Neuropeptide Y Is Critical for Bone Marrow Stromal Cells and Hematopoietic Recovery Following Injury

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 173-173
Author(s):  
Pratibha Singh ◽  
Louis M. Pelus
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Stromal Cells ◽  
Nerve Fibers ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Hematopoietic Recovery ◽  
Donor Cells ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract Bone marrow suppression is the most common limiting side-effect of conventional cancer chemo/radio therapy and is the primary cause of morbidity/mortality after accidental exposure to a high dose of ionizing radiation. The mechanisms mediating radiation-induced hematopoietic stem and stromal cell dysfunction however are not well understood. Radiation therapy causes substantial sensory neuropathy in patients. Recent studies reveal that bone marrow cells are highly innervated by sympathetic nerve fibers and that chemotherapy induced nerve-damage can impair hematopoietic regeneration, suggesting a contribution of nerve fibers in the regulation of hematopoietic stem cell and stromal cell activities. Whether irradiation- mediated nerve injury is a crucial lesion that causes deficits in hematopoietic recovery is not known. We recently discovered that differential signaling from the neuropeptide Y (NPY) receptors on bone marrow endothelial cells regulates vascular permeability and stem cell egress. NPY is an important neurotransmitter of the sympathetic nervous system and the principal adreno-medullary hormone. In this study, we found that NPY is important for reconstitution of the bone marrow niche and hematopoietic regeneration following sublethal irradiation (650 cGy). The levels of NPY were significantly reduced in bone marrow of irradiated mice suggesting damage to nerve fibers. Treatment of wild-type mice with full length NPY (1µg/mouse/day) for 3 consecutive days after irradiation markedly reduced the loss of mesenchymal stem cells (CD45-Ter119-CD31-Nestin+PDGFR+CD51+), endothelial cells (CD45-Ter119-CD31+VE-cadherin+) and hematopoietic stem and progenitor cells (SLAM LSK and LSK) in the bone marrow and promoted faster hematopoietic recovery. In addition, pharmacological NPY treatment prevented irradiation mediated nerve fiber damage. In contrast, in NPY knockout mice, regeneration of CD45neg stromal cells, SLAM LSK and LSK cells after irradiation was significantly reduced compared to wild-type controls. This reduced hematopoietic recovery in NPY deficient mice following irradiation was associated with increased apoptosis/necrosis of stromal cells and hematopoietic stem and progenitor cells. We also examined whether NPY played an intrinsic or extrinsic role in stem cell homing. Wild-type or NPY deficient BM cells were transplanted into wild-type or NPY knockout recipients. Strikingly, the homing of wild-type donor cells into NPY deficient recipients or NPY knockout donor cells into wild-type recipients were both reduced. To explore whether NPY regulates human stem cells, we treated human cord blood CD34+ cells ex vivo with NPY for 3 days and evaluated cell expansion. Long-term culture assays demonstrated that NPY treatment enhanced the clonal expansion of CD34+ cells. In conclusion, our studies suggests that NPY plays both an intrinsic and extrinsic role in hematopoiesis and that NPY-mediated protection of the sympathetic nervous system within the bone marrow can facilitate stem cell niche regeneration and enhance regenerative hematopoiesis following irradiation/injury. Disclosures No relevant conflicts of interest to declare.

scholarly journals PTPN11D61Y/+; Vavcre+ Animals Commonly Succumb to Leukemia Relapse Despite Robust Engraftment of Transplanted Wild-Type Hematopoietic Stem and Progenitor Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 496-496
Author(s):  
Stefan P. Tarnawsky ◽  
Mervin C. Yoder ◽  
Rebecca J. Chan
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Donor Cells ◽  
Stem And Progenitor Cells ◽  
Leukemia Relapse ◽  
Conditioning Regimens

Juvenile Myelomonocytic Leukemia (JMML) is a rare childhood myelodysplastic / myeloproliferative overlap disorder. JMML exhibits myeloid populations with mutations in Ras-Erk signaling genes, most commonly PTPN11, which confer growth hypersensitivity to GM-CSF. While allogeneic hematopoietic stem cell transplant (HSCT) is the treatment of choice for children with JMML, 50% of children succumb to leukemia relapse; however, the mechanism leading to this high relapse rate is unknown. We hypothesized that the hyperinflammatory nature of JMML may damage the bone marrow microenvironment, leading to poor engraftment of normal donor cells following transplant, permitting residual leukemia cells to outcompete the normal graft, and thus promoting leukemia relapse. Using Vav1 promoter-directed Cre, we generated a mouse model of JMML that conditionally expresses gain-of-function PTPN11D61Yin utero during development. While PTPN11D61Y/+; VavCre+embryos did not demonstrate in utero lethality, we observed a modest reduction of PTPN11D61Y/+; VavCre+ mice at the time of weaning compared to predicted Mendelian frequencies. Further, surviving PTPN11D61Y/+; VavCre+ mice developed elevated peripheral blood leukocytosis and monocytosis as early as 4 weeks of age compared to PTPN11+/+; VavCre+ controls. To address the hypothesis that an aberrant bone marrow microenvironment in the PTPN11D61Y/+ mice leads to poor engraftment of wild-type donor cells following transplant, we examined engraftment of wild-type hematopoietic stem and progenitor cells (HSPCs) in the PTPN11D61Y/+; VavCre+ mice and monitored animals for disease relapse. 16-24 week-old diseased PTPN11D61Y/+; VavCre+ and control PTPN11+/+; VavCre+ mice were lethally irradiated (11 Gy split dose) and transplanted with 5 x 105 CD45.1+ wild-type bone marrow low density mononuclear cells (LDMNCs), which simulates a limiting stem cell dose commonly available in a human HSCT setting. 6 weeks post-HSCT, PTPN11D61Y/+; VavCre+recipients demonstrated an unexpected elevated CD45.1+ donor cell contribution in peripheral blood compared to the control PTPN11+/+; VavCre+ recipients. However, despite superior engraftment in the PTPN11D61Y/+; VavCre+ recipients, these mice had a significantly shorter median survival post-HSCT due to a resurgence of recipient CD45.2-derived leukemic cells. We repeated the experiment using a high dose of CD45.1+ LDMNCs (10 x 106 cells) to determine if providing a saturating dose wild-type cells could prevent the relapse of recipient-derived leukemogenesis and normalize the survival of the PTPN11D61Y/+; VavCre+recipients. While this saturating dose of wild-type cells resulted in high peripheral blood chimerism in both the PTPN11D61Y/+; VavCre+ and PTPN11+/+; VavCre+ recipients, the PTPN11D61Y/+; VavCre+ animals nevertheless demonstrated significantly reduced overall survival. When we examined the cause of mortality in the HSCT-treated PTPN11D61Y/+; VavCre+mice, we found enlarged spleens, hypercellular bone marrow, and enlarged thymuses. Flow cytometry revealed that the majority of cells in the peripheral blood, bone marrow, and spleen were recipient-derived CD45.2+ CD4+ CD8+ T cells. To verify that the disease was neoplastic in origin, secondary transplants into CD45.1/.2 recipients were performed from two independent primary PTPN11D61Y/+; VavCre+and two independent primary PTPN11+/+; VavCre+ controls. Secondary recipients of bone marrow from PTPN11D61Y/+; VavCre+ animals rapidly succumbed to a CD45.2-derived T-cell acute lymphoid leukemia (T-ALL). Previous studies demonstrated that wild-type PTPN11 is needed to protect the integrity of the genome by regulating Polo-like kinase 1 (Plk1) during the mitosis of the cell cycle (Liu et al., PNAS, 2016). We now demonstrate that even when PTPN11 mutant animals are provided with saturating doses of wild-type HSCs, dysregulated residual recipient cells are able to produce relapsed disease. Collectively, these studies highlight the propensity of residual mutant PTPN11 cells to transform after being subjected to mutagenic agents that are commonly used for conditioning regimens prior to allogeneic HSCT. These findings suggest that modified pre-HSCT conditioning regimens bearing reduced mutagenicity while maintaining adequate cytoreductive efficacy may yield lower post-HSCT leukemia relapse in children with PTPN11mutations. Disclosures No relevant conflicts of interest to declare.

Muc1 Expression Identifies Human Self-Renewing Stem/Progenitor Populations and Is Elevated in AML CD34+ Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4040-4040
Author(s):  
Szabolcs Fatrai ◽  
Simon M.G.J. Daenen ◽  
Edo Vellenga ◽  
Jan J. Schuringa
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Fold Increase ◽  
Marrow Stromal Cells ◽  
Muc1 Expression ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Mucin1 (Muc1) is a membrane glycoprotein which is expressed on most of the normal secretory epithelial cells as well as on hematopoietic cells. It is involved in migration, adhesion and intracellular signalling. Muc1 can be cleaved close to the membrane-proximal region, resulting in an intracellular Muc1 that can associate with or activate various signalling pathway components such as b-catenin, p53 and HIF1a. Based on these properties, Muc1 expression was analysed in human hematopoietic stem/progenitor cells. Muc1 mRNA expression was highest in the immature CD34+/CD38− cells and was reduced upon maturation towards the progenitor stage. Cord blood (CB) CD34+ cells were sorted into Muc1+ and Muc1− populations followed by CFC and LTC-IC assays and these experiments revealed that the stem and progenitor cells reside predominantly in the CD34+/Muc1+ fraction. Importantly, we observed strongly increased Muc1 expression in the CD34+ subfraction of AML mononuclear cells. These results tempted us to further study the role of Muc1 overexpression in human CD34+ stem/progenitor cells. Full-length Muc1 (Muc1F) and a Muc1 isoform with a deleted extracellular domain (DTR) were stably expressed in CB CD34+ cells using a retroviral approach. Upon coculture with MS5 bone marrow stromal cells, a two-fold increase in expansion of suspension cells was observed in both Muc1F and DTR cultures. In line with these results, we observed an increase in progenitor counts in the Muc1F and DTR group as determined by CFC assays in methylcellulose. Upon replating of CFC cultures, Muc1F and DTR were giving rise to secondary colonies in contrast to empty vector control groups, indicating that self-renewal was imposed on progenitors by expression of Muc1. A 3-fold and 2-fold increase in stem cell frequencies was observed in the DTR and Muc1F groups, respectively, as determined by LTC-IC assays. To determine whether the above mentioned phenotypes in MS5 co-cultures were stroma-dependent, we expanded Muc1F and DTR-transduced cells in cytokine-driven liquid cultures. However, no proliferative advantage or increase in CFC frequencies was observed suggesting that Muc1 requires bone marrow stromal cells. In conclusion, our data indicate that HSCs as well as AML cells are enriched for Muc1 expression, and that overexpression of Muc1 in CB cells is sufficient to increase both progenitor and stem cell frequencies.

Multicolor Flow Cytometry Allows Quantitative Analysis of Signal Transduction in Murine and Human Hematopoietic Stem and Progenitor Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5393-5393
Author(s):  
Tamara Riedt ◽  
Claudia Lengerke ◽  
Lothar Kanz ◽  
Viktor Janzen
Keyword(s):  
Signal Transduction ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Intracellular Signaling ◽  
Hematopoietic Stem ◽  
Specific Antibodies ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract The regulation of cell cycle activity, differentiation and self-renewal of stem cells are dependent on accurate processing of intrinsic and extrinsic signals. Traditionally, signaling pathway activation has been detected by immunobloting using phospho-specific antibodies. However, detection of signal transduction in rare cells within heterogeneous populations, such as hematopoietic stem and progenitor cells (HSC) has been difficult to achieve. In a recently reported approach to visualize signaling in selected single c-Kit+ Sca-1+ Lin− (KSL) bone marrow cells, cells were sorted onto glas slides by flow cytometry and signaling was detected by confocal fluorescence microscopy, a very time consuming method that thus restricts the number of cells that can be analysed simultaneously. Moreover it permits only qualitative, but not quantitative signaling evaluation (Yamazaki et al., EMBO J. 2006). Here, we report a new protocol allowing quantitative measurement of signaling activity in large numbers of defined murine and human hematopoietic cells. The cells are stained with established surface markers and then phospho-specific antibodies are used to detect the levels of active intracellular signaling molecules. Signals are quantified by flow cytometry fluorescence measurement. Importantly, the protocol developed in our laboratory enables preservation of surface marker staining identifying the cells of interest inspite the fixation and permeabilization procedures necessary for intracellular signaling detection. This applies also for antigens previously reported to be particularly vulnerable to standard fixation and permeabilization approaches (e.g. the murine stem cell markers c-Kit and Sca1). Thus, our protocol provides an easy and reliable method for quantifying the activation degree of several intracellular signaling pathways on single cell level in defined hematopoietic (stem) cells within the heterogeous bone marrow (BM) compartment. Using cytokines known to exert a biological effect on HSCs, we have examined the susceptibility of KSL murine BM cells and human BM CD34+ cells to cytokine-induced signaling. We have performed extensive dosage titration and time course analysis for multiple cytokines (SCF, TPO, Flt-3, IL-3, IL-6, Ang-1, SDF-1α, TGF-β, and BMP-4) and signaling pathways (ERK, Akt, p38MAPK, Jak-Stat, TGF-β/BMP-Smad) in murine KSL BM cells. The activation intensity and the duration of signal activity as measured by the expression of corresponding phosphorylated proteins were cytokine specific. The obtained results can be used as a platform to explore signaling alterations in distinct compartments of the hematopoietic system, and may provide mechanistical insights for observed bone marrow defects (e.g impaired ERK signaling pathway has been detected as a possible cause of hematopoietic defects in Caspase-3 mutant murine HSCs, Janzen et al, Cell Stem Cell 2008). Furthermore, we could show that the technique is also applicable to human BM cells and that the human hematopoietic stem cell marker CD34 is also preserved by our fixation and permeabilization protocol. Preliminary results suggest that cytokines induce similar signaling activation in human CD34+BM cells collected from healthy donors. As observed in mouse KSL BM cells, stimulation of human CD34+cells with human stem cell factor (hSCF) induced activation of the ERK but not the Akt pathway. Ongoing experiments analyse the stimulatory effects of other cytokines such as thrombopoietin (TPO) and fms-related tyrosine kinase 3 (Flt-3) and their corresponding pathways. Moreover, comparative studies are underway analyzing cross-reactivity between mouse and human cytokines, aiming to provide insights into cytokine-induced biases in commonly used xenotransplantation models.

scholarly journals EBF1 As a Novel Regulator of Bone Marrow Mesenchymal Stromal Progenitors

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 96-96
Author(s):  
Marta Derecka ◽  
Senthilkumar Ramamoorthy ◽  
Pierre Cauchy ◽  
Josip Herman ◽  
Dominic Grun ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Myeloid Cells ◽  
Bone Marrow Niche ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Abstract Hematopoietic stem and progenitor cells (HSPC) are in daily demand worldwide because of their ability to replenish entire blood system. However, the in vitro expansion of HSPC is still a major challenge since the cues from bone marrow microenvironment remain largely elusive. Signals coming from the bone marrow niche, and specifically mesenchymal stem and progenitor cells (MSPC), orchestrate maintenance, trafficking and stage specific differentiation of HSPCs. Although, it is generally accepted that MSPCs are essential for hematopoietic homeostasis and generating multiple types of stromal cells, the exact transcriptional networks regulating MSPCs are not well established. Early B-cell factor 1 (Ebf1) has been discovered as lineage-specific transcription factor governing B lymphopoiesis. Additionally, it has been shown to play important role in differentiation of adipocytes, which are a niche component supporting hematopoietic regeneration. Thus, in this study we seek to examine if Ebf1 has an alternative function in non-hematopoietic compartment of bone marrow, specifically in mesenchymal stromal cells that maintain proper hematopoiesis. Here, we identified Ebf1 as new transcription regulator of MSPCs activity. Mesenchymal progenitors isolated from Ebf1-/- mice show diminished capacity to form fibroblasticcolonies (CFU-F) indicating reduced self-renewal. Moreover, cells expanded from these colonies display impaired in vitro differentiation towards osteoblasts, chondrocytes and adipocytes. In order to test how this defective MSPCs influence maintenance of HSPCs, we performed long-term culture-initiating cell assay (LTC-IC). After 5 weeks of co-culture of Ebf1-deficient stromal cells with wild type HSPCs we could observe significantly decreased number of cobblestone and CFU colonies formed by primitive HSPCs, in comparison to co-cultures with control stromal cells. Furthermore, in vivo adoptive transfers of wild type HSPCs to Ebf1+/- recipient mice showed a decrease in the absolute numbers of HSPCs in primary recipients and reduced donor chimerism within the HSCP compartment in competitive secondary transplant experiments. Additionally, Prx1-Cre-mediated deletion of Ebf1 specifically in MSPCs of mice leads to reduced frequency and numbers of HSPCs and myeloid cells in the bone marrow. These results confirm that mesenchymal stromal cells lacking Ebf1 render insufficient support for HSPCs to sustain proper hematopoiesis. Interestingly, we also observed a reduced ability of HSPCs sorted from Prx1CreEbf1fl/fl mice to form colonies in methylcellulose, suggesting not only impaired maintenance but also hindered function of these cells. Moreover, HSPCs exposed to Ebf1-deficient niche exhibit changes in chromatin accessibility with reduced occupancy of AP-1, ETS, Runx and IRF motifs, which is consistent with decreased myeloid output seen in Prx1CreEbf1fl/fl mice. These results support the hypothesis that defective niche can cause epigenetic reprograming of HSPCs. Finally, single cell and bulk transcriptome analysis of MSPCs lacking Ebf1 revealed differences in the niche composition and decreased expression of lineage-instructive signals for myeloid cells. Thus, our study establishes Ebf1 as a novel regulator of MSPCs playing a crucial role in the maintenance and differentiation of HSPCs. Disclosures No relevant conflicts of interest to declare.

scholarly journals Etoposide-mediated interleukin-8 secretion from bone marrow stromal cells induces hematopoietic stem cell mobilization

2020 ◽  
Author(s):  
Ka-Won Kang ◽  
Seung-Jin Lee ◽  
Ji Hye Kim ◽  
Byung-Hyun Lee ◽  
Seok Jin Kim ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Hematopoietic Stem ◽  
Cell Counts ◽  
Cd34 Cells

Abstract Background This study assessed the mechanism of hematopoietic stem cell (HSC) mobilization using etoposide with granulocyte-colony stimulating factor (G-CSF) and determined how it differed from that using cyclophosphamide with G-CSF or G-CSF alone.Methods The study analyzed data from 173 non-Hodgkin’s lymphoma patients who underwent autologous peripheral blood stem cell transplantation (auto-PBSCT), in vitro experiments using HSCs and bone marrow stromal cells (BMSCs), and in vivo mouse model studies.Results The etoposide with G-CSF mobilization group showed the highest yield of CD34+ cells and the lowest change in white blood cell counts during mobilization. Etoposide triggered interleukin (IL)-8 secretion from BMSCs and caused long-term BMSC toxicity, which were not observed with cyclophosphamide treatment. The expansion of CD34+ cells cultured in BMSC-conditioned medium containing IL-8 was more remarkable than that without IL-8. The expression of CXCR2, mTOR, and cMYC in HSCs was gradually enhanced at 1, 6, and 24 h after IL-8 stimulation. In animal studies, the etoposide with G-CSF mobilization group presented stronger expression of IL-8-related cytokines and MMP9 and scantier expression of SDF-1 in the bone marrow, compared to the other groups not treated with etoposide.Conclusion Collectively, the unique mechanism of etoposide with G-CSF-mediated mobilization is associated with the secretion of IL-8 from BMSCs, causing the enhanced proliferation and mobilization of HSCs in the bone marrow, which was not observed in the mobilization using cyclophosphamide with G-CSF or G-CSF alone. Moreover, the long-term toxicity of etoposide to BMSC emphasizes the need for further studies to develop more efficient and safe chemo-mobilization strategies.

Mobilized Hematopoietic Stem Cell Yield Depends on Species-Specific Circadian Timing

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3494-3494
Author(s):  
Daniel Lucas ◽  
Michela Battista ◽  
Patricia A. Shi ◽  
Luis M. Isola ◽  
Paul S. Frenette
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Transplantation ◽  
Circadian Rhythms ◽  
Molecular Clock ◽  
Marrow Transplantation ◽  
Nerve Fibers ◽  
Blood Collection ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Hematopoietic stem and progenitor cell (HSPC) recovery after mobilization remains one of the major limiting factors to perform successful bone marrow transplantation (BMT) procedures since up to 60% of cancer patients fail to mobilize enough HSPC for BMT. We have recently shown that under steady-state conditions, HSPC are released from the bone marrow (BM) in a circadian manner, peaking in the circulation 5 hours after onset of light (Zeitgeber time 5, ZT5) and reaching a nadir at ZT13. The molecular clock controls local norepinephrine activity in the BM from sympathetic nerve fibers, which downregulates CXCL12 production by BM stromal cells via the β3 adrenergic receptor (Nature2008;452:442). Here, we show that HSPC circadian rhythms could be exploited to enhance HSPC mobilization. Mice mobilized with granulocyte colony-stimulating factor (G-CSF, 250μg/kg administered daily for 4 days) have significantly more circulating colony-forming progenitors (CFU-C) at ZT5 (2598 ± 236 CFU-C/ml of blood) than at ZT13 (1604 ± 188 CFU-C/ml of blood; p<0.01). The stem cell–enriched Lin−Sca1+c-kit+ (LSK) cell recovery was also increased at ZT5 (1.4-fold more; p<0.05). To test whether circadian variations also affected rapid mobilizers, we treated mice with the CXCR4 antagonist AMD3100 (5mg/kg, 1 hour before blood collection). We found that CFU-C recovery was significantly higher at ZT5 (517 ± 79 CFU-C/ml of blood) than ZT13 (316 ± 50 CFU-C/ml of blood; p<0.05). LSK cells elicited by AMD3100 were also more abundant at ZT5 than at ZT13 (1.8-fold; p<0.05). These results suggested that synchronization of blood collection with circadian rhythms can produce greater HSPC recovery. In contrast to G-CSF-induced mobilization, which triggers CXCL12 downregulation in the bone marrow, AMD3100 results cannot be explained by changes in CXCL12 levels as the compound antagonizes CXCR4 on HSPC. Analysis of CXCR4 expression on LSK cells revealed higher levels at ZT13 than ZT5 (1.5-fold increase; p<0.05). Moreover, CXCR4 oscillations were dependent on the molecular clock as they disappeared in mice lacking the clock gene Bmal-1. These results suggest that the coordinated expression of CXCL12 in the microenviroment and CXCR4 on HSPC regulate HPSC rhythms. To determine whether similar HSPC rhythms take place in humans, we determined the number of CD34+ cells or the more primitive CD34+CD38− cells in the blood of 9 healthy human subjects at 8:00AM and 8:00PM. Both CD34+ cells (2554 ± 242 cells/ml of blood at 8:00AM vs. 5887 ± 508 cells/ml of blood at 8:00PM; p<0.001) and CD34+CD38− cells (217 ± 42 cells/ml of blood at 8:00AM vs. 497 ± 53 cells/ml of blood at 8:00PM; p<0.001) were more abundant in the early night. Analysis of CFU-C progenitor cells in the blood of the same donors showed that these cells were also increased in human peripheral blood at 8:00 PM (143 ± 12 CFU-C/ml of blood) when compared with 8:00 AM (73 ± 12 CFU-C/ml of blood; p<0.001). These results thus demonstrate that HSPC circadian rhythms take place in humans and that they are phase-shifted when compared to mice, as predicted from the nocturnal behavior of mice. To determine whether human HSPC rhythms could be used to enhance mobilization yield, we performed a retrospective data analysis of mobilization efficiency in 82 healthy donors that underwent G-CSF-induced mobilization for allogeneic bone marrow transplantation between 2000 and 2006 at the Mount Sinai Medical Center. In these donors, aphereses were performed in the morning and in the early afternoon. Hence, we divided the donors in two groups according to the collection half time: those between 10:00AM and 12:30PM and those between 12:30PM and 15:00PM. The mobilization yield (expressed as number of CD34+ cells per ml of blood processed per kg of donor weight) was significantly higher in patients mobilized in the afternoon (0.35 ± 0.02 CD34+ cells/mL/kg vs. 0.55 ± 0.05 CD34+ cells/mL/kg; p < 0.001). These results indicate that circulating human HSPC fluctuate in a phase-shifted circadian rhythm compared to that of the mouse. In both species, mobilization yield may be enhanced by synchronizing the collection time with the optimal circadian time. Although this issue needs to be tested in a prospective clinical trial, these data indicate that a simple change in the apheresis time may have a significant clinical impact.

Role of TLR4 In Acute Gvhd After Allogenetic Hematopoietic Stem Cell Transplantation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2538-2538
Author(s):  
Yi Zhao ◽  
Qiuyan Liu ◽  
Donghua He ◽  
Lijuan Wang ◽  
Jun Tian ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
Acute Gvhd ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Donor Cells

Abstract Abstract 2538 Graft-versus-host disease (GVHD) is the most common complication after hematopoietic stem cell transplantation (HSCT). Lipopolysaccharide (LPS) has been implicated in the pathogenesis of GVHD. The toll-like receptor-4 (TLR4) has been identified as a major receptor for LPS. Here arises the question whether TLR4 mutations may increase risk of microbial infection and affect acute GVHD in allogeneic HSCT recipients. In order to clarify the role of TLR4 in the occurrence of acute GVHD, we detected the interaction of TLR4 mutations in recipient and donor cells and analyzed allogeneic lymphocyte infiltration in the liver, intestine and skin of host mice by immunohistochemistry after allogeneic HSCT. Wild type C57BL/6 (TLR4+/+) and TLR4 knockout (TLR4−/−) mice were received myeloablative total body irradiation, followed by tail vein injection of donor BALB/c bone marrow cells and splenocytes to induce acute GVHD. GVHD severity was assessed using clinical scores. In vivo the proliferation activity of allogeneic donor BALB/c T cells in TLR4−/− and TLR4+/+ transplanted mice was evaluated ex vivo by flow cytometry after labeling with CFSE. Mixed lymphocyte reaction (MLR) assays were performed to evaluate the proliferation of allogeneic donor BALB/c T cells at different times of coculture with MHC class II antigen presenting cells (APCs) obtained from bone marrow of TLR4+/+ or TLR4−/− mice with or without LPS stimulation for 24 h. When myeloablative irradiated TLR4−/− mice, instead of wild-type mice, were used as graft recipients, clinical score of acute GVHD severity were decreased and survival were increased (18/30 vs 9/30 mice still alive at day 30, GVHD clinical score 6.7 vs 4.5). The decreased mortality and morbidity in TLR4−/− mice were associated with reduced proliferation of allogeneic donor cells transplanted in these mice.We evaluated the activation of spleen APCs in TLR4+/+ or TLR4−/− mice after myeloablative conditioning. Higher expression of CD80 and CD86 costimulatory molecules on MHC class II cells was detected in wide type strain at 3 d postirradiation. Ex vivo experiments CD80, CD86 and CD40 costimulatory markers on bone marrow APCs of C57BL/6 wild-type more significant up-regulation than TLR4−/− mice after LPS stimulation 24 h. TLR4−/− recipients receiving BALB/c donors developed significantly less GVHD as measured by liver, skin and intestinal of mice histopathology compared with TLR4+/+ recipients. Cytokines IL-2/IFN-γexpression in TLR4+/+ recipients mice serum was stronger but IL-4/IL-10 expression was weaker comparing to that in TLR4−/− recipients. These results suggest that TLR-4 mutation in donor cells increases the expression of Th2-related cytokines and decreases the risk of GVHD after allogeneic bone marrow transplantation.These data reveal that TLR4 mutations in recipitents is crucial in the prevention of GVHD, while responsiveness of wide type mice APC to LPS may be an important risk factor for acute GVHD. Overall together, these results suggest that the function of TLR4 has influence on the occurrence of acute GVHD, which might provide methods to reduce this complication after allogeneic hematopoietic stem cell transplantation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Multicolor Flowcytometry Analysis of Hematopoietic Stem and Progenitor Cells Subsets Among Basal and Mobilized Peripheral CD34+ Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5117-5117
Author(s):  
Valentina Giai ◽  
Elona Saraci ◽  
Eleonora Marzanati ◽  
Christian Scharenberg ◽  
Monica De Stefanis ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Healthy Volunteers ◽  
Progenitor Cells ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells ◽  
Progenitor Compartment

Abstract BACKGROUND: In the recent years, numerous studies based on multicolor flowcytometry have analyzed the different subpopulations of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) (Manz MG et al, PNAS 2002; Majeti R et al, Cell Stem Cell 2007): the common myeloid progenitors (CMPs: Lin-CD34+CD38+CD45RA-CD123+), the granulocyte-macrophage progenitors (GMPs: Lin-CD34+CD38+CD45RA+CD123+) and the megakaryocyte-erythroid progenitors (MEPs: Lin-CD34+CD38+CD45RA-CD123-) constitute the progenitor compartment, while the hematopoietic stem cells (HSCs: Lin-CD34+CD38- CD45RA-CD90+), the multipotent progenitors (MPPs: Lin-CD34+CD38- CD45RA-CD90-) and the lymphoid-myeloid multipotent progenitors (LMPPs: Lin-CD34+CD38- CD45RA+CD90-) represent the more immature HSPCs. In animal models, the progenitor compartment includes short-term repopulating cells, leading to the hematological recovery in the first 5 weeks after transplantation, whereas the stem cell compartment comprehends the long-term repopulation cells, responsible for the long-term hematological recovery. However, very little is known about the different subpopulations of HSPCs among peripheral blood (PB) CD34+ in basal state and after mobilization for harvest and transplantation. Our study was conducted to analyze PB CD34+ cells from healthy volunteers and from hematological patients during CD34+ cells mobilization. Our main aim was to understand if the proportions of different HSPCs among PB CD34+ cells were similar to those found in BM and whether the mobilizing regimens employed in chemo treated patients differently affected CD34+ cells subfractions in PB. METHODS: multicolor flowcytometry was used to analyze CD34+ cells from 4 BM samples and 9 PB samples from healthy volunteers and 32 PB samples from hematological patients prior CD34+ cells harvesting. RESULTS: Percentages of CD34+ cells subpopulations were different in basal PB compared to the BM: indeed, CMPs, GMPs and MEPs constituted respectively 27.6% ± 9.5, 23.8% ± 7.2 and 27.6% ± 16.2 of BM CD34+ cells and 47.8% ± 9.5, 10.3% ± 6.9 and 16.1% ± 7.6 of the total PB CD34+ cells. HSCs constituted 2.1% of BM and 1.5% of PB CD34+ cells. The differences between BM and circulating CMPs and GMPs were significant (p<0.005 and p<0.01). No differences in subpopulations proportions were shown comparing G-CSF mobilized and basal PB CD34+ cells. Interestingly, the 2 patients mobilized with AMD3100 (the inhibitory molecule for CXCR4) showed a higher percentage of GMPs (33.8% and 37.8% versus the average 16.3% ± 9.8 in G-CSF mobilized samples) and a lower fraction of CMPs (29.5% and 41.6% versus the average 58% ± 12 in G-CSF mobilized samples). In order to understand this result, we looked then at the CXCR4 mean fluorescence intensity among the progenitor subsets: GMPs showed significantly higher levels of this molecule compared to CMPs and MEPs. Regarding the mobilizing chemotherapy regimens, CMPs percentages were higher (61.1% versus 49.1%, p: 0.038) and GMPs’ were significantly lower (11.1% versus 27.6%, p<0.0001) in cyclophosphamide treated patients, compared to patients mobilized with other chemotherapy regimens. The percentage of HSCs did not significantly differ among bone marrow, unmobilized and mobilized PB CD34+ cells. Therefore, since an average collection of mobilized PB cells contains approximately one log more CD34+ cells than a BM harvest, a similarly higher amount of HSC are infused with mobilized CD34+ cell transplantation. A linear positive correlation between the number of mobilized CD34+ cells and the number of mobilized CMPs, GMPs, and MEPs was observed indicating that the proportions of different HSPCs did not significantly change among high- and low-mobilizers. There were no correlations between the number of mobilized subpopulations and leucocytes, hemoglobin and platelets levels. CONCLUSIONS: Our data displayed the heterogeneity of HSPC compartment between PB and BM. Many factors could contribute to this variegated scenario. These mechanisms comprehension can help us to choose the most suitable chemotherapy and cytokine administrations in order to improve clinical outcomes as infections complications, length of aplasia and transfusion requirements during an hematopoietic stem cell transplantation. Disclosures Palumbo: Bristol-Myers Squibb: Consultancy, Honoraria; Genmab A/S: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Onyx Pharmaceuticals: Consultancy, Honoraria; Array BioPharma: Honoraria; Amgen: Consultancy, Honoraria; Sanofi: Honoraria. Boccadoro:Celgene: Honoraria; Janssen: Honoraria; Onyx: Honoraria.

Prospective Isolation and Functional Characterization of Human Bone Marrow-Derived Hematopoietic Stem Cell-Supportive Mesenchymal Stromal Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3852-3852
Author(s):  
Yoshikazu Matsuoka ◽  
Yutaka Sasaki ◽  
Masaya Takahashi ◽  
Ryusuke Nakatsuka ◽  
Yasushi Uemura ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Scid Mice ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Abstract 3852 (Background) The identification of human CD34-negative (CD34−) SCID-repopulating cells (SRCs) provide a new concept for the hierarchy in the human HSC compartment (Blood 101:2924, 2003). Recently, we succeeded to highly purify these CD34-SRCs using 18 lineage specific antibodies (Blood 114:336, 2009). It has been suggested that human hematopoietic stem cell (HSC)-supportive microenvironment exist in the bone marrow (BM), which play a pivotal role in the maintenance of self-renewal capacity and dormancy of primitive HSCs. It was reported that osteoblasts and vascular endothelial cells played an important role to organize HSC niches. However, whether mesenchymal stromal cells (MSCs) contribute to organize HSC niches or not is not clearly understood, because MSCs are heterogeneous population. Therefore, it is important to clarify their origin and functional characteristics. (Objectives) The aim of this study was to prospectively isolate/identify human BM-derived MSCs and investigate their functional characteristics including HSC-supportive abilities. (Results) First, human BM-derived Lin−CD45− cells were subdivided into 4 fractions according to their expression levels of CD271 and SSEA-4 by FACS. We succeeded to isolate 3 MSC lines from these 4 fractions, including CD271+/&minus;SSEA-4+/&minus; cells. Approximately 1 out of 6 CD271+SSEA-4+ (DP) cells could form MSC-derived colony. These DP cells-derived MSCs could differentiate into osteoblasts and chondrocytes, but could not differentiate into adipocytes. In contrast, CD271+SSEA-4− cells and CD271−SSEA-4− cells-derived MSCs could differentiate into three lineages. Then, we assessed CD34− SRC-supportive activity of these 3 MSC lines. First, certain numbers of 18Lin−CD34− cells were cocultured with 3 MSC lines for 1 week, respectively. Next recovered cells were transplanted into NOD/SCID mice by intra-bone marrow injection (IBMI) to investigate SCID-repopulating cell (SRC) activity. After 8 weeks, the highest CD45+ human cell engraftments (0.1 % to 32.4 %, median 8.6 %) were observed in mice received 18Lin−CD34− cells cocultued with DP cells-derived MSCs. As recently reported (Cell Stem Cell 1:635,2007), Lin−CD34+CD38−CD45RA−CD90+ cells contained most primitive human CD34+CD38− SRCs. Very interestingly, these Lin−CD34+CD38−CD45RA−CD90+ cells were generated from the above mentioned cocultures. In order to evaluate SRC activity of these Lin−CD34+CD38−CD45RA−CD90+ cells generated from 18Lin−CD34− cells in vitro, Lin−CD34+CD38−CD45RA−CD90+/&minus; cells were sorted by FACS and then transplanted into NOD/SCID mice by IBMI. Eight weeks after transplantation, 8 out of 16 mice received Lin−CD34+CD38−CD45RA−CD90+ cells (400 to 3000 cells/mouse) were repopulated with human cells. In contrast, only 2 out of 16 mice received Lin−CD34+CD38− CD45RA−CD90− cells (1500 to 7000 cells/mouse) were repopulated. These results demonstrated that human CB-derived 18Lin−CD34− cells could generate very primitive CD34+CD38− SRCs in vitro. (Conclusion) These findings elucidate that human BM-derived DP cell-derived MSCs can support very primitive human CB-derived CD34− SRCs in vitro and suggest that these CD34− SRCs seem to be more immature than CD34+CD38− SRCs. These results provide a new concept of hierarchy in the human primitive HSC compartment. Disclosures: No relevant conflicts of interest to declare.

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