Hematopoietic Stem And Progenitor Cells Function Research Articles

Mineral and Amino Acid Profiling of Different Hematopoietic Populations from the Mouse Bone Marrow

Steady hematopoiesis is essential for lifelong production of all mature blood cells. Hematopoietic stem and progenitor cells (HSPCs) found in the bone marrow ensure hematopoietic homeostasis in an organism. Failure of this complex process, which involves a fine balance of self-renewal and differentiation fates, often result in severe hematological conditions such as leukemia and lymphoma. Several molecular and metabolic programs, internal or in close interaction with the bone marrow niche, have been identified as important regulators of HSPC function. More recently, nutrient sensing pathways have emerged as important modulators of HSC homing, dormancy, and function in the bone marrow. Here we describe a method for reliable measurement of various amino acids and minerals in different rare bone marrow (BM) populations, namely HSPCs. We found that the amino acid profile of the most primitive hematopoietic compartments (KLS) did not differ significantly from the one of their direct progenies (common myeloid progenitor CMP), while granulocyte-monocyte progenitors (GMPs), on the opposite of megakaryocyte-erythroid progenitors (MEPs), have higher content of the majority of amino acids analyzed. Additionally, we identified intermediates of the urea cycle to be differentially expressed in the KLS population and were found to lower mitochondrial membrane potential, an established readout on self-renewal capability. Moreover, we were able to profile for the first time 12 different minerals and detect differences in elemental contents between different HSPC compartments. Importantly, essential dietary trace elements, such as iron and molybdenum, were found to be enriched in granulocyte-monocyte progenitors (GMPs). We envision this amino acid and mineral profiling will allow identification of novel metabolic and nutrient sensing pathways important in HSPC fate regulation.

2009 – THE NEUROTRANSMITTER RECEPTOR GABBR1 REGULATES PROLIFERATION AND FUNCTION OF HEMATOPOIETIC STEM AND PROGENITOR CELLS

Hematopoietic stem and progenitor cells (HSPCs) have multi-lineage potential and can be used in transplants as a curative treatment for various hematopoietic diseases. HSPC function and behavior is tightly regulated by various cell types and factors in the bone marrow niche. One level of regulation comes from the sympathetic nervous system that innervates the niche and releases neurotransmitters to stromal cells. However, the direct regulation of HSPCs via cell surface expression of neural receptors has not been functionally explored. Using imaging mass spectrometry, we detected strong and regionally specific gamma-aminobutyric acid (GABA) neurotransmitter signal in the endosteal region of mouse bone marrow. GABBR1 is known to be expressed on human HSPCs, however its function in their regulation has not been investigated. Based on published mouse HSPC single cell RNA-seq data, we found that a subset of HSPCs expressed the GABA type B receptor subunit 1 (Gabbr1). We confirmed by surface receptor expression that a subset of mouse bone marrow HSPCs express Gabbr1 protein. Using the same single cell RNA-seq data as above, our own gene set enrichment analysis (GSEA) revealed positive correlation of Gabbr1 expression with genes involved in immune system processes, such as response to type I interferons. We generated a CRISPR-Cas9 Gabbr1 mutant mouse model on a C57/BL6 background for this study. Analysis of Gabbr1 mutant bone marrow cells revealed a reduction in the absolute number of Lin-Sca1+cKit+ (LSK) HSPCs, but no change in the number of long-term hematopoietic stem cells (LT-HSCs). Further hematopoietic profiling revealed reduced numbers of white blood cells in peripheral blood that was primarily due to fewer B220+ cells. We show that Gabbr1 null HSPCs display reduced proliferative capacity, as well as diminished reconstitution ability when transplanted in a competitive setting. An in vitro differentiation assay revealed the impaired ability of Gabbr1 null HSPCs to produce B cell lineages. We tested our predicted association with type I interferon response by administration of poly(I:C) and found reduced HSPC proliferation in Gabbr1 null mice. Our results indicate an important role for Gabbr1 in the regulation of HSPC proliferation and differentiation, highlighting Gabbr1 as an emerging factor in the modulation of HSPC function and behavior.

Identification of Unique mRNA and miRNA Expression Patterns in Bone Marrow Hematopoietic Stem and Progenitor Cells After Trauma in Older Adults.

Older adults have significantly worse morbidity and mortality after severe trauma than younger cohorts. The competency of the innate immune response decreases with advancing age, especially after an inflammatory insult. Subsequent poor outcomes after trauma are caused in part by dysfunctional leukocytes derived from the host's hematopoietic stem and progenitor cells (HSPCs). Our objective was to analyze the bone marrow (BM) HSPC transcriptomic [mRNA and microRNA (miR)] responses to trauma in older and younger adults. BM was collected intraoperatively <9 days after initial injury from trauma patients with non-mild injury [ISS ≥ 9] or with shock (lactate ≥ 2, base deficit ≥ 5, MAP ≤ 65) who underwent operative fixation of a pelvic or long bone fracture. Samples were also analyzed based on age (<55 years and ≥55 years), ISS score and transfusion in the first 24 h, and compared to age/sex-matched controls from non-cancer elective hip replacement or purchased healthy younger adult human BM aspirates. mRNA and miR expression patterns were calculated from lineage-negative enriched HSPCs. 924 genes were differentially expressed in older trauma subjects vs. age/sex-matched controls, while 654 genes were differentially expressed in younger subjects vs. age/sex-matched control. Only 68 transcriptomic changes were shared between the two groups. Subsequent analysis revealed upregulation of transcriptomic pathways related to quantity, function, differentiation, and proliferation of HSPCs in only the younger cohort. miR expression differences were also identified, many of which were associated with cell cycle regulation. In summary, differences in the BM HSPC mRNA and miR expression were identified between older and younger adult trauma subjects. These differences in gene and miR expression were related to pathways involved in HSPC production and differentiation. These differences could potentially explain why older adult patients have a suboptimal hematopoietic response to trauma. Although immunomodulation of HSPCs may be a necessary consideration to promote host protective immunity after host injury, the age related differences further highlight that patients may require an age-defined medical approach with interventions that are specific to their transcriptomic and biologic response. Also, targeting the older adult miRs may be possible for interventions in this patient population.

Aging-Related Reduced Expression of CXCR4 on Bone Marrow Mesenchymal Stromal Cells Contributes to Hematopoietic Stem and Progenitor Cell Defects.

Aging impairs the regenerative potential of hematopoietic stem cells (HSC) and skews differentiation towards the myeloid lineage. The bone marrow (BM) microenvironment has recently been suggested to influence HSC aging, however the mechanisms whereby BM stromal cells mediate this effect is unknown. Here we show that aging-associated decreased expression of CXCR4 expression on BM mesenchymal stem cells (MSC) plays a crucial role in the development of the hematopoietic stem and progenitor cells (HSPC) aging phenotype. The BM MSC from old mice was sufficient to drive a premature aging phenotype of young HSPC when cultured together ex vivo. The impaired ability of old MSC to support HSPC function is associated with reduced expression of CXCR4 on BM MSC of old mice. Deletion of the CXCR4 gene in young MSC accelerates an aging phenotype in these cells characterized by increased production of reactive oxygen species (ROS), DNA damage, senescence, and reduced proliferation. Culture of HSPC from young mice with CXCR4 deficient MSC also from young mice led to a premature aging phenotype in the young HSPC, as evidenced by reduced hematopoietic regeneration and enhanced myeloid differentiation. Mechanistically, CXCR4 signaling prevents BM MSC dysfunction by suppressing oxidative stress, as treatment of old or CXCR4 deficient MSC with N-acetyl-L-cysteine (NAC), improved their niche supporting activity, and attenuated the HSPC aging phenotype. Our studies suggest that age-associated reduction in CXCR4 expression on BM MSC impairs hematopoietic niche activity with increased ROS production, driving an HSC aging phenotype. Thus, modulation of the SDF-1/CXCR4 axis in MSC may lead to novel interventions to alleviate the age-associated decline in immune/hematopoietic function.

GATA2 +9.5 enhancer: from principles of hematopoiesis to genetic diagnosis in precision medicine.

By establishing mechanisms that deliver oxygen to sustain cells and tissues, fight life-threatening pathogens and harness the immune system to eradicate cancer cells, hematopoietic stem and progenitor cells (HSPCs) are vital in health and disease. The cell biological framework for HSPC generation has been rigorously developed, yet recent single-cell transcriptomic analyses have unveiled permutations of the hematopoietic hierarchy that differ considerably from the traditional roadmap. Deploying mutants that disrupt specific steps in hematopoiesis constitutes a powerful strategy for deconvoluting the complex cell biology. It is striking that a single transcription factor, GATA2, is so crucial for HSPC generation and function, and therefore it is instructive to consider mechanisms governing GATA2 expression and activity. The present review focuses on an essential GATA2 enhancer (+9.5) and how +9.5 mutants inform basic and clinical/translational science. +9.5 is essential for HSPC generation and function during development and hematopoietic regeneration. Human +9.5 mutations cause immunodeficiency, myelodysplastic syndrome, and acute myeloid leukemia. Qualitatively and quantitatively distinct contributions of +9.5 cis-regulatory elements confer context-dependent enhancer activity. The discovery of +9.5 and its mutant alleles spawned fundamental insights into hematopoiesis, and given its role to suppress blood disease emergence, clinical centers test for mutations in this sequence to diagnose the cause of enigmatic cytopenias. Multidisciplinary approaches to discover and understand cis-regulatory elements governing expression of key regulators of hematopoiesis unveil biological and mechanistic insights that provide the logic for innovating clinical applications.

Daily Ethanol Drinking Followed by an Abstinence Period Impairs Bone Marrow Niche and Mitochondrial Function of Hematopoietic Stem/Progenitor Cells in Rhesus Macaques.

Unhealthy consumption of alcohol is a major public health crisis with strong associations between immunological dysfunctions, high vulnerability to infectious disease, anemia, and an increase in the risk of hematological malignancies. However, there is a lack of studies addressing alcohol-induced changes in bone marrow (BM) and hematopoiesis as fundamental aspects of immune system function. To address the effect of chronic alcohol consumption on hematopoietic stem and progenitor cells (HSPCs) and the BM niche, we used an established rhesus macaque model of voluntary alcohol drinking. A cohort of young adult male rhesus macaques underwent a standard ethanol self-administration protocol that allowed a choice of drinking alcohol or water 22hours/day with periods of forced abstinence that elevated subsequent intakes when alcohol availability resumed. Following the last month of forced abstinence, the monkeys were euthanized. HSPCs and bone samples were collected and analyzed in functional assays and by confocal microscopy. HSPCs from alcohol animals exhibited reduced ability to formgranulocyte-monocyte and erythroidcolonies in vitro. HSPCs also displayed a decrease in mitochondrial oxygen consumption linked to ATP production and basal respiratory capacity. Chronic alcohol use led to vascular remodeling of the BM niche, a reduction in the number of primitive HSPCs, and a shift in localization of HSPCs from an adipose to a perivascular niche. Our study demonstrates, for the first time, that chronic voluntary alcohol drinking in rhesus macaque monkeys leads to the long-term impairment of HSPC function, a reduction in mitochondrial respiratory activity, and alterations in the BM microenvironment. Further studies are needed to determine whether these changes in hematopoiesis are persistent or adaptive during the abstinent period and whether an initial imprinting to alcohol primes BM to become more vulnerable to future exposure to alcohol.

The Neurotransmitter Receptor Gabbr1 Regulates Proliferation and Function of Hematopoietic Stem and Progenitor Cells

Hematopoietic stem and progenitor cells (HSPCs) have multi-lineage potential and can be used in transplants as a curative treatment for various hematopoietic diseases. HSPC function and behavior is tightly regulated by various cell types and factors in the bone marrow niche. One level of regulation comes from the sympathetic nervous system that innervates the niche and releases neurotransmitters to stromal cells. However, the direct regulation of HSPCs via cell surface expression of neural receptors has not been functionally explored. Using imaging mass spectrometry, we detected strong and regionally specific gamma-aminobutyric acid (GABA) neurotransmitter signal in the endosteal region of mouse bone marrow. GABBR1 is known to be expressed on human HSPCs (Steidl et al., Blood 2004), however its function in their regulation has not been investigated. Based on published mouse HSPC single cell RNA-seq data (Nestorowa et al., Blood 2016), we found that a subset of HSPCs expressed the GABA type B receptor subunit 1 (Gabbr1). We confirmed by surface receptor expression that a subset of mouse bone marrow HSPCs express Gabbr1 protein. Using the same single cell RNA-seq data as above, our own gene set enrichment analysis (GSEA) revealed positive correlation of Gabbr1 expression with genes involved in immune system processes, such as response to type I interferons. We generated a CRISPR-Cas9 Gabbr1 mutant mouse model on a C57/BL6 background suitable for hematopoietic studies. Analysis of Gabbr1 mutant bone marrow cells revealed a reduction in the absolute number of Lin-Sca1+cKit+ (LSK) HSPCs, but no change in the number of long-term hematopoietic stem cells (LT-HSCs). With further hematopoietic profiling, we discovered reduced numbers of white blood cells in peripheral blood that was primarily due to fewer B220+ cells. We show that Gabbr1 null HSPCs display reduced proliferative capacity, as well as diminished reconstitution ability when transplanted in a competitive setting. An in vitro differentiation assay revealed the impaired ability of Gabbr1 null HSPCs to produce B cell lineages. We tested our predicted association with type I interferon response by administration of poly(I:C) and found reduced HSPC proliferation in Gabbr1 null mice. Our results may translate well to humans, as a rare human SNP within the GABBR1 locus was found that correlates with altered leukocyte counts (Astle et al., Cell 2016). Our results indicate an important role for Gabbr1 in the regulation of HSPC proliferation and differentiation, highlighting Gabbr1 as an emerging factor in the modulation of HSPC function and behavior. Disclosures No relevant conflicts of interest to declare.

Mutant ASXL1 Disrupts Paraspeckle Formation through Aberrant Interaction with Nono in Hematopoietic Cells

Paraspeckles are sub-nuclear structures found in the interchromatin space of mammalian cells. The core paraspeckle components include a lncRNA NEAT1 and members of the DBHS family of proteins: NONO, SFPQ, and PSPC1. Paraspeckles and their components play diverse roles in gene regulatory networks, including transcription, alternative RNA splicing, nuclear retention of RNA, and DNA repair. Although a previous study showed the presence of paraspeckles in hematopoietic stem and progenitor cells (HSPCs), their roles in normal and malignant hematopoiesis remain largely unknown. ASXL1 regulates gene expression through interactions with multiple epigenetic regulators. Somatic mutations in ASXL1 gene occur frequently in myeloid neoplasms. We previously generated a hematopoietic lineage-specific conditional knockin (KI) mouse of a C-terminally truncated form of ASXL1-mutant (ASXL1-MT), and showed that ASXL1-MT inhibited repopulating capability of HSPCs. We performed deep RNA sequencing using HSPCs from ASXL1-MT-KI mice, and found aberrant alternative splicing in multiple genes involved in hematopoiesis. The altered splicing in ASXL1-MT-KI HSPCs included abnormal exon skipping or retention in Runx1, Traf6, Atm, and Dnmt3b. These findings, together with a previous report showing that ASXL1 mutations affect alternative splicing in U937 cells, strongly indicate the involvement of ASXL1 in RNA splicing machinery. Because a previous interactome analysis suggested the association between NONO and ASXL1, we hypothesized that ASXL1 may play a role in RNA maturation processes through interactions with paraspeckle proteins. To test this hypothesis, we examined physical and functional interactions between paraspeckle components and ASXL1. We found that both wild-type and mutant ASXL1 interact with NONO and SFPQ in 293T cells. Interestingly, protein and RNA immunoprecipitation (RIP) analyses revealed that coexpression of wild-type ASXL1, but not mutant ASXL1, enhanced interactions between NONO and histone H3 as well as NONO and NEAT1. These results suggest that ASXL1 acts as a scaffolding protein that assembles paraspeckle proteins and histones to promote transcription and RNA processing. Importantly, mutant ASXL1 loses this function. Next, we assessed subcellular localization of Nono in HSPCs from control and ASXL1-MT-KI mice. We observed predominant cytoplasmic expression of Nono in ASXL1-MT KI HSPCs, while Nono mainly localized in the nucleus in control cells (Figure 1). In addition, expression of NEAT1_2 isoform, which is essential for paraspeckle formation and maintenance, was substantially downregulated in ASXL1-MT-KI HSPCs. Consistent with these observations, RNA FISH against NEAT1 and immunofluorescence against NONO revealed disrupted paraspeckle formation in ASXL1-MT-KI HSPCs. These data suggest that ASXL1-MT promotes nuclear export of Nono, which results in disruption of paraspeckles in HSPCs. NONO has nuclear localization signal (NLS) at its C-terminus, and it was previously shown that a cytoplasmic C-truncated form of NONO induced senescence in fibroblasts. To assess the effect of forced expression of the cytoplasmic NONO in hematopoietic cells, we transduced vector or a NONO mutant lacking the NLS domain (NONO-ΔNLS) into c-Kit+ bone marrow cells, and transplanted these cells into recipient mice. NONO-ΔNLS induced overproduction of reactive oxygen species (ROS) and reduced engraftment of bone marrow progenitors as ASXL1-MT did. We then assessed the effect of Nono depletion in ASXL1-MT-KI HSPCs using CRISPR/Cas9 system. We crossed ASXL1-MT-KI mice with Rosa26-LSL-Cas9-KI mice, and c-Kit+ bone marrow cells derived from these mice were transduced with a non-targeting or Nono-targeting sgRNAs. This experiment revealed that Nono depletion reverted the impaired repopulation of ASXL1-MT-KI HSPCs after transplantation. Taken together, these data indicate that the cytoplasmic localization of Nono induced by ASXL1-MT has the negative impact on HSPC function. In summary, this study reveals a novel link between an epigenetic regulator ASXL1 and paraspeckle formation. The aberrant interaction between mutant ASXL1 and NONO results in NONO mislocalization, paraspeckle disruption and HSPC dysfunction. Our findings also suggest potentially important roles for paraspeckles to maintain normal hematopoiesis. Disclosures Ogawa: Qiagen Corporation: Patents &amp; Royalties; RegCell Corporation: Equity Ownership; Asahi Genomics: Equity Ownership; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; Kan Research Laboratory, Inc.: Consultancy.

Murine Fetal Bone Marrow HSPCs Undergo a Dramatic Shift in Frequency at Birth

Hematopoietic stem cells (HSCs) give rise to all cells of the hematopoietic system and are classically defined by their ability to stably engraft and reconstitute the blood system of ablated recipients after transplantation. The first transplantable HSCs arise from hemogenic endothelium at embryonic day 10.5 (E10.5) of mouse development and migrate to the fetal liver (FL), where they undergo a robust expansion followed by a second migration to the fetal bone marrow (FBM) at E15.5. The dynamics of hematopoiesis within the FBM has been largely unexplored. To gain a better understanding of FBM hematopoiesis, we catalogued the frequency, absolute numbers, phenotype and function of HSPCs in murine FBM from E15.5 through post-natal day 28 (P28). To avoid assumptions regarding HSPC location during fetal and neonatal development, we pooled bone marrow from the entire fetal skeleton for these studies. HSCs were rare in the FBM, ranging from 70-150 total HSCs at E15.5-E17.5, followed by a burst at E18.5 to 2,200 total HSCs. The frequency and absolute number of HSCs in the bone marrow steadily increased from E18.5 to P6, followed by a continual increase in the absolute number of HSCs from P6 to adulthood. This may reflect the dynamics of HSC cycling, or an influx or expansion of more differentiated progenitors in the fetal and neonatal bone marrow. We also found that the most prevalent hematopoietic stem and progenitor cell (HSPC) population within Lineage-Sca-1+c-Kit+ (LSK) cells in E15.5-E18.5 FBM was Flt3-CD48+CD150+ cells (MPP2). MPP2 cells, which are a megakaryocyte-biased multipotent progenitor population, comprised up to 75% of the LSK compartment at these time points, compared to 3% in adult bone marrow. The percentage of MPP2 cells dropped abruptly and dramatically right before birth (e.g. to 17% at E19) and continued to drop until adulthood. Transplantation of limiting numbers of MPP2 cells revealed that E16.5, E18.5, and P0 MPP2s displayed no repopulating potential, while adult MPP2s showed transient reconstitution of irradiated recipients. Fetal bone marrow MPPs displayed no colony potential in single-cell methylcellulose colony assays until E18.5 and P0, with a bias for erythroid-megakaryocyte colonies. Therefore, fetal bone marrow MPP2s are functionally distinct from their adult counterparts. Whole fetal bone marrow transplants showed that the first transplantable FBM HSCs appeared at E16.5, with up to 75% reconstitution in the peripheral blood (PB) of irradiated recipients. E16.5 FBM HSCs also displayed secondary transplantation activity, while E15.5 FBM HSCs displayed limited ability to reconstitute the PB of primary recipients. In sum, our studies reveal that until birth the predominant HSPC population in the fetal bone marrow is an immunophenotypic MPP2 that is functionally distinct from adult MPP2s, and that HSCs do not begin to accumulate in significant numbers until right before birth. These studies suggest the presence of key mechanisms during birth that influence the HSPC landscape of the fetal and neonatal bone marrow, and work is currently underway to systematically characterize global changes in the bone marrow niche during parturition. Disclosures No relevant conflicts of interest to declare.

Mutant ASXL1 Promotes Expansion of the Phenotypic Hematopoietic Stem Cell Compartment

Somatic mutations of the ASXL1 gene are recurrently detected in age-related clonal hematopoiesis (CH). However, how ASXL1 mutations causes CH are not understood. Here, using knockin (KI) mice expressing a C-terminally truncated form of ASXL1-mutant (ASXL1-MT), we investigated the effect of ASXL1-MT on physiological aging in hematopoietic stem cells (HSCs). To examine the influence of ASXL1-MT on hematopoiesis, we bred the ASXL1-MT-KI mice with Vav-Cre transgenic mice. Young ASXL1-MT-KI mice (6-12 weeks) did not show significant changes in hematological parameters and differentiation status of peripheral blood. We observed the decreased frequency of hematopoietic stem and progenitor cells (HSPCs), including long-term HSCs (LT-HSCs). Competitive transplantation assays showed the reduced repopulation ability in ASXL1-MT-KI HSPCs. Thus, ASXL1-MT decreased the number and impaired the function of HSPCs in young mice. Next, we examined age-related changes in hematopoiesis caused by ASXL1-MT. Aged ASXL1-MT-KI mice displayed a myeloid-biased differentiation and hypocellular bone marrow, indicating the dysfunction of hematopoiesis. Interestingly, ASXL1-MT markedly increased the frequency of phenotypic LT-HSCs (pLT-HSCs) in aged mice (20-24 months). Competitive transplantation assays showed the impaired repopulation potential of pLT-HSCs from aged ASXL1-MT-KI mice. These data demonstrate that the increased pLT-HSCs in aged ASXL1-MT-KI mice are not functional HSCs with long-term repopulation potential. To elucidate how ASXL1-MT drives HSPC dysfunction, we conducted RNA-Seq analysis using HSPCs from young mice. This analysis revealed upregulation of mitochondrial genes in ASXL1-MT-KI HSPCs. In addition, MitoTracker staining, extracellular flux analyses and metabolome analyses demonstrated the enhanced mitochondrial metabolism in ASXL1-MT-KI HSPCs. We also found that the aberrantly elevated mitochondrial activity induced ROS overproduction and increased DNA damage, resulting in HSPC dysfunction. As a mechanism underlying the enhanced mitochondrial activity of ASXK1-MT-KI HSPCs, we revealed that ASXL1-MT activated the Akt/mTOR pathway in HSPCs. Treatment with an Akt inhibitor perifosine or an mTOR inhibitor rapamycin normalized the mitochondrial membrane potential and ROS levels in ASXL1-MT-KI HSPCs. Moreover, rapamycin treatment improved engraftment of ASXL1-MT-KI bone marrow cells after transplantation. These data indicate that the activated Akt/mTOR signaling leads to the enhanced mitochondrial activity, elevated ROS levels, and HSPC dysfunction in ASXL1-MT-KI mice. To assess the impact of the enhanced Akt/mTOR signaling on age-related changes in ASXL1-MT-KI mice, we administered rapamycin to aged ASXL1-MT-KI mice. Intriguingly, rapamycin treatment decreased the frequency of pLT-HSCs, and normalized the bone marrow cellularity in aged ASXL1-MT-KI mice. Cell cycle analysis revealed that pLT-HSCs in G0 phase were decreased in aged ASXL1-MT-KI mice, which was normalized by rapamycin treatment. These data demonstrate that the activated Akt/mTOR pathway provokes the aberrant expansion of pLT-HSCs in aged ASXL1-MT-KI mice. We next attempted to clarify the underlying mechanism of Akt activation in ASXL1-MT-KI mice. Immunoprecipitation experiments revealed that ASXL1-MT/BAP1 complex deubiquitinated AKT in 293T cells. To determine the role of endogenous Bap1 on Akt signaling, we assessed the effect of Bap1 deletion in murine bone marrow cells transformed by combined expression of SETBP1-D868N and ASXL1-MT (cSAM cells). A time course experiments showed Akt phosphorylation induced by IL-3 stimulation was attenuated and shortened in Bap1-depleted cSAM cells. These data suggest that ASXL1-MT/BAP1 complex deubiquitinate and stabilize phosphorylated Akt. In summary, we demonstrated that ASXL1-MT cooperated with BAP1 to promote AKT deubiquitination and activation. The activated Akt/mTOR pathway led to enhanced mitochondrial metabolism, elevated ROS levels and increased DNA damage. These molecular bases underlie the age-associated expansion of the pLT-HSC compartment. Our results underscore the possibility that CH can originate from a pLT-HSC with a limited repopulation potential. A pharmacological inhibition of the Akt/mTOR pathway could be a promising therapeutic intervention to individuals with CH harboring ASXL1 mutations. Disclosures No relevant conflicts of interest to declare.

Niche Transfer of Small Non-Coding RNAs Regulates Hematopoietic Response to Stress

The bone marrow (BM) niche, is an extrinsic regulator of hematopoietic stem and progenitor cells (HSPCs) (Morrison and Scadden 2014). The niche can drive disease and loss of HSPC function (Raaijmakers et al., 2010; Kode et al., 2014; Dong et al., 2016). Of particular interest, deletion in specific niche cells of the small RNA processing enzymes, Dicer1 and Angiogenin (Ang1) resulted in hematopoietic transformation (Raaijmakers et al., 2010) and loss of HSC quiescence (Goncalves et al., 2016), respectively. We tested mechanisms by which small non-coding RNAs (sncRNA) from niche cells might contribute to HSPC regulation. The transfer of cytoplasmic contents from specific mesenchymal subsets to wild type (WT) transplanted hematopoietic cells was evaluated by GFP. Osteoblastic cell reporter mice (Ocn-GFP, Col1-GFP) transferred 40-fold more GFP to HSPC than mesenchymal stem cells (Nes-GFP) or osteoprogenitors (Osx-GFP). GFP cannot transfer through gap junctions, however 100nM extracellular vesicles (EVs) labeled with the endocytic marker TSG-101 and GFP were found in Ocn-GFP bone marrow plasma. Granulocyte macrophage progenitor (GMPs) cells were the major hematopoietic recipients of osteoblast derived EVs (Fig. 1A). Small RNA analysis of mouse BM EVs demonstrated &gt;85% were tRNA (Fig. 1B) while Northern blot (NB) revealed both tRNAs and stress induced RNAs (tiRNAs) (Fig. 1C). tRNAs were evident in EVs and in EV generating and recipient cells, while tiRNAs were highly enriched in EVs compared to cells (Fig. 1C). Extracellular vesicle recipient and non-recipient GMPs (GMP[GFP+] and GMP[GFP-]) respectively demonstrated a 2-fold enrichment in reads mapping to tRNAs in GMP[GFP+]. Principle component analysis demonstrated that the two populations are distinct in their tRNA content. In addition, twelve specific tRNAs were present at a significantly higher levels in GMP[GFP+]. Gene expression analysis of GMP[GFP+]and GMP[GFP-] revealed an enrichment of protein translation and cell cycle related functions in GMP[GFP+] which were validated using an in vivo protein translation assay and cell cycle analyses. Synthetic oligos corresponding to the sequence of top ten tiRNAs that are enriched in GMP[GFP+] were transfected into primary GMPs. 5'-Pro-CGG-1 and 5'-Cys-GCA-27 increased the rate of protein translation and cellular proliferation in transfected GMPs (Fig. 1D-E). The physiologic relevance of EV transfer was assessed using two stress states, systemic Candida albicans infection and low dose gamma irradiation. The frequency of GMP[GFP+] increased in both states in concert with increased cell proliferation. Therefore, EV transfer of sncRNAs from niche to hematopoietic progenitors in vivo is a rapid means of conveying information between cells. It alters progenitor protein translation and proliferation in a manner responsive to stress. The transfer of 5'tiRNA EV from specific niche cells to specific hematopoietic cells is an additional mechanism for niche regulation of blood cell production. Disclosures Sykes: Clear Creek Bio: Equity Ownership, Other: Co-Founder. Scadden:Clear Creek Bio: Consultancy, 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; Fog Pharma: Consultancy; Agios Pharmaceuticals: 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; Editas Medicine: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bone Therapeutics: Consultancy; Novartis: Other: Sponsored research; Fate Therapeutics: Consultancy, Equity Ownership; Red Oak Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

CRISPR/Cas9-Targeted De Novo DNA Methylation Is Maintained and Impacts the Colony Forming Potential of Human Hematopoietic CD34+ Cells

Introduction The epigenome is significantly perturbed in hematological malignancies with global DNA hypomethylation and localized hypermethylation of gene promoter CpG islands. Whether specific gene promoter hypermethylation can contribute to the clonal expansion of hematopoietic stem and progenitor cells (HSPCs) in humans by affecting HSPC biology, independently of genetic mutations, has not previously been investigated due to the lack of appropriate tools. We show for the first time that it is possible to target de novo DNA methylation using CRISPR/Cas9 in human CD34+ cells isolated from cord blood (CB). DNA methylation targeted to key cell cycle control gene promoters, INK4b (p15) and ARF (p14), is permanently maintained after dCas9 3A3L degradation and inherited as cells differentiate; inhibiting gene expression and affecting the colony forming potential of CD34+ cells. This demonstrates that specific DNA hypermethylation events can permanently change HSPC biology and impact differentiation, potentially contributing to pre-malignant processes. Methods Human CD34+ HSPCs were isolated from human CB and maintained in liquid culture for 24 hours before nucleofection with mRNA encoding an adapted form of CRISPR/Cas9 which has no nuclease activity (dCas9) and is fused to the catalytic domain of DNA methyltransferase 3A (DNMT3A) and 3L (3A3L). The nucleofection cocktail contained dCas9 3A3L or dCas9 3A3L-mut (lacks methyltransferase activity) and 1 to 3 guide RNAs to target DNA methylation to combinations of the INK4a-ARF-INK4b locus. Cells were then seeded into methylcellulose for a primary colony forming assay (CFU). Colonies were scored after 14 days and cells were either harvested and pooled or individual colonies were picked for single-colony molecular analyses. The DNA was extracted and methylation at the INK4a-ARF-INK4b promoters was quantified using targeted bisulfite sequencing; target gene expression was measured using qPCR. The remaining cells from the primary CFU were re-plated a second (secondary CFU) and third (tertiary CFU) time and colonies were again scored after 14 days. Results and Conclusions Targeting DNA methylation to the INK4a-ARF-INK4b locus or INK4b individually in human CD34+ cells resulted in maintenance of hypermethylation at ARF and/or INK4b gene promoters in individual BFU-E (burst-forming unit-erythroid) and CFU-GM (granulocyte, macrophage) colonies as measured by single-colony targeted bisulfite sequencing after the primary CFU; causing heritable repression of INK4b gene expression in the differentiated cells. Some CpGs were up to 90% methylated, indicating that DNA methylation added at these gene promoters is highly stable as cells differentiate. Hypermethylation of ARF and INK4b was found in some colonies even after the tertiary CFU, demonstrating long-term maintenance of promoter hypermethylation. Unexpectedly, no DNA hypermethylation was detected at INK4a in differentiated cells, but whether this is the case for all subpopulations of HSPCs (i.e. HSCs or lymphoid progenitors) is under investigation. Hypermethylation of INK4b and ARF increased the colony forming potential of CD34+ cells in primary, secondary and tertiary CFUs, compared to the control. Conversely, methylation targeted to INK4b alone did not significantly affect the number of colonies in the first CFU, and decreased the number of colonies in the secondary CFU. This suggests a complex interplay between key cell cycle regulators ARF and INK4b in CD34+ cells and during differentiation which can be disrupted by DNA hypermethylation and gene repression. These findings demonstrate the novel insights we can gain by using CRISPR/Cas9 tools to target DNA methylation and these investigations will reveal how gene promoter hypermethylation can impact HSPC function. Furthermore, studying this locus may uncover an important role for DNA hypermethylation in the development of myeloid malignancies, since INK4b is frequently hypermethylated, but rarely mutated, in myeloid dysplastic/proliferative neoplasms and acute myeloid leukemia. Disclosures Gribben: Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Acerta/Astra Zeneca: Consultancy, Honoraria, Research Funding.

Zinc Finger Nuclease-Mediated Disruption of the BCL11A Erythroid Enhancer Results in Enriched Biallelic Editing, Increased Fetal Hemoglobin, and Reduced Sickling in Erythroid Cells Derived from Sickle Cell Disease Patients

High fetal hemoglobin (HbF) levels are associated with decreased severity and mortality in sickle cell disease (SCD) and beta thalassemia (BT). We have developed a novel gene-edited cell therapy using autologous hematopoietic stem and progenitor cells (HSPCs) that have been genetically modified with zinc finger nucleases (ZFNs) to reactivate HbF expression. The ZFNs target the binding motif of GATA1 (GATAA) within an intronic erythroid-specific enhancer (ESE) of BCL11A, which encodes a major transcriptional repressor of HbF. Previously, we reported successful ZFN-mediated editing of the BCL11A ESE and reactivation of HbF in both dual (granulocyte colony-stimulating factor (G-CSF) and plerixafor) and single plerixafor mobilized HSPCs(Holmes 2017, Moran 2018). Both related drug candidates, ST-400 and BIVV003, are currently in phase 1/2a clinical trials for transfusion-dependent BT (NCT03432364) and SCD (NCT03653247), respectively. Here, we performed extensive genetic and phenotypic characterization of ZFN-edited HSPCs from healthy and SCD donors. We performed single-cell characterization of BCL11A ESE-edited HSPCs from 4 healthy donors. Briefly, individual HSPCs were sorted and cultured in erythroid differentiation medium. Genomic DNA and protein lysate were collected at day 14 and 20, respectively. In total, we successfully genotyped 961 single-cell derived colonies by next-generation sequencing. The distribution was highly skewed towards biallelic-edited cells (P&amp;lt;3x10-149) representing 94% of edited clones, suggesting that ZFN-expressing cells are likely to become edited at both alleles. We found that each edited allele contributed additively to an increase in HbF% of 15% (P=1x10-80) as measured by UPLC. Clones harboring GATAA-disrupting indels on both alleles displayed on average 34% more HbF% than WT clones (P=1x10-112). In contrast, clones with biallelic indels that left the motif intact displayed a more modest increase (13%, P=1x10-6). Overall, our data revealed that &amp;gt;90% of edited cells were biallelic, displaying on average 27-38% more HbF% despite variation in donor baseline levels. We observed a strong enrichment of biallelic-edited homozygotes (same indel pattern at both alleles) compared to an expected random distribution (161 vs 24; P&amp;lt;1x10-5). These clones may harbor larger deletions not captured by sequencing, as reported previously using CRISPR/Cas9 (Kosicki 2018). To address this question, we used a combination of a small amplicon sequencing assay design covering an informative SNP and a 12kb amplicon Nextera assay. We found that 27% of initially assigned homozygote clones were bona fide homozygotes (44/161) with the remaining harboring indels not originally captured. Nevertheless, most indels remained small, with 91% of indels &amp;lt;50bp, and deletions and insertions &amp;gt;1kb together consisting of less than 1% of alleles. The largest deletion was 4kb, but no indel extended outside the enhancer region of BCL11A or altered the coding region (&amp;gt;26 kb away). Moreover indels &amp;gt;50bp were not associated with enucleation levels (P=0.77), suggesting that they did not alter erythroid function. Overall, these results are consistent with previous data showing that ZFN-mediated gene editing does not impair HSPC function in vitro based on colony forming unit (CFU) production, and that injection of BIVV003 into immune-deficient NBSGW mice results in robust long-term engraftment with no impact on the number of HSPCs or their progeny, including erythrocytes. Finally, BCL11A ESE editing in HSPCs mobilized from one SCD donor resulted in a 3-fold HbF increase consistent across technical duplicates, without impacting CFU production or erythroid enucleation. Importantly, clonal analysis revealed a similar enrichment of biallelic editing (P=6x10-4) and additive HbF up-regulation, with biallelic edited cells reaching 28% more HbF% than unedited cells (50% vs 22%, P=7x10-5). Furthermore, enucleated cells differentiated from edited HSPCs showed attenuation of sickling under hypoxic conditions supporting the potential efficacy of BIVV003. Experiments in HSPCs from additional SCD donors are ongoing. Overall, our data have shown that ZFN-mediated disruption of BCL11A ESE results in enriched biallelic editing with on-target small indels, reactivates HbF and reduces sickling, supporting the potential efficacy and specificity of BIVV003 as a novel cell therapy for SCD. Disclosures Lessard: Sanofi: Employment. Rimmele:Sanofi: Employment. Ling:Sanofi: Employment. Moran:Sanofi: Employment. Vieira:Sanofi: Employment. Lin:Sanofi: Employment. Hong:Sanofi: Employment. Reik:Sangamo Therapeutics: Employment. Dang:Sangamo Therapeutics: Employment. Rendo:Sanofi: Employment. Daak:Sanofi: Employment. Hicks:Sanofi: Employment.

Functional Impact of MacroH2A Histone Variants in Acute Myeloid Leukemia

Histone variants are emerging as key regulatory molecules in cancer. Recent data provided first evidence of how transcriptional deregulation and changes in the deposition of histone variants may affect malignant transformation. H2A variants are highly conserved and consist of 8 members. Among all histone variants, macroH2A variants hold a unique C-terminal macro domain, which is approximately twice the size of its H2A-like histone domain. In mammals, there are 3 macroH2A variants encoded by H2AFY (macroH2A1.1, macroH2A1.2) and H2AFY2 (macroH2A2). MacroH2A variants occupy large repressive domains throughout the genome, exerting a repressive role on transcription. So far, the function of macroH2A proteins in hematopoiesis and leukemic transformation is incompletely understood. Here, we report on a functional role of macroH2A histone variants that may influence cell competition in leukemia and normal hematopoietic stem and progenitor cell (HSPC) function. In published gene expression datasets, macroH2A variants H2AFY and H2AFY2 are highly expressed in hematopoietic stem cells (HSCs) compared to mononuclear cells (both p&lt;0.0001****). Moreover, expression of H2AFY and H2AFY2 appeared even higher in acute myeloid leukemia (AML) (p=0.0750 and 0.0037**, respectively) when compared to the expression level in HSCs. This high expression is independent of different genetic subtypes and AML risk groups. To assess for the functional dependency of leukemic cells on the expression of macroH2A variants, we performed an in vitro CRISPR-Cas9 dropout screen focusing on macroH2A variants in MOLM-13 cells. Genetic inactivation of H2AFY but not H2AFY2 resulted in outcompetition of infected cells against non-infected competitors. This finding could be recapitulated in 3 additional AML cell lines (OCI-AML3, THP-1, HL-60), indicating a broader dependency on H2AFY irrespective of the oncogenic background or underlying driver mutation. To validate the functional impact of H2AFY on leukemia development in vivo, we generated a novel conditional knockout mouse model for H2afy and induced leukemia through retroviral infection of sorted Lineage-Sca1+cKit+ (LSK) cells with the oncogene MLL-AF9. Results of these functional studies will be presented. To understand the functional impact of macroH2A variants on normal HSPC function in vivo, we performed competitive repopulation studies. We used conventional knockout mouse models for the 2 H2afy isoforms: macroH2A1.1 and macroH2A1.2 In addition, for inactivation of both isoforms, we used the conditional macroH2A knockout mouse model. H2afy2 was either inactivated by RNAi or conditionally knockout through Mx1-Cre recombinase activation. In brief, whole bone marrow cells (WBMCs) were transplanted into primary recipient mice in a 1:1 competitive manner and compared to the respective wildtype littermate controls. Peripheral blood (PB) chimerism was monitored on a monthly basis 4 weeks post-transplantation for a total of 16 weeks. We observed no significant difference in the recipients of macroH2A1.1 (n=5/5) or macroH2A1.2 (n=10/9), suggesting both macroH2A isoforms are dispensable for intrinsic functions of HSCs. Remarkably, we found macroH2A2-deficient cells (n=6) competed significantly worse than macroH2A2+/+ (n=5) controls in PB (p=0.0043**) and in WBM (p=0.0043**), indicating a requirement for macroH2A2 in maintenance of HSPCs. These findings could be recapitulated by RNA-mediated inactivation of H2afy2 in sorted HSCs. We observed significant reduction of macroH2A2-depleted hematopoiesis after 16 weeks in WBM (p=0.0286*) and PB (p=0.0079**). Importantly, this functional impairment was less pronounced when investigating multipotent progenitor and committed progenitor cell function in spleen colony-forming unit (CFU) assay in vivo or CFU assay in vitro. Taken together, our data indicate distinct requirements for macrohistone variants in leukemic and normal hematopoietic cells. Dependency of leukemic cells but not normal HSPCs on macroH2A may indicate a potential therapeutic index. Disclosures No relevant conflicts of interest to declare.

ESGCT 27th Annual Congress In collaboration with SETGyc Barcelona, Spain October 22–25, 2019 Abstracts

Xenotransplantation of transgenic porcine pancreatic islets offers a promising alternative source to circumvent current limitations posed by the scarcity of allogeneic donors. We have investigated the feasibility to generate tissue engineered SLA silenced islet cell clusters (ICC) to decrease xenogeneic immune responses Pancreatic islets single cell suspensions were generated by enzymatic digestion of porcine ICCs. Single cells were silenced for SLA expression by lentiviral vectors encoding for Nanoluciferase as reporter gene and for short hairpin RNAs targeting beta2-microglobulin or class II transactivator, respectively. SLA transcripts were evaluated by real-time PCR and protein levels by flow cytometry and fluorescence microscopy analyses. The effect of SLA silencing was evaluated in human T and NK cell cytotoxicity assays. SLA-silenced pancreatic beta-cells were then reassembled into ICCs in stirred bioreactors. SLA class I silencing reached a level of up to 84% and class II by up to 50% on pancreatic islet cell. Silencing SLA expression did not affect cell viability and the insulin-producing beta-cell phenotype. Xenogeneic T-cell immune responses (p < 0.05) as well as antibody-mediated cellular-dependent immune responses (p < 0.01) were significantly decreased. Silencing SLA class I expression did not increase susceptibility to NK-cell cytotoxicity. In stirred bioreactors, tissue engineered islets showing the typical 3D-structure and morphology of ICC were assembled from SLA-silenced pancreatic cell suspensions. These data shows the feasibility to generate low immunogenic porcine ICC from transgenic pigs after single cell engineering and post-transduction islet reassembling that might serve as a robust alternative to allogeneic pancreatic islet cell transplantation.

RNA EDITOME ANALYSIS OF HEMATOPOIETIC CELLS REVEALS ANTIZYME INHIBITOR 1 AS A FUNCTIONAL REGULATOR IN HEMATOPOIETIC STEM AND PROGENITOR CELLS

RNA editing, adenosine (A)-to-inosine (I), plays a vital role in many biological processes. Our previous study has demonstrated that the hematopoietic stem and progenitor cells (HSPCs) deficient in adenosine deaminase acting on RNA 1 (Adar1), an RNA-editing enzyme, cannot reconstitute the irradiated recipients in vivo and form colonies in vitro (Xufeng R et al, PNAS 2009). However, the overall profile of RNA editome in hematopoiesis has not been established and the underlying mechanism how RNA editing governs the function of HSPCs is poorly defined. In this study, we sorted 12 murine adult hematopoietic cell populations and performed RNA sequencing. We depicted the landscape of RNA editome in hematopoietic cells and identified 30,796 editing sites in total. By charactering specific editing sites in different hematopoietic cell subsets at different stages, we found that the antizyme inhibitor 1 (Azin1) was highly edited in coding region in HSPCs as opposed to other cell types. To understand whether edited Azin1 was required for functioning of HSPCs, we transduced c-Kit+ HSPCs with the lentivirus carrying Azin1 cDNAs with distinct editing frequencies. c-Kit+ cells transduced with fully edited Azin1 showed enhanced reconstitution, compared to that transduced with partially edited or non-edited Azin1. Specifically, inability of RNA editing in Azin1 blocked the differentiation of hematopoietic stem cells (HSCs) in vivo. Moreover, a similar finding was obtained when Azin1 was knocked down. In summary, for the first time, this study provides a landscape of specific editing sites in hematopoietic cascade and demonstrates a pivotal role of the RNA-editable gene, Azin1, in hematopoietic stem and progenitor cells.

Abstract 1194: Adoptive cellular therapy overcomes tumor-induced dysregulation of myelopoiesis

Abstract INTRODUCTION: Hematopoietic stem and progenitor cell (HSPC) transfer during adoptive T cell immunotherapy (ACT) prolongs median survival and generates 30% long-term cures for malignant brain tumors. We recently demonstrated during ACT that intratumoral HSPCs differentiate into immune-activating dendritic cells (DCs) through T cell-released IFN-γ. We also determined that CCR2+ HSPCs are the key immune-activating progenitor population. We previously utilized syngeneic transfer of naive HSPCs that were not endemic to the recipient tumor-bearing host. This is different from the clinical paradigm of autologous transfers. This is important because in peripheral cancers tumor-bearing host (TB) HSPCs possess considerable immunosuppressive potential. It remains unknown if this is true in brain tumor-bearing hosts, and it remains unknown how this would impact HSPC+ACT immunotherapy. We therefore evaluated the immunologic function of naïve or TB HSPCs during immunotherapy. METHODS: We utilized KR158B and GL261 intracranial gliomas to generate TB HSPCs. HSPC culture experiments were performed in RPMI alone or conditioned with T cell supernatants to study HSPC differentiation. In vivo treatment models utilized intracranial gliomas and intravenous transfer of HSPCs with ACT. Data from the immgen.org database was utilized for hypothesis generation. RESULTS: Brain tumors promoted a 20% expansion of HSPCs including granulocyte monocyte precursors (GMP) but a loss of DC progenitors. The immgen database revealed that GMPs express high levels of IFN-γR1 while DC progenitors express high levels of IFN-γR2, CCR2, and CXCR4, the chemotactic receptor for HPSC homing. We next determined by flow cytometry that TB HSPCs express 40% more IFN-γR1 and 90% more IFN-γR2 on DC progenitors. To determine the differentiation preference of these cells, we performed in vitro culture. After a 3 day culture, naïve HSPCs differentiated into 30% myeloid-derived suppressor cells (MDSCs; CD11b+Ly-6G/6C+) while TB HSPCs differentiated into 60% MDSCs. When both cells types were cultured in activated tumor-specific T cell supernatants containing IFN-γ, both HSPC types differentiated into 30% MDSCs, 80% MHCII+ antigen-presenting cells, and 20% CD11c+MHCII+ DCs. In vivo, ACT rescued intratumoral TB HSPCs to prolong median survival and generate long-term cures. Additionally, TB HSPC-derived cells in brain tumors maintained higher IFN-γR2 and displayed non-inferior differentiation into DCs when compared naïve HSPCs. We are now investigating the impact of PD-1 blockade on TB HSPC function. CONCLUSIONS: Gliomas exert an immunosuppressive pressure on HSPCs. However, ACT can overcome dysregulated TB HSPC programming and promote generation of DCs instead of MDSCs. A phase I trial evaluating the impact of HSPC transfer on adoptive immunotherapy in pediatric high-grade gliomas is underway at our center (ACTION; NCT03334305). Citation Format: Tyler J. Wildes, Catherine T. Flores, Bayli DiVita Dean, Adam Grippin, Kyle Dyson, Duane A. Mitchell. Adoptive cellular therapy overcomes tumor-induced dysregulation of myelopoiesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1194.

Control of Hematopoietic Stem and Progenitor Cell Function through Epigenetic Regulation of Energy Metabolism and Genome Integrity.

SummaryIt remains largely unclear how stem cells regulate bioenergetics and genome integrity to ensure tissue homeostasis. Here, our integrative gene analyses suggest that metabolic and genotoxic stresses may underlie the common functional defects of both fetal and adult hematopoietic stem and progenitor cells (HSPCs) upon loss of DPY30, an epigenetic modulator that facilitates H3K4 methylation. DPY30 directly regulates expression of several key glycolytic genes, and its loss in HSPCs critically impaired energy metabolism, including both glycolytic and mitochondrial pathways. We also found significant increase in DNA breaks as a result of impaired DNA repair upon DPY30 loss, and inhibition of DNA damage response partially rescued clonogenicity of the DPY30-deficient HSPCs. Moreover, CDK inhibitor p21 was upregulated in DPY30-deficient HSPCs, and p21 deletion alleviated their functional defect. These results demonstrate that epigenetic mechanisms by H3K4 methylation play a crucial role in HSPC function through control of energy metabolism and protecting genome integrity.

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.