Neonatal Recipients Offer Permissive Hematopoietic Microenvironment for Engraftment of Embryonic Murine Hematopoietic Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2344-2344
Author(s):  
Natasha Arora ◽  
Shannon McKinney-Freeman ◽  
Garrett C Heffner ◽  
Il-Ho Jang ◽  
Pamela L. Wenzel ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Board Of Directors ◽  
Early Embryo ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Hematopoietic Microenvironment ◽  
Equity Ownership

Abstract Abstract 2344 The first hematopoietic stem cells (HSC) that give rise to robust, long-term, multi-lineage reconstitution in irradiated adult recipients arise in the murine embryo at embryonic day 11.5 (E11.5). However, long-term multi-lineage engraftment in neonatal recipients has been observed from E9.0 yolk sac, suggesting that the neonatal hematopoietic microenvironment is more permissive for engraftment of embryonic HSCs. To resolve the apparent discrepancy between the numbers of candidate HSCs detected by direct visualization in the early embryo, relative to the numbers that can be measured by limiting dilution, we sought to characterize engraftment of neonatal recipients versus adult recipients with hematopoietic populations dissected from the aorta-gonad-mesonephros (AGM) region of the early embryo, the first putative site of intraembryonic origin of definitive HSCs. We dissected whole AGM from E11.5 embryos and injected cell dilutions from 2 embryo equivalents (ee) to 0.25 ee into the facial vein of day 1–2 neonatal recipients that had received sublethal conditioning with 350 rad irradiation. In neonatal recipients we detected robust, long-term, multi-lineage hematopoietic engraftment from as little as 0.25 ee. From less than 1 ee of whole AGM, the engraftment chimerism ranged from 5–20%. With 2 ee, chimerism was as high as 70%. Most animals showed balanced donor derived myeloid and lymphoid contribution by 10 weeks post-transplant. Interestingly, some animals had predominantly myeloid reconstitution for as long as 18 weeks, suggesting the presence of a novel long-term, myeloid-restricted, embryonic HSC. We also explored the neonatal engraftment potential of VE-cadherin+ CD45+ and VE-cadherin+ CD45− populations. As expected from the literature, only the VE-cadherin+ CD45+ population engrafted the neonatal recipients. Our data indicate that the neonate harbors a more permissive hematopoietic microenvironment that enables more robust engraftment of early embryonic hematopoietic populations, thereby allowing us to identify potentially novel classes of embryonic hematopoietic progenitors. We are currently exploring the neonatal engraftment potential of E9.5 and E10.5 embryonic populations, additional FACS-purified populations, and hematopoietic populations derived from pluripotent stem cells in vitro. 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.

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

Blood ◽  
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.

Msi2 Maintains Normal Hematopoietic Stem Cell Self-Renewal

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 222-222 ◽  
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.

Chd7 Is a Cell Autonomous Regulator of Chromatin In Hematopoietic Stem Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1596-1596
Author(s):  
Hsuan-Ting Huang ◽  
Katie Kathrein ◽  
Yue-Hua Huang ◽  
Zachary Gitlin ◽  
Abby Barton ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Board Of Directors ◽  
Cell Formation ◽  
Charge Syndrome ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Knock Down ◽  
Cell Embryo ◽  
Equity Ownership

Abstract Abstract 1596 Hematopoietic stem cells (HSC) are specified during embryogenesis, and the induction process involves not only transcription factors but also epigenetic factors that modulate chromatin to regulate the hematopoietic transcriptional programs. Here, we performed a reverse genetic screen to identify all the chromatin factors that are required for HSC induction in zebrafish. The zebrafish homologs of 350 human chromatin factors were identified by reciprocal BLAST and knocked down by injecting morpholinos designed against each homolog into the single cell embryo. Morphants were then analyzed for changes in blood formation by in situ hybridization for β-globin e3 expression in primitive erythrocytes at 16 somite stage and for c-myb and runx1 in definitive stem cells at 36 hours post fertilization. From the screen, we have identified known regulators of hematopoiesis such as bmi1, in which knock down results in loss of stem cell formation. We have also identified one novel HSC regulator chd7. Chd7 is a member of the chromodomain helicase DNA-binding domain family that functions at gene enhancer elements and in ribosomal RNA synthesis. Zebrafish embryos injected with chd7 morpholino had higher levels of β-globin e3 and c-myb/runx1 expression. Additional markers such as scl, gata1, fli1, and lmo2 were also upregulated, although vascular markers flk1 and ephrinB2 were downregulated. Early mesodermal markers eve1 and ntl expression appeared normal, suggesting that the effects of chd7 knock down occurs when the mesodermal precursor cell population becomes an HSC. Transplants of chd7 deficient Tg(c-myb:GFP) blastomeres into Tg(lmo2:DsRed) blastulas resulted in more chimeric embryos compared to controls, demonstrating that the phenotype is cell autonomous. In humans, haploinsufficiency for CHD7 is the main cause of CHARGE syndrome, and it has been recognized more recently that these patients are immunodeficient, though the etiology remains unknown. Our studies indicate a new role for chd7 in hematopoiesis in which it functions to repress HSC formation during embryogenesis. Disclosures: Zon: FATE, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Stemgent: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

2020 ◽  
Vol 15 ◽  
Author(s):  
Fatima Aerts-Kaya
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Stress Conditions ◽  
Stress Factors ◽  
Hematopoietic Stem ◽  
Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

scholarly journals Hlf Expression Marks Early Emergence of Hematopoietic Stem Cell Precursors With Adult Repopulating Potential and Fate

2021 ◽  
Vol 9 ◽  
Author(s):  
Wanbo Tang ◽  
Jian He ◽  
Tao Huang ◽  
Zhijie Bai ◽  
Chaojie Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mouse Model ◽  
Hematopoietic Stem Cells ◽  
Fetal Liver ◽  
Lineage Tracing ◽  
Genetic Lineage ◽  
Hematopoietic Stem ◽  
Genetic Lineage Tracing

In the aorta-gonad-mesonephros (AGM) region of mouse embryos, pre-hematopoietic stem cells (pre-HSCs) are generated from rare and specialized hemogenic endothelial cells (HECs) via endothelial-to-hematopoietic transition, followed by maturation into bona fide hematopoietic stem cells (HSCs). As HECs also generate a lot of hematopoietic progenitors not fated to HSCs, powerful tools that are pre-HSC/HSC-specific become urgently critical. Here, using the gene knockin strategy, we firstly developed an Hlf-tdTomato reporter mouse model and detected Hlf-tdTomato expression exclusively in the hematopoietic cells including part of the immunophenotypic CD45– and CD45+ pre-HSCs in the embryonic day (E) 10.5 AGM region. By in vitro co-culture together with long-term transplantation assay stringent for HSC precursor identification, we further revealed that unlike the CD45– counterpart in which both Hlf-tdTomato-positive and negative sub-populations harbored HSC competence, the CD45+ E10.5 pre-HSCs existed exclusively in Hlf-tdTomato-positive cells. The result indicates that the cells should gain the expression of Hlf prior to or together with CD45 to give rise to functional HSCs. Furthermore, we constructed a novel Hlf-CreER mouse model and performed time-restricted genetic lineage tracing by a single dose induction at E9.5. We observed the labeling in E11.5 AGM precursors and their contribution to the immunophenotypic HSCs in fetal liver (FL). Importantly, these Hlf-labeled early cells contributed to and retained the size of the HSC pool in the bone marrow (BM), which continuously differentiated to maintain a balanced and long-term multi-lineage hematopoiesis in the adult. Therefore, we provided another valuable mouse model to specifically trace the fate of emerging HSCs during development.

Long-Term Ex Vivo Maintenance and Expansion of Transplantable Human Hematopoietic Stem Cells

Blood ◽  
1999 ◽  
Vol 94 (5) ◽  
pp. 1623-1636 ◽  
Author(s):  
Chu-Chih Shih ◽  
Mickey C.-T. Hu ◽  
Jun Hu ◽  
Jeffrey Medeiros ◽  
Stephen J. Forman
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
In Vitro Culture ◽  
Culture System ◽  
Ex Vivo ◽  
Human Stem Cells ◽  
Hematopoietic Stem ◽  
In Vitro Culture System

Abstract We have developed a stromal-based in vitro culture system that facilitates ex vivo expansion of transplantable CD34+thy-1+ cells using long-term hematopoietic reconstitution in severe combined immunodeficient-human (SCID-hu) mice as an in vivo assay for transplantable human hematopoietic stem cells (HSCs). The addition of leukemia inhibitory factor (LIF) to purified CD34+ thy-1+ cells on AC6.21 stroma, a murine bone marrow–derived stromal cell line, caused expansion of cells with CD34+ thy-1+ phenotype. Addition of other cytokines, including interleukin-3 (IL-3), IL-6, granulocyte-macrophage colony-stimulating factor, and stem cell factor, to LIF in the cultures caused a 150-fold expansion of cells retaining the CD34+ thy-1+ phenotype. The ex vivo–expanded CD34+ thy-1+ cells gave rise to multilineage differentiation, including myeloid, T, and B cells, when transplanted into SCID-hu mice. Both murine LIF (cannot bind to human LIF receptor) and human LIF caused expansion of human CD34+ thy-1+ cells in vitro, suggesting action through the murine stroma. Furthermore, another human HSC candidate, CD34+ CD38− cells, shows a similar pattern of proliferative response. This suggests thatex vivo expansion of transplantable human stem cells under this in vitro culture system is a general phenomenon and not just specific for CD34+ thy-1+ cells.

scholarly journals Gene transfer into hematopoietic stem cells: long-term maintenance of in vitro activated progenitors without marrow ablation.

1994 ◽  
Vol 91 (1) ◽  
pp. 350-354 ◽  
Author(s):  
D. Bienzle ◽  
A. C. Abrams-Ogg ◽  
S. A. Kruth ◽  
J. Ackland-Snow ◽  
R. F. Carter ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Transfer ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem ◽  
Marrow Ablation ◽  
Term Maintenance

Reversible Expression of CD34 by Murine Hematopoietic Stem Cells

Blood ◽  
1999 ◽  
Vol 94 (8) ◽  
pp. 2548-2554 ◽  
Author(s):  
Takashi Sato ◽  
Joseph H. Laver ◽  
Makio Ogawa
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Cell Populations ◽  
Hematopoietic Stem ◽  
Adult Mice ◽  
Cd34 Expression ◽  
Marrow Cells

We used a mouse transplantation model to address the recent controversy about CD34 expression by hematopoietic stem cells. Cells from Ly-5.1 C57BL/6 mice were used as donor cells and Ly-5.2 mice were the recipients. The test cells were transplanted together with compromised marrow cells of Ly-5.2 mice. First, we confirmed that the majority of the stem cells with long-term engraftment capabilities of normal adult mice are CD34−. We then observed that, after the injection of 150 mg/kg 5-fluorouracil (5-FU), stem cells may be found in both CD34− and CD34+ cell populations. These results indicated that activated stem cells express CD34. We tested this hypothesis also by using in vitro expansion with interleukin-11 and steel factor of lineage−c-kit+ Sca-1+ CD34− bone marrow cells of normal mice. When the cells expanded for 1 week were separated into CD34− and CD34+ cell populations and tested for their engraftment capabilities, only CD34+ cells were capable of 2 to 5 months of engraftment. Finally, we tested reversion of CD34+ stem cells to CD34− state. We transplanted Ly-5.1 CD34+post–5-FU marrow cells into Ly-5.2 primary recipients and, after the marrow achieved steady state, tested the Ly-5.1 cells of the primary recipients for their engraftment capabilities in Ly-5.2 secondary recipients. The majority of the Ly-5.1 stem cells with long-term engraftment capability were in the CD34− cell fraction, indicating the reversion of CD34+ to CD34−stem cells. These observations clearly demonstrated that CD34 expression reflects the activation state of hematopoietic stem cells and that this is reversible.

scholarly journals The chemokine GROβ mobilizes early hematopoietic stem cells characterized by enhanced homing and engraftment

Blood ◽  
2007 ◽  
Vol 110 (3) ◽  
pp. 860-869 ◽  
Author(s):  
Seiji Fukuda ◽  
Huimin Bian ◽  
Andrew G. King ◽  
Louis M. Pelus
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem ◽  
Platelet Recovery ◽  
And Function ◽  
Stimulating Factor ◽  
Cxcr4 Axis

Abstract Mobilized peripheral blood hematopoietic stem cells (PBSCs) demonstrate accelerated engraftment compared with bone marrow; however, mechanisms responsible for enhanced engraftment remain unknown. PBSCs mobilized by GROβ (GROβΔ4/CXCL2Δ4) or the combination of GROβΔ4 plus granulocyte colony-stimulating factor (G-CSF) restore neutrophil and platelet recovery faster than G-CSF–mobilized PBSCs. To determine mechanisms responsible for faster hematopoietic recovery, we characterized immunophenotype and function of the GROβ-mobilized grafts. PBSCs mobilized by GROβΔ4 alone or with G-CSF contained significantly more Sca-1+-c-kit+-lineage− (SKL) cells and more primitive CD34−-SKL cells compared with cells mobilized by G-CSF and demonstrated superior competitive long-term repopulation activity, which continued to increase in secondary and tertiary recipients. GROβΔ4-mobilized SKL cells adhered better to VCAM-1+ endothelial cells compared with G-CSF–mobilized cells. GROβΔ4-mobilized PBSCs did not migrate well to the chemokine stromal derived factor (SDF)-1α in vitro that was associated with higher CD26 expression. However, GROβΔ4-mobilized SKL and c-Kit+ lineage− (KL) cells homed more efficiently to marrow in vivo, which was not affected by selective CXCR4 and CD26 antagonists. These data suggest that GROβΔ4-mobilized PBSCs are superior in reconstituting long-term hematopoiesis, which results from differential mobilization of early stem cells with enhanced homing and long-term repopulating capacity. In addition, homing and engraftment of GROβΔ4-mobilized cells is less dependent on the SDF-1α/CXCR4 axis.

Epoxyeicosatrienoic Acids Regulate Hematopoietic Stem/Progenitor Cell Fate Decision During Stress Response and Embryonic Hematopoiesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 860-860
Author(s):  
Pulin Li ◽  
Emily K Pugach ◽  
Elizabeth B Riley ◽  
Dipak Panigrahy ◽  
Garrett C Heffner ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Board Of Directors ◽  
Marrow Transplantation ◽  
Mouse Bone Marrow ◽  
Zebrafish Embryos ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Equity Ownership ◽  
Runx1 Expression

Abstract Abstract 860 During bone marrow transplantation, hematopoietic stem/progenitor cells (HSPCs) are exposed to various stress signals, and undergo homing, rapid proliferation and differentiation in order to achieve engraftment. To explore how fate decisions are made under such stress conditions, we developed a novel imaging-based competitive marrow transplantation in zebrafish. The feasibility of handling hundreds of zebrafish for transplantation per day allowed us to screen a library of 480 small molecules with known bioactivity, aimed at identifying new drugs and pathways regulating HSPC engraftment. Two structurally related eicosanoids, 11,12-epoxyeicosatrienoic acid (EET) and 14,15-EET, were able to enhance GFP+ marrow engraftment compared to DsRed2+ engraftment in zebrafish. This remarkable effect of EETs on adult marrow prompted us to study the effect of EETs in embryonic hematopoiesis. Treating zebrafish embryos with 11,12-EET during definitive hematopoiesis increased the HSPC marker Runx1 expression in the AGM (Aorta-Gonad-Mesonephros), resulting in a significant increase of HSPC in the next hematopoietic site, caudal hematopoietic tissue, the equivalent of fetal liver/placenta in mammals. The same treatment condition also induced ectopic Runx1 expression in the tail mesenchyme, a non-hematopoietic tissue. Microarray analysis on EET-treated zebrafish embryos revealed an upregulation of genes involved in stress response, especially Activator Protein 1 (AP-1) family members. Genetic knockdown experiments confirmed AP-1 members, especially JunB and its binding partners, cFos and Fosl2, are required for Runx1 induction. Motif analysis also predicted several conserved AP-1 binding sites in the Runx1 enhancer regions. To understand how EETs induced AP-1 expression, a suppressor screen was performed in zebrafish embryos. The screen revealed that activation of both PI3K/Akt and Stat3 are required for induced AP-1 expression, and therefore Runx1 upregulation. Similarly, ex vivo treatment of mouse whole bone marrow with 11,12-EET resulted in a 2-fold increase of long-term repopulating units. Microarray data had previously shown that Cyp2j6, one of the cytochrome P450 enzymes involved in EET biosynthesis from arachidonic acid, is enriched in quiescent mouse long-term HSCs. To further increase the EET levels in HSPCs, human CYP2C8 enzyme was over-expressed in transgenic mice using the Tie2 promoter. These transgenic mice have a 4-fold increase of long-term multi-lineage repopulating unit compared to their wild-type siblings. In purified mouse HSPCs, EETs directly and cell-autonomously activate PI3K/AKT pathway. Co-treatment of mouse bone marrow with EET and a PI3K inhibitor, LY294,002, completely blocked EET-induced enhancement of mouse bone marrow engraftment. In conclusion, we performed the first competitive marrow transplantation-based chemical screen, leading to the discovery of arachidonic acid-cytochrome P450-EETs as a novel modulator of HSC cell fate decision. PI3K/Akt and Stat3 pathways activated by EETs are required for adult HSPC engraftment and/or embryonic HSC specification, partially through transcriptional regulation of AP-1. We also demonstrated the requirement of AP-1 family members for Runx1 expression during embryonic development. This discovery may have clinical application in marrow or cord blood transplantation. 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. Zon:Fate Therapeutics: Founder; Stemgent: Consultancy.

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