scholarly journals The Inhibition of CD39 and CD73 Cell Surface Ectonucleotidases by Small Molecular Inhibitors Enhances the Mobilization of Bone Marrow Residing Stem Cells by Decreasing the Extracellular Level of Adenosine

2019 ◽  
Vol 15 (6) ◽  
pp. 892-899 ◽  
Author(s):  
Mateusz Adamiak ◽  
Kamila Bujko ◽  
Katarzyna Brzezniakiewicz-Janus ◽  
Magda Kucia ◽  
Janina Ratajczak ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Surface ◽  
Progenitor Cells ◽  
Purinergic Signaling ◽  
Cell Trafficking ◽  
Hematopoietic Stem ◽  
Extracellular Adenosine ◽  
Small Molecular Inhibitors

Abstract We have recently demonstrated that purinergic signaling in bone marrow (BM) microenvironment regulates mobilization of hematopoietic stem progenitor cells (HSPCs), mesenchymal stroma cells (MSCs), endothelial progenitor cells (EPCs), and very small embryonic like stem cells (VSELs) into the peripheral blood (PB). While extracellular adenosine triphosphate (ATP) promotes mobilization, its metabolite extracellular adenosine has an opposite effect. Since ATP is processed in extracellular space to adenosine by ectonucleotidases including cell surface expressed CD39 and CD73, we asked if inhibition of these enzymes by employing in vivo small molecular inhibitors ARL67156 and AMPCP of CD39 and CD73 respectively, alone or combined could enhance granulocyte stimulating factor (G-CSF)- and AMD3100-induced pharmacological mobilization of stem cells. Herein we report that pre-treatment of donor mice with CD39 and CD73 inhibitors facilitates the mobilization of HSPCs as well as other types of BM-residing stem cells. This data on one hand supports the role of purinergic signaling in stem cell trafficking, and on the other since both compounds are not toxic against human cells, they could be potentially employed in the clinic to enhance the mobilization of BM residing stem cells for clinical purposes.

Novel In Vivo Evidence That Not Only Androgens But Also Pituitary Gonadotropins and Prolactin Directly Stimulate Murine Bone Marrow Stem Cells – Implications For Potential Treatment Strategies In Aplastic Anemias

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2476-2476
Author(s):  
Kasia Mierzejewska ◽  
Ewa Suszynska ◽  
Sylwia Borkowska ◽  
Malwina Suszynska ◽  
Maja Maj ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Sex Hormones ◽  
Peripheral Blood ◽  
Hematopoietic Stem ◽  
Clonogenic Potential

Abstract Background Hematopoietic stem/progenitor cells (HSPCs) are exposed in vivo to several growth factors, cytokines, chemokines, and bioactive lipids in bone marrow (BM) in addition to various sex hormones circulating in peripheral blood (PB). It is known that androgen hormones (e.g., danazol) is employed in the clinic to treat aplastic anemia patients. However, the exact mechanism of action of sex hormones secreted by the pituitary gland or gonads is not well understood. Therefore, we performed a complex series of experiments to address the influence of pregnant mare serum gonadotropin (PMSG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), androgen (danazol) and prolactin (PRL) on murine hematopoiesis. In particular, from a mechanistic view we were interested in whether this effect depends on stimulation of BM-residing stem cells or is mediated through the BM microenvironment. Materials and Methods To address this issue, normal 2-month-old C57Bl6 mice were exposed or not to daily injections of PMSG (10 IU/mice/10 days), LH (5 IU/mice/10 days), FSH (5 IU/mice/10 days), danazol (4 mg/kg/10 days) and PRL (1 mg/day/5days). Subsequently, we evaluated changes in the BM number of Sca-1+Lin–CD45– that are precursors of long term repopulating hematopoietic stem cells (LT-HSCs) (Leukemia 2011;25:1278–1285) and bone forming mesenchymal stem cells (Stem Cell & Dev. 2013;22:622-30) and Sca-1+Lin–CD45+ hematopoietic stem/progenitor cells (HSPC) cells by FACS, the number of clonogenic progenitors from all hematopoietic lineages, and changes in peripheral blood (PB) counts. In some of the experiments, mice were exposed to bromodeoxyuridine (BrdU) to evaluate whether sex hormones affect stem cell cycling. By employing RT-PCR, we also evaluated the expression of cell-surface and intracellular receptors for hormones in purified populations of murine BM stem cells. In parallel, we studied whether stimulation by sex hormones activates major signaling pathways (MAPKp42/44 and AKT) in HSPCs and evaluated the effect of sex hormones on the clonogenic potential of murine CFU-Mix, BFU-E, CFU-GM, and CFU-Meg in vitro. We also sublethally irradiated mice and studied whether administration of sex hormones accelerates recovery of peripheral blood parameters. Finally, we determined the influence of sex hormones on the motility of stem cells in direct chemotaxis assays as well as in direct in vivo stem cell mobilization studies. Results We found that 10-day administration of each of the sex hormones evaluated in this study directly stimulated expansion of HSPCs in BM, as measured by an increase in the number of these cells in BM (∼2–3x), and enhanced BrdU incorporation (the percentage of quiescent BrdU+Sca-1+Lin–CD45– cells increased from ∼2% to ∼15–35% and the percentage of BrdU+Sca-1+Lin–CD45+ cells increased from 24% to 43–58%, Figure 1). These increases paralleled an increase in the number of clonogenic progenitors in BM (∼2–3x). We also observed that murine Sca-1+Lin–CD45– and Sca-1+Lin–CD45+ cells express sex hormone receptors and respond by phosphorylation of MAPKp42/44 and AKT in response to exposure to PSMG, LH, FSH, danazol and PRL. We also observed that administration of sex hormones accelerated the recovery of PB cell counts in sublethally irradiated mice and slightly mobilized HSPCs into PB. Finally, in direct in vitro clonogenic experiments on purified murine SKL cells, we observed a stimulatory effect of sex hormones on clonogenic potential in the order: CFU-Mix > BFU-E > CFU-Meg > CFU-GM. Conclusions Our data indicate for the first time that not only danazol but also several pituitary-secreted sex hormones directly stimulate the expansion of stem cells in BM. This effect seems to be direct, as precursors of LT-HSCs and HSPCs express all the receptors for these hormones and respond to stimulation by phosphorylation of intracellular pathways involved in cell proliferation. These hormones also directly stimulated in vitro proliferation of purified HSPCs. In conclusion, our studies support the possibility that not only danazol but also several other upstream pituitary sex hormones could be employed to treat aplastic disorders and irradiation syndromes. Further dose- and time-optimizing mouse studies and studies with human cells are in progress in our laboratories. Disclosures: No relevant conflicts of interest to declare.

Reversibility of CD34 expression on human hematopoietic stem cells that retain the capacity for secondary reconstitution

Blood ◽  
2003 ◽  
Vol 101 (1) ◽  
pp. 112-118 ◽  
Author(s):  
Mo A. Dao ◽  
Jesusa Arevalo ◽  
Jan A. Nolta
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Surface ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Cd34 Expression

Abstract The cell surface protein CD34 is frequently used as a marker for positive selection of human hematopoietic stem/progenitor cells in research and in transplantation. However, populations of reconstituting human and murine stem cells that lack cell surface CD34 protein have been identified. In the current studies, we demonstrate that CD34 expression is reversible on human hematopoietic stem/progenitor cells. We identified and functionally characterized a population of human CD45+/CD34− cells that was recovered from the bone marrow of immunodeficient beige/nude/xid (bnx) mice 8 to 12 months after transplantation of highly purified human bone marrow–derived CD34+/CD38− stem/progenitor cells. The human CD45+ cells were devoid of CD34 protein and mRNA when isolated from the mice. However, significantly higher numbers of human colony-forming units and long-term culture-initiating cells per engrafted human CD45+ cell were recovered from the marrow of bnx mice than from the marrow of human stem cell–engrafted nonobese diabetic/severe combined immunodeficient mice, where 24% of the human graft maintained CD34 expression. In addition to their capacity for extensive in vitro generative capacity, the human CD45+/CD34− cells recovered from thebnx bone marrow were determined to have secondary reconstitution capacity and to produce CD34+ progeny following retransplantation. These studies demonstrate that the human CD34+ population can act as a reservoir for generation of CD34− cells. In the current studies we demonstrate that human CD34+/CD38− cells can generate CD45+/CD34− progeny in a long-term xenograft model and that those CD45+/CD34− cells can regenerate CD34+ progeny following secondary transplantation. Therefore, expression of CD34 can be reversible on reconstituting human hematopoietic stem cells.

Fluorescence-Based Selection of Gene-Corrected Hematopoietic Stem and Progenitor Cells From Acid Sphingomyelinase-Deficient Mice: Implications for Niemann-Pick Disease Gene Therapy and the Development of Improved Stem Cell Gene Transfer Procedures

Blood ◽  
1999 ◽  
Vol 93 (1) ◽  
pp. 80-86 ◽  
Author(s):  
Shai Erlich ◽  
Silvia R.P. Miranda ◽  
Jan W.M. Visser ◽  
Arie Dagan ◽  
Shimon Gatt ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Fetal Liver ◽  
Acid Sphingomyelinase ◽  
Hematopoietic Stem

Abstract The general utility of a novel, fluorescence-based procedure for assessing gene transfer and expression has been demonstrated using hematopoietic stem and progenitor cells. Lineage-depleted hematopoietic cells were isolated from the bone marrow or fetal livers of acid sphingomyelinase–deficient mice, and retrovirally transduced with amphotropic or ecotropic vectors encoding a normal acid sphingomyelinase (ASM) cDNA. Anti–c-Kit antibodies were then used to label stem- and progenitor-enriched cell populations, and the Bodipy fluorescence was analyzed in each group after incubation with a Bodipy-conjugated sphingomyelin. Only cells expressing the functional ASM (ie, transduced) could degrade the sphingomyelin, thereby reducing their Bodipy fluorescence as compared with nontransduced cells. The usefulness of this procedure for the in vitro assessment of gene transfer into hematopoietic stem cells was evaluated, as well as its ability to provide an enrichment of transduced stem cells in vivo. To show the value of this method for in vitro analysis, the effects of retroviral transduction using ecotropic versus amphotropic vectors, various growth factor combinations, and adult bone marrow versus fetal liver stem cells were assessed. The results of these studies confirmed the fact that ecotropic vectors were much more efficient at transducing murine stem cells than amphotropic vectors, and that among the three most commonly used growth factors (stem cell factor [SCF] and interleukins 3 and 6 [IL-3 and IL-6]), SCF had the most significant effect on the transduction of stem cells, whereas IL-6 had the most significant effect on progenitor cells. In addition, it was determined that fetal liver stem cells were only approximately twofold more “transducible” than stem cells from adult bone marrow. Transplantation of Bodipy-selected bone marrow cells into lethally irradiated mice showed that the number of spleen colony-forming units that were positive for the retroviral vector (as determined by polymerase chain reaction) was 76%, as compared with 32% in animals that were transplanted with cells that were nonselected. The methods described within this manuscript are particularly useful for evaluating hematopoietic stem cell gene transfer in vivo because the marker gene used in the procedure (ASM) encodes a naturally occurring mammalian enzyme that has no known adverse effects, and the fluorescent compound used for selection (Bodipy sphingomyelin) is removed from the cells before transplantation.

SDF-1 involvement in endothelial phenotype and ischemia-induced recruitment of bone marrow progenitor cells

Blood ◽  
2004 ◽  
Vol 104 (12) ◽  
pp. 3472-3482 ◽  
Author(s):  
Elena De Falco ◽  
Daniele Porcelli ◽  
Anna Rita Torella ◽  
Stefania Straino ◽  
Maria Grazia Iachininoto ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Time Course ◽  
Limb Ischemia ◽  
Artery Dissection ◽  
Hematopoietic Stem ◽  
Bone Marrow Progenitor Cells ◽  
Bone Marrow Progenitor

Chemokine stromal derived factor 1 (SDF-1) is involved in trafficking of hematopoietic stem cells (HSCs) from the bone marrow (BM) to peripheral blood (PB) and has been found to enhance postischemia angiogenesis. This study was aimed at investigating whether SDF-1 plays a role in differentiation of BM-derived c-kit+ stem cells into endothelial progenitor cells (EPCs) and in ischemia-induced trafficking of stem cells from PB to ischemic tissues. We found that SDF-1 enhanced EPC number by promoting α2, α4, and α5 integrin–mediated adhesion to fibronectin and collagen I. EPC differentiation was reduced in mitogen-stimulated c-kit+ cells, while cytokine withdrawal or the overexpression of the cyclin–dependent kinase (CDK) inhibitor p16INK4 restored such differentiation, suggesting a link between control of cell cycle and EPC differentiation. We also analyzed the time course of SDF-1 expression in a mouse model of hind-limb ischemia. Shortly after femoral artery dissection, plasma SDF-1 levels were up-regulated, while SDF-1 expression in the bone marrow was down-regulated in a timely fashion with the increase in the percentage of PB progenitor cells. An increase in ischemic tissue expression of SDF-1 at RNA and protein level was also observed. Finally, using an in vivo assay such as injection of matrigel plugs, we found that SDF-1 improves formation of tubulelike structures by coinjected c-kit+ cells. Our findings unravel a function for SDF-1 in increase of EPC number and formation of vascular structures by bone marrow progenitor cells.

STS1 and STS2 Are Important Regulators Of Hematopoietic Stem Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1186-1186
Author(s):  
Jing Zhang ◽  
Hubert Serve ◽  
Christian H. Brandts
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Surface ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Wild Type ◽  
Short Term ◽  
Double Knockout ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Abstract The receptor tyrosine kinases FLT3 and KIT are highly expressed on the surface of leukemic blasts in most patients with acute myeloid leukemia. Although about one third of patients display activating mutations in FLT3 (and more rarely in KIT), the majority of patients have no mutations in FLT3 or KIT. Previously, we demonstrated that Cbl functions as the E3 ligase for both FLT3 and KIT, and that ligase-inactivating mutations of Cbl stabilize FLT3 and KIT on the cell surface by preventing endocytosis and degradation (Sargin et al, Blood 2007). Furthermore, we demonstrated that expression of E3-ligase deficient Cbl mutants led to the development of a myeloproliferative disease in a murine bone marrow transplantation model (Bandi et al, Blood 2009). However, Cbl mutations are rarely found in AML. Here, we investigated the role of the Cbl regulators suppressors of T-cell signaling 1 and 2(STS1 and STS2) in stabilizing wild-type FLT3 and KIT on the cell surface of hematopoietic stem and progenitor cells (HSPCs). STS1 is ubiquitously expressed, while STS2 expression is restricted to the hematopoietic tissue. STS1 and STS2 constitutively bind to Cbl, while their binding to FLT3 and KIT is dependent on ligand-activation by FL and SCF, respectively. Interestingly, STS1 (but not STS2) functions as a tyrosine phosphatase for both ligand-activated FLT3 and KIT. This required the PGM domain of STS1, as PGM point mutant of STS1 did not dephosphorylate FLT3 or KIT. In line with this, knockdown of STS1 using stably expressing shRNA constructs showed a significant hyperphosphorylation of FLT3 and KIT. By using STS1/STS2 single and double knockout mice, we analyzed the effects of STS1 and STS2 on hematopoietic stem and progenitor cells in vivo. We found that deficiency of STS1 causes an increase of both absolute number and frequency of LSK (lineage marker-, KIT+, Sca1+) cells, which contain HSPCs. This phenotype was even more pronounced in STS1 and STS2 double knockout (dKO) mice, and is mainly attributable to the short term hematopoietic stem cell (ST-HSC) and multipotent progenitor (MPP) cell population, as defined by both standard and SLAM markers. Colony assays using primary bone marrow cells revealed a significantly higher colony forming ability in STS1-KO and dKO cells compared to wild type (wt) cells, particularly after serial replating. A careful analysis of the cells derived from methylcellulose culture revealed an increased proportion of immature (Mac1- CD48+ CD16/32-) cells in STS1-KO and dKO cells. Competitive repopulation assays showed an advantage for dKO cells when compared to wt, suggesting that the LT-HSC compartment is also affected. Even more pronounced were the differences in CFU-S assays (colony forming units spleen), displaying significantly more colonies of dKO compared to wt donor cells, functionally demonstrating a significantly increased ST-HSC / MPP population in dKO donors. A detailed analysis of the downstream signaling events demonstrated that loss of STS1 specifically causes an activated PI3-Kinase / AKT pathway. In summary, our data demonstrates that STS1 functions as a phosphatase of FLT3 and KIT and, using genetic mouse models, indicates a critical role in the maintenance and proliferation of long-term and short-term hematopoietic stem cells. This may also affect sensitivity to kinase inhibitors. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Interleukin-8 induces rapid mobilization of hematopoietic stem cells with radioprotective capacity and long-term myelolymphoid repopulating ability

Blood ◽  
1995 ◽  
Vol 85 (8) ◽  
pp. 2269-2275 ◽  
Author(s):  
L Laterveer ◽  
IJ Lindley ◽  
MS Hamilton ◽  
R Willemze ◽  
WE Fibbe
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Y Chromosome ◽  
Mononuclear Cells ◽  
White Blood Cells ◽  
Interleukin 8 ◽  
Hematopoietic Stem ◽  
Myeloid Progenitor Cells

Interleukin-8 (IL-8) belongs to a family of chemoattractant cytokines involved in chemotaxis and activation of neutrophils. As in vivo administration of IL-8 induces granulocytosis and the release of immature white blood cells into the circulation, we assessed a possible mobilizing effect of IL-8 on myeloid progenitor cells. IL-8 was administered at intraperitoneal doses ranging from 0.1 to 100 micrograms per mouse to female Balb/C mice (aged 8 to 12 weeks; weight, 20 to 25 g). Animals were killed at time intervals ranging from 1 to 240 minutes after IL-8 administration, and blood, bone marrow, and spleen cells were harvested. Injection of 30 micrograms IL-8 resulted in an increment from 25 +/- 9 to 418 +/- 299 granulocyte-macrophage colony-forming units (CFU-GM) per milliliter blood at 15 minutes after a single intraperitoneal injection. Sixty minutes after the injection of IL-8, the numbers of circulating CFU-GM per milliliter blood had almost returned to pretreatment values (82 +/- 39 CFU-GM per milliliter). A dose of 100 micrograms IL-8 per animal did not result in a further increment in the number of circulating CFU-GM. Transplantation of 5 x 10(5) blood-derived mononuclear cells (MNC) obtained at 30 minutes after IL-8 injection (30 micrograms) resulted in 69% survival of lethally irradiated (8.5 Gy) recipients at 60 days versus 22% for animals transplanted with an equal number of nonprimed blood-derived MNC. Transplantation of 1.5 x 10(6) MNC obtained from IL-8-treated donors resulted in 100% survival. Six months after transplantation, female recipients of MNC derived from IL-8-treated male donors were killed, and chimerism was determined in bone marrow, spleen, and thymus using a Y chromosome-specific probe and fluorescent in situ hybridization (FISH). The majority of bone marrow, spleen, and thymus cells (83% +/- 25%, 89% +/- 5%, and 64 +/- 28%, respectively) consisted of Y chromosome-positive cells, showing that the IL-8-mobilized cells had myelolymphoid repopulating ability. We conclude that IL-8 is a cytokine that induces rapid mobilization of progenitor cells and pluripotent stem cells that are able to rescue lethally irradiated mice and that are able to completely and permanently repopulate host hematopoietic tissues.

CCR2 Mediates Hematopoietic Stem and Progenitor Cell Trafficking to Inflammation Sites to Enhance Organ Repair.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2547-2547
Author(s):  
Yue Si ◽  
Israel Charo ◽  
Chia-Lin Tsou
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Chemokine Receptor ◽  
Conflicts Of Interest ◽  
Cell Trafficking ◽  
Hematopoietic Stem ◽  
Peripheral Tissues ◽  
Chemokine Receptor Ccr2

Abstract Abstract 2547 Poster Board II-524 Hematopoietic stem cells (HSCs) are bone-marrow derived, self-renewing pluripotent cells that give rise to terminally differentiated circulating blood cells. Recent work has implicated HSCs in the repair of parenchymal tissue in the setting of inflammation. In response to the antagonist of the chemokine receptor CXCR4, HSCs and their progenitors migrate from bone marrow to the blood. However, little is known about the signals that mediate their trafficking from the blood into peripheral tissues. Recently, we showed that mice genetically deficient in chemokine receptor CCR2 (CCR2–/– mice) have a marked decrease in the number of circulating “inflammatory” (7/4+, Ly6c+) monocytes, but no decrease in myeloid progenitor cells in the bone marrow (Tsou et al, J Clin Invest, 2007, 902). These data indicated that although CCR2 is not necessary for HSCs to differentiate into mature monocytes, it does play a role in monocyte egress from bone marrow to blood. In the current study, we extend this work and investigate the expression of CCR2 on hematopoietic stem cells and progenitor cells (HSPCs), and tested the hypothesis that CCR2 is critical for the recruitment of circulating HSPCs to sites of inflammation. Here we report that the chemokine receptor CCR2 is expressed on subsets of primitive hematopoietic stem cells as well as some early myeloid progenitors but not on the progenitors dedicated to megakaryocyte and erythroid differentiation. CCR2 mediates the chemotaxis of c-Kit+Lin- bone marrow derived cells to MCP-1 (CCL2) and MCP-3 (CCL7), which are known CCR2 ligands. In unchallenged mice, CCR2 appears to play a minimal role in HSCs trafficking. However, following instillation of thioglycollate to cause aseptic inflammation HSPCs were actively recruited to the peritoneum, as demonstrated by both FACS and functional colony formation assays. That these cells were true HSCs was demonstrated by their engraftment into the bone marrow of irradiated recipients. In contrast, mice genetically deficient in CCR2 (CCR2−/−) were profoundly impaired in the recruitment of HSCs to the inflamed peritoneum. Furthermore, in human disease models of acetaminophen induced liver damages, hematopoietic stem and progenitors were increased dramatically at site of injury in WT mice but not in CCR2−/− mice. CCR2 recruited HSPC play a tissue protective role by increase the numbers of M2 macrophages in injured liver. Taken together, these findings document a novel role for CCR2 in the specific homing of primitive hematopoietic stem and progenitors to sites of inflammation and injury, and suggest a new role for chemokines in increasing the numbers of differentiated leukocytes at sites of inflammation. Recruited HSPCs hasten the resolution of the inflammatory response, and promote repair of injured tissue, at least partially due to locally increase the number of tissue reparative macrophages. Disclosures: No relevant conflicts of interest to declare.

NPM1 Haploinsufficiency Results in Increased Numbers of Hematopoietic Stem Cells and Progenitor Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 738-738
Author(s):  
Aparna Raval ◽  
Christopher Y. Park ◽  
Wendy W Pang ◽  
Brenda Kusler ◽  
Kunju Joshi Sridhar ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Functional Role ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Expression Levels ◽  
Equity Ownership

Abstract Abstract 738 Myelodysplastic syndromes (MDS) are characterized by defective hematopoietic stem/progenitor cell maturation, resulting in ineffective hematopoiesis. This group of disorders is characterized by cytogenetic abnormalities and approximately 25% of cases progress to acute myeloid leukemia (AML). NPM1 is frequently mutated in AML and translocations involving NPM1 occur in a number of hematopoietic malignancies including MDS. NPM1 heterozygous mice (NPM1 +/−) have been shown to have a MDS-like phenotype. Taken together, these data suggest an important role for NPM1 in the function of hematopoietic stem cells (HSC) and/or committed progenitors. In order to evaluate NPM1 function in early hematopoiesis, we have evaluated NPM1 expression in both the mouse and human hematopoietic systems. Using quantitative RT-PCR, we show that NPM1 expression levels are 2-3-fold higher in normal CD34+ bone marrow progenitor cells compared to total bone marrow in humans. Furthermore, NPM1 expression levels are decreased by ∼50% in 9/37 MDS CD34+ cells when compared to normal controls. Of interest, NPM1 expression is reduced primarily in patients with poor or intermediate prognosis. Consistent with a functional role for NPM1 in HSC, NPM1 +/− mice (developed by gene trapping and obtained from the MMRRC at UC-Davis) contained significantly increased numbers of HSC (Lin-cKit+Sca+CD34-CD150+) within the Lin-cKit+Sca+ population compared to those from the littermate controls (52 ± 2.6% vs,74 ± 12%, p < 0.01). Consistent with prior reports, NPM +/− mice contained significantly fewer mature erythrocytes (Ter119+CD71lo) in the bone marrow compared to WT controls (6.5 ± 1.8% vs 10 ± 0.5% p < 0.01). In order to study NPM +/− HSC function, we tested the ability of these HSCs to form colonies in methylcellulose. NPM1 +/− HSCs formed increased numbers of both CFU-GM and CFU-GEMM colonies and decreased numbers of CFU-E colonies compared to WT HSC. Flow cytometric analysis of pooled day 14 colonies from individual mice revealed a >2 fold increase in cKit+ progenitor cells from NPM1 +/− colonies (2.0 ± 1.0% vs. 0.2 ± 0.2%, p = 0.02), suggesting that the differentiation potential of NPM+/− HSCs is impaired. To characterize HSC function in vivo, equal numbers of double-sorted HSCs from WT and NPM1 +/− mice were transplanted in triplicate into lethally irradiated C57B6 (CD45.2) recipients. Analysis of peripheral blood donor chimerism (CD45.1+CD45.2+) 21 days post-transplantation showed that NPM1 +/− HSC-transplanted recipients exhibited markedly lower granulocyte chimerism than WT HSC recipients (5.5 fold reduction, 2 ± 2% vs. 11 ± 5%, p < 0.01). This finding suggests that although NPM1 +/− mice have increased numbers of HSC, these HSC exhibit either altered myeloid fate decisions or decreased bone marrow homing capacity. We are currently investigating long-term engraftment potential to further elucidate the function of HSC in NPM1 +/− mice in vivo. In aggregate, these data demonstrate a functional role for NPM1 in early myeloid differentiation and strongly suggest that NPM effects may be exerted as early as at the level of the HSC. Disclosures: Weissman: Amgen: Equity Ownership; Cellerant Inc.: ; Stem Cells Inc.: Equity Ownership, Founder; U.S. Patent Application 11/528,890 entitled “Methods for Diagnosing and Evaluating Treatment of Blood Disorders.”: Patents & Royalties.

Fluorescence-Based Selection of Gene-Corrected Hematopoietic Stem and Progenitor Cells From Acid Sphingomyelinase-Deficient Mice: Implications for Niemann-Pick Disease Gene Therapy and the Development of Improved Stem Cell Gene Transfer Procedures

Blood ◽  
1999 ◽  
Vol 93 (1) ◽  
pp. 80-86 ◽  
Author(s):  
Shai Erlich ◽  
Silvia R.P. Miranda ◽  
Jan W.M. Visser ◽  
Arie Dagan ◽  
Shimon Gatt ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Fetal Liver ◽  
Acid Sphingomyelinase ◽  
Hematopoietic Stem

The general utility of a novel, fluorescence-based procedure for assessing gene transfer and expression has been demonstrated using hematopoietic stem and progenitor cells. Lineage-depleted hematopoietic cells were isolated from the bone marrow or fetal livers of acid sphingomyelinase–deficient mice, and retrovirally transduced with amphotropic or ecotropic vectors encoding a normal acid sphingomyelinase (ASM) cDNA. Anti–c-Kit antibodies were then used to label stem- and progenitor-enriched cell populations, and the Bodipy fluorescence was analyzed in each group after incubation with a Bodipy-conjugated sphingomyelin. Only cells expressing the functional ASM (ie, transduced) could degrade the sphingomyelin, thereby reducing their Bodipy fluorescence as compared with nontransduced cells. The usefulness of this procedure for the in vitro assessment of gene transfer into hematopoietic stem cells was evaluated, as well as its ability to provide an enrichment of transduced stem cells in vivo. To show the value of this method for in vitro analysis, the effects of retroviral transduction using ecotropic versus amphotropic vectors, various growth factor combinations, and adult bone marrow versus fetal liver stem cells were assessed. The results of these studies confirmed the fact that ecotropic vectors were much more efficient at transducing murine stem cells than amphotropic vectors, and that among the three most commonly used growth factors (stem cell factor [SCF] and interleukins 3 and 6 [IL-3 and IL-6]), SCF had the most significant effect on the transduction of stem cells, whereas IL-6 had the most significant effect on progenitor cells. In addition, it was determined that fetal liver stem cells were only approximately twofold more “transducible” than stem cells from adult bone marrow. Transplantation of Bodipy-selected bone marrow cells into lethally irradiated mice showed that the number of spleen colony-forming units that were positive for the retroviral vector (as determined by polymerase chain reaction) was 76%, as compared with 32% in animals that were transplanted with cells that were nonselected. The methods described within this manuscript are particularly useful for evaluating hematopoietic stem cell gene transfer in vivo because the marker gene used in the procedure (ASM) encodes a naturally occurring mammalian enzyme that has no known adverse effects, and the fluorescent compound used for selection (Bodipy sphingomyelin) is removed from the cells before transplantation.

Sign in / Sign up

Export Citation Format

Share Document