Canonical and Non-Canonical Wnt Signaling Regulate the Balance between HSC Self-Renewal and Differentiation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 862-862
Author(s):  
Michael Nemeth ◽  
Yingzi Yang ◽  
David Bodine
Keyword(s):  
Bone Marrow ◽  
Wnt Signaling ◽  
Bone Marrow Cells ◽  
Retroviral Vectors ◽  
Canonical Wnt Signaling ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Serum Free ◽  
Canonical Wnt ◽  
Marrow Cells

Abstract We and others have implicated activation of canonical Wnt signaling by Wnt3a in promoting hematopoietic stem cell (HSC) self-renewal. Since Wnt5a can inhibit canonical Wnt signaling (Topol, et al. 2003), we hypothesized that Wnt3a and Wnt5a act as antagonists on HSC function. To examine the effect of Wnt3a and Wnt5a on canonical Wnt signaling in HSCs, we isolated HSCs (lin−, c-kitHI, Sca-1HI, IL-7Rα−) that contain a lacZ reporter gene that is induced by canonical signaling and cultured them in serum-free media in the presence of 30 ng/ml SCF and Flt3L and recombinant Wnt3a (3a; 100 ng/ml) with and without recombinant Wnt5a (5a; 500 ng/ml). We observed a significant 50% reduction (p < .05) in lacZ mRNA in HSCs cultured with 3a + 5a compared to 3a alone, indicating that Wnt5a inhibits canonical signaling in HSCs. Treatment with 3a, 5a, and 3a + 5a caused the percentage of actively cycling HSCs to decrease 1.4 to 1.6-fold compared to control (SCF + Flt3L) (p < .02). To examine the role of Wnt signaling in HSC self-renewal, wild-type CD45.1+ HSCs were cultured for 6 days before being transplanted into lethally irradiated recipients (1:100 ratio HSCs to CD45.2+ whole bone marrow cells; total number of CD45.1+ HSCs transplanted ranged from 3 to 5 × 103). There was no difference in long-term repopulation between control HSCs (6.7 ± 5.5% CD45.1+ bone marrow cells, n = 14) and HSCs cultured with 3a (4.1 ± 3.7%, n = 8, p = .24). However, HSCs cultured with 5a (24.1 ± 21.2%, n = 10) or 5a + 3a (38.5 ± 36.4%, n = 11) showed significant increases in repopulation compared to control (p < .01; Mann-Whitney test). To determine if additional survival signals were necessary for 3a to induce HSC self-renewal, we transplanted cultured HSCs isolated from transgenic mice that overexpressed the anti-apoptotic gene Bcl2. We observed that control HSCs engrafted 7/7 mice (average repopulation: 17.8 ± 6.8% CD45.1+ bone marrow) whereas when 3a was added, only 1/7 mice showed engraftment (1.2%). Together, these data suggest that induction of canonical signaling by Wnt3a results in decreased HSC expansion and self-renewal and that Wnt5a positively regulates HSC self-renewal by non-canonical Wnt signaling pathways independent of its ability to inhibit canonical signaling. We examined the effects of Wnt3a and Wnt5a on Hoxb4, Notch1, and c-myc expression in HSCs. We observed that adding 3a to serum-free cultures caused no change in Hoxb4 mRNA, a 2-fold reduction in Notch1 mRNA (p < .01) and a 2.4-fold increase in the canonical Wnt pathway target gene c-myc (p < .05; n = 3) compared to control. No changes in Hoxb4, Notch1, or c-myc mRNA were observed in HSCs cultured with 5a or 5a + 3a. Since overexpression of c-myc has been linked to loss of HSC self-renewal, we hypothesized that overexpression of Wnt3a would inhibit HSC self-renewal and repopulation. We transduced CD45.1+ bone marrow with Wnt3a-IRES-GFP or IRES-GFP retroviral vectors and sorted GFP+ cells were transplanted with equal numbers of CD45.2+ mock-transduced cells. Four months post transplantation, 3.4 ± 1.8% of bone marrow cells in IRES-GFP recipients were GFP+ (n = 8). In contrast, no GFP+ cells were detected in Wnt3a-GFP recipients (n = 8). Wnt3a-GFP retroviral DNA was detected by PCR, suggesting that vector silencing was required for transduced cell survival. We propose that imbalanced canonical Wnt signaling in HSCs deregulates hematopoiesis by inducing pro-differentiation genes such as c-myc and that additional signals, e.g. Wnt5a, are required to maintain the balance between HSC differentiation and self-renewal.

Wnt5a Inhibits Wnt3a-Mediated HSC Differentiation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2271-2271
Author(s):  
Michael Nemeth ◽  
David Bodine
Keyword(s):  
Bone Marrow ◽  
Formation Control ◽  
Bone Marrow Cells ◽  
Wnt Signaling Pathway ◽  
Retroviral Vectors ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Canonical Wnt ◽  
Cells Cultured ◽  
Marrow Cells

Abstract Activation of the canonical Wnt signaling pathway by Wnt3a has been implicated in hematopoietic stem cell (HSC) self-renewal (Reya et al., Nature, 2003). Wnt5a has been observed to inhibit Wnt3a signaling (Topol et al., J Cell Biol, 2004). We hypothesized that Wnt3a and 5a act as antagonists on HSC function. 1 x 106 lineage negative cells (lin−) were cultured for 4 days in the presence of 50 ng/ml SCF and Flt3L (control) plus 100 ng/ml rmWnt3a and/or 500 ng/ml rmWnt5a (all factors added on day 0 and day 2). Control lin− cell numbers expanded more than lin− cells cultured with Wnt3a, 5a, or both (control 8.3 ± 0.3-fold; Wnt3a 6.9 ± 0.2-fold (p < .01); Wnt5a 4.8 ± 0.2-fold (p < .001); Wnt3a and 5a 2.6 ± 0.6-fold (p < .001); n = 3). After 4 days, cells were analyzed for myeloid colony formation. Control cells and cells cultured in Wnt3a had similar numbers of CFU-GM/5000 lin− cells (control 13.1 ± 11.1; Wnt3a 21.8 ± 15.3; p = .21; n = 8), while cells cultured in Wnt5a and Wnt3a and 5a had 2-fold and 5.9-fold more CFU-GM/5000 lin− cells than control (Wnt 5a 26.8 ± 13.3 (p = .04); Wnt3a and 5a 77.9 ± 48.3 (p < .01); n = 8). To analyze repopulating ability, 4 x 105 lin− Ly5.1 cells, cultured under the same conditions, were transplanted with 2 x 106 Ly5.2 bone marrow cells into lethally-irradiated Ly5.2 recipients. 16 weeks after transplant, repopulation by control lin− cells increased 2-fold compared to lin− cells cultured in Wnt3a or Wnt5a (control 7.3 ± 3.8%; Wnt3a 3.37 ± 1.2% (p < .01); Wnt5a 3.6 ± 1.1% (p < .01); n = 9-10). However, lin− cells cultured in Wnt3a and 5a showed normal repopulating activity (n = 10; 8.7 ± 5.3%; p = .52). 1 x 104 HSCs (lin−, c-kitHI, Sca-1HI, IL-7Rα −) were cultured for 6 days with SCF, Flt3L, Wnt3a and 5a (factors added on day 0 and day 3) as described above. Control HSC numbers expanded more than HSCs cultured with Wnt3a, Wnt 5a, or both (control 20.7 ± 10.4-fold; Wnt 3a 7.0 ± 4.1-fold (p = .05); Wnt5a 1.7 ± 1.7-fold (p = .01); Wnt3a and 5a 1.2 ± 1.0-fold (p < .01); n = 4). Similar numbers of control HSCs and HSCs cultured with Wnt3a or 5a were lin+ (control 21.7 ± 0.2%; Wnt 3a 15.4 ± 5.3% (p = .10); Wnt5a 14.4 ± 5.2% (p = .07); n = 3). However, culturing HSCs with Wnt3a and 5a resulted in a 50% decrease in the number of lin+ cells compared to control (12.3 ± 2.0% (p = .001)). Cultured Ly5.1 HSCs were transplanted with Ly5.2 bone marrow cells at a 1:100 ratio. There was no difference in repopulation between control HSCs and HSCs cultured with Wnt3a (control 5.8 ± 6.1%; Wnt3a 3.6 ± 0.4%; p = .43; n = 5). To examine the effects of enforced expression of Wnt ligands in HSCs, 5-FU treated bone marrow was transduced with Wnt3a-IRES-GFP, Wnt5a-IRES-dsRED, or IRES-GFP retroviral vectors. Sorted IRES-GFP+, Wnt3a-GFP+ and Wnt5a-dsRed+ cells (Ly5.1) were transplanted with equal numbers of mock-transduced cells and 3 x 105 Sca-1− bone marrow cells (Ly5.2) into lethally-irradiated Ly5.2 mice. 16 weeks later, recipients of IRES-GFP+ and Wnt5a-dsRed+ cells contained a similar number of engrafted cells expressing the vector (3.4 ± 1.8% GFP+ Ly5.1 and 3.5 ± 0.4% dsRed+ Ly5.1 respectively; n = 8). In contrast, no GFP+ Ly5.1 cells were detected in Wnt3a-GFP+ recipients (n = 8). 33.4 ± 3.7% of bone marrow cells were Ly5.1+ indicating successful engraftment and retroviral DNA was detected by PCR, suggesting that transduction had occurred but that only cells in which the vector was silenced survived. We conclude that activation of the canonical Wnt pathway in HSCs promotes differentiation of primitive hematopoietic cells and that other signals, such as Wnt5a, are required to maintain the balance between HSC differentiation and self-renewal.

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.

Hmgb3 Deficiency Inhibits Down-Regulation of c-kit in Hematopoietic Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 372-372
Author(s):  
Michael J. Nemeth ◽  
Stacie M. Anderson ◽  
Lisa J. Garrett-Beal ◽  
David M. Bodine
Keyword(s):  
Bone Marrow ◽  
Hind Limb ◽  
Bone Marrow Cells ◽  
Es Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Bone Marrow Cellularity ◽  
Marrow Cellularity ◽  
Marrow Cells

Abstract Hmgb3 is an X-linked member of a family of sequence-independent chromatin-binding proteins that is expressed in HSC-enriched lin−, c-kitHI, Sca-1HI, IL-7Rα− (KSIL) cells and Ter119+ erythroid cells. To define Hmgb3 function, we generated hemizygous mice (Hmgb3−/Y) using 129/SvJ ES cells. Hmgb3−/Y mice contain normal numbers of KSIL cells that are capable of normal repopulation and self-renewal. However, these mice have 1.6-fold fewer common lymphoid progenitors (CLP) and 3-fold fewer common myeloid progenitors (CMP) (p < 0.05). We hypothesized that the role of Hmgb3 in early hematopoiesis involves c-kit regulation. We observed that the level of c-kit mRNA in Hmgb3−/Y HSCs increased 30% compared to wild-type (WT) (p = 0.05). We used 5-fluorouracil (5-FU), which has been shown to down-regulate c-kit on HSCs, to characterize the interaction between Hmgb3 and c-kit. We monitored Hmgb3 expression in KSIL and lin−, Sca-1+, c-kit− cells before and after 5-FU treatment (150 mg/kg) using phenotypically normal transgenic mice containing an IRES-GFP cassette knocked into the 3′ UTR of Hmgb3. Prior to 5-FU treatment, 27% of KSIL cells were GFP+ (these cells were absent 4 days post-injection {p.i.}). In contrast, 1.8% of lin−, c-kit−, Sca-1+ cells were GFP+ before 5-FU treatment whereas 26% of lin−, c-kit−, Sca-1+ cells were GFP+ 4 days p.i. The increased proportion of GFP+ lin-, c-kit−, Sca-1+ cells after 5-FU treatment is consistent with previous findings that repopulating activity resides within the c-kit−/LO population in 5-FU treated bone marrow and our finding that Hmgb3 serves as a marker for long-term repopulating activity. To determine the time course of c-kit regulation, we compared bone marrow from 5-FU injected Hmgb3−/Y and WT mice for analysis at 2, 4, and 6 days p.i. Two days p.i., both WT and Hmgb3−/Y mice contained similar numbers of bone marrow cells (7 x 106 cells/hind limb) and the KSIL population was absent. By four days p.i., the bone marrow cellularity of WT mice declined to 5.5 ± 0.9 x 106 cells/hind limb and KSIL cells were still absent. However, in Hmgb3−/Y mice 4 days p.i., bone marrow cellularity stabilized at 7.9 ± 0.8 x 106 cells/hind limb, an increase of 43% compared to WT (p < 0.01), along with the re-emergence of the KSIL population. To determine whether the Hmgb3−/Y lin−, c-kit−, Sca-1+ population contains repopulating HSCs after 4 days of 5-FU treatment similar to WT mice, we performed repopulation assays using KSIL and lin−, c-kit−, Sca-1+ cells sorted from 4 day p.i. 5-FU treated Hmgb3−/Y mice. Recipients received either 2 x 104 KSIL or 2 x 105 lin−, c-kit−, Sca-1+ cells (Ly 5.2) from 5-FU treated Hmgb3−/Y mice along with a radioprotective dose of 3 x 105 congenic (Ly 5.1) bone marrow cells. FACS analysis performed on control recipients transplanted with congenic marrow exhibited < 1% Ly 5.2 cells in the bone marrow 16 weeks after transplant. Pre-5-FU treatment, 88% of bone marrow cells were donor derived in recipients of Hmgb3−/Y KSIL cells. There was no detectable engraftment of Hmgb3−Y lin−, c-kit−, Sca-1+ cells. In contrast to WT mice, both KSIL and lin−, c-kit−, Sca-1+ cells from 5-FU treated Hmgb3−/Y mice were capable of long-term repopulation (62–82% donor derived cells). We conclude that Hmgb3 deficiency facilitates the reemergence of c-kitHI HSCs following 5-FU treatment. Mechanisms involving either enhanced HSC self-renewal or delayed differentiation into CLPs and CMPs are both consistent with our results.

DNMT3b’s Role in Hematopoietic Stem Cell Self-Renewal.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1406-1406
Author(s):  
Matthew J Boyer ◽  
Feng Xu ◽  
Hui Yu ◽  
Tao Cheng
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Transplantation ◽  
Peripheral Blood ◽  
Marrow Transplantation ◽  
Bone Marrow Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract DNA methylation is an epigenetic means of gene regulation and is carried out by a family of methyltransferases of which DNMT1 acts to maintain methylation marks following DNA replication and DNMT3a and DNMT3b methylate DNA de novo. DNMT3b has been shown to be essential for mammalian development and necessary for differentiation of germline and neural progenitor cells. Mutations of DNMT3b in humans lead to a rare autosomal recessive disorder characterized by immunodeficiency, centromeric instability, and facial abnormalities. We have shown by real-time, RT-PCR that DNMT3b mRNA is uniquely over-expressed by approximately 30-fold in immunophenotypically-defined longterm repopulating hematopoietic stem cells (HSCs) that are CD34−lineage−c-kit+Sca-1+ as compared to progenitor and differentiated cell types within the bone marrow and with respect to the other members of the DNMT family, namely DNMT1 and DNMT3a. To determine DNMT3b’s function in HSCs competitive bone marrow transplantation was undertaken. Isolated lineage− enriched bone marrow cells were transduced with a retroviral backbone based on the Murine Stem Cell Virus (MSCV) carrying either GFP and a short, hairpin RNA (shRNA) targeting DNMT3b or GFP alone. Following transduction 1×105 GFP+ cells along with 1×105 competitor cells were transplanted into 9.5 Gray irradiated congenic recipients. Two months following transplantation mice receiving bone marrow cells transduced with DNMT3b shRNA showed a significantly lower engraftment of donor cells as a percentage of total competitor cell engraftment in the peripheral blood as compared to those receiving cells transduced with GFP alone (24.8 vs 3.7, p&lt;0.05) which persisted at 3 months (22.8 vs 1.5, p&lt;0.05). Similarly, within the donor derviced cells in the peripheral blood there was a lower percentage of myeloid (CD11b+) cells at 2 and 3 months in the recipients of DNMT3b shRNA transduced cells as compared to controls. However there was no observed difference in the percentage of peripheral B (CD45R+) or T (CD3+) cells within the donor-derived cells. To determine the mechanism behind the observed engraftment defect with DNMT3b knockdown we cultured GFP+ transduced bone marrow cells in vitro with minimal cytokine support. As a control for our targeting methodology we also transduced bone marrow cells from mice harboring two floxed DNMT3b alleles with a MSCV carrying Cre recombinase and GFP. While lineage− bone marrow cells transduced with GFP alone increased 10-fold in number over two weeks of culture, cells in which DNMT3b was down regulated by shRNA or Cre-mediated recombination only doubled. Culture of lineage− bone marrow cells in methylcellulose medium by the colony-forming cell (CFC) assay revealed increases in the granulocytic and total number of colonies with DNMT3b knockdown or Cre-mediated recombination of DNMT3b similar to the increased myeloid engraftment of DNMT3b shRNA transduced cells observed 1 month following competitive bone marrow transplantation. However when 5,000 of these cells from the first CFC assay were sub-cultured there was a significant loss of colony forming ability within all lineages when DNMT3b was targeted by shRNA or Cre-mediated recombination. Taken together with the decreased engraftment of DNMT3b shRNA cells following competitive bone marrow transplantation, the observed limited proliferation in liquid culture and loss of colony forming ability during serial CFC assays is suggestive of a self-renewal defect of HSCs in the absence of DNMT3b, that was previously only reported in the absence of both DNMT3a and DNMT3b. Further elucidation of this proposed self-renewal defect is being undertaken and results of ongoing studies including long-term culture initiating cell (LTC-IC) assays and identification of genomic sites of DNA methylation within different hematopoietic subsets will also be presented.

Non-Canonical NF-Kb Signaling Regulates Hematopoietic Stem Cell Self-Renewal and Microenvironment Interactions

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 859-859 ◽  
Author(s):  
Chen Zhao ◽  
Yan Xiu ◽  
John M Ashton ◽  
Lianping Xing ◽  
Yoshikazu Morita ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Wild Type ◽  
Double Knockout ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Physiological Processes ◽  
Marrow Cells

Abstract Abstract 859 RelB and NF-kB2 are the main effectors of NF-kB non-canonical signaling and play critical roles in many physiological processes. However, their role in hematopoietic stem/progenitor cell (HSPC) maintenance has not been characterized. To investigate this, we generated RelB/NF-kB2 double-knockout (dKO) mice and found that dKO HSPCs have profoundly impaired engraftment and self-renewal activity after transplantation into wild-type recipients. Transplantation of wild-type bone marrow cells into dKO mice to assess the role of the dKO microenvironment showed that wild-type HSPCs cycled more rapidly, were more abundant, and had developmental aberrancies: increased myeloid and decreased lymphoid lineages, similar to dKO HSPCs. Notably, when these wild-type cells were returned to normal hosts, these phenotypic changes were reversed, indicating a potent but transient phenotype conferred by the dKO microenvironment. However, dKO bone marrow stromal cell numbers were reduced, and bone-lining niche cells supported less HSPC expansion than controls. Further, increased dKO HSPC proliferation was associated with impaired expression of niche adhesion molecules by bone-lining cells and increased inflammatory cytokine expression by bone marrow cells. Thus, RelB/NF-kB2 signaling positively and intrinsically regulates HSPC self-renewal and maintains stromal/osteoblastic niches and negatively and extrinsically regulates HSPC expansion and lineage commitment through the marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

Dnmt3a is Required For Aberrant Self-Renewal Driven by PML-RARA

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 477-477
Author(s):  
Christopher B Cole ◽  
Angela M. Verdoni ◽  
David H Spencer ◽  
Timothy J. Ley
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Myeloid Cells ◽  
Cd11b Expression ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Marrow Cells

We previously identified recurrent mutations in the DNA methyltransferase DNMT3A in patients with acute myeloid leukemia (AML). DNMT3A and the highly homologous gene DNMT3B encode the two methyltransferases that are primarily responsible for mediating de novo methylation of specific CpG residues during differentiation. Loss of Dnmt3a in hematopoietic stem cells impairs their ability to differentiate into committed progenitors (Challen et al Nat Gen 44:23, 2011). Importantly, DNMT3A mutations are mutually exclusive of the favorable prognosis AML-initiating translocations, including the t(15;17) translocation (which creates the PML-RARA fusion gene), and translocations involving MLL. PML-RARA has been shown to interact with DNMT3A in vitro (Di Croce et al Science 295:1079,2002), and to require DNMT3A to induce methylation and transcriptional silencing of a subset of specific target genes. These findings, and the lack of DNMT3A mutations in APL patients, suggest that PML-RARA may require functional DNMT3A to initiate leukemia. To investigate this possibility, we utilized a well-characterized transgenic mouse model (in a pure B6 background) in which expression of PML-RARA is driven in hematopoietic stem/progenitor cells by the mouse Cathepsin G locus (Ctsg-PML-RARA+/- mice). These mice spontaneously develop acute promyelocytic leukemia (APL) with high penetrance and long latency, and also exhibit a preleukemic phenotype marked by the accumulation of myeloid cells in bone marrow and spleen. In addition, myeloid progenitor cells derived from these mice have the ability to serially replate in methylcellulose cultures, demonstrating aberrant self-renewal. We generated Ctsg-PML-RARA+/- mice lacking Dnmt3a (PML-RARA+/- x Dnmt3a-/-) as well as mice in which conditional ablation of Dnmt3b in hematopoietic cells is driven by Vav-Cre (PML-RARA+/- x Dnmt3b fl/fl x Vav-Cre+). Loss of Dnmt3a completely abrogated the ex vivo replating ability of PML-RARA bone marrow (Figure 1). Although colonies from both PML-RARA+/- and PML-RARA+/- x Dnmt3a-/- mice appeared similar in morphology and number on the first plating, PML-RARA+/- x Dnmt3a-/- marrow ceased to form colonies with subsequent replating (see Figure), and cultured cells lost the expression of the myeloid marker CD11b. The same phenotype was also observed using bone marrow from both genotypes that was secondarily transplanted into wild type recipients, indicating that it is intrinsic to transplantable hematopoietic progenitors. Reintroduction of DNMT3A into bone marrow cells derived from PML-RARA+/- x Dnmt3a-/- mice with retroviral transduction restored replating ability and CD11b expression. Competitive repopulation experiments with PML-RARA+/- x Dnmt3a-/- marrow revealed a decreased contribution to peripheral lymphoid and myeloid cells at 4 weeks, relative to PML-RARA+/- or WT control animals. Finally, 12 weeks after transplantation, recipients of PML-RARA+/- x Dnmt3a-/- bone marrow did not display an accumulation of myeloid cells in the bone marrow and spleen. Importantly, bone marrow from PML-RARA+/- x Dnmt3b fl/fl x Vav-Cre+/- mice displayed no replating deficit or loss of CD11b expression ex vivo, indicating different functions for Dnmt3a versus Dnmt3b in this model. Finally, we interrogated the effect of Dnmt3a loss on bone marrow DNA methylation patterns using a liquid phase DNA capture technique that sampled ∼1.9 million mouse CpGs at >10x coverage. Loss of Dnmt3a caused a widespread loss of DNA methylation in whole bone marrow cells, with 36,000 CpGs that were highly methylated (methylation value >0.7) in the PML-RARA+/- and WT mice, but hypomethylated (methylation value <0.4) in Dnmt3a-/- and PML-RARA+/- x Dnmt3a-/- mice. Characterization of the effect of Dnmt3a loss on leukemia latency, penetrance, and phenotype in PML-RARA+/- mice is currently being defined in a tumor watch. In summary, we have demonstrated that PML-RARA requires functional Dnmt3a (but not Dnmt3b) to drive aberrant self-renewal of myeloid progenitors ex vivo, and that loss of Dnmt3a leads to widespread DNA hypomethylation in bone marrow cells, and abrogates preleukemic changes in mice expressing PML-RARA. This data may explain why DNMT3A mutations are not found in patients with APL initiated by PML-RARA. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Thrombopoietin Does Not Induce Lineage-Restricted Commitment of Mpl-R Expressing Pluripotent Progenitors But Permits Their Complete Erythroid and Megakaryocytic Differentiation

Blood ◽  
1997 ◽  
Vol 89 (10) ◽  
pp. 3544-3553 ◽  
Author(s):  
Frédérique Goncalves ◽  
Catherine Lacout ◽  
Jean-Luc Villeval ◽  
Françoise Wendling ◽  
William Vainchenker ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Constitutive Expression ◽  
Retroviral Vectors ◽  
Granulocytic Differentiation ◽  
Hematopoietic Stem ◽  
Infected Cells ◽  
Marrow Cells

In this study, we examined the in vitro and in vivo effects of forced expression of Mpl-R (the thrombopoietin receptor) on the progeny of murine hematopoietic stem cells. Bone marrow cells from 5-FU–treated mice were transduced with retroviral vectors containing the human Mpl-R cDNA, or the neomycine gene as a control. After 7 days cocultivation on virus-producer cells, GpE86-Mpl-R or Gp86-Neo, the types of hematopoietic progenitor cells responding to thrombopoietin (TPO) were studied by clonogenic assays. Mpl-R–infected cells gave rise to CFU-GEMM, BFU-E, CFU-MK, but not CFU-GM while Neo-infected cells produced only megakaryocytic colonies. In addition, when nonadherent cells from GpE86-Mpl-R cocultures were grown with TPO as the only stimulus for 7 days, a marked expansion of CFU-GEMM, BFU-E, and CFU-MK was observed, while no change in CFU-GM number was seen. Erythroid and megakaryocytic maturation occurred in the presence of TPO while a block in granulocytic differentiation was observed at the myeloblast stage. The direct effects of TPO on Mpl-R–transduced progenitor cells were demonstrated by single cell cloning experiments. To analyze the effects of the constitutive expression of Mpl-R on the determination of multipotent progenitors (CFU-S) and long-term repopulating stem cells, Mpl-R– or Neo-infected cells were injected into lethally irradiated recipient mice. No difference was seen in (1) the number of committed progenitor cells contained in individual CFU-S12 whether colonies arose from noninfected or Mpl-R–infected CFU-S; (2) the mean numbers of progenitor cells per leg or spleen of mice reconstituted with Mpl-R– or Neo-infected cells, 1 or 7 months after the graft; and (3) the blood parameters of the two groups of animals, with the exception of a 50% reduction in circulating platelet counts after 7 months in mice repopulated with Mpl-R–infected bone marrow cells. These results indicate that retrovirus-mediated expression of Mpl-R in murine stem cells does not modify their ability to reconstitute all myeloid lineages of differentiation and does not result in a preferential commitment toward the megakaryocytic lineage.

Mutant p53 Drives the Development of Pre-Leukemic Hematopoietic Stem Cells through Modulating Epigenetic Regulators

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 307-307
Author(s):  
Sarah C Nabinger ◽  
Michihiro Kobayashi ◽  
Rui Gao ◽  
Sisi Chen ◽  
Chonghua Yao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Leukemia Stem Cells ◽  
Mutant P53 ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Tp53 Mutations ◽  
Marrow Cells

Abstract AML is thought to arise from leukemia stem cells (LSCs); however, recent evidence suggests that the transforming events may initially give rise to pre-leukemic hematopoietic stem cells (pre-leukemic HSCs), preceding the formation of fully transformed LSCs. Pre-leukemic HSCs have been shown to contribute to normal blood development and harbor a selective growth advantage compared to normal HSCs. Pre-leukemic HSCs can acquire subsequent mutations, and once differentiation capacity is impaired, leukemia emerges. Recently, acquired somatic TP53 mutations, including p53R248W and p53R273H, were identified in healthy individuals as well as AML patients, suggesting that TP53 mutations may be early events in the pathogenesis of AML. We found that p53R248W HSCs showed a multi-lineage repopulation advantage over WT HSCs in transplantation experiments, demonstrating that mutant p53 confers a pre-leukemic phenotype in murine HSCs. Although TP53 mutations are limited in AML, TP53 mutations do co-exist with mutations of epigenetic regulator, ASXL-1, or receptor tyrosine kinase, FLT3, in AML. Mutations in Asxl-1 are present in ~10-30% of patients with myeloid malignancies and confer poor prognosis. Loss of Asxl-1 in the hematopoietic compartment leads to a myelodysplastic-like syndrome in mice and reduced stem cell self-renewal. Internal tandem duplications in Flt3 (Flt3-ITD) occur in ~30% of AML patients and are associated with adverse clinical outcome. Flt3-ITD-positive mice develop a myeloproliferative neoplasm (MPN) and HSCs expressing Flt3-ITD have decreased self-renewal capabilities. We hypothesize that mutant p53 drives the development of pre-leukemic HSCs with enhanced self-renewal capability, allowing clonal expansion and subsequent acquisition of Asxl-1 or Flt3 mutations leading to the formation of fully transformed leukemia stem cells. To define the role of mutant p53 in Asxl-1+/- HSCs, we generated p53R248W/+ Asxl-1+/- mice and performed in vitro serial replating assays as well as in vivo competitivebone marrow transplantation experiments. We found that p53R248W significantly enhanced the serial replating ability of Asxl-1-deficient bone marrow cells. Interestingly, while bone marrow from Asxl-1+/- mice had very poor engraftment compared to wild type bone marrow cells 16 weeks post-transplantation, the expression of p53R248W in Asxl-1+/- bone marrow rescued the defect. To examine the role of mutant p53 in Flt3-ITD-positive HSCs, we generated p53R248W/+ Flt3ITD/+ mice. We found that p53R248W enhanced the replating ability of Flt3ITD/+ bone marrow cells. Despite the fact that Flt3ITD/+ bone marrow cells displayed decreased repopulating ability compared to wild type cells 16 weeks post-transplant, expression of p53R248W in Flt3ITD/+ cells rescued the defect. We are monitoring leukemia development in primary and secondary transplant recipients as well as in de novo p53R248W/+ Asxl-1+/- and p53R248W/+ Flt3ITD/+ animals and predict that mutant p53 may cooperate with Asxl-1 deficiency or Flt3-ITD in the formation of LSCs to accelerate leukemia development in Asxl-1 deficient or Flt-ITD-positive neoplasms. Mechanistically, dysregulated epigenetic control underlies the pathogenesis of AML and we discovered that mutant p53 regulates epigenetic regulators, including Ezh1, Ezh2, Kdm2a, and Setd2, in HSCs. H3K27me3 is catalyzed by EZH1 or EZH2 of the Polycomb repressing complex 2 (PRC2). Both Ezh1 and Ezh2 are important for HSC self-renewal. SETD2 is a histone H3K36 methyltransferase and mutations in SETD2 have been identified in 6% of patients with AML. SETD2 deficiency resulted in a global loss of H3K36me3 and increased self-renewal capability of leukemia stem cells. We found that there were increased levels of H3K27me3 and decreased levels of H3K36me3 in p53R248W/+ HSCs compared to that of the WT HSCs. In ChIP experiments, we found that p53R248W, but not WT p53, was associated with the promoter region of Ezh2 in mouse myeloid progenitor cells, suggesting that p53R248W may directly activate Ezh2 expression in hematopoietic cells. Given that Asxl-1 has been shown to regulate H3K27me3 in HSCs, the synergy between mutant p53 and Asxl-1 deficiency on LSC self-renewal could be due to changes in histone modifications. Overall, we demonstrate that mutant p53 promotes the development of pre-leukemic HSCs by a novel mechanism involving dysregulation of the epigenetic pathways. Disclosures No relevant conflicts of interest to declare.

scholarly journals Serial transplantation of methotrexate-resistant bone marrow: protection of murine recipients from drug toxicity by progeny of transduced stem cells

Blood ◽  
1990 ◽  
Vol 75 (2) ◽  
pp. 337-343 ◽  
Author(s):  
CA Corey ◽  
AD DeSilva ◽  
CA Holland ◽  
DA Williams
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Retroviral Vectors ◽  
Hematopoietic Stem ◽  
Genetic Sequences ◽  
Marrow Cells

Recombinant retroviral vectors have been used to transfer a variety of genetic sequences into hematopoietic stem cells. Although transfer and expression of foreign genetic sequences into reconstituting stem cells is one approach to somatic gene therapy, few studies have shown long lasting phenotypic changes in recipient mice in vivo. In this study, we show successful transfer of a methotrexate-resistant cDNA (DHFRr) into reconstituting hematopoietic stem cells using a retroviral vector, FrDHFRr, in which the DHFR cDNA is expressed off a hybrid Friend/Moloney long term repeat. Both primary and secondary recipients transplanted with bone marrow cells infected with this recombinant retrovirus show improved survival and protection from methotrexate- induced marrow toxicity when compared with control animals. These data suggest that retroviral-mediated gene transfer of DHFRr cDNA leads to a stable change in the phenotype of hematopoietic stem cells and progeny derived from those cells in vivo after bone marrow transplantation. Gene transfer using recombinant retroviral vectors seems to be one rational approach to establishing chemotherapy-resistant bone marrow cells.

scholarly journals Β-Catenin-Tcf/Lef Signaling Promotes Steady State and Emergency Granulopoiesis through G-CSF Receptor Upregulation

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1193-1193
Author(s):  
Petr Danek ◽  
Miroslava Kardosova ◽  
Lucie Janeckova ◽  
Vladimir Korinek ◽  
Touati Benoukraf ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
Wnt Signaling ◽  
Signaling Pathway ◽  
Target Genes ◽  
Bone Marrow Cells ◽  
Genetic Manipulation ◽  
Wild Type ◽  
Canonical Wnt Signaling ◽  
Canonical Wnt

The canonical Wnt signaling pathway is mediated by interaction of β-catenin with the Tcf/Lef transcription factors and subsequent transcriptional activation of Wnt-target genes. This pathway acts as an essential regulator of differentiation and cell fate decisions in various tissues. In the hematopoietic system, the function of the pathway has been investigated mainly by genetic manipulation of β-catenin. However, this approach does not allow to discriminate between Tcf/Lef dependent or independent β-catenin activity. In order to specifically identify the function of β-catenin-Tcf/Lef signaling in hematopoietic cells, we employed a transgenic mouse model expressing a dominant negative form of the human TCF4 transcription factor (dnTCF4). dnTCF4, a truncated protein lacking the β-catenin binding domain, abrogates activation of Wnt target genes, even when β-catenin is stabilized and translocated into the nucleus. In our model, expression of dnTCF4 is activated from the Rosa26 locus only in cells producing Cre recombinase (driven by Vav-iCre). Importantly, all components of Wnt signaling, including endogenous Tcf/Lef proteins and β-catenin, are intact in Cre-expressing cells. We observed that dnTCF4 transgenic mice have reduced numbers of granulocytes together with accumulation of short-term hematopoietic stem cells (ST-HSC) and common myeloid progenitors (CMPs) in bone marrow. Accordingly, dnTCF4-expressing bone marrow consistently showed a block of granulocytic differentiation and retention of an immature phenotype in colony forming assays. This differentiation arrest and accumulation of immature cells was also observed when wild type cells were cultured in semi-solid medium in the presence of a cell-penetrating peptide that disrupts β-catenin-Tcf/Lef interaction. Together, these results indicate that disruption of the β-catenin/Tcf-Lef interaction, either by genetic manipulation or a drug based approach, alters steady-state hematopoiesis. To identify a mechanism through which β-catenin-Tcf/Lef signaling affects granulopoiesis, wild type and dnTCF4 expressing ST-HSCs were subjected to RNA sequencing. Several genes related to myeloid development were differentially expressed in dnTCF4 expressing cells, including downregulation of Csf3r, the gene encoding for the G-CSF receptor. Publicly available datasets from ChIP-seq experiments on human cell lines confirmed TCF4 enrichment in the distal promoter of the CSF3R gene, suggesting that CSF3R is directly regulated by canonical Wnt signaling. Using flow cytometry we verified reduced levels of G-CSF receptor on the cell surface of dnTCF4 progenitor cells, and attenuation of downstream Stat3 phosphorylation after G-CSF treatment. Finally, when grown in the presence of G-CSF, dnTCF4-expressing bone marrow cells showed impaired differentiation abilities and reduced granulocytic counts compared to wild type bone marrow cells. These results encouraged us to investigate the role of the β-catenin-Tcf/Lef signaling pathway during emergency granulopoiesis by challenging mice with lipopolysaccharide (LPS). Remarkably, dnTCF4 mice showed defects upon LPS stimulation, and completely failed to maintain and expand myeloid progenitor populations, a critical step during emergency granulopoiesis. Altogether, we showed that β-catenin-Tcf/Lef signaling axis is crucial for proper differentiation of myeloid progenitors into granulocytes in steady-state and emergency granulopoiesis. Mechanistically, we demonstrated that the β-catenin-Tcf/Lef interaction controls expression of genes involved in myeloid differentiation, and directly enhances expression of the G-CSF receptor, a crucial molecule for proper development of granulocytes. Disclosures No relevant conflicts of interest to declare.

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