significant difference was observed between Shp-2+/− and WT engraftement in secondary transplants, eight weeks following tertiary transplantation, engraftment of the Shp-2+/− cells is significantly lower than that of the WT cells (WT 68.9% +/− 9.5 v. Shp-2+/− 26.1% +/− 11.7, n=6, p<0.0001) suggesting that a self-renewal defect contributes to the decreased HSC activity of the Shp-2+/− cells. These data demonstrate that Shp-2 function is not only necessary within the progenitor compartment to support proficient hematopoiesis, but is also needed within the HSC compartment to support normal HSC self-renewal. These findings provide insight into how oncogenic Shp-2 potentially may contribute to the dysregulation of hematopoiesis and the pathogenesis of childhood leukemias.

Differential Role for VLA-5 in Mobilization of Heamotopoieic Stem Cells Toward Peripheral Blood and Homing to Bone Marrow and Spleen.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2190-2190 ◽  
Author(s):  
Pieter K. Wierenga ◽  
Ellen Weersing ◽  
Bert Dontje ◽  
Gerald de Haan ◽  
Ronald P. van Os
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Hematopoietic Stem ◽  
Late Antigen ◽  
Cell Mobilization

Abstract Adhesion molecules have been implicated in the interactions of hematopoietic stem and progenitor cells with the bone marrow extracellular matrix and stromal cells. In this study we examined the role of very late antigen-5 (VLA-5) in the process of stem cell mobilization and homing after stem cell transplantation. In normal bone marrow (BM) from CBA/H mice 79±3 % of the cells in the lineage negative fraction express VLA-5. After mobilization with cyclophosphamide/G-CSF, the number of VLA-5 expressing cells in mobilized peripheral blood cells (MPB) decreases to 36±4%. The lineage negative fraction of MPB cells migrating in vitro towards SDF-1α (M-MPB) demonstrated a further decrease to 3±1% of VLA-5 expressing cells. These data are suggestive for a downregulation of VLA-5 on hematopoietic cells during mobilization. Next, MPB cells were labelled with PKH67-GL and transplanted in lethally irradiated recipients. Three hours after transplantation an increase in VLA-5 expressing cells was observed which remained stable until 24 hours post-transplant. When MPB cells were used the percentage PKH-67GL+ Lin− VLA-5+ cells increased from 36% to 88±4%. In the case of M-MPB cells the number increased from 3% to 33±5%. Although the increase might implicate an upregulation of VLA-5, we could not exclude selective homing of VLA-5+ cells as a possible explanation. Moreover, we determined the percentage of VLA-5 expressing cells immediately after transplantation in the peripheral blood of the recipients and were not able to observe any increase in VLA-5+ cells in the first three hours post-tranpslant. Finally, we separated the MPB cells in VLA-5+ and VLA-5− cells and plated these cells out in clonogenic assays for progenitor (CFU-GM) and stem cells (CAFC-day35). It could be demonstared that 98.8±0.5% of the progenitor cells and 99.4±0.7% of the stem cells were present in the VLA-5+ fraction. Hence, VLA-5 is not downregulated during the process of mobilization and the observed increase in VLA-5 expressing cells after transplantation is indeed caused by selective homing of VLA-5+ cells. To shed more light on the role of VLA-5 in the process of homing, BM and MPB cells were treated with an antibody to VLA-5. After VLA-5 blocking of MPB cells an inhibition of 59±7% in the homing of progenitor cells in bone marrow could be found, whereas homing of these subsets in the spleen of the recipients was only inhibited by 11±4%. For BM cells an inhibition of 60±12% in the bone marrow was observed. Homing of BM cells in the spleen was not affected at all after VLA-5 blocking. Based on these data we conclude that mobilization of hematopoietic progenitor/stem cells does not coincide with a downregulation of VLA-5. The observed increase in VLA-5 expressing cells after transplantation is caused by preferential homing of VLA-5+ cells. Homing of progenitor/stem cells to the bone marrow after transplantation apparantly requires adhesion interactions that can be inhibited by blocking VLA-5 expression. Homing to the spleen seems to be independent of VLA-5 expression. These data are indicative for different adhesive pathways in the process of homing to bone marrow or spleen.

Enrichment of CD34+ Hematopoietic Stem and Progenitor Cells from Human Bone Marrow Using a P-Selectin-Coated Microtube.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1219-1219
Author(s):  
Srinivas D. Narasipura ◽  
Jane L. Liesveld ◽  
Joel C. Wojciechowski ◽  
Nichola Charles ◽  
Karen Rosell ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Adhesion Molecule ◽  
Mononuclear Cells ◽  
Single Cells ◽  
Bone Marrow Mononuclear Cells ◽  
Cell Capture ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Coated Surfaces

Abstract Enrichment and purification of hematopoietic stem and progenitor cells (HSPCs) is important in transplantation therapies for hematological disorders and for basic stem cell research. Primitive CD34+ HSPCs have demonstrated stronger rolling adhesion than mature CD34- mononuclear cells on selectins (Blood2000; 95:478–486). We have exploited this differential rolling behavior to capture and purify HSPCs from bone marrow, by perfusing mononuclear cells through selectin-coated microtubes. Bone marrow mononuclear cells were perfused through the cell capture microtubes coated with adhesion molecules. These utilized a parallel plate flow chamber (Glycotech), and the P-selectin was adsorbed with laboratory tubing of appropriate lengths attached to the inlet, outlet, and vacuum ports of the gasket chamber. After perfusion, the device lumen was washed and captured cells were visualized and estimated by video microscopy. “Rolling” cells were defined as cells translating at less than 50% of the calculated hydrodynamic free stress velocity. Velocities of single cells were determined using a MATLAB program designed to measure the change in position of the cell centroid in a given time period. Adherent cells were eluted by high shear, calcium free buffer and air embolism. Immunofluorescence staining followed by flow cytometry was used to analyze CD34+ HSPCs. CD34+ HSPC purity of cells captured in adhesion molecule-coated devices was significantly higher than the fraction of CD34+ cells found in bone marrow- mononuclear cells (2.5 ± 0.8%). P-selectin coated surfaces yielded 16–20% CD34+ cell purity, while antibody coated surfaces yielded 12–18%. Although the CD34+ cell purities were comparable between selectin and antibody surfaces, the total number of CD34+ HSPCs captured was significantly higher in P-selectin devices (∼5.7–7.1 × 104) when compared to the antibody device (∼1.74–2.61 × 104). Furthermore, analysis for cells positive for CD133, a surface marker for more primitive HSPCs, depicted approximately 10–14 fold enrichment in P-selectin samples over control bone marrow mononuclear cells. The captured cells were viable and exhibited in vitro colony forming capabilities. Thus, P-selectin can be used in a compact flow device to capture and enrich HSPCs. This study supports the hypothesis that flow-based adhesion molecule-mediated capture may be a viable physiologic approach to the capture and purification of HSPCs.

G-CSF Mediated Decrease in Bone-Marrow SDF-1 Level Is Neutrophil Dependent but CD26 Independent

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3469-3469
Author(s):  
Pratibha Singh ◽  
Seiji Fukuda ◽  
Janardhan Sampath ◽  
Louis M. Pelus
Keyword(s):  
Bone Marrow ◽  
Magnetic Beads ◽  
Critical Role ◽  
Alternative Mechanism ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Osteoblast Activity

Abstract Interaction of CXCR4 expressed on hematopoietic stem and progenitor cells (HSPC) with bone-marrow stromal SDF-1 is believed to play a central role in retention or mobilization of HSPC. Recently, a mobilization regimen of G-CSF was shown to decrease osteoblast number resulting in reduced levels of bone-marrow SDF-1, however the detailed mechanism leading to this reduction is currently unknown. It is unlikely that G-CSF directly regulates osteoblast SDF-1 production since osteoblasts do not express G-CSF receptor. Proteolytic cleavage of SDF-1 by peptidase CD26 in the bone-marrow may be an alternative mechanism responsible for reduction of SDF-1 level. Although CD26 can cleave SDF-1 in vitro, direct evidence of SDF-1 cleavage by CD26 in vivo during G-CSF induced HSPC mobilization has not been demonstrated. We previously demonstrated that neutrophils are required for G-CSF induced HSPC mobilization and that CD26 expression on neutrophils, rather than HSPC, is critical for mobilization. To more fully understand the role of CD26 in altering SDF-1 protein/activity during G-CSF induced HSPC mobilization, we quantitated bone-marrow SDF-1 levels in CD26−/− and wild-type CD26+/+ mice by ELISA during G-CSF administration. A standard 4 day G-CSF mobilization regimen (100 μg/kg bid, sc × 4 days) decreased bone-marrow total SDF-1 from 4.55±0.3 to 0.52±0.06 ng/femur in wild-type CD26+/+ mice (8.7-fold) and from 4.51±0.3 to 0.53±0.05 ng/femur (8.5-fold) in CD26−/− mice. However, despite an equivalent decrease in SDF-1, total CFU mobilization and the absolute number of mobilized SKL cells were decreased (3.1 and 2.0 fold lower, respectively) in CD26−/− mice compared to wild-type CD26+/+ controls. These results suggest that the decrease in total SDF-1 level in marrow seen following G-CSF treatment is independent of CD26. Cytological examination of bone-marrow smears showed that the reduction in SDF-1 levels in bone-marrow of both wild-type CD26+/+ and CD26−/− mice following G-CSF administration correlated with an increase in total absolute bone-marrow neutrophil cell number, suggesting a role for neutrophils in modulation of SDF-1 protein. To determine if neutrophils affect osteoblast SDF-1 production, bone marrow Gr-1+ neutrophils from wild-type CD26+/+ and CD26−/− mice were purified using anti-Ly6G magnetic beads and co-cultured with MC3T3-E1 preosteoblasts in vitro. Gr-1+ neutrophils from both wild-type and CD26−/− mice decreased pre-osteoblast SDF-1 production by similar amounts (15.4-fold vs 14.8-fold respectively), while Gr-1 neg cells from both wild-type CD26+/+ or CD26−/− were without effect on SDF-1 levels. Similarly, Gr-1+ neutrophils from both wild-type and CD26−/− mice decreased SDF-1 produced by MC3T3-E1-derived osteoblasts from 1.85±0.3 to 0.52±0.06 ng/ml (3.5 fold) and 0.56±0.07 ng/ml (3.3 fold) respectively, with Gr-1neg cells having no effect. Gr-1+ neutrophils either from wild-type or CD26−/− mice, but not Gr-1neg cells, significantly induced apoptosis of MC3T3-E1 cells as measured by Annexin-V staining (70.5%±10.2 vs 71.2%±12.5 for wild-type CD26+/+ and CD26−/− neutrophils respectively) and significantly inhibited osteoblast activity (20-fold vs 20.6-fold for CD26+/+ and CD26−/− neutrophils respectively) as measured by osteocalcin expression. Furthermore, irrespective of G-CSF treatment, an inverse correlation between absolute neutrophil number and SDF-1 protein levels was observed, suggesting that G-CSF induces neutrophil expansion but does not directly affect SDF-1 production. Collectively, these results provide additional support for the critical role of neutrophils in G-CSF induced mobilization and strongly suggested that neutrophils directly regulate bone-marrow SDF-1 levels independent of CD26 activity.

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