scholarly journals Bisecting GlcNAc structure is implicated in suppression of stroma-dependent haemopoiesis in transgenic mice expressing N-acetylglucosaminyltransferase III

1998 ◽  
Vol 331 (3) ◽  
pp. 733-742 ◽  
Author(s):  
Masafumi YOSHIMURA ◽  
Yoshito IHARA ◽  
Tetsuo NISHIURA ◽  
Yu OKAJIMA ◽  
Megumu OGAWA ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transgenic Mice ◽  
Stromal Cells ◽  
Bone Marrow Cells ◽  
Adherent Cell ◽  
Wild Type ◽  
Spleen Cells ◽  
Bisecting Glcnac ◽  
Marrow Cells

Several sugar structures have been reported to be necessary for haemopoiesis. We analysed the haematological phenotypes of transgenic mice expressing β-1,4 N-acetylglucosaminyltransferase III (GnT-III), which forms bisecting N-acetylglucosamine on asparagine-linked oligosaccharides. In the transgenic mice, the GnT-III activity was elevated in bone marrow, spleen and peripheral blood and in isolated mononuclear cells from these tissues, whereas no activity was found in these tissues of wild-type mice. Stromal cells after long-term cultures of transgenic-derived bone marrow and spleen cells also showed elevated GnT-III activity, compared with an undetectable activity in wild-type stromal cells. As judged by HPLC analysis, lectin blotting and lectin cytotoxicity assay, bisecting GlcNAc residues were increased on both blood cells and stromal cells from bone marrow and spleen in transgenic mice. The transgenic mice displayed spleen atrophy, hypocellular bone marrow and pancytopenia. Bone marrow cells and spleen cells from transgenic mice produced fewer haemopoietic colonies. After lethal irradiation followed by bone marrow transplantation, transgenic recipient mice showed pancytopenia compared with wild-type recipient mice. Bone marrow cells from transgenic donors gave haematological reconstitution at the same level as wild-type donor cells. In addition, non-adherent cell production was decreased in long-term bone marrow cell cultures of transgenic mice. Collectively these results indicate that the stroma-supported haemopoiesis is compromised in transgenic mice expressing GnT-III, providing the first demonstration that the N-glycans have some significant roles in stroma-dependent haemopoiesis.

Loss of P-Selectin Promotes the Function of Aged Hematopoietic and Leukemic Stem Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1577-1577
Author(s):  
Yaoyu Chen ◽  
Sullivan Con ◽  
Yiguo Hu ◽  
Linghong Kong ◽  
Cong Peng ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Leukemic Stem Cells ◽  
Wild Type ◽  
Post Transplant ◽  
Marrow Cells

Abstract Abstract 1577 Hematopoiesis is a tightly regulated biological process that relies upon complicated interactions between the blood cells and their microenvironment. Adhesion molecules like P-selectin are essential to hematopoiesis, and their dysregulation has been implicated in leukemogenesis. We have previously shown a role for P-selectin in chronic myeloid leukemia and demonstrated that in its absence the disease process accelerates. Recently, there has also been speculation that P-selectin may play a role in the aging hematopoietic stem cells (HSCs), as its expression in upregulated as a mouse ages. In this study, we show that the loss of P-selectin function dysregulates the balance of stem cells and progenitors and that these differences become more pronounced with age. We compared the percentages of HSCs, long-term (LT)-HSCs, short-term (ST)-HSCs, multipotent progenitors (MPPs), CMPs, GMPs and MEPs in bone marrow by flow cytometry between wild type (WT) and Selp-/- mice. An age-dependent LT-HSC expansion was observed in WT mice. However, this expansion was prevented by the loss of Selp as observed in Selp-/-mice. Further, we demonstrate that with age LT-HSCs in particular express more elevated levels of P-selectin. LT-HSCs and ST-HSC/MPPs were isolated from the bone marrow of young (2 months old) and old (15 months old) WT mice and examined P-selectin expression by FACS. A significant increase in P-selectin expression was observed in LT-HSCs of old mice, and this increase was not observed in the ST-HSC+MPP subpopulations. We also show that the loss of P-selectin gene has profound effects of stem cell function, altering the capacity of these cells to home. Despite impaired homing capacity, stem cells lacking P-selectin possess a competitive advantage over their wild type counterparts. Using a stem cell competition assay, HSCs derived from Selp-/- mice (CD45.2+) and WT control mice (CD45.2+GFP+) were mixed in 1:1 ratio and transplanted into irradiated WT recipients (CD45.1). The initial findings were potentially indicative of the ability of cells derived from GFP mice to more efficiently home and engraft. Despite this initial advantage, cells derived from Selp-/- eventually exhibited a competitive and statistically significant advantage over the cells derived from GFP mice. At 30 days post-transplant, 49.9±1.4% of the CD45.2 subpopulation was GFP+. At 86 days post-transplant, 25.7±3.3 % of the CD45.2 cells derived from the peripheral blood were GFP+. Similarly, 23.0±3.7% of the CD45.2 cells derived from the bone marrow of these mice were GFP+. Indeed, we demonstrate that recipients of P-selectin deficient bone marrow cells more efficiently repopulate the bone marrow than controls and that this advantage extends and expands in the long-term. Finally, we demonstrate that recipients of leukemic cells lacking P-selectin develop a more accelerated form of leukemia accompanied by significant increases in stem and progenitor cells. Bone marrow cells from donor WT and Selp-/- mice were infected with retrovirus expressing BCR-ABL-GFP, and irradiated WT recipients were transplanted with 2×105 of these transduced donor cells. At 14 days post-transplant, recipient mice from each of the groups were sacrificed, and bone marrow cells were harvested and analyzed by flow cytometry. Recipients of leukemic Selp-/- cells possessed 3.5-fold more LSCs than recipients of wild-type cells. There were 3.1-fold more LT-LSCs and 3.8-fold more ST-LSCs and MPPs in recipients of Selp-/- cells than WT cells. In addition, recipients of leukemic Selp-/- cells possessed significantly more CMP (16.9-fold) and MEP (4.5-fold) cells. Because P-selectin expression increases with age on LT-HSCs, we sought to determine the role that age plays in CML development and progression. Bone marrow cells derived from 15-month-old donor Selp-/- and WT mice were transduced with BCR-ABL, respectively, followed by transplantation of the transduced cells into recipient mice. All recipients of BCR-ABL transduced Selp-/- cells died by 23 days after induction of CML and had a median survival of 19 days, whereas recipients of the transduced WT cells survived significantly longer. This pro-leukemic role for cells lacking P-selectin expression is leukemic stem cell-specific rather than stromal cell-specific and supports an essential role for P-selectin on leukemic stem cells. Disclosures: No relevant conflicts of interest to declare.

Functional Hematopoietic Cells Derived From Mouse Embryonic Stem Cells.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2304-2304
Author(s):  
Cheng Li ◽  
Daniel R. George ◽  
Nichole M. Havey ◽  
Jeffery M. Klco ◽  
Timothy J. Ley
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Embryonic Stem ◽  
Post Injection ◽  
Spleen Cells ◽  
Short Term ◽  
Marrow Cells

Abstract Abstract 2304 Despite two decades of effort, deriving long-term repopulating hematopoietic stem/progenitor cells (HSPCs) from embryonic stem cells (ESCs) has proven to be extremely difficult. Both embryoid body (EB)-based and stroma-based methods have been extensively explored. However, robust production of HSPCs from C57BL/6J-derived mouse ESCs (mESCs) has not yet been reported. Furthermore, in vivo engraftment of mES-derived HSCs (from any strain) has been achieved only with forced expression of HoxB4 or related oncogenes, which creates significant limitations for most studies. Here, we describe a stroma-based co-culture method to differentiate HSCs and progenitor populations from C57BL/6J-derived mESCs. After simple co-culture on OP9 stroma cells for one week, C57BL/6J-derived mESC lines differentiate into cells that mark as HSCs, CMPs, GMPs, and MEPs (by immunophenotyping); these cells are capable of giving rise to erythrocytes, monocytes, and mast cells (by morphology and immunophenotyping) after another week of culture in methylcellulose with hematopoietic cytokines (SCF, IL-3, IL-6, and Epo). Similar in vitro hematopoietic differentiation has been achieved in several different C57BL/6J-derived mESCs (B6/Blu, B6-GFP, LK1, and B6 albino), B6/SVJ129 mESCs (R1), various SVJ129-derived mESCs (SWT16, EDJ22, and SCC10), and five independent C57BL/6J mouse embryonic fibroblast (MEF)-derived induced pluripotent stem cell (iPSC) lines. C57BL/6J ESCs derived from CAGGS-GFP transgenic mice (B6-GFP ESCs, which express high levels of GFP in all hematopoietic lineages) were used to determine whether we could obtain long-term engraftment of the OP9 differentiated ESCs. B6-GFP ESCs cultured for 7 days on OP9 cells were sorted by Kit+ surface staining. Sorted cells (1×105, 2×105, 4×105) were transferred into immunocompromised NSG mice via retro orbital injection (n=1 mouse per dose). Peripheral blood from the recipients injected with 2×105 and 4×105 cells showed 5% GFP positivity in the peripheral blood at weeks 12 and 16 post-transplant, while recipients injected with 1×105 cells had no detectable GFP+ cells in the periphery. Bone marrow cells and spleens were harvested at week 22. The recipient injected with 4×105 cells showed 5% GFP positivity in the bone marrow and 20% in the spleen. Engraftment was multi-lineage. Myeloid compartments (CD34+, CD11b+, Kit+, and Gr-1+) showed similar or less GFP positivity than overall bone marrow and spleen cells. Lymphoid (CD3+ and B220+) and erythroid (Ter119+) compartments also showed similar GFP positivity compared to overall bone marrow cells. However, lymphoid and erythroid compartments contained significantly higher GFP positivity (30–60%) than overall spleen cells. We have now modified the procedure to transfer 1×106 unfractionated B6-GFP ESCs grown for 7 days on OP9 stroma directly into NSG recipients. We have detected short-term engraftment 4 weeks post-injection in the peripheral blood of one recipient and multilineage splenic engraftment 8 weeks post-injection in two recipients, confirming that short-term repopulating cells are indeed generated by this method. Secondary transplants using the GFP+ bone marrow cells from the long-term engrafted mouse have been performed. This approach could be a valuable tool for studying the hematopoietic development of a variety of mESC lines, and potentially, iPSC lines as well. Disclosures: No relevant conflicts of interest to declare.

Formation of Bone and Foci of Ectopic Hemopoiesis at Joint Application of Calcium Scaffolds with Bone Marrow Cells or Cultivated Mesenchymal Stromal Cells

2009 ◽  
Vol 16 (2) ◽  
pp. 85-90 ◽  
Author(s):  
Irina Nikolaevna Shipunova ◽  
D A Svinareva ◽  
T V Petrova ◽  
M M Ryashentsev ◽  
V E Mamonov ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Bone Growth ◽  
Bone Marrow Cells ◽  
Great Influence ◽  
Tissue Growth ◽  
Adherent Cell ◽  
Joint Application ◽  
Marrow Cells

Potential application of calcium scaffolds in combination with either bone marrow or adherent cell layers from long-term cultures containing mesenchymal stem cell for the induction of bone tissue growth was studied in mice. Two scaffolds, i.e. Osteoset® and Prodens® were tested on the model of ectopic grafting under the skin and renal capsule of mice. It was demonstrated that Prodens® and less effectively Osteoset® could be used for the induction of bone growth in combination with bone marrow cells but even more effectively in combination with cultivated mesenchymal stromal cells. Both the site of transplantation and preliminary induction of bone differentiation of stromal cells exerted a great influence upon the process of bone formation.

Effects of the Homeodomain Gene Pitx2 on Self-Renewal of Hematopoietic Stem Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 95-95 ◽  
Author(s):  
Hui Z. Zhang ◽  
Svetlana Rogulina ◽  
Wendy Chen ◽  
Barbara A. Degar ◽  
Bernard G. Forget
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Leukemic Cells ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Colony Forming Units ◽  
Marrow Cells

Abstract Pitx2, a homeodomain gene preferentially expressed in murine hematopoietic stem/progenitor cells, is also a downstream target of genes important for hematopoiesis such as MLL and Wnt/Dvl/β-Catenin. We have previously reported that Pitx2 null hematopoietic stem cells (HSCs) can contribute to multi-lineage hematopoiesis under physiologic conditions. We have now carried out serial bone marrow transplantation experiments and demonstrated that after the 3rd round of serial transplantation, Pitx2 null cells reconstituted only 28.6% of the recipient hematopoietic cells as compared to 60% in the case of wild type cells (P<0.001). There were no Pitx2 null donor-derived cells in recipient mice after the 4th round of transplantation, whereas donor-derived chimerism was 57% with wild type cells (P<0.001), and 26% with Pitx2 +/− cells (P<0.001). Therefore, Pitx2 null HSCs have decreased self renewal capacity. To further study the function of Pitx2 in HSC, we constitutively overexpressed the Pitx2 gene in murine bone marrow cells following transduction using a MSCV/IRES/GFP retroviral vector, and analyzed the effects on hematopoiesis in vitro and in vivo. Bone marrow cells overexpressing Pitx2 were isolated on the basis of their GFP expression and analyzed for their colony forming ability in vitro. Retrovirally transduced bone marrow cells were also transplanted into lethally irradiated mice, and the transplanted mice were observed for long-term reconstitution. Colony-forming unit assays showed that Pitx2 overexpressing bone marrow cells, compared to control cells transduced with vector only, had increased numbers of GM colony forming units and reduced numbers of megakaryocytic colony forming units. Pitx2-overexpressing cells continued to form GM colonies after more than eight serial replatings. When these cells were cultured in liquid medium containing SCF, IL-3 and IL-6, they gave rise to cells that stained positively either for alpha naphthyl butyrate, indicating monocytic differentiation, or for peroxidase, indicating neutrophilic differentiation. The ability of these GM-colony forming cells to cause leukemia is currently under investigation. Long-term reconstitution of hematopoiesis in mice by Pitx2 over-expressing HSCs was demonstrated by identifying GFP positive multi-lineage peripheral blood cells four months following transplantation. One of these mice manifested leukemia at this time, as evidenced by a markedly elevated WBC count and other hematologic abnormalities. The leukemic WBCs had very high levels of GFP and Pitx2 expression and were shown to contain two retroviral integration sites, neither of which involved a known oncogene or overexpression of the gene at the integration site. Immunophenotyping by flow cytometry demonstrated that the majority of the leukemic cells were c-kit positive and expressed the megakaryocytic marker CD41, as well as the common myeloid progenitor marker, CD16/32. Some of the cells expressed the erythroid marker Ter119. The leukemic cells did not express any lymphoid markers, including CD3ε, B220, CD19, and IL7R3. This Pitx2-overexpression-associated leukemia was transplantable. Experiments are under way to characterize the leukemia initiating cells. Taken together, our results provide evidence that the homeodomain gene Pitx2 plays a role in the self-renewal of hematopoietic stem/progenitor cells.

β-Catenin Expression in Bone Marrow Stromal Cells Is Required for Normal Proliferation and Differentiation of Primitive Hematopoietic Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 608-608
Author(s):  
Michael Nemeth ◽  
Yingzi Yang ◽  
David Bodine
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Wnt Signaling ◽  
Stromal Cells ◽  
Bone Marrow Cells ◽  
Wild Type ◽  
Canonical Wnt Signaling ◽  
Proliferation And Differentiation ◽  
Canonical Wnt ◽  
Marrow Cells

Abstract Normal function of adult hematopoietic stem cells (HSC) is dependent upon the bone marrow microenvironment, which is comprised of multiple cell types, including fibroblasts, endothelial cells, osteoblasts and stromal progenitors. We hypothesized that canonical Wnt signaling, which is necessary for the development of mesenchymal tissue, regulates the cellular structure and function of the microenvironment. We tested this hypothesis using an in vitro model of bone marrow stroma that is deficient in β-catenin, the critical transactivator of canonical Wnt signals. β-catenin−/ − Dexter stroma cultures were established from whole bone marrow harvested from β-cateninlox/lox Mx-cre+/cre mice treated with 7–10 doses of 300 μg pIpC. PCR analysis of stromal cell DNA showed nearly 100% deletion of β-catenin. Confluent stroma cultures were irradiated and seeded with 4 x 104 lin cells/cm2. We have reported (Nemeth, et al, Blood, 108, 29a) that β-catenin−/ − stroma exhibit decreased ability to support CFU formation and generate osteoblasts. The reduction in CFU-C correlates with a 75% increase in the percentage of lin− progenitors cultured on β-catenin−/ − stroma undergoing apoptosis (23.6 ± 3.4%) compared to wild-type (13.6 ± 1.3% ; n = 3; p < .01). To determine the mechanism by which canonical Wnt signaling regulates microenvironment function, we used a cytokine antibody array to analyze protein levels of 30 different hematopoietic growth factors and adhesion molecules. We observed decreased amounts of the soluble factors bFGF (3.3 ± 0.6-fold) and SCF (2.3 ± 0.3-fold), and the adhesion factor VCAM-1 (2.7 ± 0.3-fold) (n = 3; p < .01) in β-catenin−/ − stroma. The decrease in VCAM-1 corresponded with decreased percentages of VCAM-1+ osteoblasts (26.8 ± 0.9% vs. 43.9 ± 5.7%) and endothelial cells (31.6 ± 5.7% vs. 76.5 ± 10.9%) in β-catenin−/ − stroma compared to wild-type (n = 4, p < .01). From these data, we hypothesized that β-catenin is necessary for maintaining the stromal cells that support HSCs in vivo. We tested this hypothesis by transplanting 2 x 106 wild-type whole bone marrow cells (CD45.1) into lethally-irradiated β-cateninlox/lox Mx-cre+/cre mice and littermate controls. 8 weeks later, transplanted mice were treated with pIpC, resulting in mice with a wild-type hematopoietic system and a β-catenin−/ − microenvironment. Two weeks after the final treatment, we observed a 2.7 ± 0.1-fold reduction in the percentage of long- and short-term HSCs (lin−, c-kitHI, Sca-1HI) in bone marrow from mice with a β-catenin−/ − microenvironment compared to wild-type (n = 4, p < .05). We performed a competitive repopulation assay, transplanting 1 x 106 whole bone marrow cells harvested from primary recipients with a wild-type or β-catenin−/ − microenvironment with 1 x 106 CD45.2 whole bone marrow cells into lethally-irradiated secondary recipient mice. After 16 weeks, there was no difference in mean repopulation by bone marrow cells from the β-catenin−/ − microenvironment (9.2 ± 2.8%) compared to wild-type (10.7 ± 0.6%), indicating that self-renewal was unaffected. However, we did observe a significant 4-fold increase in variability of repopulation by bone marrow cells from the β-catenin−/ − microenvironment (F-test = .01). Since smaller numbers of HSCs will yield greater variability in repopulation than larger numbers, this is consistent with the observation that the β-catenin−/ − microenvironment supports fewer HSCs. From these data, we propose a model in which canonical Wnt signaling in the microenvironment is necessary for hematopoietic proliferation and differentiation.

Parathyroid Hormone (1–34) Treatment Induces Hematopoietic Precursor Cell Expansion in Murine Long-Term Bone Marrow Culture.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4164-4164
Author(s):  
Nina Drize ◽  
Daria Svinareva ◽  
Irina Nifontova ◽  
Joseph Chertkov
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Parathyroid Hormone ◽  
Cell Expansion ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Bone Marrow Culture ◽  
Adherent Cell ◽  
Marrow Cells

Abstract Parathyroid hormone (PTH) induces activation and increases the number of osteoblasts. An increase the stem cells number (Lin-Sca1+cKit+) was observed in mice after 4 weeks of PTH treatment, that suggest osteoblasts participation in regulation of hematopoiesis (Calvi, et al., 2003, Nature, 425, 841). Long-term bone marrow culture (LTBMC) was used for the study of PTH influence on hematopoietic progenitors of different stages of maturation. Rat PTH (1–34) (final concentration, 10−7M) was supported during whole culture period. Cell number, colony-forming units in culture (CFU-GM, CFU-C-21 days) and cobblestone area forming cells (CAFC) were measured during 10 weeks. The number of mature cells in culture did not changed during PTH treatment. The number of studied progenitors did not changed significantly during 3 and 6 weeks of PTH treatment. The number of CFU-GM, CFU-C-21 days and CAFC 14 days, CAFC 21 days, CAFC 28 days increased 7 –10 fold in suspension fraction of LTBMC after 10 weeks of PTH treatment. Table 1. Number of different precursor cells in suspension fraction of LTBMC after PTH treatment. To evaluate the possibility of stem cells expansion by using of PTH treated adherent cell layers (ACL) of LTBMC, the PTH-treated for 4 and 8 weeks cultures, were irradiated with 40 Gy and seeded with 2 × 106 bone marrow cells depleted of adherent cells. Following 24 hours the number of survived CAFC 8 – 28 was analyzed. On PTH-treated ACL the number of CAFC 21 increased 2,7 ± 0,4 fold as compared with fresh bone marrow or culture with non-treated ACLs. The number of CAFC 28, which characterizes the number of marrow repopulating cells, increased 2 fold only after cultivation on ACL treated with PTH for 8 weeks. Table 2. CAFC number in bone marrow cultivated 24 hours on PTH treated ACL of LTBMC The data suggest possibility to increase stem cell expansion ex vivo on pharmacologically manipulated microenvironment. Time of cultivation, weeks Specificity of progenitor Number of precursors, per 100000 cells Control Control PTH treatment 3 CFU-GM 50 ± 5.2 77 ± 5.6 6 CFU-GM 33 ± 4 31 ± 5.9 10 CFU-GM 20 ± 4.8 147.5 ± 3.5 3 CFU-C-21 55 ± 5.9 76.6 ± 11.2 10 CFU-C-21 11.5 ± 1.7 102.0 ± 2.0 3 CAFC-7 21.6 20.3 6 CAFC-7 25.9 26.9 10 CAFC-7 0.9 3.0 3 CAFC-21 1.01 0.69 6 CAFC-21 0.43 0.48 10 CAFC-21 0.28 3.11 3 CAFC-28 0.32 0.55 6 CAFC-28 0.12 0.14 10 CAFC-28 0.1 0.82 Age of CAFC (days) Number of CAFC per 2 x 10^6 bone marrow cells not treated PTH (4 weeks) PTH (8 weeks) CAFC-7 424.1 804.5 647.5 CAFC-14 262.5 529.8 295.2 CAFC-21 93.5 272.7 226.2 CAFC-28 38.3 30.1 79.2

CREB Overexpression In Vivo Results in Increased Proliferation, Blast Transformation, and Earlier Engraftment of Myeloid Progenitor Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3548-3548
Author(s):  
Kentaro Kinjo ◽  
Deepa B. Shankar ◽  
Jerry Cheng ◽  
Samuel Esparza ◽  
Noah Federman ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transgenic Mice ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Blast Transformation ◽  
Mouse Bone Marrow ◽  
Wild Type ◽  
Facs Analysis ◽  
Self Renewal ◽  
Marrow Cells

Abstract CREB or cAMP responsive element binding protein is a 43-kDa-basic/leucine zipper (bZip) transcription factor that regulates gene expression through the activation of cAMP-dependent or -independent signal transduction pathways. CREB promotes growth and survival in a variety of cell types and is overexpressed in the bone marrow of greater than 60% of AML patients. To understand the role of CREB in myelopoiesis, we characterized the effects of CREB overexpression in transgenic mice. We created mice in which CREB expression was targeted to the myeloid lineage using the hMRP8 promoter. CREB transgenic mice showed evidence of monocytosis, compared to age-matched littermate controls. We performed colony assays with methylcellulose containing SCF, IL-6, and IL-3. Bone marrow cells from CREB transgenic mice formed robust colonies earlier and had increased numbers of colony forming units (CFU-GM) when compared to control mice. Cytospin analysis of these cells showed the presence of more immature myeloid cells compared to controls. At day 12, cells from colonies were 50% c-Kit positive, 83% Gr-1 positive, and 67% Mac-1 positive by FACS analysis. To assess self-renewal of progenitors from CREB transgenic mice, serial replating experiments were performed. Bone marrow cells from transgenic mice were highly successful in repopulating the methylcellulose containing SCF, IL-6, and IL-3, in contrast to the control cells, which were unable to grow after serial replating. Following tertiary replating of the CREB transgenic mouse bone marrow, we observed that the colonies (96+3.5) appeared more homogeneous with immature cells that were >99% c-Kit positive and <1% GR-1, Mac-1 positive. These results suggest that persistent expression of CREB leads to a blast-like phenotype in the absence of differentiation. To determine whether increased CREB expression confers growth factor-independence, we cultured bone marrow cells in methylcellulose that did not contain cytokines. We observed a 10-fold increase in the numbers of cells from CREB transgenic mice (two different founder lines) compared to normal bone marrow. When cultured in methylcellulose containing M-CSF, the bone marrow cells from CREB transgenic mice formed larger and significantly greater numbers of colonies. However, these cells did not grow in the presence of G-CSF or EPO alone. To determine if the myeloproliferative (monocytosis) phenotype was transplantable into wild type recipient mice we injected 4x106 bone marrow cells from CREB transgenic mice into wild type C57/BL6 recipient mice. Serial analysis of the peripheral blood counts and cell surface markers by FACS analysis showed earlier myeloid engraftment at 6 weeks following transplantation compared to normal control mice. The transgenic recipients showed increased monocytes and neutrophils in the peripheral blood with a corresponding increase in Mac-1 positive, Gr-1 positive cell populations at 8 weeks after transplantation. At the same time, the lymphocyte count was significantly lower in CREB transgenic recipient mice than controls. Our results suggest that CREB plays a critical role in the regulation of normal hematopoiesis and stem cell self-renewal.

SIRT1 Deacetylase Is Essential for Hematopoietic Stem Cell Activity Via Regulation of Foxo3.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2315-2315 ◽  
Author(s):  
Pauline Rimmele ◽  
Carolina L. Bigarella ◽  
Valentina d'Escamard ◽  
Brigitte Izac ◽  
David Sinclair ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Bone Marrow Cells ◽  
Leukemic Stem Cells ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract Abstract 2315 SIRT1 is a member of the NAD-dependent family of sirtuin deacetylases with critical functions in cellular metabolism, response to stress and aging. Although SIRT1 is clearly a regulator of embryonic stem cells, reports on the function of SIRT1 in adult hematopoietic stem cell (HSC) have been conflicting. While SIRT1 was positively associated with HSC activity on a genetic screen, using a germline deletion of SIRT1 three groups found SIRT1 to be dispensable for adult HSC. Here, we first showed that nuclear SIRT1 expression is enriched in bone marrow-derived Lin−Sca1+cKit+ (LSK) cells, as compared to total bone marrow cells. Germline deletion of SIRT1 is associated with developmental defects and high perinatal mortality resulting in only 10% of mice reaching adulthood. To circumvent the potential developmental adaptation of these mice, we used an adult-tamoxifen inducible SIRT1 knockout mouse model. Full-length SIRT1 protein was nearly undetectable in the bone marrow and spleen of SIRT1−/− mice. Analysis of wild type and SIRT1−/− bone marrow cells, 4 weeks after tamoxifen treatment, showed that loss of SIRT1 increased the size and frequency of the LSK compartment. Interestingly, this was associated with a significant decrease in the frequency of long-term repopulating HSC as determined by SLAM markers (CD48−CD150+LSK) within LSK cells. This decrease was even more pronounced with time. In agreement with these results, the long-term repopulation ability of CD48−CD150+LSK cells is severely compromised in SIRT1−/− mice as measured 16 weeks after transplantation, strongly suggesting that SIRT1 is essential for long-term HSC function. Thus, loss of SIRT1 results in loss of long-term repopulating stem cells in favor of total LSK cells that is a more heterogeneous population of stem cells. SIRT1 has several substrates with a potential function in HSC. Among these, we focused on Foxo3 Forkhead transcription factor which is essential for the maintenance of hematopoietic and leukemic stem cell pool. Despite the importance of Foxo3 to the control of HSC function, mechanisms that regulate Foxo3 activity in HSC remain unknown. Negative regulation of FoxOs by AKT phosphorylation promotes their cytosolic localization in response to growth factors stimulation. Interestingly, Foxo3 is constitutively nuclear in bone marrow LSK and in leukemic stem cells, strongly suggesting that negative phosphorylation may not be the sole Foxo3 regulatory mechanism in these stem cells. FoxO proteins are regulated by several post-translational modifications including acetylation in addition to phosphorylation, although the impact of acetylation on Foxo3 function remains unresolved. Therefore, we asked whether regulation of adult HSC activity by SIRT1 deacetylase is mediated by Foxo3. The in vivo injection of sirtinol, a SIRT1 inhibitor, for 3 weeks compromised significantly the long-term repopulation capacity of wild type but not Foxo3−/− HSC as measured by the repopulation ability of CD48−CD150+LSK cells in lethally irradiated mice after 16 weeks. These results suggest that Foxo3 is likely to be required for SIRT1 regulation of HSC activity. In agreement with this, we showed that in contrast to wild type LSK cells, Foxo3 is mostly cytoplasmic in SIRT1−/− LSK cells, indicating that loss of SIRT1 is sufficient to translocate Foxo3 to the cytosol and presumably inhibit its activity. We further showed that ectopically expressed acetylation-mimetic mutant of Foxo3 where all putative acetyl-lysine residues are mutated to glutamine, in bone marrow mononuclear cells, is mostly localized in the cytosol in contrast to wild type Foxo3 protein and results in significant decrease of colony-forming unit-spleen (CFU-S) activity. Using pharmacological antagonism as well as conditional deletion of SIRT1 in adult HSC, we identified a critical function for SIRT1 in the regulation of long-term HSC activity. Our results contrast with previously published data obtained from germline deleted SIRT1 mice, and suggest that the use of a conditional approach is essential for unraveling SIRT1 function in adult tissues. Our data also suggest that SIRT1 regulation of HSC activity is through activation of Foxo3. These findings are likely to have an important impact on our understanding of the regulation of hematopoietic and leukemic stem cells and may be of major therapeutic value for hematological malignancies and disorders of stem cells and aging. Disclosures: No relevant conflicts of interest to declare.

DNMT3A R882H Can Cooperate with FLT3-ITD to Cause AML in Mice

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2458-2458
Author(s):  
Angela Maria Verdoni ◽  
Celia Venezia ◽  
Jeffery Klco ◽  
Timothy J. Ley
Keyword(s):  
Bone Marrow ◽  
Transgenic Mice ◽  
Competitive Advantage ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Myeloid Differentiation ◽  
Wild Type ◽  
One Year ◽  
Over Time ◽  
Marrow Cells

Abstract Somatic mutations in the DNA methyltransferase, DNMT3A, have been identified in approximately 22% of de novo AML cases and in ~10% of patients with MDS. To understand how mutations in DNMT3A lead to hematopoietic abnormalities, we generated transgenic mice capable of overexpressing wild type human DNMT3A, or the most common AML mutation (R882H). The system used allows for the inducible expression of DNMT3A upon the expression of a coactivator and addition of Doxycycline (Dox) in the feed. A single founder line with the WT DNMT3A allele (overexpressed ~4 fold over endogenous WT murine Dnmt3a), and two founder lines with the R882H DNMT3A allele (one line overexpressing at ~16 fold level over endogenous mouse Dnmt3a and the other at ~4.5 fold overexpression) were established in a pure C57Bl6/J background. All three lines have been shown to overexpress the transgene in bone marrow when crossed to coactivator transgenic mice carrying the Rosa26-rtTA allele, and eating Dox chow. There is significant DNA hypomethylation in the bone marrow cells of the high expressing R882H line after the mice have been on Dox chow for 3 months. To determine the effect of R882H DNMT3A on hematopoiesis, we performed competitive transplantation studies where we mixed bone marrow derived from R882H DNMT3A Tg x rtTA doubly transgenic mice (Ly5.2+) with bone marrow from wild type mice (Ly5.1/5.2+) at a 1:1 ratio, and transplanted it into lethally irradiated recipients. After a 1 month engraftment period, mice were placed on Dox chow and monitored for 1 year. All control genotypes were included. After 6 months of dox chow administration, neither R882H Tg x rtTA line displayed a competitive advantage in the peripheral blood. However, there was a trend towards myeloid skewing in R882H DNMT3A expressing cells. At one year post-transplant, mice were sacrificed. In the spleen and peripheral blood, there were trends towards a myeloid differentiation bias in low-expressing R882H Tg x rtTA mice, but a significant myeloid bias in the bone marrow of high expressing R882H Tg x rtTA mice. This demonstrated that R882H DNMT3A expression leads to a myeloid differentiation bias over time, providing a possible explanation for the observation that R882H mutations are enriched for myeloid leukemias. A tumor watch of mice with all genotype combinations demonstrated that mice expressing R882H DNMT3A do not develop AML, even after 1.5 years on Dox chow. To address whether a second mutation is required to cooperate with R882H DNMT3A to cause AML, we performed a study using donor mice derived from the above competitive transplantation experiments (aged for one year) that were chimeric for R882H Tg x rtTA marrow and wild type marrow at a ~50:50 ratio. This chimeric marrow sample was transduced with a retroviral vector that expresses a human FLT3-ITD allele, one of the most common cooperating mutations with R882H DNMT3A. Ten recipient mice have been analyzed for a minimum of 4 months. Between 6 and 9 weeks after transplantation, two mice developed AML derived entirely from Ly5.2 expressing cells, demonstrating that the leukemic clone arose and rapidly expanded in R882H expressing bone marrow cells. Peripheral blood analysis in these mice and five others (7/10 total) demonstrated that FLT3-ITD expressing cells preferentially expand in the R882H expressing cells, while 1/10 mice showed no preference. We were able to analyze a single cohort (n=4) of mice at 3 months post engraftment, and found >25% enrichment of FLT3-ITD positive cells in R882H expressing cells. Two of ten mice did not display peripheral blood expansion of cells expressing FLT3-ITD. An additional two mice in this study developed Ly5.2/R882H-derived hematopoietic diseases with a longer latency, which are currently being assessed for leukemic properties by transplantation. Irrespective of the presence of FLT3-ITD expression, R882H expressing cells showed either stable engraftment or expansion over time. Six of ten mice displayed an expansion of R882H expressing cells (no FLT3-ITD allele) demonstrating a clonal advantage, 3/10 mice displayed stable engraftment, and only 1/10 showed a loss. Taken together, our results show that expression of the R882H allele can confer a competitive advantage independently after serial transplantation, but when combined with the common cooperating FLT3-ITD mutation, the FLT3-ITD expressing cells preferentially expand in R882H expressing cells, and can synergize to cause AML. Disclosures No relevant conflicts of interest to declare.

scholarly journals CD34+ human marrow cells that express low levels of Kit protein are enriched for long-term marrow-engrafting cells

Blood ◽  
1996 ◽  
Vol 87 (10) ◽  
pp. 4136-4142 ◽  
Author(s):  
I Kawashima ◽  
ED Zanjani ◽  
G Almaida-Porada ◽  
AW Flake ◽  
H Zeng ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Marrow Cell ◽  
In Utero ◽  
Fetal Sheep ◽  
Hematopoietic Stem ◽  
Show Evidence ◽  
Marrow Cells

Using in utero transplantation into fetal sheep, we examined the capability of human bone marrow CD34+ cells fractionated based on Kit protein expression to provide long-term in vivo engraftment. Twelve hundred to 5,000 CD34+ Kit-, CD34+ Kit(low), and CD34+ Kit(high) cells were injected into a total of 14 preimmune fetal sheep recipients using the amniotic bubble technique. Six fetuses were killed in utero 1.5 months after bone marrow cell transplantation. Two fetuses receiving CD34+ Kit(low) cells showed signs of engraftment according to analysis of CD45+ cells in their bone marrow cells and karyotype studies of the colonies grown in methylcellulose culture. In contrast, two fetuses receiving CD34+ Kit(high) cells and two fetuses receiving CD34+ Kit- cells failed to show evidence of significant engraftment. Two fetuses were absorbed. A total of six fetuses receiving different cell populations were allowed to proceed to term, and the newborn sheep were serially examined for the presence of chimerism. Again, only the two sheep receiving CD34+ Kit(low) cells exhibited signs of engraftment upon serial examination. Earlier in studies of murine hematopoiesis, we have shown stage-specific changes in Kit expression by the progenitors. The studies of human cells reported here are in agreement with observations in mice, and indicate that human hematopoietic stem cells are enriched in the Kit(low) population.

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