Evaluation of Primitive Murine Hematopoietic Stem and Progenitor Cell Transduction In Vitro and In Vivo by Recombinant Adeno-Associated Virus Vector Serotypes 1 Through 5

2006 ◽  
Vol 17 (3) ◽  
pp. 321-333 ◽  
Author(s):  
Li Zhong ◽  
Weiming Li ◽  
Yanjun Li ◽  
Weihong Zhao ◽  
Jianqing Wu ◽  
...  
Keyword(s):  
Progenitor Cell ◽  
Virus Vector ◽  
Adeno Associated Virus ◽  
Hematopoietic Stem

Evaluation of Primitive Murine Hematopoietic Stem and Progenitor Cell Transduction In Vitro and In Vivo by Recombinant Adeno-Associated Virus Vector Serotypes 1 Through 5

2006 ◽  
Vol 0 (0) ◽  
pp. 060301061832003
Author(s):  
Li Zhong ◽  
Weiming Li ◽  
Yanjun Li ◽  
Weihong Zhao ◽  
Jianqing Wu ◽  
...  
Keyword(s):  
Progenitor Cell ◽  
Virus Vector ◽  
Adeno Associated Virus ◽  
Hematopoietic Stem

Evaluation of Primitive Murine Hematopoietic Stem and Progenitor Cell Transduction In Vitro and In Vivo by Recombinant Adeno-Associated Virus Vector Serotypes 1 Through 5

2006 ◽  
Vol 0 (0) ◽  
pp. 060125023345001
Author(s):  
Li Zhong ◽  
Weiming Li ◽  
Yanjun Li ◽  
Weihong Zhao ◽  
Jianqing Wu ◽  
...  
Keyword(s):  
Progenitor Cell ◽  
Virus Vector ◽  
Adeno Associated Virus ◽  
Hematopoietic Stem

Involvement of the Integrin Alpha 6 Receptor in the Homing and Engraftment of Hematopoietic Stem and Progenitor Cells to Bone Marrow.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1293-1293
Author(s):  
Hong Qian ◽  
Sten Eirik W. Jacobsen ◽  
Marja Ekblom
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Functional Blocking

Abstract Within the bone marrow environment, adhesive interactions between stromal cells and extracellular matrix molecules are required for stem and progenitor cell survival, proliferation and differentiation as well as their transmigration between bone marrow (BM) and the circulation. This regulation is mediated by cell surface adhesion receptors. In experimental mouse stem cell transplantation models, several classes of cell adhesion receptors have been shown to be involved in the homing and engraftment of stem and progenitor cells in BM. We have previously found that integrin a6 mediates human hematopoietic stem and progenitor cell adhesion to and migration on its specific ligands, laminin-8 and laminin-10/11 in vitro (Gu et al, Blood, 2003; 101:877). Using FACS analysis, the integrin a6 chain was now found to be ubiquitously (>95%) expressed in mouse hematopoietic stem and progenitor cells (lin−Sca-1+c-Kit+, lin−Sca-1+c-Kit+CD34+) both in adult bone marrow and in fetal liver. In vitro, about 70% of mouse BM lin−Sca-1+c-Kit+ cells adhered to laminin-10/11 and 40% adhered to laminin-8. This adhesion was mediated by integrin a6b1 receptor, as shown by functional blocking monoclonal antibodies. We also used a functional blocking monoclonal antibody (GoH3) against integrin a6 to analyse the role of the integrin a6 receptor for the in vivo homing of hematopoietic stem and progenitor cells. We found that the integrin a6 antibody inhibited the homing of bone marrow progenitors (CFU-C) into BM of lethally irradiated recipients. The number of homed CFU-C was reduced by about 40% as compared to cells incubated with an isotype matched control antibody. To study homing of long-term repopulating stem cells (LTR), antibody treated bone marrow cells were first injected intravenously into lethally irradiated primary recipients. After three hours, bone marrow cells of the primary recipients were analysed by competitive repopulation assay in secondary recipients. Blood analysis 16 weeks after transplantation revealed an 80% reduction of stem cell activity of integrin a6 antibody treated cells as compared to cells treated with control antibody. These results suggest that integrin a6 plays an important role for hematopoietic stem and progenitor cell homing in vivo.

Integrin alpha 6 Is Essential for Fetal Hematopoietic Progenitor, but Not Fetal Stem Cell Homing to Bone Marrow.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1387-1387
Author(s):  
Hong Qian ◽  
Sten Eirik W. Jacobsen ◽  
Marja Ekblom
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Fetal Liver ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Integrin Alpha 6

Abstract Homing of transplanted hematopoietic stem cells (HSC) in the bone marrow (BM) is a prerequisite for establishment of hematopoiesis following transplantation. However, although multiple adhesive interactions of HSCs with BM microenviroment are thought to critically influence their homing and subsequently their engraftment, the molecular pathways that control the homing of transplanted HSCs, in particular, of fetal HSCs are still not well understood. In experimental mouse stem cell transplantation models, several integrins have been shown to be involved in the homing and engraftment of both adult and fetal stem and progenitor cells in BM. We have previously found that integrin a6 mediates human hematopoietic stem and progenitor cell adhesion to and migration on its specific ligands, laminin-8 and laminin-10/11 in vitro (Gu et al, Blood, 2003; 101:877). Furthermore, integrin a6 is required for adult mouse HSC homing to BM in vivo (Qian et al., Abstract American Society of Hematology, Blood 2004 ). We have now found that the integrin a6 chain like in adult HSC is ubiquitously (>99%) expressed also in fetal liver hematopoietic stem and progenitor cells (lin−Sca-1+c-Kit+, LSK ). In vitro, fetal liver LSK cells adhere to laminin-10/11 and laminin-8 in an integrin a6b1 receptor-dependent manner, as shown by function blocking monoclonal antibodies. We have now used a function blocking monoclonal antibody (GoH3) against integrin a6 to analyse the role of the integrin a6 receptor for the in vivo homing of fetal liver hematopoietic stem and progenitor cells to BM. The integrin a6 antibody inhibited homing of fetal liver progenitors (CFU-C) into BM of lethally irradiated recipients. The number of homed CFU-C in BM was reduced by about 40% as compared to the cells incubated with an isotype matched control antibody. To study homing of long-term repopulating stem cells, BM cells were first incubated with anti-integrin alpha 6 or anti-integrin alpha 4 or control antibody, and then injected intravenously into lethally irradiated primary recipients. After three hours, BM cells of the primary recipients were analysed by competitive repopulation assay in secondary recipients. Blood analysis up to 16 weeks after transplantation showed that no reduction of stem cell reconstitution from integrin a6 antibody treated cells as compared to cells treated with control antibody. In accordance with this, fetal liver HSC from integrin a6 gene deleted embryos did not show any impairment of homing and engraftment in BM as compared to normal littermates. These results suggest that integrin a6 plays an important developmentally regulated role for homing of distinct hematopoietic stem and progenitor cell populations in vivo.

scholarly journals Cell-nonautonomous function of Id1 in the hematopoietic progenitor cell niche

Blood ◽  
2009 ◽  
Vol 114 (6) ◽  
pp. 1186-1195 ◽  
Author(s):  
Hyung Chan Suh ◽  
Ming Ji ◽  
John Gooya ◽  
Michael Lee ◽  
Kimberly D. Klarmann ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cell ◽  
Hematopoietic Progenitor Cell ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Cytokine Levels ◽  
Cell Niche ◽  
Marrow Cellularity

Abstract Development of hematopoietic stem cells (HSCs) and their immediate progeny is maintained by the interaction with cells in the microenvironment. We found that hematopoiesis was dysregulated in Id1−/− mice. Although the frequency of HSCs in Id1−/− bone marrow was increased, their total numbers remained unchanged as the result of decreased bone marrow cellularity. In addition, the ability of Id1−/− HSCs to self-renew was normal, suggesting Id1 does not affect HSC function. Id1−/− progenitors showed increased cycling in vivo but not in vitro, suggesting cell nonautonomous mechanisms for the increased cycling. Id1−/− HSCs developed normally when transplanted into Id1+/+ mice, whereas the development of Id1+/+ HSCs was impaired in Id1−/− recipients undergoing transplantation and reproduced the hematologic features of Id1−/− mice, indicating that the Id1−/− microenvironment cannot support normal hematopoietic development. Id1−/− stromal cells showed altered production of cytokines in vitro, and cytokine levels were deregulated in vivo, which could account for the Id1−/− hematopoietic phenotypes. Thus, Id1 is required for regulating the hematopoietic progenitor cell niche but is dispensable for maintaining HSCs.

Coagulation Factor Thrombin Regulates Hematopoietic Stem and Progenitor Cell Egress and Mobilization Via PAR-1 & CXCR4 Upregulation, SDF-1 Secretion and EPCR Shedding

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2341-2341 ◽  
Author(s):  
Shiri Gur-Cohen ◽  
Tomer Itkin ◽  
Aya Ludin ◽  
Orit Kollet ◽  
Karin Golan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Progenitor Cell ◽  
Single Injection ◽  
Hematopoietic Stem ◽  
Epcr Shedding

Abstract Abstract 2341 Hematopoietic stem and progenitor cell (HSPC) egress from the bone marrow (BM) to the circulation is tightly regulated and is accelerated during stress conditions. The G-protein-coupled receptor protease-activated receptor-1 (PAR-1) and its activator thrombin play an important role in coagulation following injury and bleeding. We report that a single injection of thrombin induced rapid HSPC mobilization within one hour, increasing circulating leukocytes, predominantly CFU-C and primitive Lin−/Sca-1+/c-Kit+ (SKL) progenitor cells. This rapid mobilization was preceded by a dramatic decrease of SDF-1 (CXCL12) in BM stromal cells, including rare Nestin+ mesenchymal stem cells (MSC) which functionally express PAR-1 and release SDF-1. Thrombin injection also increased expression of PAR-1 and CXCR4 by BM HSPC. These results suggest involvement of the coagulation cascade of thrombin & PAR-1 in rapid SDF-1 secretion from niche supporting BM stromal cells as part of host defense and repair mechanisms. Administration of a PAR-1 specific antagonist (SCH79797) upregulated BM SDF-1 levels and significantly reduced the amounts of circulating CFU-C and primitive SKL progenitor cells. In vitro stimulation of BM mononuclear cells with thrombin for 1 hour led to increased CXCR4 expression by Lin−/c-Kit+ progenitors, accompanied by enhanced spontaneous and SDF-1 induced migration. Of note, specific PAR-1 inhibition in vitro significantly reduced SDF-1-directed migration of Lin-/c-Kit+ progenitors. Mechanistically, we found that thrombin - activated PAR-1 induced the downstream p38 MAPK and eNOS (nitric oxide synthase) signaling pathways. Long term repopulating hematopoietic stem cells (HSC) in murine BM highly express endothelial protein C receptor (EPCRhigh) (Balazs & Mulligan et al Blood 2006; Kent & Eaves et al Blood 2009). EPCR is expressed primarily on endothelial cells (EC) and has anti coagulation and anti inflammatory roles. Surface EPCR expression on EC is downregulated by many factors, including PAR-1 activation by thrombin, a process which is termed shedding and is not fully understood. Importantly, we found that over 90% of BM CD45+/EPCRhigh long-term HSC express PAR-1 and that circulating primitive HSPC in the blood and spleen lack EPCRhigh expression. In addition, in-vivo thrombin administration downregulated EPCR from BM HSC via eNOS signaling, thus allowing the release of stem cells from their BM microenvironment anchorage to the circulation. Correspondingly, in eNOS deficient mice, thrombin failed to induce PAR-1 upregulation, EPCR shedding, and HSPC mobilization. Recently, we reported that the antioxidant NAC inhibits G-CSF induced mobilization (Tesio & Lapidot et al Blood 2011). Co-administration of G-CSF with NAC prevented PAR-1 upregulation, concomitantly with reduced HSPC mobilization and increased levels of EPCRhigh HSC in the BM. Treatment of PAR-1 antagonist with G-CSF inhibited PAR-1 and CXCR4 upregulation on BM leukocytes and immature Lin−/c-Kit+ cells accompanied by increased levels of BM EPCRhigh HSC and reduced HSPC mobilization. Tissue factor (TF) is the main initiator of the coagulation system via the formation of an enzymatic “prothrombinase complex” that converts prothrombin to active thrombin. Unexpectedly, we found a unique structure of cell clusters expressing TF, located preferentially in the trabecular-rich area of the femoral metaphysis in murine bone tips, a region highly exposed to osteoclast/osteoblast bone remodeling. In vitro, immature osteoclasts exhibited increased TF expression in cell fusion areas, suggesting that in vivo osteoclast maturation activates the coagulation thrombin/PAR-1 axis of HSPC migration to the circulation. Finally, mimicking bacterial infection a single injection of Lipopolysaccharide (LPS), rapidly and systemically upregulated TF in the murine BM. LPS treatment prompted an increase in thrombin generation and subsequently HSPC mobilization, which was blocked by the PAR-1 antagonist. In conclusion, our study reveals a new role for the coagulation signaling axis, which acts on both hematopoietic and stromal BM cells to regulate steady state HSPC egress and enhanced mobilization from the BM. This thrombin/PAR-1 signaling cascade involves SDF-1/CXCR4 interactions, immature osteoclast TF activity, Nestin+/PAR-1+ MSC secretion of SDF-1 and EPCR shedding from hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.

Dendritic Cells Direct Specific Stimulation of Hematopoietic Stem/Progenitor Cells Generating a Potentially Therapeutic Cell Population

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 483-483
Author(s):  
Yael Porat ◽  
Efrat Assa-Kunik ◽  
Michael Belkin ◽  
Shlomo Bulvik
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Hind Limb ◽  
Progenitor Cell ◽  
Limb Ischemia ◽  
Hematopoietic Stem ◽  
Equity Ownership ◽  
Medium Group

Abstract Abstract 483 Background: Recent data show that dendritic cells (DCs) are important component of stem cell niches in the bone marrow and spleen, and as such may have a role in stem/progenitor cell homeostasis and in pro- and anti-angiogenic processes (Gabrilovich, 1996; Dikov, 2005; Sozzani, 2007). For the first time we report a process in which human Hematopoietic Stem/Progenitor Cells (HSPC) are specifically stimulated by activated DCs. This newly developed process makes it possible to use even unmobilized blood cells as a source for sufficient numbers of potentially therapeutic stem/progenitor cells, thus eliminating the need for surgical bone marrow harvesting and G-CSF mobilization. Goal: To show that DCs can direct the generation of an Enriched Endothelial Progenitor Cell (EnEPC) population, which includes Endothelial Progenitor Cells (EPC) and HSPCs, addressed to treat blood vessel malfunction. Methods: Samples of 250 ml blood from both healthy and diabetic patients were collected under hospital's IRB (Bulvik 15/150109) and used as the cell source. Selected immature plasmacytoid and myeloid DCs were alternatively activated for 2–24 hours in order to induce pro-angiogenic signals before being co-cultured with HSPCs. Cultures of up to 66 hours resulted in the generation of EnEPC in a formulation named BC1. BC1 was tested in-vitro by FACS, tube formation, colony forming units (CFU) and cytokine secretion tests. In-vivo BC1 was tested in the hind limb ischemia model (Goto, 2006; Kang, 2009) of critical limb ischemia (CLI) in order to evaluate its therapeutic potential, dosing levels and bio-distribution following intramuscular transplantation (IM). The study applied a genetically modified SCID/Nude mice model supporting evaluation of both safety and efficacy of BC1 treatment. A 21-day controlled blinded experiment included a control medium group (N=10); unprocessed cells (PreBC1, N=5); two BC1 groups of 2.5×10^6/mouse, BC1-1 (N=10) cultured for 1day and BC1-3 (N=10) for 3 and a lower cell dose group of 0.5×10^6/Mouse BC1-31 (N=5). Results: DC directed BC1 containing 70 ±5×10^6 cells with a viability of 96.9±1.9% is composed of a mixture of 40.2±11.9% EPC (expressing Ulex-lectin and uptake of AcLDL, CD202b (Tie2), CD309 (VEGGFR-2; KDR), CD31 and VEGFR1) and 29.8±14.3% HSPC (co-expressing CD34 and the migration/homing marker CD184 /CXCR4-R). In-vitro functional tests demonstrated angiogenic and hematopoietic potential and secretion of IL-8, VEGF, and IL-10 but not TNF and IFN. In-vivo BC1 was found efficient and safe in the hind-limb ischemia model. Evaluation of clinical signs revealed an improvement in limb function and score in all BC1 treated groups over the control medium group. BC1 treatment doubled the blood flow (BF) to the legs from an average of 23±5% after injury to an average of 51±3.1% on day 21 after treatment (p<0.005). Conclusions: The presented data show that activated DCs can direct in-vitro cellular interactions resulting in a potentially therapeutic EnEPC population after a short-term culture of HSPC. This process makes it possible to use unmobilized blood as the raw material for generating stem/progenitor cell products. The method described here is far safer for patients and much more convenient for clinicians compared to existing methods, such as G-CSF mobilization or bone marrow and fat cells harvesting. Further research needs to be done in order to test the safety and efficacy of these cells in patients suffering from cardiovascular diseases and blood vessel malfunctions. Disclosures: Porat: BioGenCell: Employment, Equity Ownership, Research Funding; Laniado Hospital: Consultancy. Assa-Kunik:BioGenCell: Employment; Laniado Hospital: Employment. Belkin:BioGenCell: Consultancy, Equity Ownership.

Myxoma Virus Targets Primary Human Leukemic Stem and Progenitor Cells While Sparing Normal Hematopoietic Stem and Progenitor Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 14-14 ◽  
Author(s):  
Christopher R. Cogle ◽  
Manbok Kim ◽  
Masmudur Rahman ◽  
Edward W Scott ◽  
Grant McFadden ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Progenitor Cell ◽  
Cell Function ◽  
Human Cancer ◽  
Myxoma Virus ◽  
High Dose ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Abstract Abstract 14 High dose chemotherapy followed by autologous blood and marrow transplantation (ABMT) has been used to treat patients with acute myeloid leukemia (AML), but leukemia relapse rates remain high. One reason is the potential contamination of marrow with leukemic stem and progenitor cells (LSPCs). Purging autologous hematopoietic grafts of LSPCs prior to transplant serves as a viable strategy for increasing transplant efficacy in these cases; however, previous attempts using cytotoxic agents and cell culture techniques have generally resulted in loss of normal stem and progenitor cell numbers and/or functionality. Oncolytic poxviruses, such as myxoma virus (MYXV), are promising new instruments in targeting human cancer. MYXV has normal tropism towards European rabbits (Oryctolagus cuniculus) while remaining nonpathogenic for all other vertebrate species tested including humans and mice. Despite this host specificity, we have shown that MYXV is capable of infecting and killing a wide variety of human cancer cell lines. In light of these observations, we investigated whether MYXV could specifically target and eliminate LSPCs from primary AML using an ex vivo purging technique as assessed using both in vitro and in vivo functional analyses. Using a MYXV construct that expresses GFP upon cell infection, we observed GFP+ cells in leukemia exposed to MYXV at a concentration of 10 MOI over a 3-day period. No GFP expression was observed in normal bone marrow (BM) or mock (vehicle only) treated controls. GFP+ AML cells also began to undergo apoptosis shown by positive Annexin V staining. For myxoma to be a viable therapeutic for leukemia, it must not only target primary leukemia but also spare normal hematopoietic stem and progenitor cells (HSPCs). To test normal progenitor cell function following exposure to MYXV, normal BM cells were incubated with and without MYXV and tested for colony forming cell (CFC) content. Following incubation with MYXV, we observed differentiated colonies forming after 14 days indicating that the CFC potential of normal HSPCs was not adversely affected by MYXV. The frequency of the different colonies formed was also similar between mock and MYXV treated groups. When AML cells were mock treated pleomorphic colonies formed consistent with AML-colony forming units (AML-CFUs). Conversely, when exposed to MYXV, AML cells did not form recognizable AML-CFU colonies and instead remained heterodispersed suggesting impairment of progenitor cell function in vitro. To assess functional effects of MYXV on leukemia engraftment, sublethally irradiated NOG mice were transplanted with either mock treated primary AML (n=7) or primary AML pre-treated with MYXV for 3 hours (n=10). After 8 weeks, the percentage of engrafted mice was 100% after mock treated AML transplant but dropped to 10% after MYXV treatment. Significantly lower mean engraftment was observed in the group that received MYXV treated AML in comparison to mock treated samples (4.5% vs. 24% respectively; p < 0.05). Moreover, we show susceptibility of a primary AML specimen harboring an activating internal tandem duplication (ITD) mutation in FLT3, which represents an aggressive malignancy well-known for insensitivity to conventional chemotherapy. In animals showing leukemia engraftment by FACS, PCR was positive for the FLT3 ITD mutation. However, molecular remissions were evident in mice receiving MYXV treated samples. Efficacy against this leukemia signifies opportunity for disease eradication in an otherwise grim clinical setting. Finally, to assess functional effects of MYXV on normal HSPC engraftment, sublethally irradiated NOG mice were transplanted with either mock treated normal BM (n=10) or MYXV treated BM (n=9). After 8 weeks, there was no difference in the numbers of mice that engrafted between mock treated or MYXV treated groups (70% vs. 78% respectively; p = 0.72). There was also no difference in mean levels of engraftment per animal (1% vs. 2%; p = 0.41) suggesting that MYXV does not adversely affect the in vivo engraftment potential of normal HSPCs. In these studies, primary human LSPCs were targeted by MYXV purging, while normal human HSPCs showed no response maintaining both in vitro and in vivo functional potential. Given this demonstrated efficacy and safety, ex vivo purging of autologous hematopoietic grafts with MYXV may be feasible in cancer patients undergoing high dose chemotherapy followed by ABMT. Disclosures: No relevant conflicts of interest to declare.

Oncogenic Wilms Tumor 1 (WT1) Mutation Augments Hematopoietic Progenitor Cell Clonogenicity and Promotes Expansion Of The Long-Term Hematopoietic Stem Cell (LT-HSC) Compartment: Implications For WT1-Mediated Leukemogenesis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1269-1269
Author(s):  
Colleen E. Annesley ◽  
Rachel E. Rau ◽  
Daniel Magoon ◽  
David Loeb ◽  
Patrick Brown
Keyword(s):  
Progenitor Cells ◽  
Progenitor Cell ◽  
Wilms Tumor ◽  
Wild Type ◽  
Hematopoietic Progenitor ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Exon 9

Abstract Background The WT1 gene encodes for a zinc finger-containing transcription factor involved in differentiation, cell cycle regulation and apoptosis. WT1 expression is developmentally regulated and tissue-specific, with expression maintained in the kidney and in CD34+ hematopoietic progenitor cells. Inactivating mutations of this tumor suppressor gene are well-described in sporadic Wilms tumor and as germline mutations in Wilms tumor predisposition syndromes. WT1 mutations have been reported in approximately 10% of both adult and pediatric patients with cytogenetically-normal acute myeloid leukemia (CN-AML), and have been associated with treatment failure and a poor prognosis. These reported mutations consist of insertions, deletions or point mutations. Many are frameshift mutations in exon 7, can occur as biallelic double mutations, and result in truncated proteins which may alter DNA-binding ability. Missense mutations in exon 9 have also been identified, and reports suggest that these may act in a dominant-negative manner, resulting in a loss of function. Despite these observations, the functional contribution of WT1 mutations to leukemogenesis is still largely undetermined. Methods/Results We obtained a novel knock-in WT1 mutant mouse model, which is heterozygous for the missense mutation R394W in exon 9, and homologous to exon 9 mutations seen in human AML. We hypothesized that WT1 mutations may have an aberrant effect on hematopoiesis, and specifically, could alter progenitor cell differentiation or proliferation. To investigate this, we collected lineage-negative bone marrow (lin- BM) cells from two-month old WT1 mutant (WT1mut) and wild-type (wt) mice. We performed methylcellulose colony-forming assays, serially replating cells every 10-12 days. Strikingly, WT1mut progenitor cells showed higher in vitro colony-forming capacity and an increased ability to serially replate, suggesting aberrantly enhanced self-renewal capability. Furthermore, WT1mut colonies from secondary and tertiary passages were larger and more cohesive than wild-type colonies, demonstrating increased proliferation and morphology consistent with blast colony-forming units (CFU-blast). Flow cytometric analysis of these WT1mut cells at tertiary replating revealed an immature, largely c-Kit+ population. Next, in order to study the effects of WT1mut on HSCs in vivo, we performed serial competitive transplantation of HSC-enriched, lineage-depleted BM into lethally irradiated mice. At 14 weeks post-transplant, the donor bone marrow cells were harvested and analyzed by flow cytometry. We observed a significant expansion of the LT-HSC compartment in the WT1mut mice compared to wild-type mice. These data provide new insight into the biology and functional role of WT1 mutations in the aberrant regulation of hematopoietic stem and progenitor cell expansion. Conclusion Oncogenic WT1 mutations confer enhanced proliferation and renewal of myeloid progenitor cells in vitro and expansion of LT-HSCs in vivo. Our findings suggest that WT1 mutations enhance stem cell self-renewal, potentially priming these cells for leukemic transformation upon acquisition of cooperative events. Disclosures: No relevant conflicts of interest to declare.

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