Human Hematopoietic Stem/progenitor Cells Research Articles

Genome editing using CRISPR/Cas9 to treat hereditary hematological disorders.

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system is a versatile and convenient genome-editing tool with prospects in gene therapy. This technique is based on customized site-specific nucleases with programmable guiding RNAs that cleave and introduce double-strand breaks (DSBs) at the target locus and achieve precise genome modification by triggering DNA repair mechanisms. Human hematopoietic stem/progenitor cells (HSPCs) are conventional cell targets for gene therapy in hematological diseases and have been widely used in most studies. Induced pluripotent stem cells (iPSCs) can be generated from a variety of somatic cells and hold great promise for personalized cell-based therapies. CRISPR/Cas9-mediated genome editing in autologous HSPCs and iPSCs is an ideal therapeutic solution for treating hereditary hematological disorders. Here, we review and summarize the latest studies about CRISPR/Cas9-mediated genome editing in patient-derived HSPCs and iPSCs to treat hereditary hematological disorders. Current challenges and prospects are also discussed.

Correction of β-thalassemia by CRISPR/Cas9 editing of the α-globin locus in human hematopoietic stem cells

Abstractβ-thalassemias (β-thal) are a group of blood disorders caused by mutations in the β-globin gene (HBB) cluster. β-globin associates with α-globin to form adult hemoglobin (HbA, α2β2), the main oxygen-carrier in erythrocytes. When β-globin chains are absent or limiting, free α-globins precipitate and damage cell membranes, causing hemolysis and ineffective erythropoiesis. Clinical data show that severity of β-thal correlates with the number of inherited α-globin genes (HBA1 and HBA2), with α-globin gene deletions having a beneficial effect for patients. Here, we describe a novel strategy to treat β-thal based on genome editing of the α-globin locus in human hematopoietic stem/progenitor cells (HSPCs). Using CRISPR/Cas9, we combined 2 therapeutic approaches: (1) α-globin downregulation, by deleting the HBA2 gene to recreate an α-thalassemia trait, and (2) β-globin expression, by targeted integration of a β-globin transgene downstream the HBA2 promoter. First, we optimized the CRISPR/Cas9 strategy and corrected the pathological phenotype in a cellular model of β-thalassemia (human erythroid progenitor cell [HUDEP-2] β0). Then, we edited healthy donor HSPCs and demonstrated that they maintained long-term repopulation capacity and multipotency in xenotransplanted mice. To assess the clinical potential of this approach, we next edited β-thal HSPCs and achieved correction of α/β globin imbalance in HSPC-derived erythroblasts. As a safer option for clinical translation, we performed editing in HSPCs using Cas9 nickase showing precise editing with no InDels. Overall, we described an innovative CRISPR/Cas9 approach to improve α/β globin imbalance in thalassemic HSPCs, paving the way for novel therapeutic strategies for β-thal.

Renin-angiotensin system is involved in embryonic emergence of hematopoietic stem/progenitor cells.

Angiotensin-converting enzyme (ACE), a key element of the renin-angiotensin system (RAS), has recently been identified as a new marker of both adult and embryonic human hematopoietic stem/progenitor cells (HSPCs). However, whether a full renin-angiotensin pathway is locally present during the hematopoietic emergence is still an open question. In the present study, we show that this enzyme is expressed by hematopoietic progenitors in the developing mouse embryo. Furthermore, ACE and the other elements of RAS-namely angiotensinogen, renin, and angiotensin II type 1 (AT1) and type 2 (AT2) receptors-are expressed in the paraaortic splanchnopleura (P-Sp) and in its derivative, the aorta-gonad-mesonephros region, both in human and mouse embryos. Their localization is compatible with the existence of a local autocrine and/or paracrine RAS in these hemogenic sites. in vitro perturbation of the RAS by administration of a specific AT1 receptor antagonist inhibits almost totally the generation of blood CD45-positive cells from dissected P-Sp, implying that angiotensin II signaling is necessary for the emergence of hematopoietic cells. Conversely, addition of exogenous angiotensin II peptide stimulates hematopoiesis in culture, with an increase in the number of immature c-Kit+ CD41+ CD31+ CD45+ hematopoietic progenitors, compared to the control. These results highlight a novel role of local-RAS during embryogenesis, suggesting that angiotensin II, via activation of AT1 receptor, promotes the emergence of undifferentiated hematopoietic progenitors.

Modeling, optimization, and comparable efficacy of T cell and hematopoietic stem cell gene editing for treating hyper‐IgM syndrome

Precise correction of the CD40LG gene in T cells and hematopoietic stem/progenitor cells (HSPC) holds promise for treating X‐linked hyper‐IgM Syndrome (HIGM1), but its actual therapeutic potential remains elusive. Here, we developed a one‐size‐fits‐all editing strategy for effective T‐cell correction, selection, and depletion and investigated the therapeutic potential of T‐cell and HSPC therapies in the HIGM1 mouse model. Edited patients’ derived CD4 T cells restored physiologically regulated CD40L expression and contact‐dependent B‐cell helper function. Adoptive transfer of wild‐type T cells into conditioned HIGM1 mice rescued antigen‐specific IgG responses and protected mice from a disease‐relevant pathogen. We then obtained ~ 25% CD40LG editing in long‐term repopulating human HSPC. Transplanting such proportion of wild‐type HSPC in HIGM1 mice rescued immune functions similarly to T‐cell therapy. Overall, our findings suggest that autologous edited T cells can provide immediate and substantial benefits to HIGM1 patients and position T‐cell ahead of HSPC gene therapy because of easier translation, lower safety concerns and potentially comparable clinical benefits.

Editing the LRF Repressor Binding Site in the γ-Globin Promoters Induces Therapeutically Relevant Fetal Hemoglobin Levels for the Treatment of β-Hemoglobinopathies

β-hemoglobinopathies are genetic anemias caused by a reduced or abnormal synthesis of the adult β-globin chain. In β-thalassemia, the reduced (β+) or absent (β0) production of adult β-chains causes α-globin precipitation and death of red blood cell (RBC) precursors. In sickle cell disease (SCD), a single amino acid change (β6Glu→Val) in the adult hemoglobin (Hb) βS-chain causes Hb polymerization with consequent red blood cell (RBC) sickling, vaso-occlusive crises, organ damage and reduced life expectancy. The co-inheritance of genetic mutations causing a sustained fetal γ-globin chain production in adult life (hereditary persistence of fetal hemoglobin, HPFH) reduces the clinical severity of β-hemoglobinopathies. HPFH mutations in the promoter of the two γ-globin genes, HBG1 and HBG2 disrupt the binding sites (BS) for transcriptional repressors (e.g., BCL11A and LRF). Recently, we demonstrated that CRISPR/Cas9-mediated disruption of the LRF BS in the HBG promoters via non-homologous end joining and microhomology-mediated end joining (MMEJ) repair mechanisms mimics the effect of HPFH mutations by impairing the LRF binding and re-activating the γ-globin expression (Weber, Frati et al., Science Advances, 2020). Efficient editing of the LRF BS (≥ 3 γ-globin promoters in >70% of SCD hematopoietic stem/progenitor cells (HSPCs)) resulted in a robust HbF reactivation and a concomitant reduction in βS-globin levels recapitulating the phenotype of asymptomatic SCD-HPFH patients. RBCs derived from edited HSPCs displayed HbF levels sufficient to correct the SCD cell phenotype. Similarly, LRF BS targeting in β0-thalassemic cells results in HbF reactivation potentially correcting the α/β-like globin imbalance. Xenotransplantation of human HSPCs edited using several gRNAs targeting the LRF BS showed a robust engraftment of edited cells that were capable to differentiate into multiple lineages. HBG editing in engrafted cells ranged from 26% to 76% with a decrease (-33%) of editing events compared to the input HSPCs, partially due to a reduced occurrence of MMEJ-mediated events in hematopoietic stem cells (HSCs). Erythroid progenitors (BFU-E) obtained ex vivo from engrafted human cells, showed a relevant γ-globin expression (~40% of the total β-like chain) despite of the reduction in the number of edited promoters per BFU-E after transplantation. Moreover, mature RBCs ex vivo differentiated from edited human cells ensure therapeutically relevant HbF levels. Sequencing of top-scoring off-targets identified by GUIDE-seq showed a relatively high off-target activity within an intergenic site devoid of known regulatory elements both in vitro and in vivo in primary human cells treated with one of the gRNAs targeting the LRF BS. Although the occurrence of this off-target event in repopulating cells suggests that it has no detrimental effect on HSC engraftment and multilineage differentiation, we tested high fidelity Cas9 variants to reduce off-target activity in primary HSPCs. Finally, we used CAST-Seq assay to evaluate the potential chromosomal rearrangements in edited primary human cells in vitro and in vivo. To minimize the potential genotoxicity associated to Cas9 nuclease-mediated double-strand breaks (DSBs) in the genome, we explored the base editing system to introduce C>T point mutations in the LRF BS without generating DSBs. The absence of the canonical SpyCas9 NGG PAM close to the LRF BS, prompted us to use known and novel base editors containing non-NGG Cas9 variants that allowed the editing of up to 6 out of 8 cytosines of the LRF BS in erythroid cell lines and in primary HSPCs from SCD patients. These C>T conversions include not only known HPFH mutations but also mutations that can further impair LRF binding. In the majority of cases, we detected no insertions or deletions in base-edited samples, as compared to nuclease-edited samples. The 4.9-kb deletion that can be generated upon cleavage of the two identical HBG promoters by the Cas9 nuclease was barely detected in base-edited samples. Importantly, disruption of the LRF BS by non-NGG enzymes led to HbF de-repression, without affecting erythroid differentiation. This work identifies the LRF BS as an effective and safe therapeutic target for the treatment of β-hemoglobinopathies. Disclosures Casini: Alia Therapeutics: Current Employment, Current equity holder in publicly-traded company. Thrasher:Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Generation bio: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership; 4Bio Capital: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership.

A Single Metabolite which Modulates Lipid Metabolism Alters Hematopoietic Stem/Progenitor Cell Behavior and Promotes Lymphoid Reconstitution.

SummaryFatty acid β-oxidation (FAO), the breakdown of lipids, is a metabolic pathway used by various stem cells. FAO levels are generally high during quiescence and downregulated with proliferation. The endogenous metabolite malonyl-CoA modulates lipid metabolism as a reversible FAO inhibitor and as a substrate for de novo lipogenesis. Here we assessed whether malonyl-CoA can be exploited to steer the behavior of hematopoietic stem/progenitor cells (HSPCs), quiescent stem cells of clinical relevance. Treatment of mouse HSPCs in vitro with malonyl-CoA increases HSPC numbers compared with nontreated controls and ameliorates blood reconstitution capacity when transplanted in vivo, mainly through enhanced lymphoid reconstitution. Similarly, human HSPC numbers also increase upon malonyl-CoA treatment in vitro. These data corroborate that lipid metabolism can be targeted to direct cell fate and stem cell proliferation. Physiological modulation of metabolic pathways, rather than genetic or pharmacological inhibition, provides unique perspectives for stem cell manipulations in health and disease.

Abstract 489: Buoyant targeted microbubble technology provides efficient and superior quality of hematopoietic stem cells isolated from umbilical cord blood

Abstract Purposes: Various separation methods to isolate and enrich hematopoietic stem/progenitor cells (HSPC) have been developed in the past several decades. However, there is still growing interest in clinical trial settings to purify CD34+ cells for ex vivo HSPC expansion and for gene therapy. We have recently developed BUBLES (Buoyancy Enabled Separation), an innovative cell sorting technology using the lipid shell microbubble that is self-molding to external forces. In conjunction with a gas core, it is a very gentle material for cell isolation. This patented technology has the potential to overcome some key hurdles of cell therapy in humans, including targeted multi-parametric cell isolation in bulk for clinical applications. In this study, we aimed to demonstrate that the BUBLES technology is able to efficiently enrich CD34+ HSPC from umbilical cord blood (UCB) and preserve the unique characteristics of stem cells by both in vitro cell assays and in vivo animal study. Methods: Human mononuclear cells (MNCs) were first isolated from fresh UCB samples using a Ficoll-based density gradient method. Subsequently, MNCs were divided to two groups for side-by-side comparisons of CD34+ HSPC isolation using either BUBLES system or a current marketed magnetic nanobead-based CD34+ isolation kit, EasySep. Flow cytometry (FACS) assays were carried out to characterize the isolated HSPC and determine the efficiency of cell enrichment. To assess the quality and functionality of isolated HSPC, in vitro assays, including CD34+ cell expansion with StemSpan™ and colony formation experiments, were performed. Furthermore, isolated CD34+ HSPC were transplanted to immunodeficient NSG-S mice via tail vein and periodic blood screens were conducted to evaluate the engraftment efficiency and differentiation outcomes. Results: FACS analysis of the isolated human HSPC (n = 6) revealed that the average purity and recovery rate of CD34+ cells using the microbubble-based BUBLES technology were 89.8% ± 3.6% and 51.0% ± 13.9%, respectively, whereas 49.5% ± 19.6% and 72.7% ± 16.9% were observed with EasySep. The BUBLES-isolated CD34+ HSPC were expanded 32-43 fold and 301-1188 fold, higher than those isolated by EasySep at day 7 and day 14, respectively (n=2). An average of 50 colony-forming units per 100 CD34+ cells were observed in the BUBLES group, compared to 31 units in the ES group (n=4). Moreover, as early as 5 weeks post-transplantation, 1.26%-2.22% of human CD45+ cells were detected in the peripheral blood of transplanted mice with BUBLES-isolated CD34+ cells (n = 2) whereas no engraftment was observed in those with EasySep. Normal B and T cell differentiation was observed in both groups of transplanted mice after 8 weeks. Conclusion: Based on both in vitro and in vivo results, we demonstrate that BUBLES technology effectively isolates CD34+ HSPC from UCB and preserves the stemness quality. Citation Format: Guixin Shi, Christina C.N. Wu, Sean Darmadi, Zhiyong Wang, Dennis Carson, Yu-Tsueng Liu, Yu-Tsueng Liu. Buoyant targeted microbubble technology provides efficient and superior quality of hematopoietic stem cells isolated from umbilical cord blood [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 489.

Ex vivo editing of human hematopoietic stem cells for erythroid expression of therapeutic proteins

Targeted genome editing has a great therapeutic potential to treat disorders that require protein replacement therapy. To develop a platform independent of specific patient mutations, therapeutic transgenes can be inserted in a safe and highly transcribed locus to maximize protein expression. Here, we describe an ex vivo editing approach to achieve efficient gene targeting in human hematopoietic stem/progenitor cells (HSPCs) and robust expression of clinically relevant proteins by the erythroid lineage. Using CRISPR-Cas9, we integrate different transgenes under the transcriptional control of the endogenous α-globin promoter, recapitulating its high and erythroid-specific expression. Erythroblasts derived from targeted HSPCs secrete different therapeutic proteins, which retain enzymatic activity and cross-correct patients’ cells. Moreover, modified HSPCs maintain long-term repopulation and multilineage differentiation potential in transplanted mice. Overall, we establish a safe and versatile CRISPR-Cas9-based HSPC platform for different therapeutic applications, including hemophilia and inherited metabolic disorders.

Optimized culture medium for enhanced ex vivo expansion of human hematopoietic stem cells

Background & Aim Development of ex vivo culture systems to expand human hematopoietic stem-progenitor cells (HSPCs) remains a critical translational research goal that would enhance clinical hematopoietic stem cell (HSCs) transplantation and gene therapies. To address the problem that HSCs generally die or differentiate rapidly in current ex vivo culture media, we developed a xeno-free, serum-free medium – CTS™ StemPro™ HSC Expansion Medium. Methods, Results & Conclusion In a Design of Experiments approach, media constituents were systematically varied and iterations were evaluated with the goal to maximize ex vivo expansion of HSCP immunophenotypes. Culture of normal primary human CD34+ cells immunopurified from cord blood, mobilized peripheral blood and bone marrow in HSC Expansion Medium supplemented with FLT3L, SCF, TPO, IL3, and IL6 (FKT36), resulted in higher numbers of immunophenotype-defined HSPCs, as compared to either uncultured day 0 cells or cells cultured in industry-standard culture media containing FKT36. For example, culture of primary human CD34+ cells from mobilized peripheral blood (mPB) for 7 days in FKT36-containing HSC Expansion Medium resulted in ∼100-fold increased numbers of CD34+CD45+Lin- cells and ∼2000-fold increased numbers of CD34+Lin- CD90+CD45RA- cells (an early HSPC immunophenotype), as compared to uncultured day 0 cells. CD34+ cells expanded in this medium maintained differentiation capacity in in-vitro colony forming assays, forming erythroid and non-erythroid cell colonies. In addition, using a directed differentiation method we showed that CD34+ cells were able to differentiate into erythroid lineage which generated red blood cells at > 80% efficiency as indicated by co-expression of CD71 and CD235a markers. Further, we demonstrated CTS™ StemPro™ HSC Expansion Medium enabled transduction of CD34+ cells with lentiviral vector generated using CTS™ LV-MAX System, and can be gene edited with CRISPR-Cas9 Gene editing tools that are suitable for cell and gene therapies CD34+ cells expanded with CTS™ StemPro™ HSC Expansion Medium exhibited long-term engraftment potential and multilineage chimerism at 6 months post-transplant. We demonstrated that ex vivo-cultured CD34+ HSPCs generate transplanted cell dose-dependent engraftment in NRG mouse bone marrows and spleens at 26 weeks post-transplant. Thus, CTS™ StemPro™ HSC Expansion Medium will be an impactful tool to enhance expansion of HSPCs that are suited for translational purposes such as transplantation, and gene therapies.

Human hematopoietic stem/progenitor cells display reactive oxygen species-dependent long-term hematopoietic defects after exposure to low doses of ionizing radiations.

Hematopoietic stem cells are responsible for life-long blood cell production and are highly sensitive to exogenous stresses. The effects of low doses of ionizing radiations on radiosensitive tissues such as the hematopoietic tissue are still unknown despite their increasing use in medical imaging. Here, we study the consequences of low doses of ionizing radiations on differentiation and self-renewal capacities of human primary hematopoietic stem/progenitor cells (HSPC). We found that a single 20 mGy dose impairs the hematopoietic reconstitution potential of human HSPC but not their differentiation properties. In contrast to high irradiation doses, low doses of irradiation do not induce DNA double strand breaks in HSPC but, similar to high doses, induce a rapid and transient increase of reactive oxygen species (ROS) that promotes activation of the p38MAPK pathway. HSPC treatment with ROS scavengers or p38MAPK inhibitor prior exposure to 20 mGy irradiation abolishes the 20 mGy-induced defects indicating that ROS and p38MAPK pathways are transducers of low doses of radiation effects. Taken together, these results show that a 20 mGy dose of ionizing radiation reduces the reconstitution potential of HSPC suggesting an effect on the self-renewal potential of human hematopoietic stem cells and pinpointing ROS or the p38MAPK as therapeutic targets. Inhibition of ROS or the p38MAPK pathway protects human primary HSPC from low-dose irradiation toxicity.

A Novel Source of Endogenous DNA Damage That Requires Repair By the Fanconi Anemia Pathway

Fanconi anemia (FA) is the most common inherited bone marrow failure (BMF) syndrome. Impaired DNA interstrand crosslink (ICL) repair is the underlying mechanism for BMF in FA. FA patients usually develop BMF during the first decade of life, prior to any known exposure to exogenous crosslinking agents. Therefore, endogenous sources of DNA damage are most likely to play an important role in the pathogenesis of FA. Metabolic by-products, such as reactive aldehydes, have been implicated in the acceleration of BMF or leukemia in both humans and mice lacking the ICL repair pathway. However, the potential contribution of other detoxifying metabolic enzymes to genome maintenance has not been systematically investigated. Identification of all sources of endogenous DNA damage will allow us to develop novel strategies to prevent DNA damage from arising, and possibly prevent BMF and leukemia in FA as well as other BMF syndromes. To determine whether detoxifying enzymes other than ALDH2 or ADH5 play a role in the protection of HSPC, we performed a metabolism-focused CRISPR/Cas9 synthetic lethality screen (3000 metabolism genes, 10 sgRNA per gene), using wild-type and FANCD2-/- Jurkat cells. From the screen, we identified ALDH9A1 as the most significantly depleted gene in FANCD2-/- Jurkat cells compared with wild-type. Eight out of ten sgALDH9A1 were significantly depleted in FANCD2-/- Jurkat cells, indicating robust effect of the ALDH9A1 knockout. ADH5, a known synthetic lethal gene with FA, was also depleted in FANCD2-/- Jurkat cells, but to a lesser degree. In vitro fluorescence-based competition assay confirmed synthetic lethal interaction between the two genes, in two independent FANCD2-/- Jurkat clones. To determine whether ALDH9A1 deficiency also caused cell death in FA-deficient human hematopoietic stem progenitor cells (HSPCs), we performed an in vitro validation assay using human umbilical cord blood (UCB). UCB CD34+ cells were edited by ribonucleoprotein delivery of Cas9 and sgRNA, in which either of sgCTRL, sgFANCD2 or sgALDH9A1, or both sgFANCD2 and sgALDH9A1 were used. Edited cells were grown on methylcellulose for 10 to 14 days, after which individual colonies were scored and harvested for sequencing. While CD34+ cells that were targeted by both sgFANCD2 and sgALDH9A1 (double KO) achieved lower editing efficiency for each gene compared with cells targeted by single guides, they produced the fewest hematopoietic colonies and the lowest frequency of GEMM (Granulocyte, Erythrocyte, Macrophage and Megakaryocyte) colonies. We observed fewer colonies targeted for both genes (biallelic double KO; observed to expected ratio 0.33) as compared to either single gene KO. These results suggest that loss of ALDH9A1 is deleterious in FANCD2-deficient HSPC. Lastly, we generated a Fanca-/-Aldh9a1-/- mouse model to examine the in vivo hematopoietic phenotype due to increased endogenous aldehydes. These mice were born at the Mendelian ratio without significant anomalies except rare cases of eye abnormalities. At three months of life, Fanca-/-Aldh9a1-/- mice had lower platelet counts than wild-type, Fanca-/- or Aldh9a1-/- control mice, but total white blood counts and hemoglobin levels were similar between groups. A follow-up result of this mouse model will be presented at the meeting. In conclusion, we identified that cells with ALDH9A1 deficiency require the FA pathway for survival. ALDH9A1 may protect human and mouse HSPC that are deficient in the FA pathway from DNA damage and cell death. Disclosures No relevant conflicts of interest to declare.

Homeobox Transcription Factor Hhex Promotes Myeloid Leukemia in Cooperation with Mutant ASXL1

Additional sex combs-like 1 (ASXL1) mutations are frequently found in myeloid malignancies such as AML, MDS and MPNs. Our previous study on mutant Asxl1 expressing knock-in (Asxl1-MT KI) mice revealed that Asxl1-MT impaired normal hematopoiesis (Nagase R et al. J. Exp. Med. 2018). However, Asxl1-MT alone was insufficient to develop myeloid leukemia. Thus, additional factors are required for myeloid transformation in ASXL1-mutated cells. Retrovirus-mediated insertional mutagenesis approaches demonstrated susceptibility of Asxl1-MT KI cells to myeloid leukemia, and helped identifying Hematopoietically expressed homeobox (Hhex) gene as a common retrovirus integration site. Therefore, we here investigated the potential cooperation between ASXL1-MT and HHEX in myeloid leukemogenesis. We first assessed the effects of ASXL1-MT and HHEX on proliferation and differentiation of murine and human hematopoietic stem progenitor cells (HSPCs). Expression of HHEX enhanced proliferation and blocked differentiation of HSPCs expressing ASXL1-MT, while it showed only a modest effect in normal HSPCs. Some of the mice transplanted with cells expressing ASXL1-MT and HHEX developed myeloid leukemia. Expression of ASXL1-MT and HHEX dramatically promoted the growth of RUNX1-ETO-expressing Cord Blood (CB) cells in vitro by promoting cell cycle and inhibiting apoptosis. Moreover, ASXL1-MT and HHEX synergistically accelerated development of two distinct types of myeloid leukemia driven by RUNX1-ETO9a or FLT3-ITD in vivo. These data indicate that ASXL1-MT and HHEX cooperatively work in leukemic transformation of myeloid cells. Next, we evaluated the role of endogenous HHEX in ASXL1-MT-expressing leukemia cells. We here used two murine mutant ASXL1-expressing leukemia cells (cSAM cells: cells with combined expression of SETBP1 and ASXL1 mutations, cRAM cells: cells with combined expression of RUNX1 and ASXL1 mutations). Depletion of Hhex using CRISPR-Cas9 system profoundly attenuated the colony-forming ability and leukemogenicity of cSAM and cRAM cells. Similarly, depletion of HHEX attenuated the growth of human leukemia cell lines harboring an ASXL1 mutation including MEG-01 and Kasumi-1 by inducing apoptosis and differentiation. In contrast, the growth of ASXL1 wildtype cell lines such as THP-1 and U937 was unaffected by HHEX depletion. Thus, endogenous HHEX promotes survival of ASXL1-mutated leukemia cells. To elucidate the underlying molecular mechanisms, we performed RNA-seq using RUNX1-ETO expressing CB cells. Gene set enrichment analysis revealed that genes related to leukemia stem cells were more enriched in cells expressing both ASXL1-MT and HHEX than control cells. In combination with this RNA-seq data and ChIP-seq using 293T cells, we identified Myb and Etv5 as candidate genes upregulated by HHEX expression. We confirmed that ASXL1-MT and HHEX upregulated Myb and Etv5 in murine HSPCs by RT-qPCR and HHEX bound to promoter regions of MYB and ETV5 genes in HL-60 cells by ChIP-qPCR. Conversely, depletion of Hhex reduced expression of Myb and Etv5 in cSAM and cRAM cells. Luciferase reporter assay revealed that co-expression of ASXL1-MT and HHEX cooperatively enhanced promoter activity of MYB. In addition, preliminary expression analyses of primary cells showed that HHEX/ETV5 mRNA expression levels were significantly and MYB mRNA expression tended to be higher in primary AML samples including ASXL1 mutations compared to the ones of healthy controls (Beat AML). Finally, we assessed the role of MYB or ETV5 in leukemogenesis driven by ASXL1 mutations. Depletion of Myb or Etv5 reduced colony-forming activity in cSAM and cRAM cells by promoting apoptosis or differentiation, respectively. Furthermore, ectopic expression of MYB or ETV5 significantly reversed the reduced colony-forming activity of Hhex-depleted cSAM cells. Taken together, our study demonstrates that ASXL1-MT and HHEX are cooperative events promoting myeloid leukemogenesis through upregulation of MYB and ETV5. Disclosures Maciejewski: Novartis: Consultancy; Alexion: Consultancy.

Novel Lymphoid Pathway of Human Plasmacytoid and Conventional Dendritic Cells

[Introduction] Dendritic cells (DCs) play a central role in initiation and regulation of immune response. Human plasmacytoid DCs (pDCs) as well as conventional DCs (cDCs) were shown to differentiate from multi-lymphoid progenitors (MLPs) as well as myeloid progenitors via common DC progenitors. However, lymphoid pathway of DCs remained clarified. Here we investigated lymphoid origin of DCs, using a novel co-culture system which supports differentiation of various lineages of lymphoid and DCs (Br J Haematol. 157:674, 2012; J Immunol. 199:2343, 2017). [Methods and Results] CD34+CD38-CD45RA-CD10-CD7- human hematopoietic stem/progenitor cells (HSPCs) and various lymphoid progenitors including CD34+CD38-CD45RA+CD10-CD7-lymphoid-primed multipotent progenitors (LMPPs), CD34+CD38-CD45RA+CD10+ MLPs,and CD34+CD38+CD45RA+CD10+CD7-CD19- (10SPs), generally though as B/NK progenitors, were isolated from cord blood and cocultured with telomerized human stromal cells with SCF, flt3L, TPO, and GM-CSF. After 17 to 21 days, generation of CD45RA+CD19+ proB, CD56+CD3- NK cells, HLA-DR+IL-3RhighCD303+CD304+pDCs, and CD1c+cDCs was assessed. We observed that not only immature HSPCs, LMPPs and MLPs but also 10SPs gave rise to pDC and cDCs in addition to proB, NK cells. 10SPs were found to be subdivided by expression pattern of c-kit and IL-7 receptor (R), and similarly cocultured with stromal cells. pDCs, cDCs, proB, and NK cells were generated from c-kit+IL-7R- fraction, while pDC, cDCs, and proB, but few or no NK cells were generated from c-kit+IL-7R+ fractions. c-kit-IL-7R+ fraction produced a significantly lower number of cells than c-kit+IL-7R+or- fractions and mainly differentiated into proB cells. By single cell assay of 10SPs, progenitors with differentiation potential for pDC, cDC, and/or proB were detected. These data revealed a differentiation pathway of pDCs and cDCs from relatively mature lymphoid progenitors and suggested the presence of DC and B common progenitors. [Summary] The present study uncovered a closed relationship between B-lymphoid and DC differentiation pathway. We are further attempting to delineate their relationship and differentiation process toward pDCs, cDCs, and proB. Disclosures Nagaharu: kyowa hakko kirin: Research Funding; Astellas Pharma: Research Funding; Nippon Shinyaku: Research Funding; Ono Pharmaceutical: Research Funding. Ohishi:kyowa hakko kirin: Research Funding; Nippon Shinyaku: Research Funding; Astellas Pharma: Research Funding; Ono Pharmaceutical: Research Funding. Katayama:Sysmex: Honoraria; Taisho Toyama Pharma: Honoraria; Celgene: Honoraria; Pfizer: Honoraria; Alexion Pharmaceuticals: Honoraria; Chugai: Honoraria; Nippon Shinyaku: Honoraria, Research Funding; Sumitomo Dainippon Pharma: Honoraria; Ono Pharmaceutical: Research Funding; Novo Nordisk: Honoraria; Shionogi Pharmaceutical: Honoraria; Shire: Honoraria; Novartis: Honoraria; Astellas Pharma: Honoraria, Research Funding; Takeda: Honoraria; Bristol-Myers Squibb: Honoraria; kyowa hakko kirin: Honoraria, Research Funding.

Optimized Culture Medium for Enhanced Ex Vivo expansion of Human Hematopoietic Stem Cells

Increasing the numbers of donor hematopoietic stem cells (HSCs) would accelerate hematopoietic recovery in many HSC transplant recipients, thereby reducing mortality, morbidity and costs. HSC gene therapies would further benefit from high transplant donor HSC numbers, due to inefficiencies in genetic modification of HSCs and losses during transfection protocols. Unfortunately, worldwide attempts to optimize culture parameters, such as hematopoietic growth factor combinations, feeder cells and bioengineered chambers, have failed to result in the substantial HSC self-renewal needed clinically, although it was reported recently that substitution of polyvinyl alcohol for albumin resulted in massive expansion of mouse HSCs (Wilkinson et al. Nature 571:117;2019).Since the same few base hematopoietic culture media have been used for decades, we set out to develop a culture medium specifically for ex vivo expansion of human HSCs. In a Design of Experiments approach, media constituents were systematically varied and each iteration evaluated with the goal to maximize ex vivo expansion of hematopoietic stem-progenitor cell (HSPC) immunophenotypes. After 1 week (wk) culture of human mPB-, CB- or BM-derived CD34+ cells in optimized xeno-free, serum-free StemPro™ HSC Expansion Medium (Prototype) (SPHSC), supplemented with FLT3L, KITL, TPO, IL3, and IL6 (FKT36), there were on average 96-, 178- or 80-fold, respectively, greater numbers (yields) of viable nucleated cells, as compared to day (d)0 (Fig 1). Average yields of the CD34+CD45+Lin- HSPC immunophenotype were increased similarly, by 80-, 104- or 42-fold, respectively. Average yields of the CD34+CD45+CD45RA+CD90+ early HSPC immunophenotype were much more highly increased, by 2300-, 6047- or 1248-fold, respectively. CB-derived CD34+ cells typically generated greater fold increases in these parameters than did CD34+ cells from mPB or BM. In addition, we consistently observed (a) the presence of very early CD34+++CD90+ cells and (b) few Lin+ cells in the ex vivo cultured populations. Furthermore, compared to 2 different FKT36-containing commercial standard media, SPHSC supported statistically greater yields of nucleated cells, CD34+CD45+Lin- HSPCs and CD34+CD45+CD45RA+CD90+ early HSPCs. We focused on the challenge of expanding HSCs from mPB, the most common clinical source. Yields of nucleated cells, CD34+CD45+Lin- HSPCs, and CD34+CD45+CD45RA+CD90+ early HSPCs expanded progressively from d4-d10. Transplant of 1 wk-cultured mPB CD34+ HSPCs generated donor cell dose-dependent increases in %hCD45+mCD45- cells in sublethally-irradiated NRG mouse bone marrows and spleens at 26 wks post-transplant. Human HSPCs and CD33+ myeloid and CD19+ lymphoid progeny were present in hematopoietic organs of the transplanted mice (Fig 2). The long-term (LT)-HSC engraftment capacity of the entire 1 wk-cultured cell population was 10-fold greater than at d0. These results for 1 wk ex vivo expansion of mPB CD34+ cells in SPHSC are similar or superior to reported 12d or 15d expansions of CB CD34+ cells in cytokine-supplemented standard media containing UM171 or SR1 (Fares et al. Science 345:1509;2014; Cohen et al. Biol Blood Marrow Transplant 24:S190;2018; Wagner et al.Cell Stem Cell 18:144;2016). Thus, SPHSC medium may by itself be impactful in clinical transplantation and gene therapy and would also be an optimized platform medium for additional approaches to further enhance ex vivo expansion of human HSCs for transplant and gene therapies. Disclosures Sei: Thermo Fisher Scientific Inc: Employment. Harris-Becker:Thermo Fisher Scientific Inc: Employment. Kaur:Thermo Fisher Scientific Inc: Employment. Vemuri:Thermo Fisher Scientific Inc: Employment.

Phase II Study of Eltrombopag in Subjects with Fanconi Anemia

Background. Fanconi anemia (FA) is a rare genetic disorder that often presents with progressive bone marrow failure (BMF) due to an impaired DNA damage response and chronic exposure to elevated levels of proinflammatory cytokines. To date, hematopoietic stem/progenitor cell (HSPC) transplantation remains the only curative treatment for FA-associated BMF. However, donor availability, graft failure, and FA-specific transplant toxicities remain significant hurdles. Androgens have been successfully used but side effects often prevent prolonged therapy. Attempts at genetic correction of FA are underway but clinical efficacy has not yet been demonstrated. In this clinical trial, we investigate whether eltrombopag (EPAG), an FDA-approved mimetic of thrombopoietin that promotes trilineage hematopoiesis in subjects with acquired BMF (Olnes, NEJM 2012; Townsley, NEJM 2017), may offer a novel therapeutic modality for subjects with FA. Our pre-clinical studies indicate that EPAG evades blockade of signal transduction from c-MPL induced by inflammatory cytokines (Alvarado, Blood 2019). Additionally, we found that EPAG enhances DNA repair activity in human HSPCs (Guenther, Exp Hematol 2019). Thus, EPAG may positively influence two of the main known mechanisms leading to BMF in FA. Study Design. This is a non-randomized, phase II study of EPAG given to subjects with FA (NCT03204188). Subjects receive EPAG for 6 months at an oral daily dose adjusted for age and ethnicity. Subjects who cannot tolerate the medication or fail to respond by 6 months are taken off study drug. Subjects who respond at 6 months are invited in the extension phase for an additional 3 years. They continue on the same dose of EPAG until they reach robust count criteria (platelets > 50K/μL, hemoglobin (Hgb) > 10 g/dL in the absence of transfusions, and absolute neutrophil count (ANC) > 1K/uL for > 8 weeks) or until they reach steady state response (defined as stable counts for 6 months). Drug dose is tapered slowly to the lowest dose that maintains a stable platelet count and eventually discontinued until they meet off study criteria or the study is closed. Eligibility Assessment. Inclusion criteria: (1) Confirmed diagnosis of FA by a biallelic mutation in a known FANC gene and/or by positive chromosome breakage analysis in lymphocytes and/or skin fibroblasts; (2) One or more of the following cytopenias: platelets ≤ 30K/μL or platelet transfusion dependence in the 8 weeks prior to study entry, ANC ≤ 500/μL, Hgb ≤ 9.0 g/dL or red blood cell (RBC) transfusion dependence in the 8 weeks prior to study entry; (3) Failed or declined treatment with androgens; 4) Age > 4 years. Exclusion criteria: (1) Evidence of MDS or AML; (2) Cytogenetic abnormalities associated with poor prognosis in FA; (3) Known biallelic mutations in BRCA2; (4) Active malignancy or likelihood of recurrence of malignancies within 12 months; (5) Treatment with androgens ≤ 4 weeks prior to initiating EPAG. Primary Endpoints. The primary efficacy endpoint is the proportion of drug responders at 6 months. Response to EPAG is defined by one or more of the following criteria: (1) Platelets increase by 20K/μL above baseline, or platelet transfusion independence; (2) Hgb increase by > 1.5g/dL or a reduction in the units of RBC transfusions by at least 50%; (3) At least a 100% increase in ANC for subjects with a pretreatment ANC of < 0.5 x 109/L, or an ANC increase > 0.5 x 109/L. The primary safety endpoint is the toxicity profile assessed at 6 months using the CTCAE criteria. Sample Size and Statistical Methods. Simon's Two-Stage Minimax Design is used, with a response probability of ≤ 20% to terminate the treatment. In the first stage, 12 subjects will be accrued. The study will be stopped if no more than 2 subjects respond to the treatment within 6 months. If 3 or more subjects respond within 6 months in the first stage, then an additional 13 subjects will be accrued, for a total of 25 subjects. Enrollment. Two subjects have been enrolled to date. No drug-related adverse events have been observed. Subject #1 (7YO female) did not respond to 6 months of EPAG, likely due to limited HSPC reserve in the context of profound cytopenias (ANC = 100/µL, Hgb = 6g/dL, Plt = 0K/µL). In contrast, subject #2 (49YO female) showed response to EPAG at 3 months and will continue on the extension phase of the study. Conclusion. This study will provide important clinical information on safety and efficacy of EPAG in subjects with FA. Disclosures Winkler: Agios: Employment.

Gata2 as a Crucial Regulator of Stem Cells in Adult Hematopoiesis and Acute Myeloid Leukemia.

SummarySubversion of transcription factor (TF) activity in hematopoietic stem/progenitor cells (HSPCs) leads to the development of therapy-resistant leukemic stem cells (LSCs) that drive fulminant acute myeloid leukemia (AML). Using a conditional mouse model where zinc-finger TF Gata2 was deleted specifically in hematopoietic cells, we show that knockout of Gata2 leads to rapid and complete cell-autonomous loss of adult hematopoietic stem cells. By using short hairpin RNAi to target GATA2, we also identify a requirement for GATA2 in human HSPCs. In Meis1a/Hoxa9-driven AML, deletion of Gata2 impedes maintenance and self-renewal of LSCs. Ablation of Gata2 enforces an LSC-specific program of enhanced apoptosis, exemplified by attenuation of anti-apoptotic factor BCL2, and re-instigation of myeloid differentiation––which is characteristically blocked in AML. Thus, GATA2 acts as a critical regulator of normal and leukemic stem cells and mediates transcriptional networks that may be exploited therapeutically to target key facets of LSC behavior in AML.

StemPro™ HSC expansion medium (Prototype) supports superior expansion of human hematopoietic stem-progenitor cells

Background & Aim Since numbers of harvested hematopoietic stem cells (HSCs) continues to limit not only laboratory research but also clinical HSC transplantation and gene therapies, development of ex vivo culture systems to expand harvested human hematopoietic stem-progenitor cells (HSPCs) remains a critical translational research quest. To address the barrier that HSCs generally die or differentiate in current ex vivo culture media, we have developed an xeno-free, serum-free medium - StemPro™ HSC Expansion Medium (Prototype) by extensive evaluation of iterative modification of medium constituents. Methods, Results & Conclusion Cultures of primary human CD34+ HSPCs immunopurified from cord blood, mobilized peripheral blood and bone marrow harvests generated greatly increased numbers of immunphenotyped-defined HSPC in StemPro™ HSC Expansion Medium (Prototype) supplemented with FLT3L, KITL (also known as SCF), TPO, IL-3, and IL-6 (FKT36), as compared to either uncultured day 0 cells or cells cultured in industry-standard culture media containing FKT36. For example, culture of primary human CD34+ cells from mobilized peripheral blood for 7 days in FKT36-containing StemPro™ HSC Expansion Medium (Prototype) resulted in ∼100-fold increased numbers of CD34+CD45+Lin− cells and ∼2000-fold increased numbers of CD34+Lin−CD90+CD45RA− cells (an early HSPC immunophenotype), as compared to uncultured day 0 cells. The ex vivo-cultured cells contained high frequencies of aldehyde dehydrogenase-containing cells and formed erythroid and non-erythroid hematopoietic colonies in vitro. In an ongoing in vivo hematopoietic chimera experiment, ex vivo-cultured mPB CD34+ HSPCs harbored robust in vivo-engrafting capacity at the 8-week post-transplant short-term HSC time point evaluated to date. Thus, it appears that StemPro™ HSC Expansion Medium (Prototype) supports superior HSPC expansion that includes self-renewal of short-term-HSCs. Evaluation of long-term-HSC capacity is in progress.

Eltrombopag promotes DNA repair in human hematopoietic stem and progenitor cells

A causal link between hematopoietic stem/progenitor cell (HSPC) dysfunction and DNA damage accrual has been proposed. Clinically relevant strategies to maintain genome integrity in these cells are needed. Here we report that eltrombopag, a small molecule agonist of the thrombopoietin (TPO) receptor used in the clinic, promotes DNA double-strand break (DSB) repair in human HSPCs. We found that eltrombopag specifically activates the classic nonhomologous end-joining (C-NHEJ) DNA repair mechanism, a pathway known to support genome integrity. Eltrombopag-mediated DNA repair results in enhanced genome stability, survival, and function of primary human HSPCs, as demonstrated in karyotyping analyses, colony-forming unit assays and after transplantation in immunodeficient NSG mice. Eltrombopag may offer a new therapeutic modality to protect human HSPCs against genome insults.

Aging Healthy, or with Diabetes, is Associated with ACE2/ACE Imbalance in the Hematopoietic Stem Progenitor Cells

Bone marrow‐derived hematopoietic stem progenitor cells (HSPCs) have vasoreparative functions and are physiologically mobilized into circulation. Aging or diabetes increases risk for cardiovascular diseases and are known to be associated with impaired HSPC functions. Angiotensin converting enzyme (ACE) and ACE2 are primary enzymes of cardiovascular detrimental and protective axes of renin‐angiotensin system (RAS). Angiotensin‐(1‐7) is generated largely by degradation of Ang II by ACE2 and produces vasoprotective functions by activating Mas receptor. We have shown that Ang‐(1‐7) reverses diabetic dysfunction in murine and human HSPCs. This study tested the hypothesis that aging healthy or with diabetes is associated with imbalance in the vascular protective and detrimental axes of RAS in HSPCs. Peripheral blood samples were obtained from either male or female healthy (n=23) or diabetic (n=21), both type 1 and type 2, individuals of age ranging from 25 to 87 years. Lineage‐negative (Lin−) or CD34+ HSPCs were enriched from mononuclear cells. ACE and ACE2 activities in the cell lysates were determined by using enzyme‐specific fluorogenic substrates. Enzyme activities are expressed as arbitrary fluorescence units that is inhibitable by the enzyme‐selective inhibitors of ACE or ACE2, captopril and MLN‐4760, respectively. ACE2/ACE activity ratios in HSPCs in different individuals are plotted against the respective ages. ACE2/ACE is negatively correlated with the age in healthy (Pearson's r=−0.88, P<0.0001) and in diabetic individuals (Pearson's r=−0.82, P<0.0001). As predicted, a negative correlation was observed with ACE2/ACE vs HbA1C (Pearson's r=−0.899, P<0.0001). These results imply that vascular protective axis of RAS is dysfunctional in HSPCs of aging individuals either healthy or diabetic, which is likely to be an underlying mechanism of the impaired reparative functions in HSPCs. Molecular or pharmacological approaches that enhance ACE2 activity or expression could be of great promise in reversing the age‐ or diabetes‐associated dysfunctions and restore cardiovascular health.Support or Funding InformationNational Institute of General Medical Sciences (NIGMS), and National Institute of Aging (AG056881)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Induction and Therapeutic Targeting of Human NPM1c+ Myeloid Leukemia in the Presence of Autologous Immune System in Mice

Development of targeted cancer therapy requires a thorough understanding of mechanisms of tumorigenesis as well as mechanisms of action of therapeutics. This is challenging because by the time patients are diagnosed with cancer, early events of tumorigenesis have already taken place. Similarly, development of cancer immunotherapies is hampered by a lack of appropriate small animal models with autologous human tumor and immune system. In this article, we report the development of a mouse model of human acute myeloid leukemia (AML) with autologous immune system for studying early events of human leukemogenesis and testing the efficacy of immunotherapeutics. To develop such a model, human hematopoietic stem/progenitor cells (HSPC) are transduced with lentiviruses expressing a mutated form of nucleophosmin (NPM1), referred to as NPM1c. Following engraftment into immunodeficient mice, transduced HSPCs give rise to human myeloid leukemia, whereas untransduced HSPCs give rise to human immune cells in the same mice. The de novo AML, with CD123+ leukemic stem or initiating cells (LSC), resembles NPM1c+ AML from patients. Transcriptional analysis of LSC and leukemic cells confirms similarity of the de novo leukemia generated in mice with patient leukemia and suggests Myc as a co-operating factor in NPM1c-driven leukemogenesis. We show that a bispecific conjugate that binds both CD3 and CD123 eliminates CD123+ LSCs in a T cell-dependent manner both in vivo and in vitro. These results demonstrate the utility of the NPM1c+ AML model with an autologous immune system for studying early events of human leukemogenesis and for evaluating efficacy and mechanism of immunotherapeutics.