Proportion Of Hematopoietic Stem Cells Research Articles

Gestational diabetes mellitus affects the differentiation of hematopoietic stem cells in neonatal umbilical cord blood.

There are abundant hematopoietic stem cells (HSCs) in cord blood. It is known that HSCs continue to differentiate to CLP, CMP and erythroid progenitor cells (EPC), EPC ultimately differentiated to platelets and erythrocytes. It has been reported that the proportion of HSCs in cord blood was higher than that in healthy pregnant women, so as the incidence of neonatal polycythemia in gestational diabetes mellitus (GDM) patients. We aimed to investigate whether the hyperglycemic and/or hyperinsulin environment in GDM patients has effects on the differentiation of HSCs into erythrocytes in offspring cord blood. In this study, we collected cord blood from 23 GDM patients and 52 healthy pregnant women at delivery. HSCs, CLP, CMP and EPCs in cord blood of the two groups were identified and quantified by flow cytometry. HSCs were sorted out and treated with glucose and insulin, respectively, and then, the changes of HSCs proliferation and differentiation were detected. Compared to healthy controls, HSCs, CMP and EPC numbers in cord blood from GDM group were significantly increased, while CLP cell number was decreased. The differentiation of HSCs into EPC was promoted after treatment with glucose or insulin. There were more HSCs in the cord blood of GDM group, and the differentiation of HSCs to EPCs was increased. These findings were probably caused by the high-glucose microenvironment and insulin medication in GDM patients, and the HSCs differentiation changes might be influencing factors of the high incidence of neonatal erythrocytosis in GDM patients.

The Effect of SIRT5 Deletion on Recovery of Hematopoietic Stem Cells after Injury in Mouse

To investigate the effect of deacylase Sirtuin 5 in the recovery of hematopoietic stem cells (HSCs) after treated by 5-FU in mouse. Flow cytometry was used to analyze the effect of SIRT5 deletion on the proportion of hematopoietic stem/progenitor cells (HSPCs) in bone marrow (BM), the proportion of T cells, B cells and myeloid cells (TBM) in peripheral blood (PB) and spleen, and the development of T cells in thymus. Mouse were treated with 5-FU to study the effect of SIRT5 deletion on the cell cycle, apoptosis and the proportion of HSPCs in BM. The effect of SIRT5 deletion on the proliferation of HSCs was analyzed by flow sorting in vitro. SIRT5 deletion did not affect the development of T cells in thymus and the proportion of TBM cells in PB and spleen compared with wild type mice. SIRT5 deletion increased proportion of HSPCs in BM. After 5-FU treatment, the proportion of HSCs in SIRT5 deletion mice was significant decreased (P < 0.05), the HSPC in SIRT5 deletion mice was activated from G0 to G1 phase (P < 0.05), and the proportion of early apoptosis increased (P < 0.05). By monoclonal culture in vitro, the ability of HSCs to form clones in SIRT5 deletion mice was decreased significantly (P < 0.05). SIRT5 deletion lead to a decreased the ability of HSCs to clone in vitro. SIRT5 deletion is not conducive to the recovery of HSPCs injury in mice under hematopoietic stress.

Hyperactivity of platelets and increased megakaryopoiesis in COVID-19 patients with acute respiratory distress syndrome

The Severe Acute Respiratory Syndrome (SARS)-CoV-2-induced Coronavirus Disease 2019 (COVID-19) pandemic has caused an overwhelming influence on public health because of its high morbidity and mortality. Critical-illness cases often manifest as acute respiratory distress syndrome (ARDS). Previous evidence has suggested platelets and thrombotic events as key mediators of SARS-CoV-2-associated ARDS. However, how the balance of platelet regeneration from the hematopoietic system is changed in ARDS remains elusive. Here, we reported a more severe inflammation condition and hyperactivity of platelets in COVID-19 ARDS patients compared with those infected but without ARDS. Analysis of peripheral blood revealed an increased proportion of hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs), megakaryocyte-erythrocyte progenitors (MEPs), and megakaryocyte progenitors (MkPs) in ARDS patients, suggesting a megakaryocytic-differentiation tendency. Finally, we found altered gene expression pattern in HSCs in COVID-19 ARDS patients. Surprisingly, genes representing platelet-primed HSCs were downregulated, indicating these cells are being stimulated to differentiate. Taken together, our findings shed light on the response of the hematopoietic system to replenish platelets that were excessively consumed in COVID-19 ARDS, providing a mechanism for disease progression and further therapeutic development.Graphical

The effect of platelet infusion on the repair of bone marrow hematopoietic niche damage in mice induced by (60)Co radiation and hematopoietic reconstruction after bone marrow transplantation

Objective: To observe the effect of platelets on hematopoietic stem cell (HSCs) implantation in mice with radiation-induced bone marrow injury and bone marrow transplantation models. Methods: ①Male C57BL/6 mice were divided into a single irradiation group and a radiation infusion group after receiving (60)Co semimyeloablative irradiation for 18-10 weeks. The irradiation infusion group received 1×10(8) platelets expressing GFP fluorescent protein. ② The allogeneic bone marrow transplantation model was established. The experimental groups included the simple transplantation group (BMT) and the transplantation infusion group (BMT+PLT). The BMT group was infused through the tail vein only 5 × 10(6) bone marrow cells, the BMT+PLT group needs to be infused with bone marrow cells at the same time 1× 10(8) platelets. ③ Test indicators included peripheral blood cell and bone marrow cell counts, flow cytometry to detect the proportion of hematopoietic stem cell (HSC) and hematopoietic progenitor cells, bone marrow cell proliferation and apoptosis, and pathological observation of vascular niche damage and repair. Results: ①On the 3rd, 7th, 14(th), and 21st days after irradiation, the bone marrow cell count of the infusion group was higher than that in the single irradiation group (P<0.05), and the peripheral blood cell count was also higher. A statistically significant difference was found between the white blood cell count on the 21st day and the platelet count on the 7th day (P<0.05). In the observation cycle, the percentage of bone marrow cell proliferation in the infusion group was higher, while the percentage of apoptosis was lower. ② The results of bone tissue immunofluorescence after irradiation showed that the continuity of hematopoietic niche with red fluorescence was better in the irradiation infusion group. ③The chimerism percentage in the BMT+PLT group was always higher than that in the BMT group after transplantation.④ The BMT+PLT group had higher bone marrow cell count and percentage of bone marrow cell proliferation on the 7th and 28th day after transplantation than that in the BMT group, and the percentage of bone marrow cell apoptosis on the 14th day was lower than that in the BMT group (P<0.05). After the 14th day, the percentage of stem progenitor cells in the bone marrow cells of mice was higher than that in the BMT group (P<0.05). ⑤The immunohistochemical results of bone marrow tissue showed that the continuity of vascular endothelium in the BMT+PLT group was better than that in the BMT group. Conclusion: Platelet transfusion can alleviate the injury of vascular niche, promotes HSC homing, and is beneficial to hematopoietic reconstruction.

Exploring Microbes in Breast Tumors From Women of Different Races

Exploring Microbes in Breast Tumors From Women of Different Races

Effect of Recombinant Human Thrombopoietin (rhTPO) on Long-term Hematopoietic Recovery in Mice with Acute Radiation Sickness and Relative Mechanism

To investigate the effect and relative mechanism of Recombinant Human Thrombopoietin (rhTPO) on long-term hematopoietic recovery in mice with acute radiation sickness. Mice were intramuscularly injected with rhTPO (100 μg/kg) 2 hours after total body irradiation with 60Co γ-rays (6.5 Gy). Moreover, six months after irradiation, peripheral blood, hematopoietic stem cells (HSC) ratio, competitive transplantation survival rate and chimerization rate, senescence rate of c-kit+ HSC, and p16 and p38 mRNA expression of c-kit+ HSC were detected. Six months after 6.5 Gy γ-ray irradiation, there were no differences in peripheral blood white blood cells, red blood cells, platelets, neutrophils and bone marrow nucleated cells in normal group, irradiated group and rhTPO group (P>0.05). The proportion of hematopoietic stem cells and multipotent progenitor cells in mice of irradiated group was significantly decreased after irradiation (P<0.05), but there was no significant changes in rhTPO group (P>0.05). The counts of CFU-MK and BFU-E in irradiated group were significantly lower than that in normal group, and rhTPO group was higher than that of the irradiated group(P<0.05). The 70 day survival rate of recipient mice in normal group and rhTPO group was 100%, and all mice died in irradiation group. The senescence positive rates of c-kit+ HSC in normal group, irradiation group and rhTPO group were 6.11%, 9.54% and 6.01%, respectively (P<0.01). Compared with the normal group, the p16 and p38 mRNA expression of c-kit+ HSC in the irradiated mice were significantly increased (P<0.01), and it was markedly decreased after rhTPO administration (P<0.01). The hematopoietic function of mice is still decreased 6 months after 6.5 Gy γ-ray irradiation, suggesting that there may be long-term damage. High-dose administration of rhTPO in the treatment of acute radiation sickness can reduce the senescence of HSC through p38-p16 pathway and improve the long-term damage of hematopoietic function in mice with acute radiation sickness.

Frizzled-6 promotes hematopoietic stem/progenitor cell mobilization and survival during LPS-induced emergency myelopoiesis

SummaryEmergency hematopoiesis involves the activation of bone marrow hematopoietic stem/progenitor cells (HSPCs) in response to systemic inflammation by a combination of cell-autonomous and stroma-dependent signals and leads to their release from bone marrow and migration to periphery. We have previously shown that FZD6 plays a pivotal role in regulating HSPC expansion and long-term maintenance. Now we sought to better understand the underlying mechanisms. Using lipopolysaccharide (LPS)-induced emergency granulopoiesis as a model, we show that failed expansion was intrinsic to FZD6-deficient HSPCs but also required a FZD6-deficient environment. FZD6-deficient HSPCs became more strongly activated, but their mobilization to peripheral blood was impaired and they were more susceptible to inflammatory cell death, leading to enhanced release of pro-inflammatory cytokines in the marrow. These studies indicate that FZD6 has a protective effect in the bone marrow to prevent an overactive inflammatory response and further suggest that mobilization improves HSPC survival during bone marrow inflammation.

Factor analysis of effective platelet-producing ability of fetal liver-derived cells

Objective: To study the different factors affecting platelet production post transplantation of hematopoietic stem cells (HSCs) isolated from different sources in order to explore novel options for treating platelet depletion following HSCs transplantation. Methods: HSCs and their downstream derivatives including myeloid and lymphoid cells (i.e., collective of mononuclear cells (MNCs)), were isolated from E14.5 fetal liver (FL) and bone marrow (BM) of 8-week-old mice by Ficoll separation technique. These cells were subsequently transplanted into the tibia bone marrow cavity of recipient mice post lethal myeloablative treatment in order to construct the FL-MNCs and BM-MNCs transplantation mouse model. Routine blood indices were examined in these recipient mice. The chimeric rate of donor cells in recipient peripheral blood cells were determined by flow cytometry. Different groups of cells involved in platelet reconstruction were analyzed. CD41+megakaryocytes were sorted from fetal liver or bone marrow using magnetic beads, which were then induced to differentiate into platelets in an in vitro assay. Quantitative RT-PCR was used to detect the expression of platelet-related genes in CD41+megakaryocytes from the two sources. Results: Both the FL-MNCs and the BM-MNCs transplantation groups resumed normal hematopoiesis at the 4th week after transplantation, and the blood cells of the recipient mice were largely replaced by the donor cells. Compared with the mice transplanted with BM-MNCs, the platelet level of mice transplanted with FL-MNCs recovered faster and were maintained at a higher level. At week 4, the PLT level of the FL-MNCs group was (1.45±0.37)×1012/L, and of the BM-MNCs group was (1.22±0.24)×1012/L, P<0.05. The FL-MNCs contain a higher proportion of hematopoietic stem cells (Lin-Sca-1+c-Kit+)(7.60%±1.40%) compared to the BM-MNCs (1.10%±0.46%), P<0.01; the proportion of the megakaryocyte progenitor cells (Lin-Sca-1-c-Kit+CD41+CD150+) and mature megakaryocyte cells (CD41+CD42b+), also differ significantly between the FL-MNCs (3.05%±0.22%, 1.60%±0.06%, respectively) and the BM-MNCs (0.15%±0.02%, 0.87%±0.11%, respectively) groups, both P<0.01. In vitro functional studies showed that FL-MNCs-CD41+megakaryocytes could produce proplatelet-like cells more quickly after induction, with proplatelet-like cells formation on day 3 and significant platelet-like particle formation on day 5, in contrast to bone marrow-derived BM-MNCs-CD41+megakaryocytes that failed to form proplatelet-like cell on day 5. In addition, FL-MNCs-CD41+cells expressed higher levels of platelet-related genes, Mpl (3.25-fold), Fog1 (3-fold), and Gata1 (1.5-fold) (P<0.05). Conclusion: Compared with the BM-MNCs group, the FL-MNCs transplantation group appears to have a more efficient platelet implantation effect in the HSCs transplantation recipient in vivo, as well as a higher platelet differentiation rate in vitro. This might be related to a higher proportion of megakaryocytes and higher expression levels of genes such as Mpl, Fog1, and Gata1 that could be important for platelet formation in FL-MNCs-CD41+cells. Further exploration of the specific functions of these genes and the characteristics of the different proportions of the donor cells will provide valuable clues for the future treatment of platelets reconstitution after HSCs transplantation clinically.

Nuclear Protein DEK Governs Quiescence and Metabolic Homeostasis of Hematopoietic Stem Cells By Shaping Chromatin Accessibility

Hematopoietic stem cells (HSCs) must achieve a balance between quiescence and activation that fulfils the demands for hematopoiesis without compromising long-term maintenance of HSCs. DEK, a chromatin architectural factor, is involved in chromatin remodeling, transcriptional regulation and DNA replication, and is implicated in genetic and epigenetic regulation of gene expression. Here, we identified that DEK is a critical regulator of HSCs quiescence. Deletion of DEK in mice resulted in abnormal hematopoiesis with an obvious decreased HSC pool size (~3700 to ~1700 cells/mouse), associated with apparent reduction in the proportion of HSCs in G0 phase as compared to control HSCs (~72% to ~57%). As shown by serial bone marrow transplantation and competitive repopulation assays, deletion of DEK impaired the self-renewal capacity of HSCs. Mechanistically, deficiency of DEK in HSC altered chromatin accessibility landscape, resulting in increased transcription of activation-specific genes (including Akt1/2,Ccnb2, and Rps6) and decreased transcription of quiescence-specific genes (including p21 and Gata2), leading to excessively activated Akt-mTOR signaling and elevated metabolism of HSC. Targeting the Akt-mTOR pathway efficiently abrogated the impaired quiescence and the increased metabolism of HSC in DEK-deficient mice, and partially rescued the long-term functions of HSC. Further, DEK regulated chromatin accessibility of HSC by recruiting the co-repressor NCoR1 to repress acetylation of histone 3 at lysine 27. Collectively, our findings revealed crucial functions of DEK in HSC quiescence maintenance and disclosed a new link between chromatin remodelers, epigenetic modification, gene transcription, and HSC homeostasis. Disclosures No relevant conflicts of interest to declare.

ZRSR1 Cooperates with ZRSR2 in Regulating Splicing of U12-Type Introns in Murine Hematopoietic Cells

Mutations in genes encoding components of the RNA splicing machinery constitute the leading class of genetic alterations in myelodysplastic syndromes (MDS). Somatic inactivating alterations of splicing factor, ZRSR2, are observed in ~10% of MDS patients. Mutations/loss of ZRSR2 in human myeloid cells causes aberrant splicing, primarily impairing splicing of the U12-type introns. However, the precise role of ZRSR2 in splicing and hematopoietic development needs further exploration. To understand the function of ZRSR2 in hematopoietic development and splicing, we generated Zrsr2 knockout mice. RNA-Seq of sorted myeloid precursor populations (common myeloid progenitors, granulocyte monocyte progenitors and megakaryocyte erythrocyte progenitors) revealed aberrant retention of U12-type introns in ZRSR2-deficient murine cells, similar to our previous observation in ZRSR2 mutant MDS bone marrow cells (Madan et al, Nat Commun 2015). However, despite complete loss of ZRSR2 in our mouse model, the effect on splicing of U12-type introns was modest compared to the ZRSR2 mutant MDS bone marrow and ZRSR2 knockdown human AML cells (K562 and TF1). Moreover, our comprehensive analyses of hematopoietic compartment in ZRSR2-deficient (Zrsr2Δ/Y) compared to WT (Zrsr2+/Y) male mice, indicated that ZRSR2 was dispensable for hematopoietic differentiation. We did not observe any significant difference in peripheral blood counts, bone marrow cellularity, proportion of hematopoietic stem cells (HSCs) and myeloid precursors (CMP, GMP, MEP) in the bone marrow of both young (7-10 weeks) and old (&amp;gt;1 year) mice of either genotypes. Competitive and non-competitive reconstitution assays also demonstrated that loss of ZRSR2 does not affect repopulation potential of HSCs. Collectively, our data demonstrate that ZRSR2 is not essential for hematopoietic development in mice. Further, to understand the underlying determinant for our unexpected observation that deletion of Zrsr2 in mice did not impact hematopoietic development and affected modestly splicing of U12-type introns, we investigated the role of a closely-related homolog, ZRSR1, in splicing of U12-type introns. We utilized shRNA-mediated silencing of ZRSR1 in murine myeloid precursors (Lin−Kit+ bone marrow cells), and assessed splicing using RNA-Sequencing. Wildtype myeloid cells expressing Zrsr1 shRNA did not display significant mis-splicing of the U12-type introns. However, knockdown of Zrsr1 in ZRSR2-deficient myeloid cells exacerbated mis-splicing of the U12-introns. This suggested that ZRSR1 contributes to regulation of U12-spliceosome in murine hematopoietic cells. Increased mis-splicing of U12-type introns in ZRSR1/ZRSR2-deficient cells was validated using quantitative RT-PCR. Further to verify the compensatory role of murine ZRSR1 in splicing of U12-type introns, we used CRISPR/Cas9 technology to generate 32D cells lacking either one or both ZRSR proteins. Quantitative RT-PCR analysis showed notable aberrant retention of U12-type introns in Zrsr1/Zrsr2 double-deficient 32D cells compared to either WT or single KO cells. We also demonstrated that aberrant retention of U12-type introns of MAPK9 and MAPK14 caused by deficiency of ZRSR proteins led to their reduced protein expression in both human and murine myeloid cells. Taken together, our study highlights functional role of murine ZRSR1 in splicing of U12-type introns in murine hematopoietic cells where it can partially compensate for the deficiency of ZRSR2. Therefore, deficiency of ZRSR2 alone is insufficient to impact extensively RNA splicing in mice, and further studies with concurrent deficiency of ZRSR1 and ZRSR2 are warranted to replicate complete loss of ZRSR activity. Disclosures Chng: Novartis: Honoraria; Abbvie: Honoraria; Amgen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.

PEDF Can Rescue the Inhibition of Injured Endothelial Cells on Hematopoietic Stem Cell Expansion Ex Vivo

Introduction: Endothelial cells (ECs) provide a fertile niche for hematopoietic stem cell (HSC) maintenance, differentiation, and migration.Several studies have indicated that bone marrow (BM) vascular niche was impaired after HSC transplantation and severely inhibited hematopoietic reconstruction. Pigment epithelium-derived factor (PEDF) is an important potential cytoprotection and therapeutic agent for injured cells. The direct role of the injured endothelial cells on hematopoietic stem cells and whether PEDF has protective effect in this system remain unknown. This study aims to observe the influence of enjured ECs on HSCs and to explore the role of PEDF in endothelial-HSC coculture system. Methods: Injury of Endothelial cells by two important preparative regimenconditioning radiation and Busulfan respectively was evaluated with CCK8 assay. The expression of endothelial tight junctions(TJs),adherent junctions related molecules and endothelial to Mesenchymal Transition molecules such as ZO-1, Occludin,VE-cadherin, ICAM, α-SMA, CD31 and VCAM were detected by RT-qPCR, flow cytometry, immunofluorescence and western blot. The effects of injured endothelial cells on HSC self-renewal, differentiation, cell cycle and apoptosis were evaluated by flow cytometry, photography, viable cell count and clone formation assay. Hematopoiesis regulation factors SCF, IL-6, TGF-β and TNF-α were detected by ELISA. The protective effect of PEDF was also explored. Results: Both radiation and Busulfan could decrease cell viability of endothelial cells. The expression level of ZO-1, Occludin, VE-cadherin, ICAM, CD31 and VCAM were decreased and α-SMA was increased when EC exposed to radiation or Busulfan suggesting endothelial activation, impaired EC permeability and endothelial to Mesenchymal Transition after EC injured. Compared with normal endothelial cells and hematopoietic stem cell co-culture group, the HSC% of injured endothelial cells and hematopoietic stem cells co-cultured group were significantly decreased, the cell colony formation ability was decreased, the proportion of mature cells increased, and the damage of endothelial cells could not maintain the characteristics of HSC, weakened the self-renewal and multidirectional differentiation potential of HSC and promoted the maturation of HSC. After the administration of PEDF, endothelial to Mesenchymal Transition of EC was suppressed and the EC permeability was improved. Most importantly, the proportion of HSC was significantly increased, and the proportion of mature cells decreased in the coculture system. Conclusion: Injured endothelial cells can inhibit proliferation of hematopoietic stem cells, self-renewal and promote HSC differentiation. PEDF could ameliorate endothelial injury and promote HSC expansion by suppressing endothelial-mesenchymal transition and protecting TJs and AJs. Disclosures No relevant conflicts of interest to declare.

Ripk3 Signaling Regulates Hematopoietic Stem Cell Number and Function during Stress

Background: Among all tissues, bone marrow (BM) is the most sensitive tissue to ionizing radiation (IR)-induced acute tissue damage (ATD) and chronic long-term residual damage (LT-RD). BM failure and a significant reduction in blood cells (pancytopenia) often occurs within days after exposure to IR due to the massive death of proliferative hematopoietic progenitor cells (HPCs). However, due to their quiescent cell cycle status and reduced fidelity of DNA repair feature, many hematopoietic stem cells (HSCs) cannot fully eliminate such damage and enter senescence; this results in LT-RD. Abnormal dysplastic hematopoiesis is the most common LT-RD in most victims of IR, followed by an increased risk of leukemia/lymphoma development. Thus IR exposure is an established cause of BM failure and leukemia. A significant increase in the production of inflammatory cytokines is induced by IR which contributes to the pathogenesis of both ATD and LT-RD. Such inflammatory cytokines induce the activation of Ripk3-Mlkl-mediated necroptotic signaling in HSCs. However, the role of Ripk3-Mlkl signaling in IR-induced damage has not studied. Experimental procedures: The self-renewal capacity of HSCs among Ripk3-/-, Mlkl-/- and WT mice were examined and compared by serial transplantation assay. The phenotypes of ATD and LT-RD induced by different dosages of IR were compared among Ripk3-/-, Mlkl-/- and WT mice. The mechanism by which Ripk3 signaling prevents IR-induced leukemia development was studied. Results: Ripk3-Mlkl signaling is not required for hematopoiesis during homeostatic condition. However, during serial transplantation, inactivation of such signaling prevents stress-induced loss of HSCs. Interestingly, Ripk3 signaling also induces an Mlkl-independent ROS-p38-p16-mediated senescence in HSCs. Thus Ripk3-/- HSCs showed better competitive hematopoietic ability compared to Mlkl-/- and WT HSCs during serial transplantation. A sub-lethal dosage of IR (6Gy) induces Ripk3-dependent NF-κB activation and pro-survival gene expression in HSCs, which is necessary for the survival of damaged HSCs. After 6Gy IR, although DNA damage is repaired in most HSCs within 2 days, a proportion of HSCs in WT and Mlkl-/- mice fail to fully repair the damage and undergo p53-p21-dependent senescence. However such cells in Ripk3-/- mice die from apoptosis. Thus the remaining HSCs in Ripk3-/- mice should be functionally normal, while a proportion of the remaining HSCs in Mlkl-/- and WT mice remain damaged but senescent, all as demonstrated by competitive hematopoietic reconstitution assay. Multiple low-doses of IR (1.75Gy once week × 4) induce HSC exhaustion in WT mice but not in Ripk3-/- and Mlkl-/- mice. Interestingly, almost all Ripk3-/- mice develop acute lymphoblastic leukemia within 200 days after such low dose IR, while 45% of WT and 60% of Mlkl-/- mice develop thymomas within 360 days (see Figure). Mechanistically, such low-dose IR stimulates chronic inflammatory cytokine production. Such cytokines induce Ripk3-Mlkl-mediated necroptosis in response to HSC exhaustion observed in WT mice. These cytokines also induce Ripk3-ROS-p38-p16-mediated senescence in response to impaired HSC functioning observed in both WT and Mlkl-/- mice. In Ripk3-/- mice, due to the lack of both necroptotic and senescent signaling, mutant HSCs accumulate and leukemia development is accelerated. Conclusion: Ripk3 signaling plays distinct roles in HSCs in response to different doses of IR. High-dose IR induces Ripk3-dependent NF-κB/survival signaling, which is required for the survival of HSCs which fail to repair the damage. Thus temporal inhibition of Ripk3-NF-κB signaling might help to remove the damaged HSCs thus preventing the occurrence of LT-RD. However multiple low-doses of IR induces Ripk3 activation in HSCs which represses leukemia development by inducing both ROS-p38-p16-mediated senescence and Ripk3-Mlkl-mediated necroptosis. Induced activation of Mlkl-necroptosis might help to repress leukemia development by removing damaged HSCs. Disclosures No relevant conflicts of interest to declare.

Reversible TET Inhibitor As a Hematopoietic Stem Cell Booster: A Novel Strategy to Improve Stem Cell Function

Hematopoietic stem cells (HSCs) are responsible for the adaptation capacity in times of need but are also subjected to disease processes, natural or iatrogenic damage, and age-related attrition. The latter can lead to deficient production, decreased compensatory capacity and degenerative diseases such as MDS. In childhood, hereditary BMF predominates but increasingly with age, acquired idiopathic AA is a leading cause of HSC failure. Throughout life iatrogenic cases and cumulative exposures to environmental toxicities may lead to failure of the HSC compartment. Pharmacologic HSC boosters capable of expanding HSCs would have a wide range of clinical applications in acquired and inherited BMF states, including reconstitution of exhausted hematopoiesis after chemotherapy, aging, or HSC grafting. Currently, hematopoietic growth factors (HGF) are used but lead to progenitor rather than HSC expansion despite their success in clinical application. Depletion of TET2 in murine models leads to impairment of cellular differentiation and increases the proportion of HSCs and progenitors suggesting that TET2 is a key regulator of hematopoietic homeostasis and HSC self-renewal. Alterations of TET2 via somatic mutations and/ or deletion are frequent in MDS whereby HSC and progenitor expansion may be a key component of neoplastic evolution. We hypothesized that chemical agents reversibly inhibiting TET2 activity might phenocopy HSC expansion due to mutations and be applied as HSC boosters. Using structure guided approaches, we designed, synthesized and subsequently optimized bioavailable TET inhibitors (TETi). Among them, the one of the most effective TETi, named TETi76, showed dose-dependent inhibitory activity against TET dioxygenases in in vitro cell-free and cell culture systems with 5hmC production as a read out. We developed esterified forms of TETi76 and used in our cellular experimental models. Esterified TETi is bioavailable and non-toxic to normal bone marrow (BM) cells in therapeutically effective doses. Treatment of TETi76 resulted in a 44+15% and 42+17% increase in the clonogenic potential of human and murine BM cells consistent with the proliferative advantage gained by loss of TET activity in HSCs. We then performed serial replating experiments to determine the effect of TET inhibition on immature hematopoietic progenitor cells. Over 3 consecutive passages, TETi76 treatment prolonged the durability and capacity of human HSCs to maintain colony-forming cells (155+24 vs.107+14 colonies per 1x105 P1 cells). As long-term culture initiating cells (LTC-IC) are the best in vitro surrogates of HSCs, we also investigated the effect of TETi76 in LTC-IC cultures (n=3). The weekly addition of 1μM-TETi76 resulted in nearly 2-fold increase in LTC-IC numbers at the end of the culture (116±27 vs. 64 ±26 colonies per 2x106 P1 cells, p=.011). Expansion of grafts e.g., in the setting of umbilical cord HSC transplant (UCHSC) could be an important medical area of application of TETi76. We performed suspension cultures (n=3) with an optimal cocktail of hematopoietic growth factors (HGF) in the presence or absence of TETi76 (1μM). In control cultures, total cellular output peaked on day 14, but in the presence of TETi, growth continued beyond day 28. Cumulatively, total cellular output per 106 input was 27+2.61 x106cells/mL in control cultures and 32+0.642 x106 cells/mL in TETi treated cultures on day 28. CD34+ cell output was significantly higher in cultures treated with TETi vs. vehicle (2.5x105vs. 0.9X106)CD34+ cells per 1x104 CD34+ cell input). Similar effects were observed in murine BM suspension cultures. BM cells from C57BL/6 mice (n=3) were supplemented with HGF± TETi76 (1μM). TETi treatment led to a 5-fold HSC expansion compared to vehicle treated cells at 20 days of culture. The effect of TETi76 was reversed by treatment with ascorbic acid (50 μM), a known TET activator. Cumulatively our in vitro results suggest that the presence of TETi prevents exhaustion of immature cells, observed with growth factor driven expansion. In summary, our study indicates that novel agents modulating TET activity prevent exhaustion of HSC and may help expand the HSC in vitro. In vivo experiments examining the effects of TETi on hematopoietic recovery following radiation-induced aplasia, and competitive transplant experiments of grafts exposed in vivo and in vitro to TETi are underway. Disclosures Sekeres: Millenium: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.

689. The Content of the More Immature Hematopoietic Stem Cells (HSCs) Is Dependent from the Underlying Genetic Diseases: Consequence on Transduction Efficiencies

Ex vivo gene transfer into hematopoietic CD34+ cells provides therapeutic benefits in primary immunodeficiencies. However, the CD34+ cell population is heterogeneous and is mainly composed of progenitors (CD34+lin-CD38+) and multilineage progenitors (CD34+lin-CD38-). This latter population can be divided in 3 subtypes regarding to the CD45RA and CD90 expression. CD45RA+ define cells with lymphoid potential, CD45RA-CD90- defined cells with myeloid potential and CD45RA-CD90+ define cells with long term repopulating capability (HSCs) which constitutes the relevant target for gene correction. We found that in bone marrow (BM) CD34+linneg, addition of CD133 helps to delimitate CD38neg population and we have analysed HSC, MLP and MPP population in BM from SCID-X1 (n=4), Wiskott Aldrich Syndrome (WAS) (n=2), Artemis (n=2), Chronic Granulomatous Disease (CGD) patients (n=3), sickle cell disease (SCD) (n=3) and age matched healthy controls (n=6).As expected, mean percentage of HSC represent 1% of CD34+ in healthy donors and similar results are obtained in CD34+ from SCID-X1 (1.76% ± 0.72), Artemis (1.98% ± 0.15), and SCD (2.16% ± 0.6) patients. On the contrary HSC percentage are lower in CD34+ from WAS (0.35% ± 0.15) and nearly undetectable in CGD patients. Moreover in multilineage progenitors, proportion of HSC, MLP and MPP varies from one pathology to another as shown in table1table1.Table 1Mean percentage of HSC, MLP and MPP in the linnegCD133+CD38neg CD34+ cellsHSCsMLPMPPHealthy Donor42%33%24%SCIDX137%45%28%WAS35%35%30%Artemis30%48%20%SCD36%19%44%Our aim was then to determine the ability of vectors used for gene therapy to transduce these different progenitors. We first analysed transduction of bulk CD34+ and HSCs by SIN retroviral vector for IL2RG in 2 SCIDX-1 patients enrolled in the clinical trial. After transduction, the 3 cell types were purified by flow cytometry and transduction determined in CFU after 14 days of culture in methyl cellulose in order to avoid detection of non-integrated vector. We show that in both patients, transduction efficiency was lower in HSCs than in bulk CD34+ cells. We were not able to recover enough CFU to determine VCN from MPP fraction and as expected, no CFU were obtained from MLP fraction. These results will be extended to transduction of CD34+ from WAS and SCD patients and efforts will be concentrated on transduction protocols in order to improve transduction of HSCs. Nevertheless low HSCs contents observed in CD34+ from CGD patients also represents a major difficulty to obtain long term reconstitution after gene therapy.

Hyperinflammation in patients with chronic granulomatous disease leads to impairment of hematopoietic stem cell functions

Defects in phagocytic nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) function cause chronic granulomatous disease (CGD), a primary immunodeficiency characterized by dysfunctional microbicidal activity and chronic inflammation. We sought to study the effect of chronic inflammation on the hematopoietic compartment in patients and mice with X-linked chronic granulomatous disease (X-CGD). We used immunostaining and functional analyses to study the hematopoietic compartment in patients with CGD. An analysis of bone marrow cells from patients and mice with X-CGD revealed a dysregulated hematopoiesis characterized by increased numbers of hematopoietic progenitor cells (HPCs) at the expense of repopulating hematopoietic stem cells (HSCs). In patients with X-CGD, there was a clear reduction in the proportion of HSCs in bone marrow and peripheral blood, and they were also more rapidly exhausted after in vitro culture. In mice with X-CGD, increased cycling of HSCs, expansion of HPCs, and impaired long-term engraftment capacity were found to be associated with high concentrations of proinflammatory cytokines, including IL-1β. Treatment of wild-type mice with IL-1β induced enhanced cell-cycle entry of HSCs, expansion of HPCs, and defects in long-term engraftment, mimicking the effects observed in mice with X-CGD. Inhibition of cytokine signaling in mice with X-CGD reduced HPC numbers but had only minor effects on the repopulating ability of HSCs. Persistent chronic inflammation in patients with CGD is associated with hematopoietic proliferative stress, leading to a decrease in the functional activity of HSCs. Our observations have clinical implications for the development of successful autologous cell therapy approaches.

The Nutraceutical Alpha Lipid ColostemTMModulates Peripheral Blood and Bone Marrow Stem Cell Subsets following Oral Supplementation

Alpha Lipid Colostem TM is a proprietary blend of colostrum and other natural ingredients, designed to enhance stem cell activity following consumption. Previous animal feeding studies demonstrated that oral supplementation increased the frequency of hematopoietic stem cells in the bone marrow. To further characterise the steady-state effect of this supplement on stem cell activity, a feeding study was conducted in groups of mice (n=5 per group) to investigate circulating as well as bone marrow hematopoietic and mesenchymal/stromal stem cell subsets using multi-colour flow cytometry-based methodologies. The results demonstrated that supplementation increased the number and proportion of hematopoietic stem cells (HSC) in the blood, suggesting there was effective release of stem cells from the bone marrow niche into the circulation. Analysis of bone marrow cells showed a concomitant reduction in the proportion of total HSC, however within the HSC compartment there was a relative increase of CD34 + c-Kit + and reduction in CD34 + Sca-1 + HSC subsets. Mesenchymal/stromal stem cell (CD105 + CD29 + ) numbers showed a decrease in the peripheral blood, with an increased proportion detected in the bone marrow. Alpha Lipid Colostem supplementation also correlated with increased the number of blood granulocytes demonstrating enhancement of hematopoietic stem cell differentiation. These findings further strengthen the hypothesis that dietary supplementation with a synergistic combination of bovine colostrum and immune stimulatory carbohydrates promotes coherent activation of the stem cell niche.

Deletion of Nono, a Novel Double-Strand Break Repair Factor, Leads to Hematopoietic Stem Cell Defects

Three genes, SFPQ, NONO, and PSPC1 encode for a small family of tandem RRM domain-containing proteins with diverse functions in RNA processing, transcription, and DNA repair. Our previous work has shown that an SFPQ•NONO complex promotes a distinct sub-pathway of non-homologous end joining (NHEJ) in vitro, implicating it as a novel double-strand break (DSB) repair factor. Consistently, attenuation of NONO function in human fibroblasts leads to partial radiosensitivity, delayed resolution of DNA repair foci, and an increase in radiation-dependent chromosome aberrations. To investigate the in vivo function of this complex, we knocked out Nono, the mouse homolog of the human NONO gene. We hypothesized that a DSB repair deficiency would lead to stem cell exhaustion, sensitivity to DNA damaging agents, or both. First, we investigated hematopoietic stem cells (HSCs), which are known to be sensitive to impairment of DNA repair. Nono-deficient (gt/0) mice showed reduced bone marrow and spleen cellularity, potentially due to reduced body size. The proportion of HSCs (LSK-SLAM) in relation to total bone marrow cells was similar in Nono-deficient and wild type (WT) mice, and bone marrow cell cultures yielded similar numbers and types of colonies. However, HSCs from Nono-deficient mice showed severe impairment in competitive repopulation assays in primary and secondary recipients. HSCs from gt/0 mice also displayed significantly higher levels of ROS and proliferation (accessed via BrdU incorporation), as well as higher percentage of Ki-67 positivity when compared to HSCs from WT mice. Together, these results indicate that Nono-deficient HSCs exhibit impaired capacity for self-renewal that may be caused by excessive HSC proliferation, a primary defect in response to genotoxic stress or a combination of both. To further elucidate this phenomenon, we investigated potential differences in sensitivity to the DNA crosslinking agent mitomycin C (MMC) and radiation. As previously observed in other genomic instability models (e.g., Fanconi anemia), colony-forming-cells from Nono-deficient mice were highly sensitive to MMC and X-rays (2 Gγ). Utilizing FACS-sorted HSCs, we determined that NONO protein levels are induced after X-ray treatment, and that Nono-deficient HSCs show delayed resolution of γ-H2AX foci and increased apoptosis. We also evaluated changes in the testis, another organ frequently affected by DNA repair gene mutations. Accordingly, testes of Nono-deficient mice showed growth retardation that became apparent between 18 and 26 days of postnatal development. The co-occurrence of hematopoietic and germ cell defects is reminiscent of other DNA repair gene defects, including those seen in Fanconi anemia. Collectively, our data identify NONO as a novel and important regulator of both HSC maintenance and germ cell function. Its potential effect in other organ systems and mechanism of action are under investigation. DisclosuresNo relevant conflicts of interest to declare.

Impairment of Hematopoietic Stem Cell (HSC) Niche by G-CSF Is Associated with Rapid Mobilization of Serially Reconstituting HSC and Reduced Competitive Repopulation of Mobilized Bone Marrow

Abstract 1889We have previously shown that G-CSF administration impairs HSC niches in the mobilized bone marrow (BM). G-CSF causes rapid suppression (within 2 days) of endosteal osteoblasts and bone formation with concomitant down-regulation of Kit ligand, CXCL12 and angiopoietin-1. This effect is mediated by the depletion of specific populations of BM macrophages1. Considering the very rapid impairment of HSC niches in response to G-CSF, we hypothesized that 1) the most primitive HSC should also mobilize at this very early stage within the first 48 hours of G-CSF treatment, and 2) that down-regulation of HSC niche function should also alter the number or function of HSC remaining in the mobilized BM. To test this, 125μg/kg rhuG-CSF was injected twice daily to C57BL/6 mice; blood and BM harvested at days 2 and 5 of G-CSF treatment to be transplanted into congenic recipients in a long-term competitive repopulation assay (LT-CR).Transplantation of 25μL blood showed a gradual increase in the number of LT-CR cells mobilized in response to G-CSF as measured by donor chimerism in myeloid and B lineages at 16 weeks post-transplant. Expectedly repopulating units (RU) per mL blood progressively increased from 0.2 ± 0.0 (n=6) in steady-state to 2.9 ± 1.6 (n=9) and 82.6 ± 40.4 (n=9) at days 2 and 5 of G-CSF treatment respectively. At 16 weeks post-transplant, BM from primary recipients were transplanted into secondary recipients. Surprisingly, secondary recipients of blood samples collected after 2 and 5 days of G-CSF treatment had equivalent levels of donor chimerism (37.2% ± 6.6% for 2 days G-CSF and 47.1% ± 7.8% for 5 days G-CSF, n = 8 per group). Therefore, although the absolute number of RU mobilized at day 2 of G-CSF was 28-fold lower than at day 5 of G-CSF administration, more primitive serially reconstituting HSC were mobilized at equivalent levels at days 2 and 5 of G-CSF treatment. This supports our hypothesis that most potent serially reconstituting HSC are mobilized as early as day 2 of G-CSF treatment consistent with the disappearance of osteoblasts1.To test the potential of HSC remaining in the BM, BM cells from G-CSF mobilized mice were transplanted in competition with BM cells from congenic mice in steady-state. Donor chimerism at 16 weeks post-transplant showed that competitive repopulation of BM cells was severely impaired at day 5 of G-CSF treatment with the number of RU per 200,000 BM cells decreasing from 4.1 ± 1.4 in steady-state and 5.2 ± 1.6 at day 2 of G-CSF treatment, to only 0.14 ± 0.05 at day 5 of G-CSF treatment. To test whether this 29-fold decrease in competitive repopulation was due to increased HSC proliferation, we measured BrdU incorporation for the last 2.5 days prior to BM harvest as well as cell cycle analysis with Ki67 and Hoechst33342. The proportion of quiescent Lin- Sca1+ Kit+ CD48- phenotypic HSC in G0 phase decreased from 62.8 ±4.0% in steady-state to 43.5±8.2% at day 2 of G-CSF, but surged back to 80.5±1.9% and 75.1±3.5% at days 3.5 and 5 of G-CSF treatment. The proportion of HSC in G1 and S/G2/M phases followed the opposite pattern, up at day 2, down at days 3.5 and 5. This was confirmed by BrdU incorporation for 2.5 days with the number of BrdU+ cells among Lin- Sca1+ KIT+ CD48- cells rising from 35.1±4.0% in steady-state, to 51.2±4.5% at day 2 of G-CSF and going down to 18.1±1.9% at day 3.5 and 23.3±5.5% at day 5 of G-CSF. Therefore, G-CSF recruits phenotypic HSC into cell cycle within the first 2 days of administration, but HSC return to quiescence despite continuing G-CSF. Therefore decreased repopulation potential at day5 of G-CSF is not due to increased cycling. Finally, we noted that the number of Lin-Sca1+KIT+CD48-CD150+ HSC and Lin-Sca1+KIT+CD48-CD150- multipotent progenitors were reduced 2.4- and 2.8-fold respectively (p<.05) in G-CSF-mobilized BM. In conclusion, administration of G-CSF rapidly disrupts HSC niches resulting in rapid mobilization of serially-reconstituting LT-CRC as early as day 2 of G-CSF administration. Secondly, the marked reduction of competitive reconstitution potential of mobilized BM was not due to increased HSC cycling but rather to decreased number of HSC remaining in mobilized BM. Disclosures:No relevant conflicts of interest to declare.

Hematopoietic stem cells from poor and good mobilizers are qualitatively equivalent

Marrow damage from chemo- and radiation therapies has been suggested to affect quality and quantity of hematopoietic stem cell (HSC) products. We tested the hypothesis that CD34+ cells (HSCs) from low mobilizers are qualitatively inferior to HSCs from high mobilizers. HSC quality was defined by proportion of primitive HSC subsets (CD34+CD38-, CD34+HLA-DR-, and CD34+ in G0 stage of cell cycle), the proportion of HSCs that express CXCR4 and CD26 homing proteins, and days to neutrophil and platelet (PLT) engraftments after transplant. HSC content and CD34 subsets analyses were performed using flow cytometry following the ISHAGE protocol. We evaluated the HSC quantity and quality of 139 autologous filgrastim-mobilized HSC products. Patients were categorized into low, moderate, and high mobilizers if their total HSC collection was less than 3 × 10(6), 3 × 10(6) or more and less than 5 × 10(6), and 5 × 10(6)/kg or more, respectively. The median number of primitive CD34 subsets increases with increasing HSC numbers and this association was significant (p = 0.001). However, when the ratios of the primitive CD34 subsets to total HSC counts were compared among the mobilization groups, the ratios were not significantly different. Coexpression of neither CD26 nor CXCR4 with CD34 antigen correlated with HSC mobilization. Evaluation of days to neutrophil engraftment among the mobilization groups did not show a significant difference (p = 0.1). However, days to PLT engraftment among the mobilization groups was significantly different (p = 0.05). The quality of HSCs from low mobilizers was comparable to HSCs from high mobilizers.

Cathepsin X is secreted by human osteoblasts, digests CXCL-12 and impairs adhesion of hematopoietic stem and progenitor cells to osteoblasts

Hematopoietic stem cells are retained within discrete bone marrow niches through the effects of cell adhesion molecules and chemokine gradients. However, a small proportion of hematopoietic stem cells can also be found trafficking in the peripheral blood. During induced stem cell mobilization a proteolytic microenvironment is generated, but whether proteases are also involved in physiological trafficking of hematopoietic stem cells is not known. In the present study we examined the expression, secretion and function of the cysteine protease cathepsin X by cells of the human bone marrow. Human osteoblasts, bone marrow stromal cells and hematopoietic stem and progenitor cells were analyzed for the secretion of cathepsin X by western blotting, active site labeling, immunofluorescence staining and activity assays. A possible involvement of cathepsin X in cell adhesion and CXCL-12-mediated cell migration was studied in functional assays. Matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) analysis revealed the digestion mechanism of CXCL-12 by cathepsin X. Osteoblasts and stromal cells secrete cathepsin X, whereas hematopoietic stem and progenitor cells do not. Using a cathepsin X-selective substrate, we detected the catalytic activity of cathepsin X in cell culture supernatants of osteoblasts. Activated cathepsin X is able to reduce cellular adhesive interactions between CD34(+) hematopoietic stem and progenitor cells and adherent osteoblasts. The chemokine CXCL-12, a highly potent chemoattractant for hematopoietic stem cells secreted by osteoblasts, is readily digested by cathepsin X. The exo-peptidase cathepsin X has been identified as a new member of the group of CXCL-12-degrading enzymes secreted by non-hematopoietic bone marrow cells. Functional data indicate that cathepsin X can influence hematopoietic stem and progenitor cell trafficking in the bone marrow.