Cloning and Functional Analysis of the Rhesus Macaque ABCG2 Gene: Forced Expression Confers an SP Phenotype among Hematopoietic Stem Cell Progeny in Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3219-3219 ◽  
Author(s):  
Takahiro Ueda ◽  
Sebastian Brenner ◽  
Harry Malech ◽  
Saskia Langemeijer ◽  
Martha Kirby ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Rhesus Macaque ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Human Peripheral Blood ◽  
Hematopoietic Stem ◽  
Cell Counts ◽  
Abcg2 Gene

Abstract Hematopoietic cells can be highly enriched for repopulating ability based upon efflux of the fluorescent Hoechst 33342 dye by sorting for side population (SP) cells, a phenotype attributed to expression of ABCG2, a member of the ABC transporter superfamily. Intriguingly, murine studies suggest that forced ABCG2 expression prevents hematopoietic differentiation. We sought to determine the effects of forced expression of the ABCG2 gene in hematopoietic stem cells in the nonhuman primate model, a model with proven relevance to human hematopoiesis. We cloned the full-length rhesus ABCG2 (rh-ABCG2) cDNA using a series of primers spanning the entire sequence designed using the published human sequence. Sequence homology was greater than 96%. The rh-ABCG2 gene was then introduced into an MFGS based retroviral vector pseudotyped with the RD114 envelope. Mobilized human peripheral blood CD34-positive cells were transduced with either rh-ABCG2 or human GP91-phox vector with no other payload. All transductions were initiated with 4 x10e5 cells using X-VIVO10/1%HSA/4mM/L L-glutamine supplemented with 100ng/ml_FLT3L, 100ng/ml SCF, 100ng/ml TPO and polybrene (5 ug/ml). RD114 vector was concentrated by ultracentrifugation (83,000g 90minutes 4°C). Gene transfer rates to CFU of greater than 80% were achieved using both vectors with similar gene transfer rate estimated by flow cytometry. ABCG2-transduced human peripheral blood progenitor cells (PBPCs) acquired the SP phenotype, but showed significantly reduced growth compared to control (Day 8: cell counts 7.67+/− 2.54 vs. 17.83+/−6.64 x10e5 for ABCG2 and GP91-phox transduced cells, respectively p=0.0024, n=5). We then examined the engraftment of ABCG2-expressing stem and progenitor cells in the rhesus macaque autologous transplant model. GCSF/SCF mobilized PBPCs were collected from 2 animals and the CD34+ cells were divided and transduced with either vector and infused after lethal irradiation. In vivo marking levels post transplant measured in mononuclear cells and granulocytes from peripheral blood and bone marrow ranged initially from 0.5–4% by Realtime PCR, declined equally over time, and were similar between transduced fractions, with no discrepancy between bone marrow and peripheral blood marking. Furthermore, peripheral blood T cells, B cells and granulocytes expressed ABCG2 at levels predicted by vector copy number long term, and the differential of such cells within the SP gate matched that of the non-SP fraction demonstrating no block to differentiation in the large animal. In vitro studies showed selective protection against mitoxantrone among ABCG2-transduced rhesus PBPCs. Our results confirm the existence of rhesus-ABCG2, support its importance in conferring the SP phenotype, suggest no detrimental effect of its overexpression upon hematopoiesis, and imply a potential role for its overexpression as an in vivo selection strategy for gene therapy applications.

Prostaglandin E2 Inhibits Apoptosis in Hematopoietic Stem and Progenitor Cells and Enhances Their Survival Following Sub-Lethal Radiation Injury,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3401-3401
Author(s):  
Rebecca L Porter ◽  
Mary A Georger ◽  
Laura M Calvi
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Radiation Injury ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Cox 2 ◽  
Apoptotic Genes

Abstract Abstract 3401 Hematopoietic stem and progenitor cells (HSPCs) are responsible for the continual production of all mature blood cells during homeostasis and times of stress. These cells are known to be regulated in part by the bone marrow microenvironment in which they reside. We have previously reported that the microenvironmentally-produced factor Prostaglandin E2 (PGE2) expands HSPCs when administered systemically in naïve mice (Porter, Frisch et. al., Blood, 2009). However, the mechanism mediating this expansion remains unclear. Here, we demonstrate that in vivo PGE2 treatment inhibits apoptosis of HSPCs in naïve mice, as measured by Annexin V staining (p=0.0083, n=6–7 mice/group) and detection of active-Caspase 3 (p=0.01, n=6–7 mice/group). These data suggest that inhibition of apoptosis is at least one mechanism by which PGE2 expands HSPCs. Since PGE2 is a local mediator of injury and is known to play a protective role in other cell types, we hypothesized that it could be an important microenvironmental regulator of HSPCs during times of injury. Thus, these studies explored the role of PGE2 signaling in the bone marrow following myelosuppressive injury using a radiation injury model. Endogenous PGE2 levels in the bone marrow increased 2.9-fold in response to a sub-lethal dose of 6.5 Gy total body irradiation (TBI)(p=0.0004, n=3–11 mice/group). This increase in PGE2 correlated with up-regulation of microenvironmental Cyclooxygenase-2 (Cox-2) mRNA (p=0.0048) and protein levels at 24 and 72 hr post-TBI, respectively. Further augmentation of prostaglandin signaling following 6.5 Gy TBI by administration of exogenous 16,16-dimethyl-PGE2 (dmPGE2) enhanced the survival of functional HSPCs acutely after injury. At 24 hr post-TBI, the bone marrow of dmPGE2-treated animals contained significantly more LSK cells (p=0.0037, n=13 mice/group) and colony forming unit-spleen cells (p=0.037, n=5 mice/group). Competitive transplantation assays at 72 hr post-TBI demonstrated that bone marrow cells from irradiated dmPGE2-treated mice exhibited increased repopulating activity compared with cells from vehicle-treated mice. Taken together, these results indicate that dmPGE2 treatment post-TBI increases survival of functional HSPCs. Since PGE2 can inhibit apoptosis of HSPCs in naïve mice, the effect of dmPGE2 post-TBI on apoptosis was also investigated. HSPCs isolated from mice 24 hr post-TBI demonstrated statistically significant down-regulation of several pro-apoptotic genes and up-regulation of anti-apoptotic genes in dmPGE2-treated animals (3 separate experiments with n=4–8 mice/group in each), suggesting that dmPGE2 initiates an anti-apoptotic program in HSPCs following injury. Notably, there was no significant change in expression of the anti-apoptotic gene Survivin, which has previously been reported to increase in response to ex vivo dmPGE2 treatment of bone marrow cells (Hoggatt et. al., Blood, 2009), suggesting differential effects of dmPGE2 in vivo and/or in an injury setting. Additionally, to ensure that this inhibition of apoptosis was not merely increasing survival of damaged and non-functional HSPCs, the effect of early treatment with dmPGE2 post-TBI on hematopoietic recovery was assayed by monitoring peripheral blood counts. Interestingly, dmPGE2 treatment in the first 72 hr post-TBI significantly accelerated recovery of platelet levels and hematocrit compared with injured vehicle-treated mice (n=12 mice/group). Immunohistochemical analysis of the bone marrow of dmPGE2-treated mice also exhibited a dramatic activation of Cox-2 in the bone marrow microenvironment. This suggests that the beneficial effect of dmPGE2 treatment following injury may occur, both through direct stimulation of hematopoietic cells and also via activation of the HSC niche. In summary, these data indicate that PGE2 is a critical microenvironmental regulator of hematopoietic cells in response to injury. Exploitation of the dmPGE2-induced initiation of an anti-apoptotic program in HSPCs may represent a useful method to increase survival of these cells after sub-lethal radiation injury. Further, amplification of prostaglandin signaling by treatment with PGE2 agonists may also represent a novel approach to meaningfully accelerate recovery of peripheral blood counts in patients with hematopoietic system injury during a vulnerable time when few therapeutic options are currently available. Disclosures: No relevant conflicts of interest to declare.

Gene Transfer to Hematopoietic Stem/Progenitor Cells As a Novel Approach for Immunotherapy Against B-Lineage Malignancies: In Vivo Xenograft Model,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4168-4168
Author(s):  
Satiro N. De Oliveira ◽  
Francesca Giannoni ◽  
Cinnamon Hardee ◽  
Arineh Sahaghian ◽  
Laurence J N Cooper ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Malignant Cells ◽  
Hematopoietic Stem ◽  
Effector Cells ◽  
Nsg Mice ◽  
Novel Approach ◽  
B Lineage

Abstract Abstract 4168 Chimeric Antigen Receptors (CAR) against CD19 have been shown to direct T cells to specifically target B-lineage malignant cells in animal models and clinical trials, with efficient tumor cell lysis. But, there has been insufficient persistence of effector cells, limiting the clinical efficacy. We propose gene transfer to hematopoietic stem/progenitor cells (HSPC) as a novel approach to ensure persistent production of effector cells targeting B-lineage malignant cells, exponentially increasing the number of effectors that may be generated against tumor cells. Experiments were performed using NOD-SCID-IL2 receptor gamma chain null (NSG) mice engrafted with human CD34+ HSPCs transduced with lentiviral vectors carrying first and second generations of CD19-specific CAR. There was efficient and stable transduction with 1–2 copies of CAR/cell as determined by qPCR. Differentiation of modified HSPC in vivo was not impaired by gene transfer, as observed in vitro. Results of in vivo studies showed that CAR-transduced human HSPC successfully differentiated into all lineages, with CAR-expressing T, NK and myeloid cells populating bone marrow, spleen and peripheral blood. The human CD19+ B cell populations normally formed in the xenografted NSG mice were significantly reduced when the transplanted HSPC were transduced with the anti-CD19 CAR, demonstrating in vivo biological activity. Cells harvested from bone marrow and spleen of mice engrafted with modified HSPC lysed CD19-positive cell targets ex vivo. Leukemic challenges of engrafted mice are in progress. Our results provide evidence for the feasibility and efficacy of the modification of HSPC with CAR as a protocol for generation of effector cells for immunotherapy against B-lineage malignancies. Disclosures: No relevant conflicts of interest to declare.

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.

The Common Marmoset as a Target Preclinical Primate Model for Cytokine and Gene Therapy Studies

Blood ◽  
1999 ◽  
Vol 93 (9) ◽  
pp. 2839-2848 ◽  
Author(s):  
Hitoshi Hibino ◽  
Kenzaburo Tani ◽  
Kenji Ikebuchi ◽  
Ming-Shiuan Wu ◽  
Hajime Sugiyama ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Nonhuman Primate ◽  
Peripheral Blood ◽  
Common Marmoset ◽  
Transduction Efficiency ◽  
Hematopoietic Stem ◽  
The Common

Nonhuman primate models are useful to evaluate the safety and efficacy of new therapeutic modalities, including gene therapy, before the inititation of clinical trials in humans. With the aim of establishing safe and effective approaches to therapeutic gene transfer, we have been focusing on a small New World monkey, the common marmoset, as a target preclinical model. This animal is relatively inexpensive and easy to breed in limited space. First, we characterized marmoset blood and bone marrow progenitor cells (BMPCs) and showed that human cytokines were effective to maintain and stimulate in culture. We then examined their susceptibility to transduction by retroviral vectors. In a mixed culture system containing both marmoset stromal cells and retroviral producer cells, the transduction efficiency into BMPCs and peripheral blood progenitor cells (PBPCs) was 12% to 24%. A series of marmosets then underwent transplantation with autologous PBPCs transduced with a retroviral vector carrying the multidrug resistance 1 gene (MDR1) and were followed for the persistence of these cells in vivo. Proviral DNA was detectable by polymerase chain reaction (PCR) in peripheral blood granulocytes and lymphocytes in the recipients of gene transduced progenitors up to 400 days posttransplantation. To examine the function of the MDR1 gene in vivo, recipient maromsets were challenged with docetaxel, an MDR effluxed drug, yet the overall level of gene transfer attained in vivo (<1% in peripheral blood granulocytes) was not sufficient to prevent the neutropenia induced by docetaxel treatment. Using this model, we safely and easily performed a series of in vivo studies in our small animal center. Our results show that this small nonhuman primate, the common marmoset, is a useful model for the evaluation of gene transfer methods targeting 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.

scholarly journals In vivo gene transfer into rat bone marrow progenitor cells using rSV40 viral vectors

Blood ◽  
2005 ◽  
Vol 106 (8) ◽  
pp. 2655-2662 ◽  
Author(s):  
Bianling Liu ◽  
Judy Daviau ◽  
Carmen N. Nichols ◽  
David S. Strayer
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Transgene Expression ◽  
Viral Vectors ◽  
Ex Vivo ◽  
Simian Virus 40 ◽  
Hematopoietic Stem ◽  
Entire Study

AbstractHematopoietic stem cell (HSC) gene transfer has been attempted almost entirely ex vivo and has been limited by cytokine-induced loss of self-renewal capacity and transplantation-related defects in homing and engraftment. Here, we attempted to circumvent such limitations by injecting vectors directly into the bone marrow (BM) to transduce HSCs in their native environment. Simian virus 40 (SV40)–derived gene delivery vectors were used because they transduce resting CD34+ cells very efficiently. Rats received SV-(Nef-FLAG), carrying FLAG marker epitope—or a control recombinant SV40 (rSV40)—directly into both femoral marrow cavities. Intracellular transgene expression by peripheral blood (PB) or BM cells was detected by cytofluorimetry. An average of 5.3% PB leukocytes expressed FLAG for the entire study—56 weeks. Transgene expression was sustained in multiple cell lineages, including granulocytes (average, 3.3% of leukocytes, 20.4% of granulocytes), CD3+ T lymphocytes (average, 0.53% of leukocytes, 1% of total T cells), and CD45R+ B lymphocytes, indicating gene transfer to long-lived progenitor cells with multilineage capacity. An average of 15% of femoral marrow cells expressed FLAG up to 16.5 months after transduction. Thus, direct intramarrow administration of rSV40s yields efficient gene transfer to rat BM progenitor cells and may be worthy of further investigation.

A Novel Observation That Heme Oxygenase-1 (HO-1) Deficient Mice Are Easy Mobilizers and That HO-1 Plays An Important Role in Maintaining Expression of SDF-1 in Bone Marrow (BM) Stroma and Promotes Retention of Hematopoietic Stem/Progenitor Cells (HSPCs) in the Bone Marrow Microenvironment

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 315-315
Author(s):  
Marcin Wysoczynski ◽  
Janina Ratajczak ◽  
Gregg Rokosh ◽  
Roberto Bolli ◽  
Mariusz Z Ratajczak
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Heme Oxygenase ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Mrna Level ◽  
Heme Oxygenase 1 ◽  
Hematopoietic Stem ◽  
Normal Peripheral Blood ◽  
Cell Counts

Abstract Abstract 315 Background. Heme oxygenase (HO) is an enzyme that catalyzes the degradation of heme. Two distinct HO isoforms have been identified: HO-2, which is constitutively expressed, and HO-1, which is stress-responsive and plays an important function in various physiological and pathophysiological states associated with cellular stress. HO-1 plays a role in ischemic/reperfusion injury, atherosclerosis, and cancer. It has also been reported that HO-1 regulates expression of a-chemokine stromal derived factor-1 (SDF-1) in myocardium (J Mol Cell Cardiol.2008;45:44–55). Aim of study. Since SDF-1 plays a crucial role in retention and survival of hematopoietic stem cell/progenitor cells (HSPCs) in BM, we become interested in whether deficiency of HO-1 affects normal hematopoiesis and retention of HSPCs in BM. Experimental approach. To address this issue, we employed several complementary strategies to investigate HO-1−/−, HO+/–, and wild type (wt) mouse littermates for i) the expression level of SDF-1 in BM, ii) the number of clonogenic progenitors from major hematopoietic lineages in BM, iii) peripheral blood (PB) cell counts, iv) chemotactic responsiveness of HSPCs to an SDF-1 gradient, iv) adhesiveness of clonogenic progenitors, v) the number of circulating HSPCs in PB, and vi) the degree of mobilization in response to granulocyte-colony stimulating factor (G-CSF) or AMD3100 assessed by enumerating the number of CD34–SKL cells and clonogeneic progenitors (CFU-GM) circulating in PB. Results: Our data indicate that under normal, steady-state conditions, HO-1−/− and HO+/– mice have normal peripheral blood cell counts and numbers of circulating CFU-GM. Interestingly, lack of HO-1 leads to an increase in the number of erythroid (BFU-E) and megakaryocytic (CFU-GM) progenitors in BM. Next, BMMNCs from HO-1−/−have normal expression of the SDF-1-binding receptor, CXCR4, but a 5-times lower level of CXCR7, which is another SDF-1-binding receptor. Of note, we observed that the mRNA level for SDF-1 in BM-derived fibroblasts was ∼4 times lower. This corresponded with the observation in vitro that HSPCs from HO-1−/−animals responded more robustly to an SDF-1 gradient, and HO-1−/−animals mobilized a higher number of CD34–SKL cells and CFU-GM progenitors into peripheral blood in response to G-CSF and AMD3100. Conclusions: Our data demonstrate for the first time that heme oxygenase plays an important and underappreciated role in BM retention of HSPCs and may affect their trafficking. Since small non-toxic molecular inhibitors of HO-1 have been developed for clinical use (e.g., metaloporhirins), blockage of HO-1 could be a novel strategy for mobilizing HSPCs. Our recent in vivo mobilization studies lend support to this hypothesis. Disclosures: No relevant conflicts of interest to declare.

Further Evidence That HO-1 Regulates SDF-1 Expression in the Bone Marrow Microenvironment and That HO-1-Deficient Mice Show a Defect in the SDF-1–CXCR4 Retention Axis of Hematopoietic Stem/Progenitor Cells in Bone Marrow and Thus Are Easy Mobilizers - Studies in HO-1 Mutant Mice, Irradiation Chimeras, and the Effect of in Vivo Pharmacological Inhibition of HO-1

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 344-344
Author(s):  
Marcin Wysoczynski ◽  
Janina Ratajczak ◽  
Gregg Rokosh ◽  
Roberto Bolli ◽  
Mariusz Z Ratajczak
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Crucial Role ◽  
Conflicts Of Interest ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Cell Counts ◽  
Deficient Mice

Abstract Abstract 344 Background: Stromal derived factor-1 (SDF-1), which binds to the CXCR4 receptor expressed on the surface of hematopoietic stem/progenitor cells (HSPCs), plays an important role in the retention of HSPCs in BM niches. Heme oxygenase (HO-1) is a stress-responsive enzyme that catalyzes the degradation of heme and plays an important function in various physiological and pathophysiological states associated with cellular stress, such as ischemic/reperfusion injury, atherosclerosis, and cancer. Interestingly, it has also been reported that HO-1 regulates the expression of SDF-1 in myocardium (J Mol Cell Cardiol. 2008;45:44–55). Aim of study: Since SDF-1 plays a crucial role in retention and survival of HSPCs in BM, we become interested in whether HO-1 is expressed by BM stromal cells and whether deficiency of HO-1 affects normal hematopoiesis and retention of HSPCs in BM. Experimental approach: To address this issue, we employed several complementary strategies to investigate HO-1–/–, HO-1+/–, and wild type (wt) mouse littermates for i) the expression level of SDF-1 in BM, ii) the number of clonogenic progenitors from major hematopoietic lineages in BM, iii) peripheral blood (PB) cell counts, iv) the chemotactic responsiveness of HSPCs to an SDF-1 gradient as well as to other chemoattractants, including sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), and extracellular nucleotiodes (ATP, UTP), iv) the adhesiveness of clonogenic progenitors to immobilized SDF-1 and stroma, v) the number of circulating HSPCs in PB, and vi) the degree of mobilization in response to granulocyte-colony stimulating factor (G-CSF) or AMD3100, assessed by enumerating the number of CD34–SKL cells and clonogeneic progenitors (CFU-GM) circulating in PB. We also exposed mice to the small HO-1 molecular inhibitor tin protoporphyrin IX (SnPP) and studied the effect of this treatment on G-CSF- or AMD3100-induced mobilization of HSPCs. Finally, to prove an environmental HSPC retention defect in HO-1-deficient mice, we created radiation chimeras, wild type mice transplanted with HO-1-deficient BM cells, and, vice versa, HO-1-deficient mice reconstituted with wild type BM cells. Results: Our data indicate that under normal, steady-state conditions, HO-1–/– and HO+/– mice have normal PB cell counts and numbers of circulating CFU-GM, while a lack of HO-1 leads to an increase in the number of erythroid (BFU-E) and megakaryocytic (CFU-GM) progenitors in BM. However, while BMMNCs from HO-1–/– have normal expression of the SDF-1-binding receptor, CXCR4, we observed that the mRNA level for SDF-1 in BM-derived fibroblasts was ∼4 times lower. This corresponded with the observation in vitro that HSPCs from HO-1–/– animals respond more robustly to an SDF-1 gradient, and HO-1–/– animals mobilized a higher number of CD34–SKL cells and CFU-GM progenitors into PB in response to G-CSF and AMD3100. Both G-CSF and AMD3100 mobilization were also significantly enhanced in normal wild type mice after in vivo administration of HO-1 inhibitor. Finally, mobilization studies in irradiation chimeras confirmed the crucial role of the microenvironmental SDF-1-based retention mechanism of HSPCs in BM niches. Conclusions: Our data demonstrate for the first time that HO-1 plays an important and underappreciated role in modulating the SDF-1 level in the BM microenvironment and thus plays a role in retention of HSPCs in BM niches. Furthermore, our recent data showing a mobilization effect by a small non-toxic molecular inhibitor of HO-1 (SnPP), suggest that blockage of HO-1 could be a promising strategy to facilitate mobilization of HSPCs. Further studies are also needed to evaluate the role of HO-1 in homing of HSPCs after transplantation to BM stem cell niches. Disclosures: No relevant conflicts of interest to declare.

The Common Marmoset as a Target Preclinical Primate Model for Cytokine and Gene Therapy Studies

Blood ◽  
1999 ◽  
Vol 93 (9) ◽  
pp. 2839-2848 ◽  
Author(s):  
Hitoshi Hibino ◽  
Kenzaburo Tani ◽  
Kenji Ikebuchi ◽  
Ming-Shiuan Wu ◽  
Hajime Sugiyama ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Nonhuman Primate ◽  
Peripheral Blood ◽  
Common Marmoset ◽  
Transduction Efficiency ◽  
Hematopoietic Stem ◽  
The Common

Abstract Nonhuman primate models are useful to evaluate the safety and efficacy of new therapeutic modalities, including gene therapy, before the inititation of clinical trials in humans. With the aim of establishing safe and effective approaches to therapeutic gene transfer, we have been focusing on a small New World monkey, the common marmoset, as a target preclinical model. This animal is relatively inexpensive and easy to breed in limited space. First, we characterized marmoset blood and bone marrow progenitor cells (BMPCs) and showed that human cytokines were effective to maintain and stimulate in culture. We then examined their susceptibility to transduction by retroviral vectors. In a mixed culture system containing both marmoset stromal cells and retroviral producer cells, the transduction efficiency into BMPCs and peripheral blood progenitor cells (PBPCs) was 12% to 24%. A series of marmosets then underwent transplantation with autologous PBPCs transduced with a retroviral vector carrying the multidrug resistance 1 gene (MDR1) and were followed for the persistence of these cells in vivo. Proviral DNA was detectable by polymerase chain reaction (PCR) in peripheral blood granulocytes and lymphocytes in the recipients of gene transduced progenitors up to 400 days posttransplantation. To examine the function of the MDR1 gene in vivo, recipient maromsets were challenged with docetaxel, an MDR effluxed drug, yet the overall level of gene transfer attained in vivo (&lt;1% in peripheral blood granulocytes) was not sufficient to prevent the neutropenia induced by docetaxel treatment. Using this model, we safely and easily performed a series of in vivo studies in our small animal center. Our results show that this small nonhuman primate, the common marmoset, is a useful model for the evaluation of gene transfer methods targeting 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

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.

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