Long-term Hematopoietic Stem Cells Function Research Articles

STAT3 protects hematopoietic stem cells by preventing activation of a deleterious autocrine type-I interferon response.

Hematopoietic stem and progenitor cells (HSPCs) maintain blood-forming and immune activity, yet intrinsic regulators of HSPCs remain elusive. STAT3 function in HSPCs has been difficult to dissect as Stat3-deficiency in the hematopoietic compartment induces systemic inflammation, which can impact HSPC activity. Here, we developed mixed bone marrow (BM) chimeric mice with inducible Stat3 deletion in 20% of the hematopoietic compartment to avoid systemic inflammation. Stat3-deficient HSPCs were significantly impaired in reconstitution ability following primary or secondary bone marrow transplantation, indicating hematopoietic stem cell (HSC) defects. Single-cell RNA sequencing of Lin-ckit+Sca1+ BM cells (LSKs) revealed aberrant activation of cell cycle, p53, and interferon (IFN) pathways in Stat3-deficient HSPCs. Stat3-deficient LSKs accumulated γH2AX and showed increased expression of DNA sensors and type-I IFN (IFN-I), while treatment with A151-ODN inhibited expression of IFN-I and IFN-responsive genes. Further, the blockade of IFN-I receptor signaling suppressed aberrant cell cycling, STAT1 activation, and nuclear p53 accumulation. Collectively, our results show that STAT3 inhibits a deleterious autocrine IFN response in HSCs to maintain long-term HSC function. These data signify the importance of ensuring therapeutic STAT3 inhibitors are targeted specifically to diseased cells to avoid off-target loss of healthy HSPCs.

CPT1a Controls the Function of Long-Term Hematopoietic Stem Cells By Regulating the Balance between Fatty Acid Oxidation and Oxphos in Mitochondria

Hematopoietic stem cells (HSCs) rely on the balance in place of quiescence and self-renewal to sustain stem cell potential and differentiation to generate mature blood cells. Appropriate regulation of cellular and mitochondrial metabolic processes such as glycolysis, OXPHOS, fatty acid metabolism, and amino acid metabolism are required for HSC maintenance. However, the mechanisms by which cellular and mitochondrial metabolisms regulate HSC quiescence, proliferation, and differentiation remain to be fully understood. Fatty acid oxidation (FAO) is a metabolic process involving the breakdown of fatty acids into acetyl-CoA, which is then transported into mitochondria by carnitine palmitoyltransferase 1a (CPT1a). It has been reported that inhibiting FAO using non-specific inhibitors of CPT1a impairs HSC maintenance. Interestingly, CPT1a forms a complex with the Fanconi anemia complementation group D2 (FANCD2) protein (T Zhang et al., Scientific Reports, 2017). Given that Fanconi anemia is associated with HSC defects, this CPT1a complex may play a critical role in maintaining fatty acid dependent mitochondrial metabolism that mediates HSC dormancy and stemness. However, the exact role, in HSCs, of mitochondrial CPT1a, the rate-limiting enzyme in FAO, remains unknown. Here, we find that CPT1a is highly enriched in hematopoietic stem cells. Further, we investigated the role of CPT1a in hematopoiesis utilizing a CPT1aconditional knockout in mice driven by Vav1-Cre. The CPT1a knockout ( Cpt1aΔ/Δ) displays profound HSC defects, including loss of HSC self-renewal and accelerated differentiation. Mechanistically, we find that loss of Cpt1a results in increased ATP production, mitochondrial membrane potential (Δφm), and mitochondrial ROS accumulation in long-term HSCs (LT-HSCs), as well as defective LT-HSC function indicated by bone marrow transplantation and 5-FU genotoxic stress assays. Furthermore, utilizing mitochondrial complex formation assays and measurements of mitochondrial complex activity, BM cells of Cpt1aΔ/Δ mice display enhanced mitochondrial complex formation and up-regulation of mitochondrial complex activity. Our results suggest that Cpt1a regulates FAO transport and that loss of Cpt1a deregulates mitochondrial metabolisms including oxidative phosphorylation (OXPHOS). Taken together, our study suggests that CPT1a plays a key role in LT-HSC maintenance by controlling the balance between FAO and OXPHOS in mitochondria. Because of the noncanonical relationship between CPT1a and FANCD2 for the regulation of mitochondrial metabolism, correction of fatty acid metabolism and OXPHOS may present a novel therapeutic avenue for Fanconi anemia.

B-lineage acute lymphoblastic leukemia causes cell autonomous defects in long-term hematopoietic stem cell function.

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STAT3 Protects Hematopoietic Stem and Progenitor Cell Function in Non-Inflamed Conditions

The transcriptional regulator STAT3 has well established anti-inflammatory functions in mature myeloid cells by preventing excessive pro-inflammatory factor production in response to Toll-like receptor agonists or the gut microbiota. However, this role has precluded understanding of STAT3 function in maintaining hematopoietic stem and progenitor cell (HSPC) homeostasis, as Stat3 deletion in the hematopoietic system leads to systemic inflammation and ineffective hematopoiesis. Therefore, novel experimental approaches are needed to uncouple STAT3 function in HSPCs from the effects of systemic inflammation. To understand how STAT3 regulates hematopoietic homeostasis in a non-inflamed environment, we developed competitive mixed bone marrow (BM) chimeric mice with conditional Stat3 deletion in approximately 20% of hematopoietic cells (Figure 1a). We demonstrated successful engraftment of test (CD45.2+ CreER or CD45.2+ CreER Stat3f/f) and competitor (CD45.1+CD45.2+) BM cells at a 1:4 ratio, respectively, in lethally-irradiated CD45.1+ recipient mice. Then, we induced Stat3 deletion by tamoxifen treatment (Figure 1a). We found a significant impairment in the hematopoietic activity of Stat3-deficient HSPCs, as shown by ~50% reduction of peripheral blood CD45.2+ cells after Stat3 deletion, which suggests a crucial role of STAT3 in sustaining hematopoiesis (Figure 1b). Serum cytokine levels and colon pathology scores were similar between mice with Stat3-deficient cells and corresponding CreER controls, which confirms a non-inflammatory environment. We also observed loss of a majority of CD45.2+ immune lineages in spleen, colon, and BM upon Stat3 deletion. To evaluate the self-renewal capability of Stat3-deficient hematopoietic stem cells (HSCs), we performed secondary BM transplantation experiments. These experiments showed that CD45.2+Stat3-deficient HSCs could not reconstitute peripheral blood populations. Moreover, we found a striking decline in Stat3-deficient lin-Sca-1+ckit+ (LSK) cells and HSCs (LSK CD48-CD150+) in secondary BM transplants (Figure 1c). These results suggests that STAT3 is required to sustain a functional HSC compartment in non-inflamed conditions. To investigate how loss of STAT3 affects the transcriptional landscape of HSPCs, we performed scRNA-sequencing (scRNA-seq) in phenotypically defined LSKs isolated from primary BM transplant recipients. This analysis identified 12 distinct transcriptional states in the LSK compartment (Figure 1d). Compared with CreER control cells, Stat3-deficient LSKs were enriched in myeloid-related clusters at the expense of lymphoid clusters. Differential expression analysis among the clusters revealed that Stat3-deficient HSCs (Cluster 3) and multipotent progenitors (Cluster 2) upregulated Myc and Interferon (IFN) Alpha Response pathways, including genes involved in cell cycle activation, IFN-stimulated genes (Ifi203, Mndal), and IFN transcriptional regulators (Irf1, Irf2, Irf2bp2) (Figure 1e). In addition, Stat3-deficient LSKs acquired γH2AX modifications over time. Collectively, these data suggest that Stat3 deletion induces excessive DNA damage and intrinsic activation of an IFN transcriptional signature in HSPCs, which leads to impairment of HSC function. In conclusion, our results reveal a cell-autonomous role for STAT3 in preserving hematopoietic stem and progenitor cell homeostasis, sustaining long-term HSC function, and preventing induction of an intrinsic IFN response and DNA damage (Figure 1f). Our study highlights STAT3-driven protective mechanisms in HSPCs under non-inflammatory conditions. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Acetyl-CoA Carboxylase 1 Is Essential for Hematopoietic Stem Cell Homeostasis

Though fatty acid oxidation has been shown to be essential for hematopoietic stem cell maintenance, the importance of fatty acid uptake from the bone marrow microenvironment vs. de novo lipogenesis has not been elucidated. The process of de novo lipogenesis begins with the generation of malonyl-CoA from acetyl-CoA and is catalyzed by the enzyme acetyl-CoA carboxylase 1 (ACC1), encoded by Acaca. In leukemia, both ACC1 activation and degradation of ACC1 have been shown to enhance disease progression, suggesting an important role in leukemogenesis. The function of ACC1 in normal hematopoiesis remains unknown. To characterize the role of ACC1 in normal hematopoiesis, we bred mice that harbored an Acaca allele in which exons 22 through 26 were flanked by loxp sites. We crossed these animals to a mouse line harboring the Mx1-Cre transgene, generating Acaca fl/fl; Mx1-Cre + (ACC1 KO) and Acaca fl/fl; Mx1-Cre -(ACC1 WT) mice. Animals were then treated with intraperitoneal poly(I:C) injection to drive Mx1-Cre expression and Acaca excision. By 4-6 weeks after poly(I:C) injection, ACC1 KO animals developed expansion of hematopoietic stem cells and progenitors, including Lineage - Sca-1 +cKit hi (LSK) and the LSK CD48 -CD150 + long-term hematopoietic stem cell (LT-HSC) populations. In addition, these animals developed splenomegaly with extramedullary LSK expansion and enhanced myelopoiesis. ACC1 KO LSKs showed increased cell cycle activity and LT-HSCs demonstrated a reduced quiescent fraction compared to ACC1 WT controls. These data suggest that ACC1 deficiency results in increased cell cycle activity among primitive hematopoietic progenitors and biases cell fate to the myeloid lineage. To further test the impact of ACC1 deletion on LT-HSC function, we established competitive chimerism maintenance assays in which equal numbers of CD45.2 +Acaca fl/fl; Mx1-Cre +or CD45.2 +Acaca fl/fl; Mx1-Cre - bone marrow cells were mixed with CD45.1 + competitor bone marrow and transplanted into lethally irradiated recipient mice. Transplants were then given 8 weeks to establish stable engraftment. After documenting comparable engraftment, 5-poly(I:C) injections were administered every other day. Over the course of 16 weeks of tracking, the ACC1 KO graft showed a significant reduction in trilineage hematopoietic output as indicated by a reduction in B cell, T cell, and myeloid populations in the peripheral blood. After 16 weeks, animals were sacrificed and the LT-HSC compartment was analyzed, showing a significant reduction in ACC1 KO LT-HSCs. Together, these data suggest that the loss of ACC1 significantly impairs the ability of LT-HSCs to maintain long term hematopoiesis in competitive transplants. Collectively, these data are the first report of a critical role for ACC1 in hematopoiesis and LT-HSC function. Mechanistically, in the absence of ACC1, FACS sorted LSK demonstrated increased ATP content compared to WT controls, suggesting an altered metabolic state in these progenitor cells. Further studies are ongoing to determine if these findings are due to an essential function for ACC1 within the context of de novo lipogenesis, or if ACC1 participates in an as of yet uncharacterized tumor suppressive role. DisclosuresNo relevant conflicts of interest to declare.

Bone marrow remodeling supports hematopoiesis in response to immune thrombocytopenia progression in mice

AbstractImmune thrombocytopenia (ITP) is an acquired autoimmune condition characterized by both reduced platelet production and the destruction of functionally normal platelets by sustained attack from the immune system. However, the effect of prolonged ITP on the more immature hematopoietic progenitors remains an open area of investigation. By using a murine in vivo model of extended ITP, we revealed that ITP progression drives considerable progenitor expansion and bone marrow (BM) remodeling. Single-cell assays using Lin–Sca1+c-Kit+CD48–CD150+ long-term hematopoietic stem cells (LT-HSCs) revealed elevated LT-HSC activation and proliferation in vitro. However, the increased activation did not come at the expense of LT-HSC functionality as measured by in vivo serial transplantations. ITP progression was associated with considerable BM vasodilation and angiogenesis, as well as a twofold increase in the local production of CXCL12, a cytokine essential for LT-HSC function and BM homing expressed at high levels by LepR+ BM stromal cells. This was associated with a 1.5-fold increase in LepR+ BM stromal cells and a 5.5-fold improvement in progenitor homing to the BM. The increase in stromal cells was transient and reverted back to baseline after platelet count returned to normal, but the vasculature changes in the BM persisted. Together, our data demonstrate that LT-HSCs expand in response to ITP and that LT-HSC functionality during sustained hematopoietic stress is maintained through an adapting BM microenvironment.

Signal-Transducing Adaptor Protein-2 Blocks B Cell Recovery Under Hematological Stress at Pre-B Stage Via TLR4 Signaling

All lineages of blood cell production from hematopoietic stem cells (HSC) are maintained for an entire lifetime. That is consistent under homeostatic condition, while hematopoiesis is changed in response to various types of stress, including infections and aging. It has been reported that hematopoietic stem and progenitor cells (HSPC) are targets of pathogen products and danger signals. After the exposure to Gram-negative lipopolysaccharide (LPS) that is the ligand of Toll-like receptor (TLR) 4, HSC enter cell-cycle and differentiate into myeloid lineage cells while B lymphopoiesis is suppressed. Although the regulation of hematopoiesis by HSC is well studied, many questions still remain regarding how progenitors contribute the response to hematopoietic demands. Signal-transducing adaptor protein-2 (STAP-2) was cloned as a c-fms/M-CSFR interacting protein in 2003, and the interaction with a variety of signaling or transcriptional molecules such as STAT5, MyD88 and IκB kinase (IKK) in macrophages or T cells has been reported. The function as an adaptor protein in various cell types and signaling pathways, and its ubiquitous expression promoted us to investigate the effect of STAP-2 on HSPC under hematopoietic stress using gene-modified mice.We previously reported that hematopoiesis in STAP-2 knock out mice (KO) is normal under homeostatic condition. First in this study, competitive bone marrow (BM) transplantation experiment was conducted using KO. Four months after intravenous injection of Lineage- Sca1+ cKithigh (LSK) HSPC (CD45.2+) derived from KO or wild-type mice (WT) with competitor BM mononuclear cells (CD45.1+), the engraftment and lineage distribution were comparable, indicating that STAP-2 does not affect the function of long-term HSC. Interestingly, we found that chimerism in peripheral B cells at 1 month after transplantation in KO LSK transplanted mice was higher than control with statistical significance. In BM, STAP-2 deficiency increased pre-B and immature B cell cells, while there were no differences in numbers of LSK HSPC or Lin- Sca1+ cKitlow Flk2high common lymphoid progenitor (CLP) between WT and KO donor. To evaluate the role of STAP-2 in early B lymphopoiesis, we generated transgenic mice (Tg) that overexpress STAP-2 under the control of Eμ enhancer and Lck proximal promoter. The promoter could drive expression of the inserted cDNA in lymphoid lineage cells from CLP stage. In Tg, we observed significant decrease of B progenitors compared to WT BM. When HSPC (LSK or CLP) derived from Tg were cultured with OP9 stromal cells under B cell condition in the presence of IL-7, Flt3-ligand and SCF, the production of B220+ B cells were significantly suppressed. The same was true in stromal-cell free cultures. In colony-forming unit (CFU) assays, we found that STAP-2 overexpression decreased the ability to generate pre-B colonies, while the deficiency promoted high recovery with statistical significance (recovered colony number; 40.0 ± 1.4 in WT, 7.0 ± 1.4 in Tg, and 89.0 ± 5.7 in KO). To elucidate the detailed mechanisms, RNA-seq experiment with WT and Tg under steady-state as well as WT and KO 1 month after transplantation was conducted. Data among them were compared, and bioinformative approach with Ingenuity Pathway Analysis revealed that STAP-2 upregulates TLR4 signaling in pre-B cells during stress hematopoiesis. Consistent with the results, when LPS was added into CFU cultures, the recovered pre-B colony was suppressed. Moreover, deletion of STAP-2 partially cancelled the inhibitory effect of LPS on pre-B cell proliferation.In summary, we found that STAP-2 regulates the early B lymphopoiesis under hematopoietic stress. While STAP-2 did not influence stemness of HSC or homeostatic hematopoiesis, short-term recovery of B cells after transplantation was promoted by STAP-2 deficiency. This effect was induced by the activated proliferation of pre-B cells. Analyses with Tg mice confirmed the inhibitory effect of STAP-2 on early B lymphopoiesis. RNA-seq analyses have shown that activation of TLR4 signaling at pre-B stage occurs 1 month after transplantation as well as in Tg mice, and STAP-2 deficiency partially cancelled the inhibitory effects of LPS in CFU pre-B assays. Our study unveiled the critical role of lineage progenitors in short-term response to hematological demands and developmental stage-specific regulation of TLR4-STAP-2 pathway. DisclosuresOritani:Celgene: Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; MOCHIDA PHARMACEUTICAL: Speakers Bureau; Novartis Pharma: Consultancy, Speakers Bureau. Shibayama:Takeda Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Fujimoto Pharmaceutical Co.: Honoraria, Research Funding; Jansen Pharmaceutical K.K.: Honoraria; Bristol-Meyer Squibb K.K.: Honoraria, Research Funding; Ono Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Novartis Pharma K.K.: Honoraria, Research Funding; Mundipharma K.K.: Honoraria, Research Funding; Celgene K.K.: Honoraria, Research Funding. Kanakura:Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding; Bristol Myers: Research Funding; Fujimotoseiyaku: Research Funding; Chugai Pharmaceutical: Research Funding; Astellas: Research Funding; Pfizer: Research Funding; Nippon Shinyaku: Research Funding; Toyama Chemical: Research Funding; Shionogi: Research Funding; Eisai: Research Funding; Kyowa Hakko Kirin: Research Funding.

Decreased IGF-1 Expression in the Bone Marrow Microenvironment Drives Hematopoietic Aging

One factor responsible for poor hematopoietic health-span is overproduction of myeloid lineage cells at the expense of lymphoid cells, which is associated with increased risk of myeloid leukemia and reduced immune responsiveness with aging. This lineage imbalance is caused by declining ability of hematopoietic stem cells (HSC) to regenerate and sustain proper lineage-balanced production of mature hematopoietic cells. We identify the hematopoietic decline to become significant in C57BL/6 mice as young as 8 months old, suggesting that by middle age the initiation events of myeloid overproduction have already begun to occur. Placing young (2mo) long-term hematopoietic stem cells (LT-HSC) into a 2mo or middle-aged (9-12mo) bone marrow (BM) microenvironment is sufficient to induce myeloid-biased cell production (2mo into 2mo: 12.3% myeloid ± SEM (n= 3) vs. 2mo into 9-12mo: 26.5% myeloid ± SEM (n= 11) ** P < 0.01), clearly implicating components of the bone marrow microenvironment in the age-related overproduction of myeloid cells. Data utilizing RNA-Seq and ELISA analysis identify a decrease in insulin-like growth factor (IGF)-1 expression as candidate signaling factor produced by cells in the middle- aged microenvironment that drive myeloid-biased hematopoiesis from HSC. LT-HSC expression of IGF-1 signaling receptor, IGF1R, is increased with age identifying that the balance of IGF signaling is altered in the bone marrow microenvironment. In vitro studies show that the stimulation of the middle-aged (12-14mo) Lin-/c-kit+/Sca-1+(LSK) population by IGF-1 leads to decreased myeloid production, rescuing the aging phenotype (12-14mo: 126.7 CFU vs. 12-14mo + IGF1: 79.9 CFU ± SEM *** P < 0.001 by one-way ANOVA ± SEM of n= 3 replicates). Phospho-flow analysis of middle-aged LT-HSC from mice indicate increased IGF1R and AKT signaling response to IGF-1 stimulation suggesting the decrease of IGF-1 bioavailability in the bone marrow microenvironment seen during aging is influencing LT-HSC function and lineage output. Our ongoing studies aim to define how aging-induced alterations in the BM microenvironment initiate myeloid lineage-biased hematopoiesis from HSC and how this is mediated through IGF signaling. Understanding these mechanisms may enable new therapeutic approaches to improve hematopoietic health-span. DisclosuresNo relevant conflicts of interest to declare.

Mitochondrial Potentiation Ameliorates Age-Related Heterogeneity in Hematopoietic Stem Cell Function.

Aging is associated with reduced fitness and increased myeloid bias of the hematopoietic stem cell (HSC) compartment, causing increased risk of immune compromise, anemia, and malignancy. We show that mitochondrial membrane potential (MMP) can be used to prospectively isolate chronologically old HSCs with transcriptional features and functional attributes characteristic of young HSCs, including a high rate of transcription and balanced lineage-affiliated programs. Strikingly, MMP is a stronger determinant of the quantitative and qualitative transcriptional state of HSCs than chronological age, and transcriptional consequences of manipulation of MMP in HSCs within their native niche suggest a causal relationship. Accordingly, we show that pharmacological enhancement of MMP in old HSCs invivo increases engraftment potential upon transplantation and reverses myeloid-biased peripheral blood output at steady state. Our results demonstrate that MMP is a source of heterogeneity in old HSCs, and its pharmacological manipulation can alter transcriptional programs with beneficial consequences for function.

Caudal Type Homeobox 2 Is Required for Leukemogenesis but Not for Normal Hematopoieisis

Caudal Type Homeobox 2 (CDX2) is a caudal-related homeobox family transcription factor known to be developmental regulator involved in murine axial elongation and anteroposterior vertebral patterning and cell fate determination in the intestine. In adults, CDX2 expression is restricted to the small intestine and the colon. Interestingly, CDX2 is overexpressed in ~90% of human acute myeloid leukemias and the majority of acute lymphoblastic leukemias. Hence, aberrant expression of CDX2 may play a contributing role in leukemogenesis. The role of CDX2 in normal hematopoietic development is unknown. To assess the role of CDX2 in normal hematopoiesis we characterized hematopoietic development in Cdx2 fl/fl Mx1-Cre+ mice after poly (I:C)-mediated gene deletion compared to wildtype littermates. Under steady state conditions, no significant defects were identified in long-term hematopoietic stem cells (LT-HSCs), multipotent progenitors, myeloid progenitors, or mature cell lineages. To test if long-term hematopoietic stem cells were functionally impaired following Cdx2 deletion, competitive transplantation assays were performed. We found that CDX2 was not required for long-term hematopoietic reconstitution in the competitive transplant setting. Together, these data establish that CDX2 is not required for adult steady state hematopoiesis or LT-HSC function. We then asked whether or not CDX2 was essential for leukemogenesis. To interrogate this, Cdx2 fl/fl Mx1-Cre+, Cdx2 fl/+ Mx1-Cre+, and wildtype littermates were treated with poly (I:C) to facilitate gene deletion or for control purposes. Bone marrow cells from the mice were then transformed using well-established HOXA9/MEIS1 or MLL-AF10 leukemogenesis models. In both systems, Cdx2-deficiency prevented leukemia development, whereas wildtype controls succumbed to leukemia within 200 days. Cdx2 heterozygous knockout animals had a delayed onset of leukemia compared to wildtype animals. Taken together, these data indicate that CDX2 is not required for steady state adult hematopoiesis or LT-HSC function, but is necessary for the development of leukemia. Our findings suggest CDX2 has potential as a novel therapeutic target that could be exploited in the majority of AML and ALL patients across a broad mutational spectrum. The nomination of CDX2 as a key target for the majority of leukemias is significant, as current targeted treatment strategies are limited to only particular patient subsets. Disclosures No relevant conflicts of interest to declare.

Extracellular vesicles impose quiescence on residual hematopoietic stem cells in the leukemic niche.

Progressive remodeling of the bone marrow microenvironment is recognized as an integral aspect of leukemogenesis. Expanding acute myeloid leukemia (AML) clones not only alter stroma composition, but also actively constrain hematopoiesis, representing a significant source of patient morbidity and mortality. Recent studies revealed the surprising resistance of long-term hematopoietic stem cells (LT-HSC) to elimination from the leukemic niche. Here, we examine the fate and function of residual LT-HSC in the BM of murine xenografts with emphasis on the role of AML-derived extracellular vesicles (EV). AML-EV rapidly enter HSC, and their trafficking elicits protein synthesis suppression and LT-HSC quiescence. Mechanistically, AML-EV transfer a panel of miRNA, including miR-1246, that target the mTOR subunit Raptor, causing ribosomal protein S6 hypo-phosphorylation, which in turn impairs protein synthesis in LT-HSC. While HSC functionally recover from quiescence upon transplantation to an AML-naive environment, they maintain relative gains in repopulation capacity. These phenotypic changes are accompanied by DNA double-strand breaks and evidence of a sustained DNA-damage response. In sum, AML-EV contribute to niche-dependent, reversible quiescence and elicit persisting DNA damage in LT-HSC.

Septin 6 regulates engraftment and lymphoid differentiation potential of murine long-term hematopoietic stem cells

Septins are a family of filament-forming GTP-binding proteins that serve as scaffolds and diffusion barriers in various cellular processes. Septin 6 is known as a fusion partner of mixed-lineage leukemia in infant acute myeloid leukemia. The occurrence of the fusion gene is associated with a reduced expression of septin 6 itself. The role of septin 6 in hematopoiesis and whether it is involved in scaffolds within hematopoietic cells is not known. Septin 6-deficient hematopoietic stem cells (HSCs) present with an increased engraftment potential but altered lymphoid differentiation with a reduced contribution to the T-cell compartment and an increased B-cell contribution. Although multipotent progenitor cells showed a very distinct septin 6 filament organization and intracellular distribution, their function was not impaired by septin 6 deficiency. Our data therefore suggest a regulatory role for septin 6 in long-term HSC function and lymphoid lineage differentiation.

Systemic Virus Infections Differentially Modulate Cell Cycle State and Functionality of Long-Term Hematopoietic Stem Cells In Vivo

Quiescent long-term hematopoietic stem cells (LT-HSCs) are efficiently activated by type I interferon (IFN-I). However, this effect remains poorly investigated in the context of IFN-I-inducing virus infections. Here we report that both vesicular stomatitis virus (VSV) and murine cytomegalovirus (MCMV) infection induce LT-HSC activation that substantially differs from the effects triggered upon injection of synthetic IFN-I-inducing agents. In both infections, inflammatory responses had to exceed local thresholds within the bone marrow to confer LT-HSC cell cycle entry, and IFN-I receptor triggering was not critical for this activation. After resolution of acute MCMV infection, LT-HSCs returned to phenotypic quiescence. However, non-acute MCMV infection induced a sustained inflammatory milieu within the bone marrow that was associated with long-lasting impairment of LT-HSC function. In conclusion, our results show that systemic virus infections fundamentally affect LT-HSCs and that also non-acute inflammatory stimuli in bone marrow donors can affect the reconstitution potential of bone marrow transplants.

The Class IA PI3 Kinase Isoforms Have Distinct but Overlapping Roles in Adult Hematopoiesis

The PI3 kinase (PI3K) signaling pathway is activated by most hematopoietic growth factors and chemokines that orchestrate normal adult hematopoiesis. Hematopoietic cells express four different catalytic isoforms of PI3K (p110α, β, δ, and γ), each encoded by a different gene (pik3ca, pik3cb, pik3cd and pik3cg, respectively). The Class IA (p110 α, β, δ) and Class IB (p110γ) enzymes use different regulatory subunits and are activated by different sets of upstream signals. While pik3ca and pik3cb are expressed ubiquitously, pik3cd is enriched in leukocytes. The roles of PI3K in adult hematopoiesis have been difficult to investigate using gene targeting in mice, since two of the isoforms (p110α and p110β) are required during embryogenesis. However, mice with germline deletion of pik3cd are viable and fertile with normal blood counts (Jou et al, MCB, 2002. 22(24):8580-91). Using the Mx1-Cre system to conditionally delete pik3ca in adult hematopoietic stem cells (HSCs), we previously showed that p110α is dispensable for adult HSC function (Gritsman et al, J Clin Invest 2014;124(4):1794-1809). We now report that conditional deletion of pik3cb using Mx1-Credoes not affect blood counts, bone marrow cellularity, survival, bone marrow HSC or progenitor numbers, or hematopoietic reconstitution in the transplantation setting. Therefore, none of the individual Class IA PI3K isoforms have critical roles in HSCs. However, it is still unclear whether these isoforms can substitute for each other in adult HSCs, or whether they are truly dispensable for HSC function.To distinguish between these two possibilities and to uncover potentially redundant roles for PI3K isoforms in hematopoiesis, we have generated compound conditional knockout mice with the genotypes: pik3ca-lox/lox;pik3cd-/-;Mx1-Cre ("p110α-/-;p110δ-/-") and pik3ca-lox/lox;pikcb-lox/lox;Mx1-Cre ("p110α-/-;p110β-/-"). Interestingly, compound deletion of p110α and p110δ in HSCs results in significantly reduced white cell counts and anemia (Gritsman et al, Blood (ASH Annual Meeting Abstracts), Nov 2012; 120: 2322). In addition, p110α-/-;p110δ-/- mice have reduced numbers of bone marrow multipotential progenitors (MPPs), common myeloid progenitors (CMPs), and common lymphoid progenitors (CLPs), while HSC numbers are preserved. Furthermore, deletion of both p110α and p110δ leads to reduced B cell repopulation in competitive repopulation assays. This suggests that p110α and δ have redundant roles in HSC differentiation and B cell specification.In contrast, we found that compound deletion of p110α and p110β has no effect on long-term competitive repopulation of any lineages for up to 20 weeks. We also observed normal donor chimerism in the HSC, MPP, and progenitor populations in transplant recipients of p110α-/-;p110β-/- bone marrow. After non-competitive transplantation, recipients of p110α-/-;p110β-/- bone marrow maintained normal blood counts over time. This indicates that, while p110δ is sufficient to support hematopoietic reconstitution of all lineages in the transplantation setting, p110β cannot support normal HSC differentiation by itself, particularly into the B cell lineage. However, p110α-/-;p110δ-/- mice do not develop pancytopenia, and repopulation of the myeloid and T cell lineages is still largely preserved, suggesting that long-term HSC function is intact. To determine whether HSC self-renewal and myeloid reconstitution after transplantation are possible in the absence of all three Class IA PI3Ks, we have generated triple knockout mice with the genotype pik3ca-lox/lox;pikcb-lox/lox;pik3cd-/-;Mx1-Cre ("TKO"). Analysis of the hematopoietic phenotype of TKO mice will be presented. These results will be valuable in predicting future hematologic toxicity in ongoing clinical trials with isoform-selective PI3K inhibitors or pan-PI3K inhibitors. DisclosuresNo relevant conflicts of interest to declare.

The Runx-PU.1 pathway preserves normal and AML/ETO9a leukemic stem cells

AbstractRunx transcription factors contribute to hematopoiesis and are frequently implicated in hematologic malignancies. All three Runx isoforms are expressed at the earliest stages of hematopoiesis; however, their function in hematopoietic stem cells (HSCs) is not fully elucidated. Here, we show that Runx factors are essential in HSCs by driving the expression of the hematopoietic transcription factor PU.1. Mechanistically, by using a knockin mouse model in which all three Runx binding sites in the −14kb enhancer of PU.1 are disrupted, we observed failure to form chromosomal interactions between the PU.1 enhancer and its proximal promoter. Consequently, decreased PU.1 levels resulted in diminished long-term HSC function through HSC exhaustion, which could be rescued by reintroducing a PU.1 transgene. Similarly, in a mouse model of AML/ETO9a leukemia, disrupting the Runx binding sites resulted in decreased PU.1 levels. Leukemia onset was delayed, and limiting dilution transplantation experiments demonstrated functional loss of leukemia-initiating cells. This is surprising, because low PU.1 levels have been considered a hallmark of AML/ETO leukemia, as indicated in mouse models and as shown here in samples from leukemic patients. Our data demonstrate that Runx-dependent PU.1 chromatin interaction and transcription of PU.1 are essential for both normal and leukemia stem cells.

Femoral microarchitecture and endosteal niche alterations in protein malnutrition: possible effects on hematopoiesis?

Hematopoiesis is a dynamic process that leads to blood cells production. At the centre of this process sits the hematopoietic stem cells (HSC), which preferentially reside along the trabecular metaphysis of long bones. Evidences suggest that specific microenvironments could support HSC self-renewal and differentiation. The endosteal niche hosts HSC in quiescence or self-renewing state. Protein malnutrition (PM) is associated with pancytopenia, bone marrow (BM) hypoplasia and changes in bone microenvironment. In this way, we hypothesized that PM may change the endosteal niche with a consequent impairment in the number and function of long-term HSC (LT-HSC). To test this hypothesis, C57Bl/6J mice were randomly assigned in control and malnourished groups which received normoproteic and hypoproteic diets (12% and 2% protein, respectively) over a 5 weeks period. We characterized femoral bone tissue changes in vivo by measuring bone mineral density (BMD) and ex vivo, using computed microtomography. Compared to controls, malnourished animals (MA) presented a decrease in the BMD of different regions of the femur and a significant decrease in the trabecular bone volume (BV/TV), and trabecular number (Tb.N) of the distal metaphysis of the femur, followed by an increase in the trabecular separation (Tb.Sp) and a prevalence of rod-like trabeculae. Furthermore, osteoblasts and LT-HSC were isolated from the femoral region by immunomagnetic depletion and immunophenotyped by flow cytometry. We found a smaller amount of LT-HSC in the BM of MA. Osteoblasts from MA showed decreased mRNA expression of type I collagen and osteocalcin, however no difference was found on CXCL-12, angiopoetin, Jagged-2 or N-cadherin mRNA expression. Therefore, we may conclude that PM compromises trabecular bone structure and reduces the number of LT-HSC cells in the BM. Hematopoiesis is a dynamic process that leads to blood cells production. At the centre of this process sits the hematopoietic stem cells (HSC), which preferentially reside along the trabecular metaphysis of long bones. Evidences suggest that specific microenvironments could support HSC self-renewal and differentiation. The endosteal niche hosts HSC in quiescence or self-renewing state. Protein malnutrition (PM) is associated with pancytopenia, bone marrow (BM) hypoplasia and changes in bone microenvironment. In this way, we hypothesized that PM may change the endosteal niche with a consequent impairment in the number and function of long-term HSC (LT-HSC). To test this hypothesis, C57Bl/6J mice were randomly assigned in control and malnourished groups which received normoproteic and hypoproteic diets (12% and 2% protein, respectively) over a 5 weeks period. We characterized femoral bone tissue changes in vivo by measuring bone mineral density (BMD) and ex vivo, using computed microtomography. Compared to controls, malnourished animals (MA) presented a decrease in the BMD of different regions of the femur and a significant decrease in the trabecular bone volume (BV/TV), and trabecular number (Tb.N) of the distal metaphysis of the femur, followed by an increase in the trabecular separation (Tb.Sp) and a prevalence of rod-like trabeculae. Furthermore, osteoblasts and LT-HSC were isolated from the femoral region by immunomagnetic depletion and immunophenotyped by flow cytometry. We found a smaller amount of LT-HSC in the BM of MA. Osteoblasts from MA showed decreased mRNA expression of type I collagen and osteocalcin, however no difference was found on CXCL-12, angiopoetin, Jagged-2 or N-cadherin mRNA expression. Therefore, we may conclude that PM compromises trabecular bone structure and reduces the number of LT-HSC cells in the BM.

Analyzing gene function in adult long-term hematopoietic stem cells using the interferon inducible Mx1-Cre mouse system.

Long-term hematopoietic stem cells (LT-HSCs) have the ability to self-renew and differentiate into all blood cell lineages. Understanding the genetic networks that regulate LT-HSC function in the adult bone marrow requires inducible gene targeting and bone marrow transplantations. In this chapter we describe the use of the inducible Mx1-Cre mouse model to delete genes in LT-HSCs and methodologies for examining the function of LT-HSCs following deletion.

Autophagy Is Required For Long-Term Hematopoietic Stem Cell (HSC) Function and G-CSF-Induced HSC Mobilization

Hematopoiesis originates from a rare pool of hematopoietic stem cells (HSC) that are uniquely capable of both self-renewal and terminal differentiation into lineage-committed progenitor cells. Autophagy is a process of cytoplasmic protein recycling which maintains cellular homeostasis and protects the cell during periods of metabolic stress and nutrient deprivation and has an established role in the survival and function of immunological cells. Recent publications have linked autophagy with preservation of normal long term HSCs (LT-HSCs) during aging (Nature; 2013: 494:323-7). We therefore sought to determine the role of autophagy in LT-HSC function at homeostasis and during the clinically relevant stress of G-CSF-induced HSC mobilization.Using single cell imaging flow cytometry to monitor autophagosome formation in LC3-GFP transgenic mice (LC3-GFP punctae formation is a reporter of autophagy activity) we demonstrated autophagic activity in HSC populations but not in committed myeloid progenitors. In line with this, inhibition of autophagy degradation by chloroquine administration resulted in the accumulation of autophagy related protein p62 in purified HSCs compared to myeloid progenitors. To determine the contribution of autophagy to HSC development and function, we analyzed mice deficient in Atg5, a protein essential for autophagosome formation. Autophagy was not required for fetal liver (FL) HSC development, however, Atg5-/- FL HSCs showed mildly reduced long-term repopulating function in bone marrow (BM) transplantation assays (16 weeks peripheral blood (PB) engraftment Atg5-/- 83.45% vs. WT 93.10%, n=12, p<0.001). Importantly, Atg5-/- LT-HSCs (Lin-Sca-1+c-kit+Flk2-CD150+CD48-) were markedly reduced in congenic recipients compared to WT FL LT-HSCs (Atg5-/- 0.02% vs. WT 0.04%, n=11, p<0.001). Secondary competitive transplantation was used to determine the effect of autophagy loss on LT-HSC function in vivo. Atg5-/- LT-HSCs exhibited a profound impairment in the repopulation of secondary congenic recipients (Atg5-/-4.65% vs. WT 32.3%, n=5, p<0.01).Mechanistically, microarray analysis of purified LT-HSCs from Atg5-/- vs. WT FL chimeras demonstrated clear differences in gene expression by unsupervised hierarchical clustering. Differentially expressed genes included Cxcl12, Sdc2 and Apex1, regulators of HSC fate. Gene Ontology enrichment analysis demonstrated that autophagy deficient LT-HSCs had impaired metabolism, enhanced cellular differentiation, enforced proliferation and increased apoptosis. Validating these findings, there was a loss of quiescence in the Atg5-/- compared to WT LT-HSC (quiescent Atg5-/- 30% vs. WT 39%, n=16 p<0.05) and Atg5-/- FL HSCs exhibited enhanced apoptosis after culture in cytokine enriched media (Atg5-/-21.2% vs. WT 14.2%, n=5, p<0.01).Given the requirement for autophagy in homeostasis of LT-HSC and its role in proliferation and metabolic stress, we next investigated whether autophagy participated in the HSC response to G-CSF. G-CSF is commonly used to ameliorate neutropenia in patients treated with chemotherapy and is also used to mobilize HSCs for patients undergoing HSC transplantation. Using single cell imaging flow cytometry, autophagosome formation was enhanced in HSCs after 6 days of G-CSF mobilization (10mcg/day). G-CSF treatment efficiently mobilized PB neutrophils and colony forming units (CFU) in WT chimeras, however Atg5-/- chimeras showed a striking reduction in G-CSF-induced neutrophil (Atg5-/- 4.95 x106/mL vs. WT 10.46 x106/mL, n=20, p<0.0001) and PB CFU mobilization post-G-CSF (Atg5-/- 47/100µL vs. WT 126/100µL, n=5, p<0.01). BM CFU numbers were similar in Atg5-/- and WT FL chimeras both pre- and post-G-CSF mobilization. Atg5-/-neutrophils demonstrated increased apoptosis after G-CSF treatment suggesting that autophagy limits PB neutrophil survival, but not BM neutrophil development during G-CSF mobilization.These data demonstrate that autophagy is an active process in LT-HSCs and that genetic deletion of Atg5 results in the failure of adult LT-HSC maintenance and function. Autophagic activity is augmented by G-CSF-induced HSC stress and is required for G-CSF-induced HSC mobilization. These findings are particularly relevant to HSC transplantation and hematopoietic function in the context of a rapid rise in the clinical use of agents that have modulatory effects on autophagy. Disclosures:No relevant conflicts of interest to declare.

Heterogeneity Of Leukemic Stem Cell Capacity Of BCR-ABL+ Long-Term Hematopoietic Stem cells In CML Is Associated With Variability In MPL Expression

In chronic myelogenous leukemia (CML), in vivo long-term repopulating and leukemia stem cell (LSC) capacity is restricted to a small population of BCR-ABL+ long-term hematopoietic stem cells (LTHSC). Using an inducible transgenic SCL-tTA/BCR-ABL mouse model of CML, we have shown that leukemic cells with long-term repopulating and leukemia-initiating capacity have the Lin-Sca-1+Kit+Flt3-CD150+CD48- phenotype, also characteristic of normal LTHSC. Limiting dilution transplantation studies show that frequency of cells with LTHSC phenotype with long-term engraftment capacity (1:6) is considerably higher than those with leukemia-initiating capacity (1:80) suggesting that only some LTHSC may have LSC capacity (Cancer Cell 21:577, 2012). To further evaluate the basis for heterogeneity in LSC potential of BCR-ABL+ LTHSC, SCL-tTA/BCR-ABL mice were crossed with GFP expressing mice to allow tracking of donor cells, and a cohort of mice were transplanted with limiting numbers of GFP+LTHSC (200 per mouse) and followed for engraftment of GFP+ cells and development of CML (WBC>10,000/ul). Only 11 of 20 mice developed CML, whereas 9 mice showed long term engraftment without development of CML. GFP+ LTHSC selected from primary recipients were transplanted into secondary recipients (200 per mouse). Seven of 17 mice receiving cells from mice with CML also developed CML after the second transplant, whereas none of the mice receiving cells from non-CML mice developed CML, suggesting the distinction between leukemogenic versus non-leukemogenic LTHSC was maintained after transplantation. LTHSC isolated from primary recipients were also analyzed for expression of several HSC-regulatory genes by multiplex Q-PCR using the Fluidigm system. On hierarchical clustering, LTHSC from mice developing CML clustered separately from LTHSC from mice without CML. Amongst cell surface expressed genes, expression of the thrombopoietin (TPO) receptor MPL (p=0.006) and CD47 (p=0.006) was significantly increased in LTHSC from mice developing CML. We did not see significant differences in BCR-ABL expression in LTHSC from mice with or without CML. We further analyzed the relationship of MPL expression with CML LTHSC function. CML LTHSC (n=6) expressing high levels of MPL (MPLhi, top 10% based on MPL expression) showed significantly increased cell growth (p<0.0001) and CFC potential (p=0.0007) when cultured with TPO (10ng/ml) compared to LTHSC expressing low levels of MPL (MPLlo, lowest 10% based on MPL expression), as well as significantly increased cell growth (p=0.005) and CFC (p=0.03) compared to normal MPLhi LTHSC. Following transplantation, MPLhi LTHSC (200 per mouse) generated significantly higher short-term (4 wks, p=0.008) and long-term (16 wks, p=0.003) engraftment of donor cells compared to MPLlo LTHSC. Seven of 16 mice receiving MPLhi LTHSC developed CML compared to only 1 out of 17 mice receiving MPLlo LTHSC. We next evaluated heterogeneity of MPL expression in LTHSC (CD34+CD38-CD90+ cells) from CML patients and normal subjects. As was seen in murine studies, human CML MPLhi LTHSC cultured with TPO (10ng/ml) showed increased cell growth (p<0.0001) and CFC frequency (p=0.02) compared to CML MPLlo LTHSC, and significantly increased cell growth (p<0.0001) and CFC generation (p=0.02) compared to normal MPLhi LTHSC. Both baseline and TPO stimulated p-Stat3/5 levels were significantly higher in human CML MPLhi LTHSC compared with MPLlo LTHSC (p<0.0001), and in CML compared to normal MPLhi LTHSC. Interestingly p-Stat5 response peaked at 1 hour in CML LTHSC compared to 20 minutes in normal LTHSC, further indicating alterations in MPL signaling in CML LTHSC. Transplantation of CML MPLhi LTHSC (3x104 cells/mouse) into NSG mice resulted in higher engraftment of human myeloid cells in BM at both 4 and 16 weeks (p<0.05) compared with MPLlo LTHSC. Normal MPLhi LTHSC also showed higher engraftment in NSG mice at 4 and 16 weeks compared with MPLlo cells. Our studies indicate that CML LTHSC represent a heterogeneous population with varying LSC capacity. Heterogeneity in LSC capacity is associated with variability in expression of MPL. Higher levels of MPL expression in CML LTHSC are associated with significantly increased Stat3/5 signaling, in vitro and in vivo growth, and LSC capacity. These results identify MPL as a key regulator of LSC potential of BCR-ABL+ LTHSC and a potential target for LSC-directed therapeutics. Disclosures:No relevant conflicts of interest to declare.

Intermittent in Vivo Parathyroid Hormone (PTH) Treatment Results in Jagged 1 (Jag 1) Dependent and Independent Effects On Hematopoietic Stem Cells (HSCs)

Abstract 642HSCs are rare immature cells capable of reconstituting all blood cell lineages throughout the life of an individual. We have previously shown that intermittent treatment with PTH is sufficient to increase the number of HSCs in the marrow of mice. This PTH effect is blocked in vitro with inhibition of gamma-secretase, the mediator of a required step in Notch signaling. Osteoblastic cells are a critical component of the HSC niche and are likely mediators of the PTH-induced increase in HSCs. Specifically the Notch ligand Jag 1 is expressed on osteoblasts and is therefore implicated as a mechanism through which PTH acts on HSCs. Therefore we investigated in vivo the role of osteoblastic Jag 1 in the PTH-dependent increase in HSCs. We utilized the 2.3kb collagen 1 promoter driven cre recombinase to specifically excise Jag 1 from osteoblastic cells in mice (OBJag1 mice). As we previously reported treatment of wild type (WT) controls with PTH 3 times daily for 10 days resulted in a significant increase in phenotypic HSC populations including Lin-Sca1+cKit+CD48-CD150- short-term HSCs (ST-HSCs) (VEH/PTH 0.0405±0.001 vs 0.0650±0.0038, p≤0.0001) and Lin-Sca1+cKit+CD48-CD150+ long-term HSCs (LT-HSCs) (VEH/PTH 0.0077±0.0008 vs 0.0125±0.00096, p≤0.01) as determined by flow cytometric analysis. In contrast treatment of OBJag1 mice did not result in a phenotypic increase in these populations. Despite the lack of a phenotypic increase in HSCs in OBJag1 mice, when HSC function was assessed by competitive repopulation assay, OBJag1 marrow cells demonstrated the same increased repopulating ability as WT mice (WT: VEH/PTH 12.16±2.7 vs 22.32±2.4, p≤0.01, OBJag1: VEH/PTH 13.6±1.8 vs 31.6±5.9, p≤0.01). Upon secondary transplantation however, HSCs from OBJag1 donors treated with PTH resulted in a lower engraftment rate than VEH treated controls (VEH/PTH 14.61±3.8 vs 4.38±0.9, p≤0.05). This result suggests that osteoblastic Jag 1 is necessary for the increase in phenotypic HSCs resulting from PTH treatment and is required to maintain LT-HSC self-renewal. However these data also suggest an osteoblastic Jag 1 independent mechanism that mediates a transient increase in repopulating ability. Decreased apoptosis is a potential mechanism by which PTH may functionally increase HSCs in the absence of increased self-renewal. To determine if PTH treatment decreases the apoptosis rate of HSCs, WT mice were treated intermittently with PTH once a day for 7 days. Despite a lack of increased HSCs by phenotypic analysis at 7 days, marrow from PTH treated mice displayed an increase in LT-HSC function as measured by competitive transplantation. We determined to measure the effect of PTH on apoptotic rates of HSCs using Annexin V membrane expression. By the 7th day of PTH treatment, LT-HSC apoptotic rates were decreased in the PTH treated group (VEH/PTH 10.482±2.25 vs. 6.27±1.93, p≤0.01) suggesting that changes in apoptotic rate of LT-HSCs precedes the HSC increase. These results were confirmed by flow cytometric measurement of activated caspase 3. PTH treatment decreased the percentage of LT-HSCs that were positive for activated caspase 3 (VEH/PTH 4.3±0.5 vs. 2.4±0.3, p≤0.01). PTH induced micro-architectural changes in trabecular bone at day 7 of treatment suggesting bone involvement despite the lack of an increase in bone volume. These results suggest for the first time that PTH may exert its beneficial effect on bone marrow reconstitution through both Jag 1 dependent and independent effects. Additionally, HSCs demonstrate decreased apoptotic rates and increased reconstitution ability prior to a demonstrable phenotypic increase, mimicking the effect seen in the absence of osteoblastic Jag 1. Together these results suggest that the decreased apoptotic rate may be mediated by an osteoblastic Jag 1 independent mechanism. Whether osteoblasts are required for the observed osteoblastic Jag 1 independent effects remains to be seen as these effects could be mediated by a Jag 1 independent osteoblastic mechanism or by an altogether different cellular component of the HSC niche. Further, since stressful manipulation of HSCs ex vivo is essential for their use in transplantation, defining factors regulating and decreasing their apoptosis may improve their engraftment efficiency, expanding their clinical use when their numbers are limited. Disclosures:No relevant conflicts of interest to declare.