scholarly journals The identification in adult bone marrow of pluripotent and restricted stem cells of the myeloid and lymphoid systems

1977 ◽  
Vol 145 (6) ◽  
pp. 1567-1579 ◽  
Author(s):  
S Abramson ◽  
RG Miller ◽  
RA Phillips
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
T Lymphocytes ◽  
Chromosome Aberrations ◽  
Bone Marrow Cells ◽  
Hemopoietic Stem Cell ◽  
Lymphoid System ◽  
Unique Chromosome ◽  
Marrow Cells

The precise relationship between the stem cells for the lymphoid system and those for the blood-forming system is unclear. While it is generally assumed that the hemopoietic stem cell, the spleen colony-forming unit (CFU-S), is also the stem cell for the lymphoid system, there is little evidence for this hypothesis. To investigate the stem cells in these two systems, we irradiated bone marrow cells to induce unique chromosome aberrations in the stem cell population and injected them at limiting dilution into stem cell-deficient recipients. Several months (between 3 and 11) were allowed for the injected cells to repopulate the hemopoietic system. At that time, the bone marrow, spleen, and thymus were examined for a high frequency of cells having the same unique chromosome aberration. The presence of such markers shows that the marker was induced in a cell with extensive proliferative capacity, i.e., a stem cell. In addition, the splenic lymphocytes were stimulated with phytohemagglutinin (PHA) or lipopolysaccharide (LPS) to search for unique chromosomes in dividing T and B cells, respectively. Finally, bone marrow cells were injected into secondary irradiated recipients to determine if the marker occurred in CFU-S and to determine whether or not the same tissue distributions of marked cells could be propogated by bone marrow cells in a second recipient. After examination of 28 primary recipients, it was possible to identify three unique patterns of stem cell regeneration. In one set of mice, a unique chromosome marker was observed in CFU-S and in PHA- and LPS-stimulated cultures. These mice provide direct evidence for a pluripotent stem cell in bone marrow. In addition, two restricted stem cells were identified by this analysis. In three recipients, abnormal karyotypes were found only in myeloid cells and not in B and T lymphocytes. These mice presumably received a marked stem cell restricted to differentiate only into myeloid progeny. In three other recipients, chromosome aberrations were found only in PHA-stimulated cells; CFU-S and cells from LPS cultures did not have cells with the unique chromosome. This pattern suggests that bone marrow contains cells committed to differentiation only into T lymphocytes. For each of the three types of stem cells, secondary recipients had the same cellular distribution of marked cells as the primary recipients. This observation provides further evidence that unique markers can be induced in both pluripotent and restricted stem cells.

scholarly journals Demonstration of Thy-1 antigen on pluripotent hemopoietic stem cells in the rat.

1978 ◽  
Vol 148 (5) ◽  
pp. 1351-1366 ◽  
Author(s):  
I Goldschneider ◽  
L K Gordon ◽  
R J Morris
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Cell Activity ◽  
Hemopoietic Stem Cell ◽  
Hemopoietic Stem Cells ◽  
Stem Cell Activity ◽  
Series Of Experiments ◽  
Marrow Cells

Three approaches were used to demonstrate the presence of Thy-1 antigen on the surface of pluripotent hemopoietic stem cells in the rat. In the first, stem cells from fetal liver, neonatal spleen, and adult bone marrow were prevented from forming hemopoietic colonies in the spleens of irradiated recipients spleen (colony-forming unit assay) by incubation with antibodies to Thy-1 antigen. Highly specific rabbit heteroantiserum to purified rat brain Thy-1 antigen and mouse alloantisera to Thy-1.1-positive thymocytes were equally effective. This inhibition was neutralized by purified Thy-1 antigen. In a second series of experiments, Thy-1-positive and Thy-1-negative populations of nucleated bone marrow cells were separated by the FACS. All of the hemopoietic stem cell activity was recovered in the Thy-1-positive population. The stem cells were among the most strongly positive for Thy-1 antigen, being in the upper 25th percentile for relative fluorescence intensity. The relationships of Thy-1 antigen to the rat bone marrow lymphocyte antigen (BMLA) was shown in a third series of experiments. Rabbit anti-BMLA serum, which is raised against a null population of lymphocyte-like bone marrow cells, has been shown to have anti-stem cell activity. Here we demonstrate by double immunofluorescence, cocapping, and differential absorption studies that Thy-1 and BMLA are parts of the same molecule.

scholarly journals THE EFFECT OF HETEROLOGOUS ANTI-LYMPHOCYTE SERUM ON MOUSE HEMOPOIETIC STEM CELLS

1968 ◽  
Vol 127 (4) ◽  
pp. 731-748 ◽  
Author(s):  
Tom R. DeMeester ◽  
Norman D. Anderson ◽  
Charles F. Shaffer
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Cell Function ◽  
Bone Marrow Cells ◽  
Hemopoietic Stem Cell ◽  
Hemopoietic Stem Cells ◽  
Marrow Cells

The present study has demonstrated that rabbit anti-mouse lymphocyte serum (RAMLS) has the capability of destroying bone marrow cells and suppressing hemopoietic stem cell function. The in vitro incubation of bone marrow suspensions with RAMLS caused extensive cell lysis with an apparent preferential destruction of lymphoid, erythroid, and blastoid elements. Using the spleen colony assay, the number of functional hemopoietic stem cells was found to be markedly reduced in bone marrow populations exposed to RAMLS in vitro. Further, this loss of stem cell function could be produced by exposing marrow suspensions to small concentrations of antiserum which did not produce detectable cytotoxic effects on the general marrow population. A similar effect of RAMLS upon hemopoietic stem cells was found in vivo. The intravenous injection of RAMLS into lethally irradiated mice immediately after the infusion of isogeneic marrow cells reduced the number of spleen colonies formed, indicating that the antiserum could exhibit a deleterious effect upon stem cells in the bloodstream of the intact animal. Normal animals treated with daily subcutaneous injections of RAMLS for 3 wk had a significantly reduced marrow content of functional hemopoietic stem cells, suggesting that RAMLS can affect stem cells located in situ in the bone marrow. The experiments indicate that RAMLS possesses potential marrow toxicity.

scholarly journals Long-term monoclonal reconstitution of erythropoiesis in genetically anemic W/Wv mice by injection of 5-fluorouracil-treated bone marrow cells of Pgk-1b/Pgk-1a mice

Blood ◽  
1987 ◽  
Vol 70 (6) ◽  
pp. 1758-1763 ◽  
Author(s):  
T Nakano ◽  
N Waki ◽  
H Asai ◽  
Y Kitamura
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Electrophoretic Pattern ◽  
Phosphoglycerate Kinase ◽  
X Chromosomes ◽  
Hematopoietic Stem ◽  
Colony Forming Units ◽  
Marrow Cells

Abstract The spleen colony-forming assay does not represent the number of hematopoietic stem cells with extensive self-maintaining capacity because five to 50 spleen colony-forming units (CFU-S) are necessary to rescue a genetically anemic (WB X C57BL/6)F1-W/Wv(WBB6F1-W/Wv) mouse. We investigated which is more important for the reconstitution of erythropoiesis, the transplantation of multiple CFU-S or that of a single stem cell with extensive self-maintaining potential. The electrophoretic pattern of hemoglobin was used as a marker of reconstitution and that of phosphoglycerate kinase (PGK), an X chromosome-linked enzyme, as a tool for estimating the number of stem cells. For this purpose, we developed the C57BL/6 congeneic strain with the Pgk-1a gene. Bone marrow cells were harvested after injection of 5- fluorouracil from C57BL/6-Pgk-1b/Pgk-1a female mice in which each stem cell had either A-type PGK or B-type PGK due to the random inactivation of one or two X chromosomes. When a relatively small number of bone marrow cells (ie, 10(3) or 3 X 10(3] were injected into 200-rad- irradiated WBB6F1-W/Wv mice, the hemoglobin pattern changed from the recipient type (Hbbd/Hbbs) to the donor type (Hbbs/Hbbs) in seven of 150 mice for at least 8 weeks. Erythrocytes of all these WBB6F1-W/Wv mice showed either A-type PGK alone or B-type PGK alone during the time of reconstitution, which suggests that a single stem cell with extensive self-maintaining potential may sustain the whole erythropoiesis of a mouse for at least 8 weeks.

Enforced P-glycoprotein pump function in murine bone marrow cells results in expansion of side population stem cells in vitro and repopulating cells in vivo

Blood ◽  
2000 ◽  
Vol 96 (3) ◽  
pp. 902-909 ◽  
Author(s):  
Kevin D. Bunting ◽  
Sheng Zhou ◽  
Taihe Lu ◽  
Brian P. Sorrentino
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Side Population ◽  
Murine Bone Marrow ◽  
Stem Cell Expansion ◽  
Myeloproliferative Syndrome ◽  
P Glycoprotein ◽  
Marrow Cells

Abstract The human multidrug resistance-1 (MDR1) gene product, P-glycoprotein (P-gp), is well known for its ability to confer drug resistance; however, recent evidence suggests that P-gp expression can have more general effects on cellular development. In support of this idea, it was previously shown that retroviral-mediated MDR1 expression in murine bone marrow cells resulted in the expansion of stem cells in culture and in the development of a myeloproliferative syndrome in transplanted mice. It is now reported that MDR1-mediated stem cell expansion is associated with an increase in side population (SP) stem cells, defined by Hoechst dye staining. Transduction of murine bone marrow cells with an MDR1 retroviral vector resulted in an almost 2 log increase in SP cell numbers over 12 days in culture, whereas there was a rapid loss of SP cells from control cultures. Stem cell amplification was not limited to ex vivo expansion cultures but was also evident when MDR1-transduced cells were directly transplanted into irradiated mice. In these cases, stem cell expansion was associated with relatively high vector copy numbers in stem cell clones. As previously reported, some cases were associated with a characteristic myeloproliferative syndrome. A functionally inactive MDR1 mutant cDNA was used to show that P-gp pump function was required both for amplification of phenotypically defined SP cells and functionally defined repopulating cells. These studies further support the concept that ABC transporter function can have important effects on hematopoietic stem cell development.

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

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

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

scholarly journals Retroviral gene transfer of human adenosine deaminase in murine hematopoietic cells: effect of selectable marker sequences on long-term expression

Blood ◽  
1991 ◽  
Vol 78 (2) ◽  
pp. 310-317 ◽  
Author(s):  
JF Apperley ◽  
BD Luskey ◽  
DA Williams
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Gene Transfer ◽  
Bone Marrow Cells ◽  
Selectable Marker ◽  
Retroviral Vector ◽  
G418 Selection ◽  
Marrow Cells

Retroviral-mediated gene transfer of human adenosine deaminase (hADA) provides a model system for the development of somatic gene therapy as a therapy for diseases of bone marrow-derived cells. We have previously demonstrated that hADA can be observed in all hematopoietic lineages in a minority of mice transplanted with bone marrow cells infected with a simplified retroviral vector, ZipPGK-ADA. Here we report a majority of mice (six of eight) demonstrate expression of hADA in the peripheral blood at least 6 months after transplantation with bone marrow infected with this simplified retroviral vector, which contains no selectable marker. The failure to express hADA in two of eight mice was associated with the absence of the recombinant retroviral provirus in DNA prepared from bone marrow cells of these mice apparently due to failure to efficiently infect the reconstituting hematopoietic stem cell. In an effort to preselect bone marrow stem cells containing proviral integrations, we incorporated the selectable marker neo phosphotransferase (NEO) into a retroviral vector encoding hADA, N2/ZipPGK-ADATKNEO, and used G418 selection of infected bone marrow cells before transplantation. In contrast to the simplified retroviral vector, hADA expression in these recipients was short lived (less than 8 weeks), despite the continued presence of intact provirus in DNA prepared from bone marrow of these mice. To determine whether the preselection of bone marrow using G418 was responsible for the lack of sustained hADA expression, we repeated the infection with the N2/ZipPGK- ADATKNEO vector but omitted the G418 selection step. Again, the majority of recipient mice failed to express hADA long term, although the continued presence of provirus in DNA prepared from peripheral blood cell mononuclear cells was clearly demonstrated. Finally, we demonstrate clonal fluctuation of infected stem cells, and observe a temporal correlation between cessation of expression of hADA and the emergence of a dominant stem cell clone between 14 and 20 weeks posttransplantation in one recipient. These data suggest that inclusion of a second transcriptional unit that includes neo phosphotransferase sequences in this simplified vector is associated with decreased expression of the nonselectable ADA sequences.

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

Blood ◽  
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.

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

Blood ◽  
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.

Dnmt3a-Deletion Accelerates FLT3-ITD Malignancies In Mice By Hypomethylation Of Enhancer Sites and Activating Stem Cell Programs; Implications For Therapy

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 595-595 ◽  
Author(s):  
Liubin Yang ◽  
Benjamin Rodriguez ◽  
Min Luo ◽  
Mira Jeong ◽  
David Ruau ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Mouse Model ◽  
Acute Leukemia ◽  
Bone Marrow Cells ◽  
Self Renewal ◽  
Expression Of Genes ◽  
Marrow Cells

Abstract The de novo DNA methyltransferase (DNMT) 3A is mutated in 50% of patients with mixed phenotype acute leukemia, 20% with acute myeloid leukemia (AML) and 18% with T-cell acute lymphoblastic leukemia (T-ALL). The mechanisms through which mutant DNMT3A contributes to hematologic malignancy are poorly understood. In mice, deletion of Dnmt3a in hematopoietic stem cells (HSCs) leads to abnormal DNA methylation and inhibition of differentiation, but is insufficient for leukemic transformation. To study the role of Dnmt3a in leukemia, we combined Dnmt3a-deletion with the activated FLT3 proto-oncogene (FLT3-ITD), a frequent co-mutation with DNMT3A in AML patients, to establish a murine model of Dnmt3a-associated malignancy. In mice transplanted with Dnmt3a-knockout (KO) or wild-type (WT) bone marrow cells transduced with a FLT3-ITD retrovirus, Dnmt3a-loss dramatically impacted the disease phenotype. Dnmt3aKO/ITD transplanted mice had significantly shortened survival (79 days vs. 116 days) and increased rate of acute leukemia compared to mice with ITD alone. The mice developed CD4+CD8+ Notch activation-associated T-ALL or myeloproliferative disease (MPD), or concurrently both, consistent with previous studies of FLT3-ITD in mice. To determine the leukemia-initiating population, we transplanted sorted HSC, myeloid, and lymphoid progenitors transduced with FLT3-ITD. All mice transplanted with HSC and myeloid progenitors succumbed to both malignancies. To uncover the mechanisms by which Dnmt3a-deletion accelerated acute leukemia, we analyzed changes in DNA methylation in T-ALL blasts by whole genome bisulfite sequencing. Compared to Dnmt3aWT/ITD, Dnmt3aKO/ITD blasts exhibited global hypomethylation, particularly at distal enhancer sites. These hypomethylated enhancer sites were associated with genes in signaling pathways, transcription regulators, and metabolic pathways in cancer (KEGG and GO Analysis). Transcriptome analysis showed that relative to Dnmt3aWT/ITD, the Dnmt3aKO/ITD blasts had 1577 significantly differentially expressed genes positively related to cancer, cellular growth, and proliferation, and negatively to apoptosis by Ingenuity Pathway Analysis (IPA). Surprisingly, we observed increased expression of genes related to HSCs and myeloid function and decreased expression of genes related to lymphocyte function. Human AML signature genes (Oncomine) were also upregulated in our mouse model. Predicted activated pathways include Myc, Nfe2l2, Eif4e, E2f1, Csf2, Cebpb, Vegf, Rxra, Ezh2, and Brd4 and inhibited pathways include tumor suppressors Rb, let7, Cdkn2a, and Tob1 (IPA). We did not observe changes in genomic copy number variation by chromosomal comparative hybridization (cCGH). To test whether Dnmt3a-deletion could functionally bestow stem cell properties on pre-leukemic cells, we examined self-renewal capabilities of malignant cells of Flt3+/ITD knock-in mouse (an ITD mutation knocked in to the endogenous murine Flt3 allele causing MPD). Remarkably, when Dnmt3aKO; Flt3+/ITD bone marrow cells were serially transplanted, MPD was seen in all recipients, compared to none in Dnmt3aWT; Flt3+/ITD transplanted mice (n=7). Further, we transplanted sorted CLP, CMP, GMP, MPP, ST-HSC, LT-HSC populations and observed myeloproliferation in transplanted non-stem (CMP, GMP, ST-HSC) and stem cell (LT-HSC) populations. This strongly suggests that Dnmt3aKO synergized with Flt3-ITD to confer stem cell self-renewal abilities to transformed progenitor and stem cells. Increasingly, decitabine is being used to treat patients with AML and MDS, but whether patients with DNMT3A mutations could benefit is unclear, so we examined the impact of decitabine treatment on the retroviral transduced Dnmt3aKO/ITD mice. Monthly treatment led to significantly increased survival of Dnmt3aKO/ITD mice from T-ALL and MPD and reduced presence of ITD-transduced KO cells. Together, we demonstrate that Dnmt3aKO accelerated malignancies induced by FLT3-ITD in mouse and may shed light on how DNMT3A mutations contribute to lymphoid and myeloid disease in patients. Dnmt3a deletion ignited multilineage and stem cell programs at the expense of lymphoid programs to accelerate disease, but was extinguishable by decitabine therapy. The findings from our mouse model can be used for the development and testing of targeted epigenetic therapy for DNMT3A-associated malignancies. Disclosures: No relevant conflicts of interest to declare.

Enforced P-glycoprotein pump function in murine bone marrow cells results in expansion of side population stem cells in vitro and repopulating cells in vivo

Blood ◽  
2000 ◽  
Vol 96 (3) ◽  
pp. 902-909 ◽  
Author(s):  
Kevin D. Bunting ◽  
Sheng Zhou ◽  
Taihe Lu ◽  
Brian P. Sorrentino
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Side Population ◽  
Murine Bone Marrow ◽  
Stem Cell Expansion ◽  
Myeloproliferative Syndrome ◽  
P Glycoprotein ◽  
Marrow Cells

The human multidrug resistance-1 (MDR1) gene product, P-glycoprotein (P-gp), is well known for its ability to confer drug resistance; however, recent evidence suggests that P-gp expression can have more general effects on cellular development. In support of this idea, it was previously shown that retroviral-mediated MDR1 expression in murine bone marrow cells resulted in the expansion of stem cells in culture and in the development of a myeloproliferative syndrome in transplanted mice. It is now reported that MDR1-mediated stem cell expansion is associated with an increase in side population (SP) stem cells, defined by Hoechst dye staining. Transduction of murine bone marrow cells with an MDR1 retroviral vector resulted in an almost 2 log increase in SP cell numbers over 12 days in culture, whereas there was a rapid loss of SP cells from control cultures. Stem cell amplification was not limited to ex vivo expansion cultures but was also evident when MDR1-transduced cells were directly transplanted into irradiated mice. In these cases, stem cell expansion was associated with relatively high vector copy numbers in stem cell clones. As previously reported, some cases were associated with a characteristic myeloproliferative syndrome. A functionally inactive MDR1 mutant cDNA was used to show that P-gp pump function was required both for amplification of phenotypically defined SP cells and functionally defined repopulating cells. These studies further support the concept that ABC transporter function can have important effects on hematopoietic stem cell development.

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