MT1-MMP Plays a Critical Role in Hematopoiesis by Regulating HIF-Mediated Chemo-/Cytokine Gene Transcription within Niche Cells.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2351-2351
Author(s):  
Chiemi Nishida ◽  
Kaori Sato-Kusubata ◽  
Yoshihiko Tashiro ◽  
Ismael Gritli ◽  
Aki Sato ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Gene Transcription ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Stem Cell Differentiation ◽  
Cell Phenotype ◽  
Hematopoietic Stem

Abstract Abstract 2351 Stem cells reside in a physical niche. The organization of cellular niches has been shown to play a key role in regulating normal stem cell differentiation, stem cell maintenance and regeneration. Various stem cell niches have been shown to be hypoxic, thereby maintaining the stem cell phenotype of e.g. hematopoietic stem cells (HSCs) or cancer stem cells. The bone marrow (BM) niche is a rich reservoir of tissue-specific pluripotent HSCs. Proteases such as matrix metalloproteinases (MMPs) have been implicated in cell movement, partly due to their proteolytic function, and they have been linked to cellular processes such as cell proliferation and differentiation. The proteolytic function of Membrane-type 1 MMP (MT1-MMP/MMP-14) is essential for angiogenesis, arthritis and tumour growth. Recently, it has been reported that MT1-MMP is highly expressed in HSCs and stromal/niche cells. However the clear function of MT1-MMP in hematopoiesis is not well understood. To reveal the functional consequences of MT1-MMP deficiency for post-natal hematopoiesis in vivo, we have taken advantage of MT1-MMP−/− mice to demonstrate that MT1-MMP deficiency leads to impaired steady state hematopoiesis of all hematopoietic cell lineages. In a search for factors whose deficiency could cause this hematopoietic phenotype, we found not only reduced protein release, but also reduced transcription of the following growth factors/chemokines in MT1-MMP−/− mice: erythropoietin (Epo), stromal cell-derived factor-1 (SDF-1a/CXCL12), interleukin-7 (IL-7) and Kit ligand (KitL, also known as stem cell factor). All of these factors, except for Epo, are typical stromal cell-derived factors. To ensure that impaired gene transcription in vivo was not due to a lower number of stromal cells in vivo, we demonstrated that MT1-MMP knockdown in stromal cells in vitro also reduced transcription of the stromal cell derived factors SDF-1a/CXCL12, IL-7 and KitL. In contrast, overexpression of MT1-MMP in stromal cells enhanced gene transcription of these factors. All genes, whose transcription was altered in vitro and in vivo due to MT1-MMP deficiency, had one thing in common: their gene transcription is regulated by the hypoxia inducible factor-1 (HIF-1) pathway. Further mechanistic studies revealed that MT1-MMP activates the HIF-1 pathway via factor inhibiting HIF-1 (FIH-1) within niche cells, thereby inducing the transcription of HIF-responsive genes, which induce terminal hematopoietic differentiation. Thus, MT1-MMP in niche cells regulates postnatal hematopoiesis by modulating hematopoietic HIF-dependent niche factors that are critical for terminal differentiation and migration. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Maintenance of high levels of pluripotent hematopoietic stem cells in vitro: effect of stromal cells and c-kit

Blood ◽  
1993 ◽  
Vol 81 (2) ◽  
pp. 365-372 ◽  
Author(s):  
JP Wineman ◽  
S Nishikawa ◽  
CE Muller-Sieburg
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Line ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Hematopoietic Stem ◽  
Stem Cell Maintenance ◽  
Stromal Cell Line ◽  
Cell Maintenance

We show here that mouse pluripotent hematopoietic stem cells can be maintained in vitro on stroma for at least 3 weeks at levels close to those found in bone marrow. The extent of stem cell maintenance is affected by the nature of the stromal cells. The stromal cell line S17 supported stem cells significantly better than heterogeneous, primary stromal layers or the stromal cell line Strofl-1. Stem cells cultured on S17 repopulated all hematopoietic lineages in marrow-ablated hosts for at least 10 months, indicating that this culture system maintained primitive stem cells with extensive proliferative capacity. Furthermore, we demonstrate that, while pluripotent stem cells express c-kit, this receptor appears to play only a minor role in stem cell maintenance in vitro. The addition of an antibody that blocks the interaction of c-kit with its ligand essentially abrogated myelopoiesis in cultures. However, the level of stem cells in antibody-treated cultures was similar to that found in untreated cultures. Thus, it seems likely that the maintenance of primitive stem cells in vitro depends on yet unidentified stromal cell-derived factor(s).

scholarly journals Native associations of early hematopoietic stem cells and stromal cells isolated in bone marrow cell aggregates

Blood ◽  
1994 ◽  
Vol 83 (2) ◽  
pp. 361-369 ◽  
Author(s):  
PE Funk ◽  
PW Kincade ◽  
PL Witte
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Factor C

In suspensions of murine bone marrow, many stromal cells are tightly entwined with hematopoietic cells. These cellular aggregations appear to exist normally within the marrow. Previous studies showed that lymphocytes and stem cells adhered to stromal cells via vascular cell adhesion molecule 1 (VCAM1). Injection of anti-VCAM1 antibody into mice disrupts the aggregates, showing the importance of VCAM1 in the adhesion between stromal cells and hematopoietic cells in vivo. Early hematopoietic stem cells were shown to be enriched in aggregates by using a limiting-dilution culture assay. Myeloid progenitors responsive to WEHI-3CM in combination with stem cell factor (c-kit ligand) and B220- B-cell progenitors responsive to insulin-like growth factor-1 in combination with interleukin-7 are not enriched. We propose a scheme of stromal cell-hematopoietic cell interactions based on the cell types selectively retained within the aggregates. The existence of these aggregates as native elements of bone marrow organization presents a novel means to study in vivo stem cell-stromal cell interaction.

scholarly journals Native associations of early hematopoietic stem cells and stromal cells isolated in bone marrow cell aggregates

Blood ◽  
1994 ◽  
Vol 83 (2) ◽  
pp. 361-369 ◽  
Author(s):  
PE Funk ◽  
PW Kincade ◽  
PL Witte
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Factor C

Abstract In suspensions of murine bone marrow, many stromal cells are tightly entwined with hematopoietic cells. These cellular aggregations appear to exist normally within the marrow. Previous studies showed that lymphocytes and stem cells adhered to stromal cells via vascular cell adhesion molecule 1 (VCAM1). Injection of anti-VCAM1 antibody into mice disrupts the aggregates, showing the importance of VCAM1 in the adhesion between stromal cells and hematopoietic cells in vivo. Early hematopoietic stem cells were shown to be enriched in aggregates by using a limiting-dilution culture assay. Myeloid progenitors responsive to WEHI-3CM in combination with stem cell factor (c-kit ligand) and B220- B-cell progenitors responsive to insulin-like growth factor-1 in combination with interleukin-7 are not enriched. We propose a scheme of stromal cell-hematopoietic cell interactions based on the cell types selectively retained within the aggregates. The existence of these aggregates as native elements of bone marrow organization presents a novel means to study in vivo stem cell-stromal cell interaction.

scholarly journals Maintenance of high levels of pluripotent hematopoietic stem cells in vitro: effect of stromal cells and c-kit

Blood ◽  
1993 ◽  
Vol 81 (2) ◽  
pp. 365-372 ◽  
Author(s):  
JP Wineman ◽  
S Nishikawa ◽  
CE Muller-Sieburg
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Line ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Hematopoietic Stem ◽  
Stem Cell Maintenance ◽  
Stromal Cell Line ◽  
Cell Maintenance

Abstract We show here that mouse pluripotent hematopoietic stem cells can be maintained in vitro on stroma for at least 3 weeks at levels close to those found in bone marrow. The extent of stem cell maintenance is affected by the nature of the stromal cells. The stromal cell line S17 supported stem cells significantly better than heterogeneous, primary stromal layers or the stromal cell line Strofl-1. Stem cells cultured on S17 repopulated all hematopoietic lineages in marrow-ablated hosts for at least 10 months, indicating that this culture system maintained primitive stem cells with extensive proliferative capacity. Furthermore, we demonstrate that, while pluripotent stem cells express c-kit, this receptor appears to play only a minor role in stem cell maintenance in vitro. The addition of an antibody that blocks the interaction of c-kit with its ligand essentially abrogated myelopoiesis in cultures. However, the level of stem cells in antibody-treated cultures was similar to that found in untreated cultures. Thus, it seems likely that the maintenance of primitive stem cells in vitro depends on yet unidentified stromal cell-derived factor(s).

Isolation and characterization of human CD34−Lin− and CD34+Lin− hematopoietic stem cells using cell surface markers AC133 and CD7

Blood ◽  
2000 ◽  
Vol 95 (9) ◽  
pp. 2813-2820 ◽  
Author(s):  
Lisa Gallacher ◽  
Barbara Murdoch ◽  
Dongmei M. Wu ◽  
Francis N. Karanu ◽  
Mike Keeney ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells

Recent evidence indicates that human hematopoietic stem cell properties can be found among cells lacking CD34 and lineage commitment markers (CD34−Lin−). A major barrier in the further characterization of human CD34− stem cells is the inability to detect this population using in vitro assays because these cells only demonstrate hematopoietic activity in vivo. Using cell surface markers AC133 and CD7, subfractions were isolated within CD34−CD38−Lin− and CD34+CD38−Lin− cells derived from human cord blood. Although the majority of CD34−CD38−Lin− cells lack AC133 and express CD7, an extremely rare population of AC133+CD7− cells was identified at a frequency of 0.2%. Surprisingly, these AC133+CD7− cells were highly enriched for progenitor activity at a frequency equivalent to purified fractions of CD34+ stem cells, and they were the only subset among the CD34−CD38−Lin− population capable of giving rise to CD34+ cells in defined liquid cultures. Human cells were detected in the bone marrow of non-obese/severe combined immunodeficiency (NOD/SCID) mice 8 weeks after transplantation of ex vivo–cultured AC133+CD7− cells isolated from the CD34−CD38−Lin− population, whereas 400-fold greater numbers of the AC133−CD7− subset had no engraftment ability. These studies provide novel insights into the hierarchical relationship of the human stem cell compartment by identifying a rare population of primitive human CD34− cells that are detectable after transplantation in vivo, enriched for in vitro clonogenic capacity, and capable of differentiation into CD34+ cells.

Donor Origin of Multipotent Adult Progenitor Cells in Radiation Chimeras.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1395-1395
Author(s):  
Morayma Reyes ◽  
Jeffrey S. Chamberlain
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Y Chromosome ◽  
Stromal Cells ◽  
Mononuclear Cells ◽  
Multipotent Adult Progenitor Cells

Abstract Multipotent Adult Progenitor Cells (MAPC) are bone marrow derived stem cells that can be extensively expanded in vitro and can differentiate in vivo and in vitro into cells of all three germinal layers: ectoderm, mesoderm, endoderm. The origin of MAPC within bone marrow (BM) is unknown. MAPC are believed to be derived from the BM stroma compartment as they are isolated within the adherent cell component. Numerous studies of bone marrow chimeras in human and mouse point to a host origin of bone marrow stromal cells, including mesenchymal stem cells. We report here that following syngeneic bone marrow transplants into lethally irradiated C57Bl/6 mice, MAPC are of donor origin. When MAPC were isolated from BM chimeras (n=12, 4–12 weeks post-syngeneic BM transplant from a transgenic mouse ubiquitously expressing GFP), a mixture of large and small GFP-positive and GFP-negative cells were seen early in culture. While the large cells stained positive for stroma cell markers (smooth muscle actin), mesenchymal stem cell makers (CD73, CD105, CD44) or macrophages (CD45, CD14), the small cells were negative for all these markers and after 30 cell doublings, these cells displayed the classical phenotype of MAPC (CD45−,CD105−, CD44−, CD73−, FLK-1+(vascular endothelial growth factor receptor 2, VEGFR2), Sca-1+,CD13+). In a second experiment, BM obtained one month post BM transplant (n=3) was harvested and mononuclear cells were sorted as GFP-positive and GFP-negative cells and were cultured in MAPC expansion medium. MAPC grew from the GFP-positive fraction. These GFP positive cells displayed the typical MAPC-like immunophenotypes, displayed a normal diploid karyotype and were expanded for more than 50 cell doublings and differentiated into endothelial cells, hepatocytes and neurons. To rule out the possibility that MAPC are the product of cell fusion between a host and a donor cell either in vivo or in our in vitro culture conditions, we performed sex mismatched transplants of female GFP donor BM cells into a male host. BM from 5 chimeras were harvested 4 weeks after transplant and MAPC cultures were established. MAPC colonies were then sorted as GFP-positive and GFP- negative and analyzed for the presence of Y-chromosome by FISH analysis. As expected all GFP-negative (host cells) contained the Y-chromosome whereas all GFP-positive cells (donor cells) were negative for the Y-chromosome by FISH. This proves that MAPC are not derived from an in vitro or in vivo fusion event. In a third study, BM mononuclear cells from mice that had been previously BM-transplanted with syngeneic GFP-positive donors (n=3) were transplanted into a second set of syngeneic recipients (n=9). Two months after the second transplant, BM was harvested and mononuclear cells were cultured in MAPC medium. The secondary recipients also contained GFP-positive MAPC. This is the first demonstration that BM transplantation leads to the transfer of cells that upon isolation in vitro generate MAPCs and, whatever the identity of this cell may be, is eliminated by irradiation. We believe this is an important observation as MAPC hold great clinical potential for stem cell and/or gene therapy and, thus, BM transplant may serve as a way to deliver and reconstitute the MAPC population. In addition, this study provides insight into the nature of MAPC. The capacity to be transplantable within unfractionated BM transplant renders a functional and physiological distinction between MAPC and BM stromal cells. This study validates the use of unfractionated BM transplants to study the nature and possible in vivo role of MAPC in the BM.

Partial Characterization and In Vitro Expansion of Putative CLL Precursor/Stem Cells Which Are Dependent on Bone Marrow Microenvironment for Survival

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2433-2433
Author(s):  
Medhat Shehata ◽  
Rainer Hubmann ◽  
Martin Hilgarth ◽  
Susanne Schnabl ◽  
Dita Demirtas ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Healthy Persons

Abstract Abstract 2433 Chronic lymphocytic leukemia (CLL) is characterized by the clonal expansion of B lymphocytes which typically express CD19 and CD5. The disease remains incurable and recurrence often occurs after current standard therapies due to residual disease or probably due to the presence of therapy-resistant CLL precursors. Based on the growing evidence for the existence of leukemia stem cells, this study was designed to search for putative CLL precursors/stem cells based on the co-expression of CLL cell markers (CD19/CD5) with the hematopoietic stem cell marker (CD34). Forty seven CLL patients and 17 healthy persons were enrolled in the study. Twenty four patients had no previous treatment and 23 had pre-therapy. Twenty two patients were in Binet stage C and 25 patients in B. Twenty two patients had unmutated and 18 mutated IgVH gene (7: ND). Cytogenetic analysis by FISH showed that 14 patients had del 13q, 8 had del 11q, 4 had del 17p and 9 had trisomy 12. Peripheral blood and bone marrow mononuclear cells were subjected to multi-colour FACS analysis using anti-human antibodies against CD34, CD19 and CD5 surface antigens. The results revealed the presence of triple positive CD34+/CD19+/CD5+ cells in CLL samples (mean 0.13%; range 0.01–0.41) and in healthy donors (0.31%; range 0.02–0.6) within the CD19+ B cells. However, due to the high leukocyte count in CLL patients, the absolute number of these cells was significantly higher in CLL samples (mean: 78.7; range 2.5–295 cells /μL blood) compared to healthy persons (mean: 0.45: range 0.04–2.5 cells/μl)(p<0,001). These triple positive “putative CLL stem cells” (PCLLSC) co-express CD133 (67%), CD38 (87%), CD127 (52%), CD10 (49%), CD20 (61%), CD23 (96%), CD44 (98%) and CD49d (74%). FISH analysis on 4 patients with documented chromosomal abnormalities detected the corresponding chromosomal aberrations of the mature clone in the sorted CD34+/CD5+/CD19+ and/or CD34+/CD19-/CD5- cells but not in the CD3+ T cells. Multiplex RT-PCR analysis using IgVH family specific primer sets confirmed the clonality of these cells. Morphologically, PCLLSC appeared larger than lymphocytes with narrow cytoplasm and showed polarity and motility in co-culture with human bone marrow stromal cells. Using our co-culture microenvironment model (Shehata et al, Blood 2010), sorted cell fractions (A: CD34+/19+/5+, B: CD34+/19-/5- or C: CD34-/CD19+/5+) from 4 patients were co-cultured with primary autologous human stromal cells. PCLLSC could be expanded in the co-culture to more than 90% purity from fraction A and B but not from fraction C. These cells remained in close contact or migrated through the stromal cells. PCLLSC required the contact with stromal cells for survival and died within 1–3 days in suspension culture suggesting their dependence on bone marrow microenvironment or stem cell niches. RT-PCR demonstrated that these cells belong to the established CLL clone. They also eexpress Pax5, IL-7R, Notch1, Notch2 and PTEN mRNA which are known to play a key role in the early stages of B cells development and might be relevant to the early development of the malignant clone in CLL. Using NOD/SCID/IL2R-gamma-null (NOG) xenogeneic mouse system we co-transplanted CLL cells from 3 patients (5 million PBMC/mouse) together with autologous bone marrow stromal cells (Ratio: 10:1). The percentage of PCLLSC in the transplanted PBMC was 0.18% (range 0.06–0.34%). Using human-specific antibodies, human CD45+ cells were detected in peripharal blood of the mice (mean 0.9 % range 0.47–1.63%) after 2 months of transplantation. More than 90% of the human cells were positive for CD45 and CD5. Among this population, 26% (range 15–35%) of the cells co-expressed CD45, CD19, CD5 and CD34 and thus correspond to the PCLLSC. In conclusion, our data suggest the existence of putative CLL precursors/stem cells which reside within the CD34+ hematopoietic stem cell compartment and carry the chromosomal aberrations of the established CLL clone. These cells could be expanded in vitro in a bone marrow stroma-dependent manner and could be engrafted and significantly enriched in vivo in NOG xenotransplant system. Further characterization and selective targeting and eradication of these cells may pave the way for designing curative therapeutic strategies for CLL. Disclosures: No relevant conflicts of interest to declare.

The Role of Nucleophosmin In Hematopoietic Stem Cells and the Pathogenesis of Myelodysplastic Syndrome

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 95-95 ◽  
Author(s):  
Keisuke Ito ◽  
Paolo Sportoletti ◽  
John G Clohessy ◽  
Grisendi Silvia ◽  
Pier Paolo Pandolfi
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Myelodysplastic Syndrome ◽  
Cell Biology ◽  
De Novo ◽  
Stem Cell Biology ◽  
Hematopoietic Stem

Abstract Abstract 95 Myelodysplastic syndrome (MDS) is an incurable stem cell disorder characterized by ineffective hematopoiesis and an increased risk of leukemia transformation. Nucleophosmin (NPM) is directly implicated in primitive hematopoiesis, the pathogenesis of hematopoietic malignancies and more recently of MDS. However, little is known regarding the molecular role and function of NPM in MDS pathogenesis and in stem cell biology. Here we present data demonstrating that NPM plays a critical role in the maintenance of hematopoietic stem cells (HSCs) and the transformation of MDS into leukemia. NPM is located on chromosome 5q and is frequently lost in therapy-related and de novo MDS. We have previously shown that Npm1 acts as a haploinsufficient tumor suppressor in the hematopoietic compartment and Npm1+/− mice develop a hematologic syndrome with features of human MDS, including increased susceptibility to leukemogenesis. As HSCs have been demonstrated to be the target of the primary neoplastic event in MDS, a functional analysis of the HSC compartment is essential to understand the molecular mechanisms in MDS pathogenesis. However, the role of NPM in adult hematopoiesis remains largely unknown as Npm1-deficiency leads to embryonic lethality. To investigate NPM function in adult hematopoiesis, we have generated conditional knockout mice of Npm1, using the Cre-loxP system. Analysis of Npm1 conditional mutants crossed with Mx1-Cre transgenic mice reveals that Npm1 plays a crucial role in adult hematopoiesis and ablation of Npm1 in adult HSCs leads to aberrant cycling and followed by apoptosis. Analysis of cell cycle status revealed that HSCs are impaired in their ability to maintain quiescence after Npm1-deletion and are rapidly depleted in vivo as well as in vitro. Competitive reconstitution assay revealed that Npm1 acts cell-autonomously to maintain HSCs. Conditional inactivation of Npm1 leads to an MDS phenotype including a profoundly impaired ability to differentiate into cells of the erythroid lineage, megakaryocyte dyspoiesis and centrosome amplification. Furthermore, Npm1 loss evokes a p53-dependent response and Npm1-deleted HSCs undergo apoptosis in vivo and in vitro. Strikingly, transfer of the Npm1 mutation into a p53-null background rescued the apoptosis of Npm1-ablated HSCs and resulted in accelerated transformation to an aggressive and lethal form of acute myeloid leukemia. Our findings highlight the crucial role of NPM in stem cell biology and identify a new mechanism by which MDS can progress to leukemia. This has important therapeutic implications for de novo MDS as well as therapy-related MDS, which is known to rapidly evolve to leukemia with frequent loss or mutation of TRP53. Disclosures: No relevant conflicts of interest to declare.

Novel In Vivo Evidence That Not Only Androgens But Also Pituitary Gonadotropins and Prolactin Directly Stimulate Murine Bone Marrow Stem Cells – Implications For Potential Treatment Strategies In Aplastic Anemias

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2476-2476
Author(s):  
Kasia Mierzejewska ◽  
Ewa Suszynska ◽  
Sylwia Borkowska ◽  
Malwina Suszynska ◽  
Maja Maj ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Sex Hormones ◽  
Peripheral Blood ◽  
Hematopoietic Stem ◽  
Clonogenic Potential

Abstract Background Hematopoietic stem/progenitor cells (HSPCs) are exposed in vivo to several growth factors, cytokines, chemokines, and bioactive lipids in bone marrow (BM) in addition to various sex hormones circulating in peripheral blood (PB). It is known that androgen hormones (e.g., danazol) is employed in the clinic to treat aplastic anemia patients. However, the exact mechanism of action of sex hormones secreted by the pituitary gland or gonads is not well understood. Therefore, we performed a complex series of experiments to address the influence of pregnant mare serum gonadotropin (PMSG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), androgen (danazol) and prolactin (PRL) on murine hematopoiesis. In particular, from a mechanistic view we were interested in whether this effect depends on stimulation of BM-residing stem cells or is mediated through the BM microenvironment. Materials and Methods To address this issue, normal 2-month-old C57Bl6 mice were exposed or not to daily injections of PMSG (10 IU/mice/10 days), LH (5 IU/mice/10 days), FSH (5 IU/mice/10 days), danazol (4 mg/kg/10 days) and PRL (1 mg/day/5days). Subsequently, we evaluated changes in the BM number of Sca-1+Lin–CD45– that are precursors of long term repopulating hematopoietic stem cells (LT-HSCs) (Leukemia 2011;25:1278–1285) and bone forming mesenchymal stem cells (Stem Cell & Dev. 2013;22:622-30) and Sca-1+Lin–CD45+ hematopoietic stem/progenitor cells (HSPC) cells by FACS, the number of clonogenic progenitors from all hematopoietic lineages, and changes in peripheral blood (PB) counts. In some of the experiments, mice were exposed to bromodeoxyuridine (BrdU) to evaluate whether sex hormones affect stem cell cycling. By employing RT-PCR, we also evaluated the expression of cell-surface and intracellular receptors for hormones in purified populations of murine BM stem cells. In parallel, we studied whether stimulation by sex hormones activates major signaling pathways (MAPKp42/44 and AKT) in HSPCs and evaluated the effect of sex hormones on the clonogenic potential of murine CFU-Mix, BFU-E, CFU-GM, and CFU-Meg in vitro. We also sublethally irradiated mice and studied whether administration of sex hormones accelerates recovery of peripheral blood parameters. Finally, we determined the influence of sex hormones on the motility of stem cells in direct chemotaxis assays as well as in direct in vivo stem cell mobilization studies. Results We found that 10-day administration of each of the sex hormones evaluated in this study directly stimulated expansion of HSPCs in BM, as measured by an increase in the number of these cells in BM (∼2–3x), and enhanced BrdU incorporation (the percentage of quiescent BrdU+Sca-1+Lin–CD45– cells increased from ∼2% to ∼15–35% and the percentage of BrdU+Sca-1+Lin–CD45+ cells increased from 24% to 43–58%, Figure 1). These increases paralleled an increase in the number of clonogenic progenitors in BM (∼2–3x). We also observed that murine Sca-1+Lin–CD45– and Sca-1+Lin–CD45+ cells express sex hormone receptors and respond by phosphorylation of MAPKp42/44 and AKT in response to exposure to PSMG, LH, FSH, danazol and PRL. We also observed that administration of sex hormones accelerated the recovery of PB cell counts in sublethally irradiated mice and slightly mobilized HSPCs into PB. Finally, in direct in vitro clonogenic experiments on purified murine SKL cells, we observed a stimulatory effect of sex hormones on clonogenic potential in the order: CFU-Mix > BFU-E > CFU-Meg > CFU-GM. Conclusions Our data indicate for the first time that not only danazol but also several pituitary-secreted sex hormones directly stimulate the expansion of stem cells in BM. This effect seems to be direct, as precursors of LT-HSCs and HSPCs express all the receptors for these hormones and respond to stimulation by phosphorylation of intracellular pathways involved in cell proliferation. These hormones also directly stimulated in vitro proliferation of purified HSPCs. In conclusion, our studies support the possibility that not only danazol but also several other upstream pituitary sex hormones could be employed to treat aplastic disorders and irradiation syndromes. Further dose- and time-optimizing mouse studies and studies with human cells are in progress in our laboratories. Disclosures: No relevant conflicts of interest to declare.

Stem Cell Factor Sall4 Regulates Normal and Malignant Hematopoiesis In an Isoform-Specific Manner

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3728-3728
Author(s):  
Samuel Milanovich ◽  
Jeremy Allred ◽  
Jonathan Peterson ◽  
Cary Stelloh ◽  
Sridhar Rao
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem ◽  
Empty Vector ◽  
Bmi1 Expression ◽  
Colony Forming Units ◽  
Colony Forming Capacity

Abstract Stem cells play key roles in early normal development (e.g. embryonic stem cells (ESCs)), maintenance of adult organs (e.g. hematopoietic stem cells (HSCs)) and in some cancers (e.g. leukemia stem cells). To what degree these different types of stem cells rely upon shared versus distinct transcriptional programs remains controversial. Sall4 is a zinc finger transcription factor that exists in two distinct splice isoforms, Sall4a (long) and Sall4b (short). Sall4 has been implicated in embryonic, hematopoietic and malignant stem cell transcriptional regulation. Additionally, Sall4 has been proposed as a potential means of ex-vivo hematopoietic stem cell expansion prior to transplantation. Sall4 isoform-specific differences have been described in ESCs, with Sall4b shown to be critical for maintaining ESC “stemness”. Here we investigate the role of Sall4 isoforms in pediatric acute myeloid leukemia (AML) and murine hematopoiesis to unravel shared versus unique transcriptional programs across different stem cell types. Quantitative real time PCR shows that Sall4b is the predominant Sall4 isoform in murine HSCs and lin-, Sca1+, cKit+ (LSK) cells. Sall4b expression decreases in early lineage-committed progenitors, while Sall4a expression is minimal to absent across murine HSCs and progenitors. Next, we evaluated seven pediatric AML samples and found highly variable Sall4 expression across AML cases. All samples had measurable Sall4a and Sall4b; in 3/7 cases Sall4a and Sall4b expression was similar to that of ESCs, in the other 4 cases Sall4 expression was minimal (<3% of ESCs). To study overexpression of Sall4, we used a murine stem cell retrovirus system to express Sall4a or Sall4b. Bone marrow was harvested from C57/BL6 mice and lineage-committed cells were removed by magnetic column separation. Lineage-negative bone marrow was infected with either empty vector, Sall4a or Sall4b. Transduced bone marrow was then cultured in methylcellulose media to assess colony forming capacity and proliferation in vitro or transplanted in syngeneic mice to assess engraftment and hematopoietic reconstitution in vivo. Sall4a or Sall4b overexpression caused diminished colony forming capacity and cellular proliferation in vitro compared to bone marrow transduced with empty vector (Figure 1). In bone marrow transplant assays, all mice (4/4) transplanted with Sall4b-transduced bone marrow following lethal irradiation succumbed to bone marrow failure within 10 days of transplant. Transplantation of Sall4b-transduced bone marrow into sublethally irradiated mice failed to contribute to hematopoiesis as measured by peripheral blood leukocyte GFP expression (encoded by the viral vector). Together, this data shows that Sall4b-transduced hematopoietic cells fail to engraft and reconstitute hematopoiesis in vivo. We postulated that this phenotype might be mediated through the interaction of Sall4 with Bmi1. Bmi1 is a member of the polycomb complex necessary for normal hematopoiesis, and is known to be bound by Sall4. In preliminary experiments, we have found that overexpression of Sall4 leads to decreased Bmi1 expression at 48 hours post-infection compared to bone marrow infected with empty vector.Figure 1Lin- bone marrow expressing Sall4a, Sall4b or empty vector was cultured in methylcellulose; plates were flushed and replated out to three generations. Colony forming units were assessed (A) and viable cells were counted (B) after 7-10 days in culture.Figure 1. Lin- bone marrow expressing Sall4a, Sall4b or empty vector was cultured in methylcellulose; plates were flushed and replated out to three generations. Colony forming units were assessed (A) and viable cells were counted (B) after 7-10 days in culture. In conclusion, our data shows that Sall4b is expressed in murine hematopoietic stem cells and progenitors, suggesting that Sall4b but not Sall4a influences a hematopoietic cell fate. Additionally, Sall4 expression is variable in AML specimens, implicating a potential pathogenic role in some leukemias, while others are Sall4-independent. Lastly, Sall4 overexpression is associated with decreased expression of the critical hematopoietic gene Bmi1. Together this data suggests that hematopoiesis is dependent upon appropriately regulated Sall4 expression with alterations leading to impaired proliferation and self-renewal. These effects on hematopoiesis appear to be mediated at least in part through a dose-dependent effect on Bmi1 expression. Future studies will evaluate other genes targeted by Sall4 in hematopoiesis and leukemia to define Sall4-dependent gene signatures in normal versus malignant hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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