A Network Of Epigenetic Regulators Guide Developmental Hematopoiesis In Vivo

Abstract Long-term hematopoietic stem cells (HSCs) are capable of self-renewal and differentiation into all mature hematopoietic lineages. This process is regulated by transcription factors interact with co-factors to orchestrate chromatin structure and facilitate gene expression. To generate a compendium of factors that establish the epigenetic code in HSCs, we have undertaken the first large-scale in vivo reverse genetic screen targeting chromatin factors. We have designed and injected antisense morpholinos to knockdown expression of 488 zebrafish orthologs of conserved human chromatin factors. The resultant morphants were analyzed by whole embryo in situ hybridization at 36 hours post fertilization for expression of two HSC marker genes, c-myb and runx1, which are expressed in the developing blood stem cells. Morphants were categorized into five groups based on HSC marker expression, ranging from no change to mild, intermediate, or strong reduction in expression or an increase in expression. 29 morpholinos caused a complete or near complete knockdown of HSC marker expression, while 4 were found to increase HSC marker expression. As ubiquitous knockdown of chromatin factors could interfere with vascular development and the establishment of proper arterial identity, a crucial upstream event for HSC formation, we subsequently analyzed morphants with the most robust HSC phenotypes using two vascular markers: kdr for overall vasculogenesis and ephrinb2a for arterial formation. We found that of the 29 morpholinos that caused reduced marker expression, only 9 showed reduced overall vascular or arterial marker staining, suggesting that the majority of morphants with HSC phenotypes are specific to HSC formation. For the 4 morphants with increased HSC marker expression, vasculature appeared normal. These factors likely function as potent negative regulators of HSC development. Several genes known to be essential for HSC self-renewal and maintenance were identified in the screen. For example, knockdown of Mll or Dot1, which are also present in leukemia fusion proteins, fail to specify HSCs, as indicated by a nearly complete reduction in expression of the HSC markers in embryos tested. Of the remaining hits, many represent factors with no previous function ascribed in hematopoiesis. By incorporating protein interaction data, we have defined a handful of complexes necessary for HSC specification, including the SWI/SNF, ISWI, SET1/MLL, CBP/P300/HBO1/NuA4, HDAC/NuRD, and Polycomb complexes. As chromatin factors associated with the same complex likely share target binding sites, we analyzed 34 published ChIP-seq datasets in K562 erythroleukemia cells of chromatin factors tested in the screen, including hits from our screen: SIN3A, CHD4, HDAC1, TAF1, and JARID1C associated with the HDAC/NuRD complex and RNF2, SUZ12, CBX2, and CBX8 from the Polycomb complexes. We ranked triplet combinations of these factors together with all other groups of three factors based on the percent overlap of target genes. The HDAC/NuRD and PRC1/2 complex combinations predicted from our screen fell within the top 20% of all possible combinations of 3 factors, suggesting that our screen has identified chromatin factors that function in distinct complexes to regulate hematopoietic development. Our work has been compiled into a web-based database that will be made publicly available upon publication. Within this database, users can search by gene names and aliases, chromatin domain names and human or zebrafish genes. All experimental data, including experimental design, materials, protocols, images, and all further analyses of the 33 most robust morphants is included. Our large-scale genetic analysis of chromatin factors involved in HSC development provides a comprehensive view of the programs involved in epigenetic regulation of the blood program, offering new avenues to pursue in the study of histone modifications in HSCs and for therapeutic alternatives for patients with blood disorders and leukemia. Disclosures: Zon: FATE Therapeutics, Inc: Consultancy, Equity Ownership, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Stemgent, Inc: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Stocks, Stocks Other; Scholar Rock: Consultancy, Equity Ownership, Founder, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

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The Role of Chromatin Factors in Hematopoietic Development

Blood ◽

10.1182/blood.v122.21.sci-48.sci-48 ◽

2013 ◽

Vol 122 (21) ◽

pp. SCI-48-SCI-48

Author(s):

Leonard I. Zon

Keyword(s):

Progenitor Cells ◽

Board Of Directors ◽

Large Scale ◽

Erythroid Progenitors ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Conditional Allele ◽

Stem And Progenitor Cells ◽

Equity Ownership

Abstract The initiation of blood-specific programs is orchestrated by key transcription factors. To generate a complete compendium of chromatin factors that establish the epigenetic code during developmental hematopoiesis, a large-scale reverse genetic screen was conducted targeting orthologs of 425 human chromatin factors in zebrafish. A set of chromatin regulators was identified that function at distinct steps of primitive and definitive blood formation, including factors not previously implicated in blood development. We identified 15 factors that regulate development of primitive erythroid progenitors and 29 factors that regulate development of definitive stem and progenitor cells. These chromatin factors are associated with SWI/SNF and ISWI chromatin remodeling, SET1/MLL methyltransferase, CBP/P300/HBO1/NuA4 acetyltransferase, Sin3A/NuRD deacetylase, and Polycomb repressive complexes. Knockdown of a class of chromatin factors led to an expansion of hematopoietic stem cells (HSCs). In collaboration with Nancy Speck’s laboratory, we have investigated the activity of one of these chromatin factors, CHD7, that led to an expansion of hematopoietic stem and progenitor cells in the aorta. Using a T-cell line, a CBFβ protein pulldown, and a mass spectrometric sequencing approach led to the finding that CBFβ immunoprecipitated RUNX1 and CHD7. By studying a conditional allele of murine Chd7, inactivation does not have an effect on peripheral blood counts, but Chd7-deleted HSCs purified based on phenotypic markers contain an increased number of functional HSCs. Our studies suggest that CHD7 suppresses hematopoiesis, and provides a novel control mechanism for the regulation of HSCs. Our work provides a comprehensive view of how specific chromatin factors and their associated complexes play a major role in the establishment of hematopoietic cells in vivo. Disclosures: Zon: Scholar Rock: Consultancy, Equity Ownership, Founder, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Stemgent, Inc: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Stocks, Stocks Other; FATE Therapeutics, Inc: Consultancy, Equity Ownership, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

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Msi2 Maintains Normal Hematopoietic Stem Cell Self-Renewal

Blood ◽

10.1182/blood.v118.21.222.222 ◽

2011 ◽

Vol 118 (21) ◽

pp. 222-222 ◽

Cited By ~ 1

Author(s):

Michael G Kharas ◽

Christopher Lengner ◽

Fatima Al-Shahrour ◽

Benjamin L. Ebert ◽

George Q. Daley

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Board Of Directors ◽

Bone Marrow Cells ◽

Advisory Committees ◽

Self Renewal ◽

Hematopoietic Stem ◽

Equity Ownership ◽

Marrow Cells

Abstract Abstract 222 Genes that regulate normal hematopoietic stem cells are commonly dysregulated in hematopoietic malignancies. Recently we published that the Msi2 RNA binding protein is an important modulator in both normal hematopoietic stem cells and leukemia (Kharas et al, Nat. Medicine 2010). The closely related Msi1 protein has been shown to regulate mRNA translation through binding to the 3'UTR. Based on the high homology in the RNA recognition motifs, Msi2 has been considered to have similar functions. Moreover, increased MSI2 expression in chronic myelogenous leukemia blast crisis and acute myeloid leukemia predicts a worse clinical prognosis. Previous studies have mainly utilized shRNAs to functionally assess the role of Msi2 in the hematopoietic compartment. However, it remains unclear how Msi2 affects hematopoietic stem cells (HSC) and what are its critical mRNA targets. To develop a model focusing on the HSC compartment and to avoid potential compensatory mechanisms during development, we created Msi2 conditional knockout mice and crossed them with Mx1-Cre mice. We induced excision with poly(I):poly(C), (pIpC), and tested the peripheral blood, bone marrow cells and splenocytes by Southern blotting and QPCR analysis to verify Msi2 deletion. Loss of Msi2 mRNA was confirmed in the Lineagelo, Sca1+ and c-Kit+ (LSK) population. Msi2 deleted bone marrow contained reduced myeloid colony forming capacity and replating efficiency. Mice conditionally deleted for Msi2 had normal white blood cell counts but smaller spleens. In addition, we observed normal percentages of the mature hematopoietic populations, including the myeloid and lymphoid compartments. Nevertheless, absolute numbers of long-term HSCs in the bone marrow were reduced by 3-fold. Bone marrow cells non-competitively transplanted into primary and secondary recipient mice showed a dramatic reduction in HSC chimerism. This defect was also observed when bone marrow was transplanted first to allow engraftment followed by Msi2 deletion. Furthermore, we were able to recapitulate this defect in vitro using the cobblestone-forming activity assay. These results indicate that Msi2 is both an important regulator of normal HSC maintenance and required for efficient engraftment. Most interestingly, Msi2 HSCs failed to maintain a normal quiescent HSC population. We performed microarrays to identify the pathways altered in the LSK population. The Msi2 deficient LSKs showed a reduced self-renewal and increased differentiation gene signature. Gene expression analysis indicates a defective self-renewal program in Msi2-deficient HSCs that is identical to the program gained in leukemic stem cells. These data suggest that MSI2 is a critical modulator of HSCs and may help explain its requirement in the most aggressive myeloid leukemias. Disclosures: Daley: iPierian, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Epizyme, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Verastem, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Solasia, KK: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; MPM Capital, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees.

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Epigenetic Activation of the pH Regulator MCT4 in Acute Myeloid Leukemia Exploits a Fundamental Metabolic Process of Enhancing Cell Growth through Proton Shifting

Blood ◽

10.1182/blood-2019-123604 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3765-3765

Author(s):

Cheuk-Him Man ◽

David T. Scadden ◽

Francois Mercier ◽

Nian Liu ◽

Wentao Dong ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Growth ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Monocarboxylate Transporter ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Equity Ownership ◽

Acute Myeloid

Acute myeloid leukemia (AML) cells exhibit metabolic alterations that may provide therapeutic targets not necessarily evident in the cancer cell genome. Among the metabolic features we noted in AML compared with normal hematopoietic stem and progenitors (HSPC) was a strikingly consistent alkaline intracellular pH (pHi). Among candidate proton regulators, monocarboxylate transporter 4 (MCT4) mRNA and protein were differentially increased in multiple human and mouse AML cell lines and primary AML cells. MCT4 is a plasma membrane H+and lactate co-transporter whose activity necessarily shifts protons extracellularly as intracellular lactate is extruded. MCT4 activity is increased when overexpressed or with increased intracellular lactate generated by glycolysis in the setting of nutrient abundance. With increased MCT4 activity, extracellular lactate and protons will increase causing extracellular acidification while alkalinizing the intracellular compartment. MCT4-knockout (MCT4-KO) of mouse and human AMLdid not induce compensatory MCT1 expression, reduced pHi, suppressed proliferation and improved animal survival. Growth reduction was experimentally defined to be due to intracellular acidification rather than lactate accumulation by independent modulation of those parameters. MCT4-KOmetabolic profiling demonstrated decreased ATP/ADP and increased NADP+/NADPH suggesting suppression of glycolysis and the pentose phosphate pathway (PPP) that was confirmed by stable isotopic carbon flux analyses. Notably,the enzymatic activity of purified gatekeeper enzymes, hexokinase 1 (HK1), pyruvate kinase M2 isoform (PKM2) and glucose-6-phosphate dehydrogenase (G6PDH) was sensitive to pH with increased activity at the leukemic pHi (pH 7.6) compared to normal pHi (pH 7.3). Evaluating MCT4 transcriptional regulation, we defined that activating histonemarks, H3K27ac and H3K4me3, were enriched at the MCT4 promoter region as were transcriptional regulators MLL1 and Brd4 by ChIP in AML compared with normal cells. Pharmacologic inhibition of Brd4 suppressed Brd4 and H3K27ac enrichment and MCT4 expression in AML and reduced leukemic cell growth. To determine whether MCT4 based pHi changes were sufficient to increase cell proliferation, we overexpressed MCT4 in normal HSPC and demonstrated in vivo increases in growth in conjunction with pHi alkalization. Some other cell types also were increased in their growth kinetics by MCT4 overexpression and pHi increase. Therefore, proton shifting may be a means by which cells respond to nutrient abundance, co-transporting lactate and protons out of the cell, increasing the activity of enzymes that enhance PPP and glycolysis for biomass generation. Epigenetic changes in AML appear to exploit that process by increasing MCT4 expression to enforce proton exclusion thereby gaining a growth advantage without dependence on signaling pathways. Inhibiting MCT4 and intracellular alkalization may diminish the ability of AML to outcompete normal hematopoiesis. Figure Disclosures Scadden: Clear Creek Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Sponsored research; Editas Medicine: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bone Therapeutics: Consultancy; Fog Pharma: Consultancy; Red Oak Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; LifeVaultBio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Consultancy, Equity Ownership.

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Genome-Wide RNA Tomography of the Hematopoietic Stem Cell Niche in Zebrafish Reveals Unexpected Functional Macrophage-Stem Cell Interactions

Blood ◽

10.1182/blood.v128.22.3882.3882 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3882-3882

Author(s):

Elliott J Hagedorn ◽

Julie R Perlin ◽

Clara Mao ◽

Brian Li ◽

Christopher D'Amato ◽

...

Keyword(s):

Stem Cell ◽

Board Of Directors ◽

Cell Interactions ◽

Cell Populations ◽

Rna Seq ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Equity Ownership

Abstract The challenges of visualizing the mammalian bone marrow have precluded a rigorous analysis of the dynamic cell-cell interactions that control hematopoietic stem and progenitor cell (HSPC) engraftment. The transparent zebrafish embryo provides an unparalleled opportunity to directly visualize HSPC-niche cell interactions in live animals. To identify genes expressed in the zebrafish caudal hematopoietic tissue (CHT) - an embryonic niche akin to the mammalian fetal liver - we employed a new technique called tomo-seq (RNA tomography). By pairing cryosectioning with RNA-seq, this technology permits spatial analysis of transcriptome-wide gene expression. Using tomo-seq we identified ~300 genes showing enriched expression in the CHT. In situ hybridization for 75 of 107 tested genes confirmed CHT expression. In parallel we performed RNA-seq on isolated cell populations, including endothelial cells, macrophages, neutrophils and erythrocytes, sorted from whole embryos. By cross-referencing these datasets we determined the cell types in which many of the 300 CHT-enriched genes were expressed. This analysis revealed several cell surface adhesion receptors enriched on macrophages in the CHT, including the integrin heterodimers itgam/itgb2, itgae/itgb7, itga4/itgb1b and itga4/itgb7. We examined whether known ligands for any of these integrins were present on HSPCs. In situ hybridization to vcam1 (ligand for itga4/itgb1b)showed punctate HSPC-like staining in the CHT. We then generated a vcam1:GFP promoter fusion, which we found was expressed in HSPCs. Using spinning disk confocal microscopy we imaged HSPCs and macrophages in the CHT and observed direct and specific physical interactions that preceded the engraftment of HSPCs. In a grooming-like behavior that lasts for 30-45 minutes, the HSPC is engaged by the macrophage, which moves all over the surface of the cell, before disengaging the HSPC, which then remains in the CHT. Between 48-72 hours post fertilization (hpf), 20% of HSPCs were engaged in this behavior with a macrophage. To evaluate the specificity of these interactions we established in vitro co-cultures using purified cell populations. In co-cultures between macrophages (mpeg1:mCherry) and HSPCs (cd41:GFP) we observed cell-cell interactions that were strikingly similar to those observed in vivo. In macrophage-HSPC co-cultures, 25% of cells were found to interact, whereas only 5% of cells were found to interact in macrophage-erythrocyte co-cultures. To functionally evaluate the macrophage-HSPC interactions in vivo, we depleted macrophages from zebrafish embryos at 55 hpf using clodronate liposomes and observed circulating HSPCs with a significant reduction in HSPC engraftment in the CHT (11/15 embryos, compared to the control where 14/14 embryos showed normal CHT engraftment). Together these studies establish a role for macrophages in promoting the niche engraftment of HSPCs. The results of this work could have important implications for the design of new therapies to improve engraftment during stem cell transplantation. Disclosures Zon: Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Fate, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Marauder Therapeutics: Equity Ownership, Other: Founder.

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High Resolution Imaging Reveals Hematopoietic Stem Cells in the Perivascular Niche Are Anchored to Mesenchymal Stromal Cells That Orient Their Divisions

Blood ◽

10.1182/blood.v124.21.770.770 ◽

2014 ◽

Vol 124 (21) ◽

pp. 770-770

Author(s):

Owen J. Tamplin ◽

Ellen M. Durand ◽

Logan A. Carr ◽

Sarah J. Childs ◽

Elliott H. Hagedorn ◽

...

Keyword(s):

Stem Cells ◽

Endothelial Cells ◽

Stem Cell ◽

High Resolution ◽

Board Of Directors ◽

Stromal Cells ◽

Stromal Cell ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Equity Ownership

Abstract Hematopoietic stem cells (HSC) reside in a highly structured microenvironment called the niche. There is two-way communication between a stem cell and its niche that determines important cell fate decisions. HSC must remain quiescent to persist throughout life but also divide and contribute progenitors that will replenish the blood supply. Although there have been a number of elegant studies that have imaged the mammalian bone marrow, we still lack a high-resolution real-time view of endogenous HSC behaviors and interactions within the niche. To overcome these challenges, we developed a transgenic zebrafish line that expresses GFP or mCherry in HSC. We generated this line using the previously described mouse Runx1 +23 kb intronic enhancer. We confirmed the purity of these stem cells by adult-to-adult limiting dilution transplantation with as few as one cell. Based on long-term multi-lineage engraftment, we estimated a stem cell purity of approximately 1/35, which is similar to the KSL (Kit+Sca1+Lin-) population in mouse. Using a novel embryo-to-embryo transplantation assay that is unique to zebrafish, we estimated an even higher stem cell purity of 1/2. These experiments have defined the most pure HSC population in the zebrafish. Using this novel transgenic reporter we have tracked HSC as they migrate in the live zebrafish embryo. This allowed us to image HSC as they interact with other cell types in their microenvironment, including endothelial cells and mesenchymal stromal cells. We have shown that a small group of endothelial cells remodel around a single HSC soon after it lodges in the niche. Recently, we have also found that a single stromal cell can anchor an HSC as it divides. In most cases, we observed that an HSC divides perpendicular to the stromal cell, with one daughter cell remaining attached to the stromal cell and the other migrating away. To gain a much higher resolution view of these cellular events than is possible with confocal microscopy we looked for an alternative approach. A combined method is called “Correlative Light and Electron Microscopy” (CLEM), and involves identification of cells by confocal microscopy, followed by processing of the same sample for EM scanning. We have applied this method by: 1) tracking endogenous HSC in the live embryo; 2) fixing the same embryo for serial block-face scanning EM; 3) reconstructing 3D models from high resolution serial EM sections. We used easily visible blood vessels as anatomical markers that allowed us to pinpoint a single cell in a relatively large block of scanned tissue. As expected, the identified HSC was round, had a distinctive large nucleus, scant cytoplasm, and ruffled membrane. The HSC was surrounded by a small group of 5-6 endothelial cells, as predicted from our confocal live imaging. However at this very high resolution (10 nm/pixel), we could see that only part of the HSC surface was contacted and wrapped by an endothelial cell. Other regions of the HSC surface were contacted by small endothelial cell protrusions. Much of the HSC surface was surrounded by a narrow extracellular space with endothelial and stromal cells lying opposite. Strikingly, we were able to identify the firm anchored attachment between a single stromal cell and HSC that we showed previously oriented the plane of division. By combining confocal live imaging of a novel zebrafish HSC reporter, and serial block-face scanning EM, we have created the first high-resolution 3D model of an endogenous stem cell in its niche. Disclosures Tamplin: Boston Children's Hospital: Patents & Royalties. Zon:FATE Therapeutics, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other; Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other; Stemgent: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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Gene-Correction Rescues Reprogramming of Fanconi Anemia Fibroblasts and Enables Hematopoietic Differentiation of FA Induced Pluripotent Stem Cells in Vitro and In Vivo

Blood ◽

10.1182/blood.v118.21.672.672 ◽

2011 ◽

Vol 118 (21) ◽

pp. 672-672

Author(s):

Lars Mueller ◽

Michael D. Milsom ◽

Chad Harris ◽

Rutesh Vyas ◽

Kristina Brumme ◽

...

Keyword(s):

Stem Cells ◽

Board Of Directors ◽

Hematopoietic Differentiation ◽

Gene Correction ◽

Advisory Committees ◽

Median Percentage ◽

Equity Ownership ◽

Induced Pluripotent

Abstract Abstract 672 Fanconi anemia (FA) is a recessive syndrome characterized by progressive fatal bone marrow failure and chromosomal instability. FA cells have inactivating mutations in a signaling pathway that is critical for maintaining genomic integrity and repairing DNA damage caused by cross-linking agents. Transgenic expression of the implicated genes corrects the phenotype of hematopoietic cells but previous attempts at gene therapy failed largely due to inadequate numbers of hematopoietic stem cells available for gene correction and autologous engraftment. Induced pluripotent stem cells (iPSC) constitute an alternate source of autologous cells, which are amenable to ex vivo expansion and genetic correction. While fibroblasts from a limited number of FA patients have been reported to fail to undergo reprogramming (Raya et al., Nature, 2009), reproducible observations and mechanistic studies ascertained in an extended panel of patient cells and murine knock-out models are lacking to date. We undertook direct reprogramming of ten unique human FA primary fibroblast samples of the FA-A, FA-C, FA-G, and FA-D2 complementation groups. Using standard four-factor reprogramming, no human FA iPSC colonies were obtained in cells defective in the FA pathway. By contrast, reprogramming of gene-corrected patient samples, augmented by hypoxia (5%O2), yielded multiple pluripotent iPSC lines, confirming a critical cell-intrinsic role of the FA pathway in reprogramming. To determine if gene-corrected FA iPSC could be therapeutically useful, we performed karyotype analyses and evaluated in vitro hematopoietic differentiation in three FA-A iPSC lines. These FA patient iPSC lines were karyotypically normal and showed a robust multilineage hematopoietic differentiation potential, resulting in erythroid and myeloid hematopoietic colony forming units to a similar degree as compared to normal donor iPSC controls. We hypothesized that the reprogramming resistance of FA cells is due to defective DNA repair and genomic instability. To explore the mechanisms of the reprogramming defect, we transduced wild type (wt) tail-tip fibroblasts (TTF) with the reprogramming vectors. We observed significantly increased FANCD2 foci formation during reprogramming (median percentage of FANCD2 foci: mock-transduced TTF 2.5%, reprogrammed TTF 20.5%, n=8, p<0.01) indicating activation of the FA pathway. Next, we examined reprogramming in FA-deficient mouse cells. We observed a significantly higher incidence of reprogramming-induced double-strand DNA breaks and senescence in Fanca−/− TTF as compared to wt controls (γH2AX foci: wt 13%, Fanca−/− 19%; senescence: wt 47%, Fanca−/− 62%, median percentage, p<0.01). To evaluate whether these changes contribute to the reprogramming resistance of FA cells, we quantified the reprogramming efficiency of Fanca−/−, Fancc−/− and littermate wt TTF. The efficiency was 0.06% for Fanca−/− (n=8) and 0.38% for Fancc−/− (n=12) as compared to 0.55% for wt controls (n=13; p<0.01 and <0.05, respectively). To directly test the role of the FA pathway in reprogramming, TTF were transduced with retroviral vectors co-expressing FANCA and enhanced green fluorescent protein (eGFP) or encoding only eGFP as a control. Under hypoxic conditions, gene-correction of the Fanca−/− TTF with FANCA resulted in a significant reduction of senescence and rescued the reprogramming efficiency of Fanca−/− TTF to wt levels. While significant chromosomal aberrations were observed in uncorrected Fanca−/− iPCS, gene-corrected Fanca−/− iPSC did not show any significant chromosomal imbalances when analyzed by comparative genomic hybridization. To evaluate the capacity of FA iPSC to form blood cells in vivo, we injected wt, control transduced or gene-corrected Fanca−/− iPCS (CD45.1+) into wt blastocysts (CD45.2+) and analyzed the contribution of iPSC-derived hematopoietic cells in embryonic day 14.5 fetal livers. We observed 1.8–4% wt iPSC chimerism (n=15), 0.4–0.9% Fanca−/− iPSC chimerism (n=3) and 1.5 to 2.5% chimerism in gene-corrected Fanca−/− iPSC (n=11). Our data demonstrate that reprogramming activates the FA pathway. Gene-correction rescues the reprogramming block of FA cells and protects FA iPSC from genomic instability, thus yielding an expandable source of autologous stem cells with hematopoietic differentiation capacity that may be explored for future use in regenerative medicine. Disclosures: Daley: iPierian, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Epizyme, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Verastem, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Solasia, KK: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; MPM Capital, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees.

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Inhibition Of Microenvironmental Interleukin-1 Signaling Enhances TKI-Mediated Targeting Of Chronic Myelogenous Leukemia Stem Cells

Blood ◽

10.1182/blood.v122.21.512.512 ◽

2013 ◽

Vol 122 (21) ◽

pp. 512-512 ◽

Cited By ~ 1

Author(s):

Bin Zhang ◽

Yin Wei Ho ◽

Tessa L. Holyoake ◽

Ravi Bhatia

Keyword(s):

Stem Cells ◽

Board Of Directors ◽

Chronic Myelogenous Leukemia ◽

Interleukin 1 ◽

Myelogenous Leukemia ◽

Proliferation Index ◽

Leukemia Stem Cells ◽

Advisory Committees ◽

Hematopoietic Stem

Abstract BCR-ABL tyrosine kinase inhibitors (TKI), although highly effective in inducing remission and improving survival in chronic myelogenous leukemia (CML) patients, fail to eliminate leukemia stem cells (LSC), which remain a potential source of relapse. Most CML patients need continued TKI treatment to prevent disease relapse, and new strategies to eliminate residual leukemia stem cells are required to enhance possibility of achieving treatment-free remission. In previous studies we have shown that increased several cytokines expressed by leukemia cells may provide a selective growth advantage to CML compared with normal long term hematopoietic stem cells (LTHSC) within the CML BM microenvironment. Studies evaluating the effects of individual factors indicated that exposure to Interleukin-1α/β (IL-1α/β) at concentrations similar to those observed in CML BM resulted in significantly increased growth of CML compared with normal LTHSC (Cancer Cell 2012, 21:577). Consistent with previous reports (PNAS 2010, 107:16280), we observed that expression of the IL-1 receptor-associated protein (IL-1RAP), an important IL-1 signaling component, was increased in primitive CML cells, potentially explaining enhanced IL-1 sensitivity. To further evaluate the role of microenvironmental IL-1 in maintenance of CML LTHSC, we used recombinant IL-1 receptor antagonist (IL-1RA) to block IL-1 receptor signaling. IL-1RA is clinically approved for the treatment of rheumatoid arthritis. Purified LTHSC (Lin-Sca-1+Kit+Flt3-CD150+CD48- cells) from the SCL-tTA/BCR-ABL inducible mouse model of CML (CD45.1) and from congenic FVBN mice (CD45.2) were mixed in a 1:1 ratio and cultured with CML BM plasma, with and without IL-1RA. Culture with CML BM plasma for 7 days results in significantly increased growth of CML compared to normal LTHSC. The ratio of CML to normal cells was significantly reduced in the presence of IL-1RA (2.5μg/ml) (3.6:1 without IL-1RA, 1.7:1 with IL-1RA, p=0.0002), indicating that inhibition of IL-1 signaling reduced the growth advantage of CML LTHSC cultured in CML BM plasma. We next investigated the effect of IL-1RA on CML hematopoiesis in vivo. BM cells from CML mice (CD45.1) were transplanted into congenic FVBN mice (CD45.2) to generate CML-like disease in recipient mice. Four weeks after transplantation mice were treated with Nilotinib (NIL, 50mg/kg/d, gavage), IL-1RA (150mg/kg/d s.c.), the combination of NIL and IL-1RA, or vehicle (control) for 3 weeks. Treatment with NIL plus IL-1RA resulted in significantly greater reduction in CD45.1+ CML cells in blood, and in CML LTHSC, MPP, CMP and GMP in BM, compared with NIL alone (CML LTHSC/2 femurs: control 738±122, NIL 486±94, IL-1RA 525±49, combination 360±33, P=0.01 combination vs. Nilotinib). Mice treated with NIL plus IL-1RA also showed significantly prolonged survival after completion of treatment compared to mice treated with NIL alone (median survival 6 days for NIL alone versus 45 days for combination, p=0.02). Following transplantation of BM cells from treated mice into 2nd recipients (CD45.2), significantly lower CML cell engraftment in BM and reduced development of leukemia was seen after transplantation of cells from mice treated with the combination compared with NIL or untreated controls (8 out of 8 mice developed leukemia for control, 6 out of 8 for NIL, 5 out of 8 for IL-1RA, 3 out of 8 for the combination). We also studied the effect of treatment with NIL (5μm), IL-1RA (5μg/ml), NIL+IL-1RA, or vehicle for 72 hours on human CML and normal CD34+CD38+ and CD34+CD38- cells cultured with CML BM conditioned medium (CM). The combination of NIL and IL-1RA significantly reduced CML CD34+CD38+ and CD34+CD38- cell growth compared to Nilotinib alone (CD38- cells: NIL 23.7±10.1%, combination 13.1±8.9% of control, p<0.05), cell division (measured by CFSE labeling) (CD38- proliferation index: NIL 3.3±1.0, combination 2.4±0.6, p=0.06) and CFC frequency in methylcellulose progenitor assays (CD38- cells: NIL 67±22 per 1000 cells, combination 39±26, p<0.05); and moderately increased apoptosis of CML CD34+CD38- cells. We conclude that inhibition of microenvironmental IL-1 signaling using IL-1RA significantly increases inhibition of self-renewing murine and human CML stem cells in combination with NIL. Our results support further evaluation of IL-1 inhibition as a strategy to enhance elimination of CML LSC in TKI-treated patients. Disclosures: Holyoake: Novartis: Membership on an entity’s Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity’s Board of Directors or advisory committees; Ariad: Membership on an entity’s Board of Directors or advisory committees.

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Hematopoietic Stem Cells Develop in the Absence of Endothelial Cadherin 5 Expression

Blood ◽

10.1182/blood.v126.23.1165.1165 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1165-1165

Author(s):

Heidi Anderson ◽

Taylor Patch ◽

Pavan Reddy ◽

Elliott Hagedorn ◽

Owen J. Tamplin ◽

...

Keyword(s):

Stem Cells ◽

Endothelial Cells ◽

Hematopoietic Stem Cells ◽

Board Of Directors ◽

Research Funding ◽

Hemogenic Endothelium ◽

Employment Equity ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Equity Ownership

Abstract Rare endothelial cells in the aorta-gonad-mesonephros (AGM) transition into hematopoietic stem cells (HSCs) during embryonic development. Lineage tracing experiments indicate that HSCs emerge from Cadherin 5 (Cdh5, VE-cadherin)+ endothelial precursors, and isolated populations of Cdh5+ cells from mouse embryos and embryonic stem (ES) cells can be differentiated into hematopoietic cells. Cdh5 has also been widely implicated as a marker of AGM-derived hemogenic endothelial cells. Since Cdh5-/- mice embryos die before the first HSCs emerge, it is unknown if Cdh5 has a direct role in HSC emergence. Our previous genetic screen yielded malbec (mlbbw306), a zebrafish mutant for cdh5, with normal embryonic and definitive blood. Utilizing time-lapse imaging, parabiotic surgical pairing of zebrafish embryos, and blastula transplantation assays, we show that HSCs emerge, migrate, engraft, and differentiate in the absence of cdh5 expression. By tracing Cdh5-/- GFP+/+ cells inchimeric mice, we demonstrated that Cdh5-/- GFP+/+ HSCs emerging from E10.5 and E11.5 AGM or derived from E13.5 fetal liver not only differentiate into hematopoietic colonies but also engraft and reconstitute multi-lineage adult blood. These data establish that Cdh5, a marker of hemogenic endothelium in the AGM, is dispensable for the transition of hemogenic endothelium to HSCs. Disclosures Bauer: Biogen: Research Funding; Editas Medicine: Consultancy. Zon:FATE Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Scholar Rock: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder. Orkin:Editas Medicine: Membership on an entity's Board of Directors or advisory committees; Biogen: Research Funding; Pfizer: Research Funding; Sangamo Biosciences: Consultancy.

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Angiogenin Is a Niche-Secreted RNase Which Regulates Hematopoietic Regeneration

Blood ◽

10.1182/blood.v128.22.171.171 ◽

2016 ◽

Vol 128 (22) ◽

pp. 171-171

Author(s):

Lev Silberstein ◽

Kevin Goncalves ◽

Nicholas Severe ◽

Guo-fu Hu ◽

David T. Scadden

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Board Of Directors ◽

Advisory Committees ◽

Self Renewal ◽

Cellular Source ◽

Equity Ownership ◽

Ng2 Cells

Abstract Background. Identification of novel niche factors is critical for understanding of regulatory mechanisms which control HSPC cell fate decisions and design of novel pro-regenerative therapies. We have developed a proximity-based differential single cell analysis approach to the study of the bone marrow niche, which showed that individual osteolineage cells located in close proximity to transplanted HSPC are enriched for expression of niche factors, and have previously reported identification of IL18 and Embigin as regulators of HSPC quiescence. Here we describe the results of in vivo validation of Angiogenin (ANG) - the third molecule highlighted using the above strategy - as a potent regulator of HSPC quiescence and regeneration. Results. ANG is a secreted RNase which is known to promote angiogenesis, proliferation of cancer cells and also enhance cell survival in response to stress. Analysis of primitive cells subsets in the ANG knock-out mice (AngKO mice) revealed a 1.4-fold increase in the frequency and absolute number of long-term hematopoietic stem cells (LT-HSCs). Subsequent BrdU incorporation and cell cycle studies demonstrated increased proliferative activity in the primitive HSPC compartment indicating that ANG regulates HSPC quiescence. To confirm these findings functionally and to assess the effect of ANG on self-renewal, we exposed AngKO animals to weekly 5-FU injections and performed serial transplantation experiments of WT LT-HSCs into AngKO hosts. We noted significantly increased mortality of AngKO mice in both experimental settings; in a competitive transplant assay, we observed almost complete absence of engraftment by WT cells in the secondary hosts, in keeping with the exhaustion phenotype. Consistently, exposure of AngKO animals to a different type of hematopoietic stress, such as ageing, resulted in development of peripheral blood cytopenias and marked reduction in the number and frequency of HSPC. ANG is expressed in multiple non-hematopoietic cell types in the bone marrow, including osteoprogenitors, mature osteoblasts and nestin-positive mesenchymal stem cells and NG2-positive arteriolar sheath cells. To establish the predominant cellular source of ANG in the niche, we crossed Ang "floxed" mice with the animals in which tamoxifen-inducible Cre-recombinase was driven by the promoters targeting these cell subsets and examined the effect on hematopoiesis. We found that deletion of ANGfrom Osx+, Nes+ and NG2+ cells resulted in an increase of the number of LT-HSC and more active cycling of LT-HSC, short-term HSC (ST-HSC) and multi-potent progenitors (MPP) while ANGdeletion in mature osteoblasts had no effect on these cell populations, but was associated with an increase in number and more active cycling of common lymphoid progenitors (CLP), as was also seen upon ANGdeletion from Nes+ and NG2+ cells. These results indicate that the target cell population which is regulated by ANG depends on the cellular source. Interestingly, transplantation of WT bone marrow into the animals with Osx-specific ANG deletion resulted in development of macrocytic anemia and neutropenia at 6 months, thus indicating that Angiogenin deficiency in the niche is sufficient for the development of the hematopoietic failure. Impaired long-term reconstitution was also observed when ANG was deleted from Nestin+ mesenchymal stem cells but not col1+ mature osteoblasts in the recipient mice. Our findings that the absence of ANG negatively impacts HSPC self-renewal prompted us to investigate whether exposure of HSPC to recombinant ANG protein will have the opposite effect and enhance hematopoietic regeneration. We therefore treated mouse LT-HSCs with recombinant ANG or vehicle control ex-vivo for 2 hours and competitively transplanted them into lethally irradiated WT recipients. We found that ANG-treated LT-HSCs displayed a significantly higher long-term reconstituting ability, which persisted into the secondary transplants. Similar data were obtained following treatment of CD34+ cord blood cells with human ANG. Conclusion. Our work defines ANG as a previously unrecognized regulator of HSPC quiescence and self-renewal and suggests that it can be explored as a potential therapeutic agent to promote hematopoietic regeneration. Disclosures Scadden: Teva: Consultancy; Apotex: Consultancy; Bone Therapeutics: Consultancy; GlaxoSmithKline: Research Funding; Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Fate Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Dr. Reddy's: Consultancy.

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Hematopoietic Stem Cell Niche Interactions In The Embryo Can Modulate The Size Of The Stem Cell Pool Into Adulthood

Blood ◽

10.1182/blood.v122.21.585.585 ◽

2013 ◽

Vol 122 (21) ◽

pp. 585-585

Author(s):

Owen J. Tamplin ◽

Ellen M. Durand ◽

Logan A. Carr ◽

Pulin Li ◽

Leonard I. Zon

Keyword(s):

Stem Cell ◽

Board Of Directors ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Cell Pool ◽

Hsc Niche ◽

Equity Ownership ◽

Stem Cell Pool

Abstract Hematopoietic stem cells (HSC) reside in the bone marrow niche and sustain the production of blood throughout life. The entire pool of these rare and important cells is generated during a brief window of embryonic development. HSC are produced by the hemogenic endothelium of the dorsal aorta, migrate to and expand in the fetal liver, and then migrate again to seed the bone marrow. The zebrafish is a highly conserved and well-established model for HSC development. Similar to mammals, HSC emerge from the dorsal aorta, but then colonize a vascular plexus in the tail of the embryo—the caudal hematopoietic tissue (CHT). It is difficult to directly observe the interactions between an endogenous HSC and its niche, so we have developed the CHT as a model for HSC-niche interactions. To track HSC in vivo we have generated a transgenic reporter using the previously described mouse Runx1 +23 kb intronic enhancer. The purity of the stem cell pool marked by this reporter was determined. Using adult-to-adult limiting dilution transplantation with as few as one Runx1+23 positive cell, we have estimated the HSC purity to be approximately 1/35 (without immune matching), or similar to Kit+Sca1+Lin- (KSL) in mouse. This is the most pure stem cell population defined in the zebrafish system. Using embryo-to-embryo transplantation, a technique that is unique to zebrafish, we sorted Runx1+23 positive cells from one group of embryos and transplanted them to another by injection directly into circulation. Embryos are then grown to adulthood and marrow is tested for long-term engraftment between 3 and 5 months. This transplantation technique precedes formation of the thymus, thereby removing any chance of immune rejection. Highly stringent dilution of HSC in our embryo-to-embryo transplants has estimated a stem cell purity of one in two cells. Next, we applied our highly specific reporter to visualize HSC migration to the CHT niche. After arrival of the HSC, we have described 5 distinct steps during colonization: 1) adherence; 2) extravasation; 3) abluminal migration; 4) endothelial niche formation (“cuddling”); and 5) cell fate decisions. Live imaging analysis of HSC together with endothelial and stromal transgenic reporters has allowed us to quantify the relationship between different cell types within the CHT. For example, we observe preferential localization of HSC in close proximity to cxcl12a positive stromal cells. Lastly, we have sought to identify the molecular mechanisms involved in interactions between HSC and their niche. A chemical genetic screen identified the natural product lycorine as a small molecule that increases hematopoiesis in the CHT and promotes HSC-endothelial cell interactions. Combined chemical treatment and live imaging revealed that lycorine significantly increased the residence time of HSC in the niche. To test if treatment during the window of CHT colonization (2-3 days post fertilization) had long-term effects on HSC and the stem cell pool, the compound was washed off at 3 days and the Runx1+23 positive population was quantified by FACS. At 7 days post fertilization, after colonization of the marrow, there was a sustained and significant increase in Runx1+23 positive HSC. Strikingly, after 3 months, when treated embryos were raised to adulthood, we discovered that the increased HSC-endothelial cell interactions we observed in the CHT niche had in fact had an impact on the number of HSC in the adult. Our studies establish that the Runx1+23 transgenic is a highly specific reporter of HSC both in the embryo and adult, and that we can use this reporter for in vivo observation of an endogenous HSC niche. Furthermore, we show that the size of the adult stem cell pool can be altered by a transient signal during development. Disclosures: Tamplin: Boston Children's Hospital: Patents & Royalties. Zon:FATE Therapeutics, Inc: Consultancy, Equity Ownership, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Stemgent, Inc: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Stocks, Stocks Other; Scholar Rock: Consultancy, Equity Ownership, Founder, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

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