Change in the number of CD117+ stem cells, cytogenetic and cytokinetic parameters under the use of candesartan, candesartan cilexetil and resveratrol in vitro

Due to the increase in cardiovascular disease, it is urgent to research new effective and safe drugs and their combinations. Candesartan cilexetil, an angiotensin II receptor antagonist, is a precursor to the active form of candesartan. However, these antiischemic drugs have a cytotoxic effect, affecting the antioxidant system. Therefore, to prevent the cytotoxic effect is the need to use antioxidants. To study the effect of candesartan cilexetil, candesartan and resveratrol antioxidant in various doses and combinations on CD117+ stem cell mobilization, on the number of apoptotic and micronucleated cells and cell cycle parameters in vitro. Bone marrow cells isolated from C57Bl / 6 mice were selected for experiments. After incubation for 2 days with the means in different concentrations and combinations, the biological characteristics of the stem cells were determined. Flow cytometry was used to analyze the number of CD117 + stem cells, the ratio of apoptotic cells, cells with micronuclei and cell cycle parameters when using candesartan cilexetil, candesartan, and resveratrol in vitro. It was found that using candesartan cilexetil with resveratrol and candesartan with resveratrol promotes the formation of CD117 + stem cells from 1.2 times to almost 2 times compared with controls and 1.5 and 2.5 compared with cytostatics. Candesartan cilexetil and candesartan were cytotoxic, while resveratrol reduced the adverse effects of the substances in combination. Combination of candesartan cilexetil with resveratrol; Candesartan with resveratrol significantly increased CD117+ stem cell count and was not cytotoxic.

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Brief Note: Effect of Erythropoietin on Polycythemic Mouse Spleen in Vitro IV. Response of the Colony-forming Cells to Erythropoietin

Blood ◽

10.1182/blood.v32.2.271.271 ◽

1968 ◽

Vol 32 (2) ◽

pp. 271-277 ◽

Cited By ~ 1

Author(s):

HIDEAKI MIZOGUCHI ◽

YASUSADA MIURA ◽

FUMIMARO TAKAKU ◽

KIKU NAKAO

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Bone Marrow Transplantation ◽

Marrow Transplantation ◽

In Vitro System ◽

Cell Pool ◽

Stem Cell Pool

Abstract It is shown that an in vitro system of assaying the size of an erythropoietin-responsive stem cell pool could be applied to the spleens of polycythemic mice after irradiation and bone marrow transplantation. With this method, the presence of erythropoietin-responsive cells in the spleen was first detected on the second day after transplantation. Therefore, it is considered probable that colony-forming cells and erythropoietin-responsive cells are at different stages of maturation or cell cycle. Furthermore, necessity of erythropoietin for further differentiation of transplanted stem cells into erythroblasts is also suggested.

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

Blood ◽

10.1182/blood.v106.11.2190.2190 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2190-2190 ◽

Cited By ~ 1

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.

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Long Term Maintenance of Myeloid Leukemia Stem Cell-Like Populations Cultured with Mesenchymal Stromal Cells (MSC)

Blood ◽

10.1182/blood.v120.21.3546.3546 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3546-3546

Author(s):

Sawa Ito ◽

A. John Barrett ◽

Andre Larochelle ◽

Nancy F. Hensel ◽

Keyvan Keyvanfar ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Stem Cell ◽

Culture Conditions ◽

Myelogenous Leukemia ◽

Cell Cycle Analysis ◽

Remission Induction ◽

Leukemia Stem Cell

Abstract Abstract 3546 Because MSC support the growth and the differentiation of normal hematopoietic stem cells we hypothesized that MSC might also support leukemia cells, in particular leukemia stem cells (LSC) in vitro. We cultured blast cells from patients with acute myelogenous leukemia (AML) in liquid medium to study persistence of stem-cell-like and differentiated leukemia cell populations by flow cytometry, with and without MSC and additional growth factors. Cryopresrerved peripheral blood mononuclear cells (PBMC) were obtained from 6 AML patients (mean Age 47, range 23–74). Leukemia blasts were isolated by sorting live (propidium iodide (PI)-negative) CD34+ lineage (CD2+, CD3+, CD14+ and CD19+) -negative cells using a FACS ARIA II cell sorter (BD). Sorted blasts (2.5 ×105 cells) were co-cultured with an equal number of irradiated MSC derived from healthy donor bone marrow in RPMI medium supplemented with 10% human serum, with or without a human cytokine (CYTO) mixture (50 ng/ml interleukin 3, 150 ng/ml stem cell factor, and 150ng/ml Flt-3 ligand). MSC were replenished every two weeks. The phenotype of cultured cells was analyzed weekly using fluorescently-conjugated monoclonal antibodies against CD34, CD38, and CD45, plus the lineage panel and a dead cell exclusion dye Cell cycle analysis with Hoeschst 33342 and Pyronin Y was performed on cells co-stained with CD34, CD45 and PI. Primary leukemia samples were phenotypically heterogeneous with respect to proportions of cells (co-)staining for CD34 and CD38 as previously reported: three samples showed CD34+CD38- predominance (LSC-like leukemia), and three were CD34+CD38+ (common myeloid progenitor (CMP)-like leukemia). LSC-like leukemia maintained viable CD34+CD38- cells for at least 6 weeks when co-cultured with MSC alone, in contrast to cultures with cytokines or medium only which showed rapid decline in the LSC populations and no prolonged maintenance of viable cells (p=0.0005) (Figure, left panel). CMP-like leukemia maintained their CD34+CD38+ phenotype when co-cultured with MSC alone but persistence of this subset was not significantly different from the other culture conditions (p=0.5) and no culture remained viable after 4 weeks (Figure, right panel). Cell cycle analysis showed that co-culture with MSC maintained CD34+ blasts in G0 significantly more than other culture conditions (P<0.0001). We conclude that MSC support the maintenance of a leukemia stem cell phenotype in a long- term (6 week) in vitro culture system. The differential capacity of MSC to support LSC- like and CMP- like leukemia may be associated with the different frequency of leukemia initiating cells within each leukemic blast population. NSG mice xenotranplant model experiments are ongoing to confirm this hypothesis. Co-culture of LSC with MSC represents a simple approach to maintain LSC in vitro and could be utilized to screen the drug targeting LSCs. Further study of the effect of MSC on LSC would elucidate a potential mechanism whereby the marrow microenvironment serves as a reservoir of persisting leukemia after remission induction chemotherapy. Disclosures: No relevant conflicts of interest to declare.

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In vivo and in vitro stem cell function of c-kit- and Sca-1-positive murine hematopoietic cells

Blood ◽

10.1182/blood.v80.12.3044.bloodjournal80123044 ◽

1992 ◽

Vol 80 (12) ◽

pp. 3044-3050 ◽

Cited By ~ 2

Author(s):

S Okada ◽

H Nakauchi ◽

K Nagayoshi ◽

S Nishikawa ◽

Y Miura ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Cell Function ◽

Bone Marrow Cells ◽

Synergistic Effects ◽

Single Cells ◽

Hematopoietic Stem

c-kit is expressed on hematopoietic stem cells and progenitor cells, but not on lymphohematopoietic differentiated cells. Lineage marker- negative, c-kit-positive (Lin-c-kit+) bone marrow cells were fractionated by means of Ly6A/E or Sca-1 expression. Lin-c-kit+Sca-1+ cells, which consisted of 0.08% of bone marrow nucleated cells, did not contain day-8 colony-forming units-spleen (CFU-S), but 80% were day-12 CFU-S. One hundred cells rescued the lethally irradiated mice and reconstituted hematopoiesis. On the other hand, 2 x 10(3) of Lin-c- kit+Sca-1- cells formed 20 day-8 and 11 day-12 spleen colonies, but they could not rescue the lethally irradiated mice. These data indicate that Lin-c-kit+Sca-1+ cells are primitive hematopoietic stem cells and that Sca-1-cells do not contain stem cells that reconstitute hematopoiesis. Lin-c-kit+Sca-1+ cells formed no colonies in the presence of stem cell factor (SCF) or interleukin-6 (IL-6), and only 10% of them formed colonies in the presence of IL-3. However, approximately 50% of them formed large colonies in the presence of IL-3, IL-6, and SCF. Moreover, when single cells were deposited into culture medium by fluorescence-activated cell sorter clone sorting system, 40% of them proliferated on a stromal cell line (PA-6) and proliferated for more than 2 weeks. In contrast, 15% of the Lin-c- kit+Sca-1-cells formed colonies in the presence of IL-3, but no synergistic effects were observed in combination with SCF plus IL-6 and/or IL-3. Approximately 10% proliferated on PA-6, but most of them degenerated within 2 weeks. The population ratio of c-kit+Sca-1+ to c-kit+Sca-1- increased 2 and 4 days after exposure to 5-fluorouracil (5-FU). These results are consistent with the relative enrichment of highly proliferative colony-forming cells by 5-FU. These data show that, although c-kit is found both on the primitive hematopoietic stem cells and progenitors, Sca-1+ cells are more primitive and respond better than Sca-1- cells to a combination of hematopoietic factors, including SCF and stromal cells.

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CD34+ Subclasses of Blood Stem Cell Grafts Collected After Plerixafor Injection in Non-Hodgkin Lymphoma (NHL) Patients Mobilizing Poorly with Chemotherapy Plus G-CSF

Blood ◽

10.1182/blood.v118.21.2990.2990 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2990-2990

Author(s):

Esa Jantunen ◽

Ville Varmavuo ◽

Piia Valonen ◽

Taru Kuittinen ◽

Tapio Nousiainen ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Stem Cell Mobilization ◽

High Dose ◽

In Vitro Growth ◽

Control Groups ◽

Cd34 Cells ◽

Cell Mobilization

Abstract Abstract 2990 Background: Mobilization of blood stem cells is difficult in a subgroup of patients with standard methods. Plerixafor, a CXCR4 antagonist, has been used for stem cell mobilization in combination with G-CSF for some years. Mobilization method used may affect not only efficacy of stem cell mobilization and collection but also graft content which on the other hand may have effect on post-transplant outcomes. No data is available on CD34+ subclasses in grafts collected after plerixafor administration in patients who mobilize poorly with chemotherapy plus G-CSF. Patients and Methods: Altogether blood stem cell grafts collected from 26 NHL patients were studies. Thirteen patients (8 M, 5 F, median age 51 yrs) were mobilized with a combination of chemotherapy and G-CSF ad received plerixafor due to poor mobilization followed by stem cell apheressis. Thirteen patients (10 M, 3 F, median age 56 yrs) were mobilized with chemotherapy plus G-CSF without plerixafor and served as controls. Samples from the first collection after plerixafor and from the first apheresis of control patients were studied by flow cytometry using the following antibodies: CD34, CD38, CD 117, CD133, CD19 and CD45. Viability of CD34+ cells after freezing was assessed with 7-aminoactinomycin D staining. Also in vitro growth of granulocyte/macrophage progenitors (GM-CFU) were assessed from all grafts. Patients were followed after high-dose chemotherapy in regard to hematopoietic reconstitution. Results: The number of viable cells in the grafts was comparable between the plerixafor and the control groups (Table 1). The number of the most primitive stem cells (CD34+CD38−CD133+) was higher in plerixafor mobilized grafts (Table 1). Most of the CD34+ cells were myeloid progenitors, as defined by their CD117 antigen co-expression. No differences in GM-CFU were observed between the groups. All except one patient had received high-dose therapy. The median number of CD34+ cells collected from the patients was comparable (3.1 vs. 3.3 × 106/kg). The median time to reach neutrophils > 10 × 109/L was 10 days from the stem cell infusion in both groups and time to unsupported platelets was also comparable (16 d vs. 13 d). Platelet counts at 1 month, 3 months and 6 months were comparable between the groups. Absolute lymphocyte counts were higher in plerixafor group but the differences were not statistically significant. One early toxic death occurred in the plerixafor mobilized group and one death due to disease recurrence in both groups with a median follow-up of 301 and 348 days from stem cell infusion in prelixafor and control groups, respectively. Conclusions: Plerixafor added to chemomobilization in NHL patients resulted in higher number of the most primitive CD34+ cells in the graft with comparable in vitro growth and engraftment potential after BEAM chemotherapy when compared to patients mobilized without plerixafor. Longer follow-up of higher patient numbers are needed to evaluate whether differences in graft content have an effect on patient outcomes. Disclosures: Jantunen: Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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EPCR Limits Nitric Oxide Levels, Mediating Human and Murine Stem Cell Adhesion and Retention In The Bone Marrow, By Conjugating PAR1 and CXCR4 Signaling

Blood ◽

10.1182/blood.v122.21.795.795 ◽

2013 ◽

Vol 122 (21) ◽

pp. 795-795

Author(s):

Shiri Gur Cohen ◽

Tomer Itkin ◽

Aya Ludin ◽

Sagarika Chakrabarty ◽

Orit Kollet ◽

...

Keyword(s):

Nitric Oxide ◽

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Protein C ◽

Cxcr4 Signaling ◽

Cell Mobilization ◽

Epcr Shedding

Abstract Long term repopulating hematopoietic stem cells (LTR-HSC) in the murine bone marrow (BM) highly express endothelial protein C receptor (EPCR), yet the function of EPCR in HSC is incompletely defined. EPCR is expressed primarily on endothelial cells and has anti coagulation and anti inflammatory roles. While physiological stress due to injury or bleeding is a strong inducer of HSC mobilization and leukocyte production, a role for the coagulation protease thrombin, and its major receptor PAR1 in regulation of HSC has not yet been identified. We hypothesized that thrombin plays a role in HSC mobilization in the context of injury and that, conversely, signaling involving EPCR and its ligand activated protein C (aPC) play a regulatory role in HSC maintenance. Herein, we report that murine BM EPCRhigh stem cells display enhanced CXCL12 mediated adhesion and reduced migration capacitie, while motile circulating HSC in the murine blood and spleen lack high EPCR expression. Mechanistically, we found that EPCR is a negative regulator of nitric oxide (NO) levels. EPCRhigh stem cells display low intracellular NO levels, low motility, and increased adhesion to BM stroma. Furthermore, EPCRlow transgenic mouse cells displayed reduced stem cell adhesion to BM stroma and increased motility, manifested by reduced EPCRlow HSC in the BM and their corresponding increased levels in the blood. In vitro stimulation with the EPCR ligand, aPC, which we found to be physiologically expressed adjacent to small murine BM blood vessels, augmented EPCRhigh HSC adhesion and further limited their intracellular NO content by increasing eNOS phosphorylation at Thr495 in BM HSC, causing reduced production of NO. Conversely, administration of the pro-coagulant protease thrombin to mice induced PAR1 mediated EPCR shedding from BM HSC, followed by CXCR4 upregulation on HSC, and PAR1-mediated CXCL12 secretion by BM stromal cells. Together, these events lead to loss of retention and rapid stem cell mobilization to the blood. Interestingly, shedding of EPCR was found to be mediated by elevation of intracellular NO content, leading to EPCR co-localization with Caveolin. Correspondingly, thrombin failed to induce EPCR shedding and mobilization in eNOS and PAR1 deficient mice. Additionally, we found that BM LTR-HSC functionally express the metalloproteinase TACE (ADAM17) on the cell membrane, and that in- vitro inhibition of TACE activity by a newly developed selective inhibitor, reduces thrombin- mediated EPCR shedding, suggesting the involvement of TACE in EPCR shedding and HSC mobilization. Moreover, EPCR shedding was also CXCR4 dependent, revealing a crosstalk between EPCR, PAR1 and CXCR4. HSPC mobilized by thrombin possessed increased long-term repopulation capability following transplantation into lethally irradiated recipient mice and re-synthesis of EPCR by donor HSC in the engrafted host BM. In addition, EPCR expression was re-induced on circulating stem cells following in vitro treatment with eNOS inhibitor. Interestingly, bypassing eNOS by directly injecting NO donor, induced EPCR shedding, CXCR4 upregulation and rapid HSPC mobilization in both wild type and eNOS KO mice. Importantly, we found that similar to mice, EPCR was selectively and highly expressed by primitive human BM CD34+CD38- HSC, but not in the blood circulation of clinical G-CSF mobilized stem cells or in motile cord blood stem cells. Human BM CD34+/CD38- HSC are functionally EPCRhigh cells, maintaining low levels of intracellular NO which mediates their increased adhesion, while EPCR shedding was important for their migration and mobilization. In the functional pre-clinical NOD/SCID mouse model, G-CSF mobilization induced EPCR shedding, up-regulation of PAR1 and CXCR4 on human stem and progenitor cells, while NO signaling inhibition blocked G-CSF induced mobilization and increased both murine and human EPCRhigh stem cell accumulation in the murine BM. Our results define functional roles for EPCR, on both human and murine HSC, and suggest that regulation of EPCR expression is linked to NO, PAR1 and CXCR4 signaling as a pivotal mechanism determining HSC localization and function. Our study reveals that activation of coagulation in the context of cell injury controls stem cells retention and motility, and suggests that targeting this system may be useful in improving clinical stem cell mobilization and transplantation protocols. Disclosures: No relevant conflicts of interest to declare.

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Hematopoietic Stem Cell Expansion by HOXB4 Is Greatly Enhanced in p21 Deficient Stem Cells.

Blood ◽

10.1182/blood.v104.11.1688.1688 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1688-1688 ◽

Cited By ~ 1

Author(s):

Noriko Miyake ◽

Ann C.M. Brun ◽

Mattias Magnusson ◽

David T. Scadden ◽

Stefan Karlsson

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem Cell ◽

Bone Marrow Cells ◽

Ex Vivo ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Hox transcription factors have emerged as important regulators of hematopoiesis. In particular, enforced expression of HOXB4 is a potent stimulus for murine hematopoietic stem cell (HSC) self-renewal. Murine HSCs engineered to overexpress HoxB4 expand significantly more than control cells in vivo and ex vivo while maintaining a normal differentiation program. HSCs are regulated by the cell proliferation machinery that is intrinsically controlled by cyclin-dependent kinase inhibitors such as p21Cip1/Waf1(p21) and p27Kip1 (p27). The p21 protein restricts cell cycling of the hematopoietic stem cell pool and maintains hematopoietic stem cell quiescence. In order to ask whether enhanced proliferation due to HOXB4 overexpression is mediated through suppression of p21 we overexpressed HOXB4 in hematopoietic cells from p21−/− mice. First, we investigated whether human HOXB4 enhances in vitro expansion of BM cells from p21−/− mice compared to p21+/+ mice. 5FU treated BM cells from p21−/− or p21+/+ mice were pre-stimulated with SCF, IL-6, IL-3 for 2 days followed by transduction using retroviral vector expressing HOXB4 together with GFP (MIGB4) or the control GFP vector (MIG). The cells were maintained in suspension cultures for 13 days and analyzed for GFP positive cells by flow-cytometry. Compared to MIG transduced BM cells from p21+/+ mice (MIG/p21+), the numbers of GFP positive cells were increased 1.1-fold in MIG/p21−, 3.2-fold in MIGB4/p21+ and 10.0-fold in MIGB4/p21− respectively (n=5). CFU assays were performed after 13 days of culture. The numbers of CFU were increased 4.8-fold in MIG/p21−, 19.5-fold in MIG/p21+ and 33.9 -fold in MIGB4/p21− respectively. Next, we evaluated level of HSCs expansion by bone marrow repopulation assays. After 12-days of culture, 1.5 x 105 MIGB4 or MIG-transduced cells (Ly5.2) were transplanted into lethally irradiated mice in combination with 8 x 105 fresh Ly5.1 bone marrow cells. Sixteen weeks after transplantation, no Ly5.2 cells could be detected in MIG/p21+ or MIG/p21− transplanted mice (n=6). In contrast, Ly5.2 positive cells were detected in both MIGB4/p21+/+ and MIGB4/p21−/− cells. The % of Ly5.2 positive cells in MIGB4/p21− transplanted mice was 9.9-fold higher than MIGB4/p21+ transplanted mice. (38.4 % vs 3.9 %, P<0.02, n=5). These Ly5.2 positive cells differentiated into all lineages, as determined by proportions of Mac-1, B-220, CD3 and Ter119 positive populations. Currently, we are enumerating the expansion of HOXB4 transduced HSCs in p21 deficient BM cells using the CRU assay. Our findings suggest that HOXB4 increases the self-renewal of hematopoietic stem cells by a mechanism that is independent of p21. In addition, the findings demonstrate that deficiency of p21 in combination with enforced expression of HOXB4 can be used to rapidly and effectively expand hematopoietic stem cells.

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Beta Glucan Enhances Hematopoietic Stem Cell Mobilization.

Blood ◽

10.1182/blood.v108.11.1348.1348 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1348-1348

Author(s):

Stephanie Wagner ◽

Daniel Cramer ◽

Richard Hansen ◽

Ryan Reca ◽

Mariusz Ratajczak ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Mechanism Of Action ◽

Stem Cell Mobilization ◽

Combination Group ◽

Beta Glucan ◽

Hematopoietic Stem ◽

Cell Mobilization

Abstract Background: Peripheral blood stem cell infusion is the preferred method for establishing hematopoiesis in transplantation. Use of G-CSF is now the most commonly used mobilizing agent. Despite advances in stem cell techniques and agents, studies have shown that up to 20–25% of patients exhibit poor mobilization and are not able to proceed with autotransplantation. Strategies to improve mobilization include using chemotherapy alone or in conjunction with growth factor or novel agents such as AMD3100. β-glucan PGG is a soluble yeast beta glucan with a molecular mass of 150kD comprised of a β-D-(1–3)-linked glucopyranosyl backbone with a β-D-(1–6)-linked β(1–3) side chains. In previous studies, β-glucan PGG has been shown to induce hematopoietic stem and progenitor cell (HSPC) mobilization to the periphery. In this study, we examined β-glucan PGG’s ability to mobilize HSPC alone and in conjuction with G-CSF and explored its mechanism of action. Methods: Prior to our study, dose kinetic studies were done and showed peak stem cell mobilization at 24 hours and maximum results using the 9.2 mg/kg dose with β-glucan PGG alone. In this study, four groups of wildtype (WT) C57BL/6 mice (6 mice/group) were used; control (saline injection × 4 days), G-CSF only (125ug/d × 4 days), PGG only (4.8 or 9.2mg/kg × one dose), and G-CSF/PGG combination. In the combination group, G-CSF injections were given daily for four days and one PGG injection on day three. Four hours after the last G-CSF injection, the mice were sacrificed and final white blood cell count were collected. Blood was assayed for in vitro mobilization in methycellulose culture. Peritoneal macrophage were stimulated with PGG and supernatant was harvested at times indicated and concentration of MMP-9 was determined using ELISA (R&D Systems). Results: All treated groups showed increased mobilization of all major cell lines (CFU-GM, CFU-M, and CFU-G). β-glucan PGG alone was able to mobilize peripheral stem cells at both doses (4.8mg/kg–9.3 CFU/200000 PBL and 9.2mg/kg–14 CFU/200000 PBL). The combination group (G-CSF/PGG-4.8mg/kg) showed an almost two-fold increase in CFU compared to G-CSF alone (G-CSF-30.42 CFU/200000 PBL, G-CSF/PGG(4.8)-57 CFU/200000 PBL, p=0.008). Initial in vitro chemotaxis assays revealed β-glucan PGG induces HPSC mobilization independent of SDF-1 (stromal derived factor) gradient. Our previous studies have demonstrated that β-glucan can enhance bone marrow engraftment via a CR3 dependent mechanism. However, our current study indicated that β-glucan mobilized stem cells via a CR3 independent mechanism and did not induce appreciable levels of cytokine secretion. To further explore its mechanism of action, we stimulated peritoneal macrophages with β-glucan PGG. Strikingly, β-glucan PGG stimulated macrophages to produce significant amounts of matrix metalloproteinase-9 (MMP-9). Similarly, β-glucan PGG also stimulated bone marrow-derived macrophages to secrete MMP-9. Conclusion: β-glucan PGG is an agent that enhances stem cell mobilization alone and has a synergistic effect when used in conjunction with G-CSF. The mechanism of mobilization by β-glucan PGG involves MMP-9, which results from release of pro-MMP-9 from marrow macrophages. The efficacy of β-glucan PGG and lack of proinflammatory activity make it an attractive agent to supplement mobilization with G-CSF.

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The Sooner the Better: Rapid Transduction Enhances the Engraftment/Selection of Gene Corrected Fanconi Anemia HSC.

Blood ◽

10.1182/blood.v110.11.194.194 ◽

2007 ◽

Vol 110 (11) ◽

pp. 194-194 ◽

Cited By ~ 1

Author(s):

Lars U.W. Muller ◽

Michael Milsom ◽

Chad E. Harris ◽

Jeff Bailey ◽

David A. Williams

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Bone Marrow ◽

Stem Cell ◽

Reporter Gene ◽

Fanconi Anemia ◽

Peripheral Blood ◽

Bone Marrow Cells

Abstract Fanconi anemia (FA) is amenable to genetic correction of hematopoietic stem cells (HSCs). However, as demonstrated in previous clinical gene therapy trials, successful extension of murine studies into human therapies is limited by low numbers of target HSC and poor engraftment of transduced FA HSC (Kelly et al., Mol Ther, 2007). To examine the potential biological consequences/benefits of shortened transduction we used a FA mouse model in which HSC are deficient and prone to excessive loss during in vitro manipulation. We applied a rapid transduction protocol (Mostoslavsky et al., Mol Ther, 2005) utilizing lentiviral vectors and demonstrate that this shortened transduction preserves engraftment of FA HSC to the level of C57BL/6 wt cells. Lin− Sca-1+ c-Kit+ bone marrow cells were isolated from Fanca−/− CD45.2 mice and underwent 4-hr rapid (RT) vs. 96-hr conventional (CT) transduction. An equivalent number of transduced cells were transplanted into lethally irradiated CD45.1 BoyJ mice. Analysis of engraftment chimerism three months post transplantation revealed a significantly higher level of engraftment in animals receiving RT vs. CT cells (90% +/− 14% vs. 26% +/− 31%, respectively, p=<0.01). Rapid transduction also resulted in a significant reduction of engraftment failure (0/36 animals RT vs. 20/36 animals CT). Importantly--emphasizing the FA disease-specific stem cell phenotype, RT vs. CT of C57BL/6 wt cells was associated with no significant difference in engraftment of these cells (93% +/− 1.2% RT vs. 84 +/− 19% CT, p=0.33). Analysis of peripheral blood cells expressing the proviral enhanced green fluorescent protein (eGFP) reporter gene revealed a normal distribution of B-lymphocytes (B220), T-lymphocytes (CD3 epsilon), and granulocytes (MAC-1), indicating multi-lineage engraftment of gene modified cells. In spite of this engraftment advantage, transduction efficiency was low (<30%) using RT. The 6-benzylguanine (6-BG) resistant P140K mutant of O6-methylguanine DNA methyltransferase (MGMTP140K) confers a selective advantage to tranduced HSC treated with alkylating drugs. Following RT with a MGMTP140K/ eGFP expressing lentivirus, 5/6 mice treated with 6-BG and the alkylating drug temozolomide showed a significant rise in the percentage of GFP reporter gene expression in peripheral blood. We extended this approach to the FA model by generating a tri-cistronic lentiviral vector expressing the FANCA cDNA, MGMTP140K, and eGFP. Despite modest in vivo gene marking with this vector, up to 37-fold selection (85% GFP-positive cells) was achieved following exposure of bone marrow of transplant recipients to 6-BG and the alkylating drug temozolomide in vitro. Concurrently, phenotypic correction of mitomycin C hypersensitivity of transduced Fanca−/− bone marrow cells was observed. These data suggest that RT improves stem cell engrafting capacity of FA stem cells in a relevant animal model of stem cell gene therapy. The combination of RT and in vivo selection may allow more successful reconstitution of the lympho-hematopoietic system in gene therapy applications.

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Differentiation Hotspots on a Cell Cycle Related Continuum.

Blood ◽

10.1182/blood.v110.11.3703.3703 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3703-3703

Author(s):

Gerald A. Colvin ◽

David Berz ◽

Mark S. Dooner ◽

Gerri J. Dooner ◽

Kevin Johnson ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Stem Cell ◽

Stem Cell Differentiation ◽

S Phase ◽

Differentiation Potential ◽

Steel Factor ◽

A Cell ◽

Inductive Signaling

Abstract Directed differentiation is defined as the ability to program a stem cell at the most primitive level while it still has its reproductive and full proliferative potential. This is in contrast to ex-vivo expansion where the stem cells are forced into specific lineage commitments, limiting the overall therapeutic utility. We have demonstrated differentiation “hotspots” on a cell cycle continuum (Exp Heme35:96, 2007). In this work we showed marked but reversible increases in differentiation potential to megakryocyte and granulocytes at different phases of a single cytokine induced cell cycle passage of highly purified quiescent murine lineagenegative rhodaminelowHoeschtlow (LRH) marrow stem cells. We have reproducibly induced directed stem cell differentiation by capitalizing on inherent changes in sensitivities to inductive cytokine signals in the context of cell cycle position. These cells, when exposed to thrombopoietin, FLT3-ligand and steel factor, synchronously pass through cell cycle. We have found that using a differentiation cytokine cocktail of G-CSF at 0.075ng/ml, GM-CSF at 0.0375ng/ml and steel factor at 50ng/ml, we were able to see enhanced megakaryopoiesis occurring 14-days after culture in those LRH stem cells that were in early to mid S-phase at time of inductive signaling. We have now shown that a megakaryocyte hotspot clusters around 32 hours; the G1/S interface, and that dramatic reversible changes in differentiation potential occur over one hour time intervals. We have confirmed this data by looking at LRH cells through cell cycle transit after initial cell division showing that a megakaryocyte hotspot occurs in two sequential cell cycles and still tied to S-phase at time of inductive signaling of the daughter cells. This hotspot has been demonstrated on a clonal basis, although the kinetics of the hotspot shifts when clonal as opposed to population studies are carried out. An important issue is whether in vitro cytokine exposure, separate from cell cycle status, determines the existence of the hotspot. To address this, we used Hoechst 33342 dye content to assist in separation of different cell cycle fractions (G0–1, early, mid and late components of S, G2/M) of lineage negative Sca-1+ stem cells, a cycling stem/progenitor cell population in which approximately 20% of the cells are in S-phase at isolation. These cells were only exposed to the differentiation cytokines and showed a megakaryocyte hotspot present in only early S-phase cells after 14-days of culture, showing that in vitro cell cycle phase determined the presence of the hotspot, separate from cytokine exposure. These data indicate that differentiation potential of marrow stem cells exists on a cell cycle related continuum and that this potential can be demonstrated on a single cell basis. This suggests a continuum model of stem cell regulation at the stem cell level as opposed to a pure hierarchical model.

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