scholarly journals Niche displacement of human leukemic stem cells uniquely allows their competitive replacement with healthy HSPCs

2014 ◽  
Vol 211 (10) ◽  
pp. 1925-1935 ◽  
Author(s):  
Allison L. Boyd ◽  
Clinton J.V. Campbell ◽  
Claudia I. Hopkins ◽  
Aline Fiebig-Comyn ◽  
Jennifer Russell ◽  
...  
Keyword(s):  
Stem Cells ◽  
Human Leukemia ◽  
Dependent Manner ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Bm Niche ◽  
Stem And Progenitor Cells ◽  
A Cell ◽  
Niche Displacement

Allogeneic hematopoietic stem cell (HSC) transplantation (HSCT) is currently the leading strategy to manage acute myeloid leukemia (AML). However, treatment-related morbidity limits the patient generalizability of HSCT use, and the survival of leukemic stem cells (LSCs) within protective areas of the bone marrow (BM) continues to lead to high relapse rates. Despite growing appreciation for the significance of the LSC microenvironment, it has remained unresolved whether LSCs preferentially situate within normal HSC niches or whether their niche requirements are more promiscuous. Here, we provide functional evidence that the spatial localization of phenotypically primitive human AML cells is restricted to niche elements shared with their normal counterparts, and that their intrinsic ability to initiate and retain occupancy of these niches can be rivaled by healthy hematopoietic stem and progenitor cells (HSPCs). When challenged in competitive BM repopulation assays, primary human leukemia-initiating cells (L-ICs) can be consistently outperformed by HSPCs for BM niche occupancy in a cell dose-dependent manner that ultimately compromises long-term L-IC renewal and subsequent leukemia-initiating capacity. The effectiveness of this approach could be demonstrated using cytokine-induced mobilization of established leukemia from the BM that facilitated the replacement of BM niches with transplanted HSPCs. These findings identify a functional vulnerability of primitive leukemia cells, and suggest that clinical development of these novel transplantation techniques should focus on the dissociation of L-IC–niche interactions to improve competitive replacement with healthy HSPCs during HSCT toward increased survival of patients.

CITED2 Modulates Differentiation and Proliferation of Normal and Leukemic Hematopoietic Stem and Progenitor Cells Downstream of PU.1

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 922-922
Author(s):  
Hein Schepers ◽  
Patrick M. Korthuis ◽  
Jan J. Schuringa ◽  
Gerald de Haan ◽  
Edo Vellenga
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Binding Sites ◽  
Myeloid Differentiation ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Cell Numbers ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract Abstract 922 Recently, it was demonstrated that the transcriptional co-activator CITED2 has a conserved role in the maintenance of normal adult hematopoiesis. Little is known regarding the regulation of CITED2, its expression levels in leukemic stem cells (LSCs) and whether CITED2 expression contributes to leukemogenesis. Using microarray data, we found variable CITED2 expression in ∼90% of sorted CD34+ AML cells (n=46). Q-PCR on 12 of these samples indicated that 50% of the leukemic samples displayed higher expression of CITED2 as compared to mobilized peripheral blood (PB) or cord blood (CB)-derived CD34+ hematopoietic stem and progenitor cells (HSCPs). To verify whether this expression of CITED2 in leukemic cells is functional, we performed RNAinterference on primary CD34+ cells from acute myeloid leukemia (AML) patients (n=9). AML samples with high CITED2 expression were susceptible to lentiviral downregulation of CITED2 as evidenced by the loss of transduced cells from long-term leukemic cultures. To investigate a potential role of CITED2 in leukemogenesis, lentiviral gain-of-function experiments were performed with CB-derived CD34+ HSCPs. These experiments indicate that CITED2 expression increases cell numbers up to 5-fold in long-term culture-initiating cell (LTC-iC) experiments. This cell expansion on MS5-stromal cultures was paralleled by a short-term maintenance of progenitors. Colony-forming-cell (CFC) assays demonstrated a 3.5 fold higher output of CFC colonies compared to control cultures up to 2 weeks of MS-5 co-culture. CITED2-expressing colonies were larger than control colonies, verifying the increased cell numbers in LTC-iC experiments. To further dissect the effects of CITED2, transduced CD34+CD38− HSCs and CD34+CD38+ progenitors were sorted for single-cell liquid cultures and cell-divisions were followed for 6 days. Analysis of 360 single CD34+CD38− HSCs indicated that CITED2 overexpression leads to an enhanced quiescence (cells with no division) and decreased proliferation (cells that divide). However, tracking the divisions of 360 single CD34+CD38+ progenitor cells demonstrated the opposite: Cells overexpressing CITED2 divided more than control cells. These data are consistent with a role for CITED2 in leukemic stem cells (LSCs), where LSCs are thought to be more quiescent than leukemic progenitors. Since leukemias are also characterized by a differentiation block, we subsequently analyzed the differentiation of CB-derived CD34+ HSCPs upon overexpression of CITED2. In LTC-iC experiments, a shift in myelo-monocytic differentiation was observed. Enhanced CITED2 expression led to an increased percentage of CD15-positive cells (64%) at the expense of CD14-positive cells (9%) compared to control cultures (35% and 32% respectively). This bias towards granulocytic differentiation was also observed on May-Grunwald-Giemsa (MGG) stains and was furthermore confirmed in CFC assays. Furthermore, when analyzing erythroid differentiation, a clear reduction in CD71bright GPA+ cells could be observed in CITED2 expressing cells, compared to control cells (2% vs. 12% respectively), which was confirmed by CFC assays and MGG stains. Towards clarifying the regulation of CITED2, we scanned the CITED2 promoter for transcription factor binding sites and identified several PU.1 binding sites. Gene expression comparison between PU.1 and CITED2 in a panel of primary AML samples indicated that, apart from FAB M2 AMLs, PU.1 expression is inversely correlated with CITED2 expression. To functionally investigate this inverse correlation, we performed chromatin immunoprecipitations (ChIP) and demonstrated that PU.1 is indeed able to bind to the PU.1 binding sites in the CITED2 promoter of CB CD34+ cells. Subsequent overexpression of PU.1 in CB CD34+ HSPCs led to a 2-fold reduction in CITED2 expression. Taken together, we propose a model in which PU.1 tightly regulates CITED2 expression during normal myeloid differentiation. In certain AML subsets, this model would predict that low PU.1 expression results in failure to lower CITED2 expression below a certain threshold, which subsequently results in maintenance of LSC quiescence. Furthermore, the enhanced CITED2 levels result in an increased proliferation of downstream leukemic progenitors, where together with low PU.1 levels the normal myeloid differentiation program is perturbed contributing to leukemic development. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Maintenance of Hematopoietic Stem Cells Through Regulation of Wnt and mTOR Pathways.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2309-2309
Author(s):  
Jian Huang ◽  
Peter S. Klein
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Signaling Pathways ◽  
Glycogen Synthase ◽  
Dependent Manner ◽  
Mtor Inhibition ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Abstract 2309 Hematopoietic stem cells (HSCs) maintain the ability to self-renew and to differentiate into all lineages of the blood. The signaling pathways regulating hematopoietic stem cell (HSCs) self-renewal and differentiation are not well understood. We are very interested in understanding the roles of glycogen synthase kinase-3 (Gsk3) and the signaling pathways regulated by Gsk3 in HSCs. In our previous study (Journal of Clinical Investigation, December 2009) using loss of function approaches (inhibitors, RNAi, and knockout) in mice, we found that Gsk3 plays a pivotal role in controlling the decision between self-renewal and differentiation of HSCs. Disruption of Gsk3 in bone marrow transiently expands HSCs in a b-catenin dependent manner, consistent with a role for Wnt signaling. However, in long-term repopulation assays, disruption of Gsk3 progressively depletes HSCs through activation of mTOR. This long-term HSC depletion is prevented by mTOR inhibition and exacerbated by b-catenin knockout. Thus GSK3 regulates both Wnt and mTOR signaling in HSCs, with opposing effects on HSC self-renewal such that inhibition of Gsk3 in the presence of rapamycin expands the HSC pool in vivo. In the current study, we found that suppression of the mammalian target of rapamycin (mTOR) pathway, an established nutrient sensor, combined with activation of canonical Wnt/ß-catenin signaling, allows the ex vivo maintenance of human and mouse long-term HSCs under cytokine-free conditions. We also show that combining two clinically approved medications that activate Wnt/ß-catenin signaling and inhibit mTOR increases the number of long-term HSCs in vivo. Disclosures: No relevant conflicts of interest to declare.

The AML1-ETO fusion protein promotes the expansion of human hematopoietic stem cells

Blood ◽  
2002 ◽  
Vol 99 (1) ◽  
pp. 15-23 ◽  
Author(s):  
James C. Mulloy ◽  
Jörg Cammenga ◽  
Karen L. MacKenzie ◽  
Francisco J. Berguido ◽  
Malcolm A. S. Moore ◽  
...  
Keyword(s):  
Stem Cells ◽  
Fusion Protein ◽  
Progenitor Cells ◽  
Protein Interactions ◽  
Dominant Negative ◽  
Myelogenous Leukemia ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

The acute myelogenous leukemia–1 (AML1)–ETO fusion protein is generated by the t(8;21), which is found in 40% of AMLs of the French-American-British M2 subtype. AML1-ETO interferes with the function of the AML1 (RUNX1, CBFA2) transcription factor in a dominant-negative fashion and represses transcription by binding its consensus DNA–binding site and via protein-protein interactions with other transcription factors. AML1 activity is critical for the development of definitive hematopoiesis, and haploinsufficiency of AML1 has been linked to a propensity to develop AML. Murine experiments suggest that AML1-ETO expression may not be sufficient for leukemogenesis; however, like the BCR-ABL isoforms, the cellular background in which these fusion proteins are expressed may be critical to the phenotype observed. Retroviral gene transfer was used to examine the effect of AML1-ETO on the in vitro behavior of human hematopoietic stem and progenitor cells. Following transduction of CD34+ cells, stem and progenitor cells were quantified in clonogenic assays, cytokine-driven expansion cultures, and long-term stromal cocultures. Expression of AML1-ETO inhibited colony formation by committed progenitors, but enhanced the growth of stem cells (cobblestone area-forming cells), resulting in a profound survival advantage of transduced over nontransduced cells. AML1-ETO–expressing cells retained progenitor activity and continued to express CD34 throughout the 5-week long-term culture. Thus, AML1-ETO enhances the self-renewal of pluripotent stem cells, the physiological target of many acute myeloid leukemias.

Malignant Myeloma Cells Impair Phenotype and Function of stem and Progenitor Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1799-1799
Author(s):  
Ingmar Bruns ◽  
Sebastian Büst ◽  
Akos G. Czibere ◽  
Ron-Patrick Cadeddu ◽  
Ines Brückmann ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Plasma Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Healthy Donors ◽  
Stem And Progenitor Cells

Abstract Abstract 1799 Poster Board I-825 Multiple myeloma (MM) patients often present with anemia at the time of initial diagnosis. This has so far only attributed to a physically marrow suppression by the invading malignant plasma cells and the overexpression of Fas-L and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by malignant plasma cells triggering the death of immature erythroblasts. Still the impact of MM on hematopoietic stem cells and their niches is scarcely established. In this study we analyzed highly purified CD34+ hematopoietic stem and progenitor cell subsets from the bone marrow of newly diagnosed MM patients in comparison to normal donors. Quantitative flowcytometric analyses revealed a significant reduction of the megakaryocyte-erythrocyte progenitor (MEP) proportion in MM patients, whereas the percentage of granulocyte-macrophage progenitors (GMP) was significantly increased. Proportions of hematopoietic stem cells (HSC) and myeloid progenitors (CMP) were not significantly altered. We then asked if this is also reflected by clonogenic assays and found a significantly decreased percentage of erythroid precursors (BFU-E and CFU-E). Using Affymetrix HU133 2.0 gene arrays, we compared the gene expression signatures of stem cells and progenitor subsets in MM patients and healthy donors. The most striking findings so far reflect reduced adhesive and migratory potential, impaired self-renewal capacity and disturbed B-cell development in HSC whereas the MEP expression profile reflects decreased in cell cycle activity and enhanced apoptosis. In line we found a decreased expression of the adhesion molecule CD44 and a reduced actin polymerization in MM HSC by immunofluorescence analysis. Accordingly, in vitro adhesion and transwell migration assays showed reduced adhesive and migratory capacities. The impaired self-renewal capacity of MM HSC was functionally corroborated by a significantly decreased long-term culture initiating cell (LTC-IC) frequency in long term culture assays. Cell cycle analyses revealed a significantly larger proportion of MM MEP in G0-phase of the cell cycle. Furthermore, the proportion of apoptotic cells in MM MEP determined by the content of cleaved caspase 3 was increased as compared to MEP from healthy donors. Taken together, our findings indicate an impact of MM on the molecular phenotype and functional properties of stem and progenitor cells. Anemia in MM seems at least partially to originate already at the stem and progenitor level. Disclosures Off Label Use: AML with multikinase inhibitor sorafenib, which is approved by EMEA + FDA for renal cell carcinoma.

scholarly journals The Role of Notch in Endothelial Cell-Mediated Protection of AML Precursors from Targeted Therapy

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3947-3947
Author(s):  
Quy Le ◽  
Brandon Hadland ◽  
Soheil Meshinchi ◽  
Irwin D. Bernstein
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Notch Signaling ◽  
Liquid Culture ◽  
Critical Role ◽  
Common Mutation ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Inhibitory Antibodies

Abstract Background: AML is an aggressive hematologic malignancy that remains difficult to treat. A common mutation found in AML is FLT3-ITD, occurring in 15% of childhood AML. Although chemotherapy has successfully induced remission, patients with a high FLT3 ITD:WT allelic ratio (FLT3-AR) exhibit a high relapse rate, requiring hematopoietic stem cell transplantation to increase the chance of long-term remission. In this study, we demonstrate the requirement of ECs for survival of FLT3-ITD progenitors from primary pediatric AML specimens in the presence of AC220, a potent and selective inhibitor of FLT3. We further show that the Notch pathway plays a role in EC-mediated protection amongst patient samples with high FLT3-AR, suggesting the potential therapeutic use of Notch blockade in the treatment of this high-risk subset. Results: To determine whether ECs confer protection to FLT3-ITD progenitors, we quantified the number of CFC present after 2 weeks of liquid culture or EC co-culture with AC220 (added at days 0, 3 and 7) from four AML specimens with high FLT3-AR (≥1). We used PCR to determine the presence of FLT3-ITD in individual CFC. We found that the numbers of FLT3-ITD CFC (p=0.007) and FLT3-WT CFC (p=0.044) were reduced in liquid culture compared to EC co-culture, suggesting that ECs mediate the survival of FLT3-ITD hematopoietic progenitors against the therapeutic treatment of AC220. Previously, we demonstrated that ECs are critical for the growth and expansion of hematopoietic stem cells, which is dependent on the activation of Notch signaling. We asked whether Notch plays a role in EC-mediated protection of AML progenitors against AC220, using RNA-seq analysis on three FLT3-ITD-harboring AML. Among the significantly altered genes (FDR<0.05), we found an enrichment of Notch target genes that were expressed at significantly higher levels in AC220-treated cells compared to DMSO-treated cells, including HES1, HES4, NRARP, CDKN1A, CCND1, andGATA3, suggesting that Notch signaling may facilitate EC-mediated protection against AC220. Next, we assessed the effect of inhibiting Notch signaling on AML progenitor survival during AC220 treatment in EC co-culture, using inhibitory antibodies specific to the Negative Regulatory Region (NRR) of both Notch1 and Notch2 receptors (anti-NRR1 and anti-NRR2; kindly provided by Chris Siebel, Genentech). We co-cultured bone marrow cells from eight patient specimens with low FLT3-AR (<1) and five patient specimens with high FLT3-AR (≥1), with ECs and briefly treated the co-cultures with Notch inhibitory antibodies or IgG1 antibody for 3 days. AC220 was added to the cultures at days 0, 3 and 7. We assessed CFC numbers present after 2 weeks of culture. Patient samples with low FLT3-AR did not exhibit changes in the numbers of FLT3-ITD CFC (p = 0.735) and FLT3-WT CFC (p = 0.489) in response to Notch inhibition relative to IgG1 control. In contrast, patient samples with high FLT3-AR showed reduction in the number of FLT3-ITD CFC (p=0.019) but the number of FLT3-WT CFC remained unaffected (p=0.874). These results suggest a critical role for Notch in EC-mediated protection in AML with high FLT3-AR. Conclusion: Our studies suggest that inhibiting Notch signaling may have therapeutic potential for overcoming drug resistance induced by the tumor microenvironment in a subset of AML with high FLT3-AR. We have previously shown that a high FLT3-AR is associated with the presence of FLT3-ITD in the least mature hematopoietic subset (CD34+ CD33- precursors), which is thought to contain leukemic stem cells, and this association is correlated with poorer outcome. Additionally, AML cells that give rise to CFC after long-term co-culture with bone marrow stroma or ECs are derived from the CD34+CD33- AML precursors. Ongoing studies aim to determine whether Notch signaling plays a role in the survival of AML CD34+CD33- cells with the goal of eliminating leukemic stem cells responsible for relapse. Disclosures No relevant conflicts of interest to declare.

scholarly journals Fluorouracil selectively spares acute myeloid leukemia cells with long- term growth abilities in immunodeficient mice and in culture

Blood ◽  
1996 ◽  
Vol 88 (6) ◽  
pp. 1944-1950 ◽  
Author(s):  
W Terpstra ◽  
RE Ploemacher ◽  
A Prins ◽  
K van Lom ◽  
K Pouwels ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Severe Combined Immunodeficiency ◽  
Scid Mice ◽  
Leukemic Cells ◽  
Human Leukemia ◽  
Hematopoietic Stem ◽  
Acute Myeloid

A subset of leukemic cells is assumed to maintain long-term growth of acute myeloid leukemia (AML) in vivo. Characterization of these AML progenitor cells may further define growth properties of human leukemia. In vitro incubations with 5-fluorouracil (5-FU) have been used for enrichment of normal primitive hematopoietic stem cells. By analogy to normal hematopoiesis, it was hypothesized that primitive leukemic stem cells might be kinetically more inactive than colony- forming cells (colony-forming units-AML [CFU-AML]). To examine this hypothesis, conditions were established for incubation with 5-FU that eliminated all CFU-AML. These conditions selected a 5-FU-resistant AML fraction that was evaluated for its capacity for long-term growth by transplantation into mice with severe combined immunodeficiency (SCID) and long-term culture in the quantitative cobblestone area-forming cell (CAFC) assay. Transplantation of the 5-FU-resistant fraction of four cases of AML into SCID mice resulted in growth of AML. Whereas no CFU- AML survived, 31% to 82% of primitive (week-6) CAFC were recovered from the 5-FU-treated cells. Hematopoietic cells proliferating in the CAFC assay were shown to be leukemic by cytologic, cytogenetic, or molecular analysis. The reduction of AML growth as determined by outgrowth of AML in SCID mice was in the same order of magnitude as the primitive (week- 6) CAFC reduction. This indicates that both assays measure closely related cell populations and that the CAFC assay can be used to study long-term growth of AML. These results show a hierarchy of AML cells that includes 5-FU-resistant progenitors. These cells are characterized as primitive (week-6) CAFC and as leukemia-initiating cells in SCID mice.

Inactivation of PUMA Protects Hematopoietic Stem Cells and Confers Long Term Survival in Response to High Dose γ-Irradiation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 612-612 ◽  
Author(s):  
Hui Yu ◽  
Hongmei Shen ◽  
Feng Xu ◽  
Xiaoxia Hu ◽  
Yanxin Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Radiation Injury ◽  
Γ Irradiation ◽  
Hematopoietic Stem ◽  
Cycle Arrest ◽  
Stem And Progenitor Cells ◽  
Radiation Induced

Abstract Radiation injury remains a significant health problem. New medical intervention to prevent or manage radiation damage is highly dependent on a deeper understanding of how radiation-induced cell death is accomplished in the irradiated tissue cells such as stem and progenitor cells. To date, relatively specific or untainted molecular mediators in apoptosis of tissue stem and progenitor cells upon radiation injury have not been clearly defined. The p53 pathway is known as a major molecular mechanism for cell apoptosis, upon the exposure of lethal radiation. Targeting p53 confers a radioprotective effect, but may increase tumorigenesis due to impaired cell cycle arrest for DNA repair. In our current study, we have examined the specific role of PUMA (p53 up-regulated mediator of apoptosis) in the radiosensitivity of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). By quantitative RT PCR, we found that the level of PUMA mRNA was relatively low in the most primitive long-term repopulating hematopoietic stem cells (LT-HSC, isolated based on the immnunophenotype “CD34−LKS”) as compared to other hematopoietic cell populations from mice, but it was significantly elevated in response to γ-irradiation. In the mice lacking PUMA, while neither HSC number nor HSC function was altered under homeostatic conditions, the PUMA−/− HSCs appeared to be resistant to radiation damage in vivo as retrospectively quantified in a competitive HSC transplant model. Our further direct measurement with a single cell culture system for HSC growth in vitro, demonstrated that PUMA, but not p21 (the chief mediator of p53 in cell cycle arrest), is primarily responsible for the radiosensitivity of HSC in the p53 pathway (200 LT-HSCs analyzed for each cell type). Together, these data provide definitive evidence for PUMA as an essential mediator in radiation-induced apoptosis of tissue stem cells. We finally focused on the beneficial effects of targeting PUMA in HSCs and HPCs on the animal survival upon the exposure of lethal irradiation. Strikingly, the wild-type mice reconstituted with PUMA−/− hematopoietic cells exhibited a significant survival advantage after two rounds of 9-Gy γ-irradiation (18 Gy in total) as compared to the mice reconstituted with PUMA+/+ hematopoietic cells (95 % vs. 0 % survival in 20 days, n=21/each group; 50% vs. 0 % survival in 180 days, n=20 or 11/each group, respectively) as shown in the figure below. Moreover, unlike the p53−/− mice, those PUMA−/− reconstituted mice did not have an increased incidence of hematopoietic malignancies (n=20) within 180 days. Therefore, our current study establishes PUMA as an attractive molecular target for the development of therapeutic agents for the prevention and treatment of radiation injury.

DNA Methyltransferase 1 Is Essential for and Uniquely Regulates Hematopoietic Stem and Progenitor Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 392-392 ◽  
Author(s):  
Jennifer J. Trowbridge ◽  
Jonathan W. Snow ◽  
Jonghwan Kim ◽  
Stuart H. Orkin
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Progenitor Cells ◽  
Dna Methyltransferase ◽  
Adult Stem Cells ◽  
Global Gene Expression ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors ◽  
Stem And Progenitor Cells

Abstract Abstract 392 DNA methylation is essential for development and plays crucial roles in a variety of biological processes. The DNA methyltransferase Dnmt1 serves to maintain parental cell methylation patterns on daughter DNA strands in mitotic cells, however, the precise role of Dnmt1 in regulation of quiescent adult stem cells is not known. To examine the role of Dnmt1 in adult hematopoietic stem cells (HSCs), we crossed Dnmt1fl/fl mice with Mx1-Cre transgenic mice, and by injection of poly(I)-poly(C) we selectively deleted Dnmt1 in the hematopoietic system (Dnmt1Δ/Δ). In Dnmt1Δ/Δ mice, peripheral blood counts and mature multilineage composition of the bone marrow was found to be normal. Interestingly, specific defects were observed in Dnmt1Δ/Δ HSC self-renewal as assessed by long-term and secondary competitive transplantation, in retention of Dnmt1Δ/Δ HSCs within the bone marrow niche, and in the ability of Dnmt1Δ/Δ HSCs to give rise to multilineage hematopoiesis. Loss of Dnmt1 also had unique impact on myeloid progenitor cells (including common myeloid progenitors, granulocyte-macrophage progenitors, and megakaryocyte-erythrocyte progenitors), regulating their cycling and transcriptional lineage fidelity. To determine the molecular mechanisms underlying these defects, we performed global gene expression microarray analysis and bisulfite sequencing of select loci (IAP, Car1, and Gata1) in purified populations of control and Dnmt1Δ/Δ long-term HSCs, short-term HSCs/multipotent progenitor cells, and myeloid restricted progenitor cells. Through this approach, we demonstrate that loss of Dnmt1 has cell type-specific molecular consequences. For example, demethylation of the Car1 and Gata1 loci in Dnmt1Δ/Δ long-term HSCs is not sufficient to activate gene transcription, whereas demethylation of these genes in Dnmt1Δ/Δ short-term HSCs is associated with activation of transcription. In Dnmt1Δ/Δ myeloid restricted progenitor cells, we observed increases in DNA methylation at specific gene loci such as Car1, indicating that methylation can be established by other methyltransferases in the absence of Dnmt1. Our global gene expression microarray analysis clearly demonstrates that Dnmt1 regulates expression of distinct gene families in these closely related, primitive hematopoietic populations. We were unable to attribute specific functional defects in Dnmt1Δ/Δ hematopoietic stem and progenitor cells to alterations in expression of previously characterized genes, supporting the existence of novel, uncharacterized regulators of HSC and progenitor cell function to be explored from candidates in our data set. We conclude that maintenance methylation induced by Dnmt1 appears to be especially important for HSC and progenitor cell state transitions, such as the stepwise differentiation of long-term HSCs to multipotent progenitors, multipotent progenitors to myeloid restricted progenitors, stem cell mobilization, and regulating cell cycle entry. These findings establish a unique and critical role for Dnmt1 in the primitive hematopoietic compartment. Furthermore, our evidence suggests that epigenetic regulation, at least with respect to DNA methylation, of adult stem cells is distinct from embryonic stem cells and other somatic cell types. Disclosures: No relevant conflicts of interest to declare.

Single Cell Proteomics Reveals Novel Cytokine-Producing Function of Hematopoietic Stem and Progenitor Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 26-26
Author(s):  
Jimmy L. Zhao ◽  
Chao Ma ◽  
Ryan O'Connell ◽  
Dinesh S. Rao ◽  
James Heath ◽  
...  
Keyword(s):  
Stem Cells ◽  
Immune Response ◽  
Single Cell ◽  
Progenitor Cells ◽  
Cytokine Production ◽  
Conflicts Of Interest ◽  
Severe Neutropenia ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Abstract Abstract 26 During infection, hematopoietic stem and progenitor cells (HSPCs) are called upon to proliferate and differentiate to produce more innate and adaptive immune cells to combat infection. Traditionally, HSPCs are thought to respond to depletion of downstream hematopoietic cells during infection. More recent evidence suggests that HSPCs may respond directly to infection and pro-inflammatory cytokines. However, little is known about the direct immune response of HSPCs and the molecular signaling regulating this response upon sensing an infection. In this study, we have combined transgenic and genetic knockout mouse models with a novel single cell barcode proteomics microchip technology to tackle these questions. We show that although long-term hematopoietic stem cells (HSCs) (defined by Lineage-cKit+Sca1+CD150+CD48-) do not secrete cytokines upon toll-like receptor (TLR) stimulation, short-term HSCs and multipotent progenitor cells (MPPs) (defined by Lineage-cKit+Sca1+, referred to as LKS thereafter) can produce copious amounts of cytokines upon direct TLR-4 and TLR-2 stimulation, indicating that LKS cells can directly participate in an immune response by producing a myriad of cytokines, upon a bacterial infection. Within the population of LKS cells we detect multiple functional subsets of cells, specialized in producing myeloid-like, lymphoid-like or both types of cytokines. Moreover, we show that the cytokine production by LKS cells is regulated by the NF-κB activity, as p50-deficient LKS cells show reduced cytokine production while microRNA-146a (miR-146a)-deficient LKS cells show significantly increased cytokine production. As long-term HSCs differentiate, they start to gain effector immune function much earlier than we had originally anticipated. In light of this finding, we should start to view the stepwise differentiation scheme of HSCs, and perhaps all other stem cells, as a strategy to sequentially gain functional capacity, instead of simply losing stemness and self-renewal ability. The remarkable ability of LKS cells to produce copious amounts of cytokines in response to bacteria may provide some protective immunity during severe neutropenia and lymphopenia or in the early stage of HSC transplantation. This study further extends the functions of NF-κB to include the regulation of primitive hematopoietic stem and progenitor cells and provides direct evidence of the bacteria-responding ability of HSPCs through the TLR/NF-κB axis. The single cell barcode proteomics technology can be widely applied to study proteomics of other rare cells or heterogeneous cell population at a single cell level. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document