New Role of the Regulatory Gene SOX2 in Hematopoiesis.

Abstract Several genes encoding transcription factors of different families have been implicated in the development and differentiation of multiple cell systems. The Sry-type high-mobility-group box 2 gene (Sox2) encodes a transcription factor that is expressed in very early cells such as embryonic stem cells and neural stem cells, where it plays important functional roles (Genes and Dev.17:126, 2003; Development131:3805, 2004). To investigate whether Sox2 plays a role also in blood cell production, we first analyzed its expression in murine hematopoietic cells. Results indicate that the gene is transcriptionally active at low levels in primitive progenitors. Furthermore, in order to address the functional implication of Sox2 in hematopoiesis we analyzed mature and precursor cells in mutant mice compound heterozygotes for a null Sox2 allele and for the deletion of a Sox2 5′ enhancer, as the complete inactivation of the gene in homozygosis is embryonic lethal. At the peripheral blood level we did not detect significant variations in the mutants. However analysis of bone marrow precursors in clonogenic assays showed that Sox2 knock-down mice exhibited a significant increase in the number of multipotent precursors, as compared to wild type animals. Moreover, bone marrow cells of wild type and mutant mice were analyzed for the expression of a panel of regulatory genes involved in the control of different somatic stem cells. Preliminary evidence suggests that some of these genes are modulated in the mutant cells. These observations support the view that Sox2 plays a role at early stages of blood cell production, providing further evidence that common molecular mechanisms may be involved in the regulation of several different types of multipotent cells.

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Enforced Expression of the GATA-2 Transcription Factor Blocks Normal Hematopoiesis

Blood ◽

10.1182/blood.v93.2.488 ◽

1999 ◽

Vol 93 (2) ◽

pp. 488-499 ◽

Cited By ~ 157

Author(s):

Derek A. Persons ◽

James A. Allay ◽

Esther R. Allay ◽

Richard A. Ashmun ◽

Donald Orlic ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Transcription Factor ◽

Blood Cell ◽

Fluorescent Protein ◽

Bone Marrow Cells ◽

Hematopoietic Cells ◽

Immunoblot Analysis ◽

Marrow Cells

Abstract The zinc finger transcription factor GATA-2 is highly expressed in immature hematopoietic cells and declines with blood cell maturation. To investigate its role in normal adult hematopoiesis, a bicistronic retroviral vector encoding GATA-2 and the green fluorescent protein (GFP) was used to maintain the high levels of GATA-2 that are normally present in primitive hematopoietic cells. Coexpression of the GFP marker facilitated identification and quantitation of vector-expressing cells. Bone marrow cells transduced with the GATA-2 vector expressed GFP as judged by flow cytometry and GATA-2 as assessed by immunoblot analysis. A 50% to 80% reduction in hematopoietic progenitor-derived colony formation was observed with GATA-2/GFP-transduced marrow, compared with marrow transduced with a GFP-containing vector lacking the GATA-2 cDNA. Culture of purified populations of GATA-2/GFP-expressing and nonexpressing cells confirmed a specific ablation of the colony-forming ability of GATA-2/GFP-expressing progenitor cells. Similarly, loss of spleen colony-forming ability was observed for GATA-2/GFP-expressing bone marrow cells. Despite enforced GATA-2 expression, marrow cells remained viable and were negative in assays to evaluate apoptosis. Although efficient transduction of primitive Sca-1+Lin- cells was observed with the GATA-2/GFP vector, GATA-2/GFP-expressing stem cells failed to substantially contribute to the multilineage hematopoietic reconstitution of transplanted mice. Additionally, mice transplanted with purified, GATA-2/GFP-expressing cells showed post-transplant cytopenias and decreased numbers of total and gene-modified bone marrow Sca-1+ Lin−cells. Although Sca-1+ Lin− bone marrow cells expressing the GATA-2/GFP vector were detected after transplantation, no appreciable expansion in their numbers occurred. In contrast, control GFP-expressing Sca-1+Lin− cells expanded at least 40-fold after transplantation. Thus, enforced expression of GATA-2 in pluripotent hematopoietic cells blocked both their amplification and differentiation. There appears to be a critical dose-dependent effect of GATA-2 on blood cell differentiation in that downregulation of GATA-2 expression is necessary for stem cells to contribute to hematopoiesis in vivo.

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

Blood ◽

10.1182/blood.v116.21.1577.1577 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1577-1577

Author(s):

Yaoyu Chen ◽

Sullivan Con ◽

Yiguo Hu ◽

Linghong Kong ◽

Cong Peng ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Flow Cytometry ◽

Stem Cell ◽

Bone Marrow Cells ◽

Leukemic Stem Cells ◽

Wild Type ◽

Post Transplant ◽

Marrow Cells

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

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ФЕНОТИПОВІ ТА МОРФОЛОГІЧНІ ЗМІНИ КУЛЬТУРИ КЛІТИН КІСТКОВОГО МОЗКУ ЩУРІВ В ПРОЦЕСІ ЇХ КУЛЬТИВУВАННЯ

Scientific Messenger of LNU of Veterinary Medicine and Biotechnology ◽

10.15421/nvlvet6626 ◽

2016 ◽

Vol 18 (2(66)) ◽

pp. 126-132

Author(s):

A.I. Mazurkiewicz ◽

V.V. Kovpak ◽

O.S. Kovpak

Keyword(s):

Stem Cells ◽

Cell Culture ◽

Bone Marrow ◽

Bone Marrow Cells ◽

Morphological Changes ◽

Embryonic Stem ◽

In Vitro Cultivation ◽

Rat Bone ◽

Marrow Cells

Bone marrow is the only adult tissue which normally consists of immature undifferentiated and low differentiated cells which called stem cells and they are similar in structure to embryonic stem cells. But literature data analysis doesn't give an unambiguous answer regarding phenotypic and morphological changes of bone marrow cells culture of rats during their in vitro cultivation which necessitated further research.Investigate phenotypic and morphological changes of bone marrow cells culture of rats during their in vitro cultivation from first to fourth passage.We were used in these research bone marrow cells of rats from the first to the fourth passages. Microscopic analysis and evaluation morphological changes of bone marrow cells culture of rats during cultivation were carried out using inverted microscope Axiovert 40. Control of changes phenotype was performed by detecting CD markers (CD10, CD38, CD34, CD45, CD48, CD54, CD56, CD66e, CD96, CD227, CD326, pan–keratin). The evaluation was performed by the semi– quantitative method (H–Score).The research of primary culture of rat bone marrow cells showed that it morphologically heterogeneous, noted the small number of cells polygonal shape, surrounded by the fibroblast cells. During the cultivation cell culture becomes more homogenous at the expense of fibroblast–like cells. As a result of occurred the transition process from heterogeneous culture in zero passage to the most homogeneous culture in 4 passage. Immunophenotyping population of cell culture derived from rat bone marrow, revealed a high level of expression of pan–keratin; moderate level – CD34, CD48, CD66e, CD95; low level – CD38, CD45, CD56, CD227, CD326; lack of expression – CD10, CD54. Change of the expression of surface markers varies in each passage CD48, CD66e, CD95 increased significantly; CD38, SD45, SD326, pan–keratin reduced significantly. The markers CD34, CD 56, CD 227 were expressed on the one level from the first to the fourth passage. The expression of the CD10, CD54 markers during the study period was not identified.

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Hematopoietic Stem Cell Engraftment in Bone Marrow Is Dependent upon Gsα.

Blood ◽

10.1182/blood.v108.11.857.857 ◽

2006 ◽

Vol 108 (11) ◽

pp. 857-857

Author(s):

Gregor B. Adams ◽

Ian R. Alley ◽

Karissa T. Chabner ◽

Ung-il Chung ◽

Emily S. Marsters ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Bone Marrow Cells ◽

Fetal Liver ◽

Conditional Knockout ◽

Wild Type ◽

Hematopoietic Stem ◽

Cell Engraftment

Abstract During development, hematopoietic stem cells (HSCs) translocate from the fetal liver to the bone marrow, which remains the site of hematopoiesis throughout adulthood. In the bone marrow the HSCs are located at the endosteal surface, where the osteoblasts are a key component of the stem cell niche. The exogenous signals that specifically direct HSCs to the bone marrow have been thought to include stimulation of the chemokine receptor CXCR4 by its cognate ligand stromal derived factor-1α (SDF-1α or CXCL12). However, experiments in which CXCR4−/− fetal liver hematopoietic cells were transplanted into wild-type hosts demonstrated efficient engraftment of the HSCs in the bone marrow. In addition, treatment of HSCs with inhibitors of Gαi-coupled signaling, which blocks transmigration towards SDF-1αin vitro, does not affect bone marrow homing and engraftment in vivo. Therefore, we examined whether Gsα-coupled mechanisms play a key role in the engraftment of the HSCs in the bone marrow environment. Utilizing an inducible-conditional knockout of Gsα, we found that deletion of the gene in hematopoietic bone marrow cells did not affect their ability to perform in the in vitro primitive CFU-C or LTC-IC assay systems. However, Gsα−/− cells were unable to establish effective hematopoiesis in the bone marrow microenvironment in vivo in a competitive repopulation assay (41.1% contribution from wild-type cells versus 1.4% from knockout cells). These effects were not due to an inability of the cells to function in the bone marrow in vivo as deletion of Gsα following establishment of hematopoiesis had no effects on the HSCs. Examining the ability of the HSCs to home to the bone marrow, though, demonstrated that deletion of Gsα resulted in a marked impairment of the ability of the stem cells to localize to the marrow space (approximately 9-fold reduction in the level of primitive cell homing). Furthermore, treatment of BM MNCs with an activator of Gsα augmented the cells homing and thus engraftment potential. These studies demonstrate that Gsα is critical to the localization of HSCs to the bone marrow. Which receptors utilize this pathway in this context remains unknown. However, Gsα represents a previously unrecognized signaling pathway for homing and engraftment of HSCs to bone marrow. Pharmacologic activation of Gsα in HSC ex vivo prior to transplantation offers a potential method for enhancing stem cell engraftment efficiency.

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Loss of Gfi1 Impedes Development of T-Cell Lymphoma upon Exposure to N-ethyl-N-nitrosourea but Predisposes to Severe Myleodysplastic Changes.

Blood ◽

10.1182/blood.v110.11.2221.2221 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2221-2221

Author(s):

Cyrus Khandanpour ◽

Ulrich Duehrsen ◽

Tarik Möröy

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Dna Damage ◽

T Cell ◽

Bone Marrow Cells ◽

Cell Lymphoma ◽

Wild Type ◽

Hematopoietic Stem ◽

Deficient Mice

Abstract Exogenous toxic substances often cause the initiation and development of leukemia and lymphoma by acting as mutagens. N-ethyl-N-nitrosourea (ENU) is a paradigmatic example for such a substance, which introduces point mutations in the genome through DNA damage and repair pathways. ENU is widely used to experimentally induce T-cell lymphomas in mice. We have used ENU to investigate whether the hematopoietic transcription factor Gfi1 is required for lymphomagenesis. The Gfi1 gene was originally discovered as a proviral target gene and a series of experiments with transgenic mice had suggested a role of Gfi1 as a dominant oncogene with the ability to cooperate with Myc and Pim genes in the generation of T-cell lymphoma. In addition, Gfi1 deficient mice showed a defect in T-cell maturation but also aberration in myeloid differentiation and an accumulation of myelomonocytic cells. ENU was administered i.p. once a week for three weeks with a total dose of 300mg/kg to wild type (wt) and Gfi1 null mice. Wild type mice (12/12) predominantly developed T-cell tumors and rarely acute myeloid leukemia, as expected. However, only 2/8 Gfi1 −/− mice succumbed to lymphoid neoplasia; they rather showed a severe dysplasia of the bone marrow that was more pronounced than in wt controls. These changes in Gfi1 null mice were accompanied by a dramatic decrease of the LSK (Lin-, Sca1- and c-Kit+) bone marrow fraction that contains hematopoietic stem cells and by a higher percentage (18%) of bone marrow cells, not expressing any lineage markers (CD4, CD 8, Ter 119, Mac1, Gr1, B220, CD3). In particular, we found that the LSK subpopulation of Gfi1 deficient mice showed a noticeable increase in cells undergoing apoptosis suggesting a role of Gfi1 in hematopoietic stem cell survival. In addition, Gfi1−/− bone marrow cells and thymic T-cells were more sensitive to DNA damage such as radiation and exposure to ENU than their wt counterparts pointing to a role of Gfi1 in DNA damage response. Our results indicate that Gfi1 is required for development of T-cell tumors and that a loss of Gfi1 may sensitize hematopoietic cells and possibly hematopoietic stem cells for programmed cell death. Further studies have to show whether interfering with Gfi1 expression or function might represent a tool in the therapy of leukemia.

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The Tumor Suppressor Role of the Msr1 Gene in Cancer Stem Cells of Chronic Myeloid Leukemia.

Blood ◽

10.1182/blood.v114.22.188.188 ◽

2009 ◽

Vol 114 (22) ◽

pp. 188-188

Author(s):

Yaoyu Chen ◽

Con Sullivan ◽

Shaoguang Li

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Chronic Myeloid Leukemia ◽

Tumor Suppressor ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Leukemia Cells ◽

Wild Type ◽

Marrow Cells

Abstract Abstract 188 We have previously shown that the arachidonate 5-lipoxygenase gene (Alox5) functions as a critical regulator of leukemia stem cells (LSCs) in BCR-ABL-induced chronic myeloid leukemia (CML) in mice (Chen Y, Hu Y, Zhang H, Peng C, Li S. Loss of the Alox5 gene impairs leukemia stem cells and prevents chronic myeloid leukemia. Nature Genetics 41:783-792, 2009). We believe that the Alox5 pathway represents a major molecular network in LSCs. Therefore, we decided to further dissect this pathway by comparing gene expression profiles between wild type and Alox5−/− LSCs from CML mice using the DNA microarray analysis. We identified a small group of candidate genes that were changed in expression in the absence of Alox5. Among these genes, we have identified the Msr1 gene and chosen to test the function of this gene in regulating LSC function, because this gene was up-regulated, indicating that it might play a tumor suppressor role in LSCs. In our CML mouse model, we observed that recipients of BCR-ABL transduced Msr1−/− bone marrow cells developed CML much rapidly than recipients of BCR-ABL transduced wide type bone marrow cells. To test whether this accelerated CML is related to abnormal function of LSCs, we carried out a serial transplantation assay by transferring bone marrow cells from primary recipients of BCR-ABL-transduced wild type or Msr1−/− donor bone marrow cells into secondary and next-generation of recipient mice to biologically assess the effect of Msr1 on LSCs. BCR-ABL-expressing wild type leukemia cells from bone marrow of CML mice were only able to transfer CML once, whereas BCR-ABL-expressing Msr1−/− leukemia cells were able to transfer lethal CML for five genrations. This observation indicates that BCR-ABL-expressing Msr1−/− LSCs have markedly increased stem cell function. To further compare the stem cell function, we performed the leukemia stem cell competition assay by 1:1 mixing wild type (CD45.1) and Msr1−/− (CD45.2) bone marrow cells from CML mice. At day 25 or 30 after transplantation, more than 60% and 95% of GFP+Gr-1+ cells in peripheral blood of the mice were CD45.2+Msr1−/− myeloid leukemia cells, and all these mice developed CML and died of CML derived from Msr1−/− LSCs. To confirm the tumor suppressor role of Msr1 in CML development, we co-expressed BCR-ABL and Msr1 in MSR1−/− bone marrow cells by retroviral transduction, followed by transplantation of these cells into recipient mice. The ectopically-expressed Msr1 in MSR1−/− bone marrow cells rescued the accelerated CML phenotype, and some recipient mice did not even develop the CML. Together, these results demonstrate that Msr1 plays a tumor suppressor role in LSCs. The Msr1 pathway is a novel molecular network in LSCs, and it will be important to fully study this pathway for developing curative therapeutic strategies for CML. Disclosures: No relevant conflicts of interest to declare.

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Nrf2, a Critical Regulator of Oxidative Stress, Is Required for HSC Function and Cytokine Response.

Blood ◽

10.1182/blood.v114.22.1492.1492 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1492-1492

Author(s):

Akil Merchant ◽

Anju Singh ◽

Giselle Joseph ◽

Qiuju Wang ◽

Ping Zhang ◽

...

Keyword(s):

Oxidative Stress ◽

Stem Cells ◽

Bone Marrow ◽

Transcription Factor ◽

Peripheral Blood ◽

Bone Marrow Cells ◽

Cytokine Signaling ◽

Wild Type ◽

Increased Sensitivity ◽

Marrow Cells

Abstract Abstract 1492 Poster Board I-515 Previous studies have established an important role for reactive oxygen species (ROS) in regulating the function and life-span of hematopoietic stem cells (HSC). Nuclear factor erythroid-2–related factor 2 (Nrf2) is a redox-sensitive transcription factor that regulates cellular responses to ROS and detoxification pathways implicated in chemoresistance, however, its role in normal stem cells is unknown. We analyzed Nrf2null mice and found increased total bone marrow cellularity, cKit+Sca1+Lin− (KSL) stem-progenitor cells, and long-term quiescent HSC (CD34−KSL) compared to wild type mice (p<0.05). Transplantation of equal numbers of KSL cells from Nrf2wt and Nrf2null resulted in a five-fold decrease in peripheral blood chimerism from Nrf2null derived cells at 16 weeks (15% wild type vs. 3% null, p<0.05). Unlike other models of deficiencies in genes associated with ROS handling, such as ATM or the FoxO family of transcription factors, basal ROS levels were not elevated in Nrf2null HSC. However, Nrf2null bone marrow cells demonstrated increased sensitivity to induced oxidative stress and in vitro treatment with H2O2 resulted in a 2 fold decrease in colony formation in methylcellulose. We also examined the in vivo sensitivity of Nrf2null cells to oxidative stress by irradiating (400 rads) stably chimeric mice 20 weeks following transplantation with either Nrf2wt or Nrf2null HSC. Mice receiving Nrf2null HSC demonstrated a 50% decrease in peripheral blood chimerism at 4 months following radiation compared to no change in Nrf2wt recipients (p<0.05) confirming that loss of Nrf2 leads to increased sensitivity to oxidative stress. Microarray gene expression analysis from Nrf2wt and Nrf2null mice revealed down regulation of the G-CSF cytokine receptor in Nrf2null HSC and suggested that defective cytokine signaling may contribute to the HSC dysfunction seen in Nrf2null bone marrow cells. To test this hypothesis, we attempted to rescue the function of Nrf2null HSC by treating mice with exogenous G-CSF. Nrf2wt and Nrf2null mice were treated with one week of daily G-CSF and then HSC were harvested and transplanted. In contrast to the defects in engraftment of untreated Nrf2null HSC, there was no significant difference in peripheral blood chimerism following transplantation of G-CSF treated Nrf2wt or Nrf2null HSC, thus demonstrating that G-CSF treatment could rescue the HSC defect in mutant mice. In conclusion, the Nrf2 transcription factor appears to be a novel and essential regulator of normal HSC function through the modulation of oxidative stress response and cytokine signaling. Disclosures: No relevant conflicts of interest to declare.

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Roles of Histone Acetyltransferase MORF and MOZ in Hematopoiesis.

Blood ◽

10.1182/blood.v114.22.2525.2525 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2525-2525

Author(s):

Takuo Katsumoto ◽

Issay Kitabayashi

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Fetal Liver ◽

Liver Cells ◽

Wild Type ◽

Self Renewal ◽

Hematopoietic Stem ◽

Fetal Liver Cells

Abstract Abstract 2525 Poster Board II-502 MOZ (MOnocytic leukemia Zinc finger protein) and MORF (MOz Related Factor), Myst-type histone acetyltransferases, are involved in chromosome translocations associated with FAB-M4/5 subtypes of acute myeloid leukemia. We have reported that MOZ is essential for hematopoietic cell development and self-renewal of hematopoietic stem cells. To explore the possibility MORF also plays important roles in hematopoiesis, we generated Morf-deficient mice with homologous recombination methods. Morf−/− mice were smaller than their wildtype littermates and died within 4 weeks after birth on C57BL/6 background. In MORF−/− fetal liver, Flt3-negative KSL (c-Kit+ Sca-1+ Lineage-) cells containing hematopoietic stem cells were decreased. When fetal liver cells were transplanted into irradiated recipient mice, MORF−/− cells less efficiently reconstituted hematopoiesis than wild-type cells. Additionally, bone marrow cells reconstituted with MORF−/− cells rarely contributed to hematopoiesis in secondary transplants. To reveal relationship between MORF and MOZ in hematopoiesis, we generated double heterozygous (Moz+/− Morf+/−) mouse. Double heterozygous mice were smaller than wild-type littermates and died at least 4 weeks after birth. Numbers of KSL cells, especially Flt3- KSL cells and common myeloid progenitors were decreased in the double heterozygous embryos. The double heterozygous fetal liver cells also displayed less activity to reconstitute hematopoiesis than MOZ+/− or MORF+/− cells. Since MORF−/− mice and MOZ/MORF double heterozygous mice were alive at adult on a mixed C57BL/6/DBA2 genetic background, we investigated adult hematopoiesis in these mice. MORF−/− or MOZ/MORF double heterozygous mice were smaller than their wild-type littermates and had small numbers of thymocytes and splenocytes. However, there were no significant differences in number of bone marrow cells and hematopoietic lineage population in MORF−/− or MOZ/MORF double heterozygous mice. These results suggest that MORF as well as MOZ plays important roles in self-renewal of hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.

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Runx1 Haploinsufficiency Permits Lineage-Commitment but Impairs Activation of a Key Late Differentiation Gene

Blood ◽

10.1182/blood.v118.21.2442.2442 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2442-2442

Author(s):

Zhenbo Hu ◽

Kwok Peng Ng ◽

Quteba Ebrahem ◽

Yogen Saunthararajah

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Bone Marrow Cells ◽

Cpg Methylation ◽

Lineage Commitment ◽

Wild Type ◽

Differentiation Gene ◽

Wild Type Control ◽

Marrow Cells

Abstract Abstract 2442 Inactivating or dominant-negative RUNX1 mutations are a frequent first-hit in the multi-hit process of leukemogenesis. First-hits that target RUNX1 occur in stem cells, or in familial acute myeloid leukemia (AML), in the germ-line. However, self-renewing AML cells (leukemia-initiating cells, LIC) frequently display surface markers of lineage-committment, and AML cells appear lineage-committed in critical aspects, expressing high levels of key lineage-specifying transcription factors (TF) such as CEBPA with promoter CpG methylation patterns resembling mature hematopoietic cells (HSC) (Negrotto et al, Leukemia 2011). To investigate a possible basis for differentiation advancement of LIC from stem cell origins, hematopoietic stem cells from wild-type and Runx1 haploinsufficient (+/−) mice were cultured with granulocyte-colony stimulating factor (G-CSF). Repression of Hoxb4 (a stem cell associated factor) and activation of Cebpa by G-CSF was similar in wild-type and Runx1+/− lineage-negative hematopoietic precursor cells. However, the expression of Cebpe, a key late-differentiation driving TF upregulated with the transition from proliferating pro-myelocytes to non-proliferating myelocytes, and which terminates proliferation in myeloid and AML cells, was significantly decreased in the Runx1+/− cells (>2-fold, t-test p<0.01), even after 15 days of culture with G-CSF. This pattern of Cebpa and Cebpe expression was also documented at the protein level. High CEBPA, but relatively low HOXB4 and CEBPE expression, is also characteristic of human primary AML cells, including CD34+ subsets (Negrotto et al, Leukemia 2011) and leukemia initiating cells (analysis of Geo Database GSE24006 and GSE17054). Indicating epigenetic repression of Cebpe, methylation of CpG in the Cebpe promoter was significantly increased in Runx1+/− compared to wild-type cells (p<0.05). Consistent with partial maturation of Runx1+/− cells, expression of the granulocyte-lineage markers Ly6G and CD11b increased during culture with G-CSF, but to a much lesser extent than in wild-type control (30 v 40% and 11 v 30% respectively). After 15 days of culture with G-CSF, in morphological analyses, Runx1+/− cells included granulocytes (with less neutrophilic granulation than observed in wild-type), some cells with high nuclear-cytoplasmic ratio, and mitotic figures, in comparison to mostly mature granulocytes in wild-type control. Repression of Cebpe might confer a growth advantage after lineage-commitment. In liquid culture with and without G-CSF, Runx1+/− cells demonstrated more rapid and persistent proliferation than wild-type control (cumulative increase in cell numbers >2-fold, p<0.001), with this advantage more prominent in the presence of G-CSF. In semi-solid media supplemented with G-CSF, Runx1+/− lineage-negative cells produced a greater number and larger-sized colonies than wild-type control. CEBPE promoter CpG that become less methylated during G-CSF-induced differentiation of normal human CD34+ precursors into granulocytes were identified by mass-spectrometry. These CpG were in proximity to RUNX1 and CEBPA consensus binding sequences. Two of the three CpG sites were significantly hypermethylated in AML (n=27) compared to normal (n=11) and/or remission bone marrow cells (n=6) (1.5 to >2-fold, p<0.01, Wilcoxon test). In contrast to the CEBPE promoter CpG, methylation levels at LINE-1 repetitive DNA element CpGs were similar in normal, remission, and AML bone marrow cells. In conclusion, Runx1+/− abnormality present in stem cells is permissive of lineage-commitment but represses a key late-differentiation gene, hence conferring a proliferative advantage to lineage-committed daughter cells. This mechanism could explain differentiation advancement of LIC from stem cell origins, and likely contributes to the terminal maturation observed upon treatment of AML cells (including LIC) with corepressor antagonists (histone deacetylase and DNMT1 inhibitors). In contrast, treatment of normal HSC with these drugs has the opposite effect, maintaining self-renewal, possibly by preventing repression of stem cell genes by differentiation stimuli (Hu et al, Mol Ca Ther 2010). Hence, the difference in maturation level of LIC and normal HSC can potentially be exploited for AML selective therapy. Disclosures: No relevant conflicts of interest to declare.

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Functional Hematopoietic Cells Derived From Mouse Embryonic Stem Cells.

Blood ◽

10.1182/blood.v120.21.2304.2304 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2304-2304

Author(s):

Cheng Li ◽

Daniel R. George ◽

Nichole M. Havey ◽

Jeffery M. Klco ◽

Timothy J. Ley

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Peripheral Blood ◽

Bone Marrow Cells ◽

Embryonic Stem ◽

Post Injection ◽

Spleen Cells ◽

Short Term ◽

Marrow Cells

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

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