Homing and Engraftment of Mobilized Hematopoietic Stem Cells: Involvement of VLA-5.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4943-4943
Author(s):  
Pieter K. Wierenga ◽  
Gerald de Haan ◽  
Bert Dontje ◽  
Ellen Weersing ◽  
Ronald van Os
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Hematopoietic Reconstitution

Abstract VLA-5 has been implicated in the adhesive interactions of stem and progenitor cells with the bone marrow extracellular matrix and stromal cells and is therefore considered to play an important role in the hematopoietic reconstitution 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-GSF, the number of VLA-5 expressing cells in mobilized peripheral blood cells (MPB) decreases to 38±3%. Despite this low frequency of VLA-5+ cells, however, even when equal numbers of progenitor cells are transplanted MPB cells provide a much faster hematopoietic recovery compared to BM cells. To shed more light on the role of VLA-5 in the process of homing and engraftment, we investigated whether differences in homing potential of the stem cell subsets might be responsible for this enhanced reconstitution. At 3 hours post-transplant, however, no differences in homing efficiency of progenitor and stem cells from MPB and BM grafts in both bone marrow and spleen could be detected. It should be realized that MPB and BM grafts demonstrate different ratios of stem/progenitor cells which might be another explanation for the observed differences in repopulation potential. Furthermore, MPB cells migrating in vitro towards SDF-1α showed potent reconstitution while VLA-5 expression was reduced on these cells. In fact, in vitro treatment with SDF-1α showed further decrease in VLA-5 expressing cells (from 38% to 4%) in the lin- fraction. When equal numbers of MPB were transplanted with and without SDF-1α pretreatment, no difference in hematopoietic reconstitution was observed suggesting a minor role of VLA-5 in homing and engraftment. On the other hand, after VLA-5 blocking an inhibition of 59±7% in the homing of MPB progenitor cells in the bone marrow could be found, whereas homing in the spleen of the the recipients is only inhibited by 11±4%. To elucidate whether the observed enhanced reconstitution could be explained by a selective homing of VLA-5+ cells or a rapid upregulation of VLA-5 expression, cells were labelled with PKH67-GL and transplanted in lethally irradiated recipients. It could be demonstrated that at 3 hours post-transplant cells from MPB grafts showed a rapid increase from 38±3% up to 66±9% of VLA-5+ cells in the bone marrow of the recipient. In the spleen no significant increase in VLA-5+ cells was observed. When MPB cells were transplanted after pretreatment with SDF-1α an increase from 2±1% up to 33±5% of VLA-5+ cells in the bone marrow was detected. When calculating the number of cells recovered from bone marrow, a selective homing of VLA-5+ cells cannot be excluded. Therefore, we also assessed the number of VLA-5+ cells in the PKH+ fraction in peripheral blood from the recipient immediately (½-1 hour) after transplantation but found no increase during that time period. So far it can be concluded that MPB cells show low number of VLA-5+ cells but these cells possess an enhanced hematopoietic reconstitution potential. Homing of progenitor cells to the spleen seems to be less dependent on VLA-5 expression than homing to the bone marrow. A rapid upregulation of VLA-5 expression on engrafting MPB cells early after transplantation does not occur and hence our data are suggestive for the preferential homing of VLA-5+ cells in the bone marrow after transplantation.

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.

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.

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.

Homeostatic Role of the Krüppel-Like Factor 4 (KLF4) in Proliferation and Differentiation of Primitive Hematopoietic Progenitor Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1497-1497 ◽  
Author(s):  
Chun Shik Park ◽  
Takeshi Yamada ◽  
H. Daniel Lacorazza
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Proliferative Potential ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
Deficient Mice

Abstract Abstract 1497 Poster Board I-520 KLF4 is a tumor suppressor in the gastrointestinal tract known to induce cell cycle arrest in a cell context dependent manner. We recently reported that KLF4 maintains quiescence of T lymphocytes downstream of T-cell receptor signaling (Yamada et al., Nature Immunology, 2009). The role of KLF4 in reprogramming adult somatic cells into pluripotent stem cells along with Oct3/4, c-Myc and Sox2 suggests that KLF4 restricts proliferation of undifferentiated cells. In spite of a redundant role of KLF4 in fetal liver hematopoietic stem cells (HSC), its role in the maintenance of adult bone marrow HSCs has not been studied yet. To study the role of KLF4 in the hematopoietic system we used gain- and loss-of-function mouse models. Retroviral transfer of KLF4 into wild type bone marrow (BM) cells led to significant reduction of colony forming units (CFU) in methylcellulose cultures due to increased apoptosis and lower proliferation. Then, Mx1-Cre was used to induce deletion of Klf4-floxed mice by polyI:C administration. Analysis of peripheral blood cells up to 6-9 months post polyI:C administration showed significant reduction of monocytes, as previously reported, and expansion of CD8+CD44+ T cells due to their increased proliferative potential. BM cells from Klf4-deficient mice exhibited increased number of myeloid progenitor cells measured by flow cytometry (Lin-Sca-1-c-kit+FcRII/III+CD34+ cells), CFU and CFU-S8. Cytoablation with 5-fluorouracil (5-FU) showed lower nadir of peripheral white blood cells in Klf4-deficient mice compared to control mice. In spite of normal multilineage reconstitution in BM transplants experiments, competitive reconstitution with Klf4-deficient and normal BM cells resulted in reduced contribution of Klf4-deficient cells to peripheral blood, likely due to homing and proliferative differences. Collectively, our data shows that KLF4 has an important role in function of hematopoietic stem and progenitor cells. Disclosures: No relevant conflicts of interest to declare.

Dual Role Of MERIT40 In Hematopoietic Stem and Progenitor Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 797-797
Author(s):  
Krasimira Rozenova ◽  
Jing Jiang ◽  
Chao Wu ◽  
Junmin Wu ◽  
Bernadette Aressy ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Bone Marrow Failure ◽  
Adaptor Protein ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract The balance between self-renewal and differentiation of hematopoietic stem cells (HSCs) is maintained by cell intrinsic and extrinsic mechanisms, including tight regulation of signaling pathways such as Tpo-Mpl and SCF-ckit. Posttranslational modifications, such as phosphorylation and ubiquitination, regulate these pathways. While the role of protein phosphorylation is well established, the importance of ubiquitination in HSC self-renewal has not been well addressed. It is known that of the seven different lysines on ubiquitin, Lys48 polyubiquitination is a marker for protein degradation, and Lys63 polyubiquitination is associated with regulation of kinase activity, protein trafficking, and localization. In this study, we provide evidence that the adaptor protein MERIT40 has multiple roles in hematopoietic stem/progenitor cells (HSPCs). MERIT40 is a scaffolding protein shared by two distinct complexes with Lys63 deubiquitinase (DUB) activities: the nuclear RAP80 complex with a known role in DNA damage repair in breast/ovarian cancer cells, whereas the functions of the cytoplasmic BRISC remains less characterized. MERIT40 is important for integrity of both complexes, and its deficiency leads to their destabilization and a >90% reduction in deubiquitinase activity. By using MERIT40 knockout (M40-/-) mice, we found that lack of MERIT40 leads to a two-fold increase in phenotypic and functional HSCs determined by FACS and limiting dilution bone marrow transplantation (BMT), respectively. More importantly, M40-/- HSCs have increased regenerative capability demonstrated by increased chimerism in the peripheral blood after BMT of purified HSCs. The higher self-renewal potential of these HSCs provides a survival advantage to M40-/- mice and HSCs after repetitive administration of the cytotoxic agent 5-flurouracil (5FU). MERIT40 deficiency also preserves HSC stemness in culture as judged by an increase in peripheral blood chimerism in recipient mice transplanted with M40-/- Lin-Sca1+Kit+ (LSK) cells cultured in cytokines for nine days compared to recipient mice receiving cultured wildtype (WT) LSK cells. In contrast to the increased HSC homeostasis and superior stem cell activity due to MERIT40 deficiency, M40-/- mice are hypersensitive to DNA damaging agents caused by inter-cross linking (ICL), such as Mitomycin C (MMC) and acetaldehydes that are generated as side products of intracellular metabolism. MMC injection caused increased mortality in M40-/- mice compared to WT controls attributable to DNA damage-induced bone marrow failure. MMC-treated M40-/- mice showed marked reduction in LSK progenitor numbers accompanied by increased DNA damage, in comparison to WT mice. Consistent with the in vivo studies, M40-/- progenitor cells are hypersensitive to MMC and acetaldehyde treatment in a cell-autonomous manner in colony forming assays. ICL repair is known to require Fanconi Anemia (FA) proteins, an ICL repair network of which mutations in at least 15 different genes in humans cause bone marrow failure and cancer predisposition. Thus, M40-/- mice represent a novel mouse model to study ICL repair in HSPCs with potential relevance to bone marrow failure syndromes. Taken together, our data establishes a complex role of MERIT40 in HSPCs, warranting future investigation to decipher functional events downstream of two distinct deubiquitinating complexes associated with MERIT40 that may regulate distinct aspects of HSPC function. Furthermore, our findings reveal novel regulatory pathways involving a previously unappreciated role of K63-DUB in stem cell biology, DNA repair regulation and possibly bone marrow failure. DUBs are specialized proteases and have emerged as potential “druggable” targets for a variety of diseases. Hence, our work may provide insights into novel therapies for the treatment of bone marrow failure and associated malignancies that occur in dysregulated HSCs. Disclosures: No relevant conflicts of interest to declare.

CXCR4-SDF-1 Signaling Mediated up-Regulation of Survivin Expression In Mesenchymal Progenitor Cells Is Critical for Their Proliferation and Maintenance of Osteoblastic Niche

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 941-941
Author(s):  
Pratibha Singh ◽  
Jennifer Speth ◽  
Peirong Hu ◽  
Louis M. Pelus
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Survivin Expression ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Cxcr4 Signaling

Abstract Abstract 941 Hematopoietic stem cells reside in osteoblastic and vascular niches within the bone marrow. The osteoblastic niche is composed of mesenchymal stem cell derived progenitor cells (MPC) and osteoblasts and are the main sources of the CXC chemokine CXCL12/SDF-1 in the bone marrow microenvironment. Several published studies suggest that the interaction between CXCR4 expressed on hematopoietic stem cells with SDF-1 produced in the bone marrow microenvironment is important for their retention in the bone-marrow. However, the role of SDF-CXCR4 signaling in formation and maintenance of osteoblastic niches in the bone marrow is not known. In this study, we examined the role of CXCR4 signaling in MPC proliferation and differentiation and its effects on hematopoietic stem cell (HSC) function. Flow cytometry analysis demonstrated that CXCR4 is expressed on the phenotypically defined MPC. Deletion of CXCR4 in tamoxifen cre inducible CXCR4flox-flox mice (verified by PCR and flow cytometry; 90% gene deletion and surface CXCR4 expression) results in significantly decreased numbers of Lin- CD45- CD31- Sca-1+ ALCAM- MPC (39±4.2%) and Lin- CD45- CD31- Sca-1-CD51+ osteoblasts (25±2.6%) in bone marrow 15 days after tamoxifen treatment. SDF-1 induced proliferation of CXCR4 deficient MPC was decreased by 4-fold compared to control, measured by the colony forming unit-fibroblast (CFU-F) assay. To determine, whether CXCR4 deficiency in bone marrow stromal cells affects SDF-1 induced HSC proliferation, we cultured FACS sorted wild-type SLAM SKL (103 cells) on CXCR4 deficient stroma for 5 days and total SLAM SKL cell numbers were counted by flow-cytometey analysis. CXCR4 deficient stroma failed to support optimal HSC proliferation and 48±5.2% less SLAM KSL cells was observed on CXCR4 deficient stroma compared to wild-type stroma. To investigate the mechanisms through which CXCR4-SDF-1 signaling regulates MPC proliferation, we evaluated the effect of SDF-1 treatment on expression of the anti-apoptotic and cell-cycle regulator protein, Survivin, in MPC. Multivariate intracellular flow cytometry demonstrated that Survivin expression increased by 23±4.2% in wild-type MPC after SDF-1 treatment (50ng/ml), however no significant increased was demonstrated in CXCR4 deficient MPC cells. CFU-F formation was reduced by 2.5 fold when the Survivin gene was conditionally deleted in MPC. Moreover, fewer SLAM SKL cells were detected on Survivin deficient stroma compared to wild-type stroma after SDF-1 treatment for 5 days. In conclusion, our data suggest that CXCR4-SDF-1 signaling mediated Survivin expression in MPC is important for their proliferation and maintenance of the bone-marrow hematopoietic niche. Disclosures: No relevant conflicts of interest to declare.

A Possible Role Of The Tetraspanin CD9 In Bone Marrow Retention and Engraftment Of Human CD34+ Hematopoietic Stem/Progenitor Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3237-3237 ◽  
Author(s):  
Kam Tong Leung ◽  
Karen Li ◽  
Kam Sze Kent Tsang ◽  
Kathy Yuen Yee Chan ◽  
Pak Cheung Ng ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Cell Motility ◽  
Progenitor Cells ◽  
Neutralizing Antibody ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Cxcr4 Axis

Abstract The stromal cell-derived factor-1 (SDF-1)/chemokine C-X-C receptor 4 (CXCR4) axis plays a critical role in homing, engraftment and retention of hematopoietic stem/progenitor cells. We previously demonstrated that expression of CD9 is a downstream signal of the SDF-1/CXCR4 axis, and that CD9 regulates short-term (20 hours) homing of cord blood (CB) CD34+ cells in the NOD/SCID mouse xenotransplantation model (Leung et al, Blood, 2011). Here, we provided further evidence that pretreatment of CB CD34+ cells with a CD9-neutralizing antibody significantly reduced their long-term (6 weeks) engraftment, as indicated by the presence of human CD45+ cells, in the recipient bone marrow and spleen by 70.9% (P = .0089) and 87.8% (P = .0179), respectively (n = 6). However, CD9 blockade did not bias specific lineage commitment, including the CD14+ monocytic, CD33+ myeloid, CD19+ B-lymphoid and CD34+ stem/progenitor cells (n = 4). We also observed an increase of the CD34+CD9+ subsets in the bone marrow (9.6-fold; P < .0001) and spleens (9.8-fold; P = .0014) of engrafted animals (n = 3-4). These data indicate that CD9 possesses important functions in regulating stem cell engraftment and its expression level on CD34+ cells is up-regulated in the target hematopoietic organs. Analysis of paired bone marrow (BM) and peripheral blood (PB) samples from healthy donors revealed a higher CD9 expression in BM-resident CD34+ cells (57.3% ± 8.1% CD9+ cells in BM vs. 29.3% ± 5.8% in PB; n = 5, P = 0.0478). Consistently, CD34+ cells in granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood (MPB) expressed lower levels of CD9 (33.8% ± 3.0% CD9+ cells, n = 24), when compared with those in BM (56.4% ± 4.9% CD9+ cells, n = 8, P = 0.0025). In vitro exposure of MPB CD34+ cells to SDF-1 significantly enhanced CD9 expression (1.55-fold increase, n = 4, P = 0.0103), concomitant with a 75.2% reduction in the CD34+CXCR4+ subsets (P = 0.0118). Treatment of NOD/SCID chimeric mice with G-CSF increased the frequency of circulating CD45+ cells (3.4-fold) and CD34+ cells (3.3-fold), and substantially decreased the CD34+CD9+ subsets in the BM from 75.8% to 30.8%. Importantly, the decline in CD9 levels during G-CSF mobilization was also observed in the CD34+CD38-/low primitive stem cell subpopulation. Interestingly, in vitro treatment of BM CD34+ cells with G-CSF did not affect CD9 expression (n = 3), suggesting that a signaling intermediate is required for G-CSF-mediated CD9 down-regulation in vivo. Transwell migration assay revealed a significant enrichment of CD9- cells that were migrated towards a SDF-1 gradient (n = 4 for BM CD34+ cells, P = 0.0074; n = 7 for CB CD34+ cells, P = 0.0258), implicating that CD9 might negatively regulate stem cell motility. In contrast, pretreatment with the CD9-neutralizing antibody inhibited adhesion of CD34+ cells to the osteoblastic cell line Saos-2 by 33.5% (n = 2). Our results collectively suggest a previously unrecognized role of CD9 in stem cell retention by dual regulation of cell motility and adhesion, and reveal a dynamic regulation of CD9 expression in the BM microenvironment, which might represent an important event in controlling stem cell homing and mobilization. Disclosures: No relevant conflicts of interest to declare.

Hematopoietc Stem Cell Targeted Neonatal Gene Therapy Cures oc/oc Mice from Osteopetrosis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 456-456
Author(s):  
Johan Richter ◽  
Maria Johansson ◽  
Teun J. de Vries ◽  
Mats Ehinger ◽  
Vince Everts ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Bovine Bone ◽  
Wild Type ◽  
Hematopoietic Stem

Abstract Infantile malignant osteopeterosis (IMO) is a progressive, rare autosomal recessive disorder affecting osteoclast function. 50% of the affected children have a mutation in the Tcirg1 gene coding for one subunit of an osteoclast specific proton pump, OC116. The non-resorbed dense, sclerotic bones cause symptoms including pancytopenia and progressive visual loss and ultimately death. So far, the only curative treatment is hematopoietic stem cell (HSC) transplantation. The oc/oc mouse has a mutation in the gene homologous to Tcirg1 giving rise to similar symptoms as in patients leading to death of the mice at the age of 3–4 weeks. We have previously shown that the oc/oc mouse can be successfully treated with neonatal transplantation of normal HSC leading to prolonged survival and reversal of osteopetrosis (M. Johansson et al., Exp. Hematology34;242, 2006). In the current study we set out to develop HSC directed gene therapy for osteopetrosis in the oc/oc mouse model. As the bone marrow compartment is severely reduced in the oc/oc mouse fetal liver (FL) cells depleted of Ter119+ erythroid cells were used as a source of hematopoietic stem cells. We first established that wild type Ter119 depleted FL cells marked with a GFP vector and transplanted to newborn oc/oc mice i.p. could correct the osteopetrotic phenotype just as was shown for fresh bone marrow cells previously. Subsequently, Ter119 depleted FL cells from oc/oc mice were transduced with a retroviral vector expressing OC116 and GFP. In vitro transduction efficiency was 60–85%. One-day-old oc/oc mice were irradiated (400cGy) and transplanted i.p. with the transduced FL cells (1–3.5x106). 7 out of 14 mice survived past the expected lifespan and had 8–53% GFP+ cells in the peripheral blood at 3, 6 and 12 weeks. Analysis of bone structure with X-ray and histopathology showed an improvement at 8 weeks and an almost normal structure at 18 weeks, indicating induction of osteoclast activity. In vitro culture of osteoclasts from bone marrow from transplanted animals on bovine bone slices showed GFP marked osteoclasts and bone resorption, albeit at lower levels than for wild type cells. In the oc/oc mouse there is a block in B-lymphopoiesis leading to a reduced number of B-lymphocytes in the peripheral blood. In treated mice a reversal of this deficiency was observed. In summary we have demonstrated that the osteoclast defect seen in oc/oc mice can be successfully corrected by neonatal transplantation of gene modified hematopoietic stem cells and that this can lead to long-term survival of treated mice. This represents a significant step towards the development of gene therapy for osteopetrosis.

Mobilization of Hematopoitic Stem/Progenitor Cells by a Cdc42 Activity-Specific Inhibitor

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 68-68 ◽  
Author(s):  
Wei Liu ◽  
Lei Wang ◽  
Xun Shang ◽  
Fukun Guo ◽  
Marnie A. Ryan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Actin Polymerization ◽  
Specific Inhibitor ◽  
Hematopoietic Stem

Abstract Hematopoietic stem cell transplantation has become a standard of care for the treatment of many hematological diseases. Transplantation of mobilized peripheral blood stem cells has replaced bone marrow (BM) transplantation as the preferred method for hematopoietic recovery. To date, G-CSF mobilized hematopoietic stem/progenitor cell (HSPC) harvest is the main FDA-approved preparative regiment for transplantation protocols, but this application has several limitations in utilities including diverse individual variability and potential side effects in several patient populations. Although AMD3100, a chemical CXCR4-blocker, has been found effective for HSPC mobilization, the development of additional HSPC mobilization agents that work through well defined molecular mechanisms remains in need. Previously our laboratory has shown in a conditional knockout mouse model that deficiency of the Rho GTPase Cdc42 in the BM causes impaired adhesion, homing, lodging and retention of HSPCs, leading to massive egress of HSPCs from BM to the peripheral blood without compromising their proliferative potential. From an array of small molecule inhibitors of PIP2-induced actin-polymerization discovered in a high throughput screening, we identified CASIN, a novel Cdc42 Activity-Specific Inhibitor, that is effective in suppressing Cdc42 activity in a dose-dependent manner in murine fibroblasts and low density bone marrow (LDBM) cells and human CD34+ umbilical cord blood (HCB) cells in vitro, and in murine LDBM cells in vivo. The inhibitory effect by CASIN appears to be specific to Cdc42 and is reversible. We subsequently tested the hypothesis that pharmacological targeting Cdc42 by CASIN may transiently mimic the Cdc42 knockout phenotype leading to HSPC mobilization. In the dose range of 5–10 uM, CASIN does not show detectable toxicity in wild type or Cdc42 knockout HSPCs in cell survival and colony-forming unit activity assays. CASIN treatment of 32D murine myeloid progenitor cells or freshly isolated progenitor cells results in a reversible inhibition of F-actin polymerization induced by SDF-1α and blockade of α5β1 integrin mediated adhesion to fibronectin fragment CH296. Its effects on actin organization and adhesion are associated with an inhibition of directional migration of the colony-forming cells toward SDF-1α. In contrast, CASIN does not show a detectable effect on the adhesion and migration activities of Cdc42 knockout HSPCs, suggesting that it works specifically through Cdc42 to affect cell actin structure and adhesion. Upon injection into mice (5mg/Kg, intraperitoneally), CASIN is effective in stimulating mobilization of progenitor activity into the peripheral blood (~ 6-fold increase compared to control at 40 hrs post injection). Subsequent serial transplantation experiments show that the PB harvested from CASIN treated mice could reconstitute various lineages of blood cells in primary, secondary, and tertiary recipients, indicating that long-term hematopoietic stem cells were mobilized from the BM of CASIN-treated donor mice. Consistent with the mobilization phenotype, FACS analysis shows that intravenous injection of CASIN can cause transient reduction of long-term hematopoietic stem cells (IL7Ra−Lin−Sca-1+c-Kit+CD34−) and short-term hematopoietic stem cells (IL7Ra−Lin−Sca-1+c-Kit+CD34+) from BM. Similar to the effects on murine HSPCs, CASIN is active on CD34+ HCB cells in transiently suppressing F-actin assembly, adhesion to fibronectin, and SDF-1α induced migration without detectable toxicity in vitro. Whether CASIN is effective in mobilizing HCB-engrafted NOD/SCID mice is currently under investigation. Our studies suggest that the novel concept of pharmacological targeting of Cdc42, that transiently and reversibly mimics the effect of Cdc42 knockout, may be developed into a mobilization regiment with a well defined molecular and cellular mechanism.

Characterization of the Novel Function of Cyclin A1 to Influence the Stem Cell Niche and Microenvironment Signaling in Mouse Model

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2338-2338
Author(s):  
Regina R Miftakhova ◽  
Andreas Hedblom ◽  
Anders Bredberg ◽  
Debra J Wolgemuth ◽  
Jenny L Persson
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Radiation Exposure ◽  
Progenitor Cells ◽  
Cyclin A1 ◽  
Hematopoietic Stem ◽  
Stem Cell Niches

Abstract Abstract 2338 The molecules and cellular mechanisms that regulate pool size of hematopoietic stem cells and its association with stem cell niches to protect HSC from cell cycle-dependent injury are unclear. The cell cycle regulatory factor, cyclin A1 is overexpressed in patients with hematopoietic malignancies. Further, targeted overexpression of cyclin A1 in myeloid progenitor cells initiated acute myeloid leukemia in transgenic mice. In the present study, we investigated the role of cyclin A1 in controlling the HSC pool and its functional association with key molecules that regulate stem cell niches under steady-state conditions or following the cytokine stimulation or radiation exposure in vivo and in vitro. We reported that cyclin A1 null bone marrow displayed a significant increase in the frequency of stem cells (P<0,01) and increased expression of P27kip and increased phosphorylation of Akt at ser-473 site in HSCs and hematopoietic progenitors. We further showed that increased frequency and number of cyclin A1 null HSCs was associated with the increased expression BMP receptor type IA that is known as a key molecule controlling the HSC niche. In addition, cyclin A1 null HSCs exhibited increased ability to migrate as determined by in vitro migration assay, and bone marrow transplantation assay, and this correlated with the increased expression of MMP9, that is known for controlling the osteoblast cell expansion, and the accumulated nuclear localization of angiogenic and vascularization factor VEGFR2 in cyclin A1 null bone marrow cells. We also observed that IRSp53 that is a regulator for extracellular matrix signaling, was present in the nuclear compartments of cyclin A1 null bone marrow progenitor cells, but was absent in that of the wild-type controls. Further, flow cytometry and immunoblot analyses showed that cyclin A1 null HSCs and progenitor cells exhibited relatively resistant to TNF stimulation and the radiation exposure, and this was associated with the great increase in the expression of phosporylated of ser-473 Akt. Our findings suggest that the microenvironment may be altered in bone marrows from cyclin A1 null mice. Thus cyclin A1 may have important function in the decision of maintaining the HSC pools and protecting the HSCs and progenitors from exposure to the external agents by regulating the interaction between the HSCs/progenitor cells and bone marrow environment. Disclosures: No relevant conflicts of interest to declare.

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